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The expression profile of cathepsin L in developmental stages of Fascioloides magna
Šašková, Romana ; Kašný, Martin (advisor) ; Mareš, Michael (referee)
Our experimental organism Fascioloides magna is a digenetic liver fluke from Fasciolidae family which parasitizes in domestic and free-living ruminants of North America and Central Europe including Czech Republic. In Czech Republic this highly pathogenic worm causes a severe liver damage to cervids and bovids and the prevalence locally reaches up to 95%. The biology of F. magna including e.g. the characteristics of host-parasite molecular interaction and the functions of particular molecules produced by the parasite are not fully understood. According to results of our previous research the excretory-secretory products of F. magna adults contain number of molecules which play the crucial role in host tissue invasion, digestion and evasion of the host immune response. One of the most abundant is cysteine peptidase cathepsin L (FmCL). FmCL is supposed to play various key roles in biological processes of all stages during a life cycle and therefore we can suppose its different expression level in particular life stages. In order to define the expression level of FmCL we performed the pilot study with miracidia and adults where the qPCR method was applied. The results of this experiment revealed much higher expression level of FmCL1 in adults than in miracidia. The attempt to in situ localize the mRNA...
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Cloning, epression and purification of mycobacterial dihydrofolate reductase
Šedivá, Kateřina ; Novotná, Eva (advisor) ; Malátková, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Kateřina Šedivá Supervisor: Mgr. Eva Novotná, Ph.D. Title of diploma thesis: Cloning, expression and purification of mycobacterial dihydrofolate reductase Dihydrofolate reductase is an enzyme essential for the metabolism of folic acid - it catalyzes the reduction of dihydrofolate to tetrahydrofolate. Tetrahydrofolate is an important cofactor involved in one-cabron transfer reactions. Dihydrofolate reductase plays a key role in the synthesis of DNA, RNA and proteins. Dihydrofolate reductase was also found in M. tuberculosis. This bacterium is the most common causative agent of tuberculosis in humans. Thus dihydrofolate reductase could be a potential target for the design of new antituberculotics. The recombinant protein dihydrofolate reductase was prepared in several steps. The coding sequence of the protein was first amplified by polymerase chain reaction. A recombinant plasmid, obtained by the ligation of an amplified segment of DNA with plasmid pET-28b(+), was transformed into competent cells E. coli strain BH101 by the heat shock method. Cells E. coli strain BL21(DE3) were used for the protein expression. The expression was induced by the addition of isopropyl-β-D-...
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Stability of the catalytic domain of neutral trehalase studied by differential scanning fluorimetry
Šmídová, Aneta ; Obšilová, Veronika (advisor) ; Kacířová, Miroslava (referee)
This bachelor thesis is part of a project aiming for the crystallization of trehalase domain of neutral trehalase Nth1 from Saccharomyces cerevisiae. The main goal of this thesis is to optimize the purification protocol of two different constructs of yeast Nth1 and determine the optimal buffer for crystallography of these constructs using the differential scanning fluorimetry DSF. Trehalases are important, highly conserved enzymes found in many organisms. These enzymes belong to the class of hydrolases, subgroup of glycosidases and their common feature is hydrolytic cleavage of trehalose molecule into two glucose subunits. Trehalose is a naturally occurring non-reducing disaccharide which serve in yeast cells as storage carbohydrate and energy and stress metabolite. Trehalose and its hydrolysis is very important in the life cycle of the insect, as it is present as the main sugar component of insect hemolymph, so some trehalase inhibitors could be used as insecticides. By this time, however, only one structure of trehalase from a prokaryotic organism Escherichia coli was solved. For this reason it is very important to solve the structure of trehalase from a eukaryotic organism. Keywords: Nth1, expresion, purification, DSF Key subjects: Protein biochemistry, biophysical chemistry
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Aesthetic Factor in John Dewey's Conception of an Experience Aesthetic Relevance demonstrated by Example of Snowbording
Vyhnánková, Klára ; Kaplický, Martin (advisor) ; Dadejík, Ondřej (referee)
This diploma thesis examines John Dewey's concept of experience, focusing especially on his notion that an aesthetic factor is present in every complete and consummatory experience. It deals with the differences between a common experience and "an experience" and examines how an aesthetic factor works in them. The indispensable role of the aesthetic factor is intentionally demonstrated on the non-artistic example of experiences of snowboarding. The most important findings are developed through the approach of Richard Shusterman's somaesthetics. The theories of these two philosophers support the main idea of this thesis, namely that dynamic movements of the body can bring aesthetic experiences to their actor. Thus, some of the reasons that explain the attractiveness of this modern sport come to light that are not apparent at first glance.
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Impact of Toll-like receptor 4 polymorphism on pro-inflammatory responsiveness in great tit (Parus major)
Vinklerová, Jitka ; Bryja, Josef (advisor) ; Černý, Jan (referee)
Toll-like receptor 4 (TLR4) belongs among chief bacteria-sensing Pattern recognition receptors. Endotoxin (lipopolysaccharide, LPS) recognition by TLR4 triggers signalling leading to release of cytokines that direct leukocyte infiltration into the inflammatory site and cause swelling. Effector mechanisms that ensure pathogen elimination include phagocytosis and oxidative burst. It has been repeatedly reported that the polymorphism in TLR4 may affect host resistance to various diseases. TLR4 may be, therefore, an important molecule in host-parasite co-evolution. Herein, I focused on TLR4 amino acid substitution Q549R which is associated with ornamentation in great tits. In tits I describe immune responsiveness to LPS stimulation on morphological and molecular level and examine effects of the Q549R substitution on inflammation and general body condition. In LPS- treated individuals I found decrease in heterophil-lymphocyte ratio (H/L) that might be caused by attraction of the blood-borne cells into the inflamed tissue. This is in striking contrast with increase in H/L in PBS-treated animals resulting from the stress response. There was no effect of Q549R on general condition and haematological parameters but I revealed a significant effect of the interaction between host Q549R genotype and the type...
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Immunogenetic and hormonal markers of predisposition to systemic rheumatic diseases particularly systemic lupus erythematosus
Fojtíková, Markéta ; Pavelka, Karel (advisor) ; Hrnčíř, Zbyněk (referee) ; Rovenský, Jozef (referee)
Fojtikova 2011 INTRODUCTION: Several factors like genetic susceptibility is required for systemic rheumatic diseases development. Immunomodulatory PRL effect supports autoimmunity. AIMS: 1. To detect the immunogenetic background (alleles HLA class I, II and microsatellite polymorphism of the transmembrane part exon 5 of MIC-A gene) of SLE and PsA. 2. To detect PRL serum and synovial fluid with regard to clinical and laboratory RA activity. 3. To find the role of the functional polymorphism -1149G/T SNP PRL of extrapituitary promoter of PRL gene in SLE, RA, PsA, SSc and inflammatory myopathies development. METHODS: Genetic analyses of pateints with SLE (n=156), RA (n=173), PsA (n=100), SSc (n=75), PM (n=47) a DM (n=68) and 123 healthy individuals: PCR-SSP (HLA clase I and II), PCR-fragment analysis (MIC-A) a PCR-RFLP (-1149 G/T SNP PRL). In 29 RA a 26 OA PRL serum and synovial fluid concentrations were detected using immunoradiometric assay. RESULTS: 1. The allele HLA-DRB1*03 (pc=0.008; OR 2.5) and haplotype HLA-DRB1*03-DQB1*0201 (pc <0.001; OR 4.54) were determined as risk immunogenetic markers for SLE in Czech population. In SLE versus controls allele MIC-A5.1 was increased (pc =0.005; OR 1.88). MIC-A5.1 together with HLA-DRB1*03 increases the risk for SLE development, pc <0.000001; OR 9.71....
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Locomotor expression of poems with pre-school children
Hujerová, Václava ; Hník, Ondřej (advisor) ; Klumparová, Štěpánka (referee)
Dissertation deal about integration poetry with locomotion in kinderkarten. I investigate measure of interest the children about poetry and integration poetry with locomotion. I suppose, the children will be prefer the integration of activities because of congenital needed and komplex perceive. I engage charakteristic of poetry and locomotion, relation the children to poetry and importance poetry and locomotion for children. I implement the exploration in Prague 1 with twenty children. They are five years old. The method of exploration is observation and experiment. The exploration remain ten days. I investigace measure of interest about poetry and integration poetry with locomotion. I discovered, that I connect poetry and locomotion, the measure of interest increase to sewenteen percent. Thanks to, the children love poetry and get to poetry deap emotional relation. Integration poetry and locomotion attend to harmonious development of personality. I think, that it ensure the best start for life.
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Analysis of BK virus life cycle
Bakardjieva - Mihaylova, Violeta ; Drda Morávková, Alena (advisor) ; Mindlová, Martina (referee)
Polyomaviruses are small unenvelope DNA viruses, whose replication take place in cell nucleus. Despite its small genome size, these viruses can cause significant changes in the host cell, one of the most significant is cell transformation. Most studies of human pathogens from this family is the focus of clinical research, but do not provide enough information about the individual events of the life cycle of viruses. This thesis mainly aims to determining the exact time when the creation of the individual viral products and generate a timeline of events during natural infection in cells that are targets for BKV in the human body. It was found that the time course of the life cycle of BKV is very similar to those for model polyomaviruses MPyV and SV40 and in permissive cells takes about 40 - 50 hours.
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