Whilst the recruitment of phagocytic leukocytes is fundamental to the innate response against pathogenic infection, the inappropriate mobilisation of their cytotoxic potential can also lead to fatal tissue injury. To determine the contribution of Csk, a negative regulator of Src family kinases, to the regulation of phagocyte recruitment and activation in vivo, mice lacking Csk in the myeloid lineage were generated using conditional mutagenesis (cre/loxP). This Csk deficiency resulted in acute multifocal inflammation in skin and lung, accompanied by extramedullary haematopoiesis in the spleen and liver, and increased myelopoiesis in bone marrow. Animals were protected from the disease in a microbiologically controlled environment, but remained hypersensitive to LPS-induced shock. Csk-deficient granulocytes showed enhanced spontaneous and ligand-induced degranulation accompanied by hyperinduction of integrins. Hyperresponsiveness was associated with hyperadhesion and impaired migratory responses in vitro. Biochemical studies revealed spontaneous accumulation of tyrosine-phosphorylated proteins, including hyperphosphorylation of key signalling proteins including Syk and paxillin. These data support a breakdown of the activation threshold set by Csk. Thus, Csk is critical in preventing premature granulocyte recruitment through enforcing the requirement for ligand engagement while supporting the migratory capacity of activated cells through negative regulation of cell adhesion. To address the incomplete Cre mediated deletion of floxed genes in vivo, a genetic approach to elevate Cre recombinase gene expression was developed. Whilst manipulation of regulatory elements including promoter, enhancer, and untranslated regions has yielded enhanced and sustained expression in vitro, this has been difficult to achieve in vivo. Here, it is reported that construction of artificial exons through insertion of short heterologous intron sequences into the open reading frames of the Cre recombinase and enhanced green fluorescent protein results in functional expression accompanied by a 30-fold increase in transcription levels in vitro. Furthermore, green fluorescence levels were enhanced five-fold in cell lines and enhanced considerably in the rat brain after transduction with a herpes simplex virus-based vector. These data define a method of improving both the level and duration of recombinant gene expression, in addition to and independently of surrounding regulatory elements. Significantly, the method should help to increase Cre recombinase expression from weak or transiently expressed promoters thus overcoming an important limitation of Cre/loxP technology incomplete deletion. Furthermore, this method may also be applicable in gene therapy to obtain sustained and effective expression of recombinant proteins in vivo.

A thesis is presented which outlines investigations conducted to determine issues which may be faced should attempts be made to produce large plasmids (>3-10kb) for use in areas such as DNA vaccination and gene therapy. Initial studies attempted to manufacture a series of large Bacterial Artificial Chromosomes (116kb - 242kb) and centered on the fermentation and downstream purification steps required to produce the plasmid material. It was determined that these constructs were not suitable models due to the low yields at which they were propagated. Additionally, many of the analytical protocols employed were found to be inadequate for monitoring the concentration and form of large plasmids. In response to these issues large plasmids were constructed by the insertion of large fragments of S.cerevisiae DNA in to the relaxed, high-copy number vector pGEMl 1. Plasmids ranging from 3kb to 50kb were created and used to probe the upstream and downstream steps of a plasmid production process. Efforts were also made to develop more suitable analytical methods. The production of these plasmids was studied during pilot-scale fermentations at 37 C where it was discovered that the plasmids demonstrated a poor stability profile. In an attempt to improve plasmid stability, reduced temperature (25 C) fermentations were conducted which increased the stability and yields obtained from fermentations of these constructs. Cell paste from the fermentations was subject to a purification process involving alkaline lysis and ultrafiltration steps. Described are balances of plasmid DNA, genomic DNA, RNA and contaminating protein. The topology changes displayed by the plasmids are also described. Finally, studies are outlined describing the binding behaviour of the large plasmids to chromatographic resins used for plasmid purification.

The emergence of multiple-antibiotic resistance among clinical pathogens has created an urgent requirement for the development of new antibiotics. The current lack of new antibiotics has not only renewed interest in traditional natural product screening approaches, but also prompted efforts to develop alternate antimicrobial strategies. Antisense RNA based silencing provides a strategy for developing whole cell screening assays, whereby antisense RNA induction leads to target protein depletion and subsequently the increased sensitivity of test organisms to target specific inhibitors. The development of synthetic derivatives to expressed antisense RNA such as peptide Nucleic Acids (PNA), has also been explored for use as bacterial inhibitors. This thesis aims to examine two novel antimicrobial strategies, firstly by comparing mRNA and protein based techniques to evaluate essential gene requirement in bacteria, to identify novel targets for antibiotic screening assays. Secondly, to evaluate the potential use of peptide peptide-PNA’s as antimicrobials capable of targeting individual bacterial species. To successfully develop either approach requires the identification and validation of suitable gene encoded molecular targets. Essential genes may provide potential candidates, yet a suitable system is necessary for characterisation to enable genes to be ranked, so that the most suitable targets can be prioritized. A disproportionate growth requirement (stringency) is known to exist among essential genes, which provides a means to delineate between essentially required targets, yet is based upon the measurement of mRNA abundance. Due to post-transcription and translation mechanisms, mRNA does not provide a reliable indicator of expressed protein, which represents the ultimate output of gene expression. This study demonstrates the use of a quantitative proteomics strategy for evaluating essential gene stringency at the protein level, using the E.coli gene fabI. Using expressed antisense RNA silencing to deplete target protein concentration and to reduce normal growth rate to 50%, absolute protein determinations were used to define a Minimum Protein Level (MPL50), for the quantitative characterisation of essential gene stringency. To support the justification of evaluating gene stringency using expressed protein abundance, the stringency of operon based genes fusA and rplE using antisense RNA silencing was investigated and revealed transcript profiles that contradict the use of Minimum Transcript Level (MTL50) previously used to define gene stringency. Finally, to demonstrate a potential application that would benefit from the characterisation of essential gene stringency, the species-specificity of a peptide-PNA targeting the essential gene ftsZ was evaluated. Exposing a mixed culture of S.typhimurium and E.coli to a peptide-PNA conjugate, incorporating a 2 base pair mismatch demonstrated the capacity to inhibit translation of ftsZ in S.typhimurium but not E.coli. This study highlights how characterising essential genes using the MPL50 can be used to delineate stringently required gene targets to support antimicrobial screening and the development of species specific antimicrobials. Furthermore the applications of evaluating gene stringency may be extended further, to provide a tool for standardising genetic components in synthetic biology approaches.

Bacteria surround their cytoplasmic membrane with an essential, stress-bearing macromolecule, the peptidoglycan (PG) layer. During growth and division cells extend this layer through the action of membrane-anchored peptidoglycan synthases. Presumably, sacculus growth is facilitated by dynamic multiprotein complexes which are guided by cytoskeletal elements. These complexes contain all the necessary peptidoglycan synthases and hydrolases for the enlargement of the sacculus, along with their regulators. Biochemical and genetic data gathered in recent years provides evidence for the existence of these complexes, but the molecular mechanisms they employ, and how cell wall synthesis is coordinated with the synthesis of other cell envelope layers, remain largely unknown. In this work we have elucidated key biochemical features of a regulator of PG synthesis, LpoB. Its high resolution structure was solved by NMR spectroscopy and the interaction interface of LpoB with the major peptidoglycan synthase active during cell division in Escherichia coli, PBP1B, was determined. We show that LpoB interacts with the non-catalytic UB2H domain of PBP1B, situated between the glycosyltransferase and transpeptidase domains. Several other proteins have previously been implicated in the regulation of PBP1B. Here we optimised an in vitro glycosyltransferase assay and investigated the effect of interacting proteins on peptidoglycan synthesis. We have shown the first evidence that multiple interaction partners, LpoB and FtsN, exert a simultaneous synergistic regulatory effect on PBP1B GTase. We also identified novel, functional interactions of PBP1B. YbgF and TolA are both members of the Tol-Pal complex which is required for outer membrane stability and its proper invagination during cell division. TolA was shown to interact with PBP1B via its transmembrane region (domain I) and moderately stimulates the GTase activity of the synthase. YbgF also interacts with PBP1B and is the first example of a negative modulator of PG synthetic activity, inhibiting the regulation of PBP1B TPase by Lpo, which is relieved by TolA. We propose that YbgF and TolA function to fine tune the regulation of PBP1B during division which allows for a proper coordination between cell wall synthesis and constriction of the OM during division in E. coli. In summary, this work significantly enhances our understanding of the regulation of the bi-functional PG synthase active during cell division, PBP1B.

Mitochondrial DNA deletions are a primary cause of inherited and sporadic mitochondrial disease, whilst somatic mtDNA deletions contribute to the focal respiratory chain deficiency observed in post-mitotic cells associated with ageing and neurodegenerative disorders. As mtDNA deletions only cause cellular pathology at high levels of heteroplasmy, an mtDNA deletion formed within a cell must accumulate by a process known as clonal expansion to levels which result in biochemical dysfunction. The mechanism driving clonal expansion remains uncertain; this research aimed to investigate clonally expanded mtDNA deletions in sporadic and inherited mitochondrial myopathies in order to elucidate this mechanism. A number of different approaches were taken to assess the mechanism driving accumulation of mtDNA deletions. The effect of the mtDNA deletion size on clonal expansion was first investigated by assessing the longitudinal spread of mtDNA deletions in single muscle fibres isolated from patients presenting with mtDNA maintenance disorders; no relationship was found to exist between mtDNA deletion size and the area over which the mutation has accumulated. A longitudinal study was carried out using tissue acquired over a 13-year period from a single patient with a sporadic mtDNA deletion, to identify whether the mtDNA deletion heteroplasmy level continued to increase over time, as would be expected if the mutation displayed a selective advantage over wildtype mtDNA – however, both the genetic and biochemical defect were found to be stable over time in this patient. A subsequent study aimed to identify a correlation between mtDNA deletion size and heteroplasmy levels at the whole tissue level in a cohort of patients with sporadic single mtDNA deletions, but no evidence was found to suggest that larger mtDNA deletions accumulate to higher levels of heteroplasmy. Finally, single cytochrome c oxidase- deficient muscle fibres were investigated using single-molecule PCR to assess whether clonal expansion of multiple mtDNA deletions could be observed in single cells. Evidence of multiple clonally expanded mtDNA species was found in approximately 40% of all examined fibres, with no correlation between mtDNA deletion size and level of accumulation. Each of these four studies has highlighted accumulation by random genetic drift to be the most likely mechanism for clonal expansion of mtDNA deletions in human muscle; no evidence has been found to support the presence of a selective advantage for mtDNA deletion species over wildtype mtDNA.

The destruction of articular cartilage is a central feature of arthritis. The activity of collagenase enzymes, induced by pro-inflammatory cytokines, is a key step in this process. Collagenases have been targeted therapeutically but trials have not proven beneficial due to off-target effects. Understanding the signalling consequences which drive the expression of these proteolytic enzymes is therefore a major area of research. The aim of the present study was to elucidate the signalling events that regulate collagenase expression, focusing on the role of a small family of serine/threonine kinases termed protein kinase D (PKD). To understand the role of the individual PKD isoforms in the modulation of collagenase gene expression, each isoform was selectively silenced. Using a model of the pro-inflammatory milieu prevalent in arthritic disease, the consequences of isoform specific gene silencing on the expression of MMP-1 and MMP-13 was studied in primary chondrocytes stimulated with IL-1 in combination with OSM. Data suggest PKD1 to be ‘anabolic’, with gene silencing leading to increased collagenase gene expression. PKD2 was shown not to significantly modulate the collagenase expression, whereas, PKD3 silencing markedly reduced the collagenase gene expression. To understand the signalling consequences orchestrated by each isoform of PKD, the role each isoform in the regulation of signalling pathways known to modulate collagenase gene expression was examined. PKD1 and PKD3 silencing both abrogated the phosphorylation of the MAPK signalling pathways (ERK, JNK and p38). PKD3 silencing also led to decreased STAT-1 and STAT- 3 serine phosphorylation. This contrasted with the effects of PKD1 silencing, in which STAT-1 serine and tyrosine phosphorylation increased, as well as increased Akt and p65 phosphorylation being observed. These opposing roles may explain the differences in the regulation of collagenase gene expression between each isoform. To further understand this modulation the expression of the AP-1 components, Fos and Jun were examined, along with other recently identified post-AP-1 factors (ATF3, EGR2 NFATc1, and BMP-2). Data showed PKD3 silencing to reduce their expression, suggesting a potential mechanism by which PKD3 signalling can modulate MMP expression. In conclusion, this work identifies the distinct individual roles of PKD isoforms in the modulation of collagenase gene expression, illustrating the need to assess individual kinases within a family.

Novel DNA-based conductive polymer nanowires formed from thienyl-pyrrole derivatives have been synthesized and characterised by high-resolution ES-MS, ¹H and ¹³C NMR spectroscopy. Bulk DNA-templating of these materials is demonstrated by FTIR, while relative control over nanowire dimensions and deposition is shown by AFM. The electronic properties of these materials were investigated by Scanned Conductance Microscopy (SCM) and two-point I-V measurements. The resistance of the DNA/polymer nanowires, determined from variable temperature I-V measurements, was found to be in the range of 10¹²-10¹⁴Ω. Nanowire conductivity values were calculated to be in the range of 1.9x10-⁷-3.75x10-⁴S cm-¹ at 303K. FTIR data demonstrates the availability of the alkyne group in bulk DNAtemplated materials for subsequent nanowire functionalisation using ‘click’ chemistry. Efforts to couple 3-azido propanol and ssDNA probe DNA is also presented.

Human retrotransposons are genetic elements that copy themselves into new locations in the genome by way of an RNA intermediate. They are extremely numerous making up at least 45% of human DNA. Retrotransposon insertions are a major source of inter-human genetic variation, and have been known to cause disease. They are also intrinsically difficult to analyse in genomes due to their highly repetitive nature. In humans there are three currently active retro-transposable elements: LINE-1, Alu and SVA. LINE-1 is an independent element and Alu and SVA parasitise the LINE-1 retrotransposition machinery. There are experimental ways of discovering and analysing such elements, but they require significant investment, while human sequence datasets containing potentially usable data are multiplying at an ever increasing rate. In particular there are now many assembled human genome sequences as well as new sources of whole genome high throughput sequencing data, such as the 1000 Genomes Project. For this reason this study is devoted to using bioinformatic approaches to extract new knowledge about human retrotransposons from the existing datasets. Previous efforts, by past members of this research group, have been devoted to analysing the genomic variation of the LINE-1 element itself. However this study focuses on the extraction of presence / absence variation in the LINE-1 -dependent elements, Alu and SVA. In addition to building software to extract this information from a wide variety of data sources, this project has also involved making the information data available to non-specialist researchers in the form of a website. The tools developed and described here utilise generic design principles, enabling rapid, largely automated updating, necessary with the constant expansion of the underlying data.

Many of the world’s crop species are recent polyploids. The various genomes from the diploid ancestors (known or, often, unknown) interact, with variable effects on genome packaging and nuclear organization (together the nuclear architecture), chromosome stability and gene expression. This project used a comparative approach to understand the genome composition in polyploids, focusing on millets in the Panicum group, saffron Crocus, Brassica and Nicotiana. In situ hybridization using DNA probes was used to identify the chromosomes and antibodies to synaptonemal complex, DNA repair and chromatin structure proteins including histones, which allow the understanding of the modulation of chromosome behaviour depending on the ancestral origin of the chromosomes, were used. The ancestors of proso millet, P. miliaceum (2n=4x=36), were identified as P. capillare and being the same as one genome in the 4x P. repens by in situ hybridization and ITS sequencing. A cell fusion hybrid of Nicotiana x sanderae + N. debneyi was confirmed, with demonstration of chromosome loss, by IRAP markers and in situ hybridization. Saffron Crocus, Crocus sativus 2n=3x=24, was shown to not be an autopolyploid, but to include three genomes with somewhat different chromosomal and sequence characteristics. The alien lines of Brassica and Raphanus with the fertility restorer genes were identified with B. rapa carrying the two chromosomes of Raphanus carrying the fertility restorer genes. Furthermore, the meiotic pairing basis of the alien lines of Brassica and Orychophragmus was also observed which gives an insight into the meiotic pairing between two different species. The water stress resistant genes could be identified from Panicum and thus be utilized in better water usage of plants. It would be possible in future to develop a synthetic C. sativus and thus rescue its declining production around the world, thus improving its economic potential. The fertility restorer gene can now be introduced into the B. rapa species using various mutagens.

Hybrid polyketide-nonribosomal peptides are one of the important classes of fungal natural products. The aim of this work was to investigate the programming behaviour of the iterative enzymes of PKS-NRPS and delineate biosynthetic steps of PKS-NRPS compounds. This work involves isolation and structural elucidation of compounds obtained from heterologous expression of selected tenellin 87, desmethyl bassianin 88 . and aspyridone 84 PKS-NRPS genes in Aspergillus oryzae (M-2-3). Transformants obtained from gene silencing in the fungus Beauveria bassiana strain 110.25 were also chemically analysed. The tenS gene encodes a hybrid polyketide synthase nonribosomal-peptide synthetase (PKS-NRPS) in tenellin 87 and requires trans-acting ER encoding gene tenC for correct programming of tenellin compounds. Silencing of enoyl reductase gene tenC in B. bassiana 110.25 by P amyB was unsuccessful because variation of the carbon source in the original TPM media failed to produce tenellin compounds. We isolated two new A. 07yzae wild type compounds 146 and 147 from A. oryzae tenSPKS-dmbC clone but did not observe the anticipated product of TENSPKS expressed in A. oryzae without the NRPS portion. Aspyridone A 84 and aspyridone B 226 were reported as final products of a putative PKS-NRPS gene cluster in Aspergillus nidulans. We carried out heterologous expression of aspyridone PKS-NRPS encoding gene apdA and enoyl reductase encoding gene apdC in different combinations with aspyridone tailoring genes in A. oryzae. The heterologous expression produced eight different compounds in addition to aspyridone A 84. Our study showed: that the cytochrome P450 ApdE catalyses oxidative ring expansion; tetramic acid benzylic hydroxylation and an exclusive dephenylation activity; that ApdB, a second cytochrome P450 enzyme in the apd cluster catalyses an unusual N-hydroxylation of dephenylated 2-pyridones; that ApdC enoyl reductase perform different stereoselectivities during different cycles of polyketide chain synthesis. We did not observe any apparent role for ApdD and ApdG and our heterologous expression of apd genes did not produce aspyridone B 226, one of the previously reported final products of the apd pathway. This study unveiled the high biosynthetic potential of the apd pathway and diverse chemical capabilities of cYtochrome P450 enzymes in this fungus.