Radiotherapy is a fundamental cancer treatment and plays a pivotal role in the improving outcomes for the disease. Depending on the cancer site and organ at risk rates of moderate to severe acute toxicity range between 15 to 30 percent and rates of late toxicity between 5 and 15 percent. If a patient’s individual risk of radiotherapy toxicity could be predicted then their treatment could be tailored appropriately. In this thesis two cohorts have been used to analyse predictive measures for acute and late radiotherapy toxicity: the REQUITE cohort (prospective international observational study of breast, prostate and lung cancer patients) and the LeND cohort (retrospective local study of breast cancer patients). Three main areas have been examined to establish whether they can be used to predict for radiotherapy reactions. In the prostate and lung patients associations between clinical and treatment variables and acute toxicity were reviewed. The second area was predictive assays: DNA damage and repair were assessed using the comet and γ-H2AX assays and apoptosis in lymphocytes using the RILA (radiation induced lymphocyte apoptosis assay). Finally the effect of circadian rhythm and its underlying genetics on radiotherapy toxicity were assessed. Many of the variables were significantly associated with increased toxicity on univariate analysis. Three were significantly associated with toxicity on multivariate analysis. Acute toxicity in prostate patients was associated with intended duration of hormones (p=0.05) and V50 bladder (p=0.01)). Morning radiotherapy was associated with increased overall bivariate STAT score (p=0.03) in the LeND volunteers. The results of this study indicate clinical and genetic variables and the use of predictive assays can be utilised to create more personalised radiotherapy treatments that strive for better cancer and quality of life outcomes for patients.

In recent years a significant amount of biological research has been focussed on the cytoplasmic membrane of the cell. This has emerged as a complex structure, participating in cell biosynthetic activities in addition to osmoregulation. However the functional organisation of the membrane is little understood, and in this may lie the key to several biological problems. An approach to the study of the coordination of biosynthetic processes by the cell membrane may be through the use of agents interacting specifically with the membrane. One such class of compounds may well be the natural protein antibiotics, colicins. Colicins may be distinguished from conventional antibiotics by their unique mode of action with sensitive cells, whereby lethality is promoted from specific fixation sites in the bacterial cell wall. The colicin molecule neither penetrates the cell surface layer, nor causes gross cell membrane damage, and it has been postulated that interaction of the colicin protein with proteins of the cell membrane lead eventually to cell death. The studies described in this Thesis have been concerned in particular with the interaction of colicin E2, one of the E-group of colicins, with the bacterial cell, and the mode of action of this colicin. Colicin E2 is characterised by the induction of a rapid degradation of cellular DNA, and the inhibition of cell division in sensitive bacteria. It was hoped that the genetical and physiological characterisation of bacterial mutants specifically refractive to colicin E2 would indicate the nature of the altered component, normally participating in the transmission of the effect of the extracellular colicin to the intracellular DNA, and/or the cell division machinery. Mutants refractive to colicin E2, designated Ref-II, were therefore isolated from a number of strains of E. coli K12. These mutants were systematically examined, and it was found that although the majority were relatively insensitive to UV irradiation, approximately 33% did display significantly increased sensitivity. Further-more, these Ref-II UVs strains were sensitive to the detergent, Sodium Deoxycholate, and grew slowly in complex medium, properties indicative in the first instance of an altered membrane component in this class of mutant, and in the second, of a normal role of the component in some aspect of cellular metabolism. These Ref-II UVs mutants could also be distinguished from Ref-II UVr mutants by possession of various other pleiotropic characters. For example, the majority of Ref-II UVs mutants were defective in recombination, some were "resistant" to bacteriophage lambda, one grew in long filaments, and one produced excessive polysaccharide particularly when cultured on minimal agar, properties which may all be related to defective DNA metabolism in the mutants. Genetic analysis revealed that pleiotropic characteristics shown by the Ref-II UVs mutants were 100% co-transducible with the refII locus. Moreover, if two or more cistrons were present, mutations in these producing either Ref-II or Ref-II UVs mutants respectively, they must be adjacent on the chromosome. The refII locus was mapped and found to be co-transducible with serB, and to lie within four genes of the hsp locus on the bacterial chromosome. Reversion studies confirmed that the complex pleiotropy displayed by Ref-II UVs mutants did in fact arise from mutation in a single gene, and not from a series of closely linked, multisite mutations arising from the action of the mutagen employed. Moreover, the site of the reversion mutation proved to be at the refII locus, and not at a second locus, mapped and found to be closely linked to thr, potentiating the expression of colicin E2-refractivity. Different hypotheses may account for the occurrence of each individual pleiotropic character of the Ref-II UVs mutants. However, collectively, it becomes apparent that the altered component in this class of mutants normally participates in DNA metabolism in the cell, and either (a) the enzymes participating in the repair and recombination of bacterial DNA, and possibly even the DNA molecule itself, may be bound to the altered component; or (b) the altered component may be one of an aggregate of proteins concerned specifically with chromosomal metabolism. Direct evidence that the altered component, at least in Ref-II UVs mutants, is in fact structural, and presumably membrane, is received from the delayed expression of UV resistance after the receipt of the UVr gene in Ref-II UVr/UVs heterogenotes. Future experiments involving the isolation and comparison of membrane fractions from mutant and non-mutant strains will now be critical in the confirmation of this hypothesis.

The importance of cell membranes in maintaining the structural integrity of living organisms has been known for some time. A very complex and important role for membranes as the spatial and temporal organizers of the cell's numerous metabolic functions is presently being unfurled. Although overall membrane structure may appear to be similar amongst the vast range of living organisms, the basic framework is necessarily adorned with specialized coordinated systems relevant to each cell's functions. The mechanism of colicin E2 interaction with the cell surface of Escherichia coli is examined and discussed. Colicin action upon the bacterial cell's biochemical processes is both indirect and highly specific, and is mediated by some transmission system in the bacterial cell membrane. Fluorescein labelled colicin E2 and ferritin labelled anti-E2 Y globulin have been prepared and used in an attempt to locate the positions of colicin E2 molecules in or on the cell surface of sensitive bacteria. Generalized disturbance of cell envelope integrity subsequent to colicin action has been sought but not found. The highly specific nature of colicin action upon the membrane is supported. Treatments designed to interfere uith bacterial cell envelope integrity perturb the action of colicin E2. Hembrane fractionation procedures suitable for studying bacterial membrane structure are presently becoming available. An additional membrane fractionation procedure is presented, based upon the interaction of cadmium N-lauroyl sarcosinate with bacterial cell extracts. Sodium dodecyl sulphate acrylamide gel electrophoretic separation of E. coli envelope proteins from mutants refractory to the action of colicin E2 has shoun that these membrane-containing structures have an altered protein component compared to the non-refractory parental strain. Hypotheses explaining a certain type of colicin E2 refractivity are presented. Future studies involving the use of colicins and genetic analysis as effective tools in probing the complexities of the E. coli cell membrane structure are outlined.

A re-investigation of the strain Colonia of the Myxomycete Physarum polycephalum showed it to be homothallic. All amoebae can give rise to plasmodia within clones. Using genetic analysis it was shown that this ability was due to an allele of mating-type (mt), mt, which allowed (1) crossing of amoebae both carrying mt, (2) outcrossing with amoebae carrying heterothallic mating-types, Micro-spectrophotometric measurements on amoebal and plasmodial nuclei showed a change in DNA content consistent with a change in ploidy. Both results independently eliminate apogamy as an explanation for these results. Many interesting observations were made on amoebal growth during progress towards synthesising an axenic medium for amoebae. New mutagenic techniques devised to mutagenise amoebae all failed to increase mutagenic rate. An independent check on the work of Haugli using his method confirmed that (1) mutation rate could be enhanced from 10 to 100 fold by ultraviolet light with the synergistic effect of caffeine, (2) the maximum expression of mutations under these conditions occumed after 30 hours. Using this method 4 mutants affecting the amoebal-plasmodial transition were isolated. These amoebae fail to undergo the necessary developmental switch and do not produce plasmodia in clones. Genetic analysis showed them to be recessive, functionally different and, in the detailed analysis of one of these mutants, freely recombining with all other genetic markers including mt. This groundwork showed the feasibility of genetic analysis of a developmental process in great detail. A review of developmental genetics was made. The processes of cell fusion, nuclear fusion and mitosis in other organisms was discussed. Cell fusion seems to be a rare but important biological event. Nuclear fusion of interphase nuclei is restricted to gametic cells. All syncytial cells have a closed mitosis but not all cells with a closed mitosis are syncytial. The relevance of these processes to the amoebal-plasmodiel transition in other Myxomycstss was discussed.

Four different actinidin gene constructions have been created, each consisting of different functional parts of the actinidin gene: (1) the mature actinidin-coding DNA, (2) the amino-terminal extension, but without the secretion signal, plus the mature actinidin-coding DNA, (3) the mature actinidin plus the carboxy-terminal extension-coding DNA, and (4) the full-length precursor actinidin-coding DNA. The first three constructions were fused to the yeast MFa1 promoter and secretion leader sequence, while the fourth was coupled to the CYC1-GAL UAS promoter. Upon expression in yeast, no protein product was detected in the culture supernatant. Analysis of intracellular proteins showed that actinidin protein was detected only from the actinidin gene constructions which have the carboxy-terminal extensions, suggesting that the carboxy-terminal extension is required for the stability of the protein. Comparison of the actinidin proteins produced in protease-proficient and protease-deficient strains suggests that the processing of the protein requires the activity of vacuolar protease(s) and indicates that the actinidin was translocated into the yeast vacuole. Examination of the amino acid sequence suggested that actinidin possesses potential vacuolar and peroxisomal targeting signals. Since the actinidin precursor was glycosylated it must have entered the secretory pathway before being translocated into a specific cellular compartment. The HSP26 gene promoter has been shown to be induced by heat-shock and upon entry into stationary phase, thus it is potentially useful for heterologous gene expression in yeast.

Bacillus sphaericus produces a sporulation associated toxin specific for the larvae of dipteran insects: mosquitoes that are vectors for the transmission of human diseases such as malaria and filariasis. Two dissimilar DNA sequences conferring larvicidal activity on recombinant Escherichia coli had previously been cloned from B. sphaericus 1593M (Souza at al., (1988), J. Biotechnol., 7, 71-82). Proteins encoded by one of these DNA sequences are studied in this thesis. The DNA sequence cloned from B. sphaericus 1593M is here shown to encode two proteins of 41 and 59KDa that are together required for toxicity to mosquito larvae. These proteins were previously hypothesized to exist in the crystal of B. sphaericus as high molecular weight oligomers (Baumann et al., (1985), J. Bacteriol., 163, 738-747). I now show that these two proteins are distinct from the high molecular weight protein which appears to constitute the majority species of the crystal. I have identified this protein as the Surface layer protein of B. sphaericus. I also show that the 59KDa species is a distinct and separate constituent of the crystal. As a prerequisite for raising antibodies to study regulation of synthesis of these proteins, purification following overproduction or secretion was investigated. Thus, attempts were made to purify the 41 and 59KDa proteins from E. coli cells using the C-terminal signal sequence of Haemolysin, a protein normally secreted from E. coli. Furthermore, attempts were made to express the gene encoding the 4lKDa protein in E. coli to a high level from PR and PL promoters. I show that this protein could not be overexpressed in this way. This was attributed to a lack of transcript termination on the vector due to the presence of a faulty fd transcription terminator downstream of the gene. The nucleotide sequence of the gene encoding the 59KDa protein was determined. This sequence was then used to design an approach which led to the overexpression of the gene from the T7 RNA polymerase-recognized 10 promoter on the plasmid pET3a. This in turn allowed the production of antibodies. The expression of the mRNA transcripts of the genes encoding the 41 and 59KDa proteins were also studied in B. sphaericus and B-galactosidase fusions were constructed to serve as "reporters" for the expression of the 41 and 59KDa proteins in E. coli and B. subtilis. I propose that the regulation of expression of the genes is complicated, and depends upon the use of two, temporally regulated promoters.

The collagens are a family of related proteins which contain at least one triple-helical domain, a structure which is determined by the unique amino acid sequence of the polypeptide subunits. These proteins each have a specific function in the extracellullar matrices of connective tissues of multicellular organisms. In humans, type I collagen, the most abundant collagen type, is the major structural component of skin, tendon and bone. Mutations in the two genes encoding the subunits of type I collagen, COL1A1 and COL1A2, result in heterogeneous phenotypes. Some mutations result in osteogenesis imperfecta, the "brittle bone disorder", which itself has a wide range of phenotypes from mild to lethal in utero and others result in Ehlers-Danlos syndrome type VII, which is characterized by skin hyperextensibility and joint laxity. In this study, mutations were detected in these two genes using an adaptation of a recently-developed technique, single strand conformation polymorphism analysis, and DNA sequencing. Several silent changes in COL1A1 were identified and characterized. Two of these, an intronic HaeIII restriction fragment length polymorphism and an AciI polymorphism, are both of sufficient frequency to be potentially useful as markers in linkage analysis studies. The AciI polymorphism, resulting in substitution of an alanine residue with threonine, and a proline to alanine change were both found in normal individuals and therefore the altered amino acid residues do not appear to have any crucial role in the type I collagen molecule. Four patients with Ehlers-Danlos syndrome were studied but no mutations were identified. Several different mutations, assumed to result in the disorder, were found in coding sequences of COL1A1 or COL1A2 in patients with lethal and non-lethal forms of osteogenesis imperfecta. The discovery of these mutations has extended the knowledge of the relationship between the type and position of the mutation and the resulting phenotype.

Repetitive DNA sequences (tandemly repeated and dispersed elements) vary in abundance, composition and organization between individuals, breeds, and related species. Here, we aimed to define the repetitive DNA landscape in sheep (Ovis aries). Whole genome sequence reads (38Gbp; 10x genome coverage) from five Kurdistani sheep individuals were investigated by graph-based read clustering (RepeatExplorer), frequency analysis of short motifs (k-mers), alignment to reference genome assemblies, de novo assembly and fluorescent in situ hybridization. To show genes in the sequences, the scrapie locus was identified and found to be associated with intermediate susceptibility. Mitochondrial genomes of breeds Hamdani and Karadi were assembled and grouped with known sheep haplogroups. Notably, abundant nuclear mitochondrial DNA segments (numts) were found at centromeres of chromosomes, and included mitochondrial sequences from ancestral species. The tandemly repeated DNA satellite I sequence represented 6% of the genome and satellite II was 2%. Meiotic analysis showed a loose chromatin loop organization of satellite I, while satellite II sequences were tightly organized and attached to the synaptonemal complex along with telomere repeats. Novel species-specific tandem sequences (1% of the genome) were also found. Non-LTR retrotransposons including LINEs and derived SINEs represented more than 20% of the genome, while DNA transposons comprise a lower proportion (< 0.05%). Complete genomes of endogenous beta-retroviruses (enJSRV) plus three classes of endogenous retroviruses (ERVs) were identified. In total, repetitive sequences covered 30% of the genome, with tandemly repeated sequences at centromeres, and non-LTR retroelements families showing a centromeric to dispersed distribution with some being amplified on sex or submetacentric chromosomes. ERVs showed centromeric to dispersed distribution. Our results provide informative DNA markers within Bovidae lineages. Rapidly evolving repetitive sequences allow us to study processes of chromosome or genome evolution, homogenization or diversification in sheep, and more broadly across the Bovidae.

Neisseria meningitidis is one of the main causes of bacterial meningitis and septicaemia but colonizes the upper respiratory tract of humans asymptomatically as a normal commensal. Phase variation (PV) in the surface antigens is proposed as an effective mechanism to enable these bacteria to adapt and persist in the human host. Opa proteins are expressed on the outer surface of meningococcal cells playing an important role in the pathogenicity by mediating the adhesion to and invasion of human cells. These proteins are encoded by three/four loci and each locus is phase variable due to pentameric repeats within the coding region. The phase variability in Opa proteins was investigated in meningococcal isolates from 19 carriers and time points representing up to six months of asymptomatic carriage. Changes in repeat tracts were analyzed by GeneScan, and a high frequency of PV was observed in at least two loci with a rate of 0.06 mutations/gene/month during colonization. The expression state of Opa was confirmed by Western blotting indicating expression of a limited number of Opa variants. Around 70% of the isolates expressed only one Opa and none simultaneously expressed four Opa. Intergenic and intragenic recombination was detected in two carriers, leading to new opa alleles with functions differing from the previous alleles. These results revealed that persistent carriage was correlated with a high rate of variation and switching between different Opa variants with stable expression of one or more alleles that may maintain Opa-mediated adhesion. The study also highlights the role of Opa proteins in mediating in vitro escape of N. meningitidis strain MC58 from anti-Opa bactericidal antibodies while selection for populations expressing other Opa variants was also observed in vivo indicating the importance of switching between the different variants for immune evasion and maintaining the function of this protein. Four recombinant Opa proteins were generated in this study and used for developing bactericidal polyclonal antibodies that can be used for further investigations of in vitro and in vivo immune escape.

Some strains of E. coli secrete a low molecular weight compound, aerobactin, with a high affinity for ferric iron. After complexing ferric iron aerobactin may be transported back into the cell and used as a source of iron. The ability to secrete aerobactin and utilize iron derived from this source has been demonstrated to be an important virulence determinant for bacteria causing extraintestinal disease. The genes encoding the aerobactin system are often associated with large plasmids and are arranged in an operon consisting of five genes. Four genes encode enzymes responsible for the biosynthesis of aerobactin, while the fifth encodes an outer membrane receptor specific for ferric aerobactin. Antibodies against the outer membrane aerobactin receptor protein were raised in an attempt to evaluate the prospect of using the aerobactin receptor as a protective antigen. Antiserum was shown to react specifically in ELISA assays with whole cells of E. coli K-12 expressing the receptor and to inhibit uptake of [14C]aerobactin. The antiserum did not, however, react with the aerobactin receptor protein in a range of E. coli isolates from representative E. coli serotypes isolated from human extraintestinal infections. This was shown to be due to shielding of the receptor protein by lipopolysaccharide molecules on the surface of these strains. The presumed role of a number of chromosomally encoded gene products in the uptake of aerobactin across the outer and cytoplasmic membranes of E. coli has never been directly demonstrated. Their functions were confirmed experimentally, and entry of aerobactin into the cytoplasm of an uptake proficient cell was demonstrated for the first time. Inhibition of uptake into the cytoplasm of aerobactin, but not aerobactin-derived iron, was demonstrated in the presence of endogenous aerobactin. The aerobactin receptor protein also acts as a receptor for the bacteriocin cloacin DF13. No other envelope-associated proteins were known to be involved in sensitivity to cloacin DF13. The major outer membrane protein OmpF was demonstrated to be required in addition to the aerobactin receptor for sensitivity to cloacin DF13. It was shown not to be required for initial binding of the bacteriocin to the receptor and a role in subsequent translocation across the outer membrane was suggested. Studies on a number of mutant aerobactin receptor proteins revealed that structural information necessary for stable association with the outer membrane was present throughout most of the length of the polypeptide. A region involved in binding and/or translocation of aerobactin was identified and another region was shown to be involved in cloacin DF13 binding and/or translocation. A mutation which altered the signal sequence such that it more closely resembled the consensus structure for signal peptides increased the efficiency of processing of the precursor protein.