A PCR strategy showed that a number of total mtDNA molecules was significantly decreased (~30%) in the striatum (no reduction in the cortex and cerebellum) of 24-month old HD mice, but not a 15 months of age, when compared to wild-type mice, suggesting mtDNA depletion is a progressive rather than a developmental phenomenon. In light of the ~30% reduction of total mtDNA in the striatum, expression levels of the mitochondrial DNA-encoded respiratory complex enzymes, cytochrome b(Cytb), cytochrome c oxidase I (COI) and cytochrome c oxidase II (COII) were investigated in different brain regions of HD mice. At ~25 months of age, there were no significant differences in mRNA levels of CoII and Cytb in any brain region (striatum, cortex and cerebellum) studied when compared to normal littermates. However, HD mice showed significantly decreased CO-I protein levels and marginally decreased CoI mRNA levels in the striatum. Reduced levels of mtDNA may be caused by decreased replication of mtDNA or increased oxidative damage of mtDNA. Increased levels of 8-OHdG, a marker of increased oxidative stress, were detected in the dorsomedial, dorsolateral and ventromedial striatum, but not in the cortex of 24-month old HD mice providing direct evidence that increased oxidative stress specifically occurs in the striatum of HD mice. As no alterations in the mitochondrial transcription factor (mtTFA) in the striatum of HD mice could be detected, it is likely that mtDNA depletion in the HD mice is caused by increased levels of oxidative stress rather than decreased replication. The results provide a basis for further studies investigating how mutant huntingtin causes increased levels of oxidative stress and for identifying novel therapeutic targets.

Malaria, caused by infection with intracellular protozoan parasites of the genus Plasmodium, is responsible for 300 to 600 million clinical cases annually (Snow et al., 2005), resulting in the deaths of up to three million people every year (Breman, 2001, Breman et al., 2004). There is a clear need for further research aimed at identifying novel drug targets (Ridley, 2002). Reversible phosphorylation of proteins is a major regulatory mechanism in most cellular processes, and protein kinases are considered promising drug targets, comprising as much as 30% of all protein targets under investigation (Cohen, 2002). The divergences between human and plasmodial protein kinases suggest that specific inhibition of the latter is an achievable goal (Doerig, 2004, Doerig and Meijer, 2007). This study investigates protein kinase CK2 of Plasmodium falciparum, seeking to establish by reverse genetics and biochemical approaches whether it represents a possible antimalarial drug target. Protein-kinase CK2, formerly known as Casein Kinase II, is a dual-specificity (Serine/Threonine and Tyrosine) protein kinase ubiquitously expressed in eukaryotes. It has over 300 cellular substrates catalogued to date (Meggio and Pinna, 2003). Consistent with its multiple substrates, the enzyme plays a crucial role in many cellular processes, and is essential to viability in yeast and slime mould (Padmanabha et al., 1990, Kikkawa et al., 1992). The human CK2 holoenzyme consists of two catalytic a or a’ subunits and two regulatory b subunits, and recent evidence indicates that the latter interact with several protein kinases in addition to CK2a (reviewed in (Bibby and Litchfield, 2005)), pointing to a likely role in the integration of numerous signalling pathways. A putative CK2a orthologue and two predicted CK2b subunits were identified in the P. falciparum genome (Ward et al., 2004, Anamika et al., 2005). Here we present the biochemical characterisation of the PfCK2a orthologue and both PfCK2b orthologues, and demonstrate by using a reverse genetics approach that each of the three subunits is essential for completion of the erythrocytic asexual cycle of the parasite, thereby validating the enzyme as a possible drug target. Recombinant PfCK2a possesses protein kinase activity, exhibits similar substrate and co-substrate preferences to those of CK2a subunits from other organisms, and interacts with both of the PfCK2b subunits in vitro. PfCK2a is amenable to inhibitor screening, and we report differential susceptibility between the human and P. falciparum CK2a enzymes to a small molecule inhibitor. Taken together, the data indicate that PfCK2a is an attractive, validated target for antimalarial chemotherapeutic intervention.

Trypanosoma brucei is a protozoan parasite of major public health and economic importance in sub-Saharan Africa, where it is the causative agent of sleeping sickness in man and Nagana in cattle. The complete genome sequence of T.brucei is now available and the diploid genetic system has recently been demonstrated to be Mendelian. This opens up the possibility of using a classical genetic approach to identify genetic loci that determine important phenotypic traits in this parasite, such as host specificity, drug resistance, and pathogenicity. A genetic map of the non human-infective subspecies, T.b.brucei, has already been assembled and successfully used in quantitative trait analysis of a number of traits specific to this pathogen. This thesis describes the construction of a separate genetic map for the sub-species responsible for > 90% of human African trypanosomiasis infections, T.b.gambiense, which differs significantly from T.b.brucei in many key phenotypes. The genetic linkage map was constructed from the analysis of 119 polymorphic microsatellite markers in a population of 38 F1 progeny, obtained from the genetic cross of a T.b.gambiense group 2 strain, STIB 386, with a T.b.brucei strain, STIB 247. Eleven major linkage groups were resolved, one for each of the megabase chromosomes, resulting in a total genetic map length of 733 cM, and an average map unit size of 24 Kb/cM. The map provides a 90% probability of a marker being within 268 Kb of any genetic locus. A comparative analysis of the T.b.gambiense and T.b.brucei genetic maps revealed synteny and marker order to be conserved between the two sub-species. However, variation was observed in the location of regions of high and low recombination frequency (hot and cold spots) in the two maps. The genetic linkage map presented here is the first available for T.b.gambiense and can now be utilised to find the location within the genome of genes responsible for phenotypic traits in this clinically important sub-species. These traits include human infectivity, tsetse transmissibility and virulence, in addition to sensitivity to the trypanocidal drug, pentamidine, for which phenotypic variation between the parents was characterised both in vitro and in vivo in this thesis. The ability of the T.brucei genetic maps to pinpoint loci underlying phenotypic variation is limited by the number of recombination events, and therefore progeny, available for analysis. To increase the utility of this approach for future studies, an improved method for progeny isolation from uncloned genetic cross populations was also developed. This in vitro bloodstream cloning procedure is scalable and efficient, and replaces a time consuming and technically demanding in vivo method. Twelve new progeny clones were isolated by this approach during the trial and incorporated into the analysis, representing a step toward a higher resolution second-generation genetic map. Finally, whilst undertaking genotyping analysis with microsatellite markers the development of spontaneous chromosome 10 abnormalities was observed. A detailed investigation identified seven laboratory-adapted T.brucei lines in which loss of heterozygosity appeared to have occurred. These alterations to the karyotype significantly exceeded the well-characterised genomic rearrangements of subtelomeric regions that are frequently associated with antigenic variation in African trypanosomes. Microsatellite analysis, pulsed field gel electrophoresis and Illumina next generation sequencing demonstrated these changes to be the product of mitotic recombination events in the chromosome core, resulting in an extensive loss of heterozygosity of up to 75% of the chromosome and correlated with an improved growth phenotype. Further work is now required to determine the extent and frequency with which these abnormalities might occur, however these findings do highlight the potential instability of the molecular karyotype of T.brucei in prolonged in vitro culture.

The gene cluster encoding production of oxytetracycline (OTC) by Streptomyces rimosus (the commercial producer) has been studied in this laboratory. The topic of this thesis was the region of the otc cluster including and upstream of the OTC-resistance gene otrB, which has been shown previously to be responsible for reduced accumulation of the antibiotic. The otrB gene was sequenced. The sequence revealed some discrepancies with previously-published data on tet347 (Reynes et al., 1988; Journal of General Microbiology 134: 585-598), an OTC-resistant determinant from another strain of S.rimosus. The deduced gene product of the otrB showed considerable identity with efflux proteins from other Gram-negative and Gram-positive bacteria. These proteins contain conserved functional motifs, and OtrB was analysed in this context. The transcriptional start of otrB was identified. Several short investigations were undertaken into the physiology of antibiotic production. (1) A transcriptional fusion vector (pIJ2843) using catechol oxygenase as a reporter was used to monitor the response of transcription of various regions of the otc cluster (cloned from a high-producing strain) to changes in external phosphate concentration. These data were compared in relation to antibiotic production by the wild-type strain. (2) A transposon mutagenesis strategy was used to attempt to generate novel mutations within the otc cluster. (3) The presence of genetically-engineered haemoglobin cloned into S.rimosus production strains was investigated. The relationship between expression of the recombinant protein, antibiotic production and the aeration of the S.rimosus cultures is discussed.

Oxytetracyline, (OTC), is a secondary metabolite antibiotic produced by the actinomycete Streptomyces rimosus. All of the structural genes for OTC synthesis are clustered on the S. rimosus chromosome and flanked by resistance determinants, otrA and otrB. OtrA mediates resistance through non-covalent modification of the bacterial ribosome whilst otrB, the subject of this thesis, acts by efflux of the drug from the cell. At the outset of this thesis otrB had previously been cloned and sequenced. During the course of the work some discrepancies with previously published data were found, parts of the gene were then re-sequenced and a revised otrB sequence submitted to the NCBI database. The OtrB protein is a transmembrane protein belonging the Major facilitator Superfamily (MFS). Sequence comparison with other members of the family shows OtrB to be part of a subfamily of antibiotic transporters and multi drug resistance proteins made up from 14 transmembrane helices (6+2+6) arrangement. OtrB contains many conserved motifs typical of the subfamily and of the MFS. otrB was cloned into E. coli and expressed. The functional activity of the cloned gene was assessed by growth on various concentrations of OTC. Substrate specificity was investigated using TET and CTC in the growth media. Isolation of OtrB from E. coli as a polyhistidine fusion was attempted, reasons why this was not successful are discussed. Tetracycline exporters from Gram-negative bacteria generally contain 12 transmembrane helices (6+6). The relevance of the two "extra" helices present in OtrB was investigated by the construction and expression in E. coli, of a deletion mutant in which the two putative central helices were absent.

Raf Kinase Inhibitor Protein (RKIP) was originally described as an inhibitor of the Ras-Raf-MEK-ERK pathway, exerting its action by the physical inhibition of the interaction of Raf with MEK. It has subsequently been shown to play important roles in a number of other signalling pathways, including the NFκB pathway and in the stability of the mitotic spindle. Not surprisingly given that it impacts on many important signalling pathways RKIP levels have been shown to be important in the progression of a number of different cancers. RKIP expression is lost or decreased in a number of common human cancers and decreased still further in tumour metastases. One of the tumours in which RKIP is downregulated is colorectal cancer (CRC). Importantly it has been shown that not only is RKIP depleted in tumour tissue when compared with normal tissue but that the level of RKIP within a tumour is inversely correlated with the likelihood of metastatic relapse and with patient prognosis. Although we already have a number of very good prognostic indicators in CRC, one group of patients for whom new prognostic indicators would be useful are patients with Dukes B CRC. These are patients with locally advanced but non-metastatic disease and at present there is no firm consensus on their correct post-operative management. Therefore we set out to examine whether RKIP is a useful prognosticator in this particular group using a tissue microarray (TMA) with samples from over 200 patients with Dukes B CRC. The analysis revealed a strong inverse correlation between RKIP levels and disease specific survival. Moreover, in a multivariate analysis RKIP emerged as an independent prognostic indicator along with lympho-vascular invasion and peritoneal invasion, two well-known and powerful prognosticators. This allowed for the generation of a simple prognostic index, using information from the different independent indicators, allowing for improved patient risk stratification. This led us to examine whether RKIP could also function as a predictive marker in CRC. To do this we again used a TMA, this time consisting of a much larger cohort of patients across the whole range of tumour stages. The results confirmed the prognostic utility of RKIP and indicated that patients whose tumours have low levels of RKIP may derive a greater benefit from chemotherapy than those patients whose tumours have high levels, although this result did not reach statistical significance. In the second part of the thesis I have examined the effect of RKIP in previously characterised mouse models of CRC. To do this I have used a germline RKIP knockout mouse and in the first instance crossed it to the APC580S mouse. In this mouse APC is lost conditionally within the intestine and liver. RKIP knockout did not have any effect on the rate of tumourigenesis or on the invasiveness of tumours in this model. However, in the setting of acute homozygous deletion of APC, RKIP knockout resulted in a decrease in apoptoses in the small intestine and an increase in aberrant mitotic activity in the liver. To follow this up I have examined the effect of RKIP knockout in a mouse model of superficially invasive CRC, specifically to see if RKIP knockout can promote invasive and metastatic behaviour. In this model the APC580S mouse is crossed to mice which conditionally express oncogenic KRas. Although RKIP knockout did not result in an increase in invasive tumours in this model there was a shift in tumour location from the small intestine to the colon. This shift appeared to be due, at least in part to an increase in chromosomal instability in the tumours. The final aim of the thesis was to develop a mouse model of CRC which more closely recapitulates the late stages of the human disease, specifically invasion and metastasis. To do this we have crossed the APC580S mouse with either a conditional p53 knockout or with a mouse that conditionally expresses a point mutation of p53 (p53R172H). In human tumours the majority of abnormalities of p53 are point mutations that result in the production of mutant protein that accumulates in tumour cells. There is evidence that this mutant protein may have oncogenic properties beyond the simple loss of normal p53 protein function. Therefore we have also used this model to study the differing effects of p53 loss and point mutation in CRC. We found that mice homozygous for p53 deletion (p53fl/fl) and those expressing a single copy of the mutant allele with loss of the second copy (p53R172H/fl) developed invasive tumours with nearly 100% penetrance and indeed metastasis was observed. Remarkably, although mice that were heterozygous for p53 deletion (p53fl/+) only rarely developed invasive tumours almost 100% of mice expressing a single copy of the mutant allele (p53R172H/+) developed invasive tumours. We went on to show that the increase in invasion seen in this model is related to an increase in Wnt signalling, which is associated with increased expression of pro-invasive Wnt targets such as fascin. We also showed a novel pro-invasive role for ARF in this process. This is also an excellent model of Dukes B CRC and therefore the ideal model to test the effect of RKIP deletion on invasion and metastasis. These studies led us to examine the differences in effect between knockout and mutant p53 in another tumour model. In this we used a novel model of the aggressive tumour pleomorphic rhabdomyosarcoma to demonstrate that mutant p53 can both promote both tumourigenesis and metastasis more potently than p53 knockout. These studies have demonstrated the value of RKIP in the clinically important Dukes B CRC population and shown its possible utility as a predictive marker in this group. Although we have not seen an effect of RKIP knockout in traditional mouse models of CRC we have developed a novel model which closely recapitulates Dukes B CRC and may be useful in elucidating the effect of RKIP knockout. We have also used this model to gain novel insights into the invasive process, in particular into the role played by mutant p53.

Plasmodium and Toxoplasma nuclear genomes possess an ORF for a putative protein which contains domains homologous to the T7 bacteriophage primase-helicase like Twinkle enzyme and the prokaryotic family A polymerase enzyme. It has been hypothesised that this nuclear encoded protein may be responsible for the replication and repair of the apicoplast genome. Thus it was named PREX (Plastidic DNA Replication/Repair Enzyme complex). In Plasmodium falciparum the ORF (Pfprex) is 6,051 bp with no introns. RT-PCR data revealed that prex is present as a single transcript but western blot analysis of Plasmodium falciparum asexual parasite extracts revealed smaller size proteins indicating post-translational cleavage of the protein. Gene knock out studies have shown that the Pfprex genome locus is recombinogenic although parasites with a disrupted Pfprex locus appear to be unable to survive in culture. The analysis of the recombinant polymerase domain confirmed the polymerase property of the protein. In Toxoplasma gondii, the gene (Tgprex) is 7,740 bp long and interrupted by 19 introns as identified by RT-PCR. The polymerase functionality of the PREX protein was also confirmed by a study on recombinant protein from Toxoplasma gondii. The recombinant protein can be inhibited by known family A polymerase inhibitors. Other related apicomplexan parasites including Theileria, Babesia and Eimeria also possess this prex homologous gene in their nuclear genome. This PREX protein, apparently an amalgamation of functions derived from viral and bacterial origins, is probably important for maintenance of genomic integrity of the apicoplast. The recombinant protein and the assay system may provide the platform for screening of compounds for future drug search against the apicomplexan parasites.

A considerable industry has grown-up around genealogical inference from genetic testing, supplementing more traditional genealogical techniques but with very limited quantification of uncertainty. In many societies Y-chromosomes are co-inherited with surnames and as such passed down from father to son. This thesis seeks to explore what the correlation can say about ancestry. In particular it is concerned with estimation of the time to the most recent common paternal ancestor (TMRCA) for pairs of males who are not known to be directly related but share the same surname, based on the repeat number at short tandem repeat (STR) markers on their Y-chromosomes. We develop a model of TMRCA estimation based on the difference in repeat numbers in pairs of male haplotypes using a Bayesian framework and Markov-Chain Monte-Carlo techniques, such as adaptive Metropolis-Hastings algorithm. The model incorporates the process of STR discovery and the calibration of mutation rates, which can differ across STRs. In simulation studies, we find that the estimates of TMRCA are rather robust to the ascertainment process and the way in which it is modelled. However, they are affected by the site-specific mutation rates at the typed STRs. Indeed sequencing the fastest mutating STRs yields a lower error in the estimated TMRCA than random STRs. In the British context, we extend our model to include additional information such as the haplogroup status (as determined from single nucleotide polymorphisms, SNPs) of the pair of males, as well as the frequency and origin of the surname. In general, the effect of this is to reduce estimates of the TMRCA for pairs of males with an older TMRCA, typically outwith the period of surname establishment (about 500-700 years ago). In the genealogical context, incorporating surname frequency (within the prior distribution) results in lower estimates of TMRCA for pairs of males who appear to have diverged from a common male ancestor since the period of surname establishment. In addition, we include uncertainty in the years per generation conversion factor in our model.

The aim of this thesis was to further our knowledge about the contribution of silent alleles on megabase chromosomes to the late stages of trypanosome infection and test the hypothesis that this contribution takes shape in a hierarchy of expression due to differences between alleles in terms both of flanking regions and coding sequence. This was achieved through a combination of bioinformatics and molecular studies. The initial approach was to undertake an extensive manual curation of the available VSG archive; this endeavour resulted in establishment of a fertile collaboration with the Trypanosoma brucei genome sequencing project, and in creation, with the aid of P. Ward and S. Menon, of a dedicated web-based tool to handle and query curated VSG genes. Out of an updated estimate of ~1600 VSG genes, 940 (between half and three quarters) were annotated and shown to be arranged in subtelomeric arrays and to be largely present as pseudogenes (~90%). By considering separately the hypervariable N-terminal domain (three types, A, B and C) and the more conserved C-terminal domain (types 1 to 4, with two additional types identified in this study), it appeared that most of the degeneracy lay in the C-terminal domain. This suggested that N-terminal domains (one third of them being intact) would be utilised by a process of segmental gene conversion yielding hybrid genes, by recombination with functional C-terminal ends resident at the expression site. Under the assumption that “order” within the genome (the presence of patterns within the VSG archive) helps inform “order” in VSG expression (a hierarchy based on different activation probabilities), it was somewhat surprising to detect little evidence of clear substructuring within the archive: no “classes” of VSGs could be identified, based on coding sequence and flanking sequence features. In keeping with the observed high level of divergence within the VSG archive, clear orthologue groups (here defined as alleles sharing >60% amino acid sequence identity) were found not to include more than three to four members and to be scattered at random across the arrays. Putative functional genes could not be separated into groups based on expected differences in activation probabilities, such as a different number of upstream 70-bp repeats, shown to be involved in copying silent alleles to the expression site.

This thesis is concerned with the study of a Bayesian clustering algorithm, proposed by Heard et al. (2006), used successfully for microarray experiments over time. It focuses not only on the development of new ways of setting hyperparameters so that inferences both reflect the scientific needs and contribute to the inferential stability of the search, but also on the design of new fast algorithms for the search over the partition space. First we use the explicit forms of the associated Bayes factors to demonstrate that such methods can be unstable under common settings of the associated hyperparameters. We then prove that the regions of instability can be removed by setting the hyperparameters in an unconventional way. Moreover, we demonstrate that MAP (maximum a posteriori) search is satisfied when a utility function is defined according to the scientific interest of the clusters. We then focus on the search over the partition space. In model-based clustering a comprehensive search for the highest scoring partition is usually impossible, due to the huge number of partitions of even a moderately sized dataset. We propose two methods for the partition search. One method encodes the clustering as a weighted MAX-SAT problem, while the other views clusterings as elements of the lattice of partitions. Finally, this thesis includes the full analysis of two microarray experiments for identifying circadian genes.