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Proteins undergo post-translational modifications, such as ubiquitination and phosphorylation, which can alter their activity, localisation and stability, making a cell responsive to its internal and external environment. Ubiquitin ligases and kinases comprise large enzyme families which catalyse such reactions. The SCF-type E3 ubiquitin ligase sub-family utilise F-box proteins as the substrate targeting component. However, alongside promoting ubiquitination, the F-box protein Fbxo7 can also function as a scaffold and stabilise a subset of proteins, including Cdk6. Cdk6 is activated through binding the D-type cyclins, and historically, a key role has been as a cell cycle regulatory kinase that inactivates G1 checkpoint proteins. More recently, wider roles for Cdk6 have been identified, including as an inhibitor of glycolysis. Notably, Cdk6 has pro-survival activity in T acute lymphoblastic leukaemia (T-ALL) cells due to the phosphorylation and inhibition of glycolytic enzymes, including the rate-limiting gatekeeper, phosphofructokinase (PFKP). Our screens for Fbxo7-interacting partners identified a set of candidates that overlapped as cyclin D/Cdk6 substrates and included PFKP. Further study revealed that Fbxo7 promotes two post-translational modifications on PFKP, ubiquitination and phosphorylation, and specifically promotes Cdk6 activity. Analysis in T-ALL cells suggest that Fbxo7 inhibits the assembly of active PFKP complexes to ultimately inhibit glycolysis. This is confirmed in a murine model of reduced Fbxo7 expression, whose CD4+ T cells show higher levels of glycolytic flux, alongside various other metabolic defects including altered nucleotide biosynthesis and arginine metabolism. This places Fbxo7 as a negative regulator of glycolysis and unveils other diverse roles in metabolism, which may contribute to viability and activation defects observed in these Fbxo7-deficient murine T cells. Given that Fbxo7 negatively regulates glycolysis via PFKP, I also investigated how glucose regulates Fbxo7, as feedback loops in glucose signalling are commonplace in metabolic networks. I discovered Fbxo7 is a dose-dependent, glucose responsive protein in numerous cell types, which is both transcriptionally downregulated and targeted for autophagy in response to glucose starvation. Moreover, data suggest that Fbxo7 is responsive to other stresses, including oxidative stress, placing Fbxo7 as a nexus to link various cellular stress responses to metabolic reprogramming. In addition to PFKP, Fbxo7 has also been shown to recognise a protein called cereblon (CRBN), which is another E3 ubiquitin ligase. CRBN is of clinical relevance because its expression is required for the efficacy of immunomodulatory drugs (IMiDs) in multiple myeloma (MM), which primarily enable the recognition of neo-substrates by CRBN to elicit their anti-cancer effects. We sought to investigate a role for Fbxo7 in MM cells. We show that Fbxo7 promotes CRBN ubiquitination and propose that this targets CRBN for proteasomal degradation, which may have relevance for IMiD sensitivity. Together, these data identify two novel substrates for Fbxo7 ubiquitination and reveal a role for Fbxo7 in lymphocytes. We demonstrate that Fbxo7 expression is responsive to cellular stress and propose that Fbxo7 levels may fine-tune metabolism under different physiological and pathological conditions.

Histones are subject to a wide range of modifications which are differentially regulated throughout the cell cycle to modulate protein recruitment, chromatin structure, and gene expression. During mitosis, several prominent histone phosphorylation events occur. One such event is histone H3 threonine 3 phosphorylation (H3T3ph), which is enriched at centromeres to recruit the chromosomal passenger complex, a key mitotic regulator. In addition, deposition of H3T3ph on chromatin arms during mitosis is proposed to displace transcription factors that recognise the adjacent histone modification, H3 lysine 4 trimethylation (H3K4me3). This mechanism is known as a phospho-methyl switch. However, the exact localisation of H3T3ph is unclear and whether these two modifications actually colocalise in vivo is unknown. Questions also remain regarding the H3T3 kinase, haspin, such as how its kinase activity is regulated to act during mitosis and whether it may have other functions during interphase. Haspin is also known to protect centromeric cohesion during mitosis via an interaction with the cohesin regulator PDS5. However, whether both PDS5A and PDS5B are equally important in this role is unclear. To address some of these questions we defined the localisation of H3T3ph and other histone marks at the sequence level using ChIP-seq. We show that H3T3ph and H3K4me3 do not colocalise, arguing against the proposed phospho-methyl switch. We also use proximity-based labelling (BioID) to assess the environment in which haspin is found both in interphase and mitosis. Our data suggest that it may specifically be PDS5B which regulates haspin and that this also occurs during interphase, implicating haspin in cohesin regulation throughout the cell cycle. We also identify proteins involved in chromatin organisation and nuclear formation. Overall, our results suggest that haspin is enriched in heterochromatic regions at centromeres and plays a role in the regulation of these chromatin domains in both interphase and mitosis.

Introduction: There is an appreciable undiagnosed burden of Chronic liver disease within our society with mortality rates increasing over recent decades. The reasons for this include the rise in lifestyle related risk factors (e.g. hazardous alcohol use, type 2 diabetes and obesity), the asymptomatic nature of the disease which ensures patients do not present to a clinician at an early stage, and the ineffectiveness of current diagnostic tests available to primary care physicians. By transforming the diagnostic pathways we may be able to start proactively identifying and risk stratifying patients with liver disease. Implementation of a risk stratification pathway into a primary healthcare setting would allow a large number of patients to be stratified and identify those who would benefit from a referral to specialist services or further follow up in secondary care. The studies within this thesis have aimed to focus on determining the optimal approach to achieve this and whether this may be cost effective. Methods: The first part of the thesis was to understand and document the current evidence on liver disease diagnosed by a non-invasive test in a community setting focusing on those patients at risk of non-alcoholic fatty liver disease and alcoholic liver disease. In addition to the prevalence of disease within the studied populations the aim was to identify which non-invasive tests for liver fibrosis have been used to stratify patients at risk and to evaluate the difference between unselected and targeted populations. A systematic review of the literature was performed to address these aims. The second part of the thesis was to improve our understanding of which risk factors were associated with significant liver disease. A prospective diagnostic study using transient elastography was performed to investigate this. The focus was on a raised body mass index but the interaction with alcohol and type 2 diabetes was also investigated along with the performance of the portable transient elastography device within an overweight subgroup of the study cohort. The third part of the thesis was to gain an understanding of the cost effectiveness of implementing a new pathway for liver disease in a community setting. A health economic evaluation of this alternative approach compared with current standard care from an NHS England perspective was completed using Markov modelling to estimate long-term health and economic effects. Results: From the systematic review, transient elastography and Fibrotest, were identified to be the most frequently used tests for liver fibrosis within a community setting and had their results compared against histological findings. Subsequently these were the most validated non-invasive tests in a general population setting. The prevalence of advanced liver fibrosis and cirrhosis in the general population was identified to be between 0.9%-2% and 0.1%-1.7% respectively with studies which targeted patients with risk factors for liver disease (e.g. hazardous alcohol use or type 2 diabetes) reporting a higher prevalence of disease (advanced liver fibrosis (0%-27.9%), cirrhosis (2.4%-4%)). In our prospective study, implementation of a risk stratification pathway based on the risk factors of hazardous alcohol use and/or type 2 diabetes and/or a raised body mass index saw 703 patients attend of which 82 (11.7%) had an elevated transient elastography reading consistent with clinically significant liver disease. Seventy seven percent of the cohort had a single risk factor, whilst 21.3% had a combination of two and 1.4% had all three risk factors. An elevated transient elastography reading was approximately as common in patients with just a raised body mass index (≥27.5kg/m2) (8%) as it was in patients with more recognised solitary risk factors (Type 2 diabetes, 10%; Hazardous alcohol use 3.5%). A raised body mass index in combination with other risk factors further increased the proportion of patients with an elevated transient elastography reading and therefore demonstrated that a raised body mass index as a single or combined risk factor for chronic liver disease is important. Of the patients who attended the pathway 477 had a body mass index ≥ 28kg/m2 and had a transient elastography reading with both the M and XL probes. Twenty one percent of these patients had no valid measurements with the M probe. The XL probe significantly increased the number of valid (M vs XL probe: 66.2% vs 90.2%, p < 0.001) and reliable (M vs XL probe: 77.4% vs 98.5%, p=0.028) readings that were obtained and re-stratified 5.2% of patients to have a normal transient elastography reading. The XL probe is therefore not an optional extra but a necessity if transient elastography is to be utilised within a community setting where a raised body mass index is becoming common. Lastly, results from an economic evaluation suggested that the risk stratification pathway was more cost effective than standard care with a cost of £2,138 per extra quality-adjusted life-year (QALY) for patients diagnosed with non-alcoholic fatty liver disease and £6,537 per QALY for patients diagnosed with alcoholic liver disease. The models of the risk stratification pathway demonstrated ≥85% probability of cost-effectiveness at the UK willingness-to-pay threshold of £20,000/QALY. Conclusion: The feasibility of implementing a community based risk stratification pathway for chronic liver disease is clearly shown in the systematic review. Furthermore, the burden of undiagnosed liver disease revealed in the systematic review and the prospective study challenges our current clinical pathways for liver disease. Obesity is a risk factor, in isolation and in combination with alcohol and type 2 diabetes and therefore needs to be considered in any targeted case finding strategy that is proposed. The economic evidence demonstrates that a risk factor approach is likely to be cost effective which is highly relevant in the current financial climate. This research has direct implications on the implementation of care pathways for liver disease but further research is required to refine the strategy and enable adoption into clinical practice.