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Phosphorylation of the adaptor protein PSTPIP2 and its role in neutrophilic granulocytes
Dvořáček, Tomáš ; Brdička, Tomáš (advisor) ; Ballek, Ondřej (referee)
PSTPIP2 is an adaptor protein of the F-BAR family, which is an important regulator controlling the effector mechanisms of innate immune cells. The regulatory functions of this protein were discovered thanks to the CMO mouse strain, which lost the expression of this protein. As a result of PSTPIP2 deficiency, mice of the CMO strain develop an autoinflammatory disease affecting bone tissue and skin. The main mechanism that drives its pathology is the loss of regulation of the neutrophil granulocyte activity. These cells then produce excessive amounts of the pro-inflammatory cytokine IL-1β and reactive oxygen species. However, the exact molecular mechanism of action of the PSTPIP2 protein is unknown. When the PSTPIP2 protein is activated, it is phosphorylated and interacts with other proteins, which mediate its regulatory function. Interaction partners described so far in neutrophil granulocytes include phosphatases of the PEST family, the lipid phosphatase SHIP1 and the non-receptor tyrosine kinase CSK. In this thesis, we identified kinases from the SRC family as kinases that phosphorylate PSTPIP2. Furthermore, we found that the main phosphorylation sites of PSTPIP2 are tyrosines at positions 323 and 329. Finally, we proved that SHIP1 can bind to the phosphotyrosine motif around the tyrosine at...
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The role of protein tyrosine phosphatase CD45 and Src-family kinases in murine model of chronic autoinflammatory osteomyelitis
Ilievová, Kristýna ; Brdička, Tomáš (advisor) ; Černý, Jan (referee)
The development of autoinflammatory diseases is caused by the dysregulation of innate immune mechanisms. This leads to the development of spontaneous inflammation. Mice lacking adaptor protein PSTPIP2 develop chronic autoinflammatory osteomyelitis due to higher activity of neutrophil granulocytes and their increased production of IL-1β. .β. PSTPIP2 interacts with PEST phosphatases and kinase CSK. These proteins are impor- tant negative regulators of Src family kinases. In this diploma thesis, the role of Src family kinases and the role of their positive regulator phosphatase CD45 in the development of chronic autoinflammatory osteomyelitis was studied. For this purpose, a mouse model of chronic autoinflammatory osteomyelitis (CMO) lacking CD45 was used. These mice deve- lop the disease with delayed kinetics. Bone marrow cells isolated from these mice produce less IL-1β. upon silica activation and have lower phosphorylation of ERK MAP kinase. It isβ. probably caused by higher phosphorylation of the inhibitory tyrosine of Src family kinases resulting in their lower activity. The presence of different immune cell populations in the bone marrow, spleen and blood of these mice was also monitored in these mice. The re- sults of this work contribute to a better understanding of the role of Src family...
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The role of protein tyrosine phosphatase CD45 in neutrophil granulocytes
Ilievová, Kristýna ; Brdička, Tomáš (advisor) ; Černý, Jan (referee)
Strict regulation of the immune response is critical for appropriate protection against in- fection, preventing tissue damage, and maintaining homeostasis. A significant part of this regulation is mediated at the level of signaling pathways in which tyrosine phosphorylati- on plays a key role. It is regulated by the action of protein tyrosine kinases and protein tyrosine phosphatases (PTP). An important PTP expressed on all nucleated hematopoie- tic cells is the CD45. Its role has been studied primarily in T- and B-lymphocytes. There CD45 plays an important role in antigen-induced signaling and signaling triggered by other stimuli. It becomes apparent that also in neutrophils CD45 plays an importat role in many mechanisms that contribute to appropriate protection against infection. These include, for example, adhesion, extravasation, chemotaxis, phagocytosis, production of cytokines and oxidative burst. In many cases, CD45 affects these processes by regulating Src family kinases. Other means of CD45 participation in specific pathways are often not clear. This thesis summarizes our current understanding of role of CD45 in neutrophil granulocytes and its effects on the function of these cells. 1
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The use of neutrophils in cancer immunotherapy
KOVÁŘOVÁ, Markéta
The aim of this thesis was to investigate the possible role of neutrophil granulocytes in antitumor reactions. Most of our experiments were focused on in vitro studies assessing the cytotoxic effect of mouse neutrophils on B16-F10 melanoma cells labelled with PAMPs. We put an emphasis on activation and generating a prime state of neutrophils. Moreover, a release rate of enzyme myeloperoxidase from azurophil granules was detected as a marker of neutrophil degranulation. We also attempted to attract neutrophils into tumor microenvironment using thioglycolate medium and its main compound casein.
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