Whereas most G-protein-coupled receptors (GPCRs) are monomeric in structure, gamma-Aminobutyric acid type B (GABAB) receptors are heterodimers of two seven transmembrane protein subunits, GABAB(1) and GABAB(2). GABAB receptor function is dependent upon the co-expression of both these proteins which, when individually expressed, are devoid of receptor activity. Desensitisation of cell surface receptors allows tissues to rapidly adjust their response to agonist. A conserved mechanism ensures that GPCR signalling is closely followed by desensitisation. This entails the phosphorylation of activated receptors enabling interaction with arrestin proteins and subsequent internalisation. It is not known if heterodimeric GABAB receptors employ this method of desensitisation. Data presented here result from experiments to determine whether GABAB receptor cell surface stability is controlled in a similar manner to that of other GPCRs. Also documented is the study of a putative interaction between the protein kinase AMPK and the GABAB(1) subunit. Initial whole cell labelling studies demonstrated that both GABAB(1) and GABAB(2) are basally phosphorylated. Dissimilar to other GPCRs, agonist did not increase levels of phosphorylation and this remained true upon overexpression of G protein receptor kinases. Because GABAB receptors lacked the internalisation promoting increase in phosphorylation upon agonist, it was predicted that they might have enhanced surface stability. This was confirmed in heterologous systems where GABAB receptors did not demonstrably internalise after agonist application even when arrestins were overexpressed. GABAB receptors in cultured cortical neurones showed a similar lack of internalisation in response to agonist. Biotinylation of neuronal surface receptors demonstrated that GABAB receptors reside for an unusually long time at the plasma membrane. Chronic agonist decreased the surface receptor half-life, but this did not correlate with an increase in internalised receptor. Interestingly, chronic agonist did not significantly reduce total receptor protein levels, suggesting GABAB receptors may not downregulate. Protein kinase A (PKA) stimulation, both exogenously and through intracellular pathways, counteracted the agonist-induced degradation and demonstrated that this particular kinase can control GABAB receptor surface numbers. Protection from degradation was correlated with increased phosphorylation at serine 892 within GABAB(2), a residue previously demonstrated to be a PKA substrate. Subsequent experiments were carried out to identify kinases capable of phosphorylating GABAB(1). Affinity purification assays isolated a kinase from brain able to interact with and phosphorylate a twenty amino acid stretch of the carboxy-terminal domain of GABAB(1). Yeast two-hybrid studies identified the catalytic subunit of AMPK as a putative interacting protein with GABAB(1). AMPK was found to phosphorylate GABAB(1) at serines 917 and 923 within the carboxy-terminal domain. Phosphorylation of serine 917 was further confirmed with a phospho-specific antibody raised to this residue. AMPK affinity purifies with GABAB(1) carboxy-terminal domain GST fusion proteins and also co-immunoprecipitates with GABAB receptors from brain. Preliminary investigations indicate that AMPK activation increases surface GABAB receptor levels in cortical neurones and may affect the protein protein interactions of GABAB(1). The results presented in this thesis suggest GABAB receptors are highly stable at the neuronal surface. The activation of PKA and AMPK may be mechanisms by which neurones are able to regulate plasma membrane GABAB receptors.

This project proposes a novel and simple technique to pattern glia and neurons. This study investigates various microfabrication friendly patterning systems and develops a highly consistent, straightforward and cost effective cell patterning scheme based on two common ingredients: the polymer parylene and horse serum. The first part of this work describes the new patterning method and highlights its capabilities. Stripes of parylene on silicon thermal oxide (SiO­­­₂) were fabricated in the cleanroom, cleaned and subsequently activated by a simple deep and rinse process in horse serum. Glia and neurons from dissociated rat hippocampi were cultured on the patterns and imaged after 7 DIV. Standard cell culture and immunofluorescent imaging protocols were used. A comparison between experimental and control samples indicate that the protein load in horse return serum is able to alter parylene into a cytophylic substrate. The second part of this project focuses on the optimisation of the parylene based patterning method. Aspects, such as the sustainability of the cell patterns beyond the first week and the effect of glia division on the quality of the patterns are investigated. Furthermore, the reduction of the immersion time of the substrates in the horse serum is explored while a behavioural model for the cells during their first week in culture is proposed. Submersion in an alternative serum is examined while the effects of UV radiation on the parylene are revealed not to be contributing to cell patterning. In the last part of this study an attempt is made to analyse the parylene and thermal oxide surfaces before and after immersion in the horse serum. An initial theory explaining the preference of cells towards parylene is disproved, while three probable hypotheses are proposed. Evidence from XPS analysis and electrophoresis gels suggests that the amount and conformation of proteins on the parylene and thermal oxide substrates might be responsible for inducing glial and neuronal patterning.

The mammalian HPRT gene is a housekeeping gene. It is expressed constitutively at a low level in all cell types with the exception of the brain where expression is elevated. It has been shown in human brains that HPRT expression is particularly high in the basal ganglia. Total HPRT deficiency in man is the cause of Lesch-Nyhan syndrome, a severe neurological disorder. Partial HPRT deficiency leads to gouty arthritis. In the mouse it has been shown that the elevated level of HPRT activity in the brain is accompanied by an elevation in the steady state level of HPRT mRNA. The aim of the work presented in this thesis was to elucidate the mechanism of this elevation. The role of both 5' and 3' sequences has been explored. Expression of the mouse HPRT gene in cultured cells has identified promoter elements essential for a basal level of transcription. This work has been extended to animals by using gene targeting in embryonic stem cells. A HPRT mutant gene has been corrected by gene targeting, introducing a 35 bp core promoter which is capable of directing expression in cell culture. This has enabled the minimal elements of the promoter sequence to be identified which can direct brain specific elevation <i>in vivo</i>. The role of the 3' untranslated region (UTR) in determining tissue specific elevation of expression has also been investigated. The possibility that differential polyadenylation might be responsible has been excluded. Gene targeting has been used to substitute the native 3' UTR with the 3' UTR of the human growth hormone gene. This has demonstrated the potential of gene targeting as a way of manipulating the mammalian genome to study the expression of endogenous genes.

The aim of this work was to predict protein secondary structure using neural network models. Initially a Hopfield network was used but abandoned in favour of a layered network trained using the back propagation algorithm. In the early stages of this work an exploration of the many different approaches to this problem was undertaken. These included attempts to predict boundaries between secondary structures, the secondary structures of individual residues, and the secondary structures of sequences wholly within a particular secondary structure. Results indicated the latter to be the best approach to continue with. In addition two coding schemes were investigated: a coding scheme based on occurrences of pairs of residues and one based on the positions of residues. It was found that this positional coding scheme was the natural coding scheme for this problem. On segments of whole alpha-helix and whole non-alpha-helix 10 residues in length a prediction success of around 80% with a correlation coefficient of 0.52 was achieved with the positional coding scheme. On whole proteins, where predictions are made for individual residues, alpha-helix prediction drops to 73% with a correlation coefficient of 0.34. The relative predictability of alpha-helices of above and below average accessibility was also investigated showing that those of above average accessibility are more predictable than those with below average accessibility. The main body of this work concerns the apparent limit of predictability of alpha-helices. It was found that test set prediction did not depend on the number of hidden nodes. In fact, a single layer network performed as well as those with hidden nodes showing that the probolem is basically linearly separable. In addition, prediction success plateaus well below that of perfect prediction success. During training, test set prediction is seen to peak. The decrease in prediction success was found to be due to non-alpha-helix sequences that the network was unable to distinguish from real alpha-helix sequences. These regions of non-alpha-helix were shown to occur adjacent to actual alpha-helices with statistical significance. It is proposed that potential alpha-helices are disrupted by global constraints during the formation of tertiary structure. The effect of window size was also investigated as was beta-sheet prediction, but this was found to be limited by the small number of examples available with our approach. However, its distribution in the input space in relation to alpha-helix and coil was determined.

Serine proteases in the central nervous system modulate developmental and synaptic plasticity and are implicated in the pathophysiology of Alzheimer's disease. This thesis aims to characterise the spectrum of serine proteases expressed in the brain. Degenerate primers were designed from regions conserved among the major chymotrypsin clan of serine proteases; these were used for the polymerase chain reaction amplification of family members represented in cDNA form adult rat hippocampus. 10 different members of the family were uncovered. The most abundant products corresponded to tissue plasminogen activator (t-PA) and RNK-Met-1, a lymphocyte protease not previously reported in brain. Evidence is provided to suggest that the major t-PA substrate, plasminogen, is absent from brain, arguing that the target for t-PA in brain is unlikely to be plasminogen, Other enzymes represented include elastase IV, proteinase3, complement C2, Hageman factor, chymotrypsin B, chymotrypsin-like protein and two novel family members, BSP-1 (brain serine protease-1) and BSP-2. Full length sequences of BSP-1 and BSP-2 are reported and sequence motifs and homologies suggest they represent trypsin-like proteases. The expression patterns for each of the serine proteases amplified, as determined by Northern and in-situ hybridization analysis, are presented. BSP-2 is expressed in the hippocampus and the cerebral cortex whereas BSP-1 is confined in its expression to the CA fields of the hippocampus. A gene restricted in expression to the hippocampus has considerable potential for transgenic experimentation into the role of this brain region. Directing expression of suitably modified components of the synaptic signalling pathway within this brain region would allow the relationship between hippocampal synaptic plasticity and learning and memory events to be assessed. To investigate the feasibility of such an approach, the murine BSP-1 locus has been targeted with an IRES-lacZ reporter cassette. Preliminary results indicate reporter expression is active from the murine BSP-1 locus.

No description available.

Hippocampal mossy fibres (MFs) have been studied intensely in vitro. While many properties of the MFs are well characterised, other findings are debated, particularly in relation to MF long-term potentiation (MF-L TP). MF-L TP is widely accepted as being expressed presynaptically in vitro; however, the induction mechanisms remain unclear. Although kainate receptors (KARs) are generally considered to have a role in MF-L TP induction, the identity of the subtype is debated. Furthermore, the contribution of metabotropic glutamate receptors (mGlu receptors) in this form of plasticity is also controversial. The MFs also exhibit unusual short-term plasticity, including frequency facilitation (FF) which is observed to variable extents in vitro and in conscious rats. An aim of this study was to circumvent the methodological variations, which can affect experimental outcomes in vitro. This was achieved by assessing MF synaptic plasticity in vivo, in anaesthetised rats. A slow-onset MF-L TP was reliably induced and saturated by one train of tetanisation at 100 Hz. MF field excitatory postsynaptic potentials (fEPSPs) were depressed by a group II mGlu receptor agonist, DCG-IV. MF-L TP was also shown to be inducible independently of NMDAR activation. The slow-onset profile of MF-L TP was further investigated and found to be unaffected by altering anaesthetic and tetanisation parameters. Depression, as opposed to facilitation, of the MF fEPSPs occurred in anaesthetised rats during increased frequencies of stimulation. However, facilitation of the fEPSPs was noted in the hippocampus, most likely at the associational/commissural pathway following contralateral or ipsilateral stimulation. MF-L TP persisted in the presence of KAR antagonists or mGlu receptor antagonists. However, MF-L TP was abolished by intrahippocampal co-injection of a KAR antagonist with specific mGlu receptor antagonists. Use of group I mGlu receptor antagonists indicated roles for both mGlu1 and mGlu5 receptors. This study suggests that KARs and mGlu receptors play interchangeable roles in the induction of MF-LTP in vivo.

No description available.

No description available.

No description available.