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Abstract(s)
Para além dos 23 pares de cromossomas que uma célula contem, o genoma humano também é constituído pelo genoma mitocondrial. A principal função da mitocôndria é a produção de níveis altos de energia (ATP) usada pelas células por via da respiração aeróbia. Como consequência desta função, a produção de espécies reativas de oxigénio pode danificar o mtDNA, organelos celulares e a própria célula. Comparativamente com o nDNA, a taxa de mutações no mtDNA é superior devido à falta de mecanismos de proteção e de reparação. Como resultado, ocorrem danos em células e tecidos que despendem níveis mais elevados de energia como é o caso do cérebro, coração e fígado. Atualmente estão associadas doenças que resultam das mutações ocorridas no genoma mitocondrial, como é o caso de MELAS, MERRF e SL.
Desta forma, é necessário a introdução de novos genes mitocondriais com vista a restabelecer as funções perdidas pelo organelo, como é o caso da terapia de genes mitocondrial.
Esta dissertação de mestrado tem como base o desenvolvimento de um transportador incorporando o gene mitocondrial ND1 que consiga entregar eficazmente o DNA plasmídico (pDNA) à mitocôndria e que possa, futuramente, ser usado como uma alternativa para a terapia génica mitocondrial. A introdução de compostos fluorescentes às nanopartículas torna-se a principal novidade deste projeto.
Este projeto foi dividido em 2 etapas:
1. Síntese e caracterização de compostos fluorescentes;
2. Síntese e caracterização das nanopartículas à base de carbonato de cálcio pelo método de co precipitação.
As nanopartículas à base de carbonato de cálcio, incorporadas com os compostos fluorescentes e o plasmídeo, oferecem qualidades únicas como biocompatibilidade, baixo custo e facilidade de produção. Parâmetros como a morfologia, tamanho, potencial zeta, capacidade de encapsulamento, degradabilidade e biocompatibilidade foram testados neste trabalho.
Os resultados demonstraram que as nanopartículas têm capacidade e características ideais para futuros ensaios in vitro e expressão da proteína correspondente ao gene ND1.
Em suma, através do conhecimento já adquirido de outros protocolos de terapia génica mitocondrial e a realização deste vetor não viral com afinidade pela mitocôndria traz avanços importantes no tratamento a diversas patologias associadas à mitocôndria.
Beyond of 23 pairs of chromosomes that cell contains, the human genome is also comprises by mitochondrial genome. The main function of mitochondrion is the production of high levels of energy (ATP) used by cells via aerobic respiration. As consequence of this function, the production of reactive oxygen species can cause damage in mtDNA, in cell organelles and in cell itself. Comparing to nDNA, the rate of mutation in mtDNA is higher due to the lack of protection and repair mechanisms. As result, damage can occur in cells and tissues that expend higher energy levels like brain, heart and liver. Nowadays, are associated diseases as effect of mutations occured in mitochondrial genome as MELAS, MERRF and LS. So, is necessary an introduction of new mitochondrial gene in order to restore the lost functions by the organelle, as is the case of mitochondrial gene therapy. The main objectives of this master dissertation are the development of a carrier incorporating a mitochondrial ND1 gene that can deliver effectively pDNA into mitochondrion and, from now on, can be used as an alternative for mitochondrial gene therapy. The introduction of fluorescents compounds into nanoparticle, became novelity of this project. This project is divided in 2 parts: 1. Synthesis and characterization of fluorescent compounds; 2. Synthesis and characterization of calcium carbonate based nanoparticles by co-precipitation method. Calcium carbonate based nanoparticles, incorporated with fluorescents compounds and pDNa, provides qualities like biocompatibility, low cost and easier production. Parameters such as morphology, size, zeta potential, encapsulation ability, degradability and biocompatibility were tested. The results demonstrate that nanoparticles have capacity and promising characteristics for further tests in vitro and protein expression of ND1 gene. Briefly, the knowledge already acquired concerning others protocols of mitochondrial gene therapy and the achievement of this non-viral vector with affinity for the mitochondria can provides new insights in the treatment of various diseases associated with mitochondria.
Beyond of 23 pairs of chromosomes that cell contains, the human genome is also comprises by mitochondrial genome. The main function of mitochondrion is the production of high levels of energy (ATP) used by cells via aerobic respiration. As consequence of this function, the production of reactive oxygen species can cause damage in mtDNA, in cell organelles and in cell itself. Comparing to nDNA, the rate of mutation in mtDNA is higher due to the lack of protection and repair mechanisms. As result, damage can occur in cells and tissues that expend higher energy levels like brain, heart and liver. Nowadays, are associated diseases as effect of mutations occured in mitochondrial genome as MELAS, MERRF and LS. So, is necessary an introduction of new mitochondrial gene in order to restore the lost functions by the organelle, as is the case of mitochondrial gene therapy. The main objectives of this master dissertation are the development of a carrier incorporating a mitochondrial ND1 gene that can deliver effectively pDNA into mitochondrion and, from now on, can be used as an alternative for mitochondrial gene therapy. The introduction of fluorescents compounds into nanoparticle, became novelity of this project. This project is divided in 2 parts: 1. Synthesis and characterization of fluorescent compounds; 2. Synthesis and characterization of calcium carbonate based nanoparticles by co-precipitation method. Calcium carbonate based nanoparticles, incorporated with fluorescents compounds and pDNa, provides qualities like biocompatibility, low cost and easier production. Parameters such as morphology, size, zeta potential, encapsulation ability, degradability and biocompatibility were tested. The results demonstrate that nanoparticles have capacity and promising characteristics for further tests in vitro and protein expression of ND1 gene. Briefly, the knowledge already acquired concerning others protocols of mitochondrial gene therapy and the achievement of this non-viral vector with affinity for the mitochondria can provides new insights in the treatment of various diseases associated with mitochondria.
Description
Keywords
Citopatias Mitocondriais Compostos Fluorescentes Dnamt Mitocôndria Nanopartículas Pdna Terapia Génica Mitocondrial