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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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Recognition of microbial patterns in earthworms
Škanta, František ; Bilej, Martin (advisor) ; Kopečný, Jan (referee) ; Šimek, Miloslav (referee)
Survival of earthworms in the environment depends on their ability to recognize and eliminate potential pathogens. Two closely related earthworm species Eisenia andrei and Eisenia fetida inhabit different environment with specific microbiota. Both species can be reliably determined using of species-specific primers for cytochrome c oxidase I (COI) and stringent PCR conditions. Whereas, we did not observed any substantial differences in the expression and activity of CCF and lysozyme upon microbial challenge, the expression as well as the hemolytic activity of fetidin/lysenins was considerably higher in E. andrei as compared to E. fetida. Genomic DNA analyses revealed significantly higher level of fetidin/lysenins in E. andrei compared to E. fetida suggesting hypothetical gene duplication. Earthworms live in permanent close contact with microbial environment. Coelom cavity as well as the gut of E. andrei earthvworm differs in the number of bacteria. The number of bacteria in the gut is more than six time higher than in coelomic fluid. High microbial load of E. coli O55, B. subtilis W23, and S. cerevisiae S288 in the earthworm environment, resulted in an increase of microorganisms in both, the coelom and the gut. The changes in mRNA levels of defense molecules (pattern recognition receptors CCF,...
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Lymnaeid snails: hemocyte activities and their changes caused by Trichobilharzia infections
Jindrová, Zuzana ; Horák, Petr (advisor) ; Bilej, Martin (referee)
Molluscs as well as all other invertebrates rely on innate immune response only. Their internal defense system is capable of destroying most pathogens. However, there are some exceptions, e.g. some snails serve as intermediate hosts for some trematodes. Trematodes are able to develop inside these snails due to intervention in the snail internal defense system. The submitted thesis describes hemocyte activities of two lymnaeid snails, Lymnaea stagnalis a Radix lagotis, and the influence of Trichobilharzia regenti infection on R. lagotis hemocytes. Hemocytes of both species exposed to various chemicals produced different amounts of H2O2 and NO. The response varied between both lymnaeid species. The amount of circulating hemocytes was elevated in R. lagotis snails due to T. regenti infection. However, the infenction attenuated hemocyte activities monitored by us. Hemocyte basal NO production was decreased as well as phagocytosis of bacteria, cell adherence and pseudopodia formation. Toxicity of L. stagnalis plasma against T. regenti miracidia was also described. Mechanisms used by trematodes to interact with the snail internal defense system will help us to understand why one species is suitable for the develepment of the trematode whereas another closely related species kills it. Powered by TCPDF (www.tcpdf.org)
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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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Enzymes and other factors affectiing leishmania development in sand flies
Ječná, Lucie ; Volf, Petr (advisor) ; Bilej, Martin (referee) ; Kopáček, Petr (referee)
This Ph.D. thesis focuses on enzymes and other molecules that affect leishmania development in their vectors. It summarizes results of three published project and one publication in preparation. It has been proposed that cysteine peptidase inhibitor (ICP) of Leishmania mexicana protects this protozoan parasite from insect proteolytic enzymes, and therefore promotes their survival in the sand fly vector. To test this hypothesis, L. mexicana mutants deficient in ICP were evaluated for their ability to develop in the sand fly Lutzomyia longipalpis. The experiments showed that ICP of L. mexicana has no major role in promoting leishmania survival in the vectorial part of its life cycle. In addition, recombinant L. mexicana ICP did not inhibit peptidase activity of L. longipalpis midgut extracts in vitro. Another objective of this thesis was to study the attachment of leishmania to the midgut epithelium of their vectors. Laboratory studies examining the development of different Leishmania species in a range of phlebotomine species suggest that sand flies fall into two groups: specific and permissive vectors. In the specific vectors, successful parasite development is mediated by the parasite surface lipophosphoglycan (LPG), while this LPG is not required for the attachment in permissive vectors. Our...
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The development of Leishmania in the insect vector of the genus Phlebotomus
Kvapilová, Kateřina ; Volf, Petr (advisor) ; Bilej, Martin (referee)
The work was focused on the study of various aspects of development of Leishmania in vectors of genus Phlebotomus and can be divided into two main parts. In the first part, we studied the competition of Leishmania major and L. turanica during their development in the sand flies who are natural vectors of L. major using the experimental co-infections of fluorescently marked promastigots of these two species. While both leishmania species developed similar in the intestine of Phlebotomus papatasi, L. turanica prevailed in P. duboscqi in the late stages of infection. The fluorescent marking of Leishmania should allow us also to study possible genetic exchange between species at different stages of Leishmania infection (2nd, 9th and 14th day after the infective feeding). Using the flow cytometry (FACS) we have repeatedly identified dozens of objects emitting red and green signals in the intestinal homogenates of co-infected sand flies, however further analysis with confocal microscope disproved these objects as the hybrid promastigots of Leishmania. In the second part of this thesis we investigated the role of L. major genes HASP and SHERP, which lies on the locus LmcDNA16 and are expressed exclusively in metacyclics. We used mutant lines KO (lacking the locus LmcDNA16) and HASPB (KO line with gene...
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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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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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Antimicrobial lytic factors of the coelomic fluid of Earthworms
Josková, Radka ; Bilej, Martin (advisor) ; Horák, Petr (referee) ; Prokešová, Ludmila (referee)
I. SUMMARY lnvertebratesare widely distrbuted animals.They can be found in almostany krnd ofhabitat. Their successfulsurvival shategiesare basedon short life span combinedwith numerousoffspring and, more importantly,all invertebratespecieshavedevelopeda wriety ofdefensemechanismselficientlyrecognizingand respondingto non-selfsubstances. The defensemechadsmsof earthwoms were studiedover the pastfou decades.It becameappdent thattheearthworms.aswell asotherinvertebrates,lackspecificimmunoglobulins,lymphoc)4esor otherfeatures ofthe adaptiveimmunesystemdescribedin verteblates,but possessirmatedefensecomponents In this thesis,we foousedon the detaildescriptionof somedefensemoleculesinvolvedin imate immunityof earthworms. l. Lysozyme is an erzyme with strong antibacterialactivity describedin many organisms. We characterizedthelysozymeof Erieniaandrei(formerly E.fetida andrei)earthwormbothstruchmlly and functionally.Moleoularcharactefiationof lyso4rne providesa new tool for monitoringof innateimmunity in earthworms. 2. A cltol''tic effect of the coelomicfluid of E. fetida was observedin experimentswith TNF-sensitive tumorL929 cell line. Subsequentisolationof l)tic proteinsled to theidentificationof 42-kDaprotein,which wasnamedcoelomiccytolyticfactor- CCF.CCFwasshom to bepresentalsoin coelomicfluid of mother...
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