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Serological diagnosis of borreliosis deseases
Sližová, Ivana ; Chmelař,, Dittmar (referee) ; Lochman,, Ivo (advisor)
The aim of present master’s thesis was to compare the results of serological methods for diagnosing borreliosis that are commonly used in Spadia laboratories (ELISA, immunoblots) in terms of recommendation on how and when to indicate and interpret them. The theoretical part is focusing on the characteristics and history of borreliosis, microbiological description of Borrelia, immune system and pathogenesis of the disease as well as the therapy and prevention. The experimental part is focusing on the analysis of results obtained from common examinations of antibodies to Borrelia made in Spadia Lab laboratories from January 1st 2014 to December 31st 2015. Screening of antibodies to Borrelia made by ELISA in IgM and IgG was done for all samples according to recommendation of CDC. In 2014 the ELISA screening was done using ELISA kits from Euroimmun and Evolis sample processors whereas in 2015 it was done using DiaSorin’s CLIA kits on Liaison analyser. Positive results were then confirmed by Westernblot or lineblot alternatively if the physician did not ask otherwise. It must be remembered that ELISA and Westernblot belong among serological methods that are using antibodies, i.e. substances produced by the immune system. The immune system plays the key role in protecting the body against infection and the antibodies are its important tool. Serological methods belong among immunoassay methods, which is still not standardized. Diagnosis of infections cann‘t be based only on antibody testing. It is necessary to assess the results in the context of the entire clinical picture, history and in the case of antibodies it is recommended retesting with an interval.
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Experimental Infection of Mice and Ticks with the Human Isolate of Anaplasma phagocytophilum NY-18
KALINOVÁ, Eliška
Anaplasma phagocytophilum is a significant tick-borne pathogen that colonizes granulocytic cells in vertebrate hosts. It is found in North America and in several European countries. It causes severe diseases in domestic animals and potentially fatal disease in humans called human granulocytic anaplasmosis (HGA). The implementation of animal models is necessary due to the increasing prevalence of A. phagocytophilum. This study addresses the transmission of the human isolate of the bacterium A. phagocytophilum NY-18 by two tick species (Ixodes ricinus and Ixodes scapularis) and implements a laboratory model for the transmission of A. phagocytophilum by the tick I. ricinus in mice. The thesis also compares both transmissions and infection dynamics.
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Analýza slinných serpínů klíšťat Ixodes ricinus z hlediska strukturní biologie
KAŠČÁKOVÁ, Barbora
The knowledge of the detailed structure of proteins and their complexes with other proteins, such as serpins, helps to understand the mechanism of action. Serpins, a large protein group of protease inhibitors that possess almost identical secondary-structural folds, represent the perfect example of expanding the knowledge of their inhibition process through detailed structural analyses. The universal process of serpin inhibition is known, but also is known that serpins are structurally similar whereas their functional diversity is significant. Therefore, each serpin will have some properties specific to its own and knowledge of the serpin structures can explain high functional diversity. X-ray crystallography was one of the most common tools used for serpin structural analysis. This thesis describes the structural information of serpins found in Ixodes ricinus ticks. Serpins of this species are mainly responsible for the modulation of the host immune response via inhibiting involved proteases. Serpins with proteases form covalent complexes. This process leads to a suicide mechanism that inactivates the protease as well as serpin. Here are presented results of the X-ray structural analysis of four I. ricinus serpins named Iripin-3, Iripin-5, Iripin-4, and Iripin-1. All of them help the tick in different ways to stay attached to the host for sufficient time for feeding by inhibiting the proteases involved in host immune defense responses to a tick bite.
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Detekce glyoxylátové dráhy v klíštěti \kur{Ixodes ricinus}
PAVLASOVÁ, Veronika
Ticks are important blood-feeding ectoparasites and vectors of human and animal diseases. The development of rational anti-tick vaccines and drugs is strongly dependent on the identification of biochemical differences between ticks and vertebrates. The glyoxylate cycle was so far found only in bacteria, plants, fungi, and nematodes. Surprisingly, one article described an increase of glyoxylate, a product of the glyoxylate cycle, during embryogenesis in ticks. Aim of my work was to confirm the presence of the glyoxylate cycle (isocitrate lyase) in ticks, mainly in the developing eggs of Ixodes ricinus, by setting-up the biochemical test coupled with a specific inhibitor.
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Touha po cukru; fyziologické procesy klíštěte \kur{Ixodes ricinus} v závislosti na hostitelské glukóze
PLAČKOVÁ, Barbora
Blood-feeding arthropods, Ixodes ricinus acquire and digest vast amounts of the host blood meal, which often exceeds their body volume up to 100 times. It seems to be an only source of energy which they need for the reproduction. I identified homologues of six sugar transporters genes encoding sugar transporters in the I. ricinus tick. Transcripts encoding sugar transporters were found in digestive tract of I. ricinus. The results of RT PCR analysis characterised tissue expression profiles of the sugar transporters while fed by blood. The sugar transporters were determined as a drug target by RNAi. In this master thesis, the use of RNAi and available sugar inhibitors did not confirm glucose importance in physiology of ticks. Membrane feeding showed that blood meal with 2-deoxy-D-glucose had a significant impact on the reproduction and viability of the ticks.
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Selected proteolytic aspects as targets to combat ticks and tick borne pathogens
HARTMANN, David
Ticks and tick-borne diseases (TBD) represent a growing global burden for both human and animal health. Tick-host-pathogen interactions have evolved through dynamic processes that accommodated the genetic traits of the hosts, pathogens transmitted, and the vector tick species that mediate their development and survival. As in other parasites, proteases and proteolysis have been found as one of the key factors in this interaction triangle. This thesis is focused on selected proteolytic aspects of tick and tick-borne diseases: (i) processing of host blood as a source of nutrients and energy (hematophagy) as a continuum of the long-term goal of the Laboratory of Vector Immunology, that established the currently accepted model of multienzyme degradation of host blood proteins by ticks (ii) proteases in innate immunity (iii) validation of Babesia proteasome as a potential therapeutic target against the tick transmitted apicomplexan parasites.
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Prolyl endopeptidase from the tick Ixodes ricinus
Petrvalská, Olívia ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
The ticks are important blood-feeding parasites and vectors of pathogens. The hard tick Ixodes ricinus is the most common species in the Czech Republic that transmits Lyme disease and tick-borne encephalitis. Proteases of the ticks are potential drug targets for the development of new vaccines against these parasites. This work is focused on biochemical analysis of a prolyl endopeptidase from I. ricinus, which has not been studied so far. The prolyl endopeptidase was identified in the extract from the tick gut tissue by the measurement of enzyme activity and by visualization on SDS-PAGE after labelling with activity-based probe. The tick prolyl endopeptidase is probably involved in the proteolytic digestion of host blood proteins based on the highest specific activity found in the gut tissue and its upregulation during the blood-feeding period. Biochemical analysis showed that the enzymatic activity of prolyl endopeptidase is (1) dependent on a free cysteine residue in a close proximity of the active site, (2) optimal at a pH range between 8 and 9, and (3) selectively inhibited by peptide inhibitors Z-Ala-Pro-CMK and Z-Pro-Pro-CHO. Key words: prolyl endopeptidase, proteolysis, enzyme activity, substrate specificity, tick (In Czech)
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Cathepsin L from the hard tick Ixodes ricinus
Talacko, Pavel ; Konvalinka, Jan (advisor) ; Entlicher, Gustav (referee)
Ticks are globally important parasites involved in transmission of a wide variety of infectious agents. The most common tick species found in Europe is the hard tick Ixodes ricinus, which transmits bacterium Borrelia burgdorferi (a causative agent of Lyme disease) or tick-borne encephalitis virus. Cathepsin proteases are important in the process of digestion of blood proteins in the tick gut. This work is focused on cathepsin L, an important digestive cysteine protease of ticks. Recombinant I. ricinus cathepsin L was expressed in Pichia pastoris and separated from the culture medium by chromatographic purification. N-terminal protein sequencing and labeling by activity-based probe Green-DCG-04 were used for characterization of purified cathepsin L. Substrate and inhibitor specificity were analyzed using peptide substrates and inhibitors. This analysis showed that Z-FR-AMC is a suitable substrate with pH optimum 3.5, and that Z-FF-DMK is an efficient inhibitor. It was demonstrated that cathepsin L cleaves protein substrates in strongly acidic environment (pH 3.5-4.5). Cathepsin L-like proteolytic activity was demonstrated in salivary gland extract and in saliva of the I. ricinus tick. The presence of a cathepsin protease in tick saliva is reported here for the first time. This finding suggests that...
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