Bipolar affective disorder has a strong genetic heritability. In the UCL laboratory, a locus on chromosome 1p36 was found to be linked to bipolar and related unipolar affective disorders with a log of the odds score above 3.00. This region was subjected to fine mapping using tests of allelic association in a case-control sample as part of this thesis in order to identify the genes involved in bipolar disorder. In addition, a GWAS was also employed to fine map other bipolar affective disorder susceptibility genes. The tests of allelic association found evidence for the involvement of the PRKCZ gene. Markers D1S243 and rs3128396 at the PRKCZ gene were significantly associated with bipolar disorder with empirical P = 0.037 and P = 0.040, respectively. Other loci encoding brain expressed proteins found to be associated in the UCL GWAS sample were the genes - GRM3 and GRM7. Therefore, these genes were sequenced using PCR-based genomic sequencing. A 3'-UTR base pair change (rs56173829) in the GRM7 gene was found to be significantly associated with the disorder, although the minor allele was more frequent in controls. A base pair mutation (rs148754219) was found in the GRM3 exon 1 two bases before the translation start codon (forming part of Kozak sequence) of a GRM3 receptor isoform. The mutation was associated with bipolar disorder (P = 0.0046, odds ratio 4.2 (95% CI 1.42-12.37)). Transcription factor binding assays and cloning experiments comparing the gene expression activity of wild-type and mutant alleles showed that the mutant allele strongly affected the reporter gene activity in SH-SY5Y and HEK293 cells. If the GRM3 Kozak sequence mutation is confirmed as an important mutation in the aetiology of bipolar disorder, then it could be used as a marker for personalised treatment for a genetic subtype of affective disorders.

Background: Neonatal encephalopathy is a common clinical condition affecting 2-3 per 1,000 neonates in the developed world. 10-15% of cases will die in the neonatal period. Therapeutic hypothermia has become the standard of care in the developed world for infants with moderate to severe neonatal encephalopathy only recently, after 2 decades of experimental and clinical studies. Approximately half the infants who receive therapeutic hypothermia still have an abnormal outcome. Research is now being focused on pre-clinical and Phase II clinical studies of drugs, which act synergistically or additively with therapeutic hypothermia. Magnetic resonance spectroscopy (MRS) techniques provide translational biomarkers, which may be used to speed up the development of safe and effective neuroprotective interventions. The precise relation between MRS and brain histology is unknown and it is unclear whether cooling itself alters the prognostic efficacy of MRS. Aim: (i) To explore the relation between MRS biomarkers and the degree of brain pathology observed in our pre-clinical piglet model of perinatal asphyxia; (ii) To assess the predictive value of MRS biomarkers in infants with neonatal encephalopathy Methods: In our piglet study, data from 2 large piglet asphyxia studies investigating neuroprotective agents were analysed. 1. The first was a study of Xenon-augmented hypothermia. Following transient hypoxia-ischemia, 36 piglets were randomised into 4 groups (each n=9), with intervention from 2-26 h: Group (i) normothermia (38.5oC); Group (ii) normothermia (38.5oC) + 24 h 50% inhaled xenon; Group (iii) 24 h therapeutic hypothermia (rectal temperature (Trectal) 33.5oC) or Group (iv) 24 h 50% inhaled xenon +24 h therapeutic hypothermia (Trectal 33.5oC). 2. The second was a study investigating Amiloride as a neuroprotective agent. Following transient hypoxia-ischemia, 18 male piglets (<24 h of age) were randomized to 2 groups (each n=9) (1) normothermia; or (2) 2.5mg/kg of methyl isobutyl amiloride (MIA) at 10 minutes after resuscitation and 8 hourly thereafter. Both studies were performed on a Bruker 4.7 Tesla MR system. Following resuscitation after hypoxia-ischemia, proton (1H) magnetic resonance (MR) spectra were acquired with repetition time (TR) 5 sec, 128 summed transients, and echo times (TE) 25 ms, 144 ms, and 288 ms. (we measured the following peak area ratios: Lactate/N acetyl aspartate (Lac/NAA) and Lactate/Creatine (Lac/Cr) in both white matter and thalamus). Phosphorus (31P) MR spectra were acquired from whole brain using single-pulse acquire with TR 10 s (we measured inorganic phosphate (Pi)/exchangeable phosphate pool (EPP = Pi + PCr + (2γ +β)-NTP), NTP/EPP). Immunohistochemistry was performed on brain sections for activated Caspase 3, TUNEL positive cells and Iba I (activated microglia) to quantify cell death and microglial activation. For the clinical study, we assessed 45 infants (median gestational age – 40 weeks) with moderate to severe neonatal encephalopathy admitted over a 3 year period to the neonatal unit at UCH. Their neurodevelopmental outcome was assessed at 18 months. Results: (i) Experimental Study - Early Biomarkers Early biomarkers (acquired between 2 and 4 hours after hypoxia-ischemia) in particular thalamic Lac/NAA, predicted the 1H MRS area under the curve values from 0-48h and quantitative immunohistochemistry at 48 hours. Late Biomarkers: WM Lac/Cr at 40-48h after HI demonstrated the highest positive correlation with Tunel positive cell death (R20.98, p = 0.01) and NTP/EPP with microglial ramification (R20.68, p = 0.006); this correlation was present in both treated and untreated piglets. (ii) In the clinical study, deep gray matter Lac/NAA was the most accurate predictor of long term adverse neurological outcome following NE; importantly the predictive accuracy of Lac/NAA was unaltered by preceding therapeutic hypothermia. Conclusions: (i) Early thalamic Lac/NAA predicted the subsequent trajectory of energy disruption; this has importance in understanding the relevance of very early MRS studies in babies. White matter lactate/Cr at 40-48h correlated best with quantitative immunohistochemistry (especially TUNEL positive cells) and this relationship was present with and without preceding therapeutic intervention. (ii) In babies with neonatal encephalopathy, the predictive accuracy of Lac/NAA was unaltered by therapeutic hypothermia.

Pesticides can be harmful to the reproductive process and even low dose exposures can lead to miscarriage, developmental delays and birth defects. Huichol indians from the Sierra Madre highlands in northern Mexico supplement their subsistence lifestyle with annual migration to coastal tobacco farms, where they are exposed to the many pesticides that are used in the production process. The specific working and living conditions that they experience combined with cultural, economic and social factors ensure that this group of workers are particularly at risk to the effects of pesticides. This thesis will discuss how these migrant labourers understand their reproductive, maternal and child health outcomes considering the context within which they live and work, in particular their exposure to pesticides, traditional beliefs about health and their knowledge and practices relating to maternal and child health. Using ethnographic data and drawing largely on interpretative and critical medical anthropology this thesis will explore how supernatural understandings of illness causality, experiences as migrant labourers and their indigenous world view affect their understanding of the risks of pesticides. Underlying the central problems of their health are social, political, racial and economic conditions that have structured the way in which this population lives and works, ensuring that they are in harm’s way both as migrants and while living at home in the highlands. The thesis concludes that the greatest determinants of their knowledge and practices are historical and on-going forms of structural and everyday violence, mediated through their beliefs in supernatural causality. ce

Control of cell shape is crucial to the proper development of tissues and organs throughout all metazoan phyla. During organogenesis, patterning molecules are responsible for coordinating tissue remodelling that is in part mediated by cell shape changes, based on tightly controlled rearrangements of the cell cytoskeleton. These coordinated cell shape changes are in turn important for the formation of cell–cell contacts, triggering signal transduction cascades and influencing the patterning information. Precisely how cell shape is regulated in response to higher order patterning signals, and how this shape change itself subsequently impacts upon global tissue patterning, remain intriguing questions. This thesis examines and characterises at the cell-biological level a striking example of cell shape change; the morphogenetic furrow of the developing eye in the fruit fly Drosophila melanogaster. In the furrow, cells show a marked decrease in their apical surface area, and contract along their apico-basal axis. The role of polarity determinants, cytoskeletal machinery and the conserved patterning molecules involved in effecting this cell response are investigated. In particular, this work demonstrates a crucial role for the hedgehog signaling pathway in effecting the cell response. This is achieved through the alleviation of Ci75-mediated transcriptional repression, resulting in the activation of non-muscle Myosin II and the formin diaphanous, together with the F-actin effectors Profilin and Cofilin. It is shown that activation of the hedgehog pathway leads to a range of cell behaviours that appear dependent upon the level of Myosin II activity, ranging from shallow grooves to tube-like structures. These results are discussed with regard to wider developmental contexts such as cell constriction in the vertebrate neural tube, other instances of cell constriction, such as cytokinesis, and in relation to the evolution of eye development in dipterans.

No description available.

Background. Gilles de la Tourette Syndrome (GTS) is a chronic neuropsychiatric disorder characterised by multiple motor and phonic tics and associated behavioural problems, with a serious impact on the health-related quality of life (HR-QOL) of patients. However, little is known on the perception of HR-QOL by patients with GTS, and no patient reported HR-QOL measures have been proposed for this population. Aim. The objective of this study is the development and validation of a new scale for the quantitative assessment of HR-QOL in patients with GTS. Methods. A pool of 40 potential scale items was generated based on interviews with 120 GTS outpatients, literature review, and consultation with experts. These items were administered, in the form of a questionnaire, to a sample of 192 patients attending the Tourette Clinic, National Hospital for Neurology and Neurosurgery, London, along with standardised clinical scales. Validated psychometric methods were used to develop a rating scale satisfying standard criteria for reliability and validity. Results. Response data analysis and item reduction methods led to a final 27-item GTS-specific HR-QOL scale (GTS-QOL) with four subscales, addressing the psychological, physical, obsessional, and cognitive domains, respectively. The psychometric properties of the GTS-QOL were further tested in a second sample of 136 subjects recruited through the UK-Tourette Syndrome Association. The GTS-QOL demonstrated satisfactory scaling assumptions and acceptability; both internal consistency reliability and test-retest reliability were high (Cronbach's alpha ≥0.8 and intraclass correlation coefficient ≥0.8); validity was supported by interscale correlations (range 0.5-0.7), repeated factor analysis, and correlation patterns with other rating scales and clinical variables. Conclusion. The GTS-QOL is proposed as a new disease-specific patient-reported scale for the measurement of HR-QOL in patients with GTS, taking into account the complexity of the clinical picture of GTS.

Parallel-hole collimators are the most widely used collimators in clinical SPECT imaging. However the performances of these collimators are limited in terms of efficiency and resolution, 0.01% and 1cm at 10cm distance from the detector face, respectively. Simultaneous optimisation is not possible due to existence of a fundamental trade-off between resolution and efficiency. Hence an alternative method of increasing efficiency without compromising resolution is necessary. Slit-slat collimator provides a better alternative to parallel-hole, due to their magnification power. The aim and objectives of this PhD work was to design and optimise a clinical brain SPECT system based on slit-slat collimation and a novel detector with silicon drift diodes (SDDs) readout providing 1mm intrinsic resolution. The system efficiency can be improved by increasing the number of slits, however as the number of slits increases, so does the multiplexing, which is the overlapping of the projections on the detector plane. We hypothesize that the reconstruction of multiplexed (MX) projections can be improved by addition of non-MX projections. We have proved this using simulation and shown that a gain in signal to noise ratio (SNR) (25.5%) corresponding to 43% reduction in acquisition time was achievable when non-MX projections were available. Several novel system designs that utilise the SDDs technology with mixed degrees of multiplexing have been evaluated. To enable experimental validation of the simulation work, a prototype slit-slat collimator was designed and built for a conventional gamma camera. Full calibration of the system was performed, the efficiency and resolution profiles were measured and studies of point sources, Jaszczak and Striatal phantoms were completed. The results of these studies confirmed the potential for multiplexing to improve image quality when used in conjunction with non-multiplexed projections with a maximum gain in SNR equivalent to of 50.6%.

The circulating, pro-thrombotic glycoprotein von Willebrand factor (VWF) is essential for haemostasis, but has also been implicated in the development of atherosclerosis. The majority of plasma VWF is secreted from endothelial cells lining the blood vessels and it is unclear what influence attenuation of the endothelial cell secretory pathway might have on the physiology or pathology of VWF. Statins are inhibitors of cholesterol biosynthesis and are widely used in the treatment of cardiovascular diseases due to their cholesterol lowering ability. However, they have also been shown this research and in the literature to have 'pleiotropic effects' caused by inhibition of the synthesis of biosynthetic intermediates (such as prenyl-lipids used for modification of small GTPases) rather than cholesterol itself. As a result this research has studied the pleiotropic actions of statins on the VWF secretory pathway in cultured human umbilical vein endothelial cells (HUVEC). As well as confirming an inhibitory effect of statins on the stimulated secretion of VWF, we found that treatment of HUVEC with statins led to a decrease in the intracellular storage of both VWF and its non-covalently associated propolypeptide (Pro-region), while the intra- and extra-cellular levels of the VWF precursor, Pro-VWF, significantly increased. All these effects were rescued by addition of the geranylgeranyl lipids used for prenylation of the Rho and Rab GTPases, but not the farnesyl lipids used for the majority of Ras family members. Using specific inhibitors of the two disitinct enzymes responsible for either Rho or Rab geranylgeranylation we have uncovered a complex picture with neither inhibitor able to reproduce all of the effects of statins of VWF. In addition to VWF, we have also discovered Rab GTPase associated effects of statins on the transferrin receptor and on the recyling of a trans Golgi network associated protein (TGN-46). In conclusion this work highlights that statins do more to vasculature than simply lowering circulating cholesterol levels and extends the complex pleiotropic effects of these drugs to influencing other cellular pathways.

Strong over-expression of Glucagon-like Peptide-1 receptors (GLP-1-R) in human pancreatic β cells and in insulinoma provide an attractive target for imaging. This is relevant since the preoperative localisation of insulinomas remains a challenge whilst serial measurements of functional β cell mass (BCM) may improve monitoring of novel diabetes therapies such as islet cell replacement. The aim of the thesis was to develop and evaluate GLP-1-R imaging probes and translate these into clinical applications, including insulinoma and β cell imaging. After extensive preclinical evaluation (internalization, externalization, biodistribution, pharmacokinetic, imaging and detailed dosimetry studies) of different GLP-1-R imaging tracers, the most promising probe, [Lys40(Ahx-DOTA-111In)NH2]-exendin-4, was selected for a first in man study. This novel probe was then prospectively evaluated in the management setting of benign and malignant insulinoma. An additional proof of concept study was performed in a patient after intramuscular transplantation of islet cells. The GLP-1-R planar imaging study showed focally raised tracer uptake in functional β cells transplanted into the brachioradialis muscle of a 48 years old woman. In patients with benign insulinomas GLP-1-R imaging was superior to conventional imaging (CT, MRI, somatostatin receptor imaging and endoscopic ultrasound) and detected the insulinoma in all 6 patients pre- and intra-operatively. This is in contrast to patients with malignant insulinoma where conventional imaging (CT and somatostatin receptor imaging) was superior to GLP-1-R imaging, which detected the primary tumour and metastases in only 4/11 patients. In conclusion, these data, based on the development and evaluation of a novel probe, strongly suggest that GLP-1-R imaging offers a new approach to localise benign insulinomas. With further refinements of the labelling agent and the imaging methodology, it is expected that GLP-1-R imaging may have the potential for non-invasive imaging of pancreatic islets and the early diagnosis of β cell graft rejection. This thesis has led to six original publications and one review article. These are listed at the end of each chapter. For a full list please see appendix.

Pantegrals – washable pant with built-in pad – are popular among lightly incontinent women for their good aesthetics and low per-use cost but their poor leakage performance relative to disposable products stops many women from using them [1, 2, 3]. This project aimed to develop a new pantegral with improved leakage performance – specifically, to match the reference menstrual pad in an earlier study [3] – while retaining the good aesthetics and low cost of existing pantegrals. The project involved collaborating with two other universities and five companies. Published clinical and laboratory data characterizing existing products were analysed to produce a design specification for an improved product. Subsequent laboratory bench-marking with a selection of existing products established the primary technical challenges as developing novel nonwoven fabrics with increased volume at leakage and improved wicking properties to enhance the leakage performance of the product. These challenges were addressed through systematically studying the impact of fabric structure (anisotropy of fibre alignment, fabric density variation, fibre geometry, fibre finish and use of composite structures) and fibre blend on fluid handling properties. There were four development phases: the novel fabrics from each phase were characterised in the laboratory (using absorption time, absorption capacity, retention at height, lateral spreading, vertical wicking, wicking anisotropy and spreading on the curved rig test methods). The best fabrics from each phase were built into prototype products for clinical evaluation to check that improvements seen in the laboratory translated into real use. Analysis of data at the end of each phase informed the next. The two main conclusions from the laboratory characterisation of the novel fabrics were that: (i) the use of trilobal viscose fibres improved the wicking properties; and (ii) the combination of high and low density areas (through fabric and in-plane) provided the best from both high and low density fabrics; that is, promoting wicking (high capillary pressure) helping in the redistribution of fluid and greater void volume contributing to a higher absorption capacity. The clinical leakage performance of the final prototype product was substantially better than that of the reference pantegral and close to matching the target performance of the reference menstrual pad. The aesthetics and projected per-use cost of the new product matched those of the existing pantegral. Two patent applications have been filed and two of the commercial partners are now working on commercializing the product.