Glial cell line-derived neurotrophic factor (GDNF) is a growth factor promoting survival, proliferation and differentiation of neural crest cells. Neural crest cells play an important role within mesenchymal tissues during dental pulp and calvarial bone development. GDNF also has a role within non-neuronal tissues and is expressed during dental development. GDNF null mutations prevent the formation of the mineralised hard tissues of the tooth. The hypothesis for this study was that GDNF affects mesenchymal dental pulp cells (DPC), promoting the regenerative responses of mineralised tissues. This study utilised cell culture models to investigate the direct effects of GDNF on the proliferation and differentiation of dental pulp cells, bone marrow mesenchymal stromal/stem cells (BMSC) and calvarial osteoblasts. This research demonstrated that these culture models expressed GDNF and its receptors GFR\(\alpha\)1 and RET. GDNF was shown to directly stimulate DPC and osteoblast-like cell proliferation and differentiation. Moreover, GDNF was cytoprotective when DPCs were cultured under conditions reflecting aspects of inflammation, which may occur during repair. These conditions included supplementation with the pro-inflammatory cytokine TNF\(\alpha\) and culture under serum-starved conditions. It is proposed that GDNF may play an important regulatory role in dental pulp homeostasis and bone metabolism.

Streptococcus pneumoniae, also known as the pneumococcus, is an important human pathogen, with high burdens of disease and mortality worldwide. There are over 90 serotypes of this pathogen, demonstrating the vast amounts of diversity present. Currently, there are two pneumococcal vaccines, both targeting the polysaccharide capsule. However, one vaccine is ineffective in the paediatric population, whilst the other only targets a minority of disease-causing serotypes, and has increased disease caused by serotypes not present in the vaccine. One solution is a new pneumococcal vaccine targeting a protein virulence factor possessed by all pneumococci, which would afford cross-serotype protection. As a result, it is important to assess the diversity of pneumococcal virulence factors in order to determine their potential as vaccine candidates, as excess diversity present may prevent full serotype-independent protection of a vaccine. Furthermore, diversity studies offer important insight on pneumococcal biology, epidemiology and pathogenesis. The diversity in the toxin pneumolysin (Ply) was greater than previously thought, with 14 protein alleles discovered. However, diversity remained significantly lower than surface-exposed virulence factors, indicating this toxin may be more suitable as a vaccine candidate. Furthermore, all 14 alleles were recognised by polyclonal antibodies, showing the potential cross-serotype protection of a vaccine targeting this toxin. A novel non-haemolytic Ply allele was associated with clones recently expanding in the pneumococcal population, as well as serotypes associated with outbreaks of pneumococcal disease. The non-haemolytic toxin may therefore play a role in driving clonal expansion in certain genetic backgrounds, or be involved in establishing outbreaks of pneumococcal disease. The diversity in the neuraminidase A (NanA) enzyme was significantly higher than in Ply, with many point mutations, mosaic blocks and insertions regions present in 18 divergent alleles. This level of diversity should not be prohibitive to the use of this protein as a vaccine candidate, as polyclonal antibodies recognised 4 NanA alleles of significant diversity, indicating the possibility of cross-serotype protection. The role of NanA in pathogenesis of pneumococcal haemolytic uraemic syndrome (p-HUS) was investigated, although there was no correlation between p-HUS and NanA allele or activity. The novel discovery that pneumococcal NanA was inhibited by viral neuraminidase inhibitors of influenza allowed insight into the synergistic relationship between these two deadly pathogens, and showed for the first time that treatment with these drugs acts on both the primary and secondary pathogen. One of these inhibitors, Oseltamivir, was found to have potential in treating secondary pneumococcal pneumonia, which may help decrease the significant burden of this disease, as well as reducing the over-reliance on antibiotics for treating pneumococcal diseases. Homologues of Ply and NanA were identified and characterised in the related species Streptococcus mitis and Streptococcus pseudopneumoniae, giving insight into the evolutionary relationships between these species. Furthermore, the presence of these homologues in related species gives rise to the possibility of pneumococci acquiring altered genes through horizontal gene transfer. The selective pressure of a vaccine targeting these proteins may give evolutionary advantage to these pneumococci, resulting in evasion of a vaccine. Microarray studies have been used to assess pneumococcal diversity at a genome-wide level. Gene expression studies identified candidate genes which may play a role in p-HUS pathogenesis. Further studies into this area will improve the diagnosis and treatment of this disease. Furthermore, a large number of established pneumococcal virulence factors, many of which are vaccine candidates, were found to have homologues in closely related commensal species. These results must be taken into consideration for future protein-based pneumococcal vaccine design.

This thesis examines the development of a novel in vitro biological caries model and its suitability in testing the efficacy of anti-caries approaches. Dental caries remains a public health concern worldwide; with extensive treatment costs and impacts on quality of life. Ineffective removal of all dental plaque from tooth surfaces after brushing, the insufficient delivery of anti-caries therapies; along with continuing shifts towards high frequency, sucrose-rich food consumption, expedites the caries disease process. It is, therefore, important to explore caries risk and development at these sites, particularly when representatively assessing the efficacy of a test agent in preventing caries. This caries model enabled the study of the anti-caries effects of fluoride to assess its efficacy in conditions simulating the modern diet. The current methodology employed the Constant Depth Film Fermenter (CDFF) to investigate the caries disease process in response to fluoride delivered continuously; twice and thrice daily; and at different concentrations. The approach is the first in CDFF research modelling caries inclusive of a biologically relevant microcosm biofilm in addition to enamel demineralisation. Specific members of multispecies biofilm were selectively enumerated using traditional microbiological culture techniques whilst caries was simultaneously quantified with Transverse Microradiography (TMR), Quantitative Light-Induced Fluorescence (QLF), and Non-Contact Surface Profilometry (NCSP). The fluorescence of biofilm illuminated by QLF was also investigated. Results indicated that quantities of total or specific members of the microbial community are not direct indicators of caries risk and turning focus towards the metabolism of oral biofilm bacteria, and how it may be affected, is vital in caries research. TMR and QLF agree when quantifying caries whilst NCSP shows promise in studying surface changes. At 0.05 ppm, fluoride was unable to exert a significant anti-caries effect despite being continuously present during and between sucrose exposures. Laminated lesions confirmed the importance of maintaining elevated levels of fluoride in the oral environment throughout the day. At higher concentrations (1,450 and 228 ppm fluoride) the anti-caries efficacy of fluoride when supplied in frequent applications throughout the day was confirmed. A third application of fluoride did not appear to additively benefit enamel, since all strategies were effective after 10 days regardless of frequency. Nonetheless the increased plaque fluoride reservoir and subtle antimicrobial effects than in twice daily pulsed biofilm, mean the benefit of a third application is likely more discernible in the long term. Scanning Electron Microscopy (SEM) and Energy-Dispersive X-ray Spectroscopy (EDX) elucidated significant calcium fluoride deposits of enamel surfaces beneath biofilm exposed to 1,450 ppm fluoride continuously. In conclusion, the CDFF can produce multispecies biofilm under conditions similar to those of the oral milieu and investigate its cariogenicity in response to diet and experimental anti-caries agents. The model could be examined using an array of techniques to obtain information about aspects of the biofilm, the substratum, and to validate upcoming methods in an orally representative environment. In this regard, the current study contributes not only to enamel caries research but to biofilm research in general by minimising variation.