O carcinoma de cabeça e pescoço (HNSCC) é um dos tipos de câncer que mais acomete as pessoas ao redor do mundo, sendo responsável por 300 mil mortes anualmente. Essa alta taxa de mortalidade está diretamente associada ao diagnóstico tardio, ausência de biomarcadores e tratamento inespecífico. Diversos estudos mostraram que a expressão desregulada da classe de serino proteases do tipo II está intimamente relacionada com a etiologia de diversos tipos de carcinomas, como é o caso da via proteolítica da matriptase. Ainda, a matriptase, no contexto normal da descamação epitelial, ativa as KLKs 5 e 7, as quais são inibidas por LEKTI. A proteína LEKTI, codificada pelo gene SPINK5, possui 15 domínios diferentes, os quais são secretados de forma individual para a matriz extracelular, onde participa da inibição das KLKs 5 e 7. Resultados ainda não publicados do nosso laboratório mostraram que LEKTI é capaz de inibir o fenótipo pré-maligno causado pela superexpressão da matriptase na camada basal do epitélio de camundongos, quando superexpresso nessa mesma região. Ainda, marcações imunohistoquímicas de LEKTI, em amostras de carcinomas orais de humanos, mostraram que essa proteína está também presente nos carcinomas de cabeça e pescoço. Dessa forma, a hipótese levantada neste estudo é a de que LEKTI possui papel inibitório durante a carcinogênese de cabeça e pescoço. Sendo assim, para testar nossa hipótese, a presente dissertação buscou nocautear LEKTI, por meio da tecnologia de edição gênica CRISPR/Cas9, em linhagens de queratinócito imortalizado, através da dissecção por biologia celular, da função de LEKTI nestas células. Os resultados, obtidos por meio de Western Blot, imunofluorescência e sequenciamento das células potencialmente nocauteadas, mostram as dificuldades e desafios de nocautear células hipotetraplóides, como é o caso da linhagem celular utilizada neste estudo. / Head and neck squamous cell carcinoma (HNSCC) is one of the most common type of cancers around the world and it\'s responsible for 300.000 deaths every year. This high mortality rate is directly related with late diagnosis, lack of biomarkers and specific treatment. A fair amount of studies have shown that the deregulated expression of type II serine proteases, such as the matriptase proteolytic pathway, is intimately related with the etiology of a large number of carcinomas. Still, in a normal epithelial desquamation context, matriptase is able to activate KLKs 5 and 7, which are inhibited by LEKTI. The protein LEKTI, encoded by SPINK5 gene, has 15 different domains, which are secreted individually to the extracellular matrix, where acts inhibiting KLKs 5 and 7. Non published results from our laboratory has shown that LEKTI is able to inhibit the pre-malignant phenotype caused by the super-expression of matriptase in the epithelia\'s basal layer of mice, when super-expressed in the same region. Moreover, immunohistochemistry staining of LEKTI in human oral carcinomas showed that this protein is also present in head and neck carcinomas. In this way, the hypothesis of this study is that LEKTI has an inhibitory role during carcinogenesis of head and neck carcinomas. Therefore, to test our hypothesis, this dissertation aimed to knockout LEKTI in an immortalized keratinocyte cell line, using CRISPR/Cas9 editing technology, through cell biology dissection of LEKTI function in those cells. The results, obtained by Western Blot, immunofluorescence and sequencing of the cells, shows the difficulties and challenges to knockout cells that are hypo-tetraploids, just like HaCaT cell line.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22728号 / 医博第4646号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊達 洋至, 教授 萩原 正敏, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Epidermal homeostasis, including proper turnover of keratinocytes, plays important role in the barrier function and serine proteases and their inhibitors are the key players. Activated proteases cleave desmosomes in uppermost layer and thus shed the cells from the epidermal surface. Therefore the serine protease inhibitors are secreted in lower epidermal layers to prevent premature activation of proteases and consequent disruption of epidermal barrier. The most studied inhibitors in epidermis belong to Serine proteases inhibitors Kazal-type family (SPINK). This diploma thesis is aimed to investigate function of murine SPINK6 in epidermal compartment in vivo. To achieve this, the transgenic mice overexpressing mSPINK6 under modified human involucrin promoter was generated. Two of five transgenic lines exhibited higher expression of mSPINK6 at mRNA and protein levels. The mSPINK6 transgenic mice are viable with no apparent phenotype. The small but in most cases not significant differences were observed on microscopic level among mSPINK6 transgenic and wild type animals In conclusion, this work showed that mSPINK6 does not play major role in skin homeostasis but gains significant importance under specific challenges of epidermal barrier. Therefore mSPINK6 transgenic mice, in combination with other deletion or...