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Title: Cytoskeletal-like assemblies within model protocells : en route towards synthetic cellular constructs
Author: Kumar , Ravinash Krishna
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2013
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The aim of this thesis was to rationally design, construct and test novel protocells equipped with primitive cytoskeletons, which could be produced by integration of simple chemical systems. This was achieved by the non-covalent assembly of a functionalised amino acid, N-fluorenylmethyloxycarbonyl-tyrosine (FMOC-Tyr), to yield highly structured nanofilament morphologies within the aqueous interiors of micron-sized, membrane-bounded compartments. Initially. the formation of the non-covalently assembled FMOC-Tyr nanofilaments was studied in isolation. Significantly, at a critical filament concentration and appropriate pH, the formation of supramolecular hydrogels was ascertained. Moreover, by substituting FMOC-Tyr with the hydrophilic analogue N-fluorenylmethyloxycarbonyl- tyrosine-( O)-phosphate (FMOC-Tyr-POH), cleavage of the phosphate group by alkaline phosphatase or cerium oxide nanopal1icles resulted in the formation of gel networks with tuneable (PH or temperature) gel-sol and sol-gel transitions. After the experimental design parameters for hydrogel formation were determined, the FMOC-Tyr nanofilaments were grown within two contrasting model compartments: phospholipid vesicles and inorganic colloidosomes. Significantly, FMOC-Tyr supramolecular hydrogel-containing vesicles showed enhanced resistance to mechanical deformation, osmotic pressure and increased structural persistence. Moreover, primitive cytoskeletal function was further demonstrated via heat-induced structural deformations of vesicles, where morphological transformations were driven by disassembly of the internalised nanofilaments. Growth of FMOC-Tyr cytoskeletal-like filaments within inorganic colloidosomes resulted in silica membrane-directed nanofilament growth from the interior of the inorganic surface towards the centre of the microcapsule. In this instance, it was possible to demonstrate that temperature-dependent filament assembly and disassembly could be used to modulate the interior viscosities of colloidosomes, which in tum regulated intra-protocellular enzyme kinetics. The work presented in this thesis describes the first examples of protocells equipped with functional artificial cytoskeletons comprising self-assembled synthetic building blocks. The incorporation of these bio-inspired scaffolds within protocells not only provides the structural persistence that is central in all living cells, but also a mechanism to re-structure interior environments and control compartment shape.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available