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Previous work in the group identified a novel gene, designated myocyte stress 1 (ms1), which is up-regulated within 1 hour in the left ventricular following aortic banding in the rat, suggesting a possible role for ms1 in the initial signalling of the hypertrophic response. ms1 is also expressed during cardiac development and is transiently up-regulated during ischaemia-reperfusion in vitro. This suggests that ms1 may play a more widespread role in cardiac physiology.;The aim of the work in this thesis was to better understand the role of ms1 in cardiac physiology and disease through a combination of in vitro and in vivo approaches. It was demonstrated that transient over-expression of a c-Myc-ms1 fusion protein into a heart-derived rat cell line, H9c2, colocalised with actin and altered gene expression of known hypertrophic and cardioprotective markers that are target genes of the myocardin-related transcription factor (MRTF)/serum response factor (SRF) transcriptional pathway. The size of cells over-expressing ms1 significantly increased by 47% when compared to untransfected cells and over-expression of ms1 markedly inhibited staurosporine-induced apoptosis by 88%. A Cre/loxP system based construct was developed to assess the in vivo of increased ms1 expression and was confirmed to work in a cell-based system. However, two independent attempts to make a transgenic mouse over-expressing ms1 were unsuccessful despite successful integration of the transgene.;Overall the findings suggest that ms1 induces a hypertrophic response and provides cardioprotection via a MRTF-SRF signalling mechanism. The findings provide for the first time direct evidence of the involvement of ms1 in hypertrophy and cardioprotection.

Placental growth factor (PIGF) binds to VEGFR1 and is known to play a role in pathological angiogenesis, but its mechanism of action remains unclear. Endothelial cell migration in response to angiogenic stimuli requires coordination of adhesive function with VEGFR signalling, and I have studied the intracellular trafficking of integrins and VEGFRs in primary cultured human umbilical vein endothelial cells (HUVECs). VEGFR2 and alphavbeta3 integrin cycled rapidly between the plasma membrane and EEA1/Rab4-positive early endosomes, whereas VEGFR1 remained at the plasma membrane and was not subject to rounds of endo- exocytosis. Treatment of HUVECs with PIGF or VEGF-A promoted the rapid and coordinate mobilisation of both VEGFR2 and alphavbeta3 from these endosomes to the plasma membrane, via a mechanism that was developed on inactivation of GSK-3beta but did not require the activity of PKD1, nor the tyrosine kinase activity of VEGFRs. Furthermore, RNAi of Rab4a and pharmacological inhibition of alphav integrin signalling opposed PIGF-promoted endothelial cell vessel branching and cross-bridge formation in an organotypic tube formation assay.;Taken together these data show that PIGF can influence endothelial cell function by controlling the endocytic recycling of alphavbeta3 integrin. This recycling pathway is required to induce vessel branching and the formation of a complex endothelial cell vessel network. In addition, regulation of VEGFR2 recycling by VEGFR1 represents a novel mechanism for mediating receptor cross talk during angiogenesis. Therefore, I have identified a novel pathway by which PIGF/VEGFR1 is able to positively influence the angiogenic response.

The initial aims of this thesis were to investigate whether the myocardium contains resident progenitor cells that contribute to myocardial renewal and whether extra-cardiac bone marrow derived cells contribute to myocardial regeneration. I reveal that the myocardium has the capacity to produce humoral factors that enable extra-cardiac progenitors to survive in vitro though this was insufficient to induce cardiac differentiation. I have shown that the myocardium has the capacity to produce a heterogeneous population of cells in vitro, some of which express cardiac related markers but do not adopt a full cardiac phenotype. When these cells are transplanted into a normal or injured heart they integrate into the myocardium but fail to develop a full mature functional cardiac phenotype. I have set up the frame work for demonstrating and defining the qualitative histological structure of the myocardium using lineage tracing techniques and strengthening the criteria for defining various cell lineages in the heart and therefore demonstrated the deficiencies of seminal studies that claimed that adult stem cells had the capacity to differentiate into cardiomyocytes and secondly that cultured heart explants produce cardiac progenitors. From this work it is clear that more needs to be done to identify the various cell lineages and roles of endogenous cardiac cells. The identification of clusters of perivascular cells expressing cardiac markers using 3 dimensional confocal imaging by two photon molecular excitation provided a different approach for identifying putative cardiac progenitors. This in combination with lineage tracing techniques and cell isolation is now required to identify the role of these interesting perivascular cells in cardiac homeostasis.

Autonomically-mediated baroreflex control of heart rate has been extensively studied (cardiac BRS, cBRS), but peripheral control of blood pressure is less well known. Total peripheral resistance (TPR) was derived from pulse contour stroke volume (SVPC) using non-invasive blood pressure (Finapres). The beat-to-beat variability of Finapres SVPC was evaluated in cardiac catheterisation patients against aortic blood pressure SVPC. Correlations were generally good (mean R = 0.75), but regression slopes tended to be less than unity and several aortic recordings were significantly affected by the dynamic response of the measurement system. Finapres SVPC was also compared to Doppler stroke distance (SD) in healthy volunteers. Both measures followed respiratory movements well, although SVPC had higher coherence with respiration. Discrepancies between the results were considered to be mainly due to errors in the Doppler method. Coherence thresholds for spectral cBRS were determined as a function of the number of subrecords available for ensemble averaging and the effect of ventricular ectopics on cBRS estimates was investigated. Pulse contour TPR data was then used to determine peripheral BRS (pBRS) in healthy controls and neurocardiogenic syncope patients (NCS) using methods adapted from cardiac BRS analysis. Diastolic pressure produced greater pBRS estimates than systolic pressure and pBRS was generally higher in patients and fainters than in controls and non-fainters. Tilt did not have a consistent effect on pBRS and it was not linearly related to age or resting blood pressure, although it may be increased in hypertension and the elderly. pBRS was able to discriminate between the subject groups when cBRS methods showed no difference.

Endothelial progenitor cells (EPC) are found circulating in adults and preferentially incorporate into growing vessels at sites of active angiogenesis. The mechanisms for their homing to angiogenic vessels, factors that regulate their differentiation into an endothelial-like phenotype and extent to which these cells become endothelial are not known. Understanding these mechanisms would be valuable for therapeutically directing vascularisation, especially where normal compensatory angiogenic response is lacking or impaired. In this study, we have examined the possibility that ex vivo expanded EPC migration and adhesion could be regulated by Angiopoietic-1 (Ang1) and -2 (Ang2), a soluble ligand expressed by endothelial cells at sites of vessel remodelling and angiogenesis and Vascular Endothelial Growth Factor (VEGF). We show VEGF and Ang1 stimulate EPC migration and show for the first time the same effect is seen with Ang2. This was specific for EPC as the ligand failed to affect endothelial cell migration. Ang2 stimulated EPC migration was inhibited by soluble Tie2 ectodomain. Furthermore, these ligands were found to stimulate adhesion between EPC and endothelial monolayers using cell:cell adhesion assays. The Angiopoietin stimulated increase EPC:endothelial cell interaction was again inhibited by the soluble Tie2 ectodomain. The roles of these soluble mediators and adhesion in signalling differentiation were defined by examining EPC marker expression. These results showed a phenotypic overlap between EPC and monocytes and allowed definition of a moncyte sub-population with endothelial characteristics.

Two tag-based gene expression technologies (serial analysis of gene expression, SAGE, and massively parallel signature sequencing, MPSS) were used to profile a variety of lymphocyte transcriptomes. By combining these libraries with publicly available genome and transcriptome data, both immunological and general aspects of gene expression could be considered. Unexpectedly, analysis of the expression of currently known cell surface components and the proteins corresponding to "immune specific" tags in a cytotoxic T-cell (CTL) library suggested that the current knowledge of the immune specific composition of the cell surface components of a resting CTL is largely complete. An analysis of the "immune specific" tags in a natural killer cell library revealed that a small number of tags could not be matched to any previously sequenced transcripts, suggesting the presence of functionally important but previously uncharacterised transcripts or exons in these cells. To examine the entire transcriptome large libraries are required, implying that MPSS would be the most appropriate technology. A comparison of libraries produced by the two tag-based technologies to characterise CD4⁺ T-cells revealed a relatively poor correlation, suggesting bias in the two techniques. Investigations into this bias led to the conclusion that despite its great depth, the random sampling events involved in the production of a library limit the breadth of MPSS sampling, making it inappropriate for characterising entire transcriptomes. Finally, with the availability of large LongSAGE libraries it is now possible to examine weakly expressed transcript classes. A panel of LongSAGE libraries was used to conduct the first large scale quantitative study of the expression of cis-natural antisense transcripts (cis-NATs). cis-NATs were found to be expressed at approximately one tenth of the level of sense transcripts and across the panel of libraries cis-NATs were found for approximately two thirds of all observed sense transcripts. This suggests antisense transcription is more widespread than previously thought.

This thesis asks two questions. First, is there an efferent control system of the arterial chemoreceptors other than by control of the vasculature? Secondly, do single chemoreceptors show an oscillation in their discharge with respiration and, if so, what is the natural history of the oscillations? The general methods for the recording of the discharge of single chemoreceptor fibres of the sinus nerve of the cat are described. Specific experimental details are described in the appropriate chapters. <strong>Efferent control</strong> Microelectrodes were used to record intracellular potentials from the carotid body in vitro and in vivo in an attempt to link efferent inhibition arising from electrical stimulation of the sinus nerve with changes in the membrane potentials of the cells. 176 cells were recorded from in vitro and 24 cells in vivo. These were probably Type I cells. Membrane potentials ranged from 15-60 mV with a mean of about 30 mV. No 'physiological' or pharmacological stimulus that was used affected these potentials. Potassium chloride solution depolarised the cells reversibly. Stimulation of the sinus nerve at intensities sufficient for maximal excitation of the 'C' fibres within it had no effect on the recorded potentials. Neither transient junctional-type potentials nor slow drifts of potential were seen. In parallel experiments the sinus nerve was stimulated electrically whilst recording the discharge in single chemoreceptor fibres peeled from the nerve distal to the stimulating electrodes. Stimulation could cause adventitious excitation of the recorded afferent resulting in antidromic depression, which mimicked a true physiological inhibition of the discharge. Some of the features of this adventitious excitation and the ease with which it occurred were investigated using an isolated segment of vagus, baroreceptor fibres, and chemoreceptor fibres. Low concentrations of local anaesthetic could raise the threshold for adventitious excitation whilst not affecting the normal passage of impulses. When looking for true inhibition, a continuous check for adventitious excitation was kept, utilizing the fact that chemoreceptors show a minimum interspike interval of about 10 msec. With this check, stimulation of the sinus nerve still caused a reduction in discharge in afferent fibres, to about 70% of control, though this was rather variable. In four out of five fibres tested, the inhibition was reduced or abolished by close intra-arterial injection of atropine. In conclusion: the true inhibition of discharge would seem not to be synaptic but vasomotor in origin. <strong>Oscillations in discharge</strong> Most single fibres of the cat and dog showed oscillations in discharge in phase with respiration when summed by computer over many respiratory cycles. The trough in the oscillation followed inspiration with a delay equal to the lung to carotid body circulation time. The amplitude of these oscillations was increased by decreasing respiratory frequencies or by increasing metabolism by poisoning with dinitrophenol. Hypercapnia or hypoxia decreased the relative amplitude of the oscillations but the absolute amplitude remained approximately constant. Oscillations from pairs of fibres recorded simultaneously were similar in phase and relative amplitude. The oscillations were not affected by sectioning or stimulating the cervical sympathetic trunk, nor by sectioning the sinus nerve whilst recording from an afferent from the otherwise intact nerve. The most likely source of the oscillation is thought to be the respiratory oscillation of P_a,CO₂ in the carotid blood. It is proposed that the responses can be explained in terms of families of parallel transient P_a,CO₂-response curves that are steeper than the fans of response curves described for the steady state by other workers and cut these curves at the mean P_a,CO₂. A non-sympathetic efferent fibre that was recorded from showed no oscillation in the discharge with respiration, and interval distribution histograms of its discharge did not have the simple exponential form shown by afferent discharge. Earlier workers had described the discharge of single chemoreceptors as random and this was seemingly at variance with an oscillating discharge. In an attempt to resolve this it was assumed that the mean frequency of the random process varied over the respiratory cycle, and statistical tests showed that, with this assumption, the impulses were random with respect to each other although they were not random with respect to the respiratory cycle. That is, the probability of the occurrence of an impulse varies with a respiratory periodicity but not with the time since the occurrence of the previous impulse. The technique of recording from two fibres simultaneously on separate electrodes was originally used to see if it would be justifiable to apply the results of summing the discharge of one fibre over many cycles to the response of the whole nerve, i.e. many fibres over one cycle. This recording method allowed farther comparisons between fibres, when the arterial stimulus must have been the same for both receptors, by going back over the experimental records and comparing the frequencies of discharge of the pair of fibres at any time. In some experiments specific manoeuvres were performed to obtain further information. The frequencies of discharge of fibres were compared: at steady mean rates; during the responses to various ventilatory stimuli; at the intra-arterial injection of 100% CO₂-saline; as well as in their oscillatory behaviour over the respiratory cycle. If allowances were made for differences in characteristic mean frequencies of discharge, the sensitivities to any change of arterial stimulus proved remarkably similar, that is the discharge of one receptor could give as much information as that of the whole population. There were indications that receptors might differ in threshold, peak response, and adaptation. The most striking finding was that the sensitivity of a receptor could change with time, although, at any one moment, the chemoreceptors are a surprisingly homogeneous population. The results suggest that there is a single mechanism responsible for the sensitivity to hypoxia and hypercapnia.