Accurate chromosome segregation during meiosis requires the reciprocal exchange of DNA between homologous chromosomes, via a process called homologous recombination, resulting in the formation of crossovers (COs). This process begins with the formation of programmed DNA double-strand breaks (DSBs). Certain genomic loci, called hotspots, are more likely than others to produce DSBs. This is thought to be determined by various factors, which include post-translational histone modifications, such as H3K4 trimethylation. The histone methyl-transferase AtSDG2 is largely responsible for the deposition of this histone mark. This research shows that CO frequency and distribution are altered in an \(Atsdg2\) background. Study of a mutant allele of a gene which codes for a subunit of a histone-acetyl transferase complex, called AtMRG2, revealed a strongly reduced fertility phenotype and failure to produce DSBs. Further study revealed that the defects were due to mutation to the \(AtPRD3\) gene, known to be essential for DSB formation, and that the mutation to \(AtMRG2\) was not responsible. During meiosis, homologous recombination takes place in the context of specific structural arrangement of DNA organised as an array of loops emanating out from a proteinaceous axis, a major component of which is AtASY1. My studies demonstrate that the dynamics of AtASY1 are affected by mutation to AtPCH2, an AAA+ ATPase, and that formation of the synaptonemal complex is perturbed. \(Atpch2\)mutants initiate DSB formation and CO designation normally, but defects occur in CO maturation, causing a reduced CO frequency and formation of univalent chromosomes at metaphase I. Finally, the effects of temperature on the structure of meiotic chromosomes and homologous recombination were studied by cytological analysis of Col 0 and different meiotic mutants subjected to a range of temperatures for the duration of meiosis. I have demonstrated that certain mutations, such as \(Atsdg2\) and \(Atpch2\), confer some resistance to the effects of high (32˚C) temperature treatment, which causes various meiotic defects in Col 0.

CD248 is a pericyte-associated, mesenchymal stem cell (MSC) marker that is highly expressed during embryological life. This expression is down regulated during development, becoming restricted on lymphoid stroma to the capsule, but reappearing during inflammation, as well as in a number of disease states (Lax et al., 2007). CD248 has been shown to play a role in controlling the differentiation ofMSC to osteoblasts, both in vitro and in vivo, achieving this effect by modulating PDGFRsignalling, as treatment with the PDGFRinhibitor imatinib mesylate phenocopies the effects seen in the CD248·;. mouse (Naylor et al., 2012). Here we present evidence that CD248 is involved in the differentiation of MSC, via PDGFRsignalling, into lymphoid stroma progenitors both in vitro and in vivo. In adult mice expression of CD248 is detected on FDCs following immunisation. Using CD248·1- mice, we observe that FDC networks in CD248·1- mice do not form normally and lack the reticular, dendrite-like structure typical ofFDCs. This defect associates with a reduction in the functionality of the germinal centres. Embryonic development of lymph node stroma occurs in a stepwise manner with progressive upregulation of VCAM and ICAM on resident mesenchyme. In the adult stroma, recent work has established links between different stromal cell subtypes; Jarjour eta/. (2014) used a fate mapping technique to discover that marginal reticular cells are able to differentiate to follicular dendritic cells in response to immune challenge. Contrasting evidence shows that FDC in the spleen derive from ubiquitous perivascular precursors, likely to be pericytes (Krautler et al., 2012).

RNA polymerase (RNAP) \(\sigma\) factor must recognise and bind to specific DNA elements, usually AT-rich, in order to initiate transcription. At AT-rich regulatory regions or with more than one \(\sigma\) factor binding site; RNAP has to distinguish between different targets to initiate transcription correctly. At two regulatory regions: i) cbpA regulatory DNA with overlapping binding sites for \(\sigma\)70 and 38 associated RNAP and ii) regulatory region for ehxCABD operon with AT content of 71 %, I examined how correct RNAP binding is ensured. For cbpA regulatory region it was found that the shared promoter spacer region played a key role. I identified a location in spacer region that differently affected overlapping cbpA promoters. The base change at this position is sensed by \(\sigma\)70 side chain R451. Alterations in spacer sequence modulate conformation, making it easier, or more difficult, for R451-DNA interactions. Using tethered particle motion analysis, DNA compaction properties of cbpA gene product; CbpA was measured. ehxCABD regulatory region contains many sequences resembling \(\sigma\) factor binding elements. RNAP is capable of binding to the correct promoter elements in this region only in the presence of a chromosome folding protein, H-NS which binds AT-rich DNA. H-NS “coats” ehxCABD regulatory region and enables specific RNAP binding. Finally, many intragenic promoters within ehxCABD operon were identified. We thus propose that H-NS plays a role in silencing this pervasive intragenic transcription.

Since the human genome project was completed in 2003, extraordinary progress has been made in the field of genomics with the development of new sequencing technologies and the widespread introduction of next generation sequencing (NGS). The application of NGS initiated a new era in genomics by massively increasing the number and diversity of the sequenced genomes at lower cost. Human Molecular Genetics has greatly benefited from the use of NGS-based strategies to identify human disease genes. In this thesis, I investigated the application of genetic techniques to investigate the molecular basis of autosomal recessively inherited disorders of unknown etiology. A range of disease phenotypes, including oligodontia and fetal akinesia/multiple pterygium syndrome (FA/MPS), were investigated in patient cohorts that included many cases with parental consanguinity. Using an autozygosity linkage analysis-based approach and Sanger sequencing of candidate genes resulted in the identification germline RYR1 mutations in FA/MPS. Subsequently, using exome sequencing techniques, the molecular basis of FA/MPS was further elucidated by the identification of germline mutations in RYR1, NEB, CHRNG, CHRNA1 and TPM2. The application of NGS in genetically heterogeneous disorders such as fetal akinesia/multiple pterygium syndrome can enable better and less expensive molecular diagnostic services aimed at specific mutation spectra, though more extensive sequencing can lead to the identification of larger numbers of variants of uncertain significance.

Drug toxicities represent a major problem in drug discovery and development; therefore there is a push to develop new technologies to detect these early on. In this thesis I investigated the utility of zebrafish embryos and larvae in evaluating the biological activity of novel compounds and developed new methods for assaying the potential toxic effects of drugs in vivo. An electrocardiogram (ECG) recording set-up for zebrafish embryos and larvae was developed to assay drug-induced cardiotoxicity. The set-up was validated by testing drugs known to induce cardiotoxicity in humans in zebrafish larvae. The results obtained were in agreement with those documented in humans demonstrating the utility of the zebrafish larva in detecting drug-induced cardiotoxicity. The zebrafish embryo was also found to be a useful model for probing the biological activity of novel and marketed compounds providing an insight into the relationship between chemical properties and biological effects. Additionally, the assessment of the anti-inflammatory activity of a set of reference drugs revealed that the zebrafish larva also presents a promising model for therapeutic drug screens. Overall, the results described in this thesis show that the zebrafish presents an effective, reliable and rapid model for assessing the biological activity of drugs in vivo.

This thesis investigated reconstitution of CMV-specific T-cells in two cohorts of HSCT patients and studied the potential role of Tumour Necrosis Factor Receptor 2 (TNFR2) in regulation of CMV-specific T-cell expansion post HSCT. The first cohort included patients of a randomized phase II trial of adoptive cellular therapy for CMV-specific CD8\(^+\) T-cells. Cellular therapy resulted in earlier and greater expansion of CMV-specific CD8\(^+\) T cells and also reconstitution of CMV-specific CD4\(^+\) and non-infused CMV-specific CD8\(^+\) T-cells. The number of infused therapeutic T-cells and circulating levels of Alemtuzumab were found to influence immunotherapy. Additionally, reconstitution of CMV-specific CD4\(^+\) T-cells was studied using HLA-class II tetramers. CMV-specific CD4\(^+\) T-cell count of >0.7x10\(^3\)/ml was found to protect from recurrent CMV reactivation. One third of specific CD4\(^+\) T-cells were perforin and granzyme-B positive indicating cytotoxic potential, whilst the majority expressed T-bet. Expression of CD57 molecule on CD4\(^+\) T-cells was demonstrated as a potential biomarker of immune response to CMV. Also, distinct cytokine receptor expression patterns in naïve versus memory T-cells were observed. The results showed rapid decrease in IL-6R and increase in expression of TNFR2 after T-cell differentiation from naïve to effector cells and engagement of TNFR2 led to the apoptosis of CMV-specific T-cells.

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.

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.

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 chloroplast protein CP12 is present in almost all photosynthetic organisms. This protein has been shown to regulate the activity of two enzymes of the Calvin-Benson cycle, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK). The regulation of these enzymes is achieved by the reversible formation of this multiprotein complex in response to a change in light intensity. In Arabidopsis, there are three CP12 genes, CP12-1, CP12-2 and CP12-3. Expression analysis of these genes suggested that they may have a wider role in non-photosynthetic plastids through the plants’ life cycle and that their function may not be restricted to the Calvin-Benson cycle. The main aim of this study was to determine the functional significance of having three CP12 isoforms and to explore the importance of each individual isoform in vivo. This was done by using Arabidopsis thaliana T-DNA mutant and RNAi transgenic lines with a reduced level of CP12. Our results revealed that single mutant lines did not develop a severe growth phenotype. However, a reduction in the transcript of more than one CP12 gene, in a number of multiple lines, led to a significant reduction in photosynthetic capacity at early stages of development and a severe growth phenotype, including reduced fresh and dry weight, number of leaves and seed yield, as well as affected lateral roots formation. Complementation analysis of CP12-1 in the triple mutant revealed that two out of the three lines rescued the phenotype by showing normal growth and development, confirming the importance of CP12. Our results suggest that the CP12 protein family is essential for normal growth and development and that these proteins are likely to have additional functions apart from the regulation of Calvin-Benson cycle enzymes.