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Pulmonary arterial hypertension (PAH) is a progressive disease characterised by vasoconstriction and remodelling of the pulmonary vasculature. The consequence of this is increased pulmonary arterial pressure, right heart failure and eventual death if left untreated. Even in those patients receiving advanced PAH therapy, mortality rates remain high. Therefore, the development of novel therapeutic approaches in the treatment of PAH is urgently required. A better understanding of PAH pathogenesis is critical to achieving this. Gender differences exist in human PAH, with females up to three-fold more likely to present with disease than males. Limited evidence suggests that estrogens may be accountable for these differences. For example, the use of oral contraceptives has been associated with the development of PAH. Paradoxical to this however, experimental models of PAH exhibit male susceptibility which has limited research into the role of estrogens in the pathogenesis of PAH. Serotonin is implicated in both experimental and human PAH. Activity of the serotonin transporter (SERT) modulates the development of PAH and mice over-expressing SERT (SERT+ mice) exhibit PAH and exaggerated hypoxia-induced PAH. In the central nervous system, estrogens regulate expression of several serotonin signalling components including tryptophan hydroxylase (TPH), the 5-HT receptors and SERT. One hypothesis is that similar estrogen effects on serotonin signalling may also be apparent in the pulmonary vasculature, and this is one hypothesis for the increased female susceptibility observed in PAH. To examine this, the influence of gender and estrogen on the development of PAH in SERT+ mice was investigated. This was repeated following exposure to chronic hypoxia. Pulmonary vascular reactivity was determined using small vessel myography. The genotypic differences in SERT+ mice were also assessed via microarray analysis. Genes of interest were validated by qRT-PCR analysis and immunoblotting. To translate clinical relevance to any findings, the effects of female hormones were also investigated in human pulmonary artery smooth muscle cells (PASMCs) derived from both non-PAH (control) and idiopathic pulmonary arterial hypertension (IPAH) patients. PAH was assessed via measurement of right ventricular systolic pressure (RVSP), pulmonary vascular remodelling and right ventricular hypertrophy (RVH). Male SERT+ mice do not develop PAH. Female SERT+ mice exhibited increased RVSP and pulmonary vascular remodelling. This increased RVSP and pulmonary vascular remodelling were completely prevented following ovariectomy in SERT+ mice. The chronic administration of 17β estradiol (1.5mg/kg/day), which is the pre-dominant circulating female hormone in pre-menopausal women, fully re-established PAH as assessed by increased RVSP and pulmonary vascular remodelling. Pulmonary vascular reactivity to serotonin was unaffected in these mice. In chronic hypoxia, female SERT+ mice exhibited exaggerated hypoxia-induced PAH whereas male SERT+ mice remained unchanged compared to their respective wildtype (WT) controls. This exaggerated hypoxia-induced PAH phenotype was attenuated in SERT+ mice following ovariectomy, as assessed by a reduction in RVSP and pulmonary vascular remodelling. The chronic administration of 17β estradiol completely re-established exaggerated hypoxia-induced PAH in ovariectomized SERT+ mice. Similar to normoxia, serotonin-induced pulmonary vascular contraction was also unaffected in these mice. The stimulation of PASMCs with 17β estradiol increased tryptophan hydroxylase-1, SERT and 5-HT1B receptor expression. Consistent with our in vivo findings, physiological concentrations of 17β estradiol (1nmol/L) stimulated PASMC proliferation whereas estrone, estriol and progesterone had no effect. This proliferation was successfully blocked by both the tryptophan hydroxylase inhibitor para-chlorophenylalanine and the 5-HT1B receptor antagonist SB224289. Microarray analysis in the pulmonary arteries of female SERT+ mice confirmed the dysregulation of multiple pathways with relevance to PAH including those associated with metabolism, cell differentiation and contraction. A large number of PAH-related pathways were also dysregulated in female SERT+ mice exposed to chronic hypoxia. In contrast, pathways were altered in normoxic and chronically hypoxic male SERT+ mice to a much lesser extent. For microarray validation, qRT-PCR analysis was performed in ten selected genes (FOS, CEBPB, CYP1B1, MYL3, HAMP2, LTF, PLN, NPPA, UCP1 and C1S) and 100% concordance was reported. Three genes were selected for further investigation (FOS, CEBPB and CYP1B1). Immunoblotting confirmed that protein expression of c-FOS, C/EBPβ and CYP1B1 was increased in the pulmonary arteries of female SERT+ mice compared against female WT mice. With relevance to human PAH, we also confirmed that mRNA expression of FOS, CEBPB and CYP1B1 was increased in PASMCs derived from IPAH patients. Protein validation with immunoblotting confirmed that c-FOS, C/EBPβ and CYP1B1 expression was also increased in IPAH PASMCs. In summary of these findings, we have identified that females may be at an increased risk to the development of PAH via the effects of 17β estradiol. Specifically, 17β estradiol appears to increase expression of several key serotonin pathway mediators including tryptophan hydroxylase-1 (the rate-limiting enzyme in serotonin synthesis), SERT and the 5-HT1B receptors, all of which have been previously implicated in the pathogenesis of PAH. Our results suggest that 17β estradiol stimulates PASMC proliferation via activation of the serotonin pathway. Microarray analysis in the pulmonary arteries of SERT+ mice confirmed that a large number of genes associated with PAH-related pathways were differentially expressed in females. RNA and protein validation further confirmed these differences in selected genes (CEBPB, CYP1B1 and FOS). With relevance to human PAH, we also confirmed the dysregulation of several key genes at both mRNA and protein level in PASMCs derived from IPAH patients. Taken together, these findings describe the critical role of 17β estradiol in PAH, and this may offer an explanation for the increased susceptibility observed in females.

The chronic treatment of patients with a β-adrenoceptor antagonist is associated with prolongation of the atrial cell action potential duration (APD), potentially contributing to the ability of these drugs to prevent atrial fibrillation (AF). The mechanisms underlying this APD prolongation are not fully understood but may involve pharmacological remodelling of atrial K+ currents and underlying ion channel subunits. This project aimed to test the hypothesis that various characteristics of human atrial K+ currents, including voltage, time and rate dependency, differ between patients treated and not treated with a β-blocker as a result of altered expression of ion channel pore-forming and accessory subunits. Human atrial myocytes were isolated enzymatically from right atrial appendage tissue obtained from consenting patients, in sinus rhythm, undergoing cardiac surgery. Using whole cell patch clamping, K+ currents were recorded at physiological temperature. Treatment of patients with β-blockers for a minimum of 4 weeks duration was associated with a significant, 34% reduction in the transient outward K+ current (ITO) density but no change in the sustained outward current (IKSUS). There was a reduction in the Ba2+-sensitive, inwardly rectifying K+ current (IK1) but only at -120 mV and the physiological significance of this is unclear. The reduction in ITO density was not secondary to changes in the voltage dependency of the current, as determined by Boltzmann curve fits. There was no difference in the time dependent inactivation or re-activation of ITO between cells from non β-blocked and β-blocked patients, indicating these current characteristics were not contributing to β-blocker induced APD prolongation. The density of ITO decreased significantly with increasing stimulation rate in cells from both patient groups but remained significantly reduced in β-blocked patients at all rates studied. To determine a possible mechanism underlying the reduction in ITO density, the expression of Kv4.3 mRNA, the pore-forming subunit responsible for this current, was compared in right atrial appendage tissue from non β-blocked and β-blocked patients using real-time RT-PCR. mRNA levels were normalised to the expression of both 28S, a marker of total RNA, and the housekeeping gene GAPDH. The levels of mRNA for the accessory subunits KChIP2, KChAP, Kvβ1 and 2 and Frequenin, which modify Kv4.3 expression and function, were also measured. No change was found in the relative mRNA levels of any of these ion channel subunits in association with chronic β-blockade. mRNA for the pore-forming subunits Kir 2.1 and 2.2 and Kv1.5 which are responsible for IK1 and IKSUS respectively, in addition to mRNA for the pore-forming subunits underlying the L-type calcium current and sodium-calcium exchanger were also measured. Again, no significant changes in expression were found in association with chronic β-blockade. The possibility of ion channel remodelling at a translational level was investigated by measuring Kv4.3 protein levels using Western blotting with a monoclonal anti-Kv4.3 antibody. Kv4.3 protein levels were normalised to GAPDH which was used as a loading control. Chronic β-blockade did not change the ratio of the level of Kv4.3 protein relative to GAPDH. In conclusion, chronic treatment of patients with a β-blocker is associated with a reduction in atrial ITO density which may contribute to the APD prolongation reported in cells from these patients. However, this cannot be explained by changes in the expression of Kv4.3 or by changes in the expression of its regulatory accessory subunit genes.

The work presented in this thesis describes the influence of the endothelium on smooth muscle cells, and how the structure of the internal elastic lamina (IEL) affects this relationship in mesenteric and saphenous arteries. This was enabled by the study of functional and confocal microscopy dye transfer experiments. Normotensive (WKY) and hypertensive (SHR) rats of 12 weeks and 6 months of age were used to assess the effect of hypertension and ageing on endothelial and smooth muscle cell communication. The endothelium-derived hyperpolarising factor (EDHF) response in mesenteric arteries was investigated using wire myography, and the involvement of myoendothelial gap junctions (MEGJs) was assessed using the putative gap junction inhibitor carbenoxolone. Carbenoxolone attenuated the EDHF response in the WKY, suggestive of the involvement of myoendothelial gap junctions in EDHF. In the saphenous artery, incubation with L-NAME and indomethacin abolished the relaxation to ACh, indicating that there was no EDHF response in this artery. Dye transfer experiments using luminally loaded calcein-AM and the gap junction blocking peptides (GJi) 37,43Gap27 and 40Gap27 in mesenteric arteries demonstrated that the dye moved from endothelial cells to smooth muscle cells by means of gap junctional transfer in the WKY. In the SHR, calcein staining in the smooth muscle showed a significant reduction to that observed in the WKY at both age points (with endothelial cell staining remaining constant between strains), and GJi incubation did not reduce smooth muscle cell staining from that of the control. Dye transfer was correlated with the EDHF function of the vessels, and was consistent with the increasing evidence supporting the role of MEGJS in the EDHF response. No conclusive evidence could be obtained from saphenous artery imaging of dye transfer or fenestrae area due to its unsuitability for this approach. However, functional experiments illustrated this artery (reported to have a lack of MEGJs) to be devoid of an EDHF response, thereby making it a potential control artery for the mesenteric artery. Differences in the organisation of elastin in the IEL were assessed by measurement of fenestrations in the IEL, which are the only points through which endothelial and smooth muscle cells can connect. In WKY, the fenestrae appeared to fuse with age, causing a parallel increase in fenestrae area and decrease in fenestrae number which was not apparent in the SHR due to the inability of fenestrae to fuse in this strain. The smaller fenestrae area in the 6 month old SHR is thought contribute to the reduced EDHF response compared with WKY. Collectively, the results of this study imply that there is a relationship between the EDHF response and myoendothelial coupling within an artery. EDHF response and smooth muscle cell staining following luminal calcein-AM application were reduced in the 6 month old SHR compared with aged-matched WKY. The smaller fenestrae in the IEL of the 6 month old SHR is a possible explanation as to why there is reduced cellular coupling, as this would restrict the passage of substances or charge between the endothelium and smooth muscle, be it by gap-junctional transfer or by diffusion.

Cortical networks comprise excitatory principal cells and interneurons (IN); the latter showing large neurochemical, morphological and physiological heterogeneity. GABA release from IN axon terminals activates fast ionotropic GABAA or slow metabotropic GABAB receptors (GABABR); ionotropic GABA mechanisms are well described in INs, whereas GABABR activity is less well understood. The primary aim of this thesis is to ascertain GABABR mediated inhibition in different IN types containing the neurochemicals parvalbumin (PV), cholecystokinin (CCK) or somatostatin (SSt). Using immunocytochemical techniques, at light and electron microscopic levels, we examined the cellular and subcellular expression of GABAB1 receptor subunits in these INs. Application of whole-cell patch clamp techniques in acute slices, allowed analysis of GABABR effects pre- and postsynaptically; in response to endogenous GABA release or pharmacological activation. Light microscopy showed GABAB1 expression in INs containing CCK or SSt, equivalent to CA1 pyramidal cells; with low expression in PV INs. Using electron microscopy, we detected GABAB1 receptor subunits in dendrites of CCK and PV INs, with densities equivalent or higher than CA1 pyramidal cell dendrites. Unexpectedly, SSt containing dendrites showed a lower density of GABAB1 receptor subunits. In axon terminals of CCK and PV containing INs, we found comparable densities of GABAB1 receptor subunits. Electrophysiological recordings confirmed the presence of functional postsynaptic GABABR in PV and CCK INs. GABABR-mediated slow inhibitory postsynaptic currents (IPSCs) had typically large amplitudes, but with high cell-to-cell variability in both IN types. Morphological separation of PV or CCK INs revealed slow IPSC amplitudes which were large in perisomatic inhibitory (PI)cells (30.8 ± 8.6 pA and 39.2 ± 5.5 pA, respectively) and small in dendritic inhibitory (DI) cells (4.0 ± 1.7 pA and 11.6 ± 2.4 pA, respectively). Consistently, SSt-immunoreactive DI INs exhibited very small IPSCs (1.5 ± 0.2 pA). Pharmacological activation of GABAB R by the selective agonist baclofen revealed variable amplitude whole-cell currents, confirming differences between IN subtypes. Examining presynaptic GABABR activity; we minimally stimulated str. pyramidale evoking monosynaptic IPSCs in CA1 pyramidal cells. IPSCs mediated by CCK or PV PI axons were pharmacologically isolated by CB1 or M2 receptor activation. Both monosynaptic responses were reduced by baclofen, albeit differentially so. To further investigate this effect we performed paired-recordings from PV or CCK INs coupled synaptically to CA1 pyramidal cells. Baclofen inhibited PV and CCK basket cell mediated IPSCs by 51% and 98%, respectively; with a smaller effect in DI INs. In summary, we have shown that functional GABABRs are expressed pre- and postsynaptically in hippocampal GABAergic INs; with distinct populations of INs under differential GABABR control. Postsynaptic inhibition was strong in PI INs, but weak or absent in DI INs, a relationship conserved presynaptically. The observed differential expression of GABABRs is likely to play a fundamental role in regulating the excitability and activity of GABAergic INs, regulating synaptic output and potentially contributing to network and oscillatory activity. Consequentially, during periods of high GABA release, GABABR activation could act as a switch, allowing DI INs to play a greater role in network inhibition, due to GABABR mediated inhibition of perisomatic-targeting INs.

The objectives of The Identification and Prevention of Dietary and Lifestyle Induced Health Effects in Children and Infants study (child sample) and the Kenya Adolescent Physical Activity study (adolescent sample) conducted in this thesis was firstly, to assess the impact of methodological and practical decisions such as the appropriate epoch length and cutpoints to use in accelerometry studies involving children and adolescents across the physical activity continuum and the reliability of these accelerometer outcomes in predicting habitual physical activity. Secondly, the performance of uniaxial ActiTrainer accelerometry with heart rate (HR) monitoring was compared to triaxial GT3X accelerometry against indirect calorimetry during structured activities in the predominantly active Kenyan adolescent cohort. Similarly, the performance of uniaxial ActiTrainer accelerometry with HR monitoring vs. triaxial 3DNX accelerometry was compared against DLW under free living conditions in both children and adolescent cohorts. Finally, the validated uniaxial ActiTrainer was used to assess the impact of physical activity and the environment on energy expenditure and indices of adiposity in the two cohorts. The main findings of the thesis were: (a) that 15 s epoch reports significantly higher engagement in physical activity compared to a 60 s epoch in both the children and adolescents cohorts (b) choice of cutpoints significantly affected classification of physical activity and sedentary behaviour in both cohorts (c) a minimum of 6 h for 7 - 9 days in the cohort involving children and a minimum of 6 h for 4 - 5 days in the adolescents were required to reliably measure physical activity (d) triaxial accelerometry reported better predictive validity compared to uniaxial accelerometry during structured activities. In addition, HR monitoring did not improve the predictive validity of either accelerometer during structured activities (e) during free living activities, however, uniaxial and triaxial accelerometry reported comparable predictive validity. The addition of HR monitoring improved the predictive validity of uniaxial accelerometry by approximately 4% in both cohorts (f) in both cohorts, total volume of physical activity and patterns (time engaged in light and moderate to vigorous physical activity) were significantly associated with energy expenditure (g) physical activity and sedentary behaviour were significantly related to indices of adiposity in both cohorts (h) the environment was a significant predictor of physical activity and indices of adiposity in the adolescents but not children. The findings of this thesis have important implications on strategies to standardise accelerometry field protocols and future studies on the validation of accelerometers and the association between physical activity, the environment and health.

The work described in this thesis investigated the role of the sarcoplasmic reticulum (SR) in the regulation of normal mechanical function of the isolated heart, with the focus of the work resting on the calcium release channel, the ryanodine receptor (RyR) and the SR calcium pump, the sarco-endoplasmic reticulum calcium ATPase (SERCA). This was performed by characterising left ventricular mechanical function in an isolated working heart model in the face of pharmacological manipulation of the above intracellular SR proteins. The following were the aims of this project. 1. Characterisation of ex vivo working heart left ventricular mechanical function using miniaturised single and multi-segment pressure-conductance technology 2. Characterisation of working heart mechanical function under conditions where SR calcium cycling is altered; i) perfusion with elevated extracellular calcium and beta-adrenergic stimulation to induce SR calcium loading and ii) in a rabbit coronary artery ligation model of left ventricular dysfunction. Following this, the influence of acute perfusion of the compound K201 in healthy hearts and in the above experimental conditions was investigated. 3. Investigation of the specific contribution of SR calcium uptake to normal left ventricular contractility and relaxation by specific pharmacological blockade of SERCA in the isolated working heart. Hearts from male New Zealand White rabbits were cannulated via the aorta and a pulmonary vein ostium and perfused in working heart mode. Contractile function and changes in left ventricular volume during the cardiac cycle were assessed using a 3F pressure-conductance catheter inserted into the left ventricle via the aorta. The isolated working heart was found to produce a maximal stroke volume under identical loading conditions and pacing frequency using a volume of air within the compliance chamber of 15 ml of air. Using an atrial filling pressure of 7.4 mmHg and an afterload of 60 mmHg the preparation was also found to have stable function in working mode for 60-90 min with <10% decline in cardiac output. To assess the effect of the compound K201 on cardiac mechanical function isolated working hearts were perfused with increasing concentrations of K201 (0.3, 1.0 and 3.0) for 4 min at each concentration. When normalised to control hearts paced at equivalent rates 0.3 microM K201 led to a significant increase in end diastolic pressure (EDP) but did not alter other indices of mechanical function. 1.0 and 3.0 microM K201 led to a significant decline in a number of indices of cardiac contractility and relaxation. At 3.0 microM depression of mechanical function was of a sufficient extent as to cause cessation of forward flow (aortic flow) in 3 of the 8 hearts studied. In a subset of experiments where heart rate was kept constant via electrical stimulation of the right atrium, exposure to 1.0 microM K201 led to a decline in mechanical function that was of a similar degree to that of unpaced hearts. High calcium loading conditions were simulated by raising extracellular calcium concentrations from 2.5 mM to 4.5 mM, followed after 5 min by addition of 150 nM isoproterenol (ISO). Addition of ISO led to a transient improvement in all indices of mechanical function lasting approximately 20s. In hearts perfused with 4.5 mM extracellular calcium and ISO only (n=11) mechanical function declined thereafter such that by 600s post-ISO addition aortic flow had ceased in all but 5 of the 11 hearts in this group. In contrast, hearts perfused with K201 30s after addition of ISO (n=9) all maintained aortic flow and demonstrated significantly improved contractile function post-ISO in comparison to hearts not perfused with K201. K201 also led to a preservation of left ventricular peak pressure and stroke volume when applied 300s post-ISO. Despite being detrimental to mechanical function in healthy hearts K201 preserved function in a pharmacological model of high calcium loading. Left ventricular dysfunction was surgically induced in rabbits by coronary artery ligation. Ex vivo mechanical function was assessed 8 weeks post-op using a 3 segment multi-segmental pressure-conductance catheter inserted into the left ventricle with each segment delineating 3 discrete functional regions within the ventricular chamber; apex, mid and base. Hearts were paced at 200 beats/min throughout. 8 week ligation hearts exhibited a significantly reduced ejection fraction and higher end systolic volumes in comparison to sham operated controls. Assessment of regional mechanical function in 8 week ligation hearts revealed that a significantly greater proportion of total stroke volume and stroke work was performed by the mid segment vs. the apex, while regional function was similar between all segments in the sham operated controls. Treatment with 1.0 microM K201 led to a decline in systolic function and impairment of relaxation (decreased dP/dtmin and increased tau) in sham operated control hearts similar to that shown in chapter 3. In contrast, aside from dP/dtmax which was significantly reduced from baseline, K201 had no significant effect on mechanical function in 8 week ligation hearts. No region specific effects of K201 were detected, as the percentage contribution of each region to total stroke volume and stroke work remained unchanged in both groups after treatment with K201. The effects of K201 on mechanical function in hearts with left ventricular dysfunction appear to be altered in comparison to both healthy and sham operated hearts. SR calcium uptake was inhibited in an isolated working rabbit heart model using the specific pharmacological SERCA2a inhibitor thapsigargin (TG). Hearts were paced at 220 beats/min throughout the investigation. The experimental endpoint for TG treated hearts was reached when hearts no longer produced an aortic flow. Hearts perfused in working heart mode with the thapsigargin vehicle for 60 min acted as controls. At the end of each experiment left ventricular tissue samples were homogenised in a protease-phosphatase inhibitor cocktail and stored for subsequent analysis. A significant decline in systolic and diastolic functional parameters was evident 5 min after addition of TG, with no significant changes demonstrable in the vehicle control group at this timepoint. In TG treated hearts function had declined even further by the endpoint. In a subset of TG treated hearts end systolic and end diastolic volumes were shown to decline over time, indicative of imparied left ventricular filling and diastolic abnormalities. Analysis of SERCA uptake by Elliott et al. (2009) using tissue homogenates from the current investigation revealed that at the time of aortic flow cessation SERCA activity was <10%, suggesting that SR calcium uptake may decrease to very low levels before working heart function fails.

Mitochondria are known as the powerhouse of the cell due to their central role in energy generation and as the site of key metabolic pathways. Over the past 15 years, it has become unequivocally clear that most pro-apoptotic stimuli require a mitochondria-dependent step, involving the permeabilisation of the mitochondrial outer membrane to apoptogenic factors, such as cytochrome c and Smac/DIABLO. The release of these factors into the cytosol is tightly regulated by proteins of the Bcl-2 family and results in the activation of the caspase cascade, leading to cell death. This event is considered as a point of no return in the apoptotic pathway and is often inhibited in cancer. Cardiolipin (CL) is a mitochondria-specific phospholipid that contains four acyl-chains. CL has been implicated in many of the mitochondria-dependent steps that lead to the release of apoptogenic factors including interaction with the Bcl-2 family protein tBid, Bax-dependant mitochondrial outer membrane permeabilization and cytochrome c release. Despite this growing body of evidence, the mechanism by which CL and its fatty acyl chain composition regulate mitochondrial apoptotic pathways remains unresolved, mostly due to the lack of cellular model. Tafazzin is a mitochondrial enzyme, which is mutated in Barth syndrome (BTHS) and is involved in the maturation process of CL. In BTHS, loss of tafazzin activity results in a decrease in mature CL, making it a good model to investigate the role of CL in apoptosis. Using BTHS patients-derived lymphoblastoid cells and HeLa cells in which tafazzin was stably knocked-down using RNA interference, this study provides the first evidence that mature CL are required for an efficient extrinsic apoptotic pathway in type II cells. Further investigation of the impaired apoptotic pathway revealed that the major block is in the activation of caspase-8. In this work, mature CL was identified as a crucial component of a mitochondrial platform required for caspase-8 translocation, oligomerization and activation following Fas signalling in type II cells. These results support a model in which once the first cleavage of procaspase-8 occurs at the DISC, the p43/p10 heteromer product translocates and inserts into the mitochondrial membrane in a CL-dependant manner. In the mitochondria, caspase-8 further oligomerizes and auto-cleaves to adopt its fully active form p18/p10. Additionally, it is shown here that mature CL is required for the physiological association of full-length Bid, the major caspase-8 cleavage substrate, with mitochondria. Thus, Bid is directly available for active caspase-8 on the mitochondrial surface where it cleaves into tBid, which in turn inserts into the mitochondrial outer membrane and induces cytochrome c release. Therefore, by tethering full-length Bid on mitochondria and by providing an activation site for caspase-8 following Fas signalling, CL brings together both the enzyme and its substrate and provides a platform from which the mitochondrial phase of apoptosis is launched. In summary, the data presented in this thesis provide the first evidence that mature CL participates in a new mitochondrial associated platform, called the “mitosome”, required for the activation of caspase-8 in type II cells.

The availability of fully sequenced genomes of the model organisms including Drosophila, and their subsequent annotation has afforded seamless opportunities for reverse genetics in a complex model organism. With the advent of DNA microarrays to assay the levels of tens of thousands of genes in a single sample, functional genomics has been significantly aided to understand the functions in systems context. These microarrays have been employed predominantly on the RNA samples that are extracted from the whole animals for example at different developmental stages or in response to external stimuli. However, these approaches relied on the expression patterns that represent the sum of transcription coming from all the organs, which do not estimate the tissue-specificity of transcription. The purpose of this thesis is to provide tissue-specific transcriptomes of Drosophila melanogaster that were generated as part of the large FlyAtlas project using Affymetrix Drosophila GeneChips® (or microarrays). These chips, one at a time interrogate the levels of 18,500 transcripts (that represent all known genes) using 18,880 distinct probe sets in a single, total RNA sample. For each tissue, four biological replicates were analysed using the chips and the normalised signal intensities were obtained that represent the relative levels of mRNA expression. Using the transcriptomes, a general analysis was performed for potential novel insights into tissue-specific functions (Chintapalli et al., 2007) (Chapter 3). Then, a comparative analysis of epithelial tissues was performed to understand how the epithelia are organised in terms of their transcriptomes (Chapter 4). The Malpighian tubules are the Drosophila epithelial counterparts of the human kidney. They show asymmetric organisation in the body cavity. FlyAtlas segment-specific tubule transcriptomes allowed the comparison of their potential functional similarities and differences, thus to understand the asymmetry in function (Chapter 5)(Chintapalli, 2012). This identified a human Best vitelliform macular dystrophy (BVMD) disease homolog, Best2 in only the anterior pair of tubules that have the morphologically and functionally distinct enlarged initial (or distal) segment, a storage organ for Ca2+. Bestrophins were accordingly selected as candidate genes to analyse organismal functions, and thus to validate previous two theories that implicated bestrophins as Ca2+-activated Clˉ channels and/or Ca2+ channel regulators (Chapter 6). The confocal microscopy analysis of bestrophin YFP fusion proteins revealed interesting and novel localisations of bestrophins, in that Best1 was found in the apical plasma membranes, Best2 localised to peroxisomes, Best3 and Best4 were found intracellular. The salt survival analysis showed that Best1 is essential in regulating extra salt levels in the body. Furthermore, the fluid secretion analysis showed Best1’s potential role in Ca2+-dependent Clˉ function. Interestingly, the flies with reduced levels of Best2 expression showed increased ability to survive on extra salt food; the basis for this was investigated further in Chapter 7. Best2 was also found abundant in the eyes than anywhere else in the head. A comparative analysis of anterior tubule- and eye-specific transcriptomes revealed a potential overlap of Ca2+ signaling components, in that the PLCβ signaling was one. A neuropeptide Ca2+ agonist, capa1 evoked secondary cytosolic Ca2+ responses were found high in Best2 knockdowns. A quantitative PCR (qPCR) analysis of candidate Ca2+ signaling and homeostasis genes in Best2 mutants revealed their gene expression upregulation, under control-fed and salt-fed conditions than their wildtype controls, fed on similar diet regimes. The norpA that encodes PLCβ was found significantly enriched in the mutants. Cyp6a23 is another gene that was highly upregulated in Best2 mutants; it is a Drosophila homologue of human Cyp11b, a Ca2+-responsive gene implicated in renal salt wasting. Upon the downregulation of Cyp6a23, flies became sensitive to salt diet feeding. Other genes investigated and found to be upregulated in the mutants include transient-receptor-potential (trp) Ca2+ channel and retinal degeneration C (rdgC). Together, these results strongly suggest Best2 as a potential Ca2+ channel regulator, and provide fascinating insight into bestrophin function. Peroxisomal localisation of Best2 in line with the implication that peroxisomes act as dynamic regulators of cell Ca2+ homeostasis led to another aspect of the project (Chapter 8). This study identified two peroxins that are most abundant in the tubules and play essential roles in the novel cyclic nucleotide-regulated peroxisomal Ca2+ sequestration and transport pathway and that are detrimental for peroxisome biogenesis and proliferation.

Abnormalities in adrenal steroid production have been implicated in certain forms of hypertension. Mutations in the CYP11B1 gene which result in complete loss of 11-hydroxylase function cause 11-hydroxylase deficiency and hypertension due to abnormally high levels of mineralocorticoid, DOC. Mutations have been identified which destroy aldosterone synthase 18-hydroxylase activity or 18-oxidase activity or both, resulting in lack of aldosterone. Structure-function studies have identified aldosterone synthase residues specifically involved in 18-hydroxylation and 18-OHDOC production respectively. Analogous mutations in the human CYP11B2 gene in exons 3 and 4 which result in amino acid substitutions, E136D and K251R have been shown to increase aldosterone production. In essential hypertension adrenal steroids have been implicated as a contributing factor in some cases and it is possible that mutations in aldosterone synthase and 11-hydroxylase may be responsible in part for abnormalities in steroid production. The studies reported in this thesis have investigated some of the residues which may be responsible for the special properties of these enzymes and also the effects of potential inhibitors on enzyme steroid production in vitro. This thesis presents new studies on the relationship between structure and function of aldosterone synthase and 11-hydroxylase. Artificially induced changes, some relatively conservative and distant from centres of known functional importance, have been shown to alter activity significantly. A number of variations from consensus sequences of these enzymes have been identified in subjects with essential hypertension; whether these affect enzyme activity in such a way as to explain the clinical observations of mild 11-hydroxylase deficiency or suppressed renin or whether they might be used as diagnostic markers remains to be evaluated.