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This thesis aimed at searching for new effective agents against Multidrug-Resistant Enterobacteriaceae. This is necessitated by the urgent need for new and innovative antibacterial agents addressing the critical priority pathogens prescribed by the World Health Organization (WHO). Among the available means for antibiotics discovery and development, nature has long remained a proven, innovative, and highly reliable gateway to successful antibacterial agents. Nevertheless, numerous challenges surrounding this valuable source of antibiotics among other drugs are limiting the complete realization of its potential. These include the availability of good quality data on the highly potential natural sources, limitations in methods to prepare and screen crude extracts, bottlenecks in reproducing biological potentials observed in natural sources, as well as hurdles in isolation, purification, and characterization of natural compounds with diverse structural complexities.
Through an extensive review of the literature, it was possible to prepare libraries of plant species and phytochemicals with reported high potentials against Escherichia coli and Klebsiella pneumnoniae. The libraries were profiled to highlight the existing patterns and relationships between the reported antibacterial activities and studied plants’ families and parts, the type of the extracting solvent, as well as phytochemicals’ classes, drug-likeness and selected parameters for enhanced accumulation within the Gram-negative bacteria. In addition, motivations, objectives, the role of traditional practices and other crucial experimental aspects in the screening of plant extracts for antibacterial activities were identified and discussed.
Based on the implemented strict inclusion criteria, the created libraries grant speedy access to well-evaluated plant species and phytochemicals with potential antibacterial activities. This way, further studies in yet unexplored directions can be pursued from the indicated or related species and compounds. Moreover, the availability of compound libraries focusing on related bacterial species serves a great role in the ongoing efforts to develop the rules of antibiotics penetrability and accumulation, particularly among Gram-negative bacteria. Here, in addition to hunting for potential scaffolds from such libraries, detailed evaluations of large pool compounds with related antibacterial potential can grant a better understanding of structural features crucial for their penetration and accumulation. Based on the scarcity of compounds with broad structural diversity and activity against Gram-negative bacteria, the creation and updating of such libraries remain a laborious but important undertaking.
A Pressurized Microwave Assisted Extraction (PMAE) method over a short duration and low-temperature conditions was developed and compared to the conventional cold maceration over a prolonged duration. This method aimed at addressing the key challenges associated with conventional extraction methods which require long extraction durations, and use more energy and solvents, in addition to larger quantities of plant materials. Furthermore, the method was intended to replace the common use of high temperatures in most of the current MAE applications. Interestingly, the yields of 16 of 18 plant samples under PMAE over 30 minutes were found to be within 91–139% of those obtained from the 24h extraction by maceration. Additionally, different levels of selectivity were observed upon an analytical comparison of the extracts obtained from the two methods. Although each method indicated selective extraction of higher quantities or additional types of certain phytochemicals, a slightly larger number of additional compounds were observed under maceration. The use of this method allows efficient extraction of a large number of samples while sparing heat-sensitive compounds and minimizing chances for cross-reactions between phytochemicals.
Moreover, findings from another investigation highlighted the low likelihood of reproducing antibacterial activities previously reported among various plant species, identified the key drivers of poor reproducibility, and proposed possible measures to mitigate the challenge. The majority of extracts showed no activities up to the highest tested concentration of 1024 µg/mL. In the case of identical plant species, some activities were observed only in 15% of the extracts, in which the Minimum Inhibitory Concentrations (MICs) were 4 – 16-fold higher than those in previous reports. Evaluation of related plant species indicated better outcomes, whereby about 18% of the extracts showed activities in a range of 128–512 μg/mL, some of the activities being superior to those previously reported in related species.
Furthermore, solubilizing plant crude extracts during the preparation of test solutions for Antibacterial Susceptibility Testing (AST) assays was outlined as a key challenge. In trying to address this challenge, some studies have used bacteria-toxic solvents or generally unacceptable concentrations of common solubilizing agents. Both approaches are liable to give false positive results. In line with this challenge, this study has underscored the suitability of acetone in the solubilization of crude plant extracts. Using acetone, better solubility profiles of crude plant extracts were observed compared to dimethyl sulfoxide (DMSO) at up to 10 %v/v. Based on lacking toxicity against many bacteria species at up to 25 %v/v, its use in the solubilization of poorly water-soluble extracts, particularly those from less polar solvents is advocated.
In a subsequent study, four galloylglucoses were isolated from the leaves of Paeonia officinalis L., whereby the isolation of three of them from this source was reported for the first time. The isolation and characterization of these compounds were driven by the crucial need to continually fill the pre-clinical antibiotics pipeline using all available means. Application of the bioautography-guided isolation and a matrix of extractive, chromatographic, spectroscopic, and spectrometric techniques enabled the isolation of the compounds at high purity levels and the ascertainment of their chemical structures.
Further, the compounds exhibited the Minimum Inhibitory Concentrations (MIC) in a range of 2–256 µg/mL against Multidrug-Resistant (MDR) strains of E. coli and K. pneumonia exhibiting diverse MDR phenotypes. In that, the antibacterial activities of three of the isolated compounds were reported for the first time. The observed in vitro activities of the compounds resonated with their in vivo potentials as determined using the Galleria mellonella larvae model. Additionally, the susceptibility of the MDR bacteria to the galloylglucoses was noted to vary depending on the nature of the resistance enzymes expressed by the MDR bacteria. In that, the bacteria expressing enzymes with higher content of aromatic amino acids and zero or positive net charges were generally more susceptible. Following these findings, a plausible hypothesis for the observed patterns was put forward.
The generally challenging pharmacokinetic properties of galloylglucoses limit their further development into therapeutic agents. However, the compounds can replace or reduce the use of antibiotics in livestock keeping as well as in the treatment of septic wounds and topical or oral cavity infections, among other potential uses.
Using nature-inspired approaches, a series of glucovanillin derivatives were prepared following feasible synthetic pathways which in most cases ensured good yields and high purity levels. Some of the prepared compounds showed MIC values in a range of 128 – 512 μg/mL against susceptible and MDR strains of Klebsiella pneumoniae, Methicillin-Resistant Staphylococcus aureus (MRSA) and Vancomycin-Resistant Enterococcus faecium (VRE). These findings emphasize the previously reported essence of small molecular size, the presence of protonatable amino groups and halogen atoms, as well as an amphiphilic character, as crucial features for potential antibacterial agents.
Due to the experienced limited success in the search for new antibacterial agents using purely synthetic means, pursuing semi-synthetic approaches as employed in this study are highly encouraged. This way, it is possible to explore broader chemical spaces around natural scaffolds while addressing their inherent limitations such as solubility, toxicity, and poor pharmacokinetic profiles.
The recent pandemic has reminded the public that basic research in virology is pivotal for human health. Understanding the mechanisms of successful viral replication and the role of host factors can help to combat viral infections and prevent future pandemics.
Our lab has published the first SARS-CoV-2 RNA-protein interaction atlas, laying the foundation to investigate the interplay between viral RNA and host RNA binding proteins (RBP). Based on this, my project created the largest collection of binding profiles of host and viral RBPs on SARS-CoV-2 RNA to date. This revealed the host protein SND1 as the first human RBP that specifically binds negative sense viral RNA at the 5´ end, a region associated with viral transcription initiation. The binding profile shares similarities with the viral RBP nsp9, which binds the 5´ ends of positive and negative sense SARS-CoV-2 RNA. Depletion of SND1 shows reduced levels of viral RNA revealing it as a proviral host factor. To decode the underlying molecular mechanism, I characterized the protein-protein interactions of SND1 in SARS-CoV-2 infected and uninfected cells. Infection remodels the protein interactors of SND1 from general RNA biology to membrane association and viral RNA synthesis. Upon infection, SND1 specifically interacts with nsp9, the RBP that shares the same binding region on the negative strand of SARS-CoV-2 RNA. Recent work demonstrates that nsp9 is NMPylated in vitro suggesting a functional role of nsp9 in priming of viral RNA synthesis. I was able to show that nsp9 is covalently linked to the 5´ ends of SARS-CoV-2 RNA during infection of human cells. Analysing the covalent bond of nsp9 with the viral RNA on nucleotide level shows close proximity to the initiation sites of viral RNA synthesis, suggesting that nsp9 acts as a protein-primer of SARS-CoV-2 RNA synthesis. SND1 modulates the distribution of nsp9 on the viral RNA, since depletion of SND1 results in imbalanced occupancy of nsp9 at the 5´ends of viral RNA.
This study is the first to provide evidence for the priming mechanism of SARS-CoV-2 in authentic viral replication and further reveals how this mechanism is modulated by the host RBP SND1. Detailed knowledge about priming of viral RNA synthesis can help to find targeted antivirals that could be used to fight coronaviral infections.
Untersucht wurde der Einfluss mehrerer Chemotherapeutika auf den Chemokinrezeptor CXCR4 in
Myelomzelllinien auf Ebene des Promotors, der mRNA und der Rezeptorverteilung, wobei drei
Substanzen (Etoposid, Bortezomib und Dexamethason) als potenzielle Suppressoren des Promotors ausgemacht werden konnten. Abhängig vom Myelom-Zelltyp und der Dosierung können so evtl.
Rückschlüsse auf die beobachtete Suppression von CXCR4 bei erkrankten Patienten mit hoher CXCR4-Aktivität (hier: Malignes Myelom) durch die begleitende Chemotherapie gezogen werden, welche eine Diagnostik und Therapie bei diesen Patienten erschwert.
Hintergrund: Hintergrund für diese Arbeit waren Beobachtungen in klinischen Fallstudien von Lapa et al. am Universitätsklinikum Würzburg, die sich auf CXCR4 bezogen, welches u.a. bei Patienten mit
Multiplem Myelom überexprimiert wird und dadurch bereits als Target für Diagnostik und Therapie in der Klinik Anwendung findet. Dabei konnte bei PET-CT Untersuchungen in der Nuklearmedizin beobachtet werden, dass es durch die begleitende Chemotherapie der Patienten zu einer Suppression des markierten CXCR4-Signals kam, so dass es nicht mehr zur Verlaufsbeobachtung und
vor allem nicht mehr zur Radiotherapie und Therapiekontrolle verwendet werden konnte.
Um den Einfluss und mögliche Interaktionen der Chemotherapeutika auf CXCR4 zu untersuchen, war es Ziel dieser Arbeit, ein vergleichbares Szenario in-vitro nachzustellen und Einflüsse messbar zu
machen, um so mögliche Ansätze und Verbesserungsvorschläge für die klinische Anwendung zu
liefern.
Methoden/Ergebnisse: Hierfür wurden im ersten Teil INA-6 (Myelomzellen) und Mesenchymale
Stammzellen (MSC) kultiviert, in Ko-Kultur gebracht und nach einer bestimmten Zeit wieder getrennt, um anschließend den gegenseitigen Einfluss in Bezug auf CXCR4 zu messen. Zudem wurde der Einfluss von Dexamethason untersucht. Es zeigte sich eine enge Bindung zwischen INA-6 und MSC
sowie eine hohe CXCR4-Aktivität bei INA-6, jedoch konnte keine Induktion der CXCR4-Aktivität in MSC durch INA-6-Kontakt oder Dexamethason quantifiziert werden. Die Immunzytologie erwies sich aufgrund einer schweren Anfärbbarkeit von CXCR4 – auch mit verschiedensten Antikörpern und sogar Liganden-gekoppeltem Farbstoff– als kaum auswertbar, wobei eine Darstellung von CXCR4
generell aber gelang.
Der CXCR4-Promotor wurde mittels Software genauer analysiert, wobei einige relevante Bindestellen, u.a. für Glukokortikoide und NFkB gefunden wurden. Die Herstellung eines CXCR4-
pGl4.14-Promotor-Konstrukts war erfolgreich, ebenso dessen Einschleusung in Myelomzellen. Auch gelang die Herstellung stabiler transfizierter INA-6, sodass mit diesen anschließend konstantere Ergebnisse erzielt werden konnten.
Im größten Teil der Arbeit wurden geeignete Chemotherapeutika-Konzentrationen ermittelt und in Viabilitäts- und Apoptose-Versuchen überprüft. Die Stimulationsversuche mit diesen zeigten variable
Effekte abhängig vom Zelltyp (INA-6, MM1S), jedoch konnten Bortezomib, Etoposid und
Dexamethason konzentrationsabhängig als starke Suppressoren der CXCR4-Aktivität ausgemacht
werden, was sich v.a. auf Ebene der Promotoraktivität – gemessen mittels Luciferase - zeigte. Interpretation: In-vitro konnten somit drei potenzielle Suppressoren der CXCR4-Aktivität ausgemacht
werden: Etoposid, Bortezomib und Dexamethason. Zumindest beim INA-6-Zelltyp fiel dieser Effekt deutlich aus, wobei in der Klinik der entsprechende Zelltyp sowie die Dosierung der Medikamente berücksichtigt werden müssen. Hinzu kommen weitere Einflussfaktoren des menschlichen Körpers,
die nicht berücksichtig werden konnten. Die genauen Mechanismen der Suppression könnten sich aus den Bindestellen des Promotors erklären, die von uns analysiert wurden, aber auf die in weiteren Arbeiten noch näher eingegangen werden muss.
Single-molecule dynamics at a bottleneck: a systematic study of the narrow escape problem in a disc
(2023)
Diffusion facilitates numerous reactions within the biological context of a cell. It is remarkable how the cost-efficient random process of Brownian motion promotes fast reactions. From the narrow escape theory, it is possible to determine the mean first passage time of such processes based on their reaction space and diffusion coefficient. The narrow escape theory of Brownian particles is characterized by a confining domain with reflective boundaries and a small reaction site. In this thesis, the mean first passage time was systematically tested in a disc as a function of the escape opening size in vitro and in silico. For the in vitro experiments, a model system of patterned supported-lipid bilayers (SLB) was established. Such a model is prepared by a combined colloid metalization approach, where a gold scaffold on glass facilitates assembly of SLB patches of distinct sizes through vesicle fusion. The model setup was evaluated and found to match all necessary requirements to test the nar- row escape problem in vitro. In particular, the reflectivity of the boundaries, the unhindered, free diffusion of the tracer lipids, and the distinct area were assessed. Observed results of the mean first passage time agreed with the theory of the narrow escape problem. There was excellent agreement in both absolute values and across a range of small escape opening sizes. Additionally, I developed a straightforward method, a correction factor, to calculate the mean first passage time from incomplete experimental traces. By re-scaling the mean first passage time to the fraction of particles that escaped, I was able to overcome the lifetime limitations of fluorescent probes. Previously inaccessible measurements of the mean first passage time relying on fluorescent probes will be made possible through this approach. The in vitro experiments were complemented with various in silico experiments. The latter were based on random walk simulations in discs, mimicking the in vitro situation with its uncertainties. The lifetime of single particles was either set sufficiently long to allow all particles to escape, or was adjusted to meet the lifetime limitations observed in the in vitro experiments. A comparison of the mean first passage time from lifetime-unlimited particles to the corrected, lifetime-limited particles did support the use of the correction factor. In agreement with the narrow escape theory, it was experimentally found that the mean first passage time is independent of the start point of the particle within the domain. This is when the particle adheres to a minimum distance to the escape site. In general, the presented random walk simulations do accurately represent the in vitro experiments in this study. The required hardware for the establishment of an astigmatism-based 3D system was installed in the existing microscope. The first attempts to analyze the obtained 3D imaging data gave insight into the potential of the method to investigate molecule dynamics in living trypanosome cells. The full functionality will be realized with the ongoing improvement of image analysis outside of this thesis.
SPRED 2 wirkt inhibitorisch auf den Ras/ERK-MAPK-Signalweg. Im Knockout Mausmodell
zeigen sich einige schwerwiegende phänotypische Eigenschaften, unter anderem zeigen sich
ein genereller Minderwuchs, veränderte hormonelle Regelkreise, neurologische Auffälligkeiten,
eine deutlich verringerte Lebenserwartung, sowie kardiale Veränderungen. Besonders
schwerwiegende SPRED 2 KO typische Ausprägungen im Herzen sind hierbei eine myokardiale
Fibrosierung, eine myokardiale Hypertrophie und Herzrhythmusstörungen.
In dieser Arbeit wurden insbesondere kardiale Veränderungen auf Zell- und Proteinebene
untersucht. Zur Proteinanalyse der Kardiomyozyten wurden Western Blots und eine Schnittbildgebung
angefertigt. Für eine funktionelle Untersuchung wurden isolierte vitale Kardiomyozyten
mittels Fluoreszenzfarbstoffen untersucht und unter elektrischer Stimulation beobachtet.
Desweiteren wurden isolierte Mitochondrien auf ihren Stoffwechsel und eventuelle
Defekte hin analysiert. Hierbei konnte gezeigt werden, dass junge SPRED2 KO Mäuse keine
wesentlichen hämodynamischen Einschränkungen aufweisen und eine gute Kompensationsfähigkeit
gegenüber einer Nachlaststeigerung aufweisen. Auch gezeigt werden konnte, dass
Veränderungen im Rahmen der Zellkontraktion beim Kalziumhaushalt und Membranpotential
existieren und im Zusammenhang mit einer verminderten Expression von SERCA und CaV1.2
stehen. Bei der Untersuchung von Mitochondrien konnten keine wesentlichen Defizite der
mitochondrialen Funktion der SPRED 2 KO Mäuse gefunden werden. In diesem Zusammenhang
ist die bekannte Störung der Autophagie am ehesten Ursache für eine gesteigerte Fibrosierung,
sowie der gesteigerten Apoptose der Kardiomyozyten. In Folge dessen könnten die
oben beschriebenen Veränderungen des Kalziumhaushaltes der Kardiomyozyten stehen und
letztendlich über maligne Herzrhythmusstörungen zum vorzeitigen Versterben führen.
Allogenic hematopoietic stem cell transplantation (allo-HCT) is a curative therapy for the treatment of malignant and non-malignant bone marrow diseases. The major complication of this treatment is a highly inflammatory reaction known as Graft-versus-Host Disease (GvHD). Cyclosporin A (CsA) and tacrolimus are used to treat GvHD which limits inflammation but also interferes with the anticipated Graft-versus-Leukemia (GvL) effect. These drugs repress conventional T cells (Tcon) along with regulatory T cells (Treg), which are important for both limiting GvHD and supporting GvL. Both of these drugs inhibit calcineurin (CN), which dephosphorylates and activates the nuclear factor of activated T-cells (NFAT) family of transcription factors. Here, we make use of our Cd4cre.Cas9+ mice and developed a highly efficient non-viral CRISPR/Cas9 gene editing method by gRNA-only nucleofection. Utilizing this technique, we demonstrated that unstimulated mouse T cells upon NFATc1 or NFATc2 ablation ameliorated GvHD in a major mismatch mouse model. However, in vitro pre-stimulated mouse T cells could not achieve long-term protection from GvHD upon NFAT single-deficiency. This highlights the necessity of gene editing and transferring unstimulated human T cells during allo-HCT. Indeed, we established a highly efficient ribonucleoprotein (RNP)-mediated CRISPR/Cas9 gene editing for NFATC1 and/or NFATC2 in pre-stimulated as well as unstimulated primary human T cells. In contrast to mouse T cells, not NFATC1 but NFATC2 deficiency in human T cells predominantly affected proinflammatory cytokine production. However, either NFAT single-knockout kept cytotoxicity of human CD3+ T cells untouched against tumor cells in vitro. Furthermore, mouse and human Treg were unaffected upon the loss of a single NFAT member. Lastly, NFATC1 or NFATC2-deficient anti-CD19 CAR T cells, generated with our non-viral ‘one-step nucleofection’ method validated our observations in mouse and human T cells. Proinflammatory cytokine production was majorly dependent on NFATC2 expression, whereas, in vitro cytotoxicity against CD19+ tumor cells was undisturbed in the absence of either of the NFAT members. Our findings emphasize that NFAT single-deficiency in donor T cells is superior to CN-inhibitors as therapy during allo-HCT to prevent GvHD while preserving GvL in patients.
In this work, dRNA-seq (differential RNA sequencing) and RNAtag-seq were applied to first define the global transcriptome architecture of C. difficile, followed by Hfq RIP-seq (RNA immunoprecipitation followed by RNA-seq) and RIL-seq (RNA interaction by ligation and sequencing) to characterize the Hfq-mediated sRNA interactome on a transcriptome-wide scale. These approaches resulted in the annotation of > 60 novel sRNAs. Notably, it not only revealed 50 Hfq-bound sRNAs, but also > 1000 mRNA-sRNA interactions, confirming Hfq as a global RNA matchmaker in C. difficile. Similar to its function in Gram-negative species, deletion of Hfq resulted in decreased sRNA half-lives, providing evidence that Hfq affects sRNA stability in C. difficile. Finally, several sRNAs and their function in various infection relevant conditions were characterized. The sRNA nc085 directly interacts with the two-component response regulator eutV, resulting in regulation of ethanolamine utilization, an abundant intestinal carbon and nitrogen source known to impact C. difficile pathogenicity. Meanwhile, SpoY and SpoX regulate translation of the master regulator of sporulation spo0A in vivo, thereby affecting sporulation initiation. Furthermore, SpoY and SpoX deletion significantly impacts C. difficile gut colonization and spore burden in a mouse model of C. difficile infection.
Forkhead box O transcription factors are a family of proteins involved in cellular processes downstream of the Insulin-PI3K-PKB pathway. In response to extra- or intracellular stresses, for example starvation or oxidative stress, FoxOs are required to direct cell cycle progression and apoptosis. In endothelial cells, they induce apoptosis, and their deregulation is linked to diseases involving the insulin pathway, such as diabetes. FoxOs also exhibit a complex role in tumour transformation: here their main function is to suppress tumorigenesis. In both physiological and cancer contexts, FoxO activation leads to the transcription of some general targets, such as p27kip1 or IGFBP1. The FoxOs can also induce tissue-specific genes, as ANGPT2 and BIM in the endothelium.
In endothelial cells, another pathway with a pivotal function is the MEK5/ERK5 MAPK signalling way. Its activation promotes cell survival and proliferation in stressful conditions, e.g., when blood vessels are exposed to the shear forces exerted by the blood stream. Furthermore, recent data described ERK5 as a kinase directing tumour resistance upon therapy-induced stress.
Comparing their reported roles in various tumours and in the endothelium, FoxO proteins and the MEK5/ERK5 MAPK cascade appear to exert opposite functions. First non-published data confirmed the hypothesis that FoxO factors are subject to a negative modulation by the MEK5/ERK5 pathway. Hence, one goal of this PhD project was to further characterise this crosstalk at molecular level. The major mechanism of FoxO regulation is the balance among several post translational modifications, such as phosphorylation, acetylation, and ubiquitination. Most importantly, the PKB dependent phosphorylation of FoxOs negatively controls their activity, and it is critical for their subcellular localization. Therefore, the regulation of FoxO localization as mechanism of ERK5 dependent suppression was studied, but the results presented in this thesis argue against this hypothesis. However, additional experiments are required to explore the impact of ERK5 activity on FoxO post-translational modifications.
FoxO activity can also be modulated by the interaction with other proteins, which in turn could explain general- and tissue-specific gene expression. Thus, another objective of this work was to investigate FoxO3-interactome in endothelial cells and the impact of MEK5/ERK5 activation on it. As published in (Fusi et al. 2022) and presented here, this analysis unveiled TRRAP as new FoxO bound protein in several cell types. Moreover, the interaction did not rely on the capacity of the FoxOs to bind their consensus DNA sequences at the promoter of target genes. Functional data demonstrated that TRRAP is required for FoxO-dependent gene transcription in endothelial and osteosarcoma cells. In addition, TRRAP expression in the endothelium is important for FoxO induced apoptosis. In summary, the interaction between FoxO factors and TRRAP revealed a new regulatory mechanism of FoxO-dependent gene transcription. It remains to be analysed whether the MEK5/ERK5 cascade may exert its suppressive effect on FoxO activity by interfering with their binding to TRRAP and whether such a mechanism may be relevant for tumorigenesis.
Regulatory T cells (Tregs) are the masters of immune regulation controlling inflammation and tolerance, tissue repair and homeostasis. Multiple immunological diseases result from altered Treg frequencies and Treg dysfunction. We hypothesized that augmenting Treg function and numbers would prevent inflammatory disease whereas inhibiting or depleting Tregs would improve cancer immunotherapy.
In the first part of this thesis, we explored whether in vivo activation and expansion of Tregs would impair acute graft-versus-host disease (aGvHD). In this inflammatory disease, Tregs are highly pathophysiological relevant and their adoptive transfer proved beneficial on disease outcome in preclinical models and clinical studies. IL-2 has been recognized as a key cytokine for Treg function. Yet, attempts in translating Treg expansion via IL-2 have remained challenging, due to IL-2s extremely broad action on other cell types including effector T cells, NK cells, eosinophils and vascular leakage syndrome, and importantly, due to poor pharmacokinetics in vivo. We addressed the latter issue using an IL-2-IgG-fusion protein (irrIgG-IL-2) with improved serum retention and demonstrated profound Treg expansion in vivo in FoxP3-luciferase reporter mice. Further, we augmented Treg numbers and function via the selective-TNF based agonists of TNFR2 (STAR2). Subsequently, we tested a next-generation TNFR2 agonist, termed NewSTAR, which proved even more effective. TNFR2 stimulation augmented Treg numbers and function and was as good as or even superior to the IL-2 strategy. Finally, in a mouse model of aGvHD we proved the clinical relevance of Treg expansion and activation with irrIgG-IL-2, STAR2 and NewSTAR. Notably, the TNFR2 stimulating constructs were outstanding as we observed not the IL-2 prototypic effects on other cell populations and no severe side effects.
In the second part of this thesis, we explored Tregs in pancreatic ductal adenocarcinoma (PDAC) and developed targeting strategies. Among several tumor entities in which Tregs impact survival, preclinical and clinical data demonstrated their negative role on PDAC. In our studies we employed the orthotopic syngeneic Panc02 model in immunocompetent mice. Based on flow cytometric analysis of the tumor microenvironment we propose TIGIT and TNFRSF members as novel therapeutic targets. Surprisingly, we found that blocking TNFR2 did not interfere with intratumoral Treg accumulation. However, we decreased the highly abundant intratumoral Tregs when we disrupted the tumor extracellular matrix. In PDAC, Treg manipulation alone did not lead to tumor regression and we propose that an additional immune boost may be necessary for efficient tumor immune surveillance and cancer clearance. This contrasts with aGvHD, in which Treg manipulation alone was sufficient to improve disease outcome.
Conclusively, we demonstrated the enormous medical benefit of Treg manipulation. Our promising data obtained with our newly developed powerful tools highlight the potential to translate our findings into clinical practice to therapeutically target human Tregs in patients. With novel TNFR2 agonists (STAR2, NewSTAR) we augmented Treg numbers and function as (or even more) effectively than with IL-2, without causing adverse side effects. Importantly, exogenous in vivo Treg expansion protected mice from aGvHD. For the therapy of PDAC, we identified novel targets on Tregs, notably TIGIT and members of the TNFRSF. We demonstrated that altering the extracellular tumor matrix can efficiently disrupt the Treg abundance in tumors. These novel targeting strategies appear as attractive new treatment options and they may benefit patients suffering from inflammatory disease and cancer in the future.
Many arthropods such as mosquitoes, ticks, bugs, and flies are vectors for the transmission of pathogenic parasites, bacteria, and viruses. Among these, the unicellular parasite Trypanosoma brucei (T. brucei) causes human and animal African trypanosomiases and is transmitted to the vertebrate host by the tsetse fly. In the fly, the parasite goes through a complex developmental cycle in the alimentary tract and salivary glands ending with the cellular differentiation into the metacyclic life cycle stage. An infection in the mammalian host begins when the fly takes a bloodmeal, thereby depositing the metacyclic form into the dermal skin layer. Within the dermis, the cell cycle-arrested metacyclic forms are activated, re-enter the cell cycle, and differentiate into proliferative trypanosomes, prior to dissemination throughout the host.
Although T. brucei has been studied for decades, very little is known about the early events in the skin prior to systemic dissemination. The precise timing and the mechanisms controlling differentiation of the parasite in the skin continue to be elusive, as does the characterization of the proliferative skin-residing trypanosomes. Understanding the first steps of an infection is crucial for developing novel strategies to prevent disease establishment and its progression.
A major shortcoming in the study of human African trypanosomiasis is the lack of suitable infection models that authentically mimic disease progression. In addition, the production of infectious metacyclic parasites requires tsetse flies, which are challenging to keep. Thus, although animal models - typically murine - have produced many insights into the pathogenicity of trypanosomes in the mammalian host, they were usually infected by needle injection into the peritoneal cavity or tail vein, bypassing the skin as the first entry point. Furthermore, animal models are not always predictive for the infection outcome in human patients. In addition, the relatively small number of metacyclic parasites deposited by the tsetse flies makes them difficult to trace, isolate, and study in animal hosts.
The focus of this thesis was to develop and validate a reconstructed human skin equivalent as an infection model to study the development of naturally-transmitted metacyclic parasites of T. brucei in mammalian skin. The first part of this work describes the development and characterization of a primary human skin equivalent with improved mechanical properties. To achieve this, a computer-assisted compression system was designed and established. This system allowed the improvement of the mechanical stability of twelve collagen-based dermal equivalents in parallel through plastic compression, as evaluated by rheology. The improved dermal equivalents provided the basis for the generation of the skin equivalents and reduced their contraction and weight loss during tissue formation, achieving a high degree of standardization and reproducibility. The skin equivalents were characterized using immunohistochemical and histological techniques and recapitulated key anatomical, cellular, and functional aspects of native human skin. Furthermore, their cellular heterogeneity was examined using single-cell RNA sequencing - an approach which led to the identification of a remarkable repertoire of extracellular matrix-associated genes expressed by different cell subpopulations in the artificial skin. In addition, experimental conditions were established to allow tsetse flies to naturally infect the skin equivalents with trypanosomes.
In the second part of the project, the development of the trypanosomes in the artificial skin was investigated in detail. This included the establishment of methods to successfully isolate skin-dwelling trypanosomes to determine their protein synthesis rate, cell cycle and metabolic status, morphology, and transcriptome. Microscopy techniques to study trypanosome motility and migration in the skin were also optimized. Upon deposition in the artificial skin by feeding tsetse, the metacyclic parasites were rapidly activated and established a proliferative population within one day. This process was accompanied by: (I) reactivation of protein synthesis; (II) re-entry into the cell cycle; (III) change in morphology; (IV) increased motility. Furthermore, these observations were linked to potentially underlying developmental mechanisms by applying single-cell parasite RNA sequencing at five different timepoints post-infection.
After the initial proliferative phase, the tsetse-transmitted trypanosomes appeared to enter a reversible quiescence program in the skin. These quiescent skin-residing trypanosomes were characterized by very slow replication, a strongly reduced metabolism, and a transcriptome markedly different from that of the deposited metacyclic forms and the early proliferative trypanosomes. By mimicking the migration from the skin to the bloodstream, the quiescent phenotype could be reversed and the parasites returned to an active proliferating state. Given that previous work has identified the skin as an anatomical reservoir for T. brucei during disease, it is reasonable to assume that the quiescence program is an authentic facet of the parasite's behavior in an infected host.
In summary, this work demonstrates that primary human skin equivalents offer a new and promising way to study vector-borne parasites under close-to-natural conditions as an alternative to animal experimentation. By choosing the natural transmission route - the bite of an infected tsetse fly - the early events of trypanosome infection have been detailed with unprecedented resolution. In addition, the evidence here for a quiescent, skin-residing trypanosome population may explain the persistence of T. brucei in the skin of aparasitemic and asymptomatic individuals. This could play an important role in maintaining an infection over long time periods.
Adrenocortical carcinoma (ACC) is a rare, but highly aggressive endocrine malignancy. Tumor-related hypercortisolism is present in 60 % of patients and associated with worse outcome. While cancer immunotherapies have revolutionized the treatment of many cancer entities, the results of initial studies of different immune checkpoint inhibitors in ACC were heterogeneous. Up to now, five small clinical trials with a total of 121 patients have been published and demonstrated an objective response in only 17 patients. However, one of the studies, by Raj et al., reported a clinically meaningful disease control rate of 52 % and a median overall survival of almost 25 months suggesting that a subgroup of ACC patients may benefit from immunotherapeutic approaches. Following the hypothesis that some ACCs are characterized by a glucocorticoid-induced T lymphocytes depletion, several studies were performed as part of the presented thesis. First, the immune cell infiltration in a large cohort of 146 ACC specimens was investigated. It was demonstrated for the first time, and against the common assumption, that ACCs were infiltrated not only by FoxP3+ regulatory T cells (49.3 %), but also that a vast majority of tumor samples was infiltrated by CD4+ TH cells (74 %) and CD8+ cytotoxic T cells (84.3 %), albeit the immune cell number varied heterogeneously and was rather low (median: 7.7 CD3+ T cells / high power field, range: 0.1-376). Moreover, the presence of CD3+-, CD4+- and CD8+ ACC-infiltrating lymphocytes was associated with an improved recurrence-free (HR: 0.31 95 % CI 0.11-0.82) and overall survival (HR: 0.47 96 % CI 0.25-0.87). Particularly, patients with tumor-infiltrating CD4+ TH cells without glucocorticoid excess had a significantly longer overall survival compared to patients with T cell-depleted ACC and hypercortisolism (121 vs. 27 months, p = 0.004). Hence, the impact of glucocorticoids might to some extent be responsible for the modest immunogenicity in ACC as hypercortisolism was reversely correlated with the number of CD4+ TH cells. Accordingly, CD3+ T cells co-cultured with steroidogenic NCI-H295R ACC cells demonstrated in vitro an enhanced anti-tumoral cytotoxicity by secreting 747.96 ±225.53 pg/ml IFN-γ in a therapeutically hormone-depleted microenvironment (by incubation with metyrapone), versus only 276.02 ±117.46 pg/ml IFN-γ in a standard environment with glucocorticoid excess.
Other potential biomarkers to predict response to immunotherapies are the immunomodulatory checkpoint molecules, programmed cell death 1 (PD-1) and its ligand PD-L1, since both are targets of antibodies used therapeutically in different cancer entities. In a subcohort of 129 ACCs, expressions of both molecules were heterogeneous (PD-1 17.4 %, range 1-15; PD-L1 24.4 %, range 1 - 90) and rather low. Interestingly, PD-1 expression significantly influenced ACC patients´ overall (HR: 0.21 95 % CI 0.53-0.84) and progression- free survival (HR: 0.30 95 % CI 0.13-0.72) independently of established factors, like ENSAT tumor stage, resection status, Ki67 proliferation index and glucocorticoid excess, while PD-L1 had no impact.
In conclusion, this study provides several potential explanations for the heterogeneous results of the immune checkpoint therapy in advanced ACC. In addition, the establishment of PD-1 as prognostic marker can be easily applied in routine clinical care, because it is nowadays anyway part of a detailed histo-pathological work-up. Furthermore, these results provide the rationale and will pave the way towards a combination therapy using immune checkpoint inhibitors as well as glucocorticoid blockers. This will increase the likelihood of re-activating the immunological anti-tumor potential in ACC. However, this will have to be demonstrated by additional preclinical in vivo experiments and finally in clinical trials with patients.
Infectious diseases caused by pathogenic microorganisms are one of the largest socioeconomic burdens today. Although infectious diseases have been studied for decades, in numerous cases, the precise mechanisms involved in the multifaceted interaction between pathogen and host continue to be elusive. Thus, it still remains a challenge for researchers worldwide to develop novel strategies to investigate the molecular context of infectious diseases in order to devise preventive or at least anti-infective measures. One of the major drawbacks in trying to obtain in-depth knowledge of how bacterial pathogens elicit disease is the lack of suitable infection models to authentically mimic the disease progression in humans. Numerous studies rely on animal models to emulate the complex temporal interactions between host and pathogen occurring in humans. While they have greatly contributed to shed light on these interactions, they require high maintenance costs, are afflicted with ethical drawbacks, and are not always predictive for the infection outcome in human patients. Alternatively, in-vitro two-dimensional (2D) cell culture systems have served for decades as representatives of human host environments to study infectious diseases. These cell line-based models have been essential in uncovering virulence-determining factors of diverse pathogens as well as host defense mechanisms upon infection. However, they lack the morphological and cellular complexity of intact human tissues, limiting the insights than can be gained from studying host-pathogen interactions in these systems.
The focus of this thesis was to establish and innovate intestinal human cell culture models to obtain in-vitro reconstructed three-dimensional (3D) tissue that can faithfully mimic pathogenesis-determining processes of the zoonotic bacterium Campylobacter jejuni (C. jejuni). Generally employed for reconstructive medicine, the field of tissue engineering provides excellent tools to generate organ-specific cell culture models in vitro, realistically recapitulating the distinctive architecture of human tissues. The models employed in this thesis are based on decellularized extracellular matrix (ECM) scaffolds of porcine intestinal origin. Reseeded with intestinal human cells, application of dynamic culture conditions promoted the formation of a highly polarized mucosal epithelium maintained by functional tight and adherens junctions. While most other in-vitro infection systems are limited to a flat monolayer, the tissue models developed in this thesis can display the characteristic 3D villi and crypt structure of human small intestine.
First, experimental conditions were established for infection of a previously developed, statically cultivated intestinal tissue model with C. jejuni. This included successful isolation of bacterial colony forming units (CFUs), measurement of epithelial barrier function, as well as immunohistochemical and histological staining techniques. In this way, it became possible to follow the number of viable bacteria during the infection process as well as their translocation over the polarized epithelium of the tissue model. Upon infection with C. jejuni, disruption of tight and adherens junctions could be observed via confocal microscopy and permeability measurements of the epithelial barrier. Moreover, C. jejuni wildtype-specific colonization and barrier disruption became apparent in addition to niche-dependent bacterial localization within the 3D microarchitecture of the tissue model. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D host environment deviated from those obtained with conventional in-vitro 2D monolayers but mimicked observations made in vivo. Furthermore, a genome-wide screen of a C. jejuni mutant library revealed significant differences for bacterial factors required or dispensable for interactions with unpolarized host cells or the highly prismatic epithelium provided by the intestinal tissue model. Elucidating the role of several previously uncharacterized factors specifically important for efficient colonization of a 3D human environment, promises to be an intriguing task for future research.
At the frontline of the defense against invading pathogens is the protective, viscoelastic mucus layer overlying mucosal surfaces along the human gastrointestinal tract (GIT). The development of a mucus-producing 3D tissue model in this thesis was a vital step towards gaining a deeper understanding of the interdependency between bacterial pathogens and host-site specific mucins. The presence of a mucus layer conferred C. jejuni wildtype-specific protection against epithelial barrier disruption by the pathogen and prevented a high bacterial burden during the course of infection. Moreover, results obtained in this thesis provide evidence in vitro that the characteristic corkscrew morphology of C. jejuni indeed grants a distinct advantage in colonizing mucous surfaces.
Overall, the results obtained within this thesis highlight the strength of the tissue models to combine crucial features of native human intestine into accessible in-vitro infection models. Translation of these systems into infection research demonstrated their ability to expose in-vivo like infection outcomes. While displaying complex organotypic architecture and highly prismatic cellular morphology, these tissue models still represent an imperfect reflection of human tissue. Future advancements towards inclusion of human primary and immune cells will strive for even more comprehensive model systems exhibiting intricate multicellular networks of in-vivo tissue. Nevertheless, the work presented in this thesis emphasizes the necessity to investigate host-pathogen interactions in infection models authentically mimicking the natural host environment, as they remain among the most vital parts in understanding and counteracting infectious diseases.
Fabry disease (FD), an X-linked lysosomal storage disorder, is caused by variants in the gene α-galactosidase A (GLA). As a consequence, the encoded homonymous enzyme GLA is not produced in sufficient amount or does not function properly. Subsequently, globotriaosylceradmide (Gb3), the target substrate of GLA, starts accumulating in several cell types, especially neurons and endothelial cells. FD patients suffer from multiorgan symptoms including cardiomyopathy, nephropathy, stroke, and acral burning pain. It is suggested that the impact of pathological Gb3 accumulation, inflammatory and hypoxic processes, and vasculopathy are contributing to the specific FD pain phenotype. Thus, we investigated the role of inflammation, hypoxia, and vasculopathy on molecular level in dorsal root ganglia (DRG) of the GLA knockout (KO) mouse model. Further, we investigated pain-like characteristics of GLA KO mice at baseline (BS), after capsaicin administration, and after repeated enzyme replacement therapy (ERT) administration for a period of 1.5 years. Acquired data showed disturbances in immune response markers represented by downregulated inflammation-associated genes and lower numbers of CD206+ macrophages in DRG of GLA KO mice. Hypoxic mechanisms were active in DRG of GLA KO mice reflected by increased gene expression of hypoxia- and DNA damage-associated targets, higher numbers of hypoxia-inducible factor 1α-positive (HIF1α+) and carbonic anhydrase 9-positive (CA9+) neurons in DRG of GLA KO mice, and DRG neuronal HIF1α cytosolic-nuclear translocation in GLA KO mice. Vascularization in DRG of GLA KO mice was reduced including lower numbers of blood vessel branches and reduced total blood vessel length. Pain-like behavior of the GLA KO mouse model revealed no mechanical hypersensitivity at BS but age-dependent heat hyposensitivity, which developed also age-matched wild type (WT) mice. Capsaicin administration under isoflurane anesthesia did not elicit the development of nocifensive behavior in GLA KO mice after mechanical or heat stimulation. Repeated ERT administration did not show a clear effect in GLA KO mice in terms of restored heat hyposensitivity to BS paw withdrawal latencies. In summary, we demonstrated the impact of disturbed immune response markers, active hypoxic mechanisms, and reduced vascularization on molecular FD pathophysiology.
Für die Verwendung von zellbasierten Therapeutika ist vor allem die korrekt Identifikation
sowohl vom Ausgangsmaterial wie auch dem produziertem Material von
zentraler Wichtigkeit. In dieser Arbeit wurde eine Methodik entwickelt, welche eine
nicht-invasive Klassifizierung von Zellen und zellulärer Entwicklung aufgrund ihrer
zweidimensionalen Magnetresonanz-Korrelationsspektren ermöglichte.
Hierzu wurde ein mobiler MR-Scanner mit einer Feldstärke von 0.5T und einem Isozentrum
von 1 cm3 verwendet. Aufgrund der kompakten und leichten Bauweise war
es möglich, das System in normalen Zellkulturlaboren zu verwenden. Von den Proben
wurde ein zweidimensionales T1/T2 -Korrelationsspektrum aufgenommen, anhand
dessen die Zellen klassifiziert werden sollten. Mithilfe von Agarose-Dotagraf® -Zell-
Phantomen konnte die Stabilität und Reproduzierbarkeit des Messsystems und der
verwendeten Sequenz validiert werden.
Aufgrund der unter Umständen recht langen Messzeiten der MR-Technologie war
auch die Handhabung und Kultur der Zellproben während des Messprozesses von
großer Bedeutung. Um hierfür den Durchsatz an Proben zu erhöhen, wurde eine kostengünstige
und ebenfalls mobile Robotikanlage entwickelt. Diese basierte auf dem
kommerziell erhältlichen Roboterarm Braccio, welcher durch einen Arduino Mega
Mikrocontroller gesteuert wurde. Mit bis zu 24 Proben pro Tag konnte durch die
Automatisierung der Durchsatz an Proben um den Faktor 3 – 4 gesteigert werden.
Durch den entwickelten Prozess war es möglich, eine umfangreiche Datenbank –
bestehend aus 362 unabhängigen Messungen (biologische Replikate) – aufzubauen.
Die Datenbank enthielt Messungen von zehn unterschiedlichen Zelllinien. Zusätzlich
wurden T1/T2 -Korrelationsspektren von mesenchymalen Stromazellen (MSCs)
vor und nach deren Differenzierung zu Adipocyten aufgenommen, um ihre zelluläre
Entwicklung nicht-invasiv charakterisieren zu können.
Die aufgenommenen Daten wurden mithilfe einer geeigneten Support Vector Machine
wie auch angepassten künstlichen neuronalen Netzwerken klassifiziert. Mithilfe
dieser Methoden konnten die Zelllinien und MSCs anhand ihrer aufgenommenen
Korrelationsspektren mit einer Genauigkeit von bis zu 98% klassifiziert werden.
Diese hohe Treffsicherheit legte den Schluss nahe, dass die Kombination aus nichtinvasiver,
zweidimensionaler T1/T2 -MR-Relaxometrie und der Verwendung von geeigneten
Methoden des machine learning und der künstlichen Intelligenz eine effiziente
Methodik für die nicht-invasive Klassifizierung von Zellen sowie zellulärer
Entwicklung darstellt.
Regulatory T cells (Treg) are critical immune cells to ensure immune homeostasis. Treg do so by establishing tolerance to self-antigens as well as food-derived antigens. Additionally, they fine-tune immune responses to limit the damage caused by inevitable inflammation during the resolution of an ongoing infection or anti-tumor response. Despite countless efforts to gain a detailed understanding of the mechanisms Treg utilize to regulate adaptive immune responses, in vivo evidence is rather limited. We were interested in the cell-cell interactions of Treg and their spatio-temporal dynamics during a viral infection. We sought to address Interleukin-2 (IL-2) competition as a viable mechanism to control anti-viral CD8 T cell responses. We used intra-vital 2-photon imaging to analyze the interactions between Treg and activated T cells during viral infection. Additionally, we performed multiple loss- and gain-of-function experiments, addressing the IL-2 active signaling of CD8, CD4, and regulatory T cells to understand the competitive sensing of IL-2. Finally, we performed single-cell RNA sequencing to understand the cell-intrinsic differences in Treg caused by infection. We found that IL-2 competition by Treg limits the CD8 T cell response and can alter the differentiation of CD8 T cells. Furthermore, we show that Treg do not arrest in proximity to CD8 T cells for prolonged periods and therefore are unlikely to regulate CD8 T cells via contact-dependent mechanisms previously proposed. Our data support an area control model in which Treg scavenge IL-2 while actively migrating through the LN, constantly limiting access to IL-2. Establishing CD4 T cells as the major source of IL-2 during the later phases of infection, we provide direct evidence that Treg compete with CD8 T cells for CD4-derived IL-2. Finally, we show that IL-2 limitation is in correlation with CD25 expression levels and has an impact on the differentiation of CD8 T cells. Altering the differentiation of CD8 T cells to increase effector or memory functions has huge implications in clinical treatments, e.g ’checkpoint immunotherapy’. Especially in scenarios like checkpoint immunotherapy, where an efficient expansion of CD8 T cells is vital to the success of the treatment, it is invaluable to understand the spatio-temporal dynamics of Treg. Not only can the expansion phase be optimized, but also side effects can be better controlled by ensuring the adequate timing of treatments and boosting the anti-inflammatory response after the initial establishment of CD8 T cells. On top of this, the gained understanding of the regulatory mechanism of Treg can help to enhance the efficacy of autoimmune disorder treatments. Overall, this study addressed highly relevant questions in the Treg field and answered aspects of Treg regulation, refining their mode of action and the spatio-temporal dynamics during viral infection, providing evidence for IL-2 competition as a major regulatory mechanism controlling antiviral CD8 T cell responses.
Platelets have a key physiological role in haemostasis however, inappropriate thrombus formation can lead to cardiovascular diseases such as myocardial infarction or stroke. Although, such diseases are common worldwide there are comparatively few anti-platelet drugs, and these are associated with an increased risk of bleeding. Platelets also have roles in thrombo-inflammation, immuno-thrombosis and cancer, in part via C-type lectin-like receptor 2 (CLEC-2) and its ligand podoplanin. Although CLEC-2 contributes to these diseases in mice, as well as to thrombus stability, it is unclear whether CLEC-2 has similar roles in humans, particularly as human CLEC-2 (hCLEC-2) cannot be investigated experimentally in vivo.
To investigate hCLEC-2 in vivo, we generated a humanised CLEC-2 mouse (hCLEC-2KI) model, as well as a novel monoclonal antibody, HEL1, that binds to a different site than an existing antibody, AYP1. Using these antibodies, we have provided proof of principle for the use of hCLEC-2KI mice to test potential therapeutics targeting hCLEC-2, and shown for the first time that hCLEC-2 can be immunodepleted, with little effect on haemostasis. However, our results have also suggested that there are species differences in the role of CLEC-2 in arterial thrombosis. We further confirmed this using human blood where blocking CLEC-2 ligand binding had no effect on thrombosis, whereas we confirmed a minor role for mouse CLEC-2 in thrombus stability. We also investigated the effect of blocking CLEC-2 signalling using the Bruton’s tyrosine kinase inhibitor PRN473 on CLEC-2 mediated immuno-thrombosis in a Salmonella typhimurium infection model. However, no effect on thrombosis was observed suggesting that CLEC-2 signalling is not involved.
Overall, our results suggest that there may be differences in the role of human and mouse CLEC-2, at least in arterial thrombosis, which could limit the potential of CLEC-2 as an anti-thrombotic target. However, it appears that the interaction between CLEC-2 and podoplanin is conserved and therefore CLEC-2 could still be a therapeutic target in immuno-thrombosis, thrombo-inflammation and cancer. Furthermore, any potential human specific therapeutics could be investigated in vivo using hCLEC-2KI mice.
Ein Schlüsselereignis, welches dem prognosebestimmenden Organversagen bei systemi-schen Entzündungsprozessen und Sepsis vorangeht, ist die Entwicklung einer mikrovas-kulären endothelialen Schrankenstörung. Das vaskuläre endotheliale (VE-) Cadherin als mechanischer Stabilisator der Endothelbarriere spielt dabei eine wichtige Rolle. In der Inflammation werden Spaltprodukte von VE-Cadherin (sVE-Cadherin) gebildet. Ge-genstand der vorliegenden Arbeit war die Untersuchung der Hypothese ob diese Spalt-produkte selbst an der Störung der endothelialen Barrierefunktion beteiligt sind.
Es wurde hierfür humanes sVE-Cadherin bestehend aus den extrazellulären Domänen EC1-5 (sVE-CadherinEC1-5) generiert. In Messungen des transendothelialen elektrischen Widerstands (TER), mit Immunfluoreszenzfärbungen und Western Blot Analysen wird gezeigt, dass sVE-Cadherin dosisabhängig die Barriere Integrität in primären humanen dermalen Endothelzellen stört. Dies führt zu einer Reduktion von VE-Cadherin und den assoziierten Proteinen α-, γ- und δ-Catenin und ZO-1, die nach der Applikation von sVE-Cadherin an den Zellgrenzen reduziert sind. Die Interaktion zwischen VE-PTP und VE-Cadherin wird durch sVE-CadherinEC1-5 reduziert. Durch pharmakologische Hem-mung der Phosphataseaktivität von VE-PTP mittels AKB9778 wird der durch sVE-CadherinEC1-5-induzierte Verlust der Endothelbarriere aufgehoben. Dagegen zeigt die direkte Aktivierung von Tie-2 mittels Angiopoetin-1 keinen protektiven Effekt auf die durch sVE-CadherinEC1-5 gestörte Endothelbarriere. Weitere Analysen zeigen eine erhöh-te Expression von GEF-H1 durch sVE-CadherinEC1-5. Diese ist ebenfalls durch AKB9778 hemmbar.
Zusätzlich zu diesen Untersuchungen wurden die Konstrukte EC1-4 und EC3-5 in ver-schiedene Vektoren kloniert, um zu bestimmen, ob die extrazelluläre Domäne 5 von VE-Cadherin die dominante Rolle bei den sVE-Cadherin-vermittelten Effekten spielt.
Zusammenfassend zeigen diese Untersuchungen zum ersten Mal, dass sVE-CadherinEC1-5 unabhängig von proinflammatorischen Auslösern über die Aktivierung des VE-PTP/RhoA-Signalweges den Zusammenbruch der Endothelbarriere mitversursacht. Dies stellt einen neuen pathophysiologischer Mechanismus dar, der zum Gesamtverständnis der entzündungsinduzierten Barriereveränderungen des Endothels beiträgt.
RNA is one of the most abundant macromolecules and plays essential roles in numerous biological processes. This doctoral thesis consists of two projects focusing on RNA structure and RNA-RNA interactions in viral genome packaging. In the first project I developed a method called Functional Analysis of RNA Structure (FARS-seq) to investigate structural features regulating genome dimerization within the HIV-1 5’UTR. Genome dimerization is a conserved feature of retroviral replication and is thought to be a prerequisite for binding to the viral structural protein Pr55Gag during genome packaging. It also plays a role in genome integrity and evolution through recombination, and is linked to a structural switch that may regulate genome packaging and translation within cells. Despite its importance for HIV-1 replication, the RNA signals regulating genome dimerization, and the molecular mechanism leading to the selection of the genome dimer over the monomer for packaging are incompletely understood. The FARS-seq method combines RNA structural information obtained by chemical probing with single nucleotide resolution profiles of RNA function obtained by mutational interference. In this way, we found nucleotides that were critical for dimerization, especially within the well-characterized dimerization motif within stem-loop 1 (SL1). We also found stretches of nucleotides that enhanced genome dimerization upon mutation, suggesting their role in negatively regulating dimerization. A structural analysis identified distinct structural signatures within monomeric and dimeric RNA. The dimeric conformation displayed the canonical transactivation response (TAR), PolyA, primer binding site (PBS), and SL1-SL3 stem-loops, and contained a long range U5-AUG interaction. Unexpectedly, in monomeric RNA, SL1 was reconfigured into long- and short-range base-pairings with PolyA and PBS, respectively. Intriguingly, these base pairings concealed the palindromic sequence needed for dimerization and disrupted the internal loop in SL1 previously shown to contain the major packaging motif for Pr55Gag. We therefore rationally introduced mutations into PolyA and PBS, and showed how these regions regulate genome dimerization, and the binding of Pr55Gag in vitro, as well as genome packaging into virions. These findings give insights into late stages of the HIV-1 life cycle and a mechanistic explanation for the link between RNA dimerization and packaging.
In the second project, I developed a proximity ligation and high-throughput sequencing-based method, RNA-RNA seq, which can measure direct (RNA-RNA) and indirect (protein-mediated) interactions. In contrast to existing methods, RNA-RNA seq is not limited by specific protein or RNA baits, nor to a particular crosslinking reagent. The genome of influenza A virus contains eight segments, which assemble into a “7+1” supramolecular complex. However, the molecular details of genome assembly are poorly understood. Our goal is to use RNA-RNA seq to identify the sites of interaction between the eight genomic RNAs of influenza, and to use this information to define the quaternary RNA architecture of the genome. We showed that RNA-RNA seq worked on model substrates, like the HIV-1 Dimerization Initiation Site (DIS) RNA and purified ribosome, as well as influenza A virus infected cells.
Die akute Graft-versus-Host Erkrankung (GvHD), insbesondere die Darm GvHD, stellt weiterhin eine der Hauptursachen für Mortalität und Morbidität nach allogener SZT dar. Aktivierte, alloreaktive Spender T-Zellen infiltrieren dabei über die Blutbahn die intestinale Lamina Propria. Erst kürzlich konnten wir zeigen, dass neben der vaskulären Migration ein Teil der Spender T-Zellen auch direkt aus den PP in die angrenzende Lamina Propria migrieren. Um Faktoren, die diese direkte Migration fördern, zu untersuchen und die direkt migrierenden T-Zellen genauer zu charakterisieren, verwendeten wir ein MHC-inkompatibles Mausmodell zur Induktion einer akuten GvHD.
Durch RNA Sequenzierung und Massenspektrometrie lasermikrodissezierter Darmschleimhautproben konnte eine starke Expression der Chemokine CXCL9, CXCL10, CXCL11, CCL3, CCL4 und CCL5 während der akuten intestinalen GvHD aufgezeigt werden. Neben CCL4 und XCL1 wiesen verschiedene Faktoren der T-Zellaktivierung, wie CD3ζ, LAT, Lck und ZAP70, sowie Faktoren der zytoskelettalen Reorganisation, wie Dock2, Coro1α und Parvin-γ, eine vermehrte Expression insbesondere nahe der PP auf. Die Expression der migrationsfördernden Faktoren Coro1α und Parvin-γ in Spender T-Zellen nahe der PP konnte anschließend mittels histologischen Immunfluoreszenzfärbungen bestätigt werden. Durchflusszytometrische Analysen konnten weiterhin eine vermehrte Expression von CCR5, CCR9 und Intgerin α4β7 auf den vornehmlich Tbet+ Spender T-Zellen nahe der PP nachweisen. Funktionelle in vitro Migrationsversuche zeigten abschließend, dass in vivo aktivierte Spender T-Zellen eine gerichtete Migration in Richtung auf CXCL11 und zu späterem Zeitpunkt auch auf CCL4 vollziehen können.
Zusammenfassend zeigt diese Arbeit die Bedeutung zahlreicher Chemokine für das sequenzielle T-Zell-Homing während der akuten intestinalen GvHD. Neben der insbesondere durch Faktoren der zytosekeletalen Reorganisation vermittelten amoeboiden Migration kann auch eine mesenchymale Fortbewegung über Faktoren wie CCR5, CCR9 und Integrin α4β7 die direkte Migration der T-Zellen fördern. Den direkt migrierenden vornehmlich TH1 polarisierten Zellen folgen weitere, CD27 und Integrin αLβ2 exprimierende, zytotoxische T-Zellen aus der Blutbahn. Die direkt migrierenden Zellen könnten als Initiator und Potentiator der intestinalen T-Zell Infiltration wirken und müssen für zukünftige therapeutische Strategien nicht nur der Darm GvHD, sondern der intestinalen Inflammation im Allgemeinen mitberücksichtigt werden.
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.
Machine-Learning-Based Identification of Tumor Entities, Tumor Subgroups, and Therapy Options
(2023)
Molecular genetic analyses, such as mutation analyses, are becoming increasingly important in the tumor field, especially in the context of therapy stratification. The identification of the underlying tumor entity is crucial, but can sometimes be difficult, for example in the case of metastases or the so-called Cancer of Unknown Primary (CUP) syndrome. In recent years, methylome and transcriptome utilizing machine learning (ML) approaches have been developed to enable fast and reliable tumor and tumor subtype identification. However, so far only methylome analysis have become widely used in routine diagnostics.
The present work addresses the utility of publicly available RNA-sequencing data to determine the underlying tumor entity, possible subgroups, and potential therapy options. Identification of these by ML - in particular random forest (RF) models - was the first task. The results with test accuracies of up to 99% provided new, previously unknown insights into the trained models and the corresponding entity prediction. Reducing the input data to the top 100 mRNA transcripts resulted in a minimal loss of prediction quality and could potentially enable application in clinical or real-world settings.
By introducing the ratios of these top 100 genes to each other as a new database for RF models, a novel method was developed enabling the use of trained RF models on data from other sources.
Further analysis of the transcriptomic differences of metastatic samples by visual clustering showed that there were no differences specific for the site of metastasis. Similarly, no distinct clusters were detectable when investigating primary tumors and metastases of cutaneous skin melanoma (SKCM).
Subsequently, more than half of the validation datasets had a prediction accuracy of at least 80%, with many datasets even achieving a prediction accuracy of – or close to – 100%.
To investigate the applicability of the used methods for subgroup identification, the TCGA-KIPAN dataset, consisting of the three major kidney cancer subgroups, was used. The results revealed a new, previously unknown subgroup consisting of all histopathological groups with clinically relevant characteristics, such as significantly different survival. Based on significant differences in gene expression, potential therapeutic options of the identified subgroup could be proposed.
Concludingly, in exploring the potential applicability of RNA-sequencing data as a basis for therapy prediction, it was shown that this type of data is suitable to predict entities as well as subgroups with high accuracy. Clinical relevance was also demonstrated for a novel subgroup in renal cell carcinoma. The reduction of the number of genes required for entity prediction to 100 genes, enables panel sequencing and thus demonstrates potential applicability in a real-life setting.
Das maligne Melanom nimmt als Tumorerkrankung mit hoher Metastasierungsrate und steigenden Inzidenzraten bei höchster Mortalität aller Hauttumoren eine zunehmende Bedeutung in der modernen Onkologie ein. Frühzeitige Diagnosemöglichkeiten und moderne Behandlungen konnten das Überleben der Patienten bereits erheblich verbessern. Jedoch besteht nach wie vor Bedarf an geeigneten Modellen, um die Melanomprogression vollständig zu verstehen und neue wirksame Therapien zu entwickeln. Hierfür werden häufig Tiermodelle verwendet, diese spiegeln jedoch nicht die menschliche Mikroumgebung wider. Zweidimensionalen Zellkulturen fehlen dagegen entscheidende Elemente der Tumormikroumgebung. Daher wurde in dieser Arbeit ein dreidimensionales epidermales Tumormodell des malignen Melanoms, welches aus primären humanen Keratinozyten und verschiedenen Melanomzelllinien besteht, entwickelt. Die eingesetzten Melanomzelllinien variieren in ihren Treibermutationen, wodurch das Modell in der Lage ist, Wirkstoffe zu untersuchen, die spezifisch auf diese Mutationen wirken. Mit Techniken des Tissue Engineerings konnte ein dreidimensionales Hautmodell aufgebaut werden, das alle charakteristischen Schichten der Epidermis aufweist und im Bereich des stratum basale Melanomcluster ausbildet. Diese reichen je nach Größe und Ausdehnung bis in suprabasale Epidermisschichten hinein. Die Tumor-Histopathologie, der Tumorstoffwechsel sowie tumorassoziierte Proteinsekretionen ließen sich im in vitro Modell nachweisen. Darüber hinaus konnte ein Protokoll entwickelt werden, mit dem einzelne Zellen aus den Modellen reisoliert werden können. Dies ermöglichte es, den Proliferationszustand innerhalb des jeweiligen Modells zu charakterisieren und die Wirkung von Antitumortherapien gezielt zu bewerten. Die Anwendbarkeit als Testsystem im Bereich der Tumortherapeutika wurde mit dem in der Klinik häufig verwendeten v-raf-Maus-Sarkom-Virus-Onkogen-Homolog B (BRAF)-Inhibitor Vemurafenib demonstriert. Der selektive BRAF-Inhibitor reduzierte erfolgreich das Tumorwachstum in den Modellen mit BRAF-mutierten Melanomzellen, was durch eine Verringerung der metabolischen Aktivität, der proliferierenden Zellen und des Glukoseverbrauchs gezeigt wurde. Für die Implementierung des Modells in die präklinische Therapieentwicklung wurde B-B-Dimethylacrylshikonin, ein vielversprechender Wirkstoffkandidat, welcher einen Zellzyklusarrest mit anschließender Apoptose bewirkt, im Modell getestet.
Bei einer Anwendung der Modelle im Bereich der Testung topischer Behandlungen ist eine Barrierefunktion der Modelle notwendig, die der in vivo Situation nahe kommt. Die Barriereeigenschaften der Hautäquivalente wurden durch die Melanomzellen nachweislich nicht beeinflusst, sind aber im Vergleich zur in vivo Situation noch unzureichend. Eine signifikante Steigerung der Hautbarriere konnte durch die Bereitstellung von Lipiden und die Anregung hauteigener Regenerationsprozesse erreicht werden. Über den Nachweis des transepidermalen Wasserverlusts konnte eine Messmethode zur nicht-invasiven Bestimmung der Hautbarriere etabliert und über den Vergleich zur Impedanzspektroskopie validiert werden. Hierbei gelang es, erstmals die Korrelation der Hautmodelle zur in vivo Situation über ein solches Verfahren zu zeigen. Das entwickelte epidermale Modell konnte durch die Integration eines dermalen Anteils und einer Endothelzellschicht noch weiter an die komplexe Struktur und Physiologie der Haut angepasst werden um Untersuchungen, die mit der Metastierung und Invasion zusammenhängen, zu ermöglichen. Die artifizielle Dermis basiert auf einem Kollagen-Hydrogel mit primären Fibroblasten. Eine dezellularisierte Schweinedarmmatrix ließ sich zur Erweiterung des Modells um eine Endothelzellschicht nutzen. Dabei wanderten die primären Fibroblasten apikal in die natürliche Schweindarmmatrix ein, während die Endothelzellen basolateral eine geschlossene Schicht bildeten.
Die in dieser Arbeit entwickelten Gewebemodelle sind in der Lage, die Vorhersagekraft der in vitro Modelle und die in vitro - in vivo Korrelation zu verbessern. Durch die Kombination des Melanommodells mit einer darauf abgestimmten Analytik wurde ein neuartiges Werkzeug für die präklinische Forschung zur Testung von pharmazeutischen Wirkstoffen geschaffen.
Chronic pain conditions are a major reason for the utilization of the health care system. Inflammatory pain states can persist facilitated by peripheral sensitization of nociceptors. The voltage-gated sodium channel 1.9 (NaV1.9) is an important regulator of neuronal excitability and is involved in inflammation-induced pain hypersensitivity. Recently, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphatidylcholine (OxPAPC) was identified as a mediator of acute inflammatory pain and persistent hyperalgesia, suggesting an involvement in proalgesic cascades and peripheral sensitization. Peripheral sensitization implies an increase in neuronal excitability. This thesis aims to characterize spontaneous calcium activity in neuronal compartments as a proxy to investigate neuronal excitability, making use of the computational tool Neural Activity Cubic (NA3). NA3 allows automated calcium activity event detection of signal-close-to-noise calcium activity and evaluation of neuronal activity states. Additionally, the influence of OxPAPC and NaV1.9 on the excitability of murine dorsal root ganglion (DRG) neurons and the effect of OxPAPC on the response of DRG neurons towards other inflammatory mediators (prostaglandin E2, histamine, and bradykinin) is investigated. Using calcium imaging, the presence of spontaneous calcium activity in murine DRG neurons was established. NA3 was used to quantify this spontaneous calcium activity, which revealed decreased activity counts in axons and somata of NaV1.9 knockout (KO) neurons compared to wildtype (WT). Incubation of WT DRG neurons with OxPAPC before calcium imaging did not show altered activity counts compared to controls. OxPAPC incubation also did not modify the response of DRG neurons treated with inflammatory mediators. However, the variance ratio computed by NA3 conclusively allowed to determine neuronal activity states. In conclusion, my findings indicate an important function of NaV1.9 in determining the neuronal excitability of DRG neurons in resting states. OxPAPC exposition does not influence neuronal excitability nor sensitizes neurons for other inflammatory mediators. This evidence reduces the primary mechanism of OxPAPC-induced hyperalgesia to acute effects. Importantly, it was possible to establish an approach for unbiased excitability quantification of DRG neurons by calcium activity event detection and calcium trace variance analysis by NA3. It was possible to show that signal-close-to-noise calcium activity reflects neuronal excitability states.
Dendritische Zellen (DC) spielen eine Schlüsselrolle im Immunsystem. Sie dienen als professionelle antigenpräsentierende Zellen und können eine antigenspezifische Immunantwort initiieren, indem sie naive T-Zellen primen.
DC können auch verwendet werden, um T-Zellen im Kontext der onkologischen Immuntherapie zu stimulieren. In vitro können sie leicht aus Monozyten differenziert werden. Die daraus resultierenden unreifen DC können bereits Antigene phagozytieren und präsentieren, sie aktivieren jedoch noch keine Immunantwort solange keines der aufgenommenen Antigene als pathogen erkannt wird. Die Ausreifung einer unreifen, tolerogenen DC zu einer immunogenen reifen DC kann, neben anderen Methoden, durch einen Cocktail aus TLR-Liganden oder Zytokinen erreicht werden. Die Auswahl der Substanzen in diesem Cocktail bestimmt den Phänotyp und die funktionellen Eigenschaften der resultierenden reifen DC. Einige der benötigten Fähigkeiten der DC in der Tumorimmuntherapie, wo sie aus Patientenmonozyten generiert, mit Tumorantigen beladen und dem Patienten wieder zugeführt werden sollen, umfassen die Migration zu den T-Zell-Zonen der Lymphknoten, Antigenpräsentation auf sowohl MHC-I- als auch MHC-II-Molekülen, Zytokinproduktion für die Direktion der T-Zell-Antwort wie IL-12p70, und die Expression von Oberflächenmarkern wie der kostimulatorischen Moleküle CD80 und CD86.
In der Vergangenheit wurde gezeigt, dass durch Zugabe von Prostaglandin E2 (PGE2) zu einem Cocktail mit dem synthetischen TLR3-Liganden poly-I:C und dem TLR7/8-Liganden R848 (Resiquimod) sowohl eine gute migratorische Fähigkeit als auch eine erhöhte IL-12p70-Produktion erreicht werden kann, während etwa die Fähigkeit zur Antigen-Kreuzpräsentation reduziert erschien. Anhand von Monozyten anonymer gesunder Spender beleuchtet diese Arbeit daher den Effekt von PGE2 auf monozytenderivierte DC näher, indem seine konzentrationsabhängige Wirkung auf deren Phänotyp untersucht wird. In den durchgeführten Versuchen wurde dabei die Expressionsdichte der Oberflächenmarker CD83, CD80 und CD86, HLA-DR und CCR7 sowie der monozytäre Marker CD14 durchflusszytometrisch analysiert. Die Ergebnisse zeigen bei Exposition mit PGE2 dosisabhängig eine Heraufregulation von CD80, CD83, CD86 und CCR7 in der Population reifer DC, deren Maximum in unteren mikromolaren Konzentrationen erreicht wird. Gleichzeitig induzierte PGE2 dosisabhängig auch die Entstehung einer zweiten Zellpopulation mit anderen Eigenschaften, die stattdessen den monozytären Marker CD14 re-exprimierte. Dies ist für künftige Studien eine interessante Beobachtung, da sie eine differenzierte Betrachtung beider resultierender Subpopulationen anregt.
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.
Bei der Atherosklerose handelt es sich um eine chronische inflammatorische Erkrankung, die sich an der arteriellen Gefäßinnenwand abspielt. Ihre Haupt-Manifestationsformen Schlaganfall und Herzinfarkt zählen zu den häufigsten Todesursachen weltweit. Eine chronische Endothelbelastung und -funktionsstörung, beeinflusst durch Risikofaktoren wie Diabetes, arterieller Bluthochdruck, Rauchen und Entzündungszustände, führen zur Permeabilitätserhöhung des Endothels, zur Zelleinwanderung, subendothelialen Lipidanreicherung, Migration glatter Muskelzellen und der Ausbildung atherosklerotischer Läsionen. Es kommt zu Aktivierung des Immunsystems und fortschreitender Entzündungsreaktion, schließlich zur Ausbildung eines nekrotischen Kerns und zunehmender Vulnerabilität des Plaques.
Epigenetische Veränderungen betreffen klassischerweise das Chromatingerüst. Durch DNA-Methylierung und -Demethylierung sowie verschiedene Modifikationen der Histon-Proteine kann die DNA in ihrer Zugänglichkeit verändert werden. So kann die Transkription eines bestimmten Genes direkt und potenziell längerfristig beeinflusst werden, ohne dass Alterationen der DNA-Basenfolge selbst stattfinden. Das Enzym SET7 nimmt hierbei eine Sonderrolle ein, da es neben einer Methylierung von Histon 3 auch verschiedene zelluläre Zielstrukturen posttranslational direkt methylieren kann.
Epigenetische Veränderungen im Kontext der Atherosklerose sind bereits vereinzelt beschrieben. Auch sind sie relevant in der Reaktion auf Umwelteinflüsse und bei inflammatorischen Vorgängen. Der Frage, ob epigenetische Mechanismen im atherosklerotischen Geschehen eine Rolle spielen, sollte in dieser Arbeit nachgegangen werden. Dazu wurde in Zellkulturversuchen für Makrophagen und glatte Muskelzellen geprüft, ob die einzelnen pro-atherosklerotischen Stimuli oxLDL, IL-1β, TNFα und LPS bereits zu relevanten Veränderungen epigenetischer Enzyme führen. Dies erfolgte über Vergleich der entsprechenden mRNA mittels qPCR. Zur Untersuchung der genaueren Dynamik wurde für die Enzyme SET7 und DNMT1 der zeitliche Ablauf dieser Reaktion auf TNFα-Stimulation in Makrophagen genauer betrachtet. Unter gleichen Versuchsbedingungen wurde außerdem die Änderung der mRNA-Expression einiger Matrixmetalloproteasen, TIMP-Enzyme, Zytokine und Transkriptionsfaktoren analysiert,um zukünftig kausale Zusammenhänge weiter aufdecken zu können. Auch die Frage nach Veränderungen epigenetischer Enzyme in der Ldlr-/--Maus nach fettreicher Diät im Vergleich zu Ldlr-/--Mäusen ohne Diät sollte hier beantwortet werden. Dazu wurde die mRNA der Zellsuspensionen aus Milz, Aortenwurzel und gesamter Aorta der Tiere mithilfe der qPCR verglichen. Schließlich sollte ein effizienter Weg für einen individuellen und flexiblen SET7 knock-out etabliert werden, um weitere Studien dieses Enzyms zu ermöglichen. Hierzu wurde die Methode des CRISPR/Cas9 Systems gewählt und abschließend die Funktionalität des Systems überprüft.
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.
SPECT as a representative of molecular imaging allows visualization of metabolic processes in vivo. In clinical practice, single photon emission imaging is an established modality for myocardial perfusion imaging or the diagnosis of adrenal or neuroendocrine tumors, to name a few. With technical advances in scanner design and data processing leading to improved spatial resolution and image quality, SPECT has become a serious contender in small animal preclinical imaging. With multi-pinhole collimation, submillimeter spatial resolutions are achieved without limiting sensitivity, which has led to a significant increase of interest in SPECT for preclinical research in recent years.
In this dissertation, the potential of a two-detector system through an analysis of three dedicated mouse collimators with multi-pinhole configurations was demonstrated. For this, sensitivity, spatial resolution, and uniformity as key parameters were determined. In the second part of the present work, an evaluation of the image quality at different activity concentrations to allow prediction of the system performance related to in vivo studies was performed. Therefore, a visual evaluation, as well as a calculation of the contrastto-noise ratio, was performed using mini Derenzo phantoms for the respective three mouse collimators. To better classify the results, the study was extended by a comparison with the predecessor system.
Due to the absence of the third bottom detector, sensitivity and uniformity are slightly compromised. All three collimators were able to achieve a spatial resolution in the submillimeter range, XUHR-M offers a peak resolution of up to 0.35 mm. In terms of resolution, both evaluated systems performed on an equal level. Visual assessment of image quality indicates a slight advantage of the new two-detector system, and the contrast-to-noise ratio seems to benefit from the improved SROSEM algorithm. However, the differences between the two systems are marginal.
The U-SPECT5/CT E-Class is proven to be state-of-the-art for small animal imaging and is a powerful instrument for preclinical molecular imaging research. Improvements in system design compensate well for the reduction in the detection area, allowing excellent imaging even with low activity concentrations.
Molecular imaging of rats is of great importance for basic and translational research. As a powerful tool in nuclear medicine, SPECT can be used to visualize specific functional processes in the body, such as myocardial perfusion or bone metabolism. Typical applications in laboratory animals are imaging diagnostics or the development of new tracers for clinical use. Innovations have enabled resolutions of up to a quarter of a millimeter with acceptable sensitivity. These advances have recently led to significantly more interest in SPECT both clinically and preclinically.
The objective of this thesis was to evaluate the performance of the new U-SPECT5/CT E-Class by MILabs with a dedicated ultra-high resolution multi-pinhole collimator for rats and its potential for in vivo imaging of rats. The unique features of the U-SPECT are the large stationary detectors and the new iterative reconstruction algorithm. In addition, compared to the conventional system, the "E-Class" uses only two detectors instead of three.
First, the sensitivity, maximum resolution, and uniformity were determined as performance parameters. Thereafter, CNRs for different activity levels comparable to those of typical in vivo activities were examined. Finally, two example protocols were carried out for imaging with 99mTc-MIBI and 99mTc-HMDP in healthy rats to evaluate the in vivo capabilities. For this purpose, CNR calculations and an image quality assessment were performed. The focus was on image quality as a function of scan time and post-reconstruction filter across a wide range of realistically achievable in vivo conditions.
Performance was reasonable compared to other systems in the literature, with a sensitivity of 567 cps/MBq, a maximum resolution of 1.20 mm, and a uniformity of 55.5%. At the lower activities, resolution in phantom studies decreased to ≥1.80 mm while maintaining good image quality. High-quality bone and myocardial perfusion SPECTs were obtained in rats with a resolution of ≥1.80 mm and ≥2.20 mm, respectively. Although limited sensitivity remains a weakness of SPECT, the U-SPECT5/CT E-Class with the UHR-RM collimator can achieve in vivo results of the highest standard despite the missing third detector. Currently, it is one of the best options for high-resolution radionuclide imaging in rats.
Studies on the role of cytoskeletal-regulatory and -crosslinking proteins in platelet function
(2023)
Cytoskeletal reorganization in platelets is highly regulated and important for proper platelet function during activation and aggregation at sites of vascular injury. In this thesis, the role of three different cytoskeletal-regulatory and -crosslinking proteins was studied in platelet physiology using megakaryocyte- and platelet-specific knockout mice. The generation of branched actin filaments is regulated by nucleation promoting factors (NPF) and the Arp2/3 complex.
(1.) The WAVE complex is a NPF, which upregulates the Arp2/3 complex activity at the plasma membrane. As shown in this thesis, the loss of the WAVE complex subunit Cyfip1 in mice did not alter platelet production and had only a minor impact on platelet activation. However, Cyfip1 played an essential role for branching of actin filaments and consequently for lamellipodia formation in vitro. The importance of lamellipodia for thrombus formation and stability has been controversially discussed. Cyfip1-deficient platelets were able to form a stable thrombus ex vivo and in vivo and a hemostatic plug comparable to controls. Moreover, Cyfip1-deficient mice maintained vascular integrity at the site of inflammation. These data show that platelet lamellipodia formation is not required for hemostatic function and pathophysiological thrombus formation.
(2.) The WASH complex is another NPF, which mediates actin filament polymerization on endosomal vesicles via the Arp2/3 complex. Loss of the WASH complex subunit Strumpellin led to a decreased protein abundance of the WASH protein and to a 20% reduction in integrin αIIbβ3 surface expression on platelets and megakaryocytes, whereas the expression of other surface receptors as well as the platelet count, size, ex vivo thrombus formation and bleeding time remained unaltered. These data point to a distinct role of Strumpellin in maintaining integrin αIIbβ3 expression and provide new insights into regulatory mechanisms of platelet integrins.
(3.) MACF1 has been described as a cytoskeletal crosslinker of microtubules and F-actin. However, MACF1-deficient mice displayed no alterations in platelet production, activation, thrombus formation and hemostatic function. Further, no compensatory up- or downregulation of other proteins could be found that contain an F-actin- and a microtubule-binding domain. These data indicate that MACF1 is dispensable for platelet biogenesis, activation and thrombus formation. Nevertheless, functional redundancy among different proteins mediating the cytoskeletal crosstalk may exist.
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.
Die Rolle transposabler Elemente in der Genese des malignen Melanom im Fischmodell Xiphophorus
(2023)
Der Name der transposablen Elemente beruht auf ihrer Fähigkeit, ihre genomische Position verändern zu können. Durch Chromosomenaberrationen, Insertionen oder Deletionen können ihre genomischen Transpositionen genetische Instabilität verursachen. Inwieweit sie darüber hinaus regulatorischen Einfluss auf Zellfunktionen besitzen, ist Gegenstand aktueller Forschung ebenso wie die daraus resultierende Frage nach der Gesamtheit ihrer biologischen Signifikanz. Die Weiterführung experimenteller Forschung ist unabdingbar, um weiterhin offenen Fragen nachzugehen. Das Xiphophorus-Melanom-Modell stellt hierbei eines der ältesten Tiermodelle zur Erforschung des malignen Melanoms dar. Durch den klar definierten genetischen Hintergrund eignet es sich hervorragend zur Erforschung des bösartigen schwarzen Hautkrebses, welcher nach wie vor die tödlichste aller bekannten Hautkrebsformen darstellt. Die hier vorliegende Arbeit beschäftigt sich mit der Rolle transposabler Elemente in der malignen Melanomgenese von Xiphophorus.
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.
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.
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.
While the healthy brain works through balanced synaptic communication between
glutamatergic and GABAergic neurons to coordinate excitation (E) and inhibition (I), disruption
of E/I balance interferes with synaptic communication, information processing, and ultimately
cognition. Multiple line of evidence indicates that E/I imbalance represents the
pathophysiological basis of a wide spectrum of mental disorders. Genetic screening
approaches have identified Cadherin-13 (CDH13). as a risk gene across neurodevelopmental
and mental disorders. CDH13 regulates several cellular and synaptic processes in brain
development and neuronal plasticity in adulthood. In addition to other functions, it is specifically
localized at inhibitory synapses of parvalbumin- and somatostatin-expressing GABAergic
neurons. In support of CDH13’s function in moderating E/I balance, electrophysiological
recordings of hippocampal slices in a CDH13-deficient mouse model revealed an increase in
basal inhibitory but not excitatory synaptic transmission. Moreover, the search for genetic
variants impacting functional expression of the CDH13 gene identified SNP (single nucleotide
polymorphism)) rs2199430 in intron 1 to be associated with differential mRNA concentrations
in human post-mortem brain across the three genotypes CDH13G/G, CDH13A/G and CDH13A/A
.
This work therefore aimed to further validate these findings in a complementary human model
by using induced pluripotent stem cells (iPSCs). The application of human iPSCs in research
has replaced the use of embryonic cells, resolving the ethical conflict of destructive usage of
human embryos. Investigating CDH13’s mode of action in inhibitory synapses was predicted
to facilitate mechanistic insight into the effects of CDH13 gene variants on E/I network activity,
which can then be targeted to reinstate balance.
Genome-wide association studies have identified rare copy number variants (CNVs) resulting
in a deletion (or duplication) of CDH13. To reduce genetic background variance, a set of
isogenic iPSC lines with a gene dose-dependent deficiency of CDH13 (CDH13-/- and CDH13+/-
) was generated by using the Clustered Regulatory Interspaced Short Palindromic
Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. These CRISPRed iPSCs
carrying a single or two allele(s) with CDH13 inactivation facilitate investigation of CDH13
function in cellular processes, at inhibitory synapses and in neuronal network activity. In
addition, iPSCs carrying allelic SNP rs2199430 variants were used to study the effects of
common genetic variation of CDH13. These cell lines were differentiated into pure
glutamatergic and GABAergic neurons and co-cultured to generate neuronal networks allowing
its activity to be measured and correlated with electrophysiological signatures of differential
CDH13 genotypes. The work towards assessment of neuronal network activity of the iPSC
lines was subdivided into three major steps: first, generating rtTA/Ngn2 and rtTA/Ascl1-positive
iPSCs via a lentivirus-mediated approach; second, differentiating pure glutamatergic and
GABAergic neurons from the genetically transduced iPSCs and co-culturing of pure
glutamatergic and GABAergic neurons in a pre-established ratio (65:35) by direct
differentiation upon supplementation with doxycycline and forskolin on a microelectrode array
(MEA) chip; and, finally, recording of neuronal network activity of iPSC lines after 49 days in
vitro, followed by extraction and analyses of multiple MEA parameters.
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Based on the MEA parameters, it was confirmed that complete CDH13 knockout as well as
heterozygous deficiency influence E/I balance by increasing inhibition. It was further revealed
that common SNP variation alters the signature of neuronal network activity. Specifically,
CDH13 deficiency resulted in a significant reduction in network burst duration (NBD), reduced
number of detected spikes within a network burst and reduction in network burst rate (NBR)
compared to the control (CDH13G/G). CDH13A/G and CDH13A/A showed similarities with the
CRISPRed CDH13-deficient networks by showing a significant reduction in the NBD and a
reduced number of detected spikes within a network compared to CDH13G/G. Strikingly. there
was a significant increase in the NBR of the CDH13A/G and CDH13A/A compared to CDH13G/G
networks. CDH13A/G networks exhibited significant differences in both parameters. At the
cellular level, this indicates that signalling pathways which determine the length and frequency
of network bursts differ among allelic variants of SNP rs2199430, thus confirming functional
relevance of this intronic SNP.
In summary, CDH13-deficient isogenic iPSC lines were generated using CRISPR/Cas9, iPSCs
were genetically transduced via a lentivirus approach, direct differentiation of
glutamatergic/GABAergic neurons derived from transduced iPSCs were used to establish a
scalable co-culture system, and network activity was recorded by MEA using pre-established
parameters to extract and analyze activity information. The results indicate that iPSC-derived
neuronal networks following CRISPR/Cas9-facilitated CDH13 inactivation, as well as networks
with allelic SNP variants of CDH13, moderate E/I balance, thus advancing understanding of
CDH13 function at inhibitory synapses and elucidating the effects of rare and common CDH13
gene variation.
Biological Substrates of Waiting Impulsivity in Children and Adolescents with and without ADHD
(2023)
Focus of the present work were the questions whether and how the concept of waiting impulsivity (WI), defined as the ability to regulate a response in anticipation of reward and measured by the 4-choice serial reaction time task (4-CSRTT), may contribute to our understanding of Attention-Deficit/Hyperactivity Disorder (ADHD) and its neurobiological underpinnings.
To address this topic, two studies were conducted: in a first study, the relationship be-tween 4-CSRTT behavioral measures, neural correlates and ADHD symptom domains, i.e. inattention (IA) and hyperactivity/impulsivity (H/I) was explored in a pooled sample of 90 children and adolescents with (n=44) and without (n=46) ADHD diagnosis. As ex-pected, IA was associated with dorsolateral prefrontal brain regions linked with executive functions and attentional control, which was evident on the structural and the functional level. Higher levels of both IA and H/I covaried with decreased activity in the right ven-trolateral prefrontal cortex (PFC), a central structure for response inhibition. Moderation analyses revealed that H/I-related decreased activation in this region did not map linearly on difficulties on the behavioral level: brain activation was a significant predictor of task accuracy only, when H/I symptoms were low/absent but not for clinically relevant ADHD symptoms. Further, H/I was implicated in dysfunctional top-down control of reward eval-uation. Both symptom domains correlated positively with hippocampus (HC) activity in anticipation of reward. In addition, for high H/I symptoms, greater activation in the HC was found to correlate with higher motivation on the behavioral level, indicating that rein-forcement-learning and/or contingency awareness may contribute to altered reward pro-cessing in ADHD patients.
In a second study, the possible serotonergic modulation of WI and the ADHD-WI relation-ship was addressed in a sub-sample comprising 86 children and adolescents of study I. The effects of a functional variant in the gene coding for the rate-limiting enzyme in the synthesis of brain serotonin on behavior and structure or function of the WI-network was investigated. Moderation analyses revealed that on the behavioral level, a negative corre-lation between accuracy and IA was found only in GG-homozygotes, whereas no signifi-cant relationship emerged for carriers of the T-allele. This is in line with previous reports of differential effects of serotonergic modulation on attentional performance depending on the presence of ADHD symptoms. A trend-wise interaction effect of genotype and IA for regional volume of the right middle frontal gyrus was interpreted as a hint towards an involvement of the PFC in this relationship, although a more complex mechanism includ-ing developmental effects can be assumed. In addition, interaction effects of genotype and IA were found for brain activation in the amygdala (AMY) und HC during perfor-mance of the 4-CSRTT, while another interaction was found for H/I symptoms and geno-type for right AMY volume. These findings indicate a serotonergic modulation of coding of the emotional value of reward during performance of the 4-CSRTT that varies de-pending on the extent of psychopathology-associated traits.
Taken together, it was shown that the 4-CSRTT taps distinct domains of impulsivity with relevance to ADHD symptomatology: (proactive) response inhibition difficulties in relation with anticipation of reward. Furthermore, the two symptom domains, IA and H/I, contrib-ute differently to WI, which emphasizes the need to distinguish both in the research of ADHD. The results of study II emphasized the relevance of serotonergic transmission especially for attentional control and emotional processing. Although the present findings need replication and further refinement in more homogenous age groups, the use of the 4-CSRTT with a dimensional approach is a very promising strategy, which will hopefully extend our understanding of impulsivity-related mental disorders in the future.
Echinococcosis is an important zoonosis. The causative agent of Alveolar Echinococcosis (AE) is Echinococcus multilocularis. The treatment of human AE is limited to surgery and chemotherapy with albendazole (ABZ). However, ABZ works only parasitostatically and it needs to be taken for long periods, although it causes adverse side effects. Thus, development of new, parasiticidal drug with selective toxicity is required. Because undifferentiated stem cells of E. multilocularis play key role in its longevity and regenerative capacity, targeting stem cells is especially important.
In vitro screening of protein kinases inhibitors demonstrated that human PIM kinases inhibitors have detrimental effects on E. multilocularis. Through yeast two hybrid assay, the interaction of parasite PIM kinase (EmPIM) and its CDC25 (EmCDC25) was indicated. Through in situ hybridization, expression of EmPIM in the stem cells was observed. Therefore, EmPim is likely to be a positive regulator of cell cycle progression, the same as human Pim1. In addition, 20 compounds against EmPIM were selected through in silico screening and synthesized. One of them has a detrimental effect on E.multilocularis comparable to human pan-PIM inhibitors, but has much weaker toxicity on human cell lines.
Furthermore, triclabendazole (TCBZ) and its metabolite TCBZSX, which are approved for another flatworm disease, Fascioliasis were tried on E. multilocularis. With two stem cell markers, damage to stem cells by TCBZSX was shown. In addition, primary cells from treated vesicles never regenerated and the damage to stem cells proved to be irreversible.
Our in silico screening method used in EmPIM research has potential to identify compounds which overcome the side effect problem in ABZ-based chemotherapy. On the other hand, it is expected that my research of TCBZ can lead to development of a practical parasiticidal chemotherapy by combining TCBZ, which damages stem cells, and ABZ, which damages differentiated cells.
Predictability of threat is one of the key modulators of neural activity in fear and anxiety-related threat processes and there is a considerable number of studies focusing on the exact contribution of centromedial amygdala and Bed nucleus of stria terminalis (BNST) in animals as well as in humans. In this research field, some studies already investigated the differential involvement of both areas during temporally predictable and unpredictable threat processes in humans. However, these studies showed several limitations e.g. small sample size, no predictable threat conditions, no separation of anticipation and confrontation processes, which should be addressed in future studies. Furthermore, evidence for group-based inter-individual differences of amygdala and BNST activity during predictable and unpredictable threat processes have not been studied extensively.
Several studies suggest a relevant role of the amygdala and BNST activity in phobic processes in patients with specific phobia, but no study so far has investigated the exact contribution of centromedial amygdala (CM) and BNST during temporally predictable and unpredictable threat processes in specific phobia.
This thesis consisted of three studies and aimed to evaluate the exact contribution of CM and BNST during temporally predictable and unpredictable threat anticipation and confrontation with the use of an optimized functional magnetic resonance imaging (fMRI) paradigm, which aimed to solve methodological limitations of recent studies. Study 1 used a large sample of healthy participants who were grouped based on NPSR1 genotype, and study 2 and study 3 used a sample of patients with spider phobia. In sum, the results of all three studies indicated, that BNST is more relevant for anticipation processes as compared to the CM. Contrary, during the confrontation phase the CM displays a greater relevance for threat confrontation processes.
In recent years, various studies have investigated the extent to which treatment success can be predicted in patients with anxiety disorders based on pre-treatment fMRI activity. Therefore, this was investigated for the first time in study 3 in patients with spider phobia during temporally predictable and unpredictable threat processes. Results indicated that independent of temporal predictability lower anterior cingulate cortex (ACC) activity during threat anticipation and engaged BNST during threat confrontation might be benefitting factors for successful therapy response in spider phobia.
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.
Parkinson’s disease (PD) is the second most common neurodegenerative disease with still no cure available. The prominent feature of PD is the loss of dopaminergic neurons at the Substantia nigra (SN). Genetic and environmental insults affecting the SNCA gene encoding the alpha-Synuclein (alpha-Syn) protein result into an aberrant form of the protein with higher propensity towards oligomerization becoming part of insoluble inclusions called Lewy Bodies (LB). LB impart cytotoxicity leading to neurodegeneration, activate resident microglia and escape to the periphery where they get captured by dendritic cells and presented to naïve T cells. Proliferating effector T lymphocytes invade the brain releasing proinflammatory cytokines and performing a cytotoxic effect on neurons.
In this study, we examine the hypothesis that the expansion of regulatory T cells (Treg) could exert an anti-inflammatory effect that averts neurodegeneration in the AAV1/2-A53T-alpha-Syn mouse model for PD.
Mice brains were transfected by a unilateral stereotaxic injection at the SN region with a chimeric Adeno-Associated Viral vector of serotypes 1 and 2 (AAV1/2) carrying the A53T-mutated human SNCA gene encoding the readily aggregating aberrant alpha-Syn (AAV1/2-A53T-alpha-Syn). One week after injection, mice were treated with the CD28 superagonistic antibody (CD28SA), known to significantly expand the Treg population. Mice were then analyzed by behavioral analysis using the Rotarod performance test and the Cylinder test. The impact of CD28SA on the immune system was examined by flow cytometry. The integrity of the nigrostriatal system was assessed by stereological quantification of Tyrosine hydroxylase (TH)-stained dopaminergic neurons in SN and optical density measurements of TH-stained striatum. The mechanism of action of CD28SA was analyzed by treating PD mice alternatively with a Treg adoptive transfer, while CD28SA effect on levels of neurotrophic factors was quantified by ELISA.
We observed an expansion of Treg by FACS analyses three days after CD28SA treatment, demonstrating target engagement. CD28SA treatment of AAV1/2-A53T-alpha-Syn mice provided neuroprotection evident through elevated numbers of dopaminergic neurons in the SN and higher optical density of TH-staining in the striatum, in CD28SA-treated mice compared to PBS-treated control mice, and that was reflected in an enhanced performance in behavioral studies. Additionally, brain infiltration of proinflammatory activated T lymphocytes (CD4+CD69+ and CD8+CD69+ cells), that were obvious in PBS-treated AAV1/2-A53T-alpha-Syn control mice, was augmented in PD mice receiving CD28SA. The alternative treatment with Treg adoptive transfer did replicate the beneficial effects of CD28SA indicating that Treg expansion is the main effector mechanism by which it exerts its neuroprotective effect. CD28SA treatment of PD mice led to an increase of GDNF and BDNF in some brain structures that was not observed in untreated mice.
We conclude that in the AAV1/2-A53T-alpha-Syn PD mouse model, CD28SA suppresses proinflammation, reverses behavioral deficits and is neuroprotective on SN dopaminergic cells.
The human body has very good self-healing capabilities for numerous different injuries to a variety of different tissues. This includes the main human mechanical framework, the skeleton. The skeleton is limited in its healing without additional aid by medicine mostly by the defect size. When the defect reaches a size above 2.5 cm the regeneration of the defect ends up faulty. Here is where implants, defect fillers and other support approaches developed in medicine can help the body to heal the big defect still successfully.
Usually sturdy implants (auto-/allo-/xenogenic) are implanted in the defect to bridge the distance, but for auto- and allogenic implants a suitable donor site must be found and for all sources the implant needs to be shaped into the defect specific site to ensure a perfect fit, the best support and good healing. This shaping is very time consuming and prone to error, already in the planning phase. The use of a material that is moldable and sets in the desired shape shortly after applying negates these disadvantages. Cementitious materials offer exactly this property by being in a pasty stage after the powder and liquid components have been mixed and the subsequently hardening to a solid implant. These properties also enable the extrusion, and therefore may also enable the injection, of the cement via a syringe in a minimal invasive approach.
To enable a good injection of the cement modifications are necessary. This work aimed to modify commonly used calcium phosphate-based cement systems based on α-TCP (apatitic) and β-TCP (brushitic). These have been modified with sodium phytate and phytic acid, respectively. Additionally, the α-TCP system has been modified with sodium pyrophosphate, in a second study, to create a storable aqueous paste that can be activated once needed with a highly concentrated sodium orthophosphate solution.
The powder phase of the α-TCP cement system consisted of nine parts α-TCP and one part CDHA. These were prepared to have different particle sizes and therefore enable a better powder flowability through the bimodal size distribution. α-TCP had a main particle size of 20 μm and CDHA of 2.6 μm. The modification with sodium phytate led to an adsorption of phytate ions on the surface of the α-TCP particles, where they started to form complexes with the Ca2+ ions in the solution. This adsorption had two effects. The first was to make the calcium ions unavailable, preventing supersaturation and ultimately the precipitation of CDHA what would lead to the cement hardening. The second was the increase of the absolute value of the surface charge, zeta potential, of the powder in the cement paste. Here a decrease from +3 mV to -40 mV could be measured. A strong value for the zeta potential leads to a higher repulsion of similarly charged particles and therefore prevents powder agglomeration and clogging on the nozzle during injection. These two modifications (bimodal particles size distribution and phytic acid) lead to a significant increase in the paste injectability. The unmodified paste was injectable for 30 % only, where all modified pastes were practically fully injectable ~90 % (the residual paste remained in the nozzle, while the syringe plunger already reached the end of the syringe).
A very similar observation could be made for the β-TCP system. This system was modified with phytic acid. The zeta potential was decreased even stronger from -10 ± 1.5 mV to -71.5 ± 12 mV. The adsorption of the phytate ions and subsequent formation of chelate complexes with the newly dissolved Ca2+ ions also showed a retarding effect in the cements setting reaction. Where the unmodified cement was not measurable in the rheometer, as the reaction was faster than the measurement setup (~1.5 min), the modified cements showed a transition through the gel point between 3-6 min. This means the pastes stayed between 2 and 4 times longer viscous than without the modification. Like with the first cement system also here the effects of the phytate addition showed its beneficial influence in the injectability measurement. The unmodified cement was not injectable at all, due to the same issue already encountered at the rheology measurements, but all modified pastes were fully injectable for at least 5 min (lowest phytate concentration) and at least 10 min (all other concentrations) after the mixing of powder and liquid.
The main goal of the last modification with sodium pyrophosphate was to create a paste that was stable in aqueous environment without setting until the activation takes place, but it should still show good injectability as this was the desired way of application after activation. Like before also the zeta potential changed after the addition of pyrophosphate. It could be lowered from -22 ± 2mV down to -61 to -68 ± 4mV (depending on the pyrophosphate concentration). The pastes were stored in airtight containers at room temperature and checked for their phase composition over 14 days. The unmodified paste showed a beginning phase conversion to hydroxyapatite between 7 and 14 days. All other pastes were still stable and unreacted. The pastes were activated with a high concentrated (30 wt%) sodium orthophosphate solution. After the activation the pastes were checked for their injectability and showed an increase from -57 ± 11% for the unmodified paste to -89 ± 3% (practically fully injectable as described earlier) for the best modified paste (PP005).
It can be concluded that the goal of enabling full injection of conventional calcium phosphate bone cement systems was reached. Additional work produced a storage stable paste that still ensures full injectability. Subsequent work already used the storable paste and modified it with hyaluronic acid to create an ink for 3D extrusion printing. The first two cement systems have also already been investigated in cell culture for their influence on osteoblasts and osteoclasts. The next steps would have to go more into the direction of translation. Figuring out what properties still need to be checked and where the modification needs adjustment to enable a clinical use of the presented systems.
The behavior of honeybees and bumblebees relies on a constant sensory integration of abiotic or biotic stimuli. As eusocial insects, a sophisticated intraspecific communication as well as the processing of multisensory cues during foraging is of utter importance. To tackle the arising challenges, both honeybees and bumblebees have evolved a sophisticated olfactory and visual processing system.
In both organisms, olfactory reception starts at the antennae, where olfactory sensilla cover the antennal surface in a sex-specific manner. These sensilla house olfactory receptor neurons (ORN) that express olfactory receptors. ORNs send their axons via four tracts to the antennal lobe (AL), the prime olfactory processing center in the bee brain. Here, ORNs specifically innervate spheroidal structures, so-called glomeruli, in which they form synapses with local interneurons and projection neurons (PN). PNs subsequently project the olfactory information via two distinct tracts, the medial and the lateral antennal-lobe tract, to the mushroom body (MB), the main center of sensory integration and memory formation. In the honeybee calyx, the sensory input region of the MB, PNs synapse on Kenyon cells (KC), the principal neuron type of the MB. Olfactory PNs mainly innervate the lip and basal ring layer of the calyx. In addition, the basal ring receives input from visual PNs, making it the first site of integration of visual and olfactory information. Visual PNs, carrying sensory information from the optic lobes, send their terminals not only to the to the basal ring compartment but also to the collar of the calyx. Receiving olfactory or visual input, KCs send their axons along the MB peduncle and terminate in the main output regions of the MB, the medial and the vertical lobe (VL) in a layer-specific manner. In the MB lobes, KCs synapse onto mushroom body output neurons (MBON). In so far barely understood processes, multimodal information is integrated by the MBONs and then relayed further into the protocerebral lobes, the contralateral brain hemisphere, or the central brain among others.
This dissertation comprises a dichotomous structure that (i) aims to gain more insight into the olfactory processing in bumblebees and (ii) sets out to broaden our understanding of visual processing in honeybee MBONs.
The first manuscript examines the olfactory processing of Bombus terrestris and specifically investigates sex-specific differences. We used behavioral (absolute conditioning) and electrophysiological approaches to elaborate the processing of ecologically relevant odors (components of plant odors and pheromones) at three distinct levels, in the periphery, in the AL and during olfactory conditioning. We found both sexes to form robust memories after absolute conditioning and to generalize towards the carbon chain length of the presented odors. On the contrary, electroantennographic (EAG) activity showed distinct stimulus and sex-specific activity, e.g. reduced activity towards citronellol in drones. Interestingly, extracellular multi-unit recordings in the AL confirmed stimulus and sex-specific differences in olfactory processing, but did not reflect the differences previously found in the EAG. Here, farnesol and 2,3-dihydrofarnesol, components of sex-specific pheromones, show a distinct representation, especially in workers, corroborating the results of a previous study. This explicitly different representation suggests that the peripheral stimulus representation is an imperfect indication for neuronal representation in high-order neuropils and ecological importance of a specific odor.
The second manuscript investigates MBONs in honeybees to gain more insights into visual processing in the VL. Honeybee MBONs can be categorized into visually responsive, olfactory responsive and multimodal. To clarify which visual features are represented at this high-order integration center, we used extracellular multi-unit recordings in combination with visual and olfactory stimulation. We show for the first time that information about brightness and wavelength is preserved in the VL. Furthermore, we defined three specific classes of visual MBONs that distinctly encode the intensity, identity or simply the onset of a stimulus. The identity-subgroup exhibits a specific tuning towards UV light. These results support the view of the MB as the center of multimodal integration that categorizes sensory input and subsequently channels this information into specific MBON populations.
Finally, I discuss differences between the peripheral representations of stimuli and their distinct processing in high-order neuropils. The unique activity of farnesol in manuscript 1 or the representation of UV light in manuscript 2 suggest that the peripheral representation of a stimulus is insufficient as a sole indicator for its neural activity in subsequent neuropils or its putative behavioral importance. In addition, I discuss the influence of hard-wired concepts or plasticity induced changes in the sensory pathways on the processing of such key stimuli in the peripheral reception as well as in high-order centers like the AL or the MB. The MB as the center of multisensory integration has been broadly examined for its olfactory processing capabilities and receives increasing interest about its visual coding properties. To further unravel its role of sensory integration and to include neglected modalities, future studies need to combine additional approaches and gain more insights on the multimodal aspects in both the input and output region.
More than 150 different RNA modifications have been detected in all kingdoms of life and 60 are known to decorate bacterial RNA. Among them, pseudouridine is universally conserved and one of the most abundant modifications present in bacterial stable RNAs such as tRNAs and rRNAs. In bacteria, the nucleotide is posttranscriptionally generated by dedicated enzymes called pseudouridine synthases (PUSs). With the advent of sophisticated deep-sequencing technologies, this modification has been identified in different types of RNA classes (tRNAs, rRNAs, mRNAs, snRNAs, and lncRNAs) in diverse eukaryotic organisms. However, these techniques have never been applied to bacteria, generating a knowledge gap about the location of the modified nucleotide in prokaryotic RNAs. Mutations or deletions of specific eukaryotic PUS enzymes are linked to human diseases and therefore their absence is deleterious for the correct function of the cell. However, deletion of tRNA or rRNA PUS enzymes in the bacterial model organism E. coli have not revealed any such drastic phenotypes, suggesting a different role and function of the modification itself and of the enzymes in different kingdoms of life.
Since the roles of tRNA PUS enzymes in bacteria is still poorly understood, a functional characterization of these proteins is pursued in the Epsilonproteobacteria Campylobacter jejuni and Helicobacter pylori. While C. jejuni is the leading cause of bacterial foodborne gastroenteritis in humans, infection with H. pylori is associated with the development of gastric cancer. In particular, phenotypes were explored for the tRNA PUS enzymes TruA, TruB, and TruD in C. jejuni as well as TruA and TruD in H. pylori. Upon deletion of truD, a severe growth defect is observed for C. jejuni but not for H. pylori, highlighting a potential difference in function of the enzyme in the two related bacterial pathogens.
Moreover, a genome-wide approach called Pseudo-seq is established and applied for RNA of these two pathogens, which allows, for the first time, the global identification of pseudouridine modifications at single-nucleotide resolution in the bacterial transcriptome. Applying Pseudo-seq in RNAs of wildtype and diverse PUS enzyme deletion mutants enabled the identification of the distinct RNA substrates of tRNA PUS enyzmes in C. jejuni and H. pylori. Hereby, the tRNA-Glu was determined to be the major tRNA substrate of TruD in C. jejuni. Interestingly, the tRNA-Glu is expressed as a single copy in the C. jejuni genome. To link the growth defect observed for a C. jejuni ∆truD mutant strain to the pseudouridine modification of the tRNA-Glu, a catalytically inactive TruD complementation was generated. This strain is unable to restore the tRNA-Glu modification but surprisingly, was able to complement the growth defect. The same observation was made for a cross-complementation with a copy of H. pylori TruD. This indicates that there is a potential additional function of the TruD PUS enzyme in C. jejuni that is independent of the pseudouridine modification. Using a combination of deep-sequencing technologies (RIP-seq, RNA-seq, Ribo-seq, and CLIP-seq), the dual function of TruD is investigated.
Overall, this study provides the first in-depth investigation into pseudouridylation of bacteria in general and the bacterial pathogens C. jejuni and H. pylori in particular. The work presented in this thesis reveals not only a global map of pseudouridine in tRNAs and rRNAs of the two bacteria but it also explores the function of the responsible tRNA PUS enzymes. In addition, this study provides evidence for a dual function of the C. jejuni PUS enzyme TruD that goes beyond its RNA modifying function. Future research could focus on unravelling the function of TruD and its potential interaction partners and thus reveal new mechanisms of regulation of a protein previously only described as an RNA modification enzyme.
CRISPR-Cas systems are highly diverse and canonically function as prokaryotic adaptive immune systems. The canonical resistance mechanism relies on spacers that are complementary to the invaders' nucleic acids. By accidental incorporation or other mechanisms, prokaryotes can also acquire self-targeting spacers that are complementary to their own genome. As self-targeting commonly leads to lethal autoimmunity, the existence of self-targeting spacers poses a paradox. In Chapter 1, we provide an overview of the prevalence of self-targeting spacers, summarize how they can be incorporated, and which means can be employed by the host to evade lethal self-targeting. In addition, we outline alternative functions of CRISPR-Cas systems that are associated with self-targeting spacers. Whether CRISPR-Cas systems can efficiently target their own genome depends heavily on the presence of protospacer adjacent motifs (PAMs) next to the target region. In Chapter 2, we developed a method to determine PAM requirements. Thereby, we specifically focused on type I systems that engage multi-protein complexes, which are challenging to assess. Using the cell-free transcription-translation (TXTL) system, we developed an enrichment-based binding assay and validated its reliability by examining the well-known PAM requirements of the E. coli type I-E system. In Chapter 3, we applied the TXTL-based PAM assay to assess 16 additional CRISPR-Cas systems. These 16 systems included three CRISPR-Cas associated transposons (CASTs). CASTs are recently discovered transposons that employ CRISPR-Cas systems in a non-canonical function for the directed integration of the transposon. To further characterize CASTs in TXTL outside their PAM requirements, we reconstituted the transposition of CASTs in TXTL. In Chapter 4, we turned to non-canonical self-targeting CRISPR-Cas systems, which were already discussed in Chapter 1. While investigating how the plant pathogen Xanthomonas albilineans survives self-targeting by its two endogenous CRISPR-Cas systems, we identified multiple putative anti-CRISPR proteins (Acrs) in the genome of X. albilineans. Two of the Acrs, named AcrIC11 and AcrIF12Xal, inhibited degradation by their respective CRISPR-Cas systems but still retained Cascade-binding ability, and appear responsible for the lack of autoimmunity in X. albilineans. In summary, we developed new technologies that eased the investigation of non-canonical multi-component systems and, if applied to additional systems, might reveal unique properties that could be implemented in new CRISPR-Cas based tools.
Humans spontaneously blink several times a minute. These blinks are strongly modulated during various cognitive task. However, the precise function of blinking and the reason for their modulation has not been fully understood. In the present work, I investigated the function of spontaneous blinks through various perceptual and cognitive tasks. Previous research has revealed that blinks rates decrease during some tasks but increase during others. When trying to understand these seemingly contradictory results, I observed that blink reduction occurs when one engages with an external input. For instance, a decrease has been observed due to the onset of a stimulus, sensory input processing and attention towards sensory input. However, for activities that do not involve such an engagement, e.g. imagination, daydreaming or creativity, the blink rate has been shown to increase. To follow up on the proposed hypothesis, I distinguished tasks that involve the processing of an external stimulus and tasks that involve disengagement.
In the first part of the project, I explored blinking during stimulus engagement. If the probability of blinking is low when engaging with the stimulus, then one should find a reduction in blinks specifically during the time period of processing but not during sensory input per se. To this end, in study 1, I tested the influence of task-relevant information duration on blink timing and additionally manipulated the overall sensory input using a visual and an auditory temporal simultaneity judgement task. The results showed that blinks were suppressed longer for longer periods of relevant information or in other words, blinks occurred at the end of relevant information processing for both the visual and the auditory modality. Since relevance is mediated through top-down processes, I argue that the reduction in blinks is a top-down driven suppression. In studies 2 and 3, I again investigated stimulus processing, but in this case, processing was triggered internally and not based on specific changes in the external input. To this end, I used bistable stimuli, in which the actual physical stimulus remains constant but their perception switches between different interpretations. Studies on the involvement of attention in such bistable perceptual changes indicate that the sensory input is reprocessed before the perceptual switch. The results revealed a reduction in eye blink rates before the report of perceptual switches. Importantly, I was able to decipher that the decrease was not caused by the perceptual switch or the behavioral response but likely started before the internal switch. Additionally, periods between a blink and a switch were longer than interblink intervals, indicating that blinks were followed by a period of stable percept. To conclude, the first part of the project revealed that there is a top-down driven blink suppression during the processing of an external stimulus.
In the second part of the project, I extended the idea of blinks marking the disengagement from external processing and tested if blinking is associated with better performance during internally directed processes. Specifically, I investigated divergent thinking, an aspect of creativity, and the link between performance and blink rates as well as the effect of motor restriction. While I could show that motor restriction was the main factor influencing divergent thinking, the relationship between eye blink rates and creative output also depended on restriction. Results showed that higher blink rates were associated with better performance during free movement, but only between subjects. In other words, subjects who had overall higher blink rates scored better in the task, but when they were allowed to sit or walk freely. Within a single subject, trial with higher blink rates were not associated with better performance. Therefore, possibly, people who are able to disengage easily, as indicated by an overall high blink rate, perform better in divergent thinking tasks. However, the link between blink rate and internal tasks is not clear at this point. Indeed, a more complex measurement of blink behavior might be necessary to understand the relationship.
In the final part of the project, I aimed to further understand the function of blinks through their neural correlates. I extracted the blink-related neural activity in the primary visual cortex (V1) of existing recordings of three rhesus monkeys during different sensory processing states. I analyzed spike related multi-unit responses, frequency dependent power changes, local field potentials and laminar distribution of activity while the animal watched a movie compared to when it was shown a blank screen. The results showed a difference in blink-related neural activity dependent on the processing state. This difference suggests a state dependent function of blinks.
Taken altogether, the work presented in this thesis suggests that eye blinks have an important function during cognitive and perceptual processes. Blinks seem to facilitate a disengagement from the external world and are therefore suppressed during intended processing of external stimuli.
Gene expression in eukaryotic cells is regulated by the combinatorial action of numerous gene-regulatory factors, among which microRNAs (miRNAs) play a fundamental role at the post-transcriptional level. miRNAs are single-stranded, small non-coding RNA molecules that emerge in a cascade-like fashion via the generation of primary and precursor miRNAs. Mature miRNAs become functional when incorporated into the RNA induced silencing complex (RISC). miRNAs guide RISCs to target mRNAs in a sequence-specific fashion. To this end, base-pairs are usually formed between the miRNA seed region, spanning nucleotide positions 2 to 8 (from the 5' end) and the 3'UTR of the target mRNA. Once miRNA-mRNA interaction is established, RISC represses translation and occasionally induces direct or indirect target mRNA degradation. Interestingly, miRNAs are expressed not only in every multicellular organism but are also encoded by several viruses, predominately by herpesviruses. By controlling both, cellular as well as viral mRNA transcripts, virus-encoded miRNAs confer many beneficial effects on viral growth and persistence. Murine cytomegalovirus (MCMV) is a ß-herpesvirus and so far, 29 mature MCMV-encoded miRNAs have been identified during lytic infection. Computational analysis of previously conducted photoactivated ribonucleotide-enhanced individual nucleotide resolution crosslinking immunoprecipitation (PAR-iCLIP) experiments identified a read cluster within the 3' untranslated region (3'UTR) of the immediate early 3 (IE3) transcript in MCMV. Based on miRNA target predictions, two highly abundant MCMV miRNAs, namely miR-m01-2-3p and miR-M23-2-3p were found to potentially bind to two closely positioned target sites within the IE3 PAR-iCLIP peak. To confirm this hypothesis, we performed luciferase assays and showed that activity values of a luciferase fused with the 3'UTR of IE3 were downregulated in the presence of miR-m01- 2 and miR-M23-2. In a second step, we investigated the effect of pre-expression of miR-m01-2 and miR-M23-2 on the induction of virus replication. After optimizing the transfection procedure by comparing different reagents and conditions, plaque formation was monitored. We could demonstrate that the replication cycle of the wild-type but not of our MCMV mutant that harbored point mutations in both miRNA binding sites within the IE3-3'UTR, was significantly delayed in the presence of miR-m01-2 and miR-M23-2. This confirmed that miR-m01-2 and miR-M23-2 functionally target the major transcription factor IE3 which acts as an indispensable regulator of viral gene expression during MCMV lytic infection. Repression of the major immediate early genes by viral miRNAs is a conserved feature of cytomegaloviruses. The functional role of this type of regulation can now be studied in the MCMV mouse model.