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Leukaemia is one of the types of cancer where treatment resistance is prevalent. Better understanding of leukemic cells metabolism opens possibilities for new therapeutic strategies and better prognosis stratification. Leukaemia arises from many different genetic alterations, that affect distinct cellular processes, all driving leukemogenesis. Many of these result in a phenotype that grants metabolic advantages amidst the hypoxic conditions of the haematopoietic niche of the bone marrow, the point of origin of all types of this cancer. In order to understand the metabolic reprogramming that grants these advantages, we performed metabolic characterization in vitro of cell lines of acute monocytic leukaemia (THP1) and chronic myeloid leukaemia (HAP1), with focus on the role of Transketolase-like 1 (TKTL1) and ten-to-eleven Methylcytosine dioxygenase 2 (TET2), respectively, on both normoxic and hypoxic experimental settings. We revealed that TKTL1 is a key enzyme in the metabolic reprogramming of the hypoxia adaptation, driving proliferation, higher glycolytic rates, higher glutamine consumption and subsequent glutamate production. Our results also showed the altered metabolism of many amino acids and biogenic amines, that grant more substrates for nucleotides synthesis, higher stress tolerance and manipulation of the immune response of the microenvironment. It also highlighted the vulnerabilities that arise from focusing on targeting TKTL1 for therapy and possible secondary targetable metabolic pathways for future therapies. Additionally, we demonstrated the effects of the loss of TET2 in the leukemic cells through a tracer-based metabolomics approach, showing how its mutation primes the cells to shift their metabolism at a higher cost for their ROS homeostasis, a known hallmark of cancer which increases the risk of more mutations occurring due to genomic instability. This allowed us to create a metabolic map of the changes induced by the loss of TET2, as a blueprint for new therapeutic venues in leukaemias that have this mutational hit. Together, the thesis presented here contributes to the knowledge of the mechanisms underlying metabolic reprogramming of leukaemia according to specific mutational genotypes and how they open new possible therapies for patients that develop resistance.

The detection of moving objects is a trivial task when performed by vertebrate retinas, yet a complex computer vision task. This PhD research programme has made three key contributions, namely: 1) a multi-hierarchical spiking neural network (MHSNN) architecture for detecting horizontal and vertical movements, 2) a Hybrid Sensitive Motion Detector (HSMD) algorithm for detecting object motion and 3) the Neuromorphic Hybrid Sensitive Motion Detector (NeuroHSMD) , a real-time neuromorphic implementation of the HSMD algorithm. The MHSNN is a customised 4 layers Spiking Neural Network (SNN) architecture designed to reflect the basic connectivity, similar to canonical behaviours found in the majority of vertebrate retinas (including human retinas). The architecture, was trained using images from a custom dataset generated in laboratory settings. Simulation results revealed that each cell model is sensitive to vertical and horizontal movements, with a detection error of 6.75% contrasted against the teaching signals (expected output signals) used to train the MHSNN. The experimental evaluation of the methodology shows that the MH SNN was not scalable because of the overall number of neurons and synapses which lead to the development of the HSMD. The HSMD algorithm enhanced an existing Dynamic Background subtraction (DBS) algorithm using a customised 3-layer SNN. The customised 3-layer SNN was used to stabilise the foreground information of moving objects in the scene, which improves the object motion detection. The algorithm was compared against existing background subtraction approaches, available on the Open Computer Vision (OpenCV) library, specifically on the 2012 Change Detection (CDnet2012) and the 2014 Change Detection (CDnet2014) benchmark datasets. The accuracy results show that the HSMD was ranked overall first and performed better than all the other benchmarked algorithms on four of the categories, across all eight test metrics. Furthermore, the HSMD is the first to use an SNN to enhance the existing dynamic background subtraction algorithm without a substantial degradation of the frame rate, being capable of processing images 720 × 480 at 13.82 Frames Per Second (fps) (CDnet2014) and 720 × 480 at 13.92 fps (CDnet2012) on a High Performance computer (96 cores and 756 GB of RAM). Although the HSMD analysis shows good Percentage of Correct Classifications (PCC) on the CDnet2012 and CDnet2014, it was identified that the 3-layer customised SNN was the bottleneck, in terms of speed, and could be improved using dedicated hardware. The NeuroHSMD is thus an adaptation of the HSMD algorithm whereby the SNN component has been fully implemented on dedicated hardware [Terasic DE10-pro Field-Programmable Gate Array (FPGA) board]. Open Computer Language (OpenCL) was used to simplify the FPGA design flow and allow the code portability to other devices such as FPGA and Graphical Processing Unit (GPU). The NeuroHSMD was also tested against the CDnet2012 and CDnet2014 datasets with an acceleration of 82% over the HSMD algorithm, being capable of processing 720 × 480 images at 28.06 fps (CDnet2012) and 28.71 fps (CDnet2014).

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Approximately 50% of patients with UM develop metastatic disease, usually occurring in the liver. Patient survival is directly related to the presence of hepatic metastases and how they affect liver function. After the identification of metastases, most patients die within a year, and the differing forms of existing treatment rarely extend life considerably. It has been proposed that prognostication can improve the quality of life, even when the probability of survival is reduced. Certain prognostic factors have been identified in UM that are associated with an increased risk of metastatic disease: these are clinical parameters as well as the histomorphological and genetic features of the primary UM. A team from Liverpool designed in 2011 a multiparameter algorithm called the "Liverpool Uveal Melanoma Prognostication Online" (LUMPO), to establish the prognosis for UM patients, stratifying them according to their relative risk of metastatic death. This was based on data collected over 20 years, and was validated externally by other ocular oncology centres. It was later updated to LUMPO3, and included new parameters and functions. The objective of this thesis was to perform a retrospective study, analysing the liver scan reports of patients with UM; examining whether LUMPO3 is able to predict the appearance of metastases in patients in Liverpool, and to determine the cost analysis of liver screening for the detection of metastases in patients with UM; and also to validate LUMPO3 externally. Following the Introductory chapter, the characteristics of the scan reports of 615 patients diagnosed with UM were analysed in Chapter 2. The data were collected over an eleven-year period (2008-2018). Data of the characteristics of liver scanning and the metastases detected were analysed, and later combined with the demographic, histological, genetic and patient outcome data. It was estimated that 37% of UM patients treated in Liverpool developed metastases at different stages of the disease. The analysis of the characteristics of the primary UM demonstrated that increasing tumour size and hence TNM staging category of the UM, was associated with an increasing risk of metastatic disease and reduced survival. Most metastatic UM were associated with monosomy 3 and gain of chromosome 8q. Many previous studies have identified different risk groups (low, intermediate and high) for UM patients to develop metastases: in this study, I identified 3 groups of patients that were divided according to when and whether they developed metastatic disease. Liver surveillance was most frequent in patients who ultimately developed metastases and the most frequent modality employed was magnetic resonance imaging (MRI). In chapter 3, the same cohort of 615 UM patients were analysed, here focussing on the estimated costs for all scans performed for which both the minimum and maximum costs were calculated using the NICE costing for clinical imaging. A feature of LUMPO3 called "linear predictor of death due to metastases" (lpmd) was extracted to predict the onset of liver metastases and to determine the hypothetical costs of liver surveillance in UM patients. Model performance was determined in terms of discrimination and calibration. Results showed consistent discrimination performances over a 5-year time period from date of initial treatment. Calibration performance was determined visually with good agreement between observed and predicted onset of UM metastases up to 10 years following initial treatment. The results suggested that unnecessary radiological examinations could be avoided in the UM patients with low metastatic risk, without any adverse effects to patient management. These results suggested that this lpmd could save costs by minimizing the number of examinations for metastases screening, and likely decrease the patient angst associated with liver screening. Chapter 4 study was a multicentre project that allowed the recruitment of a sufficient number of patients from 7 external collaborative centres, using the existing clinical network within the Ocular Oncology Group and the USA. Data were collected from patients diagnosed and / or treated for UM at these collaborating centres. The results showed that LUMPO3 is a reasonably accurate and valuable tool for predicting all-cause mortality in UM patients, despite the differences in the recording of clinical, histopathological and genetic data between the centres and hence the various cohorts studied. The calibration graphs presenting the expected probabilities of actuarial survival showed a good agreement between the observed and the predicted probabilities. In conclusion, I examined in detail "real world" data of UM patients treated in various ocular oncology centres that allowed the construction of the LUMPO3 model, and its validation in Liverpool. These data are valuable when considering the costs of surveillance programs, as well as potential modifications and revisions to the predictive algorithms for UM.

Baculoviruses such as Autographa californica nucleopolyhedrovirus (AcMNPV) are insect-specific viruses that usually kill the host. However, many species harbour persistent, non-lethal covert baculovirus infections although these have rarely been observed in vitro. Hence, little is known about the mechanisms for establishment or maintenance of persistent infections. Re-infection with a homologous baculovirus (known as superinfection) has been shown to activate the persistent infection to an overt form. In vitro studies have shown that infected cells can be resistant to superinfection although the mechanisms involved are largely unknown. In 2011, a serendipitous event led to the creation of a persistently infected Trichoplusia ni cell line (Hi5) with an AcMNPV p10-deletion mutant (AcUW1.lacZ). From this culture, a clonal cell line (C20) was established that has been maintained to this day. This thesis aimed to use C20 to further our knowledge of the fine balance between host and virus (designated AcC20) that promotes the establishment and maintenance of a persistent infection. The cell line also enabled study of the superinfection exclusion phenomenon. C20 cells demonstrated remarkable viability and diversity of cell size/shape compared to the parental cells. The persistent AcC20 virus was maintained at low levels but budded virus (BV) titres varied considerably within and between different cultures. Presence of BV and the major membrane protein GP64 confirmed C20 harboured a persistent rather than latent infection. Analysis indicated that only a small proportion (<7%) of cells were producing BV whilst superinfection studies demonstrated that the majority of cells were resistant to a secondary infection. Microscopy studies indicated that superinfection may be blocked as early as the cell binding stage although virus uptake was also affected. Whole genome analysis of AcC20 showed four major deletions or insertions with other mutations in 34 coding regions affecting eight essential genes and a homologous region. Transcriptome analysis showed that all virus genes were expressed in C20 cells although the regulated phases of gene expression observed in a typical lytic infection were absent. Host cell transcriptome analysis also revealed differences between the cell response to a lytic and a persistent infection. In particular, endocytic pit formation and host defence pathways appeared to be compromised. The former may help explain resistance to superinfection and the latter may help maintain the persistent infection. Attempts to create further persistent cell lines proved difficult. However, using viruses with a mutation in lef2, in which BV production is severely reduced, overcame this problem. These persistent infections were also used to evaluate the possibility of continuous recombinant protein production by expressing dsred and urokinase under strong promoters. DsRed but not urokinase was produced over several cell passages. This thesis demonstrated establishment and maintenance of persistent infections in cell culture. Further research is needed to understand this process and why these cells are resistant to superinfection.

Degradation of DNA can lead to either poor quality, imbalanced, or even no profiles. Therefore, appropriate collection and storage methods are critical to minimize its impact. If the DNA is degraded prior to sample collection, then the degradation process can only be arrested and other methods have to be employed to try to improve the quality of the DNA profile. The major aims of this thesis were to assess alternative methods for molecular preservation of muscle tissue samples and to obtain better DNA profiles from degraded samples. Assessment of DNA degradation was undertaken using an in-house PCR assay which amplifies four amplicons from 70 bp to 384 bp. DNA degradation was evaluated in whole pig carcasses exposed to hot and humid environmental conditions. A full DNA profile could be generated for 24 hours, but some full profiles were obtained from samples taken as late as 72 hours. It was determined that when collecting tissue samples from partially decomposed bodies, those should be preferentially from the surface of the body in touch with the ground, as the results show that DNA persistence is improved. In order to compare field and laboratory degradation patterns, muscle tissue samples were incubated in the laboratory at 25 °C and 37 °C. The persistence of DNA was increased when compared to field, most likely due to the lack of insect activity and of variations in temperature and humidity. Partially degraded muscle samples were preserved with 96% ethanol, cell lysis solution, or cell lysis solution with 1% sodium azide, which had been stored at room temperature for seven years. Samples were re-extracted to assess the long-term efficacy of these storage solutions. The results show that ethanol and cell lysis solution with 1% sodium azide were successful in preserving DNA for this period. Fresh muscle tissue samples were stored at 25 °C and 37 °C for up to 42 days using vodka and 37.5% ethanol as preservatives. Complete amplification profiles were obtained up to the last time point from samples that had any preservative solution, while samples left untreated had dropouts after 14 days. It is recommended that the use of drinking ethanol should be considered in situations where the stock of absolute ethanol is limited. The possibility of using vacuum for preservation was tested on fresh muscle tissue samples incubated at 25 °C and 37 °C. The results show that even if there was a limited amount of air inside the storage bag, and not complete vacuum, DNA persistence was enhanced when compared to samples incubated at the same conditions in plastic tubes. Some approaches were attempted to improve degraded DNA profiles. First, degraded DNA was selectively extracted from agarose gels to manipulate the proportion of longer and smaller DNA fragments present. Despite promising preliminary results, this technique showed no usefulness in improving DNA profiles. Purification columns were used with the same aim, but when comparing the original sample with the processed samples, the best results obtained were of equivalence. As an alternative approach, a protocol of DNA Capture was developed in an attempt to preferentially extract the fragments to be analysed in a degraded DNA sample in equal amounts. Whilst the DNA capture method worked in preliminary experiments, it was not applied to degraded profiles. The results obtained have allowed recommendations around collection (i.e. how long samples could be viable for DNA analysis) and storage to be refined. Attempts to rebalance already degraded profiles were not successful. Future field experiments planned as a follow up to the work presented involve testing collection methods and the effectiveness of vacuum body bags.

In this thesis I set out to write the first in-depth study of Pessoa's detective stories. I approached this task in three steps: firstly, by tracing Pessoa's interest in the genre of crime fiction, his readings and influences. Secondly, by analysing the themes and structure of the Quaresma stories. Thirdly, by placing them in the context of Pessoa's written output. The first step is addressed in the first two chapters of the thesis, where I study the connections between Pessoa and Anglo-American detective fiction, as well as how he adapted foreign models to a Portuguese context. The second step of my approach is developed in chapters 3 to 5. In the first of these I focus on the construction of Quaresma as a literary character. My key finding is that the texts featuring him are composed by two kinds of writing: on the one hand narrative prose, including descriptions, actions and elements that further the plot; on the other, an essayistic prose which consists of Quaresma's long speeches expounding his theories on criminal investigation, philosophy, psychology, and reasoning. Chapters 4 and 5 study several of the Quaresma stories from the point of view of gender relations and how these shape the construction of plot and character. At this juncture I use Lacanian and Derridean readings on Poe's 'The Purloined Letter', having previously established that author's influence on Pessoa. The third and final step of my thesis is an attempt to interpret Pessoa's detective fiction in relation to his wider work: I propose a reading of the Quaresma stories, other prose texts and heteronymity as parts of a literary project of creating non-narrative fictions.

This thesis examines the contemporary practice of the digital audiovisual essay, which is defined as a material form of thinking at the crossroads of academic textual analysis, personal cinephilia, and popular online fandom practices, to suggest that it allows rich epistemological discoveries not only about individual films and viewing experiences, but also about how cinema is perceived in the context of digitally mediated audiovisual culture. Chapter one advances five key defining tensions of the digital audiovisual essay: its object is the investigation of specific films and cinephiliac experiences; it uses a performative research methodology based on the affordances of digital viewing and editing technologies; it exists primarily in Web 2.0 and takes advantage of its collaborative and dialogical modes of production; it is a “rich text object” that continuously tests the different contributions of both verbal and audiovisual forms of communication to the production of knowledge about the cinema; and finally, the digital audiovisual essay has an important pedagogical potential, not only for those who watch it, but especially for those who practice it. Chapter two presents the theoretical framework of the dissertation, challenges the ‘newness’ of the digital audiovisual essay, and suggests that any investigation of this cultural practice must address its ideological implications and its role in the context of contemporary audiovisual culture. Accordingly, it relates the editing and compositional techniques of the digital audiovisual essay with modernist montage and suggests that the audiovisual essay has not only inherited, but has also updated and enhanced the dialectical interdependency between critical and consumerism drives that shaped modernism’s ambiguous relation to mass culture. The final chapter examines four case studies (David Bordwell, Catherine Grant, ::kogonada, and Kevin B. Lee) that showcase the contradictory tensions of this cultural practice and broad our understanding of the politics of the digital audiovisual essay.

This thesis, which focusses on the relationship between Lord Holland and Portugal, investigates aspects of political, diplomatic and cultural history. It covers the period between 1793 and 1840 and traces the evolution of Holland's views on Portugal from the time of his first visit to Spain to his later contribution to the establishment of a constitutional regime in Portugal. Particular attention is given to the Hollands' visits to Portugal in 1804-5 and 1808-9. Their journals and correspondence reveal their impressions of the people, culture and history of Portugal. On their travels, they met a number of prominent Portuguese, notably Palmela, who were to remain in contact with Holland House - especially during periods of exile - for many years into the future. The Portuguese journeys and the continuing contact with people like Palmela were to play an important part in the development of Lord Holland's views, not only on Portugal but also on broader political and constitutional issues. Thus the thesis investigates Lord Holland's influence on ' the establishment of a constitutional regime in Spain in 1809-10 and - indirectly and unintentionally - in Portugal in 1820-23, It includes a study of Holland's contribution to the settlement of a government in Brazil in 1808 - that is at the time the Bragancas moved from Portugal to Rio de Janeiro - and his indirect influence on the establishment of the United Kingdom of Portugal, Brazil and the Algarves in 1815, as well as his role in the abolition of the Atlantic Slave Trade and the effects of abolition on Anglo-Portuguese relations. Lord Holland's contribution to the establishment of a Liberal regime in Portugal in 1834 is examined at some length. It includes a study of the extent of Holland's support for the Portuguese Liberal Cause after Dom Miguel's usurpation of the throne in 1828 and of his subsequent role in the 'Liberal invasion' of Portugal. To this end it investigates relations between Portuguese emigres and the Holland House Circle, Holland's role in the triangular diplomacy between Lisbon, St James and South Audley Street in 1828 and later. Finally, it considers Holland's contribution to the end of the Portuguese Civil War in 1834 and to the subsequent establishment of a constitutional regime in that country.

Portugal has traditionally had the highest HIV incidence in Western Europe, currently standing at 0.6% (UNAIDS, 2011). In addition, men in Portugal are disproportionately infected with HIV. Portuguese men are traditionally expected to initiate sexual activity earlier than women and not to worry about safer sex. However, little is known about how prevailing norms of masculinity may influence their experiences of living with HIV. Informed by an interpretivist epistemology and utilising multiple methods, data were gathered from in-depth interviews with 20 men living with HIV and 10 professionals involved in their care, as well as observation of clinical and social support spaces. A number of structural issues impact on men's experiences of living with HIV. In particular, for some men there was a sense of social death, one that drew on the apparent invisibility of HIV, overall ignorance regarding the virus and its effects, reduced government HIV prevention efforts, and feelings of rejection towards people living with HIV. Despite an apparent move from HIV being a fatal disease to a chronic illness globally, participants indicated that HIV in Portugal is still regarded as a dangerous disease at both social and institutional levels. Concomitantly, some men successfully adapted to living with HIV in positive and meaningful ways. Adaptation was facilitated if there had been previous experience of biographical disruption: in particular among gay men or men from ethnic minorities. The close focus, qualitative methods employed allowed for deeper insights into the complexities of structural factors associated with men's experiences of living with HIV. In particular, this study captured some of the struggles, tensions and challenges inherent to living with HIV in a developed country today.

The thesis investigates the extent to which acting as a male provider remains an open avenue for coming of age and displaying gender belonging for the shabāb (lads) of the Shatila Palestinian Refugee Camp, in the southern suburbs of Beirut, Lebanon. The literature on Palestinians prior to 1948 suggests that a man would come of age by marrying at the appropriate age and bearing a son. For the Palestinian diaspora in Lebanon, and throughout the 1970s, acting as a fidāʾī (fighter) worked as an alternative mechanism for coming of age and displaying gender belonging. Accordingly, the central question of this thesis is how the shabāb today come of age and display their gender belonging, when on the one hand, Lebanese legislation, through forms of institutional violence, bars their free access to the labour market, forcing them to postpone marriage plans, and on the other hand, participation in the Palestinian Resistance Movement, at least in its military version, is not an option anymore. Through a plethora of investigative techniques – participant observation, questionnaires, focus groups, and open-ended interviews – I have registered the differences between the fidāʾiyyīn and their offspring in their coming of age and gender display. While the fidāʾiyyīn bore pure agency – understood as resistance to domination – and displayed their maturity through the fight to return to their homeland, their offspring have a far more nuanced relation to Palestine and articulate their coming of age and gender belonging in different ways, such as building a house and getting married. Effectively, by observing how the shabāb do their gender, it is not only the full historicity and changeability in time and space of masculinity that come to the fore, but also the scholarly concepts of agency and gender that can be transformed and undone. The tendency in studies of the Middle East to define gender strictly in terms of power and relations of domination fails to grasp the experiences of those, like the Shatila shabāb, with very limited access to power. It is not that the shabāb are emasculated, but rather that defining agency only in terms of resistance to domination and gender in terms of relations of power alone is rather restrictive. Throughout my fieldwork, I have also become acutely aware of anti-state forces at play in Shatila. Accordingly, this study portrays the (dangerous) liaisons between gender and agency as concepts and state machines. Thus, I reflect on what happens to gender (and agency) when state effects organizing and attempting to solidify a sex-gender system at the local level are of limited purchase. Ultimately, this ethnography points to an economics, a politics, a citizenship and sexes-and-genders of another kind, beyond the state.

Learning induces functional and structural plasticity. This thesis used a range of neuroimaging approaches in both humans and rodents to address three main questions: (1) Can we predict learning performance using baseline imaging measures? (2) To what extent do performance outcomes or training amount determine experience-dependent plastic changes? (3) What biological mechanisms underlie white matter plasticity detected using MRI? Effects of performance and amount of practice on brain structure were studied by varying the amount of juggling practice. Brain structure was found to predict performance on a complex juggling task before learning acquisition. Both performance and practice were found to affect brain structure after learning. Overall, participants that achieved higher performances had higher grey matter (GM) and WM matter change. Also, participants that trained juggling for longer had higher positive brain changes than participants that practiced less. The effects of juggling performance and practice in functional connectivity and GABA levels as measured by MR spectroscopy (MRS) were also investigated. High intensity training was found to decrease the motor resting-state network strength while lower intensity increased the network strength. The increase in strength was associated with a decrease in GABA concentration. A correlation was also found between motor resting-state strength change and GABA concentration change after learning. Finally, since WM plasticity has not been thoroughly investigated and to understand which cellular events underlie WM change, an animal model of motor learning was combined with diffusion tensor imaging (DTI) and immunohistochemistry. Learning a novel motor task increased WM fractional anisotropy, an indirect measure of WM microstructure, in the contralateral hemisphere to the used paw. Immunohistochemistry staining with myelin basic protein (MBP) antibody of this region revealed higher myelin stain intensity for the learning group that correlated with performance in the task.

Cardiomyopathies and arrhythmias are major causes of death in untreated hereditary haemochromatosis, acute iron poisoning and during secondary iron overload resulting from repeated blood transfusions in β-thalassaemia. Iron overload cardiomyopathies are associated with systolic and diastolic dysfunction, suggesting that Ca2+ homeostasis is impaired. However, the cellular mechanisms of these dysfunctions are unknown. The data presented in this thesis establishes for the first time iron effects on cardiomyocyte Ca2+ handling, as well as the potential cellular substrates responsible for this impairment during iron overload. Exposure of isolated rat ventricular cardiomyocytes to 200μM iron led to biphasic changes in systolic Ca2+ release. Phase 1: an initial reduction of systolic Ca2+ release followed by; Phase 2: increased Ca2+ release with arrhythmogenic spontaneous Ca2+ release, cell contracture and cell death. There is evidence that Fe2+ enters cardiomyocytes via L-type Ca2+ channels (LTCC) and reduces the Ca2+ trigger. The close apposition of LTCCs to cardiac ryanodine receptors (RyR2) suggests RyR2 may be a first target. Indeed RyR2 activity was drastically reduced on exposure to nanomolar [Fe2+] in single channel studies. Together with evidence that Fe2+ may reduce the Ca2+ trigger from LTCC, this is consistent with iron reducing sarcoplasmic reticulum (SR) Ca2+ release during Phase 1. In Phase 2, the presence of spontaneous Ca2+ release events is consistent with SR Ca2+ overload. Indeed, in single rat ventricular cardiomyocytes SR Ca2+ content was found to be increased by 27% during Phase 2. The cellular substrates responsible for this increased SR Ca2+ content were 2-fold: 1) through reduced extrusion via both the Na+ Ca2+ Exchanger (NCX) and Plasmalemmal Ca2+ ATPase (PMCA) and 2) through increased resequestration via the SR Ca2+ ATPase. Iron catalyses the production of reactive oxygen species (ROS) during the Fenton reaction. To investigate whether iron effects might be due to ROS, I used the cell permeant ROS scavenger Tempol. Tempol attenuated Phase 2 effects but Phase 1 effects were not affected. This is consistent with the hypothesis that Phase 1 effects were due to direct effects of Fe2+ affecting LTCC trigger and RyR2 function. The attenuation of Phase 2 effects suggests that ROS damage to key Ca2+ handling mechanisms, such as NCX and PMCA might account for a reduced Ca2+ extrusion and subsequent SR Ca2+ overload.

Apparently balanced chromosome rearrangements (ABCRs), mainly reciprocal translocations and inversions, are common in our species and are present both in patients with clinical abnormalities and in phenotypically normal individuals. The four main features that are thought to explain clinical abnormalities in patients with ABCRs are: (i) breakpoint-mediated gene disruption, (ii) breakpoint-associated genomic imbalances, (iii) additional chromosomal complexity and (iv) genomic imbalances unrelated to the breakpoints. The work presented in this thesis represents one of the first systematic studies to ascertain the occurrence of the above four features in both phenotypically normal and abnormal carriers of ABCRs. Molecular cytogenetic analyses by FISH and/or array painting and by array CGH were applied in the characterisation of ABCRs in 31 phenotypically normal individuals (control cohort) and in 16 phenotypically abnormal patients (patient cohort). The occurrence of the above four features was assessed in both cohorts and the results were compared in an attempt to determine if the ABCRs in these two groups are molecularly distinct. Genomic imbalances both at the breakpoints and unrelated to the breakpoints and additional chromosomal complexity were present in 25% of the cases in the patient cohort, but in none of those in the control cohort. Surprisingly, breakpoint-mediated gene disruption was equally frequent in both cohorts. However, there was a difference in the type of genes involved, with those of the patient cohort being more commonly involved in development and function of the nervous system. These observations suggest that there are molecular differences in the two groups of carriers. Furthermore, among the four features analysed, genomic imbalances both at the breakpoints and elsewhere in the genome appear to be the main cause of phenotypic abnormalities in carriers of ABCRs; nevertheless disease candidate genes have also been identified and future studies will assess their contribution to the abnormal phenotypes. In summary, the application of molecular techniques is an invaluable approach to characterise genomic regions involved in chromosome rearrangements and other genomic regions unrelated to the breakpoints, thus aiding in the understanding of the contribution of these genomic regions to human disease and to human variation.

This study contributes to the IS literature with a distinctive explanation of the process of institutionalisation of technology in organizations. The research analyses the role of micro level processes of interplay in embedding an intranet in the formal functioning of an organisation and in the habits and routines of its employees. Findings identify two types of processes of interplay underpinning this process of institutionalisation. The first operates at the level of constitutive expectations and refers to mutual changes to the governance, policy and control mechanisms which foster the perception that the intranet is part of the expected formal functioning of the organisation. The second operates at the level of background expectations and refers to mutual changes that make the intranet look more familiar, functional and easier to use, fostering its embedding in the routines and habits of the employees. The study unravels processes of mutual transformation to an intranet and its hosting organisation, a bank in the UK, by following their evolution over a period of five years. It uses the single longitudinal case study research strategy and is informed by Markus (1983) to support the longitudinal reconstruction of the intranet in the bank. Institutional-based trust theory (Zucker 1986) is used to inform the interpretation of data. This theory is enhanced by the work of Schutz (1962) in developing the concept of background expectations and Garfinkel (1967) in developing the concept of constitutive expectations. The study aims to motivate more research on institutionalisation as a micro level process of ongoing interplay and gradual development of institutionalised behaviour.

Type-B natriuretic peptide (BNP) is an important hormone abundantly present as a pro-peptide in cardiomyocytes. Its release is triggered by exercise, hypoxia and myocardial ischaemia, in addition to chronic haemodynamic cardiac overloading states. BNP's main endocrine actions of vasodilation and natriuresis are mediated by a particulate receptor guanylyl cyclase, natriuretic peptide receptor-A (NPR-A), with subsequent elevation of intracellular cGMP. The role and mechanisms of action of BNP in cardiac ischaemia are not known. We hypothesised that BNP mediates cardioprotection during acute ischaemia-reperfusion, via guanylyl cyclase and cGMP elevation, and examined the role of KATP channel opening in the protective mechanism. The role of nitric oxide (NO) in BNP's signal transduction was also evaluated. Pharmacological studies were carried out in the Langendorff perfused rat heart. Endogenous BNP release was assessed by radio-immunoassay of coronary effluent samples following global normothermic ischemia. Peak concentrations in the first min of reperfusion were markedly elevated following 2 min, 5 min and 20 min of ischaemia. In rat hearts subjected to 35 min regional ischaemia, exogenous BNP limited infarct size in a concentration-dependent manner. The protective action of BNP was sensitive to inhibition by glibenclamide and 5-hydroxydecanoate, blockers of the mitochondrial KATP channel, but not by HMR1098, a blocker of the sarcolemmal KATP channel. Radio-immunoassayed cGMP in cardiac tissue showed a proportionate rise when hearts were subjected to graded durations of ischaemia and when perfused with BNP. Hearts perfused with varying concentrations of 8-bromo-cGMP, a cell-permeable cGMP analogue, were protected against infarction at the lower concentration. L-NAME a blocker of nitric oxide synthase (NOS), and ODQ a blocker of soluble guanylyl cyclase (sGC), both abolished cardioprotection when co-perfused with BNP, suggesting that NO and its activation of sGC play key roles in the protective effect of BNP. To evaluate the source of NOS, studies were undertaken using Western Blot techniques to probe the involvement of endothelial isoform of NOS (eNOS) known to be partially responsible for BNP's vasodilation. However, we found no evidence for acute phosphorylation of eNOS at serine 1177, following BNP or acute ischaemia. Finally, together, our findings indicate a previously-unrecognised cardioprotective action of exogenous BNP via opening of the putative mitochondrial KATP channel with the involvement of basal nitric oxide in the NO/sGC, and cGMP release in the signal transduction. The work contained in this thesis thus confirms a cytoprotective role for BNP in myocardial ischaemia-reperfusion injury and requires further studies in other species and in transgenic models.

Flavonoid dyes were analysed by negative ion mode electrospray ionisation quadrupole ion trap mass spectrometry (ESI QIT MS). New structure-dependent breakdown pathways were identified and the mechanisms were supported by deuterium labelling experiments. The results were extended to provide important structural information on unidentified flavonoids in natural yellow dye extracts. Samples of alum mordanted wool dyed with pure flavonoids or with natural dyes were exposed to accelerated light ageing. Under these conditions photooxidation was found to be the main degradation process. The photodegradation products were analysed by photodiode array high performance liquid chromatography (PDA HPLC) and by liquid chromatography electrospray ionisation quadrupole ion trap mass spectrometry (LC-ESIMS). In general the photodegradation products of flavonols retain information on the nature of substituents of the B-ring and therefore provide partial structural information related to the original dye molecule. The nature of the light source was found not to influence the nature of the photodegradation products detected. No photodegradation products could be identified from flavone or flavonol 3-O-glycosides dyed textiles under the light ageing conditions used. The acid hydrolysis dye extraction process was found to influence the chemical profile of the dye extracts. The identification of the degradation produces allowed their quantification and hence the kinetics of the photooxidation process of flavonoid dyes on wool fibres could be studied. It was found that experimental data did not fit a consecutive first order reaction model. A rapid initial rate of decay of the flavonol was followed by a slower process. The results suggest that a model where the dye population is divided into two groups with different decay rates fits the experimental data adequately. The rates of decay were found to be influenced by the nature of the light source.

A flow-sorted chromosome 1 cosmid library was used to produce a cosmid pocket map spanning two regions in chromosome 1p - approximately 10 Mb in 1p31.1 and 1.7 Mb in 1p13.1. Loss of heterozygosity (LOH) in human breast cancer has been described at these regions. In chromosome 1p13.1 a more detailed physical map consisting of overlapping genomic inserts contained within bacterial recombinant clones was constructed. To the present moment no genomic sequence is yet available at this chromosomal region. For the identification of novel genes, exon trapping and cDNA selection were performed, on 9 overlapping clones from the physical map. The resulting putative exons/transcripts were analysed and compared with non-redundant, cDNA and protein databases. The DNA sequence of seven clones was identical to human cDNA clones. Two highly significant similarities of translations of these clones were found with a human alpha-mannosidase and a rat prostaglandin associated regulatory protein. Both genes were located in a smallest region of overlapping deletion in tumour DNA from patients with breast cancer. Analysis of the coding sequence of the genes using genomic DNA from patients with breast cancer showing LOH at this region will be presented in the thesis.

The first section of this thesis reviews such signal dependent techniques and describes a method of inversion for the Reassigned Spectrogram through use of its link to the phase of the Short Time Fourier Transform (ST-FT). In order to achieve this inversion, signal reconstruction is required using only knowledge of the phase of the ST-FT. Such an inversion is shown to be possible to within an overall amplitude constant given mild conditions upon the degree of overlap used in the computation of the ST-FT. Complimentary to signal reconstruction from knowledge of the phase of the ST-FT, is reconstruction from its magnitude. Two distinct approaches are described. The first previously given in the literature, iteratively applies the Minimum Least Squares (MLS) inversion of the complete (amplitude and phase) ST-FT. The second uses an analogous approach to that used for signal reconstruction from phase. Although the first approach proves more robust to non-valid input TF, both of these techniques require a similar degree of overlap to the phase case, and both reconstruct the signal to within an overall phase constant. The description of the theory concludes with a discussion of the set of Generalised Wavelet Transforms (GWTs), of which both the SF-FT and WT are members. After defining the set of GWTs, descriptions are given for the MLS inversion for complete GWT information, and for signal reconstruction from either phase or amplitude. The thesis concludes by using the MLS based technique to create signals from modified or synthetic spectrograms generated using heart sounds. The first application of this is to extend the duration of heart sounds. Temporal extension in this fashion has the ability to extend the signals in time without affecting the spectrum of the sound. In addition, it does not require the use of a model as in matching pursuit based methods described in the literature. The second application is to create at time-series from a synthetic spectrogram constructed by averaging the spectrograms of a patient's heart murmur. Such averaging cannot take place in the time-domain owing to the random nature of the flow noise in a murmur.

Although a unifying view of literacy development is already implicit within several studies, much of the knowledge is still fragmented. Hence, practitioners lack a comprehensive theoretical framework within which to articulate their practice. This thesis contributes to this framework by investigating whether children's conceptions of the alphabetic system: 1) determine the quality of their orthographic representations and their ability to make inferences about graph-phonetic segments, 2) are affected by adults' explanations of how scripts represent speech and by the characteristics of the particular orthography that children are trying to learn. Sixty two monolingual Brazilian children (mean age 6 years) and 28 bilingual Portuguese children attending two schools in London (mean age 6:7 years), participated in this study, which involved a brief intervention (20 daily sessions). The findings suggested that children's full understanding of the alphabetic principle is not affected by orthographic transparency and that it is the result of a process involving two levels of conceptual change: 1) The characteristics of written words are not related to their meaning - letters represent sub-lexical phonological units. This allows children to detect phonological identity of the initial syllable and to produce syllabic spellings by collating letters that represent syllables. Explicit information about letter-sound correspondences is not essential for this understanding. 2) Adding up the sounds of letters does not produce a word - letters within words or syllables do not sound the same as in isolation. This discovery triggers the use of partial phonological recoding, the production of syllabicalphabetic spellings, the use of analogies and the detection of phonological identity based on articulatory cues. Explicit information about the role of the letters within words may facilitate this understanding and enables the children to work out the grapheme-phoneme correspondence, which is the last step towards grasping the alphabetic principle.

American adult adoptees may possess limited information about their biological families and turn to direct-to-consumer personal genomic testing (PGT) for genealogical and medical information. I investigated the motivations and outcomes of adoptees undergoing PGT using data from the Impact of Personal Genomics (PGen) Study. The PGen Study surveyed new 23andMe and Pathway Genomics customers before and 6 months after receiving PGT results. Exploratory analyses compared adoptees’ and non-adoptees’ PGT attitudes, expectations and experiences. I evaluated the association of adoption status with motivations for testing and post-disclosure actions using logistic regression models. Of 1,607 participants, 80 (5%) were adopted. As compared with non-adoptees, adoptees were more likely to cite limited knowledge of family health history (OR = 10.1; 95% CI = 5.7–19.5) and the opportunity to learn genetic disease risks (OR = 2.7; 95% CI = 1.6–4.8) as strong motivations for PGT. Of 922 participants who completed 6-month follow-up, there was no significant association between adoption status and PGT-motivated health-care utilisation or health-behaviour change. PGT allows adoptees to gain otherwise inaccessible information about their genetic disease risks and ancestry, helping them to fill the void of an incomplete family health history.