Using the cervical carcinoma cell line, HeLa, molecular mediators of selenium-induced apoptosis were investigated. These studies were later extended to biopsies of normal human oral mucosa cells (NOMCs) and human oral carcinoma cells (SCCs), using a primary culture system. Two selenium compounds were tested: selenodiglutathione (SDG), the primary metabolite of selenite, the most commonly used cancer-protective selenium compound in animal models, and the synthetic compound, 1,4-phenylenebis (methylene)selenocyanate (pXSC), one of the most potent chemopreventive selenium compounds. Both compounds induced apoptosis in the cells tested and this was associated with a strong induction of Fas ligand (Fasl). Blocking Fas activation resulted in inhibition of apoptosis. Activities of stress kinases INK (c-jun N-terminal kinase) and p38 were also induced on selenium treatment and intervention studies using specific chemical inhibitors and/or dominant negative mutants suggested that INK, but not p38, was functionally important for the induction of apoptosis and Fasl. The relative levels of induction of INK activity and Fasl also correlated with the extent of apoptosis observed. On treatment with SDG, but not pXSC, SCCs were found to preferentially activate the JNK/Fasl pathway compared to NOMCs and this correlated with enhanced sensitivity of SCCs to SDG-induced apoptosis. Both SDG and pXSC also appeared to modulate activities of other signalling proteins like extracellular regulated kinases (ERKs) 1,2 & 5 and Akt. However, functional interference with these pathways using specific chemical inhibitors or constitutively activated mutants revealed either none or a small effect on apoptosis-induction. One exception was the activation of Akt by SDG in SCCs: inhibiting its activity sensitised the cells to apoptosis significantly. Finally, preliminary xenograft studies have confirmed the induction of apoptosis in vivo, using chemopreventive dietary levels of selenium.

Azadirachtin was isolated from neem seeds, by flash column chromatography, with a purity of > 95%. Yields from Ghanaian seeds were higher than those from Pakistani seeds (0.04% w/w and < 0.0015% w/w respectively). An unsuccessful attempt was made to produce 14C-Labelled azadirachtin for use in systemic movement studies. Qualitative and preliminary quantitative chromatographic analyses of the leaf extracts of tobacco seedlings (Nicotiana clevelandii) whose roots had been immersed in azadirachtin solutions showed systemic movement of the compound from the roots to the aerial parts. The electrical penetration graph (EPG) method was used to analyse the feeding behaviour of apterous, adult Myzus persicae (Homoptera : Aphididae) on N.clevelandii seedlings, treated systemically with azadirachtin. The percentage of the 9h recording period devoted to non-penetration activities and to stylet pathway patterns, the number of probes inititated and the numbers of sieve tube penetrations all increased with increasing azadirachtin concentration. Azadirachtin treatment significantly reduced the percentage of probes that reached sieve elements and increased non-penetration activity before and after the first period of ingestion from the sieve elements. The percentage of the recording period spent in the EPG pattern associated with sieve tube penetration was significantly reduced by an azadirachtin concentration of 300ppm, and the duration of each individual sieve tube penetration was significantly reduced by an azadirachtin concentration of 100ppm. A novel quantitative method, based on honeydew production, was used to measure aphid feeding on artificial liqued diets containing various concentrations of azadirachtin. During a 52h period on diets containing azadirachtin at concentrations of 100 and 300ppm, adult apterous M.Persicae produced 4-5 times less honeydew than those feeding on control diets and aphids on diets containing 500 and 1000ppm azadirachtin produced no more honeydew than aphids which were starved for the same period. During this period the rate of nymph production by aphids on all treated diets fell to less than half that of aphids on control diet. When the aphids were subsequently transferred to untreated diets for 44h, a large proportion of the nymphs produced by aphids which had been on diets treated with 100-500ppm azadirachtin were born dead with undeveloped appendages.

The main part of the work was to develop PCR techniques for identification and typing of avian S. aureus. Different primer sets were applied in order to generate reproducible profiles of the PCR amplimers. Of the 86 avian S. aureus studied, 12, 7, 4 and 5 groups were recognised by the use of ribosomal, REP, coagulase gene and protein A gene primers respectively. PCR-ribotyping (method 1) produced a relatively simple pattern which was used for rapid identification of these isolates and for differentiation of these isolates from each other. These PCR fingerprinting methods were simple to perform and reproducible. The majority (80%) of the strains even from different geographical locations had similar “typical avian” profiles whereas 20% had atypical avian profiles. Results showed that many of the virulence factors characterised in this study in avian S. aureus were likely to be regulated by the agr and sar loci. Sequencing of the agrD region in selected strains suggested that 80% of avian (typical avian) isolates belonged to agr specificity group II and the other (20%) (atypical avian) stains belonged to agr specificity groups I or III. An investigation by multiplex PCR for eight toxin genes (encoding enterotoxins A-E, exfoliative toxins A-B and toxic shock syndrome toxin-1) revealed that only 5% of the avian isolates produced one or more of these toxin genes and all were atypical by other criteria. A multiplex PCR developed to detect haemolysin (a, g and d) genes showed that all the 86 avian S. aureus isolates had the a and d-haemolysin genes and a majority (86%) of the strains also had the g-haemolysin gene. Many of those that lacked the g-haemolysin gene were atypical by other criteria. Only 5% of the avian S. aureus strains possessed the b-haemolysin gene and, again, these isolates were atypical by other criteria.

There is an urgent need for new chemotherapies against human African trypanosomiasis (HAT), caused by the protozoan parasite Trypanosoma brucei. It is anticipated that the parasites’ divergent biochemistry will enable development of novel therapies. To study the behaviour of a complex network as metabolism, one can employ mathematical models. In this thesis, metabolism of bloodstream form T. brucei was investigated. Cellular metabolism consists as a complex system connecting enzymes with metabolites, and to study such a network one can construct mathematical models that describe the connections within the biological system. A previously published, and well-curated model of glycolysis in bloodstream form T. brucei (Bakker BM, et al. (1997) J Biol Chem 272:3207 15), was extended here with the pentose phosphate pathway (PPP), the second major pathway in glucose metabolism in most life forms. Several hypotheses were derived during the model building process and these were tested experimentally. It became apparent that the glycosomal bound-phosphate balance is essential for the parasite. Extension of the glycolytic model with the PPP introduced the risk of a so-called ’phosphate leak’, where bound-phosphates are depleted in the glycosome. Two hypotheses were investigated in silico, while one hypothesis could also be tested experimentally; (i) a glycosomal ATP:ADP antiporter was proposed, but in silico analysis indicated that the activity of such an antiporter requires tight regulation. (ii) A glycosomal ribokinase was investigated both in silico and experimentally. Genetic mutants indicated that ribokinase is essential to bloodstream form T. brucei, albeit at low levels. Additional analysis of the generated models indicated that ablation of 6-phosphogluconate dehydrogenase (6PGDH) in T. brucei is lethal by a different mechanism as seen in other organisms. Overall, extension of the glycolytic model with the PPP demonstrated the fragility of the model regarding the bound-phosphate balance and indicated that future analysis on glycosomal metabolism should be focused on this. Important in the use of mathematical models of metabolism is that the underlying stoichiometry of the model reflects (albeit with simplification) the in vivo system. It is therefore paramount to know what enzyme activities are present in the organism of interest. In this thesis a metabolomics approach was used to elucidate the function of three T. brucei genes. These genes were putatively annotated as arginase (ARG), N-acetylornithine deacetylase (NAO) and nicotinamidase (NAM). The results suggested that ARG has catalytic activity as tryptophan monooxygenase (EC 1.3.12.3), while substrate promiscuity was indicated for NAO and NAM. The work presented in this thesis has provided us with new insights on trypanosomal metabolism. The extended model now allows us to research a larger part of T. brucei metabolism with mathematical modelling, and will thereby aid in the identification and further investigation of (proposed) drug targets.

Neisseria meningitidis is an important cause of meningitis and bacteraemia worldwide and is associated with high case-fatality rates. Meningococcal disease continues to remain a public health issue in Scotland and the rest of Europe. Typing methods are used for epidemiological purposes to investigate outbreaks and the spread of meningococci and to examine the population structure of the organism in order to better understand its variation and evolution. Reference institutes have employed such methods for a number of decades for the diagnosis and detection of meningococci. However, phenotypic methods for serogrouping, serotyping and serosubtyping meningococci, although providing good strain information, can lead to endemic strains appearing identical using these methods when they are in fact quite different. More recently methods have been developed to further characterise bacteria. These methods have included PCR for the detection of meningococcal disease within blood, serogrouping and sequencing of housekeeping genes (MLST) and antigen genes such as PorA. These molecular epidemiological methods were used for the retrospective typing of invasive meningococci in Scotland, 1972-1998, using a fully automated procedure. The results of these were then analysed using statistical packages to examine the population structure of the organism. In total there were 2517 invasive isolates, received by the Scottish Meningococcus and Pneumococcus Reference Laboratory (SMPRL) from the start of 1972 to the end of 1998. Serogroup distribution changed from year to year during the time period 1972-1998 but serogroups B and C were dominant throughout this period. Serogroup B was the dominant serogroup throughout the seventies and early eighties until serogroup C became dominant during the mid 1980s. This increase in dominance of serogroup C has been found in this study not to be associated with one particular sequence type (ST) but is associated with a number of STs, which include ST-8, ST-11, ST-206 and ST-334. This is in contrast to the increase in serogroup C disease in the 1990s that was due to the ST-11 clonal complex. While there was much diversity in the STs (309 different STs among the 2517 isolates), only ten accounted for 1562 isolates (59.9%). These were ST-11, ST-8, ST-41, ST-153, ST-1, ST-32, ST-33, ST-269, ST-334 and ST-60. There were 177 new STs found during the time period. The STs were further differentiated into 31 clonal complexes, with 57 singleton types. As with the STs, although there was much diversity in the clonal complexes, only seven accounted for 1993 isolates. It was found that with PorA variable region (VR) types there were certain combinations significantly more common than others. There was a strong link with PorA type and ST and more so with clonal complex. This link was evident with the PorA type 5, 2-1, 36-2, which occured in 70 isolates representing the ST-11 complex and in all but two isolates representing ST-11. Similarly PorA type 18-3, 1, 35-1 was associated with 15 isolates belonging to the ST41-44 complex. However, this was not the case with all PorA combinations as the PorA type 19, 15, 36 was associated with 10 different complexes. There was some association between serogroup and PorA VR types. There was strong evidence of certain VR1, 2 and 3 regions being associated with certain serogroups, although this was not definitive. For example, of 192 isolates with PorA type 19, 15, 36, 85.4% were associated with serogroup B. Genosubtyping of the porA gene has been shown to increase the power of differentiation within clonal meningococcal populations. For, example, seven isolates that had the same serogroup, ST, VR1 and VR2 could be differentiated by their VR3 type. Using cluster detection software SaTScan to analyse all isolates, it was found there were 29 clusters in Scotland, from 1972-1998. These clusters included 63 cases, which accounted for 2.5% of all cases. A range of different strains caused the clusters that were identified in this study, some caused by hypervirulent strains. These strain types were responsible for a number of cases throughout the world as well as in Scotland during the period of this study. However it was also shown that there were clusters identified in this study caused by lesser-known strain types that were not responsible for many cases and that appear to be unique to Scotland or the UK. This study is the first to look at the detection of clusters over a time period of 26 years and to identify clusters that would have previously been unidentified due to lack of suitable characterisation techniques. The results in this study indicate that the multivalent preparation produced by the Netherlands Vaccine Institute (Nonavalent vaccine) had the potential, based on the PorA types that it contains, to prevent the majority of serogroup B infection that had occurred in Scotland, from 1972-1998. It also had the potential, although not to the same extent as serogroup B, to protect against other serogroups. For the age groups that would potentially have been the first to be immunised with any vaccine as part of the childhood vaccination programme, the 0-4 years old group, the potential coverage was over 92% which is comparable with the coverage seen with the serogroup C meningococcal conjugate (MCC) vaccine, of approximately 90%.

Protein translocation systems are invaluable to pathogenic bacteria, facilitating the display of virulence factors on their surface or their release into the extracellular environment. Some protein export systems are ubiquitous and essential to cell survival whereas others are horizontally acquired on prophages or pathogenicity islands (PAI), in many cases providing the bacterium with pathogenic advantages. For the majority of the known protein export systems, their structure, function and secreted substrates have been characterised, yet some proteins have been identified that are secreted via unknown mechanisms. Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is an important cause of human foodborne disease worldwide. The pathogenesis of this bacterium is mainly attributed to the secretion of toxins and the presence of a Type III Secretion System (T3SS). The T3SS can translocate bacterial proteins, known as effectors, into the host cell which mediate an effect culminating in the formation of a characteristic attaching and effacing (A/E) lesion. This system is encoded on a horizontally acquired PAI termed the locus of enterocyte effacement (LEE). The LEE not only encodes the T3SS apparatus but also several effectors secreted by the system and transcription factors which regulate its expression. However, it was recently found that T3SS not only secretes LEE encoded effectors but can also secrete proteins encoded on other prophages present in the EHEC genome. Characterisation of these non-LEE encoded effectors is ongoing and this study investigates the expression, regulation and function of non-LEE encoded effector H1 (NleH1) and H2 (NleH2). NleH1 and NleH2 are secreted by the T3SS but are encoded on different prophages. This study demonstrates that expression of NleH1 and NleH2 is induced in the same in vitro conditions which stimulate the expression of the LEE but is diminished upon initial host cell contact in vitro. Transcription of nleH1 and nleH2 is dependant upon factors specific to E. coli O157:H7 and these factors are regulated by LEE encoded regulators Ler and GrlA, as they have a positive effect on nleH transcription. NleH1 and H2 are predicted serine/threonine protein kinases and are able to autophosphorylate. Yeast two hybrid screening and 2D differential gel electrophoresis did not elucidate a eukaryotic protein binding partner of NleH1 or NleH2. Transfection assays show that they do not have a significant effect upon NF-κB activation in vitro. Determining the expression, regulation and function of non-LEE encoded effectors contributes towards further understanding of how this pathogen causes disease. Streptococcus pneumoniae, also known as the pneumococcus, is another globally important human pathogen. It is a very diverse pathogen, with over 90 capsular serotypes and is naturally competent for DNA uptake. Pneumococcal pathogenesis is facilitated by the production of a pore-forming toxin, pneumolysin. Pneumolysin’s activities in pneumococcal pathogenesis extend beyond its cytolytic function as it can also activate the complement pathway and modulate the host cytoskeleton. Pneumolysin is a member of a conserved family of toxins known as the cholesterol dependant cytolysins but differs due to the lack of a secretion signal peptide within its sequence. This indicates that it is not secreted from the bacterium however it has been reported that some strains can release pneumolysin in a cell lysis-independent manner. Additional to this, pneumolysin can also localise to the cell wall, and this localisation is not strain dependent. This study characterised codon-optimised N-terminally labelled pneumolysin constructs and applied them to assess the localisation of pneumolysin. In addition, the importance of autolysin and genes which are co-transcribed with Ply upon the localisation/secretion of pneumolysin was investigated by construction of a pneumococcal strain carrying an autolysin-pneumolysin fusion which naturally occurs in equine strains. These genes were not required for the translocation of pneumolysin or its association with the cell wall. Growth of this strain, and its isogenic parent, in vitro at a low density and low temperature resulted in the pneumolysin being detected in the broth culture. This indicates that pneumolysin can be released from the cell wall and that this action is not dependant upon the genes which were deleted in the mutant. The distribution of pneumolysin on the pneumococcal surface was assessed with immunofluorescence, and LumioTM substrate fluorescence, microscopy and found to have a general distribution. As a contribution to future pneumococcal research, codon-optimised fluorescent protein reagents were developed and can be used as reporters for gene expression and protein localisation.

Brachyspira hyodysenteriae is an anaerobic intestinal spirochaete and the aetiological agent of swine dysentery (SD). Throughout the UK and Europe, pathogenic and potential non-pathogenic isolates of B. hyodysenteriae have been recovered from pig herds, creating major obstacles for the detection and control of this economically important pathogen. Therefore, the main aim of this research was to compare one representative of virulent (P8544) and one representative of avirulent (P7455) strains of B. hyodysenteriae using genomic and proteomics approaches with a view to identify distinctive genes or proteins. The B. hyodysenteriae draft genomes of P8544 and P7455 consisted of a circular 3.0 Mb chromosome and a 31,469-34,822 bp circular plasmid that is also present in the only published B. hyodysenteriae genome, WA1. A considerable number of genes (~27-35) were identified in both the virulent and avirulent strains that shared high sequence homology with genes found in other spirochaetes, such as B. murdochii and B. intermedia, as well as in other species of bacteria; these may have been acquired via horizontal gene transfer. Comparative genomics of the two pathogenic genomes P8544 and WA1 versus the non-pathogenic genome P7455 revealed that the gene encoding for the methyltransferase type 11 (Bhyoa7455_20) was identified as being unique to the P7455 plasmid sequence and was successfully PCR amplified in a greater number of avirulent than virulent strains. However, as this was only just statistically significant (P=0.049), screening of a much larger strain set would clearly be required to support this gene as a suitable genetic marker to distinguish virulent and avirulent B. hyodysenteriae strains. Bacterial acquisition of iron in-vivo is crucial for successful colonisation and persistence in the host. A further aim of this study was to compare the growth phenotype of B. hyodysenteriae isolates P8544 and P7455 grown under iron-limiting conditions; such as would be found in-vivo in the large intestine of the host. Analysis of P8544 and P7455 growth rate in iron-sequestered media (containing 0.1 mM of the iron-chelator dipyridyl) demonstrated that both these isolates could replicate in this media although with an extended lag-phase of approximately 32-34 hrs; growth rate was on par with the iron-replete conditions. qRT-PCR analysis of eight putative iron-acquisition genes under iron-sequestered and iron-replete conditions revealed a difference in transcription for a number of ABC-transporter genes in P8544 and P7455, however, none of these were classified as statistically significant. Non-quantitative shotgun proteomic based approach was used to analyse outer-membrane protein (OMPs) expression of P8544 and P7455 under low-iron and iron-replete growth conditions and revealed alteration in the OM expression profiles between the isolates and conditions using KEGG analysis. The majority of expressed proteins under iron-replete conditions were categorized in membrane transport (11%) and carbohydrate metabolism (7%). Under iron-restriction the OM profile changed most obviously in a decreased percentage of proteins particularly assigned in the categories energy metabolism and membrane transport. The percentage of proteins assigned no predicted function increased by 19% under iron-limited conditions highlighting the fact that biological functions of the majority of these expressed proteins in such an environment remains to be determined. Two-dimensional gel-electrophoresis (2-DGE) of whole cell fraction indicated that the alkyl-hydrogen peroxide reductase protein (AhpC) in P7455 and the non-haem iron-containing ferritin (Bhyov8544_1528) in P8544 were significantly (P<0.05; 1.5-fold) more expressed under iron-restricted conditions than under iron-replete conditions. These data confirmed the importance of iron to virulent and avirulent B. hyodysenteriae. The so far identified significantly expressed proteins may serve as a potential biomarker for global diagnostic purposes for B. hyodysenteriae infections rather than a tool for differentiation for virulent and avirulent isolates. However, further work is required to prove if these candidates are expressed in-vivo and conserved in a wider panel of field isolates. In conclusion, this research has contributed to the scientific knowledge regarding B. hyodysenteriae stress responses induced by iron-starvation and has provided further insight into the genetic and proteomic make up of this spirochaete. This work should also aid future investigations concerning the biology and pathogenicity of this important and grossly understudied swine pathogen.

A sequence encoding a cathepsin B-like cysteine peptidase of the clan CA, family C1 was identified in the genome database of E. tenella. The sequence corresponded to a single copy gene, and did not carry any introns. The E. tenella enzyme, sharing 42% identity with the Toxoplasma gondii toxopain-1, which is reportedly involved in the invasion of host cells, was expressed as a soluble inactive zymogen in E. coli. Recombinant cathepsin B of E. tenella was functionally expressed as a mature enzyme in the Pichia pastoris inducible system as a glycosylation protein, but the glycosylations did not apparently affect the enzyme’s activity. The biochemical characteristics of the recombinant enzyme were consistent with what has been reported in the literature for cathepsin Bs. The cathepsin B of E. tenella was detected in oocyst and sporozoite extracts, and, more specifically, in microneme preparations. In the sexual stages, the enzyme localised to mature macrogametes in discrete granules, the size and location of which suggest that they are wall-forming bodies, organelles involved in the oocyst wall formation in Eimeria. These data suggest that the cathepsin B may play roles in both cell invasion by sporozoites and the formation of the protective wall of the oocyst. A single copy gene encoding a PPI was identified in the genome database of L. major. Active recombinant enzyme was successfully produced in E. coli and its biochemical properties coincided with those of mammalian PPIs. The active site catalytic triad E101, C210, and H234 (L. major PPI numbering) was confirmed by mutagenesis. The PPI activity was detected in L. major promastigotes, and the enzyme localised to the parasite cytoplasm. PPI knockout mutants were generated, and knock-out of the PPI activity did not seem to induce a phenotype in L. major. The parasites retained the properties in vivo and in vitro of L. major wild type cells. The over-expression of the active PPI in L. major promastigotes seemed to impair metacyclogenesis and in vivo infectivity, while the over-expression of the C210A mutant did not have any detrimental effect.

Neisseria meningitidis is an important cause of meningitis and septicaemia worldwide. Invasive meningococcal disease (IMD) is cyclical and varies by age group, being more common in children, especially those under 5 years. However, IMD is a rare outcome relative to asymptomatic carriage of the organism by the host and disease-causing isolates represent the tip of the iceberg in terms of the overall meningococcal population biology. The emphasis of previous research into N. meningitidis has rested firmly on the study of IMD isolates. In more recent times, however, there has been a more conscious effort to re-dress this balance to aid in our understanding of the relationship between carriage and disease. The Scottish Haemophilus, Legionella, Meningococcus and Pneumococcus Reference Laboratory (SHLMPRL) have accrued an extensive isolate archive dating back to the 1960s. Only recently with the expansion of molecular techniques has the SHLMPRL begun to investigate this valuable resource. For around a decade or more the SHLMPRL have routinely used genotyping techniques to characterise all IMD isolates it receives. However, these methods have hitherto not been employed to investigate isolates of the archive not obtained from cases of IMD. This study sought to characterise a collection of carried meningococci assembled from the isolate archive of the SHLMPRL that was obtained during the 31-year period, 1974 – 2004. Multi-locus sequence typing (MLST), porA Variable Region (VR) subtyping and a panel of genogrouping PCRs was used to characterise 791 carriage isolates. Temporal analyses of the data revealed how the presence of individual serogroups, clonal complexes, Sequence Types (STs), PorA subtypes and individual strain types in carried meningococci had changed in Scotland. Furthermore, these data were used in a comparison with a previously characterised collection of IMD isolates obtained during the same 31-year period to investigate the association of individual serogroups, clonal complexes, STs, PorA subtypes and individual strain types either with a carriage phenotype or with invasive disease. Nongroupable isolates [Odds Ratio: OR 17.66; 95% Confidence Interval: CI (12.71 to 24.54)] and those of serogroups W135 [OR 2.49; 95% CI (1.72 to 3.60)], Y [OR 6.26; 95% CI (4.18 to 9.39)], X [OR 3.13; 95% CI (1.20 to 8.14)], Z [OR 131.89; 95% CI (18.00 to 960.76)] and 29E [OR 21.30; 95% CI (4.76 to 95.36)] were significantly associated with a carriage phenotype. In contrast, serogroups A [OR 3.64; 95% CI (1.96 to 6.76)], B [OR 2.65; 95% CI (2.25 to 3.12)] and C [OR 1.92; 95% CI (1.58 to 2.33)] were significantly associated with invasive disease. The carriage strain collection reported herein was also observed to be highly diverse with the majority of STs identified only once. This diversity observed within the carriage strain collection [0.981; 95% CI (0.955, 1.006)] was significantly greater than the diversity within an IMD strain collection [0.938; 95%CI (0.934, 0.942)] from the same period. Temporal changes in the most prevalent clonal complexes (ccs) were observed throughout the 31-year period with increases in cc22, cc41/44 and cc269 and decreases in cc1, cc5, cc8, cc11, cc32, cc35, cc37, cc254, cc334 and cc364. Furthermore, for several ccs a significant association with a carriage phenotype (cc22, cc23, cc35, cc92, cc167, cc174, cc212, cc213, cc254, cc461, cc750, cc1157 and meningococci unassigned to a clonal complex) or with invasive disease (cc1, cc8, cc11, cc32, cc41-44 and cc269) was observed. Four lineages were identified amongst capsule null locus-containing meningococci, two of which, cc53 and cc1117, contained a unique allele (cnl-8) that was distinct from isolates of these lineages reported elsewhere. Despite significant associations at the level of cc, distinct differences in those associations were apparent for individual STs within a given clonal complex; most notably the significant association of ST41 and both ST43 and ST44 with invasive disease and a carriage phenotype, respectively. A feature of the carriage strain collection was the concentration of cc8 isolates during the period 1984 – 1986 and the high proportion of isolates obtained from individuals resident in Lanarkshire at a time when an episode of increased disease was experienced within that region. In this study, cc8 was found to be significantly associated with invasive disease. Furthermore, whilst ST8 was also significantly associated with invasive disease the most common strain type within cc8, C:8:8:5,2,36-2, [OR 1.68; 95% CI (1.25, 2.27)] was however, significantly associated with a carriage phenotype. The strain types B:213:22,14,36 [OR 2.38 (95% CI 1.40, 4.07)] and B:43:19,15-1,36 [OR 2.88 (95% CI 1.42, 5.88)] were also significantly associated with a carriage phenotype. Due to the heterogeneity of PorA subtypes in meningococci in Scotland the potential coverage by experimental or licensed PorA-based OMV vaccines would be limited. Therefore the introduction of monovalent or multivalent PorA-based vaccines in Scotland may be of little benefit. Improved strain coverage as a whole, not just against those of serogroup B, may require the addition of other vaccine antigens. Several other vaccine targets, including factor H-binding protein, are currently under investigation to improve coverage. Surveillance of these antigens and of the different lineages and serogroups in carriage and IMD isolates is essential to accurately monitor the effects that future vaccines will have on the meningococcus. We must remain vigilant despite a downward trend in cases of meningococcal disease in more industrialised countries.

Malaria remains a devastating disease with respect to both mortality and the constraints it places on the economic development of the countries in which it is endemic. Our laboratory is seeking new antimalarial targets, by characterising the protein kinases of the most lethal human malaria parasite, Plasmodium falciparum. As central components of many diverse signalling pathways, protein kinases are crucial for the control of proliferation and differentiation in other eukaryotes; we hypothesise that they play similar roles in P. falciparum. The life cycle of P. falciparum is complex, consisting of a series of tightly controlled stages of division and differentiation. In the related apicomplexan parasite Toxoplasma gondii, stress stimuli have been implicated in an important differentiation step, from rapidly dividing tachyzoites, to quiescent bradyzoites (which enable immune evasion). Evidence suggests that stress may also contribute to an essential differentiation stage, gametocytogenesis, in P. falciparum. In yeast and metazoans, part of the stress response is mediated through phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha), which results in selective translation of mRNAs encoding stress response proteins. Post-transcriptional control of gene expression is suspected to play an important role in P. falciparum. Importantly, the Goldberg laboratory recently demonstrated that similarly, in P. falciparum the eIF2alpha orthologue is phosphorylated in response to starvation. Here we identify the P. falciparum orthologue of the translation initiation factor eIF2alpha and provide bioinformatic evidence for the presence of three eIF2alpha kinases in P. falciparum; PfeIK1, PfeIK2 and PfPK4, only one of which (PfPK4) has been described previously (Mohrle et al., 1997). We show that one of the novel eIF2alpha kinases, PfeIK1, is able to phosphorylate P. falciparum eIF2alpha in vitro. In addition, initial experiments support previous observations that PfPK4 is indeed an active protein kinase (Mohrle et al., 1997). We present evidence that PfPK4 is essential for asexual growth, which precludes straightforward reverse genetics studies aiming to determine its possible role in gametocytogenesis. In contrast, transgenic parasites allowed us to show that neither PfeIK1 nor PfeIK2 are required for asexual growth, or sexual development of the parasite in the mosquito vector. However, preliminary evidence (requiring confirmation) may indicate that parasites lacking PfeIK1 over-express PfPK4, which would suggest that PfeIK1 may play an important function in the parasite. This study strongly suggests that a mechanism for versatile regulation of translation by several kinases with a similar catalytic domain, but distinct regulatory domains, is conserved in P. falciparum.