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Study of phosphorylation of inorganic pyrophosphatase from Streptococcus pneumoniae
Štechová, Michaela ; Doubravová, Linda (advisor) ; Svobodová, Jaroslava (referee)
The human patogen Streptococcus pneumoniae encodes a single copy of eukaryotic-like Ser/Thr protein kinase StkP. StkP regulates virulence, competence, stress resistence, gene expression and plays a role in the regulation of cell division cycle. Analysis of phosphoproteome maps of the wild type and stkP mutant strain of S. pneumoniae showed that in vivo StkP phosphorylates several putative substrates including Mn-dependent inorganic pyrophosphatase PpaC. Mass spectrometry analysis identified two phosphorylation sites in an active site of the protein. Pyrophosphatases are essential enzymes that catalyze hydrolysis of inorganic pyrophosphate produced during various biosynthetic reactions that utilize ATP. Changes in pyrophosphatase activity have been described to have global effects on cell metabolism, growth and division of bacteria. The aim of this thesis was to investigate the phosphorylation of inorganic pyrophosphatase PpaC in S. pneumoniae. Gene ppaC was cloned, expressed in E. coli and protein was purified via affinity chromatography. Phosphorylation of PpaC by StkP was examined in a kinase assay but we did not confirm that PpaC is a direct substrate of StkP in vitro. Further we prepared a set of mutants in ppaC gene. We replaced two presumable phosphoaminoacids identified by mass-spectrometry with...
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Stress proteins in the cytoplasmic membrane fraction of Bacillus subtilis
Šemberová, Lenka ; Svobodová, Jaroslava (advisor) ; Krásný, Libor (referee)
Stress proteins in the cytoplasmic membrane fraction of Bacillus subtilis A primary habitat of the Gram-positive bacterium Bacillus subtilis is the upper layer of the soil. Within this ecosystem, B. subtilis experiences a wide variety of environmental challenges and nutrient limitations that induce several mechanisms to help the cells to survive. A process known as a general stress response belongs among them. Expression of about 150 genes is enhanced and their products called general stress proteins (GSP) minimize the cell damage. Although the size and structure, as well as the regulation, of many stress proteins have been fairly well elucidated the information about stress membrane proteins is very limited. We characterized the membrane proteome of Bacillus subtilis 168 trp2- exposed to acidic pH and ethanol. Cells were 1) grown under optimum conditions in complex medium (pH 7.0) at 40řC with aeration; 2) grown in complex medium at pH 5.0 for 20 hours or 3) challenged with 3% v/v ethanol for 30 minutes during exponential growth. The cultures were harvested in the mid-exponential phase by rapid filtration. Isolated membrane fractions were analysed by an optimized two-dimensional gel electrophoresis. Two alternative methods of protein detection were compared: silver staining and radioactive labeling with...
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Study of regulation of branched-chain amino acid biosynthesis in C.glutamicum and construction of valine producing strain
Holátko, Jiří ; Pátek, Miroslav (advisor) ; Petříček, Miroslav (referee) ; Svobodová, Jaroslava (referee)
L-valine is an essential amino acid with a great medical and industoial importance and the need for efficient bacterial producersof this amino acid is increasing. Corynebacteriumglutamicum is a suitable organism for construction of such productionstrainsandthusbiosynthesisofvaline and expressionofgenes involved in its pathway are intensively studed. In this PhD. thesis, analysis of regulationand modulationof expressionwas carriedout with: (l) genes,which code for common enzymesof all branched-chainamino acids; (2) genes,whose products are involved in the competing biosynthesis pathways of L-isoleucine and L-leucine; and (3) the genethatdeterminesthe key enzyme in the catabolism of pyruvate. Results were used for construction of C. glutamicum production shains with deregulatedbiosynthesisof valine, restrictedbiosynthesisof isoleucine and leucine and optimum distributionof intermediatesof valine. The production strains were manipulated via modification of promoters in the chromosome. Promoters of the ilvA, leuA, ilvD, ilvE, and aceE genes were modified by site-directedmutagenesisof their -10 regions. All these alterationsincreased production of valine by the modified strains. Introduction of weak P-ilvA and P-leuA promotersinto the C. glutamicumchromosomeresultedin an intrinsic lowJevel biosynthesisof...
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Analysis of lincomycin biosynthetic genes usable for the preparation of hybrid compounds
Ulanova, Dana ; Janata, Jiří (advisor) ; Varečka, Ľudovít (referee) ; Svobodová, Jaroslava (referee)
Analysis of lincomycin biosynthetic genes usable for the preparation of hybrid compounds Lincosamide antibiotics have relatively simple molecular structure compared with other types of antibiotics. However, these molecules contain so called "hot spots" whose modification leads to the development of more effective drugs. Combinatorial biosynthesis appears to be a promising way to design and preparation of lincosamides' derivatives. For this purpose a perfect knowledge of the natural biosynthetic pathway is needed. The submitted doctoral thesis analyzed genes lmbIH, K, N, Q, T, U and V with unknown function from the lincomycin biosynthetic gene cluster. Mutation analysis of lmbN has proven its bifunctional character, 5 'end of the gene is coding for a potential N-demethyllincomycin synthetase subunit, while the rest of the gene encodes an enzyme for synthesis of sugar precursor methylthiolincosamide (MTL). Gene lmbT was also assigned to the biosynthesis of the sugar precursor. In the case of other genes the determination of function was not so clear. It is assumed that their roles in the biosynthetic pathway are more regulatory or supporting. The mutasynthetic approach was used for the preparation of lincomycin derivatives with longer alkyl residues. Activity of obtained lincomycin derivatives was tested on a...
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The influence of the level and type of postural load on parameters of dynamic plantography during standing and walking.
Svobodová, Jaroslava ; Opálková, Michaela (advisor) ; Vachovcová, Sylva (referee)
Bibliographic record SVOBODOVÁ, Jaroslava. The influence of the level and type of postural load on parameters of dynamic plantography during standing and walking. Prague: Charles University, 2nd Faculty of Medicine, Department of Rehabilitation and Sports Medicine, 2018, 124 p. Supervisor Mgr. Michaela Opálková. Abstract The thesis deals with the reaction of body to postural loading during stance and walking. The theoretical part of this thesis is focused on postural control and postural reaction during stance and walking. Particular attention is paid to gait cycle, dynamic plantography and Zebris FDM-T Rehawalk system, which has been used in the practical part of this thesis. Two chapter deal with postural loading, the first is focused on symmetrical loading (e.g. backpack) and second on asymmetrical loading (laptop back - ipsilateral, contralateral and held in hand). The practical part presents the examination of 32 volunteers who were either standing or walking on a dynamic plantograph. The volunteers were exposed to either symmetrical or asymemtrical 5 or 10 kg load. Subsequently, statistical tests were used to search for any change of dynamic plantography parameters depending on the level of loading, on the type of loading and in the comparison of the situation with and without loading. The results...
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Trunk muscle activity and walkin
Svobodová, Jaroslava ; Opálková, Michaela (advisor) ; Davídek, Pavel (referee)
Bakalářská práce Aktivita trupového svalstva při chůzi Abstract This thesis refers to the anatomy and the kinesiology of trunk, spine and pelvis. In addition, it deals with the control of gait and gait biomechanics. Nevertheless, the main aim of this thesis is to summarize the information on the activity of trunk muscles during walking. This includes also trunk muscle activity during modified walking. This part reviews the changes of trunk muscle activity in reaction to low back pain and to the changes in walking speed. Thesis is focused on the activity of abdominal and back muscles such as m. rectus abdominis, m. obliguus externus abdominis, m. obliguus internus abdominis, m. transversus abdominis and m. erector trunci. The thesis also includes a case study of a patient who suffers from chronic low back pain. The activity of some of her trunk muscles was recorded by surface electromyography. In addition, the patient was tested using the zebris Rehawalk FDM-T system.
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