Circadian rhythms are present in all living organisms; daily oscillations of biological process from the expression of a gene to the number of times that an animal displays a given behaviour. The light/dark cycle is the primary cue that entrains these rhythms and the suprachiasmatic nuclei, within the hypothalamus are the central pacemaker which synchronises peripheral body clocks. Mice are useful circadian biology models and two peripheral circadian outputs were studied, locomotor activity and the rhythm of body temperature in a common inbred strain, the C57BL/6 mouse. The use of individually ventilated cages to house mice increases biocontainment, enabling the maintenance of high health status colonies and reducing the risk of allergies for laboratory personnel. The effect of these sealed units on ambient light levels was examined, using locomotor activity as a marker of entrainment to the light/dark cycle. Mice housed closer to the overhead light source experienced greater levels of illumination than those at the lower levels, yet all entrained to the light/dark cycle. Mice housed lower on the rack showed more activity during light hours when they normally rest and the onset of activity was advanced in relation to the time the lights turned off. Individually ventilated cages do not therefore compromise circadian entrainment but cage position may alter the distribution of rest and activity in relation to the light cycle. Measuring the rhythm of body temperature of animals is often confounded by the stress associated with immobilisation and restraint. A novel non-invasive method, a thermal imaging camera was trialled against an indwelling intraperitoneal implant, to compare the relationship between peripheral and core body temperature under different light cycles. A stable relationship was found between the two methods (average R² value = 0.92) and this persisted in conditions of constant darkness, where lack of light cues resulted in free-running of the rhythm, assuming a shorter period length of oscillation. This novel method has potential for use in circadian phenotyping studies and to improve welfare, following experimental interventions where the mouse, a small, metabolically active animal is at risk of hypothermia.

In vitro studies of drug permeability are traditionally carried out using cultured monolayers of epithelial cell lines grown on semi permeable membranes. Caco-2 cells, which are colon-derived, spontaneously form polarised cell layers when cultured in vitro which are akin to an epithelium of enterocyte-like cells. The Caco-2 model has been developed as a powerful in vitro tool in the early assessment of human drug permeability and is even approved by regulatory agencies for biowaver applications (i.e. in vitro tests in lieu of in vivo animal experiments). As Caco-2 cells are derived from colon tissue they represent a more formidable barrier to drug absorption than the upper regions of the intestine which is where the majority of oral drugs permeate into the body. Whilst the Caco-2 model, alongside other in vitro methods, has provided a significant means to understand the mechanics of drug permeability. Many researchers have sought to improve upon the existing unicellular model; it is hoped that this will result in a more relevant and predictive model for researchers to test new drugs but also to dissect cellular cross talk and to probe cell-matrix interactions. Myofibroblasts are a niche cell type located subjacent to epithelial tissues which regulate the integrity, growth and differentiation of the overlying epithelium. In this study the co-culture of human epithelial cell lines with a myofibroblast cell line, CCD-18co, were investigated to study how myofibroblasts influence the barrier integrity of epithelia in vivo. Additionally, nanofibre scaffolds produced by electrospinning were explored as 3-dimensional and topographically relevant cell scaffolds to support the growth of intestinal cells in vitro. In a traditional transwell format, cultured epithelial lines Caco-2 (intestinal), HT-29 (intestinal) and Calu-3 (airway) in co-cultures with CCD-18co revealed cell-line specific response with respect to the modulation of barrier integrity. The mechanism of the modulation was confirmed to be mediated through paracrine signalling by using myofibroblast conditioned media. Fibre scaffolds which mimic the fibrillar nature of the extra cellular matrix and basement membrane were produced by electrospinning using the polymer, poly (ethylene terephthalate). Nanofibre scaffolds were characterised and further optimised for cell culture with surface coating with collagen to achieve adequate cell attachment and confluence. Work was also conducted to incorporate villi architectures into the fibre scaffolds; the potential of this ambition was investigated by using models produced by rapid prototyping. These models, which demonstrated good fidelity with the actual villi dimensions found in vivo, were used during the electrospinning process to shape the polymer scaffolds towards the geometry found in intestinal tissue. A number of molecular tracers and model drug compounds were used to evaluate the permeability profiles of Caco-2 monocultures, Caco-2/CCD-18co co-cultures cultured on the two different culture substrates in addition to the assessment of resected porcine intestinal tissue sections. Caco-2 cells and Caco-2/CCD-18co co-cultures grown on nanofibre substrates were found to have lower electrical resistance and higher permeability properties than their transwell equivalents. Caco-2/CCD-18co co-cultures on conventional transwell inserts demonstrated a permeability profile closer to the resected porcine tissue and reported human tissue values than the conventional Caco-2 model whilst maintaining p-glycoprotein assay sensitivity. This work forms a solid foundation for further research into the role of myofibroblasts in epithelial cell function and in the development of more predictive in vitro cell models for widespread scientific research.

The process of soft tissue injury repair at the cellular lew I can be decomposed into three phases: acute inflammation including coagulation, proliferation and remodelling. While the later phases are well understood the early phase is less so. We produce a series of new mathematical models for the early phases coagulation and inflammation. The models produced are relevant not only to soft tissue injury repair but also to the many disease states in which coagulation and inflammation play a role. The coagulation cascade and the subsequent formation of the enzyme thrombin are central to the creation of blood clots. By focusing on a subset of reactions that occur within the coagulation cascade, we develop a model that exhibits a rich asymptotic structure. Using singular perturbation theory we produce a sequence of simpler time-dependent model which enable us to elucidate the physical mechanisms that underlie the cascade and the formation of thrombin. There is considerable interest in identifying new therapeutic targets within the coagulation cascade, as current drugs for treating pathological coagulation (thrombosis) target multiple factors and cause the unwelcome side effect of excessive bleeding. Factor XI is thought to be a potential therapeutic target, as it is implicated in pathological coagulation but not in haemostasis (the stopping of bleeding), but its mechanism of activation is controversial. By extending our previous model of the coagulation cascade to include the whole cascade (albeit in a simplistic way) we use numerical methods to simulate experimental data of the coagulation cascade under normal as well as specific-factor-deficient conditions. We then provide simulations supporting the hypothesis that thrombin activates factor XI. The interest in inflammation is now increasing due to it being implicated in such diverse conditions as Alzmeimer's disease, cancer and heart disease. Inflammation can either resolve or settle into a self-perpetuating condition which in the context of soft tissue repair is termed chronic inflammation. Inflammation has traditionally been thought gradualIy to subside but new biological interest centres on the anti-inflammatory processes (relating to macrophages) that are thought to promote resolution and the pro-inflammatory role that neutrophils can provide by causing damage to healthy tissue. We develop a new ordinary differential equation model of the inflammatory process that accounts for populations of neutrophils and macrophages. We use numerical techniques and bifurcation theory to characterise and elucidate the physiological mechanisms that are dominant during the inflammatory phase and the roles they play in the healing process. There is therapeutic interest in modifying the rate of neutrophil apoptosis but we find that increased apoptosis is dependent on macrophage removal to be anti-inflammatory. We develop a simplified version of the model of inflammation reducing a system of nine ordinary equations to six while retaining the physical processes of neutrophil apoptosis and macrophage driven anti-inflammatory mechanisms. The simplified model reproduces the key outcomes that we relate to resolution or chronic inflammation. We then present preliminary work on the inclusion of the spatial effects of chemotaxis and diffusion.

All organisms studied so far respond to heat shock by inducing the synthesis of a number of proteins called heat shock proteins(LISPs). This universal response can also be induced by a variety of stressors, including heavy metal ions and organic and organo-metallic compounds. As a result, the stress response has recently attracted the attention of ecotoxicologists for use in environmental biomonitoring. In the present study, we have investigated the stress responses of two different organisms ; namely the free-living soil nematode Caenorhiabdities elegans(both wild-type and transgenic strains) and the freshwater crustacea Asellus aquazicus. We have also explored the possible use of these model systems in environmental monitoring using different techniques which include metabolic labelling with subsequent one-dimensional electrophoresis and autoradiography, and one- or two-dimensional western blotting using antibodies specific to stress protein 70. The study with A. aquaticus shows that this organism exhibits a classical stress response. The exposure of asellids to heat shock-treatment (26°C ; 13°C above the standard maintenance temperature) or to sublethal concentrations of metal ions (Cd++ and Cu++) resulted in the induction of at least 5 putative HSPs which belong to several major HSP families (HSP100, HSP90 and possibly HSP60). An increase in the synthesis of smaller sizes of polypeptides (25-35 kD) should be also noted. Moreover, the time-course of heat versus heavy metal stress-response in this organism suggests that the pattern of stress-protein synthesis changes considerably with increasing exposure time ; notably the response to heat is more transient than that to heavy metals. However, HSP70 does not appear to be the major stress protein induced in this organism. The presence of low molecular weight (LMW) proteins which react with anti-HSP70 antibodies and the apparent deficiency of classical 70 kD stress proteins in A. aquaticus, both suggest that HSP70s in this organism are for some reason prone to degradation. In the nematode C .elegans, shifting the culture temperature from 20°C to 34°C induces the synthesis of a set of HSPs corresponding to the HSP90, HSP70 and small HSP families. There are at least nine members of the hsp7O multigene family in C. elegans ; some members are expressed constitutively while others are stress inducible. W e have studied the effects of heat and heavy metal (cadmium) stress on the expression patterns of the HSP70 protein family in the nematodes by one- and two-dimensional Western blotting using a monoclonal anti-HSP70 antibody that recognises a conserved epitope shared by most HSP70 family members. Constitutive C. elegans HSP70s (expressed at 20°C) are almost undetectable on one-dimensional immunoblots, but chemiluminescent probing of two-dimensional blots reveals a complex pattern of several HSP70s pots .Mild heats hock at 31° C induces a doublet HSP70 band on one-dimensional blots, of which the heavier component (75 kD) is more prominent than the lighter (73 kD). On two-dimensional blots, this pattern is shown to be more complex with a prominent 75 kD spot newly induced and several other spots intensified. Severe heat shock at 34°C strongly induces both 75 and 73 kD bands on one-dimensional blots; two dimensional analysis reveals a series of novel and/or elevated 73 and 75 kD spots. Treatment with cadmium( 16 ppm) at 31° C gives a different pattern of spots as compared with 31 °C alone ; several spots show enhanced while some are newly expressed, and not all of these are present at 34°C. These results indicate that related members of the HSP70 protein family in C. elegans are independently regulated in response to different forms of stress. The possible significance of these findings is discussed in relation to the possible use of stress responses s environmental biomonitors. We have also utilised a stress-inducible C. elegans strain (CB4027) for monitoring environmental contamination. This transgenic strain carries integrated copies of the Drosophila hsp70 promoter fused to an E.coli lacZ reporter gene. When exposed to heat shock or to several environmentally relevant stressors, the transgenic strain expresses the reporter product, 3-galactosidase, which can easily be quantified or localised in situ in stained worms or on Western blots (apparently enzymatically active as a 170 kD form). We have exposed transgenic worms to a variety of toxicants at an elevated temperature (32°C) just below that required for heat shock (34°C), in order to obtain optimal transgene induction. Exposure of nematodes to several heavy metals (e. g. Cd+, Hg++, Zn+, Sn++, Mn++ and Ag+), organometallic toxicants (tributyltin) or organic pollutants (lindane) induces ß-galactosidase expression in a dose-dependent manner. Cadmium is found to be by far the strongest inducer of transgene activity amongst the agents tested, although tributyltin is an effective inducer at ppb levels. The effects of mixtures of divalent metal ions (Cd++/Ca++, Cd++/Zn++ and Cd++/Hg++) on ß-galactosidase expression have been also investigated. All three divalent ions tested in combination with cadmium significantly inhibit cadmium-induced transgene activity in comparison to cadmium alone. In the case of Cd++/C++ mixtures, a marked inhibition of Cd++ accumulation by worm tissues has also been demonstrated, directly related to the Ca++ concentration. These effects may represent competition for metal-ion uptake through calcium channels. Our results show that this transgenic system works well within strictly defined assay conditions, and can detect clear responses over a 7h exposure period to environmentally relevant toxicants at sublethal concentrations well below the 24 or 48h LCSO values. However, there is a need for careful characterisation and containment of any transgenic organism if it is to be used as environmental monitoring tool.

Humans are ubiquitously exposed to peroxisome proliferators including hypolipidemic agents, industrial solvents and atural products. Because of this and the fact that peroxisome proliferators cause non-genotoxic hepatocarcinogenesis in rodents, it is of importance to elucidate the mechanism of action of the peroxisome proliferators in order to provide an assessment of the hazard, if any, of these compounds to humans. It is also known that the peroxisome proliferators begin their actions by inducing hepatic DNA synthesis. Thus, the aim of this thesis was to find genes that could be responsible for triggering the induction of hepatic DNA synthesis caused by peroxisome proliferators, specifically ciprofibrate. First, it was important to indicate when the induction of hepatic DNA synthesis actually happens. This was done with BrdU immunohistochemical procedures. The induction of hepatic DNA synthesis with ciprofibrate in mice was observable only after 4 days making it difficult to specify when the induction actually happened. In rats the induction of hepatic DNA synthesis was found to peak at 24 hours and this system gave the better opportunity to find the genes responsible. The difference in the timing of induced hepatic DNA synthesis betweenmice and rats implied that there could be a species difference in the mechanism of each species’ response to PPAR. With immunohistochemistry it was noticed that there was a difference in the lobular localization of hepatic DNA synthesis in the liver tissues of rats and mice dosed with different inducers, with the rat livers exhibiting periportal distribution while hepatic DNA synthesis in the mice seemed to be distributed throughout the liver tissue. The effects of ciprofibrate or cyproterone acetate on liver gene expression in rats were studied, using cDNA microarrays, transcriptome sequencing and quantitative real- time PCR. A 1- 5 hour treatment period was chosen to detect the immediate early gene response, while a 24 hour time point was chosen to elucidate the confounding effects from the hepatic DNA synthesis seen during the 24 hour stimulation. The results showed that ciprofibrate altered the expression of numerous genes including previously known PPARa agonist-responsive genes involved in processes such as PPAR signalling pathways, fatty acid metabolic pathway, cell cycle, palmitoyl-CoA hydrolase activity, lipid metabolism, inflammatory responses, and stress responses, in addition to a large number of novel candidate genes. Three novel induced genes G0s2, Ccnd1 and Scd1, (and two marker genes CYP4A1 and CYP3A1) were confirmed with quantitative real- time PCR. The G0s2, Ccnd1 and Scd1 were found to be up-regulated at the hours 1 and 3 after dosing and not 24 hours, and the G0s2 and Scd1 were specific for the ciprofibrate suggesting they were involved in a distinct PPARa pathway responsible for the hepatic DNA synthesis. The complete database of the transcriptional response provided here opens doors of opportunity for further research to identify genes responsible for the liver growth induced by peroxisome proliferators.

Background. Necator americanus is one of the major causes of human hookworm infection, affecting over 800 million people worldwide. Hookworm infections cause gastro-intestinal bleeding, anaemia and iron deficiency, and are associated with high rates of morbidity, especially in children. Although chemotherapy has proven effective, high rates of reinfection are reported in socioeconomically developing countries, possibly due to the short-term efficacy of anthelmintic drugs in addition to individual predisposition to these infections, raising interests in developing suitable alternatives to chemotherapy which are capable of providing complete, long-term protection against hookworms. Understanding of the molecular mechanisms used by Necator americanus larvae to penetrate the human skin and the vasculature would therefore aid the development of effective vaccines against this important pathogen. Methods. First, Necator americanus larval exsheathing fluid (EF) and excretory/secretory products (ES) were profiled using gel electrophoresis and enzyme assays. Protease inhibitors against the main protease classes were used to determine which proteases are present in larval products. Second, the interaction of larval EF and ES products with human skin and extracellular matrix (ECM) macromolecules including collagens I, III, IV and V, fibronectin and laminin was investigated using western blots and protein separation by gel electrophoresis. Third, the impact of Necator americanus larval EF and ES on the endothelial barrier was examined using human umbilical vein endothelial cells (HUVEC). Permeability, an essential endothelial barrier function, was assessed during treatment with larval products, using transendothelial electrical resistance (TEER), and post-treatment using albumin-tracer flux. Finally, at the cellular level, responses to treatment with larval products were assessed by investigating molecular changes at cell-cell vascular endothelial (VE)-cadherin junctions and actin filaments, and by determining levels of secreted inflammatory cytokines, IL-6 and IL-8, and vascular endothelial growth factor (VEGF) in the culture medium. Results. It would appear that a repertoire of larval proteases, including serine, cysteine, aspartyl and metalloprotinases, caused partial degradation of skin macromolecules, collagens I, III, IV and laminin while fibronectin was fully degraded. Proteolysis of skin- and ECM macromolecules was related to the characteristic presence of proteolytic enzymes in larval products. The presence of transglutaminase activity was confirmed in both EF and ES products. Larval proteases caused a dose related increase in endothelial permeability, characterised by a decrease in monolayer resistance (TEER) with increased permeation of albumin tracer, which was minimal in the presence of a cocktail of protease inhibitors. These barrier changes were associated with disruption of junctional VE-cadherin and F-actin, the formation of intercellular gaps and an increase in endothelial secretion of IL-6 and IL-8. Conclusions. Necator americanus larvae produce a repertoire of proteolytic enzymes which could play an important role in negotiating the skin and breaching the endothelium to gain access to the host’s blood circulation.

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.

This thesis investigated the impact of sodium acetate infusion on muscle fuel metabolism during low and high intensity exercise and demonstrated a decrease in fat oxidation during the former and a decrease in muscle lactate production during the latter. We propose that the rate of fat oxidation decreased during low intensity exercise due to acetate being preferentially metabolised over fat and possibly reduced muscle free carnitine availability. During high intensity exercise, muscle lactate accumulation decreased due to higher acetyl-CoA supply to the TCA cycle. This thesis also investigated the impact of chronic carnitine supplementation on overall glucose disposal and demonstrated a decrease in blood glucose and serum insulin concentrations following an OGTT in the carnitine group post-supplementation compared to baseline, coupled with a decrease in muscle 2-deoxyglucose accumulation in the carnitine group post-supplementation compared to control. We propose that carnitine-induced increased fat oxidation caused a decrease in hepatic fat deposition which in turn resulted in increasing glucose disposal by the liver. We also propose that increased hepatic glucose disposal resulted in reduced hepatic glucose release and hence diminished serum insulin concentrations. The impact of a chronic lifestyle intervention protocol involving either oral supplementation with carnitine (carnitine group) or placebo (control group) combined with carbohydrate and protein, together with regular exercise and a prescribed diet on muscle fuel metabolism and body composition in overweight volunteers was investigated in this thesis. This study demonstrated that increased muscle carnitine content caused an increase in fasting fat oxidation and a decrease in carbohydrate oxidation at rest. The carnitine group did not show any significant difference in body mass and body fat mass losses compared to the control group. Whether increased carnitine content, in combination with caloric restriction and exercise, has any impact on body composition seems likely but needs further investigations.

Overconsumption of foods is thought to be one of the main causes of the rising number of global obesity. This thesis aims to investigate the role of food flavour in human appetite and eating behaviour through three studies. The first study investigated whether the sweetness intensity of a milkshake affected ad libitum intake of the milkshake and sensory-specific satiety (SSS). In a crossover single-blinded design, 24 participants consumed ad libitum high, ideal and low sweetness (HS, IS or LS) milkshakes over three visits. After milkshake intake, participants consumed ad libitum one, or both of a sweet and a savoury snack. All milkshake consumption was similar, suggesting that the sweetness intensity did not affect the ad libitum intake of the milkshake. After intake of all sweet milkshakes, ratings of desire for something sweet decreased, and subsequent savoury snacks were consumed more than subsequent sweet snacks. The sweetness intensity of milkshakes did not affect the change in the desire for something sweet or the subsequent snack intake. Ratings of desire for something savoury increased after the intake of HS milkshake and were higher than the ratings collected following the intake of IS milkshake. Therefore, this study suggested that a sweeter milkshake did not affect the magnitude of SSS for sweet foods, but increased a stronger sensory-specific appetite (SSA) for savoury foods. The second study examined the effects of aroma, taste and their interaction on subjective appetite sensation and subsequent lunch intake. In a crossover design, 26 females consumed 1 of the 4 test drinks as a preload: 1) water; 2) strawberry aroma in water; 3) sucrose and citric acid in water; 4) strawberry aroma, sucrose and citric acid in water. The subsequent lunch intake did not differ after all drink preloads. The drink with only aroma or only taste were not different from water in affecting appetite sensation. A drink with both aroma and taste reduced hunger ratings greater than water or a drink with only taste or aroma, during 15 min drinking and up to 30 min post drinking. Meanwhile, the drink with both taste and aroma was the highest in perceived flavour intensity. This suggests enhancing flavour perception of a drink through aroma-taste cross-modal interaction can increase the satiating effect of a drink. The third study investigated effects of sweetness, thickness and caramel flavour perception of custards on expected satiation and expected satiety of the custards. 90 participants (65 females, 25 males) tasted 18 custard samples over two sessions. Ingredients of custards were different only in the concentrations of caramel aroma, Truvia sweetener and carboxymethyl cellulose (CMC), based on an experimental design. Thickness enhanced both expected satiation and expected satiety. Sweetness enhanced expected satiation but not expected satiety. Caramel flavour did not affect expected satiation or expected satiety. The cognitive expectation on satiation and satiety has previously been shown to determine self-selected portion size. Therefore, the current study suggests that manipulating sweetness and thickness perception of a food without changing its energy content might help portion size control, via manipulating consumers’ cognitive expectation of the food. In conclusion, manipulating food flavour is a promising area to explore with the respect to hunger suppression and fullness enhancing, limiting the intake of eaten foods while promoting intake of other foods via SSS or SSA, and contributing to the cognitive control of portion size. Therefore, manipulation food flavour might be helpful for appetite control and supporting an energy-restrict diet; however, it seems challenging to reduce actual food energy intake through manipulating only the flavour properties of foods.

Lung diseases affect the lives of millions of people across the UK and result in the thousands of deaths every year. It is therefore vitally important to continue to develop a wide range of diagnostic techniques to improve our understanding of lung diseases and how they can be treated. This thesis provides an overview of the main methods of assessing lung condition before focussing on developments in two specific areas: Forced Oscillation Technique (FOT) and Hyperpolarised (HP) gas MRI. FOT is an inexpensive, non-invasive lung function test that measures the acoustic impedance of the airways by applying an oscillating waveform via a mouthpiece. FOT cannot be used to image the lung but instead provides information on a variety of other physiological parameters. Two FOT studies are presented in this thesis: a multi-site phantom study and a patient based study. The phantom study confirmed the validity of the Nottingham FOT system used in the patient study and investigated the effects of lung stiffness and airway obstruction on measured FOT parameters using a 3D printed lung phantom, as well as comparing phantom results between three different FOT devices (an in-house device from the University of Nottingham, an Erich Jaeger Master-Screen IOS and a tremoFlo C-100 airwave oscillometry system) at two sites (the University of Nottingham and Glenfield Hospital, Leicester). It was found that changes in lung stiffness and airway obstruction are observable in the reactive and resistive (respectively) components of measured impedance. A difference was seen between the Jaeger IOS system and the other two devices. The patient based study was undertaken to investigate the efficacy of FOT, in comparison to spirometry, in differentiating between three patient groups, healthy, asthmatic and chronic obstructive pulmonary disease (COPD), with a particular focus on investigating the effect of a bronchodilator on measured FOT parameters. It was found that both FOT and Spirometry were effective at differentiating between the patient groups, however, they provided different information about patient response to bronchodilator thus demonstrating that both techniques should be performed to obtain the maximum information about a patient's disease state. HP gas MRI uses isotopes of noble gases, such as 3He and 129Xe, to either image the lungs or perform non-imaging measurements of parameters such as the Apparent Diffusion Coeffcient (ADC). A short study using 3He was performed comparing ADC measurements at two different time scales between two sites (the University of Nottingham, 13ms, and the University of Sheffield, 2ms) with a secondary aim of investigating the effect of age on ADC. A study on HP 129Xe MRI is presented covering developments that have been made in various imaging techniques including breathing protocols and scanning techniques. The objective of this study is to establish a reliable scanning protocol using healthy volunteers before expanding the study to investigate different disease states including COPD and idiopathic pulmonary Fibrosis (IPF). Although progress has been made in testing the validity of various imaging techniques, with ventilation images at 25mm and 10mm slice thicknesses obtained, more development is still needed to improve the quality of the images in order for them to be useful in a clinical setting.