The Notch pathway is evolutionary conserved and involved in several key cellular functions that ensure tissue homeostasis in the adult organism. Such an important pathway requires a fine regulation which was found to be orchestrated by different regulatory mechanisms. Our group and others showed that the endocytic trafficking of Notch contributes to its regulation by sorting Notch into activation or degradation routes. Genetic alterations in Notch have been found in a number of diseases, such as different types of cancer. However, very often it is not known how mutations affect the Notch pathway thus making it difficult to specifically target Notch mutants. It is possible that a number of Notch mutants might alter specific regulatory mechanisms and it would be easier to specifically manipulate such mutants by targeting the regulatory steps they rely on. The study presented in this thesis aimed to functionally analyse mutations in the Abruptex (Ax) domain of Notch which spans EGF-like repeat 24-29 of the extracellular domain. The function of Ax domain and how Ax mutations affect Notch pathway have never been elucidated. Using Drosophila melanogaster as an in vitro and in vivo model of study, we showed a potential mechanism by which Drosophila Ax mutants alter the endocytic trafficking of Notch, ultimately affecting Notch signalling. We also showed that Ax mutants found in human cancers share Ax-like features, when reproduced in Drosophila Notch, and they can be functionally classified depending on their position in the Ax domain. Finally, we proposed that the Ax domain might mediate the binding of Notch with other proteins and we tested the effect of Ax mutants on potential binding partners. These findings provide new insights into the mechanism of Ax mutants, the function of the Ax domain and the relevance of Ax mutants in cancer.