velikost textu

New signaling pathways involved in mast cell activation and cell membrane repair

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Název:
New signaling pathways involved in mast cell activation and cell membrane repair
Typ:
Disertační práce
Autor:
Gouse Mohiddin Shaik, Ph.D.
Školitel:
doc. RNDr. Petr Dráber, DrSc.
Oponenti:
doc. RNDr. Jan Černý, Ph.D.
Ing. Jiří Hašek, CSc.
Id práce:
112816
Fakulta:
Přírodovědecká fakulta (PřF)
Pracoviště:
Katedra buněčné biologie (31-151)
Program studia:
Fyziologie a imunologie (P1511)
Obor studia:
Imunologie (XIMUN)
Přidělovaný titul:
Ph.D.
Datum obhajoby:
25. 11. 2008
Výsledek obhajoby:
Prospěl/a
Jazyk práce:
Angličtina
Abstrakt:
    RESULTS       RESULTS AND DISCUSSION Vacuolin-1-induced changes in mast cells morphology In pilot experiments we determined the effect of vacuolin-1 on mast cell morphology. Incubation of RBL-2H3 cells or BMMCs with 10 μM vacuolin-1 in complete culture media for 3 h resulted in appearance of numerous vacuoles in cytosolic compartment of the cells (Fig 1A-D). Size of the vacuolin-1-treated cells rose (Fig 1E and F) as reflected by mean increase in diameter of RBL-2H3 cells from 14.8 ± 0.2 μm (mean ± S.D., n = 3) to 18.8 ± 0.3 μm, and BMMCs from 14.6 ± 0.2 μm to 17.3 ± 0.2 μm. To decide whether formation of vacuoles depends on specific metabolic pathways we examined cells exposed to various concentrations of pharmacological inhibitors and 10 μM vacuolin-1. Data presented in Table 1 show that most of the drugs tested, including Cl- and/or K+ channel blockers [indanyloxyacetic acid 94 (IAA-94), (dihydroindenyl)oxy/alkanoic acid (DIOA), 5- Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'- disulphonate (DIDS) and glybenclamide], protein kinase C (PKC) inhibitor (tamoxifen), phosphoinositid-3 kinase (PI3K) inhibitor (wortmannin), Src and Syk family kinase inhibitors (PP2 and piceatannol) and non-muscle myosin (NMM) II ATPase activity inhibitor (blebbistatin) had no effect on vacuolin-induced formation of vacuoles even at such high doses tested that were often toxic. The only exception was macrolide antibiotic bafilomycin A1, a potent inhibitor of vacuolar H+-ATPase (Bowman et al., 1988). Treatment with 0.01 μM bafilomycin completely inhibited vacuolin-1-induced formation of vacuoles in both RBL-2H3 cells and BMMCs without any decrease in cell viability, as confirmed by trypan blue staining (not shown); toxic effects were only observed at >50-fold higher concentrations of bafilomycin. These data indicate that vacuolar H+-ATPase is involved in vacuolin-1-induced formation of vacuoles. In its sensitivity to bafilomycin A1, vacuolin-1 resembles the Vac A toxin produced by Helicobacter pylori, which mediates an influx of anions into endosomes leading, in turn, to increased activity of vacuolar H+-ATPase, osmotic swelling and formation of vacuoles (Papini et al., 1993; Cover and Blanke, 2005). Vacuolin-1 could thus also enhance the influx of anions into lysosomes and endosomes resulting in their osmotic swelling. Inhibition of vacuole production was also observed in cells pretreated with nonlethal doses of myosin light chain (MLC) kinase inhibitors ML-7 and ML-9.       54         RESULTS       These two inhibitors were effective only in BMMCs, suggesting that MLC kinase could be responsible at least in part for the observed differences in vacuolin-1 sensitivity between RBL- 2H3 cells and BMMCs (see below). Sensitivity of exocytosis to vacuolin-1 To check whether vacuolin-1 inhibits secretory response in mast cells, RBL-2H3 and BMMCs were simultaneously sensitized with IgE and treated with 10 μM vacuolin-1. After 3 h incubation unbound IgE and vacuolin-1 were washed out, cells were activated with antigen in the presence of freshly added vacuolin-1, and the amount of β-glucuronidase released was determined. Control cells were sensitized and activated in the same way except that vacuolin-1 was replaced by vehicle [(dimethylsulfoxide (DMSO)], which at concentrations used (<1%) had no effect on secretory response (not shown). Fig 2A shows that antigen (TNP-BSA)-induced secretory response was more profound in BMMCs than in RBL-2H3 cells at both time intervals, 5 and 30 min. Vacuolin-1 enhanced slightly, but significantly, the FcεRI-mediated secretory response in RBL-2H3 cells. Unexpectedly, activation of vacuolin-1 treated BMMCs under the same conditions completely inhibited the secretory response. This inhibition was not due to a decreased amount of FcεRI on the surface of the cells determined by flow cytometry analysis (not shown). Furthermore, the cells did not bind annexin-V-fluorescein isothiocyanate (FITC) conjugate (not shown), indicating that their membranes remained intact (Smrz et al., 2008). Importantly, vacuolin-1 also induced inhibition of exocytosis in BMMCs activated in BSA-free buffered salt solution (BSS). This implies that the molecular mechanism of inhibition is more complex then expected (Steinhardt, 2005). To check whether the observed inhibitory effect was confined to FcεRI-mediated activation, we also examined the secretory response in cells activated by Ca2+ ionophore A23187 or thapsigargin. Data in Fig 2B indicate that vacuolin-1 inhibited A23187-induced secretion both in BMMCs and in RBL-2H3-cells. This inhibitory effect could be related to known multiple effects of A23187 on plasma membrane, such as decreased membrane packing measured by MC540 binding (Smrz et al., 2007), or increased plasmalemmal Ca2+ ATPase activity resulting in a decline in ATP content of the cells (Gmitter et al., 1996). It should be noted that this inhibition was independent of the presence of BSA in BSS (not shown). Interestingly, when vacuolin-1-treated cells were activated by endoplasmic reticulum ATPase inhibitor, thapsigargin, (Thastrup et al., 1989), secretory response was slightly       55         RESULTS       enhanced in RBL-2H3 (significantly at 5 min after triggering) but decreased only insignificantly in BMMCs (Fig 2C). To find out whether the observed effect of vacuolin-1 on antigen-activated cells was confined just to certain time intervals or concentrations of vacuolin, time- and dose- responses were studied. Pretreatment of RBL-2H3 cells for 15 h with 10 μM vacuolin-1 had no significant effect on spontaneous release of β-glucuronidase (Fig 2D). Enhanced secretory response in antigen-activated cells was observed at all time intervals of vacuolin-1 treatment, peaking at 12 h. In BMMCs, secretory response was inhibited in cells treated for 3 - 15 h, confirming that these cells are extremely sensitive to 10 μM vacuolin-1. To construct the dose- response curves, cells were first incubated with vacuolin-1 for 3 h and then activated with antigen. Pretreatment with various concentrations of vacuolin-1 (from 0.5 to 90 μM) enhanced in a dose-dependent manner the FcεRI-induced secretory response in RBL-2H3 cells but decreased it in BMMCs (Fig 2E). These data suggest that FcεRI-signaling pathways might be affected by vacuolin-1 in different ways, depending on cell type studied. Vacuolin-1 and tyrosine phosphorylation in BMMCs The earliest biochemically defined step in FcεRI triggering is tyrosine phosphorylation of FcεRI β and γ subunits, followed by phosphorylation of other substrates, such as linker for activation of T cells (LAT), non-T cell activation linker (NTAL), Akt and MAP kinase Erk. In further experiments we therefore tested whether the observed inhibition of exocytosis in vacuolin-1-treated and antigen-activated BMMCs could reflect an inhibition of those early signaling events. Fig 3A, documents strong tyrosine phosphorylation of FcεRI β and γ subnits in FcεRI-activated cells pretreated with vehicle alone and almost the same response in vacuolin-1- pretreated cells. Similarly, tyrosine phosphorylations of LAT and NTAL (Fig 3B), Akt (Fig 3C) and Erk (Fig 3D) were comparable in vehicle- or vacuolin-1-treated cells. All these data indicate that early receptor-mediated tyrosine phosphorylations are not affected by vacuolin-1 and are not responsible for the observed inhibitory effect of vacuolin-1 on antigen-induced exocytosis in BMMCs.       56         RESULTS       Vacuolin-1 and Ca2+ uptake in BMMCs Early FcεRI-triggered activation events are followed by release of Ca2+ from intracellular stores, and subsequent enhanced uptake of extracellular Ca2+. Because these changes are essential for exocytosis, we next examined the uptake of extracellular Ca2+ in control and vacuolin-1-treated cells. When IgE-sensitized RBL-2H3 cells or BMMCs were activated by antigen, the enhanced 45Ca2+ uptake of was comparable in both vehicle- and vacuolin-1-treated cells (Fig 4A). For comparison we also determined 45Ca2+ uptake in cells activated by A23187 and again, the uptake was comparable in both control and vacuolin-treated cells (Fig 4). A small yet significant inhibition of Ca2+ uptake was only observed in vacuolin-pretreated and thapsigargin-activated BMMCs (Fig 4C), which could explain some decrease in exocytosis in cells activated by thapsigargin (Fig 2C). The combined data indicate that signaling pathways from FcεRI to Ca2+ uptake are not affected by vacuolin-1 in FcεRI-activated BMMCs. Effect of vacuolin-1 on filamentous (F) actin formation Translocation of secretory granules and their exocytosis in mast cells is negatively regulated by F- actin (Frigeri and Apgar, 1999; Tolarová et al., 2004). We therefore tested the effect of vacuolin-1 on F-actin formation in activated cells. Our data show that the amount of F-actin in antigen-activated RBL-2H3 cells is enhanced and that vacuolin-1 significantly inhibits this rising at both time intervals chosen, 2 and 8 min (Fig 5). The observed inhibition of F-actin polymerization could explain enhanced exocytosis in vacuolin-1 treated cells. No increase, but rather decrease, in F-actin polymerization was observed in activated BMMCs and no evidence of the effect of vacuolin-1 on F-actin polymerization was obtained. Different properties of F-actin in activated RBL-2H3 cells and BMMCs and the observed differences in sensitivity of antigen- mediated exocytosis to vacuolin-1 could be related to the fact that RBL-2H3 cells are adherent and BMMCs grow in suspension. To address this issue, we used RBL-2H3 cells grown in suspension for 24 h and found that FcεRI-induced exocytosis is also enhanced in this case by vacuolin-1 (not shown). These data indicate that differences in adhesion properties of RBL-2H3 and BMMCs are not responsible for the observed differences between these two cell types.       57         RESULTS       Effect of vacuolin-1 on membrane repair Recent experiments with bacterial toxin SLO showed that membrane resealing and removal of SLO-containing pores requires Ca2+-dependent endocytosis (Idone et al., 2008). To determine whether vacuolin-1-sensitive structures are involved in membrane resealing we compared repair of SLO-permeabilized plasma membranes in control and vacuolin-1-treated cells. When RBL- 2H3 cells were permeabilized with SLO an increase in the number of propidium iodide (PI)- positive cells was observed with rising concentrations of SLO (Fig 6A, +Ca2+ and summary data in C). Pretreatment with 10 μM vacuolin-1 resulted in a significant decrease in the count of PI- positive cells at all concentrations of SLO examined, which is in accord with observed enhanced exocytosis in vacuolin-1-treated RBL-2H3 cells (Fig 2A and D). When experiments were repeated in calcium-free solutions, more RBL-2H3 cells were PI positive with the same concentrations of SLO, and vacuolin had no significant effect on PI staining (Fig 6A, -Ca2- and summary data in C). These findings support previous data showing that removal of SLO- containing membrane pores is Ca2+ dependent (Walev et al., 2001; Idone et al., 2008) and complement them by showing that vacuolin-1 promotes this process. BMMCs showed higher resistance to SLO and, importantly, treatment with vacuolin-1 did not enhance removal of membrane pores, but rather reduced it (reflected in higher percentage of PI-positive cells; the increase was significant at 3 U SLO/ml). As expected, the number of PI-positive BMMCs was enhanced in the absence of Ca2+and again vacuolin had no effect on this parameter. Thus vacuolin-1-induced inhibition of FcεRI exocytosis in BMMCs has its counterpart in the inability of vacuolin-1 to promote membrane repair after SLO permeabilization. Correlation between vacuolin-1-modulated exocytosis and membrane repair in SLO-permeabilized cells suggest a coupling of lysosomal exocytosis and endocytosis (compensatory endocytosis) in mast cells.       58    
Abstract v angličtině:
    RESULTS       RESULTS AND DISCUSSION Vacuolin-1-induced changes in mast cells morphology In pilot experiments we determined the effect of vacuolin-1 on mast cell morphology. Incubation of RBL-2H3 cells or BMMCs with 10 μM vacuolin-1 in complete culture media for 3 h resulted in appearance of numerous vacuoles in cytosolic compartment of the cells (Fig 1A-D). Size of the vacuolin-1-treated cells rose (Fig 1E and F) as reflected by mean increase in diameter of RBL-2H3 cells from 14.8 ± 0.2 μm (mean ± S.D., n = 3) to 18.8 ± 0.3 μm, and BMMCs from 14.6 ± 0.2 μm to 17.3 ± 0.2 μm. To decide whether formation of vacuoles depends on specific metabolic pathways we examined cells exposed to various concentrations of pharmacological inhibitors and 10 μM vacuolin-1. Data presented in Table 1 show that most of the drugs tested, including Cl- and/or K+ channel blockers [indanyloxyacetic acid 94 (IAA-94), (dihydroindenyl)oxy/alkanoic acid (DIOA), 5- Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'- disulphonate (DIDS) and glybenclamide], protein kinase C (PKC) inhibitor (tamoxifen), phosphoinositid-3 kinase (PI3K) inhibitor (wortmannin), Src and Syk family kinase inhibitors (PP2 and piceatannol) and non-muscle myosin (NMM) II ATPase activity inhibitor (blebbistatin) had no effect on vacuolin-induced formation of vacuoles even at such high doses tested that were often toxic. The only exception was macrolide antibiotic bafilomycin A1, a potent inhibitor of vacuolar H+-ATPase (Bowman et al., 1988). Treatment with 0.01 μM bafilomycin completely inhibited vacuolin-1-induced formation of vacuoles in both RBL-2H3 cells and BMMCs without any decrease in cell viability, as confirmed by trypan blue staining (not shown); toxic effects were only observed at >50-fold higher concentrations of bafilomycin. These data indicate that vacuolar H+-ATPase is involved in vacuolin-1-induced formation of vacuoles. In its sensitivity to bafilomycin A1, vacuolin-1 resembles the Vac A toxin produced by Helicobacter pylori, which mediates an influx of anions into endosomes leading, in turn, to increased activity of vacuolar H+-ATPase, osmotic swelling and formation of vacuoles (Papini et al., 1993; Cover and Blanke, 2005). Vacuolin-1 could thus also enhance the influx of anions into lysosomes and endosomes resulting in their osmotic swelling. Inhibition of vacuole production was also observed in cells pretreated with nonlethal doses of myosin light chain (MLC) kinase inhibitors ML-7 and ML-9.       54         RESULTS       These two inhibitors were effective only in BMMCs, suggesting that MLC kinase could be responsible at least in part for the observed differences in vacuolin-1 sensitivity between RBL- 2H3 cells and BMMCs (see below). Sensitivity of exocytosis to vacuolin-1 To check whether vacuolin-1 inhibits secretory response in mast cells, RBL-2H3 and BMMCs were simultaneously sensitized with IgE and treated with 10 μM vacuolin-1. After 3 h incubation unbound IgE and vacuolin-1 were washed out, cells were activated with antigen in the presence of freshly added vacuolin-1, and the amount of β-glucuronidase released was determined. Control cells were sensitized and activated in the same way except that vacuolin-1 was replaced by vehicle [(dimethylsulfoxide (DMSO)], which at concentrations used (<1%) had no effect on secretory response (not shown). Fig 2A shows that antigen (TNP-BSA)-induced secretory response was more profound in BMMCs than in RBL-2H3 cells at both time intervals, 5 and 30 min. Vacuolin-1 enhanced slightly, but significantly, the FcεRI-mediated secretory response in RBL-2H3 cells. Unexpectedly, activation of vacuolin-1 treated BMMCs under the same conditions completely inhibited the secretory response. This inhibition was not due to a decreased amount of FcεRI on the surface of the cells determined by flow cytometry analysis (not shown). Furthermore, the cells did not bind annexin-V-fluorescein isothiocyanate (FITC) conjugate (not shown), indicating that their membranes remained intact (Smrz et al., 2008). Importantly, vacuolin-1 also induced inhibition of exocytosis in BMMCs activated in BSA-free buffered salt solution (BSS). This implies that the molecular mechanism of inhibition is more complex then expected (Steinhardt, 2005). To check whether the observed inhibitory effect was confined to FcεRI-mediated activation, we also examined the secretory response in cells activated by Ca2+ ionophore A23187 or thapsigargin. Data in Fig 2B indicate that vacuolin-1 inhibited A23187-induced secretion both in BMMCs and in RBL-2H3-cells. This inhibitory effect could be related to known multiple effects of A23187 on plasma membrane, such as decreased membrane packing measured by MC540 binding (Smrz et al., 2007), or increased plasmalemmal Ca2+ ATPase activity resulting in a decline in ATP content of the cells (Gmitter et al., 1996). It should be noted that this inhibition was independent of the presence of BSA in BSS (not shown). Interestingly, when vacuolin-1-treated cells were activated by endoplasmic reticulum ATPase inhibitor, thapsigargin, (Thastrup et al., 1989), secretory response was slightly       55         RESULTS       enhanced in RBL-2H3 (significantly at 5 min after triggering) but decreased only insignificantly in BMMCs (Fig 2C). To find out whether the observed effect of vacuolin-1 on antigen-activated cells was confined just to certain time intervals or concentrations of vacuolin, time- and dose- responses were studied. Pretreatment of RBL-2H3 cells for 15 h with 10 μM vacuolin-1 had no significant effect on spontaneous release of β-glucuronidase (Fig 2D). Enhanced secretory response in antigen-activated cells was observed at all time intervals of vacuolin-1 treatment, peaking at 12 h. In BMMCs, secretory response was inhibited in cells treated for 3 - 15 h, confirming that these cells are extremely sensitive to 10 μM vacuolin-1. To construct the dose- response curves, cells were first incubated with vacuolin-1 for 3 h and then activated with antigen. Pretreatment with various concentrations of vacuolin-1 (from 0.5 to 90 μM) enhanced in a dose-dependent manner the FcεRI-induced secretory response in RBL-2H3 cells but decreased it in BMMCs (Fig 2E). These data suggest that FcεRI-signaling pathways might be affected by vacuolin-1 in different ways, depending on cell type studied. Vacuolin-1 and tyrosine phosphorylation in BMMCs The earliest biochemically defined step in FcεRI triggering is tyrosine phosphorylation of FcεRI β and γ subunits, followed by phosphorylation of other substrates, such as linker for activation of T cells (LAT), non-T cell activation linker (NTAL), Akt and MAP kinase Erk. In further experiments we therefore tested whether the observed inhibition of exocytosis in vacuolin-1-treated and antigen-activated BMMCs could reflect an inhibition of those early signaling events. Fig 3A, documents strong tyrosine phosphorylation of FcεRI β and γ subnits in FcεRI-activated cells pretreated with vehicle alone and almost the same response in vacuolin-1- pretreated cells. Similarly, tyrosine phosphorylations of LAT and NTAL (Fig 3B), Akt (Fig 3C) and Erk (Fig 3D) were comparable in vehicle- or vacuolin-1-treated cells. All these data indicate that early receptor-mediated tyrosine phosphorylations are not affected by vacuolin-1 and are not responsible for the observed inhibitory effect of vacuolin-1 on antigen-induced exocytosis in BMMCs.       56         RESULTS       Vacuolin-1 and Ca2+ uptake in BMMCs Early FcεRI-triggered activation events are followed by release of Ca2+ from intracellular stores, and subsequent enhanced uptake of extracellular Ca2+. Because these changes are essential for exocytosis, we next examined the uptake of extracellular Ca2+ in control and vacuolin-1-treated cells. When IgE-sensitized RBL-2H3 cells or BMMCs were activated by antigen, the enhanced 45Ca2+ uptake of was comparable in both vehicle- and vacuolin-1-treated cells (Fig 4A). For comparison we also determined 45Ca2+ uptake in cells activated by A23187 and again, the uptake was comparable in both control and vacuolin-treated cells (Fig 4). A small yet significant inhibition of Ca2+ uptake was only observed in vacuolin-pretreated and thapsigargin-activated BMMCs (Fig 4C), which could explain some decrease in exocytosis in cells activated by thapsigargin (Fig 2C). The combined data indicate that signaling pathways from FcεRI to Ca2+ uptake are not affected by vacuolin-1 in FcεRI-activated BMMCs. Effect of vacuolin-1 on filamentous (F) actin formation Translocation of secretory granules and their exocytosis in mast cells is negatively regulated by F- actin (Frigeri and Apgar, 1999; Tolarová et al., 2004). We therefore tested the effect of vacuolin-1 on F-actin formation in activated cells. Our data show that the amount of F-actin in antigen-activated RBL-2H3 cells is enhanced and that vacuolin-1 significantly inhibits this rising at both time intervals chosen, 2 and 8 min (Fig 5). The observed inhibition of F-actin polymerization could explain enhanced exocytosis in vacuolin-1 treated cells. No increase, but rather decrease, in F-actin polymerization was observed in activated BMMCs and no evidence of the effect of vacuolin-1 on F-actin polymerization was obtained. Different properties of F-actin in activated RBL-2H3 cells and BMMCs and the observed differences in sensitivity of antigen- mediated exocytosis to vacuolin-1 could be related to the fact that RBL-2H3 cells are adherent and BMMCs grow in suspension. To address this issue, we used RBL-2H3 cells grown in suspension for 24 h and found that FcεRI-induced exocytosis is also enhanced in this case by vacuolin-1 (not shown). These data indicate that differences in adhesion properties of RBL-2H3 and BMMCs are not responsible for the observed differences between these two cell types.       57         RESULTS       Effect of vacuolin-1 on membrane repair Recent experiments with bacterial toxin SLO showed that membrane resealing and removal of SLO-containing pores requires Ca2+-dependent endocytosis (Idone et al., 2008). To determine whether vacuolin-1-sensitive structures are involved in membrane resealing we compared repair of SLO-permeabilized plasma membranes in control and vacuolin-1-treated cells. When RBL- 2H3 cells were permeabilized with SLO an increase in the number of propidium iodide (PI)- positive cells was observed with rising concentrations of SLO (Fig 6A, +Ca2+ and summary data in C). Pretreatment with 10 μM vacuolin-1 resulted in a significant decrease in the count of PI- positive cells at all concentrations of SLO examined, which is in accord with observed enhanced exocytosis in vacuolin-1-treated RBL-2H3 cells (Fig 2A and D). When experiments were repeated in calcium-free solutions, more RBL-2H3 cells were PI positive with the same concentrations of SLO, and vacuolin had no significant effect on PI staining (Fig 6A, -Ca2- and summary data in C). These findings support previous data showing that removal of SLO- containing membrane pores is Ca2+ dependent (Walev et al., 2001; Idone et al., 2008) and complement them by showing that vacuolin-1 promotes this process. BMMCs showed higher resistance to SLO and, importantly, treatment with vacuolin-1 did not enhance removal of membrane pores, but rather reduced it (reflected in higher percentage of PI-positive cells; the increase was significant at 3 U SLO/ml). As expected, the number of PI-positive BMMCs was enhanced in the absence of Ca2+and again vacuolin had no effect on this parameter. Thus vacuolin-1-induced inhibition of FcεRI exocytosis in BMMCs has its counterpart in the inability of vacuolin-1 to promote membrane repair after SLO permeabilization. Correlation between vacuolin-1-modulated exocytosis and membrane repair in SLO-permeabilized cells suggest a coupling of lysosomal exocytosis and endocytosis (compensatory endocytosis) in mast cells.       58    
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Stáhnout Text práce Gouse Mohiddin Shaik, Ph.D. 1.06 MB
Stáhnout Abstrakt v českém jazyce Gouse Mohiddin Shaik, Ph.D. 129 kB
Stáhnout Abstrakt anglicky Gouse Mohiddin Shaik, Ph.D. 129 kB
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Stáhnout Posudek oponenta doc. RNDr. Jan Černý, Ph.D. 837 kB
Stáhnout Posudek oponenta Ing. Jiří Hašek, CSc. 1.92 MB
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