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Clonagem e expressão de recetores de citotoxidade expresso por células natural killer
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Abstract(s)
As células Natural Killer (CNK) expressam uma série de recetores codificados pela linha
germinativa capazes de desencadear citotoxicidade. As células NK tendem a expressar
muitos membros de famílias específicas de moléculas sinalizadoras. A presença de muitos
recetores ativadores e de muitos membros de famílias específicas de moléculas sinalizadoras
pode permitir que as células NK detetem diferentes tipos de células-alvo e iniciem diferentes
tipos de resposta. Desta forma, este sistema contribui para a robustez da resposta das células
NK. Na ausência de moléculas sinalizadoras selecionadas, a função citotóxica das células NK
permanece frequentemente inalterada.
Esta dissertação teve como como principal objetivo a sub-clonagem dos recetores de
citotoxicidade natural NCR1, NCR2 e NCR4 expressos por células Natural Killer.
Seguidamente, e através de transformação de células competentes pretendia-se a obtenção
de estirpes bacterianas estáveis capazes de expressar as proteínas recombinantes,
identificáveis através de amplificação por PCR dos genes de interesse, e tendo como objetivo
final a purificação e caracterização das proteínas finais expressas.
Sendo este um estudo exploratório, o planeamento de atividades e tarefas foram moldadas
tendo em conta a obtenção de resultados e por esse motivo,uma vez que os resultados não
foram conclusivos, e apesar de tentadas abordagens distintas, não foi possível realizar os
passos de purificação e caracterização das proteínas recombinantes.
Natural Killer (CNK) cells express a series of germline-encoded receptors capable of triggering cytotoxicity. NK cells tend to express many members of specific families of signaling molecules. The presence of many activating receptors and many members of specific families of signaling molecules may allow NK cells to detect different types of target cells and initiate different types of responses. In this way, this system contributes to the robustness of the NK cell response. In the absence of selected signaling molecules, the cytotoxic function of NK cells often remains unchanged. This dissertation had as its main objective the sub-cloning of the natural cytotoxicity receptors NCR1, NCR2, and NCR4 expressed by Natural Killer cells. Subsequently, and through the transformation of competent cells, the aim was to obtain stable bacterial strains capable of expressing recombinant proteins, identifiable through PCR amplification of the genes of interest, and with the ultimate objective of purifying and characterizing the final expressed proteins. As this is an exploratory study, the planning of activities and tasks was shaped taking into account the achievement of results for this reason, since the results were not conclusive, and despite trying different approaches, it was not possible to carry out the purification steps. and characterization of recombinant proteins.
Natural Killer (CNK) cells express a series of germline-encoded receptors capable of triggering cytotoxicity. NK cells tend to express many members of specific families of signaling molecules. The presence of many activating receptors and many members of specific families of signaling molecules may allow NK cells to detect different types of target cells and initiate different types of responses. In this way, this system contributes to the robustness of the NK cell response. In the absence of selected signaling molecules, the cytotoxic function of NK cells often remains unchanged. This dissertation had as its main objective the sub-cloning of the natural cytotoxicity receptors NCR1, NCR2, and NCR4 expressed by Natural Killer cells. Subsequently, and through the transformation of competent cells, the aim was to obtain stable bacterial strains capable of expressing recombinant proteins, identifiable through PCR amplification of the genes of interest, and with the ultimate objective of purifying and characterizing the final expressed proteins. As this is an exploratory study, the planning of activities and tasks was shaped taking into account the achievement of results for this reason, since the results were not conclusive, and despite trying different approaches, it was not possible to carry out the purification steps. and characterization of recombinant proteins.
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Keywords
Clonagem Sub-clonagem PCR Recetor de citotoxicidade natural Células Natural Killer Cloning Sub-cloning Natural cytotoxicity receptor Natural Killer cells