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Identifier

000334467

Title

Μελέτη του σηματοδοτικού μοναπατιού του μετασχηματίζοντα αυξητικού παράγοντα β (TGF-β) in vivo, με χρήση ανασυνδυασμένων αδενοϊών

Alternative Title

Study of the TGF-β signaling pathway in vivo, using recombinant adenoviruses

Author

Σκούρτη-Σταθάκη, Κωνστντίνα

Thesis advisor

Καρδάσης, Δημήτριος

Abstract

Transforming growth factor-β (TGF-β) is an important growth-inhibitor of epithelial cells and belongs to the large family of pleiotropic cytokines that regulate diverse biological functions during development and adult tissue homeostasis. TGF-β signals via membrane bound Ser/Thr kinase receptors which transmit their signals via the intracellular signalling molecules Smad2, Smad3 and Smad4. These Smad proteins contain conserved MH1 and MH2 domains separated by a flexible linker domain. Smad complexes regulate gene expression and determine the biological response to TGF-β. Recent work has demonstrated that the linker domain of Smad3 contains a Smad transcriptional activation domain. This domain is capable of recruiting the p300 transcriptional co-activator and is required for Smad3-dependent transcriptional activation. The identification of mutations, generated in our lab in previous studies, in the conserved residues P229 and Q222 of the linker domain has led to the conclusion that these mutations play suppressive roles on the Smad3 molecule. The first aim of this Thesis was the study of these mutations in vivo and the introduction of the mutated forms to eukaryotic cells using recombinant adenoviruses. To determine the role of these mutations on Smad3 protein, we examined their effect on the transcriptional activation of two known gene-targets of the TGF-β signalling pathway, ΡΑΙ-1 (Plasminogen Activator Inhibitor-1) and p21 (CDKN1A), in the presence of adenoviruses that express the mutated forms, Smad3(P229A) and Smad3(Q222A). We showed that the AdSmad3(P229A)-6myc adenovirus suppresses the transcriptional activation of gene-targets when the cells were treated with ΤGF-β for 1h and when they were infected with the AdALK5ca adenovirus. The second aim of this Thesis was the development of a protein biotinylation system in vivo using recombinant adenoviruses in order to study protein-protein interactions in living organisms. To address this purpose, we generated recombinant adenoviruses that express the Smad3bio protein (that expresses the wt Smad3 protein in frame with the Bio epitope) and the biotine ligase BirA and we showed that using this system the Smad3 protein can be biotinylated in HaCaT cells.

Language

Greek

Subject

BCAR Breast Cancer Anti-estrogen Resistance

PAI Plasminogen Activator Inhibitor

Smad

ευκαρυωτικά κύτταρα

Issue date

2008-12-04

Collection

School/Department--School of Sciences and Engineering--Department of Biology--Post-graduate theses

Type of Work--Post-graduate theses