Craniofacial assessment for diagnosis, treatment planning and outcome has traditionally relied on imaging techniques that provide a static image of the facial structure. Objective measures of facial movement are however becoming increasingly important for clinical interventions where surgical repositioning of facial structures can influence soft tissue mobility. These applications include the management of patients with cleft lip, facial nerve palsy and orthognathic surgery. Although technological advances in medical imaging have now enabled three-dimensional (3D) motion scanners to become commercially available their clinical application to date has been limited. Therefore, the aim of this study is to determine normal and abnormal lip shapes during movement for use as a clinical outcome measure using such a scanner. Lip movements were captured from an average population using a 3D motion scanner. Consideration was given to the type of facial movement captured (i.e. verbal or non-verbal) and also the method of feature extraction (i.e. manual or semi-automatic landmarking). Statistical models of appearance (Active Shape Models) were used to convert the video motion sequences into linear data and identify reproducible facial movements via pattern recognition. Average templates of lip movement were created based on the most reproducible lip movements using Geometric Morphometrics (GMM) incorporating Generalised Procrustes Analysis (GPA) and Principal Component Analysis (PCA). Finally lip movement data from a patient group undergoing orthognathic surgery was incorporated into the model and Discriminant Analysis (DA) employed in an attempt to statistically distinguish abnormal lip movement. The results showed that manual landmarking was the preferred method of feature extraction. Verbal facial gestures (i.e. words) were significantly more reproducible/repeatable over time when compared to non-verbal gestures (i.e. facial expressions). It was possible to create average templates of lip movement from the control group, which acted as an outcome measure, and from which abnormalities in movement could be discriminated pre-surgery. These abnormalities were found to normalise post-surgery. The concepts of this study form the basis of analysing facial movement in the clinical context. The methods are transferrable to other patient groups. Specifically, patients undergoing orthognathic surgery have differences in lip shape/movement when compared to an average population. Correcting the position of the basal bones in this group of patients appears to normalise lip mobility.

Endogenous electric fields (EFs) are present during a variety of physiologic and pathologic events, including penetrating injury to epithelial barriers. An applied electric field with strength within the physiologic range can induce directional cell migration of epithelial cells, endothelial cells, fibroblasts, and immune cells suggesting a potential role in controlling cell behaviours during wound healing. Dendritic cells (DCs) and dermal fibroblasts were used to explore the molecular mechanisms underlie EF-induced cell activities during two aspects of wound healing: immune response and remodelling. In this study, we investigated the effects of applied EFs on several types of DCs in response to IL18. DC progenitor cells KG-1 shows dose dependently response to EFs stimulation to increase IFN-γ expression. Moreover, the migration of KG-1-derived DCs and Langerhans cells (LCs) in mouse skin showed increased response to IL18 with directional migration when exposed to EFs in vitro and ex vivo. Furthermore, the in vivo investigation suggested that pharmacologically increased trans-epithelial potential difference (TEPD) induced LCs to emigrate from skin to draining lymph node. The sensitization of DCs to IL18 can be strengthened by EFs through redistribution of IL18 receptors and phosphorylation of p38 MAPK. We also comparatively studied the responses of human chronic wound fibroblast (CWF) and chronic matched fibroblast (CMF) to applied EFs with addition of platelet derived growth factor (PDGF). The results indicate that 1) EFs induce human dermal fibroblast directional migration in a voltage dependent manner. 2) CWF shows impaired sensitivity in response to EFs compared to CMF and HF. 3) Activation of PDGFR and PI3K are both required for EF-induced directional migration. 4) PDGF attenuates EF-induced migration directedness through PDGFR-ROCK other than PI3K pathway. 5) Optimised concentration of PDGF plus physiological EFs enhance chronic wound healing. We propose that the EF-induced re-distribution of the receptors on the cell surface results in a shift of membrane receptors between the cathode-facing and the anode-facing membrane of the cell. There would be a higher probability to overcome the threshold of signal transduction at the higher density receptor side. The downstream IV signalling cascade therefore can be ignited. Understanding the signalling pathways underlying guidance cues (EFs, cytokines, chemokines) will help to optimise future therapies for immunomodulation, vaccination, wound healing and regeneration.

The remaining debris and biofilm in the anatomical complexities of root canal systems can affect treatment outcomes. Files with asymmetric cross-section design may improve debris and biofilm removal from these difficult spaces during canal preparation. Tooth opacity and different densities of the remaining materials prevent the direct systematic assessment of the preparation process. The present study assessed remaining debris and biofilm using a novel transparent root canal model with novel approaches. Natural and simulated root canal samples with isthmus space were evaluated. Canal preparation by ProTaper Next and Revo-S asymmetric systems was evaluated in comparison to the standard ProTaper Universal symmetric system. The root canals were investigated by microcomputed tomography (micro-CTL confocal laser scanning microscopy (CLSML and optical coherence tomography (OCT))) imaging tools. Data analysis was undertaken with SPSS (V. 24). Files with asymmetric cross-section and constant taper removed more debris and biofilm from the complex root canal system. The model allowed direct assessment of remaining materials and confirmed the novel imaging approach with the OCT. In conclusion, the asymmetric design improves debris and biofilm removal especially when used with a constant taper. The model was verified as an ideal system for assessing root canal treatment in vitro.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that affects about 1% of the world population. This common disease is characterized by chronic inflammation of the synovium that leads to destruction of cartilage and bone in the join, and the cause of this exacerbated inflammatory reaction remains unknown. Periodontitis (PD) is also a chronic inflammatory disease characterized by destruction of bone and other connective tissue that shares notable similarities with RA. Over the last 20 years, numerous studies have found an epidemiological connection between RA and periodontitis. However the biological mechanisms that explain the interrelations between the two conditions are not known. The aim of this thesis was to investigate the role of periodontitis in RA and the effect of periodontal therapy on immunological and microbiological parameters. To do that, different biological samples were collected from two pilot studies, comparing RA and periodontitis patients to the appropriate controls and from a selected group of randomized RAPD patients before and after periodontal therapy. The antibody response and subgingival microbiome of patients with RA and periodontitis were compared to the appropriate controls (no RA no PD, RA no PD, no RA PD). The effect of periodontal therapy on these parameters and on the cytokine changes in gingival crevicular fluid was also investigated. The findings from this thesis lend further credence to the link between RA and the oral microbiome, with RA patients having a disrupted and more anaerobic microflora and an exacerbated immunological reaction against periodontal bacteria and citrullinated proteins.

Dental Health Care has been provided by the Grenfell Association for the people of Northern Newfoundland and Labrador since the late 1800's. The capability to provide dental care has been influenced by many factors including a small population living in isolated communities over an enormous geographical area with adverse climatic conditions and long lines of communication. Transportation alone presented a formidable challenge. Based on his early experience with the Association between 1964-69, the author realised, on his return to Canada in 1976 that there was need to identify major problems, adapt existing facilities and change established philosophies, as well as to introduce new systems and methods to improve dental health-care delivery. The main areas of concern were the high prevalence of dental disease, the lack of adequate manpower, appalling working conditions and lack of overall policies. Initially, one of the major tasks was to establish basic equipment at each clinic. Small portable light-weight dental units were designed and constructed by the author, for each dentist with a travelling commitment, these units being readily connected to on-site compressors. Thereafter, a longterm policy of equipment upgrading and replacement was pursued to enable dental staff to undertake a complete range of clinical duties. Later, when new Health Centres and Nursing Stations were established, the author had the opportunity to become involved in their design and layout. The method of dental service financing also contributed to many of the problems. Thus the existing monetary arrangements had to be studied which, in turn, required an understanding of the sources of funding. Hence, following analysis of financial data collected after only the first few months of 1976, it became clear that several issues required urgent action and, as monetary considerations appeared to be the crux or the problem, the author decided to introduce a standard billing system which was applied throughout the region. This was primarily operated by dental assistants to provide continuity between dental staff changes. To reduce financial hardship, fee increases to conform with general dental fees charged elsewhere in Newfoundland were introduced gradually over a five year period. When sufficient data on the work being undertaken via the Provincial Dental Programme became available, Department of Health funding was evaluated. This led to a report and submission to the Department of Health in 1977, which was intended to exert pressure for fairer remuneration of the Grenfell Dental Service. The effect of the measures taken by the author to increase manpower, improve facilities, reduce caries prevalence, introduce preventive measures and modify unfavourable treatment patterns has been evaluated continuously throughout his tenure as Co-ordinator and subsequently as Chief of Dental Services. The establishment of administrative pathways and centralised procedures has also provided evidence of change. In addition, further recommendations regarding future priorities have been formulated to ensure continuous staff recruitment and supporting facilities required to achieve the W.H.O. dental goals set for the year 2000. Happily, with 10 years of the Century remaining, a further mean DMFT reduction of only 0.82 per child is now required to meet the target of 3.0 for 12-yr-olds.

In Malaysia, the public water supply has been artificially fluoridated since 1972 at an optimum level of 0.7 ppm fluoride as a public health measure to control dental caries. However, concerns arose that a fluoride concentration of 0.7 ppm was too high given increasing exposure to other sources of fluoride. That prompted a downward adjustment of the fluoride concentration from 0.7 to 0.5 ppm in 2005. In addition to Malaysia, there has recently been a movement towards the downward adjustment of fluoride concentration in the water in the United States, Hong Kong, Singapore and Ireland. However, little is known about the impact of such adjustments on oral health. This thesis aimed to evaluate the outcome of the downward adjustment of fluoride concentration in the Malaysian public water supply from 0.7 to 0.5 ppm in relation to dental fluorosis and dental caries. Two projects were conducted. The first project comprised a systematic review to critically appraise the literature on stopping the addition of fluoride or reducing the level of fluoride in public water supply on dental caries and fluorosis. This review highlighted the gaps in knowledge and several methodological issues such as lack of examiner blinding and control of confounders. The second project was a cross sectional survey involving life-long residents aged 9 and 12 year-olds in fluoridated and non-fluoridated areas in Malaysia (n=1155). In the fluoridated area, children aged 12 years and 9 years were exposed to 0.7 and 0.5 ppmF respectively at the times when maxillary central incisors enamel developed. Fluoride exposures were assessed by questionnaire. Standardized photographs of maxillary central incisors were blind scored for fluorosis using Dean’s Index. Caries prevalence was examined using ICDAS-II criteria. The key findings indicated that the change in fluoride iv level from 0.7 to 0.5 ppm has reduced fluorosis and maintains caries preventive effect. The change in fluoridation concentration has also had a significant impact on caries prevalence at different thresholds of severity. The findings support the policy initiative of a lower fluoride concentration in the Malaysian public water supply. It also highlights the need for modification of oral health advice with regards to fluoride exposure in maximising caries prevention while minimising fluorosis.

Mechanically ventilated (MV) patients are at risk of ventilator-associated pneumonia. During mechanical ventilation, it has been proposed that the mouth becomes colonised by respiratory pathogens (RP) and the endotracheal tube (ETT) facilitates leakage of oropharyngeal secretions to the lower airways, whilst also supporting a biofilm. These are likely contributory risk factors for VAP. This research aimed to further establish the relationship between oral microorganisms and RP in colonisation of dental plaque and ETT biofilms. The study also investigated intervention strategies to limit RP colonisation. The microbial composition of dental plaque, ETT biofilms, and non-directed bronchial lavages (NBLs) from MV patients was characterised using culture and molecular approaches. RPs were frequently present at all these sites, with oral microorganisms also occurring in ETTs and NBLs. Isolates from these sites in a single patient also were determined to be the same strains based on molecular typing. Additionally, NGS showed no significant difference between dental plaque and ETT biofilm microbiomes. In vitro biofilms revealed that oral microorganisms increased RP colonisation and associated gene expression in biofilms. In in vivo studies, toothbrushes and foam swabs were found to be equally efficient at removing dental plaque and improving oral hygiene in MV patients. In vitro investigation found Chlorhexidine to be the most effective mouthwash in combatting ETT biofilms, despite high tolerance by P. aeruginosa. No difference between ETT biomaterials in supporting biofilms was evident. The work highlights the importance of dental plaque as a reservoir of RP in MV patients, and these RP also colonise ETT biofilms. The synergistic effect of oral microorganisms in promoting RP colonisation reinforced the need to adequately manage oral care in MV patients. For the first time, equal effectiveness of achieving improved oral care by toothbrushes and foam swabs was demonstrated.

Introduction Modern society is passionate about beauty and aesthetics. According to a 2016 survey by the International Society of Aesthetic and Plastic Surgery, the demand for aesthetic surgery is more than ever. People’s perception and awareness of facial aesthetics has increased. Orofacial cleft is the most common facial dysmorphology, with prevalence about 1.46:1000 in Scotland. The aim of the initial surgical repair of cleft lip is to improve facial aesthetics and function, without interrupting facial growth. Nevertheless, facial asymmetry is a stigma in cleft patients, and revision surgery due to a patient or their parents’ dissatisfaction with the outcomes is not uncommon. Objective evaluation of facial asymmetry after primary surgical repair is valuable. It is an indication of the success of surgery, and it informs the surgeon of the magnitude and location of residual asymmetry. The evaluation of facial asymmetry has evolved significantly from landmark-based assessment to surface-based analysis. The latter provides a comprehensive evaluation of facial asymmetry by superimposing the original 3D model on its mirror copy. This permits the quantification and the visualisation of the disparity between the two halves of the face. Many studies evaluated facial asymmetry a few years after primary surgical repair. Longitudinal monitoring and quantifying of facial shape changes can potentially guide the surgeon to the optimal surgical technique. Only a few studies evaluated facial asymmetry before and after primary surgery, and their analysis was dependent on a set of facial landmarks that did not describe the asymmetry of facial surfaces between these landmarks. Unfortunately, the existing literature does not provide comprehensive longitudinal evaluation of facial asymmetry of cleft patients, and the impact of facial expression on residual facial asymmetry has not been fully investigated. Aim The aim of this study was the longitudinal evaluation of facial asymmetry of UCLP patients using an advanced facial analysis tool, and to compare the postoperative residual asymmetry with the control group. The study was carried out to assess the impact of growth and facial expression on residual facial asymmetry. Methodology This study was carried out on 30 UCLP patients. All the patients were Caucasian and underwent the same surgical protocol, which was carried out by the same surgeon at the Royal Hospital of Sick Children, Edinburgh. 3D facial images were captured for the patients, before surgery, at about 4 months after surgery and at four-year follow-up, at rest and at maximum smile using 3dMDface system. Historical data of 70 3D facial images of six-month-old non-cleft infants were also analysed in this study. Facial asymmetry was evaluated using a generic mesh. A generic facial mesh is a mathematical facial mask that consists of 7,190 vertices. The mesh was conformed on each 3D facial image. The conformed meshes were utilised to evaluate facial asymmetry using two methods: the average asymmetry, the total and regional facial asymmetry. The average asymmetry method involved the creation of four average faces for cleft patients: an average preoperative face, an average postoperative face, and two average faces at the four-year follow-up (one at rest and one at a maximum smile). The fifth average face was that of six-month-old non-cleft infants. A mirror copy for each average 3D facial model was created by reflecting it on a lateral arbitrary plane. The original and mirror models were superimposed, the absolute distances between corresponding points on the two surfaces were calculated and analysed in three directions (mediolateral, vertical and anteroposterior), to quantify facial asymmetry. The results were displayed in colour-coded maps. Asymmetry scores were obtained by calculating the median of the absolute distances between corresponding points for the total face, upper lip and nose. The asymmetry scores in the mediolateral, vertical and anteroposterior directions were also quantified. Statistical tests were applied to detect significant differences in asymmetry scores of the total face and each facial region between study groups (before surgery, after surgery and at four-year follow-up), and between surgically managed cleft group and the control group. The correlations of asymmetry scores of the total face, nose and upper lip before surgery, after surgery and at four-year follow-up were also investigated. Results Facial asymmetry in cleft patients was dramatically improved after surgery. However, the postoperative residual asymmetry of UCLP patients was significantly higher than the non-cleft infants in the three directions. Furthermore, facial asymmetry increased during growth, with main impact on the nose. Facial expressions accentuated the residual asymmetry. Specifically, there was considerable shifting of the upper lip toward the scar tissue of the affected side. The residual asymmetry of the nose at the four-year follow-up was correlated to initial nasal asymmetry and residual nasal postoperative asymmetry. The anteroposterior deficiency of the upper lip, nose and paranasal area was pronounced in the cleft group at all time intervals due to insufficient bony support of the cleft maxilla. Conclusions Cleft patients and their parents should be informed of the likelihood of residual asymmetry following surgery. Refinements in primary surgery are necessary. The superficial and deep fibres of the orbicularis muscle have to be accurately repaired according to the direction of the muscle fibres to avoid the shifting of the philtrum of the upper lip toward the scar tissue on the affected side. The orbicularis oris muscle has to be adequately dissected and rotated in the downward direction to eliminate the residual vertical deficiency at the corner of the mouth on the affected side. An incision in the internal lateral side of the nose should be considered to reduce this deficiency. The levator labii superioris alaeque nasi muscle of the cleft side has to be reflected and sutured to the corresponding muscle fibres on the other side, to avoid the residual shifting of the nose to the non-cleft side, and to eliminate the residual vertical deficiency of the alar base on the cleft side during smiling. Revision surgery should be delayed until completion of growth. Before lip revision surgery, it is necessary to evaluate the residual asymmetry when the face is at rest and during facial expressions. Consideration should be given to initial nasal asymmetry and residual postoperative nasal asymmetry. Patients should be informed about the expected need for revision surgery including rhinoplasty. We were able for the first time to quantify facial asymmetry in three directions which provided an insight into the cause of the residual facial asymmetry at rest and at maximum smile. The generic mesh is an innovative tool for the assessment of facial asymmetry.

The overall aim of this study, was to investigate the jaw-opening reflexes in TMD patients and bruxists in order to get a better understanding of these disorders. At the beginning, it was decided to study the reflexes evoked in the masseter muscle by electrical stimulation across the lip in healthy subjects. The aim was to find the threshold at which each of the significant responses occurred and to investigate the effect of reversing the polarity of the stimulus electrode on the pattern of reflexes. A sequence of inhibitor, excitatory, inhibitory and excitatory responses could be produced in the muscle by both polarities of stimuli. It was found that stimulation of nerves supplying the skin outside the mouth evokes predominantly long-latency jaw reflexes whereas short-latency responses can be evoked by stimulating nerves supplying oral mucosa. Furthermore, long-latency excitatory reflexes seem to be the most easily evoked by stimulation of the lip. Another aim of the study was to investigate whether difference could be observed between bruxists and non-bruxists in response to electrical stimulation of the lip. In both groups, there were significant differences in the thresholds of the different responses. Also, there were differences between the two groups in the presence of the short-latency excitation and the long-latency inhibitory responses. These findings suggest that long-latency inhibitory responses evoked by electrical stimulation of the lip are weaker in bruxists than in non-bruxists. Finally, jaw reflexes in TMD patients were investigated to determine whether differences could be detected in electrically-evoked inhibitory and excitatory responses. Moreover, the occlusal splint is one of the most universally accepted forms of therapy in TMD patients and it was possible that these splints might have an effect on the pattern of jaw reflexes in view of their therapeutic effect.

The adhesion G protein-coupled receptor 56 (GPR56) plays a major role in early brain development. Mutations in Gpr56 cause the developmental brain disease bilateral frontoparietal polymicrogyria (BFPP), which is recapitulated in Gpr56-/- mice. GPR56 interacts with collagen III in the brain pial basement membrane and with tissue transglutaminase (TG2) in melanoma, where it potentially acts as a tumour suppressor by antagonising TG2-related functions. In glioblastoma, however, GPR56 is highly overexpressed and might play an important role for the invasive behaviour of these cells, which could be regulated by TG2 in the tumour stroma. The main aim of this thesis was to analyse GPR56 signalling in response to TG2, thus exploring a potential link to cancer development and progression. Identifying downstream signalling pathways activated by GPR56 in response to TG2 could provide valuable information regarding potential targets for future therapeutic intervention in the context of anti-cancer therapies. In order to investigate GPR56 signalling, a cell-based assay was established that measures GPR56 activation as metalloproteinase-dependent ectodomain shedding of alkaline phosphatase-tagged amphiregulin (AP-AR) in HEK293 cells. The assay was used to demonstrate for the first time activation of GPR56 by TG2. RhoA/Rho-associated protein kinase (ROCK)are activated by GPR56, which likely requires Gα12/13 coupling to GPR56. RhoA/ROCK activity is required for the activation of a disintegrin and metalloproteinase 17 (ADAM17), the main metalloproteinase responsible for GPR56-dependent AP-AR shedding. Shedding of EGF-like ligands such as amphiregulin leads to the activation of epidermal growth factor receptors, inducing cellular responses such as cell proliferation and migration. Further investigations using different GPR56 mutants revealed that the Nterminal domain of GPR56 is required for activation by TG2. The crosslinking activity of TG2 is dispensable for GPR56 activation and the C-terminal β-barrel domains of TG2 are sufficient to stimulate GPR56 signalling. Moreover, two novel potential GPR56 ligands, TG6 and TG7, were shown to stimulate GPR56-dependent AP-AR shedding. Using confocal microscopy, GPR56-dependent internalisation of TG2 via clathrin-coated pits was demonstrated, a mechanism that is well known for agonist-activated GPCRs. Finally, the potential role of GPR56 in glioblastoma was investigated by generating stable GPR56 knockdown glioma cells. Analysis of GPR56 knockdown cells indicated that GPR56 may play a role for glioblastoma migration and invasion. These results present a novel signalling pathway activated by GPR56 in response to TG2 that is involved in cell proliferation, growth and migration, potentially providing an explanation for the supposed tumour promoting functions of GPR56 in glioblastoma.