Background: Obesity in Qatar is amongst the highest globally and constitutes a serious health risk. Obese individuals are at greater risk of vascular disease compared to the lean, especially in the insulin resistant state. The impact of obesity on endothelial vasomotor function is also adipose tissue depot-dependent, with the visceral environment being more pathogenic. It is, however, unclear how severe the impact of vascular dysfunction is in a relatively young and obese population. Further, it is becoming apparent that obesity-associated vascular dysfunction is heterogeneous. Recent research has focussed on different groups of obese subjects in order to elucidate the mediators of the differential cardiometabolic risk. In pathological obese (PO) subjects, sub-cutaneous adipocyte dysfunction and inflammation have been reported along with adipocyte hypertrophy. This led to the following hypotheses: 1. The enlarged adipocytes are more susceptible to hypoxia due to decreased capillary density of the depot and changes in vascular tone. Hypoxia leads to cell necrosis and the formation of immunological foci. 2. The more hypoxic and inflamed fat depot secretes less adiponectin, which then may determine the increased systemic insulin resistance and dyslipidaemia seen in the PO. Specifically differences between metabolically healthy but obese (MHO) and pathologically obese (PO) in relation to capillary density and vascular function was determined. To facilitate the overall objectives, in this study: 1. A cohort of MHO and PO subjects were identified. 2. Vascular differences in the adipose tissue of PO versus MHO by functional studies (myography) and histological assessment of vascular density were carried out, and, 3. Mechanisms that underlie these differences were investigated. Methods: Patients were recruited from the local hospital and blood and adipose tissue (Omental, OM; Subcutaneous, SC) samples collected. Fasting plasma glucose and insulin were assayed to determine insulin resistance status. Vascular function was assessed by wire myography. Cumulative concentration-response curves were generated for various vasoconstrictors (e.g. noradrenaline, potassium chloride), and vasodilators (e.g. acetylcholine, Sodium nitroprusside (SNP), and prostaglandin E2). Relaxation to acetylcholine was recorded in the absence or presence of Nω-Nitro-L¬arginine methyl ester (L-NAME), indomethacin, diclofenac, BaCl2, apamin+charybdotoxin, and arginine. mRNA expression of hypertension associated genes in stromal vascular fractions (SVFs) of both depots was assessed by real time RT-PCR. Paraffin-embedded tissues were used for histological studies. Results: OM arterioles were less sensitive to noradrenaline-mediated vasoconstriction compared with SC (log EC50 -5.9±0.2 vs. -6.5±0.1, p<0.05). Vasorelaxation to acetylcholine was attenuated in OM vessels compared with SC vessels (p<0.01). In contrast, relaxation to SNP was greater in OM compared with SC vessels (p<0.01). Acetylcholine curves for insulin-sensitive patients were less attenuated compared with insulin-resistant patients. L-NAME, apamin, charybdotoxin, and BaCl2 caused right-ward shifts of the acetylcholine curves, while indomethacin, diclofenac and arginine produced the reverse. In the whole group, COX2 mRNA, but not eNOS and COX1, were up regulated in OM compared with SC SVFs. However, when analyzed separately, in the OM compared to SC SVFs, of the MHO several genes were unregulated (AGT, ARG2, CLIC5, EPHX2, ITPR1 and PRKG1), while in the PO only two were significantly different between the depots (CLIC5 and PDE3B). Conclusions: Hyperinsulinaemia in adipose tissue microvessels was associated with і) vasocontractile insensitivity to noradrenaline and іі) to changes in NO-mediated vasodilation, at least partially mediated through components of the COX2 pathway.

Background: Managing child and adult undernutrition is a global public health priority. In poor settings, improved specialised products are needed for treatment and prevention, including for chronic disease/HIV. Objective: To develop a method for the design and pre-clinical testing of novel, low-cost Ready-to-Use Therapeutic Foods (RUTF), to be also applied to supplementary/complementary feeding interventions. A method was developed and tested, using four sequential studies, with HIV-positive Kenyan adults with severe acute malnutrition (case-study). A qualitative study explored adherence and consumption barriers with the current UN standard peanut/milk-powder-based therapeutic formulation (P‐RUTF). A study using Linear Programming (LP) designed an improved, cheaper formulation soy/maize/sorghum-based (SMS-RUTF), considered accurate if: its manufactured prototype, compared to calculated values; it had a measured energy density difference (EDD) < 10%; a protein or lipid difference (P/LD) < 5g/100g. An acceptability study (4-weeks-cross-over design; washout one-week) compared use of SMS-RUTF against P-RUTF (n=41), using 18 consumption/safety/preference criteria. Based on a literature review (28 randomized controlled trials of micronutrient supplementation; outcomes: increased survival and CD4 cell count, reduced viral load), four criteria to determine micronutrient specifications for the SMS-RUTF fortification were developed and applied. The reported compliance with the prescribed RUTF was relatively low, and informed the necessary formulation improvements. The LP-determined formulation was accurate (EDD: 7%; PD and LD: 2.3 and 1.0g/100g). The LP-based prototype was acceptable and safe, but with an average number of days of nausea and vomit (0.16 and 0.04 d) occurred with a higher frequency (P < 0.05) than in the control (0.09 and 0.02 d). The existing evidence for determining micronutrient specifications for SMS-RUTF posed some challenges for the development of manufacturing specifications. Twelve of the micronutrient specifications developed for SMS-RUTF fortificant premix were equivalent to the UN minimum standards; eleven were 2 to 10 times higher. Conclusions: The proposed set of methods can be used to design and pre-clinically test improved/cheaper RUTF products, targeting malnourished adults. Novel formulations should be clinically trialled before widespread-use.

Fetal growth is influenced by the in utero environment and genetic factors inherited from both parents. Poor fetal growth leading to low birth weight is associated with insulin resistance and type-2 diabetes in later life. The fetal programming of adult disease hypothesis suggests that growth-restricted fetuses make enduring physiological adaptations that predispose to diabetes in later life. The fetal insulin hypothesis suggests that poor fetal growth and diabetes are two phenotypes of genetically determined insulin resistance. Under these circumstances, an insulin resistant fetus cannot optimise insulin-mediated growth and is predisposed to diabetes in later life. Environmental and genetic influences come together through epigenetic modifications, for example DNA methylation, that alter gene expression without altering the nucleotide sequence. The first aim of this thesis was to investigate whether men who fathered pregnancies complicated by fetal growth restriction had an insulin resistant phenotype at the time of the index pregnancy. A case-control study showed that men who fathered growth-restricted offspring have pre-clinical insulin resistance and are more likely to smoke than fathers of normal grown offspring. This observation supports the concept that an insulin resistant genotype inherited from a father could manifest as poor fetal growth in offspring. I then investigated the mechanisms through which paternal insulin resistance might be inherited by a growth-restricted fetus. I studied DNA extracted from the cord blood of growth-restricted offspring using whole exome sequencing to identify novel gene variants and those known to be associated with type-2 diabetes. I validated findings with Sanger sequencing and Taqman genotyping in all family members. Using the Illumina Human 450 BeadChip, I found marked differences in genome wide DNA methylation of fetal cord blood and placental samples from growth restricted compared with normal grown offspring. Future work is aimed at investigating the functional consequences of genetic and epigenetic differences to identify targets for treatment and prophylaxis against fetal growth restriction and diabetes.

5-Methyltetrahydrofolate (5-MTHF) is involved in over 100 metabolic reactions. Potentially treatable cerebral folate deficiency (CFD) of 5-MTHF in cerebrospinal fluid (CSF) is associated in mitochondrial disease. The prevalence and significance of CSF 5-MTHF deficiency in mitochondrial disease was initially investigated. Prevalence of CSF 5-MTHF deficiency in skeletal muscle mitochondrial respiratory chain enzyme (RCE) defects was 15% and the minimum population prevalence of mitochondrial disease with CSF 5-MTHF deficiency was at least one in 30,000. This suggests under-diagnosis of the condition. The most common RCE defect in CSF 5-MTHF deficient patients was isolated complex IV deficiency. Severe CSF 5-MTHF deficiencies (<10 nmol/L) were confined to Kearns-Sayre syndrome or FOLR1 mutations. A novel homozygous missense mutation in FOLR1 exon 5 was observed. Oral folinic acid supplementation restored CSF 5-MTHF levels to within the age-related reference range in the majority of cases. Measurement of CSF 5-MTHF and, where appropriate, FOLR1 mutation analysis, in suspected mitochondrial disease patients is recommended. The mechanisms responsible for CFD in mitochondrial disease are unclear. The potential role of oxidative stress as a contributing mechanism was also investigated. CSF conveyed antioxidant properties towards 5-MTHF, which were overcome by hydroxyl radicals. CSF antioxidants may include ascorbic acid (AA). A CSF AA reference range was established and a significant positive correlation between CSF 5-MTHF and AA demonstrated. In SH-SY5Y cells, inhibition of mitochondrial complex I caused increased mitochondrial superoxide generation and significantly increased loss of 5-MTHF from the extracellular medium. Selenium has been reported to be elevated in CFD. The latter observation was also seen following treatment of cells with the selenium compound selenite; selenite has previously been implicated in ROS generation. Addition of AA prevented 5-MTHF degradation. Oxidative stress may be a factor in the development of CFD. Co-supplementation of folinic acid and AA may be of therapeutic benefit.

Introduction: Cotrimoxazole prophylaxis (CTX-p) is a cost-effective intervention that saves lives of HIV positive individuals. It is recommended by WHO for all infants born to HIV positive women (HIV-exposed infants). Despite this it is poorly implemented in resource limited countries including Zimbabwe. This project aimed to explore health system and patient-level factors that affect implementation of CTX-p among HIV-exposed infants in Harare, Zimbabwe. Methods: In the first phase of the study, policy and implementation procedures for CTX-p were studied at national and health care centre level through document review and key informant interviews. In the second phase, a detailed study of implementation procedures was conducted at Mbare Clinic, Harare, to explore challenges to CTX-p at various points in the prevention of mother to child transmission (PMTCT) cascade. This involved 1) a survey among post-partum women, 2) qualitative interviews with women who delayed/did not seek antenatal care (ANC), 3) follow-up of HIV positive women at six-weeks postpartum to investigate initiation of CTX-p, and 3) follow-up of HIV-exposed infants until six months to explore adherence. In addition, a systematic review was conducted to investigate the magnitude of loss to follow-up (LTFU) of HIV exposed infants from real-life PMTCT programs. Results: CTX-p is recognised as important by the Zimbabwe Ministry of Health; it has been incorporated into guidelines and treatment procedures for HIV-exposed infants. Health systems face challenges implementing CTX-p due to lack of human resources and poor supply chain management. For women, the first hurdle is seeking ANC, where user fees, fear of HIV testing, unsupportive husbands/partners, nurses’ discourteousness and long queues are barriers. Lack of knowledge of the importance of a six-week visit is the main barrier to sixweek visit attendance. Adherence challenges include: unsupportive husbands/partners, drug stock-outs and fear of unwanted HIV disclosure and associated stigma. The systematic review revealed that there is unacceptable LTFU of HIV-exposed infants along various points of the PMTCT cascade. Conclusion: Health care systems need to put in place measures to ensure optimum implementation of life-saving interventions and retention of HIV-exposed infants in care.

Proton therapy is one of the most advanced modalities for cancer treatments based on radiation, offering finite penetration depth, low energy deposition at the entrance and sharp dose fall-off. However some of its benefits may become a risk for the patient due to the uncertainties during treatment planning and dose delivery. This project investigates the feasibility of implementing a tool for the real time proton beam range verification in order to improve targeting accuracy and help to spare vital organs and healthy tissues as much as possible. The study is addressed to eye proton treatments. Here it is of key importance to obtain a good local control on the tumour while sparing the optical nerve and preserving the functionality of the eye. This involves surgically implanting a metal marker in the back of the eye between the tumour and the optic nerve and detecting the proton induced x-ray emissions (PIXE) generated by a therapeutic proton beam in the target. Preliminary experiments and Monte Carlo modelling were performed in an attempt to identify the parameters that will lead to the design of an ideal system. We focused on reducing the experimental background noise and op- timising the detection limit. PIXE signals were successfully acquired with a Cadmium Telluride (CdTe) detector at the Clatterbridge Cancer Centre (UK) and at the CATANA proton beam line (Italy). It was found that PIXE has a linear dependence with the proton fluence but it is energy dependent. This makes it unfeasible to be used for in vivo dosimetry. The most suitable metal was investigated and the minimum detectable residual range for gold and silver was assessed, even at clinical conditions. Statistical models for the multivariate analysis of the acquired data were implemented and a pos- sible use of PIXE was suggested in combination with the current treatment planning tools, to double check the correctness of a treatment delivery.

Babies that are born more than 8 weeks premature or those deprived of Oxygen during the perinatal period are susceptible to brain injury, particularly in conjunction with maternal or fetal infection, leading to neurological deficits later in life. Multiple studies have shown that even brief exposure to hypoxic conditions will cause rapid and selective increase in specific mitogen-activated protein kinases including extracellular signal - related kinase 1 and 2 (ERK1/2) and C-Jun N-terminus Kinases 1 to 3 (JNK1-3) as well as the JNK downstream effector: C-Jun. To gain insight into the in vivo function of some of these intracellular pathways that contribute to neuronal damage in ischemic postnatal brains, we examined the effects of global or cell type specific as single or combined deletions of ERK 1 and 2 and both cell type specific or functional deletions of C-Jun. In addition, ERK phosphorylation was prevented by the administration of the selective MEK inhibitor SL327. This was observed with both pre- and post-injury application. Lastly, to explore the effect of endotoxin as a sensitising agent prior to neonatal hypoxia ischemia: lipopolysaccharide given to neonatal mutant mice, with sensitising effects noted at a dosage of 0.6mg/kg of LPS and a time interval between endotoxin administration and hypoxia-ischemia of 12hr. The Rice-Vannucci mouse model is a well-established experimental paradigm for hypoxic ischemic injury, providing insights into molecular signals that determine both white and grey matter tissue loss. Normal pERK is detectable in periventricular white matter axons (15-45min post HI), followed by white and grey matter glia and cortical neurons (1-4h post HI), returning to normal by 8hr. Mice with double mutation of global ERK1 and neuronal ERK2 deletion showed a lack of pERK expression through the forebrain following HI. In LPS-sensitised HI, we observed a strong decrease in infarct size, histological brain injury and microglial activation in cortex, striatum, and thalamus. A more discreet effect was seen in subcortical white matter and hippocampus. ERK1 deletion attenuated the effect of neuronal ERK2 deletion. These results were reproduced following severe HI insult alone. Astrocytic ERK mutation exhibited a polar response with a 3-4fold increase in microglia activation and the number of dying cells within grey matter regions. Global inactivation of ERK, through pharmacological ERK inhibitor SL327 significantly reduced cell death, and associated microglial activation in both grey and white matter at 48h following HI insult. Application of SL327 even as late as 1hr post insult significantly reduced brain damage induced by mild HI exposure. Systemic administration 1hr after severe HI dramatically increased the survival rate of pups at 48hr post insult by 80% compared to sham-treated controls. Deletion of C-Jun in all neural-epithelial lineage cells resulted in a strong increase in injury following severe and LPS-sensitised insult. In contrast, neuron-specific deletion of C-Jun resulted in neuroprotection. Tunel positive cell death was significantly reduced compared to control groups, in white matter, cortex and thalamus. Microglial activation, and infarct volume loss was more discreetly decreased with a notable effect in cortex. C-Jun expression in astrocytes is not a major contributor to ischemic damage response, with very mild reduction in markers for cell death and microglia recruitment following severe hypoxia, and no change observed between mutants and controls after endotoxin-sensitised HI. Replacement of the four JNK-dependent C-Jun phosphorylation sites (jun4A) resulted in a mild decrease in cell death and microglial activation compared to littermate controls, which did not reach the level of statistical significance. Overall, the data points to an important role of neuronal ERK and C-Jun in both a cellular and biochemical response to HI in the neonatal cerebral brain, but also argues against the involvement of JNK-dependent C-Jun phosphorylation in mediating neural damage. In addition, inhibition of ERK via pharmaceutical agents shows promise in decreasing morbidity and mortality caused by mild to moderate HI exposure. Lastly, neuroepithelial C-Jun expression appears to be a critical factor in normal cortical development and employment of endogenous neuroprotective mechanisms against postnatal insult.

The work in this thesis documents the optical modelling and design of Fabry Perot Interferometer (FPI) sensors for the detection of ultrasonic waves. The ultrasonic waves modulate the optical phase of the beam through a change in the cavity spacing of the FPI sensor. The optical sensitivity is defined as the change in the reflected light per unit change in the cavity thickness. An optical model to simulate Interferometer Transfer Functions (ITFs) for a Gaussian beam propagating in a Fabry Perot Interferometer is implemented. An understanding of the Gaussian beam phase propagation in a Fabry Perot Interferometer is presented to help in explaining the shape of ITFs simulated. The model is experimentally validated. The model is applied as a design tool for the purpose of optimising FPI sensors. This is achieved by choosing the beam radii, mirror reflectivities and cavity spacing’s which lead to high optical sensitivity. A FPI sensor with high optical sensitivity and pressure linearity is achieved. A high pressure linearity can be achieved by creating a highly asymmetric ITF, by a combination of a highly diverging beam and aperturing the reflected beam. The understanding presented in this work helps in designing optimised FPI sensors for ultrasound detection, as well as in providing a general understanding of the effects of Gaussian beams or other types of divergent beams illuminating FPIs.

The risk of cardiovascular-related death is several-fold higher in patients with chronic kidney disease (CKD) compared with the general population. Arterial calcification (AC) is extremely common in patients with CKD and strongly associates with cardiovascular-related mortality, however, there are currently no specific treatments to prevent its development and/or progression. Abundant evidence now suggests that AC is cell-mediated and actively-regulated, involving mechanisms linked to bone homeostasis, production of calcification inhibitors and vascular smooth muscle cell (VSMC) function. The P2X7 receptor (P2X7R) is an ATP-sensitive cation channel which has been implicated in several biological processes, in non-vascular contexts, thought to be important in the aetiology of AC. In addition, disruption to the normal function of some enzymes involved in ATP metabolism has been shown to contribute to AC, although little is known about their role in CKD-related arterial calcium deposition. The work in this thesis tested the primary hypothesis that P2X7R contributes to the pathogenesis of CKD-associated AC. Preliminary work was also conducted to examine the expression of components of the ATP-metabolising system in this clinical setting. P2X7R expression was confirmed in human and rodent vascular smooth muscle but was un-affected by calcification. In vitro, the P2X7R-specific antagonist, A438079, did not influence calcium deposition occurring in the presence of human VSMCs or segments of rat aorta exposed to ‘calcification-promoting’ medium. Calcification of cultured rat aorta was also not influenced by a second P2X7R-specific antagonist, A839977, or by BzATP (a receptor agonist). Aortic rings from mice deficient in P2X7R calcified to a similar extent to wild-type controls in vitro. A novel, adenine-based mouse model was developed to evaluate the effect of P2X7R gene deficiency on CKD-associated AC in vivo. However, the number of mice exhibiting AC in the final experiment was too low to draw any firm conclusion. Therefore, rats were fed an adenine-containing, high phosphate diet for 4 weeks (to induce CKD and AC) and administered a selective P2X7R antagonist, twice daily, throughout this period. Pharmacological blockade of P2X7R did not influence the magnitude of aortic calcification in this model. Quantification of mRNA performed on tissue obtained from the in vivo rat experiment suggested that VSMC-specific markers are down-regulated in calcified arteries, although VSMC osteogenic transformation, which is widely reported in the literature to occur in the context of AC, was not detected. Expression of the apoptosis marker, caspase-3, was increased in calcified arteries in vivo. P2X7R blockade did not influence any of these changes in mRNA expression. Expression of mRNA for ENPP-1, an ATP-metabolising enzyme responsible for the generation of the calcification inhibitor, pyrophosphate (PPi), was significantly increased in calcified arteries from CKD rats. Functional activity of ENPP-1 was also increased in these vessels. The expression of mRNA for other components of the ATP-metabolising system was also in keeping with an attempt by VSMCs to generate more PPi, possibly as an adaptive, defensive response to uraemic, calcification-promoting factors. Furthermore, an increase in ENPP-1 mRNA expression was detected in calcified inferior epigastric arteries from patients with end-stage renal disease (extracted at the time of kidney transplantation). In summary, P2X7R does not appear to contribute to the pathogenesis of CKD-associated AC, although this should be confirmed in experimental models which more closely simulate human disease. Arterial expression of enzymes involved in the metabolism of ATP does seem to change in AC. Future work should therefore focus on gauging the clinical relevance of this in order to better understand the mechanisms underlying the disease and potentially develop new therapeutic interventions.

Faecal incontinence (FI) is a debilitating disorder that affects a significant portion of the population. The research included in this thesis aimed to test the hypothesis that magnetic actuating of smooth muscle cells loaded with superparamagnetic iron oxide nanoparticles (SPION) can modify the cell phenotype, which could be used with as a future therapy. The research focused on exploring a novel method of magnetic actuation and assessing its effects on the phenotype and biocompatibility of human rectal smooth muscle cells (HRSMC). A 2D model was used to demonstrate the effects of SPION on HRSMC. Initially, the effect of incubating HRSMC with different concentrations of SPION (0, 31.25, 250 and 1000 μg/ml) for 24 hours was investigated. Transmission electron microscopy revealed that SPION were endocytosed by cells and became concentrated inside endosomes. Superconducting quantum interference device (SQUID) measurements showed that SPION loading was concentration dependent and also that saturation occurred for concentrations above 250 μg/ml. SPION loading of HRSMC led to inhibition of the gene expression of actin and calponin when incubated in differentiation medium, with or without magnetic actuation, suggesting SPION caused the cells to shift towards a more proliferative phenotype. Live cell imaging revealed actuation of SPION-loaded HRSMC with stronger magnets led to an observable movement of internalized SPION and the plasma membrane. The findings from this research indicate SPION is biocompatible and may alter the phenotype of HRSMC. Therefore, SPION may offer novel benefits for regenerating damaged muscle in the treatment of FI. Further investigation is needed to assess the effects of magnetic actuation on SPION loaded cells.