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Glycocalyx shedding by cercariae of bird schistosomes
Chaloupecká, Jana ; Mikeš, Libor (advisor) ; Štěrba, Ján (referee)
Trichobilharzia spp. are avian schistosomes related to medically important human parasites of the genus Schistosoma. Penetrating cercariae are well known as causative agent of cercarial dermatitis in humans. Cercariae actively penetrate the skin of definitive hosts and transform into schistosomula. This process is preceded by cercarial tail detachment and includes emptying of penetration glands and extensive surface changes. One of these changes is the loss of highly immunogenic glycocalyx which represents a protective coat in the aquatic environment. The glycocalyx has specific composition of saccharide molecules which are bound to lipids or proteins on the membrane of cercarial tegument. There is only limited information about the mechanism of shedding. Hypotheses based on indirect evidences suggest that peptidases or (phospho)lipases from penetration glands could be involved. This work describes the changes in surface glycosylation during transformation of cercariae into schistosomula by fluorescently labelled lectins and monoclonal antibodies against Lewis X antigen. Lectins UEA-I, LTA and PNA have been chosen as markers of transformation of T. regenti. Further, our experiments have been focused on shedding of cercarial glycocalyx. During in vitro induction of penetration gland emptying and...
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Interactions between Schistosoma spp. and their hosts at the metabolome level
Kurečka, Martin ; Kameník, Zdeněk (advisor) ; Mikeš, Libor (referee)
The blood flukes of the genus Schistosoma are important parasites that cause serious chronic diseases in mammals, including humans, in tropical and subtropical countries. Treatment of these diseases is challenging; therefore, new molecular targets are still being sought for the development of vaccines and more effective drugs. To achieve this, better understanding of interactions between the parasite and the host at the molecular level is an important prerequisite. These processes can be studied by quantitative and qualitative determination of metabolite differences in healthy and infected individuals using metabolomics. The work represents a review of low molecular weight substances in tissues and body fluids of schistosome hosts, in which a change in concentration of metabolites putatively related to the infection was observed. Only metabolites with a hypothetical or known mechanism of these changes in the context of infection are covered. The thesis also includes a brief overview of basic methods of analytical chemistry, which are used in studies based on metabolomics. Key words: Schistosoma spp.; intermediate host; definitive host; spectrometry; metabolomics; low molecular compounds
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Biologically active compounds of selected model trematodes
Kurečka, Martin ; Kameník, Zdeněk (advisor) ; Mikeš, Libor (referee)
Trematoda are parasites known for their ability to manipulate their host for survival and reproduction. They have complex life cycles with the intermediate host represented by mollusks and the definitive host, represented by vertebrates. This work focuses on three medically important genera of trematodes: Schistosoma, Fasciola and Opisthorchis. The aim of this work is to summarize biologically active low molecular weight substances that parasites modulate or produce in order to manipulate their host. The result of the work is a literature research of a comparative change in the concentration of metabolites of infected and uninfected trematode hosts with a focus on the analytical method used. Metabolomics deals with a comprehensive analysis of the metabolism of biological samples. It uses spectrometric analytical methods such as nuclear magnetic resonance and mass spectrometry combined with gas or liquid chromatography. Part of the work is also a summary of the importance, development and perspectives of metabolic profiling in parasitology. Current research in this area focuses mainly on vertebrate hosts. In addition, for vertebrate hosts, the sum of studied substances is still much broader than that in intermediate hosts. In intermediate host studies also focus on different types of substances,...
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Analysis of secretome from Trichobilharzia regenti cercariae and characterisation of selected peptidases
Konečný, Lukáš ; Kašný, Martin (advisor) ; Horn, Martin (referee)
(English): Trichobilharzia regenti is a neurotropic parasite of birds from the family Schistosomatidae. Cercariae, the invasive stages of these trematodes actively penetrate the host skin employing excretory- secretory products (ESPs), which contain proteolytic enzymes able to disrupt host tissues and thus reach the successful transmission. The most abundant secreted enzyme responsible for cercarial penetration of the human schistosome S. mansoni is a cercarial elastase. This serine peptidase is well known for the degradation of skin proteins such as elastin, keratin, collagen or laminin. However, the active expression of the orthologue of this enzyme has never been found in the genus Trichobilharzia. For this reason, it was firmly believed, that cercaria of T. regenti uses mainly cysteine peptidases for the invasion of the host, particularly cathepsins, which were repeatedly identified in this life stage. To strengthen this hypothesis, we incubated T. regenti cercariae in the apparatus with the excised duck skin stimulating the release of their glands' content. The collected ESPs were further analysed by shotgun mass-spectrometry and for the first time, the protein form of cercarial elastase was identified. Unfortunately, we failed to produce its active recombinant protein in yeast and bacterial...
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Functional morphology of schistosome miracidia
Svobodová, Kateřina ; Horák, Petr (advisor) ; Bulantová, Jana (referee)
Trematodes of the family Schistosomatidae are important pathogens invading various species of birds and mammals, including man. Miracidium is the first larval stage in the complex life cycle of these parasites. The fate of the parasite depends on the ability of the larva to find, recognize and penetrate into a suitable snail host. Miracidium locates its snail host actively. Miracidium respond to environmental cues and also by recognition of specific glycoproteins produced by snails to the water environment. Free-living miracidium has limited energy reserves, so it has to find and penetrate into a suitable host as quickly as possible. Cilia serve to fast movement in the aquatic environment. The surface of the miracidium is covered with flat ciliated epidermal plates arranged in four tiers. These plates are separated from each other by epidermal ridges. The anterior end of miracidia is cone-shaped and is called apical papilla. Apical papilla is a key tool for penetration and it contains openings of penetration glands. Miracidia display a variety of sensory receptors which help miracidium in orientation. However, there is not much known about miracidial receptor function. Miracidial morphology can serve as a suitable characteristic for schistosome species identification. The thesis summarizes the...
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Excretory-secretory proteins of schistosome intravertebrate stages in context of their biology
Konečný, Lukáš ; Mikeš, Libor (advisor) ; Kašný, Martin (referee)
Schistosomatidae is a family of blood-dwelling trematodes which cause serious disease in humans and warm blooded animals. Human schistosomiasis affects over 258 million people worldwide. The fundamental pathological manifestations of the disease are not caused by adult worms, but by responses to antigens released from eggs, which remain trapped in the tissues. All stages of these parasites release excretory-secretory products, which mostly comprise of proteins which are, to a certain extent, species- and stage-specific. These products have many functions that play roles in successful transmission of the parasite and its interactions with the host. Various approaches to the collection and analysis of these molecules do not always accurately reflect the secretory processes in vivo, because they always employ in vitro techniques. The research of these secreted molecules discovered many mechanisms, through which the parasite successfully modulates and evades host immune system. Despite the fact, that many of these molecules were identified as potential vaccine candidates, still no sufficiently effective vaccine has been developed. With great technological advances used for the identification of these proteins, this area is becoming more and more topical. This review focuses on composition of...
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Proteolytic system of blood flukes of the genus Schistosoma
Bakardjieva, Marina ; Mareš, Michael (advisor) ; Dvořák, Jan (referee)
Blood flukes of the genus Schistosoma are parasitic trematodes causing a disease called schistosomiasis, which afflicts more than 200 million people in the tropics and subtropics. Adult schistosomes live in human blood vessels and feed on blood. Critical nutrients required for growth, development and reproduction of schistosomes are obtained from the major blood protein haemoglobin. Its digestion is mediated by the proteolytic arsenal of the schistosome digestive tract, which includes enzymes with complementary specificity belonging to the classes of cysteine and aspartic proteases, and metalloproteases. Proteolytic enzymes also play an important role in other processes, such as host penetration, tissue migration, immune evasion and modulation of inflammation. Here, serine and cysteine proteases importantly participate. The proteolytic system is essential for the viability of schistosomes and is a current topic of intense research focused on the development of new vaccines and chemotherapeutics for the treatment of schistosomiasis. Powered by TCPDF (www.tcpdf.org)
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Structural and functional analysis of cathepsin B1 from the blood fluke, Schistosoma mansoni
Jílková, Adéla ; Mareš, Michael (advisor) ; Obšil, Tomáš (referee) ; Mikeš, Libor (referee)
Schistosomiasis is a serious infectious disease that afflicts over 200 million people in tropical and subtropical regions. It is caused by Schistosoma blood flukes that live in human blood vessels and obtain nutrients from host hemoglobin, which is degraded by digestive proteases. Current therapy relies on a single drug and concern over resistance necessitates new drug development. In Schistosoma mansoni, cathepsin B1 (SmCB1) is a critical digestive protease that is a target molecule for therapeutic interventions. This thesis provides a comprehensive characterization of SmCB1 focused on structure-activity relationships and inhibitory regulation based on six crystal structures solved for SmCB1 molecular forms and complexes. SmCB1 is biosynthesized as an inactive zymogen in which the N-terminal propeptide operates as a natural intra-molecular inhibitor by blocking the active site. Detailed biochemical and structural analyses have identified a new and, so far, unique mechanism of SmCB1 zymogen activation through which the propeptide is proteolytically removed and the regulatory role of glycosaminoglycans in this process has been described. A study of SmCB1 proteolytic activity has revealed that the enzyme acts in two modes, as endopeptidase and exopeptidase, which makes it an efficient tool for host...
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Humoral response of specific hosts to bird schistosome antigens
Turjanicová, Libuše ; Mikeš, Libor (advisor) ; Kolářová, Libuše (referee)
This thesis focuses on humoral immune response of specific hosts to antigens of various developmental stages of bird schistosomes T. regenti and T. szidati, and follows up on previous research of antibody response in non-specific hosts (mouse, human). Sera of experimentally infected and hunted-down wild ducks were examined using the ELISA and western blot methods. The sera samples were taken in predefined intervals. Results of the ELISA analysis show the process of humoral immune response after infection by bird schistosomes. The level of specific antibodies IgY against homogenate of T. regenti cercariae increased significantly 20 d.p.i. in ducks infected by T. regenti. Such reaction wasn't observed in ducks infected by T. szidati. Slight changes in level of specific antibodies IgM against T. szidati cercariae homogenate were observed 10 d.p.i. only in fully immunocompetent ducks and in reinfected ducks. Examination of hunted-down wild ducks didn't prove infection by bird schisosomes; this conclusion was confirmed by results of the ELISA analysis. IgY antibodies from ducks infected by T. regenti demonstrated strong reactions with 2 antigens in ranges 49-47 kDa and 47-45 kDa. Other reactions, which were recognized, have not been observed in all specimen. An Western blott with homogenate from 7 days...
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