The project aimed to identifY socio-cultural factors influencing attitudes to sickle cell disease (SCD) and decisions about a pregnancy at risk ofproducing a child with sickle cell anaemia; and to identifY differences between African and Caribbean respondents. Sample: Phase 1 - General public African and Caribbean men and women; Phase 2 Pregnant African and Caribbean women with sickle cell trait (HbAS) Phase 3 - Pregnant women from Phase 2 and their partners, who have HbAS, placing the couple at-risk ofhaving a child with HbSS.Methodology: Phase 1 and 2 - a questionnaire (appendix 1) examining - knowledge ofSCD and five attitude variables - importance ofhaving children, locus ofcontrol in genetic decision-making, prevention ofbirth ofchildren with SCD, perception ofthe burden and severity ofSCD.. Phase 3 - semi structured interview (appendix 3), which examined attitude and response ofpregnant women and their partners to their being at-risk ofhaving a child with sickle cell anaemia.

Background: The molecular mechanisms of ectopic pregnancy remain unclear. Studies from knockout mice suggest that perturbations in oviductal endocannabinoid levels, endocannabinoid receptors (CB1) or endocannabinoid degrading enzyme (fatty acid amide hydrolase, FAAH) expression result in infertility secondary to physical trapping of embryos in their oviducts. Perturbations in endocannabinoid metabolism and action may therefore underlie ectopic pregnancy. Aims: To (1) quantify plasma and tubal endocannabinoid levels (2) evaluate blood activity of FAAH and the endocannabinoid degrading enzyme N-acylphosphatidyl-ethanolamine phospholipase-D (NAPE-PLD) and relate that to β-hCG levels (3) evaluate the expression of cannabinoid receptors (CB1, CB2), FAAH and NAPE-PLD in Fallopian tubes and (4) examine the effect of endocannabinoids [N-arachidonoylethanolamine (AEA), N-oleoylethanolamide (OEA) and N-palmitoylethanolamide (PEA)] on cilia beat frequency (CBF) in tubal epithelial cells ex-vivo. Methods: Whole blood collected from women with ectopic pregnancy and suitable controls were used for quantification of endocannabinoids by UHPLCMS/MS and FAAH and NAPE-PLD activity by HPLC methods. Fallopian tubes were fixed in formalin for immunohistochemistry and had RNA and protein extracted for RT-qPCR and immunoblotting respectively. Fallopian tube explants were exposed to endocannabinoids and changes in CBF evaluated using highspeed digital camera. Results: Plasma AEA, OEA, PEA and tubal AEA were significantly higher (p < 0.05) in ectopic pregnancy compared to controls. Tubal OEA and PEA showed a similar trend though the results were not statistically significant. Blood FAAH but not NAPE-PLD activity was attenuated (p < 0.05) in ectopic pregnant women consistent with the higher endocannabinoid level observed in plasma. CB1 and FAAH were localised in Fallopian tube and showed significant attenuation (p < 0.05) in ectopic pregnancy compared to luteal phase controls. Exposure of Fallopian tube epithelial cells to OEA unlike methanandamide and PEA resulted in a significant reduction (p < 0.05) in CBF. Conclusion: The results implicate ECS dysfunction in the pathogenesis of ectopic pregnancy.

The 677C~ T polymorphism in the gene encoding the folate-metabolising enzyme MTHFR is associated with an increased risk of several disease outcomes, including cardiovascular disease (CVD). Although this polymorphism has recently been linked with higher blood pressure (BP), no previous study has investigated its role as a determinant of hypertension throughout adulthood. Riboflavin acts as a cofactor for MTHFR and previous work at this Centre has shown that intervention with riboflavin results in significant BP-Iowering specifically in patients with the homozygous mutant MTHFR 677TT genotype, but whether this gene-nutrient interaction is an important determinant of hypertension is not known. Furthermore, although this polymorphism is also linked with neural tube defects (NTDs), the potential implications for folate recommendations to prevent NTDs in women who carry this genetic risk factor have not previously been considered. The aims of this thesis therefore were to investigate the role of this polymorphism, and any relevant gene-nutrient interactions, as a determinant of BP, and to consider folate requirements for preventing NTDs specifically in this genetically at-risk group. Analysis of data from two large observational studies of Irish adults (n 5225) demonstrated, for the first time, that adults with the MTHFR 677TT genotype from aged 18yrs onwards have a significantly increased risk of developing hypertension, but this risk can be substantially reduced through optimising riboflavin (and to a lesser extent, folate) status. Preliminary results from a randomised trial provide no evidence to support a BP-Iowering effect of folic acid in adults with the TT genotype, but provide some further evidence that age influences the BP phenotype and the potential to reduce BP through targeted intervention in these individuals. Analysis of data from a pregnancy cohort investigated in this thesis highlights the particular importance for women with the TT genotype to strictly adhere to the recommendation of commencing folic acid prior to conception (as recommended) for NTD prevention. In conclusion, this thesis demonstrates a role for the MTHFR 677C T polymorphism as a determinant of hypertension and highlights the important role of optimal B-vitamin status in modulating disease risk in adults with this genetic variant. These findings have important public health implications.

Nitric oxide (NO) and hydrogen sulfide (H2S) are two gasotransmitters with important physiological functions. NO exerts many different roles: it is a vasorelaxant, an inflammatory mediator and a neurotransmitter. Similarly, it has been shown that H2S relaxes blood vessels, is involved in inflammation and is a neurotransmitter too. The two gases are also involved in redox signalling reactions, being able to interact with reactive oxygen species (ROS). Interestingly, several pathological conditions, such as cardiovascular diseases and neurological disorders, are characterised by an imbalance in both the levels of . NO and H2S. All these observations contributed to develop the hypothesis of a possible interaction between the two molecules, especially in the cardiovascular system and during inflammation. While . NO biology is very well studied and despite the increasing interest in H2S biology, the mechanisms of action of H2S have not been fully elucidated yet. The aim of this work was to characterise some aspects of the cross-talk between . NO and H2S signalling pathways, with particular attention given to signalling pathways in the cardiovascular system (e.g. NO synthesis and cGMP production) and in inflammation (e.g. inflammasome complex activation). By using . NO derived metabolites of pharmacological interest for cardiovascular diseases (nitrite (N02-) and S-nitrosoalbumin (SNOA)) it was possible to study non-enzymatic H2S-dependent . NO synthesis. N02- and SNOA were reduced to ·NO by H2S donors (such as the sulfide salt NaSH and the slow releasing GYY 4137), as assessed by electron paramagnetic resonance spectroscopy (EPR) and gas-phase ozonebased chemiluminescence. The reactions were also chemically characterised and the effects of H2S-mediated . NO synthesis on smooth muscle and endothelial cells were studied. Endogenous H2S production was shown to significantly increase cGMP synthesis in N02--treated human aortic smooth muscle cells (compared to only N02- treatment). H2S-mediated . NO production from SNOA was also shown to increase nitrosothiol transport through cell membrane and subsequently to increase SNOA antioxidant properties in human microvascular endothelial cells. The study of the cross-talk between . NO and H2S was also carried forward by characterising the post-translational modification (S-nitrosation and sulfhydration) that they cause on key Cys residues of common target protein. S-nitrosation of albumin was performed using incubation with acidified N02- solutions and the presence of the modification on the protein was characterised by mass spectrometry. It was possible to show that SNOA had increased antioxidant capacity compared to unmodified albumin controls. Similarly it was done for H2Sinduced modification. It was possible to demonstrate that H2S was causing the decysteinylation of albumin samples, inducing an increase in albumin antioxidant properties (DMPD assay). Finally, the interaction between the two gases was also studied for key inflammatory processes such as the inflammasome complex activation and induction of IL-113 production. Unfortunately, with the assay used (IL-113 ELlSA) it was not possible to find a clear interaction between . NO and H2S, but it was possible to clarify some of the mechanisms behind H2S biological properties. It was indeed demonstrated the potential of H2S as antiinflammatory mediator by inhibition of inflammasome activation and IL- 113 production. In conclusion, with this work it was possible to demonstrate that H2S and . NO have "overlapping" (at least in part) signalling pathways and that consequently H2S is able to regulate non-enzymatic . NO production in the cardiovascular system. It was also possible to characterise novel post-translational modifications induced by H2S, potentially shedding new light on its biological mechanisms of action. The field of gasotransmitters biology is very exciting and more work is certainly needed to advance our understanding of the key cellular signalling processes that gases like· NO and H2S can modulate.

Background: Although the long term effect of intrauterine growth restriction has been assessed in a number of singleton studies, they all suffer from multiple confounding effects. A model that utilises monozygotic twins may markedly reduce the effect of confounders as monochorionic twins share the same gestational age length, family background, gender and genetic influences on growth and cognition. Comparison of monochorionic twins with birth weight discordance of 20% or more could be used as a model of in utero growth constraint. This model will still involve certain limitations and assumptions nevertheless; we used this to determine the level of cognitive function of in-utero growth discordant monochorionic twins in later childhood along with any differences in auxology and behavioural problems. Methods: This was a retrospective cohort study. Eligible twins were identified from the Northern Survey of Twins and Multiple Pregnancies register. Cognitive function was assessed by a single observer using the British Ability Scales 2 to measure the general conceptual ability. Strength and Difficulties Questionnaire was used to identify behavioral problems. Height, weight, mid arm circumference, waist measurement and head circumference were also collected. Generalised estimating equations were used to determine the effect of birth weight on general conceptual ability scores. Statistical analyses were performed using SPSS v19. Results: Between 2000 and 2004, a total of 51 twin pairs were assessed (n=23 female) with mean birth weight discordance 664gm and mean gestational age 34.7 weeks. The mean difference in the general conceptual ability score between the heavier and lighter twins was 3 points. Significant association between within pair differences in birth weight and general conceptual ability scores was found. Increasing birth weight discordance was not associated with a decrease of general conceptual ability scores. The differences in the size seen at birth between the twins were still detectable at the age of 5-8 years. There was a trend to increased prevalence of behavioural problems in the lighter twin compared to the heavier twin as reported by both teachers and parents but this result was not statistically significant. Conclusions: The smaller twin of a monochorionic growth discrepant pair was statistically significantly more likely to have a lower cognitive score compared to their co-twin at 5-8 years of age. This suggests that growth restriction in-utero is associated with lower cognitive scores in later childhood.

The prevalence of major depression in women in Europe in a 12- month period has been estimated at 3.0-11.2%. Untreated depression in pregnancy is associated with significant morbidity and unhealthy lifestyle behaviours and increases the risk of poor birth outcomes. Pharmacotherapy with SSRls is a common treatment approach, including in pregnancy, but there are concerns about effects on the fetus, including an increased risk of congenital heart defects (CHDs). The aim of this thesis was to investigate the teratogenic risks of antidepressant use by pregnant women and the socio-demographic characteristics of women of childbearing age who use antidepressants in order to inform clinical practice and public health interventions. A case-malformed control study was performed with data from 12 European Countries in EUROCAT: a European network of population-based congenital anomaly (CA) registries. The results showed that infants/fetuses with CHDs were significantly more likely to have been exposed in the first trimester to SSRls compared to infants/fetuses with CAs other than CHD (Odds Ratio 1.38, 95% Confidence Interval 1.05-1.82). The association was stronger for severe CHD (OR 1.54, 95% CI 1.04-2.28) and, therefore, cannot be explained as a product of neonatal or infant examination bias. Specific associations were demonstrated for Ebstein's anomaly, and Tetralogy of Fallot. Five signals in the literature for anorectal atresia and stenosis, gastroschisis, renal dysplasia, clubfoot, and hypospadias were supported, and a new association detected for microcephaly. In all the observed associations, there was little evidence of specificity as to SSRI type.

Preterm birth is responsible for 75% of the perinatal mortality and morbidity. There is a trend towards an increase in its prevalence. Until now screening and prevention programmes have not been that successful due to limited knowledge of the basic molecular mechanisms of labour. As a result the predictive markers of preterm birth are not that effective raising the need for novel biomarkers. The endogenous endocannabinoid N-arachidonoylethanolamine (anandamide; AEA) acts as a ligand for cannabinoid receptors CB[subscript 1], CB[subscript 2].and non-CB[subscript 1] and non-CB[subscript 2] receptors. There is lack of knowledge on the role of anandamide in pregnancy and labour in humans. However the fact that smoking of exogenous cannabinoids such as marijuana is associated with preterm birth suggests a role for the endocannabinoids in labour. Plasma AEA was shown to increase 3.7 fold with labour. The aim of this thesis was to develop an improved method for the measurements of plasma AEA and to investigate further its role in labour and to measure its level in a cohort of women at high risk of delivering preterm. This led to the development of UPLC-MS/MS method of measuring plasma AEA. In women undergoing induction of labour, plasma AEA level was found to be elevated 1.5 fold in active labour (1.82±0.87nM) in comparison to levels in the non-labouring phase (1.20 ± 0.57nM) (P<0.0001). In women at high risk of delivering preterm the plasma AEA levels were significantly elevated in those who delivered within 6 weeks of sampling compared to women who delivered at term (P=0.01). A plasma AEA level of 1.10nM, predicted preterm delivery at <37 weeks of gestation with a sensitivity of 66.6% and specificity of 81.8%. Plasma AEA levels were not found to be significantly elevated in women delivering preterm amongst women presenting with threatened preterm labour. However a small sample size could have contributed to this result. The mean plasma AEA level was significantly elevated in women who had a positive fetal fibronectin test when compared to those who tested negative suggesting a relationship between the two. These data provide additional evidence that plasma AEA plays a significant role in labour and it could be used as a predictor of preterm delivery.

The resting fore-arm blood flow rates were measured at four weekly intervals throughout pregnancy and at increasing intervals up to 40 weeks after delivery. Twenty-six subjects were normotensive as defined on page 5 , twenty-four subjects were included in the hypertension I group and thirty-six subjects were included in the hypertension II group. Control groups of twenty-two normotensive and ten hypertensive non-pregnant subjects were also studied. The resting hand blood flow rates were measured in the same intervals during and after pregnancy in three normotensive subjects, six subjects in the hypertension I group and nine subjects in the hypertension II group. The non-pregnant control group consisted of nine subjects who were normotensive and six subjects w'ho were hypertensive. At the same time, measurements were made of blood volume by means of Evans Blue Dye and the haemoglobin, red cell count and packed cell volume estimated. Assessment of hormonal function was made in all subjects by measurement of the cornification index and in 34 subjects by pregnanediol excretion.

Cephalopelvic disproportion, a major cause of perinatal death and morbidity in Africa, is notoriously difficult to assess both ante-natally and in labour. Recognising the limitations of medical personnel and equipment in Africa, a simple method of diagnosis and management is required. Professor Philpott with his introduction of the partogram and the "Alert Line" (1972), to African obstetrics has produced a simple screening device. This enables even a remote and inexperienced midwife to detect the primigravid patient with cephalopelvic disproportion early, and to refer her into the nearest hospital. Once in hospital, with the cervicograph across the "Action Line" (Philpott and Castle, 1972) and in the absence of complications other than dysfunctional labour and possible disproportion, active management should be instituted and an Oxytocin augmented Trial of Labour commenced. This "Trial of Oxytocin" must be closely controlled and the infant delivered at the correct time and by the correct method.

This study explored the ways in which women experienced non-facilitative communication with medical staff during a complicated pregnancy. Complicated pregnancy has been associated with a higher risk of mental health difficulties. The research was carried out in Ireland and focused on women’s relationships with medical hospital staff during this time as they are ideally placed to offer relational support, and potentially prevent longer term problems. In-depth interviews were conducted with six women. The qualitative methodology of interpretive phenomenological analysis was used to understand their experiences. Four superordinate themes emerged. These were ‘Information difficulties' ‘Disempowerment', ‘Empathic failure’ and ‘Relational impacts’. Crucial information was withheld, private details were discussed in public spaces and key aspects of women’s experiences were omitted from their hospital notes. Participants spoke of feeling disempowered and manipulated by staff. All participants referred repeatedly to feeling that the majority of staff failed to demonstrate empathy. Women felt isolated and unseen, with staff focused on tasks rather than holistic treatment and their infant’s needs but not those of the women. Finally, women described how their relationships with themselves, partners, and crucially, their infants had been impacted. Participants’ accounts may represent a gap in the care of women who experience the trauma of serious pregnancy complications. Findings suggest a role for counselling psychologists in providing training and consultation for medical staff in order that they might develop the capacity to offer improved support to women and infants during this time when they are more susceptible to mental health difficulties.