The islets of Langerhans are multicellular aggregates within the pancreas containing beta cells that sense changes in blood glucose level and respond by secreting insulin. The cells within the islets communicate with each other through gap junctions. The 3D structure of the islets is crucial for their metabolic function. Artificial reconstruction of isolated beta cells into 3D cell aggregates has led to the generation of Pseudoislets (PIs) which mimic islets of Langerhans and can produce insulin. However, the lifespan of PIs is generally a few days in culture after which, central cell death occurs. The overall aim of this project was to improve PI viability and biofunction through development of two new techniques. The first technique being developed was a series of coating solutions consisting of Pluronic F127 (F127) and gelatin type A (Gelatin), which systematically altered the substrate properties of cell culture plates where PIs were formed by suspension culture. The second technique was generation of gelatin beads, functioning as microchannels inside of PIs, and drug carriers when loading with anti-inflammation agents to reduce inflammation and central necrosis. The beta cell line, BRIN-BD11, was used to form PIs. Coating solutions with different ratios of gelatin and F127 (90%, 95%, 98% and 100% of Gelatin in the mixture) have been designed. Surfaces coated with these coating solutions generated different ATR-FTIR spectra, contact angle (CA) values and protein adsorption capacities and the changes were dependent on the original substrate chemistry. The coating of suspension culture plates with gelatin alone failed to generate suspended PIs, while coating with F127 alone resulted in the generation of large, irregular PIs. On the other hand, coating suspension culture plates with a combination of gelatin and F127 solutions formed suspended PIs of differing sizes. The lower the percentage of gelatin in the mixture for the coating, the higher the size of PIs. The higher the F127 content, the more hydrophilic the surface was, which led to lower CA values of the substrate. Coating solutions containing 95-98% Gelatin produced homogenous and smaller PIs than on F127 coating. PIs formed on 98% Gelatin coated plates had larger size but more homogenous morphology (268 ± 10 μm) than PIs produced on ULA plate (235 ± 30 μm). These larger PIs exhibited comparable cell viability, the same level of insulin expression in GSIS and Western blotting assays and higher gene expression of key beta cell markers than smaller PIs formed on ULA plates. Furthermore the larger PIs expressed more of the gap junction protein, connexin 36, in comparison to PIs generated on ULA plates, revealing the optimised configuration of cells in these PIs. Altogether, regulating the hydrophicility of suspension culture plates by F127 and gelatin ratio in the coating solution changed the physical properties of PI and altered the expression of key beta cell markers. Gelatin beads (GBs) of 30-40 μm sizes were produced by Water/Oil emulsion techniques. The degree of swelling and stability of GBs was controlled by crosslinking with 5% glutaraldehyde (GA) vapour for 6 hours. The incorporation of 40 μm GBs into PIs (25-50 GB/per PI) significantly increased the proliferation and cellular viability of the PIs for both large and small sized PIs. Loading of anti-inflammatory cytokines, IL-10 and anti-IL-1β into GBs allowed for sustained release of these agents over time. Incorporation of IL-10 and anti-IL-1β loaded GBs to PIs revealed a synergetic effect in the improvement of the proliferation/viability of PIs regardless of PIs size (large or small). LDH release from the PIs was decreased dramatically and insulin release was increased significantly. Thus GBs played active roles as microchannels and drug carriers which improved the viability and function of PIs.

With the advent of powerful gene editing tools such as CRISPR/Cas9, advances in gene therapy have gained a second wind. Despite this, disease therapy still has not progressed beyond clinical trials due to limitations in current delivery methods. The work presented in this thesis studies the development of a non-viral gene delivery method which is the nanomagnetic transfection method, which is the delivery of genes to cells using magnetic nanoparticles (MNPs) with a cationic surface charge and an external magnetic _eld. The advantage of nanomagnetic transfection over other non-viral chemical methods is the low dosage required to transfect cells coupled with a short transfection time. The presence of an external magnet provides targeting functionality, whereby the MNPs carrying the gene of interest are pulled towards the cells, thus increasing the e_ciency of cell to MNP contact. The research looks at the synthesis of MNPs using thermal decomposition to obtain particles with a narrow size distribution and exhibiting a combination of Brownian and N_eel relaxation. The MNPs were coated with polyethyleneimine (PEI), which binds and condenses DNA to deliver into cells for protein expression. PEI is known to be toxic to cells at high concentrations, hence PEI not bound to MNPs were removed using dialysis. A unique study observing the gradual loading of PEI coating on MNPs using AC susceptometry (ACS) is described. ACS provided information on the MNP coating and aggregation process that was not accessible through dynamic light scattering (DLS) due to the additional presence of non-magnetic polymer particulates in the suspensions. In combination with complementary structural characterization techniques, a simple method was derived to obtain dense, uniform PEI coatings a_ording high-stability suspensions without excessive quantities of unbound PEI to reduce cytotoxic e_ects. This method can be used for improving coating and functionalization therefore advancing MNP-drug/gene delivery studies. The PEI-coated MNPs were subsequently studied for their transfection capabilities in HeLa cells and compared to commercial MNP transfection agents. It was found that nanomagnetic transfection had higher GFP reporter expression compared to Lipofectamine and PEI. The parameters a_ecting transfection activity were determined in order to improve transfection rates of synthesized MNPs. A trade-o_ between transfection e_ciency and cytotoxicity was observed, where the presence of unbound PEI improved transfection but a_ected cell viability. To overcome this, polymers and block-copolymers with a lower charge density should be developed. The proton-sponge e_ect, which is the mechanism of MNP-PEI escape from the endolysosome was studied by measuring the AC susceptibility of MNP-PEI in live cells. However, the low transfection e_ciency of MNP-PEI and low sensitivity of the AC susceptometer made it di_cult to obtain conclusive evidence. A novel study using Raman spectroscopy to obtain _ngerprint spectra of the MNP-PEI complexes and to determine their localization in cells is reported. Individual spectra of MNP and PEI were obtained, as well as the area map of the cell, however the localization of MNPs within the cell was not possible due to the limited sensitivity of the Raman spectrometer. Finally, the e_ect of the MNP-PEI transfection agents on cells were identi_ed. It was observed that MG-63 and HeLa cells expressed increased cell stress with the formation of actin stress _bres and increased cell adhesion. Between the two transfection components, PEI antagonized the cell adhesion e_ect compared to MNPs. In addition, the genes associated with actin _bres and cell adhesion were identi_ed, which were ACTA2, ACTN1, MVCL, VCL, P4HA2, PCDHB12, SVIL, and TGFBI, which showed increased expression to MNP-PEI treatment. Collectively, the study conducted reports the development of an MNP transfection agent, from synthesis to application.

In this project, we have designed, constructed, and validated a human tissue-engineered blood vessel construct (TEBV) to assess whether it could be used as a new model system in which to study thrombus formation under physiologically relevant conditions. A layer-by-layer fabrication technique was adopted to fabricate the TEBV. This allowed for the biological properties of both the medial and intimal layers of the construct to be assessed individually, as well as in combination as the full TEBV. The TEBV model was shown to mimic the anatomical structure and cellular phenotype of the native human artery. In addition, a novel technique for quantitatively assessing the pro- and anti-aggregatory properties of these constructs was developed, utilising real-time measurements of cytosolic Ca2+ signalling to assess real-time human platelet activation when exposed to the tissue-engineered blood vessels constructs. The real-time measurement of cytosolic Ca2+ signalling of human platelets when exposed to the TEBV models was shown to be a sensitive technique to assess the haemostatic properties of the 3-dimensional (3D) TEBV to validate the physiological relevance of the construct. Experiments conducted with this novel methodology, alongside other traditional platelet function assays, demonstrated that our TEBV had an anti-aggregatory intimal layer and a pro-aggregatory medial layer, consistent with the haemostatic functions of this blood vessel layers in vivo. We have also established an ex vivo ferric chloride (FeCl3) arterial injury model to be used as an alternative to intravital microscopy study of in vivo thrombus formation in mice models. Treatment of the TEBV with FeCl3 elicited a significant increase in platelet aggregation upon the surface of the construct when these cells were perfused over the construct at arterial shear stresses. By using this perfusion system, experiments also provided initial evidence that the use of the general anaesthetic ketamine, in intravital microscopy experiments may interfere with thrombus formation, and therefore could affect the validity of results seen in previous in vivo studies. In conclusion, we have successfully created a TEBV that is able to replicate the functional properties of the native vessel and which may be useful as a novel model to study human thrombus formation.

The essentiality of silicon and the toxicity of aluminium within humans are both topics of much debate; the current conception is that silicon is essential in ameliorating aluminium toxicity, however, the evidence for this remains inconclusive. To be able to elucidate the connection between silicon and aluminium in neurological disorders, it is first important to understand the relationship between the elements in healthy individuals. This research was designed to investigate whether supplementing the diet with a silicic acid-rich mineral water could be a non-invasive means of reducing aluminium body burden in both healthy individuals and those suffering from multiple sclerosis. Drinking a silicic acid-rich mineral water significantly enhanced the urinary excretion of aluminium, in both healthy individuals and in multiple sclerosis. Collecting whole daily urinary excretions for healthy individuals indicated that aluminium was concomitantly excreted with silicon; this was most effective when the mineral water was consumed as a bolus, suggesting that the mineral water has greater influence when consumed in large quantities over a short time. In addition, reductions in urinary aluminium were also witnessed over time, supporting the use of silicic acid-rich mineral water in reducing and maintaining aluminium body burden at a lower level. Supplementing the diet with a silicon-rich mineral water, for a period of 12 weeks, reduced aluminium body burden in individuals with multiple sclerosis; concomitantly, in this short amount of time, disability scoring showed clinically relevant improvements in 2 out of 15 individuals. Longer-term studies, involving larger study populations, are now needed to see if these effects are long lasting; if improvements are seen over time, it could support the link between aluminium and multiple sclerosis. This research also presents preliminary evidence that sweat may be a more efficient excretory mechanism in lowering the body burden of aluminium in healthy individuals. In conclusion, the results suggest that including a silicic acid-rich mineral water into the regular diet, without the need of following strict restrictions, could be a prophylactic therapy against aluminium toxicity in healthy individuals, and in addition, could be beneficial as a chelating agent for endogenous aluminium.

The emergence and spread of resistance to antimalarial drugs has highlighted the demand to search for new drugs that target novel pathways in the human malaria parasite Plasmodium falciparum. This thesis explores the potential for targeting of the PfAtg8-PfAtg3 protein-protein interaction (PPI) that is required for lipidation of phosphatidylethanolamine (PE) within parasite autophagy pathway. Whilst P. falciparum lacks a complete cascade of the classical autophagy pathway, the homologues for key proteins such as PfAtg8 exist and have been shown to be involved in cellular processes such as vesicle trafficking and apicoplast biogenesis. Here, the antiplasmodial activity of a library of 131 compounds designed in silico to act as inhibitors of hLC3 (Atg8 homologue)-Atg3 interaction were evaluated. Two hits, SK1.47 and SK1.49, have been identified and shown to have moderate antiplasmodial activity (EC50 of 1-2 μM) against intraerythrocytic parasites, produce a rapid cytocidal activity against trophozoite stage parasites and have selectivity for the parasites over HepG2 cells. As a first proof of concept, both compounds inhibit the formation of PfAtg8-labelled vesicles, potential autophagosomes, induced by nutrient starvation. Computational modelling of SK1.47 and SK1.49 docking to PfAtg8 suggests that the naphthalene group binds to the W-pocket and the substituted phenyl binds to the L-pocket of the PfAtg3 interacting region of PfAtg8. This docking study also highlights aspects of the core structure of both molecules that should be further explored in terms of their antiplasmodial activity. Ultrastructural changes and induction of biochemical markers of apoptotic cell death appear to suggest that SK1.47 and SK1.49 treated parasites do not undergo apoptotic cell death. This study was extended to exploit a bioluminescence assay of parasite viability following exposure to a range of benchmark antimalarial drugs, which allows samples to be prepared that match a defined and titrated kill effect applied using drugs of different chemical classes. These established the conditions for a comparative study of apoptotic markers of early cell death using these different chemical classes, and also highlighted the central role of mitochondrial membrane potential collapse for the majority of drugs that explored and Ca2+ redistribution from the digestive vacuole following treatment with 4-aminoquinolines. This study also highlights the opportunity of autophagy related proteins (Atg) of P. falciparum parasite as a novel target for drug development. Although SK1.47 and SK1.49 compounds are not considered as lead compounds for drug development due to lack the required potency with unfavourable physicochemical properties (high LogP), they are available now as chemical probes to explore the contested role of autophagy in malaria parasite homeostasis and response to drugs.

Background: Consultation skills are the core competencies required at graduation of the doctor as a practitioner. Every medical school has its own system of teaching and assessing consultation skills. These are generally amalgams of previous curricula and not rigorously developed. We took the opportunity presented by a new undergraduate medical curriculum to systematically develop the consultation skills curriculum from classroom teaching to OSCE assessment and formative workplace-based assessment and feedback. Methods: The consultation skills curriculum and assessment system were developed by action research. Data were collected using mixed methods involving questionnaires, focus groups, participant interviews, student reflective summaries and routine monitoring of usage of an app which we developed for generating feedback summaries in the clinical workplace. Participants were teachers and students at Keele University school of medicine. In addition, clinical tutors from seven other UK medical schools participated in a Delphi study of undergraduate medical consultation skills competencies. Results: A case study of curriculum development by action research is presented in nine published papers. Conclusion: This work has contributed to medical education knowledge as follows: an instrument for assessment of consultation skills has been developed and validated; and a set of strategies for improvement of these consultation skills have been developed and validated. It has added to understanding about transfer of learning from the classroom to the workplace; the impact of assessment grades on medical students’ learning and self-perception; and the value of a system of formal workplace-based assessment. Additionally this work was one of the first applications of realist methods in medical education research, and it has developed guidance on feedback in the workplace for individual tutors and educational institutions.

Articular cartilage is a thin hydrated tissue, which covers articulating surfaces. In the native articular cartilage tissue, the extracellular matrix (ECM) is a fibrillar mesh of interacting proteoglycans, collagens, and other non-collagenous proteins residing in a highly aqueous environment. Chondrocytes are surrounded by a pericellular matrix (PCM) forming chondrons. The PCM, exclusively rich in collagen VI, is more easily destroyed during their extraction and subsequent in vitro culture than ECM. The retention of the PCM has a significant influence on the metabolic activity of the chondrocytes in addition to the mechanical signalling from and to the ECM via cell-matrix interactions. The ECM and the residing chondrocytes are organised into three distinct zones: superficial, middle and deep. The complex and organised structure of cartilage allows it to resist the tensile stress ‘superficial zone’, sheer stress ‘middle zone’, and compressive stress ‘middle and deep zones’ imposed by articulation. This study initially focused on the morphology and chondrogenic capacity of chondrocytes, chondrons (bovine) and mesenchymal stromal cells (MSCs, rat) alone in monolayer cultures to establish a baseline of PCM preservation and regeneration approaches. Co-culture monolayer models of cartilage cells with MSCs (20%, 50% and 80%) was established to assess the effect of MSC on PCM maintenance and ECM production by biochemical assays, immunofluorescence and histological staining. Co-culture of MSCs with chondrons enhanced ECM production, as compared to chondrocyte or chondron monocultures. The co-culture of MSCs with chondrons appeared to decelerate the loss of the PCM as determined by collagen VI expression, whilst the expression of a high temperature requirement family of serine proteases, HtrA1, demonstrated an inverse relationship to that of the collagen VI. The 50:50 ratio of MSCs: chondrons in co-culture presented the highest potential for better cartilage regeneration. For the first time, it is confirmed that MSCs directly or indirectly inhibited HtrA1 activity in the co-culture, which played a role in enhancement of ECM synthesis and the preservation of the PCM. However, PCM could not be fully preserved or regenerated in 2D culture up to 7 day culture even starting from chondron, and co-culture with MSCs. Next 3D model systems using hydrogels to improve PCM formation and maintenance were developed. Four culture conditions were compared; hyaluronic acid (HA) versus agarose hydrogel; basal medium versus chondrogenic medium; chondron or chondrocytes versus co-culture with 50% MSC. Up to 21 day culture, chondron samples in both mono- and co-culture maintained PCM at all culture conditions. The quantity and quality of regenerated PCM in chondrocyte samples were culture condition dependent. HA in combination with chondrogenic media and co-culture with MSC was the best support for PCM generation and ECM deposition. Basal and chondrogenic culture mediums influenced the expression of cartilage-specific ECM markers but did not affect collagen VI synthesis. Synchrotron microFTIR measurements assisting with PCA analysis of spectra in fingerprint and lipid regions on the 3D cultured samples have cross-validated that culturing chondrocytes in HA hydrogel up to 21 days might generate chondron-like cell morphology and composition because day 14 and day 21 samples clustered to chondron spectra, whilst day 7 samples to chondrocyte spectra. Zonal-specific 3D hybrid scaffolds have been fabricated using a combination of polylactic acid and HA to induce the generation of near-native cartilage. For the superficial and middle zones, specifically orientated or randomly arranged polylactic acid nanofibre meshes were embedded in HA. For the deep zone, vertical channels in HA were created. The aligned nanofiber mesh used in the superficial zone induced an elongated cell morphology, lower GAG and collagen II production, than the middle zone scaffold. Within the middle zone scaffold, which comprised of a randomly orientated nanofiber mesh, the cells were clustered and expressed more collagen II. The deep zone scaffold induced the highest GAG production, the lowest cell proliferation and the lowest collagen I expression of the three zones. Overall a convenient and reproducible model system which mimics the zonal organisation of articular cartilage has been developed.

Background: Serine/Threonine protein phosphatase 4 (PP4) plays a crucial role in cell signalling, proliferation and apoptosis, and its catalytic subunit has been reported as an important regulator in many types of cancer, including breast and lung cancers. Aims: Evaluate the effects of PP4c endogenous modulation in leukaemic cell lines; K562 for chronic myeloid leukaemia and HL-60 for acute promyelocytic leukaemia, on cell viability, apoptosis, and proliferation. Methods: K562 and HL-60 cells were transfected with pcDNA3.1-PP4c to over-express or with specific siRNAs to down-regulate PP4c. Following transfection, cell viability, total apoptosis percentage, proliferation rate and cell cycle were assessed. The response of these cells to four different therapeutic drugs (imatinib, cisplatin, doxorubicin and rapamycin) was assessed post PP4c silencing. Results: This study demonstrated that PP4c over-expression in K562 cells significantly reduce cell viability and cell proliferation, accompanied by a significant increase in apoptosis at 24 and 48 hours post re-plating. It also altered the cell cycle profile by increasing the number of cells in G0/G1 phase and reducing the cell number in S phase. On the other hand, PP4c silencing significantly increased cell viability in K562 cells at 48 hours. PP4c down-regulation also enhanced and promoted the action of both imatinib and rapamycin by increasing apoptosis in K562 cells. PP4c over-expression in HL-60 decreased the viable cell number at 48 hours, while PP4c down-regulation increased the viable cell number at 72 hours and altered the cell cycle profile by reducing the cell number in S phase and increasing the cells number in G2/M phase. PP4c silencing in HL-60 cells had no effect on the action of the chemotherapeutic drugs. Conclusion: The results suggested that PP4c regulates cell proliferation, cell death, and cell cycle in K562 cell line in addition to enhancing their response to imatinib and rapamycin. Such studies are important to identify molecular regulators of the pathways involved in the pathogenesis of leukaemia and will contribute to greater understanding of the mechanism(s) by which phosphatases regulate cell survival in cancer and may allow design of optimised targeted therapies in the future.

Oxygen is an essential factor for life in many organisms. Oxygen concentrations vary widely across different human tissues, but in general, are much lower than the 21% oxygen in atmospheric air. In spite of this, most research continues to be based on the culture of cells in an air oxygen (21% O2) environment. This ignores the mounting evidence of the role of physiological oxygen levels in the maintenance of survival, proliferation, stemness, genetic and epigenetic changes. The positive benefits of low oxygen tension on the maintenance of properties of several cells including embryonic and mesenchymal stem cells are well established while data describing negative impacts via genomic stability are few and conflicting. The role of cytosine modifications in cancer cells in response to hypoxia is poorly understood either in vitro or in vivo. In this study, we aimed to determine the role of low oxygen in the regulation of DNA methylation marks (5mC and 5hmC) and their associated genes including DNMT1/3A/3B/3L and TET1/2/3 in different cell lines including human embryonic stem cells, human mesenchymal stem cells and cancer cells. In addition to above, we also sought to corroborate upon and extend previous studies describing the effect of reduced oxygen on a range of cellular aspects including proliferation, metabolic activity, stemness and differentiation. To achieve these aims we cultured cells in either air oxygen, a fully defined 2% O2 environment (Hypoxycool media, tri-gas workstation), or in a multiuser tri-gas incubator with handling in a standard biological safety cabinet. Routine culture of stem cells in physiological normoxia enhanced the functional profile of stem cell populations including proliferation, metabolic activity and stemness. In contrast, culture of cancer cells in reduced oxygen caused significant decreases in growth profiles (vs. air oxygen). Quantitative RT-PCR and Western blots results of cells cultured in reduced oxygen revealed significant transcriptional translational downregulation of DNMT3B and TET1 vs. air oxygen (except for TET1 in cancer cell lines cultured in 2% O2 where it increased significantly), accompanied by significantly reduced in levels of 5mC and 5hmC (again except for 5hmC in cancer cell lines cultured in 2% O2 where significant increases were noted). Noteworthy was that the downregulation in gene expression of DNMT3B was associated with an increase in its CpG promoter methylation. Importantly, these changes observation was associated with increases HIF2A, at protein levels in most cell types investigated. The role of physiological oxygen in these changes was confirmed by transitioning cancer cell lines between 2% O2 and air oxygen and detailing the reversibility of both DNMT3B and DNMT3L expression at mRNA level and promoters CpG island methylation. Together these data suggest that the level of 5mC is reduced by HIF2A, via DNMT3B- mediated methylation. In conclusions, our cells display oxygen-sensitive methylation patterns where de novo methylation is linked to oxygen culture and correlates directly with transcriptional and translational regulation of the de novo methylase DNMT3B. Delineating cancer and stem cell biology under niche-like culture conditions will ultimately enhance our understanding of mechanisms of action promoting improvements to both tumour-targeting medicines and regenerative medicine applications.

Most analyses in stroke clinical trials assess the primary outcome at a single time point and do not consider conducting a longitudinal analysis, even when the outcome has been recorded at several time points. Efforts to improve the quality of statistical analyses have previously been made, but have been directed at techniques that compare groups at a predetermined fixed time point. The aim of this thesis was to specifically investigate the use of longitudinal techniques, such as generalised linear mixed models and latent variable methods, to model serially correlated stroke outcome data, and to consider the challenge of having a score for death within the scale. The chosen outcome was the modified Rankin scale, which is a functional assessment outcome after stroke and is one of the most popular endpoints in clinical trials; often recorded repeatedly during follow-up. Initial chapters describe the epidemiology of stroke and various stroke outcomes, with a review of what appears in the literature regarding current longitudinal modelling of mRS data. Subsequent chapters report how proportional-odds models are fitted to longitudinal repeated measures trial data, with the effect estimates compared to an analysis at a single time point. Simulation techniques assess how different patterns of drop-out affect treatment estimates derived using all the data. These are then compared to a Markov analysis that treats death as an absorbing state. Finally, clustering methods are considered in order to try and describe trajectories of the mRS, but it is highlighted that this is challenging given limited movement between states. Each of the methods have their own merits, and trials should be encouraged to consider longitudinal analyses when there is repeated measures data.