Cytoskeleton in Mast Cell Signaling

Mast cell activation mediated by the high affinity receptor for IgE (FcεRI) is a key event in allergic response and inflammation. Other receptors on mast cells, as c-Kit for stem cell factor and G protein-coupled receptors (GPCRs) synergistically enhance the FcεRI-mediated release of inflammatory mediators. Activation of various signaling pathways in mast cells results in changes in cell morphology, adhesion to substrate, exocytosis, and migration. Reorganization of cytoskeleton is pivotal in all these processes. Cytoskeletal proteins also play an important role in initial stages of FcεRI and other surface receptors induced triggering. Highly dynamic microtubules formed by αβ-tubulin dimers as well as microfilaments build up from polymerized actin are affected in activated cells by kinases/phosphatases, Rho GTPases and changes in concentration of cytosolic Ca2+. Also important are nucleation proteins; the γ-tubulin complexes in case of microtubules or Arp 2/3 complex with its nucleation promoting factors and formins in case of microfilaments. The dynamic nature of microtubules and microfilaments in activated cells depends on many associated/regulatory proteins. Changes in rigidity of activated mast cells reflect changes in intermediate filaments build up from vimentin. This review offers a critical appraisal of current knowledge on the role of cytoskeleton in mast cells signaling

Identification of new regulators of proinflammatory signaling pathways

Identifikace nových regulátorů prozánětlivých signálních drah Helena Dráberová V imunitním systému byla popsána funkce proteinu 4.1R, který reguluje migraci a adhezi buněk, ale také aktivaci T lymfocytů. Polymorfismus v genu pro ORMDL-3 představuje rizikový faktor pro vznik astmatu u dětí a souvisí se zvýšenou expresí ORMDL-3. Tato dizertační práce popisuje funkci proteinů 4.1R a ORMDL-3 v aktivaci žírných buněk po stimulaci FcεRI receptoru. IL-17 je prozánětlivý cytokin, který hraje roli v obraně organismu před kvasinkovými a houbovými infekcemi. IL-17 však přispívá k patologii autoimunitních chorob jako jsou revmatoidní artritida, psoriáza a roztroušená skleróza. IL-17 signalizace je velmi přísně regulovaná, ale přesný mechanismus regulace doposud nebyl popsán. Tato dizertační práce se soustředí na charakterizaci IL-17R komplexu pomocí hmotnostní spektrometrie a analýzu funkce jeho známých i nových komponent v buňkách deficientních v jednotlivých proteinech metodou CRISPR-Cas9. Poslední část se zabývá objevem zcela nové podjednotky IL-17RC proteinem CMTM4, jehož role v IL-17 signalizaci nebyla dosud prozkoumána. CMTM4 stabilizuje IL-17RC a je nezbytný pro IL-17RC povrchovou expresi. In vitro data jsou podpořeny daty z autoimunitního myšího modelu psoriázy. Výsledky naznačují nový potenciální terapeutický...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...Katedra buněčné biologieDepartment of Cell BiologyPřírodovědecká fakultaFaculty of Scienc

γ-Tubulin 2 Nucleates Microtubules and Is Downregulated in Mouse Early Embryogenesis

γ-Tubulin is the key protein for microtubule nucleation. Duplication of the γ-tubulin gene occurred several times during evolution, and in mammals γ-tubulin genes encode proteins which share ∼97% sequence identity. Previous analysis of Tubg1 and Tubg2 knock-out mice has suggested that γ-tubulins are not functionally equivalent. Tubg1 knock-out mice died at the blastocyst stage, whereas Tubg2 knock-out mice developed normally and were fertile. It was proposed that γ-tubulin 1 represents ubiquitous γ-tubulin, while γ-tubulin 2 may have some specific functions and cannot substitute for γ-tubulin 1 deficiency in blastocysts. The molecular basis of the suggested functional difference between γ-tubulins remains unknown. Here we show that exogenous γ-tubulin 2 is targeted to centrosomes and interacts with γ-tubulin complex proteins 2 and 4. Depletion of γ-tubulin 1 by RNAi in U2OS cells causes impaired microtubule nucleation and metaphase arrest. Wild-type phenotype in γ-tubulin 1-depleted cells is restored by expression of exogenous mouse or human γ-tubulin 2. Further, we show at both mRNA and protein levels using RT-qPCR and 2D-PAGE, respectively, that in contrast to Tubg1, the Tubg2 expression is dramatically reduced in mouse blastocysts. This indicates that γ-tubulin 2 cannot rescue γ-tubulin 1 deficiency in knock-out blastocysts, owing to its very low amount. The combined data suggest that γ-tubulin 2 is able to nucleate microtubules and substitute for γ-tubulin 1. We propose that mammalian γ-tubulins are functionally redundant with respect to the nucleation activity

Forests Evaluation in the Czech Republic

Import 04/07/2011Cílem diplomové práce je provést tržní a administrativní ocenění lesa, srovnání použitých metod a zjištěných výsledků. Administrativní ocenění je provedeno dle oceňovací vyhlášky platné ke dni oceňování lesa. Při tržním ocenění je použita metoda porovnávací a metoda výnosová. V závěru práce je provedeno zhodnocení použitých metod a jsou uvedeny návrhy změn týkající se oceňování lesů v České republice.The main aim of this thesis is market and administrative evaluation of forest and comparison of used method and calculated results. The administrative evaluation is done according to evaluation edict valid in the day of the evaluation. The market evaluation is done with the comparative method and the yield methods. In the end of the thesis is done review of used methods and there are mentioned proposals of changes relating to forests evaluation in the Czech republic.153 - Katedra veřejné ekonomikyvýborn

Identification of new regulators of proinflammatory signaling pathways

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..