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Parasite cystatins as inhibitors of cysteine proteases: structural aspects of functional specificity and their evolution
Buša, Michal ; Mareš, Michael (advisor) ; Hudeček, Jiří (referee) ; Kukačka, Zdeněk (referee)
Members of the cystatin family are important inhibitors of cathepsin-type cysteine proteases and are involved in a number of pathologies. Parasite cystatins are attractive target molecules for parasite control, but our knowledge about them is still limited. This work is focused on cystatins of two blood-feeding parasites: the common tick (Ixodes ricinus) as the main vector of Lyme disease and tick-borne encephalitis, and the liver fluke (Fasciola hepatica), the causative agent of fasciolosis. Four novel cystatins were functionally and structurally characterized to determine the structural determinants of their inhibitory specificity and describe them in the context of evolution and physiological role of cystatins. The cystatin FhCyLS-2 from F. hepatica has broad inhibitory specificity and is suggested to play a dual role in the regulation of proteolytic systems in host tissue and the parasite gut. FhCyLS-2 combines the characteristics of two cystatin subfamilies in a unique way and is a model representative of a novel evolutionary group of cystatins identified in several orders of parasitic flukes. Ricistatin and iristatin are salivary cystatins of I. ricinus with immunomodulatory effects on the host caused by an exceptionally narrow inhibitory specificity. It was explained by structural modifications of...
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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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Liver fluke - treatment and resistance
Kněžíková, Tereza ; Vokřál, Ivan (advisor) ; Lamka, Jiří (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Tereza Kněžíková Supervisor: PharmDr. Ivan Vokřál, Ph.D. Title of diploma thesis: Liver fluke - treatment and resistence Fasciola hepatica is a parasite of global importance that we find both in farm animals and in humans. This thesis aims to summarize information on the potential of drugs and treatment alternatives that are suitable for treatment of F. hepatica. Given that for a number of drugs used in the past, as well as the drugs currently administered, resistance developed, this thesis is also focused on this phenomenon, especially the mechanisms of its origin. The drugs used to treat fasciolosis are called antitrematodal drugs. They can be divided into five chemical groups, of which the most important group are currently benzimidazoles and their representative triclabendazole. Also other drugs as albendazole, clorsulon, hexachlorophene, closantel, diamphenitide, bithionol, rafoxanide are important. The rate of resistance development is affected by many factors that may be genetic, biological or functional. F. hepatica actively uses its enzymatic system, especially oxidation enzymes or efflux transporters. The influence on the development of resistance, apart from the parasite itself,...
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Secreted proteases of the liver fluke and their interaction with endogenous inhibitor
Buša, Michal ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
The liver fluke, Fasciola hepatica, is one of the most important parasites of livestock, and it also infects humans. The proteolytic system of trematodes is critical for their interaction with the host and is a potential target for the development of novel vaccines. This work is focused on proteases secreted by F. hepatica adults and on FheCy2, a new protease inhibitor from the cystatin family. The proteolytic activity of the secreted proteases was analyzed using: (a) chromogenic protein substrates and fluorogenic peptide substrates, (b) selective protease inhibitors, and (c) a fluorescent activity-based probe for visualization of proteases. The results showed that the secreted proteases are cysteine proteases of papain family belonging to cathepsins L and B. These proteases were effectivelly inhibited by FheCy2 as demonstrated by enzymological analysis. It can be assumed that FheCy2 participates in the physiological regulation of endogenous proteases secreted by F. hepatica adults, which makes it attractive candidate protein for vaccination studies. Key words: Fasciola hepatica, cathepsins, proteolytic activity, substrate specificity, protease inhibitors (In Czech)
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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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Characterization of excretory-secretory proteins of liver fluke Fascioloides magna.
Beránková, Kateřina ; Kašný, Martin (advisor) ; Horn, Martin (referee)
Fascioloides magna (the giant liver fluke) originated from North America, is known in the Czech Republic since 1930s. This pathogenic fluke invades mostly cervids, but livestock too. Excretory-secretory products (ES products) contain number of esential biomolecules which are produced by excretory and secretory system of the fluke. These molecules play key role in many biological process during the life cycle not only of fascioloid flukes (e.g. migration in the host tissues, immune evasion and digestion). Due to their antigenic properties they could be also used in immunodiagnostics. Excretory-secretory proteins from adult Fascioloides magna and comparative related species Fasciola hepatica were purified and separated by the basic biochemical methods (1D, 2D electrophoresis, ion-exchange chromatography) and their activity was confirmed by specific (fluorogenic peptide) and nonspecific (gelatine) substrates. By using the mass spectrometry methods (MALDI TOF/TOF), the most abundant peptidolytically active proteins from ES products of F. magna were identified as cathepsin L (FmCL). Recombinant analog of FmCL was expressed in Pichia pastoris expression system. The peptidolytic activity was again confirmed using the synthetic fluorogenic substrates; the specifity of recombinant FmCL active site was...
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