Murine atypical chemokine receptor 4, ACKR4, which binds to chemokines CCL19, CCL21 and CCL25, is expressed specifically in germinal centre (GC) B cells and in a layer of stromal cells surrounding the splenic marginal zone. Although ACKR4 is unable to signal as classical GPCRs, it creates gradients of its ligands. It is shown here that ACKR4 deficiency in the stroma causes infiltration of naïve B cells into the GC area. ACKR4 deficiency in the B cells causes dysregulation of the intragerminal centre distribution, with enlarged light zones and reduced dark zones when compared to ACKR4-competent mice. This skewed GC distribution is caused by the inability of ACKR4-deficient B cells to downregulate c-Myc, as they receive increased signalling though the CCR7- p-Akt - c-Myc signalling pathway. Moreover, ACKR4-deficient mice contain elevated numbers of memory B cells (MBC) in the distant lymphoid organs. MBCs are retained in the draining lymph node (drLN) by the presence of a CCL19/21 gradient towards the subcapsular sinus (SCS). When this gradient is absent, as occurs in ACKR4-deficient mice, MBCs escape the drLN easier through the SCS and appear in other sites in elevated numbers. Together, this shows a new role for ACKR4 in the GC response and in the migration of GC-derived MBCs out of the follicle and the drLN.

The expression of genes is tightly controlled, predominantly at the point of transcription. RNA polymerase (RNAP) must first bind to a deoxyribonucleic acid (DNA) promoter upstream of a gene to transcribe it. However, the ability of RNAP binding is dictated by the core promoter DNA sequence, the presence of transcription activator or repressor proteins and numerous other factors. The strength of promoters has been indirectly measured. Only a few studies have attempted to directly address the RNAP flux through transcription units, and further studies are still required. In the current study, the aim was to directly correlate RNAP gene transcription with the strength of core promoter elements. To do that, I employed the direct method of chromatin immunoprecipitation (ChIP), followed by quantification of immunoprecipitated DNA. For promoter regions, this method directly measures the occupancy by RNAP; for regions within transcription units, the flux of the RNAP was deduced. A range of semisynthetic promoters, with different combinations of core promoter elements to obtain different levels of expression, was used to validate our method. This direct method enabled the calculation of “promoter competitivity”, “promoter occupancy index” (POI), RNAP “escape index” (EI), “fragment occupancy percentage” (FOP) and the time interval between transcribing RNAPs (Tint). On the basis of Tint, the number of RNAPs crossing any DNA sequence of interest per second (polymerase per second; PoPS) was calculated. Surprisingly, the results of the present study revealed that the RNAPs are well separated during transcription of the \(lac\) operon.

In the post-genomic era the availability of genome-wide datasets has revealed an unexpected complexity of transcriptional regulation. In this context, where most enhancer predictions are based on computational analyses, functional validations are lacking. This thesis investigated the utility of the transgenic zebrafish embryo as an in vivo vertebrate model to study the function of candidate human enhancers, and detect subtle changes in enhancer function caused by disease-associated variants. Our functional validations indicated that despite the evolutionary distance between human and fish, 60% of the conserved enhancers predicted by a combination of chromatin signatures, TF binding events and bidirectional transcription, lead to reporter expression that recapitulates the patterns of either zebrafish or human genes. To improve the reliability of zebrafish transgenesis, a targeted integration system mediated by PhiC31 integrase was validated for enhancer testing. I demonstrated that this method overcomes position effect variation commonly found in transposon-based assays. However, enhancer-driven expression could not be detected when I attempted to quantitate TCF7L2-associated enhancer variants, indicating the need for further studies to understand the limitations of the zebrafish model. Taken together, my results provide strong support for zebrafish as a valuable in vivo model to study the function of mammalian transcriptional regulatory elements.

Up-frameshift protein 1 (Upf1) is required for nonsense mediated mRNA decay (NMD) across eukaryotes. It has been reported that Upf1 has also nuclear functions, which are independent of its role in NMD. However, it is unclear whether the nuclear role of Upf1 in mammalian cells is conserved in \(Schizosaccharomyces\) \(pombe\) (\(S\). \(pombe\)). In this study, I investigated whether Upf1 functions in the nucleus and its other possible roles in \(S\). \(pombe\). Similar to the previous findings in mammalian cells, I found that \(upf1\) deletion mutant of \(S\). \(pombe\) was hypersensitive to the DNA replication inhibitors hydroxyurea and methyl methanesulfonate, suggesting increased DNA damage in this mutant. Additionally, each of \(upf1\), \(upf2\) and \(upf3\) shows synthetic sick with \(rad52\), which plays a central role in DNA double-strand break repair in \(S\). \(pombe\). Moreover, the S-phase progression is slower in NMD mutants. I also found an RNA dependent association of Upf1 with highly transcribed genes by chromatin immunoprecipitation (ChIP) experiments, implying its association with nascent RNA. Furthermore, deletion of Upf1 leads to increased RNA levels of \(tf2\) and rDNA, which are bound by Upf1, suggesting it has a direct role in regulating transcription. This hypothesis will be assessed for investigating whether the loading pattern of RNA polymerase II on chromatin changes without Upf1 using ChIP-Seq. Additionally, I identified that Upf1 interacts with genes involved in nuclear activities including nucleosome remodelling, transcription and cell cycle regulation.

Within this thesis, the use of anthracene to perform various applications within biomolecules is assessed. Anthracene displays two interesting photo properties which make it an appealing molecule for incorporation; fluorescence and photodimerisation. The former is utilised to develop a single nucleotide polymorphism detection assay which is shown to allow for determination of the base present in a complementary strand of DNA. In addition to this, the photodimerisation properties of anthracene are used within a protein for the first time. This is utilised to develop a photoswitched binding protein, allowing for control of DNA binding of the protein. Further to this, the photodimerisation properties are utilised within oligonucleotides to achieve structural control of a G-quadruplex as well as photo-triggered release of single stranded DNA.

The cell wall of Mycobacterium tuberculosis has a very complex ultrastructure, consisting of mycolic acids, an array of polysaccharides and surface exposed antigenic glycolipids. These cell wall components play a vital role in pathogenicity and virulence and hence, are attractive drug targets. Recent advances in TB drug discovery have produced a plethora of candidate drug molecules, many of them, affect mycolic acid biosynthesis and transport. A major part of the thesis work consists of biochemical and structural characterisation of proteins involved in mycolic acids biosynthesis and transport using a combination of genetic and biophysical tools. Through deletion of fabH, its non-essentiality for growth and survival of mycobacteria was demonstrated, and suggested the possibility of a functional substitute. The latter part of the thesis deals with the identification and characterisation of glycosyltransferases involved in the biosynthesis of lipooligosaccharides (LOS) in M. kansasii. LOS’s are surface exposed, highly polar antigenic glycolipids, present in several mycobacterial species. Using targeted gene deletion, mutant strain defective in LOS production was obtained that has provided the first insights into LOS biosynthesis in M. kansasii.

The partitioning of low-copy number broad host range plasmids depend on a centromere binding protein (CBP) that binds to a centromere-like site on plasmid. For RK2 plasmid, the CBP is a 358 residue, multi-domain protein, KorB. KorB contains an N-terminal domain (NTD), a central DNA-binding domain (DBD), and a C-terminal dimerisation (CTD) domain and the protein binds to the O\(_B\) site on the plasmid as a dimer. Structures of central DBD and CTD have been elucidated whilst limited information is available about the N-terminal of the KorB. In this study, NTD KorB protein was expressed and purified. Size exclusion chromatography and analytical ultracentrifugation data confirm that the NTD KorB behaves as a monomer in solution. Using solution-state NMR spectroscopy data, majority of the backbone and side-chain resonances for the NTD KorB are assigned and an ensemble structure of the protein is calculated. The flexibility of the NTD KorB is studied with coarse-grain Molecular Dynamics simulation package, AWSEM. The N-terminal of the NTD KorB is mostly unstructured whilst two α-helices towards the C-terminal of the protein exhibit limited motion. Two C-terminal KorB deletion mutants capable of binding the OB DNA (CΔ100 KorB and NΔ31CΔ100 KorB) were purified and characterised using circular dichroism (CD) and mass spectrometry. DNA-binding properties of these two deletion mutants are compared to the KorB wild-type (WT) using circular dichroism, fluorescence anisotropy and microscale thermophoresis measurements, indicating weaker binding of the deletion mutants with respect to the KorB WT to its centromere-like site (O\(_B\)). Considering the current state of structural information about the KorB and homologous proteins, a DNA partitioning model for the KorB is proposed.

Intraspecific genetic, morphological and life history structuring is evident in many taxa. Where such intraspecific structuring exists, study of the nature of the patterns displayed can reveal much about the evolutionary processes that operate during the early stages of divergence. Intraspecific structuring is particularly prevalent amongst fishes that occupy recently glaciated freshwater systems. One such species, the brown trout, Salmo trutta, was the subject of the work presented in this thesis. Genetic and morphological intraspecific structuring of brown trout was examined across a single but large dendritic catchment, the River Foyle, Ireland. Structuring was examined at three spatial scales (large-scale, compared between major sub-catchments; medium-scale, compared between tributaries within sub-catchments; small-scale, compared between streams within tributaries). The two general aims of the study were to look for any structuring in either phenotype or genotype in brown trout across the catchment and, if this was found, to look for landscape or environmental gradients that might be driving such structuring. Using a suite of 21 microsatellite markers that were chose for their ability to resolve population differences in this species elsewhere, this study identified clear and distinct genetic structuring. Brown trout collected from 28 sampling sites, resolved into 21 genetically distinct and discrete populations using a hierarchical approach implemented in STRUCTURE. The structuring was evidence across all three spatial scales. There was strong evidence of isolation by distance and isolation by environment playing a role in shaping the magnitude of the genetic differences between populations. Landscape variables which are shaped by anthropogenic impacts (urbanised area (measured as the number of houses in the catchment), proximity to farmland (measured as the distance to the nearest farm) and concentration of phosphorus in the water) showed the greatest effects in shaping the genetic population structuring (chapter 2). In a parallel study, the morphological structuring of brown trout from across the Foyle catchment was investigated at three spatial scales. Morphology was measured as the shape of brown trout determined by Geometric Morphometric Analysis of fixed position landmarks identified on photographs of trout taken from 22 sampling sites across the catchment. Very clear, statistically significant differences in morphology (fish shape) were evident for all the 21 sampling sites (one sampling site was removed from the analysis due to small sample size) with Canonical Variate Analysis resolving 21 discrete phenotypic groups. Morphological structuring was evident across all spatial scales (large, medium and small). Analysis showed that genetic distance and geographic distance between morphological groups was significantly correlated with morphology of populations, with morphological groups that were most divergent from each other also being most genetically distinct and geographically more distant. The effect of landscape and environmental variables driving morphology of populations was tested. In-stream substrate composition, water pH, stream order, site elevation, river gradient and the number of houses per km2 (representative of urban area) were all found to have a significant effect on morphology of populations. However, once the effect on morphology on these environmental variables were accounted for the residual effect of genetic distance was not significant (chapter 3). To attempt to discriminate between three alternative population genetic hypotheses for the origin of two alternative life history strategies in brown trout; freshwater residency and anadromy, the genetic structuring of brown trout was examined between life history strategy (anadromy or resident), between three sites and across two years (2013/2014) for brown trout collected from the Foyle catchment. There was no evidence of population structuring being attributed to life history strategy (that is no genetic differences between anadromous or resident trout). There was however strong and clear evidence of five genetic populations based on geographical site. Two sympatric populations were identified at each of two locations. However, both populations in each river were composed of both freshwater resident and anadromous brown trout, although the frequency of each life history strategy significantly differed between these rivers. The results of this study support the concept that partial migration in brown trout is most likely driven by a quantitative threshold trait, where the threshold trait value varies both between populations and between individuals within populations (chapter 4). It is critical, for effective management of the relatively high economic value anadromous component of brown trout populations in a catchment, to be able to identify which tributaries are contributing most to their production. A Genetic Stock Identification (GSI) analytical framework was used to determine the tributary of origin for anadromous brown trout captured from a mixed stock within the River Faughan sub-catchment, River Foyle and to look for any evidence of straying. The results showed that three genetic populations from specific parts of the sub-catchment contributed disproportionately to the production of anadromous brown trout. There was also evidence of straying of anadromous trout, particularly to one tributary elsewhere in the catchment (chapter 5). Taken together this body of work has demonstrated strong genetic and morphological structuring amongst brown trout in this large dendritic catchment. Genetic structuring seems to be at its most extreme when driven by factors which could be regarded as anthropogenic. This raises questions about human effects on the process of genetic divergence. Morphological structuring was, if anything even stronger than genetic structuring. Although there was evidence of genetic divergence between populations of differing morphologies, this neutral genetic differentiation was not a significant driver of morphological variation once landscape and environmental variables, such as substrate composition, driving morphological differences were taken into account. This suggests that the environmental drivers of structuring are greater in magnitude than neutral genetic divergence. Examining genetic structuring between two common morphologies of brown trout (anadromous and freshwater resident) in more detail, revealed no genetic differentiation between life history strategies but there was evidence of differences in frequency of life history between populations. Using the genetic structuring of brown trout as a genetic baseline it was possible to determine which tributaries within the River Faughan sub-catchment produce anadromous brown trout. If some discrete populations in a catchment are contributing disproportionately to the anadromous trout population (as they are in the Foyle) there is a strong risk of over exploitation and a need for enhanced attention in the nursery areas for those populations. These results have significant implications for the management of all trout in the Foyle catchment and elsewhere.

Human essential hypertension is a highly heritable disorder with complex aetiology and is a major risk factor for cardiovascular events such as ischaemic heart disease and stroke. A combination of multiple environmental and lifestyle factors contribute to blood pressure variation alongside a strong genetic component. Only a small proportion of the genetic factors that regulate blood pressure in the population are currently known, although there is strong evidence that the adrenal cortex and the steroid hormones it produces contribute. Several research strategies have been utilised to dissect the genetic aetiology of hypertension, including candidate gene studies and association studies. Two recent genome-wide association studies aimed to identify variations associated with altered blood pressure and hypertension. A total of ten variants were identified with genome-wide significance after a combined analysis between the two consortia, including a polymorphism located within intron 3 of the CYP17A1 gene. This variant was reported to be associated with a systolic blood pressure increase of 1.16 mmHg. The CYP17A1 gene codes for a dual-function enzyme (17α-hydroxylase/17,20 lyase) expressed primarily in the adrenal cortex and gonads that plays a key role in the steroidogenic pathway. Mutations in its coding region and splice sites are known to cause a rare form of congenital adrenal hyperplasia, suggesting that more common genetic variations at this locus might result in more subtle effects on blood pressure. A detailed examination of the variation across the CYP17A1 locus was required to establish patterns of linkage disequilibrium and is presented in Chapter 3. Some information on the polymorphic variation in this region was already available in public databases but precise details on linkage disequilibrium and the corresponding haplotype blocks were lacking. The entire CYP17A1 gene was directly sequenced, including approximately 2.5kb upstream from the transcriptional start site, in 62 subjects drawn from a normotensive population. Polymorphic variations were identified mainly in the promoter and introns. Two seemingly unrelated blocks of SNPs were identified as being worthy of follow-up investigations, particularly those located in the promoter region, as these could be responsible for alterations in the transcriptional activity of the gene. A total of seven promoter polymorphisms were then genotyped in a larger hypertensive population where the relationship between SNPs was less clear. In Chapter 4 the effect of CYP17A1 genotype on intermediate corticosteroid phenotype is explored in a hypertensive population. Corticosterone, cortisol and androgen production were not significantly altered in the population when stratified by genotype for each polymorphism. However when further split by gender, increased cortisol excretion rates were found to associate with the minor allele at position -362 in males and at positions -1204 and -2205 in females. Ratios of various corticosteroid intermediary metabolites were also compared as indices of CYP17A1 enzymatic activity. Ratios of THDOC:THS were significantly reduced in the presence of the minor allele at positions -34, -1204 and -2205, suggesting increased 17α-hydroxylase efficiency. In addition, aldosterone excretion was significantly elevated in individuals with CC genotype at position -1877; an indirect genotype-dependent effect has been speculated. A bioinformatic search was conducted to identify putative transcription factor binding sites at the polymorphic locations. This is presented in Chapter 5. This confirmed the hypothesis that single base changes at each of the seven polymorphic sites could lead to altered transcriptional activity. Using reporter gene assays in vitro, the G allele at position -362 (rs248658) associated with greater transcriptional activity than the A allele. The T allele at position -1877 (rs138009835) was transcriptionally less active than its alternative C allele. Similarly, the C allele at position -2205 (rs2150927) showed lower activity than the T allele. These data provide strong evidence that common variation at this locus may be of functional significance. The studies in Chapter 6 investigate a potential regulatory role of microRNA (miRNA) at the CYP17A1 locus. MiRNAs are a class of small non-coding RNA molecules that have recently emerged as novel post-transcriptional regulators of gene expression. They function by targeting the 3’ untranslated region (3’UTR) of specific mRNAs and cause repression either through mRNA destabilisation followed by degradation, or by mRNA translational repression. Previous research utilised a siRNA approach to knock down Dicer, a protein required for miRNA maturation, and noted significantly increased CYP17A1 mRNA levels in the H295R human adrenocortical cell line. The investigation presented here cross-referenced bioinformatic analysis with microarray expression data in order to predict which adrenal miRNAs are most likely to regulate CYP17A1 expression. Predicted miRNAs also shown to be differentially expressed between normal and diseased adrenal tissue were then selected for further analysis. In vitro investigation involved artificial manipulation of the specific miRNA levels in H295R cells followed by measurement of CYP17A1 mRNA levels. Increased amounts of hsa-miR-320a significantly raised CYP17A1 mRNA levels, although subsequent reporter construct assays showed that this was not due to direct miRNA binding of the CYP17A1 3’UTR. The studies in this chapter are the first to demonstrate miRNA-mediated regulation of CYP17A1 expression. In summary, this work aimed to investigate polymorphic variation in the human CYP17A1 gene and its association with hypertension. Patterns of linkage disequilibrium across the CYP17A1 gene were identified and the association of several polymorphisms with intermediate corticosteroid phenotype examined. The functional effects of candidate polymorphisms have also been assessed in vitro. Further studies will be required to determine whether observed changes in transcriptional activity are the direct result of altered transcription factor binding at polymorphic sites. Finally, the role of miRNA in the post-transcriptional regulation of CYP17A1 has been confirmed.

Maternal antibodies to placental-specific antigens were not detected in third trimester maternal sera or in concentrated material adsorbed from maternal sera on a placental affinity column using immuno-fluorescence and gel precipitation techniques, A method was described for the dissociation of antigens of less than 100,000 daltons from soluble antibody-antigen complexes. This method was applied successfully to the dissociation of albumin-anti-albumin complexes formed in antigen excess. Application of this method to pregnancy serum followed by immunisation of rabbits with the dissociated material resulted in anti-SP1 response detectable on AACE. The molecular weight of SP1 is approx, 100,000 daltons and the bulk of SP1 was detectable in the retentate at the end of the dissociation. This argued against a conclusion that SP1 had been dissociated from immune complexes. Immunisation of rabbits uith soluble extract of a placenta which had been adsorbed over anti-human serum column induced AACE-detectable responses to very few serum proteins, PAPP-A, HPL, SP1 and several unidentified antigens found in placental tissue. Placental soluble extract which had in addition been adsorbed over anti-human lung column and anti-human amniotic fluid column induced responses to very few serum proteins, PAPP-A and HPL, Using these latter antisera two other antigens were identified in placental soluble extract (antigens a and b) which were not detectable in lung soluble extract or in non-pregnant serum. Antigen was also detected in pregnancy serum. By contrast, immunisation with unfractionated placental soluble extract induced extensive anti-serum response, anti-HPL and anti-SP1 but no detectable anti-PAPP-A response. The extent to which several serum proteins were denatured by a variety of commonly used dissociants of antibody-antigen complexes UBS assessed by changes in the protein's precipitating behaviour on one-dimensional AACE. AACE was found to be superior for this to double immunodiffusion. The smaller proteins studied were more resistant to irreversible denaturation, but the sample size was too small to seek a general relationship between molecular weight and denaturation. All proteins examined proved resistant to one hour exposure to 1o5 n KI-PBS and to pH 11 buffer. 1o5 n KI-PBS was used in the purification of PAPP-A by antibody affinity chromatography followed by DEAE-cellulose ion exchange and Sepharose 6B gel filtration. Double AACE arcs formed by PAPP-A in term maternal serum were also formed by partially-purified PAPP-A indicating that the antigenic variants had not been separated. The purification product contained PAPP-A which was 427-fold purified in terms of immunological reactivity on AACE, The high degree of purity was also suggested by studies with the radiolabelled purification product, 125I-PAPP-A was shown to have similar molecular weight and electro-phoretic mobility to maternal serum PAPP-A, Immune-precipitated [125]I-PAPP-A was analysed by 5% and 3% SDS-PAGE and found to contain a major radioactive component of approx, 180,000 daltons and a minor component of between 74,-93,000 daltons. The nature of the minor component has not been determined. It may be tentatively concluded that PAPP-A (mol. wt. 750,000) contains polypeptide subunits of approx. 180,000 daltons and may therefore be composed of up to four such subunits, A collaborative clinical study in which PAPP-A was assayed in the blood of women during the third trimester of pregnancy by AACE suggested that fetal sex may affect PAPP-A levels with males giving rise to higher levels than females. No significant difference in the levels of PAPP-A was detected when patients with babies affected with intrauterine growth retardation were compared with controls. No significant change in the mean concentration of PAPP-A was detected in the group of patients with gestational diabetes, but some very high values were found in this group. In the group of patients with insulin-controlled diabetes the mean concentration of PAPP-A was significantly reduced.