Autophagy is a fundamental degradative process, maintaining cellular homeostasis and functions in host defense against intracellular pathogens, including mycobacteria and Salmonella. The thesis... Show moreAutophagy is a fundamental degradative process, maintaining cellular homeostasis and functions in host defense against intracellular pathogens, including mycobacteria and Salmonella. The thesis investigated the function of an regulator of antibacterial autophagy, Damage Regulated Autophagy Modulator 1 (DRAM1) against infection and shows that DRAM1 restricts bacterial growth not only through canonical antibacterial autophagy (xenophagy) but also promotes an autophagy-related pathway, named LC3-associated phagocytosis (LAP). The function of DRAM1 in restricting bacterial proliferation is independent from the recognition of bacteria by xenophagy receptors. Mechanistically, DRAM1 promotes the infection-induced activation of autophagy and LAP as well as the maturation of bacteria-containing vesicles in both pathways. This maturation process, stimulated by DRAM1, involves multiple vesicle fusion steps directing bacteria to lysosomes. Through this maturation process, DRAM1 delivers the cytosolic protein Fau to bacteria-containing vesicles, where it serves as a precursor for antimicrobial peptides. The underlying mechanism may be explained by the discovery of an interaction between DRAM1 and the SNARE protein VTI1B. Overall, the work in this thesis contributes to ongoing research into the potential application of autophagy modulation as a host-directed therapy against infectious diseases. Show less
This thesis focuses on the role of chemokine receptors CXCR3 and CCR2 in the inflammatory process and infection control using the zebrafish model. It describes the regulatory interplay between an... Show moreThis thesis focuses on the role of chemokine receptors CXCR3 and CCR2 in the inflammatory process and infection control using the zebrafish model. It describes the regulatory interplay between an atypical and a conventional chemokine receptor during chemotaxis in macrophages, the role of chemotactic signaling in cell polarization and explores an in vivo screening workflow for human anti-inflammatory drugs using zebrafish. Show less
In this thesis I studied the functions of the zebrafish orthologs of the human TLR5 and TLR2 genes that were shown to be responsible for recognition of bacterial flagellin and a broad spectrum... Show moreIn this thesis I studied the functions of the zebrafish orthologs of the human TLR5 and TLR2 genes that were shown to be responsible for recognition of bacterial flagellin and a broad spectrum of bacterial cell wall components, respectively. One of the focal points of this thesis is the difference at the transcriptomic level of the downstream pathway of the TLR5 and TLR2 receptors and the roles of TLR signaling in host innate immune responses to infection by Mycobacterium marinum, a close relative to Mycobacterium tuberculosis and a natural pathogen of zebrafish. The new possibilities for analysis of transcriptomes using RNA deep sequencing make it highly attractive to analyze the responses of an entire test animal model at the system biology level. Furthermore, we used genetic knockdown and knockout tools to further analyze the function of TLR5 and TLR2 and downstream signaling partners in innate immunity, infectious disease and insulin resistance. Show less
To date, 128 mycobacterial species have been characterised, ranging from non-pathogenic to pathogenic for humans. Molecular methods contributed significantly to the identification of the species,... Show moreTo date, 128 mycobacterial species have been characterised, ranging from non-pathogenic to pathogenic for humans. Molecular methods contributed significantly to the identification of the species, replacing conventional laborious methods. In this thesis, the design and application of a genus-specific real-time PCR, for the rapid detection of non-tuberculous mycobacteria in clinical materials, was described. The technique was extremely useful for the rapid detection of the slowgrowing Mycobacterium species. Addition of species-specific probes to the ITS assay, identified M. haemophilum to be present in previously undiagnosed skin inflammation and resulted in the recognition of M. haemophilum as the second most common mycobacterial species causing lymphadenitis. Subsequent Amplified Fragment Length Polymorphism analysis of M. haemophilum isolates showed this species to posses an extremely low mutation rate. Also, M. haemophilum lymphadenitis cases are suspected to have a common source, most likely piped water, in contrast to M. avium infections, which appear to originate from variable environmental sources as was underscribed by Restriction Fragment Length Polymorphism analysis. Contamination with saprophytic mycobacterial DNA is problematic for the current NTM detection in clinical materials. This and other bottlenecks in the molecular diagnostics of NTM were addressed in this thesis as well. Show less
