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Physiological functions of muscarinic receptor M3
Urbánková, Anna ; Randáková, Alena (advisor) ; Rudajev, Vladimír (referee)
Muscarinic receptors belong to the family of G-protein coupled receptors. G-proteins are heterotrimeric GTP-binding proteins that transfer signals from receptors to effectors. Effectors change levels of second messengers in the cell. Individual subtypes of muscarinic receptors bind to various classes of G-proteins. According to the G-protein coupling, muscarinic receptors change levels of various second messengers. Individual subtypes of the muscarinic receptor also differ in location and function. The M3 muscarinic receptor subtype is located primarily on the periphery where it mediates smooth muscle contraction and endocrine and exocrine secretion. The goal of this bachelor thesis was to describe the tissue-specific signalling pathways of the M3 receptor and their physiological roles in the periphery as well as in the central nervous system. Further, the role of M3 receptors in several pathologies is described. Finally, the M3 receptors as a possible pharmacological target will be discussed. Key words: muscarinic receptors M3, cell signaling, G-proteins
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Extracellular vesicles and middle T antigen of mouse polyomavirus
Kropáček, Václav ; Šroller, Vojtěch (advisor) ; Brázdová, Andrea (referee)
This study is focused on middle tumor antigen (MT Ag) of mouse polyomavirus (MPyV), potential consequences of it's secretion via extracellular vesicles (EVs) and it's effect on cellular signaling. MT Ag is membrane bound protein able to induce cellular transformation thanks to it's ability to interfere with cellular signal transduction. Mainly due to aberrant activation of MAP kinase pathway. Firstly we followed up previous observations of our group concerning ability of MT Ag to be secreted from cells via extracellular vesicles. We were interested if MT Ag could contribute to malignant transformation in recipient cells. We performed 2 types of EVs isolation from cell lines stably expressing middle T antigen (3T6MT). We confirmed presence of MT Ag in isolated EVs. Then we characterized isolated EVs by detection of exosomal markers and cryo-electron microscopy. In next step we exposed recipient cell line (3T6) to isolated EVs and with use of flow cytometry tried to detect internalization of MT Ag. Simultaneously we tried asses levels of Erk phosphorylation in 3T6 cells exposed to EVs. Secondly we tried to confirm and analyse previous unpublished observations of elevated levels of NF-kB phosphorylation in cells stably expressing MT Ag. We used western blot and detection of NF-kB dependent secreted...
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Physiological functions of muscarinic receptor M3
Urbánková, Anna ; Randáková, Alena (advisor) ; Rudajev, Vladimír (referee)
Muscarinic receptors belong to the family of G-protein coupled receptors. G-proteins are heterotrimeric GTP-binding proteins that transfer signals from receptors to effectors. Effectors change levels of second messengers in the cell. Individual subtypes of muscarinic receptors bind to various classes of G-proteins.According to the G-protein coupling, muscarinic receptors change levels of various second messengers. Individual subtypes of the muscarinic receptor also differ in location and function. The M3 muscarinic receptor subtype is located primarily on the periphery where it mediates smooth muscle contraction and endocrine secretion. The goal of this bachelor thesis was to describe the tissue-specific signalling pathways of the M3 receptor and their physiological roles in the periphery as well as in the central nervous system. Further, the role of M3 receptors in several pathologies is described. Finally, the M3 receptors as a possible pharmacological target will be discussed. Key words: muscarinic receptors M3, cell signaling, G-proteins
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Liver cells regeneration in mammals
Ťažký, Timotej ; Tlapáková, Tereza (advisor) ; Onhajzer, Jakub (referee)
Liver cell regeneration is an important biological process that allows mammals to maintain liver function while recovering from liver damage. Liver cell proliferation serves as the primary mode of liver regeneration, which in hepatocytes is activated by the transition from the G0 to G1 phase of the cell cycle. Proliferation is also promoted by non-parenchymal liver cells among which include Ito cells, Kupffer cells, and endothelial cells of hepatic sinusoids. In a comprehensive analysis of key signaling pathways, it was clearly demonstrated that the Wnt/β catenin, Notch, Hippo, NF-κB, and Hedgehog signaling pathways play a key role in the regulation of liver cell proliferation and differentiation during regeneration. The regenerative potential of the liver is influenced by various factors such as age, extent of damage and health conditions. Additionally, the remarkable regenerative capacity of the liver has clinical implications in the context of liver transplantation, partial hepatectomy and the treatment of liver diseases such as cirrhosis, hepatitis and hepatocellular or cholangiocellular carcinoma. Modulation of key signaling pathways and identification of novel molecular targets can improve the clinical outcomes of patients with liver diseases or even accelerate the entire process of liver...
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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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Structural and functional studies of MICAL signalling in cytoskeletal dynamics
Rožová, Dominika ; Rozbeský, Daniel (advisor) ; Sulimenko, Vadym (referee)
The main focus of this project was chicken protein MICAL1, which is involved in the semaphorin-plexin signalling pathway and has a significant effect on the rearrangement of the cytoskeleton. The prominent role of the MICAL1 protein is primarily associated with axon guidance, as it destabilizes actin filaments through its oxidative activity. We focused on elucidating the molecular mechanisms of chicken MICAL1 autoinhibition using molecular and structural biology methods together with new protein structure prediction methods. Chicken MICAL1 was produced in Sf9 insect cells using a baculovirus expression system and we produced both full-length and truncated versions of chicken MICAL1 protein. We kinetically characterized the protein and determined its oligomeric state in solution. We made great efforts to solve the protein structure using crystallography, electron microscopy and protein structure prediction in Alphafold 2. Based on the results of these experiments and assays, we conclude that MICAL1 proteins are regulated through their C terminal domain, which interacts with the monooxygenase domain. The part of this interaction is the autoinhibition of chicken MICAL1. We excluded the possibility that chicken MICAL1 is regulated by changing its oligomeric state. The results of this master's thesis...
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Recombinant preparation of DNA binding domain of transcription factor TEAD1
Kúdelová, Veronika ; Novák, Petr (advisor) ; Dračínská, Helena (referee)
TEAD proteins belong to a significant family of transcription factors that contribute to the regulation of organism growth and cell differentiation during its development by activating the expression of a wide variety of genes. This family shares two highly conserved sites, the TEA DNA binding domain, after which the proteins have been named, and the domain by which transcription factors bind other coactivators. Because TEAD proteins are not able to activate transcription themselves, they interact with a number of coactivators. These coactivators allow the transcription of the gene of interest to be regulated. Failure of TEAD protein activity regulation can lead to cancer. Therefore, TEAD family proteins nowadays play an important role in the development of new anticancer drugs. One way of inhibiting these proteins is to block the active site in their DNA binding domain, thus, to block their binding to DNA. This bachelor thesis deals with recombinant expression of said DNA binding domain of transcription factor TEAD1, which is extended by amino acids in unstructured regions. After finding suitable conditions of protein production, we proceeded to large volume production which was followed by purification and protein identity verification. Finally, the ability of the produced protein to interact...
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Adenosine metabolism and its role in cell physiology
Neumannová, Kateřina ; Novotný, Jiří (advisor) ; Hansíková, Jana (referee)
Adenosine is not just a major component of important molecules such as ATP, RNA or cAMP, but also has its own signaling function. Therefore, its extracellular level is strictly maintained by balance in its formation, degradation and transport. Both inside and outside the cell adenosine is formed mainly through degradation of ATP and is eliminated by two enzymes, adenosine kinase and adenosine deaminase. Transport of adenosine through the cell membrane is provided by nucleoside transporters, which are either equilibrative or concentrative according to the mechanism of transfer. All three processes described above contribute to maintaining adenosine level under normal conditions and its increase in pathological situations. Extracellular adenosine as a signal molecule binds to adenosine receptors (subtypes A1, A2A, A2B, A3) that affect many cellular signaling pathways via G-proteins. By these pathways adenosine regulates energy homeostasis, controls the function of various organs and also modulates the nervous and immune system and thus it may participate in a number of pathological processes. Pharmacological affecting of specific adenosine receptors or enzymes involved in its metabolism can serve as an effective therapy. Some drugs based on this system are already in use, others are being tested, and many...
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Non-conventional bacterial signaling pathways
Krupička, Jiří ; Branny, Pavel (advisor) ; Beranová, Jana (referee)
Two component systems were traditionally considered as main phosphorylation systems of bacteria involved in cell signalling. Recently, attention focuses increasingly on bacterial eukaryote-like Ser/Thr protein kinases (eSTKs). These protein kinases are structurally similar to their eukaryotic counterparts. Some eSTKs possess additional domains such as extracellular PASTA domains that were discovered in a variety of gram-positive bacteria. It has been proved that these domains can act as sensors for unlinked peptidoglycan fragments. However, majority of environmental signal molecules still remains unknown. eSTKs phosphorylate a broad spectrum of substrates including proteins involved in various cell processes such as virulence, cell wall biosynthesis, cell division, and central and secondary metabolism. Cross talk between eSTKs and two component systems also occurs. In this thesis, the current knowledge about eSTKs and their significant substrates in different bacterial species is discussed.
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Mechanisms of MHCII signaling in B lymphocytes
Kotlabová, Klára ; Brdička, Tomáš (advisor) ; Černý, Jan (referee)
During the initiation of an antigen-specific immune response, peptide fragments originating from the antigen are presented in complex with MHC class II glycoproteins (MHCgpII) on the surface of the antigen presenting cells (APC). Antigen recognition by T lymphocyte is accompanied by the formation of the molecular structure at the interface with APC called immunological synapse (IS). During this contact, signal transduction is initiated at both, T lymphocyte and APC, sides of the IS. For a long time it was thought that the only function of MHCgpII is presentation of antigen. However, later it was found that stimulation of MHCgpII led to triggering of signals contributing to decision about the further fate of APC. MHCgpII do not have any signaling motifs in their cytoplasmatic domains, and so associated molecules are necessary for the transduction of the signals. This work focuses on B lymphocytes in which the associated molecules are Ig alfa/beta, MPYS, CD19 and CD20. After the stimulation of MHCgpII these proteins mediate signaling events including activation of several families of protein kinases, phospholipase C, mobilization of calcium and activation of transcriptional factors NFAT and AP-1. In B lymphocytes, activities of these pathways may result in proliferation and differentiation but also in the...
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