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No description available.

Previous work showed that mouse embryos homozygous for a null allele of the gene that encodes the glycolytic enzyme, glucose phosphate isomerase (GPI), died shortly after implantation. Although the homozygous GPI null embryos cannot produce their own GPI, because they lack the appropriate gene, they survived until 7.5 - 8.5 days (West <I>el al</I>, 1990) and some extraembryonic tissues survived until 10.5 days. A histological study was undertaken to determine when the first signs of abnormality become apparent in the dying homozygous GPI null embryos. The critical time period for these mutant embryos was found to be between 6.5 days and 7.5 days. This is after the oocyte coded GPI is exhausted so the embryo has to rely on it's own production of the enzyme. At this stage the embryo is implanting under relatively anaerobic conditions because the placenta has not yet formed. The mutant embryos fail to gastrulate properly and produce only a small amount of mesoderm. The abnormally developed egg cylinder expands to form an empty sac-like structure. The membrane that resembles the yolk sac is in fact comprised of extraembryonic ectoderm and extraembryonic endoderm. Aggregation chimeras were produced between homozygous GPI null embryos and normal embryos to examine whether homozygous GPI null cells could survive for longer when combined with normal cells. Because the homozygous GPI null embryos are embryo lethal, two heterozygotes were intercrossed to produce embryos, 25% of which should be homozygous for the null allele. All of the embryos produced from the intercrossing of the heterozygotes were aggregated to normal 8 - cell embryos.

A homogeneous population of myeloid blast cells was purified by negative selection from human fetal liver by enzyme digestion of liver tissue, ficoll fractionation, indirect erythrocyte rosette sedimentation of unwanted cells, after coating these cells with monoclonal antibodies directed against erythroblasts and macrophages, and finally by cell elutriation. Characterisation of these cells confirmed their undifferentiated status and in culture these cells generated only neutrophils and macrophages. Treatment of these cells with 10nM phorbol myristate acetate (PMA) induced rapid terminal monocyte differentiation of 62% of this population and also completely inhibited the neutrophil differentiation of the remaining cells. The undifferentiated blast cells were maintained in culture in a serum-free medium containing 100 U/ml interleukin-3 and 1mg/l inositol which permitted the equilibrium labelling of cells with [<SUP>3</SUP>H] myo-inositol and the subsequent analysis of concentrations of inositol metabolites within these cells. High concentrations of various inositol metabolites, similar to those found in HL60 cells, were observed in normal myeloid blast cells and following PMA-induced monocyte differentiation of these cells significant changes occurred - namely a decrease in inositol tetrakisphosphate (InsP<SUB>4</SUB>) and inositol pentakisphosphate (InsP<SUB>5</SUB>) and an increase in glycerophospho-inositol (GPI). These changes in response to PMA, with the exception of the rise in GPI, are similar to those reported in HL60 cells undergoing monocyte differentiation and suggest that abundant inositol polyphosphates may play an important role in myeloid differentiation.

Since the number of pre- and postimplantation human polyploid embryos available for analysis is very small and relatively few are available at any one centre, I have used appropriate experimental mouse models to facilitate a large scale investigation. One aim was to analyse the influence of ploidy on the cleavage rate of preimplantation embryos. A deviation from the optimum embryonic (diploid fertilized) cell number during this time might explain the poor development and failure of genetically abnormal embryos. Therefore, experimentally produced haploid, diploid, triploid and tetraploid embryos were analysed together with appropriate control embryos. In addition, the analysis of parthenogenetic embryos and diandric and digynic triploid and homozygous tetraploid embryos during this time would also enable the influence of the parental genomes on development to be investigated. I also wished to examine whether a predictable relationship existed between ploidy and cell size and number in the tissues of postimplantation mammalian polyploid embryos. Confirmation of such a relationship may explain the abnormal morphological features encountered in some of these embryos and the premature death of all triploid and tetraploid mouse embryos, and the majority of human embryos with similar genetic abnormalities. My results show that the duplication of one or both parental genomes leading to the triploid or tetraploid status, respectively, still allows apparently normal preimplantation development to occur. However, the loss of a haploid genome from the diploid status is invariably detrimental to normal development. My results confirm that the presence of a maternal genome is important for normal early embryogenesis since parthenogenetic diploid embryos and diandric and digynic triploid embryos developed as well as fertilised diploid embryos during the pre- and very early postimplantation period.

The secretory granules of the adrenal medulla, known as 'chromaffin granules', store catecholamines, proteins and nucleotides that afterwards will be secreted by exocytosis. The accumulation of the catecholamines within the granules occurs by a chemiosmotic mechanism involving a membrane-bound H<SUP>+</SUP>-translocating ATPase, coupled to a separate H<SUP>+</SUP>/catecholamine exchanger. This ATP belongs to a recently identified group of proteins called 'vacuolar' of V-type ATPases, that are distinct from the P-type H<SUP>+</SUP>-ATPases of fungalplasma membranes and F-type H<SUP>+4-ATPases of energy-transducing membranes. The aim of this thesis is the structural and functional characterization of the bovine chromaffin granule ATPase. First, a new and rapid method of purification and reconstitution of the enzyme was developed. The method can be accomplished in less than two hours, yielding a partially purified form of the enzyme incorporated into liposomes of defined composition. This permitted the measurement of the H</SUP>^+ -translocation activity of the enzyme by use of the fluorescent probe ACMA. The effect of lipid composition on ATP hydrolysis and H^+-translocationactivities was studied. Kinetic and regulatory studies were carried out on the reconstituted enzyme. MgATP, MgdATP, MgGTP and Mg/TP were all shown to be substrates for the enzyme and Mn^2+, Ca^2+, Co^2+ and Ni^2+ could substitute for Mg^2+. Kinetic parameters were calculated for all these cases. The inhibitory effects of ADP, GDP and IDP were also studied. The regulatory properties were consistent with the existence of several substrate and inhibitor binding sites and the kinetic data could be fitted to a model involving positive homotropic and negative heterotropic effects. Structural studies of the purified enzyme were performed. It was shown that one of the subunits (116 kDa) was a glycoprotein. A hydrophobic probe specifically labelled two subunits (116 and 16 kDa) suggesting that they form the part of the enzymic complex embedded in the granule membrane. The stoichiometry of the complex was calculated from quantitative aminoacid analysis of the subunits separated by electrophoresis in polyacrylamide gels. In order to do this a purer form of the enzyme was obtained by centrifugation through glycerol density gradients. This separated the active enzyme from a complex formed by polypeptides of molecular weights 116, 40, 19 and 16 kDa, suggesting that the enzyme contains two separable domains.

To study the genetic changes which confer resistance to oxidants, cell lines that are resistant to the redox-cycling agent, menadione, have been isolated from Chinese hamster ovary (CHO) and human transitional carcinoma (EJ) parental cell lines. They exhibit cross-resistance to chemical oxidants (hydrogen peroxide and sodium arsenite) but not to ionising radiation (in oxic conditions). The concentrations of the major sources of intracellular thiol groups, glutathione and cysteine, are two-fold greater in menadione-resistant than in the corresponding parental cell lines. Exposure to menadione results in depletion of both glutathione and cysteine but the subsequent recovery of thiols is more rapid and of greater magnitude in menadione-resistant than sensitive cell lines. <SUP>1</SUP>H spin echo nuclear magnetic resonance (NMR) spectroscopy was used to study intact cells. Using this technique the removal of menadione from suspensions of resistant and sensitive cells was observed. However, only in menadione-sensitive cells was concomitant depletion of the NMR-visible pool of glutathione observed. The acquisition of resistance to menadione was associated with significant changes in the expression of several enzymes that are implicated in the oxygen-induced stress response and in protection from redox-cycling agents. The transcription of genes encoding heme oxygenase and the glutathione-related enzymes, GST-Pi and glutathione peroxidase, increases in CHO parental cells after transient oxidative stress. These genes are constitutively induced in CHO menadione-resistant cell lines. This suggests that resistance results from perpetuation of a response that normally occurs only transiently.

A human gene, encoding a protein highly homologous to CYP2A7, has been isolated. Only thirteen base pair differences resulting in five amino acid changes were found. Genomic analysis demonstrated that the isolated gene is an allele of <I>CYP2A7</I>, designated <I>CYP2A7A</I>. The gene is about 8 kb long and contains 9 exons encoding a protein of 494 amino acids. The 0.5 kb 5' flanking region of <I>CYP2A7A</I> contained several putative promoter elements including a typical TATA box, a steroid regulatory element (SRE), and a HepG2-specific factor-1 binding sequence (HPF-1). The presence of HPF-1 motif was essential for the transcription of the gene in HepG2 cells. However, the function of SRE element is still unclear. In order to investigate the relationship between the expression levels of human <I>CYP2A</I> genes and the polymorphisms of coumarin hydroxylase activity in man, three cDNAs, <I>CYP2A6, CYP2A7</I> and an alternatively spliced version of <I>CYP2A7 (CYP2A7AS)</I> have been cloned. The later one was missing exon 2 but contained a 10 bp segment of intron 1. Translation of the <I>CYP2A7AS</I> resulted in an in frame deletion of 51 amino acids. The expression of these cDNA's in COS-7 cells showed that both CYP2A6 and CYP2A7 generated a protein <I>M</I>r 49 kDa, while the protein product of CYP2A7AS was about 44 kDa. Only the CYP2A6 had coumarin hydroxylase activity. The relative level of mRNA encoding CYP2A6 and CYP2A7 was established in six human liver samples with RT-PCR followed by PstI digestion and found to range between 1:0.5 to 1:3, respectively. These results showed that the relative level of CYP2A7 was variable.

Tissue Transglutaminase (TG2) and FXIIIa, members of the transglutaminase (TG) family, catalyses a transamidating reaction and form covalent bond between or within proteins. In bone development, both enzymes expressions correlate with the initial of the mineralisation process by osteoblasts and chondrocytes. Exogenous TG2 also promotes maturation of chondrocytes and mineralisation in pre-osteoblasts. To understand the role of endogenous TG2 in osteoblast mineralisation, the TG2 expression was examined during the human osteoblast (HOB) mineralisation. The expression of the endogenous TG2 increased during the mineralisation, yet, its expression was not essential for mineral deposition due to the compensation effect by other members in the TG family. The extracellular transamidating activity of HOBs was found increased during mineralisation and a shift from FXIIIa dominant- to TG2-dominant crosslinking activity was suggested after differentiation. However, the transamidating activity of both TG2 and FXIIIa were not critical for cell mineralisation. On the other hand, Exogenous TG2 was found to enhance wild type HOB and TG2 knockdown HOB mineral deposition. The transamidating activity of TG2 was not required but most likely a close conformation was essential for this enhancement. Results also demonstrated that exogenous TG2 may activate the ß-catenin pathway through LRP5 receptor thus contribute in cell mineralisation. This enhancement could be abolished by addition of ß-catenin inhibitors. Finally, using of TG2 crosslinked collagen gel for bone and cornea repair was evaluated. Crosslinked collagen gel showed promising results in improving HOB mineralisation, human corneal fibroblast (hCF) proliferation and migration. These effects might be resulted from the trapped TG2 within the collagen matrix and the alteration of matrix topography by TG2.

This PhD thesis contributes towards extending connectivity analyses and neurocomputational modelling of brain activities, and their applications. It starts with a brief review of different classes of neural models with different levels of realism. A main focus is the neural mass models (NMMs) that account for the collective behaviour of neuronal populations striking a balance between mathematical simplicity and biological plausibility, and hence suitable for large-scale neural circuit modelling. This is followed by a review on two popular large-scale neural connectivity frameworks, dynamic causal modelling (DCM) and Granger causal modelling (GCM), with a focus on human neuroimaging data. The thesis has led to four original research contributions. In the first contribution, a fully self-feedback model (FSM) is introduced as an extension to existing NMMs. FSM contains extra sel£feedback loops in both excitatory and inhibitory subpopulations, more consistent with the canonical cortical column architecture. It is shown that under both single- and multi-area simulations of event-related potentials (ERPs), FSM outperforms classic NMMs. In the second contribution, the DCM approach is used to provide insights into the underlying neural circuit dynamics of pattern reversal visual evoked potentials extracted from concurrent EEG-fMRI data. An optimal DCM model with recurrent forward-backward process disclosed the timing of signal propagation across ventral and dorsal connectivity pathways as well as the largescale effective connectivity in the human visual system. In the third contribution, an extension of GCM called 'partial' Granger causality (PGq is introduced to decipher the directed functional connectivity of ERPs. It is shown that time-domain PGC can detect the underlying causal connectivity relatively rapidly and robustly while capturing the accurate evolution of the directed neural connectivity dynamics over time. Lastly, PGC technique applied to EEG segments under a robot-assisted gait training reveals brain connectivity changes in fronto-centroparietal network with strong ·correlation with the behavioural performance of individuals.