Utilize este identificador para referenciar este registo: http://hdl.handle.net/10451/11459
Título: Molecular determinants of the cellular internalization of crotamine-derived peptides
Autor: Rodrigues, Margarida Filipe Dias
Orientador: Santos, Nuno C., 1972-
Palavras-chave: Teses de doutoramento - 2014
Data de Defesa: 2014
Resumo: The work described throughout this thesis was focused in the study of crotaminederived peptides’ (NrTPs) mechanism of action. Also, and due to the cell-penetrating nature of these molecules, we emphasized the importance of its use as drug delivery adjuvants. The different peptides used were designed to address important mechanistic questions. In Chapter 2 we describe experiments carried out using lipid membrane model systems. We studied the interaction, partition and extent of membrane penetration of NrTPs using large unilamellar vesicles (LUV). We prepared POPC, POPG, POPC:POPG and POPC:Cholesterol LUV and studied them by fluorescence spectroscopy techniques. Also, we conducted translocation assays for NrTP1 and NrTP5 using giant multilamellar vesicles (GMLV) and giant multivesicular liposomes (GMVL). In these experiments, conducted by confocal microscopy (and later for flow cytometry studies too), we used rhodamine B-labeled NrTPs. In Chapter 3, we describe the experiments that tested peptide’s uptake into mammalian cells. In these studies, carried out using fluorescence microscopy or flow cytometry, we evaluated the ability of NrTPs to be internalized into different types of cells, using different peptide concentrations and at different incubation temperatures. We used immortalized cells lines (BHK21, HeLa, BV-173 and MOLT-4) and primary cells (mainly PBMCs). Also, we assessed NrTPs cytotoxicity in different cell models and determined the kinetic profiles for their uptake into PBMCs (peripheral blood mononuclear cells) along with tests to unravel the internalization mechanism. In Chapter 4, we describe a very important proof-of-concept experiment: the test for NrTPs’ ability to carry and delivery a large molecule into cells. We prepared conjugates of NrTP6 covalently linked (maleimide-based chemistry) to β-galactosidase (β-gal). Conjugates were tested for their enzymatic activity in vitro and after incubated with HeLa cells. β-gal activity in the cell free extract after conjugate incubation was used as readout of the efficient cellular entry. Throughout this project we used different techniques, methodologies and models. Our key conclusions are: NrTPs interact and translocate across membrane model systems. They are successfully internalized into mammalian cells without compromising cell viability. Cytotoxicity is peptide- and concentration-dependent and the signs of cytotoxicity appeared only for the higher concentrations tested. The kinetic uptake profiles are cell-type dependent, concentration-independent and significantly different among NrTPs. The internalization inhibition at 4°C and in the presence of dynasore (dynamin inhibitor) is different among NrTPs, leading as to conclude that both endocytosis and direct translocation play a role in cell internalization of NrTPs. Still, endocytosis was the predominant mechanism. The methodology employed to follow and analyze the uptake of NrTPs using flow cytometry is a valuable comparative and quantitative method to test CPP efficiency among different peptides. NrTPs are responsible for cargo entry. They are able to deliver large molecules such as proteins (β-gal) that are then still able to perform their biological activity. The take home message: NrTP6 is an efficient tool for intracellular delivery!
Descrição: Tese de doutoramento, Ciências Biomédicas (Bioquímica Médica), Universidade de Lisboa, Faculdade de Medicina, 2014
URI: http://hdl.handle.net/10451/11459
Aparece nas colecções:FM - Teses de Doutoramento

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