Investigation of the functions of sumo in conserved biological processes in Saccharomyces cerevisiae

PhD ThesisThe small ubiquitin-like modifier (sumo) is a conserved post-translational modification found throughout eukaryotes. Over the last 25 years a large range of studies have investigated the role of sumoylation, identifying hundreds of substrates and linking sumo to a diverse range of key cellular processes, including stress responses, the response to DNA damage and cell cycle progression. Although sumoylation has also been shown to be essential in a number of eukaryotes, including the model yeast Saccharomyces cerevisiae, the fundamental role(s) of sumoylation remains unclear. Sumo dysregulation is associated with a number of human diseases, including cancer, hence it is important to understand and characterise the role(s) of sumo pathways within these diseases. In an attempt to identify the important functions of sumoylation, a recent SGA screen carried out in our lab used a S. cerevisiae strain with reduced sumo (Smt3) function to identify a number of suppressor proteins which were able to suppress the growth defects of this smt3 mutant. Excitingly, several novel cytoskeletalrelated suppressors proteins were identified which rescued the smt3 growth phenotype, including subunits of the CCT chaperonin complex, b-tubulin and branched F-actin. Hence, the aim of this thesis was to further characterise the phenotypes associated with the smt3 mutant and to investigate the relationship of other proteins in the sumo conjugation/deconjugation pathways, the role of polysumoylation and the effect of different S. cerevisiae strain backgrounds within stress responses including exposure to cold temperature, responses to oxidative stress and cell cycle progression. In addition, another aspect of this study was to investigate the relationship of the smt3 mutant with the novel suppressor proteins including subunits of the CCT complex, b-tubulin and F-actin. Excitingly, this study has revealed novel and strain specific roles for sumoylation and polysumoylation within S. cerevisiae stress responses, cell cycle progression and chromosome dynamics, including a novel, strain specific role for sumoylation during S phase. In addition, data in this study also revealed that enzymes within the sumo conjugation and deconjugation pathways respond differently when presented with different stresses. Interestingly, our studies of the relationships between smt3 and the novel cytoskeletal suppressor proteins iv revealed that although these suppressors partially suppress the smt3 growth defects, the smt3 strain also suppresses several phenotypes associated with the cytoskeletal suppressors. Furthermore, our data suggests that b-tubulin is a substrate of sumoylation in S. cerevisiae cells. Thus, these results are consistent with a model in which sumoylation is functionally linked to the cytoskeleton by the interaction of sumo with microtubules and F-actin. Given that dysregulation of sumo, the CCT complex, F-actin and microtubules are common in many human diseases, this study provides novel insights into the relationship between the mutations in these complexes, potentially identifying new routes for the development of therapeutic treatments for human diseases.BBSR

Investment formation of social capital benefits for value creation and strategy enhancement : the case of SMEs in the UK’s construction industry

Ph. D. ThesisAddressing calls for research into social capital (SC), value creation (VC), and business sustainability (BST), this study answers: What strategies do individual SMEs in the UK’s construction industry apply to generate SC benefits through network engagements? How do SMEs, in the construction industry in the UK, create value from the SC benefits generated through network engagements? and How do SC benefits enhance business sustainability (BST) strategies of SMEs in the UK’s construction industry? This exploratory and interpretive study adopts a social constructivist epistemology to examine, through abductive analysis, the ‘lived experiences’ of small and medium-sized enterprises (SMEs), when their managers and/or directors engage with networks to generate SC benefits, create value, and enhance BST strategies. Forty-one interviews are thematically analysed producing seven findings. First, SMEs deliberately undertake actions to engage with strategic stakeholders. Second, SMEs prioritise stakeholders to generate SC benefits. Third, SMEs prioritise specific actions to generate SC benefits. Fourth, there are categories and representations of SC benefits unaccounted for in the SC literature. Fifth, SMEs execute strategic actions to transform SC benefits into value. Sixth, there are value representations across six categories not accounted for in the SC-VC literature. And seventh, SC benefits trigger VC and strategy enhancement in SMEs. Findings encourage managers to adjust network engagement strategies following actions suggested in this study; nonetheless, contextual limitations demand research beyond the UK and its construction industry to better understand the SC-VC-BST strategy relationship. In summary, contributions of this study are fourfold. First, recognition of premeditated actions, supporting synergistic strategies, that SMEs adopt to generate SC benefits. Second, recognition of additional SC benefits not currently present in the management literature. Third, acknowledgement of value categories and representations transformed from SC benefits. And fourth, clarification of process and nature of relationship between SC benefits, their transformation into value, and enhancement of firms’ BST strategies

Investigation and therapeutic targeting of the metabolic relationship between pancreatic stellate cells and pancreatic ductal adenocarcinoma

Ph. D. ThesisIntroduction Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive solid organ malignancies with persistently poor survival despite advancements in chemotherapy and surgical techniques. Pancreatic stellate cells (PSCs) are key pro-­‐tumourigenic players within the inflammatory microenvironment/stroma of PDAC, and thus represent an attractive therapeutic target. This project aimed to examine the under-­‐investigated relationship of PSCs to cancer metabolism, and identify novel translational treatment approaches. Methodology Human PSC and PDAC (Panc1, Miapaca2 and Bxpc3) cell lines were utilised in numerous in vitro transwell co-­‐culture experiments to investigate the effect on activity and more specifically metabolic phenotype, including the use of qPCR, western blot, metabolic assays, and seahorse technology. The impact of drugs targeting enzymes and transporters specific to lactate metabolism were then examined. In addition immunohistochemistry (IHC) was performed on resected tumours to examine the expression patterns and prognostic relevance of certain metabolic markers within the tumour microenvironment. Results Initial co-­‐culture experimentation revealed PSCs and PDAC impact on lactate metabolism, with a significant pro-­‐glycolytic effect of PSCs on the PDAC cell lines. In keeping with this, a notable upregulation in the monocarboxylate transporters 6 MCT1 and MCT4 was also observed, indicating an impact on lactate flux. PSC interactions with the PDAC lines were influenced by the underlying genetic/metabolic phenotype of each cell line. IHC staining revealed distinct MCT expression patterns, with a positive correlation between the tumoural and stromal compartments, whilst expression of MCT1 and MCT4 within the stroma and tumour respectively correlating with reduced overall survival. The MCT1 inhibitor AZD3965 exerted a KRAS-­‐dependent de-­‐activating effect on PSCs, with a reduction in αSMA and IL6 expression. AZD3965 also exerted a KRAS-­‐ dependent anti-­‐proliferative effect on the PDAC lines, a cell line-­‐specific PSC-­‐ dependent reduction in PDAC metabolic processes, as well as a reduction in PSC lactate transporter expression. Inhibition of all LDH isoforms with Galloflavin revealed a mixed effect on both PDAC proliferation and apoptosis, although an over-­‐riding reduction in lactate acidification. Conclusion This project has proven a metabolic symbiosis exists between PDAC and PSCs, which appears to be inextricably related to lactate metabolism. Inhibition of lactate transporters or enzymes has demonstrated effects on both cancer cells and PSCs which, in combination with the correlation to poor prognosis in resected specimens, holds great promise in a potentially beneficial translational impact for patients. The heterogeneity of results observed between cell lines, postulated to be due to either KRAS status or underlying metabolic phenotype, suggests a patient/tumour-­‐specific personalized approach to MCT or LDH inhibition should be adopted in any future clinical trials

Characterising the prostate stem cell niche and its architecture in benign prostatic tissue

M. D. Thesis.Prostate cancer is the most common cancer among males in the UK with 1 in 8 men being diagnosed with the disease during their lifetimes. Despite its high prevalence and incidence, a lot about the disease process is still unknown. To understand the changes that occur in a malignant state, it is important to understand normal physiology and homeostatic mechanisms. It then becomes easier to pinpoint and understand what exactly goes wrong. Understanding the role of stem cells could also help in understanding castration-resistant prostate cancer as there could be cells that exhibit similar characteristics driving the tumour process at that point. Key among the factors in maintaining a normal physiological state is the existence of prostate stem cells and prostate stem cell niches. There was a debate about the location of these cells – and whether they were basal or luminal. Previous work done also conclusively pointed towards a basal location although there was also evidence to say that luminal stem cells existed. Further work done in the lab previously, also confirmed these findings in addition to saying that these cells were clustered at the juxta-urethral prostatic ducts. There has also been research that has pointed to the existence of stem cells by discovering two cell types that did not fit into traditional classifications of prostate cells. This study attempts to characterise the location of the stem cells and the stem cell niche within the larger context of prostate tissue. By using immunohistochemical methods to characterise each type of cell based on cell type-specific markers such as Prostate Specific Antigen and Uroplakin 1b, the aim is to paint a picture of the architecture of the stem cell niche and the surrounding microenvironment. Some positive findings from this study could only add to the evidence that there exist certain areas of the prostate tissue which do not fall under traditional categorisations of prostate epithelium or urothelium. There also exist areas of overlap between prostate and urothelium which could point towards an important overlap in their origin stories – this needs to be studied further. However, for various reasons, the methods of study need to be optimised further for better results. In conclusion, this project adds to evidence of a potential basal location for stem cells as well as talking about the various limitations with the methodologies used. In addition, there is also potential for future studies with regard to more structural as well as functional aspects of the niche including evaluating the role of stem-like cells in castration resistant prostate cancer

Factors involved in the regulation of Long Interspersed-Nuclear-Elements (L1) retrotransposons in the context of Hepatocellular Carcinoma

PhD thesisHepatocellular carcinoma (HCC) generally develops on the background of a chronic liver disease following the accumulation of genetic damage and epigenetic alterations of growth regulatory genes, leading to activation of oncogenes and loss of function of tumour suppressor genes. Recent studies indicate that epigenetic aspects play an important role in the initiation of HCC. This includes dysregulation of repeat elements belonging to the Long Interspersed Nuclear Elements (LINE1 or L1) class. The L1 elements are autonomous mobile elements and upon activation contribute towards genomic instability via insertional mutagenesis. The thesis is aimed at understanding the factors leading to aberrant activation of retrotransposons and regulators of active retrotransposition in the context of HCC. All the liver cancer cell lines (Huh7, HepG2, Hep3B, PLC-PRF/5 and SK-Hep1) supported active retrotransposition in vitro irrespective of their basal L1 expression status or TP53 status. Since, active L1 retrotransposition through ‘Target Primed Reverse Transcription’ (TPRT) involves first DNA strand nicking by ORF2 endonuclease followed by second strand cleavage, we hypothesised that the DNA damage response pathways are involved in regulating the process. To decipher the influence of individual DNA repair pathway elements on the process of active retrotransposition, small molecule inhibitors towards ATM (KU-55933), DNA-PK (NU7441), ATR (VE-821), CHK1 (SRA737) and PARP (Rucaparib) were utilised. Overall, inhibition of ATR (Ataxia Telangiectasia And Rad3-Related Protein), a serine/threonine kinase involved in DNA replication stress and DNA damage signalling increased retrotransposition rate in all the cell lines. In addition, an increase in active retrotransposition was observed in Huh7 cell in presence of subgenomic copy of Hepatitis C Virus (HCV, a prevalent cause of HCC and contributes towards hepatocarcinogenesis by inducing oxidative stress, DNA damage and epigenetic changes in hepatocytes). Interestingly, the rate of retrotransposition remained higher in cells compared to control cell lines even when they were treated with PSI7977 (antiviral agent) successfully eliminating the viral genome from the cells. Hence, HCV upregulated active retrotransposition even beyond viral clearance and thus can contribute towards hepatocarcinogenesis by a ‘hit-and-run’ mechanism. Interrogating publicly available datasets - GSE84346 (RNAseq of Chronic HCV Hepatitis (CHC) patients and controls) and RNAseq data of non-tumour liver from the Cancer Genome Atlas HCC study - confirmed upregulation of L1 transcripts in chronic hepatitis patients liver. Hence, L1s can be activatedNewcastle University NUORS fellowship and JGW Patterson special gran

Investigation of wireless power transfer-based eddy current non-destructive testing and evaluation

PhD ThesisEddy current testing (ECT) is a non-contact inspection widely used as non-destructive testing and evaluation (NDT&E) of pipeline and rail lines due to its high sensitivity to surface and subsurface defects, cheap operating cost, tolerance to harsh environments, and capability of a customisable probe for complex geometric surfaces. However, the remote field of transmitter-receiver (Tx-Rx) ECT depends on the Tx-Rx coils gap, orientation, and lift-off distance, despite each coil responding to the effect of sample parameters according to its liftoff distance. They bring challenges to accurate defect detection and characterisation by weakening the ECT probe’s transfer response, affecting sensitivity to the defect, distorting the amplitude of the extracted features, and responding with fewer feature points at non-efficient energy transfer. Therefore, this study proposed a magnetically-coupled resonant wireless power transfer (WPT)-based ECT (WPTECT) concept to build the relationship between Tx-Rx coil at maximum energy transfer response, including shifting and splitting (resonance) frequency behaviour. The proposed WPTECT system was investigated in three different studies viz., (1) investigated the multiple resonance point features for detection and characterisation of slots on two different aluminium samples using a series-series (SS) topology of WPTECT; (2) mapped and scanned pipeline with a natural dent defect using a flexible printed coil (FPC) array probe based on the parallel-parallel (PP) topology of WPTECT; and (3) evaluated five different WPTECT topologies for optimal response and extracted features and characterised entire parameters of inclined angular Rolling Contact Fatigue (RCF) cracks in a rail-line material via an optimised topology. Multiple feature extraction, selection, and fusion were evaluated for the defect profile and compared in the study, unattainable by other ECT methods. The first study's contribution investigated multiple resonances and principal component analysis (PCA) features of the transfer response from scanning (eight) slots on two aluminium samples. The results have shown the potential of the multiple features for slot depth and width characterisation and demonstrated that the eddy-current density is highest at two points proportionate to the slot width. The second study's contribution provided a larger area scanning capability in a single probe amenable to complex geometrical structures like curvature surfaces. Among the extracted individual and fused features for defect reconstruction, the multi-layer feed-forward Deep learning-based multiple feature fusion has better 3D defect reconstruction, whilst the second resonances feature provided better local information than the first one for investigating pipeline dent area. The third study's contribution optimised WPTECT topology for multiple feature points capability and its optimal features extraction at the desired lift-off conditions. The PP and combined PP and SS (PS-PS) WPTECT topologies responded with multiple resonances compared to the other three topologies, with single resonance, under the same experimental situation. However, the extracted features from PS-PS topology provided the lowest sensitivity to lift-off distances and reconstructed depth, width, and inclined angle of RCF cracks with a maximum correlation, R2 -value of 96.4%, 93.1%, and 79.1%, respectively, and root-mean-square-error of 0.05mm, 0.08mm, and 6.60 , respectively. The demonstrated magnetically-coupled resonant WPTECT Tx-Rx probe characterised defects in oil and gas pipelines and rail lines through multiple features for multiple parameters information. Further work can investigate the phase of the transfer response as expected to offer robust features for material characterisation. The WPTECT system can be miniaturised using WPT IC chips as portable systems to characterise multiple layers parameters. It can further evaluate the thickness and gap between two concentric conductive tubes; pressure tube encircled by calandria tube in nuclear reactor fuel channels.PTDF Nigeri

A biochemical and biophysical study on cell division proteins from Staphylococcus aureus and biofilm proteins from Bacillus subtilis.

PhD ThesisPart I Cell division in bacteria is tightly regulated by a multiprotein complex called the divisome. Proteins in the divisome couple cell division and growth, ensuring that a single copy of the chromosome is present in each resulting daughter cell, and preventing more than one instance of division from occurring at any one time. This thesis concerns a combination of biophysical and biochemical techniques used to study the cell division proteins DivIVA, Stk1 and GpsB from Staphylococcus aureus. A model of the solution molecular envelope of DivIVA is derived by small-angle X-ray scattering and compared to a previously proposed model of the protein from Bacillus subtilis. The molecular mechanisms of DivIVA oligomerization are probed through use of size-exclusion chromatography coupled multi-angle light scattering on various truncations of the protein. The structure of the N-terminal domain of S. aureus GpsB is solved and used to rationalise the interaction between GpsB and PBP4. Attempts are made to determine an interaction network between the cell division proteins and members of the peptidoglycan and wall-teichoic acid synthesis machinery by several biochemical and biophysical assays. Part II Biofilms are communities of sessile bacteria that form on a wide variety of natural and manmade surfaces, sometimes at a detriment to human health. Bacteria in biofilms are held together by a viscous extracellular matrix consisting of polysaccharides, lipids, proteins, and extracellular DNA (eDNA). Species of Bacillus are known to secrete two structurally similar endonucleases, Nuclease A and B (NucA & NucB), into their environment as a means of taking up eDNA either to enhance their genetic diversity, or for metabolic purposes, respectively. As a mechanism of protection from self-induced genome degradation, NucA is co-expressed with a proposed inhibitor, Nin. A combination of biophysical/chemical techniques are used to probe the interaction between NucA/B and Nin from Bacillus subtilis. In vitro studies show that NucA and NucB bind to Nin and that Nin inhibits their endonuclease activity. The affinity of the interactions between NucA or NucB and Nin are probed and found to be sub-nanomolar. The structures of NucA/NucB in complex with Nin are solved by X-ray crystallography, revealing the mechanism of inhibition by Nin, and allowing for the calculated dismantling of the complexes by site-directed mutagenesis

Optimising associations of arbuscular mycorrhizal fungi with wheat

PhD ThesisArbuscular mycorrhizal fungi (AMF) are ubiquitous symbionts of most vascular plants and essential contributors to soil health for which reason their application in agriculture has been investigated extensively. In wheat as one of the staple foods where large amounts of fertiliser and pesticides are used, the integration of mycorrhizal benefits such as increased nutrient uptake and plant health is desirable, but mutualistic outcomes of the symbiosis are determined by variety, agronomic management practices as well as nitrogen (N) and phosphorus (P) content of the soil. The present study investigated the impact of different fertiliser sources (biogas digestate, farmyard manure and mineral N) on AMF at five key development stages of two wheat varieties (Aszita and Skyfall) +/- crop protection over two cropping seasons 2017-18 and 2018-19 in a P-depleted soil. Additionally, the effect of a commercial AMF inoculum (INOQ Advantage) on plant performance, yield and grain quality was assessed. AMF-root colonisation (AMF-RC) was consistently higher in the modern variety Skyfall which also showed lower abundances of native AMF in response to AMF inoculation. Biogas digestate and farmyard manure application decreased AMF-RC in both years, whereas mineral N only reduced AMFRC when soil N was high in the first season following grass-clover, but not in the second season following wheat (i.e. 2nd wheat crop). Amplicon sequencing of the ITS1-region revealed that mycorrhizal communities in roots were dominated by Glomus spp. and were not affected by agronomic management or variety. Differential abundance analyses based on sequences of the small subunit (SSU) however indicated increased diversity of fine root endopyhtes (FRE) in response to mineral N. Although the AMF inoculum was not detected in roots using strainspecific primers in digital droplet PCR, inoculation with AMF increased biomass production of wheat without fertiliser and decreased biomass production with mineral N treatment, but these changes did not affect grain yields. A pot experiment that tested a cellulose-based seed coating with the INOQ Advantage root powder showed negative effects on plant growth, but without root colonisation. The results of this study imply a key role of N that impacts AMF-RC, FRE and the effect of biostimulants. The use of such in wheat production requires further optimisation to guarantee economic benefit for farmers while excluding side-effects of exogenous strains on native AMF.Marie Skłodowska-Curie research programme of the European Union

Optimisation of the ozone pre-treatment of agricultural residues and conversion to platform chemicals

Ph. D. ThesisBiomass in the form of agricultural residues offers clear potential for conversion into energy, especially the use of processing residues such as corn cob and rice husk etc, due to their abundance/availability and high carbohydrate content. Depending on the conversion process, pre-treatment allows easier access to the carbohydrate components (cellulose and hemicellulose) for conversion to advanced generation biofuels and platform chemicals such as 5-hydroxymethyl furfural, furfural etc. leaving lignin as a low-value residue which is often burnt to provide parasitic energy for the conversion process. The conversion of lignin into high value platform chemicals will increase the profitability and sustainability of the biorefinery process. However, to achieve complete utilisation of the lignin, it must be of high purity and lack extensive modifications. Ozone pre-treatment is recently gaining attention as a promising green alternative for cellulose isolation. However, the drawback of using ozone for large-scale industrial applications is the high costs for generating ozone in-situ as it cannot be stored due to its short lifetime and high reactivity. This study aims to pre-treat and fractionate the agricultural processing residues (corn cob and spelt husk) and convert the carbohydrate components to chemical platforms (5-hydroxymethyl furfural and furfural) that can be used in several applications i.e medicines, diesel, fuel additives and plastics. The first stage of this study developed an energy-efficient surface two-zone plasma ozone generator consisting of two stainless-steel mesh electrodes and a dielectric of quartz glass. The design offered good temperature control, which produced 2.5 times higher concentration and quantity of ozone at the same power input than a conventional single-zone plasma reactor. A maximum ozone concentration of 140g m-3 and 90g (kWh) -1 productivity was obtained from the two-zone system, comparable to commercial ozone generators but with 30 – 40% lower power consumption (11kWh kg-1 O3). Hence mitigating the drawbacks with the use of ozone in industrial applications caused by large energy demand. Optimisation of the ozone pre-treatment process was achieved by incorporating ultrasound which enhanced lignin separation by 38.5%. Following organosolv fractionation at low temperature (80⁰C), about 90% and 94% of lignin with high purity (95%) were recovered for corn cob and spelt husk respectively with guaiacyl-syringyl lignin the major fraction from corn cob and guaiacyl lignin from spelt husk. In addition, 84 - 85% cellulose was recovered with III 78% purity. The recovered cellulose had its crystallinity decreased by 19% and its degree of polymerisation (DP) decreased by 17%. In a microwave reactor, corn cob and spelt husk (untreated and pre-treated) were reacted in a DMSO-H2O media to produce HMF and furfural. HMF and furfural yields of untreated corn cob were higher than those from spelt husk due to a difference in their morphology with increased porosity of corn cob allowing easy access to cellulose. Pre-treatment led to a 58% and 74% increase in HMF yield for corn cob and spelt husk respectively, while a 10% and 66.7% increase in furfural. Reacting fractionated cellulose from corn cob and spelt husk yielded a similar HMF yield of 40mg g -1 irrespective of the feedstock used. Overall, HMF and furfural yields were influenced by changes in cellulose properties following pre-treatment such as fibre size, increased surface area, decrease in the degree of polymerisation and decrease in lignin content following lignocellulose pre-treatment. The economic efficiency and competitiveness of the conversion process for large scale co production of HMF, furfural and lignin from spelt husk was determined. The proposed approach was compared with process where HMF and furfural were co-produced without lignin. Co-production with lignin yielded a profit of $213,657 higher than without lignin for a plant size of 100 tonnes per day of spelt husk due to extra revenue obtained from lignin sales, meaning fractionation of lignin had a positive effect on the process economics than its utilisation for heat or boiler fuel. Thus, the improved pre-treatment and quality separation of biomass components enhanced downstream conversion to value-added products, thereby improving the sustainability and cost-effectiveness of the ozone pre-treatment process and conversion to HMF and furfural. In addition, the co-production of lignin will offset the cost of production of platform chemicals, thereby increasing the economics of a biorefinery.Petroleum Technology Development Fund (PTDF) Nigeri

Planning for Floods: An analysis of planning law and planning practice in flood risk management

Ph. D. Thesis.Flooding has considerable detrimental economic, social and environmental impacts. These impacts are already being felt in England and the rest of the UK and will further increase in the future with the projected increase in flood risk attributable to climate change and socioeconomic growth. It is widely agreed that urgent steps are needed to more effectively manage flood risk, and these may include changes in the regulatory and planning policy frameworks currently applied to shape development in the built environment. Using a combination of doctrinal and empirical research, this thesis examines the role of the town and country planning system in the management of flood risk, focusing on local planning authorities (LPAs) and an examination of the extent to which they can - and do - manage flood risk effectively. The doctrinal research examines the obligations on LPAs to manage flood risk and the legal planning tools they have at their disposal to do so. The empirical research uses four case studies to collate and present quantitative and qualitative data that has been used to interrogate in each case the relevant LPAs’ management of flood risk in practice. Based on the findings of the doctrinal and empirical research, the thesis makes a number of recommendations for reforms to the planning system and the legal planning tools available to LPAs that would enable them to more effectively manage flood risk.Economic and Social Research Counci