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Host-microbiota, pro-inflammatory immunity and physiological senescence in wild birds
Těšický, Martin ; Vinkler, Michal (advisor) ; Tschirren, Barbara (referee) ; Štěpánek, Ondřej (referee)
Triggered by microbial ligands, inflammation serves as a "double-edged sword" to fight infections on the one hand, but on the other hand causing tissue damage due to oxidative stress if it is dysregulated. For example, chronic inflammation can contribute to inflammaging, which is now widely regarded as one of the causes of ageing. In my interdisciplinary dissertation, my colleagues and I investigated three interrelated aspects of inflammation, using an evolutionary framework and various free-living birds as models: (1) ecological and evolutionary determinants of gut microbiota (GM) composition and diversity, a driver of wild bird immunity, (2) diversity in immune genes affecting inflammatory responses in wild birds and (3) inflammation-related physiological senescence in a free-living passerine bird, the great tit (Parus major). Firstly, using 16S rRNA gene metabarcoding, we revealed high intra- and interspecific variation in passerine gut microbiota (GM) dominated by the major phyla Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Although in mammals GM depends strongly on host phylogeny and diet, in birds we found only moderate effects of phylogeny and very limited effects of host geography and ecology on GM composition. While microbiota diverged between the upper and lower...
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Identification of new regulators of proinflammatory signaling pathways
Dráberová, Helena ; Štěpánek, Ondřej (advisor) ; Krulová, Magdaléna (referee) ; Funda, David (referee)
Identification of new regulators of proinflammatory signaling pathways Helena Dráberová Protein 4.1R has been described in immune system as regulator of migration and cell adhesion, but was also shown to play a role in activation of T lymphocytes. Polymorphism in gene ORMDL-3 is associated with asthma risk in children and correlates with increased ORMDL-3 expression. This disertation thesis describes the function of proteins 4.1R and ORMDL-3 in activation of mast cells after stimulation of FcεRI receptor. IL-17 is a proinflammatory cytokine that plays a role in immune response against fungal and yeast infections. IL-17 however also plays a role in the pathology of autoimmune diseases such as reumatoid arthritis, psoriasis and multiple sclerosis. IL-17 signaling is tightly regulated, however the exact mechanism has not been described. This disertation thesis describes the IL-17R complex by mass spectrometry and analyze the function of its known and newly discovered components in cells deficient in individual proteins by method CRISPR-Cas9. Last part focuses on the discovery of new subunit of IL-17RC protein CMTM4, which role in IL-17 signaling has not been described so far. CMTM4 stabilizes IL-17RC and is required for its surface expression. In vitro data are supported by data from autoimmune model of...
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Searching for the common function of the BBSome across the evolution and development
Mašková, Kristýna ; Štěpánek, Ondřej (advisor) ; Čajánek, Lukáš (referee)
The BBSome is a protein complex whose function is associated with ciliary trafficking. It has been found that the BBSome is evolutionarily conserved among ciliated organisms. The disruption of the BBSome leads to cilia dysfunctions and affects many signalling pathways. In humans, genetic defects in the BBSome are the cause of Bardet-Biedl Syndrome, which is a pleiotropic disease. The BBSome is studied separately in various model organisms and cell lines to understand the molecular functions of the BBSome. These studies have not been compared to see if there is a common BBSome function, which could be why the BBSome is evolutionarily conserved among ciliated organisms. In this bachelor's thesis, the knowledge about the BBSome was summarized and compared to identify a putative common function of the BBSome among various model organisms or cell types. It seems that there has not been found any BBSome function that could be identified as the common function because the BBSome has many specialized functions in different organisms and tissues. The only distant similarity is BBSome-dependent ciliary retrograde transport, which has been described in most of the studied model organisms and cell types.
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Mechanisms of the tolerance and homeostasis of immune cells
Tsyklauri, Oksana ; Štěpánek, Ondřej (advisor) ; Černý, Jan (referee) ; Froňková, Eva (referee)
The ability of the immune system to tolerate self-antigens while mounting appropriate responses to pathogens is indispensable for the survival of the organism. Despite years of research, many details of the mechanisms of self-tolerance are still not well understood. The objective of this thesis is to extend our knowledge of the mechanisms of immune tolerance. The core of the PhD thesis consists of five publications related to two main research directions. The first one addresses the mechanisms of peripheral immune tolerance established by regulatory T cells (Tregs). We showed that Tregs increase the quorum of self-reactive CD8+ T cells required for the induction of autoimmunity. In addition, we identified a novel subset of antigen-stimulated CD8+ T cells, which expand in the absence of Tregs. We called them super-effector T cells. We revealed that the administration of IL-2 phenocopies the absence of Tregs, i.e., it induces super- effector T cells, and enhances CD8+ T cell response in autoimmunity and cancer. Our results provide strong evidence that the major suppressive mechanism of Tregs is limiting IL-2 availability for CD8+ T cells. Furthermore, in a collaborative project, we have shown that MyD88 signaling in thymic epithelial cells contributes to the development of Tregs and thus to the...
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The Industry Business of Prague Municipality in the first half of 20th Century. The Industrial Factories of Capital City Prague, ltd.
Štěpánek, Ondřej ; Jakubec, Ivan (advisor) ; Drnek, Kryštof (referee)
This work describes the industrial activities of the Prague municipality in building industry in the first half of the twentieth century, especially in the years 1921-1948, during which the company "Industrial factories of the Capital City of Prague" operated successfully in which the municipality owned half of the shares. The main objective is to monitor the activities of the company running the lime and brick factories and a sawmill. I focused on its development and significance. This thesis is based on information from the Prague City Archives, from the fund of Industrial Factories. The research illustrates the quantities of produced and sold building materials, then also important buildings in which the company was involved as a supplier. The main contribution of this work is the "re-discovery" of an interesting company which significantly supported the building industry in Prague, supplied the building material not only to municipal buildings and was a competitor for cartels.
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Immune responses of naive and memory CD8+ T cells.
Cesneková, Michaela ; Štěpánek, Ondřej (advisor) ; Dobeš, Jan (referee)
Adaptive immune system plays a crucial role in effective pathogen clearance as well as establishment of immunological memory and its understanding is important for vaccine and drug development, besides cancer and autoimmune disease treatment. CD8+ T lymphocytes are able to efficiently kill infected cells and develop into antigen-specific memory cells, which are kept in a steady-state and demonstrate enhanced cytokine production and faster response upon reinfection, compared to naive T cells. Additionally, the pool of CD8+ memory T cells is more abundant, diversified and localizes to lymphoid as well as non-lymphoid tissues. On the other hand, proliferation rate, threshold of activation and CD28 costimulation independence are questionable. Even though the opposite was accepted for a long time, it seems that on a per cell basis, memory cells aren't superior to naive in these features and have decreased TCR sensitivity. Interestingly, in contrast to naive, memory CD8+ T cells can be activated independently of TCR, even in the absence of a cognate antigen, which emphasizes their increased sensitivity to inflammatory milieu and contribution to innate immune responses.
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Role of CD8- and CD4-Lck interactions in the signaling and development of T cells.
Horková, Veronika ; Štěpánek, Ondřej (advisor) ; Černý, Jan (referee) ; Hons, Miroslav (referee)
Adaptive immune response plays a key role in maintaining homeostasis of the organism. T cells use an immense repertoire of T-cell receptors (TCRs) to discriminate between self and foreign antigens with very high sensitivity. Although we have many clues outlining how an ideal TCR repertoire is selected, and a good understanding of the TCR signaling machinery, there are still some key aspects of these processes that remain controversial. The objective of this thesis is to extend our knowledge of the very proximal events of TCR signaling, with special focus on interaction of TCR coreceptors with lymphocyte-specific kinase LCK. Coreceptor-LCK interaction has been described to regulate several aspects of T- cell development and response. We observed dynamic change of this interaction in course of T-cell development. Interestingly, CD4 and CD8 coreceptors displayed differential dynamics of interaction with LCK. Our data suggest that such disparity in coreceptor- LCK interaction leads to selection of more self-reactive TCR repertoire in CD8+ T cells. Moreover, when the highly self-reactive CD8+ T cells get to the periphery, the homeostatic signals drive their differentiation towards a more tolerogenic memory-like phenotype. To finally resolve the role of coreceptor-LCK interaction in the T-cell...
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Synthesis of biologically active compounds with quinazoline scaffold
Štěpánek, Ondřej
This thesis concludes my contribution to research of HIV-1 capsid assembly inhibitors. It has been shown that 2,4-disubstituted quinazoline derivatives are able to inhibit this process both, in competitive biochemical assay based on the AlphaScreen technology as well as in tissue cultures. The main objective of the work was to prepare the aforementioned quinazolines, to design and prepare new candidates with higher activity based on results of biochemical tests, and also to try to increase the solubility of otherwise poorly soluble compounds. Disubstituted quinazolines are relatively easily accessible from the commercially available anthranilic acid derivatives. These are converted to the corresponding quinazolin- 4(3H)-ones by the condensation reactions. In this work, two methods were used for the preparation of quinazolin-4(3H)-ones: reaction of acyl chlorides with aromatic anthranilamides provided 2-arylamidobenzamides whose subsequent cyclisation under basic conditions led to derivatives of 2-arylquinazolin-4(3H)-one; reaction of anthranilic acid esters with aromatic nitriles, which afforded desired quinazolin-4(3H)-one in one reaction step. Chlorination of 2-arylquinazolin-4(3H)-ones using POCl3 then led to 2-aryl-4- chloroderivatives as key intermediates. Nucleophilic substitution of...
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The Role of Lck Kinase in T-cell Antigen Receptor Signaling
Němec, Dušan ; Štěpánek, Ondřej (advisor) ; Rösel, Daniel (referee)
LCK activity is crucial for the triggering of the entire T cell activation process. The primary function of LCK is to convert the signal of TCR:pMHC ligation into the intracellular environment. The outcome of the LCK-triggered pathway is T cell activation, cytokine production, differentiation, and clonal expansion. This thesis provides a summary of recent knowledge about the unique position of LCK in the T cell signaling machinery as well as an overview of molecules and interacting partners that regulate LCK activity. It describes the importance of the LCK-coreceptor association for optimal TCR signaling and physiological thymocyte development and mentions discussed adaptor role of LCK in the T cells. Keywords: LCK, T-cell, antigen, kinase, enzyme
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