| Summary: | Hypoxia-inducible Factor (HIF) is an oxygen-sensitive transcription factor present in all multicellular life, including humans. It is responsible for mediating the cellular response to hypoxia, a situation in which oxygen supply falls below adequate levels. In the presence of oxygen HIF is constitutively degraded and inactivated. In the absence or reduction of oxygen, HIF escapes degradation and accumulates. Once accumulated, HIF elicits an adaptive response by inducing a diverse array of genes affecting the processes of cell fate, vascularisation and red blood cell production. However, there are many instances of the pathological activation of this pathway, which is known to cause serious and maladaptive issues, as found in cancer. HIF is functional as a heterodimer comprised of an Os-sensitive alpha subunit (HIF-α), of which there are two isoforms (-lα and -2α), and an Oe-insensitive beta subunit (HIF- β). The Os-dependent destabilisation of HIF-α is carried out by the prolyl hydroxylase domain proteins (PHDs). Importantly, several of the PHDs are themselves HIF target genes, thereby forming a delayed negative feedback loop. Delayed negative feedback loops are a common signalling motif able to confer dynamic properties to the signalling network. The resulting kinetics may have the capacity to encode additional information into the system and alter the course of downstream gene expression. The dissection and understanding of this requires a high-degree of characterisation of the temporal profile of response. Live-cell imaging offers an experimental system ideally suited to this approach. To date, the dynamics of HIF-l accumulation have not been investigated in this detail and so it was a main aim of this study to depict the temporal properties of the HIF signalling system. The dynamic response of HIF -a protein to hypoxia and re-oxygenation was imaged by live-cell time-lapse confocal microscopy. Interestingly, it was observed that the hypoxic accumulation of HIF-α was heterogeneous and transient. The duration of HIF- α accumulation was found to be a distinctive and robust temporal feature of the response. This assorted data was used in building a mathematical model depicting the HIF - PHD feedback motif. Subsequently, the model was utilised to accurately predict the consequence of loss of the PHD feedback. Additionally the model provided predictions on how HIF -α dynamics are differentially responsive to varied hypoxic and re- oxygenation conditions. Live-cell imaging of the HIF-α response also provided insight into the novel subcellular localisation pattern of the HIF-2α protein, which differs distinctly from HIF-la. HIF-2α was found to reside in several types of subcellular bodies. The dynamic interplay and flux of protein in and out of these bodies was investigated. Evidence gathered supports the involvement of HIF -2α in aggresomes and PML bodies and also the involvement of the subcellular landscape in the HIF regulatory network. Overall, research arising from this thesis offers new insights into the intracellular spatial and temporal dynamics of the HIF signalling system. 11
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