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Study of the RGD role in cells patterning onto a substrate in microfluidic devices

A method of patterning cells onto glass was studied utilizing micro-fluidic channels fabricated by means of replica moulding technique. In this work 3T3 mouse fibroblast cell line was used for the immobilization thanks to property of the integrin on the cell membrane that has to bind to the peptide arginine- glycine-aspartate (RGD) sequence present in extra cellular matrix molecule such as fibronectin.
Several experiment than were ran for enhancing the strength of the bind between the peptide and the cell. Other artificial peptides previously cloned and expressed in the laboratory have been used in comparison with fibronectin. These proteins contains different repetitions of the RGD sequence (1 to 40).
The objective was to see that even small peptides containing such a sequence can compete with the big fibronectin molecule and whether the presentation of multiple simultaneous interaction can achieve an enhanced overall binding compared to that of individual ligands. This work represent only the beginning of a more detailed study about the multivalent effect, since only the comparison between fibronectin and a artificial peptide containing one RGD repetition (1-mer) was consistently.
This study may be useful for tight attachment of cells to surface of several substrates. In addition, since this methods involves mono-disperse species, it may be attractive for quantitative studies about the multivalency described above. In this work The first part of the project is focused on all the steps involved for the fabrication of these micro-fluidic devices as well as the
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materials used. The method we are considering is very effective for biological purpose and very simple for what concern the micro-fabrication techniques.
The second part aim to explain the chemistry behind the binding of the cells, the linker molecule used to bind the peptide to the glass, the preparation and the concentration of them and a shear stress setup to test how different flow rates affect the bind.
The future development of the project include a detailed comparison between the adhesion effectiveness of fibronectin molecule and different length peptides up to forty repetition of the RGD sequence.

Mostra/Nascondi contenuto.
1.3. Protein Functions The proteins play many important roles in a organism depending both on their chemical and mechanical disposition. Since they are made of a sequence of amino acids they can fold up in different three dimensional shapes showing different interactions on different binding sites 1 The property of proteins to bind to other molecules enable them to act with different function such catalysts, signal receptors, switches, motors or tiny pumps [3]. All proteins stick to other protein in a organism. Sometimes this binds are very weak and short-lived, sometimes they are thigh and lasting, but every protein show a great specificity , that is every binding sites present on its structure can bind only to a specific chemical. The fi gure 1.1 represent schematically the kind of interaction that usually happens between a protein and its ligand. 1 - Introduction 3 1 The binding site is defined in biochemistry as the region of protein that bind to a specific molecule or ion called ligand. The binding sites exhibit a specificity that is related to their capability to bind only to certain types of molecules, and an affinity that measure the strength of the bind.

Laurea liv.II (specialistica)

Facoltà: Ingegneria

Autore: Daniele Sacchi Contatta »

Composta da 110 pagine.

 

Questa tesi ha raggiunto 67 click dal 16/02/2011.

Disponibile in PDF, la consultazione è esclusivamente in formato digitale.