Implementation of mammalian two-hybrid system for highthroughput drugs screening

• Autor(a) principal: Patrício, Daniela Marques
• Data de Publicação: 2017
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/22327
• topic: Interacção celular; Espermatozóides - Mobilidade; Reprodução humana

The base of cell communication is signalling pathways which, when deregulated can lead to several diseases, from cancer to autoimmune diseases. Protein-protein interactions (PPIs) are the backbone of signalling pathways. Consequently, identifying and modulating PPIs can unravel the pathophysiological mechanisms of a disease. When considering the male reproductive system, the same is observed, hence PPIs are responsible and regulate most of the cellular functions. PPP1CC2 is a testis enriched protein, key to sperm motility acquisition. Its interaction with AKAP4, also a testis-specific protein, appears to be a potential new drug target. Therefore, the modulation of PPP1CC2/AKAP4 interaction under mammalian two-hybrid system is crucial. Two-hybrid system, particularly the mammalian two-hybrid (MTH), is an efficient technique to validate PPIs ex vivo. Thus, we will explore this tool for highthroughput drug screening through the modulation of PPIs. Consequently, it will be used to identify either a target to a specific drug or drugs. This methodology relies on the fact that in eukaryotes, gene transcription only occurs when the DNA-binding domain (BD) and DNA activation domain (AD) of a transcription factor come into close proximity. When fusing the cDNA of two interacting proteins with the BD and AD, a reporter gene is expressed and the interaction can be detected. The main goal of this thesis is to implement the MTH into the Signal Transduction laboratory and to be able to identify and modulate PPIs in a system that can mimic the natural way PPI’s occur – mammalian cells.