The aim of the work presented in this thesis was to develop vectors for gene transfer, that are based on herpes simplex virus type 1, for possible application in the field of surgery.

Purpose: Assess for an association of wait times to melanoma treatment with overall survival. Methods: Retrospective review of Ontario patients with melanoma, with descriptive and survival analyses. Results: Median wait times were 43 days (interquartile range (IQR), 24-64) for wide local excision (WLE), 59 days (IQR, 41-81) for sentinel lymph node biopsy (SNB), 63 days (IQR, 43-91) for lymph node dissection (LND), 124 days (IQR, 96-150) for medical therapy, and 130 days (IQR, 89.5-157.5) for radiation therapy. In multivariate analysis, wait times to treatment were not associated with overall survival for WLE (hazard ratio (HR), 0.97; 95% confidence interval (CI), 0.87-1.08; p=0.62), SNB (HR, 0.89; 95% CI, 0.74-1.07; p=0.21), LND (HR, 0.99; 95% CI, 0.89-1.11; p=0.92), medical therapy (HR, 0.94; 95% CI, 0.80-1.10; p=0.41) or radiation therapy (HR, 0.80; 95% CI, 0.61-1.03; p=0.08). Conclusion: Overall survival for patients with melanoma was not associated with wait times to surgical, medical or radiation therapy.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2016. / Made available in DSpace on 2016-09-20T04:10:56Z (GMT). No. of bitstreams: 1 341096.pdf: 2936966 bytes, checksum: 935577bec836eb461f4854e3cc51d7e7 (MD5) Previous issue date: 2016 / Inúmeros estudos demonstram que a inflamação em indivíduos obesos está fortemente associada com um maior risco de desenvolvimento e progressão tumoral. O fator de necrose tumoral alfa (TNF-a) é uma das citocinas responsáveis pela iniciação e progressão de tumores, como também a amiloide sérica A (SAA), proteína produzida pelo fígado e pelo tecido adiposo em condições de inflamação. Além disso, ligantes do receptor de fator de crescimento epidermal (EGFR), como o EGF, demonstraram efeitos no favorecimento da proliferação celular e metástase. Neste sentido, analisamos se o soro de pacientes obesos e meio de cultura suplementado com citocinas poderiam criar um microambiente favorável ao crescimento tumoral com células de melanoma, além de verificar alterações na expressão gênica relacionada à progressão tumoral (B-RAF e N-RAS). Realizamos o ensaio clonogênico, o ensaio de migração 2D e ciclo celular em três linhagens de melanoma, SK-mel-19, SK-mel-28 e SK-mel-147. Uma delas, a SK-mel-28, demonstrou uma resposta mais intensa em relação à migração e capacidade clonogênica quando tratada com TNF-a e EGF. Deste modo, as células com mutação em B-RAF (SK-mel-28) foram tratadas com soro de 10 pacientes com IMC acima de 30 kg/m2, e 6 indivíduos com IMC abaixo de 25kg/m2, TNF-a (25 e 50 pg/mL), EGF (100 ng/mL), IL-1ß (10 pg/mL), IL-6 (10 pg/mL), MCP-1 (25 pg/mL) e SAA (75 ng/mL) a fim de avaliar os efeitos de componentes inflamatórios na migração pelo ensaio de migração 2D, e na expressão de B-RAF e N-RAS por PCR em tempo real. Também analisamos estes tratamentos na co-cultura de SK-mel-28 e mononucleares isolados do sangue periférico (PBMC). Foi observado um aumento na capacidade migratória das células de melanoma quando tratadas com soro dos obesos, rico em SAA (soros com concentração de SAA 50 vezes maior do valor de referência), além do tratamento com TNF-a. O soro dos obesos aumentou a expressão de N-RAS nas células de melanoma, fato também observado após tratamento com IL-6. As citocinas IL-1ß e IL-6 ampliaram a expressão de B-RAF. No ensaio clonogênico, os tratamentos com EGF e TNF-a aumentaram a capacidade clonogênica. Além disso, na co-cultura de melanoma com mononucleares, as células foram capazes de liberar citocinas no sobrenadante após tratamento com proteínas inflamatórias. Os resultados demonstraram uma associação entre as citocinas envolvidas em situação de obesidade (TNF-a, EGF, IL-1ß, IL-6, MCP-1 e SAA) e progressão tumoral, e sugerem que as citocinas pró-inflamatórias podem servir como alvo secundário no tratamento do melanoma. É necessário um aperfeiçoamento no entendimento dos efeitos das citocinas nas células de melanoma a fim de desenvolver novas abordagens terapêuticas para o tratamento do câncer.<br> / Abstract : It has been shown that low-grade inflammation on obese patients is strongly associated with an increased risk of cancer development and tumor progression. Tumor necrosis factor-a (TNF-a) is one of the cytokines that are responsible for both initiation and progression of tumors, as well as serum amyloid A (SAA), a cytokine produced by both liver and adipose tissue in inflammatory conditions. In addition, epidermal growth factor receptor (EGFR) ligands, such as EGF, have also demonstrated effect on fostering cell proliferation, tumor resistance and metastasis. Therefore, we examined whether serum from obese patients and cytokine-supplemented medium could create a growth-enhancing microenvironment in melanoma cells and gene expression alterations regarding tumor progression in melanoma (B-RAF and N-RAS). We performed clonogenic assay, scratch assay and cell cycle analysis in three different melanoma cell lines. One of them (SK-mel-28) demonstrated a major improvement regarding migration and clonogenicity when treated with TNF-a and EGF. Therefore, B-RAF mutated melanoma cells (SK-mel-28) were treated with serum from 10 patients with BMI > 30 kg/m2, and 6 individuals with BMI < 25 kg/m2, TNF-a (25 and 50 pg/mL), EGF (100 ng/mL), IL-1ß (10 pg/mL), IL-6 (10 pg/mL), MCP-1 (25 pg/mL) and SAA (75 ng/mL) in order to evaluate the effect of these cytokines on 2D cell migration, along with B-RAF and N-RAS gene expression by real-time PCR. We also analyzed these treatments in co-culture of SK-mel-28 and peripheral blood mononuclear cells (PBMC). An improve in melanoma cell migration was observed on cells treated with SAA-rich serum of obese patients (sera with a 50-fold increment of SAA above reference value), along with TNF-a?treated cells. Likewise, SAA-rich serum increased N-RAS gene expression in melanoma cells, fact observed with treatment with IL-6 as well. Cytokines such as IL-1ß and IL-6 increased the expression of B-RAF. In clonogenic assay, treatment with EGF increased the colony-forming potential, as well as TNF-a. Furthermore, when in co-culture of melanoma with PBMC, cells were able to release cytokines in supernatant after treatment with inflammatory proteins. The results demonstrated an association between cytokines involved in obesity (TNF-a, EGF, IL-1ß, IL-6, MCP-1 and SAA) and tumor progression, moreover, these cytokines may be target for melanoma treatment. The improvement in the understanding of the effects of cytokines in tumor cells is important in order to pave the way for new therapeutic approaches for cancer treatment.

The metastatic process requires that tumour cells are capable of traversing various micro-environmental barriers, such as the basement membrane. There are various proteolytic mechanisms which could contribute to the process, plasminogen activation by tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) is one such mechanism. Extensive reports in the literature (reviewed in the introduction) indicate that most tumour cells synthesize uPA and that it is this enzyme, particularly when receptor-bound, which plays a role in invasion. UCT-Mel 3 is a human malignant melanoma cell line which was established in our laboratory, and has been shown to be highly metastatic in the nude mouse. This cell line is typical of many melanomas in that it synthesizes only tPA and not uPA. In part 1 of this thesis I further investigated the plasminogen activator production by these cells (at the level of mRNA as well as activity) as well as expression of plasminogen activator inhibitor PAl-1 and receptors for tPA and uPA (uPAR). UCT-Mel 3 cells expressed uPAR although uPA was not detected. I also examined cells cultured from two metastatic deposits. Interestingly, the metastatic cells produced PAl-1 which was undetected in the parent cells. After confirming that UCT-Mel 3 do not express detectable levels of uPA, I attempted (in part 2) to determine whether tPA could play a comparable role to that of uPA in the invasive process. My strategy was to inhibit the expression of tPA via two different methods, namely the use of antisense RNA and ribozyme. I then hoped to isolate clones producing no tPA, which would have been injected into nude mice in order to assay for metastasis. Unfortunately, neither of these methods proved to be successful in abrogating tPA expression. I was thus unable to achieve the ultimate aim of the project. However, during the course of the study a number of unforeseen problems arose. Firstly, the clonal variation within the cell population, and secondly, my inability to obtain antisense transfectants. I have speculated that a possible reason for the latter may be that the cells are in fact unable to grow in the absence of tPA.

No description available.

Excerpt: Approximately 1 week after having been shot in the left eye with a foam dart from a toy gun, a 37-year-old woman presented to an optometrist due to persistent pain in the eye.

No description available.

No description available.

No description available.

Yes / Metastatic melanoma, a deadly form of skin cancer, often develops resistance to the BRAF inhibitor drug vemurafenib, highlighting the need for understanding the underlying mechanisms of resistance and exploring potential therapeutic strategies targeting integrins and TGF-β signalling. In this study, the role of integrins and TGF-β signalling in vemurafenib resistance in melanoma was investigated, and the potential of combining vemurafenib with cilengitide as a therapeutic strategy was investigated. In this study, it was found that the transcription of PAI1 and p21 was induced by acquired vemurafenib resistance, and ITGA5 levels were increased as a result of this resistance. The transcription of ITGA5 was mediated by the TGF-β pathway in the development of vemurafenib resistance. A synergistic effect on the proliferation of vemurafenib-resistant melanoma cells was observed with the combination therapy of vemurafenib and cilengitide. Additionally, this combination therapy significantly decreased invasion and colony formation in these resistant cells. In conclusion, it is suggested that targeting integrins and TGF-β signalling, specifically ITGA5, ITGB3, PAI1, and p21, may offer promising approaches to overcoming vemurafenib resistance, thereby improving outcomes for metastatic melanoma patients.