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Expression of sTGFbeta RII-Fc-Jun from recombinant vaccinia virus
Samková, Zuzana ; Němečková, Šárka (advisor) ; Španielová, Hana (referee)
Expression of sTGFbetaRII-Fc-Jun from recombinant vaccinia virus TGFß has a biphasic role in tumorigenesis. In early phases it acts as tumor sup-pressor. However, in late phases when cells have escaped selectively from the antimito-genic response of TGFß, it may act as a promoter of tumor progression and invasion. One way of control tumor formation and progression is blocking of TGFß signalling pathways in late phases of tumorigenesis. We have constructed recombinant vaccinia virus P13 expressing soluble TGFbeta type II receptor fused with the Fc fragment of IgG1 and with Jun fragment (sTbetaRII-Fc-Jun). This sTbetaRII-Fc-Jun is supposed to increase the effect of antitumor vaccinia virus vaccine expressing SigE7LAMP, which is investigated for the treatment of the HPV-16 associated cervical cancer. Binding of sTbetaRII-Fc-Jun to protein G were tested by SDS-PAGE and by im-munoblotting. We found that Jun fragment and sTbetaRII fragment do not block Fc bind-ing site for protein G. sTbetaRII-Fc-Jun was characterised using SDS-PAGE and immunoblot analysis. We observed that the amount of sTbetaRII-Fc-Jun was higher in cell supernatans of in-fected cells in comparison to cell lysates. In cell lysates we observed higher amount of sTbetaRII than sTbetaRII-Fc-Jun. The expression of sTbetaRII-Fc-Jun was stronger under...
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The regulation of primary response genes by the ERK signaling pathway
Chvalová, Věra ; Vomastek, Tomáš (advisor) ; Doubravská, Lenka (referee)
The ERK signaling pathway represents an evolutionary conserved mechanism that enables cells to perceive various extracellular signals and convert them to a diverse array of biological outcomes such as proliferation, differentiation, cell cycle control, apoptosis or cell migration. Key components of this pathway are protein kinases Raf, MEK and the effector protein kinase ERK. In addition to its physiological role, continuous activation of the ERK pathway caused by somatic mutations of some of its components or upstream regulators appears to be significant cause of many human tumor diseases. That is why this pathway plays an important role also from the biomedical viewpoint. The multistep changes in gene expression are primarily responsible for these physiological and pathological events. Changes in genes expression are induced by activated kinase ERK that after translocation into the nucleus phosphorylates transcription factors (TFs) whose activation, in turn, leads to transcription of so-called immediate early genes (IEGs), many of which also code for other TFs (e.g. c-Fos, c-Jun or c-Myc). The latter TFs then regulate expression of further genes for structural and signaling proteins. This causes global changes in gene expression and leads to functional reprogramming of the cells. This thesis...
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Expression of sTGFbeta RII-Fc-Jun from recombinant vaccinia virus
Samková, Zuzana ; Španielová, Hana (referee) ; Němečková, Šárka (advisor)
Expression of sTGFbetaRII-Fc-Jun from recombinant vaccinia virus TGFß has a biphasic role in tumorigenesis. In early phases it acts as tumor sup-pressor. However, in late phases when cells have escaped selectively from the antimito-genic response of TGFß, it may act as a promoter of tumor progression and invasion. One way of control tumor formation and progression is blocking of TGFß signalling pathways in late phases of tumorigenesis. We have constructed recombinant vaccinia virus P13 expressing soluble TGFbeta type II receptor fused with the Fc fragment of IgG1 and with Jun fragment (sTbetaRII-Fc-Jun). This sTbetaRII-Fc-Jun is supposed to increase the effect of antitumor vaccinia virus vaccine expressing SigE7LAMP, which is investigated for the treatment of the HPV-16 associated cervical cancer. Binding of sTbetaRII-Fc-Jun to protein G were tested by SDS-PAGE and by im-munoblotting. We found that Jun fragment and sTbetaRII fragment do not block Fc bind-ing site for protein G. sTbetaRII-Fc-Jun was characterised using SDS-PAGE and immunoblot analysis. We observed that the amount of sTbetaRII-Fc-Jun was higher in cell supernatans of in-fected cells in comparison to cell lysates. In cell lysates we observed higher amount of sTbetaRII than sTbetaRII-Fc-Jun. The expression of sTbetaRII-Fc-Jun was stronger under...
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