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This thesis focuses on the influence of selenium supply on the expression of selenoproteins and possible mechanisms involved. It examines the effects of selenium on the expression of cytosolic glutathione peroxidase (cGSH-Px), phospholipid hydroperoxide glutathione peroxidase (PHGSH-Px) and type I iodothyronine 5'deiodinase (IDI) in liver, thyroid and heart of the rat. Nutritional studies in animals fed diets with different selenium content have shown that there is differential regulation of the three mRNAs and subsequent protein synthesis within and between organs, suggesting both that mechanisms exist to channel selenium for synthesis of a particular selenoprotein and that there is tissue-specific regulation of selenoprotein mRNAs. Since selenium supply could regulate selenoprotein expression by either transcriptional or post-transcriptional mechanisms, the effects of selenium depletion on the transcription rate of the three genes and their mRNA stability were investigated. Nuclear run-off assays with isolated liver nuclei showed selenium deficiency to have no effect on transcription of the three genes whereas actinomycin D chase experiments, in hepatoma cells, showed that selenium deficiency had no effect on the stability of PHGSH-Px mRNA but decreased the stability of cGSH-Px mRNA. Additionally, studies with hepatoma cells transfected with chimeric constructs of IDI coding region linked to different 3'UTRs showed that cGSH-Px 3'UTR is less efficient than PHGSH-PX 3'UTR in permitting Se-cysteine incorporation when selenium is limiting. In conclusion, the mechanisms of selenoprotein regulation in selenium deficiency involve post-transcriptional controls: in liver, selenium deficiency affects translation of cGSH-Px and PHGSH-Px mRNAs at different extents and differences in the 3'UTR of these two mRNAs could partially explain the differential effect of selenium deficiency on cGSH-Px and PHGSH-Px activities and mRNA abundance, stability and translation.

Rapeseed pomace (RSP) is the waste/by-product obtained after edible oil production from Brassica napus and is currently used as animal feed. In an attempt to revalorize this by-product as a potential nutraceutical for the treatment or prevention of neurodegenerative disease, this project aimed to determine (i) a suitable extraction technique, (ii) the phytochemical composition and in vitro activity of the extract, in relation to neuroprotective properties, (iii) the neuroprotective potential of the extract in a cellular model (SH-SY5Y neuroblastoma cells) and (iv) the extract's potential to prevent/treat neurodegenerative disease in one Machado-Joseph disease (MJD) and three Parkinson's disease (PD) C. elegans (nematode) models. To achieve this aim at the start of the project, three extraction techniques (i.e. Soxhlet, ultrasonic assisted and accelerated solvent extraction) were employed, using a solvent mixture of ethanol and water (95:5) on the RSP samples obtained from the north east of Scotland. Based on the chemical composition of the extracts and their antioxidant properties, Soxhlet extraction was revealed as the most promising and practical extraction technique. Bulk extraction was carried out, to obtain enough RSP extract for the duration of the project. Thereafter, the composition of the extract was further investigated, to show sinapine to be the most abundant secondary metabolite, together with other phenolic acids, such as sinapic, ferulic, caffeic and syringic acid. The final extract showed in vitro antioxidant and acetylcholinesterase inhibition activity, the potential to protect plasmid DNA from oxidative damage, copper ion chelating potential and the inhibition of self-mediated β-amyloid (1-42) aggregation. The latter in vitro characteristics and properties could be beneficial for the protection of neurons from oxidative stress induced degeneration. In the SH-SY5Y neuroblastoma cell line, the RSP extract was found to be non-toxic up to a concentration of 1.5 mg/mL. Subsequent cellular studies using 1 mg/mL or less of the RSP extract, showed its ability to protect the cells from reactive oxygen species (ROS) and oxidative DNA strand breakage induced by hydrogen peroxide. In addition, using protein array technology, the RSP extract was able to down regulate cell stress associated proteins SIRT2 and SOD2. In the C. elegans nematode model, the RSP extract showed no toxicity up to 5 mg/mL. The RSP extract was able to improve the disease phenotype in the MJD model (motility deficiency) as well as in all three PD models (dopaminergic neuronal loss). This improvement was at least partially dependent on the activation of the antioxidant gene glutathione-s-transferase (gst-4) in C. elegans. Overall, the RSP extract showed very positive in vitro characteristics and valuable in vivo effects in 4 disease C. elegans models, thus warranting further detailed studies on the use of RSP extract to help prevent and/or treat neurodegenerative diseases.

Breast cancer is the most commonly occurring cancer in women, with incidence rates approaching 1.38 million cases per year worldwide. Over the last few decades, there have been numerous attempts to develop, synthesise and advance into the clinic novel and selective breast cancer therapies. Research work has shown that bisnaphthalimidopropyl diaminodicyclohexylmethane (BNIPDaCHM) exerts potent in vitro anti-cancer activities and strong DNA binding properties. The aim of this thesis was to synthetise novel bisnaphthalimidopropyl derivatives (BNIPs) and investigate their subsequent modes of action within two human metastatic breast cancer cell lines, MDA-MB-231 and SKBR-3. A series of novel BNIPs, bisnaphthalimidopropyl-piperidylpropane (BNIPPiProp), bisnaphthalimidopropyl- ethylenedipiperidine (BNIPPiEth) and (trans(trans))-4,4’-methylenebis-cyclohexylamine (trans,trans-BNIPDaCHM) were synthesised, characterised and studied in comparison to BNIPDaCHM for their DNA binding and anti-cancer activities against MDA-MB-231 and SKBR-3 cells. Thermal denaturation studies have shown that BNIPs can intercalate and stabilize the double helix of Calf Thymus, each BNIP can competitively displace EtBr from DNA in a dose dependent manner and by UV binding studies, high affinity was found for the three novel BNIPs. After 24 hours treatment, all novel BNIPs, exhibited strong cytotoxicity with IC50 values ranging from 1.4 μM to 3.3 μM in MDA-MB-231 cells and 0.2 - 0.7 μM in SKBR-3 cells, confirming the importance of bisnaphthalimidopropyl functionality. BNIPs were also found to increase intracellular ROS levels after 8 hours treatment and induce a significant increase in DNA strand breaks compared to endogenous levels, after 24 hour treatment in both cell lines. After cell synchronisation, cell cycle distribution was studied, revealing that trans,trans-BNIPDaCHM induces sub-G1 cell population arrest in MDA-MB-231 and SKBR-3 cells, after 24 hours treatment. In addition, BNIPs induced apoptotic phosphatidylserine exposure, after 0.5 hours treatment, inhibited Caspase-3 activity and increased autophagy, after 24 hour treatment in MDA-MB-231 and SKBR-3 cells. Moreover, BNIPs inhibited histone deacetylases (HDAC) activity after 24 hours treatment in MDA-MB-231 and SKBR-3 cells and BNIPDaCHM was identified as a potential SIRT2 inhibitor, in SKBR-3 cells. According to Proteome Profiler Arrays, BNIPDaCHM and BNIPPiEth altered the expression of cell stress-related proteins in a cell dependent manner and bioinformatic analysis revealed two novel, putative pathways for BNIP-induced oxidative stress-mediated cell death in MDA-MB-231 and SKBR-3 cells. The above findings indicate that BNIPs represent promising candidates for future breast cancer studies and cancer treatment.

Type 2 diabetes and metabolic syndrome are the metabolic disorders which constitute a major public health problem in both developed and developing countries. Various studies have suggested the genetic susceptibility to the disorders. The main aim of the thesis was to investigate the putative association of single nucleotide polymorphisms with Type 2 diabetes (T2D), metabolic syndrome (MetS) and the major components of metabolic syndrome. This study used meta‐analysis, polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP) and Sanger sequencing methods to analyse the results. The single nucleotide polymorphism rs57829442 of peroxisome proliferator‐activated receptor‐γ coactivator‐1 (PPARGC1A) gene and its relation to risk of type 2 diabetes has been studied in the United Kingdom population. A meta‐analysis of genetic variant rs8192678 (Gly482Ser) of peroxisome proliferator‐activated receptor‐γ coactivator‐1 (PPARGC1A) gene and its association with the components of metabolic syndrome has been studied. An association of the genetic variants rs8192678 (Gly482Ser) of the PPARGC1A gene, rs7903146 of Transcription Factor 7 Like 2 (TCF7L2) gene, rs9939609 of Fat mass and obesity‐associated (FTO) gene and rs1801282 (Pro12Ala) of peroxisome proliferator‐activated receptor gamma (PPARG) gene with the metabolic syndrome and its components has been studied in the Nepalese population. The results showed that variant rs57829442 of PPARGC1A is not associated with T2D in the United Kingdom population. Further investigation with increased sample size is warranted. In the meta‐analysis, the variant rs8192678 (Gly482Ser) of PPARGC1A gene was found to be significantly associated with body mass index (BMI) in Asian populations under dominant genetic model, total cholesterol (TC) in non‐Asian population under recessive genetic model and with fasting plasma glucose (FPG) under a recessive model in overall and non‐Asian populations. No significant association of the variants rs8192678 (Gly482Ser), rs7903146, rs9939609 and rs1801282 (Pro12 Ala) was found associated with MetS under dominant, recessive, co‐dominant and additive models in the Nepalese population. However, the genotypes (AG and AA) of rs8192678 (Gly482Ser) had a statistically significant protective effect on systolic blood pressure. The genotypes with the risk allele of rs9930609 of FTO gene was significantly associated with weight, waist circumference and diastolic blood pressure under dominant genetic model and with BMI under both dominant and recessive genetic models in the Nepalese population. To the best of our knowledge, this is the first study to report the findings in the Nepalese population.

Obesity has reached worldwide epidemic proportions and with this increased incidence of obesity, comes an increase in incidence of the comorbidities associated with obesity such as diabetes and cardiovascular disease (CVD). The underlying mechanisms which connect these diseases are still poorly understood. One system which has been shown to be up-regulated in the setting of obesity and diabetes is that of the G-protein coupled receptor-55/Lysophosphatidylinositol (GPR55/LPI). Despite being upregulated in the setting of obesity, the function of GPR55 in obesity and other disease states remains elusive. Therefore, the present study aimed to 1) investigate the role of GPR55 in obesity by characterising the phenotype of the GPR55 knockout (GPR55-/-) mouse when challenged with a high fat diet (HFD) intervention, 2) elucidate any effect of the GPR55 knockout and HFD intervention on the myocardial infarct size sustained following a period of ischaemia/reperfusion (I/R) and 3) make use of an in vitro model to elucidate the mechanisms by which changes occur in the adipose tissue of mice fed a HFD. GPR55-/- mice fed a HFD for 12-weeks gained significantly more weight in the form of fat mass, compared to wild-type (WT) controls and consequently become obese. Obese GPR55-/- mice displayed hypertrophic adipose tissue concurrent with the significant dysregulation of plasma lipids, increases in specific circulating LPI species, increased lipid deposition within the liver and a change in adipose tissue gene expression profile. These changes were not observed in GPR55-/- mice fed a standard diet or WT mice fed a HFD. Following a period of I/R, the myocardial infarct size in hearts from WT HFD fed mice was significantly smaller than in hearts from WT standard diet fed mice. This reduction in infarct size due to HFD intervention was not dependent on RISK-pathway activation and was not observed in hearts from GPR55-/- mice, therefore demonstrating that the cardio-protective effect of a HFD on infarct size is dependent on GPR55. In vitro studies using 3T3-L1 cells determined that the changes in adipose tissue gene expression of HFD fed mice was not due to enhanced stimulation with LPI or via hypoxic mechanisms. The results of these studies demonstrate that GPR55 has an anti-obesity function in vivo and also mediates the cardio-protective effect of a HFD on myocardial infarct size, through currently unknown mechanisms.

Surface interactions and reactivity are of critical importance in current biomedical technologies, for example, satisfactory cell attachment and long term viability are essential for optimal in vitro tissue culture and for successful implantation and stability of cardiovascular medical implants such as stents and grafts. To achieve this, the control of fundamental forces and the resulting molecular interactions between the relevant surface and absorbing or adhering species in the physiological system is compulsory. This work utilised the surface modification technique of Ultra-Violet/ Ozone to improve the polystyrene biocompatibility by oxidising the surface with additional polar oxygen functional groups without damaging the surface bulk property. UV/Ozone treatment utilised throughout this study produced controllable oxygen functional groups and led to an increase in surface atomic oxygen level to 41% on unwashed and 35% on washed polystyrene surfaces, washing resulted in the removal of low molecular weight oxidised materials. Surface energy was increased by the addition of oxygen functional groups with the combination of alcohol (C-OR), carbonyl (C=O) and carboxyl (O-C=O); Saturation state was reached after 300s of UV/Ozone treatment where no more oxygen functionalities were incorporated to the surface. Moreover, UV/ozone treatment did not show an effect on the surface roughness studied by atomic force microscopy. The biological responses of human endothelial umbilical vein cells (HUVECs) were studied at the different level of UV/Ozone treated surfaces. HUVEC adhesion, proliferation and migration were significantly improved by the treatment compared to untreated and tissue cultures plastics (TCPs). Among the levels of UV/ozone treatment studied, 120s and 180s were found to be the most effective and HUVEC proliferation did not seem to be affected by the high level of oxygen. Similarly, the surface oxygen level did not affect the migration over UV/Ozone treated over 60s. Hypoxic condition significantly increased HUVECs migration on UV/Ozone treated, TCPs and untreated surfaces compared to normoxia, the oxygen rich surface did not favour to HUVECs that underwent regulatory process to enable the cells to increase migration. Under laminar flow conditions, HUVECs did not only grow, proliferate and migrate but also showed standard responses on UV/Ozone treated polystyrene surface. A decrease in cell size was observed at all shear stress intensities studied (1 dyn/cm2, 9 dyn/cm2 and 25 dyn/cm2) and the decrease was more obvious at higher shear stress. High shear stress intensity also induced high cell turnovers, which may be related to air bubbles induced at high flow rate. The overall findings of this study clearly illustrate that UV/Ozone surface treatment can be applied on polystyrene to improve human endothelial cells functionalities in term of adhesion, proliferation and migration in both static and laminar flow environment.

Obesity is a multi-factorial metabolic disease, resulting in increased adipose tissue acquisition by the host. This disease increases the risk for developing co-morbidities, including Metabolic Syndrome and other disorders such as breast cancer. Obesity, and particularly abdominal obesity, is characterised by metabolic changes, including chronically elevated insulin concentrations and aberrant secretion of cytokines released from fat tissue, called adipokines. Epidemiologically, the risk of developing postmenopausal breast cancer is increased in obese individuals. The molecular link between obesity and breast cancer however is not well understood. The study presented here aimed at identifying the molecular mechanisms involved in this link, by testing the hypothesis that high insulin concentration and certain adipokines may promote breast cancer progression and/or breast cancer aetiology. A cell culture system of breast cancer cells and breast epithelial cells was employed to investigate changes in cell proliferation, activation of cell signalling pathways, cell cycle progression and apoptosis after treatment with insulin, leptin, TNF-α, adiponectin and IL-6. In MDA-MB-231 breast cancer cells, insulin treatment did not affect cell proliferation, cell cycle or apoptosis. Conversely, IR-phosphorylation, AKT-phosphorylation and ERK1/2-phosphorylation were all significantly increased. Microarray analysis indicated several important changes in gene expression with insulin treatment. Leptin treatment increased proliferation by 21%. Additional analyses of the effect of leptin indicated that neither the PI3-kinase pathway nor the MAP-kinase pathway was involved in mediating this effect. Treatment with TNF-α increased apoptosis, but did not affect cell proliferation or activation of cell signalling pathways. In MCF-10A breast epithelial cells, cell proliferation increased after insulin treatment by 180%. IR-phosphorylation, AKT-phosphorylation and ERK1/2 phosphorylation were all significantly increased while early apoptosis decreased after insulin treatment. Analysis of cell cycle however did not indicate a change in progression. Microarray analysis indicated that insulin treatment may increase expression of genes related to cancer growth. Leptin treatment increased cell proliferation and also increased ERK1/2-phosphorylation, while AKT-phosphorylation was not affected. Leptin did not change cell cycle progression. TNF-α treatment increased cell proliferation and also increased ERK1/2 phosphorylation, while AKT-phosphorylation was not changed. TNF-α treatment tended to increase apoptosis, the change however was not statistically significant. In SK-BR-3 breast cancer cells, cell proliferation did not change after insulin treatment. IR-phosphorylation and AKT-phosphorylation increased after insulin treatment, while ERK1/2-phosphorylation decreased. Gene expression of cyclin D and cyclin E increased with insulin treatment, while apoptotic rate and cell cycle profile were also not affected. Cell proliferation increased by 115% after treatment with 100 ng/ml leptin. ERK1/2-phosphorylation however decreased, while AKT-phosphorylation tended to increase, but the change was not statistically significant. Cell cycle profile was not affected by leptin treatment, G1-phase however tended to increase, but the change was again not statistically significant. Cell proliferation increased by 59% after 48 h treatment with 10 ng/ml TNF-α. AKT-phosphorylation and ERK1/2-phosphorylation increased with TNF-α treatment. Cell cycle analysis showed a decrease in S-phase and G2-phase, indicative of a decrease in cell cycle progression. These results indicate that none of the examined obesity-related factors is convincingly identified as the main molecular link between obesity and postmenopausal breast cancer. Conversely, all treatments affected each of the cell lines in, at least, one of the examined aspects. This indicates that many of the obesity-related factors may affect breast cancer and that a single breast tumour may utilise a unique combination of those factors to promote growth. All treatments increased proliferation in MCF-10A breast epithelial cells, with additional analysis generally supporting growth promotion. Insulin treatment particularly increased cell proliferation, while leptin and TNF-α increased MAP-kinase signalling. This may indicate that insulin and adipokines may have a higher impact on breast cancer aetiology than on breast cancer progression.

The synthesis and characterisation of novel bisnaphthalimidopropyl polyamine (BNIPP) derivatives, has gained pace over the last couple of years, as they have enhanced aqueous solubility, without loss of biological activity, in contrast to parent bisnaphthalimide derivatives. Recent work has shown that bisnaphthalimidopropyl spermidine (BNIPSpd) bis-intercalates to DNA, induces oxidative DNA damage, depletes polyamine levels and causes cell death, by apoptosis, in human colon cancer CaCO-2 and HT-29 cells. The aim of this thesis was to synthesise new BNIPP derivatives to highlight the important structural features required for biological activity, particularly, bisnaphthalimidopropyl functionality, and investigate their subsequent modes of action in breast cancer MDA-MB-231 and breast epithelial MCF-10A cells. Initially, work focused on determining the DNA binding affinities and biological activity of BNIPP derivatives. All BNIPP derivatives, except bisphthalimidopropyl diaminodecane (BPHPDadec) and mononaphthalimidopropylamine (NPA) (Δ Tm values of 15.8 and 10.2 °C, respectively, C50 values of > 10 μM, IC50 values of > 40 μM), exhibited strong DNA binding affinities and cytotoxic properties in both cell lines. Results indicate that BNIPP derivatives interact with DNA by bis-intercalation suggesting, therefore, that BNIPP derivatives target DNA. For the first time, an investigation into the mechanism of cellular entry, via the polyamine transport (PAT) system, was studied. However, none of the BNIPP derivatives utilised the MGBG-specific PAT system, suggesting that BNIPP derivatives utilise other modes of cellular entry. Two BNIPP derivatives, BNIPSpd and BNIPDaCHM, were further investigated, and results show that these derivatives significantly induced a dose dependent increase in DNA strand breaks from ≥ 0.1 μM, after 4 hours. BNIPSpd and BNIPDaCHM (at non toxic concentrations) also inhibited the repair of oxidative (H2O2) and methylative (MMS)-induced DNA strand breaks. Based on phosphatidylserine exposure and membrane integrity analyses, early apoptotic cell death was determined as a mode of cell death utilised by both BNIPSpd and BNIPDaCHM (5 μM), after only 0.5 hours treatment in MDA-MB-231 cells. Interestingly, BNIPDaCHM was identified, using HDAC assay kits, as a potent and selective SIRT2 enzyme inhibitor, thus, identifying, a novel structural backbone for the selective inhibition of HDAC enzymes.