Development of versatile imaging tools for mechanical and chemical interrogation of biological systems
File(s)
Author(s)
Kalyviotis, Konstantinos
Type
Thesis
Abstract
Living multicellular organisms exhibit both organisational structures and dynamic behaviours. Common to all is the necessity to preserve information for robust organism development and the need to sense and adapt to environmental changes. Hence, to understand holistically a biological system, interrogation of both its biochemical and biomechanical components is needed. Here, I detail my work on developing advanced optical precision imaging tools that enable the study of the biochemical and mechanical nature of biological systems.
First, I present my work on Piezo1, an ion channel directly gated by mechanical forces essential for cellular mechanosensation across biological kingdoms. I describe my efforts in testing, further developing, and validating both in vitro and in vivo the first genetically-encoded fluorescent biosensor for Piezo1-dependent activity, named GenEPi. Then, I describe my efforts on primed conversion, a novel method for axially confined and non-toxic photoconversion of green-to-red photoconvertible fluorescent proteins. I detail my work on improving primed conversion efficiency using a semi-rational protein engineering approach combined with X-ray crystallography and describe the evaluation of primed conversion implementation in a single-objective light-sheet microscope. Finally, I present my work on bioharmonophores, a new class of polymer-encapsulated biodegradable Second Harmonic Generation nanoprobes, which overcome inherent limitations of fluorescent probes and can be used for specific cell targeting without expected adverse effects.
Altogether, this advanced imaging toolkit, which includes a biosensor (GenEPi), improved fluorescent proteins (primed conversion), a novel microscope (primed conversion) and SHG nanoprobes (bioharmonophores) is poised to change the way we capture the intricate interplay between biochemical and mechanical signals that influence the fundamental processes of life, providing a holistic view of systems biology.
First, I present my work on Piezo1, an ion channel directly gated by mechanical forces essential for cellular mechanosensation across biological kingdoms. I describe my efforts in testing, further developing, and validating both in vitro and in vivo the first genetically-encoded fluorescent biosensor for Piezo1-dependent activity, named GenEPi. Then, I describe my efforts on primed conversion, a novel method for axially confined and non-toxic photoconversion of green-to-red photoconvertible fluorescent proteins. I detail my work on improving primed conversion efficiency using a semi-rational protein engineering approach combined with X-ray crystallography and describe the evaluation of primed conversion implementation in a single-objective light-sheet microscope. Finally, I present my work on bioharmonophores, a new class of polymer-encapsulated biodegradable Second Harmonic Generation nanoprobes, which overcome inherent limitations of fluorescent probes and can be used for specific cell targeting without expected adverse effects.
Altogether, this advanced imaging toolkit, which includes a biosensor (GenEPi), improved fluorescent proteins (primed conversion), a novel microscope (primed conversion) and SHG nanoprobes (bioharmonophores) is poised to change the way we capture the intricate interplay between biochemical and mechanical signals that influence the fundamental processes of life, providing a holistic view of systems biology.
Version
Open Access
Date Issued
2023-01-12
Date Awarded
2024-02-01
Copyright Statement
Creative Commons Attribution ShareAlike Licence
Advisor
Pantazis, Periklis
Publisher Department
Bioengineering
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)