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Continuous sampling of the same cattle was carried out on several groups of animals, at regular intervals of 3 or 5 weeks, for periods up to 22 months. Normal range of values were obtained for serum Ca, serum Mg, inorganic P, blood sugar, chlorine and. N. P.N. No change in blood composition could be attributed to lactation or to season of the year: slight seasonal differences were associated with grazing. Parturition caused decrease in serum Ca and inorganic P: the fall in inorganic P was more marked, and was even diagnostic of approaching labour. The hypocalcaemia at parturition is discussed in relation to milk fever, the hypocalcaemia of which appears to be a pathological exaggeration of a normal physiological phenomenon. Calves at birth have higher levels of serum Ca, inorganic P, blood sugar and N. P.N. and lower levels of serum Mg and chlorine than are normal in the dam. In general the blood of the calf attains the dam's normal at the age of about 6 months. Increase in P intake caused a rise in inorganic P level: when Ca intake was lowered at the same time as P intake was raised the rise in inorganic P was greater but serum Oa showed little or no alteration. 7. A diet rich in clover, when associated with shortage of water, caused over 100 % rise in N.P.N. 8. Evidence is provided of a definite diurnal rhythm in the blood sugar level of lactating cows, apparently associated with withdrawal and secretion of milk, since it did not appear in dry cattle or sheep.

Voltage-gated calcium channels (VGCC) are essential actors for many physiological functions of excitable cells. The properties of VGCC are modulated by association with auxiliary subunits: β, α2δ and in some cases γ. The α2δ subunits increase the functional expression of calcium channels, via a trafficking mechanism. The α2δ-1 and α2δ-2 subunits of VGCC are the binding sites of gabapentin, an anti-epileptic and anti-hyperalgesic drug. The goals of this study were: 1) to characterise the trafficking properties of the α2δ- 2 subunit; 2) to elucidate the controversial mechanism of action of gabapentin upon binding to α2δ-1 and α2δ-2 subunits. These questions were addressed by the biochemical and imaging study of calcium channel subunits in heterologous expression systems. A Bungarotoxin Binding Site epitope was introduced in the extracellular region of α2δ-2, and the resulting tagged subunit was used in combination with fluorescent conjugates of αBungarotoxin, in order to study the internalization and insertion into the plasma membrane of the α2δ-2 subunit. Consistent with the previous identification of α2δ-2 as a lipid raft protein, the alteration of the cholesterol content of the plasma membrane was found to modify its cell-surface expression and trafficking. Cholesterol depletion and loading were found to respectively decrease, and increase the endocytosis of α2δ-2. The chronic, but not the acute application of gabapentin caused a reduction in the cell surface expression of α2δ-2 and Cav2.1 subunits. This effect was shown to be due to a reduction in the forward trafficking of α2δ-2, by preventing its recycling from Rab11-associated endosomes to the plasma membrane. These studies have led to the identification of some of the elements regulating the trafficking of α2δ-2, and their alteration by gabapentin. These mechanisms might be crucial for the modulation of the cell surface expression of calcium channels, in physiological and pathological conditions.

á2ä-1 is an auxiliary subunit of the voltage-gated calcium channels, and it regulates the trafficking and functions of the channel complex. á2ä-1 subunits contain the binding site for gabapentin (GBP), used for the treatment of neuropathic pain. The first aim of this thesis was to determine whether important functional properties of á2ä-1, such as the ability to regulate calcium currents and binding to GBP, were altered by alternative splicing events on á2ä-1. In addition, the distribution of á2ä-1 splice variants in dorsal root ganglia (DRGs) of naive and spinal nerve ligated (SNL) rats was analysed by RT-PCR and gel electrophoresis. The results obtained from [3H]-GBP binding experiments identified two splice variants characterised by a low binding affinity to GBP. One of those, which is known to undergo up-regulation in DRGs neurons upon SNL, was found to be specifically expressed in smaller neurons, which are normally nociceptive. The interaction of endogenous ligands with á2ä-1 can also contribute to influence the the binding affinity of GBP to á2ä-1. Therefore, the other goal of this study was to analyse the interaction of endogenous ligands with á2ä-1. First, co-immunoprecipitation and radioligand binding experiments demonstrate that á2ä-1 interacts with thrombospondins (secreted proteins that bind á2ä-1 and promote synaptogenesis) and that this interaction reduces the binding affinity of GBP to á2ä-1. Secondly, it was investigated wheather another protein: the low-density lipoprotein receptor (LDLR)-related protein-1 (LRP1) could also interact with á2ä-1 and TSP. It was found that LRP1 co-immunoprecipitates with TSP4 and it also decreases the amount of GBP binding to á2ä-1, suggesting that LRP1 might be involved in promoting the interaction between á2ä-1 and TSP4. These studies have shown that the existence of alternative splice variants of á2ä-1 and the interaction of á2ä-1 with endogenous interactors like TSPs and LRP1 affect the functionality of these subunits. An altered functionality might play an important role upon nerve damage, when á2ä-1 becomes up-regulated, because GBP requires binding to á2ä-1 to exert its therapeutic effect.

Mutations in the Pit1 gene severely affect the development and function of the anterior pituitary gland in both mice and humans, resulting in deficiencies of growth hormone, prolactin and thyroid stimulating hormone. These hormone deficiencies are thought to result from the failure to specify the identity of cells, presumably through the inability to activate genes that encode hormones. Furthermore, hypoplasia of the pituitary gland may result from the inability of pituitary endocrine cells to proliferate. The aim of this project is to study the effect of dominant Pit1 gene mutations to determine the mechanism of action of the mutant proteins. The study of mutant PIT1 proteins should allow greater understanding of how wild type PIT1 protein functions. Luciferase reporter assays were used to test the transcriptional activity of dominant negative PIT1 mutant proteins on different target promoters. These assays showed that the mutations had cell and reporter specific effects on luciferase activity that may be due to the combined effects of both direct transcriptional repression and indirect off-target effects, perhaps negative effects on cell viability. Several of the mutations resulted in increased activity in a reporter and cell type specific manner. Mutation of the N-terminal transactivation domain affected protein stability and electrophoretic mobility, strongly suggesting an effect on post-translational modification. However, subsequent analysis was unable to confirm this. Additionally, two novel recessively inhertited mutations were identified in a patient with combined pituitary hormone deficiency. The IVS1+3nt (a>g) mutation severely affected splice donor site definition whereas the R265W missense mutation resulted in reduced protein expression.

The spatial distribution of ion channels in different subcellular regions is a key determinant of neuronal behaviour. Patch clamp electrophysiology allows characterisation of ion channel activity, but precise localisation is more difficult. This is particularly true for very small, specialised compartments such as synaptic terminals, which are inaccessible by conventional, direct patch recording methods. Scanning ion conductance microscopy (SICM) generates high resolution topographic images by using a precisely positioned probe to measure ion currents. The development of a new ‘hopping’ mode allows convoluted neuronal networks to be imaged using the SICM probe. Some details of the implementation of this mode are described. The geometry of SICM pipette tips is examined, and the interaction between the probe and the cell membrane is shown to differ from the standard account. Application of SICM to localised patch clamp recording has previously been demonstrated in several cell types and here is extended to record from presynaptic sites. Control experiments are performed and a model is introduced to explain how the use of fine-tipped SICM pipettes may give rise to artefacts seen in some of these experiments. The technique is then applied to synaptic boutons in primary cerebellar culture and a number of successful recordings are presented, along with some attempts to combine the advantages of SICM positioning with those of more conventional patch clamp pipettes. Experimental limitations of these approaches are discussed.

Persistence of lactase into adulthood varies in frequency worldwide and is attributable to several different single nucleotide changes in an enhancer of the LCT gene. One of these is at particularly high frequencies in Europeans and several others have been found elsewhere. However, information about their worldwide distribution is patchy. 2056 DNA samples from populations of Europe, Asia and the Middle East were sequenced to examine the distribution of allelic variants of the LCT enhancer region. It was confirmed that -13910*T is also the predominant allele around the periphery of Europe, and that this allele extends as far as the South and East of the Arabian Peninsula. Other alleles appear to have spread out of Africa or Arabia and most variation was found in the Middle East. No new common alleles were found that were likely to be causal. In previous studies four lactase persistence associated nucleotide substitutions (-13910*T, -14010*C, -13915*G and -13907*G) have been studied functionally. In this thesis four additional enhancer alleles were examined using enhancer/promoter construct transfections and electrophoretic mobility shift assays. Three enhancer variants alter transcription factor binding in vitro and/or reporter-gene expression. Bioinformatic tools and specific antibodies were used to assist in identifying the transcription factors involved. The results show that different mechanisms lead to a disruption of the normal down-regulation of lactase in adult life. Known haplotypic markers were assessed on an overlapping set of samples and a greater number of flanking markers was typed in an extended region of 1.8 Mb around LCT, in order to chart the evolutionary relationships and extent of historic recombination of the chromosomes carrying the derived alleles, as well as those th at do not. All of the functional alleles tested have longer extended haplotypes than their ancestral counterparts, but so also does the derived variant at position -958, a haplotype marker for the B haplotype. The finding of an extended region of high linkage disequilibrium in all populations, and an extended B haplotype, is discussed in relation to the methods to study selection. Because phenotypic studies suggest missing functional variants, the immediate promoter and a part of intron 2 of LCT were also selected for sequencing. No serious candidates were found in intron 2. Two alleles in the immediate promoter were studied by transfections but there was little evidence for any in vitro effect of these variants.

Voltage-gated calcium (Cav) channels are essential components of excitable cells. The function of neurons is highly dependent on the plasma membrane availability of Cav channels, which allow entry of Ca2+. Cav channels control a variety of processes, including synaptic transmission and muscle contraction. Alpha-2-delta (α2δ), an auxiliary subunit of Cav channels increases the current amplitude of by about 4-fold through supporting membrane trafficking of the ionconducting Cav alpha-1 subunit. Furthermore, α2δ modulates the biophysical channel properties. The α2δ-1 isoform has an important pharmacological role of binding antiepileptic gabapentinoid drugs. Additionally, α2δ regulation is enhanced in neuropathic pain models. Until recently, α2δ was characterized as a Type I transmembrane protein. We have identified that α2δ is instead anchored to the extracellular leaflet of the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor. This type of membrane attachment plays an important role in sorting proteins to specialized domains of the lipid bilayer, termed lipid rafts, allowing lateral movement, interactions and signal transduction. The first part of this thesis examines the regulatory properties of neuron-specific α2δ-3 in association with the N-type Cav channel. The results obtained strongly support the hypothesis that α2δ-3 is attached to the membrane via a GPI-anchor. This was demonstrated by enzymatic PI-PLC cleavage, along with proteoglycan NotumWT hydrolytic activity, both of which resulted in a significant reduction in the Ba2+ current. These findings are further corroborated through the use of recombinant constructs of α2δ-3, mutated at the GPI-attachment site. Together with immunofluorescence results, which demonstrate reduced cellular transport of the two α2δ-3 constructs, it was concluded that less channels reach the plasma membrane in the absence of an intact GPI-signal sequence. Moreover, effects of acute and chronic cholesterol modulation on Cav channels plasma membrane targeting were examined. In both instances, N-type Cav/α2δ-3 function and distribution were impaired, providing additional support for the importance of Cav channel localization to lipid raft regions. The second part of this thesis examines the prediction that the α2δ-1 isoform is GPIanchored. A C-terminal truncated form of α2δ-1 (DGPI α2δ-1) shows altered subcellular and surface localization, as well as functional P/Q-type Cav/α2δ-1 channel reduction, providing independent evidence that the GPI anchor of α2δ-1 facilitates Cav channel functional expression. In the final section of the thesis, the WT and DGPI α2δ-1 were used to assess cellular prion protein (PrPC) modulation on P/Q-type Cav/α2δ-1 trafficking using electrophysiological and immunocytochemical methods. An interaction between the WT α2δ-1 and PrPC was proposed, based on the findings that co-expression of the two proteins resulted in a strong colocalization as well as altered channel function. The implications of these results are highlighted by established findings that cellular PrP is required for susceptibility to prion infections and for prion toxicity, and that mice expressing anchorless PrP show reduced infectivity with the pathogenic scrapie PrP isoform.

Homotypic death domain (DD)-death domain interactions are important in the assembly of oligomeric signalling complexes such as the CD95/Fas DISC, PIDDosome, and the RIPoptosome. These complexes are considered as platforms for triggering apoptotic and other downstream signalling pathways. The first part of this thesis is aimed at characterisation of the PIDDosome core complex in solution using different techniques, with a specific focus on the application of methyl-TROSY NMR experiments. Analysis of the stoichiometry and molecular weight (MW) of the intact complex by AUC, SEC-MALS, and Nano-ESI confirmed the formation of high MW species (130-158 kDa) with a flexible stoichiometry of 10-12 chains that is not in complete agreement with previously reported data. Standard (15N, 1H)-HSQC titration experiments displayed global loss of signal confirming the formation of high MW species. 13C-methyl-TROSY experiments of the complex showed splitting of the cross peaks and evidence of differential line broadening and chemical shift heterogeneity that reflects the presence of non-equivalent sites within the different inter-domain interfaces within the asymmetric complex. Methyl-TROSY spectra for ILV-labelled samples titrated with unlabelled binding partners were characterised by chemical shift changes in fast/intermediate exchange in early points followed by a switch in the behaviour to slow exchange at an intermediate point. This pattern is consistent with a model for multi-step complex assembly via intermediate species formed in fast exchange followed by a co-operative ‘lock-in’ to the final state in slow exchange. The second part of the thesis describes in vitro folding and interaction studies of receptor interacting protein kinase 1-death domain (RIP1-DD) and represents the first biophysical/biochemical characterisation for this protein. The combined results from different techniques suggest that human (but not macaque) RIP1-DD adopts a molten globule state. Interaction studies between RIP1-DD and FADD-DD showed formation of a complex with molecular weight exceeding 190 kDa.

Activation of B cells is dependent on two events: the encounter of cognate antigen and the triggering of an adequate B cell receptor (BCR) signal. In vivo B cells encounter antigen within secondary lymphoid organs, such as lymph nodes (LNs). Here, specialised CD169+ macrophages, strategically located across the fenestration of the inner wall of the subcapsular sinus (SCS), trap lymph-borne antigen and present it to B cells. I have shown that within CpG-inflamed LNs, CD169+ macrophages undergo a dramatic reorganisation by retracting from the inner wall fenestration and scattering along the edges of the tissue. These events are partly caused by the arrival of dendritic cells pushing away the macrophages form the SCS fenestration to gain access to the LN. In vitro CD169+ macrophages were found to respond directly to CpG by downregulating CD169 and some scavenger receptors, while upregulating the expression of interferon-γ. Because of these structural and phenotypic changes, antigen retention at the SCS is impaired resulting in fewer B cells encountering their cognate antigen and weaker antibody responses. Antigen encounter is important because it initiates BCR signalling. Within a signalling cascade, adaptor proteins act as signal integrators and amplifiers. The adaptor protein Nck is best known for linking receptor signalling to cytoskeleton regulation. However upon B cell activation I found that Nck controls the PI3K/Akt pathway by recruiting the B-cell adaptor for PI3K (BCAP). Nck can carry out these functions by directly binding to the BCR via the non-ITAM phospho-tyrosine 204 in the Igα tail. Importantly, genetic ablation of Nck resulted in defective BCR signalling leading to hampered survival and proliferation in vivo. Antibody responses in Nck-deficient animals were thus severely impaired. Collectively these findings are important as they shed light on some of the regulatory mechanisms for antigen encounter and BCR signalling.