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Molecular characterisation of the binding of influenza virus neuraminidase with inhibitors
Hejdánek, Jakub ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
Influenza virus is responsible for seasonal epidemics among human population. Flu illness usually causes only mild symptoms and can be overcome by few days rest. However, this illness might have fatal consequences for young, elderly and immunocompromised individuals. Some viruses are able to "jump" across species resulting in rise of new types of virus that can have pandemic potential. The search for new treatment options to prevent emergence of new pandemics is thus a high priority task. Influenza virus neuraminidase is a protein located on the surface of viral particles. It has enzyme activity and catalyzes release of newly formed viral particles from cytoplasmic membrane of infected host cells. This step is crucial in virus life cycle, so the active site of this enzyme has become an important target for development of anti-influenza drugs. The neuraminidase inhibitors are currently the only effective drugs used for influenza treatment. Nowadays, there are two drugs against flu used worldwide: Tamiflu (oseltamivir) and Relenza (zanamivir). Recently, resistant influenza strains have been detected with increasing frequency. Point mutations in the active site of neuraminidase are causing decreased susceptibility of virus to inhibitors. Formerly, it was thought that these mutations would...
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The role of fibroblast activation protein and other proteases in tumour progression
Dvořáková, Petra ; Konvalinka, Jan (advisor) ; Gemperle, Jakub (referee)
Proteases are often associated with cancer and play a role of leading enzymes responsible for tumour cell invasion and metastasis. Proteolytic activity enables proteases to influence tumour progression in many ways, including cleavage of extracellular matrix, which is necessary for invasion. This thesis deals with the study of processes by which proteases regulate tumour development, either positively or negatively, and also mentions factors influencing the activity of the proteases. In connection with invasiveness of tumour cells, a major part of the thesis is dedicated to the role of fibroblast activation protein. This serine protease is expressed in stromal fibroblasts, is able to cleave collagen and has been thus established as a therapeutic target for the treatment of carcinomas. The emphasis is also put on dipeptidyl peptidase IV, a close homologue of fibroblast activation protein, especially in relation with a selective targeting of fibroblast activation protein using the inhibitors and other therapeutic agents, whose development and use are also outlined in this thesis. Powered by TCPDF (www.tcpdf.org)
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The effect of iron on the metabolism of the protozoan Trypanosoma brucei
Arbon, Dominik ; Konvalinka, Jan (advisor) ; Grantz Šašková, Klára (referee)
Iron ions are essential components of numerous cell processes. Their utilisation is strictly regulated, since any impairment can have devastating effect on the cell. In living organisms, iron ions are bound to proteins, for storage, transportation, or as a vital part of catalytic centers of enzymes. Transportation of iron ions between different compartments is important for the correct function of the cell. It was recently shown on yeast, how a mitochondrial transporter of iron ions is essential for the synthesis of iron-sulfur clusters of enzymes. This work aims to describe the localization and function of a homologous protein of a parasitical organism Trypanosoma brucei, which causes African trypanosomiasis, also known as sleeping sickness. This parasite is entirely dependent on uptake of iron ions from its host and therefore the utilization of iron ions is studied as a potential therapeutic target. This work is focused on the characterization of protein Mcp17, which is assumed to function as a transporter of iron ions into the mitochondria of T. brucei. Utilizing expression of marked Mcp17, the transporter was confirmed to be localized on the mitochondrial membrane of the cell. Measuring of enzyme activity of selected enzymes indicated that cells with inhibited expression of the gene mcp17...
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The role of iron in the metabolism of the amoeba Naegleria gruberi
Ženíšková, Kateřina ; Konvalinka, Jan (advisor) ; Hlouchová, Klára (referee)
Iron is a biogenic trace element that is vital for all organisms on the planet Earth. This element occurs in biological systems in the form of Fe3+ and Fe2+. These two forms are often incorporated in heme structures or iron-sulfur clusters. Proteins containing iron ions have a wide range of functions in organisms. The main functions include the transport of electrons in the respiratory chain (Rieske's proteins, cytochromes), DNA synthesis (ribonucleotide reductase) and the participation in the Krebs' cycle (aconitase, succinate dehydrogenase). Naegleria gruberi is a nonpathogenic amoeba known for its pathogenic relative Naegleria fowleri. This organism causes the primary amoebic meningoencephalitis. An interesting fact about Naegleria gruberi genome is that it contains genes for both aerobic and anaerobic metabolisms. The purpose of my bachelor work was to investigate the effect of availability of iron ions on metabolism in Naegleria gruberi. Changes in the activities of enzymes from different metabolic pathways were studied including lactate dehydrogenase, isocitrate dehydrogenase, Fe- hydrogenase, aconitase and fumarase. The most significant changes were observed in the activities of alcohol dehydrogenase and Fe-hydrogenase. Key words: Iron, heme, iron-sulfur clusters, availability of iron ions,...
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Functional role of HIF-1-regulated pathway in diabetic nephropathy
Nepomucká, Kateřina ; Konvalinka, Jan (advisor) ; Jonáková, Věra (referee)
Diabetic nephropathy (DN) remains the most common cause of end stage renal failure. Nearly 10% of patients with diabetes develop nephropathy. Hyperglycaemia in the kidneys leads to the activation of alternative metabolic pathways of glucose (glycation, activation of protein kinase C, and polyol pathway). These biochemical alterations lead to hypoxia and oxidative stress due to the increased formation of reactive oxygen species (ROS). Cellular response to hypoxia is controlled by hypoxia-induced factor 1 (HIF1), which is involved in the regulation of more than 800 genes. Target molecules of the HIF1 pathway participate in a wide range of physiological and pathological processes, e.g. angiogenesis, energy metabolism, apoptosis, migration, and proliferation. DN is associated with the pathological tissue remodelling process, epithelial-mesenchymal transition (EMT), and inflammation. HIF1 regulates key molecules of these pathological processes. EMT is regulated by TGFß1, CTGF, and SOX9. The progression of inflammation is regulated by VEGFA and AngII. The exact role of HIF1 signalling in the development of DN is not yet fully understood. This thesis evaluates the functional role of the HIF1 signalling pathway in the development of DN using a global heterozygous mutant with the deletion of the Hif1α gene....
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The role of D-amino acids in central nervous system
Pangrácová, Marie ; Konvalinka, Jan (advisor) ; Balík, Aleš (referee)
Only recently, the presence of D-amino acids in the mammalian central nervous system has been confirmed and their biological functions revealed. D-serine and D-aspartate, the best described D-amino acids, have been found to be the co-agonists activating NMDA receptors. In this way D-serine and D-aspartate, among other functions, affect synaptic plasticity which is the basic cellular mechanism for learning and memory. Pathological changes in the levels of these D-amino acids and their metabolical enzymes can lead to the development of epilepsy, schizophrenia, and neurodegenerative diseases such as amyotrophic lateral sclerosis, Huntington disease or Alzheimer disease. The main role in the D-serine synthesis is played by serin racemase while D-aspartate is synthetised with the help of aspartate racemase. The key enzymes for the degradation of D-amino acids are DAAO (D-amino acid oxidase) and DAspO (D-aspartate oxidase). This thesis presents an overview of available knowledge on the individual amino acids and their respective metabolical enzymes in the mammalian central nervous system, i.e. their distribution, cell localizations, metabolism and functions. Furthermore, the emphasis is put on the possibilities of inhibition and activation of the metabolical enzymes and their importance with respect to...
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Serine protease SmSP2 of Schistosoma mansoni
Leontovyč, Adrian ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
Blood fluke Schistosoma mansoni is one of the most important human parasites. Proteolytic system of schistosoma is crucial for parasite - host interactions. Therefore some of the proteases became potential therapeutic targets. This work is focused on not yet characterized serine protease SmSP2. SmSP2 is newly discovered protease of S. mansoni, whose biological role is unknown. This protease is highly expressed in developmental stages parasitizing humans. SmSP2 was recombinantly expressed in prokaryotic and eukaryotic expression system (E. coli a P. pastoris) and purified using chromatographic methods. Recombinant SmSP2 was used for polyclonal antibody production. Conditions for refolding were optimized. Basic biochemical properties of the protease were detected and substrate amino acid preferences for P1 - P4 sites for single aminoacids were identified using synthetic fluorogenic peptides for positional scanning substrate combinatorial library (PS-SCL). (In Czech)
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Structure-function study of N-terminal domain of protein SGIP1
Dvořáková, Michaela ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
The cells are communicating with each other using membrane-bound receptors. These receptors can recognize various ligands. Signalling via receptors allows the cell to control energy homeostasis, cell growth, differentiation, signalling and migration. Many of membrane-bound receptors are dynamically exchanged between plasma membrane and internal endosomal compartments by exo- and endocytosis. The most studied mechanism of endocytosis is clathrin-mediated endocytosis. There are many proteins involved in the sophisticated endocytic machinery. So called adaptor proteins allow and/or facilitate proper selection of cargo, which should be internalized. Some of them help to curve the membrane and form a vesicle, some of them may have opposite effect. "Src Homology 3-Domain Growth Factor Receptor-Bound 2-Like (Endophilin) Interacting Protein 1" (SGIP1) might fall in this category. This protein influences endocannabinoid signalling probably via its effect on cannabinoid receptors endocytosis. SGIP1 was recently identified as a gene involved in regulation of energy metabolism with overexpression leading to obesity. The aim of this work is structural and functional analysis of SGIP1 membrane phospholipid-binding domain (MP-domain). This domain shares no sequence homology with any of known proteins. In this...
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New analogues of prolactin-releasing peptide with prolonged effect on food intake
Tichá, Anežka ; Ryšlavá, Helena (advisor) ; Konvalinka, Jan (referee)
Prolactin-releasing peptide (PrRP) is a member of the family of RF-amide peptides. These peptides have typical C-terminal sequence -Arg-Phe-NH2 and similar biological effects. PrRP was discovered as an endogenous ligand of an orphan receptor GPR10 while searching for a factor responsible for a prolactin secretion. This effect was not later confirmed and nowadays, PrRP is mainly considered as an anorexigenic peptide. This is supported by a fact that PrRP and GPR10 deficient mice suffer from hyperphagia and late-onset obesity. Besides GPR10, PrRP is bound to NPFF2 receptor whose endogenous ligand is neuropeptide FF (NPFF). In this study, the PrRP's analogues modified at the N-terminus with fatty acids of different lenghts were tested in vitro on binding and activation MAPK/ERK1/2 signalling pathway. In in vivo experiments on food intake, the central anorexigenic effects of lipidized PrRP-analogues were tested provided their crossing blood brain barrier. Binding studies showed that all analogues bound to rat pituitary RC-4B/C cells with high affinity, analogues containing fatty acid with Ki of one order of magnitude lower than native PrRP. High affinity was also confirmed for binding to cells overexpressing GPR10 receptor and cell membranes with overexpressed NPFF2 receptor. All tested analogues...
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Analysis of substrate specificity and mechanism of GlpG, an intramembrane protease of the rhomboid family.
Peclinovská, Lucie ; Stříšovský, Kvido (advisor) ; Konvalinka, Jan (referee)
Membrane proteins of the rhomboid-family are evolutionarily widely conserved and include rhomboid intramembrane serine proteases and rhomboid-like proteins. The latter have lost their catalytic activity in evolution but retained the ability to bind transmembrane helices. Rhomboid-family proteins play important roles in intercellular signalling, membrane protein quality control and trafficking, mitochondrial dynamics, parasite invasion and wound healing. Their medical potential is steeply increasing, but in contrast to that, their mechanistic and structural understanding lags behind. Rhomboid protease GlpG from E.coli has become the main model rhomboid-family protein and the main model intramembrane protease - it was the first one whose X-ray structure was solved. GlpG cleaves single-pass transmembrane proteins in their transmembrane helix, but how substrates bind to GlpG and how is substrate specificity achieved is still poorly understood. This thesis investigates the importance of the transmembrane helix of the substrate in its recognition by GlpG using mainly enzyme kinetics and site-directed mutagenesis. We find that the transmembrane helix of the substrate contributes significantly to the binding affinity to the enzyme, hence to cleavage efficiency, but it also plays a role in cleavage site...
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