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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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Birds in teaching at secondary schools with new knowledge in the field
Weiser, Jiří ; Řezníček, Jan (advisor) ; Kopřivová, Kateřina (referee)
In the introduction of theoretical part of diploma thesis is tabularly summed up the systém of birds classification. Other parts are focused not only on general issues and commonly used themes in school textbooks, popular literature and educational media, but also to highlight specific features, characteristic only for the birds. These themes are historical and evolutional questions, cover morphology, the effect of the senses, body structury and function of organs. In detail is analyzed the issue of body composition and skeletal system of birds. In the final sections are mentioned specialities in the behavior and communication between birds and their habits. The practical part includes a SWOT analysis of available textbooks for the four-year gymnasium with regard to the chapters and information about the animal class of birds. The data analyzed in the textbook can only be considered as basic with need to extend and complement by the teacher. Attached to the diploma thesis are two presentations in Powerpoint, containing basic information and specific features, characteristic only for birds. The presentations are created worksheets. For reference classes was developed as a lab practical examination of selected thematic blocks, memorized theoretical interpretation. Students' knowledge of the...
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Streptomycetes surface growth and differentiation on inert microbeads- morphology and proteome study
Tesařová, Eva ; Weiser, Jaroslav (advisor) ; Lichá, Irena (referee)
Streptomyces, filamentous Gram-positive bacteria are producers of more than 70% of antibiotics used in human therapy and agriculture. They are remarkable because of their complex life cycle (morphological differentiation) which leads to a formation of dormant spores able to survive unfavorable living conditions and allowing long-term survival of the organism. Soil represents their mostly natural living environment. In laboratory conditions they are cultivated in liquid media or on agar. We have developed in our laboratory two phase cultivation system which allows quantitative and reproducible preparation of samples for proteomic, transcriptomic and metabolomic analyses of Streptomycetes differentiation. The system is composed of inert micro- beads submerged in liquid medium. We used two types of micro-beads in our studies, glass and zirconia/silica beads. We followed the surface growth and differentiation of Streptomycetes on both types of beads using optical and electron microscopy (SEM) techniques. We observed major growth and higher antibiotic production on glass beads. Another difference we observed was in size and shape of colonies. In further research, using comparative proteomics, we attempted to identify proteins which might be responsible for recognition and adhesion of Streptomycetes to...
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Mycobacterium smegmatis biofilm formation om glass and zirkonia beads-proteomic study
Sitařová, Barbora ; Weiser, Jaroslav (advisor) ; Zikánová, Blanka (referee)
Biofilms represent universal strategy for bacterial survival. Living in form of biofilms, bacteria acquire wide range of advantages over planktonically growing cultures. It can be assumed that nearly 99% of world bacterial population is living in form of biofilms. There are benefits and drawbacks associated with bacterial biofilms for mankind. Life in biofilms makes pathogens more effective and persistent through higher antibiotic resistance and helps them to hide before immune system of the host. Mycobacteria, which are capable of forming biofilms on variety of surfaces, differ from most of other bacteria by unique composition of their cell wall. It provides them with high resistance against physical or chemical damage. This is one of the reasons for considering Mycobacterium tuberculosis as a highly potent pathogen. The studies of mycobacterial biofilms are motivated by effort to improve or find new therapeutic methods. This work is aimed at morphological and proteomic comparative analyses of biofilms obtained from Mycobacterium smegmatis grown on surface of glass and silica/zirconium beads, on liquid medium surface or grown submerged in shaken planktonic culture. We have developed technique for preparation of "floating" biofilm sample to be observed in SEM. We have shown that the growth of...
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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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Monitoring of Mycobacterium smegmatis floating biofilm development - morphological and proteome analysis
Sochorová, Zuzana ; Weiser, Jaroslav (advisor) ; Zikánová, Blanka (referee)
Microorganisms grow in planktonic form, but more often they adhere to a number of surfaces and create three-dimensional structures called biofilms. Floating biofilms, which are formed at the water-air interface, are one of the life strategies, which the bacteria can take. Non-pathogenic Mycobacterium smegmatis was used as a laboratory model for the study of this kind of biofilm. The understanding of mechanisms of their formation of this species may be applicable to the pathogenic species of the genus Mycobacterium, study of which in the laboratory brings a number of disadvantages. This diploma thesis focuses on the morphological and proteome analysis of the M. smegmatis floating biofilm. Using a stereo microscope and scanning electron microscopy was observed that bacteria clump and create the "nucleation centres" at the beginning of the biofilm development. This centers grow to the surroundings and connect afterwards. In the later stages of the development the centers fuse in compact layer, which then grows into the compact and multilayer biofilm. The key method in this study was two-dimensional electrophoresis of proteins. The proteome analysis of floating biofilm was performed with this method. The preparation of protein samples and the method for protein concentration measurement was optimized....
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Růst Mycobacterium smegmatis na agarovém médiu a agarovém médiu pokrytém celofánovou folií - morfologická a proteomová studie
Ramaniuk, Volha ; Weiser, Jaroslav (advisor) ; Beranová, Jana (referee)
Biofilm formation is one of the most common bacterial survival strategies. Majority of bacterial species are able to form these three-dimensional structures, including pathogens like Mycobacterium tuberculosis. Representatives of Mycobacterium genus widely occur in the nature, although they can cause serious problems when they appear in medical equipment and artificial replacements of the human body. Non-pathogenic Mycobacterium smegmatis mc2 155 was used as a model organism in our experiments. We investigated morphology of the three- and six-day-old colonies (in fact biofilms) on agar and agar covered with cellophane using Stereo microscope and Scanning Electron Microscope. We found that a type of surface as well as a carbon source has a great influence on the morphology of the M. smegmatis colonies. We isolated proteomes from the agar and cellophane cultures and from planktonic culture. Two-dimensional electrophoresis was used as the main proteomic method. Proteomic data were analyzed using PDQuest software. Then the sets of proteins detected by qualitative and quantitative analyses were compared using Venn diagrams. As a result, we recognized 7 unique proteins that might be specific for recognition and adhesion of bacteria to the cellophane, no unique protein in agar proteome and 46 unique...
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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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