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Infectious disease is an important issue for animal breeders, farmers and governments. Solutions to control infectious disease are needed and research focused on the genetic loci determining variation in immune-related traits has the potential to deliver solutions. The primary aim of this thesis is to discover regions of the bovine genome which influence the immune response post immunisation. To accomplish this two types of immunising agents, a Foot-and-Mouth Disease Virus (FMDV) peptide (FMDV15) and a commercial vaccine for Bovine Respiratory Syncytial Virus (BRSV), were used to immunise the second generation (F2 and backcrosses) of the Roslin Bovine Genome (RoBoGen) herd, a Charolais Holstein cross population. The FMDV15 peptide consisted of two sections of the VP1 protein located on the FMDV capsid, together encompassing the major neutralising antibody sites that are known to be immunogenic. Protection against FMDV is generally believed to relate to the levels of neutralising antibody and has been correlated with IgG1 and IgG2 levels as well as interferon- . In addition it has been shown that T cell responses also play a role in protection against FMDV. Thus all of these were used as phenotypic measurements post immunisation to the FMDV15 peptide. The BRSV vaccine used was an attenuated live vaccine. Protective mechanisms against BRSV infection include IgA, IgG1, IgG2 and IgM BRSV-specific antibodies and antibody titres particularly those of the IgG isotypes are considered to be correlates of protection. Thus, IgG1 and IgG2 antibody levels were measured post vaccination with the BRSV vaccine. All phenotypes were measured across time, and allowed analysis of the primary and secondary adaptive immune responses. Both agents caused considerable variation in the phenotypes measured post immunisation, with significant responses detected two weeks post immunisation. REstricted Maximum Likelihood (REML) analysis attributed much of this variation to sire, highlighting the heritable component, and environmental effects. Significant positive correlations were detected across time within each trait for both the FMDV and BRSV responses. The FMDV and BRSV antibody levels also correlated with each other at later time points, suggesting that there may be animals which are genetically predisposed to be high or low responders in general. Initially a linkage mappingapproach was followed using 165 microsatellite markers, which detected 77 QTL in response to the FMDV peptide and 27 QTL in response to the BRSV vaccine. There were some overlapping QTL, for example QTL which spanned the Major Histocompatibility Complex. Further analysis was conducted by developing a Perl scripted program which genotyped the RoBoGen herd in two ways; 1) Single Nucleotide Polymorphism(s) (SNP) were genotyped within the confidence intervals of the previously discovered QTL and 2) SNP were genotyped via a candidate gene approach. Association study methodology, accounting for relationship stratification via principal components of the genetic relationship matrix, was used to detect significant SNP, in response to both the FMDV peptide and the BRSV vaccine. Twenty significant SNP associations were discovered across 19 traits, with some SNP located in genes with known biological relevance to an immune response, such as the Toll-Like Receptors (TLR), TLR4 and TLR8. This thesis has detected regions of the genome which are significantly associated with the immune responses elicited by two different agents, suggesting similar pathway(s)/gene(s) may be used in defence of multiple pathogens. Once regions of significance were detected, further analysis using SNP markers identified significant, non-synonymous SNP that were associated with the immunising agents. The novel markers discovered in this study may aid breeding for resistance to disease via marker assisted selection. In addition, they may also have highlighted new targets for vaccinologists to develop ‘next generation’ vaccines.

This thesis explores, develops and implements modelling strategies for studying relationships between childhood growth and later health, focusing primarily on the relationship between the development of body mass index (BMI) in childhood and later obesity. Existing growth models are explored, though found to be inflexible and potentially inadequate. Alternative approaches using parametric and nonparametric modelling are investigated. A distinction between balanced and unbalanced data structure is made because of the ways in which missing data can be addressed. A dataset of each type is used for illustration: the Stockholm Weight Development Study (SWEDES) and the Uppsala Family Study (UFS). The focus in each application is obesity, with the first examining how the adiposity rebound (AR), and the second how the adiposity peak (AP) in infancy, relate to later adiposity. In each case a two-stage approach is used. Subject-specific cubic smoothing splines are used in SWEDES to model childhood BMI and estimate the AR for each subject. As childhood BMI data are balanced, missingness can be dealt with via mUltiple imputation. The relationship between the AR and late-adolescent adiposity is then explored via linear and logistic regression, with both the age and BMI at AR found to be strongly and independently associated with late-adolescent adiposity. In the UFS, where childhood BMI data are unbalanced, penalised regression splines are used within a mixed model framework to model childhood BMI and estimate the AP for each subject. The data correlations induced by the family structure of the observations are addressed by fitting multilevel models in the second stage. Both age and BMI at AP are found to be positively associated with later adiposity. The two nonparametric modelling approaches are found to be effective and flexible. Whilst the thesis concentrates on BMI development in childhood and later adiposity, the techniques employed, both in terms the modelling of growth and the relating of the derived features to the outcomes, are far more widely applicable.

Heterotetrameric sarcosine oxidase (TSOX; EC. 1.5.3.1) from Arthrobacter sp. 1-IN is a complex flavoprotein that catalyses the oxidative demethylation of sarcosine to produce glycine, formaldehyde and hydrogen peroxide. TSOX is composed of four subunits with approximate molecular weights of 106, 43, 24 and 15 kDa the genes for which have been cloned and the recombinant protein expressed at low levels. The Arthrobacter sp. 1-IN genomic DNA containing the sox operon was sequenced and, as well as the four TSOX genes, contains genes for a serine hydroxylmethyltransferase and a formyl-tetrahydrofolate deformylase. Native and recombinant TSOX were expressed, purified and characterised. Cofactor analysis showed that TSOX contains 1 mol each of non-covalently bound FAD and NAD+ and also 1 mol of covalent flavin. The reductive half-reaction of TSOX was studied using stopped-flow spectroscopy. pH dependence of flavin reduction by sarcosine indicated no kinetically influential ionisations in the enzyme-substrate complex. Two ionisations with pKa values of 7.4 0.1 and 10.4 0.2 were identified from a klim/Kd versus pH plot. Kinetic isotope effects studies of the rate of C-H bond breakage in sarcosine indicated a ground-state quantum tunnelling mechanism for H-transfer assisted by the thermal fluctuations of the protein molecule. Anaerobic reduction experiments indicated that the two flavins of TSOX were reducible and that two electrons were required per flavin for complete reduction. The redox potentials for the two flavins were determined and indicated that one flavin had an unusually high redox potential. The inter-flavin electron transfer rate was measured using a pH jump technique between pH 7 and 9. The pH jump experiments and redox potential measurements indicated that the intramolecular electron transfer in TSOX was an endergonic process.

Treatment of Escherichia coli DNA gyrase A protein with trypsin generates two large, stable fragments of molecular masses 64 kDa and 33 kDa which are derived respectively from the N-and C-terminus of GyrA. The trypsin-cleaved A protein (A'), can support DNA supercoiling, relaxation and other reactions of gyrase. The isolated 64 kDa fragment will also catalyse DNA supercoiling but the 33 kDa fragment shows no enzymic activity. An amber mutation, introduced into gyrA near the point which corresponds to the tryptic cleavage site, yields GyrA(1-573) which shares the same properties as the 64 kDa tryptic fragment. Using genetic engineering, large numbers of 3'-gyrA deletion mutants have been produced; those encoding a protein smaller than 58 kDa (GyrA(1-523)) did not obviously overproduce truncated GyrA. GyrA(1-523) shows similar enzymic properties to GyrA(1-573) but cannot perform DNA supercoiling. Deletion of fifty C-terminal residues from GyrA(1-573) has the effect of disrupting part of the protein essential for supercoiling. I propose that the N-terminal 64 kDa represents the DNA breakage/reunion domain of the A protein, while the 33 kDa fragment contributes to gyrase-DNA complex stability. Certain N-terminal deletion mutants of the GyrA protein were also constructed. Removal of the N-terminal 6 amino acids had no effect on the properties compared to GyrA. Removal of the N-terminal 69 amino acids yields a protein with no supercoiling or cleavage ability. The start of the N-terminal breakage-reunion domain is probably located within this 63 amino acid region. The domains of GyrA were investigated by microcalorimetry. GyrA yields two unfolding transitions. GyrA(1-573) and GyrA(1-523) both yield a single unfolding transition corresponding to one of the GyrA transitions. Therefore GyrA contains two structural domains that can be assigned to the functional moieties described above. GyrA(1-573) has been crystallized, and four crystal forms identified. A diffraction pattern to 7 A has been obtained.