Cigarette smoke (CS) has many damaging effects on the body, and the chronic inhalation of cigarette smoke can change immunological functions through impact on both innate and adaptive immunity. The incidences of many diseases are affected by the adverse effects of cigarette smoke on the immune system, and the induction of an inflammatory response, which affects several tissues and organs. On this basis, a comparison of smokers′ and non-smokers′ immunoglobulin levels could provide valuable insights into the mechanisms of smoking related diseases. Although the effects of cigarette smoking on humoral and cellular immunity have been investigated previously, the results have varied between the studies, and therefore more research is still required. The aim of this study was to determine whether the levels of immunoglobulin (Ig) isotypes are different in the serum and saliva of non-smoking individuals compared to smoking individuals. An examination of serum and saliva would provide information on the effects of cigarette smoke systemically and in the oral mucosa, respectively. The effects of cigarette smoke extract on B-cell secretions were also examined to establish whether cigarette smoke components can have direct effects on immunoglobulin production by B cells. In order to determine Ig isotype levels, antibody microarray techniques were established and calibrated for determining the sample concentrations of IgM, IgG, IgA and IgD. The results showed that smoking has different effects on systemic and salivary immunoglobulin levels. In the serum, smokers had decreased levels of IgG and IgD, but increased IgM and IgA levels compared to non-smokers. However, in the saliva smokers had decreased levels of IgG, IgD, and IgM, whereas there were increased levels of IgA in smokers’ saliva. As CS has been found to influence the serum and salivary levels of Ig isotypes ex-vivo, the mechanisms underlying these effects were investigated in vitro to determine whether the changes were as a result of a direct effect of the CS on B-cells. This study has shown that CS had deleterious effects on the production and the levels, of Ig isotypes. These results support the concept that CS is related to diseases, and more research is necessary in this field.

The endocannabinoid system (ECS) comprises the endocannabinoids (ECs), including anandamide (AEA) and 2-arachidonoyl glycerol (2AG), which interact with the G protein-coupled type-1 and type-2 cannabinoid receptors(CB1 and CB2 respectively). The ECS is thought to have a role in a number of central processes including neuroinflammation, neurogenesis, neuroprotection, learning and memory. Due to its influence on a diverse number of processes, it has been suggested that modifying the ECS may be therapeutically beneficial in Alzheimer's disease (AD). AD is an age-related neurodegenerative disorder characterised by the presence of extracellular amyloid beta (Ab) plaques and intracellular neurofibrillary tangles (NFTs) resulting in impairments in learning in memory. The aim of this thesis was to determine the status of the brain ECS in the APPswe/PS18E9 mouse model of AD and wild type littermates at 4, 6 and 8 months of age and the performance of these animals in a behavioural test battery. The results of this study indicated that APPswe/PS18E9 animals were hyperactive compared to their wildtype counterparts at all ages and that they also displayed deficits in behavioural flexibility. EC levels increased with age in both wild type and APPswe/PS18E9 mice. Cannabinoid receptor coupling was increased in the frontal cortex and striatum of APPswe/PS18E9 mice relative to wildtype. This study concluded that the status of the brain ECS is altered in AD. Modifications to the performance of the ECS were made in the form of chronic administration of a CB1 receptor antagonist (SR141716A1rimonabant) and a CB2 receptor agonist (JWH133). Chronic administration of SR141716A was able to reverse some learning impairments in APPswe/PS18E9 animals. In contrast, chronic administration of JWH133 resulted in impaired memory extinction in both wildtype and APPswe/PS18E9 mice. The results support the potential benefit of modulating the endocannabinoid system in the treatment of memory impairment in AD.

Type 2 diabetes mellitus is a chronic condition caused by a deficiency in the secretion of insulin from the islets of Langerhans and/or impaired insulin signalling, resulting in hyperglycaemia. The role of the endocannabinoid system is well-recognised in the CNS and immune system, but its role in glucose homeostasis is poorly understood. The aim of this study was to define the roles of cannabinoids in insulin secretion, to provide insights into their therapeutic potential (or limitation) in the treatment of type 2 diabetes. Isolated islets were used, from Wistar rats, in static incubation studies measuring changes in insulin secretion rates. The endocannabinoid anandamide (AEA) was found to inhibit insulin secretion in a glucose- and concentration-dependent manner, with an IC50 of 1.6μM (95% CI: 227nM to 4.0μM; n= 10). Upon further analysis of the concentration-response data islet sensitivity to AEA appeared to vary, with islets either appearing to be sensative (IC50 220nM; 95% CI: 21.9nM to 2.2μM; n= 5) or less sensative (IC50 12.3μM; 95% CI: 6.8μM to 19.4μM; n= 5) to AEA. Pre-incubation of islets with a fatty acid amide hydrolase inhibitor did not affect islet responsiveness to AEA. AEA-mediated inhibition of insulin secretion was not consistently affected by cannabinoid receptor 1 (CB1) or CB2 antagonism. Surprisingly, the CB1 receptor antagonist AM251 was found to inhibit insulin secretion in a glucose- and concentration-dependent (IC50 1.6μM; 95% CI: 507nM to 3.3μM; n= 6) manner. Results from this study suggest that differences in CB-receptor signalling pathways, rather than endocannabinoid metabolism, could be responsible for the variations in the potency of AEA between islet preparations. Characterisation of cannabinoid signalling in islets was hindered as the CB receptor antagonists used in this study also affected insulin secretion. This study highlights the dynamics of endocannabinoid signalling in islets, which may be linked to their physiological function.

At the height of its popularity, mephedrone was the most common recreationally used cathinone. This is thought to be due to its perceived likeness to MDMA. Therefore the aim of this thesis was to examine mephedrone-induced changes in behaviour, body temperature or neurochemistry in the rat and to compare these changes to those observed following MDMA administration. This was achieved by assessing changes in body temperature following acute mephedrone, MDMA, cathinone or methcathinone administration. Additionally, locomotor activity and cognitive tasks were performed following chronic intermittent administration of mephedrone, MDMA or cathinone. These behaviours, as well as mephedrone-induced changes to body temperature and ‘anxiety-related’ behaviour, were also assessed following either pre-treatment with MDMA or co-administration of caffeine. Finally, locomotor activity, body temperature changes and in vivo striatal dopamine release were assessed following rapid repeated dosing of mephedrone, and the roles of dopamine, 5-HT and noradrenaline in these responses were examined. Post mortem monoamine concentrations from specific brain regions were also assessed following acute, chronic intermittent and rapid repeated dosing of mephedrone. It was found that the neurochemical, behavioural and physiological effects of mephedrone in the rat include hyperactivity, hypothermia, cognitive deficits, anxiety-related behaviour and increased striatal dopamine efflux. Pre-exposure to MDMA, or concomitant caffeine administration, caused an increase in rectal temperature following mephedrone injection while caffeine co-administration prolonged the hyperactive profile of mephedrone. Importantly, unlike MDMA, rapid repeated mephedrone administration (3 x 10 mg kg-1 at 2 h intervals) had no cumulative effect on mephedrone-induced hypothermia or hyperactivity. It is also clear that mephedrone is inducing its effects via noradrenergic, dopaminergic and serotonergic mechanisms. The cathinones and MDMA had varying effects on post mortem tissue levels of the monoamines and their metabolites. Importantly, these effects of mephedrone appear to be occurring by mechanisms that are different, but similar, to MDMA.

The initial aims of this investigation were to develop a reliable assay system for measuring serum 1,Z5-dihydroxyvitamin D [1,Z5(OH)ZD] concentrations and to establish a normal range in young healthy adults. Compared with young healthy individuals, the elderly population are indeed vitamin D deficient. Vitamin D deficiency was also demonstrated in a group of elderly osteomalacic patients. Slight improvements in osteomalacia was achieved by one month of treatment with vitamin D3' or alphacalcidiol with or without calcium supplements. The improvements were small and occured slowly. A significant increase in the strength of bone seems unlikely to occur in the short term. On the present evidence the combination of alphacalcidiol and calcium supplements seems no better than vitamin D3 or alphacalcidiol alone although it may require closer monitoring to avoid hypercalcaemia. In a group of elderly patients with osteoporosis and femoral neck fracture (FNF), serum osteocalcin concentrations rose significantly in the first week after fracture fixation. The change in osteocalcin correlated well (p < 0.001) with the change in serum 1,Z5(OH)ZD concentration. Histomorphometric measurements of the extent of osteoid correlated better with osteocalcin than alkaline phosphatase. Serum concentrations of 1,Z5(OH)ZD were also reduced in elderly patients with FNF irrespective of the presence of osteomalacia and therefore cannot be used as a screening test for osteomalacia in this patient group. Reduction of 1,Z5(OH)ZD was not due to a reduction in vitamin D binding protein. It is suggested that the low rate of bone turnover in these elderly patients reduces the requirement of vitamin D. Of the ten elderly patients who had underwent laryngo pharyngeal surgery all developed hypocalcaemia. This immediate post-operative decrease, due to a rapid reduction in circulating PTH concentrations, lead to an overall increase in urinary calcium excretion. Serum concentration of 1,25(OH)2D also fell postoperatively thus potentiating the hypocalcaemic state in these patients. Thus, it is important to give parenteral feeding supplemented with calcium and vitamin D, preferably alphacalcidiol. If delayed then profound as well as prolonged hypocalcaemia can occur. The human osteosarcoma cell 20S metabolised 25(OH)D3 in a substrate concentration and time dependent manner to produce products which were secreted into the extracellular medium. These products eluted from HPLC with a retention time coincident with 24,25(OH)2D3 and exhibited an UV absorption spectrum characteristic of a vitamin D sterol. Mass spectroscopy analysis indicated at least two products were synthesised by the cells. One was identical to 24,25(OH)2D3; the other appeared to be an unsaturated trihydroxylated derivative of vitamin D3.

Angiogenesis, a process that enables the growth of blood vessels from a pre-existing vasculature and is common to all solid tumours greater than 1 mm3 in size (Gacche and Meshram, 2014). The angiogenic process is heavily promoted by vascular endothelial growth factor (VEGF). Compounds able to inhibit VEGF signalling have been shown to reduce cancer mass (Arjaans et al., 2016). However, VEGF receptor tyrosine kinase inhibitors (RTKIs), a class of anti-VEGF treatment, have been shown to cause cardio-toxicity, with hypertension being a commonly reported, and often severe, side effect (Eskens and Verweij, 2006; Widakowich et al., 2007; Abi Aad et al., 2015). Depending on the nature of the study, the incidence of hypertension in the VEGF RTKI patient population ranges from 23% to 90% (Hamberg et al., 2010; La Vine et al., 2010; Aparicio-Gallego et al., 2011; Bible et al., 2014). Due to the increasing incidence and seriousness of hypertension observed in oncology clinics, it is clear that there are important cardiovascular issues relating to the use of RTKIs, particularly those that target VEGF, that require further exploration. This body of work set out to determine the in vitro potencies of vandetanib, pazopanib, cediranib and sorafenib at VEGFR2, alongside the in vivo cardiovascular haemodynamic and vasoactive profile of vandetanib and pazopanib, two VEGF RTKIs shown to cause hypertension in approximately 32% (Wells et al., 2012) and 33%-40% of the patient population, respectively (Bible et al., 2014). In NFAT luciferase assays cediranib, sorafenib, pazopanib and vandetanib were shown to inhibit, in a non-competitive fashion, VEGF165 mediated signalling in vitro. In haemodynamic studies, using Doppler flowmetry and telemetry methodologies, both vandetanib and pazopanib caused significant hypertension (P < 0.05, in comparision to vehicle). Pazopanib and vandetanib lead to significant vasoconstriction of the mesenteric and hindquarter vascular beds, pazopanib also produced significant vasoconstriction in the renal vascular bed (P < 0.05, in comparision to vehicle). None of the variables measured in the haemodynamic studies significantly differed between the 30 mgkg-1day-1 pazopanib and 25 mgkg-1day-1 vandetanib groups. In chronic radio-telemetric studies, vandetanib was shown to cause a significantly greater but more transient increase in mean arterial blood pressure in comparison to pazopanib (P < 0.05). Vandetanib was also shown to inhibit VEGF and ACh-mediated vessel dilatation in pressure myography experiments. Finally, vandetanib and pazopanib were shown to induce vasodilatation in the presence of a vasoconstrictor (U46619), a previously unseen finding. In conclusion, the body of work undertaken here has given novel insight into the ability of non-competative anti-VEGF RTKIs to inhibit VEGF-mediated signalling and vessel dilatation as well as produce direct effect of vessel diameter in the absence of VEGF. It has also produced a validated method of hypertension in a rat model, both in the short and long term. These models have shown that different anti-VEGF RTKIs have different regional haemodynamic and post-treatment hypertensive side effect profiles. These findings are important for understanding the mechanisms behind the therapeutic and non-therapeutic effects of VEGF RTKIs and allow for further research into the signalling mechanism involved in VEGF RKTI-mediated hypertension and the potential therapeutic treatments that could treat this.

Clostridium sporogenes is part of a highly diverse group of Gram positive, spore forming, anaerobic bacteria. C. sporogenes can be used as a delivery vehicle for chemotherapeutics in cancer treatment due to the inactive spore form of C. sporogenes only germinating in the microenvironment of the hypoxic tumour. Cancer, despite large investment in treatment and diagnosis, still remains one of the leading causes of death in the world. As such, improvements on current treatments are necessary to improve patient prognosis. Utilising C. sporogenes could be a cheap and effective way to do this by utilising their germination properties to deliver anti-cancer therapeutics directly to the hypoxic regions of solid tumours. Through introducing Prodrug Converting Enzymes (PCEs) into C. sporogenes when the spores germinate in the tumour the production of the PCE will result in the breakdown of a Prodrug into a toxic product resulting in an anti-cancer effect. This system is known as Clostridial Directed Enzyme Prodrug Therapy (CDEPT). Previous iterations of this system incorporated a nitroreductase gene, used for the breakdown of the prodrug CB1954. During this project alternative prodrugs were investigated, in this case carboxypeptidase G2. Alternatives into the prodrug and enzyme system are also being investigated in a direct action therapy in the form of the monoclonal anti-VEGF. The aims of this project were to implement the genes and optimise the activity of the drugs if necessary. It was also necessary to confirm that the bacterium was a non-pathogenic group 1 bacterium through sequencing and annotation of the genome.

Generally, most of the in vitro tests used in neurotoxicology are limited to transformed cell lines which are derived from rodent or human. For an in vitro test to have high rate of predictability of neurotoxicity and teratogenicity it should undergo the important processes of embryological development, such as cell proliferation, cell migration, and differentiation. Human neural stem cells have been proposed for this purpose, which have the ability to divide, differentiate, and migrate. In this study, it was found that double coating of laminin with either poly D lysine or poly L lysine was most suitable for growing human neural stem cells rather than coating with a single extracellular molecule. Several chemicals and drugs were then chosen to assess the utility of neural stem cells as an assay for neurotoxicity: methyl mercury and lead acetate; four anti-epileptics drugs (sodium valproate, phenytoin, carbamazepine, and phenobarbitone); anti-oxidants (folic acid and melatonin). These anti-oxidants were tested alone and when added to sodium valproate and to phenytoin (which are well known in their teratogenicity), and other drugs (lithium, diazepam, and amitriptyline), which are weak teratogens. To assess the effects of these molecules on human neural stem cells cell survival, total cellular protein, neuronal process length, neurosphere sizes, migration distance, Glial Fibrillary Acidic Protein, and tubulin III protein expression were measured. The study shows that methyl mercury caused significant reduction in most of the end points from the dose of 1µM and it led to significant increase in Glial Fibrillary Acidic Protein expression (which is a sign of reactive gliosis). Lead acetate led to a significant reduction in cell migration 48hours after treatment with 10µM. In the case of the anti-epileptics, sodium valproate appeared to reduce neurosphere size significantly from the dose of 500µM and decrease migration distance significantly 48hours after treatment with 1000µM. Moreover, phenytoin treatment resulted in significant reduction in neurosphere sizes from the dose of 25µM and reduced cell migration significantly from the dose of 50µM. However, the other anti-epileptics (carbamazepine and phenobarbitone) revealed their effect only at high doses which are above their therapeutic range. On the other hand, adding the anti-oxidants (Folic acid or Melatonin) to sodium valproate or phenytoin had to some extent beneficial effects, by making their toxic effect appear at doses which were higher than when used alone. Regarding the other drugs (lithium, diazepam, and amitriptyline), it seems that their toxic effect appeared only at doses which are higher than the therapeutic range. Therefore, it can be concluded that human neural stem cells are a sensitive model in detecting the neurotoxicity of methyl mercury and lead acetate at low doses and can predict the neurotoxicity of sodium valproate and phenytoin at their therapeutic doses.

In recent years evidence has emerged that the endocannabinoid system (ECS) may have a significant role in metabolism and energy homeostasis. Several studies have identified upregulation of the peripheral ECS in obesity and type 2 diabetes, but the mechanisms behind this and the consequences of upregulation are unclear. The aim of this thesis was to further elucidate the role of the ECS in mature adipocytes, and its activity in obesity and related metabolic dysfunction. Three adipose tissue depots were dissected from lean, obese and obese diabetic Zucker rats (n=6-8). In human studies, written informed consent was obtained from healthy volunteers within the University of Nottingham and obese surgical patients at the Royal Derby Hospital. Anthropometric measurements and venous blood samples were obtained. In these studies, subcutaneous abdominal adipose tissue was taken from all subjects (n=28 healthy study; n=27 surgical study), and visceral adipose tissue was obtained from some of the surgical patients (n=14). In all studies, collagenase was used to isolate mature adipocytes from the adipose tissue, and FAAH and MGL activities in the adipocytes were assayed using tritium labelled substrates. Human subcutaneous preadipocytes (Promocell, Germany) were cultured and differentiated. Adipocytes were cultured with high concentrations of glucose (15 mM) and/or insulin (1 μM) for 24 hours, in combination with anandamide or 2-AG for 2 or 24 hours. Adiponectin, leptin and resistin in the cell culture media were then measured using sandwich ELISAs. In another study, anandamide and 2-AG uptake were measured in differentiated adipocytes after 2 or 24 hours’ stimulation with glucose and/or insulin. FAAH and MGL activities in the cultured adipocytes were also measured in this study. In rats, FAAH and MGL activities correlated with body mass. In healthy humans, FAAH activity in subcutaneous adipocytes correlated with BMI and waist circumference, but not with other anthropometric measurements, serum glycaemic markers or adipokines. In obese patients, the enzyme activities had no relationships with any of the anthropometric or metabolic markers investigated. Furthermore, there were no differences in activity between patients with metabolic syndrome or diabetes and those without. In both rats and humans, there were no significant differences in FAAH and MGL activities between subcutaneous and visceral adipocytes. In the cell culture studies, anandamide and 2-AG did not alter adipokine secretion under normal, high glucose or high insulin conditions. Chronic insulin exposure increased anandamide uptake, but none of the other acute or chronic treatments with glucose and/or insulin affected anandamide or 2-AG uptake. Glucose and insulin were found to reduce MGL activity. These studies suggest that the rate of anandamide hydrolysis in mature adipocytes is increased in obesity. This relationship was not apparent in a morbidly obese sample. MGL activity in humans does not have relationships with adiposity or metabolic markers, and this may reflect its role as a major component of lipid metabolism, particularly lipolysis. Anandamide and 2-AG are unlikely to be direct mediators of adipokine secretion, at least in cell culture. Insulin may affect endocannabinoid signalling in adipocytes by increasing anandamide uptake and suppressing MGL activity. Overall, these results support the notion that the ECS in adipocytes is dysregulated in obesity, but this is not driven by specific factors associated with obesity.