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In recent years there has been a reported requirement by the pharmaceutical and agrochemical industry that there is a need to monitor In vitro cell toxicity in real time, and also a necessity for using cell models that mimic the In vivo situation more closely. The aim of this project was to provide a solution for these key issues set out above. This was achieved by developing, characterising and implementing the use of electrochemical micro-biosensors and micro-sensors manufactured from screen printed electrodes. These were then used for monitoring cytotoxicity in HepG2 spheroids which mimic more closely their corresponding organ in vivo. In chapter two a new method of using screen-printed carbon electrodes (SPCEs) incorporating the electrocatalyst cobalt phthalocyanine (CoPC) for the manufacture of microband electrodes for hydrogen peroxide detection is described. Tubular microband electrode (TMBEs) fabricated using an organic based ink displayed superior properties over plain microbands. TMBEs were demonstrated to display steady state currents which, is indicative of microelectrode behaviour. The current density values obtained from the voItammogram was compared to that obtained for a conventional sized CoPC-SPCE, and the values were 5618 and 35.65 μA cm -2, respectively. Cyclic voltammetry was carried out for the same electrodes; using 7 mM H202 prepared in phosphate buffer at scan rates between 1 and 50 mV s-I and no significant increase in current response was observed. The application of these tubular microband CoPC-SPCEs, to the measurement of H202 using chronoamperometry was investigated. A calibration study was performed and the plot showed a sensitivity value of 252 μA mM-1 cm-2 and a lower detection limit of70 μM. Although this data was promising for later biosensor construction it is known that the organic based ink used to screen-print the sensors would not allow for direct incorporation of the enzyme which would be later incorporated to allow for monitoring of biomarkers important in cell toxicity. Therefore, in chapter three microband electrodes screen-printed using a water based ink were investigated which would allow for enzyme incorporation in to the inks subsequent to screen-printing and thus allowing for the formation of biosensors. Plain microband electrodes fabricated from this water based carbon ink displayed superior characteristics over the TMBEs fabricated from the same ink for hydrogen peroxide monitoring, this included higher current density values, stir independence and these were more reproducible. Therefore the plain microband was the design choice for future sensor and biosensor construction. In chapter three and four screen-printed sensors and biosensors were constructed using a water based ink with the incorporation of lactate oxidase or glucose oxidase. In chapter 5, organic based carbon ink incorporating Meldola's blue was added prior to printing allowing for a one step print process. These were then cut forming microband biosensors and sensors, allowing for the monitoring of the analytes lactate, glucose and NADH respectively. Lactate microband biosensors could monitor lactate and was measured over a dynamic range of 1-10 mM which was linear up to 6mM; a calculated lower limit of detection of 289 μM was ascertained. Microband sensors for glucose determination were used and could measure glucose over the concentration range 1-14 mM. A linear range was obtained up to 6 mM glucose (y = 3.05x) giving a R2 value of 0.99 and an intra-electrode coefficient of variation over this range of 10%. A sensitivity value over this range of 3.1 nA mM-I was obtained with a theoretical lower detection limit of 258 μM. Microband sensors incorporating the electrocatalyst were used to measure NADH over a dynamic range 0.2-3.5 mM. A linear range was obtained up to 400 IlM NADH (y = 0.0076x + 0.7665) giving a R2 value of 0.999 and a typical inter-electrode coefficient of variation over this range of 18% (n=5). A sensitivity value over this range of 7.6 nA mM-I was obtained with a theoretical lower detection limit of 63 IlM. The microband screen-printed sensors for lactate, glucose and NADH were then used to monitor cytotoxicity by monitoring the release of their corresponding analyte. This was performed over a 24 hour period in which HepG2 spheroids were exposed to the model hepatotoxin galactosamine. The results provide proof of principle that the developed electrochemical sensors could follow the above biomarkers which were good indicators of cell induced toxicity caused by the galactosamine.

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Infection with murine cytomegalovirus (MCMV) induces a large population of virusspecific CDS· T-cells that is maintained at a high frequency in the peripheral organs for the lifetime of the host. This striking response to MCMV is termed "memory inflation". It has also been shown that a similar response occurs after immunization with a non-repl icating adenovirus expressing a transgene. The key features which distinguish "infiating" CDS· T-cell memory responses from classica l "non-infiating" CDS· T-cell memory responses have not been fully defined. To determine the molecular signature of memory inflation I sorted antigen-specific ce lls ex vivo and compared the gene expression profiles of infiationary CDS· T-cells to those of central memory and effector CDS+ T-cells. This data showed that memory inflation has a distinct expression profile, with high expression of KLR receptors, specific chemokine receptors such as CX3CR1, transcription factors such as T-bet and Foxk1, and survival factors, such as Bcl-XL and down regu lation of inhibitory molecules such as BTLA. These inflationary cells are functional - expressing an array of cytokines, granzymes, IFNy, and TNFa, and lack transcriptional features of T-cell exhaustion. Similar features were identified in CDS· T-cells undergoing memory inflation using both the MCMV and adenovirus models. To address the turnover of these "inflationary" CDS+ T ce lls and their role in vivo, I created tetramers bound to the toxin saporin to specifically knock out inflationary cells. I found that they contribute to the control of vira l repl ication, since depletion was followed by a rise in viral load and a subsequent rebound increase in the tetrarner+ popul ation. In the final chapter I addressed the ro le of one specific cel l surface molecule, CD73, during CDS· T-cell memory. I found that although CD73 is thought to playa role in immune regu lation, CD73-1 - mice showed normal memory inflation and class ical noninflationary memory development. Overall the data in this thesis define for the first time the transcriptional profile associated with CDS· T-cell memory and show the distinct nature of this T-cell programme in vivo. Future work using further genetically modified mouse strains should allow definition of the critical molecules and pathways involved in the development of these important memory pools.

Natural killer (NK) cells kill abnormal cells through the exocytosis of secretory lysosomes containing the cytotoxic proteins perforin and granzymes. Secretory lysosome exocytosis requires the movement of secretory lysosomes along microtubules to cluster at the microtubule organising centre (MTOC), before polarisation to the immunological synapse and fusion with the plasma membrane. The aims of this thesis were to investigate the roles of syntaxin 11, Rab7 and Arl8b in secretory lysosome exocytosis. Familial haemophagocytic lymphohistiocytosis subset 4 (FHIA) is caused by mutation of syntaxin 11, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein. NK cells from FHL4 patients have a defect in secretory lysosome exocytosis. Analysis of syntaxin 11 in the NK cell line, YTS and primary NK cells demonstrated that this SNARE partially localises to early endosomes, and that it was membrane associated and palmitoylated. Membrane localisation in YTS cells was abolished ill the syntaxin 11 FHIA mutants Gln268X, Leu194ProfsX2 and Va1l24fsX60, which may be due to loss of the potentially palmitoylated C-tenninal region of this SNARE. Consistent with this, syntaxin 11 in which C-terminal cysteine residues were mutated was mislocalised in YTS cells, whilst additional disruption of SNARE domain interactions abrogated membrane localisation. Rab7 promotes lysosome movement along microtubules such that they cluster around MTOC, whilst Arl8b promotes lysosome movement towards the cell periphery. Rab7 and Arl8b may therefore play antagonistic roles in secretory lysosome movement along microtubules in NK cells. Overexpression of Rab7 promoted secretory lysosome clustering in YTS cells, suggesting an increase in MTOC-directed movement along microtubules, although a dominant negative Rab7 mutant did not affect secretory lysosome exocytosis in primary NK cells. Overexpression of dominant negative Arl8b mutant promoted secretory lysosome clustering in YTS cells, and increased secretory lysosome exocytosis in primary NK cells. This suggests Arl8b may inhibit secretory lysosome exocytosis in NK cells.

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