E-Locus - Institutional Repository of the University of Crete

Home Collections School/Department School of Medicine Department of Medicine Post-graduate theses

Post-graduate theses

Current Record: 942 of 1238

[Add to Basket]

Identifier

000373899

Title

Κλωνοποίηση και έκφραση της πρωτείνης Foxola σε ευκαρυωτικό σύστημα.

Alternative Title

Cloning and expeddion of the Foxola protein in eukariotic system.

Author

Ιερωνυμάκη, Ελευθερία

Thesis advisor

Γαρίνης, Γεώργιος

Reviewer

Καραγωγέως, Δόμνα

Abstract

Ageing is inherently complex, involving multiple mechanisms at different levels. Recent evidence suggests that ageing is characterized by conserved patterns of adult-onset gene regulation predominantly involved in growth, stress resistance and energy metabolism. Nevertheless, it remains unknown how such universal networks are tightly orchestrated over the entire organismal lifespan and in animal species as evolutionary diverse as nematodes and mammals. Gene activity is regulated within the context of chromatin involving, among others, transcription factors and chromatin structure changes. One particular class of transcription factors are the Forkhead box O (FoxO) proteins. FoxOs are involved in a wide range of biological processes, including their conserved ability to increase lifespan. The interaction of FoxOs with multi-protein complexes likely regulates forkhead activity but also allows diversification of function efficiently coordinating distinct sets of genes with far-reaching consequences for cell fate with advancing age. In the present study, the cloning strategy of FoxO1a is described. An expression vector carrying the FoxO1a gene sequence was used to develop an in vitro biotinylation methodology. This methodology, takes advantage of the high affinity bonding between biotin and avidin/streptavidin and facilitates the isolation of protein complexes. FoxO1a was fused with an 14 aa biotinylation tag and expressed in mammalian cell lines, transfected with a BirA biotin ligase. By using Real-time PCR and Western blot analysis, it was clear that FoxO1a could be transcribed efficiently in cells, indicating the first step for the development of the in vitro biotinylation system.

Language

Greek

Subject

Ageing

Biochemistry

Cloning

Foxola

Longevity

Γήρανση

Κλωνοποίηση

Μακροζωία

Issue date