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Dimerization of Streptococcus pneumoniae eukaryotic-type SerThr protein kinase and characterization of its substrate, phosphoglucosamine mutase GlmM
Pallová, Petra ; Branny, Pavel (advisor) ; Janata, Jiří (referee) ; Španová, Alena (referee)
104 7 Závěr 7.1 Proteinkinasa StkP Připravili jsme kmen S. pneumoniae KDTM-his, který ve svém genomu kóduje epitopem značenou kinázovou doménu proteinkinasy StkP ukotvenou do membrány pomocí transmembránové domény. Imunologickou detekcí s monoklonální protilátkou proti histidinové kotvě jsme potvrdili lokalizaci proteinu v membránové frakci S. pneumoniae. V in vitro kinázových reakcích jsme prokázali, že se jedná o plně funkční protein s autofosforylační aktivitou. Pomocí in vivo reportérového systému jsme zjistili, že transmembránová doména a extracelulární doména proteinkinasy StkP tvoří stabilní dimery. Dimerizace proteinkinasy StkP byla následně potvrzena pomocí nativní elektroforézy. Je tedy velmi pravděpodobné, že proteinkinasa StkP se in vivo vyskytuje ve formě dimeru a dimerizace je nutným předpokladem její autofosforylační aktivity. Na základě těchto výsledků jsme vyslovili hypotézu, že protein kódující kinázovou doménu postrádající transmembránovou doménu není funkční, neboť není schopen dimerizace a podléhá degradaci. 7.2 Fosfoglukosaminmutasa GlmM V in vitro kinázové reakci na bezbuněčných lyzátech S. pneumoniae Cp1015, ∆stkP a ∆phpP-stkP v přítomnosti rekombinantní proteinkinasy StkP jsme prokázali fosforylaci proteinu o molekulové hmotnosti odpovídající fosfoglukosaminmutase GlmM....
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The safety of probiotics used in foods
Balažovičová, Nikola ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
The theoretical part of this thesis focuses on definition of probiotics, their health benefits, probiotic foods, safety criteria for probiotics used in foods, including methods for the identification of probiotic bacteria. Experimental part of work was focused on the identification of probiotic bacteria contained in the milk product Actimel. DNA quality suitable for the PCR was isolated by magnetic microparticles P(HEMA-co-GMA). Identification was estimated using the method of polymerase chain reaction. The presence of DNA of domain Bacteria, genus Lactobacillus and species Lactobacillus casei was confirmed by gel electrophoresis of PCR products. Specific PCR products of the sizes of 466 bp (domain Bacteria), 250 bp (genus Lactobacillus) and 136 bp (Lactobacillus casei) were detected after amplification of DNA isolated from a probiotic milk product.
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The pleiotropic effect of WD-40 domain containing proteins on cellular differentiation and production of secondary metabolites in Streptomyces coelicolor
Ulrych, Aleš ; Branny, Pavel (advisor) ; Pátek, Miroslav (referee) ; Španová, Alena (referee)
The pleiotropic effect of WD-40 domain containing proteins on cellular differentiation and production of secondary metabolites in Streptomyces coelicolor WD-40 domains, also known as beta-transducin repeats, are highly conserved repeating amino acid units, which are found in a wide variety of eukaryotic proteins that have a range of different functions. In the late 1990s, the first WD-40 containing proteins were identified in prokaryotes, however the knowledge about their function is scarce. Streptomyces coelicolor is a gram-positive bacterium with complicated morphological and physiological differentiation in the course of its life cycle. The genome of Streptomyces coelicolor encodes 6 potential genes encoding proteins with WD-repeat motifs. To determine the function of two of these WD-40 genes (wdpB and wdpC), the deletion replacement mutants in both genes were prepared. Both mutants exhibited medium-dependent phenotypes, which are markedly evident on modified R3 plates. Phenotypic studies revealed that deletion of wdpB gene resulted in substantial reduction of aerial hyphae formation and reduced production of undecylprodigiosin. In addition, the hyphae of ΔwdpB mutant were unusually branched and showed the signs of precocious lysis. Delayed spore-containing hyphae were irregularly septated....
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Functional studies of Ser/Thr protein kinases and phosphatases of Pseudomonas aeruginosa
Goldová, Jana ; Branny, Pavel (advisor) ; Nešvera, Jan (referee) ; Španová, Alena (referee)
Reversible protein phosphorylation is considered the universal language for intracellular communication in all living organisms. This process, catalysed by protein kinases and phosphatases, enables the translation of extracellular signals into cellular responses and also allows for adaptation to a constantly changing environment. In recent years, a number of bacterial eukaryotic-type Ser/Thr protein kinases and phosphatases have been identified. However, their precise functions and substrates are not yet well defined. The genome of opportunistic human pathogen Pseudomonas aeruginosa contains at least five genes encoding putative eukaryotic-type Ser/Thr protein kinases and phosphatases. In the first part of this study, we have attempted to establish the role of Ser/Thr protein kinase PpkA and phosphatase PppA, which belong to type VI secretion system H1-T6SS. Double mutant strain ∆pppA-ppkA was prepared in P. aeruginosa PAO1 background. Phenotypic studies revealed that the mutant grew slower than the wild-type strain in minimal media and exhibited reduced secretion of pigment pyocyanin. In addition, the mutant had altered sensitivity to oxidative and hyperosmotic stress conditions. Consequently, mutant cells had an impaired ability to survive in murine macrophages and an attenuated virulence in the...
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Analysis of the composition of selected probiotic products by PCR-HRM
Tomanová, Barbora ; Španová, Alena (referee) ; Trachtová, Štěpánka (advisor)
This work was focused on the detection of probiotic bacteria in four different probiotic products (probiotic cream, probiotic tampons, oral probiotics and soy beverages with probiotics). The viability of the bacteria contained in the products was verified. Complex matrices of the products were used to isolate DNA in a quality suitable for the PCR method, followed by identification of the declared bacterial genus and species. Amplification was achieved with conventional PCR and real-time PCR, genus- and species-specific primers were used. Bacteria, of the genus Lactobacillus and Bacillus and bacterial species Lactobacillus pentosus, Lactobacillus rhamnosus, Lactobacillus fermentum and Lactobacillus gasseri, were proven to be within the products. Subsequently, the DNA from mixed bacterial species in the probiotic tampon were distinguished using PCR-HRM. Five sets of primers were used to test this. Two sets of primers (primers P1V1, P2V1 and V1F-HRM, V1R-HRM) were evaluated as the most suitable for resolution.
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Application of magnetic particles for isolation and purification of DNA
Němeček, Milan ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
With a development of molecular biology methods it is an increasing interest in new procedures of DNA isolation of high quality. DNA isolation is performed on crude cell lysates by many techniques e.g. phenol extraction, salting out or adsorption on solid phase. Classical DNA isolation, such as phenol extraction is quite complicated and time consuming. New alternative methods of DNA isolation was development using reverse immobilizing DNA to a solid phase. Widespread is the use of the magnetic particles as carriers, which allow the isolation of DNA in high quality directly from crude cells lysates of complex samples. The current method of DNA adsorption onto the surface of magnetic particles does not provided sufficiently pure DNA for analysis of some comlex samples (e.g. food). Some inhibitors of the polymerase chain reaction (PCR) are apparently adsorbed onto the tube wall and the next step of DNA elution leads to their release into the solution and cpnsequent negative effect on quality of DNA (e.g. decreasing of PCR amplification). The principle of the developed procedure is design a device, which utilizes transfer of magnetic particles by paramagnetic newddle from one to another Eppendorf tube, in which further processing of the sample extends. Transfer of magnetic particles with DNA using needle prevents transmission of contaminating impurities. The proposed device allows to realize above-mentioned procedure. The functionality of the device being tested in the isolation of plasmid pUC19 DNA from crude lysates of E. Coli JM 109 (pUC19).
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Identification of microorganisms in cosmetic products with probiotics
Langová, Denisa ; Španová, Alena (referee) ; Trachtová, Štěpánka (advisor)
Probiotics products are an integral part of the current market. Products containing probiotics cultures are also cosmetic products. The first part of the study focuses on testing of bacterial survival abilities in the environment of preservatives presented in cosmetic products. Collection strains of genus Lactobacillus were used for these tests. Another part of the study focuses on isolation of bacterial DNA from probiotic cosmetic products Ryor, Yoghurt of Bulgaria, FeminaMed and Lactovit Activit in PCR-ready quality. DNA was isolated by fenol extraction and with magnetic particles. Presence of bacteria was proved by genus and species specific PCRs Lactobacillus. Species specific PCR for identification of Lactobacillus pentosus was optimalized. Species identification was in accord with data declared by producers.
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The application of magnetic particle for DNA isolation from selekted probiotic products for children
Vozárová, Petra ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
In the food industry, it is important to correctly identify the species of bacteria and thier properties so that they can be used as a probiotic in dietary supplements. This is performed using DNA diagnostics. In the experimental part, the DNA from four probiotic dietary supplements for children was isolated. Magnetic particles P(HEMA-co-GMA) were tested for isolation. Isolated DNA was amplified by PCR and the presence of DNA of genus Lactobacillus, Bifidobacterium and Bacillus was demonstrated in the products according to the data declared by the manufacturer. The presence of species L.acidophilus, B.animalis in accordance with the data on the product has been demonstrated by PCR with species specific primers. Using PCR, the presence of L.casei, which was declared by the manufacturer, has not been proven in one product at given experimental conditions.
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Effect of probiotic bacteria Lactobacillus paracasei subsp. paracasei on the development of acute colitis in BALB/c mice.
Součková, Martina ; Jílek, Petr (advisor) ; Španová, Alena (referee)
Inflammatory bowel disease - ulcerative colitis and Crohn's disease - are chronic relapsing intestinal disorders of unknown etiology. Acute ulcerative colitis was induced in BALB/c mice by 3 % dextran sulfate sodium (DSS) administrated in drinking water for 5 days. The effect of Lactobacillus paracasei subsp. paracasei RL14-P, a bacterium with potential probiotic influence, on the development of inflammation was evaluated. Lactobacillus was administrated intragastrically to mice on days 1, 4 and 8 and the administration continued for next 5 days during DSS treatment. Control groups received instead of lactobacillus MRSC medium or phosphate-buffered saline according to the same schedule. Clinical symptoms such as diarrhoea, rectal bleeding and loss in body weight were evaluated daily. Cytokine secretion (IL-6, IL-10 and TNF-α) was determined in supernatants of 48 h cultivated splenocytes, mesenteric lymph node lymphocytes and colon pieces by ELISA. The impairment of colon descendens was evaluated histologically. Lactobacillus paracasei subsp. paracasei RL14-P administrated mice remained healthy without signs of the intestinal inflammation. We did not find the shortening of colon (a sign of inflammed intestine) in lactobacillus-treated mice. We observed that administration of probiotic bacteria...
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Nucleic acid detection using magnetic microparticles
Pešková, Aneta ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
Modern and fast separation proces for nucleic acids isolation uses magnetic microparticles in routine laboratory practise. The comparision of DNA isolation efectivity by three types of magnetic microparticles P(HEMA-co-GMA) (A, B), PGMA and magnetic nanoparticles covered by poly-L-lysine (PLy) from probiotic products Pangamin was carried out. The quality and quantity of isolated DNA were verified by spektrofotometric measurments and gel electrophoresis. Possibility of amplifying of isolated DNA was verified by polymerase chain reaction (PCR) with specific primers for yeast and evaluated by agarose gel electrophoresis. The quantity of isolated DNA depended on the product and the type of magnetic particles. The highest amount of DNA was isolated by magnetic nanoparticles of poly-L-lysine (PLy) and magnetic microparticles P(HEMA-co-GMA) (A).
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