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New aspects of the cell submembrane signaling
Heneberg, Petr ; Dráber, Petr (advisor) ; Bilej, Martin (referee) ; Folk, Petr (referee)
This dissertation contributes to elucidation of some mechanisms of the mammalian cell submembrane signaling. Major part of the research was conducted on mast cells and basophils activated via the high affinity IgE receptor, FcεRI, or via the cell surface glycoprotein Thy-1. New roles of actin cytoskeleton in mast cell signaling via FcεRI and Thy-1 are described. Discovery of new transmembrane adaptor protein non-T cell activation linker, NTAL, short time before the initiation of work on the thesis led to the increased attention paid to this protein. Dramatic changes of signaling in mast cells deficient in NTAL, or with up- or down-regulated expression of this protein are described. NTAL was also found to be one of proteins phosphorylated following the Thy-1 aggregation. Spatiotemporal distribution of surface glycoprotein Thy-1 at different levels of resolution and some biochemical properties of cells activated via Thy-1 are depicted. Screen for nonreceptor hitherto unknown protein tyrosine phosphatases in mast cells and basophils was conducted and initial analysis of spatiotemporal distribution and function of phosphatase PTP20 in mast cell signaling was performed. Next, the role of reactive oxygen and nitrogen species in the regulation of mast cell protein tyrosine phosphatases was summarized. New...
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Effects of microbiota on defense system of earthworms
Dvořák, Jiří ; Bilej, Martin (advisor) ; Kopečný, Jan (referee) ; Horák, Petr (referee)
Earthworms are important soil invertebrate organisms that participate in nutrient cycling in terrestrial ecosystems and in the formation of the soil profile from the physical, chemical and also microbial point of view. Soils are considered the most microbially diverse environments on earth. All invertebrates living in soil therefore need to possess a complex immune system. Earthworms are used as a model organism in immunology for decades. Their simple body plan consists of two main body cavities: true coelom and digestive tube. Both coelomic cavity and digestive tract represent open systems with permanent contact with soil microorganisms. Eisenia andrei species is used as a standard immunological model in our laboratory for many years. E andrei earthworms live in compost, microbially abundant environment, which is reflected in their well-developed immune system. Some new mechanisms of E. andrei defense system are described in this work. Two novel pattern recognition receptors (PRRs), Toll-like receptor (TLR) and lipopolysaccharide binding protein/bactericidal permeability-increasing protein (LBP/BPI) were characterized in earthworms. These molecules are expressed in coelomocytes and their production is upregulated after microbial challenge. Moreover, both receptors were detected in digestive tract...
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Critical factors affecting pathogen development in sand flies
Jančářová, Magdaléna ; Volf, Petr (advisor) ; Ratinier, Maxime (referee) ; Bilej, Martin (referee)
This thesis deals with barriers and factors critical for development of viruses, leishmania and gregarines in sand flies. First, we focused on life cycle of sand fly-borne phleboviruses, especially possible routes of sand fly infection. As a laboratory model we chose Massilia virus (MASV), species closely related to Toscana virus, which is main causative agent of summer meningitis in Mediterranean area. We tested different ways of infection by MASV in various developmental stages of Phlebotomus perniciosus; infection of (i) first (L1) and fourth (L4) instar larvae through larval food, (ii) females by blood meal, (iii) both sexes by sugar meal. Infection of L1 and L4 by larval food and subsequent transstadial MASV transmission to adults were not efficient; from 875 adults only three were MASV-positive. Infection through bloodmeal led to high infection rate before defecation, nevertheless, post defecation the infection rate declined and only 5 out of 27 females were MASV-positive. The most efficient infection way was through the sugar meal: 72% of females (88 out of 122) and 51% of males (58 out of 113) were detected as MASV-positive. Moreover, both males and females infected by this way released MASV particles into the drop of sugar which stayed infectious for next 24 hours for other naïve sand...
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Leishmania-gregarine coinfections in sand flies
Jančářová, Magdaléna ; Volf, Petr (advisor) ; Bilej, Martin (referee)
This thesis consists of two related parts; the first one deals with the effect of gregarines Psychodiella sergenti on susceptibility of sand fly Phlebotomus sergenti to experimental infection of Leishmania tropica. Comparison of the Turkish colony of P. sergenti infected by gregarines (TRG) and the Israeli colony noninfected by gregarines (IS) revealed significantly higher intensity of L. tropica infection in TRG colony on days 2 and 10 after the infective bloodmeal. In addition, in TRG colony leishmania promastigotes significantly faster colonised the stomodeal valve. However, these differences seems to be caused not due to the presence of gregarines but due to intraspecific genetic variability of P. sergenti or different composition of gut microbiota in both colonies. In two colonies of P. sergenti originally from Israel, one infected by gregarines (ISG) and the second noninfected (IS), no significant difference was found. ISG and IS colonies did not differ in the percentage of infected females, the intensity of infection and the localization of L. tropica promastigotes. In the second part of my thesis, degenerated primers designed on the basis of sequences of immune molecules of Phlebotomus papatasi and Lutzomyia longipalpis and subsequent cloning of products by means of pGEM®-T Easy Vector...
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Molekulárna charakterizácia vybraných obranných faktorov v čeľadi Lumbricidae
Mančíková, Veronika ; Bilej, Martin (advisor) ; Král, Jiří (referee)
Earthworms belonging to oligochaete annelids have been a model for comparative immunology for over 40 years. They possess various defense mechanisms efficiently recognizing and responding to non-self substances. Among these there are molecules with many biological activities including cytolytic, antimicrobial and proteolytic. This work is aimed to compare the immunological features of two closely related earthworm species Eisenia andrei and Eisenia fetida. Due to many morphological and life cycle similarities they have been, until recently, regarded as members of subspecies. Interestingly, their natural habitat varies considerably, and it was of particular interest to investigate how these environmental differences affect the features of innate immunity of both species. Key words: annelids, innate immunity, Eisenia andrei, Eisenia fetida, CCF, fetidin, lysenin, lysozyme
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Antigenic and enzymatic properties of sand fly saliva
Hostomská, Jitka ; Volf, Petr (advisor) ; Salát, Jiří (referee) ; Bilej, Martin (referee)
i SUMMARY OF RESULTS The thesissumsup the resultsof threeprojectsI was involvedin duringmy PhD study.Specifically, I adressedthe putativeeffect of sand fly salivary hyaluronidaseon transmissionandestablishmentof kishmania infection.The secondprojectwasdealingwith thekineticsof anti-salivaantibodyresponsein dogsexposedto Lutzomyialongipalpissutd flies andwith the characterizationof salivaryantigensrecognizedby thesedogs.Finally, I constructedand annotateda cDNA library from Phlebotomusarabicas and characterized P. arabicus sďivary antigensreactingwith arrtibodiesof mice exposedto this sand fly species.Theresultsofthe projectsaÍebrieflyoutlinedhere. We detectedhyaluronidaseactivity in saliva of various bloodsuckingDipter4 includingsandflies. In coinoculationexperimentswith BALB/o mice we proveda positive conelationbetweenthe size of the cutaneouslesion causedby Leishmaniamajor andthe presenceofhyaluronidasein theinfectiveinoculum.In hyďuronidase-coinoculatedmice,the lesionsweresigniÍicantlylargerfromweek3 postinfection(infectiondosel05 parasites)or week 4 (infection dose 10"). On the other hand, hyaluronidasedid not affect early viscerďizationof L. majorat24 hrspostinfection.Thus,we demonstratedthathya|uronidase promotes Leishmania establishment in murine skin and we hypothesize that immunomodulatoryeffectsof...
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Molecular mechanisms of regulation of FcɛRI signaling in mast cells
Bambousková, Monika ; Dráber, Petr (advisor) ; Černý, Jan (referee) ; Bilej, Martin (referee)
Mast cells are critical component of the immune system. In pathological situations, they are activated and are responsible for allergic reaction. Therefore, detail understanding of mast cell activation at molecular level is important for design of new therapies of allergic diseases. Principal transmembrane receptor of mast cells is the high-affinity Fc receptor for IgE (FcεRI). FcεRI anchors IgE on mast cell surface and upon cross-linking with multivalent antigen it becomes phosphorylated at its intracellular immunoreceptor tyrosine-based activation motifs (ITAMs). This triggers signaling cascade leading to cell degranulation and cytokine production. The antigen- mediated signaling through the FcεRI is critically dependent on interplay with intracellular protein- tyrosine kinases that phosphorylate the ITAM motifs and many other components of the signaling pathway. This study was focused on better understanding of signaling events leading to mast cell activation; emphasis was put on early activation events. First, we examined the role of protein- tyrosine phosphatases (PTP) in FcεRI phosphorylation. We found that upon antigen triggering of FcεRI, PTPs undergo inhibition by oxidation of their active site located tyrosine. Studies of plasma membrane topography of inactivated PTPs showed their...
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