Background: Corynebacterium urealyticum, a pathogenic, multidrug resistant member of the mycolata, is known as causative agent of urinary tract infections although it is a bacterium of the skin flora. This pathogenic bacterium shares with the mycolata the property of having an unusual cell envelope composition and architecture, typical for the genus Corynebacterium. The cell wall of members of the mycolata contains channel-forming proteins for the uptake of solutes. Results: In this study, we provide novel information on the identification and characterization of a pore-forming protein in the cell wall of C. urealyticum DSM 7109. Detergent extracts of whole C. urealyticum cultures formed in lipid bilayer membranes slightly cation-selective pores with a single-channel conductance of 1.75 nS in 1 M KCl. Experiments with different salts and non-electrolytes suggested that the cell wall pore of C. urealyticum is wide and water-filled and has a diameter of about 1.8 nm. Molecular modelling and dynamics has been performed to obtain a model of the pore. For the search of the gene coding for the cell wall pore of C. urealyticum we looked in the known genome of C. urealyticum for a similar chromosomal localization of the porin gene to known porH and porA genes of other Corynebacterium strains. Three genes are located between the genes coding for GroEL2 and polyphosphate kinase (PKK2). Two of the genes (cur_1714 and cur_1715) were expressed in different constructs in C. glutamicum Delta porA Delta porH and in porin-deficient BL21 DE3 Omp8 E. coli strains. The results suggested that the gene cur_1714 codes alone for the cell wall channel. The cell wall porin of C. urealyticum termed PorACur was purified to homogeneity using different biochemical methods and had an apparent molecular mass of about 4 kDa on tricine-containing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Conclusions: Biophysical characterization of the purified protein (PorACur) suggested indeed that cur_1714 is the gene coding for the pore-forming protein in C. urealyticum because the protein formed in lipid bilayer experiments the same pores as the detergent extract of whole cells. The study is the first report of a cell wall channel in the pathogenic C. urealyticum.

Protein-DNA interactions are central to many biological processes and form the bedrock of gene transcription, DNA replication, and DNA repair processes. Many proteins recognize specific sequences in DNA- a restriction enzyme must only cut at the correct sequence and a transcription factor should bind at its consensus sequence. Some proteins are designed to bind to specific structural or chemical features in DNA, such as DNA repair proteins and some DNA modifying enzymes. Target-specific DNA binding proteins initially bind to non-specific DNA and then search for their target sites through different types of diffusion mechanisms. Atomic force microscopy (AFM) is a single-molecule technique that is specifically well-suited to resolve the distinct states of target-specific as well as nonspecific protein-DNA interactions that are vital for a deeper insight into the target site search mechanisms of these enzymes. In this thesis, protein systems involved in epigenetic regulation, base excision repair (BER), and transcription are investigated by single-molecule AFM analyses complemented by biochemical and biophysical experiments. The first chapter of this thesis narrates the establishment of a novel, user-unbiased MatLab-based tool for automated DNA bend angle measurements on AFM data. This tool has then been employed to study the initial lesion detection step of several DNA glycosylases. These results promoted a model describing the altered plasticities of DNA at the target lesions of DNA glycosylases as the fundamental mechanism for their enhanced efficiency of lesion detection. In the second chapter of this thesis, the novel automated tool has been further extended to provide protein binding positions on the DNA along with corresponding DNA bend angles and applied to the study of DNMT3A DNA methyltransferase. These AFM studies revealed preferential co-methylation at specific, defined distances between two CpG sites by the enzyme and when combined with biochemical analyses and structural modelling supported novel modes of CpG co-methylation by DNMT3A. In the third chapter of this thesis, the role of 8-oxo-guanine glycosylase (hOGG1) in Myc-mediated transcription initiation has been investigated. AFM analyses revealed that in the presence of oxidative damage in DNA, Myc is recruited to its target site (E-box) by hOGG1 through direct protein-protein interactions, specifically under oxidizing conditions. Intriguingly, oxidation of hOGG1 was further observed to result in dimerization of hOGG1, which may also play a role in the mechanism of transcription regulation by hOGG1 under oxidative stress.

(1) Background: During maturation of the Hepatitis B virus, a viral polymerase inside the capsid transcribes a pre-genomic RNA into a partly double stranded DNA-genome. This is followed by envelopment with surface proteins inserted into a membrane. Envelopment is hypothetically regulated by a structural signal that reports the maturation state of the genome. NMR data suggest that such a signal can be mimicked by the binding of the detergent Triton X 100 to hydrophobic pockets in the capsid spikes. (2) Methods: We have used electron cryo-microscopy and image processing to elucidate the structural changes that are concomitant with the binding of Triton X 100. (3) Results: Our maps show that Triton X 100 binds with its hydrophobic head group inside the pocket. The hydrophilic tail delineates the outside of the spike and is coordinated via Lys-96. The binding of Triton X 100 changes the rotamer conformation of Phe-97 in helix 4, which enables a π-stacking interaction with Trp-62 in helix 3. Similar changes occur in mutants with low secretion phenotypes (P5T and L60V) and in a mutant with a pre-mature secretion phenotype (F97L). (4) Conclusion: Binding of Triton X 100 is unlikely to mimic structural maturation because mutants with different secretion phenotypes show similar structural responses.

Experimental evidence has emerged that local platelet activation contributes to inflammation and infarct formation in acute ischemic stroke (AIS) which awaits confirmation in human studies. We conducted a prospective observational study on 258 consecutive patients undergoing mechanical thrombectomy (MT) due to large-vessel-occlusion stroke of the anterior circulation (08/2018-05/2020). Intraprocedural microcatheter aspiration of 1 ml of local (occlusion condition) and systemic arterial blood samples (self-control) was performed according to a prespecified protocol. The samples were analyzed for differential leukocyte counts, platelet counts, and plasma levels of the platelet-derived neutrophil-activating chemokine C-X-C-motif ligand (CXCL) 4 (PF-4), the neutrophil attractant CXCL7 (NAP-2), and myeloperoxidase (MPO). The clinical-biological relevance of these variables was corroborated by specific associations with molecular-cellular, structural-radiological, hemodynamic, and clinical-functional parameters. Seventy consecutive patients fulfilling all predefined criteria entered analysis. Mean local CXCL4 (+ 39%: 571 vs 410 ng/ml, P = .0095) and CXCL7 (+ 9%: 693 vs 636 ng/ml, P = .013) concentrations were higher compared with self-controls. Local platelet counts were lower (- 10%: 347,582 vs 383,284/µl, P = .0052), whereas neutrophil counts were elevated (+ 10%: 6022 vs 5485/µl, P = 0.0027). Correlation analyses revealed associations between local platelet and neutrophil counts (r = 0.27, P = .034), and between CXCL7 and MPO (r = 0.24, P = .048). Local CXCL4 was associated with the angiographic degree of reperfusion following recanalization (r =  - 0.2523, P = .0479). Functional outcome at discharge correlated with local MPO concentrations (r = 0.3832, P = .0014) and platelet counts (r = 0.288, P = .0181). This study provides human evidence of cerebral platelet activation and platelet-neutrophil interactions during AIS and points to the relevance of per-ischemic thrombo-inflammatory mechanisms to impaired reperfusion and worse functional outcome following recanalization.

Ranvier-Schnürringe spielen eine entscheidende Rolle bei der schnellen Weiterleitung von elektrischen Impulsen in Nervenzellen. Bei bestimmten neurologischen Erkrankungen, den Neuropathien, kann es zu Störungen in der ultrastrukturellen Organisation verschiedener Schnürring-Proteine kommen (Doppler et al., 2018, Doppler et al., 2016). Eine detailliertere Kenntnis der genauen Anordnung dieser Schnürring-Proteine und eventueller Abweichungen von dieser Anordnung im Krankheitsfall, könnte der Schlüssel zu einer vereinfachten Diagnostik von bestimmten Neuropathie- Formen sein. Ziel meiner Arbeit war es daher, die Untersuchung der ultrastrukturellen Architektur der (para-)nodalen Adhäsionsproteine Neurofascin-155 und Caspr1 unter Verwendung der super-hochauflösenden Mikroskopiemethode dSTORM (direct Stochastic Optical Reconstruction Microscopy) an murinen Zupfnervenpräparaten zu etablieren. Nach erster Optimierung der Probenpräparation für die 2-Farben-dSTORM sowie der korrelationsbasierten Bildanalyse, konnte ich mittels modellbasierter Simulation die zugrundeliegende Molekülorganisation identifizieren und mit Hilfe der Ergebnisse aus früheren Untersuchungen validieren. In einem translationalen Ansatz habe ich anschließend humane Zupfnervenpräparate von 14 Probanden mit unterschiedlichen Formen einer Neuropathie mikroskopiert und ausgewertet, um die Anwendbarkeit dieses Ansatzes in der Diagnostik zu testen. Obgleich keine signifikanten Unterschiede zwischen physiologischem und pathologischem neurologischem Gewebe hinsichtlich Neurofascin-155 und Caspr1 festgestellt werden konnten, scheint der Ansatz grundsätzlich dennoch vielversprechend zu sein, bedarf jedoch noch weiteren Anstrengungen hinsichtlich Probenpräparation, Auswertungs- und Versuchsprotokollen und einer größeren Anzahl an humanen Biopsien mit homogenerem Krankheitsbild.