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Functional characterization of selected Kunitz proteins of Eudiplozoon nipponicum
Tymich, Alexandr ; Mikeš, Libor (advisor) ; Kašný, Martin (referee)
Proteins containing the Kunitz domain are mostly 6-10 kDa inhibitors of serine proteases, but in exceptional cases they can also inhibit cysteine and aspartic proteases. The main characteristic is the presence of six cysteine residues forming three disulfide bridges creating a typical active loop, which is complementary to the active site of various proteases. The specificity of this binding is largely determined by the amino acid in the P1 position. Their functions include the regulation of a number of physiological events based on proteolysis, e.g. the blood coagulation cascade or immune reactions. However, due to their nature, they have also become a powerful tool for parasitic organisms to interact with their host, where they again target proteases involved in the host's physiological events and thus allow the parasite to survive the interaction with the host. Until recently, representatives of the class Monogenea were a neglected group from the point of view of molecular parasite-host interactions, and only a few works were devoted to their biochemistry and the description of biologically active molecules. In this work, I focused on two selected Kunitz proteins in Eudiplozoon nipponicum, a blood-sucking ectoparasite from the Monogenea class, which has become a fairly common parasite of common...
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Dominant protein antigens of Toxocara canis
Skulinová, Kateřina ; Kašný, Martin (advisor) ; Panská, Lucie (referee) ; Vadlejch, Jaroslav (referee)
Larval toxocarosis is a worldwide widespread zoonosis occurring in developed countries as well as developing countries. The disease is caused by roundworms of the genus Toxocara, primarily intestinal parasites of dogs, cats and other animals. Viable eggs released into the environment with the dog's faeces can infect not only definitive hosts, but also paratenic hosts, which include many vertebrates, some invertebrates, and also humans. In humans, larval migration can cause severe and irreversible tissue damage, which is characterized by various clinical forms of the disease. For the purposes of routine diagnosis of larval toxocarosis, the most frequently used method so far is ELISA and Western blot, which enable the reaction demonstration of specific antibodies with the larval excretory-secretory product (TES). TES is obtained for diagnostic purposes from larvae cultured in nutrient medium. The preparation of such an antigenic mixture is very laborious and may vary across the laboratories. Current research in the field of diagnosis of larval toxocarosis is therefore focused on the standardization of serodiagnostic procedures. A fundamental prerequisite is knowledge of the detailed composition of TES, especially antigenic (protein) molecules. However, the number of studies devoted to the...
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The role of UDP-glycosyltransferase in development of drug resistance in parasitic nematodes
Dimunová, Diana ; Matoušková, Petra (advisor) ; Bílková, Zuzana (referee) ; Kašný, Martin (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Diana Dimunová Supervisor: doc. Ing. Petra Matoušková, Ph.D. Title of Doctoral Thesis: The role of UDP-glycosyltransferases in drug resistance in parasitic nematodes The diseases caused by parasitic nematodes represent a serious problem, which threatens livestock's health, because pharmacotherapy is complicated by widespread anthelmintic resistance. Understanding of the mechanisms of parasite drug resistance and defense strategies is important to maintaining the effectiveness of currently used anthelmintics and developing new approaches to controlling these infections. The ability of parasites to inactivate anthelmintics through their metabolism, which is provided by biotransformation enzymes, may contribute to the development of drug resistance. The UDP-glycosyltransferases (UGT) superfamily can protect parasites from the toxic actions of anthelmintics by modifying drugs to inactive glycoside metabolites. These metabolites have been identified in benzimidazole metabolism to an increased extent in a resistant strain of H. contortus, which suggests the involvement of UGTs in anthelmintic resistance. In the genome of this parasitic nematode, 32 genes encoding UGTs divided into 15 families have...
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The role of UDP-glycosyltransferase in development of drug resistance in parasitic nematodes
Dimunová, Diana ; Matoušková, Petra (advisor) ; Bílková, Zuzana (referee) ; Kašný, Martin (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Mgr. Diana Dimunová Supervisor: doc. Ing. Petra Matoušková, Ph.D. Title of Doctoral Thesis: The role of UDP-glycosyltransferases in drug resistance in parasitic nematodes The diseases caused by parasitic nematodes represent a serious problem, which threatens livestock's health, because pharmacotherapy is complicated by widespread anthelmintic resistance. Understanding of the mechanisms of parasite drug resistance and defense strategies is important to maintaining the effectiveness of currently used anthelmintics and developing new approaches to controlling these infections. The ability of parasites to inactivate anthelmintics through their metabolism, which is provided by biotransformation enzymes, may contribute to the development of drug resistance. The UDP-glycosyltransferases (UGT) superfamily can protect parasites from the toxic actions of anthelmintics by modifying drugs to inactive glycoside metabolites. These metabolites have been identified in benzimidazole metabolism to an increased extent in a resistant strain of H. contortus, which suggests the involvement of UGTs in anthelmintic resistance. In the genome of this parasitic nematode, 32 genes encoding UGTs divided into 15 families have...
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Inhibitors of proteolytic enzymes of Trematodes
Šteiger, Vladimír ; Kašný, Martin (advisor) ; Salát, Jiří (referee)
i Abstract Trematodes are important parasites possessing various localization in the bodies of invertebrate and vertebrate hosts, including human; therefore they are subject of long time intensive worldwide research. Trematodes developed various adaptations and strategies (some of them have also molecular background) enable them to survive in the host bodies. Trematodes produce large amount of different molecules, which are involved in various physiological processes. Inhibitors of proteolytic enzymes form a large group of biologically active compounds, e.g. they regulate the activity of peptidases or modulate host immune response. Many of these inhibitors are investigated as potential candidates in chemotherapeutic fight against trematodes. This thesis reviews the information concerning the natural inhibitors produced by trematodes and also synthetic inhibitors. Key words: Inhibitor, trematode, peptidase, serpin, cystatin i
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Features and functions of glycocalyx of trematode cercariae
Chaloupecká, Jana ; Mikeš, Libor (advisor) ; Kašný, Martin (referee)
Trematodes are parasites from phylum Platyhelminthes which have compex life cycles involving two to four hosts. This work focuses especially on trematodes of the family Schistosomatidae. Their cercariae which leave the snail intermediate host, actively penetrate the skin of definitive hosts and transform into schistosomula. This is accompanied by detachment of cercarial tail and emptying of penetration glands. During transformation, cercarial bodies undergo extensive ultrastructural and molecular changes. One of these changes is the loss of surface glycocalyx which represents a protective coat in the aquatic environment. In glycocalyx shedding, participation of proteolytic enzymes from cercarial penetration glands is expected during invasion of the host. Glycocalyx has specific composition of saccharide molecules which are bound to lipids or proteins on the membrane of cercarial tegument. This work describes the origin, ultrastructure, saccharide composition, function and shedding mechanism of cercarial glycocalyx.
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Ontogenesis of trematode larval stages of the family Fasciolidae in the intermediate snail hosts.
Pankrác, Jan ; Kašný, Martin (advisor) ; Soldánová, Miroslava (referee)
The family Fasciolidae is an important group of trematodes including serious pathogens of humans and livestock. The life cycle is divided into two phases - sexual reproduction in the definitive host body (large land mammals) and an asexual reproduction in the intermediate host body (aquatic snails of the family Lymnaeidae and Planorbidae). Development within the snail host is characterized by production of large amounts of parasite larvae (sporocyst, rediae, cercariae). Cercariae released from the snail immediately start to transform into metacercariae, the larval stages infectious for definitive host. Snail phase of infection is generally accompanied by number of mostly negative symptoms (massive pathological changes, often followed by reduction of fertility). This summary reveals that current knowledge concerning the ontogenetic development of fasciolids in the intermediate host is uncomplete and unequally investigated. According to this summary is also obvious that some of the published findings are universally valid for all members of the family Fasciolidae and other are characteristic only for particular species.
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Cathepsin L by parasites - occurrence, features, functions
Perháčová, Terézia ; Mikeš, Libor (advisor) ; Kašný, Martin (referee)
Cathepsines L are lysosomal cysteine endopeptidases with an universal function in protein catabolism. This work discusses present knowledge about their characteristics in the context of their specific function in parasites. Features and function differences are described in detail on molecular level. The emphasis is on the biochemical properties with resultant use of these enzymes. Cathepsines L of kinetoplastida, aplikomplexa, entamoeba and helmints (focused on Fasciola spp and Schistosoma spp) are each discussed in appropriate chapters. Key words: hydrolase, protease, cysteine peptidase, cathepsin L, lysosome, parasite
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Peptidases of Trematodes
Kašný, Martin ; Mikeš, Libor (advisor) ; Kopáček, Petr (referee) ; Haas, Wilfried (referee)
90 3. SUMMARY The text above refers about the majority of characterized trematode peptidases; the fundamental enzymes for trematode existence, which are integrated in many physiological processes like pathogenesis, tissue invasion/migration, nutrition, immune evasion and host-parasite interactions. In the history (until 1996), the peptidase catalytic activities in trematode extracts have been monitored. During 1980s and 1990s, the information of first cloned trematode peptidase genes were published and during last three decades cca 90 trematode peptidase sequences belonging to 19 peptidase families of 5 clans have been identified. The most studied trematode peptidases have been of Schistosoma mansoni origin: the serine peptidase - cercarial elastase (of cercariae), cysteine peptidases - cathepsins B, L, F, C plus the asparaginyl endopeptidase SmAE and the aspartic peptidase - cathepsin D (of adult worms and some other life stages). The recent computational cluster analysis revealed that the sequence S. mansoni elastase (the main cercarial penetration enzyme) is quite divergent from other serine peptidases of the S1 family. Cercarial elastase gene was proved in S. mansoni, S. haematobium and Schistosomatium douthitti, but not in the related S. japonicum. Mass spectrometry analysis confirmed cercarial...
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