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Topography of signaling molecules on the plasma membrane in the course of mast cell activation
Lebduška, Pavel ; Dráber, Petr (advisor) ; Šebo, Peter (referee) ; Benada, Oldřich (referee) ; Tučková, Ludmila (referee)
presented technique of plasma membrane sheet preparation from nonadherent cells may facilitate research in this field. It must be, however, mentioned that a plastic view ofsignal transduction across the plasma membrane can be achieved only by combination of various mutually complementary approaches. Conclusions Three techniques of lsoladon of plasmr m€mbrane sh$ts from nonadherent BMMC mast cells have bmn developed. one of them, based on edsorption ofl€ukocýes to glass suďace, turned out to be very promlsing md provided many scientifrc datr(article E). Actlvation of RBL m9st c€l|s by FGRI raeptor dimerintion led to increme of Grb2 adaptor content in the Plasma membrane. Ilowever' by contřast to the case of receptor mu|t|merintion, this Grb2 did not sign|ficantly colocallz€ w|th FERI' and' by |mmuno|rbeling of membrane she€ts, distribution of FC5RI wrs not d|stinguishabl€ from the disfibution on nonact|vrted ce|ls (article A). BMMC, In contrast to RBL cells, after multimerization of FaRI did not form larger aggregat€s ofthis r€c€ptor thrn nonact|vat€d cells did. FGRI muldmer|ation led to lts int€rna|iation of comparable intensity rnd overa|l dynemics ln BMMC end RBL cel|s' but loce| redistribut|on of FaRI fundamentďly differed betwcn these two c€|| wes (article E). Established mode| oflrrg€ (8pproxim8t€|y...
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Multiple regulatory roles of the transmembrane adaptor protein NTAL in gene transcription and mast cell physiology
Polakovičová, Iva ; Dráber, Petr (advisor) ; Vyklický, Ladislav (referee) ; Hašek, Jiří (referee)
(EN) This thesis focuses mainly on understanding of the regulatory roles of the transmembrane adaptor proteins, non-T cell activation linker (NTAL) and phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), in murine mast cell signaling. There are conflicting reports on the role of NTAL in the high affinity immunoglobulin E receptor (FcεRI) activation pathways in mast cells. Studies carried out on mast cells prepared from NTAL knock-out mice have indicated that NTAL is a negative regulator of FcεRI signaling, whereas experiments performed on human mast cells and rat basophilic leukemia cells with silenced NTAL expression have suggested its positive regulatory role. To thoroughly examine the involvement of NTAL in FcεRI-mediated signaling events in mouse mast cells and to determine whether different methodologies of NTAL ablation have different physiological consequences, we utilized a broad range of assays. Using bone marrow-derived mast cells (BMMCs) as a model, we obtained cells from NTAL wild type and knock-out cells and using lentiviral delivery approach we transduced part of the wild type cells, with vector bearing NTAL shRNA or empty vector to generate NTAL knock-down cells and control cells, respectively. Comparison of all four groups of generated cells in our assays...
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Molecular mechanisms of regulation of FcɛRI signaling in mast cells
Bambousková, Monika ; Dráber, Petr (advisor) ; Černý, Jan (referee) ; Bilej, Martin (referee)
Mast cells are critical component of the immune system. In pathological situations, they are activated and are responsible for allergic reaction. Therefore, detail understanding of mast cell activation at molecular level is important for design of new therapies of allergic diseases. Principal transmembrane receptor of mast cells is the high-affinity Fc receptor for IgE (FcεRI). FcεRI anchors IgE on mast cell surface and upon cross-linking with multivalent antigen it becomes phosphorylated at its intracellular immunoreceptor tyrosine-based activation motifs (ITAMs). This triggers signaling cascade leading to cell degranulation and cytokine production. The antigen- mediated signaling through the FcεRI is critically dependent on interplay with intracellular protein- tyrosine kinases that phosphorylate the ITAM motifs and many other components of the signaling pathway. This study was focused on better understanding of signaling events leading to mast cell activation; emphasis was put on early activation events. First, we examined the role of protein- tyrosine phosphatases (PTP) in FcεRI phosphorylation. We found that upon antigen triggering of FcεRI, PTPs undergo inhibition by oxidation of their active site located tyrosine. Studies of plasma membrane topography of inactivated PTPs showed their...
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The role of ORMDL proteins in mast cell signaling
Paulenda, Tomáš ; Dráber, Petr (advisor) ; Heneberg, Petr (referee) ; Malý, Petr (referee)
1. Abstract (EN) This thesis is collection of work focused mainly on the understanding of mast cell activation and its regulation by Orm1-like (ORMDL) proteins. ORMDL family is a group of endoplasmic reticulum (ER) membrane resident proteins that are highly conserved amongst mammalian species. ORMDL proteins can be found in diverse range of organisms from plants through fungi to animals. ORMDL proteins were first discovered in yeasts and the interest in these proteins skyrocketed after the discovery that ORMDL3 is associated with childhood onset asthma in genome wide association studies. Following research connected ORMDL3 also with allergic inflammation and inflammatory bowel disease. Since mast cells are mainly known for their role in allergy and allergen induced inflammation, we decided to investigate the role of ORMDL proteins in regulation of mast cell activation and signaling. In our first study we focused on the role of ORMDL3 in mast cell activation via the high affinity IgE receptor 1 (FcεRI). We prepared bone marrow-derived mast cells with decreased (ORMDL3-KD) or increased (ORMDL3-OE) ORMDL3 expression. We showed that ORMDL3 is a negative regulator of mast cell activation events like degranulation, cytokine release and migration, without any effect on calcium mobilization. ORMDL3 was previously...
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Involvement of Asthma-associated Protein ORMLDL3 in Mast Cell Signalling
Eitler, Jiří ; Dráber, Petr (advisor) ; Konvalinka, Jan (referee)
4 Abstract Mast cells are involved in variety of immunological processes, but they are mostly known for their role in allergy and asthma. As asthma and allergy are serious diseases with spreading tendency during last decades, mast cells are subject of intensive research. It is expected that studies of mast cell signalling pathways will contribute to our understanding of the nature of these diseases and help to design efficient treatment strategies. In an attempt to identify genes responsible for asthma disease, genome-wide screening methods have been currently applied. Using these methods, mutations in ORMDL3 (Orosomucoid1-like) protein were found out as a high risk asthma factor. ORMDL3 is a member of evolutionary conserved ORMDL family, comprising in mammals also of ORMDL1 and ORMDL2. Physiological function of these proteins is poorly understood and it has not been studied in mast cells. We decided to study the role of ORMDL proteins in mast cells. Lentiviral delivery system was optimised for generation of stable knock-downs (KD) of all three members of the ORMDL family in primary mast cells. The ORMDL gene expression was measured by improved qPCR (quantitative PCR) reaction buffers. We found that all ORMDL genes are expressed in mast cells in order ORMDL3 > ORMDL2 > ORMDL1. Next, we investigated the...
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The role of ORMDL proteins in mast cell signaling
Paulenda, Tomáš ; Dráber, Petr (advisor) ; Heneberg, Petr (referee) ; Malý, Petr (referee)
1. Abstract (EN) This thesis is collection of work focused mainly on the understanding of mast cell activation and its regulation by Orm1-like (ORMDL) proteins. ORMDL family is a group of endoplasmic reticulum (ER) membrane resident proteins that are highly conserved amongst mammalian species. ORMDL proteins can be found in diverse range of organisms from plants through fungi to animals. ORMDL proteins were first discovered in yeasts and the interest in these proteins skyrocketed after the discovery that ORMDL3 is associated with childhood onset asthma in genome wide association studies. Following research connected ORMDL3 also with allergic inflammation and inflammatory bowel disease. Since mast cells are mainly known for their role in allergy and allergen induced inflammation, we decided to investigate the role of ORMDL proteins in regulation of mast cell activation and signaling. In our first study we focused on the role of ORMDL3 in mast cell activation via the high affinity IgE receptor 1 (FcεRI). We prepared bone marrow-derived mast cells with decreased (ORMDL3-KD) or increased (ORMDL3-OE) ORMDL3 expression. We showed that ORMDL3 is a negative regulator of mast cell activation events like degranulation, cytokine release and migration, without any effect on calcium mobilization. ORMDL3 was previously...
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Phosphatidylserine and phospholipid scramblase in mast cell signaling
Smrž, Daniel ; Dráber, Petr (advisor) ; Bezouška, Karel (referee) ; Šebo, Peter (referee)
6. CONCLUSIONS 1. We found that mast cell stimulation can induce PS externalization in the absence of secretory response. 2. We identified GPI-APs as molecules whose engagement can induce sustained and reversible non-apoptotic PS externalization. 3. GPI-AP-induced PS externalization was determined as non- apoptotic and distinct from the FceRl-induced PS externalization. 4. The effect of multiple triggering on PS externalization was additive and dependent on a tlpe of stimulus and cells engaged. 5. We identifred PLSCR1 as a molecule that becomes tyrosine phosphorylated in mast cells stimulated through GPI-APs. 6. We found that the PLSCR1 tyrosine phosphorylation is not associated with mast cell secretory response' and with GPI-AP- or FceRl-induced non-apoptotic PS externalization. 7. Using confocal microscopy and electron microscopy visualization of PLSCR1 in the course of mast cell activation we found that PLSCR1: (1) is not co-localized with extemalized PS, (2) is not co- localized with aggregated Thy-l.l or FceR[, and (3) does not form self-aggregates. 8. We děřelóped a modifred one-tube semi-nested PCR-ELISA. The modified assay showed higher sensitivity and specificity than the conventional hybridization-based and a modified semi-nested- based PCR-ELISA. Due to its versatility and robustness, the...
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