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Interaction of protein subunits SEC10 and SEC15 of the exocyst tethering complex
Bartáková, Anna ; Ryšlavá, Helena (advisor) ; Čermáková, Michaela (referee)
The exocyst is an evolutionarily conserved tethering complex involved in the regulation of the secretory pathway in eukaryotic cells. As an effector of Small GTPases, the exocyst contributes to efficient targeting of secretory vesicles to the sites of intense exocytosis at the plasmatic membrane through interactions with specific membrane phospholipids. The exocyst complex consists of eight subunits: SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70, and EXO84. Each subunit binds at least two other subunits, among them the interaction of SEC10 and SEC15 is crucial for the exocyst function. The question remains how this particular interaction is evolutionarily conserved across different plant taxonomic groups. SEC10 and SEC15 genes from the three evolutionary groups of plants (green algae - Klebsormidium nitens, mosses - Marchantia polymorpha, angiosperms - Arabidopsis thaliana) were cloned into yeast two-hybrid vectors for studying protein interactions. Testing the interaction of SEC10 and SEC15 exocyst subunits from different plant species in the yeast-two hybrid system showed that despite partially different protein sequences of the tested subunits, their interaction across different evolutionary groups of plants remains very well conserved.
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Role of Arginine 717 in insulin receptor respective Arginine 704 in IGF-1 receptor for the interaction with ligands
Kertisová, Anna ; Selicharová, Irena (advisor) ; Ryšlavá, Helena (referee)
Insulin and insulin-like growth factor 1 (IGF-1) are peptide hormones that are important regulators of cellular metabolism, proliferation and apoptosis. Disruptions in signalling pathways may cause a whole range of diseases from diabetes mellitus type 1 and type 2 to cancer or neurodegenerative diseases. The cellular response to these hormones is mediated by insulin (IR) and IGF-1 receptors (IGF-1R) with a tyrosin-kinase activity. Receptors are created as hetero-tetramers of two extracellular α-subunits and two intracellular β-subunits. Studies of receptor structures try to elucidate the basic principles of the interaction of receptors with their ligands. However, the role of some amino-acid residues in binding remains unclear. It was suggested that the arginine 704 of IGF-1R may interact with Glu58 IGF-1. In comparison with IGF-1R, the equivalent arginine 717 IR was not associated with an important role in insulin binding in previous studies. This thesis is focused on clarifying the role of Arg704 IGF-1R and for comparison analogically on Arg717 IR isoform A (IR-A) in ligand binding to the receptors. Therefore, mutant variants of IGF-1R in positions His697 and Arg704 and variants IR-A in positions His710 and Arg717 were created. The role of histidines 697 IGF-1R and 710 IR was already elucidated...
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The characterisation of sperm and seminal plasma glycoproteins: Their role within the tetraspanin network and male fertility
Páleníková, Veronika ; Postlerová, Pavla (advisor) ; Ryšlavá, Helena (referee) ; Pěknicová, Jana (referee)
Mgr. Veronika Páleníková only to describe the localization and heterodimerization of α3, α6, αV, β1, and β4 integrins in mentioned integrins. Importantly, we detected the presence of integrin β4 subunit in mouse sperm, and characterised that it forms functional heterodimer with α6 subunit. In tion, we described α6β1 and α3β1integrin pairs, which contribute to the integrity and complexity of sperm membranes. Moreover, we found that α6 integrin subunit interacts with proposed CD9 and CD81 as tetraspanin partners for αV integrin in tetraspanin web. Our goal
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Structural characterization of a model heme-containing oxygen sensor
Tajovská, Eva ; Martínková, Markéta (advisor) ; Ryšlavá, Helena (referee)
One subgroup of hemoproteins are heme-based gas sensors, which are able to detect biatomic gas molecules in their immediate surroundings. Upon binding of a gas molecule to the heme iron in a sensor domain of these proteins or, conversely, upon its dissociation from the heme iron, the signal is then transmitted from the sensor domain to a functional domain and subsequent regulation of important cellular functions occurs. Understanding the regulatory mechanism of gas sensors is key to potentially manipulating their function. Such knowledge would then allow the use of heme-based gas sensors as therapeutic targets for the development of next-generation antibiotics, if we take into account their presence in pathological bacteria. The diploma thesis focuses on a model heme-based gas sensor, the oxygen sensor EcDOS from E. coli, and its apoform, EcDOS His77Ala. Both proteins were prepared by recombinant expression and purification, and subsequently spectrophotometrically characterized. Using gel permeation chromatography, the oligomeric states of EcDOS Fe(III), EcDOS Fe(II)-O2 and EcDOS His77Ala were determined under different conditions (different temperatures of protein incubation, presence of c-di-GMP substrate etc.). Furthermore, the structural dynamics of EcDOS Fe(III), EcDOS Fe(II)-O2 and EcDOS...
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Selected nutritional supplements as modulators of biotransformation enzymes
Bača, Ondrej ; Hodek, Petr (advisor) ; Ryšlavá, Helena (referee)
Flavonoids are secondary metabolisms of plants, that have wide range of biological activities. Between the most known activities are antioxidant activities, antibacterial activities, antivirus activities and chemoprotective activities. They are consumed as a part of plants to varying degrees by the population. They are consumed either in their plant forms, or as a nutritional supplement, such as pills. As nutritional supplement they don't fall under strict regulations and their effect or active quantity isn't always examined properly. As a foreign substance to the body that is xenobiotics, they can fundamentally influence so called biotransformation enzymes, which apart from other handle metabolism of xenobiotics. One of those enzymes is N-acetyltransferase. This biotransformative enzyme, which among others metabolises lot of pharmaceuticals, shows number of polymorphisms, where the occurrence of some of those relates to different illnesses such as breast cancer or bladder cancer, diabetes, or Parkinson. The modulation activity of those enzyme can either hurt the organism or help it. In this paper we will focus on inhibitive effect of flavonoids myricetin and dihydromyricetin to N-acetyltransferase especially the mechanism of this inhibition. Inhibition was measured in vitro for both steps of the...
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Effect of triazole fungicides on soil properties
Račko, Ján ; Ryšlavá, Helena (advisor) ; Smrček, Stanislav (referee)
Plant protection still depends on the use of pesticides, often in mixtures that pose a potential threat to non-target organisms. Triazole fungicides (tebuconazole, penconazole and others) are widely used in agriculture against fungal pathogens, but they can affect both the metabolism of the plants themselves and the composition of soil organisms. Biochemical indicators of soil health and quality include the content of microorganisms and the activity of glycosidase, protease and dehydrogenase enzymes. In this work, it was investigated how the application of penconazole and tebuconazole and their combination by watering the soil and spraying on the leaves of edible tomato plants (Solanum lycopersicum L., cv. Cherrola) affects the activities of enzymes involved in carbon and phosphorus metabolism. Endoglycosidases involved in the degradation of plant and fungal cell walls (cellulases, chitinases) were found to have reduced activities in the soil contaminated with triazoles, while the activity of endo-β-1,3-glycanase was increased in the sPT group. The activity of exoglycosidases (β-glucosidases, β-hexosaminidases, β-galactosidases and α-mannosidases) in the soil was increased. The activity of these enzymes was also determined in the roots of tomato plants, from which enzymes can be secreted into the soil....
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Antioxidant properties of tobacco plants grown in vitro with different nitrogen sources
Vlčková, Denisa ; Ryšlavá, Helena (advisor) ; Kubíčková, Božena (referee)
The established concept of plant nutrition with NO3 − and NH4 + ions, as the only absorbable forms of nitrogen, has been expanded in recent years to find that some plants are also able to use amino acids and peptides as a nitrogen source. In this work, the content of proteins, phenolic compounds and flavonoids, antioxidant capacity and activity of antioxidant enzymes in leaf extracts of six groups of tobacco plants grown in vitro were observed. The nutrition of these plants varied in the form and concentration of nitrogenous compounds. The experimental groups included: control plants grown on complete Murashige-Skoog agar rich in NH4 + and NO3 − ions (MS), plants grown in the presence of NO3 − ions, with reduced nitrogen concentration compared to MS (AD), plants grown in the presence of casein hydrolysate as the sole nitrogen source (H), plants grown in presence of both NO3 − ions and casein hydrolysate (AD+H) and AD and H plants grown in the presence of polyethylene glycol simulating drought stress (AD+PEG and H+PEG). Of all groups, plants H showed the highest content of phenolic compounds and flavonoids and the highest values of antioxidant capacity and antioxidant enzyme activity (catalase, glutathione reductase, superoxide dismutase, total peroxidases, ascorbate peroxidase, glutathione-S-...
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Isolation and study of proteins with zona pellucida binding activity
Zigo, Michal ; Postlerová, Pavla (advisor) ; Ryšlavá, Helena (referee) ; Antalíková, Jana (referee)
Binding of sperm to the oocyte is mediated by complementary molecules on the surface of both gametes and involves the interaction of sperm protein receptors with the zona pellucida (ZP) saccharide structures. It has been shown that many proteins receptors are involved in the sperm-ZP interaction, and potential primary sperm receptors for ZP glycoproteins have been investigated in various mammals. The majority of proteins with identified sperm-ZP binding activity belong to the plasma membrane proteins. However, the exact methods for isolation of sperm membrane proteins are still to be standardized. This study is focused on investigating how employment of various isolation protocols leads to acquisition of various protein mixtures. Further in the work, two possible approaches towards identification of potential ZP-binding partners are implemented, in order to disclose novel primary ZP-binding receptor candidates. Sperm proteins of ejaculated and in vitro capacitated boar sperms were isolated by: Triton X-100, Triton X-114, acetic acid, sodium dodecyl sulphate (SDS), N-octyl-β-D- glucopyranoside (OBG), rehydration buffer (RHB), and finally by freezing-thawing extraction and they were characterized by 1-D, 2-D protein profiles, glycoprotein staining and substrate zymographic methods. The results have...
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