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Bioorthogonal labelling of surface receptors on living lymphocytes
Paldusová, Kateřina ; Cebecauer, Marek (advisor) ; Benda, Aleš (referee)
The surface of cells displays high heterogeneity on chemical and geometrical levels. To understand the function of cells, we need to pay attention to the morphological features formed at the plasma membrane. To study cell surface with molecular specificity, there are plenty of imaging methods starting with the conventional wide-field microscopy through confocal microscopy, ending with super-resolution fluorescence microscopies and electron microscopies. Super-resolution microscopy studies conducted on the fixed cells provide detailed steady-state data about the cell surface nanoscopic organisation and distribution of molecules at the morphological structures. However, since cells are parts of living organisms and constantly change their properties in time and space, the information about dynamics of cellular structures and motility of molecules remains hidden when using this approach. Live-cell compatible methods are required to study dynamic changes of molecules at the single-molecule level. In this study we are focusing on the distribution and dynamics of molecules CD2 and CD4 expressed on the surface of non-stimulated T cells. The main aim of this thesis was to develop a novel method for live-cell imaging and single-molecule tracking of membrane-bound proteins in 3D and at nanoscale. With such a...
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Critical evaluation of the current models of proximal T cell signaling
Tahtahová, Valerie ; Filipp, Dominik (advisor) ; Paprčková, Darina (referee)
The adaptive immune responses are initiated by interaction of T cells and antigen- presenting cells (APC), in which the engagement of T cell receptor (TCR) and peptide-major histocompatibility complex results in the proximal TCR signaling and subsequent T cell activation. However, how exactly the signal is transduced from outside of TCR to the interior o T cell is not completely understood. To provide resolution for this complex signaling process, various models have been proposed. Among them, the kinetic-segregation (KS) model and mechanosensing models are the most discussed in the field. Inhere, these models are assessed in the context of advances in microscopic technologies. Strikingly, T cell membrane protrusions referred to as microvilli emerged as immunomodulatory elements which harbor TCRs on the top of their tips. Microvilli can serve as a structural platform to further develop often neglected concept of T cell activation which integrates membrane topology with the initiation of TCR signaling. Furthermore, mechanical external cues, primarily tensile forces which were shown to be capable of TCR triggering are also taken into consideration. Taken together, in this thesis, the current models of T cell activation are critically evaluated in the light of technological advances which are...
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Quantitative fluorescence microscopy techniques to study three-dimensional organisation of T-cell signalling molecules.
Chum, Tomáš ; Cebecauer, Marek (advisor) ; Lánský, Zdeněk (referee) ; Brameshuber, Mario (referee)
10 SUMMARY Proteins represent one of the basic building blocks of all organisms. To understand their function at the molecular level is one the critical goals of current biological, biochemical and biophysical research. It is important to characterise all aspects that affect the localisation of proteins into different compartments with specific functions, the dynamic structure of proteins and their role in multiprotein assemblies, because altering these properties can lead to various diseases. Most of the proteomic studies are nowadays performed using biochemical approaches that allow us to study multicellular organism or tissue at once. The disadvantage of these methods is complex preparation of sample and the need for a large number of cells, which leads to the loss of information at the molecular level and in individual cells. On the contrary, microscopy can provide rather detailed information about proteins of interest and at the level of a single cell. A variety of fluorescence microscopy methods in combination with recombinant DNA techniques were applied to elucidate subcellular localisation of transmembrane adaptor proteins (TRAPs) in human lymphocytes and their nanoscopic organisation at the plasma membrane. Linker of activation of T lymphocytes (LAT), phosphoprotein associated with...
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Antigen-specific T cell ex vivo detection methods
Kolařík, Daniel ; Drbal, Karel (advisor) ; Krulová, Magdaléna (referee)
Antigen-specific T cells are an essential component of adaptive immunity. They are involved in protection of organism against extracellular and intracellular pathogens as well as against tumor cells. A defect of their function may lead to autoimmune disorders or allergies. That is why their detection and any further manipulation are essential for clinical immunology. In this thesis, I am going to describe two important methods which are useful for studies of antigen-specific T cells. It involves analysis of static snapshot using flow cytometry and kinetic single-cell detection in microwells. The information obtained by those compatible methods is potentially useful for medical practice in both prevention as well as diagnostics and therapy of relevant human pathologies.
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Localisation of CD4 coreceptor and its variants in human T cells
Glatzová, Daniela ; Cebecauer, Marek (advisor) ; Drbal, Karel (referee)
CD4 co-receptor of main T cell receptor (TCR) is essential for proper development of T lymphocytes and their function in adaptive immune responses. It is believed that CD4 stabilizes the interaction of TCR with antigenic ligand, peptide-MHC, and thereby improves T cell-dependent responses during immune reaction. CD4 is transmembrane glycoprotein with a number of structural motifs in its intracellular domain which do not dramatically affect its sorting to the plasma membrane but can influence its local organization at nanoscale. CD4 was shown to transiently accumulate in the immunological synapse formed between T cell and antigen-presenting cell. Such accumulation is rapidly followed by its internalization and/or delocalization outside the synapse. This is in contrast with TCR which accumulates strongly in the immunological synapse and is later found enriched in the central area of this structure. It is therefore unclear how TCR and its CD4 co-receptor function together when binding to their common ligand during the initiation of signaling in T cells. We aim to study localization of CD4 at nanoscale using advanced fluorescence microscopy techniques achieving significant improvements in resolution. In this work, CD4 and its mutant variants, potentially causing its different localization at the...
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