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Nanoparticles and Their Effect on the Immune System: Study of the Proinflammatory Potential of Selected Carbon Nanomaterials
Švadláková, Tereza ; Krejsek, Jan (advisor) ; Stulík, Jiří (referee) ; Roušar, Tomáš (referee)
Carbon-based nanomaterials (CNMs) have unique physical-chemical properties, which make them appropriate candidates for both industry and medicine. However along with production, there are growing concerns about their effects on human organism. For this reason, CNMs are frequent topic of toxicological studies. A key step in the clarification of their safety is to evaluate their interaction with the components of the immune system, particularly their ability to cause inflammation. For some allotropes e.g., pristine graphene derivatives, significant results are still missing or incomplete. For these reasons, this dissertation deals with the evaluation of the proinflammatory effect of two types of pristine graphene platelets (GPs), which represent common intermediate in the processing of other graphene derivatives, and which can penetrate to body via inhalation. For comparison, our work also includes an evaluation of the proinflammatory effect of multiwalled carbon nanotubes (MWCNTs). Professional phagocytes, particularly monocytes and macrophages represent key cells in processing and elimination of foreign and damaged or abnormal elements. Since phagocytes also represent main mediators of inflammation, we selected human primary monocytes and human monocytic cell line THP-1 differentiated in macrophages...
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Investigation of protein-protein interaction important for pathogenesis of tularemia infection
Prokšová, Magdaléna ; Stulík, Jiří (advisor) ; Myslivcová Fučíková, Alena (referee) ; Bouchal, Pavel (referee)
Francisella tularensis, sometimes called as a "stealth pathogen", causes zoonotic disease tularemia. Uniqueness of this intracellular bacterium is due to its ability to infect, survive and replicate within host phagocytic cells and evade the host immune response. Because of its extreme infectivity, ability to cause disease via inhalation route, and absence of a vaccine licensed for human use, F. tularensis is classified as a potent biothreat agent. The escape from phagosome plays a key role in the virulence of the bacterium. Type VI secretion system and F. tularensis pathogenicity island proteins are involved in this process; however, the exact molecular virulence mechanisms of F. tularensis are not fully characterized yet. The aim of this work was to characterize host-pathogen protein-protein interactions, which direct the infection process to F. tularensis benefits. The minimal experimental approach was selected for identification of protein-protein interaction between the host and the pathogen. The selected secreted proteins from FPI of F. tularensis subspecies novicida were fused with an epitope anchor FLAG tag and expressed in HEK 293T cell line. Interaction partners were identified by affinity purification followed by nanoLC-MS/MS analysis. The data indicate that the bacterial protein IglJ...
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Analysis of intracellular pathogen Francisella tularensis expression upon cultivation under stressfull conditions and during the interaction with a host cell
Lenčo, Juraj ; Stulík, Jiří (advisor) ; Rychlík, Ivan (referee) ; Weiser, Jaroslav (referee)
Univerzita Karlova v Praze Lékařská fakulta v Hradci Králové Analýza exprese intracelulárního patogena Francisella tularensis kultivovaného za stresových podmínek a v průběhu interakce s hostitelskou buňkou Autoreferát dizertační práce Mgr. Juraj Lenčo Hradec Králové 2007 2 Dizertační práce byla vypracována v rámci doktorského studijního programu lékařská biologie na Ústavu lékařské biologie a genetiky Lékařské fakulty v Hradci Králové, Univerzity Karlovy v Praze a Ústavu molekulární patologie Fakulty vojenského zdravotnictví Hradec Králové, Univerzita obrany Brno. Uchazeč: Mgr. Juraj Lenčo Ústav molekulární patologie, Fakulta vojenského zdravotnictví Hradec Králové, Univerzita obrany Brno. Školitel: Doc. MUDr. Jiří Stulík, CSc. Ústav molekulární patologie, Fakulta vojenského zdravotnictví Hradec Králové, Univerzita obrany Brno Školitel konzultant: Prof. MUDr. RNDr. Miroslav Červinka, CSc. Ústav lékařské biologie a genetiky, Lékařská fakulta v Hradci Králové, Univerzita Karlova v Praze Oponenti: RNDr. Ivan Rychlík, Ph.D. Oddělení bakteriologie Výzkumný ústav veterinárního lékařství Brno RNDr. Jaroslav Weiser, CSc. Mikrobiologický ústav Akademie věd České republiky Stanovisko k dizertační práci vypracovala oborová rada oboru lékařská biologie akreditovaného na Ústavu lékařské biologie a genetiky Lékařské...
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Mechanism of action of adenylate cyclase toxin on immune function of dendritic cells
Švédová, Martina ; Šebo, Peter (advisor) ; Černý, Jan (referee) ; Stulík, Jiří (referee)
The adenylate cyclase toxin (CyaA) is a key virulence factor of the whooping cough agent Bordetella pertussis. CyaA primarily penetrates CR3-expressing myeloid phagocytes and subverts cellular signaling by a rapid conversion of ATP to cAMP. In parallel, CyaA can form cation-selective pores within cellular membrane, provoking massive potassium efflux from cell cytosol. An enzymatically inactive adenylate cyclase toxoid (CyaA-AC- ) has then been abundantly used as an efficient antigen delivery tool over the past 20 years. This work focused mainly on the mechanism of action of CyaA toxin and of its toxoid on dendritic cells. We studied the potency of the CyaA toxoid to act as adjuvant, its penetration capacity and its potential use in delivery of influenza epitopes. We show that the pore-forming activity and the activation of MAP kinases JNK and p38 were crucial for the adjuvant effects of the CyaA-AC- , which provokes maturation of dendritic cells (DC) independently of Toll-like receptor (TLR) or inflammasome signaling. Furthermore, such CyaA-AC- -stimulated DC acquired the ability to induce CD8+ and CD4+ T cells responses, as was determined both in vitro and in vivo. We further showed that the first 371 amino acids are dispensable for the capacity of CyaA to deliver its AC domain with inserted...
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Structure-function relationships and use of RTX proteins of Gram-negative bacteria
Sadílková, Lenka ; Šebo, Peter (advisor) ; Stulík, Jiří (referee) ; Weiser, Jaroslav (referee)
RTX (Repeat in ToXin) superfamily consists of many proteins divided into several groups according to their different functions and characteristics: toxins, metalloproteases, lipases, proteins of the S-layer, bacteriocins and proteins with unknown function. However, all of them can be characterized by the following features: i) they contain tandemly repeated (6-50) nonapeptide glycine-rich calcium-binding consensus sequences GGXGXDX[L/I/V/W/Y/F]X (where X is any amino acid residue) in the C-terminal part of the protein. The presence of these repeats is a sine qua non condition for RTX protein family membership; ii) secretion from the cell occurs without a periplasmic intermediate by a mechanism which involves recognition of a signal sequence at the C-terminus of the protein by membrane-associated proteins that export the toxin across a channel spanning the entire bacterial envelope directly to the outside of the cell (Type I Secretion System); iii) the genes for protein synthesis, activation and secretion are mostly grouped together on the chromosome and form rtx operons. RTX toxins are the largest protein group of the RTX family. To this group belong mostly the proteins with molecular weight ranging from 100 to 200 kDa, with posttranslational fatty acid acylation mediated by a specific activating...
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Analysis og gene expression in prokaryotic and eukaryotic model organisms by proteomic gel-based separation tools
Petráčková, Denisa ; Weiser, Jaroslav (advisor) ; Nešvera, Jan (referee) ; Stulík, Jiří (referee)
This PhD thesis showed the applicability of a gel-based proteomic separation tool, 2-D electrophoresis in three independent projects. Supplemented with results obtained using different techniques the proteomic studies enabled a global imaging of proteoms in the studied biological systems. Comparing total proteoms of E. coli 61 protein changes were identified and connected with the development of the bacterial population in the presence of an antibiotic compound, erythromycin. This classic proteomic approach included sample extraction, optimization of its 2D separation followed by 2D gel analysis and protein identification by MS methods. A disadvantage of this work was an enourmously large amount of data to be analyzed by computer analysis. For the study of membrane proteom of B. subtilis during a pH induced stress, on the other hand, a modification of isolation techniques for membrane and membrane associated proteins was required first to improve the subsequent protein separation by 2-D electrophoresis. The optimalization of protein extraction included changes in detergents used for protein solubilization and a prolongation of time periods in the protein solubilization protocol. 5 relevant protein changes were then described that play a role in the bacterial response to pH stress. The proteins were...
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Analysis of intracellular pathogen Francisella tularensis expression upon cultivation under stressfull conditions and during the interaction with a host cell
Lenčo, Juraj ; Stulík, Jiří (advisor) ; Rychlík, Ivan (referee) ; Weiser, Jaroslav (referee)
Univerzita Karlova v Praze Lékařská fakulta v Hradci Králové Analýza exprese intracelulárního patogena Francisella tularensis kultivovaného za stresových podmínek a v průběhu interakce s hostitelskou buňkou Autoreferát dizertační práce Mgr. Juraj Lenčo Hradec Králové 2007 2 Dizertační práce byla vypracována v rámci doktorského studijního programu lékařská biologie na Ústavu lékařské biologie a genetiky Lékařské fakulty v Hradci Králové, Univerzity Karlovy v Praze a Ústavu molekulární patologie Fakulty vojenského zdravotnictví Hradec Králové, Univerzita obrany Brno. Uchazeč: Mgr. Juraj Lenčo Ústav molekulární patologie, Fakulta vojenského zdravotnictví Hradec Králové, Univerzita obrany Brno. Školitel: Doc. MUDr. Jiří Stulík, CSc. Ústav molekulární patologie, Fakulta vojenského zdravotnictví Hradec Králové, Univerzita obrany Brno Školitel konzultant: Prof. MUDr. RNDr. Miroslav Červinka, CSc. Ústav lékařské biologie a genetiky, Lékařská fakulta v Hradci Králové, Univerzita Karlova v Praze Oponenti: RNDr. Ivan Rychlík, Ph.D. Oddělení bakteriologie Výzkumný ústav veterinárního lékařství Brno RNDr. Jaroslav Weiser, CSc. Mikrobiologický ústav Akademie věd České republiky Stanovisko k dizertační práci vypracovala oborová rada oboru lékařská biologie akreditovaného na Ústavu lékařské biologie a genetiky Lékařské...
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