It is known that many types of leukemias and lymphomas are of viral origin. A new strain of immunologically deficient mice, the BALB/c x C57B1/6 beige nude mice, has been observed to develop spontaneous lymphomas of unknown origin at a high frequency. It is possible the tumors originate from a retroviral infection, which we attempted to show by detection of viral reverse transcriptase (RT) activity. We measured the (RT) activity in the supernatants of cocultures from the spleen and lymph node tissues of the beige nude animals by two methods, tritiated thymidine triphosphate incorporation in a standard RT assay, and the commercially available RT-DetectTM (DuPont) method. Of all supernatants tested, none showed a significant amount of RT activity compared with a cell line that was known to be actively producing the retrovirus MuLV. Upon electron microscopic analysis of the tumor-like cells grown in coculture, no viral particles were observed. Flow cytometric analysis of the tumor-like cells showed two general phenotypes; one predominately of a helper T cell type, and the other of a less differentiated immature thymocyte type. / Department of Biology

Zebrafish (Brachydanio rerio) are small, freshwater teleost fishes in the family Cyprinidae, the true minnows. They are native to the tropical latitudes of India, but have become widespread through their use as aquarium fish and as models for several branches of biological research. Their ease of rearing, short generation time, year-around egg laying potential, brief developmental period, and embryo transparency have made them especially desirable as models for developmental biology, genetics, and neurobiology. Because of their popularity, they were also the first small aquarium fish to be used as test organisms for carcinogenesis in the early 1960's. For reasons that have never been stated, their use as a model for carcinogenesis research did not continue. Due to the number of positive characteristics that this species has, the goal of this research effort was to systematically evaluate the potential of zebrafish for use as an environmental monitor, to evaluate the toxicology and carcinogenesis of surface and/or ground waters. The overall project was multidisciplinary in nature, but the focus of this thesis research was on the whole animal, dose-response to a number of well-known carcinogens, administered by multiple exposure routes, and the pathological description of the resulting lesions. Exposure to N-nitrosodiethylamine (DEN) and N-nitrosodimethylamine (DMN) in the diet was ineffective, but static water bath exposure of fry and embryos to these nitrosamines resulted in neoplasms, primarily in the liver. Embryo exposure to DEN resulted in a low response of neoplasms in several other organs as well. Dietary exposure of zebrafish to aflatoxin B₁ resulted in few hepatic neoplasms, revealing a marked resistance to this carcinogen. Dietary exposure to methylazoxymethanol acetate (MAM-Ac) produced mostly liver tumors, as did both fry and embryo water bath exposures. Each water bath exposure also produced neoplasms at other tissue and organ sites, but the embryo stage produced the greatest variety. These results demonstrate a relative resistance to neoplastic development compared to the well-known rainbow trout model. But in one comparative trial, zebrafish were similar to Japanese medaka in their response to dietary MAM-Ac. The major limitation of this species, that will prevent its use as a model for environmental monitoring, however, is its narrow range of temperature tolerance. Temperatures below 15°C produce marked sluggishness, and below 10-12°C cause anesthesia and death. Therefore, this research indicates that this species is not as versatile as some other small fish species for laboratory and especially field monitoring of environmental carcinogenic hazards. / Graduation date: 1997

Master of Medicine / Non-melanoma skin cancer (NMSC) is the most common human cancer worldwide. Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) make up almost all NMSC. SCC usually arises from actinic keratosis (AK) as a result of exposure to sunlight. SCC and AK provide a useful clinical model to investigate changes involved in the progression of NMSC. This project examines the expression of Brm, Brg-1, Snail 1 and Snail 2 in the progression of NMSC. Brm and Brg-1 are subunits of the SWI/SNF chromatin-remodelling complex which is involved in regulating the access of cell machinery to DNA by altering the structure of chromatin. It has been suggested that loss of this function is involved in carcinogenesis as the cell is unable to access to DNA normally in order to repair mutations or activate apoptosis. The loss of Brm or Brg-1 has been described in several human cancers. Snail 1 and Snail 2 are zinc-finger transcription factors that are known for their role in epithelial to mesenchymal transition (EMT), a process vital to embryological development. Increased expression of these factors leads to a loss of cell-cell adhesion and a migratory phenotype and has been described in some human cancers. In this project, double-label immunohistochemistry was used to determine the relative expression of these proteins in human SCC, BCC, AK and normal skin. The expression of Snail was unable to be determined due to poor specificity of the antibodies used. The expression of both Brm and Brg-1 proteins was found to be dramatically and consistently decreased in SCC and BCC when compared to normal skin and AK. This loss of Brm and Brg-1 occured as the tumour progressed from benign AK to malignant SCC. This finding suggests that the loss of either Brm or Brg-1 constitutes a key step in carcinogenesis. The results of this study identify Brm and Brg-1 as putative tumour suppressors involved in the progression of non-melanoma skin cancer from benign to malignant.

There are two chief molecular pathways to sporadic colorectal cancer (CRC), the chromosomal instability (CIN) and the CpG island methylator phenotype (CIMP) pathways. A third pathway, the pure microsatellite instability pathway, is important in inherited CRC specifically hereditary non-polyposis colorectal cancer. The CIN pathway is characterized by an adenomatous pathological precursor, aneuploidy and microsatellite stability. CIMP pathway cancers, however, are frequently proximal, develop from serrated rather than adenomatous polyps and are strongly associated with BRAF mutation. The CIMP pathway is driven primarily by epigenetic rather than genetic instability. These pathway-specific molecular traits are evident within the pathological precursors to these cancers and thus pathway divergence must occur at the beginning of carcinogenesis or even before. Although DNA methylation is recognized as a key mechanism in colorectal carcinogenesis, relatively little is known about its pattern, regulation and relevance in normal colorectal mucosa. This PhD thesis characterized the profile of DNA methylation in the normal human colorectum and explored its associations with luminal, environmental, dietary and pathological factors. The genes methylated in CRC are characterized as “type A” (Age-related) genes and “type C” (Cancer-specific) genes. Generally, “type A” genes are methylated in both normal and neoplastic tissue with the degree of methylation proportional to the age of the tissue. The methylation of “type C” genes, however, is more specific for neoplastic tissue. The primary study recruited 166 patients undergoing colonoscopy. At colonoscopy, mucosal biopsies were taken from the caecum, transverse colon, sigmoid colon and rectum. DNA methylation was analysed by MethyLight at “type A” (ESR1, GATA5, HIC1, HPP1, SFRP1) and “type C” methylation markers (MGMT, MLH1, CDKN2A, MINT2, MINT31, IGF2, CACNA1G, NEUROG1, SOCS1, RUNX3). LINE-1 methylation was quantified by pyrosequencing. The last 5 “type C” markers comprise a CIMP panel used to identify CIMP cancers. Mean “type A” and CIMP panel methylation Z-scores were calculated. The PMR for each of these CpG island loci was compared to patient age, gender, previous colorectal polyps, smoking history and the presence of concomitant pathology. Most “type A” genes demonstrated strong and direct correlations between methylation and patient age (e.g. ESR1, ρ=0.66, p<0.0001) and had greater methylation within the distal compared to the proximal colorectum (e.g. ESR1, p<0.0001). On multivariate analysis, the mucosal “type A” methylation Z-score had a strong, independent, inverse association with the diagnosis of colorectal adenomas (OR=0.23, p<0.001), the precursor to CIN cancers. The mean CIMP methylation Z-score in normal mucosa, however, was significantly and independently associated with advanced proximal serrated polyps (OR=5.1, p=0.009), the precursor to CIMP cancers. The luminal and epithelial associations with colorectal methylation were explored by a randomized, double-blind, placebo-controlled trial. This experiment was undertaken to determine whether dietary supplementation could modulate epithelial DNA methylation. In addition, the study was designed to evaluate intra-individual reproducibility of the MethyLight technique. The study consisted of a 4 week cross-over trial of resistant starch and Bifidobacterium lactis either alone or as a combined synbiotic preparation, in 20 human volunteers. Rectal biopsies, faeces and serum were collected. Rectal mucosal endpoints included DNA methylation at the CpG island loci and LINE-1, epithelial proliferation (Ki67 immunohistochemistry) and crypt cellularity. Faecal short-chain fatty acid concentrations, pH, ammonia and microbiological profiles (by DGGE and sequencing) were examined. The synbiotic intervention fostered a significantly different faecal stream bacterial community than either the prebiotic or the probiotic interventions alone, but did not show any significant associations with the epithelial or luminal parameters. To explore possible associations between luminal and epithelial parameters and mucosal DNA methylation, the baseline indices were further analysed. There was a strong positive correlation between baseline epithelial proliferation and “type A” marker methylation (ρ = 0.7, p = 0.0001). Thus, “type A” methylation may reflect the cellular age or mitotic burden of a tissue, which is a function of both time and cell turnover. There were consistent inverse trends evident between faecal short-chain fatty acid levels and rectal mucosal DNA methylation. This PhD project found that DNA methylation within the normal colorectal mucosa varied with patient age and region and was strongly associated with the development of pathway-specific pathology, suggesting that the background colorectal field may predict both the at-risk patients and at-risk pathways. Diet and the luminal environment more broadly may influence levels of DNA methylation in the colorectal mucosa and could help to explain regional patterns of colorectal DNA methylation.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 86-98).

Thesis (Ph.D.) -- McMaster University, 1999. / Includes bibliographical references (leaves 199-221). Also available via World Wide Web.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on November 27, 2007) Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph.D.)--University of Hong Kong, 2008. / Also available in print.

Thesis (Ph. D., Bioinformatics and Computational Biology)--University of Idaho, December 2006. / Major professor: Robert B. Heckendorn. Includes bibliographical references (leaves 66-71). Also available online (PDF file) by subscription or by purchasing the individual file.