Genetic Manipulation, Gene Silencing and Biomarker Development in Multiple Experimental Models.

De Cecco, Marco (2012) Genetic Manipulation, Gene Silencing and Biomarker Development in Multiple Experimental Models., [Dissertation thesis], Alma Mater Studiorum Università di Bologna. Dottorato di ricerca in Biotecnologie, farmacologia e tossicologia: progetto n. 1 "Biotecnologie cellulari e molecolari", 24 Ciclo. DOI 10.6092/unibo/amsdottorato/4254.
Documenti full-text disponibili:
[img]
Anteprima
Documento PDF (English) - Richiede un lettore di PDF come Xpdf o Adobe Acrobat Reader
Download (5MB) | Anteprima

Abstract

The thesis is set in three different parts, according to the relative experimental models. First, the domestic pig (Sus scrofa) is part of the study on reproductive biotechnologies: the transgenesis technique of Sperm Mediated Gene Transfer is widely studied starting from the quality of the semen, through the study of multiple uptakes of exogenous DNA and lastly used in the production of multi-transgenic blastocysts. Finally we managed to couple the transgenesis pipeline with sperm sorting and therefore produced transgenic embryos of predetermined sex. In the second part of the thesis the attention is on the fruit fly (Drosophila melanogaster) and on its derived cell line: the S2 cells. The in vitro and in vivo models are used to develop and validate an efficient way to knock down the myc gene. First an efficient in vitro protocol is described, than we demonstrate how the decrease in myc transcript remarkably affects the ribosome biogenesis through the study of Polysome gradients, rRNA content and qPCR. In vivo we identified two optimal drivers for the conditional silencing of myc, once the flies are fed with RU486: the first one is throughout the whole body (Tubulin), while the second is a head fat body driver (S32). With these results we present a very efficient model to study the role of myc in multiple aspects of translation. In the third and last part, the focus is on human derived lung fibroblasts (hLF-1), mouse tail fibroblasts and mouse tissues. We developed an efficient assay to quantify the total protein content of the nucleus on a single cell level via fluorescence. We coupled the protocol with classical immunofluorescence so to have at the same time general and particular information, demonstrating that during senescence nuclear proteins increase by 1.8 fold either in human cells, mouse cells and mouse tissues.

Abstract
Tipologia del documento
Tesi di dottorato
Autore
De Cecco, Marco
Supervisore
Dottorato di ricerca
Scuola di dottorato
Scienze biologiche, biomediche e biotecnologiche
Ciclo
24
Coordinatore
Settore disciplinare
Settore concorsuale
Parole chiave
Pig SMGT Drosophila RNAi Fibroblasts Nuclear Proteins
URN:NBN
DOI
10.6092/unibo/amsdottorato/4254
Data di discussione
27 Gennaio 2012
URI

Altri metadati

Statistica sui download

Gestione del documento: Visualizza la tesi

^