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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.

The ω-3 polyunsaturated fatty acids (ω-3 PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been shown to have cardioprotective and antiatherosclerotic actions. While it has been proposed that ω-3 PUFAs may structurally stabilise atherosclerotic lesions, the underlying mechanisms have yet to be fully elucidated. There is evidence that increased dietary intake of ω-3 PUFAs alters the concentration of endocannabinoids (ECs) in central and peripheral tissues and, since synthetic cannabinoid agonists have been shown to exert anti-atherosclerotic effects, this thesis explored whether a link exists between the endocannabinoid system and the beneficial effects of ω-3 PUFAs in the setting of experimental atherosclerosis. Therefore, the present studies were performed to (1) determine the impact of long-term intervention with ω-3 PUFAs on several physiological parameters, including the function of the endocannabinoid system, in an in vivo model of hypercholesterolaemia and (2) to examine the role, if any, of the activated endocannabinoid system in the activity of an in vitro model of macrophage lipid accumulation. At the cellular level, the present study demonstrated the interference of cannabinoid receptor signalling with intracellular uptake and accumulation of DHA, an effect which was demonstrated to be mediated, at least in part, via CB2 receptor signalling. Moreover, the study provided the novel observation that DHA treatment of activated macrophages significantly inhibited 2- arachidonoylglycerol (2-AG) synthesis. This finding is strongly suggestive of an association between the effects of ω-3 PUFAs and regulation of the endocannabinoid system and its function. In the ApoE-/- mouse model of experimental atherosclerosis, maintenance on an atherogenic diet induced significant hyperlipidaemia, an effect which was associated with endothelial dysfunction, cardiac remodelling, and significantly elevated anandamide (AEA) and 2-AG levels in brain, heart and aortic tissues. Supplementation of the atherogenic diet with the EPA, while having a negligible impact on endothelial dysfunction or cardiac remodelling, improved the hypercholesterolaemic profile and normalised the overproduction of ECs in both central (brain) and peripheral (heart and vascular) tissues. Fenofibrate, a lipid lowering control administered as a supplement to the atherogenic diet, was observed to significantly attenuate hypercholesterolaemia, endothelial dysfunction and elevated EC levels although no effect on cardiac remodelling was observed. In light of the observation that hypercholesterolaemia was associated with elevated tissue EC levels, together with the demonstration that EPA improved hypercholeseterolaemia and attenuated EC levels suggests (1) a detrimental role of the endocannabinoid system in hypercholesterolaemia in the ApoE-/- model and (2) regulation of this interaction by ω-3 PUFAs. Given the mounting evidence implicating the endocannabinoid system in various disease pathologies, the capacity of ω-3 PUFAs to influence the activation and function of this system may prove key to their beneficial effects.