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Cell cycle and differentiation in Giardia Intestinalis
Jiráková, Klára ; Nohýnková, Eva (advisor) ; Doležal, Pavel (referee) ; Müller, Norbert (referee)
Giardia is a unicellular parasitic organism; it is a worldwide cause of human diarrhea. It has minimalistic genome equipment and simplified molecular and metabolic pathways. In this respect, it is a suitable model organism for studying cell cycle regulation and to define the minimal genetic and protein equipment required for the functional reproduction of the eukaryotic cell. Its life cycle comprises of two stages; a pathogenic trophozoite and an infective cyst, which can survive in outer environment. New knowledge about encystation can be therapeutically important because this process is a target for vaccine and drug development. Since cell cycle analysis requires a synchronized population, we studied the effect of the synchronization drug aphidicolin on individual cell characteristics during the cell cycle of Giardia trophozoites. Our results showed that aphidicolin caused inhibition of DNA synthesis and trophozoites were aligned according to their DNA content in G1/S border. Subsequent inhibition of entry into mitosis and cytokinesis indicates, that Giardia has functioning DNA damage checkpoint. Extensive treatment with aphidicolin causes side effects. We detected positive signals for phosphorylated histone H2A which, in mammalian cells, is involved in a signaling pathway triggered as a reaction...
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Heme-protein interactions in cytochromes P450
Majerová, Kristýna ; Hudeček, Jiří (advisor) ; Stiborová, Marie (referee)
Cytochromes P450 (CYP) are monooxygenases participating reactions of the PhaseI in xenobiotic biotransformations. Better and more detailed knowledge of heme - protein interaction is crucial for understanding of function of these enzymes; in particular, of their regulation. We analyzed orientations of both heme vinyl side-chains in all X-ray structures of CYP enzymes available in the PDB data bank. For two mammalian forms, CYP 2A4 and 3A4, a more detailed analysis of the heme contacts with the apoprotein was performed, based on identification of the amino acid side chains in the vicinity. In addition to spatial information, dissociation constants and Coulombic interaction of polar residues in proximity to the heme were calculated using the PropKA web server. Deviations of the heme from planarity in both forms of P450 was investigated using the normal coordinate analysis (NSD server). Distribution of torsion angles of vinyls in position 2 and 4 of the heme shows that the side- chain at position 2 is conformationally more restricted in most P450 forms studied. Comparison of all forms shows that the range of ättainable values of torsional angles is very wide, practically unrestricted, and that the actual conformations of the heme moiety are probably determined more by the interaction with the protein,...
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An effect of environmental factors on foetal development in mammals
Sedláčková, Lucie ; Bartoš, Luděk (advisor) ; Bučková, Katarína (referee)
The development of fetus begins with merging of male and female sex cells, of the sperm with the egg, and with the formation of zygote. This moment triggers a complex process of prenatal development, a period very sensitive to changes and adverse conditions in the mothers surroundings. Such circumstances may include poor nutrition, sudden changes in the mothers social environment, a high occurrence of predators, stressful experiences due to inadequate handling and many other factors. All of these are connected by a frequently spelled term - stress.
It is stress that causes excessive discharge of glucocorticoid hormones, for example cortisol. This glucocorticoid is necessary for fetal growth and for induction of certain enzymes, such as lung surfactants. Under certain conditions, maternal cortisol can reach abnormal values. Consequently, the excess of cortisol impacts the fetus in high concentrations, which may negatively influence the growth of the fetus and its development.
In mammals, the mothers stress during her pregnancy not only increases her own cortisol levels, but also decreases the expression and activity of glucocorticoid barrier enzyme 11beta-HSD2 in the placenta; therefore, the fetus is less protected. On the other hand, this affects placental production and metabolism of other proteins and hormones sensitive to glucocorticoid, such as prostaglandins, progesterone, estrogens, glucose transporter and placental lactogen. Additionally, inhibition of 11beta-HSD2 by prenatal stress might contribute to low birth weight and to pregnancy diseases, such as premature birth.
Prenatal stress mainly affects brain development. The effects of prenatal stress in mammals have been observed with increased latency for games, indirect locomotion behavior, anxiety and impaired memory. Prenatal stress is also associated with greater volatility and attention disorders, which are also connected with a reduced learning ability. It has been ascertained that prenatally stressed individuals may exhibit increased aggressive behavior, demasculinized and feminized behavior has been observed in males.
The effects of prenatal stress are transmissible to the next generation and there is evidence that these are manifested to a greater extent in the following generations.
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Regulation of Cathepsin D Activity and Activation
Máša, Martin ; Mareš, Michael (advisor) ; Jonáková, Věra (referee) ; Holada, Karel (referee)
PhD Thesis ABSTRACT REGULATION OF CATHEPSIN D ACTIVITY AND ACTIVATION Martin Máša supervisor: Michael Mareš Department of Biochemistry Faculty of Science Charles University Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic PRAGUE 2009 Introduction Cathepsin D (CD) is an aspartic peptidase located in the lysosomes of all mammalian cells, its main role is catabolic degradation of proteins. More over CD is known to participate in a range of physiological processes such as apoptosis and tissue homeostasis, as well as in the regulation of angiogenesis and the production of peptidic antigens. The role of CD in pathophysiology is associated with several diseases such as Alzheimer's disease and cancer. Alzheimer's disease is a neurodegenerative disorder, which is generally considered to be the most common form of dementia. Progression of this disease is accompanied with the deposition of amyloid plaques (AP) in the brain, which leads to neurodegeneration. The AP is a fragment released from amyloid precursor protein (APP) cleaved by secretases1 . High levels of CD were found in cerebrospinal fluid of the Alzheimer's patients2 . It was demonstrated that CD is able to cleave APP and produce the pathogenic AP. A genetic polymorphism in the CD gene was reported, which changes...
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The role of Prp45p in mRNA expression and maturation
Abrhámová, Kateřina ; Půta, František (advisor) ; Pichová, Alena (referee) ; Janderová, Blanka (referee)
Prp45p of Saccharomyces cerevisiae and Snw1p of Schizosaccharomyces pombe are essential proteins, which share extensive homology with the mammalian transcription regulator and splicing factor SNW/SKIP. We have analyzed the essential function of these proteins in both yeasts and found a mutation (prp45(1-169)) that exhibited temperature sensitivity. The mutant strain harboring the corresponding chromosomal deletion shows temperature sensitive phenotype and hypersensitivity to cycloheximide, hydroxyurea, calcofluor white, and to microtubule inhibitors. At 30řC, the cells are often elongated, deformed, and larger than wt. After synchronization prp45(1-169) cells stop their growth with 2N DNA content at 37řC. We found that the temperature sensitivity is not overcome and the hypersensitivity to microtubule destabilizing drugs is only partially suppressed by the excision of intron from TUB1 gene. This distinguishes prp45(1-169) from those splicing factor's mutants that cause tubulin-dependent G2/M arrest, which can be relieved by the expression of intronless tub1. We performed analysis of splicing in vitro and found that splicing of optimal substrates is not impaired. We also compared the content and stability of RNA in wt-cells and in prp45(1-169) cells at variol temperatures using microarrays. The...
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Genome reprogramming during the first cell cycle of embryonic development
Barnetová, Irena ; Fulka, Josef (advisor) ; Pěknicová, Jana (referee) ; Strejček, František (referee)
The sperm head contains highly compacted genome. This compaction is mediated by protamines. Sperm protamines are replaced by cytoplasmic histones after the sperm entry into the oocyte. Beside the proteins replacement, also some epigenetic remodeling occurs. One of the most studied epigenetic remodeling in early zygotes is DNA demethylation. This phenomenon was observed in some mammals (mouse, rat, monkey) but not in some other mammals (sheep) and what is more, in some of them quite inconsistent data were published (pig, human, goat, rabbit). In our work we were mostly concentrated on porcine zygotes and attempted to explain the reasons of inconsistency in observed data. Three factors were evaluated in our work - the technique of embryo production, sperm factors, and the oocyte quality. In the first part of the study (the technique of embryo production) we compared the zygotes produced by conventional in vitro fertilization and zygotes produced by intracytoplasmic sperm injection. The epigenetic remodeling was evaluated by immunolabeling. There were no differences between zygotes produced by the two mentioned techniques. The paternal genome was not demethylated in any of zygotes. The labeling with anti-H3/K9- me2 (anti dimethyl group on lysine 9 of histone 3) showed the positive labeling of both...
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The Influence of eco-toxicological factors on spermatogenesis, morphology and function of mammalian sperm
Vašinová, Jitka ; Pěknicová, Jana (referee) ; Hortová, Kateřina (advisor)
An increasing infertility due to pathological changes on sperm has become a serious issue. A rising concentration of fluorides in the environment could be a potentially negative factor affecting the male fertility. Eco-toxicological effect of fluorides can be enhanced in the present of aluminium ions by forming fluoro-metallic complexes, analogues of phosphate groups that interfere with the activity of G-proteins and P-type ATPases, which are part of several signalling pathways during sperm maturation. The aim of this thesis was monitoring the effect of fluorides on mouse sperm capacitation in vitro, analysis of the effect of fluorides on male reproductive organ and on the level of mouse spermatogenesis. Fluorides were administered to mice through table water at the concentrations of 0, 1, 10, 100 ppm and at the same time we monitored their synergic action with aluminium at 10 ppm dose. The level of tyrosine phosphorylation in the sperm head was the main marker for successful capacitation in vitro. The results show that an oversized load of ions, to which mice were exposed, affected negatively spermatogenesis and ability of sperm to capacitate in vitro. Besides that, a multigenerational study in mice demonstrated that even low concentrations of aluminium fluorides can have a negative effect on...
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Mammalian circadian clock in peripheral organs, molecular mechanism and entrainment
Polidarová, Lenka ; Kuthan, Martin (referee) ; Sumová, Alena (advisor)
Mammalian circadian clock in peripheral organs, molecular mechanism and entrainment The circadian system controls timing of behavioral and physiological processes in most organisms. In mammals, central oscillator is located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. Apart from the SCN, peripheral oscillators are located in numerous organs like liver, heart, lung, muscle, intestine etc. The central and peripheral oscillators need to be synchronized by external cues (Zeitgeber). The SCN coordinates and entrains the phase of the clocks in numerous peripheral tissues via neuronal and humoral signals. For the SCN, dominant synchronizer is external light-dark cycle. Peripheral oscillators are cell-autonomous, they could work also independently of the SCN as a consequence of a feeding cycle. The basic molecular core clock mechanism responsible for generating circadian rhythms in the central and peripheral clocks is composed of transcriptional/translational feedback loops between the clock genes and their protein products. The aim of the present thesis was to ascertain whether the clock gene and protein expressions exhibit circadian rhythms in the rat intestine and whether the core clock mechanism drives expression of a cell cycle regulator rWee1. Next aim was to reveal how the circadian...
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Signalling of protein kinase B and expression of cyclooxygenase in early phases of diabetic nephropathy
Ždychová, Jana ; Komers, Radko (advisor) ; Haluzík, Martin (referee) ; Matoušovic, Karel (referee) ; Škrha, Jan (referee)
Introduction: Renal hypertrophy, extracellular matrix accumulation, aItered apoptosis as well as changes in regional hemodynamics have been implicated in the pathophysiology of nephropathy in diabetes mellitus (DM). On the molecular level the detailed mechanisms for development of diabetic nephropathy (DN) have becn intensively studied. Insulin induces a variety of biologicaI effects in a number of cell types via phosphatidylinositol-3 kinase (PI3K)/Akt kinase signaling pathway. Considering multiple function of Akt that incIude potentiaIly hannful pro-growth effects mediated by mTOR and cyclooxygenas-2 (COX-2), as well as protective effects mediated by endotheliaI nitric oxide synthase (eNOS), it is possible that aIterations in activities of Akt may play role in the pathophysiology ofDN. Renal corticaI activity and expression of Akt, its down-strearn effectors mTOR, eNOS, and "C<JIX-:Z. as well as PTEN, an endogenous Akt inhibitor, were investigated in streptozotocin (STZ): diabetic rats as a model of Type 1 DM with different levels of glycenůc control, and in Zucker ~d.iabetic fatty rats, a model ofDM2, and in nondiabetic rats as controls. Methods: Akt activity was measured by kinase assay. Protein expressions were measured by .immunoblotting and immunohistochemistry in renal cortex of 4- and 12- week old...
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