Molecular probes for elucidating biological processes

King, Lewis James (2018) Molecular probes for elucidating biological processes. PhD thesis, University of Glasgow.

Due to Embargo and/or Third Party Copyright restrictions, this thesis is not available in this service.
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The thesis is comprised of two independent strands of work, one investigating splicing the other targeting gene expression.

Splicing is a process that removes introns from a pre-mRNA to form the mature mRNA strand that is then translated into a protein. It is facilitated by the spliceosome. Alternative splicing is vital to eukaryotes and allows for gene diversity by allowing multiple proteins to be translated from a single gene but its misregulation is the cause of many human diseases making the spliceosome an important therapeutic target. A natural product, hinokiflavone, modulates splicing and leads to splice variants in in vitro and in cellulo assays. It interacts with the SENP1 enzyme and prevents deSUMOylation of key proteins in the spliceosome. Presented is a total synthesis of hinokiflavone and a functionally equivalent fluorinated analogue that is easier to synthesise. Using similar methodology eleven further analogues were also synthesised to investigate more efficient syntheses of splice modulators, to contribute to a structure activity relationship for phenotypic effects and binding SENP1, and to tag or irreversibly modify protein targets. The SAR is important for determining the structural features of hinokiflavone necessary for SENP1 binding, which could be used to develop simpler, more drug-like compounds should they be investigated as therapeutics.

Controlling gene expression is important to determining how a local effect can affect functions in a whole organism. Photoactivatable activation of gene expression is useful because it allows spatiotemporal control. Six analogues of photoactivatable caged cyclofen-OH have been synthesised for the Cre/estrogen receptor approach to controlling gene expression. The intention of these are to increase control of localisation to ensure gene activation in a specific area, improve cellular uptake of the caged precursor to allow in cellulo and in vivo experiments to be carried out at lower concentrations and to allow two photon activation, allowing light of lower energy to be used thus decreasing the damage to cells. Preliminary benchtop uncaging results indicate partial to full uncaging of all six compounds and initial in vitro results indicate full uncaging of the one compound tested in five seconds using 405 nm light.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Splicing, splice modulators, hinokiflavone, gene expression, spatiotemporal control of gene expression, light activated.
Subjects: Q Science > Q Science (General)
Q Science > QD Chemistry
Colleges/Schools: College of Science and Engineering > School of Chemistry
Funder's Name: Engineering and Physical Sciences Research Council (EPSRC)
Supervisor's Name: Hartley, Professor Richard C.
Date of Award: 2018
Embargo Date: 29 March 2022
Depositing User: Mr Lewis King
Unique ID: glathesis:2018-41115
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 29 Mar 2019 10:46
Last Modified: 30 Apr 2019 07:36

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