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Made available in DSpace on 2015-05-14T12:56:03Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2601299 bytes, checksum: 03c727557f510c297974aa71ae47105c (MD5) Previous issue date: 2014-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The oral cancer is a major cause of morbidity and mortality worldwide. In Brazil, 15,000 new cases are expected for the year 2014. Lip squamous cell carcinoma features are similar to skin lesions and have as main etiological agent, the chronic sun exposure. The galectins -1, -3 and -7 are proteins involved in tumorigenesis and have been investigated due to changes in their expressions in oral cancer. The objective of this study was to evaluate the expression by Immunohistochemistry of galectins-1, -3 and -7 in 30 cases of lip squamous cell carcinoma, making the correlation with clinical data, histological grading of malignancy through the proposed systems for Bryne (1998) and WHO (2005). The parameters analyzed and expression of galectins -1, -3 and -7 were subjected to statistical analysis (Chi-square, Fisher exact test and test for comparison of Binomial proportions). There was no expression of galectin -1 in 93.3% of cases, showing statistically significant correlation (p = 0.0356) between histological grading of malignancy proposal by WHO and the marked cell type. There was no statistically significant correlation between the galectin -3 with none of the evaluated parameters. Expression of galectin-7 was present in all cases evaluated and showed statistical significance between the marked cell type and regional metastasis (p=0,0000) and between the marked cell type and histological grading proposal for Bryne. Changes in the expression of galectinas -1, -3 and -7 suggest the participation of these proteins in carcinogenesis. We conclude, through the results of this research, that the expression of these proteins in squamous cell carcinoma of lip can act as a marker of biological behaviour of this type of lesion. / O câncer oral é uma importante causa de morbidade e mortalidade em todo o mundo. No Brasil, são esperados quinze mil novos casos para o ano de 2014. O carcinoma epidermóide de lábio apresenta características semelhantes a lesões de pele e tem como principal agente etiológico a exposição solar crônica. As galectinas -1, -3 e -7 são proteínas envolvidas na tumorigênese e têm sido investigadas devido a mudanças em suas expressões em casos de câncer oral. O objetivo deste estudo foi avaliar a expressão através da imunoistoquímica das galectinas -1, -3 e -7 em 30 casos de carcinoma epidermóide de lábio, fazendo a associação com os dados clínicos, gradação histológica de malignidade através dos sistemas propostos por Bryne (1998) e OMS (2005). Os parâmetros analisados e a expressão das galectinas -1, -3 e -7 foram submetidas a análise estatística (teste do qui-quadrado, teste exato de Fisher e teste Binomial para a comparação de proporções). Houve expressão da galectina -1 em 93,3% dos casos, apresentando associação estatisticamente significativa (p=0,0356) entre a gradação histológica de malignidade proposta pela OMS e o tipo celular marcado. Não houve associação estatisticamente significante entre a galectina -3 com nenhum dos parâmetros avaliados. A imunoexpressão da galectina -7 esteve presente em todos os casos avaliados e exibiu significância estatística entre o tipo celular marcado e metástase regional (p=0,0000) e entre o tipo celular marcado e a gradação histológica proposta por Bryne. Alterações na expressão das galectinas -1, -3 e -7 sugerem a participação dessas proteínas na carcinogênese. Concluímos, através dos resultados da presente pesquisa, que a imunoexpressão dessas proteínas em carcinoma epidermóide de lábio possa atuar como marcador do comportamento biológico deste tipo de lesão.

Les télomères sont des structures complexes associant des répétitions de séquences nucléotidiques TTAGGG à diverses protéines dont le complexe shelterin. Le TRF2 (Telomeric repeat-binding Factor 2) est un composant majeur de ce complexe, indispensable à la formation de la boucle télomérique T-loop qui permet la protection des extrémités vis-à-vis des exonucléases et systèmes de réparation de l'ADN. Ce facteur est aussi impliqué dans la tumorigenèse de nombreux cancers. Le carcinome épidermoïde oral représente 90% des cancers des Voies Aéro-Digestives Supérieures (VADS) sixième cause de cancer dans le monde avec une incidence de plus de 14000 nouveaux cas en France en 2012. Souvent diagnostiqué par des chirurgiens-dentistes, c'est un cancer de mauvais pronostic traité par chirurgie, radiothérapie, chimiothérapie et thérapies ciblant le R-EGF. Peu d'études se sont intéressées à l'expression de TRF2 dans le carcinome épidermoïde oral et leurs résultats contradictoires ne permettent pas de comprendre son rôle dans leur agressivité.L'expression de TRF2 a été analysée dans les biopsies de 62 patients atteints d'un carcinome épidermoïde oral, à l'aide d'un score de lecture en immunohistochimie. Une forte expression de TRF2 est associé une survie globale significativement plus faible ce qui en fait un facteur pronostique pertinent. In vitro, une faible expression de TRF2 induit une modification du profil sécrétoire des cellules tumorales qui se traduit in vivo par des modifications de l'architecture tissulaire de tumeurs chez la souris nude. Enfin, TRF2 est un marqueur prédictif de réponse aux thérapies ciblées, leur efficacité étant optimale lorsque TRF2 est faiblement exprimé. / Telomeres are composed of tandmely repeated TTAGGG nucleotidic sequences associated with various proteins including shelterin complex. TRF2 (Telomeric repeat-binding Factor 2) is the major component of this complex, necessary for T-loop formation which protects telomeres from DNA damage response and exonucleases. Oral Squamous Cell Carcinoma (OSCC) is the most incident subtype of head and neck cancer (90%), sixth most common cancer in the world with an incidence of 14000 cases in France in 2012. Often diagnosed by dental surgeons,this bad prognosis malignancy is treated by surgery, radiotherapy, chemotherapy and targeted therapies against EGFR. Few studies evaluated TRF2 expression in OSCC and their results are discrepant. TRF2 expression has been assessed in immunohistochemistry in 62 tumoral samples of patients with an history of OSCC. TRF2 overexpression is associated with bad prognosis. TRF2 knockdown changes cells' secretome thus modifying tumoral tissue architecture. Our results showed that TRF2 is predictive for treatment response in targeted therapies in OSCC.

A espectroscopia Raman é uma das tecnologias ópticas que tem ganho cada vez mais destaque na oncologia. Esta tecnologia fornece a composição molecular dos mais variados tipos de amostras, inclusive biofluidos, células e tecidos. Na tentativa de avaliar sua aplicação em lesões bucais, o objetivo deste trabalho foi obter um perfil espectral dos distúrbios epiteliais nãodisplásicos, displásicos, e do carcinoma espinocelular bucal, bem como verificar a possibilidade diagnosticar estas alterações pelos espectros. Inicialmente, foram usados 44 amostras de mucosa bucal em blocos de parafina, com diagnóstico de hiperplasia epitelial, hiperparaceratose, displasia epitelial e carcinoma espinocelular bucal. Da porção epitelial de cada uma destas amostras foram obtidos 10 espectros Raman. A média dos espectros de cada alteração gerou um perfil espectral correspondente à sua composição química. Os resultados desta análise apresentaram picos ou bandas espectrais referentes a moléculas usualmente encontradas nos tecidos biológicos: proteínas, lipídios e DNA. Em seguida, os dados espectrais foram submetidos a análise dos componentes principais (PCA) e análise discriminante linear (LDA), onde foi encontrada uma eficiência de 86,36% no uso da Espectroscopia Raman para o diagnóstico de alterações epiteliais. A validade do método foi comprovada pela Curva Roc resultando em 91% de sensibilidade e 96,9% de especificidade. Em um segundo momento, com a finalidade de avaliar a influência da fixação com formalina nas análises espectrais, a superfície epitelial de uma amostra de tecido bucal fresca ex vivo foi submetida a análise pela Espectroscopia Raman e armazenada em formalina tamponada a 10% por 24h para posteriormente se obter outro espectro. A análise comparativa entre os perfis espectrais de uma mesma amostra antes e após a fixação, demonstraram diferenças em relação aos picos de proteínas e DNA, que podem modificar o perfil espectral da alteração analisada e, portanto, gerar algum erro de classificação. De maneira geral, pelos resultados desta pesquisa, observou-se que a Espectroscopia Raman é uma tecnologia eficiente para identificar a composição molecular de amostras de tecido, seja imediatamente após a excisão cirúrgica, fixação em formalina ou emblocadas em parafina. Entretanto, deve-se considerar que estas a diferentes formas de condicionamento influenciarão diretamente no resultado dos espectros. / Raman spectroscopy (RS) is one of the optical technologies that has garnered a great deal of interest in oncology. This technology provides the molecular composition of various types of samples, including biofluids, cells and tissues. In an attempt to assess its application in oral lesions, the purpose of this study was to obtain a spectral profile of epithelial disorders non-dysplastic, dysplastic, and oral squamous cell carcinoma, as well as checking the ability to diagnose these changes by the spectra. Initially, 44 samples of oral mucosa formali-fixed paraffin embebed were used with epithelial hyperplasia, hyperparakeratosis, epithelial dysplasia and oral squamous cell carcinoma. From the epithelial area of each sample were obtained 10 Raman spectra. The average of the spectra of each change generated a spectral profile corresponding to its chemical composition. Results of this analysis showed peaks or spectral bands related to molecules generally found in biological tissues: proteins, lipids and DNA. Then the spectral data were submitted to principal component analysis (PCA) and linear discriminant analysis (LDA), which was found 86.36% efficiency in the use of Raman spectroscopy (RS) for diagnosis of epithelial changes. The validity of the method was confirmed by Roc curve resulting in 91% sensitivity and 96.9% specificity. In a second step, in order to evaluate the influence of fixation with formalin in the spectral analysis, the epithelial surface of a sample of freshly biopsied oral tissue was subjected to analysis by RS and stored in 10% formalin for 24 hours for later obtain another spectrum. The comparative analysis of the spectral profiles of the same sample, before and after fixation, shows differences relative to spectral peaks of proteins and DNA that can modify the spectral profile of the analyzed change and thus generate some misclassification. In general, the results of this research, it was observed that Raman spectroscopy is an efficient technology to identify the molecular composition of tissue samples, either immediately after surgical excision, either after fixation in formalin and embedded in paraffin. However, it should be considered that these different means of conditioning will influence the outcome of the spectra.

Epithelial cells are unique for their ability to strongly adhere to one another and coordinate communication across an asymmetrical, polar plasma membrane. These properties are necessary for carrying out normal epithelial function, such as absorbing/secreting molecules, repairing wounds, lining organs, etc. Cadherins, claudins, and occludins are major players of epithelial cell adhesion and polarity. Previously, transmembrane immunoglobulin domain containing-1, TMIGD1, was identified as a novel cell adhesion molecule, whose expression is downregulated in human renal carcinomas. Re-expression of TMIGD1 in renal tumor cells resulted in altered cell morphology and inhibition of tumor growth. In this study, we examined the hypothesis that TMIGD1 activity is associated with epithelial cell polarity. We demonstrated that TMIGD1 regulates actin stress fibril formation. A 3-dimensional (3D) cell culture assay was developed to examine the role of TMIGD1 in cell morphology and polarity. Our results demonstrate that TMIGD1 regulates actin fibril formation in Madin-Darby Canine Kidney (MDCK) cells, as blocking TMIGD1 activity by blocking antibody inhibited actin fibril formation in 3D cell culture system. Moreover, ectopic expression of TMIGD1 in rectal carcinoma cells, (RKO) , significantly inhibited filopodia formation. Taken together, our data identifies TMIGD1 as a possible regulator of epithelial cell morphology and polarity. / 2020-07-03T00:00:00Z

AIM & OBJECTIVES: Human papilloma virus (HPV) has been related to cervical infection, however, its part in Head and Neck Squamous Cell Carcinoma (HNSCC) is still debatable and is easy to refute. Suspicion of HPV causation is heightened when carcinomas arise in patients that are young and have never smoked. The present UK based study undertaken at Northampton NHS Trust endeavoured to determine the extent to which HPV is an entity in HNSCC in the UK. Furthermore, the study investigated whether sexual behaviour (as measured by sexual health clinic (SHC) attendance) is linked the acquisition of HPV associated HNSCC in young age groups. HNSCC incidences and sexual trends in the UK were collected from publicly available databases to identify if there were any changes at a national level in sexual behaviours and their influence on HNSCC in young age groups. MATERIALS & METHODS: PCR was used to evaluate the presence of HPV in biopsy samples from of 99 patients diagnosed with HNSCC at Northampton Hospital from 2006 to 2014. Patient demographics on age, sex, smoking, alcohol use and SHC attendance were also collected. All HPV PCR positive biopsies were further genotyped using an ABI 3130xl genetic analyser. Databases in the UK; including GLOBOCAN, NATSAL and PHE were searched for data on HNSCC prevalence, sexual behaviour trends and vaccine uptake. Multinomial regression explored the relationship between HPV positivity and sex, age, smoking, drinking, race and SHC attendance. RESULTS: PCR showed that 25.2% (25/99) of biopsies tested were positive for HPV and were all obtained from white participants. Most specimens (23, 92%) were high-risk (HR) HPV 16 positive with a mean age of 56 for HPV positivity and 72% of the cases 50-60 years old. Smokers were 11% in total (11/99) with most 88.9% participants (88/99) being non-smokers. HPV positivity was strongly linked with non-smoking history (p < 0.001); no alcohol abuse (p < 0.001); male gender (p < 0.001); young age less than 60 years (p < 0.001) and SHC attendance (p < 0.001). A Kruskal-Wallis post hoc test affirmed the impact of age on HPV positivity (p= < 0.05). GLOBOCAN and Cancer Research demonstrated a rising UK HNSCC pattern of over 200% for both sexes from 1975 to 2011. The three NATSAL surveys undertaken in 1990-1991, 1999-2001 and 2010-2012 demonstrated an overall increase in opposite and same sex partners. The UK average of individuals engaging in oral sex was in the younger age groups of between 16 and 54 with at least 70% of males and 63% females of that age engaging in oral sex. Finally, NASTAL 1, 2 and 3 surveys reported 20 vs 15; 25 vs 55; 55 vs 65 of males and females respectively with more than 10 sexual partners to have attended the SHC. The UK immunization take-up was over 90% countrywide. CONCLUSION: Few research studies have been conducted to date on HPV as a cause of HNSCC in the UK. The present research showed 25.2% of HNSCC to be caused by HPV, with the high risk (HR) genotype 16 (the leading cause of cervical cancer) accounting for 92% (23/25) of the cases. These outcomes affirmed the high prevalence of HR-HPV in HNSCC, with a rate of 25.2% similar to those reported previously. Routine HPV testing in those aged below 60 is therefore warranted. Smoking and drinking showed negative correlation; the young age of below 60 and attendance of the SHC for both sexes showed a positive correlation with HPV positive HNSCC. NATSAL data showed increased sexually risky behaviour coupled with attending the SHC in younger ages for both sexes. Increased sexually risky behaviour as shown in NASTAL surveys may be the reason why young age and SHC attendance is positively correlated with HPV HNSCC. The study highlights a conceivable relationship between HPV positive HNSCC in those under 60 years with no smoking history who attended the SHC. Smoking and drinking are known risks for HNSCC in those past 65 years of age; the negative association with HPV HNSCC in the young in the present research revealed smoking and drinking to have reduced association with HPV HNSCC. The reported HR-HPV positive HNSCC in young age groups inform future vaccination strategies and consequently decrease the quantity of HPV HNSCC's.

Testicular germ cell cancer (TGCC) has been increasing in incidence over recent decades, and is currently the most common malignancy amongst young men resulting in significant morbidity. These tumours are believed to arise from premalignant germ cell neoplasia in situ (GCNIS) cells, which originate from the aberrant germ cell differentiation from gonocyte to spermatogonia during fetal/early postnatal life. GCNIS cells remain dormant in the testis until puberty when they are activated to become tumours. Therefore, GCNIS cells remain in a pre-invasive stage during early childhood and early adulthood prior to the development of a seminoma or non-seminoma TGCC. GCNIS cells are phenotypically similar to gonocytes with expression of stem cell/early germ cell markers including OCT4, PLAP and LIN28. Furthermore, proteins which are expressed in more mature germ cells (spermatogonia) such as MAGE-A4 have also been shown to be expressed in GCNIS cells and these studies have indicated that GCNIS cells are a heterogeneous population in terms of protein expression profile. The relationship between the protein expression profile of individual GCNIS cells populations and their oncogenic potential has not been fully explored. GCNIS cells are located in the seminiferous tubules supported by somatic Sertoli cells. These cells have been previously reported to exhibit an immature protein expression profile in GCNIS tubules from patients with testis cancer, suggesting that the germ stem cell niche in GCNIS tubules resembles that of a fetal one. Associations between Sertoli cell maturation and GCNIS progression into tumour formation has not been fully investigated. Oncogenes are key players in the regulation of oncogenic potential of cancer cells. Gankyrin is an oncogene that has been shown to down-regulate OCT4, and interact with MAGE-A4 in hepatocellular carcinoma and colorectal cancer, where Gankyrin interaction with MAGE-A4 reduces the oncogenic potential of tumour cells. In this study I aimed to investigate the heterogeneity of GCNIS in relation to disease stage and Sertoli cell development. We also aimed to determine the role of Gankyrin in TGCC cell survival and invasion. The co-expression of early germ cells proteins such as OCT4, LIN28 and PLAP was characterized in GCNIS cells during childhood and adulthood pre-invasive TGCC and in invasive disease characterized by the presence of a testicular tumour. These results show that LIN28 was expressed in 95% of OCT4 GCNIS cells, whereas PLAP expression in GCNIS cells increased as the disease progressed from childhood pre-invasive disease to invasive seminoma (32.3% v 76%; p < 0.05). In contrast there was a reduction in the proportion of MAGE-A4 expressing GCNIS cells with disease progression. The MAGE-A4 expressing population was also less proliferative than the MAGE-A4 negative GCNIS population. The methylation status of GCNIS cells was then investigated. EZH2 a methyltransferase previously reported to be important for TGCC development, was expressed in GCNIS cells at all stages of disease, however the histone 3 modification H3K27me3 (mediated by EZH2) was expressed in a significantly higher percentage of the proliferative OCT4+/MAGE-A4- GCNIS cells compared with the OCT4+/MAGEA4+ population (11.7% v 1.1%; p < 0.01) which could indicate a repressive role for H3K27me3 over MAGE-A4 expression. Next, it was determined whether an association between Sertoli cell maturation status and progression of TGCC could be observed. The maturation status of Sertoli cells was studied using proteins indicative of immature (desmin, cytokeratin, fibronectin and AMH) and mature (vimentin and androgen receptor) Sertoli cells. These studies demonstrated heterogeneity of Sertoli cells maturation in GCNIS-containing tubules. Desmin, fibronectin, AMH and vimentin expression did not show any association with TGCC progression. Cytokeratin was expressed in Sertoli cells of human fetal testis up to second trimester of fetal life, absent in tubules with active spermatogenesis but heterogeneously present in GCNIS, demonstrating that cytokeratin expression is indicative of the presence of GCNIS. Androgen receptor was weakly present in Sertoli cells from human fetal testis and pre-pubertal pre-invasive TGCC testis whereas in GCNIS of adult pre-invasive testis and invasive samples, androgen receptor was abundantly expressed in Sertoli cells of GCNIS-containing tubules. These combined results for cytokeratin and androgen receptor suggest that Sertoli cells from GCNIS-containing tubules, in pre-invasive and invasive TGCC patients are partially differentiated. Gankyrin expression was characterised in fetal germ cells, GCNIS cells and TGCC tissue. In fetal testis nuclear Gankyrin was absent in OCT4+/MAGE-A4- (gonocyte) population whereas it was present in a subpopulation of OCT4-/MAGE-A4+ (spermatogonia) germ cells. In GCNIS cells from TGCC patients nuclear Gankyrin was expressed in 87%, 63.3%, 91.5% and 79% in childhood pre-invasive, adult pre-invasive, seminoma and non-seminoma GCNIS cells respectively. Finally, in seminoma cells, Gankyrin was expressed in the cytoplasm indicating a change in localisation as the GCNIS cells become invasive. We used siRNA to knockdown Gankyrin in NT2 (a TGCC cell line) cells in-vitro and demonstrated a decrease in cell number, suggesting that Gankyrin might play a role in TGCC progression and invasiveness. Gankyrin down-regulation also resulted in an increase in p53 and p21 mRNA level. Given the role of P53 and p21 in cisplatin cytotoxic effect in TGCC we went on to investigate the role of Gankyrin in cisplatin resistance using NT2 cells. We demonstrate that Gankyrin mediated cisplatin resistance through the p53/p21 pathway, upregulating apoptosis rates through BAX and FAS, whilst there was no effect on cell proliferation, cell cycle or cell migration. In conclusion, we have shown that GCNIS cells are heterogeneous and their phenotype can determine their oncogenic potential. We also show that Sertoli cells from GCNIS-containing tubules undergo partial differentiation displaying markers of immature and mature Sertoli cells, with a heterogeneous association of cytokeratin with GCNIS presence. We also demonstrate that the oncogene Gankyrin has a role in NT2 cells survival and cisplatin resistance indicating that manipulation of Gankyrin may have a role in the treatment of TGCC.

Leucoplasia bucal (LB) é uma desordem potencialmente maligna, com risco de transformação maligna que varia de 0,13% a 17,5%. Muitos estudos vêm buscando estabelecer biomarcadores capazes de predizer o potencial de transformação maligna dessa lesão. O Ki-67 é uma proteína não-histônica nuclear que tem sido amplamente utilizada para avaliar proliferação celular. O BMI-1 é uma proteína considerada um marcador essencial para a manutenção das propriedades de autorrenovação e da tumorigenicidade do carcinoma espinocelular. O objetivo principal desse estudo observacional transversal foi avaliar proliferação e imortalização celulares em LB a partir da marcação imunoistoquímica do Ki-67 e do BMI-1, comparando lesões displásicas com não displásicas. Casos de LB não displásica - LBND (n=28), LB displásica - LBD (n=33) foram selecionados a partir de prontuários de pacientes e comparados com mucosa clinicamente normal - MN (n=9), hiperplasia inflamatória - HI (n=17) e carcinoma espinocelular - CEC (n=19). Os diagnósticos histopatológicos foram confirmados a partir da revisão de cortes histológicos corados por hematoxilina e eosina. Adicionalmente, cortes histológicos foram submetidos à técnica imunoistoquímica para avaliação de Ki-67 e BMI-1. Para a quantificação foi considerado o percentual de células positivas por 1000 células para o CEC e 1500 células para os demais grupos. O percentual de imunomarcação de Ki-67 e de BMI-1, quando avaliadas todas as camadas epiteliais em conjunto, foi mais alto no CEC quando comparado aos demais grupos (Kruskal-Wallis, p<0.05). A expressão de Ki-67 foi maior em LBND, LBD e HI quando comparada com MN (Kruskal-Wallis, p<0.05). Além disso, a imunomarcação de BMI-1 foi maior em LBD quando comparada com MN, quando analisadas todas as camadas em conjunto (Kruskal-Wallis, p<0.05). A partir da avaliação das camadas epiteliais separadamente, observou-se aumento da expressão de BMI-1 na camada parabasal e suprabasal em LBND quando comparada com MN. Houve correlação positiva entre Ki-67 e BMI-1 (Correlação de Spearman, R=0.37, p<0.05). Conclui-se que a proliferação e as alterações na transição epitélio-mesênquima são eventos relacionados e que estão presentes desde estágios precoces e se acentuam nos estágios mais tardios da carcinogênese. / Oral leukoplakia (OL) is potentially malignant disorder, with a risk of malignant transformation that ranges from 0.13% to 17.5%. Many biological markers have been used as an attempt to predict malignant transformation, but no reliable markers have been established so far. The Ki-67 is a nuclear non-histone regarded as reliable marker of proliferating cells .BMI-1 is a protein considered as an essential marker for maintenance of properties self-renewability and tumorigenicity of squamous cell carcinoma. The main aim this cross-sectional observational study was to evaluate cell proliferation and immortalization in oral leukoplakia, comparing non-dysplastic and dysplastic lesions. Cases of non-dysplastic – Non-dys OL (n=28), dysplastic – Dys OL (n=33) records were selected and compared with normal oral mucosa – NOM (n=9), inflammatory hyperplasia –IH (n=17), and oral squamous cell carcinoma - OSCC (n=19). The histopathological diagnosis was confirmed by the revision of Hematoxilin and Eosin stained slides. Additionally, histological sections were submitted to immunohistochemical technique for evaluation of Ki-67 and BMI-1. The labeling index was determined by counting the labeled nuclei of 1000 cells for OSCC cases and 1500 for the others comparison groups. Ki-67 and BMI-1 immunolabeling percentage were higher in OSCC in comparison of others groups when all epithelial layers were evaluated together (Kruskal-Wallis, p<0.05). Ki-67 immunolabeling increased in Non-dys OL, Dys OL and IH when compared to NOM (Kruskal-Wallis, p<0.05). Furthermore, BMI-1 immunolabeling was higher in Dys OL relation to NOM in the analysis of all epithelial layers together (Kruskal-Wallis, p<0.05). When the evaluation considered the epithelial layer separately, an increased BMI1 expression was observed in the parabasal and suprabasal layers of Non-dys OL when compared to NOM. A significant positive correlation was found between Ki-67 and BMI-1 (Spearman correlation coefficient, R=0.37, p<0.01). In conclusion, the present findings support that proliferation and changes toward epithelial-to-mesenchymal transition increase gradually since early stages of oral carcinogenesis.

A espectroscopia Raman é uma das tecnologias ópticas que tem ganho cada vez mais destaque na oncologia. Esta tecnologia fornece a composição molecular dos mais variados tipos de amostras, inclusive biofluidos, células e tecidos. Na tentativa de avaliar sua aplicação em lesões bucais, o objetivo deste trabalho foi obter um perfil espectral dos distúrbios epiteliais nãodisplásicos, displásicos, e do carcinoma espinocelular bucal, bem como verificar a possibilidade diagnosticar estas alterações pelos espectros. Inicialmente, foram usados 44 amostras de mucosa bucal em blocos de parafina, com diagnóstico de hiperplasia epitelial, hiperparaceratose, displasia epitelial e carcinoma espinocelular bucal. Da porção epitelial de cada uma destas amostras foram obtidos 10 espectros Raman. A média dos espectros de cada alteração gerou um perfil espectral correspondente à sua composição química. Os resultados desta análise apresentaram picos ou bandas espectrais referentes a moléculas usualmente encontradas nos tecidos biológicos: proteínas, lipídios e DNA. Em seguida, os dados espectrais foram submetidos a análise dos componentes principais (PCA) e análise discriminante linear (LDA), onde foi encontrada uma eficiência de 86,36% no uso da Espectroscopia Raman para o diagnóstico de alterações epiteliais. A validade do método foi comprovada pela Curva Roc resultando em 91% de sensibilidade e 96,9% de especificidade. Em um segundo momento, com a finalidade de avaliar a influência da fixação com formalina nas análises espectrais, a superfície epitelial de uma amostra de tecido bucal fresca ex vivo foi submetida a análise pela Espectroscopia Raman e armazenada em formalina tamponada a 10% por 24h para posteriormente se obter outro espectro. A análise comparativa entre os perfis espectrais de uma mesma amostra antes e após a fixação, demonstraram diferenças em relação aos picos de proteínas e DNA, que podem modificar o perfil espectral da alteração analisada e, portanto, gerar algum erro de classificação. De maneira geral, pelos resultados desta pesquisa, observou-se que a Espectroscopia Raman é uma tecnologia eficiente para identificar a composição molecular de amostras de tecido, seja imediatamente após a excisão cirúrgica, fixação em formalina ou emblocadas em parafina. Entretanto, deve-se considerar que estas a diferentes formas de condicionamento influenciarão diretamente no resultado dos espectros. / Raman spectroscopy (RS) is one of the optical technologies that has garnered a great deal of interest in oncology. This technology provides the molecular composition of various types of samples, including biofluids, cells and tissues. In an attempt to assess its application in oral lesions, the purpose of this study was to obtain a spectral profile of epithelial disorders non-dysplastic, dysplastic, and oral squamous cell carcinoma, as well as checking the ability to diagnose these changes by the spectra. Initially, 44 samples of oral mucosa formali-fixed paraffin embebed were used with epithelial hyperplasia, hyperparakeratosis, epithelial dysplasia and oral squamous cell carcinoma. From the epithelial area of each sample were obtained 10 Raman spectra. The average of the spectra of each change generated a spectral profile corresponding to its chemical composition. Results of this analysis showed peaks or spectral bands related to molecules generally found in biological tissues: proteins, lipids and DNA. Then the spectral data were submitted to principal component analysis (PCA) and linear discriminant analysis (LDA), which was found 86.36% efficiency in the use of Raman spectroscopy (RS) for diagnosis of epithelial changes. The validity of the method was confirmed by Roc curve resulting in 91% sensitivity and 96.9% specificity. In a second step, in order to evaluate the influence of fixation with formalin in the spectral analysis, the epithelial surface of a sample of freshly biopsied oral tissue was subjected to analysis by RS and stored in 10% formalin for 24 hours for later obtain another spectrum. The comparative analysis of the spectral profiles of the same sample, before and after fixation, shows differences relative to spectral peaks of proteins and DNA that can modify the spectral profile of the analyzed change and thus generate some misclassification. In general, the results of this research, it was observed that Raman spectroscopy is an efficient technology to identify the molecular composition of tissue samples, either immediately after surgical excision, either after fixation in formalin and embedded in paraffin. However, it should be considered that these different means of conditioning will influence the outcome of the spectra.

O Carcinoma de células escamosas é o tipo histológico mais comum das neoplasia esofágicas no mundo.Tem sua origem no epitélio escamoso do esôfago e é achado com maior frequência no terço médio do órgão. A carcinogênese esofágica é um processo que resulta do acúmulo de mutações envolvendo diferentes genes, como o TP53. Este está mutado em mais de 50% dos tumores esofágicos. Inúmeras pesquisas têm sido direcionadas para restaurar a função da proteína p53, as quais resultaram na descoberta da PRIMA-1. Neste estudo, foram pesquisados possíveis efeitos protetores da PRIMA-1 no epitélio do esôfago sob efeito carcinogênico químico evitando o desenvolvimento neoplásico e induzindo involução da carcinogênese. Para testar esta hipótese, realizamos estudo, com utilização da PRIMA-1, em um modelo experimental de carcinogênese esofágica por dietilnitrosamina com indução de carcinoma de células escamosas. O estudo experimental foi realizado com 6 grupos de animais, onde os grupos I e II foram considerados controles, sendo diferenciados por uso da PRIMA-1. E os grupos III e IV, e Va e Vb, foram considerados estudos, os quais receberam dietilnitrosamina por 3 dias consecutivos semanalmente. Enquanto os grupos III e IV, foram diferenciados pelo uso de PRIMA-1 prévio ao início da carcinogênese, os grupos Va e Vb, foram diferenciados pelo uso de PRIMA-1 ao final da carcinogênese. O estudo apresentou data de eutanásia com colheita de peças esofágicas, aos 150 dias, grupos I, II, III e IV, e 180 dias, grupos Va e Vb, devido ao período de observação após uso tardio da PRIMA-1. Não foram detectadas alterações histopatológicas nos grupos controles, grupo I (DEN-/PRIMA-) e II (DEN-/PRIMA+). Não foram detectadas diferenças nas alterações histopatológicas estatisticamente significativas entre os grupos III (DEN+/PRIMA-) e IV (DEN+/ PRIMA+ inicial). Foram detectadas diferenças nas alterações histopatológicas estatisticamente significativas entre os grupos Va (DEN+/PRIMA-) e Vb (DEN+/PRIMA+ tardio). A análise imunohistoquímica do p53 evidenciou diferença estatística nos grupos que utilizaram a PRIMA-1, em relação as seus controles. A análise imunohistoquímica do Ki-67 demonstrou diferença estatística somente entre os dois grupos controles. Conclue-se que a PRIMA-1 teve efeito de redução da gravidade histopatológica em esôfago de camundongos previamente induzidos a carcinogênese com dietilnitrosamina e aumento da expressão imuno-histoquímica da proteína p53. / The esophageal squamous cell carcinoma is the most common histological type in the world. It is origin in the squamous epithelium of the esophagus and is found more often in the middle third of the organ. The esophageal carcinogenesis is a process that results from the accumulation of mutations in different genes such as TP53. This is mutate in more than 50% of esophageal tumors. Numerous researches have been directed to restore the function of the p53 protein, which resulted in the discovery of PRIMA-1. In this study, we investigated the possible protective effects of PRIMA-1 in the epithelium of the esophagus under chemical carcinogenic effect preventing neoplastic development and inducing regression of carcinogenesis. To test this hypothesis, a study with use of PRIMA-1 in an experimental model of esophageal carcinogenesis induced by diethylnitrosamine with squamous cell carcinoma. The experimental study was performed wth six groups of animals, where the groups I and II were considered controls being differentiated by the use of PRIMA-1. And the groups III and IV, and Va and Vb, were considered studies, which received diethylnitrosamine for 3 consecutive days weekly. While the groups III and IV, were differentiated by the use of PRIMA-1 prior to the beginning of carcinogenesis, the Va and Vb groups were differentiated by the use of PRIMA-1 at the end of carcinogenesis. The study presented date of euthanasia with crop esophageal parts, at 150 days, groups I, II, III and IV, and 180 days, Va and Vb groups because of the observation period after late use of PRIMA-1. Histopathological changes were detected in the control groups, Group I (DEN-/PRIMA-) and II (DEN-/PRIMA+). There were no statistically significant differences in pathological changes between the groups III (DEN+/PRIMA-) and IV (DEN+/PRIMA- first). Differences were found in statistically significant histopathological changes between Va groups (DEN+/PRIMA-) and Vb (DEN+/PRIMA+ late). Immunohistochemical analysis of p53 showed a statistically significant difference in the groups using PRIMA-1, compared to their controls. Immunohistochemical analysis of Ki-67 showed statistical difference only between the two control groups. It concludes that the PRIMA-1 was reduced by histopathological effect of gravity previously induced esophageal carcinogenesis in mice diethylnitrosamine and increased immunohistochemical expression of p53 protein.