Author(s):
Henriques, Sónia Troeira, 1980- 
Date: 2007
Persistent ID: http://hdl.handle.net/10451/1605
Origin: Repositório da Universidade de Lisboa
Subject(s): Biofísica molecular; Teses de doutoramento
Document details
Study of the molecular mechanism used by the peptidic vector pep-1 to introduce...
Description
Tese de doutoramento em Bioquímica (Biofísica Molecular), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 2008 The introduction of genetic material or proteins to originate a defined biochemical effect inside the cell has a remarkable potential for the treatment of Human diseases, however this is hampered by the cell membrane barrier for the entry of hydrophilic macromolecules. A possible strategy to overcome the membrane barrier was proposed after the discovery of basic peptidic sequences with ability to pass trough the membrane in a non-toxic and non-immunogenic manner. These peptides are commonly designated as cell-penetrating peptides (CPPs). Pep-1 is a CPP and has been successfully used to introduce several macromolecules biologically active inside cultured cells. The main goal of this thesis is to clarify the mechanism used by this peptide to pass through the membrane and to confirm if its efficiency as a carrier to introduce proteins inside cells. The interaction with membranes was followed in vitro with model membranes: large unilamellar vesicles; planar lipid membranes, giant unilamellar vesicles and supported bilayers. HeLa cells were used to follow the translocation of pep-1 associated with a protein. Fluorescence and UV-Vis spectroscopy methodologies, CD and ATR-FTIR spectroscopy, electrophysiological measurements and fluorescence microscopy were used to carry on the experimental work. It was shown that pep-1 is able to interact with and to destabilize the lipidic bilayers without evidence for pore formation at variance with other CPPs that use an endosomal pathway. Although all the evidences show that pep-1 translocates by a physically-mediated mechanism promoted by transmembrane potential with no evidences for the use of endosomal routes as an alternative pathway. Differences between this particular pep-1 and other CPPs can be related with the affinity for membrane. Peptides with higher affinity for membrane have more propensities to be internalized by a non-endocytic mechanism. Lower affinity for membranes favours endocytic uptake. Resumo alargado em português disponível no documento
Tese de doutoramento em Bioquímica (Biofísica Molecular), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 2008 The introduction of genetic material or proteins to originate a defined biochemical effect inside the cell has a remarkable potential for the treatment of Human diseases, however this is hampered by the cell membrane barrier for the entry of hydrophilic macromolecules. A possible strategy to overcome the membrane barrier was proposed after the discovery of basic peptidic sequences with ability to pass trough the membrane in a non-toxic and non-immunogenic manner. These peptides are commonly designated as cell-penetrating peptides (CPPs). Pep-1 is a CPP and has been successfully used to introduce several macromolecules biologically active inside cultured cells. The main goal of this thesis is to clarify the mechanism used by this peptide to pass through the membrane and to confirm if its efficiency as a carrier to introduce proteins inside cells. The interaction with membranes was followed in vitro with model membranes: large unilamellar vesicles; planar lipid membranes, giant unilamellar vesicles and supported bilayers. HeLa cells were used to follow the translocation of pep-1 associated with a protein. Fluorescence and UV-Vis spectroscopy methodologies, CD and ATR-FTIR spectroscopy, electrophysiological measurements and fluorescence microscopy were used to carry on the experimental work. It was shown that pep-1 is able to interact with and to destabilize the lipidic bilayers without evidence for pore formation at variance with other CPPs that use an endosomal pathway. Although all the evidences show that pep-1 translocates by a physically-mediated mechanism promoted by transmembrane potential with no evidences for the use of endosomal routes as an alternative pathway. Differences between this particular pep-1 and other CPPs can be related with the affinity for membrane. Peptides with higher affinity for membrane have more propensities to be internalized by a non-endocytic mechanism. Lower affinity for membranes favours endocytic uptake. Resumo alargado em português disponível no documento
Document Type
Doctoral Thesis
Advisor(s) Castanho, Miguel Augusto Rico Botas, 1967-
Advisor(s) Castanho, Miguel Augusto Rico Botas, 1967-
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