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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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The Role of B-cell Receptor Signaling in Lymphoid Malignancies
Kochmannová, Kateřina ; Havránek, Ondřej (advisor) ; Štěpánek, Ondřej (referee)
The aim of this thesis is to review current knowledge about tumor-specific B cell receptor (BCR) signaling and related novel therapy options in B-cell malignancies with the main focus on non-Hodgkin lymphomas (NHL). To a certain degree, the pathogenic BCR signaling mirrors normal forms of BCR signaling, antigen-induced and tonic. Differences between antigen- dependent and antigen-independent forms of BCR signaling are well characterized in two major subtypes of diffuse large B-cell lymphoma, the most common type of NHL. In addition to the conventional chemotherapy, several BCR inhibitors targeting BTK and PI3K have been approved for the treatment of lymphoid malignancies. However, improvements in the tumor specificity, toxicity profiles and patients selection are needed. A better understanding of BCR signaling deregulation and overall tumor pathogenesis is believed to further improve NHL treatment outcomes. Keywords: B-cell malignancies, non-Hodgkin lymphoma, B-cell receptor, tumor signaling, targeted therapy, inhibitors
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The role of S100A11 protein (calgizzarin) in rheumatoid arthritis pathogenesis
Navrátilová, Adéla ; Andrés Cerezo, Lucie (advisor) ; Štěpánek, Ondřej (referee)
Rheumatoid arthritis (RA) is a systemic connective tissue autoimmune disease which is characterized by persistent inflammation of the synovial tissue, joint destruction and visceral organs and vessels damage. The RA joint environment contains pro-inflammatory molecules and immune cells. Small calcium-binding proteins of the S100 family are also known to be involved in the inflammatory process of RA, and some of them have been studied in our laboratory. One member of this family, S100A11 (calgizzarin), is well described in oncological diseases but its role in autoimmune diseases has not yet been described. Neutrophil extracellular traps (NETs), which are a potential source of autoantigens and immunoactive molecules, also contribute to the chronic inflammatory process in RA. The aim of our study was to investigate S100A11 with the focus on its role in the processes of inflammation and tissue destruction and to assess its possible association with NETs. Using in vitro experiments and molecular methods (reverse transcription, quantitative polymerase chain reaction (qPCR) and multiplex assay) we have shown that S100A11 exerts its pro-inflammatory effect on mononuclear cells via toll-like receptor (TLR)-4, however there was not any significant effect of S100A11 on the production of pro-inflammatory...
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Characterization of T-cell clones from naïve and virtual memory compartment
Přibíková, Michaela ; Štěpánek, Ondřej (advisor) ; Drbal, Karel (referee)
Virtual memory (VM) CD8+ T cells represent a population of antigen-inexperienced T cells with an apparent memory phenotype. In lymphoreplete germ-free mice VM CD8+ T cells represent 10-20% of all peripheral CD8+ T cells. Their origin correlates with the levels of self-reactivity where the main factor that determinates the T-cell fate decision is the strength of homeostatic signals. In the first part of this thesis, we demonstrated that VM CD8+ T cells and naïve CD8+ T cells had distinct TCR repertoire and T-cell subsets contained different clonotypes. Moreover, 'VM clones' were enriched among VM T cells and were also present in naïve T cells. In contrast, 'naïve clones' were almost exclusively detected in naïve T cells. Next, we characterized the signaling of particular OVA-reactive TCRs from both naïve and VM subsets. We confirmed that 6 out of 8 tested TCRs were responsive to Kb-OVA. In the last part of the thesis, we developed and optimized a qPCR-based method for the relative quantification of specific T-cell clonotypes prior to and during the immune response. This method will serve as a tool for studying the biology of particular VM and naïve T-cell subsets and their role during the immune response. Keywords: T-cell receptor, homeostatic signaling, self-reactivity, virtual memory cells, T cells
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