Time-lapse system(s) (TLS) have, potentially, two benefits over standard incubation systems; an undisturbed culture environment and an enormous volume of images of the embryos within them. The current research aimed to determine if a TLS could provide a comparably stable culture environment compared to a standard incubator measured as pH, osmolality and treatment success rates. Second, the hypothesis that patient, treatment and environment specific embryo selection algorithms (ESAs) are required to improve the efficacy of a TLS as an embryo assessment tool was tested. A TLS was shown to provide a comparably stable environment when compared to a standard incubator in terms of pH and osmolality. In addition, using a strict matched-pair design, embryos cultured in a TLS resulted in a significantly higher implantation, clinical pregnancy and live birth rates. It was also concluded that, of six published ESAs, none performed with clinically relevant predictive capabilities when applied to the same cohort of known implantation embryos. Owing to this, the identification of five abnormal division events as significantly reducing an embryos implantation potential was performed providing an easily adopted, clinically relevant means to deselect embryos cultured in a TLS. A regression analysis found a number of treatment and patient parameters having a significant effect on crucial morphokinetic parameters, although no systemic effect was observed. Finally, an interim analysis of a sibling oocyte study of three, commercially available culture media revealed significant differences in the time of embryo compaction as well as embryo quality and utilisation. Together, these results highlight that a TLS provides a stable culture environment and leads to increased implantation, clinical pregnancy and live birth rates. It is also likely that the patient, treatment type and environment can significantly alter an embryos morphokinetic profile and specific ESAs are required to unlock the true potential of time-lapse technology.

In 2009 and 2010, the major drug regulatory bodies, the European Medicines Agency and the Food and Drug Administration in the USA, issued requests for the generation of information relating to the absorption, distribution, metabolism, excretion, efficacy and safety of investigational drugs in pregnant women prior to approval. In the wake of thalidomide, research involving pregnant women other than for obstetric or gynaecologic purposes became rare, and studies of investigational drugs practically unknown. Consequently, none of the legislation applicable in the UK and few of the guidelines introduced in the last 40 years properly addresses the conduct of clinical trials of investigational drugs in this population. This thesis questions whether the legal protection for the foetus is adequate in clinical trials. The answer appears to be a qualified “no”. Arguments persist regarding the moral standing of the foetus, particularly regarding abortion. That will not be the intent of such trials, and a moral case is made for the conduct of clinical trials in this population by analogy with the neonate, and the pregnant woman’s autonomy. Legally, we already recognise the foetus has ‘interests’ which crystallise upon live birth, and that compensation is recoverable for harm inflicted in utero manifesting as congenital injury. The essence of research is quite different from medical practice, and the extent to which this is understood by trial participants is unclear. The approvals processes contain a number of inadequacies which have the potential to expose the foetus to harm and affect the consent of the pregnant woman. The recovery of compensation in the event of children born injured following clinical trials during pregnancy in many ways may be more complex than other personal injury cases. The conclusions of this thesis are that the existence of a foetus does merit recognition by the law in this setting and that morally such studies are justifiable. However, the present legislation and approval processes potentially expose the foetus to avoidable risk and may not be appropriate to enable the recovery of compensation, thereby creating potential to deter future trial participants. A proposal is made regarding an approach to simplify the process for recovery of compensation, and thereby strengthen the approval and consent processes.

In a rat model of intrauterine growth restriction (IUGR) induced by maternal global undernutrition, adult offspring are obese with associated metabolic disturbances. These metabolic abnormalities are all augmented by feeding a high calorie postnatal diet and reversed by neonatal leptin treatment. Evidence is now accumulating which indicates that altered epigenetic regulation and gene expression may underpin the relationship between the early life environment and metabolic disturbances in adult life. Therefore, to determine the mechanism responsible for the alterations in energy balance in the IUGR rat, this study investigated the effect of maternal diet, neonatal leptin treatment and a postnatal high fat diet on the expression and DNA methylation of genes involved in energy balance in the liver and adipose tissue of adult offspring. These genes included the peroxisome proliferator activated receptors (PPARs) and their target genes; acyl-coA oxidase (AOX), carnitine palmitoyl transferase-1 (CPT-1) and lipoprotein lipase (LPL). Real time PCR indicated that the expression of several key genes involved in energy balance, including PPARα, PPARγ and their target genes, was not altered by maternal diet or postnatal diet in the liver or adipose tissue of these offspring. However, in adipose tissue, neonatal leptin treatment resulted in an increase in the expression of most genes tested, including PPARα, PPARγ and their target genes. The increased PPARγ and LPL would facilitate the uptake of fatty acids into the adipocyte, whilst the upregulation of PPARα and its target genes AOX and CPT-1, not normally expressed in adipocytes, would direct fatty acids taken up towards the β-oxidation pathway instead of storage. This would imply that the fat cell had transformed from a fat storing cell to a fat metabolising cell. Gene expression data therefore indicated that the phenotypic changes induced by neonatal leptin treatment, i.e. the reduced weight gain, could be due to increased expression of PPARα, PPARγ and their target genes in adipose tissue: Furthermore, the effects of this are persistent, due to the specific period of leptin administration during neonatal development. There was, however, no evidence of altered DNA methylation in the promoter regions measured which could account for these persistent effects. To investigate mechanisms underlying the regulation of the PPARα promoter by leptin, the rat PPARα promoter was mapped, cloned and characterised. As part of this process, six alternatively spliced variants were identified; one from adipose tissue (P1), two from the liver (P2, P3), one from the heart (P4) and two from the kidney (P5, P6). These transcripts were found to differ in their 5’untranslated region due to tissue specific promoter usage and alternative transcription start sites. The liver and adipose specific promoters were cloned and characterised using a reporter gene strategy. They were shown to differ in their basal activity, response to known activators of transcription and to neonatal leptin treatment. The regulation of the PPARα promoter by leptin was investigated and shown to function via a non-canonical mechanism requiring both signal transducer and activators of transcription (Stat3) and specificity protein-1 (Sp1), which act at a unique region of the liver specific P2 promoter. The adipose specific P1 promoter was shown to be unresponsive to leptin treatment. Furthermore, real time PCR with primers specific to the P1 and P2 PPARα transcripts indicated that the increased PPARα expression seen in leptin treated offspring was due to an increase in the P2 specific transcript, not the P1 transcript. This indicated that the neonatal leptin treatment facilitated a selective switch in promoter usage to increase the expression of PPARα and its target genes in a tissue in which they are not normally expressed, thus inducing an altered metabolism within the adipocytes of these offspring.

Environmental challenges during early life have been shown to result in greater risk of chronic diseases such as diabetes and coronary disease in later life. Factors such as unbalanced nutrition before birth result in metabolic and structural adaptations that lead to persistent modifications to offspring phenotype. There is evidence that respiratory disease is influenced by developmental environment. Reduced fetal growth is associated with impaired lung development, increasing risk of developing asthma and Chronic Obstructive Pulmonary Disease (COPD) in later life. To investigate the mechanisms underlying these effects, it is necessary to utilise animal models that emulate the phenotypes observed in human studies. The aim of this thesis was to investigate whether exposure to a maternal low protein diet in utero affects offspring lung morphology, bronchial hyperresponsiveness (BHR) and global methylation and/or gene expression in Wistar rats. Pregnant Wistar rats were allocated to either control (C, 18% casein) or protein restricted (PR, 9% casein) diet. Lung tissue was harvested (225 days) from male offspring (F1, (28 days, C=15; PR=10, 120 days, C=11; PR=7, 225 days: C=6; PR=6) F2, (28 days, C=24; PR=17) and F3 (21 days, C=16; PR=5)). Lungs were removed and the left lung was sectioned and haematoxylin and eosin stained, imaged at 10x magnification and using stereological methods, point counted to give volume fractionation of selected areas. Other morphological measurements were made to estimate surface area (10x magnification), and alveolar wall thickness (63x magnification). Primary bronchi were dissected out, mounted in a myograph and bronchoconstriction in response to a range of bronchoconstrictors and bronchodilators was assessed. RNA and DNA were extracted using TRIzol ® and global methylation assessed using MethylampTM Global DNA Methylation Quantification Ultra Kit. There was no significant difference in either lung volume or lung structure between the F1 or F2 offspring of control of PR exposed mothers except for a significant increase (p=0.046) in the amount of smooth muscle around blood vessels in the protein restricted group in the F1 225 day old group. However, in assessment of constrictor responses of isolated bronchi, a significant difference in response between groups was found with both carbachol, an acetylcholine mimetic, and U44619, A thromboxane mimetic, with the F1 offspring from protein restricted mothers exhibiting significantly increased BHR at 75 days of age (p=<0.001) compared to controls. No significant difference in global DNA methylation in lung tissue was found between F1 of F2 offspring of mothers exposed to the control of PR diet during pregnancy. Gene expression levels of selected candidate genes in lung tissue were then assessed using qPCR and again no differences were found between groups. In this study there is no evidence to suggest that in utero exposure to a maternal low protein diet has affected offspring lung physiology, structure, methylation patterns or gene expression. However, there could be differences in the amount of smooth muscle found around the vessels in response to this nutritional challenge as protein restricted animals appear to have more smooth muscle compared to controls. BHR was observed in the 75 day old rats indicating a possible shift towards a priming of asthma phenotype which could be induced with post natal allergen challenges.

No description available.

No description available.

Introduction: Clinical Leadership is a way of facilitating change and increasing the quality of care at the front line of practice. However, the failure of midwifery leadership and being designated an oppressed group questions the ability of midwives to practice as clinical leaders in the labour ward environment. Whilst there is some research relating to clinical leadership in nursing, no research exists that investigates the clinical leadership of midwives who are directly involved in giving care to women. Aim: The aim of this research was to explore clinical leadership on the labour ward and to develop an understanding of the associated characteristics of clinical leadership. The attributes that delineated effective clinical leadership were examined in addition to associated professional discourses and relationships of power that existed on the labour ward. Methods: A critical ethnographic approach was undertaken on the labour ward of a district general hospital and a teaching hospital in the North of England, using participant observation and semi-structured interviews. A total of sixty-nine hours of participant observation was undertaken. A purposive sample of 30 midwives were interviewed in the first instance and further interviews were undertaken with 18 midwives who were nominated as effective clinical leaders by the midwives in the initial interviews. Data were examined through the lens of Bourdieu’s Theory of Practice. Findings: Clinical leadership existed at different levels on the labour ward, however, midwives mostly identified LWCs in this role. LWCs’ clinical leadership was necessary, contradictory, gendered, socialised and unsupported within the hierarchical, high-risk and fearful labour ward. A combination of heroic and values-based clinical leadership was required to maintain safety and facilitate productivity. Heroic leadership, the high level of accountability and symbolic capital invested in the LWC led to a loss of autonomy for other midwives, a lack of dissent and difficulty initiating changes in practice. The contradictory nature of the LWCs’ work and a lack of support led to them experiencing both emotional and physical stress. Within an increasingly highrisk labour ward environment the LWC clinical leaders experienced professional misrecognition and discrimination that resulted in dysfunctional inter-professional relationships and keeping the obstetricians away from women. Conclusion: A high level of responsibility invested in the LWC combined with socialisation led to heroic leadership which fostered dependency prevented change and innovation. Inequalities of power and dysfunctional relationships were symptoms of a system failure that does not support midwifery practice or woman-centred care. Recommendations are made for policy, education, practice and future research.

Pre-eclampsia is a common hypertensive disorder of pregnancy that substantially affects maternal and neonatal morbidity and mortality worldwide. Despite decades of research, the aetiology of the disease remains poorly understood, and the clinical management of it is hampered by the lack of reliable diagnostic tests and effective therapy. Several screening tests have been suggested for the prediction of preeclampsia; however, none possess the sufficient specificity and sensitivity. Moreover, multiple pathways are known to be involved in the pathogenesis of pre-eclampsia, so it is very unlikely that a single or a small group of biomarkers will accurately predict the disease. In this thesis, two separate targeted LC-MS/MS methods were developed and validated to quantify plasma glycoproteins in pre- eclampsia to increase the pool of preeclampsia biomarker candidates and explore new pathways associated with the disease. The first method was hypothesis-driven and aimed to quantify the oxidation level of the plasma glycoprotein angiotensinogen (AGT), which has been proposed to be involved in the increased blood pressure characteristic of pre-eclampsia. The second method was hypothesis-generating and aimed to detect glycoprotein fold changes between different disease conditions using a simple and cost-effective conventional LC-MS/MS workflow. For both methods, a reproducible workflow for efficient glycoprotein/AGT extraction from human plasma was developed by coupling ConA lectin affinity chromatography with reversed-phase solid phase extraction fractionation (RP-SPE). Analysis of the enzymatically digested proteins was conducted using targeted LC-MS/MS working under the multiple reaction monitoring mode. For the quantification of the two distinct forms of AGT in the plasma (the sulphydryl-bridged oxidised form and the free thiol reduced form), a differential alkylation strategy was coupled with targeted LC-MS/MS to recognise and quantify the cysteine (Cys) peptides involved in the redox switch of AGT. The developed method enabled the reproducible detection of the two distinct forms of AGT in the plasma with CV% < 15%, and confirmation of the identity of the differentially alkylated Cys peptides was supported by LC-MS/MS. Analysis of clinical plasma samples using the developed method showed a significantly higher level of the oxidised AGT in pre-eclamptic women compared to gestational age-matched normotensive controls (P=0.008), whilst maintaining a similar total AGT level in the plasma. The research findings indicate that the elevated level of oxidised AGT rather than its total level might be a contributing factor to the hypertension characteristic of pre-eclampsia, and provide an extra line of evidence linking the oxidative state and the generation of reactive oxygen species with hypertension in pre-eclampsia. In the second part of the research, 54 clinically relevant glycoproteins were selected to be profiled by label-free targeted LC-MS/MS. Measurement of the analytical precision of the method revealed acceptable CV values for the majority of the assays (median CV 11.8%). Analysis of plasma samples collected from early- and late-onset preeclamptic women using the developed glycoprotein profiling methodology successfully identified significant changes in the level of several proteins in pre-eclampsia. Two of them, apolipoprotein D and kallikrein, are reported for the first time to be altered in the plasma of pre-eclamptic women suggesting that they could be further evaluated as novel biomarkers. Some pre-eclampsia-relevant pathways and biological processes, including iron transport and metabolism, coagulation, and lipid metabolism and oxidative stress were found to be altered in the disease. Moreover, different glycoproteins were changed in early-onset compared to late-onset pre-eclampsia which might reflect different pathophysiological mechanisms. Additionally, the method was applied to identify any altered glycoproteins in plasma samples from women with polycystic ovary syndrome (PCOS). These were subsequently compared with those found to be altered in pre-eclampsia, resulting in the proposal of possible underlying pathophysiological mechanisms that may explain the reported association between the two conditions, such as hypofibrinolysis and thrombophilia and iron overload. Moreover, the study detected, for the first time, significant changes in the plasma levels of vitronectin and insulin growth factor acid labile subunit, suggesting that these may also be further appraised as potential biomarkers for the diagnosis of PCOS. Taken together, the two targeted LC-MS/MS methods developed in this thesis provided relevant information regarding pre-eclampsia by identifying potential pre-eclampsia protein biomarkers. This sheds light on the different biochemical processes altered in the disease and points to possible pathophysiological mechanisms that might assist in explaining the link between PCOS and pre eclampsia, all of which should improve the understanding of the molecular mechanisms of the disease. The present work offers information that may play a key role in improving the health care of women with preeclampsia and serves as a foundational cornerstone for future work.

The success of any civilisation rests on a number of factors, to include their ability to procreate and produce heirs. This given, the health of women in any society is of most importance given their primary role in both birth and raising children. The study of medicine dedicated to the care of women in ancient Egypt is of vital importance and to this end, various archaeological finds have been consulted and analysed. Information in the field gynaecology shows a relatively advanced discipline with many overlaps with modern medicine and modern pharmacopoeia. Information on obstetrics is more limited with reliance on mythological texts, inscriptions, artifacts, conjecture and deductive reasoning required. A lot of areas still require exploration in the field and the study raises issues for future research / Biblical and Ancient Studies / M.A. (Ancient Near Eastern Studies)

Epithelial ovarian cancer is the deadliest gynaecological cancer: most women die within five years of their diagnoses. Moreover, survival of women with ovarian cancer (OC) has not significantly improved in the past decade. Oncolytic viruses (OVs), a new class of anti-cancer agent, infect and replicate selectively within malignant cells, whilst sparing normal cells. OVs also induce profound immune responses, for example disruption of chemokine and cytokine networks, with potential influence on therapeutic effectiveness. On the other hand, Natural Killer cells (NK cells), a key immune population that surveillance against cancers and viruses, may hinder the spread of OVs or promote anti-tumoural effects of OVs. This work investigated the role of innate immune responses, in particular NK cells and interleukin (IL)-17F, on the efficacy of oncolytic adenovirus in OC. I demonstrated that NK cells were activated by adenovirus-infected OC cells. Activated NK cells then augmented oncolytic adenovirus in eliminating OC via contact-dependent interactions between activating NK receptor DNAM-1 and adenovirus-infected malignant cells. In addition, consistent changes in chemokines and cytokines were observed after wild-type and oncolytic adenovirus infection. In particular, IL-17F, but not IL-17A, was significantly upregulated in different established and primary OC lines after adenovirus infections. Moreover, a range of inflammatory chemokines, including CCL2, CXCL1, CXCL2 and CXCL5, were down-regulated after oncolytic adenovirus infection. This work also revealed the logistical and technical challenges of the use of primary patient materials. I identified that our primary culture method for expanding OC cells was suboptimal. I subsequently evaluated a simple immunohistochemical method to screen for successful primary expansion of malignant cells from OC ascites. I showed that PAX8, but not CK7, was a specific marker of successful ex vivo expansion of HGSOC.