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The Interaction of Borrelia Outer Surface Proteins with Tick Salivary Proteins
PAKTAN, Aylin
The aim of this thesis was to investigate the interaction of the salivary gland protein, Salp15, in relation to the outer surface protein C, OspC, using in silico analysis, as well as trying to amplify Salp15 from different tick species found in same geographical areas and observing the growth of Borrelia in the midgut and salivary glands of these different tick species in vitro.
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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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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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Salivary glycoproteins of bloodsucking arthropods
Sumová, Petra ; Volf, Petr (advisor) ; Mikeš, Libor (referee)
During obtaining their blood meal, bloodsucking arthropods salivate into their host. Bloodsucking arthropods' saliva contains wide array of bioactive macromolecules. Host organism develops antibody response against many of these molecules. Due to interspecies variability in salivary protein composition, detection of antibody response may serve as a marker of the exposure to individual species of bloodsucking arthropods. Host antibody response is mostly elicited by proteins or glycoproteins. Glycoproteins contain one or more oligosaccharide chains attached to the protein. Glycoprotein's antigenicity could be caused by either both parts, or by only the protein, or the sugar part. This fact has to be taken into consideration for choice of the expression system for recombinant glycoprotein synthesis. This work summarizes current knowledge about structure, function and features of salivary glycoproteins in various species of bloodsucking arthropods.
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Characterization and antigenic properties of salivary yellow-related proteins in phlebotomine sand flies
Cikrtová, Petra
2 ABSTRACT Yellow-related proteins (YRPs) form an abundant protein family, whose members were found in salivary gland transcriptomes of all sand fly species studied up to date. This protein family belongs to the group of MRJP/Yellow proteins occurring in insects and some other organisms. Though sharing similar folding as a six-bladed β-propeller comprising central tunnel, MRJP/Yellow proteins adopted different functions. The structure and biogenic amine-binding property described for the sand fly salivary YRPs was based on a single study conducted on Lutzomyia longipalpis. In the present work, we have modelled the structures of 32 salivary YRPs belonging to 13 different sand fly species. We have shown the general structural similarity of these proteins along with both inter- and intra-specific differences in surface charge, tunnel parameters and in amino acids composition of the amine-binding motif. These modifications indicated divergence in function of individual YRPs, which was experimentally verified in the second project focused on identification of the amine-binding properties of YRPs in two important vectors of Leishmania; Phlebotomus perniciosus and P. orientalis. In each species, two YRPs differ in affinities for biogenic amines serotonin, histamine and catecholamines. However, in both sand fly...
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Characterization and antigenic properties of salivary yellow-related proteins in phlebotomine sand flies
Cikrtová, Petra
2 ABSTRACT Yellow-related proteins (YRPs) form an abundant protein family, whose members were found in salivary gland transcriptomes of all sand fly species studied up to date. This protein family belongs to the group of MRJP/Yellow proteins occurring in insects and some other organisms. Though sharing similar folding as a six-bladed β-propeller comprising central tunnel, MRJP/Yellow proteins adopted different functions. The structure and biogenic amine-binding property described for the sand fly salivary YRPs was based on a single study conducted on Lutzomyia longipalpis. In the present work, we have modelled the structures of 32 salivary YRPs belonging to 13 different sand fly species. We have shown the general structural similarity of these proteins along with both inter- and intra-specific differences in surface charge, tunnel parameters and in amino acids composition of the amine-binding motif. These modifications indicated divergence in function of individual YRPs, which was experimentally verified in the second project focused on identification of the amine-binding properties of YRPs in two important vectors of Leishmania; Phlebotomus perniciosus and P. orientalis. In each species, two YRPs differ in affinities for biogenic amines serotonin, histamine and catecholamines. However, in both sand fly...
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Correlation of Immunohistochemical and Molecular Methods in Diagnostics of Salivary Gland Tumors
Horáková, Markéta ; Skálová, Alena (advisor) ; Laco, Jan (referee) ; Dušková, Jaroslava (referee)
This doctoral thesis is dealing with the correlation of morphological, immunohistochemical and genetical findings in malignant tumors of salivary glands. The first half of the thesis comprises the summary of current knowledge about salivary malignancies. The second half is presenting the research itself. The research results are divided into three parts. The first part is presenting the method of "2-step diagnostic test" of malignant tumors. This screening test aims to find new, so far not described gene aberrations with a focus on malignant tumors of salivary glands. This method takes place in two consecutive steps. In the first step the material is examined by an immunohistochemical mixture of antibodies, which non-specifically detects aberration in the genes NTRK1-3, ALK and ROS1. In the second step all positive cases are subjected to highly sensitive and specific molecular-genetic examination by the method of next generation sequencing (NGS) using the Archer kit. In the second part of the work there has been designed the approach to the cytological diagnosis of salivary secretory carcinoma by the fine-needle aspiration (FNA). This part is describing to the details the cytomorphology of secretory carcinoma in both, Pap smears and cell blocks, from which additional immunocytochemical and genetic...
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Diferenciální exprese transkriptů slinných žláz klíštěte \kur{Ixodes ricinus}
KROPÁČKOVÁ, Sára
Ticks are using different mechanisms for host immune system suppression. In this thesis, I compared ten transcripts from ticks salivary glands, which are differentially expressed during feeding on live animal and artificial systems. Further, a tick protein anticomplement 1, AC1, was chosen and subjected to deeper investigation, because of the significant difference in expression during feeding. Serum with specific antibodies was made and AC1 expression levels were examined in nymphs and adults, during feeding on different hosts. Moreover, any potential interacting partners from host serum through covalent binding were tested. This study contributes to the understanding of regulation of expression and funtional characterisation of tick salivary proteins.
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Salivary glycoproteins of bloodsucking arthropods
Sumová, Petra ; Volf, Petr (advisor) ; Mikeš, Libor (referee)
During obtaining their blood meal, bloodsucking arthropods salivate into their host. Bloodsucking arthropods' saliva contains wide array of bioactive macromolecules. Host organism develops antibody response against many of these molecules. Due to interspecies variability in salivary protein composition, detection of antibody response may serve as a marker of the exposure to individual species of bloodsucking arthropods. Host antibody response is mostly elicited by proteins or glycoproteins. Glycoproteins contain one or more oligosaccharide chains attached to the protein. Glycoprotein's antigenicity could be caused by either both parts, or by only the protein, or the sugar part. This fact has to be taken into consideration for choice of the expression system for recombinant glycoprotein synthesis. This work summarizes current knowledge about structure, function and features of salivary glycoproteins in various species of bloodsucking arthropods.
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Salivary gland hyaluronidase of tabanids and ticks
Tothová, Viktorie ; Volf, Petr (advisor) ; Mikeš, Libor (referee)
6 1. Abstrakt Hyaluronidázy jsou významnou skupinou enzymů odpovědných za štěpení kyseliny hyaluronové, která je jednou z hlavních složek pojivové tkáně obratlovců. U většiny krevsajících členovců je tento enzym přítomen ve slinách a usnadňuje sání tím, že se podílí na průniku kůží a zvětšení potravní léze v místě bodnutí. Vzniklé fragmenty extracelulární matrix mohou navíc modulovat lokální imunitní odpověď hostitele a zvyšovat pravděpodobnost přenosu patogenů. V naší práci jsme se zaměřili na průkaz hyaluronidázové aktivity u klíštěte Ixodes ricinus (Acari: Ixodidae) a zástupců čtyř rodů ovádů (Diptera: Tabanidae). V slinných žlázách klíšťat se hyaluronidázovou aktivitu detekovat nepodařilo. Naopak, ve slinných žlázách ovádů jsme prokázali velmi silnou aktivitu u všech studovaných druhů a dále ji charakterizovali biochemickými a elektroforetickými metodami. Hyaluronidázy ovádů štěpí hyaluronan i chondroitin sulfát; enzymy jednotlivých druhů se mírně liší molekulovou hmotností, pH optimem a citlivostí k redukujícím podmínkám. Abstract (in English) Hyaluronidases are an important group of enzymes responsible for cleaving hyaluronic acid, which is a major component of the extracellular matrix of vertebrates. In bloodsucking arthropods these enzymes are frequently present in saliva. Salivary hyaluronidases...
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