Mycobacterial chaperonin 60 (Cpn60) is an intracellular molecule essential for cellular function due to its protein folding actions. However, it has recently been established that Cpn60 can be released by certain bacteria and act as an extracellular signalling protein. Mycobacterium tuberculosis has two genes coding for Cpn60 proteins: cpn60.1 and cpn60.2 (also known as hsp65). It was recently shown that Cpn60.1 and Cpn60.2 are pro-inflammatory activators of myeloid cells and that the effects of Cpn60.1, but not Cpn60.2, depend on CD14. This PhD project shows that M. tuberculosis Cpn60.1 and Cpn60.2 proteins strongly bind to human peripheral blood monocytes but their binding sites are different. Both M. tuberculosis Cpn60 proteins depend on MyD88 for the maximal activation of intracellular signalling pathways but have different TLR requirements: Cpn60.1 signals exclusively through TLR4 while Cpn60.2 needs the presence of TLR2 and TLR4 for maximal induction of pro-inflammatory cytokines. A study has recently established that an M. tuberculosis mutant with an inactivated cpn60.1 gene is unable to induce granulomas in animal models suggesting that Cpn60.1 has inflammation-modulatory effects on myeloid cells. This PhD project identified that recombinant M. tuberculosis Cpn60.1 has bipolar effects on human circulating monocytes. At high concentrations Cpn60.1 induces the synthesis of TNF-α and promotes the phosphorylation of NF-κB p65, p44/42MAPK and p38 MAPK while at picomolar concentrations, Cpn60.1 inhibits the lipopolysaccharide-induced formation of TNF-α and the phosphorylation of NF-κB p65 without affecting the activation of MAP kinases. Gene expression analysis show that low concentrations of Cpn60.1 completely suppress cell activation while higher concentrations of Cpn60.1 down-regulate the expression of major pro-inflammatory cytokine and chemokine genes in human monocytes. It is therefore concluded that M. tuberculosis Cpn60.1 is an unusual protein with the ability to induce bipolar effects which may help explain the pathology of granuloma formation in tuberculosis.

The principle of Optical Coherence Tomography (OCT) is based on the property of light coherence. OCT generates cross-sectional images of two-dimensional objects to obtain in-vitro and in-vivo images of tissues. Non–commercially available OCT systems, which have a higher resolution and scanning rate, have been previously reported. However, some clinical research has already been conducted using the first commercially available OCT device (Niris system) to image the larynx; but applications on oral and skin tissue have not been tested yet. This thesis aims to explore, compare and validate three specific types of commercially available OCT equipment for imaging head and neck tissue. An animal cancer model has been used to verify the feasibility of one system (Niris) to differentiate normal from malignant oral tissue, using in-vivo tissue samples. Since images of oral tissue samples didn’t show much structure using the Niris system, a different machine (Michelson Diagnostic bench based) with different specifications and resolution was employed. Great emphasis has been put on validating OCT structurally and histomorphometrically in comparison to the gold standard of pathology. This was tested and validated with ex-vivo oral and skin tissues using the lab based version of the machine. Use of an upgraded system (Michelson vivo sight with probe) has been tested on abnormal oral and skin biopsy tissue but with different timing for the scan (instant ex- vivo). One original study evaluated and classified tongue papilla atrophy from patients having their suspicious tongue lesions biopsied. In conclusion, this thesis concludes that the new version of this commercially approved OCT system can be applied to the diagnosis of superficial premalignant and malignant oral and skin lesions in-vitro. Furthermore, OCT holds the promise of complementing surgery to eradicate tumors and monitor the consequences.

It is widely observed that dermal wound healing results in the re-establishment of skin continuity through the formation of a scar. In comparison oral wounds heal more rapidly, with less visible scarring. The diverse nature of sub-epithelial fibroblasts has targeted them as mediators for such a distinction in phenotype. Fibroblasts and their differentiated form, the myofibroblast (characterised by the expression of α-SMA), are chief synthesisers of the ECM during wound healing. Myofibroblasts in particular play an important role in wound contracture, ECM synthesis and remodelling during normal wound healing and scar production; but are also observed in fibrosis and pathological scarring. The most potent mediator of this phenotype in the cytokine TGFβ1, which is abundantly present throughout wound healing. Here we used organotypic co-cultures and mono-cultures to assess the differences in phenotype and the relative contribution of topographically distinct fibroblasts within them. There is currently little data on the responsiveness of oral buccal mucosal and skin fibroblasts to TGFβ1, particularly when keratinocytes are present in a 3D environment. Here, for the first time, we used heterotypic organotypic co-cultures containing skin and oral buccal mucosal fibroblasts and added TGFβ1 under both resting and tethered conditions. We monitored the changes in response of these fibroblasts with and without the presence of keratinocytes, and subsequently assessed changes in phenotype upon substitution of the dermal fibroblasts with those from the reduced scarring oral buccal mucosa. Under resting conditions oral fibroblast seeded organotypics showed a lower constitutive myofibroblast expression, and were unresponsive to TGFβ1 mediated α- SMA expression, regardless of substrate compliance. These fibroblasts were found to significantly promote epithelial maturation under resting conditions, and express higher levels of active MMP-9 (and enzyme involved with keratinocyte migration) upon treatment with TGFβ1, when compared to dermal fibroblast seeded counterparts. When oral fibroblasts were introduced into a dermal organotypic co-culture environment, we observed a preferential change α-SMA phenotype, offering the potential for use of these cells in an area distinct from there origin to a favourable effect.

Chapter I: Literature review on the Temporomandibular joint (TMJ) and Temporomandibular disorders (TMD) Chapter II: Systematic review of TMD in orthognathic patients This review was conducted to investigate the prevalence of temporomandibular joint dysfunction (TMD) in orthognathic patients and to determine the effect of the surgical intervention on the status of the temporomandibular joint (TMJ). A methodological process was applied for study selection, data management and quality assessment and meta-analyses were conducted where appropriate. This review identified 53 papers for inclusion and there was heterogeneity in the diagnosis and classification of TMD between the studies. Patients undergoing orthognathic treatment for the correction of dentofacial deformity and suffering from TMD appeared more likely to see an improvement in their signs and symptoms than deterioration, particularly with respect to pain related symptoms. This information should be given to prospective patients during the consent process, but it should be stressed that no guarantees can be made. Chapter III: TMD in orthognathic patients and a control group with no skeletal discrepancies Sixty eight orthognathic patients and 72 control subjects (with no anterior-posterior, vertical or transverse discrepancies) were recruited for this section of the PhD. Self-reported symptoms and clinical signs of TMD were recorded and compared between the two groups. A significant difference in TMD prevalence was observed between the controls (27.8%) and patients (44.1%), with the patients being more susceptible to TMD. However, although orthognathic patients appear more likely to suffer from TMD, whether treatment improves their TMJ condition is highly questionable. This issue should be highlighted in any informed consent process. Chapter IV: A longitudinal study of TMD in orthognathic patients Twenty orthognathic patients were followed longitudinally throughout treatment to establish whether TMD signs and symptoms altered during the course of treatment. Although no significant differences were found when comparing the pre-treatment (T1) findings with those prior to surgery (T2), sufficient individual changes in TMD signs and symptoms were observed to question the suitability of the "prior to surgery" time point as a baseline for comparisons in future studies. When comparing pre (T1) and post-treatment (T3) TMD changes, no significant differences were observed. This study supports the theory that TMD is a dynamic condition and signs and symptoms are likely to fluctuate throughout treatment. However, the small sample size in this study was clearly a limiting factor. Chapter V: TMJ information course: Comparison of the instructional efficacy of an internet-based TMJ tutorial with a traditional face-to-face seminar A TMJ tutorial was developed on a virtual learning environment (VLE) to enable students to enhance their examination and diagnostic skills and a randomised cross-over trial was then conducted. Thirty postgraduate students were recruited as participants and the success of this mode of teaching was compared with a conventional face-to-face seminar. This study found that both modes of teaching were equally effective in delivering information to students but teaching the topic twice enhanced the retention of knowledge. In addition the students reported positive perceptions of VLE learning and the feedback for this mode of teaching was comparable with traditional methods of teaching.

Comparison of multi-lineage differentiation ability of craniofacial skeletal muscle and limb muscle progenitor cells Alqahtani, K, Wall, I, Lewis, M.P. Craniofacial Skeletal Muscles (CSkM), including masticatory muscles, have a number of properties that distinguish them from the majority of skeletal muscles. They have different embryological origin from other muscles; they have better regenerative abilities compared to other body muscles including limb muscles. In addition, they may have easier access for biopsies. These findings suggest that CSkM may contain highly active multipotent progenitor cells that may have enhanced differentiation abilities and survival rates. The aims of this study were to isolate craniofacial muscle progenitor cells based on their adhesion properties and to investigate their differentiation ability compared to cells derived from limb muscles. Material and methods: Cells derived from mouse masseter and hind-limb muscles were isolated using enzymatic digestion method, then serially plated into three different plates, pre-plate 1, 2 and 3 (PP1, PP2 and PP3), based on their adhesion properties. Cells in PP1, PP2 and PP3 adhered within the 1st hr, 24 hrs and 4 days respectively, of initial plating. Cells were investigated for their stem cell, neural crest cells (NCCs) gene expression profile, and also their differentiation along the osteogenic and myogenic lineages using osteogenic medium, or myogenic medium. Osteogenic differentiation was assessed by Alkaline Phosphatase (ALP) staining, Alizarin Red staining, Calcium deposition assay, and gene expression. The myogenic differentiation was assessed by the presence of multinucleate myotubes using bright field microscope and immune-fluorescent stating, and gene expression. Different growth behaviours and gene expression profile were seen in all isolated muscle cells. Cells in PP1 and PP2 had a fibroblastic appearance, whereas cells in PP3 were spindle-like and round in shape. Moreover, cells in PP3 had slower growth rate initially compared to cells in PP1 and PP2. The early adhered cells in both muscle groups showed higher stem cell gene expression compared to late adhered cells. Moreover, early adhered cells in CSkM showed higher expression of NCC genes compared to late adhered cells of the same group and all cells from LM. The differentiation abilities were also different among different pre-plates. In osteogenic differentiation, early adhered cells from both muscle groups had higher differentiation ability compared to late adhered cells while myotubes were less evident in early adhered cells compared to late adhered cells. Different population of cells that may have different osteogenic and myogenic differentiation abilities were isolated from craniofacial skeletal muscle (CSkM) based on their adhesion properties. There were no siginificant differences in differentiation abilities between cells isiolated from CSkM and their equivalent ceels isolated from LM. early adhered cells isolated from CSkM have higher expression of NCCs genes than cells isolated from LM.

This thesis describes the development and use of a novel microtitre plate biofilm system for testing the antimicrobial activity of test materials. The developed model is capable of high-throughput screening and furthermore the system has been shown to be stable and reproducible. The search for new antimicrobial agents for improved plaque control requires appropriate screening models. Key criteria for these models include; predictive of clinical data, orally relevant organisms (mixed species, bacteria present in biofilms), short contact time, rapid, reproducible and high throughput. The most widely used biofilm system for evaluating oral antimicrobials are the Constant Depth Film Fermentor (CDFF) and the Minimum Biofilm Eradication Concentration (MBEC) model systems. Each system has advantages for specific investingations; however neither and no other single system fulfils all of the criteria listed above. The CDFF is an orally relevant model that mimics biofilm development under constant salivary flow; typically the inoculum is derived from human dental plaque. Microbial analyses of in vitro growth show populations that are representative of in vivo plaque. However, the system is prone to contamination, is labour intensive and has limited capacity for testing multiple agents. The MBEC model investigates the antibacterial susceptibility of attached bacteria to the 96-pegs on the lid of a microtitre plate based system. Unfortunately, this model was not originally designed for use with oral bacteria; therefore, concerns exist for the use of the MBEC with oral bacteria including the development of oral biofilms on non-orally relevant surfaces such as polystyrene, as found in the MBEC pegs. The aim of this project was to develop a microtitre plate based biofilm assay that could assess the effects of antimicrobials against orally relevant biofilms grown on a relevant surface and compare it to recognised standard model systems. Biofilms derived from a defined inoculum were grown on hydroxyapatite-coated microtitre plate wells. Biofilm characteristics were assessed and were shown to be reproducible and allow for high-throughput screening. Antimicrobial testing showed a dose response and known actives were able to be 'ranked' in the same order as seen in clinical trials. This research has culminated in the development of a simple, high-throughput, reproducible 'off-the-shelf' method for the rapid screening of antimicrobial compounds against an orally relevant biofilm.

Craniofacial form is defined by a number of factors. A major contributor is the jaw musculature especially of the masseter muscle, as differences in transcription and translation of various genes have been documented from this tissue. Up to this point however, no reliable biological predictors of form have been identified. The aims of this study were therefore, to describe the transcriptome of the masseter muscle using microarray technology and to establish and correlate the expression levels of potential candidate and known “informative” genes in masseter muscle with selected clinical, radiographic and dental features of subjects with a variety of craniofacial morphologies. A total of 29 patients (18 deformity and 11 control) were selected from the orthodontic/orthognathic clinics at the Eastman Dental and Whipps Cross Hospitals, London, and Riyadh Military Hospital, Saudi Arabia. Microarray results indicated five “novel” genes not previously reported in relation to the masseter muscles of subjects with variable craniofacial morphologies. Two genes (KIAA1671 and DGCR6) were down-regulated in long face patients, one (SERGEF) was down-regulated in Class III patients and one (LOC730245) was up-regulated in Class II long faces and in all Class III subjects, compared to controls. Another gene (NDRG2) was down-regulated in Class II compared to Class III individuals. Subsequent quantitative Reverse Transcriptase PCR results strongly confirmed that the “novel” gene SERGEF was down-regulated in relation to the clinical, dental and radiographic features of subjects with Class III appearance. SERGEF gene had a positive relationship to the number of dental occlusal contacts and ANB angle. The “informative” gene MHC7 was strongly related to both vertical and horizontal facial deformities. These data suggest that the expression profiles of a number of genes can be analysed and used to make assessments as to their role in the primary aetiology and successful or unsuccessful treatment of patients with specific craniofacial morphologies.

Immune-mediated diseases (IMDs) can give rise to long standing painful oral mucosal disease which adversely affect oral function and perhaps lessens quality of life. The present series of studies, retrospectively determine the clinical presentation and long-term efficacy and safety of treatment of large groups of patients with oral lichen planus, mucous membrane pemphigoid, pemphigus vulgaris and orofacial granulomatosis. These diseases are some of the challenging disorders to be managed by oral medicine specialists. It was found that patients with oral lichen planus (OLP) rarely have extra-oral manifestations of LP. The symptoms of OLP can generally be controlled with topical corticosteroids and/or tacrolimus. While tacrolimus is not notably better than topical corticosteroids for the management of OLP, it does not seem to increase any risk of malignant transformation. Adverse side effects are uncommon with topical corticosteroids, while 21% of patients with OLP may have adverse side effects with tacrolimus, particularly unpleasant taste. In the present cohort of 49 patients with orofacial granulomatosis (OFG) the onset of disease was characterised by facial swelling in 50% and the long-term behaviour of OFG was characterised by the development of further clinical manifestations with most patients developing orofacial swelling and/or intra-oral ulceration. The response of OFG to therapy was typically remitting and although a lessening of soft tissue swelling oral ulceration could generally be achieved with topical and/or systemic therapy. Complete remission of facial swelling occurred in 50% of patients within 3 years of therapy but may be achieved quicker when intra-lesional corticosteroids are used. Spontaneous remission was rare. Significant adverse side effects to therapy were rare. In a cohort of 62 patients, mucous membrane pemphigoid typically manifested as recurrent oral mucosal ulceration and/or desquamative gingivitis and 32.3% patients had some extra-oral involvement. Treatment generally lessened painful symptoms however gingival lesions rarely resolved. Adverse side effects affected 50% of patients; however in the majority of affected individuals these were minor. In a cohort of 40 patients with pemphigus vulgaris the mouth was often the initial site of involvement but other mucocutaneous sites could be affected. Management necessitated topical and systemic therapy. Adverse side effects occurred in 50% patients and were mainly associated with systemic immunosuppressive agents (e.g. azathioprine). The results of this present study indicate that the long-term treatment of IMDs of the oral mucosal are challenging to both the patients and clinicians. While many patients do experience an improvement in their disease status, many do not. The precise impact of IMDs upon the quality of life of affected individuals remains unclear.

Objectives: To validate Abeer Cognitive Dental Anxiety Scale (ACDAS) for children and adolescents in English and Arabic and to investigate the use of video modelling in Paediatric Dentistry Methods: A total of 439 children (≥6 years) were used in the study. The main study utlilised 165 children, 84 from a London dental hospital, 81 from a primary school in Central London. For each child, 2 operators each assessed ACDAS at visit 1, and the chief investigator (AA) also assessed Child Fear Survey Schedule-Dental Subscale (CFSS-DS) at visit 1 and ACDAS two weeks later. A sample of 274 children (≥6 years) was assessed for external validity (generalisability); 184 children from Dubai and 90 children from a school in East London. For video modelling, a sample of 112 children attending for dental treatment were randomly allocated to either the control (prevention video) or the test (modelling video). Their level of anxiety was recorded before and after the video on ACDAS and their ability to cope with the subsequent procedure was assessed on a Visual Analogue Scale. Results: The ACDAS scale had substantial to almost perfect intra- and inter- examiner reliability along with concurrent validity 0.77 and discriminative validity 0.79. For convergent validity, ACDAS had a significant relationship between the Dental Anxiety (DA) scores and the cognitive status (P<0.001), Cronbach’s Alpha (α) was 0.90 which indicated a good internal consistency. Results of external validity were compared favourably with the results that were obtained from the main study. The video modelling was effective to reduce anxiety in the test group of the inhalation sedation and behaviour management RCTs (P<0.025). Conclusions: ACDAS is a valid cognitive scale to measure DA and it encompasses the required criteria for the Gold Standard DA scale for children and adolescents. The video modelling is an effective method to reduce dental anxiety in children.

Staphylococcus aureus is well-known for its ability to acquire resistance to a broad range of antimicrobial agents and a limited number of commercially available antibiotics exist that are active against multidrug resistant strains. Antimicrobial peptides have been suggested as promising alternatives to current antimicrobials due to their potent antimicrobial activity against a broad range of microorganisms including multidrug resistant bacteria, and a membrane-lytic mode of action that is thought to have low possibility of inducing bacterial resistance. This study describes the identification of S. aureus genes involved in resistance to the human cationic antimicrobial peptide LL-37, with a particular interest in the effects of a physiological concentration of bicarbonate on the resistance mechanism. Transposon mutagenesis and recombinase-based in vivo expression technology systems were designed to enable genome-wide screening. A S. aureus transposon mutant library was screened for increased resistance to LL-37 in the presence of bicarbonate. Mutants with insertions in yycH and yycI, demonstrated bicarbonate-dependent resistance to LL-37. Both yycH and yycI form part of a predicted operon yycFGHI in S. aureus, and have been shown to be suppressors of an essential two component system YycFG in B. subtilis that regulates cell wall metabolism. The resistance of S. aureus small colony variants (SCVs) to LL-37 was also investigated. SCVs defective in hemB, menD or aroD, demonstrated bicarbonate-dependent resistance to LL-37. Furthermore, SigB (a global regulator) and TcaR (an activator of protein A) were found to exert opposite effects on resistance to LL-37 in the presence of bicarbonate. Strains defective in TcaR showed bicarbonate-dependent resistance to LL-37, interestingly, this resistance was abolished by either deleting sigB or repairing tcaR in these strains. These data suggest that YycFG, SigB, TcaR and the SCV phenotype may play important roles in resistance to LL-37 under in vivo conditions where bicarbonate is present.