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Analysis of trypsin inhibitor-like cysteine-rich domain-containing peptides (TIL-domain inhibitors) from the tick Ixodes ricinus
PECHOVÁ, Hana
The master thesis deals with the analysis of trypsin inhibitor-like cysteine-rich domain-containing peptides (TIL-domain inhibitors) extracted from salivary glands of a tick Ixodes ricinus. It comprises the initial bioinformatical analysis of TIL-domain containing peptide family from I. ricinus, molecular cloning of the representative protein into expression vector, followed by production of recombinant TIL-domain inhibitor in bacteria. A representative TIL-domain gene is proved for their structure, biochemical properties as well as immunomodulatory and anticoagulation features.
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Význam signální dráhy insulinu ve fyziologii klíštěte \kur{Ixodes ricinus}
KOZELKOVÁ, Tereza
Tato diplomová práce se zabývá analýzou insulin receptorové dráhy v klíštěti Ixodes ricinus. Klíšťata jsou obligátní krev sající ektoparazité, přenášející mnoho patogenů jako jsou bakterie, viry a protozoa. Parazité jsou závislí na svých hostitelích skrze příjmu živin pro přežití, vývoj a rozmnožování. Insulin receptorová dráha je silně konzervovaná a reguluje širokou škálu fyziologických a anabolických procesů v závislosti na dostupných živinách. Cílem mé práce bylo ověření funkce komponent insulin receptorové dráhy. Tyto komponenty byly identifikovány ve střevním transkriptomu, konkrétně insulin receptor (IrInR), protein kináza B tzv. AKT (IrAKT) and IrTOR. Dále byla zhodnocena exprese jednotlivých genů v průběhu sání a po plném nasátí pomocí qRT-PCR. V této diplomové práci byl dále posouzen fenotyp po RNAi umlčení, injikování insulin receptor antagonistou (IRA) a umělém krmení klíšťat AKT a TOR inhibitory. Dále byl posouzen vliv imunizace králíků IrInR rekombinantním proteinem na následnou infestaci klíšťat.
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Hafnium Chloride, an Alternative Staining Reagent for Biological Electron Microscopy
BARANYI, Magdalena Victoria
This study focuses on the staining pattern of the non-radioactive heavy metal EM stain HfCl4, used during freeze substitution specimen preparation. HfCl4 was investigated as an alternative for the prominent uranyl acetate, since uranium based materials have been placed under heavy restrictions and bans worldwide. We have found a strong HfCl4 staining pattern of microtubules in myoepithelial cells of Ixodes ricinus salivary glands. Additionally, in several samples, HfCl4 was found to completely fill the cytoplasm of the myoepithelial cells. Nonetheless, artefact formation around granulated cells was also experienced.
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Anti-inflammatory and immunomodulatory activities of Ixodes ricinus salivary proteins
CHLASTÁKOVÁ, Adéla
Ticks are highly specialized obligate ectoparasites whose life cycle is fully dependent on obtaining a blood meal from a host. To prevent blood clotting and counteract the host's immune and wound healing responses, ticks secrete saliva, containing hundreds to thousands of proteins and non-proteins, into the feeding cavity. This thesis focuses on deciphering the function of three serpins (inhibitors of serine proteases) expressed in the salivary glands of the European tick Ixodes ricinus. Iripin-1, Iripin-3, and Iripin-8 were produced in the Escherichia coli expression system, and their effects on the immune response were evaluated using various in vitro assays and a mouse model of acute inflammation. Furthermore, the thesis involves one review article that summarizes the role of serpins in tick physiology and tick-host interaction.
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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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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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Sialic acid as a recognition motif for host-originated glycoproteins in Ixodes ricinus
SAMEK, Jakob
Glycans are found on the surface of every living cell and they are responsible for a countless number of functions. N-acetylneuraminic acid (often called sialic acid) is mostly found in vertebrate and only to a small degree in arthropods. The aim of this thesis was to find out if sialic acid is a recognition motif for the tick organism to take up and incorporate or recycle host originated proteins.
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Studium dráhy pro syntézu mastných kyselin u klíštěte \kur{Ixodes ricinus}
ZEMANOVÁ, Aneta
Cílem této práce byla charakterizace dráhy syntézy mastných kyselin u klíštěte Ixodes ricinus. Charakterizovala jsem tkáňový a stádiový profil tří genů: acc (acetyl coenzyme A carboxylase), fas (fatty acid synthetase) and fabp (fatty acid binding protein) pomocí kvantitativní PCR. Poté byly geny umlčeny pomocí RNA interference a byly sledovány fenotypy v průběhu a po sání, jak u nympf tak dospělců. Tato dráha je uázala jako klíčová pro klíšťata.
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