The Mexican axolotl Ambystoma mexicanum is a member of a family of salamanders with large nuclear genomes. The known geographical distribution and evolutionary history of the genus Ambvstoma makes it an ideal model system for the study of the evolution of repetitive DNA sequences in large genome animals. The genome of the axolotl has been examined by various methods, in particular by reassociation kinetic analysis and associated techniques on bulk DNA and by the molecular cloning of several members of the repetitive fraction of the genome. The haploid genome (37 pg) can be resolved into several distinct components. A foldback fraction (4.6%), a moderately repetitive fraction (43.3%), a slow repetitive fraction (20.4%) and a unique fraction (31.7%). Foldback regions appear to form randomly dispersed clusters. Members of the moderately repetitive fraction are predominantly interspersed amongst other members of the same fraction. At moderate fragment length only (57.5%) of the genome is arranged as moderately repetitive DNA interspersed with more slowly repeating sequences. Fragments of axolotl DNA have been cloned by recombinant DNA techniques. Clones containing axolotl DNA were selected. One clone (pAMW113l) has an apparent repetition frequency of 1480 in the axolotl genome and may be part of a larger fragment undergoing reamplification in the genome. A second clone (pAMW1184) is highly repeated with a short repeat length. Some family members appear to be scattered through the genome. Some members are transcribed on lampbrush chromosomes. A third clone (pAMW1199) has several features characteristic of bulk repetitive DNA. It has a complex organisation in the genome and may contain a number of shorter repeats present in the genome at different frequencies. These clones represent different aspects of the organisation of the axolotl genome. Together with the general genome analysis they provide a starting point for comparative evolutionary analyses of the genus Ambvstoma.

The chromatin provides a structural organization in which the DNA can be compacted up to 10,000-20,000 fold. Nevertheless, this compaction achieved by the chromatin structure has to be highly dynamic and controlled in order to allow the different vital processes of the DNA to occur such as transcription, replication, DNA repair, chromosome segregation and recombination (mitosis and meiosis). Nucleosomes are the basic unit of chromatin compaction that are positioned throughout the vast genomic DNA in higher eukaryotes. A nucleosome consists of a pair of each histone protein H2A, H2B, H3 and H4 and the associated 147 base pairs (bp) of DNA. They are important contributors to overall chromatin organization. Structure Specific Recognition Protein 1 (SSRP1) is an HMG protein that has been investigated in Human. We have also carried out a Small interfering RNA (siRNA) strategy to reduce the expression of hSSRP1 in endothelial cells. The knocked down cells showed a clear reduction of beta-tubulin microtubules in the mitotic spindle and errors in their organization that led to a poor alignment of the chromosomes and missegregation. Furthermore, DNA repair and cytokinesis were also affected in the siRNA knockdowns. Immunolocalization of hSSRP1 and hSPT16 have shown that both could be involved in DNA repair when localising to the chromatin forming the FACT complex but also they could be deeply involved in spindle formation and organization in higher eukaryotes. Especially, since hSSRP1 localises in the centrioles.

An absence of dystrophin in muscle has a massive impact throughout muscle development, and Duchene Muscular Dystrophy (DMD) is one of the consequences. The disruption of the dystrophin-glycoprotein complex (DGC) is caused by a mutation in the dmd gene, which effects muscle integrity, resulting in progressive muscle degeneration and weakness. In this study, dfd13 (dystrophin-deficient) and C2C12 (non-dystrophic) myoblasts were cultured in low mitogen conditions for 10 days to induce differentiation; however, dfdl3 myoblasts did not achieve terminal differentiation. It has been suggested that Pax7 may play a major role during myogenesis, therefore its expression pattern and transport protein were examined for any impairments. It was established that Pax7 localises in the cytoplasm of dystrophindeficient myoblasts and high expression is retained during differentiation. Colocalisation of Pax7 with subcellular markers analysis indicated that Pax7 is synthesised during the proliferative state. Pax7 was shown to possess a nuclear location signal and KPNA2 was suggested as escort protein for Pax7 translocation into the nucleus. The PTEN-PI3K/Akt signalling pathway was investigated and protein synthesis regulation and Fox03 was found to be impaired. Autophagy related genes were found to be highly expressed; however, LC3 lipidation and autophagy flux showed a reduction upon differentiation, indicating defective autophagy. The contribution of PTEN overexpression was assessed in relation to endoplasmic reticulum (ER) stress and activation of the unfolding protein response (UPR). It was established that a reduction in ER stress and changes to UPR activation lead to apoptosis. Finally, minidystrophintransfection of both types of myoblasts was utilised to examine the effect, especially in dystrophin-deficient myoblasts. Minidystrophin improved protein synthesis activation and increased autophagy (increased LC3 lipidation), suggesting that minidystrophin ameliorates dystrophic events at the level of autophagosome formation. To conclude, destabilisation of the plasma membrane owing to a dystrophin mutation causes cell signalling alterations which minidystrophin restoration can partly improve.

In the last 15 years, the increased use of high throughput biology techniques such as genome-wide gene expression profiling, fitness profiling and protein interactomics has led to the generation of an extraordinary amount of data. The abundance of such diverse data has proven to be an essential foundation for understanding the complexities of molecular mechanisms and underlying pathways within a biological system. This thesis demonstrates the capabilities and applications of using biological networks to extrapolate biological information from the wealth of data available in the yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. This study marks the first time a mutual information based network inference approach has been applied to a set of specific genome-wide expression and fitness compendia. In particular, this work has generated hypotheses in S. pombe that have led to a deeper understanding of the relationship between ribosomal proteins and energy metabolism, a recently discovered pathway termed riboneogenesis. Experimental validation of this hypothesis has led to new theories on the role of energy metabolism enzymes in controlling ribosome biogenesis in S. pombe, including the novel finding that fructose-1, 6-bisphosphatase (FBP1) may have roles in both gluconeogenesis and riboneogenesis. This thesis also demonstrates how the use of multi-level data allows for comprehensive insight into nuclear functions of the S. pombe nonsense-mediated mRNA decay protein, UPF1. This study provides substantial evidence demonstrating the role of UPF1 in DNA replication. The applicability of fitness data in identifying targets of metal and metalloid toxicity in S. cerevisiae has also been investigated.

This research aimed to investigate the molecular regulation of odontoblast behaviour during primary, secondary and tertiary (reactionary and reparative) dentinogenesis. Using gene microarray analysis and sq-RT-PCR, evidence is presented that the changes in secretory activity of odontoblasts reflect differential transcriptional control and that common regulatory processes may exist between dentine and bone. The p38 gene was shown to be highly expressed in odontoblasts during active primary dentinogenesis, but was drastically down-regulated as cells become quiescent in secondary dentinogenesis. Based on the hypothesis that parallels between development and healing processes in teeth exist, the results suggested that the p38- MAPKinase pathway may be activated during odontoblast stimulation in tertiary dentinogenesis by both p38 phosphorylation and enhanced nuclear translocation, supporting a recapitulation of events from primary dentinogenesis during tertiary dentinogenesis. The feasibility of use of the mouse as an in vivo model for studying pulpal healing in response to restorative procedures was also assessed. This approach provides a novel opportunity to exploit use of genetically modified animals to explore cellular and molecular processes during reparative events. Lastly, a transgenic mouse model was used to analyse the possible role of Msx2 transcription factor in odontoblast differentiation. The nature of the tooth phenotype in Msx2 null mutant animals was subsequently analysed. This study has increased our understanding of the regulation of dentinogenic events, which may allow translation into new therapies aimed at maintenance of the vitality of the pulp. L’objectif de ce travail a été d’explorer les régulations moléculaires au sein de l’odontoblaste au cours des dentinogénèses primaire, secondaire et tertiaire. Les résultats obtenus à partir de micro-puces et de PCR semi quantitative ont permis de mettre en évidence un contrôle différentiel transcriptionnel de l’activité sécrétrice de l’odontoblaste. Des processus de régulation commun entre la dentine et l’os sont également discutés. La forte expression du gène p38 au cours de la dentinogénèse primaire, est drastiquement diminuée dans l’odontoblaste mature. Les résultats suggèrent également que la voie de signalisation p38-MAPKinase pourrait être activée pendant la dentinogénèse tertiaire, par la phosphorylation de la protéine p38 et sa translocation nucléaire, confirmant ainsi la récapitulation d’un processus du développement initiale de la dent dans celui de la cicatrisation pulpaire. L’utilisation de la souris comme nouveau modèle de laboratoire pour étudier la cicatrisation pulpaire est également décrite. Ce nouveau modèle constitue une opportunité réelle car il permet d’utiliser les animaux génétiquement modifiés pour explorer les processus cellulaires et moléculaires impliqués dans la réparation pulpaire. Enfin, un modèle de souris transgénique a été utilisé afin d’analyser l’éventuel rôle du facteur de transcription Msx2 dans le processus de différentiation odontoblastique. Le phénotype dentaire du mutant nul Msx2 -/- est analysé en détail. Ce travail permet de compléter les connaissances sur la régulation moléculaire de la dentinogénèse, étape importante pour le développement de nouvelles thérapeutiques en terme de conservation de la vitalité pulpaire.

Histone modifications form an important part of the epigenetic landscape that controls many aspects of cellular function, including regulation of gene expression and cell differentiation. The persistence and inheritance of many of these modifications through the cell cycle and differentiation are still unknown. Here, I show global epigenetic karyotypes of metaphase chromosomes labelled to highlight specific marks. Metaphase is transcriptionally inactive and so epigenetic marks here are not simply reflective of gene transcription. I found that histone marks such as H3K27me3 are inherited through differentiation, whereas others such as H4K20me3 have re-organised distributions. FISH analysis allowed the alignment of genetic features with H3K4me3 and H3K27me3 distributions, showing that these marks are correlated with increased gene density, revealing a deeply intertwined distribution in ES cells, indicating bivalency. Focusing on the Hoxa cluster using N-ChIP in ES cells allowed the analysis of histone modification prevalence at the single gene level in ES cells. Most histone modifications remain stable between G1/S phase and G2/M phase, although H3K9ac decreases in ES cells at G2/M. Results for bivalent modifications show permissive chromatin environments denoted by high H3K4me3 and low H3K27me3 methylation at gene promoters that are expressed soon after the onset of differentiation, denoting a predictive chromatin signature. This signature was altered after five days of differentiation, where H3K4me3 increases and H3K27me3 decreases at most Hoxa promoters, concomitant with the rise in expression of some Hoxa genes, displaying the dynamic properties necessary to represent a mechanism for control of transcription during differentiation.

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).

The modification of natural nucleic acids or synthesis of novel DNA mimics can facilitate new structure, function and properties. In particular, the use of modified nucleic acids for applications in biosensing has become a popular field of study given the desire for rapid and reliable theranostic devices. The aim of the projects detailed in this thesis was to study a range of DNA modifications, with a view to gaining an enhanced understanding of their effects on DNA structure, but also on their ability to act as sensing platforms for the extraction of important biological information stored within DNA targets. The four projects discussed include: organometallic mimics of DNA based on ferrocene (FcNA) and corresponding FcNA-DNA conjugates and their effect on structure; FcNA-DNA conjugates that bind mercury; redox-active macrocycles incorporated into DNA as SNP sensors and DNA labeled virus particles that probe the presence of pathogens via a bionanoparticle supported PCR reaction which can be monitored by Linear Dichroism spectroscopy.

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.