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"DASH molecues" in local and systemic pathogenetic processes of rehumatoid arthritis
Šromová, Lucie ; Šedo, Aleksi (advisor) ; Borovanský, Jan (referee) ; Prokešová, Ludmila (referee)
The biological half-life of several pro-inflammatory mediators involved in the pathogenesis of rheumatoid arthritis (RA) is controlled by molecules exhibiting dipeptidyl peptidase-IV (DPP-IV)-like enzymatic activity (Dipeptidyl peptidase-IV activity and/or structure homologues- DASH). The aim of this thesis was to identify the molecular source of the DPP-IV-like enzymatic activity in the peripheral blood and synovial fluid in patients with rheumatoid arthritis as compared to control patients with osteoarthritis (OA), and to evaluate the association of DPP-IV with the disease activity. We found that the main source of the DPP-IV-like enzyme activity in the plasma and in the synovial fluid in patients with RA is the canonical DPP-IV. DPP-IV-like enzymatic activity and canonical DPP-IV were also detected on the cell surface of blood and synovial fluid mononuclear cells. Significantly lower DPP-IV-like enzymatic activity and DPP-IV expression in the synovial fluid mononuclear cells was found in RA as opposed to OA patients. In the synovial fluid of RA patients there was also a negative correlation between the concentration of the pro-inflammatory DPP-IV substrate SDF (stromal cell-derived factor-1 and the proportion of the DPP-IV+ T cells. The blood plasma DPP-IV-like enzymatic activity and...
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Definition of the expression pattern of DASH system in transformed glial cells, the coupled expression of fibroblast activation protein and dipeptidyl peptidase-IV.
Balážiová, Eva ; Šedo, Aleksi (advisor) ; Borovanský, Jan (referee) ; Mareš, Vladislav (referee)
Dipeptidyl peptidase-IV (DPP-IV) is a multifunctional transmembrane glycoprotein removing X-Pro dipeptide from the amino-terminus of the peptide chain. This evolutionary conserved sequence protects a number of biologically active peptides against the unspecific proteolytic cleavage. DPP-IV belongs into the group of "Dipeptidyl peptidase-IV Activity and/or Structure Homologues" (DASH), which, except the canonical DPP-IV, comprises fibroblast activation protein-α/seprase (FAP), and several other molecules. However several of DASH molecules are the enzymes, they execute at least some of their biological functions by non-proteolytic protein-protein interactions. DASH molecules, their substrates and binding partners are parts of "DASH system" which is affected in several pathological process including a cancer. Specifically DPP-IV and its closest structural relative FAP are among others expected to be involved in the development and progression of malignant glioma. In this study we showed the expression and colocalization of DPP-IV and FAP in glioma cells in vitro and in human high grade gliomas. In addition to the DPP-IV/FAP double positive transformed glial cells, we also identified a subpopulation of FAP positive mesenchymal cells located in the perivascular compartment. Moreover we described the...
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Modulation of HIV-1 Protease Activity
Pokorná, Jana ; Konvalinka, Jan (advisor) ; Šedo, Aleksi (referee) ; Ruml, Tomáš (referee)
HIV-1 protease plays a crucial role in the late state of the life cycle of HIV virus when it cleaves the viral polyprotein precursors into the structural and functional proteins. If it is effectively inhibited, HIV particles remain immature and noninfectious. The application of highly active antiretroviral therapy (HAART) including protease inhibitors can reduce plasma HIV-1 levels below the detection limit in adherent patients and thus dramatically change their life expectancy. The clinical utility of the first inhibitors was limited by severe side effects, low bioavailability, high pill burdens, and rapid development of viral resistance under the selection pressure of HIV antiretrovirals. To overcome these difficulties, second-generation inhibitors were developed. Despite an indisputable improvement they brought to antiretroviral therapy, the development of new highly active HIV-1 protease inhibitors with optimal pharmacokinetic properties, higher metabolic stability, little off-target activity, and particularly, more favorable resistance profiles is still of high importance. This thesis provides an overview of anti-HIV- drugs including development of substituted metallacarboranes, a new class of potent, unusual, nonpeptidic HIV protease inhibitors with therapeutic potential. Next, the impact of...
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Expression and function of serpin B9 in lung carcer cells
Roušalová, Ilona ; Křepela, Evžen (advisor) ; Šedo, Aleksi (referee) ; Kotyza, Jaromír (referee)
Background: Granzyme B (GrB) is a key proapoptotic secretory protease of CTLs and NK cells. Its specific proapoptotic effects in cancer cells can be blocked by increased expression of serpinB9. SerpinB9 gene expression can be transcriptionally upregulated by some interleukins and by the oestrogen activated oestrogen receptor-α (ERα) in cells which express ERα protein. The aims of my thesis were to evaluate the expression of SB9 and to examine its inhibitory activity against exogenous active GrB in non-small cell lung carcinoma (NSCLC) cell lines and tissues. To analyse the expression status of GrB mRNA in NSCLC cell lines and tissues. To investigate the role of estradiol-17β (E2), selected ILs and DNA methylation in regulation of SB9 expression in NSCLC cells. The apoptosome apparatus is a cell death signalling platform activates the initiator procaspase-9. Activation of the apoptosome apparatus is often impaired in various types of cancer but the molecular basis of its suppression is still unknown. APIP and UACA/nucling belong to the endogenous regulators of apoptosome apparatus. The aim of my thesis was to investigate whether DNA methylation is involved in the transcriptional regulation of expression of APIP and UACA genes in NSCLC cell lines. Methods: Following methods were used in this thesis:...
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Model of experimentally regulated expression of dipeptidyl peptidase IV in glioma cell lines
Němečková, Jana ; Šedo, Aleksi (advisor) ; Vaníčková, Zdislava (referee)
"Dipeptidyl peptidase IV Activity and/or Structure Homologues" (DASH) represent a newly defined group of multifunctional molecules, typically bearing dipeptidyl peptidase IV-like hydrolytic activity. Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5, identical to CD26) cleaves out X-Pro dipeptides from the N-terminus of peptides. Recent knowledge shows substantial role of DASH in cancer pathogenesis. Here we present (i) an overview of the issue of DASH molecules and their functional substrates in the neuroectodermal tumor and (ii) a preparation of stable transfected human glioblastoma cell lines with inducible gene expression of DPPIV. Vectors containing human DPPIV gene and its mutated form in the catalytic active site have been prepared to assess the importance of the enzymatic activity of the final product. This will enable us to study the biological role of DPPIV in genesis and progression of neuroectodermal tumors - cells growth, invasion and migration of transformed glial cells in vitro. Moreover, complex role of DPPIV will be studied using a model of homotopic application of transfected cells into the brain of immunodeficient mice. Prepared cell lines provide more consistent information about DPPIV from the point of view of its "autocrine" importance for the expressing cells, as well as its potential...
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The Role of Dipeptidyl Peptidase-IV in Glioma Cell Growth
Bušek, Petr ; Šedo, Aleksi (advisor) ; Trka, Jan (referee) ; Konvalinka, Jan (referee)
Dipeptidyl peptidase-IV (DPP-IV, CD26, EC 3.4.14.5) is a widely expressed serine protease that by limited proteolysis regulates a number of biologically active peptides including a number of mitogenic peptides involved in cancer development. Deranged DPP-IV expression and/or enzymatic activity has been reported in a number of tumors, and could lead to altered signaling and biological function of its substrates. Changes in DPP-IV expression were observed in glioma cell lines in association with differentiation, and recently also in malignant gliomas in vivo. In addition, DPP-IV substrates substance P (SP) and stromal cell derived factor-1 (SDF-1) that trigger growth promoting intracellular signaling cascades in glioma cells have strongly been implicated in the pathogenesis of gliomas. The aim of this study was (i) to investigate the effects of DPP-IV on the signaling of its biologically active substrates that promote the malignant phenotype of glioma cells, and (ii) to assess the effects of DPP-IV on the growth, migration and adhesion of human glioma cells. Using transfected glioma cell lines (U373CD26, T98GCD26, U87CD26) with mifepristone-inducible DPP-IV expression, we demonstrate that DPP-IV overexpressing T98GCD26 cells can cleave SP and thus abrogate its ability to trigger calcium signaling in U373...
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Dipeptidyl Peptidase-IV Activity and/or Structure Homologues : Their Role in Gliomagenesis
Stremeňová, Jarmila ; Šedo, Aleksi (advisor) ; Michalová, Kyra (referee) ; Mandys, Václav (referee)
Dipeptidyl peptidase-IV (DPP-IV, EC 3.4.14.5) together with fibroblast activation protein-alpha (FAP), DPP-7, -8 and -9 belong to the functionally defined group of "DPP-IV activity and/or structure homologues" (DASH). They hydrolyse N-terminal X-Pro dipeptides from a number of biologically active peptides like neuropeptide Y, substance P and chemokines such as stromal cell derived factor-1alpha (SDF-1). Limited proteolysis of such mediators by DPP-IV-like enzymatic activity can modify consequent biological responses of the target cells. By that, DASH molecules are supposed to be important for multiple cellular processes, including cell proliferation, malignant transformation, migration and invasion and thus involved in cancer development and progression. This study was set up to characterise DASH expression pattern and DPP-IV-like enzymatic activity in human astrocytic tumours in comparison with non-tumorous brain tissue, and to assess its context with the expression of receptors of some local mediators- DASH substrates implicated in gliomagenesis. Moreover, the possible functional relevance of DASH molecules in growth properties of transformed astrocytic cells was studied in model of primary cell cultures derived from the glioblastoma in vitro. (...) Hence we speculate that although the upregulated DPP-IV...
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