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The liver has a major role in the metabolism of both endogenous and exogenous compounds. In drug development, reliable and reproducible results regarding the toxicity of a new compound must be obtained. The gold standard is freshly isolated primary human hepatocytes; however, these are costly, difficult to obtain regularly and cannot be cultured extensively. Secondary hepatocyte cell lines are cheaper with a much longer lifespan in culture but do not accurately reflect the expression profile of a hepatocyte. The aim of this work was to promote differentiation of hepatic carcinoma cell lines towards the in vivo hepatic profile and potentially create a well defined and easily accessible model for early stage drug testing. Initial profiling of HepG2 and Huh7 for differentiation markers, transporters and enzymes was carried out by qPCR. Differentiation was attempted by use of growth factors and dimethyl sulfoxide (DMSO) for periods of up to thirty days. Analysis of differentiation markers by qPCR indicated that 1% DMSO treatment for at least 15 days promoted maturation most effectively. Cells treated with 1% DMSO were analysed for mRNA expression of selected transporters and enzymes, followed by treatment with typical inducers, Western blotting and functional assays to assess the presence and function of certain proteins. Although initial results showed potential, further analyses of the 1% DMSO treated cells were less promising. Analysis of transporter and enzyme mRNA expression revealed that many levels did not change favourably towards those observed in liver, or significantly changed from control but remained vastly removed from liver. Results from protein and induction experiments also indicated no benefit of 15-day DMSO treatment in either cell line. In conclusion, 1% DMSO treatment does not promote differentiation towards a more representative hepatic profile in either cell line; alternative methods are needed to develop a more heptocyte-like model using these cells.

Reactive oxygen species (ROS) including superoxide (O2̄), nitric oxide (NO•) and hydrogen peroxide (H2O2) are thought to play a central role in many disease processes. This thesis details the development of novel optical and electrochemical sensor platforms for the analysis of ROS. These technologies were established in response to the current limitations of existing techniques to enable greater understanding of the role of ROS in cellular pathology. The overproduction of O2̄ by mitochondria has been linked to the initiation of disease processes. Specifically, defects in the mitochondrial electron transport chain (mETC) can result in electron leakage and subsequent ROS generation. Using a gold electrode, surface-modified with cytochrome c, the amperometric detection of real-time O2̄ production from isolated mitochondria was enabled. Specific transport proteins within the mETC were chemically inhibited and the change in O2̄ flux was observed, allowing the contribution to ROS production of inhibition of mETC Complex I and Complex III to be observed. ROS-sensitive nanosensors, based on the entrapment of the fluorophore dihydrorhodamine-123 (DHR123) in a porous polyacrylamide shell, were developed. These sensors were successfully introduced into the macrophage cell line NR8383, which facilitated the analysis of intracellular ROS fluctuations following stimulation with phorbol-12-myristate 13-acetate (PMA). Nanosensors containing the pH responsive fluorophore fluorescein isothiocyanate (FITC) were also used to measure intracellular pH (pHi) in primary myoblasts derived from patients with Chronic Fatigue Syndrome (CFS). These sensors have provided new insight into the role of intracellular acidosis in this disease. Intracellular ROS-sensitive nanosensor technology was combined with custom fabricated gold microelectrode arrays to produce a novel integrated cell monitoring platform capable of reporting real-time ROS flux in both the intra- and extracellular environment. Rat macrophage cells loaded with ROS-sensitive nanosensors were seeded into wells containing functionalised, ROS-responsive, gold ring electrodes. Following stimulation of the cells with PMA it was possible to measure intracellular ROS generation using fluorescence spectroscopy. External ROS flux as a consequence of PMA stimulation was simultaneously measured amperometrically.

Much interest currently surrounds the emerging field of regenerative medicine. Cell-based therapies represent a novel therapeutic platform for the treatment of ischaemic diseases, shifting the focus clinically from the management of disease symptoms to their potential cure. Endothelial progenitor cells (EPCs) contribute to vascular repair and angiogenesis. Despite controversy surrounding the heterogeneous nature of these cells in vitro and in vivo, their vasoreparative functions suggest that EPCs have potential clinical value. The aim of this thesis is to further define the precise phenotype, angiogenic properties and potential clinical development of two EPC subtypes and to further elucidate the optimal myeloid angiogenic cell (MAC) or outgrowth endothelial cell (OEC) transplant regimen that is safe and effective prior to large-scale clinical application of these cells for therapeutic angiogenesis. EPCs were isolated from human peripheral blood and cord blood by density gradient centrifugation. Mononuclear cells were cultured on fibronectin or collagen and two different isolation protocols were utilised to obtain MACs and OECs respectively. MACs, previously referred to as early EPCs (eEPCs), were found to be M2 alternatively activated macrophages, which induced angiogenesis via release of IL8 in a paracrine manner both in vitro and in vivo. OECs were also assessed in a similar manner, and the effect of high glucose conditions and senescence on their vasoreparative function was evaluated. The effect of cell density on MAC secretome and vascular contribution was also investigated in vitro, as this represents an important Issue in the clinical and commercial development of a cytotherapy This study has provided a complete analysis of both types of EPCs and shed further light on a number of issues associated with the development of a cell-based treatment strategy for autologous or allogeneic therapy. These findings have imp011ant implications in relation to the harnessing of these cells as a cell therapy for vascular repair of ischaemic tissues.

Primary cilia are microtubule-based organelles projecting from most epithelial cells in vertebrates, with numerous essential roles chemo- and mechanosensation. A suite of inherited human conditions are caused by defects in the structure or function of primary cilia, and the study of these so-called 'ciliopathies' offers an insight into the roles of cilia in normal development and disease. Meckel-Gruber syndrome (MKS) is the most severe ciliopathy, an embryonic lethal condition affecting multiple organ systems. At the opposite end of the ciliopathy phenotypic spectrum, Leber's Congenital Amaurosis (LeA) type 5 is a ciliopathy with a phenotype restricted to the retina. These conditions provide excellent models to study the importance of cilia. The work in this project aims to elucidate the function of the primary cilium, by studying the molecular genetics of these ciliopathies, using a candidate gene approach, and by studying the proteins mutated in these diseases. Genetic screening identifies C2CD3 as a possible modifier of multi-organ ciliopathies, and suggests that LCA5 is only involved in retinal ciliopathies. Functional work focuses on three poorly-understood ciliopathy proteins: lebercilin, mutated in LCA; and meckelin and MKS1, mutated in MKS. Live-cell imaging and interaction assays suggest a role for•lebercilin in bidirectional intraflagellar transport (1FT). Meckelin is shown to interact with nesprin-2, a nuclear scaffold protein, with potential impacts on basal body positioning and non-canonical Wnt signalling. Studies of MKS1 clearly suggest that defects in the interaction between cilia proteins and the proteasome are associated with deregulation of Wnt signalling, which is causative of the ciliopathy disease state. This work will give insights into gene function and molecular mechanism of disease in complex genetic conditions. The understanding of ciliopathies at the molecular level may result in new therapeutic interventions for such conditions to modify disease progression.

The cell cytosol and the different subcellular organelles house the most important biochemical processes that control cell functions. Effective delivery of bioactive agents within cells is expected to have an enormous impact on both gene therapy and the future development of new therapeutic and/or diagnostic strategies based on single cell bioactive agent interactions. The main aim of this project was the evaluation of pH sensitive polymersomes made of poly(2-(methacryloyloxy)ethyl phosphorylcholine)-poly(2- (diisopropylamino)ethyl methacrylate) (PMPC-PDPA) block copolymer as a potential vector for intracellular delivery applications. Upon internalization through endocytosis, polymersomes were demonstrated to disassemble, triggering an increase in osmotic pressure within the endosomal compartments. This increase in pressure temporally destabilizes the endosomal membrane and facilitated the release of the polymersome payload within the cell cytosol. Biocompatibility of polymersomes and their uptake kinetics by different cells (both primary cells and cell lines) were assessed by Confocal Laser Scanning Microscopy (CLSM), Transmission Electron Microscopy (TEM), Flow Cytometry and Fluorescence Spectroscopy. The cellular-uptake kinetics was strongly dependent on the polymersomes surface chemistry, size and surface topology. The latter is controlled by the extent of polymer-polymer phase separation within the external envelope of the polymersome. Polymersomes were also successfully used as efficient vectors for the delivery of DNA, functional proteins and different imaging probes.

Transient protein production by cultured mammalian cells from transfected episomal DNA is frequently used in bioindustry to generate small quantities of candidate therapeutic products during early stages of process development. However, transient production processes typically exhibit low productivity, limiting their use at scale. In this thesis, three distinct but complementary approaches were evaluated for the de novo design of a high productivity scaleable transient production process starting with the discrete raw materials: transfection reagent, Chinese hamster ovary cell line, plasmid DNA and chemically defined medium. (1) Optimisation of CHO host cell transfection: The optimal combination of continuous basal parameters underpinning polyethylenimine (PEI) mediated transfection (relative concentrations of PEI, plasmid DNA and cells) was determined utilising Design of Experiments (DoE) methodology. Optimum transfection conditions were cell line specific - highly dependent upon resistance to PEI cytotoxicity. Comparing different CHO cell hosts operating at their unique optima, variations in specific productivity were limited by the rate of polyplex endocytosis. (2) Modulation of the cell culture environment: Combinations of environmental variables were evaluated using factorial screening to determine an optimal cell culture regime for transient production. For the CHO cells used in this study, the addition of valproic acid, recombinant insulin-like growth factor and a reduced culture temperature were found to interact synergistically to maximise recombinant product yield at an increased cell concentration. (3) Production process design: Utilising response surface modelling to determine key process interactions, transient transfection and medium environment optima were effectively combined to create an intensified, high cell density process exhibiting a five-fold increase in volumetric titre. Combining these approaches, volumetric yield for a transient monoclonal antibody production process was increased from 2 mg L-1 to > 90 mg L-1 - the highest transient volumetric titre achieved with un-genetically modified CHO cells in a chemically defined environment to date.

The correct development of multicellular organisms is dependent upon the communication of individual cells by signalling. Negative feedback loops are often key, as once a signalling pathway has been activated, and the resulting changes in gene expression implemented, the pathway must be attenuated. Misregulation of these processes can lead to diseases such as cancer. The Ras pathway is a well-characterised example, being overactive in many types of cancer. In C. elegans, it has been proposed that attenuation of Ras can occur through the action of sumoylation on chromatin factors. To this end, we performed an RNAi screen in a SUMO-sensitised background, in which we targeted predicted C. elegans chromatin factors. We identified BET-1, a double bromodomain protein, and subsequently showed that it interacts genetically and physically with the sumoylation pathway to prevent hyperactivation of Ras signalling. Loss of BET-1 caused phenotypes consistent with excessive Ras signalling in the vulva and germline, and led to increased Ras signalling at a global level in the worm. Loss of both BET-1 and SUMO led to a striking effect on fluid homeostasis, and loss of muscle mass, in adult worms. This synthetic phenotype was found to be caused in part by overexpression of the FGF receptor, EGL-15. To summarise, the RNAi screen successfully identified BET-1 and other candidates as novel regulators of Ras signalling. We have shown that BET-1 alone has a role in preventing excessive Ras signalling in C. elegans, and that BET-1 and SUMO work together to maintain fluid homeostasis and muscle mass. In conclusion, the work presented in this thesis shows that BET-1 and SUMO cooperate to prevent excessive Ras signalling, and is consistent with a model in which BET-1 and SUMO perform this function by acting as part of a negative feedback loop.