Abstract
Extracellular matrix (ECM) remodelling is heavily involved in the regulation of many biological events including tissue regeneration, wound healing and bone regeneration. Degeneration of the ECM is implicated in a wide variety of severe pathological conditions including, but not limited to tumor malignancy, auto-immune disorders, and cardiovascular diseases. Several key components of the ECM including collagen and gelatin are susceptible to degradation by matrix metalloproteinases (MMPs) from multiple cell origins. The precise roles of MMPs within normal tissue development and pathologies are still poorly understood. The primary goal of this thesis was to develop a tool for in situ imaging of cell- derived MMP extracellular activity, which will enable a better understanding of the dynamic interactions between cell-driven MMP activity and ECM remodelling. Most sensors described in the literature employ fluorescence intensity as the indicator to report MMP enzymatic activity. However, intensity-based measurement depends not only on the sensor state (cleaved vs non-cleaved) but also on the sensor concentration, which can significantly affect the measurement accuracy and resolution, besides requiring troublesome background normalization procedures. In this thesis, an MMP sensor based on Förster resonance energy transfer (FRET) was developed which responded to the extracellular MMP-mediated cleavage with alterations in the FRET donor fluorescence lifetime. Fluorescence lifetime, being independent on the sensor concentration, has the potential to provide a more reliable measurement of the MMP activity than concentration-dependentapproaches.
| Date of Award | 1 Dec 2022 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Eileen Gentleman (Supervisor), Ricardo M. P. da Silva (Supervisor) & Manuel Muller (Supervisor) |