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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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Subcellular insight into cholesterol-mediated proliferation of the tick cell line
KROPÁČKOVÁ, Sára
Arthropods, including ticks, are not able to synthetise cholesterol de novo. However, cholesterol is an essential component of their cell membranes and serves as a precursor for the synthesis of steroid hormones. In this thesis, I looked at the biological function of exogenous cholesterol on the tick Ixodes ricinus cell line IRE/CTVM19. I verified the need for cholesterol supplementation in the growth medium for proper cell proliferation. Cellular proliferation was stimulated by cholesterol and not by other sterols (ergosterol, sitosterol). We further explored the two assumed aforementioned cholesterol cellular roles, but also investigated the role of cholesterol as a signal molecule: The effect of cholesterol sensing is described on the tick cell level, as well as on the level of the whole organism, using the ex vivo feeding system of ticks. This work provides novel insights into the cholesterol biology of ticks and tick cells, increasing our understanding of tick-host blood molecules' interaction.
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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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Determination of tick-pathogen interactions during acquisition and transmission of Borrelia duttonii by Ornithodoros moubata
ĆETKOVIĆ, Ana
The goal of this thesis was to investigate the interaction between Borrelia duttonii, a causative agent of TBRF in Africa, and its tick vector Ornithodoros moubata in terms of acquisition and transmission dynamics. The infectivity of frozen sera and infected unfed O. moubata ticks was evaluated. The acquisition efficiency of B. duttonii 1120K3 and Ly isolates by O. moubata was determined and compared. The minimum acquisition time was established as well. Moreover, the sensitivity of Borrelia in the presence of different animal sera was analysed.
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Exploration of the tick-Borrelia molecular interactions by employing the transcriptomic approaches
MAHMOOD, Sazzad
Along with climate change and increased sharing of habitat, ticks are coming into more frequent contact with humans. The hard tick Ixodes scapularis and Ixodes ricinus are known disease vectors in Northern America and Europe, respectively. Along with many other pathogenic microorganisms, these ticks spread Borrelia sp. by ectoparasitic blood feeding. Borrelia afzelii is the major European Lyme disease pathogen spread by I. ricinus. Our study focuses on differential gene expression in I. ricinus salivary gland and midgut, induced in the nymphal stage by B. afzelii infection. Tick genes upregulated by infection are considered to play essential roles for the acquisition, persistence, and transmission of Borrelia. We have determined 32,897 full length sequences of tick mRNA from B. afzelii infected/noninfected tick salivary glands and the whole body. In addition, we have obtained MACEseq (Massive Analysis of cDNA Ends) from both midgut and salivary glands while the nymphs were non-infected or infected with B. afzelii during three different phases of blood-feeding. From the MACE database, we obtained 250-500 bp 3'-end sequences with raw quantitative expression values. Total reads, unique sequences and protein coding tick genes from midgut samples were 38,199,641, 88,825 and 24,276, and from salivary gland were 74,651,134, 93,096 and 26,179, respectively. After filtering, using several criteria, expression was validated by qPCR. Hence, the validated genes may most likely interact with Borrelia in its acquisition, persistence, or transmission to the vertebrate host. In our study, RNA interference approaches and vaccination were implemented in order to investigate the impact of upregulated tick midgut and salivary gland genes on Borrelia transmission to C3H mice.
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Functional analysis of tick salivary serine and cysteine protease inhibitors
KOTÁL, Jan
The proposed thesis focuses on the characterization of two protease inhibitors present in tick saliva. More specifically, the thesis presents immunomodulatory properties, biochemical specificity and structure of a cysteine protease inhibitor named Iristatin. Another characterized protein, IRS-8, comes from a serpin family (serine protease inhibitors) and inhibits blood coagulation and complement system in the host. Furthermore, the thesis provides a literature overview and discussion of tick salivary molecules in the context of tick-host-pathogen interaction, vaccination potential and medicine potential. Two review manuscripts, which are part of this thesis summarize the effects of tick saliva and protease inhibitors on host immune mechanisms.
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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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