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Molecular mechanisms of fibroblastoid cell phenotype transitions:dedifferentiation of myofibroblasts and influencing of invasiveness and metastasis of sarcoma
Kosla, Jan ; Dvořák, Michal (advisor) ; Peková, Soňa (referee) ; Reiniš, Milan (referee)
Fibroblasts are the principal cellular component of the connective tissue. They are a heterogeneous group of cells which contribute to the structure of connective tissue and wound healing by their ability to produce extracellular matrix (ECM). Fibroblasts and cells derived from them are involved in many pathological processes such as formation of malignant tumors and fibrosis. Tumor progression which finally leads to metastasis is a serious biomedical problem. There is a growing body of the recent evidence showing an important role of the tumor stroma and its interaction with cancer cells in cancer progression. Tumor stroma comprises mainly of myofibroblasts and their products, namely ECM, soluble factors, and enzymes. Myofibroblasts contribute more or less to all steps of cancer progression. Furthermore myofibroblasts play a key role in fibrosis, another serious human disease which is not efficiently treatable and which is associated with cancer progression. These facts made us to search for molecular means capable of eliminating the myofibroblastic phenotype. We succeeded to entirely dedifferentiate primary myofibroblasts by concomitant inhibition of TGFβ signaling and perturbation of MAPK signaling in a chick model that we have introduced. Malignant fibroblasts form sarcomas. ECM is the first...
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The use of CAM assay for characterization and study of cancer cell invasive properties
Vágnerová, Lenka ; Dvořák, Michal (advisor) ; Geryk, Josef (referee)
The chorioallantoic membrane (CAM) of chicken embryos belongs to the in vivo model systems frequently used for the study of angiogenesis and cell invasiveness. Using CAM assay we have tested selected chicken sarcoma cell lines characterized by different angiogenic properties and different ability to form metastasis. In addition to CAM assay, several other methods have been used to characterize the phenotype of these cell lines. We have selected a few proteins which could significantly influence the angiogenic and metastatic properties of investigated cell lines. We have established cell lines stably overexpressing these genes and compared their phenotypes with parental cell lines. We have shown that genes encoding ISL1, ARNT2, PROM1, HOXA11 proteins participate, in our experimental model, in activation of programes controlling angiogenesis and cell invasion.
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Study of extracellular nucleic acids in maternal circulation in the cases of pathological and physiological pregnancies
Žejšková, Lenka ; Hromadníková, Ilona (advisor) ; Černá, Marie (referee) ; Dvořák, Michal (referee)
In 1997, Prof. Dennis Lo discovered the presence of cell-free fetal DNA (cffDNA) in the maternal plasma and serum of pregnant women. This finding started the development of new non-invasive prenatal diagnosis methods, which are currently in the forefront of the advanced care of mother and fetus. Non-invasive genetic tests based on the detection of paternally inherited alleles, including determination of fetal sex in cases at risk of X-linked disorders or congenital adrenal hyperplasia and RHD or RHCE genotyping in alloimunized pregnancies, were quickly introduced into routine practice. This thesis focuses on the basic characteristics of cffDNA and fetal cells in maternal circulation and its usage for non-invasive prenatal diagnosis, especially in cases of placental insufficiency related complications, e.g. preeclampsia and IUGR. This severe disorder is characterized by placental dysfunction with an abnormal invasion of trophoblasts and a defect in the transformation of maternal spiral arteries, leading to placental ischemia followed by increased apoptosis of trophoblast associated with an elevated concentration of cell-free nucleic acids in maternal circulation. Until recently, cffDNA quantification studies were mostly done using amplification of SRY or DYS-14 genes localized on chromosome Y, and...
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Regulace pre-mRNA sestřihu v prostředí buněčného jádra
Hnilicová, Jarmila ; Staněk, David (advisor) ; Půta, František (referee) ; Dvořák, Michal (referee)
Eukaryotic genes contain non-coding sequences - introns that are removed during pre-mRNA splicing by the spliceosome. The spliceosome is composed of five snRNPs (U1, U2, U4/U6 and U5) which assemble on pre-mRNA in a step-wise manner and together with additional non-snRNP proteins catalyse splicing. Mutations in splicing factors can cause severe diseases, for example a point missense mutation (called AD29) in hPrp31 (U4/U6 snRNP specific protein) induces retinitis pigmentosa, disease often leading to complete blindness. In this PhD thesis we show that the hPrp31 AD29 mutant is unstable and is not properly incorporated into spliceosomal snRNPs. In addition, the expression of the mutant protein reduces cell proliferation, which indicates that it interferes with cellular metabolism (likely splicing) and could explain the induction of retinitis pigmentosa. Next, we focus on a role of nuclear environment in pre-mRNA splicing. It was shown that new U4/U6·U5 snRNPs are preferentially assembled in non-membrane nuclear structure - Cajal body. Here we expand this finding and provide evidence that Cajal bodies are also important for U4/U6·U5 snRNP recycling after splicing. In addition, we analyzed a role of chromatin and particularly histone acetylation modulates in splicing regulation. Using inhibitor of...
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From the search for new oncogenes to the effort of redefining the cancerogenesis phenomenon
Pajer, Petr ; Dvořák, Michal (advisor) ; Vodička, Pavel (referee) ; Eckschlager, Tomáš (referee)
The described experimental model of clonal tumors induced through the insertional mutagenesis with MAV-2 proved to be a valid and rich source of information describing the process of transformation of normal into tumor cell. We have mapped more than 2000 individual clonal VISs from several hundreds of tumor tissue samples. We have analyzed five tumor types of different histology and tissue of origin along with their derivative tissue cultures. Furthermore, we have discovered the industasis phenomenon and described it during the course of the study. The goal of my study was to uncover common reasons for neoplastic transformation of the cell. The results of my study led me to the paradoxical conclusion that the significance of genetic changes as the primary cause of induction of neoplastic transformation is being overestimated. Although studying the functions of individual genes and search for new tumor markers and therapeutical targets are still beneficial, I believe that the traditional perception of tumor formation as a function/result of mutation accumulation and selection is becoming a serious drawback in further investigations. These conclusions are further discussed in the last section of the presented Ph.D. thesis.
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The biological importance of CAS SH3 domain tyrosine phosphorylation
Janoštiak, Radoslav ; Brábek, Jan (advisor) ; Dvořák, Michal (referee)
Protein CAS is a major tyrosine-phosphorylated protein in cells transformed by v-crk and v-src oncogenes. It is a multidomain adaptor protein, which serves as a scaffold for assembly of signalling complexes which are important for migration and invasiveness of Src-transformed cells. A novel phosphorylation site in N-terminal SH3 domain was identified - tyrosine 12 located on binding surface of CAS SH3 domain. To study biological importance of tyrosine 12 phosphorylation, non-phosphorylable (Y12F) and phosphomimicking ( Y12E) mutant of CAS were prepared. We found that phosphomimicking mutation Y12E leads to decreased interaction of CAS SH domain with kinase FAK a phosphatase PTP-PEST and also reduce tyrosine phosphorylation of FAK. Using GFP-tagged CAS protein, we show that Y12E mutation caused delocalization of CAS from focal adhesion but has no effect on localization of CAS to podosome-type adhesion. Non-phosphorylable mutation Y12F cause hyperphosphorylation of CAS substrate domain and decrease turnover of focal adhesion and associated cell migration of mouse embryonal fibroblasts (MEFs) independent to integrin singalling. Analogically to migration, CAS Y12F decrease invasiveness of Src-transformed MEF. The results of this diploma thesis show that phosphorylation of Tyr12 in CAS SH3 domain is...
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French Muses of Jindřich Štyrský
Dvořák, Michal ; Listíková, Renáta (advisor) ; Ébert-Zeminová, Catherine (referee)
Jindřich Štýrský est né le 11 aot 1899 dans le village appelé Černá en Bohme de l'est ; ses parents y avaient une grande propriété. Štýrský avait une relation difficile avec ses parents. Son pre, enseignant atteint d'alcoolisme chronique, était assez exigeant et obligeait son fils de devenir l'enseignant. Sa mre était trs croyante et l'a peu materné. Sa soeur aînée du premier mariage de sa mre s'appelait Marie et Štýrský enfant était amoureux d'elle. Cette relation platonique a fortement marqué Štýrský et elle a évoqué chez lui de premires fantaisies érotiques. Les témoignages décrivent Štýrský comme un jeune homme élégant et bien élevé. Il était connu comme un séducteur des femmes. Il détestait ses études d'enseignant et il préférait la peinture et la littérature. Parmi ses auteurs aimés se trouvaient les écrivains français. La mort de sa mre lui a libéré et aprs ses études, un an de service mititaire et deux ans de la profession du maître, il a quitté la campagne et s'est installé Prague. Malgré l'opposition de son pre il y a fréquenté l'Académie d'art plastique et il rencontrait des artistes connus. A cause de sa personnalité compliquée il a bientôt abandonné ses études l'Académie et grâce aux moyens obtenus de la succession de sa mre il a décidé voyager. Štýrský a voulu aller en Polynésie mais il s'est...
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Cytokine expression in chemically-induced senescence
Nováková, Zora ; Hodný, Zdeněk (advisor) ; Hampl, Aleš (referee) ; Dvořák, Michal (referee)
AbstractJanderova-Rossmeislova L., Novakova Z., Vlasakova J., Philimonenko V., Hozak P., Hodny Z., 2007. PML protein association with specific nucleolar structures differs in normal, tumor and senescent human cells. J. Struct. Biol. 159, 56-70. Cellular senescence, widely recognized as a potent suppressor of tumorigenesis, represents response to cellular stress and DNA damage which results in irreversible cell growth arrest. Growing evidence signalizes crucial role of cytokine production in senescence phenomenon. Recently, many research groups have efforted to define exact character of senescence-associated secretory phenotype and its function in senescence development and maintentance. Factors secreted by senescent cells, mainly of proiflammatory character, were found to have pronounced effects on their environment as well as on its own producer. These observations were obtained preferentially on models of replicative senescence and oncogene-induced senescence. Vlasakova J, Novakova Z, Rossmeislova L, Kahle M, Hozak P, Hodny Z. 2007. Histone deacetylase inhibitors suppress IFNalpha-induced up-regulation of promyelocytic leukemia protein. Blood 109: 1373-80 Novakova Z, Man P, Novak P, Hozak P, Hodny Z. 2006. Separation of nuclear protein complexes by blue native polyacrylamide gel electrophoresis....
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Interactive flexible program - Sulfur chemistry and its impact to acquiring of curriculum
Dvořák, Michal
UNIVERZITA cká fakulta KARLOVA V PRAZE, Přírodověde Katedra učitelství a didaktiky chemie CHA ce RLES UNIVERSITY IN PRAGUE, Faculty of Scien Department of Teaching and Didactic of Chemistry Interaktivní flexibilní program - Chemie síry a jeho vliv na efektivitu osvojování učiva Interactive flexible program - Sulfur chemistry and its impact to acquiring of curriculum Mgr. Michal Dvořák Souhrn disertační práce Summary of PhD. Thesis 1 Praha/Praque 2009 2 KEYWORDS Interactive flexible program; electronic materials; digital textbook; education software; effectiveness; process of acquiring curriculum; key competencies; chemistry xperiments, personal computer; technology. e INTRODUCTION We live in an era of information technology where the revolutionary way of acquiring new information is available. The new information deepen and widen acknowledge in individual branches of science. Most of the new information contradicts the validity of the existing data. Huge increase and still rising difficulty of the new information require the fundamental changes in the organization of process of acquiring curriculum. But the existing education tools specified for self-studying pupils do not reflect these...
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