Bacteria communicate with each other by releasing chemicals called quorum sensing molecules. A common gram-negative bacteria, Pseudomonas aeruginosa, releases a number of such molecules including, N-3-(oxododecanoyl)-L-homoserine lactone (3OC12-HSL) and Pseudomonas quinolone signal (2-heptyl-3,4-dihydroxyquinoline, PQS). The quorum sensing molecules have immunomodulatory effects on mammalian cells in addition to their role in bacteria. There has been very little study of the vascular effects of quorum sensing molecules on the host. The aim of this thesis was; therefore, to investigate the effect of Pseudomonas aeruginosa quorum sensing molecules, 3OC12-HSL and PQS, on vascular function and endothelial cell permeability and to study the potential mechanisms involved. Both 3OC12-HSL and PQS caused slowly developing vasorelaxation in arterial and venous preparations isolated from pigs. PQS was slightly more potent (pIC50) as a vasorelaxant than 3OC12-HSL. The endothelium was not a prerequisite for 3OC12-HSL-induced vasorelaxation since removal of endothelium and the nitric oxide synthase inhibitor (L-NAME) did not attenuate responses. Indeed, the opposite is true since 3OC12-HSL vasorelaxation responses were larger in the absence of the endothelium and nitric oxide. The mechanism of 3OC12-HSL does not involve either prostanoids, cyclic GMP, mitochondrial targets, calcium channels, chloride channels, PPAR-γ receptors, pannexin receptors, gap junctions or cystic fibrosis transmembrane conductance regulator. However, ouabain and depolarisation induced by potassium chloride decreased 3OC12-HSL responses suggest an impact on membrane potential. Overnight exposure of the porcine coronary artery to wild type Pseudomonas aeruginosa (PAO-L), produced a selective reduction in the magnitude of contractions to potassium chloride. However, neither 3OC12-HSL nor PQS produced an inflammatory response with prolonged exposure since they did not modify tumor necrosis factor alpha or vascular tone under these conditions. Both 3OC12-HSL and PQS increased endothelial cell permeability. Both caused a fall in endothelial resistance and an increase in FITC-dextran transport across human brain microvascular endothelial cells (HBMECs). The immunoblotting data showed that the levels of adherens junction and tight junction protein expression were decreased in HBMECs exposed to 3OC12-HSL, but not PQS. Rather PQS, but not 3OC12-HSL, caused a concentration-dependent phosphorylation of p38 MAPK, suggesting these molecules may modify vascular permeability by different mechanisms. In summary, these data show that 3OC12-HSL and PQS affect mammalian vascular function by decreasing vascular tone and increasing endothelial permeability. The potential advantage of these effects of quorum sensing molecules on host cells allowing bacteria to promote increase local circulation or produce oedema or allow access to promote systemic infection.

Physical inactivity has been linked to poor health and disease progression, particularly in older people. This has led to an increasing interest in the effects of physical activity, physiological function and ageing. Importantly, negative health traits generally attributed to ageing, such as frailty, cognitive decline and brain atrophy, may in part result from decreased habitual physical activity levels, and be preventable with increased exercise. Here, we use magnetic resonance imaging (MRI) techniques to quantify the cortical haemodynamic and metabolic responses to acute low/moderate intensity exercise in healthy young and older volunteers, to investigate how this response is influenced by ageing and cardiorespiratory fitness. In addition, structural MRI is used to investigate global and regional grey matter volume, and cortical thickness in young and older adults, and to assess its association with age and cardiorespiratory fitness. In the final Chapter, methods are developed to study the impact of a 16-day limb immobilisation on brain and muscle function using ultra-high field, 7 T MRI. Functional magnetic resonance imaging (fMRI) is used to assess changes in brain motor cortex function over the course of 16 days of upper limb immobilisation, and assess whether any changes are observed associated with the loss of voluntary handgrip strength over the same time period. Muscle MRI is performed to assess changes in muscle cross sectional area, and muscle magnetic resonance spectroscopy (MRS) developed with the aim of quantifying changes in forearm muscle IMCL and EMCL content over the immobilisation period.

Magnetic Resonance Spectroscopy is a powerful non-invasive tool for investigating liver metabolism in vivo. PRESS and STEAM localized 1H MRS can be used to provide liver lipid measurements calculated from the fat to water peak ratios. Surface coils can also be used to measure other metabolites using multinuclear MRS, 13C MRS directly measures glycogen levels and 31PMRS measures ATP and other phosphate metabolites. This thesis reports on studies undertaken to develop these techniques and describes a number of in vivo investigations that explored the effects of dietary interventions on hepatic metabolism.

Increasing skeletal muscle carnitine content enhances PDC flux during 30 minutes of continuous exercise at 80% Wmax, reducing reliance on non-mitochondrial ATP production and improving work output. These studies in healthy volunteers evaluated a carnitine feeding strategy that did not rely on the high carbohydrate load previously used, then investigated whether manipulating muscle carnitine could alter the adaptations to a period of submaximal high-intensity intermittent training (HIT). The rate of orally ingested 2H3-carnitine uptake into skeletal muscle was directly quantified for the first time in vivo and increased 5-fold following ingestion of an 80g carbohydrate formulation. This positive forearm carnitine balance was entirely blunted when the carbohydrate load was supplemented with 40g of whey protein, suggesting a novel antagonisation of insulin-stimulated muscle carnitine transport by amino acids. Skeletal muscle biopsy sampling demonstrated minimal acetylcarnitine accumulation and non-mitochondrial ATP production during single-leg knee extension at 85% Wmax, suggesting that PDC flux does not limit oxidative ATP production under these conditions. Conversely, PDC flux declined over repeated bouts of cycling at 100% Wmax, as evidenced by greater non-mitochondrial ATP production in the face of similar acetylcarnitine accumulation. This suggested that muscle carnitine availability could influence oxidative ATP delivery during submaximal HIT. Manipulation of muscle carnitine content by daily carnitine/carbohydrate feeding elevated free carnitine availability and maintained PDC flux during repeated bouts of intense exercise. However, profound improvements in oxidative ATP delivery in response to HIT eclipsed any effect of this carnitine-mediated increase in PDC flux on non-mitochondrial ATP production and indeed, carnitine supplementation did not potentiate any increases in exercise capacity above submaximal HIT alone. These novel data advance our understanding of muscle carnitine transport and the interplay between carnitine metabolism, PDC flux and non-mitochondrial ATP production during intense exercise, having important implications for the development of nutritional and exercise prescription strategies to enhance human performance and health.

Introduction: Obesity and related cardio-metabolic complications have acquired global epidemic proportions. Suboptimal nutritional environment in early life induces adaptations in energy homeostasis, metabolism and adipose tissue development that may confer short-term survival advantages but are detrimental in later life, particularly if nutrient supply is restored. Brown adipose tissue (BAT) has a unique role in energy homeostasis because it can provide a potential compensatory mechanism against excess weight gain via cold or diet-induced adaptive thermogenesis. Brown adipocytes also have a potential role in lipid and glucose metabolism and BAT activation can increase clearance of lipids and glucose from the circulation. Pericardial fat, particularly epicardial adipose tissue (fat present between the myocardium and the visceral layer of the pericardium), is anatomically and clinically related to cardiac morphology and function and is believed to be a metabolically active organ that affects cardiac function and the evolution of cardiac pathologies. High expression of mRNA for uncoupling protein (UCP) 1 in adult human epicardial adipose tissue suggests that this may be a depot of BAT. Hypotheses: In my thesis, I hypothesised that pericardial adipose tissue is a depot of brown fat in humans and sheep. I also hypothesised that suboptimal nutrition in early life will affect adiposity and development of BAT in this depot. Methods: UCP1 mRNA expression and protein abundance and other BAT and white adipose tissue related genes were studied in pericardial adipose tissue. In the first study, pericardial fat was sampled from newborn and 30 day old sheep born to mothers fed with 100% or 60% of their total metabolisable energy (ME) requirement from 110 day gestation to term. In the second study, pericardial fat was sampled from near-term (140 day gestation) fetuses delivered to mothers fed 100% or 60% of total ME requirement from 28 to 80 days and then fed ad libitum. Gene expression was measured by reverse transcription-polymerase chain reaction and protein abundance by Western blotting and immunohistochemistry. To confirm the presence of BAT in the human epicardial fat depot, relative abundance of UCP1 was measured by Western Blotting in epicardial, paracardial, and subcutaneous fat samples taken from adults. In the final study, epicardial fat samples were collected from 63 children (0-18 years of age) undergoing cardiac surgery and gene expression of UCP1 and other BAT and WAT related genes identified by microarray. The presence of UCP1 was confirmed by immunohistochemistry. Results: Pericardial adipose tissue is a depot of BAT in fetal and newborn sheep. Suboptimal maternal nutrition in late gestation reduces the abundance of UCP1 and downregulates other BAT related genes whilst suboptimal maternal nutrition in early-to-mid gestation followed by ad libitum feeding to term, increases adiposity, enhances UCP1 abundance and upregulates genes involved in brown and white adipogenesis. Epicardial fat from newborn infants, children, adolescents and older adults contains UCP1 confirming that it is a BAT depot in humans. UCP1 gene expression in infancy and early childhood in humans is downregulated in children with poor nutritional states. Conclusions: I have shown that adipose tissue depots present around the heart are a repository of brown fat, at least in humans and sheep. In view of the potential role of BAT in regulation of lipid and glucose metabolism, this may have therapeutic implications for treatment of cardiovascular complications of obesity. Suboptimal nutrition in utero and during early life compromises BAT development. Although the exact mechanism of how these changes affect the propensity towards obesity and metabolic dysregulation remains to be elucidated, a reduction in thermogenesis presents a plausible mechanism for the increased metabolic efficiency associated with nutritional deprivation in early life. BAT persists beyond the neonatal period in to adult life and, therefore, presents a potential target for long lasting nutritional manipulations to promote better health.

Tissue regeneration approaches involve the recreation of biochemical and mechanical cues dictating tissue fate. Gradients of chemical cues are common in the natural microenvironment and are usually accompanied with gradual changes in cellular responses. Consequently, thorough understanding of biomolecule gradient development, their effective concentrations and the corresponding cellular responses as a function of time and space are essential for efficient design of scaffolds for biomedical applications. Here, we developed a compartmental diffusion model to study the development and measurement of biomolecule gradients. The model was validated to ensure effective spatiotemporal measurements of diffusing species within three-dimensional (3D) hydrogels. Results confirmed that the factors regulating the diffusing molecules’ behaviour in hydrogel matrices were dependant on the size of the diffusing species and the interaction with the matrix. The source compartment was subsequently replaced by polymeric particulate depots with tuneable characteristics to maintain structural protein stability and provide controlled temporal release of proteins and the diffusion through the hydrogel compartment was accordingly monitored. Glycosaminoglycan enhanced transduction (GET) technology was employed to study 3D gradient transduction of reporter protein in cell-laden hydrogels and to examine the effect of cells on the diffusion of biomolecules. Results demonstrated that cellular uptake of GET proteins altered the diffusion pattern as compared to acellular scaffolds and cells themselves acted as a sink that maintained steep GET protein gradients over the 5 mm wide scaffold. Furthermore, the synergistic combination of poly-arginine cell penetrating peptide (CPP) together with the cell membrane binding peptide using the GET technology demonstrated significant intracellular transduction in a gradient fashion in comparison to CPP alone. Employing GET technology and the compartmental diffusion model in the gradient delivery of the transcription factor MyoD to cell-laden hydrogels, resulted in directing the cells towards myogenic differentiation. However, the gradient pattern of differentiation was not clearly observed due to the limited number of genes examined. In conclusion, the model can be employed for the effective spatiotemporal gradient delivery of functional proteins to achieve the tissue complexity observed in the native tissues.

In vertebrates, haematopoiesis is maintained by self-renewing multipotent haematopoietic stem cells (HSCs) in the adult bone marrow. HSCs are specified and generated during early embryonic development from arterial endothelial cells in the ventral wall of the dorsal aorta (vDA) that become haemogenic. Examination of mib mutants and rbpja/b morphants zebrafish embryos, that have defects in Notch signalling show that both arterial specification and HSCs development require an intact Notch signalling pathway. Utilizing in-house generated zebrafish Notch reporter lines, we show that Notch signalling initiates in the migrating hEC progenitors before their arrival to the midline to form the DA. Following arterial specification, Notch activity rapidly increases in the DA cells and persists at high levels until the time point of HSC emergence from the vDA, then sharply falls to hardly detectable levels. Embryos treated with the γ-secretase inhibitor DAPT or DAPM lose HSC development but retain arterial specification. Quantification of the residual Notch activity in our reporter lines by RT-PCR, revealed that low levels of Notch signalling retained in treated embryos are sufficient for arterial gene expression, while high levels of Notch signalling are required for hECs induction. Additional to these loss of function experiments, our endothelial specific gain of function studies suggested that continuous expression of nicd in the ECs in stable transgenic lines prevent cells from differentiation into mature HSCs that can leave the vDA to seed the CHT. By contrast, transient expression of nicd in ECs appeared to expand HSC fate and allow cells to seed the CHT, suggesting that down-regulation of Notch signalling is essential to enable cells committed to the blood lineage to leave the vDA and seed the CHT. These data represent the first demonstration, to our knowledge, that cell autonomous Notch induction in ECs in vivo is sufficient to expand HSC formation in vertebrates. Ten-Eleven-Translocation (Tet) proteins are a family of enzymes known to be actively involved in DNA de-methylation by 5-mC oxidation. This process is essential for proper development and cell lineage specification. Here, we used zebrafish tet1/2/3 morpholinos to study the role of Tet enzymes in zebrafish development and organogenesis. Our results showed that individual depletion of Tet1 or Tet3 enzymes is sufficient to arrest zebrafish development at the onset of somitogenesis, whereas tet2 morphants appear normal. Moreover, analysis of mildly affected morphants shows that Tet1 knockdown leads to atypical neurogenesis in zebrafish embryos. Global 5hmC levels are dramatically reduced in both tet1 and tet3 morphants, indicating the importance of Tet proteins in vertebrate organogenesis.

NAD metabolism is increasingly implicated in a variety of biological functions, including regulation of gene transcription, lifespan, cell death, circadian rhythm and glucose home-ostasis. The three mammalian isoforms of the central NAD biosynthetic enzyme, nicotin-amide mononucleotide adenylyltransferases (NMNATs), have recently emerged as crucial players in neuronal maintenance and protection, in ageing-processes and in many neurodegenerative diseases, such as Alzheimer’s disease. The biological basis of ageing and its related pathologies are not fully elucidated and there is an urgent need to develop valid therapeutic strategies to minimize the impact that these conditions have on the society. In the present study, I used two mouse models, heterozygous Nmnat1 knockout mice (Nmnat1+/-) and mice overexpressing Nmnat1 (Nmnat1 tg), in order to assess the role of the nuclear isoform NMNAT1 in ageing and age-related diseases. First, I asked whether modulating the expression of NMNAT1 influences ageing-related mechanisms. To this aim, Nmnat1 tg and Nmnat1+/- mice were subjected to metabolic analysis and behavioral tests for measuring locomotor activity. Furthermore, NMNAT enzyme activity, NAD levels, gene expression and protein levels were analyzed with bio-chemical techniques at 6 and 24 months of age. I found that Nmnat1 tg mice had a reduced body weight and increased locomotor activity during ageing, while Nmnat1+/- weighted more at 12 months than their wild type littermates. NMNAT enzyme activity, significantly higher in Nmnat1 tg mice at 6 months, did not change during ageing. In contrast, NMNAT enzyme activity in Nmnat1+/-, which is already low in young mice, showed a trend of decrease with ageing. Second, in order to investigate the role of the nuclear isoform NMNAT1 in age-related diseases, I analyzed the effect of modulating NMNAT1 levels on behavioral and neuro-pathological traits in a mouse line which expresses non-mutant human tau isoforms (htau mouse), representing a model of tauopathy relevant to Alzheimer’s disease. To this aim, htau were crossed to Nmnat1 tg and Nmnat1+/- mice to produce experimental mouse groups with four genotypes that were all heterozygous for murine tau (mtau+/-): Nmnat1 tg/htau, Nmnat1+/-/htau and wild type littermates. Mice were subjected to a bat-tery of specific tests to assess potential behavioral abnormalities that correlate with dys-functions characteristic of AD. Furthermore, image analysis was performed to assess the integrity of the brain areas mainly impaired in AD. Finally, biochemical studies were conducted in order to test whether modulating NMNAT1 levels caused changes in NMNAT activity and in NAD levels in htau mice. I found that htau mice have an early, selective deficit in food burrowing, a behavioral task used to assess activities of daily living which are impaired early in Alzheimer’s dis-ease, and that overexpression of Nmnat1 ameliorates this defect. Despite the behavioral abnormalities, htau mice did not show neurodegenerative impairments in cortex and hippocampus. Modulating NMNAT1 levels produced a corresponding effect on NMNAT enzymatic activity but it did not alter NAD levels in htau mice. My results suggest beneficial effects of NMNAT1 on the early behavioral deficits in this mouse model of tauopathy. Finally, I asked whether modulation of Nmnat1 can influence the ischemic cell death, which is at least in part caused by an overactivation of PARP1 and NAD depletion. I hypothesized that reducing NMNAT1 levels exacerbates the ischemic brain damage, whereas increasing these levels confers protection by respectively decreasing or increas-ing NAD availability within the nucleus. To address this question, I used a stable NSC-34 clone expressing Wlds/NMNAT1, as well as wild type NSC-34 cells. I measured NAD levels and then tested cell viability af-ter genotoxic stress induced by N-Methyl-N-nitro-N-nitrosoguanidine (MNNG), which causes DNA damage and activation of PARP1, mimicking some mechanistic aspects of ischemic cell death. I first confirmed that Wlds was expressed in all cellular fractions in Wlds/NMNAT1 cells, but not in wild type cells. Furthermore, both wild type and Wlds/NMNAT1 cells showed lower NAD levels in the nuclear fraction compared to the cytoplasmic fraction. Paradoxically, NAD levels tended to be even lower in all cellular fractions of Wlds/NMNAT1 cells respect to the wild type cells. Correspondingly, they show higher susceptibility to cell death induced by MNNG. In addition to the in vitro experiments, I also started to set-up an in vivo model of cerebral ischemia. Even though time constraints did not allow analyzing the effects of Nmnat1 modulation, my study will be useful for future investigations to test whether overexpression of Nmnat1 or its downregulation may protect or exacerbate the effect of cerebral ischemia in an in vivo model. Taken together, my results suggest that modulating Nmnat1 correspondingly influences ageing and neurodegeneration processes and underline the utility of the Nmnat1 tg and Nmnat1+/- mice as a tool for future research in this field.

The nonspecific binding of proteins to various biological and non-biological surfaces has limited the potential of several detection techniques such as surface plasmon resonance (SPR), Luminex and ELISA (among others). Plasma proteins have been shown to decrease the sensitivity of instruments when working with complex fluids such as blood samples. This study examines the binding properties of several plasma proteins to a range of surfaces and utilises reagents in the media to eliminate the nonspecific binding of the plasma proteins. This has created a set of conditions that can reduce the nonspecific binding interaction, without affecting the specific interactions which the various techniques measure. These mechanisms were then applied to the assembly/disassembly of membrane microdomains. Membrane microdomains have been shown to be affected by from several factors such as acyl-chain length and temperature. This study demonstrates how reagents in the media can affect the assembly of these domains. We proposed a novel mechanism for the regulation of the domains, in which the reagents alter the intermolecular interactions between lipid head groups by altering the water network around these domains to promote domain assembly. These results that could have significant ramifications for the functional characterisation of membrane microdomains and the proteins that are known to associate with them. Finally, membrane binding affinity and kinetics of different polypeptides with various lipid membrane composition were characterised and resultant microdomains were monitored. Demonstrating how the previously uncontrolled, soluble factors in a model system can control the intermolecular interactions that occur in the system that is being measured.

As well as cognitive decline and neuropathological changes, Alzheimer’s disease (AD) is also characterized by non-cognitive behavioural symptoms like restlessness, wandering, agitation, confusion and profound disruptions of circadian rhythms. This group of symptoms is commonly referred to as ‘Sundowning’ and typically occurs in the late afternoon, evening or at night and causes significant problems for Carers. There are no specific drug treatments for these symptoms. One contributory factor is that little is known about the biological basis of these symptoms in Alzheimer’s disease. There is evidence, however, that they may arise as a consequence of abnormal circadian rhythms. Circadian rhythms characterize a number of human physiological and behavioural systems including energy metabolism, sleep-wake cycles, cardiovascular activity, body temperature and locomotor activity. Several studies have demonstrated a role for chromatin modifications in normal circadian function. In mammals, circadian rhythms are generated by a transcriptional-translational feedback loop in which the components of the positive limb (CLOCK and BMAL1), activate the components of the negative limb (the cryptochrome and period proteins). CLOCK possesses intrinsic histone acetyltransferase activity which has been implicated in the circadian control of gene expression. Disrupted rhythmic expression of the core clock genes has also been demonstrated in patients and AD mouse models. With a view to establishing a potential animal model to study the biological basis of Sundowning symptoms, we investigated whether a transgenic mouse model of AD, APPswe/PS1dE9 exhibits circadian alterations in locomotor activity, chromatin modifications and expression of clock genes. The effect of age on altering rhythms in locomotor activity was also investigated. Results show that the APPswe/PS1dE9 mutation alters levels and patterns in locomotor activity at all ages tested, most notably in the activity travelled in the last 2 hours of the dark phase, which is potentially relevant to the disturbance previously reported in AD patients. C57BL/6J and CD1 mice also showed evidence of altered circadian profile with age for locomotor activity. Biomolecular studies revealed altered rhythmic expression of the core clock genes as well as day/night rhythmic chromatin modifications. In summary, these studies reveal altered circadian regulation of locomotor activity, chromatin modification and clock gene expression in APPswe/PS1dE9 mice. They also provide strong evidence that disruption of circadian rhythms of locomotor activity has biomolecular analogies in a widely available model of AD. The APPswe/PS1dE9 model of AD demonstrates the potential to serve as a tool in understanding the neuropathology of circadian abnormalities in Alzheimer’s disease and a model system to test novel therapeutic agents.