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Title: The molecular correlates of sleep and sleep deprivation in vivo and in vitro
Author: Gee, William
ISNI:       0000 0004 7652 4492
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2018
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This thesis describes the use of in vivo and in vitro models to better understand the molecular correlates of sleep and sleep deprivation. Unlike previous studies, we utilise a timecourse based experimental design throughout, which has the advantage of identifying how the abundance of molecules return to baseline following sleep deprivation. Chapter 3 outlines the transcriptome of mouse cortex collected over 54 hours from mice subjected to varied durations of sleep deprivation. The timecourse experimental design aids in the identification of genes that are induced during both spontaneous and enforced wakefulness, and facilitates the dissociation of genes whose expression is tightly linked to the current wake state of the animal from those whose expression is linked to the total amount of wakefulness recently experienced by the animal. Like previous studies, we identify several genes involved in the unfolded protein response and synaptic function that are upregulated by sleep deprivation. We also find that increasing durations of sleep deprivation progressively reduces the total number of rhythmically expressed genes in mouse cortex, with only a handful of transcripts identified as diurnal following 12 hour sleep deprivation. Chapter 4 outlines the proteomic and metabolomic effects of 12 hour sleep deprivation. Proteomic analyses indicate that the abundance of ribosomal and nucleosomal proteins is suppressed for at least 24 hours following sleep deprivation, whilst the abundance of several phosphodiesterases are acutely increased following sleep deprivation. Metabolomic analyses of sleep deprived mouse cortex identified 3 molecular species whose abundance profile implicate them as sleep homeostats. Finally, we also set out to develop an in vitro model of sleep deprivation based on the optogenetic activation of a neuroblastoma cell line, which is outlined in Chapter 5. Following several rounds of optimisation, the stable expression of an opsin was found to induce intracellular calcium spikes and immediate early gene expression during illumination. Transcriptomic profiling of illuminated SH-SY5Y cells induced large scale transcriptomic changes, and modulated the expression of genes involved in synapses, cholesterol synthesis, the molecular clock and the unfolded protein response. Although these functional classes are reminiscent of those modulated by in vivo sleep deprivation, there was only a slight enrichment of individual genes modulated by in vivo sleep deprivation amongst the blue light sensitive genes, indicating further work is required to more closely model in vivo sleep deprivation.
Supervisor: Yeo, Giles Sponsor: MRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Sleep ; Sleep Deprivation ; Transcriptomic ; Circadian ; Mouse Cortex ; SH-SY5Y ; Optogenetic