Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains xv, 146 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 117-129).

Recent evidence suggests that leflunomide, a dihydroorotate dehydrogenase (DHODH) inhibitor, disrupts neural crest development and melanoma pathogenesis via inhibiting transcription elongation. DHODH is an enzyme in the pyrimidine biosynthetic pathway and inhibition of this enzyme by leflunomide triggers a low nucleotide state. Leflunomide effectively ablates the neural crest lineage in embryonic zebrafish, preventing the formation of mature melanocytes, among other neural crest lineages. This drug also effectively suppresses melanoma and is in a clinical trial, administered in combination with the BRAF inhibitor vemurafenib, for metastatic melanoma. Despite knowing that leflunomide targets transcription elongation, the mechanism by which low nucleotides directly regulates transcription is unknown.

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Nordihydroguaiaretic Acid (NDGA) is a polyphenol, antioxidant, natural product lignan isolated from the creosote bush (Larrea tridentata). The in vivo and in vitro Pharmacology and Toxicology of NDGA has been continuously studied for more than 36 years. The Pharmacology of NDGA has been studied in Diabetes, Alzheimer's disease and Amyotrophic Sclerosis. Most of the research on NDGA has been in anticancer and cancer prevention models. Toxicology studies reveal NDGA-mediated hepatotoxicity and nephrotoxicity. This data has influenced a recent interest in semi-synthetic derivatives of NDGA focused on modifying the phenolic groups which are responsible for in vivo toxicity. The tetra-methylether of NDGA (M4N) has shown reduction in toxicity and enhanced anti-melanoma activity when compared to NDGA.The specific aim of this project was to increase the number of NDGA analogs with anti-melanoma activity and explore a novel synthetic approach by binding the ortho-phenols together by one atom, creating a 5-membered ring as opposed to the prior work which involved tetra-substituted phenolic hydroxyl group modifications.Eleven new analogs were synthesized, characterized and evaluated for their anti-melanoma activity. The anti-melanoma activity was evaluated by 5-day colorimetric-based, and DNA, RNA and protein synthesis inhibition-based cytotoxicity assays. The cytotoxicity assays were compared to NDGA and M4N in terms of structure and activity. Selected analogs were evaluated in an in vivo, mouse, tumor growth inhibition model. In the first in vivo model, tetra acetyl NDGA (TA-NDGA) and ortho-cyclic carbonate NDGA (OCC-NDGA) were evaluated at two dose-levels, 100 mg/kg and 200 mg/kg. They both showed dose-dependent, but moderate tumor growth inhibition at the 200 mg/kg dose-level. An ortho-cyclic sulfate of NDGA showed in vivo toxicity at 100 mg/kg. In the second in vivo model, the dose was escalated to 300 mg/kg, TA-NDGA showed moderate tumor growth inhibition, but OCC-NDGA-mediated tumor growth inhibition was not repeated. However, in the second study an ortho-difluoromethylene NDGA analog did show moderate tumor growth inhibition.Structure-activity relationships indicated that the ortho-cyclic analogs are inferior to the tetra-substituted phenolic group-modified analogs in terms of anti-melanoma activity and therefore future synthesis' should be focused on generating more tetra-substituted phenolic group analogs of NDGA.

Malignant melanoma is a common type of solid tumor that causes high cancer-related mortality in young adults of Northern Europe. The incidence of melanoma increases rapidly which renders us a special responsibility to investigate this disease in depth. One recent promising approach to treat malignant melanoma is adoptive cell therapy with tumor-directed autologous T cells. This thesis aims to improve this therapy in four different studies. We first sought to establish a protocol for the assessment of melanoma-specific T-cell cultures in order to screen for optimal specificity and reactivity in a robust, reliable and simple manner. The conclusion was that reactive cells could be found in a majority of patients and could be screened for specificity by stimulation with melanoma cell lines. In the next study, 28 melanoma patients with advanced disease were treated with autologous tumor-infiltrating T cells. Objective responses (18%) including one sustained complete response were observed. This is the first study in cancer patients with autologous T cell transfer combined with low-dose s.c. IL-2 as supportive cytokine. In the following two studies we wanted to improve management and culture conditions of the T cells. When investigating methods for improved handling and preservation of large numbers of T cells, we observed that freeze-thawing of T cells could impair the metabolic activity of the T cells. Another conclusion was that rapid expansion of T cells could lead to loss of antigenic specificity and apoptosis. These adverse effects could be prevented with short time recovery. In order to improve expansion methods, mass expansion of T cells in an automated bioreactor was evaluated. We concluded that the bioreactor is suitable for this task and allows for higher cell densities and absolute cell numbers compared to traditional culturing conditions without influencing cell phenotype or reactivity. Taken together, my current studies present guiding principles and encouragement for the further development of immunotherapies for treatment of patients with malignant melanoma.

Feature extraction of segmented skin lesions is a pivotal step for implementing accurate decision support systems. Existing feature sets combine many ad-hoc calculations and are unable to easily provide intuitive diagnostic reasoning. This thesis presents the design and evaluation of a set of features for objectively detecting melanoma in an intuitive and accurate manner. We call these "high-level intuitive features" (HLIFs). The current clinical standard for detecting melanoma, the deadliest form of skin cancer, is visual inspection of the skin's surface. A widely adopted rule for detecting melanoma is the "ABCD" rule, whereby the doctor identifies the presence of Asymmetry, Border irregularity, Colour patterns, and Diameter. The adoption of specialized medical devices for this cause is extremely slow due to the added temporal and financial burden. Therefore, recent research efforts have focused on detection support systems that analyse images acquired with standard consumer-grade camera images of skin lesions. The central benefit of these systems is the provision of technology with low barriers to adoption. Recently proposed skin lesion feature sets have been large sets of low-level features attempting to model the widely adopted ABCD criteria of melanoma. These result in high-dimensional feature spaces, which are computationally expensive and sparse due to the lack of available clinical data. It is difficult to convey diagnostic rationale using these feature sets due to their inherent ad-hoc mathematical nature. This thesis presents and applies a generic framework for designing HLIFs for decision support systems relying on intuitive observations. By definition, a HLIF is designed explicitly to model a human-observable characteristic such that the feature score can be intuited by the user. Thus, along with the classification label, visual rationale can be provided to further support the prediction. This thesis applies the HLIF framework to design 10 HLIFs for skin cancer detection, following the ABCD rule. That is, HLIFs modeling asymmetry, border irregularity, and colour patterns are presented. This thesis evaluates the effectiveness of HLIFs in a standard classification setting. Using publicly-available images obtained in unconstrained environments, the set of HLIFs is compared with and against a recently published low-level feature set. Since the focus is on evaluating the features, illumination correction and manually-defined segmentations are used, along with a linear classification scheme. The promising results indicate that HLIFs capture more relevant information than low-level features, and that concatenating the HLIFs to the low-level feature set results in improved accuracy metrics. Visual intuitive information is provided to indicate the ability of providing intuitive diagnostic reasoning to the user.

The purpose of the study has been primarily to draw a profile of malignant melanoma in the different population groups inhabiting the Natal Province and Kwa-Zulu, to compare the presentation and incidence of the disease between these groups as well as with similar racial groups in different countries. The data collected then permitted to estimate which parameters were relevant in predicting the course of the disease, as well as the results of surgical and adjuvant therapy, and was utilized in a search for the aetiology of the tumour. / Thesis (M.D.)-University of Natal, Durban, 1985.

CDKN2A/p16 is a cell cycle inhibitor which blocks abnormal cell growth and proliferation. The CDKN2A gene is frequently mutated or deleted in a wide variety of tumour types. Germline mutations have also been identified in familial atypical multiple mole melanoma (FAMMM) pedigrees. However, the role of CDKN2A in hereditary cancer is uncertain. To explore the relationship between CDKN2A germline mutations and risk of cancer, 75 families with cancers at multiple sites were analysed for germline mutations in the CDKN2A gene. A Met53Ile mutation was found in a non-FAMMM kindred with multiple cancers, including one case of melanoma. The Met53Ile mutation has been previously reported in three Australian FAMMM kindreds. A known Ala148Thr polymorphism was also detected in 5 individuals. No other families were found to have CDKN2A alterations. There were no reported CDKN2A mutations in families without cases of melanoma. Analysis of microsatellite markers adjacent to CDKN2A on chromosome 9p21 revealed that this family shares a common haplotype with one other family with this mutation, suggesting that Met53Ile is a founder mutation. These results suggest that while CDKN2A mutations are not restricted to FAMMM pedigrees, they are very rare or absent in families with individuals without melanoma.

Reactivation of master regulators of epithelial to mesenchymal transition (MR-EMT) represents the molecular basis for tumour cell plasticity, malignant transformation and metastases. However, the current evidence on the specific role of MR-EMT in melanomagenesis has not been fully addressed. The purpose of this investigation was to assess the expression and regulation of these factors in malignant melanoma and to evaluate their prognostic and clinical significance. In vitro experiments indicated that a switch in MR-EMT protein expression ZEB2/SNAI2 to ZEB1/TWIST1 is RAS-RAF-MAPK signalling dependent. In addition, evidence supported a MR-EMT interactome, in which transcriptional repression of ZEB2 by Fra-1 resulted in upregulation of ZEB1, independently of miR-200 family. Further in vitro and immunohistochemical (IHC-P) analyses showed that E-cadherin and VDR protein levels were significantly reduced by the presence of ZEB1 in melanoma cells and archive tissues. Motility assays demonstrated that ZEB1 but not ZEB2 enhances cell migration. IHC-P analyses of ZEB2/SNAI2 (n=142/28) showed a statistically significant gradient of stronger staining at superficial sites compared to the deep sites in a select cohort of independent and matched melanoma tumour samples. In contrast, ZEB1 (n=142) and TWIST1 (n=133) showed higher staining in deep sites of primary melanomas and metastases. Trend analyses showed a significant MR-EMT switch in this progression series from high levels of ZEB2/SNAI2 in naevi towards high ZEB1/TWIST1 expression in melanomas. In primary melanomas these factors were also significant in Kaplan Meier survival curves and after two step cluster analysis the combined profile of ZEB1[superscript high]/TWIST1[superscript high]/ZEB2[superscript low] predicted the worse prognosis (P=0.001). Multivariate Cox regression analyses of IHC-P staining indicated that only the gain of ZEB1 (P<0.002, n=98) and superficial TWIST1 (P=0.012) were associated with poor metastasis-free survival and independent of breslow depth. In conclusion, the reversible switch between ZEB1/TWIST1 and ZEB2/SNAI2 is controlled by RAS-RAF-MAPK pathway activity and constitutes an independent factor of poor prognosis in patients with malignant melanoma.

The purpose of this thesis was to investigate the knowledge and perceptions on the sun, risks of prolonged exposure, tanning and beauty of young Canadian women. Conversations with 15 young pregnant women from the University of Ottawa were tape-recorded, transcribed, and then analyzed using thematic analysis and theories on gender and beauty. The results were divided into two articles, the first exploring the perception and knowledge young Canadian women have about the sun, tanning and its risks. In the first article, the themes generated were (a) perceptions of benefits and risks of sun exposure; (b) outdoor versus Indoor tanning; (c) conformity; (d) conflicting and ambiguous messaging; (e) self risk and other’s risk and; (f) no UV index awareness. The second article explores how the fifteen interviewees make sense of the sun safety messaging they are exposed to, and how they act on it. The themes identified were: (a) tanning as a social activity; (b) beauty; (c) base tanning; and (d) wearing SPF and reapplication. The overall conclusion to be drawn from this study is that while for the most part the group of women I interviewed was well informed when it came to sun safety and tanning, they still felt the pressure to tan from peers, society and the media. There were times when they shared that they were misinformed on the risks of engaging in harmful tanning practices. Further, the study contributed to finding that the vast majority of the participants admitted to not checking the UV index before going outdoors, either because they did not understand it or because they felt it would not make a difference to their daily practices and behaviours. This contradicted previous literature that emphasized on the connection Canadians often made with the environment and UV index. Impacts, implications, and future research directions are discussed in both articles.