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There has been a clear shift in the literature from employability as the acquisition of skills to employability as an active and socially constructed process that unfolds over time and across contexts. Yet there has been limited research on graduates' career journeys and the development of employability capitals over time using Tomlinson's (2017) graduate capital model. This study aims to add to this small body of literature by examining the development and usage of employability capitals over time among first-generation graduates, who have received little attention in the literature. Tomlinson's graduate capital model, which is positioned as a more nuanced conceptualisation of employability, was chosen as the study's main theoretical framework and the model's usefulness in the study of employability was examined. A narrative inquiry research design was employed to elicit the career stories of five firstgeneration graduates who graduated from UK universities approximately ten years ago. Schlossberg's transition theory was used to organise the central themes because career transitions were a significant feature in the participants' narratives. A main finding of the study was that, while capital accumulation and deployment can assist career transitions, it was unclear from the career stories how developing capitals can support successful and sustainable employment when professional development is not always supported in the workplace. This study offers a critique of the graduate capital model by highlighting its shortterm, static nature, which prioritises the exchange value of graduate labour over long-term employment fulfilment. The career stories indicated that employability is a fluid and organic process, in contrast to the graduate capital model's strategic approach to capital formation and utilisation. This study demonstrates the need to move away from a static, individual-centred view of employability and towards one that considers long-term working conditions and practices, rather than just a graduate's immediate entry into the workforce.

1. Gastric Ulcer. X Ray is undoubtedly superior to test meals in usefulness. 2. Duodenal Ulcer. Usefulness of X Ray with test meal is about equal. X Ray, of course, gives more accurate information. 3. Carcinoma. Test meal is more useful in diagnosing an early case. 4. Gastritis. The X Ray does not give any help other than in assisting to exclude carcinoma or ulceration, whereas the test meal is very useful, besides aiding the Physician in the diagnosis, it also helps him to exclude carcinoma definitely. 5. Functional Disease. The test meal gives, on the whole, more help to the Practitioner than the X Ray. In cases of functional disease, the X Ray does give a certain amount of help, but it is chiefly in a negative sense, namely, in excluding organic disease, although it does, to a certain extent assist in the diagnosis of hypertonicity and atony. The test meal, on the other hand, is very useful in helping the Physician to diagnose the various conditions of the gastric secretion, besides hypertonicity and atony of the stomach. I shall finish by stating that if the Physician, or Surgeon, after a careful history and clinical examination has been done, is undecided as to the diagnosis, then a test meal should be performed. If there is still doubt, or if more accurate information is desired, then a radiological examination should be carried out without delay. Finally, both the results of X Ray examination and the fractional test meal, unless they agree with the history and clinical findings, should be accepted with great caution, or ignored, or else a further examination should be carried out.

PREFAB is a political modernist project whose threefold purpose, through development of a theory- in-praxis framework, is to counter the naïve assertions of the realist aesthetic (the dominance of which serves to reproduce capitalist common sense), as well as the conformism of the 'art for art's sake' avant-garde. In the third place, PREFAB wishes to repudiate the hagiographic emphasis of liberal art history & theory in the evaluation of the arts. Contrariwise, in the world of work, no less than in the academy and culture, women and men 'make their own history, but they do not make it just as they please; they do not make it under circumstances chosen by themselves, but under circumstances directly encountered, given and transmitted from the past.' (Marx, 1968, p.96).1 On this model of structured agency, focusing on real social and economic determinants, PREFAB instead features an 'author as producer'2 frame of analysis, where the object of study is the materialised memories of the life and times, or lived experience (through the decades, circa 1930-95), of a working-class Edinburgh family. Namely my own. The intellectual formation of PREFAB's integrative synthesis of making and writing was informed, signally, by Marx's profound insight that social being determines thought, by Antonio Gramsci's counter-hegemonic inventory of traces - a knowing oneself as a product of the historical process to date3 - and by the dialectical distanciation techniques, or 'the separation of the elements',4 of Bertolt Brecht's anti-illusionist 'epic' theatre (and by affiliated political modernists in the interwar era and since). The project's primary sources also include the demythologising semiology of Roland Barthes, the cultural materialist5 formulations of Raymond Williams, Janet Wolff's Marxist sociology of art, Avery Gordon's exploration of the hauntological imagination, and Lukacs's critique of the ideological reproduction of the capitalist commodity-scape. Determined therefore (in paraphrase of Godard's slogan) not to make political art but to make art politically, (Godard : MacCabe, 1980, p.19)6 the PREFAB how-is-what case studies address the Scottish nativism grounded in invented traditions of neo-tartanry,7 the ideological distortions of the 'thing-world' of finance-capitalism, with especial interest in the fetish forms of reification8 evocative of the proletarian popular culture of my grandfather's prefab-worldview. Reproduced from indexical signs of post-war capitalist consumer culture, the PREFAB ready-mades accompanying the written thesis, comprise a family history of redux commodities, parodic fetishes of nation-state, and Marxist signage: a laboratory of didactic experiments in what may also be called critical realist method (in complex unity with critical realist theory) against the dominant conventions of bourgeois ways of seeing, knowing, and telling. References: 1 Marx, K., 'The Eighteenth Brumaire of Louis Bonaparte', Karl Marx and Frederick Engels Selected Works in One Volume, Lawrence & Wishart, 1968, p. 96. 2 Benjamin, W., Understanding Brecht, Verso, 1998. 3 Gramsci, A., Selections from Prison Notebooks, Lawrence & Wishart, 1971, p. 324. 4 Willet, J., ed., Brecht on Theatre, Methuen, 1978, p.37. 5 Bottomore defines materialism as: '(a) a denial of the autonomy, and then of the primacy, of ideas in social life; (b) a methodological commitment to concrete historiographical research, as opposed to abstract philosophical reflection; (c) a conception of the centrality of human praxis in the production and reproduction of social life and, flowing from this, (d) a stress on the significance of labour, as involving the transformation of nature and the mediation of social relations, in human history' (Bottomore, 1985, p.324). 6 'The problem is not to make political films but to make films politically.' MacCabe, C., Godard: Images, Sounds, Politics, BFI/Macmillan, 1980, p.19. 7 By coincidence, a key date in the emergence of Scottish neo-Romanticism as embodied in the invented traditions of tartanry, is marked by the visit of George IV to Scotland in August 1822. Stage-managed by Sir Walter Scott, the visit included a military review in which the British monarch, keen to improve his popularity after a series of personal scandals, appeared on Portobello beach in philabeg, sporran, and elaborate Glengarry, to inspect 3000 volunteer cavalrymen and honour the Highland clans. It is estimated that 50,000 people turned out to watch the spectacle. The event is recorded in a painting by William Turner de Lond titled George IV, 1762 - 1830. Reigned as Regent 1811 - 1820, as King 1820 - 1830 (At a military review on Portobello Sands 23 August 1822) (1828). 8 In A Dictionary of Marxist Thought (1983) Bottomore defines reification as: 'The act (or result of the act) of transforming human properties, relations and actions into properties, relations and actions of man-produced things which have become independent (and which are imagined as originally independent) of man and govern his life. Also transformation of human beings into thing-like beings which do not behave in a human way but according to the laws of the thing-world. Reification is a 'special' case of ALIENATION, its most radical and widespread form characteristic of modern capitalist society' (Bottomore, 1983, p. 411).

Early response to antipsychotic medication in schizophrenia has been found to be highly predictive of future response to pharmacological intervention. Recent research has reported that non-response as early as 2 weeks into antipsychotic treatment is associated with future non-response. Up to a third of individuals with a schizophrenia diagnosis will experience antipsychotic treatment resistance following multiple trials of antipsychotic medication with no or minimal remission of symptoms and are termed treatment resistant (TRS). Those with a resistant illness are recommended to have early intervention with clozapine, with earlier initiation associated with better clinical and functional outcomes. In comparison to those responsive to antipsychotic medication, TRS patients show greater glutamatergic abnormalities, a lack of dopaminergic abnormalities, an earlier onset of schizophrenia, are more likely to be male and have poor premorbid functioning. However, there is a need to understand other potential predictors of antipsychotic resistance and non-response, particularly early in the illness onset. Neuropsychological measures provide a cheap and non-invasive tool to examine cognitive performance and functioning in schizophrenic and other clinical populations. Cognitive deficits have long been documented in schizophrenia; in comparison to healthy controls, prior to illness onset, in unaffected relatives, and research has found deficits in adolescence to also be predictive of a future psychosis diagnosis. However, little is known about the relationship between cognitive performance and antipsychotic non-response and resistance; are those with a resistant illness more cognitively impaired than those who respond to antipsychotic medication? This thesis tackles this question by investigating the relationship between cognitive performance and antipsychotic response across the disease timeline; from secondary research, first episode samples, and known cases of antipsychotic response and non-response. In Chapter 8 I meta-analysed the results from 17 published, mostly cross-sectional, studies comparing known samples of treatment resistant schizophrenia (TRS) and responsive schizophrenia on multiple measures of cognitive performance. Five metaanalyses were performed in relation to 1) executive function, 2) general cognitive function, 3) attention, working memory and processing speed, 4) verbal memory and learning, and 5) visual-spatial memory and learning. Small to moderate effect sizes emerged for all domains, with the largest effect size observed in verbal memory and learning. A sub-analysis of language-related functions, extracted from across the primary domains, yielded a comparable effect size. These results are discussed in relation to the neurodevelopmental origins of treatment resistance and verbal functions in schizophrenia. In Chapter 9 I then investigate the relationship between cognition and antipsychotic response in an international cohort of first episode psychosis patients, with follow-up data to prospectively categorise patients as responsive or resistant to antipsychotic treatment. Baseline cognitive data, originating from multiple cognitive tasks and seven international cohorts, was categorised in the similar manner to domains in Chapter 8. On average patients who were future classified as treatment resistant reported poorer performance across most cognitive domains at baseline assessment. Univariate logistic regressions showed that antipsychotic treatment resistance cases had significantly poorer IQ/general cognitive functioning at first episode. These findings remained significant after adjusting for age, gender, cohort, duration of untreated psychosis, length of follow-up, and positive and negative symptoms. The findings and limitations are discussed, as well as the addition of cognitive performance to clinical markers of psychosis and future antipsychotic response. Chapter 10 also utilises a cohort early in their antipsychotic treatment and illness onset. Here the sample was followed up at 3 study visits over a period of 6 weeks, providing cognitive (using the Brief Assessment of Cognition in Schizophrenia) and symptom severity (Positive and Negative Syndrome Scale) data at each visit. Trajectory analyses were used to classify the participants by treatment response from symptom severity data; 84.8% of the sample were classified as treatment responsive, and the remaining 15.2% as treatment non-responsive. Unadjusted and adjusted logistic regressions observed no significant relationship between baseline cognitive performance and antipsychotic response at 6 weeks. However larger samples may have been required to observe a meaningful difference between trajectory groups. In Chapter 11, I discuss the results from a cross-sectional study with antipsychotic treatment responders and antipsychotic non-responders. Cognitive data were again collected using the Brief Assessment of Cognition in Schizophrenia, such as in Chapter 10. Univariable regression models observed no significant differences between antipsychotic responder and non-responder groups on any measure of the BACS. This pattern of findings was consistent in multivariable models. The lack of group difference in cognition in this sample is likely due to a lack of clinical distinction between groups or an absence of effect between early response and cognitive performance. Finally, in Chapter 12 I discuss the findings from this thesis in the context of the current literature, clinical settings, and the future potential of including cognitive markers in clinical and demographic prediction models of antipsychotic response. In addition to this, I also provide recommendations for future research in the field as well as discuss the strengths and limitations of my findings.

The burden of poor antipsychotic response in schizophrenia has a profound impact on patients, their caregivers and wider society. Patients who demonstrate a poor antipsychotic response may have some distinct clinical and biological features compared to those who respond well to antipsychotic treatment. Treatment-resistant schizophrenia is associated with more severe cognitive impairment and may have a distinct genetic loading to other more treatment responsive forms of illness. Most consistently, poor clinical and functional outcomes under antipsychotic treatment have been associated with elevations in glutamate metabolite concentrations in the anterior cingulate cortex. The overall aim of this work was to investigate the relationships between these features of poor antipsychotic response in schizophrenia, to better understand the aetiology of disease and ultimately aid the development of stratified treatment approaches. The introductory chapter of this thesis provides an overview of schizophrenia, treatment resistance and features of poor antipsychotic response. As an extension of this literature synthesis, Chapter 2 is a systematic review and meta-analysis of clinical and demographic variables associated with response to clozapine treatment in treatment-resistant schizophrenia. The three subsequent data chapters in this thesis examine relationships between glutamate in the anterior cingulate cortex and 1) neurocognitive function; 2) common genetic variation. I present data from STRATA-1, a multicentre cross-sectional study of patients with schizophrenia spectrum disorders displaying a good or poor response to antipsychotic treatment. Brain glutamate may contribute to both clinical outcomes and cognition in schizophrenia, but these relationships are not well understood. In Chapter 4, I find that higher levels of glutamate in the anterior cingulate cortex are associated with better cognitive function. I also demonstrate that this relationship does not differ according to the degree of antipsychotic response in participants. This result suggests that the nature of the relationship between anterior cingulate cortex glutamate and cognition is similar between good and poor antipsychotic responders, at least within the range of glutamate and cognitive function measured in our sample. Next, I investigate the common genetic architecture of anterior cingulate cortex glutamate concentrations in schizophrenia. In Chapter 5 I generate a treatment resistance polygenic score, which summarises the common genetic loading for treatment resistance. I find no association between the treatment resistance polygenic score and brain glutamate within the study sample. There was, however, some indication that the relationship between the treatment resistance polygenic score and anterior cingulate cortex glutamate was different between good and poor antipsychotic response groups. Finally, in Chapter 6 I generate pathway polygenic scores. These were made from common genetic variants associated with an increased risk of treatment-resistant schizophrenia that fall within genes implicated in NMDA receptor function. I find evidence of a negative association between the treatment resistance NMDA receptor complex pathway polygenic score and brain glutamate. In summary, I show that genetic liability for treatment-resistant schizophrenia within NMDA receptor encoding genes is associated with glutamate concentrations in the anterior cingulate cortex. In turn, these variations in glutamate are associated with verbal cognitive function in this disorder. These results are discussed within the wider context of novel drug development and personalised medicine for the treatment of schizophrenia spectrum disorders.

Background: Clozapine is the only evidence-based medication for treatment-resistant schizophrenia. However, clozapine is severely under-prescribed mainly because of clozapine-induced agranulocytosis, an adverse drug reaction of clozapine that occurs in 0.4% of clozapinetreated patients. Since there are no clinical predictors for clozapine-induced agranulocytosis, all clozapine users are required to be under strict blood test monitoring throughout the duration of their clozapine treatment. Aims of this thesis: To use data from electronic health records to test the following hypothesis: (i) Predictors for clozapine-induced agranulocytosis can be investigated using the results of the analysed electronics health records data published in (Iqbal et al., 2020); (ii) The frequency density of clozapine blood test results that indicate clozapineinduced agranulocytosis risk changes with clozapine treatment time; (iii) Clozapine-treated patients are at an increased risk of COVID-19 infection; (iv) Clozapine-treated patients are at an increased risk of severe outcomes of COVID-19 infection. Methods: All data were extracted from Clinical Record Interactive Search (CRIS), the de-identified electronic health records of South London and Maudsley NHS Foundation Trust (SLAM). All data extraction was performed using SQL. All analysis was performed using either R, python or STATA. The statistical methods used in this thesis are logistic regression, survival analysis and Cox proportional hazard models. Results: We found that the data from (Iqbal et al., 2020) was not informative for investigating predictors for clozapine-induced agranulocytosis, thus this study did not bear any significant results. However, it helped us to realise that the next step was to study the patterns of clozapine blood monitoring data. From studying the patterns in clozapine blood monitoring results, we showed that the highest risk of clozapine-induced agranulocytosis is in the early months of treatments. The Kaplan-Meier survival curve and the incidence rates analysis showed that 75% of blood test results that indicated clozapine-induced agranulocytosis risk occurred within the first 6 months of clozapine treatment. At the onset of the COVID-19 pandemic, we investigated the associations between clozapine treatment and increased risk of COVID-19. We found that clozapine-treated patients had an increased risk of COVID-19 compared with those who were on other antipsychotic medication (unadjusted HR = 2.62, 95% CI 1.73 - 3.96), which was attenuated after adjusting for potential confounders, including clinical contact (adjusted HR=1.76, 95% CI 1.14 - 2.72). We followed up on the previous study to investigate the associations between clozapine treatment and increased risk of severe outcomes of COVID-19, namely COVID-related hospitalisation, COVID-related intensive care treatment, and death. We found that even though clozapine treatment appears to increase the risk of COVID-19 infection, it does not increase the risk of the severe outcomes of COVID-19. Conclusion: In conclusion, electronic health records are a valuable resource for studying clozapine health outcomes. In particular, the CRIS data is a very informative resource for answering research questions related to mental health disorders.

This thesis critically examines the gender gap in the music industry through a practice-based enquiry. The study is based on my professional practice developing and delivering a large-scale participatory programme for early career, female musicians called Both Sides Now (BSN). Through BSN I proposed an outcomes based Theory of Change (Kirkpatrick, 1978) built around the framework of Access, Agency and Change. The thesis is structured around these three areas of enquiry which pertain to significant junctures in the personal and professional development of female musicians; educational access and early influence, artistic agency, preprofessional development and career pathways. Taking each 'key word' (MacCabe and Yanacek, 2018) in turn it tests this conceptual framing, pursuing a primary line of enquiry which asks what needs to change and how? Guiding the investigation is a commitment to the principles of feminist research and an exploration of the concept of 'affect' within the discourse of change-making. This, I argue opens up new knowledge that is felt and embodied and offers a contemporary understanding of ways in which the practices and structures of the industry are discriminatory. The practice submission offers a portfolio of documentary and archival materials and an ongoing body of work which includes a new teaching resource for the music curriculum, female-only projects and the BSN Manifesto for Change which was presented to the Culture Committee of the European Parliament in October 2019. This is presented alongside a written submission which provides a theoretically informed analysis of the initiative and its findings. Collectively these insights position BSN as an interventionist practice. They argue that change is required on multiple, intersecting levels in order to comprehensively respond to this inequality and to reshape a discipline which has for too long overlooked and under-valued the contribution of women.

This thesis critically examines the gender gap in the music industry through a practice-based enquiry. The study is based on my professional practice developing and delivering a large-scale participatory programme for early career, female musicians called Both Sides Now (BSN). Through BSN I proposed an outcomes-based Theory of Change (Kirkpatrick, 1978) built around the framework of Access, Agency and Change. The thesis is structured around these three areas of enquiry which pertain to significant junctures in the personal and professional development of female musicians; educational access and early influence, artistic agency, pre-professional development and career pathways. Taking each 'key word' (MacCabe and Yanacek, 2018) in turn it tests this conceptual framing, pursuing a primary line of enquiry which asks what needs to change and how? Guiding the investigation is a commitment to the principles of feminist research and an exploration of the concept of 'affect' within the discourse of change-making. This, I argue opens up new knowledge that is felt and embodied and offers a contemporary understanding of ways in which the practices and structures of the industry are discriminatory. The practice submission offers a portfolio of documentary and archival materials and an ongoing body of work which includes a new teaching resource for the music curriculum, female-only projects and the BSN Manifesto for Change which was presented to the Culture Committee of the European Parliament in October 2019. This is presented alongside a written submission which provides a theoretically informed analysis of the initiative and its findings. Collectively these insights position BSN as an interventionist practice. They argue that change is required on multiple, intersecting levels in order to comprehensively respond to this inequality and to reshape a discipline which has for too long overlooked and under-valued the contribution of women.

Theory and practice of psychiatric genetics transformed within a decade. Genetics has gone through a century of unsuccessfully trying to find individual specific genes, basic units of heritability, which would explain risk for complex disorders. For the past ten years, genetics has been exploring joint contributions of thousands of genes instead. Research on psychosis has been a strong focus throughout this shift. Psychosis is an umbrella term for multiple types of experience (hallucinations, voices or visions which are not there, delusions, fixed, false beliefs, changes in emotion and drive, and decline in memory and other cognitive skills). Psychosis is a spectrum: it is not distressing and negative to all who experience it and it does not always result in clinical intervention, but also, patients with psychosis live with substantial disability and have a shortened life expectancy, often due to disorders simultaneously present with psychosis (e.g., individuals with schizophrenia lose the equivalent of on average 73% of healthy life per year due to comorbid mental and substance use disorders). Thus, clinical interventions try to alleviate psychosis and improve patient outcomes. As psychosis has a genetic component (its heritability is 70% or more, depending on a psychotic disorder), the aim of psychiatric genetics is to develop measures which would incorporate genetic factors to help explain and predict psychotic symptoms to anticipate, improve, and time, optimal treatment. As it is now understood that no single candidate gene explains risk for psychosis, a measure including information on multiple genetic variants has been developed. This measure, a polygenic score, is supposed to reflect individual genetic predisposition towards a trait. Polygenic scores sum up information on thousands of common genetic variants into a single score. Each genetic variant is weighted by its effect size from a genome-wide association study (GWAS). For associations with disorder, GWAS compares common genetic variants between participants with a disorder and controls to determine which variants are more statistically likely to be related to the disorder. Polygenic scores have been primarily used to differentiate cases with psychotic disorders from controls. However, psychosis is a complex and heterogenous disorder. It can have different trajectories (from single episodes and complete remission to multiple relapses throughout life), different combinations of symptoms, and symptoms which change over time. Thus, the aim of this thesis was to explore if polygenic scores for major psychiatric disorders predict specific psychosis manifestations which may impact psychosis trajectories over time: age of onset of psychosis (age of first symptom manifestations which eventually lead to psychiatric intervention; AOO); duration of untreated psychosis (from symptom onset to either hospitalisation or first antipsychotic treatment; DUP); and longitudinal cognitive decline of memory and executive function in later life (after the age of 50). In Chapter 3, I show that polygenic scores for major psychiatric disorders (schizophrenia, bipolar disorder, major depressive disorder, autism spectrum disorder, and attention deficit hyperactivity disorder) do not predict AOO in first episode psychosis patients from different European cities. In Chapter 4, I demonstrate that these polygenic scores do not predict DUP in first episode psychosis patients. However, in both studies, research sites, from which the data come, are significantly related to AOO and DUP. Finally, in Chapter 5, I show that the polygenic score for schizophrenia predicts baseline cognitive performance, but not decline of memory and executive function, over a decade-long follow-up. These findings suggest a nuanced picture of common genetic variants in psychosis: they may be related to increased risk for specific psychosis symptoms (e.g., general cognitive deficit), but not all clinical features which are labelled as "psychosis", such as early psychotic symptoms or cognitive decline over time. Alternatively, findings in this thesis may be a result of differences between research sites in how psychosis manifestations are measured, inaccurate patient recall of their medical history, bias in diagnosis, or suboptimal methods of generating polygenic scores (these issues, and suggestions for future research, are discussed in Chapter 6).

Disturbances in sleep and circadian rhythm are commonly reported in the period preceding deterioration in mental state in people with psychosis. Sleep disturbance may therefore serve as an early sign of impending relapse, for prompting timely, preventative interventions. Also, establishing a temporal association between sleep and symptomatic deterioration would lend support to the hypothesis that sleep plays a causal role in generating psychotic symptoms, and may in itself form an important therapeutic target. I aim to explore both of these questions through a digital phenotyping approach, which harnesses continuous, real-time data from mobile and wearable devices, in real-world settings. In the first chapter of this thesis incorporating publication, I summarise the evidence for sleep-circadian disruption during the relapse prodrome in schizophrenia, and incorporate literature from the bipolar disorder field. I explore the neurobiological mechanisms by which sleep reduction and circadian misalignment may drive psychopathology, and provide an overview of how digital technologies are being harnessed as clinical tools in psychiatry. The second chapter, a systematic review and meta-analysis of case-control actigraphy studies, seeks to understand sleep-circadian dysfunction across the psychosis spectrum by comparing actigraphy parameters in schizophrenia and bipolar disorder, in the non-acute phase of illness. I show that in both disorders, patients show significantly longer total sleep time and time in bed, with lower daytime activity. However, sleep is also more disturbed, with greater sleep latency and fragmentation, pointing to a range of difficulties pervading all aspects of the 24 hour cycle. Effect sizes were greater in schizophrenia; higher antipsychotic dose was associated with longer sleep time, and greater use of sedative medication was associated with reduced sleep latency, suggesting that treatment effects play a role in these findings. This sets the scene for the subsequent examination of dynamic changes, across phases of illness. To date, sleep-circadian and psychopathology variables have not been studied for long enough to allow episodes of deterioration and relapse to be captured. Increasingly ubiquitous mobile and wearable technologies may allow continuous sampling of subjective and objective sleep-circadian variables in the home environment, over extended periods. In chapter three, I describe the findings of a pilot study, undertaken to develop and test the feasibility and acceptability of Sleepsight, a digital platform designed specifically for this objective. Although there were high rates of engagement with the technology, greater negative symptom burden was associated with poorer completion of smart-phone diaries. Importantly, when the study was conducted in a transparent, collaborative manner, concerns over privacy and security were not significant barriers to engagement and adherence. This tool was then used in a substantive study of 36 people with psychosis, each participating for 12 months, in order to capture natural fluctuations in mental state, including relapse episodes. In chapter four, I examine the association between subjective sleep-circadian variables and psychopathology data using the Differential Time Varying Effects Model (DTVEM), a novel analytic approach which elucidates the lag structure between sleep variables and mental state. The results clearly suggest that poorer subjective sleep quality and reduced subjective sleep duration precede deterioration in symptoms by 8-12 days. Although an inverse direction of effect of psychopathology on sleep was also found, this was shorter, and of smaller magnitude. These findings support the hypothesis that sleep disturbance precedes deterioration in psychopathology. In chapter five, I describe how raw digital phenotype data are classified into sleep-circadian variables using a preprocessing pipeline, and employ a range of analytic approaches to explore these data. Visualisation of rest-activity patterns demonstrated a wide range of sleep-circadian phenotypes, some with striking patterns of circadian dysfunction including relative co-ordination and non-24 hour rhythms. A second analysis using the DTVEM method, with objective data, sustained the finding that sleep reduction preceded symptomatic deterioration. Group-level comparisons between relapsing and non-relapsing participants did not demonstrate significant differences in clinical or sleep-circadian variables. However, visualising these variables in the participants who relapsed, centred on relapse event, suggested that a reduction in sleep duration and decrease in activity accompany the period before, during and after relapse. Mathematical modelling was able to simulate major circadian disturbances in a subset of participants, and crucially showed that increasing daylight in the model restored normal circadian rhythmicity. Some of the technological limitations currently inherent in the approach are also discussed. Taken together, these findings suggest that sleep reduction, fragmentation and poorer sleep quality are features of schizophrenia in remission, and are also associated with deterioration and relapse. The evidence supports these changes playing a causal role in the development of psychopathology. Despite technical challenges relating to data quality, these findings also suggest that meaningful objective data can be collected from smartphone and wearable devices, which show promise for use as decision-support tools.

Schizophrenia may be associated with elevations in glutamate levels in the anterior cingulate cortex (ACC), and this may be apparent in patients who have not responded well to conventional antipsychotic treatment. Compounds that can decrease ACC glutamate levels may have therapeutic potential for this group, however before this PhD the extent to which glutamate can be modulated pharmacologically was unknown. This PhD examined the potential of two different drugs to reduce ACC glutamate levels in schizophrenia, N-acetylcysteine (NAC), and clozapine. Study One: N-acetylcysteine. The primary aim of this study was to examine whether a single oral dose of NAC can alter brain glutamate levels, as well as regional cerebral blood flow (CBF) and resting state functional connectivity (rs-FC) in patients with schizophrenia. In the ACC, glutamate + glutamine was significantly reduced in the NAC compared to placebo condition, but this effect was not related to changes in CBF or rs-FC. The results provided preliminary evidence that NAC may reduce ACC glutamate metabolites in schizophrenia. Study Two: Clozapine. Clozapine has been shown to be effective in treating symptoms that have not responded to conventional antipsychotic medication. While the reasons for this superior efficacy is unclear, it may relate to the ability of clozapine to modulate brain glutamate. The primary aim of this study was to measure glutamate levels in the ACC and caudate in patients with TRS before and after 12 weeks of treatment with clozapine. We found that clozapine treatment was associated with a longitudinal reduction in glutamate levels in the caudate, which was associated with symptomatic improvement. Conclusions: The work described in this PhD supports the idea that brain glutamate metabolites can be reduced pharmacologically in schizophrenia. These findings can be translated to future work to determine the extent to which such reductions in glutamate may relate to clinical efficacy.

Treatment resistance (TR), affecting approximately 20-30% of patients with psychosis, has a high burden both for patients and healthcare services. I provided a general introduction to TR in Chapter 6 and highlighted the need to identify TR earlier in the course of the illness, so that an effective treatment, such as clozapine, can be offered promptly. The purpose of this thesis was to identify clinical, demographic, and genetic predictors of TR, using information identified when a patient first presents to clinical services with psychosis. First, I reported the results of a systemic literature review, which synthesised predictors of TR identified in longitudinal, observational studies (Chapter 8). Younger age of onset was the most consistent predictor of TR, but this review also indicated that, to date, studies have not used statistical methods specifically designed to identify predictors that have a high chance of predicting TR in future patients. Existing literature has primarily used methods designed to capture the magnitude of association, between predictors and TR, within the study sample. Second, I reported the results of my own analysis using a dataset created by combining existing longitudinal, first episode psychosis cohorts. This dataset, known as STRATA-G (and described in Chapter 7), included patients who had a minimum of one year follow-up, provided a DNA sample, could be classified as either TR or non-treatment resistant (NTR). I found significant associations between TR and both younger age of onset and fewer years in education (Chapter 9). I created a predictive model of TR that selected nine first episode variables and could, after internal validation, correctly classify 59% of TR patients and 65% of NTR patients (Chapter 9). Third, I reported the results of a genome-wide association study (GWAS) of treatment resistance, which was used to create a polygenic risk score for TR (Chapter 10). This polygenic risk score was significantly associated with TR in the STRATA-G sample. Fourth, I investigated whether age of psychosis onset mediated the relationship between genetic burden for TR and subsequent TR, using STRATA-G (Chapter 11). Age of onset was not associated with genetic risk for TR and there was no evidence it mediated the relationship between the polygenic risk score for TR and subsequent TR. Finally, I expanded on the discussion of the findings in the preceding chapters (Chapter 12). Here, I placed my findings in the context of the wider literature and discussed the limitations of my data. I discussed the future of prediction modelling in TR; the potential uses of a prediction model, in both clinical practice and research studies, and the importance of future work to validate the findings reporting in this thesis.

This thesis explores the need for, feasibility and value of building a whole disease model (WDM) of schizophrenia and its management in the UK. Conventional health economic models are 'piecewise' in that they typically address a single decision problem at a specific decision point in a care pathway. This can lead to inconsistencies between decisions and such economic analyses may not achieve their intended objective - the maximisation of health given a fixed budget constraint. An alternative system-level modelling approach - whole disease modelling - involves developing a health economic model of whole disease and treatment pathways which support the economic analysis of technologies and services for prevention, diagnosis, treatment and follow-up within a single consistent mathematical model. System-level modelling approaches (including WDMs) have been applied to several disease areas, including cancer, metabolic diseases and cardiovascular diseases. However, they have not been applied to mental health disorders, such as schizophrenia. A systematic literature review of existing model-based economic analyses for schizophrenia services revealed that: (1) there are no existing WDMs for schizophrenia; (2) there are few model-based economic analyses for non-pharmacological interventions; (3) existing models are generally poor quality; (4) existing models cannot support the joint assessment of options for service investment and disinvestment. To address these problems, this thesis focuses on the design, development and evaluation of a novel WDM of schizophrenia services in the UK. The feasibility and value of the schizophrenia WDM in informing resource allocation decisions was evaluated by using the model to assess multiple topics considered within existing NICE guidelines on the management of schizophrenia. The schizophrenia WDM was implemented as a patient-level discrete event simulation model. The choice of modelling technique was informed by a literature review of modelling techniques adopted by previous system-level models and the application of published model selection tools. Conceptual models underpinning the WDM were developed using a methodological framework specially adapted for this thesis. Evidence to inform the model parameters was obtained from the best available sources. Extensive validation and verification activities were undertaken to ensure that the model was robust. The WDM was used to conduct cost-effectiveness analyses for five NICE guideline topics (Topic A, B, C, D and E), covering five different components of the schizophrenia care pathway and involving 19 different interventions. Outcomes were measured in terms of quality-adjusted life years (QALYs) and costs included those relevant from the health and social care perspective. Two decision rules were adopted to assess the cost-effectiveness of alternative options: (1) a piecewise cost per QALY threshold rule (Decision Rule 1), and (2) a disease-level constrained maximisation of QALYs decision rule, which is subject to a fixed budget constraint (Decision Rule 2). The WDM results suggest that the current schizophrenia service configuration is not optimal. Cost savings and additional QALYs can be gained by: (1) replacing current interventions with more cost-effective interventions; (2) increasing the level of implementation of currently recommended interventions. Results using Decision Rule 1 suggest that the most cost-effective interventions for each topic are: cognitive behavioural therapy (CBT) for people at clinical high risk of psychosis (Topic A); a mix of hospital admission and crisis resolution and home treatment team (CRHT) for people with acute episode of psychosis (Topic B); amisulpride, risperidone or olanzapine as the first-line oral antipsychotic medication for people with first-episode psychosis (FEP) (Topic C); family intervention combined with antipsychotic medication for people with FEP (Topic D); and clozapine for people with treatment resistant schizophrenia (Topic E). The use of Decision Rule 2 leads to a different conclusion for Topic C, with quetiapine becoming the most cost-effective option. The results also suggest that the following options for improving adherence to the NICE schizophrenia guideline recommendations are likely to result in cost savings and QALY gains compared with current practice: (1) increasing the provision of CBT from current levels of 41.01% to 100% for people at clinical high risk of psychosis; (2) increasing the provision of family intervention from current levels of 30.98% to 100% for people with FEP; and (3) providing clozapine to people with treatment-resistant schizophrenia with no delay compared to current provision with a delay of 3.98 years. This thesis demonstrates that it is feasible to develop a WDM for schizophrenia, although a significant initial investment of time and resources was required; overall, the schizophrenia WDM took approximately 12 months to develop (full-time equivalent) and 4 months to run. Compared with conventional piecewise models, WDMs offer several benefits, such as addressing more decision problems using a single consistent model, flexibility around the choice of decision rules, and the inclusion of interactions between interventions across the pathway. The schizophrenia WDM shows that the use of alternative decision rules and changing the current service configuration can alter the cost-effectiveness conclusions for certain topics. Three priorities for future research are warranted: (1) improving the evidence base for schizophrenia models; (2) re-use of the WDM to evaluate other decision problems within schizophrenia services; and (3) exploring methods for reducing the development time of WDMs.

Clozapine is an atypical antipsychotic known to be uniquely effective in patients with treatment-resistant schizophrenia, but associated with numerous adverse reactions. Despite substantial research efforts, we lack a full understanding of the mechanistic processes underlying clozapine's efficacy and identification of the most relevant mechanisms. Animal studies suggest that clozapine induces changes in DNA methylation (a dynamic epigenetic modification which can regulate gene expression). However, research in humans is extremely limited. This thesis provides an original contribution to the literature as the first study to report positions of significant differences in longitudinal DNA methylation trajectories associated with clozapine exposure, therapeutic response and adverse reactions. 22 participants with schizophrenia were recruited and within-participant sampling was conducted before clozapine initiation and at three time-points over the subsequent six months. The Illumina 450k array was used to assay epigenome-wide DNA methylation in 85 samples of whole blood. I conducted multi-level regression models and pathway analysis. Across all models, I identified 33 epigenome-wide significant (p < 1*10-7) differently methylated positions (DMPS), with over-representation of DMPS indicated to have correlated methylation in brain tissue. DMPs located on genes involved in signal transduction, glutamatergic and GABAergic systems, calcium signalling, glucose homeostasis, immune function and transcription regulation were associated with both clozapine exposure and therapeutic efficacy. Additionally, DMPs located on genes involved in cell adhesion, glycine and neuronal development were associated specifically with therapeutic efficacy. DMPs located on genes involved in immune function and signal transduction were associated with drowsiness, increased appetite and weight, and cardiovascular symptoms. DMPs located on genes involved in energy metabolism were associated with increases in appetite and weight. These results align with literature suggesting that clozapine normalises schizophrenia-associated disruption in glutamatergic and GABAergic systems, calcium signalling and immune function; however, further studies of gene expression and downstream effects are required to establish the causal role of DNA methylation.

Introduction: Psychotic disorders are known for their wide variability in clinical and social outcomes beginning from illness onset and throughout their course. Our current lack of understanding of the origins for this heterogeneity is further compounded by dearth in knowledge on how patients come to the attention of mental health services and methodological incongruity across different studies. Employing samples of first episode psychosis (FEP) patients, the aims of this thesis were to: 1) look back on the pathways to care patients used to enter mental health services and the use of prodromal services in South London; and 2) examine trajectories of the psychotic disorders and potential predictors of their longitudinal outcomes. Methods: Two large samples of patients with FEP (i.e., GAP and EU-GEI) were utilised in this thesis. For the study 1, information on pathways to care undertaken prior to coming to generic services for FEP was extracted from the Biomedical Research Centre (BRC) Case Register Interactive Search (CRIS) system. For studies 2, 3 and 4, using electronic clinical records, extensive information in three domains-clinical, social and service uses-was collated over 4-5 years after contact with mental health services. Results: Only a small fraction of individuals (4.1%) who present with FEP to the main secondary mental health provider have previously been in contact with prodromal services and made a subsequent transition to psychotic disorder; 77% this sub-group of patients entered their pathway to care via referral from General Practice or other health professional. In contrast, 45% of FEP group without prior contact with the prodromal services made first contact with mental health services via emergency services and 18% of this group were referred by the criminal justice system. Further, combining the baseline schizophrenia diagnosis with five symptom dimensions (i.e., positive, negative, excited, disorganised/concrete and depressed dimensions) generated the best model fit for predicting time to first remission. During the 5-year follow up after first contact with mental health services, a higher proportion of Black African and Black Caribbean ethnicity had compulsory re-admissions and instances of police involvement during an admission to a psychiatric unit compared with White British ethnic group. Patients of Black African and Black Caribbean ethnicity did not differ from White British ethnic group in overall functional disability and illness severity, or frequency of remission or recovery during the follow up period. However, patients of Black ethnicity become increasing socially excluded as their illness progress. In a sample of first-episode schizophrenia spectrum patients, 35% of the sample met the criteria for treatment resistance (TR) at the end of the first 5 years of follow up. Of these TR patients, 70% of these were treatment resistant from illness onset. Those who subsequently developed TR were more likely to have an early illness onset (<20 years) compared to those with non-TR. The relationship between an early age of onset (<20 years) and TR was specific to patients of Black ethnicity and patients of male gender. Conclusions: Very few of those who come to FEP services come after being seen for an at-risk-mental state by prodromal services suggesting that the scope for reducing or delaying onset of psychosis by this means may still be limited. My results indicate that supplementing the baseline categorical schizophrenia diagnosis with ratings on five symptom dimensions improves the prediction of delayed treatment response as measured by time to first remission. Further, the longitudinal trajectory of psychosis in patients of Black ethnicity did not show greater clinical or functional deterioration than white patients. However, their course remains characterised by more compulsion, and longer periods of admission. Finally, I showed that for the majority of the TR group, lack of response to antipsychotic treatment is present from illness onset, necessitating a consideration for an earlier use of clozapine.

Background: Investigating long-term antipsychotic polypharmacy is key to unpacking the associations between serious mental illnesses (SMI) and detrimental outcomes, such as premature death and frequent hospital readmissions, observed in this population. However, existing research is sparse and hampered by methodological problems such as examining small and homogeneous samples and residual confounding. Objectives: 1) To identify cases on long-term antipsychotic polypharmacy (≥ 6 months) prescribing in South London and Maudsley electronic health records (EHR); 2) To identify factors that predict long-term antipsychotic polypharmacy prescribing for SMI patients in secondary mental health care; 3) To investigate whether outcomes such as hospital readmission and mortality are associated with long-term antipsychotic polypharmacy prescribing in secondary mental health care. Methods: Antipsychotic medication information was derived from the Clinical Record Interactive Search (CRIS), a de-identified electronic patient records system, for the period between 2007 and 2014. Data on mortality were extracted using existing linkages between CRIS and death certification (Office of National Statistics). Information about antipsychotic co-prescribing was extracted using a bespoke algorithm. Multivariable logistic models were built to investigate predictors of antipsychotic polypharmacy. To investigate the impact of antipsychotic polypharmacy on hospital readmission and all-cause mortality, I constructed multivariable Cox proportion hazard models. To test the association between long-term antipsychotic polypharmacy and cause-specific mortality I used competing risk regression. Implications: On a clinical level, this thesis provides an insight into factors that can predict clinical decision-making regarding antipsychotic polypharmacy prescribing in real-life clinical settings. On a patient level, the findings highlight patient burden associated with this antipsychotic regimen. In the wider treatment, service and policy context, the lack of patient benefit from antipsychotic polypharmacy highlights the need for programmes that target prescribers, to reduce antipsychotic polypharmacy.

Wireless sensor networks, in an effort to be energy efficient, typically lack the high-level abstractions of advanced programming languages. Though strong, the dichotomy between these two paradigms can be overcome. The SENSIX software framework, described in this dissertation, uniquely integrates constraint-dominated wireless sensor networks with the flexibility of object-oriented programming models, without violating the principles of either. Though these two computing paradigms are contradictory in many ways, SENSIX bridges them to yield a dynamic middleware abstraction unifying low-level resource-aware task reconfiguration and high-level object recomposition. Through the layered approach of SENSIX, the software developer creates a domain-specific sensing architecture by defining a customized task specification and utilizing object inheritance. In addition, SENSIX performs better at large scales (on the order of 1000 nodes or more) than other sensor network middleware which do not include such unified facilities for vertical integration.

Cache injection is a viable technique to improve the performance of data-intensive parallel applications. This dissertation characterizes cache injection of incoming network data in terms of parallel application performance. My results show that the benefit of this technique is dependent on: the ratio of processor speed to memory speed, the cache injection policy, and the application's communication characteristics. Cache injection addresses the memory wall for I/O by writing data into a processor's cache directly from the I/O bus. This technique, unlike data prefetching, reduces the number of reads served by the memory unit. This reduction is significant for data-intensive applications whose performance is dominated by compulsory cache misses and cannot be alleviated by traditional caching systems. Unlike previous work on cache injection which focused on reducing host network stack overhead incurred by memory copies, I show that applications can directly benefit from this technique based on their temporal and spatial locality in accessing incoming network data. I also show that the performance of cache injection is directly proportional to the ratio of processor speed to memory speed. In other words, systems with a memory wall can provide significantly better performance with cache injection and an appropriate injection policy. This result implies that multi-core and many-core architectures would benefit from this technique. Finally, my results show that the application's communication characteristics are key to cache injection performance. For example, cache injection can improve the performance of certain collective communication operations by up to 20% as a function of message size.

The Internet has developed into an important economic, military, academic, and social resource. It is a complex network, comprised of tens of thousands of independently operated networks, called Autonomous Systems (ASes). A significant strength of the Internet's design, one which enabled its rapid growth in terms of users and bandwidth, is that its underlying protocols (such as IP, TCP, and BGP) are distributed. Users and networks alike can attach and detach from the Internet at will, without causing major disruptions to global Internet connectivity. This dissertation shows that the Internet's distributed, and often redundant structure, can be exploited to increase the security of its protocols, particularly BGP (the Internet's interdomain routing protocol). It introduces Pretty Good BGP, an anomaly detection protocol coupled with an automated response that can protect individual networks from BGP attacks. It also presents statistical measurements of the Internet's structure and uses them to create a model of Internet growth. This work could be used, for instance, to test upcoming routing protocols on ensemble of large, Internet-like graphs. Finally, this dissertation shows that while the Internet is designed to be agnostic to political influence, it is actually quite centralized at the country level. With the recent rise in country-level Internet policies, such as nation-wide censorship and warrantless wiretaps, this centralized control could have significant impact on international reachability.

A hierarchical group model that decouples computation from hardware can characterize and aid in the construction of sensor network software with minimal overhead. Future sensor network applications will move beyond static, homogeneous deployments to include dynamic, heterogeneous elements. These sensor networks will also gain new users, including casual users who will expect intuitive interfaces to interact with sensor networks. To address these challenges, a new computational model and a system implementing the model are presented. This model ensures that computations can be readily reassigned as sensor nodes are introduced or removed. The model includes methods for communication to accommodate these dynamic elements. This dissertation presents a detailed description and design of a computational model that resolves these challenges using a hierarchical group mechanism. In this model, computation is tasked to logical groups and split into collective and local components that communicate hierarchically. Local computation is primarily used for data production and publishes data to the collective computation. Similarly, collective computation is primarily used for data aggregation and pushes results back to the local computation. Finally, the model includes data-processing functions interposed between local and collective functions that are responsible for data conversion. This dissertation also presents implementations and applications of the model. Implementations include Kensho, a C-based implementation of the hierarchical group model, that can be used for a variety of user applications. Another implementation, Tables, presents a spreadsheet-inspired view of the sensor network that takes advantage of hierarchical groups for both computation and communication. Users are able to specify both local and collective functions that execute on the sensor network via the spreadsheet interface. Applications of the model are also explored. One application, FUSN, provides a set of methods for constructing filesystem-based interfaces for sensor networks. This demonstrates the general applicability of the model as applied to sensor network programming and management interfaces. Finally, the model is applied to a novel privacy algorithm to demonstrate that the model isn't strictly limited to programming interfaces.

Establishing secure communication channels in sensor networks is made especially difficult because of low power resources, hostile environments, and wireless communication. The power requirements of traditional cryptographic methods create the need for alternative strategies for secure communication in sensor networks. This thesis explores key distribution techniques in sensor networks. Specifically, we study in depth one method that enables sensors to establish pairwise secure communication channels. This strategy relies on a cooperative set of peer sensors to construct a unique key between two sensors. We built a unique network simulator to test secure communication parameters in a typical deployment scenario. This research tests the strategy by which the cooperative set of sensors is chosen. The results demonstrate that a strategy favoring neighbor nodes consumes significantly less energy than other alternatives at the expense of vulnerability to geographically localized attacks.

Network servers are vulnerable to attack, and this state of affairs shows no sign of abating. Therefore security measures to protect vulnerable software is an important part of keeping systems secure. Anomaly detection systems have the potential to improve the state of affairs, because they can independently learn a model of normal behavior from a set of training data, and then use the model to detect novel attacks. In most cases, this model represents more instances than were in the training data set—such generalization is necessary for accurate anomaly detection. This dissertation describes a framework for testing anomaly detection algorithms under identical conditions. Because quality test data representative of todays web servers is not available, this dissertation also describes the Hypertext Transfer Protocol (HTTP) request data collected from four web sites to use as training and test data representing normal HTTP requests. A collection of attacks against web servers and their applications did not exist either, so prior to testing it was necessary to also build a database of HTTP attacks, the largest publicly-available one. These data were used to test nine algorithms. This testing was more rigorous than any performed previously, and it shows that the previously-proposed algorithms (character distribution, a linear combination of six measures, and a Markov Model) are not accurate enough for production use on many of the web servers in use today, and might explain the lack of their widespread adoption. Two newer algorithms (deterministic finite automaton induction and n-grams) show more promise. This dissertation shows that accurate anomaly detection requires carefully controlled generalization. Too much or too little will result inaccurate results. Calculating the growth rate of the set that describes the anomaly detector's model of normal provides a means of comparing anomaly detection algorithms and predicting their accuracy. Identification of undergeneralization locations can be automated, leading to more rapid discovery of the heuristics needed to allow an anomaly detection system to achieve the required accuracy for production use.

Since the discovery of the mouse quaking viable allele (qkv) in 1964, researchers have attempted to elucidate the function of quaking in postnatal brain myelination. More recently, a critical role for quaking in embryogenesis was revealed, but little has been determined on how the loss of quaking function leads to embryonic death. In the work presented here, we report that the defect in the embryonic lethal qkk2 allele is a T to A trans version in the KH domain, and that homozygous qkk2 mutant embryos die from cardiovascular defects. Antibodies against the vascular endothelial marker PECAM-1 reveal that qkk2 homozygous mutants completely lack the yolk sac vascular network present in wild-type embryos. Hearts of qkk2/qkk2 embryos lack the looping morphogenesis of their wild-type littennates, but Nkx2.5, a gene that functions in cardiac looping, is expressed in a wild-type pattern. Cardiac myocytes in mutant embryos also express α-sarcomeric actin protein and whole hearts in organ culture were found to beat at wild-type rates. Real-time quantitative RT-PCR reveals that cardiac developmental markers are expressed at normal levels in qkk2 mutant hearts but that expression of the vascularization gene, Vegf, is increased two-fold compared to wild-type. In addition, the cell cycle genes Ccnd1, Cdkn1a and Cdkn1c have abnormal expression compared to wild-type embryonic hearts. The neural associated genes Shh, Foxa2, Fgf8, and Pax6 as well as the notochord marker T, are all expressed in wild-type patterns in qkk2 embryos at the time of death. However, adult carriers of the qkk2 allele are significantly more susceptible to chemically induced seizures with the convulsant drug pentylenetetrazole than wild-type animals. In an effort to further dissect the function of quaking, we performed a single generation ENU mutagenesis screen to create new alleles. We report here, the generation of the qke5 allele which, unlike the previous induced alleles of quaking, is homozygous viable. The qke5 phenotype consists of postnatal quaking and seizures, which progresses to severe ataxia and early death. Ultrastructural analysis reveals an almost complete lack of myelin compared to both wild-type and qkk2/qkk2 brains. Protein expression analysis in the postnatal brain shows that like qkk2/qkk2, qke5/qke5 mice have low QKI-5 and no QKI-6 and QKI-7 proteins in developing oligodendrocytes. However, oligodendrocytes of both qke5 and qkv homozygous mice express a full range of developmental marker genes indicating that quaking may function relatively late in oligodendrocyte development. We determined by electroencephalogram recordings, that qke5/qke5 surface cortex activity is abnormal and that seizures result in electrical activity characteristic of a decrimental response. Together these results reveal a previously unsuspected and critical role for quaking in cardiovascular development as well as a more important role in postnatal brain myelination then had been determined from 40 years of studies utilizing the qkv allele.

Occupational exposure to mineral fibers, such as asbestos, has been associated with increased risk of pulmonary fibrosis, malignant mesothelioma and lung carcinoma. It has been proposed that fibers with diameters < 0.25 and lengths > 8 um, possess the greatest capacity for cytotoxicity and cell transformation. (Stanton, 1977). The present study was prompted by the limited data on the health effects of silicon carbide whiskers (SiCW), a recently marketed man-made mineral fibers. This study was designed to compare two SiCW of differing dimensions as well as a non-fibrous form. In order to examine the immediate biological effects of Sic, cultured explants of tracheal epithelium were exposed to a range of fiber concentrations (5 ug/cm2 - 100 ug/cm2) . There were no dramatic effects on ciliary function as measured by optical spectrum analysis which provided discrete ciliary frequencies. Particles were swept by ciliary activity into non-ciliated regions where foci of extensive cell damage and death were observed with whiskers penetrating epithelial layers into the underlying connective tissues. Similiar necrotic foci were observed in tracheas from rats exposed by intratracheal instillation to silicon carbide whisker (SiCW) in vivo. Investigation into the intermediate and long term pulmonary response began with intratracheal administration of SiCW-1 (0.8 um diameter), SiCW-2 (1.5 um diameter), Sic platelets (SiCP), boron carbide fiber (BC) and crocidolite asbestos. By bronchopulmonary lavage, phagocytic cells (macrophages, monocytes, lymphocytes) were harvested for examination of cell population changes. Lavage analysis revealed a significant (p < 0.05) increase in macrophages still in evidence 28 days after SiCW exposure, an early sign of chronic inflammatory processes. Lungs removed from fiber-exposed animals exhibited tissue alterations characterized by severe fibrosis and granulomatous tumors at 18 months post exposure. The highest frequencies of occurence, 88% and 91%, were noted in SiCW-1 treatment groups. Additonal pathological abnormalities included hyperplasia, metaplasia and interstitial pneumonia. Interperitoneal injections of SiCW similarly resulted in inflammatory fibrosis and benign peritoneal tumors. SiCW-1 was shown to produced the greatest cellular damage. The single whisker form of Sic may pose a serious health threat without precautionary handling measures,

To study the mechanisms associated with the carcinogenicity of natural and man- made fibers/whiskers at the cellular level, mouse fibroblast cultures were exposed to 5 µg/cm2 crocidolite asbestos (0.36 µM in diameter) and two different sizes of silicon carbide whiskers (0.8 and 1.5 µM in diameter). After 48 hours of exposure, 60 to 90 percent of metaphase spreads had chromosomes that were entangled with, attached to, or lying adjacent to fibers. Over 40 percent of isolated nuclei contained fibers, and were seen inside of, or attached to various parts of the nucleus (i.e. nuclear membrane, nuclear matrix, DNA, chromatin). Total DNA and chromosomal content were also determined up to 20 generations post exposure to test materials. After 10-12 generations postexposure (6-8 passages), all fibers/whiskers induced a 40 to 75 percent increase in cellular DNA. There was also a significant increase in chromosome number per cell 18 generations post exposure to fibers/whiskers, as compared to control. This increase in DNA and chromosome content could possibly provide information on the cellular mechanism of toxicity associated with the exposure to fibrous materials. These quantitative in vitro tests may also be useful as a tool to suggest the long-term genetic effects of a potential carcinogen.

An enigmatic feature of mammalian spermatogenesis is the transcriptional inactivation of the X chromosome in meiotic prophase. This study examines aspects and consequences of spermatogenic X-chromosome inactivation in the mouse. Since the X chromosome encodes several housekeeping proteins, serious consequence could arise for the spermatogenic cell in the absence of mechanisms to ensure continued enzymatic function throughout development. The goal of this study was to determine the spermatogenic expression of the X-linked gene for hypoxanthine phosphoribosyltransferase (Hprt) and the relevance of its expression to X-chromosome inactivation. The results demonstrated that HPRT activity is maintained in spermatogenic cells throughout development in spite of X-chromo- some inactivation; however, specific activities of HPRT in meiotic and postmeiotic germ cells were significantly lower than that in premeiotic ones. Maintenance of Hprt transcripts was also demonstrated. Interestingly, the highest level of Hprt transcripts was found in leptotene/zygotene spermatocytes, suggesting a hyper- activation of the Hprt gene and/or accumulation of Hprt transcripts in these cells. The level of Hprt transcripts also appeared to be higher in round spermatids than in pachytene spermatocytes, suggesting a reactivation of the Hprt gene after meiosis. It was also found that the relative abundance of Hprt transcripts in germ-cell-deficient testes of XXSxr mice was much higher than that in meiotic and postmeiotic germ cells even though their activities of HPRT were similar. Examination of the translational status of Hprt transcripts in testes and germ cells revealed that most of the transcript was translationally active in XXSxr testes but not in pachytene iv spermatocytes or round spermatids. Since no functional autosomal Hprt gene exists in the mouse, these data suggest a strategy for maintaining HPRT activity in germ cells throughout development that involves stabilization of gene products coupled to possible hyperactivation of the Hprt gene prior to X-inactivation and reactivation of the gene in postmeiotic cells.

The regulatory mechanisms of ciliary activity of frog palate epithelial cells are investigated in this study. Ciliary responses to electrical, mechanical and autonomic neurotransmitter are characterized with measuring ciliary frequency at single cell level. Ciliated cells of frog palate outgrowth cultures respond to electrical, mechanical and acetylcholine stimuli by an increase of ciliary beat frequency. The ciliary beat frequency shows dose response to electrical stimulation. Under effective electrical stimulation ciliary beat frequency increase is transmitted cell to cell from explant to outgrowth at a speed of 1.8 cells/second. Under mechanical stimulation ciliary response is transmitted in the same way as with electrical stimulation, but from stimulated cell to adjacent cells in all directions at a speed of 1.4 cells/second. Ciliated cells do not respond to epinephrine, but respond to acetylcholine in a dose dependent manner. The ciliary response to acetylcholine is inhibited specifically by atropine. Direct innervation of frog palate ciliated cells has been demonstrated in this study with electron microscopy. Electrical stimulation of the palate outgrowth culture can induce ciliary beat frequency increase of ciliated cells in the culture. This ciliary response results from palatine nerve endings in the explant releasing acetylcholine in response to the stimulation. Either removal of explant from a culture or atropine treatment inhibits outgrowth ciliated cells from responding to electrical stimulation. The morphology and ciliary activity of the palate outgrowth cells over three weeks in culture are characterized. Outgrowth of the primary culture is formed by migration of ciliated epithelium from the explant. The outgrowth maintains stratified epithelial characteristics of the palate epithelium, but secretory granule in basal secretory cells are gradually lost in culture. As a coincidence ciliary beat frequency gradually increases. The ciliary beat pattern changes from frequent fluctuation to a relative constant frequency with culture age. It is suggested that the basal secretory cells of the palate may inhibit ciliary activity by releasing secretory granules. Gap junctions are found between epithelial cells in both explants and outgrowths. Intercellular communication by gap junction provides an integrated control mechanism for regulation of the ciliary activity of large numbers of cells in the epithelium. Stimulation of a single cell or a few cells induces ciliary activity in many cells. Mechanical stimulation and gap junctional intercellular communication may represent a mechanism of local activation of mucociliary transport in response to exogenous particles. Electrical stimulation and gap junctional intercellular communication provides a mechanism of more general activation of mucociliary transport mediated by the palatine nerve. The effect of Ca2+ in mediation of ciliary response to stimulation is examined. It is found that extracellular Ca2+ is not necessary for initiation of ciliary response to a stimulation, but is important in maintaining the stimulated ciliary activity. It is suggested that mitochondria and nuclei are the major intracellular Ca2+ storage sites. Mobilization of Ca2+ from mitochondria and nuclei into the cytoplasm and cilia initiates ciliary beat frequency increase to various stimulations. Free Ca2+ or other signal molecules in stimulated cells passing through gap junctions into adjacent cells may cause the propagation of the ciliary response.

The ability and accuracy of an assay designed to detect antiendometrial antibodies found in samples of serum, follicular fluid, and peritoneal fluid is significant in determining whether endometriosis can be predicted. Samples were collected from 264 patients of local gynecologists and an in vitro fertilization laboratory. An antiendometrial antibody (AEA) assay was used to test for endometriosis. Collected samples were applied to normal human endometrial tissue. A second antibody labeled to fluorescein isothiocyanate (FITC) was also applied. Laparoscopy performed by gynecologists and the in vitro fertilization laboratory served as a control for the AEA assay. Serum produced the most accurate results when compared to laparoscopy while follicular fluid and peritoneal fluid produced results similar to each other, but less accurate than serum. Further refinement of the AEA assay could result in a useful, nonsurgical technique for predicting endometriosis. Also under investigation was whether human antiendometrial antibodies cross reacted with endometrial tissue from other mammals. Among the mammals tested, marmoset and porcine endometrial tissue produced results that best correlated to human endometrial tissue when exposed to antiendometrial antibodies. Due to economic practicality, porcine endometrial tissue was chosen as an alternative to human endometrial tissue for use in the AEA assay.

This study was conducted to assess the ciliotoxic potential of the organophosphorous insecticides Dursban and Lorsban, their active ingredient, chlorpyrifos and their carrier ingredients (blanks). The ciliated epithelium of the frog palate was used as the model for an innervated ciliated system, since chlorpyrifos inhibits nervous system acetylcholinesterase. Frog palates were excised and cultured to form ciliated outgrowths. Outgrowths were monitored with an inverted phase contrast microscope, and ciliary beat frequencies were determined using an optical spectrum analysis system. All compounds caused a decrease in frequency of ciliary beat over time. Concentration-response curves were constructed from data of time to inhibition of ciliary beating. The order of toxicity was Lorsban, Dursban, Lorsban Blank, Dursban Blank, and chlorpyrifos, with Lorsban being the most toxic and chlorpyrifos being the least toxic. ECs estimations were also calculated from these data; and they followed the same order of toxicity as the time to inhibition studies. Samples were examined by electron microscopy to evaluate any ultrastructural effects. All compounds were found to cause mitochondrial damage, including swelling, disruption of cristae, and loss of matrix.The response of individual cells was monitored immediately after addition of test compounds. Stimulation of ciliary beating was evident immediately after exposure to all compounds. Stimulation was ultimately followed by inhibition, within a time frame (a few minutes to hours) depending on the concentration of the compound in the test medium. Dursban, Lorsban, and both blanks elicited stimulatory effects in the presence of atropine. Atropine blocked the initial stimulatory response only with chlorpyrifos. It appears that in addition to chlorpyrifos some component (s) of the inert ingredients were initially stimulatory but ultimately inhibitory to ciliary beating in the frog palate model.

The process of gametogenesis is of paramount importance for reproductive success in mammals Gamete production includes a specialized form of cell division termed meiosis whereby chromosomally diploid cells become haploid. Although meiosis is genetically essential for the formation of a normal gamete, very little is known of the molecular and cellular events constituting this phase of gametogenesis. An initial and reasonable approach to elucidating factors related to the meiotic process is to isolate genes expressed during meiosis. An overview of the literature, as it is related to this work, is presented in Part I of this dissertation. Part II of this dissertation outlines the construction of spermatogenic cell-specific cDNA libraries in order to establish a system for the molecular analysis of meiosis in male mammals. Cytological and molecular characterization of these libraries are also described. These libraries represent an important tool for the isolation of genes that are expressed meiotically. Part III of this dissertation describes the use of the meiotic cDNA libraries in a novel screen for meiotically-enriched cDNA clones. This screen utilizes RNA from meiotically-arrested male-sterile mutant mice for the successful identification of spermatocyte-enriched cDNA clones. The isolation of these clones serves as a foundation for further investigations into the molecular basis of meiosis. Part IV of this dissertation describes the use of homology screening by PGR to identify a meiotically-expressed cDNA. The isolation and characterization of a novel meiotically-expressed cDNA is outlined in this section. This cDNA is expressed most abundantly in pachytene spermatocytes and in the brain and thymus of mice and contains a putative leucine zipper motif. Additionally, although a molecular analysis of meiosis will potentially reveal new information, cytological approaches for understanding meiosis should lead to the discovery of different information concerning this process. In this regard. Part V reports the development of a spermatocyte in vitro culture system for the analysis of meiosis and interesting observations that have come from in vitro manipulation of spermatocytes. Finally, Part VI serves to summarize the various conclusions and significance of this work and postulates future directions for continued investigation into the molecular basis of meiosis.

Two regions of programmed cell death that occur in the mesoderm of developing chick wing buds were studied in vitro. The permanence of FGF-2 rescue in the opaque patch (OP) and posterior necrotic zone (PNZ) was examined using the chromium release assay. While rescue in the OP was found to be permanent, rescue in the PNZ only delayed death while FGF-2 was present in the culture medium. Also, the OP and PNZ were examined for the presence of internucleosomal DNA degradation and for rescue by protein synthesis inhibition, two defining characteristics of apoptosis. Agarose gel electrophoresis showed that DNA from OP and PNZ tissue was cleaved into nucleosome size pieces and this cleavage was prevented by the presence of cycloheximide. Both the OP and PNZ showed rescue by protein synthesis inhibition with cycloheximide, as determined by the chromium release assay and examination of electron micrographs. This research suggests that in vitro FGF-2 rescue is permanent in the OP but is merely a delay of cell death in the PNZ, and that death in both of these regions is prevented by inhibition of protein synthesis with cycloheximide. It also shows that death in the OP and PNZ exhibits internucleosomal DNA fragmentation, biochemically characterizing this death as apoptosis.

Natural and man-made mineral fibers possess physical and chemical characteristics that have been associated with cytotoxicity. Among these are fiber length, fiber width, fiber aspect ratio, and surface chemistry. This study correlated measurements of these characteristics with their respective effects on colony forming ability of 3T3 fibroblasts in vitro. These correlations and measurements were used to develop a model for predicting the cytotoxicity of mineral fibers. A proprietary treatment that reduces the toxicity of both natural and man- made mineral fibers was tested. This proprietary treatment covalently binds a hydrophobic coating to the fibers' surfaces causing them to behave in a hydrophobic manner. Colony forming ability assays were performed on treated and untreated silicon carbide whiskers (SiCW) and crocidolite asbestos. The coating significantly decreased cytotoxicity as measured by colony forming ability. At the highest dosage levels, the fiber coating decreased the cytotoxicity of crocidolite asbestos and silicon carbide whisker by 35% and 60% respectively. A preliminary transformation assay indicated that the coating technique may reduce the fibers' ability to transform cells.

Skin of the lizard, Anolis carolinensis, changes color from green to brown when stimulated with visible light or melanocyte-stimulating hormone (MSH). MSH and light-mediated responses involve melanosome dispersion within dermal melanophores. Color change by these agents was studied reflectometrically in intact skin, and melanophore responses were visually assessed in cultured cells. The MSH response, but not the dermal photic response (DPR) , utilizes adenosine 3',5' cyclic AMP (cAMP). cAMP levels increase in skins darkened with MSH, but not light or calcium ionophore a23187. cAMP-elevating agents also caused dispersion in cultured melanophores. There is no intracellular calcium requirement since the MSH response of skin samples or cultured melanophores remains intact in the presence of lanthanum, and cultured melanophores loaded with the intracellular calcium chelator, BAPTA-AM, respond normally. The action spectrum of DPR reveals the presence of a blue-light sensitive photopigment (&ligma;=425 nm) . Western blotting and immunolocalization suggests the presence of a cone-like opsin protein present on the melanophore plasmalemma and possibly on the melanosome. Calcium is apparently released from intracellular stores during DPR. X-ray microanalysis suggests that this store may be the melanosome. Studies with 8-Bromo-cGMP, which inhibits DPR but not MSH response, suggests that cGMP may inhibit calcium release. The mechanism of DPR may have similarities to the transduction mechanism of the retinal rod cell which shares a similar embryological origin. The MSH and DPR responses converge on a common microfilament-based dispersion system. A 57 kd protein (P57) is phosphorylated by light or MSH. Phosphorylation is inhibited by 8-Bromo-cGMP in the assay buffer. Since the protein phosphatase inhibitor, okadaic acid, triggers pigment dispersion in cultured cells, P57 is hypothesized to be the molecular switch which controls pigment movement. Phosphorylation of P57 may be the point at which the two pathways converge. Finally, we report MSH-sensitivity of the iridophore. Cultured iridophores shift transmittance from red to blue when treated with MSH, cAMP-elevating agents, or colchicine, but not light. Using spectral measurements from xanthophores and iridophores, theoretical skin reflectance spectra were calculated to examine the contribution of iridophore activity to skin color and color change.

We have evaluated two in vitro ciliated epithelial systems, dog trachea and frog palate, for their potential as toxicological screens for inhaled chemicals. We examined the effects of a series of nonpolar narcotic agents (alcohols and ketones) and related observed toxicities (EC50) to hydrophobicity (log Ko/w). The baseline QSAR for the dog trachea system is log 1/EC50 = 0.80 (log Ko/w) - 2.99; n = 7, r2 = 0.982, s = 0.18, F = 275.63, Pr > F = 0.0001. The frog palate baseline QSAR is log 1/EC50 = 0.81 (log Ko/w) - 3.12; n = 7, r2 = 0.991, s = 0.13, F = 565.66, Pr > F = 0.0001. The slopes of these QSARs are equivalent to the slopes of QSARs from established systems, including in vivo mouse inhalation, and differing intercepts have been explained on the basis of method of exposure. We also examined a set of bioreactive chemicals, whose toxicities were expected to elicit greater toxicity than would be predicted by their hydrophobicity alone, and reported computed excess toxicity (Te) values greater than 2 for each bioreactive chemical. Mechanisms of toxicity represented by the set of bioreactive chemicals tested in our study include bimolecular nucleophilic substitution, Michael-type addition to a carbon-carbon double bond, Schiff base formation, uncoupling of oxidative phosphorylation, and reversible and irreversible inhibition of acetylcholinesterase. Ranking the bioreactive chemicals in increasing order of Te for the dog trachea results in: trans-2-octenal < benzyl chloride < carbaryl < malathion < heptaldehyde < 4,6-dinitro-o-cresol. In the frog palate, the increasing rank order of Te is: trans-2-octenal < heptaldehyde < benzyl chloride < 4,6-dinitro-o-cresoI < carbaryl < malathion. The differences in rank orders between dog and frog indicate that more bioreactive chemicals need to be studied with these in vitro systems in order to determine if certain chemical classes are more or less appropriate for evaluation with one or the other system. Based on the positive preliminary data in this investigation, we feel that, as one component of a battery of tests, these ciliated in vitro systems would provide valuable, usable information about potential respiratory effects of inhaled chemicals. The additional advantages of lower cost and efficiency of data collection make these systems attractive as alternatives to the current whole animal test system (mouse inhalation chamber).

Citrinin Is a highly potent nephrotoxin produced as a secondary metabolite during the growth of numerous fungi. Though citrinin may be detected in a variety of food products, it is most commonly found as a contaminant of both the large and small grains. It has been implicated in the production of renal disease and death among livestock and is implicated in a fatal renal disease in humans. Current methods used to detect citrinin such as high performance liquid chromatography (HPLC) and thin layer chromatography are either expensive, require extensive research equipment or are not satisfactory for the detection of the small, but toxic, levels of mycotoxin which may be present in a food, feed or blood sample. The objective of this study was to develop a hybridoma producing monoclonal antibody to citrinin for use in an enzyme-linked immunosorbent assay (ELISA) for the mycotoxin. Citrinin was coupled to bovine serum albumin (BSA). Analysis of buffers by HPLC following equilibrium dialysis revealed a molar binding ratio of 19 moles of citrinin per mole BSA. This citrinin/BSA conjugate was used to immunize 8-week-old male RBF mice over a period of 6-8 weeks. Analysis of sera from the Immunized mice by Indirect ELISA showed that citrinin specific antibody was formed to the Immunogen. Spleen cells from the Immunized mice were fused to FOX-NY myeloma cells In the presence of polyethylene glycol. Actively growing hybridomas were expanded and screened by ELISA. Hybridomas positive for the production of antl-citrinin antibody were cloned by a limiting dilution technique and the clones then expanded and rescreened by ELISA. Positive clones were expanded and stored In liquid nitrogen as appropriate. It Is anticipated that a monoclonal antibody developed by this method may be used In the rapid screening of agricultural commodities or In diagnosis of animals suspected to be suffering from citrinin toxicosis.

A retinol-binding protein (RBP) synthesized and secreted by bovine and ovine allantois in vitro was purified from culture medium. The protein consisted of three isoelectric variants (pI 5.3-6.1) of identical molecular weight of about 23,000 as determined by two-dimensional polyacrylamide gel electrophoresis. Thirty-one of the first 34 N-terminal amino acids of the purified protein from both sources were sequenced and shown to have complete homology with bovine plasma RBP. The UV absorption spectrum and fluorescence excitation and emission spectra of the purified bovine and ovine placental RBP indicated the presence of bound retinol. Rabbit antiserum was raised against purified bovine placental RBP. Metabolic labelling, immunoprecipitation studies demonstrated that the protein was synthesized by bovine and ovine extraembryonic membranes (allantois, chorion and amnion). The protein was also detected in allantoic and amniotic fluids by immunoblotting. With immunocytochemical procedures, the placental RBP was immunolocalized in trophectodermal cells of the chorion, endodermal cells lining the allantois and ectodermal cells lining the amnion. Results from this study suggest that bovine and ovine placental membrane epithelia synthesize and secrete REP. Transport, storage, and metabolism of retinol mediated by placental REP may be essential for normal embryonic development during pregnancy.

Bovine endometrial glandular epithelial and stromal cells were isolated by enzymatic digestion from uteri of cows harvested on days 15 - 17 of the estrous cycle. Cell types were separated by slow speed centrifugation and grown a on collagen matrix. In a separate study, isolated gland cells were grown on millicell inserts coated with Matrigel and cultured in the presence of stromal cells. Response of isolated cells and co-cultured cells to bovine serum albumin (BSA), recombinant bovine trophoblast protein-1 (rbTP-1), recombinant bovine interferon (brIFN) and oxytocin were analyzed by measuring prostaglandin (FSα) (PGF) and E2 (PGE) and protein secretion. Regulation of gene expression of prostaglandin synthase (PGS) in each cell type was also evaluated. Gland cells treated with rbTP-1 and brIFN produced less (p < 0.01) PGF (ng/μg DNA) than BSA treated cells. However, PGE was unaffected (p > 0.05) by rbTP-1 or brIFN. Oxytocin stimulated glandular cells to release significantly (p < 0.01) higher concentrations of prostaglandin F and E (p < 0.01). Recombinant bTP-1 and brIFN were ineffective in reducing (p > 0.05) oxytocin stimulated PGF secretion by gland cells. Isolated stromal cells released HGF secretion by gland cells. Isolated stromal cells released PGF in levels below the detection limits of the assay. Stromal cell secretion of PGE was unaffected by rbTP-1 and brIFN. Oxytocin reduced (P < 0.01) PGE released by stromal cells when compared to cells cultured in the absence of oxytocin. Proteins synthesized and released in the medium were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. Percent incorporation of 35S methionine into radiolabeled proteins by isolated stromal cells was significantly (p < 0.01) higher than gland cells regardless of treatment. Patterns of proteins produced by glandular cells were different when compared to stromal cells. Glandular cells produced twenty unique proteins which were absent on fluorograms obtained from stromal cells. Gland cells co-cultured with stromal cells released significantly (p < 0.01) higher concentrations of PGF (ng/μg DNA) than gland cells cultured in the absence of stromal cells. A significant (p < 0.01) reduction in PGF release occurred when co-cultured cells were incubated in the presence of rbTP-1 and brIFN. Only brIFN reduced (p < 0.01) PGE release by co-cultured cells. Secretion of PGF and PGE by co-cultured cells was unaffected (p > 0.05) by oxytocin treatment. Percent incorporation of radiolabeled amino acids into proteins were not affected by any treatment. However, protein secretion was significantly reduced as a result of glandular-stromal interactions. Differences were present in the array of proteins produced by co-cultured cells and gland cells cultured alone. Cocultured cells produced an acidic protein (45 KDa, pI 5.5-6.8) which was absent on fluorograms from gland cells cultured alone. Total RNA from glandular and stromal cells was isolated and subjected to Northern and slot blot analysis. Glandular cells contained a major PGS mRNA transcript of ≈ 2.8 Kb. Expression of PGS mRNA in stromal cells was evident but barely detectable. After correcting for total RNA loading by comparison with rat β-actin, quantitative slot blot analysis demonstrated no significant difference between treatments by either cell type. These results indicate that the major effect of the trophoblast interferon is on PGF production by uterine glandular epithelial cells. Oxytocin stimulated prostaglandin production by gland cells and the IFN’s diminished this response in some not all cases. Patterns of proteins synthesized by isolated glandular epithelial and stromal cells were distinct. However, there was no effect of the IFN’s on protein synthesis by either cell type. These data do not support the concept that trophoblast interferon modulates PGF production through alteration of PGS expression.

Collagen is by far the most prevalent vertebrate protein as it constitutes approximately 30% of all proteins present in mammals. In fibrous or solid forms, the various collagens junction primarily as the principal supporting elements in a wide variety of connective tissues. Nevertheless, more recent researches strongly suggesting that collagen may play active roles in developmental processes, cell attachment, differentiation, growth and migration. In this dissertation, a major placental protein, designated 2B9, was identified as carboxyl-terminal propeptide of bovine collagen III during early embryonic development. cDNA clone of this protein was obtained and characterized. Developmental gene expression of this protein was studied in both bovine and ovine extraembryonic tissues. Results indicated that cDNA clone of 2B9 protein contained the entire 245 amino acid coding region for the carboxyl-terminal propeptide of bovine collagen III as well as the carboxyl telopeptide region and 3' untranslated region extension. On set of expression of bovine procollagen III mRNA was paralleled by the protein production, suggesting procollagen III regulation might be under transcriptional control. Coincidental expression of the procollagen III gene with development of bovine chorioallantois suggested an important role for collagen III in the early embryonic development. Bovine procollagen III gene contained multiple polyadenylation signals, differential usage of these signals might be a characteristic of collagen genes during development.

Vitamin A (retinol) and its natural metabolites, isoforms of retinoic acid, are collectively known as retinoids and are important signaling molecules in vertebrate development and (differentiation. A growing body of evidence suggests retinoids play an important role in reproduction. Systemic and intercellular transport of retinol is mediated by retinol binding protein (RBP). The cellular binding proteins, cellular retinol binding proteins (CRBPs) and cellular retinoic acid binding proteins (CRABPs), participate in retinol storage and metabolism. The actions of retinoids are mediated through nuclear retinoic acid receptors which belong to the family of nuclear ligand inducible transcription factors. The goals of this study were to localize the retinoid binding proteins in the oviduct and ovary and examine the effect of retinoids on oocyte maturation/competence and early embryonic development. Retinol binding protein was immunolocalized to the luminal epithelia of oviductal mucosa of both the ampulla and isthmus. Retinol binding protein expression was considerably higher on day 1 than days 5 or 10 of the estrous cycle. Synthesis, secretion and gene expression of RBP was modulated by ovarian steroids. Within the ovary, RBP was localized IV primarily in the thecal cells of non atretic follicles with some diffuse staining in the granulosa cells and stromal cells. Retinol binding protein staining was also present in the cytoplasm of oocytes from some antral but not pre-antral follicles. Cellular retinol binding protein was localized in thecal cells of non atretic follicles with diffuse staining in the stromal layer. Cellular retinol binding protein was observed in large cells of the corpus luteum while RBP was identified in both large and small luteal cells. Only CRABP exhibited intense staining in nuclei of oocytes from primordial follicles, but was not observed in oocytes or follicular cells of all other size follicles. Cellular retinoic acid binding protein was absent in all cells of the corpus luteum but was present in the stromal layer encapsulating it. In cattle, ovaries from normal cycling animals were analyzed for the presence of RBP and CRBP. Retinol binding protein mRNA was present in thecal but not granulosa cells of antral follicles. However, both thecal and granulosa cells synthesized RBP in vitro. No relative differences were observed in RBP mRNA concentrations or synthesis in luteal tissue between days 2, 6, 10 or 15. Retinol binding protein and CRBP were immunolocalized exclusively to large luteal cells. Experiments were conducted to identify effects of retinoid treatments on superovulated ewes upon subsequent in vitro embryonic development. Ewes were treated with retinoids or vehicle on the first and last day of FSH treatment and embryos surgically recovered and cultured in vitro until occurrence of blastocyst formation and hatching. Treatment of ewes with retinol during superovulation resulted in a dramatic increase in both blastocyst formation and embryonic hatching in comparison with retinoic acid, 9 cis retinoic acid or vehicle. Retinol treatment also significantly improved the number of embryos that progressed through the 8-cell in vitro block. The final experiment was conducted to evaluate the effects of the retinoids, retinol (100 µM and 10 µM ROH) and retinoic acid (10 µM and 1 µM RA) on the maturation, fertilization and subsequent development of cattle oocytes/embryos in vitro. Addition of 100 µM ROH or 10 µM RA to either maturation and fertilization or embryonic culture media resulted in no blastocyst formation or reduced blastocyst formation. The presence of retinoids in the lower concentrations during oocyte maturation either had no effect or diminished blastocyst formation. Embryos cultured in the presence of 10 µM ROH or 1 µM RA, regardless of maturation and fertilization treatment, exhibited increased blastocyst formation in comparison with culture medium alone. Collectively, these experiments identify retinoid VI binding proteins in the ovary and oviduct and provide evidence that retinoids may influence oocyte development/competence and early embryonic development. These results suggest that retinol has the potential to positively impact reproductive efficiency and assisted reproduction protocols in domestic animals.