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Investigating the roles of the ubiquitin ligases Cullin2 and TRAIP during DNA replication termination
Get PDFDNA replication is a well-orchestrated process occurring throughout the S-phase of the cell cycle and unperturbed replication is the basis for genome maintenance. To understand how genome integrity is maintained, it is critical to study the underlying mechanisms and regulation of DNA replication. The defects in the first two stages of DNA replication- initiation and elongation are known to contribute to human diseases and are already targeted by cancer therapies. Recent investigations suggest that disruption of the final stage of replication-termination can also lead to genomic instability. As replication reaches completion and replication forks converge, replisomes are unloaded from chromatin. This unloading mechanism is highly regulated to prevent the unregulated unloading of active replisomes.
Using Xenopus laevis egg extracts, the Gambus lab had previously provided the first insights into replisome disassembly mediated by Cullin2 . This PhD project aimed to further understand the mechanism by which Cullin2 identifies its substrate through structural analysis. While the purification of Lrr1 proved technically challenging, we employed in silico methods to gain high confidence structural predictions for Lrr1.
Furthermore, we have also identified the existence of a backup pathway for replisome disassembly in mitosis. This mitotic pathway uses a different E3 ubiquitin ligase - TRAIP. Our findings demonstrate that catalytically active TRAIP plays an essential role in post-termination and stalled replisome disassembly in mitosis. Following this, we also explored TRAIP’s involvement in replisome disassembly during S-phase. Intriguingly, the depletion of TRAIP did not affect replisome disassembly in S-phase within Xenopus laevis egg extracts. Moreover, this PhD project also indicates that the TRAIP activity in both S-phase and mitosis is subject to regulation via post-translational modifications,
specifically phosphorylation.
Therefore, this thesis expands our understanding of the mechanisms upholding genome stability during termination of DNA replication. It uncovered dual regulation of replisome removal in mitosis versus S-phase through activation of distinct ubiquitin-dependent pathways
Development of an in-situ oil conversion process to generate clean hydrogen
Get PDFThere are huge reserves of heavy oil throughout the world that can be energy intensive to recover. Improving the energy efficiency of the recovery process and developing novel methods of cleaner recovery will be essential for the transition from traditional fossil fuel usage to net-zero. In-situ combustion (ISC) is a less used technique, with Toe-to-Heel Air Injection (THAI) and CAtalytic PRocessing In-Situ (CAPRI) being specialised novel versions of traditional ISC. They utilise a horizontal producing well and in the case of CAPRI, a catalyst. This project aims to investigate the impact that injected steam has on both the THAI and CAPRI processes for the purpose of in-situ heavy oil upgrading and will help to bridge the gap between the extant laboratory research and the unknown commercial potential. This thesis also presents a novel method for modelling the THAI-CAPRI method using CMG STARS, proposing an in-situ hydrogen production reaction scheme. THAI and CAPRI experimental-scale models were run under three conditions: dry, pre-steam and constant steam. Starting from a reservoir API (a measure of a hydrocarbon liquid’s density compared to water) of 10.5°, THAI reached an average API of ~16 points, observing no increase in API output with the use of steam injection. A decreased API output by ~0.7 points during constant steam injection was achieved due to a high temperature oxidation (HTO) dominant environment. This decreases reactant availability for thermal cracking. CAPRI dry run reached an API of 20.40 points and achieved increased API output for both pre-steaming (~21.17 points) and constant steaming (~22.13 points). The mechanics for this increased upgrading were discussed, and catalytic upgrading, as opposed to thermal cracking was shown to be the reason for the increased upgrading. Both processes produce similar cumulative oil (~3150 cm3) during dry and pre-steamed runs, only increasing to ~3300 cm3 with the constant steam injection during THAI and 3500 cm3 for CAPRI.
This thesis also investigates the possibility and extent of hydrogen generation during Toe-to-Heel Air Injection (THAI) applied to heavy oil via reservoir simulation. This was achieved by incorporation of hydrogen reactions such as coke gasification, forward and reverse water gas shift into the THAI model for ISC processes in heavy oil reservoirs. This enabled the interaction between components and reactions to be assessed. The reservoir simulation model was history matched using field data from the Kerrobert THAI field pilot; it successfully incorporated hydrogen reactions, showing good agreement. The inclusion of coke gasification reactions resulted in hydrogen production but reduced oil rates due to temperature decrease and increased gas saturation. Forward water-gas shift (FWGS) had negligible impact on oil production, due to reduced competition with cracking and oxidation reactions. Combined coke gasification and FWGS increased hydrogen production and slightly reduced oil rates. Reverse water-gas shift (RWGS) had minimal impact on hydrogen fraction. This thesis emphasises the importance of hydrogen reactions in the THAI model and their influence on the THAI process. It provides valuable insights for optimising THAI projects and underscores the role of reaction scheme choice in history matching
Investigating the role of the UbcH10 proto-oncogene product in high grade serous ovarian cancer
Get PDFHigh grade serous carcinoma (HGSOC) is the 5th leading cause of cancer related deaths in females worldwide. According to the Cancer Genome Atlas (TCGA), HGSOC is marked by early TP53 mutation and has four molecular subtypes; Mesenchymal, Immunoreactive, Proliferative and Differentiated. Despite advances in our understanding of the molecular background of HGSOC, treatment modalities have not improved patient survival. UbcH10/Ube2C is an E2-conjugating enzyme that regulates cell-cycle progression, by promoting the degradation of mitotic regulators through the ubiquitin-proteasome pathway. UbcH10 is also a proto-oncogene whose aberrant expression has been reported in numerous cancers.
Research carried throughout this thesis aimed at investigating UbcH10 as a potential biomarker for the prognostication and molecular stratification of HGSOC. We aimed to study its relative protein expression in HGSOC, relation to TP53 mutations, molecular subtypes of HGSOC and its effect on patient outcome. In addition, we aimed to identify its role in the progression of HGSOC by studying its expression in its precursor lesions; serous tubal intraepithelial carcinomas (STIC) and p53 signatures. Three experimental cohorts were investigated in the present study and the publicly available TCGA cohort of ovarian serous carcinomas was also interrogated.
Immunohistochemistry (IHC) was used to investigate UbcH10 and p53 protein expression in a training cohort which included 100 full-faced HGSOC sections and a validation cohort of 81 cases of tissue microarrays were used. UbcH10 was scored according to the IRS and H-scores, and the X-tile tool was used to generate biologically significant cut-off values. Ki-67 and CD8 IHC were used for molecular subtyping of the training cohort according to TCGA groups. Multiplex IHC was used to investigate the co-expression of UbcH10/Ki-67 as well as UbcH10/p53 in a third cohort of 24 STIC samples and their matched HGSOC.
UbcH10 overexpression was detected in 29-33% and 16-17% of the training and validation cohorts respectively. In the training cohort, UbcH10 overexpression did not significantly impact survival nor correlate with p53 mutations, prognostic, or predictive parameters. Additionally, UbcH10 expression was positively correlated with Ki-67 index and significantly associated with the aggressive and treatment resistant Proliferative molecular subtype of HGSOC.
In the validation cohort, Ubch10 negatively impacted overall survival. The trend was maintained in progression-free survival but was not significant. Results from both cohorts therefore conclude that UbcH10 is significantly related to the proliferative state of the cell and has a borderline poor prognostic significance in HGSOC.
In STIC/HGSOC cohort, like p53, UbcH10 is expressed early in STICs and is positively correlated with Ki-67 index. A heterogeneous rather than a directly proportional relationship exists between p53 and UbcH10 in STICs and their corresponding HGSOC.
Analysis on TCGA cohort confirms results from the experimental cohorts at the gene level. Ube2C is significantly co-expressed with genes involved in cell-cycle regulation and has no impact on HGSOC mortality or progression. Ube2C gene expression levels do not correlate with TP53 mutations and are significantly associated with the Proliferative molecular subtype of HGSOC. So, whilst UbcH10 is only a borderline prognostic marker in HGSOC, it is more beneficial as a predictor of treatment response when interpreted in the context of the molecular subgroup of HGSOC
The application of morphokinetic algorithms to predict ploidy status during assisted conception
Get PDFAim: To undertake a series of studies to answer 5 key questions within assisted conception:
1. Are morphokinetic variables and morphological features associated with the ploidy status of pre-implantation human embryos?
2. Are artificial intelligence or machine learning algorithms superior to logistic regression for predicting ploidy status?
3. Are morphokinetic model risk scores associated with live birth and miscarriage?
4. Should clinical factors be incorporated into embryo selection models?
5. Are morphokinetic models better at prioritising a euploid embryo for transfer over morphological selection by a senior embryologist?
Methods: The above questions were addressed throughout four studies, first, a systematic review and meta-analysis investigated the association of ploidy status and abnormal cleavage, morphokinetic variables, fragmentation, multinucleation and embryo contraction. Second, a model development study collected data on the prognostic variables investigated in the systematic review from nine IVF clinics. Here, a sample of 8148 biopsied blastocysts was used to develop and compare 12 machine learning models to predict ploidy status. This was using four different algorithms, logistic regression, random forest classifier, extreme gradient boosting and deep learning. One model for each algorithm was built with euploidy as target outcome, a second with aneuploidy and a third using a smaller dataset which incorporated embryo Gardner’s classification. Third, the best performing model was retrospectively externally tested on a total of 3587 single embryo transfers. This determined association between three different model derived aneuploidy risk scores (low, moderate and high) and live birth and miscarriage. The final study used a separate cohort of 1958 biopsied blastocysts to compare the ability of morphokinetic models to rank euploid embryos first, given that these models will not be asked to classify embryos but only prioritise.
Results: Meta-analysis demonstrated that ten morphokinetic variables were significantly delayed in aneuploid embryos. It is uncertain whether the morphological components investigated have prognostic potential. On comparing 12 different models, logistic regression performed the best (AUC=0.61). Including predictors such as age resulted in no variability in the ranking within a patient’s cohort of embryos. Incorporating morphological Gardner’s classification resulted in no improvement in the discriminatory ability of the model. A ‘morphokinetics only’ approach was therefore investigated by adjusted logistic regression analysis that demonstrated the model was not associated with miscarriage when comparing the ‘high’ to the ‘moderate risk’ (OR 0.87; 95% CI 0.63-1.63; p=0.39) or ‘high’ to ‘low risk’ embryos (OR 1.07; 95% CI 0.79-1.46, p<0.63). However, an embryo deemed ‘low risk’ was significantly more likely to result in a live birth than those embryos graded ‘high risk’ (OR 1.95; 95% CI 1.65-2.25; p<0.001). The final cohort study reported that arbitrary embryo selection would rank a euploid embryo first 37% of the time, embryologist selection 39%, and the ploidy morphokinetic model 47% of the time.
Conclusions: Morphokinetic variables and the risk scores derived from morphokinetic models are significantly associated with ploidy status. Including predictors such as age, results in a clinically ineffective model; a ‘morphokinetics only’ approach is therefore advised. Logistic regression was the best performing algorithm in this dataset for predicting ploidy status, with aneuploidy as the target variable. The application of this model resulted in an improved chance of a euploid embryo being selected for transfer over that by a senior embryologist
An unexceptional people: regional variations in Quaker occupations in eighteenth-century England and Wales c1700-1800
Get PDFPrevious scholars such as Richard Vann & David Eversley and Richard Allen & Rosemary Moore have suggested that Quakers in the seventeenth- and early eighteenth-centuries were ‘middle-class’ or of a ‘middling sort’ in their economic activity. These conclusions are based on studies which were limited either in geographic scope or to specific economic sectors. Other work such as that by Arthur Raistrick, attempts to suggest that Quakers as a group were exceptional in their achievements or position in society – the phrase ‘a peculiar people’ has been used, for example by Joseph John Gurney. This thesis suggests that Quakers were aligned with local economies in terms of occupation, except for a low representation in agriculture, and that claims for exceptional sector influence or control are not supported by the evidence. As such, in economic terms they were not such a ‘peculiar’ and exceptional people.
This study uses the digested Quaker marriage records from 1691 to 1809, located in Friends House Library, London, as the basis for an examination of the occupational and geographic location patterns of the eighteenth-century Quaker population across England and Wales. The results provide a wide perspective on Quakers in different economic sectors and highlight the patterns of geographical change in Quaker activity across the country. Additionally, it addresses the idea of exceptionalism by examining Charles Hyde’s claim of Quaker domination of the iron industry in the early eighteenth century.
The database shows that the recording of occupational data in the marriage records varied in completeness with distance from London. There were much lower recording rates in distant regions, such as Cumberland and Northumberland, which were agricultural and economically poorer than the metropolis. This finding has implications for conventional socio-economic descriptions of Quakers. The biggest groups of economically active Quakers, representing 40% of the database, were artisans and retailers. Comparisons with studies of the general population show that their work was reflective of the local economy, although Quakers were less likely to be involved in agriculture.
Such comparisons also showed that Quakers followed the eighteenth-century trend towards urbanisation and were early movers to towns. Their liking for retail activity was shown by the appearance of more food retailers in later years, which was a shift in their food-related activity away from processing. Because these artisan and retail occupations can be regarded as either working- or middle-class, the size of this combined group means that the received view of Quakers as predominantly middle-class is challenged. This challenge is potentially enhanced by the low level of recording of occupations in remote regions. Such regions were likely to contain poorer populations with a higher proportion of humbler occupations. Thus, the reduction in recording rate is likely to disproportionately affect the recording of these humbler occupations.
The connection to local economies is a challenge to the received view of Quaker exceptionality. Hyde’s claim for Quaker control of the iron industry, a specific example of such exceptionality, is investigated. An analysis of the English iron industry is presented which includes both production and distribution to examine the extent of Quaker involvement. The findings are that Quakers were more likely to be involved with iron distribution than production and that Hyde’s claim is not supported
The investigation of small heat shock protein expression under endoplasmic reticulum stress treated c2c12 myotubes
No full textSkeletal muscle function declines with age and is associated with reduced health span. Dysregulated proteostasis is a key hallmark of ageing, characterised by an imbalance between protein synthesis, breakdown, and folding within the cell. To counteract this, chaperones are expressed to prevent the accumulation of misfolded protein species. The endoplasmic reticulum (ER) is crucial for maintaining protein and ensuring the newly synthesized proteins are folded correctly to achieve their functional form, which can be disrupted under stressful conditions. Research, including our lab preliminary data, revealed that ER stress-related and unfolded protein response (UPR) signaling pathways proteins are altered in aged mouse skeletal muscle. Furthermore, another group of proteins, small heat shock protein (sHSP), which performs holdase function under general protein aggregation, also recorded an increase in the data. We hypothesized that ER stresses stimulate the expression of sHSP to prevent the formation of protein aggregates. Herein, this thesis used two different pharmacological ER stress inducers, Tunicamycin and Thapsigargin, in C2C12 myotubes to investigate the regulation of sHSP under ER stress. Our results show that HSPB1 and HSPB6 decrease throughout ER stress whilst HSPB5 and HSPB7 remain unchanged. The results show that ER stress regulates sHSP expressions throughout two types of ER stress inducers. Altogether, this thesis improves the understanding of sHSP regulation under various types of unfolded protein responses in skeletal muscle
An economic analysis of safe surgery in low- and middle- income countries: a case study of surgical site infection
Get PDFIntroduction
Surgical Site Infection (SSI) is a common postoperative complication and is associated with substantial financial and quality of life consequences. Numerous logistical challenges make accurate quantification of SSI-related costs and outcomes difficult. SSI has a higher incidence rate in low- and middle- income countries (LMICs) relative to high income countries. This along with efforts to increase the volume of surgeries to address unmet surgical need in LMIC settings gives a more pressing focus on postoperative surgical outcomes. The reduction of postoperative complications is a key outcome measure for safe surgery. For decision makers in resource-constrained environments, it is important to know whether investments in safe surgery represent a worthwhile use of health care resources given the opportunity cost of those healthcare resources.
Thesis aim
The thesis uses primary and secondary research sources to answer the research question: What is the economic rationale for investing in safe surgery interventions to reduce SSI in LMICs?
Methods
Separate systematic reviews on the costs and utility decrement of SSI were undertaken to populate a preliminary economic model to assess the cost-effectiveness of SSI prevention interventions using ‘best guess’ estimates. The model was further refined using cost and quality of life estimates from a re-analysis of an SSI Randomised Control Trial (RCT). Next, the cost and quality of life implications of SSI were ascertained from primary research by i) generating health state valuations associated with SSI, and ii) collecting costs associated with SSI in a sub study of a multinational RCT. The preliminary economic model was then updated with the primary research data to assess the economic rationale of safe surgery interventions to reduce SSI in LMICs.
Results
The base case results of the preliminary decision analytic model indicated that both exemplar interventions were cheaper and more effective than Usual Care and the results were robust to sensitivity analyses across the examined settings. Refining the preliminary model-based economic evaluation with the RCT re-analysis did not change the decision recommendation. For the primary data collection, an SSI was associated with additional postoperative costs in patients undergoing abdominal surgery across Ghana, Mexico, Nigeria, and India. Specially constructed vignettes to describe different severity levels of SSI were successfully valued with the general public remotely using different health state valuation techniques.
For the final updated model with one exception, one or more of the SSI prevention interventions were cost-effective versus Usual Care but these varied by wound contamination. For the patient group in Mexico with Clean-Contaminated wounds, the results varied by outcome measure with Alcoholic Chlorhexidine being the most cost-effective strategy under the Cost per DALY averted approach and Usual Care being the most cost-effective strategy under the Cost per QALY approach.
Conclusion
An economic rationale in investing in safe surgery interventions to reduce SSI in LMICs was uncovered. Despite possible transferability issues with the cost, utility and treatment effectiveness data, the decision recommendations of preliminary economic modelling did not change when the model was iteratively refined with more suitable LMIC data
Study on ammonia/hydrogen combustion for disruptive heat engine development
Get PDFAmmonia and hydrogen portrays a favourable potential to act as alternative fuels due to no carbon-related emissions. This thesis aims to investigate the characteristics of ammonia and hydrogen combustion, providing guidance for the development of a disruptive heat engine using ammonia and hydrogen.
Nakamura’s mechanism is found to best predict the fundamentals of ammonia and hydrogen combustion and employed in the numerical study. The premixed laminar flame speed, ignition delay and flame structure of ammonia and hydrogen combustion are explored with various parameters. It shows an equivalence ratio of 1.1 is advantageous for both combustion stability and emission control. Hydrogen enrichment and oxygen enrichment effectively promote the laminar flame speed and reduce the ignition delay time. However, they comes at the cost of higher NO emission. Elevated pressure tends to reduce NO emission.
A lab-scaled swirl-enhanced combustor rig is established to analyse the characteristics of non-premixed ammonia and hydrogen flame. The results indicate increasing hydrogen blending ratios, initial gas temperature and decreasing the swirl numbers are effective ways to broaden the flame stability range. The optimal emission control occurs at stoichiometric condition. More hydrogen involvement leads to the increase of NO and NO2 emissions and the decrease in ammonia slip. However, the effect is weakened under fuel-rich conditions.
The numerical and experimental emission results are compared with those from various ammonia-based heat engines. Most results exceeds EURO 7 regulation standards. The two-zone rich-lean neat ammonia fuelled micro gas turbine shows the best control of emission with thermal efficiency of ∼ 22%
The challenge of retaining teachers: evidence from the perspective of secondary headteachers in England
Get PDFConcerns over teacher retention in secondary schools in England are not new however, this issue has recently escalated within the education policy agenda as a result of rising pupil numbers, ongoing and chronic shortfalls in new teacher recruitment and increases in the proportion of teachers leaving the profession. There is little doubt that the quality of the teaching workforce makes a substantial difference to pupils’ schooling and their educational outcomes. Thus, in order to deliver on the economic and social agendas of successive governments, it is more important than ever that we keep our best teachers. My research aims to improve understanding of why retaining teachers continues to be a significant challenge, from a new perspective. By interviewing Headteachers I seek to capture their assessment of the problem, in terms of their struggles in retaining their teachers within their schools, combined with that of their wider perspective as to the problems within the profession as a whole, particularly as their views are rarely embraced within literature in this context, nor based on participant evidence, policy decision making.
This qualitative case study employs a two-stage approach. Firstly, an online questionnaire with twenty-five Headteachers, to provide an overview of the issues, combined with background and contextual data. Secondly, the main focus of data collection, through semi-structured in-depth interviews with thirteen of these participants. The findings were then thematically coded and analysed. These Headteachers corroborated the long-standing tensions surrounding working conditions and the damaging effects of incessant policy-driven changes instigated by successive governments. Importantly, they provided insight as to other factors, including pay, escalating levels of surveillance, quality of recruits and ITT provision, ‘parent-power’ and a societal changing attitude to career mobility. All these elements have a profound and detrimental effect on retention and are presented in my diagram ‘Retention: untying the knot’. This conceptualises retention in a different way, illustrating the substantial and important connectors within marketization, managerialism and career mobility and inspired by the work of Peter Woods, the interrelated macro, meso and micro levels. How these Headteachers navigate the issues depends largely upon their school’s funding position, with some using their advantages to ‘buy the best and keep the best’; others, less well-positioned are facing considerable challenges in retention. Yet all were of a mind that retention was critical to the success, even the very survival of their school. Overall, the data exposes the complexities and interrelated factors involved in the retention of teachers, revealing some dominant, escalating and new factors as well as strategies employed by these Headteachers in an attempt to mitigate the problem. My analysis of the data demonstrates the profound dynamics at work, comprising policy, institutional factors and the individual respectively. My findings offers an in-depth examination of how Headteachers negotiate and understand retention within the present-day, constantly evolving education system. Potentially, it has implications for current policy, the practices of Headteachers and for further academic application. This research adds to knowledge about those who leave and why they leave and why this continues to be a perennial problem
Synthesis and crystallisation-driven self-assembly of ɛ-substituted poly(ɛ-caprolactones).
Get PDFThe ability to control the morphology and dimensions of nanostructures is critical to enhance their bioavailability and biological interactions. However, the preparation of non-spherical nanoparticles that can covalently incorporate small molecules in the inner core remains challenging and has not been extensively explored. In this regard, crystallisation-driven self-assembly is a powerful strategy that uses amphiphilic block copolymers containing at least one semi-crystalline block to access a great variety of well-defined 1D and 2D nanostructures. Among these semi-crystalline polymers, poly(ɛ-caprolactone) (PCL) is of high interest owing to its biocompatibility and biodegradability. However, the lack of side group functionality prevents the modification or addition of small molecules, restricting the application of the resultant nanostructures as delivery systems. To overcome this limitation, this thesis focused on the synthesis and self-assembly of substituted PCL-containing amphiphilic block copolymers. The synthesis of semicrystalline substituted PCL was investigated via ring-opening polymerisation following three approaches: i) polymerisation of enantioenriched ɛ-allyl-ɛ-caprolactone, ii) stereoselective polymerisation using a chiral catalyst and iii) copolymerisation of ɛ-caprolactone and ɛ-allyl-ɛ-caprolactone, in which the allyl functional handle enables the attachment of small molecules post-polymerisation. Following this, crystallisation-driven self-assembly of substituted block copolymers with various degrees of functionalisation was explored on different solvent conditions and at different temperatures to give a range of anisotropic structures. Moreover, biological studies demonstrated well-defined 1D and 2D nanoparticles bearing functionalities in the core-forming block exhibited high biocompatibility and considerable internalisation within the cellular cytoplasm, highlighting the great potential of the system for drug delivery applications
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