1. The present study examined the function and regulation of cholecystokinin receptors in the rat hypothalamus, using the technique of <i>in</i> <i>vitro</i> receptor autoradiography, in conjunction with radioimmunoassay, <i>in</i> <i>vitro</i> electrophysiology and stereotaxic surgery. 2. Cholecystokinin receptors were localized in the supraoptic, paraventricular, ventromedial and dorsomedial nuclei of the hypothalamus. Competition studies revealed that cholecystokinin receptors in the supraoptic nucleus exhibited the specificity of central rather than peripheral cholecystokinin receptors. 3. Cholecystokinin receptor density was greatly elevated in the supraoptic and paraventricular nucleus of salt-loaded (2% sodium chloride), water-deprived and homozygous Brattleboro rats compared to control animals; this increase occurred preferentially in those areas containing predominantly oxytocin neurones. The increase in cholecystokinin receptor density in the supraoptic nucleus of salt-loaded rats was reversed upon rehydration. There was no change in cholecystokinin receptor binding in the supraoptic nucleus of lactating rats compared to control animals. Eledoisin receptor binding was decreased in the supraoptic and paraventricular nuclei following salt-loading. 4. The effect of cholecystokinin on the extracellular activity of neurones in the paraventricular nucleus in hypothalamic slices from control and salt-loaded animals was investigated. There was a large proportion of fast firing (possibly osmotically stimulated) cells which increased their firing rate in response to cholecystokinin in 'salt-loaded' slices, whereas no fast firing cells did in control slices. The large proportion of CCK-responsive fast firing cells in 'salt-loaded' slices may represent an electrophysiological correlate of increased CCK receptor density in the supraoptic and paraventricular nucleus of salt-loaded animals. 5. Central (i.c.v.) administration of caerulein to rats caused an increase in the plasma levels of oxytocin. This may have resulted from an interaction of caerulein with cholecystokinin receptors in the supraoptic and paraventricular nuclei. 6. Electrolytic lesions of the anteroventral third ventricle region did not attenuate the increase in cholecystokinin receptor density in the supraoptic nucleus elicited by salt-loading, although vasopressin and oxytocin release were attenuated. This implies that afferents from the anteroventral third ventricle region were not required for the osmotic upregulation of cholecystokinin receptor density in the supraoptic nucleus, and suggests that elements in this nucleus remain osmoresponsive in the absence of excitatory input from the anteroventral third ventricle region.

To investigate further the functions of astrocytes, a transgenic mouse was generated in which astrocytes can be ablated selectivity. Expression of herpes thymidine kinase (HSV-tk) was targeted to astrocyte using the GFAP promoter. HSV-tk can metabolise anti-herpetic agents, such as ganciclovir (GCV), which results in the formation of toxic moieties that induce cell death. The GFAP promoter cassette was obtained from Lennart Mucke of the Scripps Institute (now UCSF) into which the HSV-tk coding sequence was inserted. A linearised GFAP-HSV-<I>tk </I>construct was microinjected into fertilised mouse eggs to create potential transgenic mice. Four founder animals were identified of which three were capable of transgene transmission. Western blot analysis demonstrated that two lines of mice expressed HSV-tk protein. In one line (7.1), immunohistochemical analysis of brain sections showed that HSV-tk was expressed exclusively in astrocytes and was present in more than 90% of these cells. Neonatal glial cultures derived from this line, demonstrated that transgenic astrocytes were vulnerable to GCV mediated toxicity <I>in vitro. </I>GCV was administered for 14 days following a cortical stab injury. Disruption of both glial scar formation and reformation of the blood-brain barrier was observed. Areas of astrocyte ablation were co-incident with areas of astrocyte proliferation. Thus transgenic astrocytes are vulnerable to GCV mediated toxicity <I>in vivo</I>. In some animals, astrocyte loss was seen also in the hippocampus. In these areas, a secondary loss of neurons was observed consistent with the neurosupportive function of astrocytes. This model system will be useful for studying the effects of astrocyte ablation and disruption of glial scar formation on the regeneration of axons <I>in vivo. </I>

The formation of mesoderm is an important development process of vertebrate embryos, which can be broken down into several steps; mesoderm induction, patterning, morphogenesis and differentiation. Although mesoderm formation in <i>Xenopus</i> has been intensively studied, much remains to be learned about the molecular events responsible for each of these steps. Furthermore, the interplay between mesoderm induction, patterning and morphogenesis remains obscure. Here, I describe an enhanced functional screen in <i>Xenopus</i> designed for large-scale identification of genes controlling mesoderm formation. In order to improve the efficiency of the screen, I used a <i>Xenopus tropicalis</i> unique set of cDNAs, highly enriched in full-length clones. The screening strategy incorporates two mesodermal markers, <i>bra </i>and <i>myf5</i>, to assay for cell fate specification and patterning, respectively. In addition I looked for phenotypes that would suggest effects in morphogenesis, such as gastrulation defects and shortened anterior/posterior axis. To maximise the output of this screen, several other non-mesodermal phenotypes were recorded as well. Out of 1728 full-length clones I isolated 82 for their ability to alter the phenotype of tadpoles and/or the expression of <i>bra</i> and <i>myf5</i>. Many of the clones gave rise to replace the lost <i>islet1</i>⁺ expressing cells. Thus, I conclude that the major function of <i>cyclin Dx</i> is to maintain motor neuron progenitors and provides a temporal cue for cell cycle exit of pMNs in the developing <i>Xenopus</i> neural tube. These results provide the first evidence that a cell-type specific cyclin can control the timing of cell cycle exit to create diversity of differentiating motor neurons and could contribute to our understanding of how the cell cycle control can contribute to neuronal diversity in the neural tube.

I have studied the role of intracellular stores in modulating intracellular calcium using the fluorescent indicator fura-2 in voltage-clamped snail neurones in isolated ganglia. I have used caffeine as an agonist to stimulate release of calcium from the intracellular stores and cyclopiazonic acid (CPA) to prevent the stores refilling. Caffeine application (10mM) resulted in a transient rise in Ca<SUP>2+</SUP> that was followed by an undershoot. To explore the process of the undershoot, intracellular calcium was raised by inhibiting the plasma membrane Ca<SUP>2+</SUP>-H<SUP>+</SUP> ATPase (PMCA) using high extracellular pH, vanadate or Eosin B. Inhibiting the PMCA resulted in a raised Ca<SUP>2+</SUP> level and a larger undershoot. The undershoot was abolished in the presence of 50μM CPA. These results are consistent with uptake into stores pump generating the undershoot. Depolarisation of the plasma membrane to 0mV and application of 10 mM caffeine to neurones, both resulted in a calcium transient. An increase in intracellular fura-2 concentration affected the depolarisation and caffeine-induced calcium transients differently. In five out of seven experiments the caffeine-induced calcium transients were reduced to a greater extent than the depolarisation-induced transients. This may be through the calcium buffering action of fura-2 or direct interaction of fura-2 with the ryanodine receptor. Attempts were also made to measure the calcium concentration within the endoplasmic reticulum (ER). The low affinity fluorescent calcium indicator fluo-3ff (AM) was used.

One factor implicated in mediating contact-repulsive activity is the molecule F-Spondin. However, findings with regard to its effects on the sensory neurite pathfinding are contradictory. Initially, I have shown that this contradiction results from membrane sequestration by COS7 cells of full-length protein used. The bulk of the work described here concerns the identification of genes differentially expressed between anterior and posterior half-sclerotomes as this is a prerequisite for the further investigation into the molecular mechanisms of somite segmentation and development. I developed a novel amplification method to generate sufficient material from anterior and posterior half-sclerotomes from mouse embryos to allow transcriptional profiling using oligonucleotide microarrays. Computational validation indicated that differences in expression data reflected differences between anterior and posterior sclerotome-halves. Statistical approaches identified a pool of ~60 candidates as putatively differentially expressed between anterior and posterior halves. Whole mount in situ hybridisation was used to verify the potential differential localisation of these candidates, resulting in 12-32 differentially expressed genes. Seven confirmed differentially expressed candidates were chosen on the basis of temporo-spatial expression patterns for further analysis using in vitro axon guidance assays. These secreted and transmembrane proteins were cloned into a mammalian expression vector such as to allow for production of soluble protein. The supernatants from transiently transfected cultured mammalian cells were used to conduct preliminary experiments into the potential of these molecules as axon repellents in the PNS.

Reported here is the X-ray crystal structure and the solution dynamics of T70N lysozyme, as monitored by hydrogen/deuterium exchange and NMR relaxation experiments. The X-ray crystal structure shows that a substantial structural rearrangement results from the amino acid substitution, involving residues 45-51 and 68-75 in particular, and gives rise to a concomitant separation of these two loops of up to 6.5 Angstroms. A marked decrease in the magnitudes of the generalised order parameter (S²) values of the amide nitrogen, for residues 70-74, shows that the T70N substitution increased the flexibility of the peptide backbone around the site of mutation. In hydrogen/deuterium exchange experiments monitored by mass spectrometry, a transient but locally cooperative unfolding event was observed for the T70N variant (at 47°C) and the wild type protein (57°C), but at higher temperatures than for the amyloidogenic variants I56T and D67H (37°C). These findings reveal that such partial unfolding is an intrinsic property of the lysozyme structure, and suggest that the readiness with which it occurs is a critical feature determining whether or not amyloid deposition occurs <i>in vivo.</i> At physiological temperature, the protection factors for many of backbone amides in the β-domain of the T70N variant are decreased relative to those in the wild type protein, whereas those in the α-domain display wild type-like values. At the higher temperature a dramatic decrease in protection was observed for the β-domain and C-helix for the T70N variant. This clearly demonstrated that partial unfolding event affects the β-domain and C-helix is similar to that observed previously for the I56T and D67H variants.

This thesis describes the finding that changes in calcium concentration in different cellular compartments, the cytoplasm and the nucleus, activate transcription through different DNA regulatory elements in the promoter of the <I>c-fos</I> gene. Transcriptional activation following increases in nuclear calcium concentrations is mediated by the cAMP response element (CRE). A second signalling pathway activates transcription through the serum response element (SRE), is triggered by increases in cytoplasmic calcium and does not require an increase in nuclear calcium. I have also shown that <I>c-fos</I> transcriptional induction mediated through the SRE, is independent of ternary complex formation in AtT20 cells and primary hippocampal neurons. To further characterise nuclear calcium-dependent transcription of <I>c-fos</I>, I identified the transcription factors and coactivators. I showed that the CRE binding protein CREB can mediate a nuclear calcium-dependent response. It is well established that transcriptional activation by CREB requires its phosphorylation on serine 133. This phosphorylation event can occur independently of nuclear calcium transients, indicating that other regulatory steps are necessary for nuclear calcium dependent transcription. These may involve the CREB binding protein CBP. Recruitment of CBP by CREB phosphorylated on serine 133 is critical for CREB-mediated transcriptional responses, raising the possibility that a post-translational modification of CBP may represent a crucial regulatory step in calcium induced transcription. I have shown that the CBP protein when directly tethered to the promoter can confer calcium inducibility to a <I>c-fos</I> based reporter gene and CBP-mediated transcriptional activation requires nuclear calcium transients.

The hypothesis tested in this thesis was that passage of ovine scrapie isolates in isogenic hosts is associated with the formation of PrPSc with distinct molecular properties, thereby providing a molecular basis for the discrimination of ovine prion strains. The molecular profile of PrPSc associated with the mouse-adapted sheep scrapie strains RML and ME7 was investigated. Western blot analysis, together with denaturant-dependent conformational stability analysis, revealed that PrPSc associated with RML and ME7 prion strains showed distinct biochemical and biophysical properties. PrPSc associated with RML and ME7 exhibited similar resistance to Proteinase K digestion but differed significantly in the glycoform profile of the resultant PrP27-30. In addition, ME7 PrPSc showed greater conformational stability than did RML PrPSc following denaturation with guanidine hydrochloride and proteolytic cleavage with Proteinase K. These novel data demonstrated that mouse-passaged ovine scrapie strains RML and ME7 could be discriminated at the molecular level by distinct conformers of PrPSc. Subsequently, PrPSc associated with primary passage, in conventional and ovine PrP transgenic mouse lines, of different isolates of natural ovine scrapie was investigated for the presence of distinct conformers of prion protein. Transmission of ARQ homozygous scrapie isolates resulted in the accumulation of significantly greater levels of PrPSc in <i>Prnp^a, Prnp^b </i>and ovine ARQ PrP transgenic mice than did VRQ homozygous inocula. In contrast, similar levels of PrPSc were generated in ovine VRQ transgenic mice. Significantly, the degree of conformational stability of the accumulated PrPSc correlated with its glycoform profile. transmission of ARQ homozygous scrapie isolates induced the accumulation of PrPSc characterised by a predominance of di-glycosylated PrPSc that was significantly more stable than that associated with the passage of VRQ homozygous inocula, which had similar levels of di- and mono-glycosylated PrPSc. These novel observations provide a biochemical basis for ovine prion strain typing.

No description available.

Repressor Element 1 (RE1) Silencing Transcription factor (REST, also known as Neuron Restrictive Silencer Factor, NRSF) is a key regulator of gene expression in development. REST has been characterized as a repressor that silences neuronal genes in non-neuronal cells and neuronal precursors through interactions with co-repressor proteins, such as CoREST. In this thesis, I show for the first time that REST interacts with the AAA ATPase and Bromodomain containing protein, ATAD2, leading to activation of target genes. ATAD2 and REST bind in a mutually dependent manner to chromatin during a discrete window of the cell cycle. This occurs before REST is degraded and previously REST-repressed genes become transiently activated. Overexpression of ATAD2 leads to a loss of CoREST occupancy at target sites, suggesting that ATAD2 counteracts repression by displacing CoREST. As a result, the observed activation of target genes may be a result of their de-repression from the REST repressor complex. I propose that this cell cycle-coupled burst of transcriptional activity is required for differentiation, as it primes repressed genes for activation. Consistent with this hypothesis, ATAD2 promotes REST-mediated gene expression during differentiation of neuronal precursor cells. Taken together, my data reveal that gene control by REST may involve a successive interplay of repressive and activating functions. This highlights an unexpected layer of regulatory mechanism in REST function.