von Willebrand disease (VWD) is the most prevalent inherited mucocutaneous bleeding disorder in humans, and results from either quantitative or qualitative defects in circulating levels of the multimeric plasma glycoprotein, von Willebrand factor (VWF). Type 1 VWD, a partial quantitative deficiency of VWF, occurs in 50-70% of affected individuals. The genetic mechanisms involved in the pathogenesis of type 1 VWD were investigated in a European Union cohort study, with no candidate mutation identified in 30% of 150 index cases recruited. A proportion of these patients may be heterozygous for exon deletions of VWF. However, standard DNA sequencing methods undertaken in the original study can mask the presence of copy number variation (CNV) within VWF due to the presence of a wild-type allele. Recent validation of the P011-A1/P012-A1 (MLPA) VWF probe kit enabled analysis of VWF for the presence of heterozygous deletions or duplications (CNV). CNV analysis in this study led to the identification of two novel heterozygous in-frame deletions, which, along with previous deletion identification during optimisation identified 6 deletions in total. This suggested that heterozygous deletions are a significant contributor to the pathogenesis of type 1 VWD. Furthermore, identification, characterisation and in silico analysis of the deletions was undertaken to map the intronic breakpoint locations and understand the pathogenetic mechanisms of deletion formation in VWF. Additional analysis on historical VWF deletions was also undertaken and indicated that VWF was enriched for Alu repetitive elements and these repetitive element sequences are the predominant pathogenetic mechanism in generation of large deletions in VWF. In vitro expression analysis of the in-frame deletion mutants revealed a significant secretion defect of VWF into conditioned media. Furthermore, in some instances, in-frame deletions have a dominant-negative mechanism on VWF secretion, multimerisation and intracellular storage. The phenotypes of patients identified with the heterozygous deletions were found to correlate with the in vitro expression results for all deletions expressed. This provided novel insight into the effects of heterozygous deletions in type 1 VWD.

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No description available.

This thesis presents a novel sigma delta-based pulse oximeter with higher resilience against ambient light interference in comparison to the conventional pulse oximeters available in the market. The pulse oximeter reported in this thesis uses a novel cross-coupled sigma delta modulator to drive the pulse oximeter LEDs with two chaotic, non-overlapping pulse density modulated sequences. The deployment of this modulator not only provides more immunity against ambient light but it also offers more control over the pulse oximeter LEDs power consumption resulting in less power consumption under low ambient light conditions. Additionally, this thesis suggests a novel trans-impedance amplifier (TIA) topology for better detection of the physiological signal under low perfusion state and weak heart pulses. The TIA increases the physiological signal power by partially removing its DC component and providing enough headroom for the amplification of the AC physiological signal. This AC physiological signal is used to calculate the level of blood oxygenation and its accurate detection has a direct impact on the accuracy of the overall pulse oximeter measurement. The thesis also offers a detailed description about the computer model of the pulse oximeter developed in Simulink. This model is the first computer model of pulse oximeter and it is a useful tool for design and development of a pulse oximeter. Comparing the hardware measurement results and the Simulink simulation results presented in Chapter 2 and 5 reflects the capability of the model in predicting the behavior of pulse oximeter under different measurement conditions. The thesis also includes a detailed explanation about our flexible PC-based pulse oximeter hardware prototype which was used as a reference to evaluate the performance of our novel sigma delta-based pulse oximeter. This PC-based conventional pulse oximeter prototype performs the entire signal processing tasks within the Matlab environment and therefore it can be individually used as a platform to design and evaluate the new signal processing algorithms designed for pulse oximeter application. Finally the thesis reports on the standalone FPGA based fixed point implementation of the novel pulse oximeter processing chain on a Xilinx Spartan 3 FPGA Fabric, with real-time measurement results compared and contrasted against the results obtained from the Matlab based processing engine.

Despite being resident in the heart, which derives most its energy needs from the oxidation of fatty acids, cardiac stem/progenitor cells are assumed to be glycolytic. Therefore, the transition of these non-contractile progenitor cells into beating cardiomyocytes necessitates an obligatory transformation of the metabolic infrastructure with mitochondrial network expansion and a distinctive switch from glycolysis to oxidative phosphorylation. However, expansion of progenitor cells in culture may impair their ability to upregulate fatty acid metabolism and increase mitochondrial function. Therefore, the main objective of this project was to determine, for the first time, substrate and energy metabolism in cardiac stem/progenitor cells before, during and after cardiomyogenic differentiation. Control passage two cardiosphere-derived cells (P2-CDCs), created through culture on fibronectin/poly-D-lysine (FN/PDL) or collagen IV/hanging drops (ColIV/HD), have been shown to derive most of their energy needs from glycolysis with elevated lactate efflux. However, after 3-weeks of treatment with 5-Azacytidine (5-Aza) and ascorbic acid (AA), differentiated cells started to upregulate fatty acid oxidation and significantly reduced glycolytic flux and lactate production. Therefore, different metabolic mediators were used to stimulate fatty acid oxidation in differentiated cells. Various media with or without synthetic small molecule(s) were tested to optimize differentiation capacity and to stimulate aerobic oxidative phosphorylation in P2-CDCs. WY-14643+TGFβ supplemented medium was able to induce cardiomyogenic differentiation of P2-CDCs into mature, differentiated myocytes. The differentiated cells showed a significant increase in expression of both cardiac-specific genes and proteins, calcium handling genes as well as transforming glycolytic metabolism into more efficiently producing higher energy system, fatty acid, and carbohydrate oxidation in addition to glycolysis, suggesting they may be forming ventricular myocytes.

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Human angiotensin-1–converting-enzyme (ACE) plays a primary role in the regulation of blood pressure and electrolytes as part of the Renin-Angiotensin System (RAS) in humans. Renin cleaves angiotensinogen to angiotensin I and ACE regulates the vasoconstriction of blood vessels by converting angiotensin-1 to angiotensin-2 (a potent vasoconstrictor) and also by breaking down bradykinin (a potent vasodilator). Renin is regulated by a feedback loop mechanism and is inhibited by higher concentrations of angiotensin-2. A lack of or inhibition of ACE can lead to a reduction in blood pressure (BP) and may reduce the risk of diabetic nephropathy as high BP and high fluid retention can cause swelling in the kidneys. ACE inhibitors are used as treatments for these conditions as well as treating congestive heart failure (Stanley & Samson, 2002). ACE expression has been found in human adipose cells (Jonsson, et al., 1994) and ACE expression in this tissue was reduced when rats were treated with the ACE inhibitor Enalapril (Santos, et al., 2009). Currently ACE’s role in this tissue is unknown and therefore the study of the Drosophila homolog ACER, which is expressed within the fly fat body which is similar in structure to human adipose cells, may highlight a role for ACE in adipose tissue. To investigate the role of ACE-like enzymes in dietary effects on ageing-related and circadian health, function and metabolism we are studying ACER, a homologue of human ACE, in the fruit fly Drosophila melanogaster. Previous studies (Taylor, et al., 1996; Carhan, et al., 2010) have shown that Acer is expressed in the embryonic heart, adult head and adult fat body of the fly. The expression in the fat body is particularly interesting as the fly fat body acts like the human liver and adipose cells where ACE in humans is expressed. Acer’s expression in the head shows a circadian cycle and appears to be regulated by the circadian gene Clock. AcerΔ mutant flies exhibit normal circadian locomotor rhythms but show defects in the regulation of sleep, and the ACE inhibitor, Fosinopril, fed to flies disrupts night time sleep in the same way. This suggests a role for ACER in a circadian phenotypes in Drosophila and therefore a potential circadian role for ACE in humans (Carhan, et al., 2010). The present study has found the effect of the loss of Acer expression in the Acer deletion mutant (AcerΔ) was complex and was often dependent on genetic background and sex. AcerΔ mutants responded normally to dietary restriction (DR) for both sex and background therefore, Acer was not required for the DR response to lifespan. Lipid storage in the AcerΔ mutants was unaffected by the loss of Acer expression but glycogen storage was reduced on high food levels and did not show the normal increase in storage with increasing food compared to controls. Thus, indicating a role for Acer in the modulation of glycogen storage. The genetic backgrounds analysed in this study were the outbred whiteDahomey (wDah) and inbred white1118 (w1118) backgrounds which did show a difference in the effect of the loss of Acer for certain phenotypes. Fecundity in AcerΔ females was lower than in controls in the more fecund wDah background but not in the less fecund w1118 background. Sleep also showed an altered response between the backgrounds and sexes to changing diet in AcerΔ mutants. AcerΔ mutants were starvation and oxidative stress resistant but only in the wDah background and showed sensitivity when compared to controls in the w1118 background. To investigate Acer’s role in the response to nutrition the effect of the loss of Acer on drosophila-insulin-like peptides (dilps) was investigated. Altered transcript levels of dilps in the head and body of the fly in AcerΔ mutants indicated a possible link between Acer and the IIS (insulin/IGF-like signalling) nutrient-sensing pathway. In this study a role for Acer in the modulation of nutrient responsive phenotypes was established.

No description available.

No description available.