Periodontal disease encompasses a range of inflammatory infections that affect the human gingiva and underlying tissues that support the tooth. Periodontal disease, as typified by periodontitis, has a varied and highly complex polymicrobial etiology. Oral Treponema, as the only genus of spirochetes identified in the oral cavity, is one of the bacterial groups which have been found associated with the occurrence and severity of periodontal infections. Oral treponeme population has been demonstrated as a complex and diversified community. This study mainly investigated and compared the composition of treponeme and related bacterial populations present in the subgingival plaques from periodontitis subjects and periodontitis-free controls. In the first part of the study, the 16S rDNA clone library-based approach was introduced to systematically compare the diversity and composition of treponeme operational taxonomic units (OTUs) present within subgingival plaques collected from ten periodontitis subjects and ten periodontitis-free controls. The results clearly indicated that the subgingival plaque sampled from periodontitis subjects contained a significantly higher diversity and clonal abundance of oral treponeme OTUs than corresponding samples taken from the periodontitis-free controls. Community-based comparisons demonstrated that the periodontitis and control subjects contained quite distinct populations of treponeme OTUs in their corresponding periodontal niches. Most notably, the difference of treponeme populations with the diversity and clonal abundance increase in phylogroup 2 and decrease in phylogroup 6 was observed in periodontitis subjects compared to controls. Since the Spirochaetes/Synergistetes specific primer was used for 16S rDNA clone library construction, the Synergistetes sequences were also analyzed to investigate and compare the diversity and composition of members of the Synergistetes phylum in the subgingival plaques from periodontitis and control subjects. In agreement with previous studies, the results found Synergistetes clones were significantly more prevalent and more diverse in periodontitis subjects, and the presence of certain Synergistetes OTUs appeared associated with periodontitis. The major surface protein (Msp) was identified and investigated in several Treponema species, particularly phylogroup 1 and phylogroup 2 species, as a key virulence factor. In the second part of the study, the consensus primers, which cover the msp gene sequences present in a diverse set of phylogroup 1 and phylogroup 2 oral treponemes, were designed to systematically characterize the msp genotypes present in clinical treponeme communities in subgingival plaques collected from another ten periodontitis and ten periodontitis-free subjects. The results showed a great diverse of msp genotypes were identified in periodontal environment, particularly in periodontitis subgingival plaques, with majority of them have never been reported before. One of these genotypes, OTK-like NP5_2 appears to be associated with periodontitis. In the last part of the study, DNA aptamers were selected in vitro against the recombinant Msp fragment of T. denticola ATCC 35405 (rMsp405V). The preliminary study successfully selected aptamers with high binding affinity with the target protein as well as rMspNP5_2 which is the newly identified Msp homologue mentioned above. The characteristics of selected aptamers in terms of binding affinity (Kd) and predicted secondary structures were also reported. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

Doctor of Philosophy / Macrophages play a critical role in many chronic inflammatory diseases. The pleiotrophic functional capacity of these cells includes phagocytosis and killing of opsonised microorganisms, antigen presentation to T cells, cytotoxicity and the secretion of potent angiogenic growth factors, of central importance in the maintenance or restoration of tissue homeostasis. Although the pathology of the chronic inflammatory disease, periodontitis, has been studied extensively there is a paucity of data relating to the role of macrophages. Recent studies of the untreated advanced periodontitis lesion have revealed extensive vascular pathology, including alteration in blood vessel morphology with thickening of the basement membrane, the accumulation of fragments of vascular basement membrane, persistence of foci of degenerate plasma cells and a restricted capacity to develop reparative granulation tissue. In relation to these pathological features, the distribution and functional status of macrophages assumes prime importance. Macrophage populations in untreated advanced periodontitis biopsies were compared with those in biopsies of clinically healthy, minimally inflamed, gingival tissue. The immunohistochemical investigation used high specificity monoclonal antibodies including a pan-macrophage marker and probes for acute inflammatory, resident histiocytic, and reparative macrophage phenotypes. Results indicated that advanced periodontits and minimally inflamed tissues displayed similar distribution patterns and numbers for the macrophage phenotypic markers. Regionally-specific differences in the populations occurred, however. Further studies investigated macrophage expression of the functional activation markers MHC class II for antigen presentation, and acid phosphatise (AP) and tartrate-resistant acid phosphatise (TRAP) for lysosomal enzyme activity. In the advanced periodontitis lesion there was little evidence of macrophage activation for the expression of HLA-DR and TRAP, although strong expression of HLA-DR was observed in association with blood vessels. Macrophages expressing AP showed a distinct regional distribution; however this was not associated with foci of degenerate plasma cells. These data support the hypothesis that macrophages do not express appropriate activation for key functions in the unrelated lesion of periodontitis. It is postulated that the apparent failure of recruitment and activation of macrophages may in part be both a cause and a consequence of the unusual pathological features of this disease. Although periodontisis is a chronic inflammatory disease of the highly vascularised supporting tissues of the teeth, little is known about the vascular changes in untreated advanced periodontitis. Observations of vascular features of the pathology were investigated and defined, forming the basis for a study of two angiogenic growth factors in periodontitis. In the connective tissue subjacent to the altered epithelium lining the periodontal pocket, there was a significant increase in the numerical density of vascular profiles, primarily accounted for by vessels ≥25μm in diameter. In addition, vascular basement membranes were thickened and there was regional accumulation of non-vascular basement membrane remnants. The co-localisation of type IV collagen and laminin and the discrete nature of these structures was confirmed, using 3-D reconstruction. The distribution of two major angiogenic growth factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), in untreated periodontitis was studied using immunohistochemistry. Basic fibroblast growth factor was not expressed by macrophages. Although bFGF was consistently associated with blood vessels, there was no regional variation in its distribution. In contrast, there was marked regional variation in the intensity of immunostaining for VEGF, with significantly reduced staining of the pocket epithelium. Few macrophages of the Ber-MAC3 phenotype expressed VEGF, as determined by dual immunofluorescence and confocal microscopy. It is considered that the changes in the vascularity of the periodontal connective tissues in untreated advanced periodontitis are, in part, a consequence of altered expression of angiogenic activity by the epithelium and limited expression of angiogenic growth factors by macrophages. Macrophage anergy and vascular disruption observed in the described studies indicate that tissue defence, maintenance and repair are compromised in the untreated lesion of advanced periodontitis.

Thesis (Ph. D.)--University of Sydney, 2000. / Title from title screen (viewed Apr. 27, 2009) Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Oral Biology, Faculty of Dentistry. Includes bibliography. Also available in print form.

Thesis (DR. P.H.)--University of Michigan.

Thesis (DR. P.H.)--University of Michigan.

Thesis (doctoral)--University of Oslo, 1975.

Thesis--University of Helsinki, 1994. / Includes bibliographical references.

Thesis (doctoral)--University of Oslo, 1975.

Thesis--University of Helsinki, 1994. / Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains x, 104 p.; also includes graphics (some col.). Includes bibliographical references (p. 92-104). Available online via OhioLINK's ETD Center