The investigation of the mechanisms governing the control of chromosomal DNA synthesis and cell division is fundamental to the understanding of the regulation of the cell cycle in <I>Escherichia coli</I>. Research over many years has shown that two proteins are central to these processes: DnaA in initiation of chromosomal replication and FtsZ in cell division. DnaA and FtsZ are not only thought to be essential to the biochemistry of these events but appear also to be involved in their timing within the cell-cycle. For this reason expression of both the <I>DnaA</I> and <I>ftsZ</I> genes are regulated by a number of different mechanisms, presumably to ensure efficient growth and division of the organism under a wide range of environmental conditions. In addition to its aforementioned role, DnaA also functions as a regulatory protein in the expression of a number of genes. Its effect is mediated by a particular recognition sequence present at its site of action, known as the DnaA-box. Such a sequence is present in the promoter region of the <I>dnaA</I> gene and has in the past been reported to be involved in autoregulation of this gene. In this work this putative role of the DnaA protein is reassessed and cast in doubt. The promoter region of the <I>ftsZ</I> gene also contains a number of DnaA boxes and some evidence exists that DnaA may be involved in <I>ftsZ</I> regulation. In this work evidence is presented to the contrary and it is shown that the apparent role of DnaA in <I>ftsZ</I> regulation is probably due regulation of the gene by a growth-rate sensitive mechanism.

Borealin is a novel fourth subunit of the vertebrate chromosomal passenger complex containing aurora-B kinase, INCENP, and surviving, which has essential regulatory roles at centromeres and the central spindle in mitosis. Co-immunoprecipitation experiments of endogenous proteins suggest that essential all of surviving, the majority of INCENP, and approximately half of aurora-B are complexed with borealin in mitotic cells. We also detected a sub-complex containing aurora-B and INCENP, but no borealin or surviving. Results from sucrose gradient sedimentation experiments suggest that there are high molecular weight complexes containing chromosomal passengers, which may contain nucleosomes. Binding experiments <i>in vitro</i> revealed a strong direct interaction of borealin with surviving, suggesting that it is the main binding partner of borealin in mitosis. Borealin also binds itself <i>n vitro,</i> and this interaction is detectable <i>in vivo</i>. We investigated the role of borealin within the complex and present evidence that, in contrast to INCENP and surviving, borealin is unlikely to be involved in the regulation of aurora-B kinase activity, but may be the subunit that targets the aurora-B and polo-like kinase 1, another essential mitotic kinase.  Depletion of borealin by RNA interference delays mitotic progression and results in kinetochore-spindle mis-attachments and an increase in bipolar spindles associated with ectopic asters. The extra poles severely disrupt the partitioning of chromosomes in anaphase, revealing an unexpected role for the chromosomal passenger complex in the maintenance of mitotic spindle integrity. These studies have identified novel non-histone components of mitotic chromosomes, one of which, nuclear protein p30, is likely to play a role in centromeric chromatin structure.

In <i>Drosophila melanogaster </i>females, upon completion of recombination, dramatic reorganisation of the oocyte nucleus occurs and meiotic chromosomes form a compact cluster called the karyosome within the enlarged oocyte nucleus. However, little is known about the mechanism or regulation of karyosome formation. In this thesis, I describe the role of the conserved kinase nucleosomal histone kinase-1 (NHK-1) in karyosome formation. I identify a novel substrate of NHK-1, barrier-to-autointegration factor (BAF), a protein that acts as a linker between the nuclear envelope and chromatin. I find that both a reduction in NHK-1 levels and expression of non-phosphorylatable BAF in oocytes disrupt karyosome formation, resulting in the ectopic association of meiotic chromosomes with the oocyte nuclear envelope. I propose NHK-1 phosphorylates BAF to release meiotic chromosomes from tethering at the nuclear envelope, allowing karyosome formation in the oocyte nucleus. I also show that activation of the meiotic recombination checkpoint maintains the attachment of meiotic chromosomes with the oocyte nuclear envelope and delays post-recombination nuclear reorganisation. Critically, I demonstrate reduction in NHK-1 activity on meiotic recombination checkpoint activation. Therefore, I propose that NHK-1 is a target of the meiotic recombination checkpoint, and has a pivotal role in orchestrating the post-recombination reorganisation of the oocyte nucleus in <i>Drosophila </i>female meiosis.

The evolutionary significance of genetic recombination is one of the most intriguing problems in evolutionary biology, since recombination is one of the primary features of sexual reproduction. Of the vast number of questions that one can ask in relation to sex and recombination, the simplest is: what happens in their absence. It was realised long ago that the level of recombination influences the action of natural selection. Non-recombining genomes are expected to adapt more slowly than recombining ones. In addition, they are also more prone to degeneration by the accumulation of harmful mutations. This study is concerned with the latter process: the fate of a non-recombining genome or chromosome that is continuously subject to recurrent mutation to deleterious alleles. H.J. Muller argued that one major difference between a non-recombining asexual and a recombining sexual population is that, in the former, genetic drift can overwhelm selection against deleterious mutations, whereas this not so likely in the presence of recombination. In the absence of recombination, deleterious mutations can therefore accumulate by what Muller called a ratchet-like process. This study focuses on four aspects of this mechanism. First, the quantification of its rate is examined, both by simulation methods and by analytical approximations, under the simplest possible model. Second, its interaction with another phenomenon, the continuous elimination of strongly deleterious alleles, is studied. Third, its effects on neutral DNA polymorphism, and the possibility of detecting its action by measuring these effects, are studied. Fourth, the circumstances under which this process may have significance for the evolution of non-recombining Y chromosomes are analysed. It is hoped that this work represents a useful contribution to a better understanding of Muller’s ratchet. The overall conclusion is that, given the present empirical knowledge on rates and effects of deleterious mutations and on levels and patterns of variability on Y chromosomes, Muller’s ratchet may well be significant in driving their evolution, even in species with large populations.

The NuRD (Nucleosome Remodelling and Deacetylation) complex is a highly conserved and abundant transcriptional repression complex. Embryonic Stem (ES) cells lacking a central structural component of the complex, Mbd3, proliferate slowly and are unable to differentiate in the absence of LIF. We show here that in addition they show significant demethylation of DNA at a number of repetitive sequences, such as the centromeric repeats and IAP elements, as well as some single-copy DMRs and CpG islands throughout the genome. This demethylation is associated with histone hyperacetylation and aberrant transcription of these sequences or, in the case of regulatory regions, genes controlled by them. Many of the sequences demethylated here are also demethylated in <i>Dnmt3a/3b^-/-</i> ES cells, and interestingly <i>Mbd3^-/- </i>ES cells contain significantly reduced levels of the methyltransferase<i> Dnmt3b. </i>However <i>Dnmt3b^-/- </i>ES cells are demethylated to a lesser extent than <i>Mbd3^-/-</i>cells, suggesting that a decrease in Dnmt3b levels is not the sole cause of the demethylation observed here. An interaction has been detected between Dnmt3b and the NuRD components Mta2 and Mbd3 in wild type cells, leading to speculation that they may co-operate to bring about a repressive chromatin structure at target sites, but chromatin immunoprecipitation of a tagged version of Mbd3 and another NuRD component, Mi-2β, does not reveal binding of the complex at affected sequences. However immunofluorescence of Mbd3, Mta2 and Mi-2β reveals localisation to centromeric regions in approximately 30% of wild type cells, suggesting localisation is cell-cycle dependent. The degree of centromeric localisation of Mta2 is significantly reduced in <i>Mbd3^‑/-</i> cells. We propose that NuRD binds to target sequences in a cell cycle dependent manner, where it participates with DNA methyltransferases in the formation and/or maintenance of a repressive chromatin structure.

A detailed physical map of restriction endonuclease cutting sites has been determined for the region surrounding the transcription-termination site at the end of the early operon of coliphage T7. Using this information and the chain-terminating method for sequencing DNA, a nucleotide sequence 370 base-pairs in length has been determined corresponding to the DNA between 18.88 and 19.81 on the T7 physical map. By comparison with the data of others, including nucleotide sequences for regions of known physiological importance, several interesting functional sites have been tentatively identified within the sequence presented. These include: 1) the termination site (map position 18.96) for transcription of the early operon by E.coli RNA polymerase; 2) two sites (19.35 and 19.65) likely to act as promoters for the initiation of class II transcripts by T7 RNA polymerase; 3) two regions (18.89-19.31 and 19.34-19.61) each of which includes a plausible ribosome-binding site and which probably code for a pair of previously unreported class II genes tentatively designated genes 1.4 and 1.5, respectively; 4) the ribosome-binding site (position 19.65) and initial 18 codons of the class II gene 1_7. A particularly striking feature of this sequence is the frugal arrangement of several of the proposed functional sites so that two perfect, direct repeats of 21 base-pairs contain the stop-codons for the putative genes 1.4 and 1.5, the two class II promoters, and the ribosome-binding sites for genes 1.5 and 1_7i respectively.

Type 2 diabetes is an age-related condition and is characterised by a progressive decline in insulin secretion. Mitochondria play a key role in energy generation for insulin secretion. We previously reported an age-related decline in mitochondrial DNA (mtDNA) copy number in isolated human islets. TFAM, mtDNA Transcription Factor A, regulates mtDNA transcription and mtDNA copy number. Aims: We aimed to replicate the percentage decrease in mtDNA copy number that we observed with ageing in human islets, and to explore whether this affected mitochondrial function and insulin secretion. Methods: Two independent models of mtDNA depletion were created. The first model knocked down TFAM gene expression using siRNA technology. The second model subjected cells to didanosine, a nucleoside analogue of adenosine with a high affinity to POLG, a mtDNA polymerase. Results: Both models produced comparable levels of mtDNA depletion. Upon investigating the effects of partial mtDNA depletion on mitochondrial function, we found that both mtDNA depletion models displayed reduced mtDNA gene transcription and translation. However, neither model of mtDNA depletion affected ATP content or mitochondrial membrane potential. Glucose-stimulated insulin secretion was decreased following mtDNA depletion in the TFAM knock down cells which was rescued following treatment with the insulin secretagogue glibenclamide. Conversely, didanosine-induced mtDNA depleted cells showed increased insulin secretion. Conclusions: Both models generated a similar degree of mtDNA depletion, which was comparable to the percentage decrease seen in human islets with ageing. Both models were seen to impair mitochondrial function, but with opposing effects on insulin secretion. The TFAM model findings are in line with previous studies of severe mtDNA depletion, suggesting that the increase in insulin secretion seen with didanosine is due to drug off target effects. Strategies to slow islet mtDNA depletion in man could help to preserve insulin secretion and delay the development of Type 2 diabetes.

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS) is a technique by which protein profiles can be rapidly produced from biological samples. Proteomic profiling and biomarker identification using MALDI-ToF MS have been utilised widely in microbiology for bacteria identification and in clinical proteomics for disease-related biomarker discovery. To date, the benefits of MALDI-ToF MS have not been realised in the area of mammalian cell culture during bioprocessing. This thesis explores the approach of ‘intact-cell’ MALDI-ToF MS (ICM-MS) combined with projection to latent structures – discriminant analysis (PLS-DA), to discriminate between mammalian cell lines during bioprocessing. Specifically, the industrial collaborator, Lonza Biologics is interested in adopting this approach to discriminate between IgG monoclonal antibody producing Chinese hamster ovaries (CHO) cell lines based on their productivities and identify protein biomarkers which are associated with the cell line productivities. After classifying cell lines into two categories (high/low producers; Hs/Ls), it is hypothesised that Hs and Ls CHO cells exhibit different metabolic profiles and hence differences in phenotypic expression patterns will be observed. The protein expression patterns correlate to the productivities of the cell lines, and introduce between-class variability. The chemometric method of PLS-DA can use this variability to classify the cell lines as Hs or Ls. A number of differentially expressed proteins were matched and identified as biomarkers after a SwissProt/TrEMBL protein database search. The identified proteins revealed that proteins involved in biological processes such as protein biosynthesis, protein folding, glycolysis and cytoskeleton architecture were upregulated in Hs. This study demonstrates that ICM-MS combined with PLS-DA and a protein database search can be a rapid and valuable tool for biomarker discovery in the bioprocessing industry. It may help in providing clues to potential cell genetic engineering targets as well as a tool in process development in the bioprocessing industry. With the completion of the sequencing of the CHO genome, this study provides a foundation for rapid biomarker profiling of CHO cell lines in culture during recombinant protein manufacturing.

The mechanisms for DNA repair in mitochondria is an area in which there is limited knowledge in comparison to the DNA repair mechanisms that have been defined in the nucleus. Although it is understood that mitochondria have less DNA repair mechanisms than in the nucleus there is still a lot more scope for identifying new proteins involved in the repair of mitochondrial DNA (mtDNA). The main focus of this thesis was to attempt to determine whether there was presence and activity of a DNA repair enzyme in mitochondria, namely tyrosyl-DNA-phosphodiesterase 1 (TDP1), and if so what is the exact role of this enzyme in mtDNA repair. This enzyme has already been characterised as an single strand break repair (SSBR) enzyme in the nucleus, and a mutation in this gene can cause the autosomal recessive disorder spinocerebellar ataxia with axonal neuropathy 1 (SCAN1). The data in this thesis provides evidence for the presence and activity of TDP1 in mitochondria and that the function of this enzyme on mtDNA is most likely limited to the removal of mitochondrial topoisomerase 1 (TOP1mt). It has also been shown that phosphorylation of amino acid 81 of TDP1 does not facilitate its interaction with DNA ligase 3α in mitochondria and that there most probably no direct link between these enzymes in this organelle, unlike that found in the nucleus. This data indicates that there is still potential for identification of more enzymes that are involved in mtDNA repair.

The presence of DNA double-stranded breaks in a mammalian cell typically activates the Non-Homologous End Joining (NHEJ) pathway to repair the damage and signal to downstream systems that govern cellular decisions such as apoptosis or senescence. The signalling system also stimulates effects such as the generation of reactive oxygen species (ROS) which in turn feed back into the damage response. Although the overall process of NHEJ is well documented, and much is known about downstream processes that together constitute the DNA damage response (DDR), we know little of the dynamics and how the system operates as a whole. To further our understanding of this we have constructed computational models which integrate current knowledge of the DNA repair process and key downstream signalling systems. The models are coded in Systems Biology Mark-up Language and BioNetGen Language and are quantified as far as possible with experimental data generated within our own laboratories or otherwise gathered from the literature. They are designed to simulate the observed stochastic dynamics of repair by DNA Protein Kinase (DNA-PK) dependent NHEJ (D-NHEJ) and back-up NHEJ mechanisms (B-NHEJ) following damage induced by gamma irradiation in human fibroblasts and the response this causes in the p53-p21 senescence signalling pathway. We have used the models to investigate a number of issues relevant to the study of ageing cells. Our work suggests that this observed heterogeneity in the repair of DNA damage foci that is influenced by levels of damage cannot be explained solely by inherent stochasticity in the NHEJ system. We find that the presence of multiple repair mechanisms and the modulation of key repair factors by oxidation along with further damage inducing feedback triggered by p53 and changes brought about by cellular processes such as senescence all play a cumulative role in causing the differences between stressed and unstressed cells. Our model highlights the importance of Ku oxidation which leads to increased Ku dissociation rates from DNA damage foci and shifts in favour of the less efficient B-NHEJ system. Furthermore we have utilised the model to investigate the role that various levels of DNA damage and repair have on the maintenance of the important p53 oscillations in a cell. We find that, contrary to the current view, p53 levels are affected by temporal dynamics of DNA damage and have used our model to inform the design of further experimental work to investigate the effect of iii maintained low levels of DNA damage induced by frequent low pulses of γ irradiation on the p53 mediated DDR.