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Although treatment strategies for advanced and metastatic clear cell renal cell carcinoma (ccRCC) have markedly evolved with the recent use of immune-checkpoint inhibitor (ICI)-based combinations, most patients eventually develop resistance to these therapies. Therefore, there is still an urgent need for the development of effective treatment options. NUC-7738, a novel ProTide transformation of the nucleoside analogue 3ﹶ-deoxyadenosine, releases 3ﹶ-deoxyadenosine monophosphate (3'-dAMP) in cells which is then phosphorylated to the di- (3'-dADP) and tri-phosphate forms (3'-dATP). 3'-dAMP might have the ability to activate AMP-activated protein kinase (AMPK), a key cellular energy sensor, and thus disrupt metabolic homeostasis in cancer cells. Furthermore, 3'-dATP might interfere with RNA synthesis affecting protein expression and survival of cancer cells. The ability of NUC-7738 to activate AMPK through phosphorylation of Th172 was tested in ccRCC cell lines and ex vivo tissue slices of ccRCC from patients. The effect of NUC-7738 on mRNA synthesis and polyadenylation was investigated in two ccRCC cell lines, 786-O and 769-P. AMPK activation by NUC-7738 showed inter-replicate variability and inter-patient variability in ccRCC cell lines and ex vivo tissue slices, respectively, indicating the complexity of the regulation of AMPK phosphorylation. Moreover, mass spectrometry analysis showed that 3'-dATP is the main active metabolite of NUC-7738. Transcriptome data analysis showed mitochondrial gene transcripts of electron transport chain (ETC) complexes were the most significantly altered in both 786-O and 769-P cell lines, with lower expression levels in response to NUC-7738 treatment. This was accompanied by downregulation of the protein expression of ETC complexes subunits. NUC-7738 induced the intrinsic pathway of apoptosis in these cells through the release of cytochrome c from mitochondria and the subsequent activation of caspases -9 and -7. These data suggest that NUC-7738 might inhibit tumour cells growth and proliferation through the inhibition of mitochondrial respiration and the subsequent induction of apoptosis.

Clear Cell Renal Cell Carcinoma (ccRCC) is the most common form of kidney cancer and its incidence is constantly increasing. Although immunotherapy has shown promising results, the only current curative method is ablative surgery, and recurrence is observed in about one third of cases. Moreover, patient prognosis drops significantly with metastatic disease. Current prognostic tools such as Leibovich Score (LS) aim to predict the risk of recurrence by stratifying patients into high, low, and intermediate risk groups. However, this algorithm only takes into account morphological features of the tumour, such as tumour stage and nuclear grade, while it does not consider the vast molecular milieu present in the tumour microenvironment (TME). Furthermore, morphological features are manually assessed by pathologists and are therefore subject to inter- and intra- observer variability. ccRCC TME is complex and heterogeneous, consisting of cell subtypes and molecular mechanisms which may either favour or prevent disease progression and metastatic spread. In this thesis, the author focused on four main molecular mechanisms: immune evasion, T cell exhaustion, epithelial-to-mesenchymal transition (EMT) and cancer stem-like cells (CSCs). Multiplex immunofluorescence (mIF) and GeoMx digital spatial profiling (DSP) by NanoString technology have been used on 150 ccRCC tissue microarrays (TMA) and whole slides (WS) in order to investigate the prognostic role of more than 160 markers. mRNA was also extracted from primary and metastatic tissues from a subset of the cohort to evaluate the expression of 750 genes using the NanoString nCounter technology. Machine learning-based image analysis software were used to detect and quantify these markers, their co-expression at single cell level, and their spatial relationship. Moreover, some markers were chosen to better stratify patients at intermediate LS risk, and increase LS accuracy. To conclude, a semi-automated method was developed in order to investigate the ccRCC TME, reduce observer bias, and increase patients' stratification, facilitating personalised therapy.

Muscle-invasive bladder cancer (MIBC) prognosis is mainly assessed by clinical cancer stage which is codified using the Tumour-Node-Metastasis (TNM) staging system. However, recent studies have demonstrated that disease progression and thus prognosis is profoundly influenced by the immune context of the tumour microenvironment. Multiplex immunofluorescence was applied on MIBC tissue sections to capture whole slide images and quantify potential prognostic markers related to lymphocytes, macrophages, PD-L1 and tumour buds. Two independent machine learning-based methodologies were implemented and the resulting prognostic models reported that: (i) tumour budding was the most significant feature (HR=2.59, P=0.0091) for the stratification of non-metastatic patients into high or low risk of disease specific death, and (ii) the combination of image, clinical, and spatial features stratified MIBC patients into two risk groups with high statistical significance (P<1E⁻⁰⁵) and greater accuracy than the current clinical gold standard, the TNM staging system . To provide further insights into the tumour-immune microenvironment, spatially resolved differential expression of immunologically relevant proteins was quantified across entire MIBC tissues using a 31-plex spatial profiling platform. Significant alterations in the expression of proteins were identified within different compartments of the tissue related to tumour core, tumour buds, stroma and tumour infiltrating lymphocytes showing that this technology has the capability to capture immunological signatures if applied in a larger heterogeneous sample population. Lastly, to delve into the molecular causes of immune evasion by cancer cells, extracellular vesicles (EVs) were isolated by differential ultra- centrifugation from conditioned media of PD-L1 and PD-L1 knockout human bladder carcinoma cells. Co-culture assays demonstrated that EVs derived from PD-L1 bladder carcinoma cells can impair immune functions by reducing CD8 T-cell proliferation. In addition, 210 EV proteins were identified by proteomic analysis, including two newly identified proteins which are not present in known exosome databases.

Tight control of post-translational modifications such as ubiquitination is important for cell homeostasis and dysregulation of this process is a common feature in many diseases. Itch encodes an E3 ubiquitin ligase that conjugates ubiquitin to a variety of substrates such as Jun B, PKC-θ, PLC- γ1, LATS, TXNIP and p73. In this study both transient and stable knockdown techniques (siRNA and CRISPR respectively) were utilised to explore the role of Itch in both cancer progression and bone remodelling. Transient siRNA-mediated knockdown of Itch inhibited cell viability of MiaPaCa-2 and Capan-2 cell lines by ~50% and ~60% respectively compared to both scrambled and untreated controls as determined by SRB. Although siRNA did not increase cell line sensitivity to γ-radiation, doxorubicin, and gemcitabine, it did have an additive effect on resultant survivability. CRISPR-Cas9 mediated stable Itch knockdowns in the same cell lines in contrast showed increased sensitivity to the aforementioned treatment regimens however resultant data obtained from cell survival and clonogenicity assays suggested that the CRISPR⁺ MiaPaCa-2 cells divide faster than both scrambled and parental controls. SWATH-DIA analysis was performed to look for differences in proteome between CRISPR⁺ and parental MiaPaCa-2 cells. Preliminary data based on isogenic clones suggests potential differences, but additional repeats and further validation is required. Transient Itch knockdown in peripheral blood monocytes increased their capacity to differentiate into fully matured osteoclasts ex-vivo: compared to untreated controls, si-Itch treated wells showed higher mean numbers of large TRAP⁺ cells (mean = 63 and 54 for si-Itch and untreated wells respectively) and multinucleate cells (mean = 25 and 17 for si-Itch and untreated wells respectively) in a 96-well format. Meanwhile CRISPR-mediated knockdown induced a large degree of cellular toxicity in PBMCs. This contrast between stable and transient Itch knockdown prompted discussion of the strengths and weaknesses of each technique and their suitability in different experimental contexts.

Background: In the UK, kidney cancer is the most lethal urologic cancer whose major subtype is renal cell carcinoma (RCC). Surgical resection is the first line of the treatment for renal cell carcinoma (RCC). A number of different integrated staging systems such as UISS, SSIGN and Leibovich score, has been introduced and utilised in clinic as a prognostic tool or as an inclusion criterion of clinical trials. Among them, Leibovich score has been widely utilised in the UK to predict the likelihood of disease free survival for clear cell renal cell carcinoma (ccRCC). However, its prediction rate of disease relapse free for 5 years after surgery varies from 97% (low risk) to 31% (high risk) which might lead to inclusion of some ccRCC patients, who would not recur, into a clinical trial. Therefore, we aim to improve the prediction power of recurrence free (specificity) in localised clear cell renal cell carcinoma (ccRCC) patients, by either modifying Leibovich score with more precise and accurate measurement of ccRCC nuclear morphological features or by improving currently available Leibovich score with the features measured from the chromatin marker colocalisation status, chromatin marker Haralick texture features, and the tumour microenvironment using computational image analysis. Methods: To modify Leibovich score by replacing manual Fuhrman's nuclear grade with the computational image analysis measurement of the nuclear morphological features, digitised images from haematoxylin and eosin stained slides were utilised. For the chromatin marker (H3K9me3, H3K4me3 and HP1α) colocalisation analysis, Haralick texture analysis and tumour microenvironment marker (CD105 and CD3) analysis, the multiplexed immunofluorescence (IF) was performed and IF images were utilised. The image analysis was performed by using Definiens Tissue studio® (Definiens AG, Munich, Germany) and the Developer platform. Moreover, a novel statistical model was developed using AUCp (partial area under the curve) function in R studio, which defines the range of specificity between 1 and 0.8 on the basis of the binomial GLM (Generalised linear model) framework with GAM-SALSA (Generalised Additive Models - Spatially adaptive local smoothing algorithm) as a calibration tool. In order to avoid the overfitting problem, 5-fold cross validation with 100 times repetition was also added in the analysis. Results: Firstly, our statistical model replaced Fuhrman's nuclear grade with 'mean perimeter', which was named 'Modified Leibovich algorithm'. The modified Leibovich algorithm improved its overall specificity 0.86 (80 out of 93 cases) from 0.76 (71 out of 93) from the classic L score in the Scottish training cohort. In particular, the most increase in specificity was seen in Leibovich score 5 and 6, which were 57% and 40%, respectively. The modified Leibovich algorithm also increased overall specificity up to 0.94 (141 out of 150 cases), compared to the classic (original) Leibovich score whose specificity was 0.84 (126 out of 150 cases) in a Singaporean validation cohort. Moreover, specificity was dramatically increased in Leibovich score 5 from 0% to 92%. Secondly, the chromatin marker colocalisation feature significantly improved the specificity of the classic and partial Leibovich score up to 0.98 in overall. In particular, the specificity of the cases in Leibovich score 4, 5 and 6 was improved up to 100%. However, the chromatin marker Haralick texture features did not improve the specificity of the classic and partial Leibovich score as much as the chromatin marker colocalisation features. Thirdly, the tumour microenvironment features such as the density and the spatial distances of CD105 positive blood vessels and CD3 positive mature T lymphocytes augmented the specificity of the classic and partial Leibovich score up to 0.93. In particular, it improved the specificity up to 92% in Leibovich score 5 compared to the classic Leibovich score. Conclusions: Computational image analysis enabled to measure such various ranges of features not only in tumour cells, but also in tumour microenvironment. In this study, ccRCC tumour cell nuclear morphological features, chromatin marker colocalisation feature, chromatin Haralick texture features and tumour microenvironment features were visualised by the multiplexed immunofluorescence and measured by the Tissue studio and developer software: this cannot be done manually. The data from this analysis significantly augmented the specificity of the currently available prognostic tool, Leibovich score, in clinic. In particular, the very last final model developed by combining features of the chromatin marker colocalisation ('Average.HP1a.Intensity..H3K4_HP1a.Overlap.' and 'Manders.Coefficient.Nuclei..H3K4.HP1a..M2') and tumour microenvironment ('Mean.CD3.Area.percentage.of.All.Tissue' and 'Mean.No..of.CD105') selected along with the classic Leibovich score, predicted 100% (93 out of 93 cases) correctly the cases which did not experience the disease recurrence within 5 years after surgery, while the classic Leibovich score predicted 76% (71 out of 93 cases) correctly. This could have prevented 22 ccRCC patients not only to get unpleasant and unnecessary treatment after nephrectomy, but also helped them having been free from the fear of disease recurrence.

Background: Between 2009 and the end of UK combat operations in Helmand, Afghanistan (2014), each consecutive surgical procedure carried out by the multinational, military surgical team at the Medical Treatment Facility, Camp Bastion was collated. Through analysis I aim to develop the template for prioritising surgical procedures to improve surgical training and readiness for future armed conflicts and terror attacks. Methods: All surgical teams operating in Camp Bastion filled out detailed, handwritten theatre logbooks for each surgical case. I transcribed all 10,891 consecutive surgical cases, and 20,266 surgical procedures, into an electronic format. I provide for the first time the distinct, original and stand-alone surgical database for this thesis: the "Maitland Module", the largest of its kind. Results: I present a new analysis and classification of surgery by anatomical region which accounts for the impact of the multiplicity of wounding caused by explosion on surgical workload. I present the first evidenced-based skill set requirement for war, including the most frequently performed humanitarian surgical procedures and their impact on the skill set required of a surgeon in armed conflict. I show that surgical experience predicted shorter operation times for blast trauma. I found that surgical experience of >50 blast trauma cases predicted lower fatality rates. Skill atrophy in surgical competence between conflicts exists, with an associated rise in preventable loss of life at the start of conflict. Discussion: I propose that the Complex Attack Surgical Team (CAST) of surgeons, with >50 blast trauma case experience, be on standby in the event of a terrorist attack in the UK to support the currently blast-naive civilian surgeons. I hope that the template for surgical procedures may lead to standardisation and prioritisation of the skill set requirement for: trauma surgical training; NATO curriculum development; and intelligent, risk-assessed, deployment of surgeons to conflict zones.

BACKGROUND - Bipolar disorder has been postulated as an explanation for King John's inconsistencies of leadership and vagaries of character. Changes in activity, matching those in mood, are core features of the condition. METHOD - A measure of King John's activity was derived from his travelling itinerary. Change Point Analysis (CPA) was used to detect significant changes in that travelling activity and from them, to identify clinically compliant, high and low, activity time periods. The results were tested against an alternative mathematical model (Bollinger Bands™), three alternative parameters and two comparator itineraries (familial & non-familial). Using primary historical sources and published analyses, bipolar symptoms were identified and their temporal relationship to the ICD-10 compliant CPA periods evaluated. The influence of circumstances was also evaluated using primary sources and a representative sequential sample (1200-1204). RESULTS - CPA identified 83 periods of changed travelling activity. These changes were mathematically independent of the availability of the historical sources that underpin the itinerary. From these, 37 high and 22 low periods complied with current diagnostic guidelines and demonstrated descriptive and statistical similarities to those found in the bipolar literature. Analyses using alternative mathematical modelling and different parameters showed similar changes; analyses of comparator itineraries showed a possible familial trait. Of the 17 bipolar symptoms identified, all were found in CPA periods of appropriate polarity. Of the 23 sequential periods, 10 showed evidence of behaviour that was difficult to attribute to circumstances. CONCLUSIONS & OUTCOMES - The pattern of changes in King John's activity are highly suggestive of bipolar disorder with primary historical sources describing synchronous bipolar behaviour. This may alter our understanding both of King John and of Magna Carta. Change Point Analysis merits greater consideration when analysing time based data, as does the use of activity as an objective marker of human behaviour.