Anti-inflammatory drugs such as corticosteroids (CSs) and minocycline (MINO) are widely used in the treatment of a range of clinical conditions and to suppress graft rejection in stem cell transplantation therapy. However, such treatment is associated with adverse effects on brain development. The effects of anti-inflammatory drug on neural stem cells (NSCs) are largely unknown and the molecular mechanisms underlying these effects are poorly documented. The focus of this project is to systematically investigate the effects of different anti-inflammatory drug at different concentrations on the fate of NSCs using two different in vitro models. In this thesis, it is shown that all three types of CSs (dexamethasone, prednisone and methylprednisolone) affect NSCs propagated in monolayers and neurospheres. Comparison of the monolayer and neurosphere growth formats for NSCs following CS treatment revealed that CS decreased NSCs proliferation and neuronal differentiation while accelerated the maturation of oligodendrocytes without concomitant effects on cell viability and apoptosis. The findings suggest that the difference in the physical format of NSCs does not impact on CS influences on these cells with similar results obtained for both culture systems. Further, label-free quantitative proteomics was used to study methylprednisolone effects on NSCs at the cellular and molecular levels in monolayer cultures. Proteomics and bioinformatics analyses revealed that methylprednisolone induced downregulation of growth associated protein 43 and matrix metallopeptidase 16 with upregulation of the cytochrome P450 family 51 subfamily A member 1. These findings support the hypothesis that neurological deficits associated with CS treatment mediated via effects on NSCs, and highlight putative target mechanisms underpinning such effects. Finally, the organotypic spinal cord slice model was used to investigate the efficacy of MINO as a combinatorial therapy with transplanted NSCs. The data from neurosphere culture showed that MINO had no direct effect on key regenerative properties of NSCs such as proliferation and differentiation. While, the findings from organotypic spinal cord slice culture showed the astrogliosis and activated microglia were reduced and the outgrowth of the nerve fibres was increased following a combinatorial therapy. This study demonstrates the utility of the organotypic spinal cord slice model to test the efficacy of MINO as a combinatorial therapy with transplanted NSCs.

Successful gene therapy depends upon specific gene delivery into the cells and tissues of interest. Nanomagnetic gene transfection is a relatively new gene delivery technique that has attempted to meet that need and has been effectively used with both viral and non-viralvector systems. It uses magnetic nanoparticles (MNPs) in assisting and directing specific delivery of reporter or therapeutic genes on a single cell basis, in the presence of an externally introduced oscillating magnet. The novelty of the lateral oscillation further stimulates endocytosis of MNP:plasmid complexes with improved in vitro transfection efficiency compared to the static magnet application and other non-viral gene delivery approaches. This work’s purpose was to contribute in the optimisation of this tool for safe and efficient gene delivery, and to investigate the applicability of the method in a wider range of cell types used for regenerative medicine purposes, improving transfection efficiency and duration. Novel transfection experiments using commercially available MNPs coupled to a reporter gene, demonstrated higher levels of transfection efficiency (differing among cell types) and cell viability (80-94%), at the lowest reagent concentrations across all posttransfected cell types, compared with the most widely used cationic lipids (Lipofectamine) and/or electroporation. In particular, using human lung mucoepidermoid carcinoma cells (NCI-H292), the magnetic field requirements for transfection were evaluated; using human osteosarcoma fibroblasts (MG-63), a nanomagnetic transfection protocol at shorter transfection times (30 min) was established for increased transfection efficiency (53% oscillating and 49% static magnet, 7% at 30 min and 24% at 6 hr Lipofectamine, and 21% electroporation); using mouse embryonic fibroblasts (NIH-3T3), the 30 min-protocol was applied further (25% oscillating and 22% static magnet, 2% at 30 min and 22% at 6 hr Lipofectamine); using human mesenchymal stem cells (hMSCs), the ability of the method to transfect primary cells and to retain key markers for multipotency was demonstrated; using human embryonic stem cells (hESCs), the transfection capability in additional types of primary cells was shown, along with indications of retention of key markers for pluripotency and differentiation, although further work is required to confirm this finding. Nanomagnetic gene transfection shows promising results for in vitro and in vivo non-viral gene delivery and biomedical engineering applications. Data from this study could to be used for MNP drug delivery strategies, ultimately for clinical translation.

This thesis describes the biochemical, biophysical and structural characterisation of two proteins believed to play an important role in active gene silencing mechanisms present in the human malarial parasite, Plasmodium falciparum. These investigations were performed using the histone deacetylase enzyme, PfSir2a, and the DNA binding protein, PfAlba3. The initial goal of this PhD project was to obtain structural information on both PfSir2a and PfAlba3, as well as the proposed silencing complex thought to be formed by the two proteins. This information would then aid the development of novel pharmaceuticals with a perspective towards new therapeutics to combat the continued threat of malaria. Thorough biochemical and biophysical characterisation of both PfSir2a and PfAlba3 was conducted and is described in Chapter 3 of the thesis. These results could not characterise a strong interaction between PfSir2a and PfAlba3 and highlighted several properties exhibited by the proteins that, as a result, proved challenging to characterise by structural methods. Most notably concentration dependent oligomerisation and protein aggregation effects were observed for PfSir2a and PfAlba3 respectively. Nevertheless, structural studies were performed using SAXS and SANS techniques to investigate the individual proteins to obtain structural information about the solution state of PfSir2a and PfAlba3 and generate ab initio models for both proteins and these are presented in Chapter 4. The difficulties presented by the target proteins aided in the development of a new investigative method for bio-SANS experiments, SEC-SANS, and the first example of testing and validation of this technique is presented in Chapter 5. The findings of work conducted for this thesis are summarised in Chapter 6 with an outlook for future work and development.

Self-renewal and an ability to differentiate are key hallmarks of stem cells. Pluripotent stem cells (PSC) have the capacity to differentiate into all cell types found within the three germ layers; ectoderm, endoderm, mesoderm. Multipotent stem cells including mesenchymal stem cells (MSC) customarily differentiate into cell types representative of one germ layer only. Metabolomics focusses on characterising the low molecular weight organic compounds which are by-products of protein-protein interactions and metabolic enzymatic processes; volatile organic compounds (VOCs) emitted from or consumed by cells can be correlated with metabolic status. Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) has been used to identify and discriminate between metabolites (VOCs) from both undifferentiated and differentiated stem cells in specific cell culture oxygen conditions (air (21% O2) and physiological oxygen (2% O2)). The suitability of SIFT-MS for detecting and identifying VOCs from hPSCs was evaluated with SIFT-MS spectral data analysed via OPLS-DA. hPSCs cultured in 2% O2 displayed a distinct metabolic profile to those cultured in 21% O2. Metabolite markers differing between culture conditions for undifferentiated hPSCs included ethanol and acetaldehyde. Inhibition of ethanol/acetaldehyde conversion enzymes revealed mechanistic control of ethanol and acetaldehyde levels linked to environmental oxygen. hPSC differentiation-linked differences in metabolic profiles included immediate reductions in acetaldehyde and DMS/ethanethiol levels upon onset of differentiation. For hMSCs, OPLS-DA score plots indicated that hMSCs cultured in a controlled, hermetic, workstation maintained at 2% O2 were distinct to those cultured in either 2% O2 or 21% O2. The VOC profile of hMSCs varied with oxygen condition and degree of differentiation during osteogenic differentiation. In summary, this thesis demonstrates that SIFT-MS can detect and discriminate VOC profiles in two distinct stem cell populations. These profiles are sensitive to oxygen and differentiation status and therefore provide a potential valuable tool for non-invasive explorations of stem cell biology.

Oxygen is an important molecule in life and is essential for a broad spectrum of physiological reactions that include, but are not restricted to, cell metabolism, respiration and growth. Under physiological conditions, oxygen levels vary from one tissue to another ranging from 0.002% to 10% and substantially lower than the atmospheric level of 21 % O2. Hypoxia is defined as a state of reduced O2 supplied to cells or tissues when compared to their normal physiological levels. Hypoxia mimetic agents (HMAs) are chemical used to induce pharmacological hypoxia without affecting environmental oxygen levels per se. The name HMA is routinely applied as these agents will activate the family of transcription factors which respond to reduced oxygen availability, Hypoxia Inducible Factors (HIF), which is taken as a surrogate indicator for hypoxia. These agents have been proposed as a cheaper alternative to engineered oxygen control measures including tri-gas incubators and workstation approaches. Multipotent stem cells (e.g. neuronal PC12 and human mesenchymal stem cells (hMSCs)) due their ability to differentiate into various cell types provide a means to develop better understanding of tissue development, repair, and protection. In addition, they provide better therapeutic perspectives and opportunities for the treatment of many diseases. Physiological oxygen plays a key role in the maintenance of cellular proliferation, behaviour and biology both in vivo and when mimiced in vitro. Physiological oxygen and hypoxia are routinely confused creating additional complexity in defining the role of oxygen in cell behaviours. This work has therefore evaluated the role of a panel of well-established HMAs (CoCl2, DFO, DMOG) and new agent (IOX2) vs. different reduced oxygen culture conditions. PC12 and hMSCs were both used to examinine the roles of HMAs on proliferation, metabolic activity, HIFs expression, nitroreductase activity, oxidative stress, apoptosis, death/necrosis and mitochondrial dynamic (burden, action potential and genome copy number) in comparison to physiological normoxia and intermittent hypoxia. HMAs induced HIF expression, apoptosis, trapped cell at G0/G1 phase and induced both ROS formation and nitroreductase activity in a manner that was not consistent with a reduced oxygen culture condition alone. Mitochondrial burden, mitochondrial action potential, and mitochondrial genome number also changed in response to HMA exposure in a manner that was indenependent of the oxygen culture conditions tested. In summary, HMAs do not provide an accurate replication of engineered oxygen control measures in PC12 and hMSC biology. This is reflected in biological alterations impacting on cell yield, behaviour and biology.

Several long non-coding RNAs (lncRNAs) have been identified to play key, rate-limiting roles in malignancies, and the mechanisms involved are now being elucidated. This study addressed the roles of NEAT1 and MIAT lncRNAs, in breast cancer. The short isoform NEAT1 _1 was found to be significantly up-regulated in advanced stages of breast cancer samples and in the ER/PR +ve and HER –ve molecular subtype, where its expression correlated positively with that of its neighbouring gene, MALAT1. NEAT1 transcripts in breast cancer cell lines were detected in both nuclear and cytoplasmic compartments. Silencing of cytoplasmic NEAT1 led to an increase in the expression of nuclear NEAT1, where such overexpression inhibited apoptosis and increased cell survival. Consistent with this, siRNA and ASO mediated knockdown of NEAT1 transcript levels decreased cell survival and migration and promoted cell death induced by different apoptotic stimuli including chemotherapeutic agents and UV-C irradiation. Reduced NEAT1 expression levels were also associated with changes in the expression of genes involved in the regulation of cell proliferation and survival. More importantly, it was found that NEAT1_1 regulates gene expression in cis and trans. MIAT expression levels were significantly increased in triple negative breast cancer samples and its expression correlated with NEAT1 expression. In breast cancer cell lines, MIAT expression was found to correlate with the expression of the transcription factor Oct4. MIAT down-regulation in breast cancer cells enhanced the basal apoptosis level and inhibited short and long-term survival. Induction of cell death by UV-C irradiation and chemotherapeutic drugs was also augmented in cells transfected with MIAT specific siRNA. Taken together, the outcome of this study reveals important roles for NEAT1 and MIAT lncRNAs in breast cancer. Future work should explore the potential of these lncRNAs in the development of therapeutic drugs and as diagnostic and prognostic markers.

Human mesenchymal stem cells (hMSCs) have multiple potential roles in regenerative medicine. These roles revolve around the exploitation of their multipotent differentiation potential, their immunogenic privilege, or the broad range of molecules they secrete. The functions of these molecules include stimulation of cell migration during wound repair, activation of endogenous tissue-specific stem cell pools, and suppression of T-cell-based inflammatory responses. hMSCs have been exploited clinically in the treatment of graft versus host disease following bone marrow or organ transplant via systemic administration and in other indications including ischemic heart disease, Crohn’s disease, and diabetes mellitus. However, the mechanisms underlying the regenerative potential of hMSCs are still uncertain. Moreover, the effectiveness of hMSCs-based cell therapy has been doubted because the therapeutic actions have often been noticed in the absence of hMSCs homing to the target tissues. Thus, indicating that the level of improvement of diseased tissues is not reciprocal to the observed target tissue homing/differentiation, suggesting that it may play an indirect role in tissue regeneration or it may have a dualistic mode of action. Irrespective MSCs provide a great promise in the treatment of degenerative disorders and inflammatory ailments. However, there are still many challenges to overcome prior to their widespread clinical application. For instance, their ambiguous paracrine mechanism remains a matter for controversy and exploration; moreover, current in vitro cultures have limited success in reflecting many aspects of the in vivo niche – including oxygen level. This study focused on the paracrine properties of hMSCs with a particular focus on their role in immunosuppression and anti-inflammation. Coupled to this we explored oxygen-dependent regulation of the paracrine biology of MSCs focussing on a number of important paracrine factors with roles in immunomodulation. Serum free-conditioned media (SFCM) were used as a model to characterize the immune sentinel function of MSCs. Proteomic analysis indicated that SFCM of hMSCs contained various cytokines which may play an important role in the suppression of inflammation and that the composition and concentration of SFCM was oxygen dependent. Intermittent hypoxia (IH) initiated a stark upregulation of paracrine factor secretion. The therapeutic effectiveness of SFCM was reflected via its efficacy across two in vitro cell line models; SFCM suppressed T cell model activation (Jurkat) in an oxygen independent manner while the modulation of macrophage (THP-1) terminal differentiation was oxygen sensitive. Collectively, IH SFCM suppressed the immune response at T cell and antigen presentation levels while air oxygen (21% O2) cultured hMSC SFCM supressed the immune response at T cell level and maintain the antigen presentation which might elicits the immune response. These optimized in vitro-culture findings support clinical application of hMSCs and/or their secretory factors as a pharmacoregenerative modality for the treatment of non-curative diseases, such as, rheumatoid arthritis, Crohn’s disease, myocardial infarction, and advanced critical limb diseases. Moreover, this property may be harnessed to produce an optimized acellular biological product with immunomodulatory actions similar to hMSCs, leading to production of an “off-the-shelf” biological product. The production of such biological products will have important economic considerations in cell-based therapy.

Aims: To evaluate the in vitro potential of umbilical cord(UC)-derived cells as an allogeneic cell source that could be used ‘off-the-shelf’ in orthopaedics for bone and cartilage regeneration. The study also assesses the in-vivo efficacy of cell therapy in orthopaedics for the formation of de novo bone, cartilage and integration of both. Methods: - In vitro potential of cells isolated from the four structural layers of the umbilical cord were characterised according to the criteria of the International Society for Cellular Therapy (ISCT). The differentiation potentials of these cell preparations, particularly for bone and cartilage formation, were also evaluated to ascertain their efficacy as potential cell sources for orthopaedic regenerative medicine. - Efficacy of autologous bone marrow-derived mesenchymal stromal cells (BMSC) for new bone formation in vivo for patients with lower limb long bone nonunions were assessed with a self-controlled randomised trial. - Efficacy and structural outcome of simultaneous autologous bone plug graft to restore subchondral bone with Autologous Chondrocyte Implantation (ACI) were evaluated to identify the quality and integration of the repair cartilage with the subchondral bone, described as the ‘Osplug’ technique. - Efficacy of concurrent realignment with ACI in patients with underlying chondral defects and idiopathic varus or valgus malalignments of the knee joint were studied to ascertain the outcome of simultaneous correction of the mechanical axis in patients receiving biological repair of the cartilage. Results: - Potential of UC-derived cells in bone and cartilage formation: Cell preparations from four structural regions of umbilical cord were isolated via an in vitro explant culture technique. Osteogenic differentiation in these cell preparations correlated with a significant rise in alkaline phosphatase activity in the culture medium of the differentiated cells, in comparison to their respective controls. Following chondrogenic differentiation, a considerable variation in metachromasia was noted with toluidine blue staining, although type II collagen immunostaining was predominantly absent except in one sample of cells from Wharton’s Jelly. Cells from all the four layers of UC also expressed surface markers according to the ISCT criteria for Mesenchymal Stem Cells (MSC). However, it did not conform to the recommended standards quantitatively on fluorometric analysis. - New bone formation in nonunion: There was absence of significant increase in new bone formation on the side of BMSC insertion in cases with nonunion of fracture. Four predictors of successful fracture union in this study were shorter in-vitro cell doubling times of patient’s BMSC, the absence of diabetes, younger age and fewer operative procedures to treat the nonunion before the trial intervention. - Bone and cartilage healing in osteochondral defects: Significant improvement in clinical and functional outcome was found at mid-term follow-up after concurrent bone graft and ACI to restore subchondral bone and cartilage. Integration of the grafted bone had a direct correlation with the clinical outcome in these patients. - Cartilage repair with realignment: Simultaneous ACI with correction of malalignment led to significant improvement in clinical outcome, particularly in patients with varus deformity. Patients with valgus deformity were noted to fail relatively early with poor outcome. Conclusion: The current thesis extends from exploring the in vitro potential of UC to the clinical application of autologous chondrocytes and BMSC for cartilage and bone regeneration. UCderived cells were noted to have properties akin to MSC with trilineage differentiation capacity. However, regeneration of new bone with BMSC in nonunions remains challenging. Nonetheless,significant clinical improvement was noted in patients receiving ACI with underlying malalignment and subchondral bone defect when treated with concurrent realignment and bone graft respectively. Further work on the immunomodulatory effect of UC-derived cells in addition to longer-term follow-up of the patients receiving cell-based therapy is required to consolidate our understanding of future cell therapy in orthopaedics.

Association football (soccer) is a popular sport and there is a high risk of injury for participants. Within the context of professional clubs, the risk of injury is also associated with the risk of financial costs. Therefore, injury reduction processes are considered important, and previous studies have sought to identify and model injury risk factors. Although formal screening tests e.g. The Functional Movement Screen (FMS) and monitoring procedures e.g. Union of European Football Associations (UEFA) have been developed for modelling and predicting injuries, the processes in current use, lack precision or clinical usefulness. The aims of this thesis were therefore to explore why existing methods of screening, measuring and modelling are not effective in predicting injuries. In order achieve this the following things were done; Literature review to evaluate the UEFA screening process and advocated variables, Validation of the FMS, the most commonly used exercise screening test, against a 3D photogrammetric system (Vicon (©Vicon Motion Systems Ltd)) Injury modelling on a pre-established database designed in accordance with the UEFA guidelines The literature review confirmed that the established database was compliant with the UEFA screening guidelines. The most commonly used screening measure (FMS) for injury risk was found to be an invalid measure and therefore removed from the modelling process. The models developed were unable to prospectively model injuries accurately (R = 0.23), and the primary problem was a large number of false positives i.e. those predicted as having risk of injury not sustaining injury. Reasons for poor model performance could be attributed to inappropriate screening methods, inadequate datasets or inadequate modelling methods for rare events. Future work should focus on addressing the limitations in the existing UEFA screening framework and simultaneously develop better methods of rare event modelling from small datasets.

The biologically and medically important heparan sulphate and heparin polysaccharides have previously been shown to modulate the activity of an aspartyl protease, β-secretase (BACE1), implicated in the aetiology of Alzheimer’s disease. Research groups investigating the activity of heparin with BACE1 have demonstrated both inhibitory and stimulatory effects of this glycosaminoglycan, and other analogues. In an attempt to understand this relationship, a review of the available recombinant BACE1 products was conducted to determine if protein purification tag had an effect on the heparin/heparan sulphate interaction with BACE1. FLAG-tagged BACE1 was identified as a suitable proxy for native, untagged BACE1 for activity studies. In this study, the common purification tag, IgG Fc region, has been shown to interact with heparin and other glycosaminoglycans at acid pH (pH 4.0 and pH 5.0) with heparin affinity of > 800 mM. Further investigation, first of whole immunoglobulin (IgG & IgM) and then with Fab and F(ab’)2 antibody fragments, identified heparin binding at acid pH in all fragments tested. Thermal stability studies were conducted to identify heparin/HS structural requirements for this novel interaction, which suggest sulphate is necessary but not sufficient for activity. Finally, a library of chemically sulphated non-heparin polysaccharides were utilised to identify ‘hit’ BACE1 inhibitors with beneficial BACE1 therapeutic attributes such as low molecular weight, minimal charge and attenuated off-target effects, such as anti-coagulant activity. Differential scanning fluorimetry was identified as a potential high-throughput screening assay to replace fluorescence resonant energy transfer, during the initial nonheparin polysaccharide library BACE1 inhibitor screening.