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Proteinová rodina HSP70 a její role v biotických interakcích
Čičmanec, Petr
Heat shock proteins 70 (HSP70s) are ubiquitous and widespread proteins across prokaryotic and eukaryotic organisms, and accumulated evidence indicates that these proteins are involved in much more than the response to heat shock. This thesis characterizes the role of HSP70s in response to different stimuli and provides insight into the HSP70's role in regulating the interaction between biotic and abiotic factors. The theoretical part summarizes the current knowledge of HSP70, including information about its structure, activity, posttranslational modifications, and the so-called chaperone code. It describes the functions of HSP70s and pays particular attention to the HSP70s' role in immunity and biotic interactions. The experimental part is divided into several sections. First, a bioinformatic analysis provides a cross-kingdom comparison of HSP70 and its role in biotic stress. This part is supplemented with the analysis of HSP70 distribution and abundance at the proteome level. Next, the effect of different stimuli on HSP70 protein abundance in different model organisms is presented, including E. coli, S. cerevisiae, A. thaliana, Pisum sativum, and Linum usitatissimum. Finally, the effects of the hsp70 mutation are analyzed, providing insight into the complexity of the HSP70 machinery and confirming its role in the biotic interaction between a model plant and fungi.
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Heat-shock proteins and plant tolerance against high temperature stress
Ott, Kristián ; Hála, Michal (advisor) ; Konrádová, Hana (referee)
In context with ongoing climate change, the average temperature is still rising. This is a problem mainly for agriculture and production of groceries, because plants generally have difficulties when coping with ambient temperature higher than 40 řC. If there would be continuing trend in decrease of global food production, it could cause problems with feeding still growing world population. Organisms in general developed many mechanisms of stress adaptation. One of these evolutionary adaptions to high temperature stress is production of heat shock proteins (HSPs). HSPs are very conserved family of proteins present in both prokaryotes and eukaryotes. This thesis summarizes present knowledge about the HSPs and their function (not only) during the high temperature stress in plants, but some of the information presented in this thesis were obtained also from other organisms. The thesis starts with general introduction to the high temperature stress and how plants can sense the ambient temperature. Next is general mechanism of induction of HSPs and their classification, structure, and mechanism of action. The possibility of future use of HSPs in agriculture and medicine is opened in last chapter.
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Heat shock proteins - - their role in diagnosis and prognosis of pregnancy related complications
Dvořáková, Lenka ; Hromadníková, Ilona (advisor) ; Růžičková, Šárka (referee)
Heat shock proteins increase their gene expression after exposure of cells or organisms to some forms of stress, which may be high temperature, infection, inflammation, hypoxia, lack of nutrients and water. Stressful situations for the body are also pregnancy-related complications associated with placental insufficiency - preeclampsia and IUGR, as well as other pregnancy-related complications such as fetal growth restriction and gestational hypertension. Therefore, I examined whether the occurrence of pregnancy-related complications (preeclampsia, fetal growth retardation, gestational hypertension) affect the gene expression of heat shock proteins. Five hsp systems was detected, namely Hsp27, Hsp60, Hsp70, Hsp90 and HspBP1 in placental tissue samples and whole maternal peripheral blood. Samples came from women with physiological pregnancy and from women with certain pregnancy-related complications (PE, FGR, GH). RNA was isolated from the samples. Detection of hsp expression was performed by using real-time RT-PCR and the comparative Ct method. Changes in gene expression between the test samples and reference sample were examined. To assess the difference between physiological pregnancies and pregnancies with selected pregnancy- related complications, an analysis of variance (ANOVA) was used....
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Studies on the molecular mechanisms of cardioprotective effects of morphine
Škrabalová, Jitka ; Novotný, Jiří (advisor) ; Nováková, Olga (referee) ; Hlaváčková, Markéta (referee)
Acute and chronic morphine administration can significantly reduce ischemia- reperfusion injury of the rat heart. However, the molecular mechanisms mediating the protective effect of morphine are not yet fully elucidated. Concurrently, there is a lack of information about the effects of the long-term action of morphine on heart tissue. Therefore, in the first part of the project, we studied the effect of long-term administration of high doses of morphine (10 mg/kg/day, 10 days) on rat heart tissue. In the second part of the project, we investigated the effect of 1 mM morphine on viability and redox state of rat cardiomyoblast cell line H9c2 that was influenced by oxidative stress elicited by exposure to 300 μM tert-butyl hydroperoxide (t-BHP). Our experiments have shown that long-term morphine administration affected neither the amount nor the affinity of myocardial β-adrenergic receptors (β-AR), but almost doubled the number of the dominant isoforms of myocardial adenylyl cyclase (AC) V/VI and led to supersensitization of AC. At the same time, proteomic analyses revealed that long-term morphine administration was associated with significant changes in the left ventricular proteome. In particular, there was an increase in the expression of heat shock proteins (HSP). Increased expression of HSP27...
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Role of heat shock proteins in the pathogenesis of leukaemia
Kopřivová, Olga ; Hromadníková, Ilona (advisor) ; Černá, Marie (referee)
(Abstract) Some of heat shock proteins (Hsp), for example the inducible form Hsp70, are expressed on the surface of tumour cells. High Hsp expression is reflected in tumour cell features, such as ability to progression, to metastasize and resistance to apoptosis. The question is whether Hsp gene expression correlates with surface expression. The aim of this master thesis is to compare surface and gene expression of Hsp70 and observe the gene expression of some other Hsp proteins (Hsp27, Hsp60, Hsp90 and HspBP1) in leukaemia. The research was carried out on cell lines obtained from leukaemic blasts of patients with acute myeloid leukaemia: UoC-M1, HL-60, OCI/AML3, THP-1, HU-3 and TF-1 that had been cultivated in vitro. Hsp70 surface expression was detected using flow cytometry, and gene expression of each Hsp was studied using real-time RT-PCR. It was found out that high surface expression of Hsp70 did not correlate with gene expression in consequence of negative feedback applied in Hsp expression regulation. Hsp27 gene expression was increased compared to negative (healthy) control on all tumour cell lines, with the highest increase on the THP-1 line. Hsp60 gene expression was increased compared to negative (healthy) control on all tumour cell lines and there were not remarkable differences in...
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Use of biochemical methods in the study of plant metabolism with emphasis on the defense system
Bělonožníková, Kateřina ; Ryšlavá, Helena (advisor) ; Luhová, Lenka (referee) ; Plchová, Helena (referee)
As sessile organisms, plants cannot avoid being exposed to various biotic and abiotic stress factors. To defend themselves plants have developed complex and sophisticated defense mechanisms. In agriculture, pesticides are used for plant protection, but they can have undesirable side effects, and therefore attention is being paid to biological control agents. One such agent is Pythium oligandrum, a soil oomycete that has a positive effect on plants at several levels. On the one hand, it secretes elicitins, which stimulate defensive responses and prepare the plant for possible infection (i.e., priming effect), and on the other hand, it produces tryptamine, a precursor of auxin, thus supporting the plant's growth. Furthermore, P. oligandrum directly acts as pathogen mycoparasite or evokes antibiosis, or competition for nutrients and space with other microorganisms. The key research topic of this thesis was the analysis of the biochemical properties of eleven Pythium strains and their impact on plant metabolism. Our results showed that even closely related P. oligandrum strains significantly differ in the content of compounds secreted into the medium, including proteins, amino acids, tryptamine, and hydrolytic enzymes capable of degrading cell walls (endo-β-1,3-glucanase, chitinase, and cellulase),...
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Use of biochemical methods in the study of plant metabolism with emphasis on the defense system
Bělonožníková, Kateřina
As sessile organisms, plants cannot avoid being exposed to various biotic and abiotic stress factors. To defend themselves plants have developed complex and sophisticated defense mechanisms. In agriculture, pesticides are used for plant protection, but they can have undesirable side effects, and therefore attention is being paid to biological control agents. One such agent is Pythium oligandrum, a soil oomycete that has a positive effect on plants at several levels. On the one hand, it secretes elicitins, which stimulate defensive responses and prepare the plant for possible infection (i.e., priming effect), and on the other hand, it produces tryptamine, a precursor of auxin, thus supporting the plant's growth. Furthermore, P. oligandrum directly acts as pathogen mycoparasite or evokes antibiosis, or competition for nutrients and space with other microorganisms. The key research topic of this thesis was the analysis of the biochemical properties of eleven Pythium strains and their impact on plant metabolism. Our results showed that even closely related P. oligandrum strains significantly differ in the content of compounds secreted into the medium, including proteins, amino acids, tryptamine, and hydrolytic enzymes capable of degrading cell walls (endo-β-1,3-glucanase, chitinase, and cellulase),...
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Studies on the molecular mechanisms of cardioprotective effects of morphine
Škrabalová, Jitka
Acute and chronic morphine administration can significantly reduce ischemia- reperfusion injury of the rat heart. However, the molecular mechanisms mediating the protective effect of morphine are not yet fully elucidated. Concurrently, there is a lack of information about the effects of the long-term action of morphine on heart tissue. Therefore, in the first part of the project, we studied the effect of long-term administration of high doses of morphine (10 mg/kg/day, 10 days) on rat heart tissue. In the second part of the project, we investigated the effect of 1 mM morphine on viability and redox state of rat cardiomyoblast cell line H9c2 that was influenced by oxidative stress elicited by exposure to 300 μM tert-butyl hydroperoxide (t-BHP). Our experiments have shown that long-term morphine administration affected neither the amount nor the affinity of myocardial β-adrenergic receptors (β-AR), but almost doubled the number of the dominant isoforms of myocardial adenylyl cyclase (AC) V/VI and led to supersensitization of AC. At the same time, proteomic analyses revealed that long-term morphine administration was associated with significant changes in the left ventricular proteome. In particular, there was an increase in the expression of heat shock proteins (HSP). Increased expression of HSP27...
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Biological control of plant pathogens
Chudý, Michal ; Ryšlavá, Helena (advisor) ; Müller, Karel (referee)
Heat shock proteins (HSPs) play a major role in protecting cellular proteins, acting as chaperones, preventing aggregation of partially damaged molecules, and help the protein repack, repair and create its right three-dimensional structure. Their synthesis occurs mainly during the stress state of the cell or the whole plant. Biotic forms of stress induce the expression of pathogenesis-related proteins (PRs), which are often characterized by enzyme activity, such as glucanases, chitinases and peroxidases. In this work, the effect of infection with fungal pathogens Alternaria brassicicola and Verticillium longisporum on the content of HSP70, HSP90 and PR-1, PR-2, PR-3 was followed in the tomato plants (Solanum lycopersicum L. cv. Micro-Tom) and rapeseed plants (Brassica napus subsp. oleifera). The seeds were treated with isolates of oomycete Pythium oligandrum, which is one of the unique and commercially available biological control agents. HSPs and PRs proteins were detected immunochemically on a nitrocellulose membrane. Immunochemical analysis showed the interaction of primary antibodies against HSP70 and HSP90 with low relative molecular weight proteins. Infection with Alternaria brassicicola and Verticillium longisporum increased the representation of 33 kDa - 43 kDa proteins. Seed treatment with...
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Studies on the molecular mechanisms of cardioprotective effects of morphine
Škrabalová, Jitka
Acute and chronic morphine administration can significantly reduce ischemia- reperfusion injury of the rat heart. However, the molecular mechanisms mediating the protective effect of morphine are not yet fully elucidated. Concurrently, there is a lack of information about the effects of the long-term action of morphine on heart tissue. Therefore, in the first part of the project, we studied the effect of long-term administration of high doses of morphine (10 mg/kg/day, 10 days) on rat heart tissue. In the second part of the project, we investigated the effect of 1 mM morphine on viability and redox state of rat cardiomyoblast cell line H9c2 that was influenced by oxidative stress elicited by exposure to 300 μM tert-butyl hydroperoxide (t-BHP). Our experiments have shown that long-term morphine administration affected neither the amount nor the affinity of myocardial β-adrenergic receptors (β-AR), but almost doubled the number of the dominant isoforms of myocardial adenylyl cyclase (AC) V/VI and led to supersensitization of AC. At the same time, proteomic analyses revealed that long-term morphine administration was associated with significant changes in the left ventricular proteome. In particular, there was an increase in the expression of heat shock proteins (HSP). Increased expression of HSP27...
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