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Glioblastoma (GBM) is a highly aggressive and fatal brain cancer that is associated with a number of diagnostic, therapeutic, and treatment monitoring challenges. At the time of writing, inhibition of a protein called poly (ADP-ribose) polymerase-1 (PARP-1) in combination with chemotherapy was being investigated as a novel approach for the treatment of these tumours. However, human studies have encountered toxicity problems due to sub-optimal PARP-1 inhibitor and chemotherapeutic dosing regiments. Nuclear imaging of PARP-1 could help to address these issues and provide additional insight into potential PARP-1 inhibitor resistance mechanisms. Furthermore, nuclear imaging of the translocator protein (TSPO) could be used to improve GBM diagnosis, pre-surgical planning, and treatment monitoring as TSPO is overexpressed by GBM lesions in good contrast to surrounding brain tissue. To date, relatively few nuclear imaging radiotracers have been discovered for PARP-1. On the other hand, numerous tracers exist for TSPO many of which have been investigated in humans. However, these TSPO radiotracers suffer from either poor pharmacokinetic properties or high sensitivity to human TSPO polymorphism that can affect their binding to TSPO. Bearing in mind the above and the high attrition rates associated with advancement of radiotracers to the clinic, there is a need for novel radiotracers that can be used to image PARP-1 and TSPO. This thesis reports the pre-clinical discovery programme that led to the identification of two potent PARP-1 inhibitors, 4 and 17, that were successfully radiolabelled to generate the potential SPECT and PET imaging agents [123I]-4 and [18F]-17 respectively. Evaluation of these radiotracers in mice bearing subcutaneous human GBM xenografts using ex vivo biodistribution techniques revealed that the agents were retained in tumour tissue due to specific PARP-1 binding. This thesis also describes the pre-clinical in vivo evaluation of [18F]-AB5186, which is a novel radiotracer discovered previously within the research group with potential for PET imaging of TSPO. Using ex vivo autoradiography and PET imaging the agent was revealed to accumulate in intracranial human GBM tumour xenografts in good contrast to surrounding brain tissue, which was due to specific binding to TSPO. The in vivo data for all three radiolabelled compounds warrants further pre-clinical investigations with potential for clinical advancement in mind.

Molybdo-flavoenzymes (MFEs), aldehyde oxidase (AOX) and xanthine oxidoreductase (XOR) are involved in the oxidation of N-heterocyclic compounds and aldehydes, many of which are environmental pollutants, drugs and vitamins. This biotransformation generally generates more polar compounds that are more easily excreted, thus MFEs have been classed as detoxication enzymes. To date there has been scant study of the properties, substrate and inhibitor specificities of MFEs in non-mammalian vertebrate organisms. This investigation focuses on MFEs in rainbow trout (Oncorhynchus mykiss) as it belongs to a class of fish that host a single AOX (AOXβ) and one XOR. In this study the substrate specificity of rainbow trout liver AOX and XOR was investigated using HPLC and spectrophotometric assays. AOX in hepatic cytosol was found to be able to catalyse the oxidation of azanaphthalenes belonging to a group of compounds that are environmental pollutants such as phenanthridine, phthalazine and cinchonine. In addition, xenobiotic aromatic aldehydes (vanillin and dimethylaminocinnamaldehyde) and drugs such as allopurinol and pyrazinamide were substrates. Several endogenous vitamins including pyridoxal (vitamin B6), all-trans retinal (vitamin A) and N1-methylnicotinamide were also biotransformed by the rainbow trout AOX. In contrast to liver no AOX activity was detectable in kidney and gill tissue. XOR activity in rainbow trout liver was measurable with the endogenous purine xanthine, purine drug metabolites (1-methylxanthine and 6-thioxanthine) and N-heterocyclic drugs (allopurinol and pyrazinamide). Unlike mammalian XOR that can utilise both NAD+ and O2 as electron acceptors, trout XOR was exclusively NAD+-dependent with no activity being detected with O2. Eadie-Hofstee plots were using to determine the Km and Vmax of rainbow trout AOX and XOR with different substrates and it was found the Vmax of the rainbow trout enzymes were generally lower and Km generally higher than mammalian AOX and XOR. Inhibitors of mammalian AOX were tested to determine if they could interact with the piscine AOX. Environmental pollutants (17α-ethinyl estradiol and phenanthridine), an endogenous steroid (estradiol) and drugs (chlorpromazine and menadione) were found to be effective inhibitors and were classed as competitive, non-competitive and uncompetitive respectively using Lineweaver-Burk plots. The drug metabolite, oxipurinol, was a non-competitive inhibitor of rainbow trout XOR. In order to further characterise trout AOX protein purification was carried out. In contrast to mammalian AOX, the piscine enzyme was not thermotolerant at 55°C nor was it inhibited by benzamidine, thus heat treatment and affinity chromatography could not be used as a purification steps. Trout AOX was purified 210-fold using ammonium sulphate fractionation, together with ion exchange and gel filtration chromatography. The native molecular mass of the piscine AOX was 295 kDa, which is similar to mammalian AOXs. In conclusion this study yields new insight into groups of anthropogenic environmental pollutants, drugs and vitamins that are substrates and inhibitors of an ancestral vertebrate AOX. The toxicological relevance of these findings is discussed.

CD40 is a member of the tumour necrosis factor receptor (TNFR) superfamily and ligation by membrane- presented CD40 ligand (mCD40L), but not soluble agonists, causes extensive apoptosis in malignant epithelial cells, including colorectal carcinoma (CRC) cells. This thesis aimed to unravel the precise cell signalling pathways responsible for mCD40L-mediated apoptosis in CRC cells. This study has provided evidence that CRC cell death by mCD40L is rapid. mCD40L activated MOMP, cytochrome c release from mitochondria and induction of Bak/Bax within <6 hours post ligation. The pro- apoptotic role of Bax was confirmed by shRNA-mediated Bax knockdown as this attenuated apoptosis and decreased caspase 3/7 activity. mCD40L triggered rapid TRAIL induction and a caspase-dependent pathway that involved caspase-10 (but not caspase-8) and caspase-9 to cause CRC cell death. Thus CD40 cross-talks with the extrinsic pathway by inducing TRAIL-mediated, caspase-10 activation, mitochondrial disruption, tBid activation, Bak/Bax induction, and activation of caspase-9 and -3/7 to cause CRC cell death. When the signalling pathways triggered by CD40 were studied further, we found that CD40 induced both p- JNK and p-p38 in CRC cells which is necessary for apoptosis, and that JNK might be acting downstream of p38. p38 and JNK directly regulated Bak/Bax and TRAIL induction at the transcriptional level. We also showed that TRAF1, -3, and 6 were induced in CRC cells as early as 1.5 hours post ligation. Our studies not only demonstrated a novel pattern of TRAF regulation in CRC cells but revealed for the first time that TRAF3 has an essential role in CD40-mediated CRC cells death. TRAF3 is central in the induction of apoptosis as its knockdown attenuates apoptosis, by abrogating p38 and JNK activation, induction of Bak/Bax and caspase-3/7 activation. Therefore, despite the existence to a dual apoptotic pathway being engaged in CRC cells, TRAF3 appears to be central in both signalling axes. ROS are rapidly induced in CRC cells by CD40 in a Nox-dependent fashion and this plays an important role in CD40-mediated killing. More specifically, CD40 activation appears to result in TRAF3-dependent p40phox activation. CD40 also regulates directly ROS scavenging mediators, as we detected reduction in Trx-1 expression. Moreover, CD40 triggered activation of the Trx-regulated pro-apoptotic kinase ASK-1, which provided direct molecular explanation for the importance of ROS in CD40 signalling and downstream activation of MKKs and p38/JNK. Thus, the mCD40L-CD40-TRAF3-NOX axis utilises ROS for the activation of ASK-1/MKK/p38/JNK pro-apoptotic pathways in CRC cells. Based on observations in this thesis and more recent findings following completion of this work, we hypothesise that at some point the MAPK/p38/JNK pathway diverges to drive on one hand transcriptional upregulation of TRAIL, activation of tBid and cross talk to the mitochondria, whilst the other p38/JNK pathway directly induces Bak/Bax to also induce MOMP and mitochondrial death, the latter being more reminiscent of CD40-mediated cell death in UCC cells. However, unlike UCC cells were the operation of only the latter pathway takes place means apoptosis requires a minimum of 24-36 hours to occur, in CRC cells there is rapid amplification of the apoptotic signal and quick induction of death. To our knowledge, this is the first demonstration of such extensive and rapid carcinoma cell apoptosis triggered by CD40 ligation. Overall, this study has identified the intracellular signalling cascade triggered by CD40 ligation and results in extensive apoptosis in CRC cells. It has identified a TRAF3-Nox-ROS-ASK1-MKK-p38/JNK pathway (that activates caspase-10 and caspase-9) as the driving force that triggers both a TRAIL-associated extrinsic as well as the intrinsic apoptotic pathways. Thus, in CRC cells CD40 induces apoptosis by pathway cross talk which permits strikingly rapid apoptosis. These findings not only provided novel observations on the mechanisms of apoptosis triggered by the TNSRF member CD40, and also reinforced the importance of the quality of CD40 signal in determining functional outcome, but they have also raised interesting hypotheses for further biological studies. Equally importantly, the findings have also assisted in the formulation of a novel combinatorial therapeutic approach that may exploit CD40 for anticancer therapy.

The work presented herein covers a broad range of research topics and so, in the interest of clarity, has been presented in a portfolio format. Accordingly, each chapter consists of its own introductory material prior to presentation of the key results garnered, this is then proceeded by a short discussion on their significance. In the first chapter, a methodology to facilitate the resolution and qualitative assessment of very large inorganic polyoxometalates was designed and implemented employing ion-mobility mass spectrometry. Furthermore, the potential of this technique for ‘mapping’ the conformational space occupied by this class of materials was demonstrated. These claims are then substantiated by the development of a tuneable, polyoxometalate-based calibration protocol that provided the necessary platform for quantitative assessments of similarly large, but unknown, polyoxometalate species. In addition, whilst addressing a major limitation of travelling wave ion mobility, this result also highlighted the potential of this technique for solution-phase cluster discovery. The second chapter reports on the application of a biophotovoltaic electrochemical cell for characterising the electrogenic activity inherent to a number of mutant Synechocystis strains. The intention was to determine the key components in the photosynthetic electron transport chain responsible for extracellular electron transfer. This would help to address the significant lack of mechanistic understanding in this field. Finally, in the third chapter, the design and fabrication of a low-cost, highly modular, continuous cell culture system is presented. To demonstrate the advantages and suitability of this platform for experimental evolution investigations, an exploration into the photophysiological response to gradual iron limitation, in both the ancestral wild type and a randomly generated mutant library population, was undertaken. Furthermore, coupling random mutagenesis to continuous culture in this way is shown to constitute a novel source of genetic variation that is open to further investigation.

A novel screening method for detecting lignin-degradation activity on agar plates was developed using nitrated lignin. Using this method, ten lignindegrading bacteria have been isolated from environmental sources, including seven mesophilic soil bacteria and three thermotolerant strains from composted wheat straw. All of the isolates have demonstrated activity towards lignin degradation in the assays, the most active strain being a thermotolerant Sphingobacterium strain from the Bacteroidetes family. The ability of each strain to degrade a variety of aromatic carbon sources and size-fractionated Kraft lignin has been examined by laboratory-scale growth experiments and gel filtration chromatography respectively, and the bioconversion of different lignin-containing feedstocks by three of the most active strains has been examined in a series of laboratory-scale fermentation experiments. Purification of extracellular lignin-degrading enzymes from the culture supernatant of Sphingobacterium sp. has highlighted several different enzyme activities and possible lignin-degrading enzymes.

The ecophysiology of calcified macroalgal species of the genera Corallina (C. officinalis and C. caespitosa) and Ellisolandia (E. elongata) (Corallinales, Rhodophyta) was examined in intertidal rock pools of the NE Atlantic, to facilitate predictions of ocean-acidification and warming impacts on these ecosystem engineers. An initial phylogenetic study highlighted significant cryptic diversity within the genus Corallina, and demonstrated that C. officinalis is restricted predominantly to the North Atlantic, while the recently established C. caespitosa shows a cosmopolitan distribution. Three subsequent studies were performed across the NE Atlantic (Iceland to northern Spain) to examine (i) the production, respiration, calcification and growth of Corallina in relation to irradiance, water temperature, and carbonate chemistry; (ii) the photoacclimation and photoregulation strategies of Corallina and Ellisolandia; and (iii) the recent-past (1850 – 2010) and present-day skeletal mineralogy (Mg/Ca ratios) of Corallina and Ellisolandia and its relationship to sea surface temperature. Data demonstrated that species currently experience significant seasonal and tidal fluctuations in abiotic conditions that may be important when considering future responses to ocean-acidification and climate-change. Seasonality in production, calcification and growth were demonstrated, with decreasing growth observed with increasing latitude. Photoacclimation to allow maximal light utilisation during winter periods, and photoregulation via nonphotochemical quenching were highlighted as important in allowing Corallina and Ellisolandia to maintain maximal productivity while controlling for photo-stress. Seasonal cycles in skeletal Mg incorporation were demonstrated with strong relation to sea surface temperature, though no significant change in skeletal mineralogy was evident since pre-industrial times. Taken together, data indicated that Corallina and Ellisolandia have the potential to survive under future ocean-acidification and warming conditions, though loss of species at high latitudes and shifts in the relative abundances of species across the region is likely to be evident, with overall range contraction predicted for C. officinalis due to both warming and ocean-acidification impacts.