Le lymphome est l'une des tumeurs les plus communes tant chez le chien que l’humain. Chaque année, un nombre important de chiens développe ce cancer agressif. La majorité décédant un an suivant le diagnostic. Le lymphome canin est maintenant identifié comme un excellent modèle de recherche pour la tumeur chez l'homme, particulièrement en ce qui concerne la biologie moléculaire de la maladie. En conséquence, la recherche sur le lymphome canin sera bénéfique non seulement pour les chiens mais aussi pour l’oncologie humaine. Parmi les méthodes diagnostiques de choix pour dépister de façon hâtive le lymphome se trouve la mesure de marqueurs tumoraux. Ceci a l’avantage d’être peu invasive, simple et peu dispendieuse. Ainsi, dans le but d’évaluer la protéine VCP (valosin containing protein) comme biomarqueur tumoral dans les lymphomes canins à cellules B et T, nous avons évalué la protéine VCP par immunobuvardage sur sérums et tissus tumoraux de chiens atteints et par immunohistochimie sur des tumeurs de haut grade, grade intermédiaire et bas grade. Pour mieux définir l’expression de VCP dans les cellules cancéreuses, nous avons également examiné par immunobuvardage les niveaux de VCP dans 3 lignées cellulaires: CLBL-1, CL-1, et 17-71. Il s’avère que les lymphomes à cellules B de haut grade avaient une élévation significative du taux de VCP comparé aux tumeurs de bas grade (P < 0,05). De même, une accumulation importante de VCP a également été détectée dans les lignées tumorales comparées aux cellules mononucléaires du sang périphérique (P < 0,05). D’autre part, le taux sérique de VCP est resté similaire à ceux des chiens normaux. Ces résultats suggèrent une corrélation entre le taux de VCP et le degré de malignité des lymphomes à cellules B. En conclusion, la protéine VCP doit faire l’objet d’une évaluation approfondie pour déterminer son utilité comme marqueur pronostique. / Lymphoma is one of the common malignancies in both dogs and humans. Annually, an important number of canine patients develop this aggressive cancer and a majority succumbs to the disease within one year. In recent years, canine lymphoma has been increasingly recognized as an excellent model for the disease in humans, especially with regards to the molecular biology of the disease. Consequently, research targeted at canine lymphoma benefits not only dogs but the field of human oncology as well. Among the most desirable diagnostic and screening tests for lymphoma is the measurement of cancer biomarkers. They have the advantage of being minimally invasive, simple, and inexpensive. Thus, with the aim of evaluating valosin containing protein (VCP) as a cancer biomarker in canine B and T-cell lymphomas, we first performed western blots on sera and tumor tissue of dogs with lymphoma and then immunohistochemical analysis on low, intermediate and high-grade tumors. To further determine VCP expression in cancer cells, we also examined VCP levels by immunoblotting in 3 tumor cell lines: CLBL-1, CL-1, and 17-71. High-grade B-cell lymphomas had significantly increased levels of VCP compared to low-grade tumors (P < 0.05). Additionally, we detected a corresponding accumulation of VCP in tumor cells lines compared to peripheral blood mononuclear cells (PBMCs) (P < 0.05). In contrast, VCP levels were not elevated in sera of dogs with lymphoma compared to healthy controls. These results suggest that VCP positively correlates with malignancy in canine B-cell lymphomas. We conclude that VCP merits further investigation to determine its potential as a clinically useful prognosis biomarker for canine B-cell lymphoma.

O carcinoma de célula renal (RCC) subtipo célula clara é o câncer mais letal e prevalente do sistema urinário. O diagnóstico deste tipo de câncer frequentemente é tardio em conseqüência da falta de sintomas perceptíveis aos pacientes. Um dos objetivos deste trabalho é a identificação de novos marcadores moleculares para diagnóstico precoce, o que ajudaria a diminuir a mortalidade em função de complicações resultantes do avanço da doença. Outro objetivo é a identificação de um conjunto de marcadores moleculares de prognóstico, de modo à prever com acurácia a evolução clínica da doença e, por conseqüência, o tempo de sobrevida do paciente. As modificações transcricionais associadas à carcinogênese e à progressão do câncer de rim ainda não foram completamente elucidadas. Além dos oncogenes e genes supressores de tumor, RNAs não-codificadores (ncRNAs) recentemente foram apontados como importantes reguladores da expressão gênica em humanos, e podem ter um papel importante na transformação maligna do câncer de rim. Para analisar a expressão gênica de ncRNAs e de genes codificadores para proteína foram utilizados dois microarranjos desenvolvidos por nosso grupo, enriquecidos em sondas para ncRNAs. Uma das plataformas possui 4 mil sondas de cDNA, das quais 822 sondas são para ncRNAs mapeando em regiões intrônicas. Outra possui 44 mil elementos e combina sondas de oligonucleotídeos (60-mer) intrônicas e exônicas de um mesmo locus genômico. Análises estatísticas foram feitas com a ferramenta Significance Analysis of Microarrays (q &#8804; 0,05) combinadas ou com a técnica de \"patient leave-one-out\" (genes com presença em 8 100% dos subconjuntos), ou alternativamente com o teste discriminante de Golub (p &#8804; 0,01 ou p < 0,05). Com a plataforma de 4 mil sondas foram estudadas 30 amostras de tecido renal de 18 pacientes com RCC subtipo célula clara. Um conjunto de 36 ncRNAs foi identificado como diferencialmente expresso entre amostras tumorais e não-tumorais. Uma assinatura adicional de 265 genes codificadores de proteínas foi identificada, indicando possíveis novos marcadores moleculares. Uma análise estatística supervisionada com dados de 16 pacientes identificou uma assinatura de ncRNAs correlacionada com sobrevida de 5 anos, formada por 27 ncRNAs com significativa expressão alterada em pacientes livres da doença em comparação com pacientes que morreram em função da doença. Uma assinatura adicional de 64 genes codificadores de proteínas também foi identificada como significativamente correlacionada com o acompanhamento clínico dos pacientes. Com a plataforma de 44 mil sondas foram analisados 17 pacientes, com amostras pareadas de tecido renal tumoral e não-tumoral agrupadas em 8 pools, sendo 4 de amostras tumorais e 4 de não-tumorais. Um conjunto de 66 ncRNAs parcialmente intrônicos antisenso e outro de 52 ncRNAs totalmente intrônicos antisenso foram identificados como diferencialmente expressos. Identificamos um subconjunto de 28 ncRNAs totalmente intrônicos antisenso e senso cuja expressão do gene codificador de proteína do mesmo locus estava simultaneamente alterada. Estes dados apontam para possíveis redes de regulação da expressão gênica dos ncRNAs em câncer. A extensa lista de ncRNAs e de genes codificadores para proteína identificados neste estudo podem ser promissores marcadores moleculares de carcinoma renal subtipo célula clara. / Renal cell carcinoma (RCC) is the most common malignancy of the adult kidney, and the clear cell subtype is the most prevalent and lethal cancer of the urinary system. Late diagnosis for this type of cancer is frequent, usually as a consequence of the lack of symptoms. One of the objectives of the present work is the identification of new molecular markers for the early diagnosis, which would help decrease mortality that develops as a function of disease progression. Another objective is the identification of a set of prognosis molecular markers, so as to accurately predict the clinical outcome of the disease, and consequently, patient survival. Transcriptional changes associated to carcinogenesis and to kidney cancer progression have not been entirely elucidated. Besides oncogenes and tumor suppressor genes, non-coding RNAs (ncRNAs) have been recently indicated as important regulators of gene expression in humans, and could have an important role in the malignant transformation in renal cancer. In order to measure ncRNA and protein-coding gene expression we have used two microarray platforms developed by our group, which are enriched in ncRNA probes. One of the platforms has 4 thousand cDNA probes, of which 822 are for ncRNAs that map to intronic regions. Another has 44 thousand elements and combines 60-mer oligonucleotide probes for intronic and exonic regions from the same genomic locus. Statistical analyses have been performed with the Significance Analysis of Microarrays tool (q &#8804; 0.05) combined with a patient leave-one-out approach (genes present in 100% of the sub-sets), or alternatively with Golubs discriminant test (p &#8804; 0.01 or p < 0.05). 11 With the 4-thousand probes platform we studied 30 samples from renal tissue of 18 RCC patients with clear cell subtype. A set of 36 ncRNAs has been identified as differentially expressed between tumor and non-tumor tissue. An additional signature of 265 protein-coding genes has been identified, indicating possible new molecular markers. A supervised statistical analysis with data from 16 patients has identified a ncRNA signature correlated to 5-year survival outcome, comprised of 27 ncRNAs with significantly altered expression in diseasefree patients compared to patients who died from cancer within the 5-year follow-up. An additional 64-gene signature of protein-coding genes has been identified as significantly correlated to clinical outcome. With the 44-thousand probes platform we have analyzed 17 patients, with paired tumor and non-tumor samples grouped into 8 pools, of which 4 were from tumor and 4 from nontumor samples. A set of 66 partially intronic antisense ncRNAs and another of 52 totally intronic antisense ncRNAs have been identified as differentially expressed between tumor and non-tumor tissue. A sub-set of 28 totally intronic antisense or sense ncRNAs were identified as having a simultaneous change in expression of the protein-coding gene from the same locus. Overall, the data point to a possible ncRNA regulatory network in cancer. The extensive lists of ncRNAs and of protein-coding genes identified in the present study can be seen as promising molecular markers of RCC from the clear-cell subtype.

This thesis describes a set of image analysis tools developed for the purpose of quantifying the distribution of chromatin in (light) microscope images of cell nuclei. The distribution or pattern of chromatin is influenced by both external and internal variations of the cell environment, including variations associated with the cell cycle, neoplasia, apoptosis, and malignancy associated changes (MACs). The quantitative characterisation of this pattern makes possible the prediction of the biological state of a cell, or the detection of subtle changes in a population of cells. This has important application to automated cancer screening. The majority of existing methods for quantifying chromatin distribution (texture) are based on the stochastic approach to defining texture. However, it is the premise of this thesis that the structural approach is more appropriate because pathologists use terms such as clumping, margination, granulation, condensation, and clearing to describe chromatin texture, and refer to the regions of condensed chromatin as granules, particles, and blobs. The key to the structural approach is the segmentation of the chromatin into its texture primitives. Unfortunately all of the chromatin segmentation algorithms published in the literature suffer from one or both of the following drawbacks: (i) a segmentation that is not consistent with a human's perception of blobs, particles, or granules; and (ii) the need to specify, a priori, one or more subjective operating parameters. The latter drawback limits the robustness of the algorithm to variations in illumination and staining quality. The structural model developed in this thesis is based on several novel low-, med-ium-, and high-level image analysis tools. These tools include: a class of non-linear self-dual filters, called folding induced self-dual filters, for filtering impulse noise; an algorithm, based on seeded region growing, for robustly segmenting chromatin; an improved seeded region growing algorithm that is independent of the order of pixel processing; a fast priority queue implementation suitable for implementing the watershed transform (special case of seeded region growing); the adjacency graph attribute co-occurrence matrix (AGACM) method for quantifying blob and mosaic patterns in the plane; a simple and fast algorithm for computing the exact Euclidean distance transform for the purpose of deriving contextual features (measurements) and constructing geometric adjacency graphs for disjoint connected components; a theoretical result establishing an equivalence between the distance transform of a binary image and the grey-scale erosion of its characteristic function by an elliptic poweroid structuring element; and a host of chromatin features that can be related to qualitative descriptions of chromatin distribution used by pathologists. In addition, this thesis demonstrates the application of this new structural model to automated cervical cancer screening. The results provide empirical evidence that it is possible to detect differences in the pattern of nuclear chromatin between samples of cells from a normal Papanicolaou-stained cervical smear and those from an abnormal smear. These differences are supportive of the existence of the MACs phenomenon. Moreover the results compare favourably with those reported in the literature for other stains developed specifically for automated cytometry. To the author's knowledge this is the first time, based on a sizable and uncontaminated data set, that MACs have been demonstrated in Papanicolaou stain. This is an important finding because the primary screening test for cervical cancer, the Papanicolaou test, is based on this stain.

Made available in DSpace on 2014-12-17T15:32:18Z (GMT). No. of bitstreams: 1 CyntiaCPC.pdf: 2470782 bytes, checksum: 4619c7ffcab85bffa54a732d30786d99 (MD5) Previous issue date: 2010-02-24 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Currently, bone morphogenetic proteins (BMPs) have effective participation in the growth of malignancies. Knowing that there are few studies involving BMPs and oral squamous cell carcinoma, this work constitutes an immunohistochemical study of BMP-2, BMPR IA and BMPR II in squamous cell carcinomas (SCC) of the lower lip relating to the clinical and pathological aspects of this lesion. The sample consisted of 40 cases of SCC of the lower lip, being 20 cases of SCC of the lower lip with regional metastasis and 20 cases without metastasis. We evaluated the intensity of expression (score 1 to mark absent / weak, score 2 for high ) and was found the percentage of labeled cells, where the score was 1 cases with 0 to 50% of positive cells, score 2 with 51 to 75% of positive cells, and score 3 more than 75% of positive cells. The sample comprised 72.5% of men with a mean age of 65.8 years, there was a predominance of stage II and 52.5% of the carcinomas were classified as low grade, being carcinoma with metastasis presenting most cases (70%) as carcinomas of high malignancy grade (p = 0.004). The largest number of cases of SCC of the lower lip that were in stages I / II (61, 9%) were classified as carcinomas of low grade malignancy and carcinomas in stages III / IV were classified as high-grade tumors (p = 0, 024). The BMP-2 showed strong intensity of immunostaining in 82.5%, BMPR-IA showed 55% of cases with an intensity of immunostaining absent / weak and BMPR-II showed 85% of cases with an intensity of immunostaining absent / weak. Only the protein BMPR-IA were significantly associated with all clinic-pathological parameters studied, metastasis (p <0.001), TNM (p <0.001) and histological grade of malignancy with (p = 0.028). The percentage of positive cells, all markers showed the highest number of cases with more than 75% of positive cells (score 3) and only BMPR-II showed statistical difference when related to the presence and absence of metastasis (p = 0.049 ). We conclude that there is disturbance in the BMP signaling pathway in EC-mediated lower lip and that high expression of BMP-2 associated with the expression of BMPR-IA and BMPR-II are associated with metastasis in carcinoma / Atualmente as prote?nas morfogen?ticas do osso (BMPs) t?m efetiva participa??o no crescimento de neoplasias malignas. Sabendo que s?o escassos os trabalhos envolvendo BMPs e o carcinoma epiderm?ide oral, este trabalho realizou um estudo imunoistoqu?mico da BMP-2, BMPR IA e BMPR II em carcinomas epiderm?ides (CE) de l?bio inferior relacionando com os aspectos clinico-patol?gicos desta les?o. A amostra constou de 40 casos de CE de l?bio inferior, sendo 20 casos de CE de l?bio inferior com met?stase linfonodal regional e 20 casos sem met?stase. A grada??o histol?gica de malignidade foi realizada no front invasivo da les?o. Foi avaliada a intensidade de express?o (escore 1 para marca??o ausente/ fraca e escore 2 para marca??o forte), bem como foi verificado a porcentagem de c?lulas positivas, onde o escore 1 era os casos com 0 a 50% das c?lulas positivas; escore 2 com 51 a 75% das c?lulas positivas; e escore 3 com mais de 75% das c?lulas positivas. A amostra foi composta por 72,5% de homens com a m?dia de idade de 65,8 anos, houve um predom?nio do est?gio II e 52,5% dos carcinomas foram classificados como de baixo grau, sendo os carcinomas com met?stase regional apresentando a maioria dos casos (70%) como carcinomas de alto grau de malignidade (p =0,004). O maior n?mero de casos de CE de l?bio inferior que estavam nos est?gios I/ II (61, 9%) foi classificado em carcinomas de baixo grau de malignidade e os carcinomas nos est?gios III/ IV foram classificados em alto grau de malignidade (p =0, 024). A BMP-2 apresentou intensidade da imunomarca??o forte em 82,5%, BMPR-IA observou-se 55% dos casos com intensidade de imunomarca??o ausente/ fraca e a BMPR-II revelou 85% dos casos com intensidade de imunomarca??o ausente/ fraca. Apenas a prote?na BMPR-IA apresentou associa??o estatisticamente significante com todos os par?metros clinico-patol?gicos estudados, met?stase (p<0,001), TNM (p<0,001) e grada??o histol?gica de malignidade com ( p=0,028). Quanto ? porcentagem de c?lulas positivas, todos os marcadores apresentaram o maior n?mero de casos com mais de 75% das c?lulas positivas (escore 3) e apenas a BMPR-II apresentou diferen?a estat?stica quando relacionada com a presen?a e aus?ncia de met?stase (p=0,049). Conclui-se que existe dist?rbio na via de sinaliza??o BMP-mediada no CE de l?bio inferior e que a alta express?o da BMP-2 associada com a express?o da BMPR-IA e BMPR-II est?o relacionadas com a met?stase neste carcinoma

Made available in DSpace on 2014-12-17T15:32:22Z (GMT). No. of bitstreams: 1 KaruzaMAP.pdf: 544780 bytes, checksum: e0fadeed7d2d1ec7568970935306d05c (MD5) Previous issue date: 2006-02-22 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The Human Papillomavirus (HPV) has been strongly implicated on development of some cases of oral squamous cell carcinoma (OSCC). However, the immunological system somehow reacts against the presence of this virus. Among the cells involved on such mechanism of defense detaches the Langerhans cells (LC), which are responsible for processing and presenting antigens. The purpose of this study was to evaluate the immunohistochemical reactivity for Langerhans cells between HPV positive and HPV negative OSCC, as well as, the relation of the immunoreactivity for this cells and the histological grading of malignancy proposed by Bryne (1998) and modified by Miranda (2002). Additionally, HPV infection was evaluated in relation to sex, age, lesion localization and histological grading of malignancy. In the total, 27 cases of OSSC were evaluated, 09 of them HPV positive and 18 HPV negative. Anti S-100 antibody was utilized for the immunohistochemical labelling, followed by the counting of LCs in 5 highpower fields (400x). No statistically significant difference was verified between the variables sex, age, lesion localization, histological grading of malignancy and HPV presence in OSSC. There was neither association between the immunohistochemical labeling for LCs (S-100+) and HPV infection nor correlation between the quantity of LCs labeled and the histological grading of malignancy of OSSC. The results suggest that despite the absence of statistically significant difference, the presence of HPV in such cases of OSCC can alter the immunological system, particularly the Langerhans cells / O Papilomav?rus Humano (HPV) tem sido implicado fortemente no desenvolvimento de alguns carcinomas epiderm?ides orais (CEOs). Contudo, o sistema imunol?gico reage de alguma forma ? presen?a desse v?rus. Dentre as c?lulas envolvidas nesse mecanismo de defesa, destaca-se a c?lula de Langerhans (CL), por serem c?lulas processadoras e apresentadoras de ant?genos. O objetivo desse estudo foi avaliar a marca??o imuno-histoqu?mica das c?lulas de Langerhans entre os casos de CEOs HPV positivos e negativos, bem como a rela??o da imunomarca??o dessas c?lulas e a grada??o histol?gica de malignidade proposta por Bryne (1998) e modificada por Miranda (2002). Adicionalmente, a infec??o pelo HPV foi estudada com rela??o ao sexo, idade, localiza??o da les?o e a grada??o histol?gica de malignidade. Foram analisados 27 casos de CEOs, sendo 09 destes HPV positivos e 18 casos negativos. Para a marca??o imuno-histoqu?mica utilizou-se o anticorpo anti S-100, sendo as CLs quantificadas em 5 campos de maior aumento (400x). A an?lise estat?stica revelou n?o existir rela??o das vari?veis, sexo, idade, localiza??o da les?o e grada??o histol?gica, com a presen?a do HPV nos CEOs estudados. N?o existiu associa??o entre a marca??o imuno-histoqu?mica das CLs(S-100+) e a infec??o pelo HPV, e tamb?m n?o houve correla??o entre as CLs imunomarcadas e a grada??o histol?gica nos casos de CEOs analisados. Diante desses resultados, pode-se sugerir que mesmo n?o havendo diferen?a significativa, a presen?a do HPV nos casos de carcinoma epiderm?ide oral pode alterar o sistema imune, particularmente as c?lulas de Langerhans

Made available in DSpace on 2014-12-17T15:32:24Z (GMT). No. of bitstreams: 1 JoaoAVGF.pdf: 1413564 bytes, checksum: f5d8ea2da8907cd551169a4091430007 (MD5) Previous issue date: 2006-02-17 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Squamous cell carcinoma is the most common malignant neoplasm in the oral cavity, accounting for more than 90% of all malignancies in this location. Cyclooxygenases (COX s) are key enzymes on arachidonic acid metabolism and prostaglandin synthesis, being expressed basically in two forms: the constitutive (COX-1) and the inducible (COX-2). Increased levels on the expression of COX-2 have been implicated in the pathogenesis tumor progression of various forms of human cancer, including oral squamous cell carcinoma, some of what suggesting a possible interaction between COX-2 and the protein expressed by the tumor suppressor gene p53, mutated in more than 50% of all human cancers. The mean of the present research consisted in analyze the correlation between the expression of COX-2 and p53, at the protein level, as well as evaluate the difference on the expression of these two proteins with the histological grading of malignancy. 34 cases of oral squamous cell carcinoma were selected and graded according to the histological grading system proposed by Bryne (1998) and the labeling indexes (LI s) for COX-2 and p53 evaluated using immunohistochemistry method. The results revealed that COX-2 was expressed in increased levels in most of the specimens, although there was no statistic significant correlation between LI s from COX-2 and p53 (p>0.05), and there were no statistical differences on the expression of these proteins between tumors of high and low grade of malignancy (p>0.05). Interestingly, the expression of COX-2 and p53 was detected in fragments of dysplastic oral epithelium adjacent to tumor areas, on basal and suprabasal layers. The absence of statistical correlation between the expression of COX-2 and p53 proteins do not rule ot the existence of a relation between them, were it may reflect the diversity of regulatory pathways between both, different direct and indirect inhibitory effects of COX-2 over p53, as well as the wide range of activation macheenisms for COX-2 and mutational status of the p53 gene Another conclusion point that the increased expression of COX-2 observed in oral squamous cell carcinomas suggest a role for this protein in the processes of pathogenesis and tumoral evolution of this malignant neoplasm / O carcinoma epiderm?ide ? a neoplasia maligna mais comum na cavidade oral, representando mais de 90% das malignidades nesta localiza??o. As cicloxigenases (COX s) s?o enzimas chave no metabolismo do ?cido aracd?nico e s?ntese de prostaglandinas, sendo expressas basicamente sob duas formas: uma constitutiva (COX-1) e uma induzida (COX-2). N?veis elevados na express?o da COX-2 t?m sido implicados na patog?nese e progress?o tumoral em diversos tipos de c?ncer em humanos, incluindo o carcinoma epiderm?ide oral, alguns dos quais sugerindo uma poss?vel intera??o entre a COX-2 e a prote?na expressa pelo gene supressor tumoral p53, mutado em mais de 50% de todos c?nceres humanos. O prop?sito da presente pesquisa consistiu em analisar a correla??o entre a express?o de COX-2 e p53, em n?vel de prote?na, bem como avaliar a diferen?a na express?o destas duas prote?nas em rela??o ao grau histol?gico de malignidade. Para tal, foram selecionados 34 casos de carcinoma epiderm?ide oral, os quais foram classificados de acordo com o sistema de grada??o histol?gica de malignidade proposto por Bryne (1998) e cujos ?ndices de positividade para COX-2 e p53 foram avaliados atrav?s da t?cnica imuno-histoqu?mica. O resultados revelaram que a COX-2 esteve expressa em n?veis elevados na maior parte dos esp?cimes analisados, embora n?o se tenha verificado correla??o estatisticamente significativa entre os IP s da COX-2 e da p53 (p>0,05), tampouco diferen?a estatisticamente significativa entre a express?o destas prote?nas entre tumores de alto e baixo grau de malignidade (p>0,05). Interessantemente, foi detectada a express?o da COX-2 e da p53 em fragmentos de epit?lio oral displ?sico, nas camadas basal e parabasal, adjacentes ao tumor. A aus?ncia de correla??o estat?stica entre a express?o das prote?nas COX-2 e p53 n?o descarta a exist?ncia de uma rela??o entre as mesmas, podendo refletir a diversidade de vias regulat?rias entre ambas, os diferentes efeitos inibit?rios diretos e indiretos da COX-2 sobre a p53, bem como os in?meros mecanismos de ativa??o da COX-2 e o estado mutacional do gene p53. Conclui-se ainda que a elevada express?o da COX-2 observada em carcinomas epiderm?ides orais sugere um papel desta prote?na dentro dos processos de patog?nese e evolu??o tumoral desta neoplasia maligna

Made available in DSpace on 2014-12-17T15:32:28Z (GMT). No. of bitstreams: 1 MartaRP.pdf: 1904281 bytes, checksum: 2912c6b8ea9a773c553d0776245f93a5 (MD5) Previous issue date: 2009-02-27 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The Oral Epithelial Dysplasia (OED) is the lesion that precedes or co-exists with the Oral Squamous Cell Carcinoma (OSCC), presenting molecular and/or histological similar alterations. The divergences about the malignization potential of OEDs and the role of inflammation in this process make hard the early diagnosis and evaluation of OSCCs aggressiveness. Thus, it became the goal of this study to evaluate the role of inflammation in oral carcinogenesis and tumoral aggressiveness. For this purpose a morphological study was performed in 20 OED cases and 40 OSCC cases to detect the malignization potential of OEDs and the histologic malignancy grading (HMG) of OSCCs, analyzing superficial masses for dismorphism evaluation and the invasive front for evaluation of tumoral growing; and immunohistochemical, using anti-CD8, anti-FOXP3, anti-TGF?, anti-TNF? and anti-NF-?B antibodies, comparing their with the types lesion, histological degree and intensity of the inflammatory infiltrate. The results were statistically significant for the parameters: cell maturity (p=0,0001), masses presence (p=0,038) and dismorphism (p=0,037), when associated to HMG. To compare the expression of the markers with the types lesion, a significantly higher expression of CD8 (p=0,001) and NF-?B (p=0,002) in the OED, and also a smaller expression of the epithelial TGF? in the severe OEDs (p=0,011), without significant expression between OSCC degrees. By relating the expression of the studied markers with the inflammatory infiltrate intensity, a positive relation was observed with: inflammatory TNF?(p=0,003), epithelial TNF? and NF-?B (p=0,051 and p=0,004), in OEDs; and with CD8 (p=0,021) and TNF? (p=0,015) in conjunctive OSCCs; and a negative relation with epithelial TNF? (p=0,034) in OSCCs. No significant relation was found between FOXP3 with any of the studied variables. These findings lead to the conclusion that, the study of the invasive front is as important as the study of superficial masses for the evaluation of tumoral aggressiveness; the intensity of the inflammatory infiltrate has no use as a parameter for prognostic evaluation of OSCC in routine exams, but, the molecular events detected in this study may be necessary to give basis for determining the malignant potential in OEDs and aggressiveness in OSCCs / A Displasia Epitelial Oral (DEO) ? a les?o que precede ou co-existe com o Carcinoma Epiderm?ide Oral (CEO), apresentando altera??es moleculares e/ou histol?gicas semelhantes. As diverg?ncias sobre o potencial de maligniza??o das DEO e o papel da inflama??o nestes processos t?m dificultado o diagn?stico precoce e a avalia??o da agressividade dos CEO. Sendo assim, tornou-se objetivo deste estudo avaliar o papel da inflama??o na carcinog?nese oral e agressividade tumoral. Para isso foi realizado estudo morfol?gico em 20 casos de DEO e 40 casos de CEO para detectar o potencial de maligniza??o das DEO e o Grau Histol?gico de Malignidade (GHM) dos CEO, analisando as massas superficiais para avalia??o do dismorfismo e o front invasivo para avalia??o do crescimento tumoral; e imuno-histoqu?mico, utilizando os anticorpos anti-CD8, anti-FOXP3, anti-TGF?, anti-TNF-? e anti-NF-?B, para comparar a express?o dos mesmos com o tipo de les?o, grau histol?gico e intensidade do infiltrado inflamat?rio. Os resultados foram estatisticamente significantes para os par?metros, maturidade celular (p=0,0001), presen?a de massas (p=0,038) e dismorfismo (p=0,037), quando associados aos GHM. Ao comparar a express?o dos marcadores com o tipo de les?o, encontrou-se uma express?o significativamente maior do CD8 (p=0,001) e do NF-?B (p=0,002) nas DEO, assim como uma menor express?o do TGF? epitelial nas DEO severas (p=0,011), n?o tendo express?o significativa entre os graus dos CEO. Ao relacionar a express?o dos marcadores estudados com a intensidade do infiltrado inflamat?rio, observou-se uma rela??o positiva com o TNF? inflamat?rio (p=0,003), o TNF? e o NF-?B epiteliais (p=0,051 e p=0,004), nas DEO; com o CD8 (p=0,021) e o TNF? (p=0,015) no conjuntivo dos CEO; e uma rela??o negativa com o TNF? (p=0,034) epitelial dos CEO. N?o foi encontrada rela??o significativa da FOXP3 com nenhuma das vari?veis estudadas. Esses achados levaram a concluir que, o estudo do front invasivo ? t?o importante quanto o estudo das massas superficiais para avalia??o da agressividade tumoral; a intensidade do infiltrado inflamat?rio n?o pode ser utilizado como par?metro para avalia??o progn?stica do CEO no exame de rotina; mas os eventos moleculares detectados neste estudo podem ser necess?rios para embasar a determina??o do potencial de malignidade nas DEO e da agressividade nos CEO

Dans le domaine des biomatériaux, cette thèse s’intéresse à l’influence de l’élasticité du substrat sur la division et la plasticité de la chromatine de cellules épithéliales. La létalité des cellules est corrélée aux faibles rigidités des substrats. Cependant, quelques cellules tumorales SW480, incluant celles portant des anomalies de ségrégation des chromosomes, progressent en mitose. Ces anomalies seraient à l’origine de réarrangements chromosomiques, sources de nombreuses mutations. Les substrats mous conduisent à la formation d’hétérochromatine tandis que les substrats très mous induisent la nécrose des cellules PtK2. Sur ces substrats, l’euchromatine est maintenue après inhibition de HDAC, permettant aux cellules de résister à la nécrose, indépendamment de la compétence transcriptionnelle du noyau. Ces cellules s’étalent à nouveau après transfert sur un substrat rigide. Ces résultats suggèrent 1) une voie de signalisation entrante initiée par le substrat conduisant à la nécrose via la formation d’hétérochromatine 2) une voie de signalisation sortante initiée par l’euchromatine permettant la survie cellulaire. / In the biomaterials field, this PhD work is about influence of substrate elasticity on cell division and chromatin plasticity of epithelial cells. Soft substrates cause massive death.However, some SW480 tumor cells, including those bearing chromosomal segregation abnormalities progress in mitosis. These abnormalities could result in more chromosomal rearrangements, increasing mutations. Soft substrates lead to heterochromatin remodelling and very soft substrates promote necrosis of PtK2 cells. On these substrates, euchromatin could be maintained after HDAC inhibition independently of the nuclear transcriptional competence.These cells spread again after tranfer on stiff substrates. These results suggest i) outside-insignalling cascade initiated at the soft substrate surface leading to heterochromatin remodelling and ultimately necrosis, ii) inside-out signaling cascade initiated from euchromatin allowing cell to overcome necrosis on soft substrate.

O carcinoma de célula renal (RCC) subtipo célula clara é o câncer mais letal e prevalente do sistema urinário. O diagnóstico deste tipo de câncer frequentemente é tardio em conseqüência da falta de sintomas perceptíveis aos pacientes. Um dos objetivos deste trabalho é a identificação de novos marcadores moleculares para diagnóstico precoce, o que ajudaria a diminuir a mortalidade em função de complicações resultantes do avanço da doença. Outro objetivo é a identificação de um conjunto de marcadores moleculares de prognóstico, de modo à prever com acurácia a evolução clínica da doença e, por conseqüência, o tempo de sobrevida do paciente. As modificações transcricionais associadas à carcinogênese e à progressão do câncer de rim ainda não foram completamente elucidadas. Além dos oncogenes e genes supressores de tumor, RNAs não-codificadores (ncRNAs) recentemente foram apontados como importantes reguladores da expressão gênica em humanos, e podem ter um papel importante na transformação maligna do câncer de rim. Para analisar a expressão gênica de ncRNAs e de genes codificadores para proteína foram utilizados dois microarranjos desenvolvidos por nosso grupo, enriquecidos em sondas para ncRNAs. Uma das plataformas possui 4 mil sondas de cDNA, das quais 822 sondas são para ncRNAs mapeando em regiões intrônicas. Outra possui 44 mil elementos e combina sondas de oligonucleotídeos (60-mer) intrônicas e exônicas de um mesmo locus genômico. Análises estatísticas foram feitas com a ferramenta Significance Analysis of Microarrays (q &#8804; 0,05) combinadas ou com a técnica de \"patient leave-one-out\" (genes com presença em 8 100% dos subconjuntos), ou alternativamente com o teste discriminante de Golub (p &#8804; 0,01 ou p < 0,05). Com a plataforma de 4 mil sondas foram estudadas 30 amostras de tecido renal de 18 pacientes com RCC subtipo célula clara. Um conjunto de 36 ncRNAs foi identificado como diferencialmente expresso entre amostras tumorais e não-tumorais. Uma assinatura adicional de 265 genes codificadores de proteínas foi identificada, indicando possíveis novos marcadores moleculares. Uma análise estatística supervisionada com dados de 16 pacientes identificou uma assinatura de ncRNAs correlacionada com sobrevida de 5 anos, formada por 27 ncRNAs com significativa expressão alterada em pacientes livres da doença em comparação com pacientes que morreram em função da doença. Uma assinatura adicional de 64 genes codificadores de proteínas também foi identificada como significativamente correlacionada com o acompanhamento clínico dos pacientes. Com a plataforma de 44 mil sondas foram analisados 17 pacientes, com amostras pareadas de tecido renal tumoral e não-tumoral agrupadas em 8 pools, sendo 4 de amostras tumorais e 4 de não-tumorais. Um conjunto de 66 ncRNAs parcialmente intrônicos antisenso e outro de 52 ncRNAs totalmente intrônicos antisenso foram identificados como diferencialmente expressos. Identificamos um subconjunto de 28 ncRNAs totalmente intrônicos antisenso e senso cuja expressão do gene codificador de proteína do mesmo locus estava simultaneamente alterada. Estes dados apontam para possíveis redes de regulação da expressão gênica dos ncRNAs em câncer. A extensa lista de ncRNAs e de genes codificadores para proteína identificados neste estudo podem ser promissores marcadores moleculares de carcinoma renal subtipo célula clara. / Renal cell carcinoma (RCC) is the most common malignancy of the adult kidney, and the clear cell subtype is the most prevalent and lethal cancer of the urinary system. Late diagnosis for this type of cancer is frequent, usually as a consequence of the lack of symptoms. One of the objectives of the present work is the identification of new molecular markers for the early diagnosis, which would help decrease mortality that develops as a function of disease progression. Another objective is the identification of a set of prognosis molecular markers, so as to accurately predict the clinical outcome of the disease, and consequently, patient survival. Transcriptional changes associated to carcinogenesis and to kidney cancer progression have not been entirely elucidated. Besides oncogenes and tumor suppressor genes, non-coding RNAs (ncRNAs) have been recently indicated as important regulators of gene expression in humans, and could have an important role in the malignant transformation in renal cancer. In order to measure ncRNA and protein-coding gene expression we have used two microarray platforms developed by our group, which are enriched in ncRNA probes. One of the platforms has 4 thousand cDNA probes, of which 822 are for ncRNAs that map to intronic regions. Another has 44 thousand elements and combines 60-mer oligonucleotide probes for intronic and exonic regions from the same genomic locus. Statistical analyses have been performed with the Significance Analysis of Microarrays tool (q &#8804; 0.05) combined with a patient leave-one-out approach (genes present in 100% of the sub-sets), or alternatively with Golubs discriminant test (p &#8804; 0.01 or p < 0.05). 11 With the 4-thousand probes platform we studied 30 samples from renal tissue of 18 RCC patients with clear cell subtype. A set of 36 ncRNAs has been identified as differentially expressed between tumor and non-tumor tissue. An additional signature of 265 protein-coding genes has been identified, indicating possible new molecular markers. A supervised statistical analysis with data from 16 patients has identified a ncRNA signature correlated to 5-year survival outcome, comprised of 27 ncRNAs with significantly altered expression in diseasefree patients compared to patients who died from cancer within the 5-year follow-up. An additional 64-gene signature of protein-coding genes has been identified as significantly correlated to clinical outcome. With the 44-thousand probes platform we have analyzed 17 patients, with paired tumor and non-tumor samples grouped into 8 pools, of which 4 were from tumor and 4 from nontumor samples. A set of 66 partially intronic antisense ncRNAs and another of 52 totally intronic antisense ncRNAs have been identified as differentially expressed between tumor and non-tumor tissue. A sub-set of 28 totally intronic antisense or sense ncRNAs were identified as having a simultaneous change in expression of the protein-coding gene from the same locus. Overall, the data point to a possible ncRNA regulatory network in cancer. The extensive lists of ncRNAs and of protein-coding genes identified in the present study can be seen as promising molecular markers of RCC from the clear-cell subtype.

Effective prevention, or treatment, of invasive fungal infection in the neutropenic patient has hitherto been unsatisfactory because of either an inadequate anti-fungal spectrum of the agent or important toxicity. Itraconazole is effective against a broad spectrum of the opportunistic pathogens seen in Europe and North America. Prior problems with absorption, e.g. in the marrow transplant recipient, have been overcome with the introduction of an oral solution and an i.v. preparation. The deliberations of an expert meeting held in June, 1998 include recommendations on which patient requires one of these new preparations based on clinical trials, the dose and route. Important drug interactions are also detailed. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.