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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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Role of the serotonin receptor type 7 (5-HT7) in autoimmune disease
NUßBAUMER, Mia
Serotonin receptor type 7 (5-HT7) displays immunomodulatory functions and is overexpressed in individuals with Crohn's disease. Here, we set out to determine 5-HT7 mode of interaction with its cognate proteins Gs and G12. A unique type of coupling, termed 'inverse coupling' is known to occur between the 5-HT7 receptor and the Gs protein. The aim of our research was to determine whether G12 can also undergo this kind of coupling. We found that the G12 protein has a surprisingly lower mobility than Gs implying a specific interaction with a membrane domain or another membrane-localized protein. For both G proteins, we were unable to detect their inverse coupling with the 5-HT7 receptor at room temperature. Our findings indicate peculiar differences in G protein mobility and emphasize the importance of temperature for studies of interactions between G proteins and G protein-coupled receptors.
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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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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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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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Role of oxidative stress in cardioprotection induced by exercise.
Kyclerová, Eva ; Nováková, Olga (advisor) ; Kašparová, Dita (referee)
Cardiovascular diseases are the major cause of death in developed countries. It is known that heart muscle can activates endogenous protective pathways in response to stress, thereby increasing resistance against ischemia/reperfusion (I/R) injury. Protective pathways involve many signaling molecules and reactive oxygen species (ROS) play an important role among them. ROS are applied in cardioprotection induced by various stimuli, such as chronic hypoxia, preconditioning and also physical exercise. It has been demonstrated that regular physical exercise naturally leads to the positive adaptation to protect heart against injury. The balance between production of ROS and their removal by antioxidant protection system is important for the right functioning of the heart. The overproduction of ROS occurs in pathological conditions such as an I/R leading to oxidative stress contributing to subsequent damage of heart. ROS may contribute not only to the injury but in the mild concentrations, resulting for example from physical exercise, ROS are important signaling molecules involved in series of events leading to cardioprotection. Slightly increased oxidative stress protects the heart by increasing the capacity of antioxidant system, stimulates angiogenesis, activates mitochondrial biogenesis and physiological...
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Study of the unique signaling pathway of Ser/Thr protein kinase StkP and phosphatase PhpP in Streptococcus pneumoniae
Keil, Jan ; Ulrych, Aleš (advisor) ; Bobková, Šárka (referee)
The major human pathogen Streptococcus pneumoniae is a unique model for the study of eukaryotic-type serine/threonine protein kinases and its cognate phosphatases in bacteria, since it encodes only a single signaling pair composed of the StkP protein kinase and PhpP phosphatase. This signaling pair plays a role in several cellular processes, mainly in cell wall biosynthesis and cell division. StkP and PhpP proteins with a pleiotropic effect appear to regulate a complex signaling cascade by phosphorylation of many substrates. However, only a few have been characterized so far. Using MS analysis, we have identified about 90 phosphopeptides that are potential substrates for the StkP kinase and PhpP phosphatase. This diploma thesis is focused on the characterization of the new substrate Spr0929 and its role in pneumococcal physiology. One of the objectives was to investigate cell morphology of strains carrying deletion of the spr0929 gene in different genetic backgrounds. It turned out that the role of Spr0929 in cell morphology is strain specific. The growth curves of strains with this deletion were compared to that of the wild type in various physiological conditions as well. As Spr0929 contains a nucleoid-associated domain called NdpA, determination of its cell localization was an important...
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