In den letzen Jahren gab es durch epidemiologische und molekularbiologische Studien vermehrt Hinweise, dass kutane humane Papillomviren (HPV) ursächlich an der Entstehung nicht-melanozytärer Hauttumore (engl. NMSC) beteiligt sind. Ziel der vorliegenden Arbeit war die Identifizierung molekularer Mechanismen der viralen Proteine E6 und E7 kutaner HPV-Typen. Die E6 oder E7 Gene der verschiedenen HPV-Typen 1, 4, 5, 8, 20, 38 und RTRX7 wurden untersucht. Natürliche Wirtszellen dieser Viren, humane primäre Keratinozyten (HPK) der Haut, wurden mit rekombinanten, für E6 oder E7 kodierenden Retroviren infiziert. Die Analysen erfolgten in Monolayer-Kultur (undifferenzierte Keratinozyten) oder in organotypischen Hautmodellen (Induktion der Keratinozytendifferenzierung). Die Expression von E6 oder E7 führte in Monolayer-HPK zu einer Verlängerung der Lebensspanne und zu einer deutlich erhöhten Verdoppelungsrate. Eine Telomeraseaktivierung, die charakteristisch für immortale Zellen ist, wurde nur in HPV 8 E6 positiven HPK nachgewiesen. In organotypischen Hautmodellen induzierte das E7 Protein von HPV 1, 4 und 38 starke Veränderungen in der Differenzierung sowie eine Zunahme der Proliferation. Weiterhin wurde eine Aufhebung der normalen Zellzykluskontrolle in suprabasalen HPV 5 E7 oder HPV 8 E7 beobachtet. Hinweise auf ein starkes invasives Potential von E7-infizierten HPK wurden für HPV 8 E7 bestätigt und für HPV 4 E7, HPV 38 E7 und RTRX7 E7 erweitert. Molekulare Mechanismen der viralen Gene E6 und E7 kutaner HPV unterscheiden sich von mukosalen Typen. Das Mehrstufenmodell der Karzinogenese beinhaltet eine Reihe fundamentaler Zelltransformationen, die für eine Tumorgenese nötig sind. In dieser Arbeit beschriebene Mechanismen der Modulation der Zelldifferenzierung und Zellproliferation durch die kutanen HPV-Typen 4, 5, 8 und 38 können unter Umständen zur Induktion und Progression früher Stadien von Plattenepithelkarzinomen (SCC) beitragen. / In the last years epidemiologic and molecular biological studies accumulated increasing evidence that cutaneous human papillomaviruses are etiologically involved in the formation of non-melanoma skin cancer (NMSC). The presented work aims to identify the underlying molecular mechanisms of the viral proteins E6 and E7 of cutaneous HPV types. The E6 and E7 genes of the different HPV types 1, 4, 5, 8, 20, and RTRX7, which are in vivo associated with cutaneous benign or malignant lesions, were studied. Natural host cells of these viruses, human primary keratinocyts (HPK) of the skin, were infected with recombinant E6 and E7 encoding retroviruses. The following analyses were performed in monolayer culture (non-differentiated keratinocytes) or in organotypic skin culture (induction of keratinocyte differentiation). The expression of E6 and E7 elongated the life span of monolayer HPK and significantly increased the doubling rate. An activation of the telomerase, characteristic for immortalized cells, was only detected in HPV 8 E6 positive cells. In organotypic skin cultures E7 of HPV 1, 4 and 38 induced drastic changes in differentiation and proliferation. Additionally an impairment of the normal cell cycle control in suprabasale HPV 5 E7 and 8 E7 cultures was seen. Hints for a strong invasive potential of E7 infected HPK were proven for HPV 8 E7 and expanded to HPV 4 E7, HPV 38 E7 and RTRX E7. The viral E6 and E7 genes of cutaneous and mucosal HPV types exhibit different molecular mechanisms. The multistep model of carcinogenesis includes a series of fundamental cell transformations necessary for tumorigenesis. Mechanisms for the modulation of cell differentiation and proliferation by cutaneous HPV types 4, 5, 8 and 38 described in this work could potentially contribute to the induction and progression of early stages of squamous cell carcinoma.

Made available in DSpace on 2014-12-17T14:54:49Z (GMT). No. of bitstreams: 1 HelianaBS_da_capa_ate_cap4.pdf: 2361373 bytes, checksum: 3e1e43e8ba1aadc274663b8b8e3de72f (MD5) Previous issue date: 2008-02-22 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The skin cancer is the most common of all cancers and the increase of its incidence must, in part, caused by the behavior of the people in relation to the exposition to the sun. In Brazil, the non-melanoma skin cancer is the most incident in the majority of the regions. The dermatoscopy and videodermatoscopy are the main types of examinations for the diagnosis of dermatological illnesses of the skin. The field that involves the use of computational tools to help or follow medical diagnosis in dermatological injuries is seen as very recent. Some methods had been proposed for automatic classification of pathology of the skin using images. The present work has the objective to present a new intelligent methodology for analysis and classification of skin cancer images, based on the techniques of digital processing of images for extraction of color characteristics, forms and texture, using Wavelet Packet Transform (WPT) and learning techniques called Support Vector Machine (SVM). The Wavelet Packet Transform is applied for extraction of texture characteristics in the images. The WPT consists of a set of base functions that represents the image in different bands of frequency, each one with distinct resolutions corresponding to each scale. Moreover, the characteristics of color of the injury are also computed that are dependants of a visual context, influenced for the existing colors in its surround, and the attributes of form through the Fourier describers. The Support Vector Machine is used for the classification task, which is based on the minimization principles of the structural risk, coming from the statistical learning theory. The SVM has the objective to construct optimum hyperplanes that represent the separation between classes. The generated hyperplane is determined by a subset of the classes, called support vectors. For the used database in this work, the results had revealed a good performance getting a global rightness of 92,73% for melanoma, and 86% for non-melanoma and benign injuries. The extracted describers and the SVM classifier became a method capable to recognize and to classify the analyzed skin injuries / O c?ncer de pele ? o mais comum de todos os c?nceres e o aumento da sua incid?ncia deve-se, em parte, ao comportamento das pessoas em rela??o ? exposi??o ao sol. No Brasil, o c?ncer de pele n?o melanoma ? o mais incidente na maioria das regi?es. A dermatoscopia e ideodermatoscopia s?o os principais tipos de exames para o diagn?stico de doen?as da pele dermatol?gicas. O campo que envolve o uso de ferramentas computacionais para o aux?lio ou acompanhamento do diagn?stico m?dico em les?es dermatol?gicas ainda ? visto como muito recente. V?rios m?todos foram propostos para classifica??o autom?tica de patologias da pele utilizando imagens. O presente trabalho tem como objetivo apresentar uma nova metodologia inteligente para an?lise e classifica??o de imagens de c?ncer de pele, baseada nas t?cnicas de processamento digital de imagens para extra??o de caracter?sticas de cor, forma e textura, utilizando a Transformada Wavelet Packet (TWP) e a t?cnicas de aprendizado de m?quina denominada M?quina de Vetor de Suporte (SVM Support Vector Machine). A Transformada Wavelet Packet ? aplicada para extra??o de caracter?sticas de textura nas imagens. Esta consiste de um conjunto de fun??es base que representa a imagem em diferentes bandas de freq??ncia, cada uma com resolu??es distintas correspondente a cada escala. Al?m disso, s?o calculadas tamb?m as caracter?sticas de cor da les?o que s?o dependentes de um contexto visual, influenciada pelas cores existentes em sua volta, e os atributos de forma atrav?s dos descritores de Fourier. Para a tarefa de classifica??o ? utilizado a M?quina de Vetor de Suporte, que baseia-se nos princ?pios da minimiza??o do risco estrutural, proveniente da teoria do aprendizado estat?stico. A SVM tem como objetivo construir hiperplanos ?timos que apresentem a maior margem de separa??o entre classes. O hiperplano gerado ? determinado por um subconjunto dos pontos das classes, chamado vetores de suporte. Para o banco de dados utilizado neste trabalho, os resultados apresentaram um bom desempenho obtendo um acerto global de 92,73% para melanoma, e 86% para les?es n?o-melanoma e benigna. O potencial dos descritores extra?dos aliados ao classificador SVM tornou o m?todo capaz de reconhecer e classificar as les?es analisadas

Für die Entstehung nicht-melanozytärer Hauttumore sind mehrere Risikofaktoren verantwortlich: UV-Exposition, Pigmentierung, Alter, Immunsuppression und möglicherweise Humane Papillomviren (HPV). Die molekularen Mechanismen der Tumorgenese des kutanen Plattenepithelkarzinoms (SCC) sowie der Präkanzerose Aktinische Keratose (AK) sind nur lückenhaft bekannt. Fokus dieser Arbeit ist die Untersuchung von SCC-Genexpressionsprofilen sowie der Einfluss kutaner HPV-Typen während der Karzinogenese bei immunkompetenten und immunsupprimierten, organtransplantierten Patienten. Durch Genexpressionsanalyse kutaner SCC, AK und normaler Haut konnten 118 differenziell exprimierte Gene in SCC mittels cDNA-Microarrays identifiziert werden. Bestätigt wurde die Expression von 11 aus 13 ausgewählten Genen (85%) mittels quantitativer real-time RT-PCR (qPCR), dabei konnte eine Korrelation der Genexpression mit der Progression der AK zum SCC für 3 Gene nachgewiesen werden. Dazu zählen das Gen Metalloproteinase-1, kodierend für ein Enzym, das in den Umbau von extrazellulärer Matrix involviert ist, das Protoonkogen RAB31 und das Tenascin-C (Tn-C) kodierende Gen Tn-C. Tn-C war im SCC-Gewebe an der Invasionsfront in Basalzellen sowie Keratinozyten im Stratum papillare und retikulare als Protein nachweisbar, nicht aber in normaler Haut. Die im Rahmen dieser Arbeit erstmalig nachgewiesene 2243 bp-Spleißvariante von Tn-C könnte aufgrund der primären Expression in SCC–Gewebe als diagnostischer Marker für SCC dienen. Diese Daten zeigen, dass simultane, multifaktorielle Dysregulationen von Genexpression und DNA-Reparatur, Zellzyklus und Proliferation, proteolytischen Enzymen und Adhäsionsmolekülen in SCC vorliegen. Ferner wurde die Expression von HPV in SCC und damit der kausale Zusammenhang einer HPV-Infektion mit der Hauttumorgenese untersucht. Das Infektionsmuster von SCC-Gewebe und normaler Haut mit spezifischen HPV-Typen erfolgte durch den Nachweis typenspezifischer HPV-DNA. Virale E6/E7-mRNA-Transkripte der kutanen HPV-Typen 8, 9 und 15 wurden in AK und SCC nachgewiesen. Dagegen konnten in HPV-DNA positiver, gesunder Haut oder Warzen keine HPV-Transkripte gefunden werden. Die Variantenanalyse des offenen Leserahmens von E6 identifizierte eine einzelne, bislang nicht beschriebene Punktmutation mit nicht bekannter Veränderung der Proteinstruktur. Die virale Aktivität der Onkogene E6 und E7 einiger kutaner Typen in AK und SCC weisen auf eine mögliche Rolle von HPV bei der kutanen Hautkarzinogenese hin. / During development of non-melanoma skin cancer, several risk factors are involved: UV-exposition, pigmentation, age, and potentially human papilloma virus (HPV). The molecular mechanisms underlying tumourgenesis in squamous cell carcinoma (SCC) and its pre-cancerosis actinic keratosis (AK) are not fully understood. In this study, the gene expression profile and HPV-infection status were analysed in SCC from immunocompetent and organ transplanted, immunocompromised patients.By global transcriptome analysis from cutaneous SCC, AK and healthy skin, 118 genes were identified differentially expressed in a cDNA-microarray. The expression of 11 out of 13 selected genes (85%) was investigated by real-time RT-PCR (qPCR) and the expression of three genes remarkably induced in SCC correlated with the progression to AK until SCC. These genes encoded for Metalloproteinase-1, which is involved in the remodelling of extracellular matrix, and the protooncogene RAB31 and Tenascin-C (Tn-C). Tn-C protein is expressed in SCC-tissue at the invasion front in basal cells and in keratinocytes in the Stratum papillare and retikulare, but not in healthy skin. This study, the 2243 bp Tn-C-specific splice-variant has for the first time detected in SCC, but not in normal skin. Thus it might serve as diagnostic marker of SCC progression. The data of the transcriptome analysis indicates that a simultaneous dysregulation of oncogene expression and DNA-repair, cell-cycle and proliferation, proteolysis and adhesion molecules exists in SCC. Additionally, the expression of HPV in SCC and thus the causal relationship between HPV-infection and tumourgenesis of SCC in immunocompromised patients was investigated. The HPV-infection pattern in SCC-tissue and normal skin was assessed by detection of DNA from cutaneous HPV-types. Viral E6/E7-mRNA-transcripts of the cutaneous HPV-types 8, 9, 15 were expressed selectively in AK and SCC. In contrast, no HPV-specific mRNA was present in HPV-DNA positive normal skin. The analysis of the open reading frame from the respective E6-protein genes unravelled one single pointmutation, which is not been characterized so far in terms of e.g. its impact on protein structure. The viral activity of the oncogenes E6 and E7 of cutaneous HPV-types indicates a potential function of HPV in the tumourgenesis of SCC in immunocompromised individuals.