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Title: Cultural and molecular analysis of the microorganisms present within human oral squamous cell carcinoma
Author: Hooper, Samuel James
ISNI:       0000 0004 2747 5882
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2005
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There is increasing interest in the relationship between bacteria and the different stages of cancer development, yet the association of bacteria with cancer of the oral cavity has yet to be adequately examined. The main objective of this thesis was to characterise the bacteria present within oral squamous cell carcinoma (OSCC) tissue by a combination of standard culture and molecular techniques. Portions of tissue were harvested at the time of surgery from deep within the tumour mass using fresh blades for each cut. Whenever possible, "superficial" portions from the mucosa overlying the tumour and non-tumourous control specimens from at least 5 cm away from the primary tumour site were also obtained. Twenty deep tissue specimens, 19 corresponding superficial tissues and 12 control tissues were studied. Surface contamination was successfully eliminated by immersion in Betadine and washing with PBS. Viable microorganisms were isolated by culturing aseptically macerated specimens on non-selective media under both aerobic and anaerobic conditions. Concurrently, PCR using Bacteria-specific universal primers was undertaken on DNA extracted from the specimens. The products were singularised by TA cloning. All isolates, cultivated and cloned, were identified to species-level by sequence analysis of the 16S rRNA gene. Different species were detected by the culture and the culture-independent techniques, highlighting the importance of a combined approach. Both methods revealed the presence of a diversity of species within both tumourous and non-tumourous tissue, including some putatively novel taxa. Denaturing gradient gel electrophoresis (DGGE) of amplified 16S rRNA gene fragments was used to generate profiles of the bacterial populations within each tissue specimen. Differences in the banding patterns provided evidence that the composition of the microflora is significantly different in OSCC than in non-tumourous tissue. The hypothesis that bacteria are present within OSCC tissue was further supported by performing in situ hybridisation on sections from one of the specimens with a fluorescently-labeled oligonucleotide probe, designed to bind to all Bacteria. This work has demonstrated for the first time the existence of viable bacteria within OSCC tumour tissue. A diversity of bacterial species was detected and a degree of restriction in comparison to control sites demonstrated. The significance of these bacteria within the tumour tissue warrants further study.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available