Optimização de métodos de manipulação da expressão de genes em células primárias e tecidos
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2014
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As infecções cirúrgicas são um dos principais problemas a nível hospitalar, contribuindo para o sofrimento dos doentes devido à dor, atraso na cicatrização e consequentemente aumento do tempo de hospitalização. Representam 13% das infecções nosocomiais, dos quais 56,9% são devidas a infecções do local cirúrgico. Esta problemática encontra-se muitas vezes associada a microorganismos com várias resistências antimicrobianas, como a Escherichia coli, Pseudomonas aeruginosa e Staphylococcus aureus (incluindo o S. aureus meticilino-resistente), consequência da utilização de antibióticos de largo espectro. Como tal, pretendeu-se contribuir para o desenvolvimento de novas estratégias de combate a infecções cirúrgicas, através da sobreexpressão de péptidos antimicrobianos, nomeadamente, a utilização de β-defensinas humanas (HBD). Esta sobreexpressão foi efectuada utilizando vectores lentivirais, contendo os respectivos genes, que foram introduzidos no modelo cirúrgico com ratos Wistar. De modo a verificar a capacidade antimicrobiana das HBD seleccionadas, procedeu-se previamente ao estabelecimento e caracterização de culturas das bactérias Pseudomonas aeruginosa ou Staphylococcus aureus. Posteriormente estas bactérias foram usadas para infectar o modelo cirúrgico, obtendo-se diferentes graus de infecção consoante a concentração de bactérias utilizadas.
Verificou-se em resultados preliminares, uma diminuição da proliferação das bactérias presentes nos modelos cirúrgicos, sempre que se introduziam vectores lentivirais expressando os genes que codificam as HBD.
Mais estudos estão a decorrer por forma a confirmar o papel antimicrobiano das HBD no modelo de infecção.
Surgical infections are a major problem in hospital, contributing to the suffering of the patients due to pain, delayed healing and, hence increased length of hospitalization. These infections represent 13% of nosocomial infections, of which 56.9% are due to surgical site infections. This problem is often associated with antimicrobial resistance of various microorganisms, such as Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus), as a consequence of the use of broad spectrum antibiotics. In this thesis, it was intended to contribute to the development of new strategies to combat surgical infections, through the overexpression of antimicrobial peptides, in particular, the use of human β-defensins (HBD). HBD overexpression was performed using lentiviral vectors containing the respective genes which have been introduced in the surgical model with rats. In order to verify the efficacy of HBD, we first optimzed and characterized cultures of P. aeruginosa or S. aureus bacteria. Thereafter these bacteria were used to infect the surgical model, obtaining different degrees of infection depending on the concentration of bacteria used. Preliminary results, revealed a decrease in the proliferation of bacteria in surgical models, where lentiviral vectors expressing the HBD genes were introduced. Further studies are in progress in order to confirm the antimicrobial role of HBD in the infection rat model.
Surgical infections are a major problem in hospital, contributing to the suffering of the patients due to pain, delayed healing and, hence increased length of hospitalization. These infections represent 13% of nosocomial infections, of which 56.9% are due to surgical site infections. This problem is often associated with antimicrobial resistance of various microorganisms, such as Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus (including methicillin-resistant Staphylococcus aureus), as a consequence of the use of broad spectrum antibiotics. In this thesis, it was intended to contribute to the development of new strategies to combat surgical infections, through the overexpression of antimicrobial peptides, in particular, the use of human β-defensins (HBD). HBD overexpression was performed using lentiviral vectors containing the respective genes which have been introduced in the surgical model with rats. In order to verify the efficacy of HBD, we first optimzed and characterized cultures of P. aeruginosa or S. aureus bacteria. Thereafter these bacteria were used to infect the surgical model, obtaining different degrees of infection depending on the concentration of bacteria used. Preliminary results, revealed a decrease in the proliferation of bacteria in surgical models, where lentiviral vectors expressing the HBD genes were introduced. Further studies are in progress in order to confirm the antimicrobial role of HBD in the infection rat model.
Descrição
Orientação: Paula Videira ; orientação interna: Susana Santos ; coorientação: José Ramalho, Diogo Casal
Palavras-chave
MESTRADO EM ENGENHARIA BIOTECNOLÓGICA, ENGENHARIA BIOTECNOLÓGICA, BIOTECHNOLOGY ENGINEERING, INFEÇÕES, INFECTIONS, CIRURGIAS, SURGERIES, VÍRUS, VIRUS, ANTIMICROBIANOS, ANTIMICROBIAL, SISTEMA ARTERIAL, ARTERIAL SYSTEM