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Effect of small DNA viruses on function of plasmacytoid dendritic cells
Janovec, Václav ; Hirsch, Ivan (advisor) ; Růžek, Daniel (referee) ; Filipp, Dominik (referee)
Plasmacytoid dendritic cells (pDC) are a highly specialized subset of immune cells that sense viral nucleic acids by endosomal toll-like receptors 7 and 9 (TLR7/9). Activation of TLR7/9 leads to the production of type I interferons (IFN-I). Moreover, pDC contribute to the antiviral response by presenting viral antigens to T lymphocytes and link innate and adaptive immunity. pDC need to be properly regulated in order to limit excessive production of IFN-I that is associated with autoimmune diseases. Therefore, pDC possess a battery of regulatory receptors (RR) that limit TLR7/9-mediated cytokine production. This thesis focuses on the mechanism of RR-mediated inhibition of IFN-I production in pDC and explores interactions between pDC and two enveloped viruses, that possess the ability to hijack RR in pDC: hepatitis B virus (HBV) and human immunodeficiency virus (HIV). We showed, that MEK-ERK signaling pathway plays an active role in RR-mediated inhibition of IFN-I in pDC. Our results indicate that in line with other studies of our group, pharmacological targeting of MEK1/2-ERK signaling could be a strategy to re-establish immunogenic activity of pDC. Then, we investigated whether antiretroviral therapy (ART) in a cohort of 21 treatment-naive chronic HIV-infected patients has restored the number and...
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Ser/Thr protein kinases in mycobacteria
Borovcová, Taťána ; Doubravová, Linda (advisor) ; Konopásek, Ivo (referee)
The Mycobacterium tuberculosis genome encodes 11 Ser/Thr protein kinases. These protein kinases are structurally related to eukaryotic protein kinases. The phosphoproteome contains hundreds of proteins phosphorylated on Ser/Thr residues that influence all aspects of cell biology, which supports the critical role of phosphorylation in the regulation of physiology. Particularly important role in regulation belongs to protein kinases PknA, PknB, PknG and PknL, these protein kinases occur in all species of mycobacteria. Although only PknA and PknB are essencial for the M. tuberculosis, they regulate cell shape through the regulation of cell wall synthesis and cell division. Another important protein kinase is PknG, although not essential for growth it is necessary for virulence, because it promotes the survival of pathogen inside macrophages of the host. As a result, Ser/Thr protein kinases represent an interesting target for inhibitor development that could be used as drugs against tuberculosis.
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Effect of small DNA viruses on regulation of interferon production
Hofman, Tomáš ; Hirsch, Ivan (advisor) ; Elleder, Daniel (referee)
Plasmacytoid dendritic cells (pDC) represent innate immune cells capable to detect viruses in their endosomal environment via Toll-like receptors (TLRs). Viral nuclear acid recognition leads to the massive production of type I interferon (IFN I) and induction of the antiviral state in uninfected cells. Crosslinking of the surface regulatory receptors, such as BDCA-2, with monoclonal antibodies or with some viruses leads to the activation of MEK1/2- ERK signaling pathway and inhibition of IFN I production in pDC. In this study, the role of MEK1/2 kinase has been highlighted. Its inhibition reversed the inhibitory effect of BDCA-2 crosslinking and its direct activation with PMA led to the inhibition of IFN-α production. Yet an unclear role of pDC in sensing of BK polyomavirus virus (BKV) responsible for kidney transplant rejection was investigated as a major topic of this thesis. Experiments with the pDC cell line Gen2.2 and HRPTEC primary cell line showed that pDCs were not able to detect BKV particles, however, exposure of activated Gen2.2 cells to BKV inoculum dramatically upregulated production of IFN-α. Most importantly, coculture of Gen2.2 cells with BKV- infected HRPTEC cells resulted in IFN-α and TNF-α production, which was prevented by Bafilomycin. These results suggest that BKV-infected...
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Recombinant preparation of DNA binding domain of transcription factor TEAD4
Zákopčaník, Marek ; Novák, Petr (advisor) ; Šulc, Miroslav (referee)
6 Abstract Transcription factors play a key role in the management of cell growth and differ- entiation and their deregulation is associated with many cancers. TEAD proteins utilise highly conserved DNA binding domain to recognise specific DNA sequences. This domain could facilitate new drug design and development. The goal of this master thesis includes recombinant preparation of DNA binding domain of transcriptional factor TEAD4 extended by a part of an unstruc- tured variable sequence, which connects this domain with transactivation domain. Purification steps include affinity chromatography followed by size exclusion chro- matography. The characterization of produced protein was performed by mass spectrometry and finally, native gel electrophoresis was used to prove the ability of the produced protein to bind DNA. During purification steps, a fragmentation from C-terminus was observed. Based on analysis of the mass spectra, three most represented forms of produced protein were described all of which were fragmented. The most abundant form (55%) consisted of amino acids 30-131 from TEAD4 protein. Second most abun- dant form (18%) consisted of amino acids 30-144 and the third form consisted of amino acids 30-81. Native gel electrophoresis verified the ability to bind DNA, the efficiency was however lower...
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Effect of HBV protein HBx on activation of MEK1/2 signaling and inhibition of type I IFN in hepatoma cell line Huh7
Berehovska, Olena ; Hirsch, Ivan (advisor) ; Zábranský, Aleš (referee)
Hepatitis B virus (HBV) infection is one of the major causes of chronic and cancerous liver disease. Elimination of HBV from chronically infected patients by recombinant interferon α (IFNα) monotherapy shows that the mechanisms of the innate immunity play an important role in suppressing viral infection. However, the mechanisms of recognition of the HBV genome and its escape from the mechanisms of natural immunity are still little known. One of the principal factors enabling the virus to escape from cellular restriction mechanisms is the HBx viral protein. HBx is a 154 amino acid pleiotropic multifunctional protein affecting transcription, signal transduction, cell cycle, protein degradation, apoptosis, and chromosomal stability in the host cell. Previous results from our laboratory have shown that activation of the MEK1/2-ERK signaling pathway in plasmacytoid dendritic cells leads to inhibition of IFNα production. The aim of my work was to determine whether HBx activates the MEK1/2-ERK pathway and thus inhibits IFN type I production also in hepatocytes. For this purpose, I monitored HBx production in the Huh7 hepatoma cell line by transfecting the bicistronic plasmid pHBx- IRES-EGFP and Western blotting. Using the same method, I monitored activation of the MEK1/2-ERK signaling pathway by ERK...
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Effect of chronic morphine on cell survival after oxidative stress in the SH-SY5Y neuroblastoma cell line
Moutelíková, Karolína ; Hejnová, Lucie (advisor) ; Musílková, Jana (referee)
Morphine is a natural opioid which is used in medicine due to his potent analgesic and sedative effects. In the forefront of scientific interest is a chronic usage of opioids which can lead to a development of drug addiction. Morphine role in oxidative stress was described in last years. It was revealed its protective potencial by many studies. However, some studies described its pro-oxidative effect. The aim of this study was to determinate effect of chronic morphine on cell survival after oxidative stress caused by H202 analog - tBHP in the SH-SY5Y neuroblastoma cell line. The results verified morphine protective effect against oxidative stress. The highest protective effect of morphine was achieved in a concetration of 10 µM. It was desribed that morphine can induce activation of mu-opioid (MOR) and Toll-like 4 (TLR4) receptors signalling pathway on molecular level. The aim of this thesis was to evaluate the role of MOR a TLR4 in protective effect of morphine against oxidative stress by two methods. Firstly, it was used tests of oxidative stress on cell viability. The obtained results demonstrated majority role of TLR4 and minory role of MOR. Afterwards, we assesed changes in the expression of MOR a TLR4 after chronic morphine by SDS-PAGE electrophoresis. Results of these experiments did not...
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The regulation of primary response genes by the ERK signaling pathway
Chvalová, Věra ; Vomastek, Tomáš (advisor) ; Doubravská, Lenka (referee)
The ERK signaling pathway represents an evolutionary conserved mechanism that enables cells to perceive various extracellular signals and convert them to a diverse array of biological outcomes such as proliferation, differentiation, cell cycle control, apoptosis or cell migration. Key components of this pathway are protein kinases Raf, MEK and the effector protein kinase ERK. In addition to its physiological role, continuous activation of the ERK pathway caused by somatic mutations of some of its components or upstream regulators appears to be significant cause of many human tumor diseases. That is why this pathway plays an important role also from the biomedical viewpoint. The multistep changes in gene expression are primarily responsible for these physiological and pathological events. Changes in genes expression are induced by activated kinase ERK that after translocation into the nucleus phosphorylates transcription factors (TFs) whose activation, in turn, leads to transcription of so-called immediate early genes (IEGs), many of which also code for other TFs (e.g. c-Fos, c-Jun or c-Myc). The latter TFs then regulate expression of further genes for structural and signaling proteins. This causes global changes in gene expression and leads to functional reprogramming of the cells. This thesis...
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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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Effect of small DNA viruses on regulation of interferon production
Hofman, Tomáš ; Hirsch, Ivan (advisor) ; Elleder, Daniel (referee)
Plasmacytoid dendritic cells (pDC) represent innate immune cells capable to detect viruses in their endosomal environment via Toll-like receptors (TLRs). Viral nuclear acid recognition leads to the massive production of type I interferon (IFN I) and induction of the antiviral state in uninfected cells. Crosslinking of the surface regulatory receptors, such as BDCA-2, with monoclonal antibodies or with some viruses leads to the activation of MEK1/2- ERK signaling pathway and inhibition of IFN I production in pDC. In this study, the role of MEK1/2 kinase has been highlighted. Its inhibition reversed the inhibitory effect of BDCA-2 crosslinking and its direct activation with PMA led to the inhibition of IFN-α production. Yet an unclear role of pDC in sensing of BK polyomavirus virus (BKV) responsible for kidney transplant rejection was investigated as a major topic of this thesis. Experiments with the pDC cell line Gen2.2 and HRPTEC primary cell line showed that pDCs were not able to detect BKV particles, however, exposure of activated Gen2.2 cells to BKV inoculum dramatically upregulated production of IFN-α. Most importantly, coculture of Gen2.2 cells with BKV- infected HRPTEC cells resulted in IFN-α and TNF-α production, which was prevented by Bafilomycin. These results suggest that BKV-infected...
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Renal carcinoma bological therapy and the role of cell signaling checkpoints
Černá, Kristýna ; Otavová, Katarína (advisor) ; Tolde, Ondřej (referee)
Principles of targeted biological treatment of metastatic renal cell carcinoma include mainly inhibitors of the tyrosine kinase receptors VEGFR and inhibitors of intracellular mTOR kinase. Across the new healing regimes there are the blockades of immune checkpoints of the immune system cell. Detailed molecular characterization of tumor is necessary not for only aplication of medicaments, but also for the development of drugs that target specific molecular pathway of cell signalization of the carcinoma cells. The work is focused on the description of the signaling pathway mTOR and VEGF in metastatic renal cell carcinoma. It summarizes all validated clinical biomarkers which are used to diagnose and stratify patients for the treatment of mRCC. It also offers insight into the present experiments that are finding new specific molecular markers. That may be the future solution for customized approach in the treatment of renal carcinoma an tumors in general.
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