Atherosclerosis is thought to be initiated by endothelial cell dysfunction. Research described in this dissertation is focused on interactions of nutrients, cytokines and pharmaceutical compounds in the intracellular signaling pathways leading to endothelial cell activation. The flavonoid quercetin could significantly downregulate the inflammatory pathways induced by linoleic acid as determined by DNA binding assays of the proinflammatory transcription factors nuclear factor-kappaB and activator protein-1 as well as by gene expression studies of interleukin-6 and vascular adhesion molecule-1. Interestingly, quercetin and vitamin E also prevented the linoleic acid-induced activation of PPAR DNA binding - suggesting a role of oxidation in the fatty acid-mediated induction of PPAR. In addition, we studied an interaction of zinc with the antiinflammatory transcription factors, peroxisome proliferator activated receptors (PPARs) alpha and gamma. Our data suggest that PPAR alpha and gamma and their synthetic agonists require zinc for their antiinflammatory properties in endothelial cells. We could confirm the importance of zinc in PPAR gamma signaling in vivo by a decreased PPAR DNA binding activity in livers of zinc deficient mice. Furthermore, zinc had dramatic lipid lowering effects in LDL-receptor deficient mice on a diet rich in corn oil. Triglycerides, phospholipids and cholesterol levels were significantly elevated in mice receiving a zinc deficient diet when compared to control and where decreased in zinc supplemented animals. Zinc deficiency also increased oxidative stress as determined by quantitation of plasma isoprostanes and mRNA expression of glutathione reductase. In conclusion, our data show novel interactions of proinflammatory nutrients, such as linoleic acid, with antioxidant and anti-inflammatory nutrients, such as quercetin and zinc.

The main concern of my thesis is to show that sexuality, and in particular the motifs of bisexuality and sexual violence, which were generally mere modish accessories in the Austrian literature of the fin-de-siecle, have a crucial, constitutive function in the works of these two authors. It seeks thereby to redress the prevalent view of the sexual thematic in both writers as a marginal curiosity rather than as fundamental to the economy of their, in other respects, very different oeuvres. There are striking affinities; in either case a narcissistic ideal of androgynous union with the sister is set up, only to be revoked by the counter-motif of incestuous 'Lustmord'. The problem of sexual antagonism is exploited by both writers as symbolic ground for the exploration of the crisis in the sense of self. At the same time it determines the discursive structures of the works, as sensuality subverts both the characters' quest for a 'common sense' in language, and their creators' pursuit of a more authentic aesthetic discourse and form. The section on Trakl concentrates on the corruption of semiotic patterns in the poetry by sexual desire. The signs are conceived as metatexts (images of the text within the text); their disarray, and the resultant disorientation of 'readers' within the poetic landscape, at once allegorises the predicament of the poet as seer and the reader's quest for a sense of direction in the text. My discussion of the problematics of sexuality, discourse and the self will turn on the crucial motif of the mirror. The mirror coordinates all three of these themes, maintaining a multiple function as medium for narcissistic desire, as metaphor for poetic creativity, and as the space inhabited by the 'Doppelg&aacuteger'. It is also identified as a corollary of the narcissistic sister-figure; the disfigurement which both are made to suffer in the later poetry is seen as symptomatic of a more general threat to the poet's figurative enterprise. The section on Musil also centres on the twin motifs of the mirror and the double. Here the interrelation of psycho-sexuality and problems of discourse is shown to be related to theories of the psyche contemporary with Musil's work (Weininger,Freud and Rank). In its sophistication it is also seen to anticipate certain post-Freudian developments in psychoanalytic theory, with key ideas from the thought of Lacan being applied in the exegesis of the texts. In <i>Tóeβ</i> the protagonist's sexual and epistemological crises are shown to be inextricably meshed, indicating a far closer affinity with Freud's early work than critics have been willing to recognise. In the <i>Vereinigungen</i> novellas the emphasis is on the correlation between the deferral of desire and the failure of dialogue between the sexes, focusing on the partitive intervention of the 'Vorwand'. There follows an analysis of key chapters from <i>Der Mann ohne Eigenschaften</i> where the problematic association of sexual identity, discursive integrity, and the sense of self, is most acutely in evidence. My argument culminates in the proposition that the sense/sensuality dichotomy is fundamental to the problem of finding a true end for the text.

Background: Myelination is an exquisite and dynamic example of heterologous cell-cell interaction, which consists of the concentric wrapping of multiple layers of oligodendrocyte membrane around neuronal axons. Understanding the mechanism by which oligodendrocytes ensheath axons may bring us closer to designing strategies to promote remyelination in demyelinating diseases. The main aim of this study was to follow glial-axonal interactions over time both in vitro and ex vivo to visualise the various stages of myelination. Methodology/Principal findings: Two approaches have been taken to follow myelination over time i) time-lapse imaging of mixed CNS myelinating cultures generated from mouse spinal cord to which exogenous GFP-labelled murine cells were added, and ii) ex vivo imaging of the spinal cord of shiverer (Mbp mutant) mice, transplanted with GFP-labelled murine neurospheres. The data demonstrate that oligodendrocyte-axonal interactions are dynamic events with continuous retraction and extension of oligodendroglial processes. Using cytoplasmic and membrane-GFP labelled cells to examine different components of the myelin-like sheath, evidence from time-lapse fluorescence microscopy and confocal microscopy suggest that the oligodendrocytes’ cytoplasm-filled processes initially spiral around the axon in a corkscrew-like manner. This is followed subsequently by focal expansion of the corkscrew process to form short cuffs which then extend longitudinally along the axons. From this model it is predicted that these spiral cuffs must extend over each other first before extending to form internodes of myelin. Conclusion: These experiments show the feasibility of visualising the dynamics of glial-axonal interaction during myelination over time. Moreover, these approaches complement each other with the in vitro approach allowing visualisation of an entire internodal length of myelin and the ex vivo approach validating the in vitro data.

Since the introduction of glucocorticoids (GCs) in the treatment of rheumatoid arthritis in 1949, GC therapy has been associated with a number of adverse effects. Long-term use of GCs can result in growth retardation during childhood due to their actions on growth plate chondrocytes, although the exact mechanisms involved are unclear. The work of this thesis has investigated the cellular and molecular mechanisms involved in mediating GC effects at the growth plate. Affymetrix microarray has been used to identify and characterise the expression of lipocalin 2, a novel GC-responsive chondrocyte gene which may contribute to GC-induced growth retardation in the growth plate. In vitro and in vivo studies have also been used to examine the role of the cell cycle regulator, p21WAF1/CIP1 in GC-induced growth retardation. Finally, the growth plate sparing effects of a novel GC receptor modulator, AL-438, have also been identified. AL438, has reduced effects on bone growth compared to Dex, but maintains similar anti-inflammatory efficacy. This work has not only determined novel mechanisms of GC-induced growth retardation, but has also advanced the search for novel GC receptor modulators with reduced adverse effects.

There are few in depth attempts to address the question: why teach ethics to medical students? This thesis argues that, identifying moral growth and development as the primary goal in teaching medical ethics is essential. Lawrence Kohlberg's moral development theory is the starting point for this research. This is important to understand the work of the researcher at the Medial School in Kuwait. The instrument used in assessing the moral reasoning of medical students at Kuwait University is the Defining Issues Test (DIT), which was devised at the University of Minnesota. The study hypothesis is that the rigid, authoritarian medical education in Kuwait University that lacks any emphasis on medical ethics will inhibit the expected growth in moral development of medical students. With a disappointing response rate of only 27.8%, it was found that normally expected growth did not occur in the first four years of medical education, suggesting that the educational experience somehow inhibited student's moral reasoning ability rather than facilitating it. The results of this study cannot be understood in isolation from the general understanding of the fabric of the researcher society, which was detailed in Chapter Five. The implication of this study is basically that medical education that ignores the moral nature of medicine will fail its own purpose, the needs of its students and the welfare of society.

Insulin stimulates glucose transport in fat cells by inducing the movement of glucose transporters (Glucose transporter-4) from specialised storage vesicles to the plasma membrane. Insulin resistant individuals and those with Type II Diabetes exhibit impairment in the ability of insulin to stimulate glucose transport. The molecular mechanisms of glucose transporter-4 trafficking in adipocytes are an important focus in understanding the underlying etiology of this disease. Glucose transporter-4 (GLUT4) recycles between the plasma membrane and intracellular stores in the absence of insulin using a complex intracellular pathway. This involves two intracellular cycles: one is the prototypical endosomal system, the other a specialised cycle involving the trans-Golgi network and a sub-set of intracellular vesicles called GSVs (the slow cycle). Understanding the control of the entry into this second cycle is the subject of this thesis. In particular, the work in this thesis will examine the role of Syntaxin 16 and its cognate Sec1/Munc18 protein mammalian Vps45 (mVps45). The regulation of Syntaxin 16 has not been fully elucidated and understanding the role of Syntaxin 16 in SNARE complex regulation and subsequent control of GLUT4 traffic into the slow cycle requires an understanding of its cognate binding partner Sec1/Munc18 (SM) protein, mammalian Vps45 (mVps45). The absolute levels of both Syntaxin 16 and mVps45 were quantified and found to be present in 3T3-L1 adipocytes in roughly stoichiomeric amounts. IP experiments also showed the ability of mVps45 to interact with Syntaxin 16 in the absence of insulin. Using the model eukaryote Saccharomyces cerevisiae, we found that mVps45 could complement for the deletion of Vps45p. Assays for CPY secretion showed that mVps45 is able to complement for the loss of Vps45p function in the trafficking of carboxypeptidase Y (CPY). Additionally, mVps45 mutants were made that correspond to yeast mutants made previously in the lab and were tested for homology of function. Depleting 3T3-L1 adipocytes of mVps45 showed alterations in the levels of GLUT4 protein as well as the protein levels of Syntaxin 16, IRAP, and Rabenosyn. Insulin-stimulated deoxyglucose uptake was also profoundly decreased upon depletion of mVps45. Further experiments using mVps45 depleted cells show that these cells lose their sensitivity to insulin and that the loss of mVps45 in these cells causes GLUT4 to have the inability to enter the slow cycle. Taken together, these findings demonstrate that mVps45 has a role in allowing GLUT4 entry into the slow cycle.

In Scotland, stroke is the third most common cause of death behind heart disease and cancer. However, most strokes are not fatal and can cause severe disability, with one third of survivors still functionally dependent after one year. The advent of recombinant tissue plasminogen activator (rT-PA) as a thrombolytic modality revolutionised the treatment for ischaemic stroke, providing a treatment aimed to promptly restore nutritional blood flow to the ischaemic penumbra, a transient tissue state which is amenable to salvage. Crucially, patient ineligibility from a multitude of factors (including the narrow time window for benefit and the risk of intracranial haemorrhage) means that fewer than 10% of all stroke patients are thrombolysed. Positive identification of penumbra is not employed in the current intravenous rT-PA administration strategy, which is instead based on two main prerequisites: stroke patients in whom intracerebral haemorrhage has been excluded with non-contrast computed tomography (CT) and who also present within 4.5 hours of symptom onset. The technical impracticalities and limited availability of the gold standard penumbral imaging modality, multitracer 15O positron emission tomography (PET), and the lack of standardised thresholds to identify penumbra using non-contrast CT have hindered the development and inclusion of routine brain imaging in the management of acute stroke patients. An alternative research tool which may potentially be used in clinical practice is magnetic resonance imaging (MRI) which defines penumbra on the basis of diffusion-perfusion (DWI/PWI) mismatch. However, this provides an imprecise measure of penumbra and fails to identify tissue viability. Current PET-derived definitions of penumbra use metabolic indices such as oxygen extraction fraction (OEF) and the cerebral metabolic rate of oxygen (CMRO2), which are not fully incorporated into MR definitions. This thesis presents an alternative MRI method for identifying the metabolic penumbra in a rodent model of focal cerebral ischaemia. This utilises an MRI sequence similar to that used in functional MRI (fMRI) techniques, and uses 100% oxygen inhalation as a biotracer to detect penumbral tissue. Specifically, by using a blood oxygen level dependent (BOLD) T2*-weighted sequence in which changes in the deoxyhaemoglobin:oxyhaemoglobin ratio are detected - in conjunction with a transient hyperoxic challenge (Oxygen Challenge (OC) paradigm: 5 minutes breathing air followed by 5 minutes breathing 100% oxygen) - penumbral tissue can be distinguished from adjacent ischaemic core and benign oligaemia (Santosh et al, 2008). Changes in CBF, cerebral blood volume (CBV), tissue oxygenation, and oxidative metabolism can all influence the T2* signal (Ramsay et al, 1993; Corfield et al, 2001), so it was important to evaluate the possibility that factors other than tissue metabolism were influencing the signal change during OC. An initial study was performed which showed that baseline CBF did not influence T2* signal response to OC, whilst a greater increase in the percentage change in arterial oxygen saturation following OC caused an increased magnitude in T2* percentage signal change in contralateral tissue and penumbra, but not in ischaemic core. Arterial oxygen levels (PaO2) affect the magnitude of the T2* signal change to OC, with lower baseline PaO2 levels amplifying the T2* signal response in metabolically active regions, implying that careful control of physiological variables may optimise the T2*OC technique. The first validation study used [14C] 2-deoxyglucose autoradiography to determine the metabolic status of penumbra defined by T2*OC MRI. The results confirmed that glucose metabolism in the T2*OC-defined penumbra was comparable to contralateral values, whereas markedly different levels of glucose metabolism were evident in the ADC-derived ischaemic core and an adjacent region of increased 2DG phosphorylation. From this, it was concluded that metabolic information could be yielded from the ischaemic brain that may improve delineation of the penumbra using the OC technique. As penumbral tissue must fulfil the fundamental criteria of being potentially salvageable and responsive to therapy, the consequences of reperfusion on the T2*OC-defined penumbra was tested. This study confirmed that T2*OC-defined penumbra displayed a T2* signal change significantly higher than contralateral tissue during ischaemia which subsequently returned to contralateral levels following reperfusion and did not progress to infarction when assessed at day 7 following stroke. Finally, the spatiotemporal characteristics of the T2*OC-defined penumbra were investigated and compared with DWI/PWI mismatch-defined penumbra. Serial scanning demonstrated that T2*OC penumbra behaved in a similar manner to tissue defined by traditional mismatch criterion. The spatial location and tissue volumes of penumbra were similar with both methods, showing that, in animals where mismatch tissue volume reduced over time, T2*OC penumbra reduced similarly, and in animals where mismatch volume remained static over time, T2*OC-defined penumbra behaved similarly. Additionally, an interesting finding arose in the latter study which showed that ischaemic damage continues to progress beyond 4 hours following permanent MCAO, which may be relevant to the calculation of ADC and CBF thresholds used in defining DWI/PWI mismatch. Collectively, the preclinical data support the potential of T2*OC to discriminate tissue compartments in acute stroke based on metabolic status which thereby provides an alternative and improved means of defining the ischaemic penumbra.

VEZF1 is a highly conserved transcription factor that is restricted to vertebrates. A chicken homologue of this protein, BGP1 has been recently identified to function at the HS4 insulator element demarcating the 5' boundary of the chicken β-globin domain. BGP1 binding sites are required for chromatin barrier activity and are associated with protection from de novo DNA methylation. Gene targeting experiments in mice have also revealed crucial roles for Vezf1 in vascular and lymphatic development. Work described in my thesis utilises a ChIP-chip approach to map VEZF1 binding sites across 1% of the human genome, known as the ENCODE regions, in K562 erythroid cells. It was found that VEZF1 preferentially targets gene regulatory elements and promoters of actively transcribed genes in particular. A significant proportion of intergenic sites were identified at the boundary regions between active and repressive chromatin. This indicates that these may harbour insulator activities akin to HS4. In addition, a subset of binding sites were found to bind VEZF1 in tissue specific manner being restricted to haemopoietic/ erythroid lineages. Depletion of VEZF1 levels in K562 cells results in specific downregulation of alpha- and beta-globin gene RNA levels, whilst expression of more than 20 other gene targets bound by VEZF1 remains unchanged. Findings presented in this thesis provide a global view of VEZF1 mediated functions in the genome and highlight its potential roles in regulating endothelial and haemopoietic gene expression.

Decision analytic modelling (DAM) is a mathematical technique which is used to structure and synthesise evidence in order to inform decision making, given uncertainty. Decision models are an ideal tool for undertaking economic evaluations as they enable a wide range of data on costs and effects to be synthesised within the model in order to derive cost-effectiveness outcomes. The iterative framework for economic appraisal has been proposed as good practice for undertaking economic evaluations (1), and DAM plays a key role within this framework. In particular there is a role for early stage DAM prior to primary research, to provide an indication of the potential cost-effectiveness of a new health technology (2) given current evidence, and the use of value of information (VOI) techniques to help inform further research priority setting. In practice, support and funding for early stage DAM and full exploitation of VOI techniques is rare. The aim of this thesis is to examine the role for early decision analytic modelling in informing research priorities and the design of future studies in a health care setting. This thesis explores the feasibility, merits and drawbacks of undertaking early DAM and considers potential reasons as to why it has not been more widely implemented. This thesis demonstrates the value and importance of early DAM; in both an ‘ideal’ setting and also in a less desirable, time-constrained setting. Applying early DAM and VOI techniques enables researchers to provide relevant conclusions and recommendations to decision makers, who can make informed decisions as to whether a new intervention should be adopted (or rejected), or whether further information is required to help make the decision; as opposed to making decisions based on subjective reasoning. There is considerable merit with employing early DAM for health care research, such as reduced uncertainty, reduction of costs and efficiency gains, however, some drawbacks exist in terms of whether it is always viable to fully exploit VOI analyses, which may hinder widespread support both inside and out-with the health economics community.

Conduction abnormalities affect prognosis in chronic heart failure (CHF). Previous investigators have observed abnormal delay in atrioventricular (AV) conduction in a rabbit model of left ventricular dysfunction (LVD) due to apical myocardial infarction. In this model, AV conduction time increased with increasing pacing rates, suggesting the most likely site of delay is the AV node. The mechanisms by which this occurs are not fully understood. The purpose of this thesis was to confirm that the abnormal prolongation of AV conduction time originates at the AV node in a rabbit model of LVD due to apical myocardial infarction, and explore possible mechanisms underlying the observation. Using surface electrogram recording and standardised pacing techniques in an isolated AV node tissue preparation I confirmed that there is abnormal prolongation of AV nodal conduction in this rabbit model of LVD, as evidenced by prolongation of atrio-hisian (AH) interval and Wenckebach cycle length (WCL) in LVD compared to control. Furthermore, using optical mapping of electrical activation using voltage sensitive dye I observed that the prolongation of the AH interval is predominantly a consequence of conduction delay between the inputs of the AV node and the compact nodal region. Neuro-hormonal derangement in chronic heart failure has a central role in the pathogenesis of the disease, with evidence of downregulation of beta ()-adrenoceptors in the left ventricular myocardium. I therefore explored the possibility of β-adrenoceptor downregulation in the AV node as a mechanism underlying the abnormal AH interval prolongation in LVD. There was no evidence of β-adrenoceptor downregulation in the AV node in LVD compared to control to account for the observed abnormal conduction delay. Adenosine is known to have profound effects on AV nodal conduction and the possibility of tonic excess of adenosine in LVD was explored as a possible mechanism for the prolonged conduction delay. Using an exogenously applied adenosine A1 receptor antagonist there was no evidence of excess endogenous adenosine in LVD compared to control. There was, however, an increase in the sensitivity of the LVD samples compared to control to exogenous adenosine, with a significant increase in AH interval and WCL with increasing concentrations. This thesis also investigates the effect of acidosis on AV nodal conduction. There was significant prolongation of the spontaneous sinus cycle length, AH interval and WCL, as well as the AV nodal functional and effective refractory periods, proportional to the degree of acidosis. These effects were reversible with return to normal pH. Optical mapping studies showed that the spatiotemporal pattern of AV nodal delay during acidosis was similar to that observed in LVD, with the predominant delay in conduction between the AV nodal inputs and the compact AV node. In summary this thesis has confirmed that even in the absence of a direct ischaemic insult to the AV junction, conduction abnormalities in the AV node may still occur as a pathophysiological response to a myocardial infarction resulting in LVD. The mechanisms underlying this response are likely to be complex and multiple, and are not yet clear. Establishing the electrophysiological basis and the effects of neuro-hormonal modulators of atrioventricular nodal function may lead to development of targeted therapeutic strategies to improve overall survival and improve symptom control for patients with CHF.