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|Title: ||THE ROLE OF THE STEM CELL MARKER, MELANOMA CELL ADHESION MOLECULE MCAM/CD146|
|Authors: ||COSTA, ROSSELLA|
|Tutor: ||SAGGIO, ISABELLA|
|Issue Date: ||Dec-2009|
|Abstract: ||CD146 is a transmembrane Ig-like glycoprotein identified as a marker of tumor progression and metastasis. More recently, Sacchetti et al. have identified CD146 as a marker of mesenchymal cell stemness related to the ability to regenerate bone and stroma and to establish the hematopoietic microenvironment. Some aspects concerning CD146 are still to be investigated: these include the identification of CD146 natural ligand and the mechanistic relationship between the protein and its patho-physiological role. To investigate on these questions, by genetically manipulating the expression of CD146, we produced a CD146 encoding lentivector and a set of CD146 interfering vectors. By using the interfering vectors, we assessed that CD146 plays a pivotal role in supporting proliferation of human and murine primary stem cells, as its absence alters their growth.
In order to shed light on CD146 patho-physiological role we investigated its molecular ligand by screening a random phage peptide library on CD146 over expressing (through a lentiviral vector) human mesenchymal stem cells or skeletal stem cells (hSSCs). After several rounds of biopanning we randomly isolated some phage clones which show (through an ELISA assay) a strong binding ability to hSSC CD146+ and recurrent amino acid motifs.
The isolated peptides were further characterized and a blast analysis was carried on. They didn’t show any sequence homology to MCAM/CD146 suggesting i) that CD146 could be involved both in a heterophilic and homophilic (as recently reported by Staquicini et al.) interactions and ii) that the isolated peptides could represent novel ligands of human mesenchymal stem cells. In vivo, we could successfully use a CD146 interfering vector, containing a puromycin resistance coding gene, to produce a knocked down mouse model. Currently, we have a second generation of mice that were genotyped at weaning and tested for mRNA expression at two months. 100% of the mice analysed was positive for provirus integration and a large percentage (almost 62% for F1) expressed the puromycin resistance gene. Moreover, more than 50% CD146 down regulation was observed in four F1 mice. In this context, we could define i) that interfered genotype can be inherited and ii) that CD146 down regulation is compatible with the mouse survival|
|Research interests: ||Molecular Biology, cell biology, gene therapy, mouse manipulation, science journalism|
|Personal skills keywords: ||Lentiviral Vector Production, titration, Lentivirus – Mediated Transduction|
Mouse manipulation, practical skills about animal breeding and handling of mice colonies
Isolation of stromal stem cells from mouse bone marrow
|Appears in PhD:||GENETICA E BIOLOGIA MOLECOLARE|
Files in This Item:
|TESI DOTTORATO XXII CICLO A.A. 2008-2009 ROSSELLA COSTA.pdf||TESI DI DOTTORATO ||21.24 MB||Adobe PDF|
File del Curriculum Vitae:
|CurriculumVitae.pdf|| ||44.32 kB||Adobe PDF|
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