PURPOSE: To determine the accuracy of prostate biopsy Gleason score (GS) compared to prostatectomy GS. To determine whether a biopsy is a satisfactory diagnostic procedure to offer active surveillance for patients with low-grade prostate cancer. METHODS: This study was conducted in Tuft Medical Center as retrospective cohort study over the period from 2007-2010. The study included 83 patients for whom biopsy and prostatectomy GS were available. MEASUREMENTS: Gleason scores of 6, 7, and 8-10 were assigned to low, moderate, and high-grades, respectively. The kappa statistic was calculated to assess the degree of agreement between biopsy and prostatectomy. The ROC curve was used to evaluate the sensitivity and specificity of prostate biopsy for different Gleason grades. Also, compared whether the use of specific criteria for active surveillance (Johns Hopkins and UCSF) may decrease the level of up-grading in patient with low-grade prostate cancer using Chi-square test. RESULTS: The distribution of low, moderate, and high-grade cancer in biopsy (52%, 32%, 16%) and prostatectomy specimen (33%, 55%, 12%) showed fair agreement with weighted kappa 0.35. The prostate biopsy accurately predicted GS in 46%, up-graded in 38%, and down-graded in 16%. The patients with low-grade cancer and potentially eligible for active surveillance showed up-grading in 50% of cases. This up-grading reduced to 40% with the use of Johns Hopkins criteria and to 41% with the use of UCSF criteria. CONCLUSIONS: The accuracy of biopsy GS in predicting prostatectomy GS is severely limited and therefore biopsy is not enough diagnostic procedure to offer active surveillance.

PURPOSE: This study was undertaken to compare HALO™ software image analysis measurements of cellularity with visual estimations from the pathologist and to outline a protocol for future experimental determinations of cellularity using HALO™. Secondly, this study investigated the clinically challenging prostate cancers of Gleason score 7 by analyzing a large database of radical prostatectomy (RP) specimens with regard to their Gleason grade composition and percentage tumor volume composition. The importance of these values of tumor cellularity, prostate volume, and tumor volume data were discussed in terms of future diagnostic endeavors. Finally, this study provided a brief background on prostate cancer, prostate cancer epidemiology, digital pathology, and the limitations and difficulties in the technological transition to digital pathology. All work for this study was done at Dana-Farber Cancer Institute (Boston, MA). METHODS: In the first part of this study, histological slides were acquired by radical prostatectomy (RP) and contained 12 tumor foci of varying degrees and sizes. These slides were scanned and imported into the HALO™ image analysis software. The tumor foci, previously demarcated by a pathologist, were annotated by hand in HALO™. An algorithm for image analysis was created by training classifiers to recognize and differentiate between epithelial tissue, stromal tissue, glass, and other. This process was accomplished by classifying 62 regions which were tested for accuracy before becoming the components of an algorithm to analyze the entire annotation layer. Each tumor focus was analyzed individually, and the results were exported into Microsoft® Excel from which relevant data were extracted. Cellularity was calculated by the percentage of tumor area that the algorithm characterized as epithelial. Cellularity values derived from HALO™ measurements for each tumor focus were compared with the visual estimations of cellularity provided by the pathologist using Pearson's correlation analysis. In the second part of this study, a database of 1386 slides containing tumors with Gleason scores between 6 and 9 was compiled from 140 RP cases. The average percentages of Gleason grades 3, 4, and 5 in each case were determined. The percentage of each slide that was occupied by the tumor was also averaged for each case, yielding an average percentage of tumor volume for each case. The average Gleason grade 3, 4, or 5 percentage for each case was plotted against the associated average tumor volume percentage of that case. The cases of Gleason score 7 (3+4, 4+3) were then isolated and plotted in a similar manner. Pearson’s correlation analysis was used to determine the degree of linear correlation between the two variables in each plot. Results: In the first part of this study, a statistically significant positive correlation between the cellularity estimations of the pathologist and the HALO™ cellularity measurements was found (r = 0.92, p < 0.01, n =12). In the second part of this study, there was a statistically significant negative correlation between average Gleason grade 3 percentage per case and average tumor volume percentage per case (r = -0.55, p <0.001, n = 140). There was also a statistically significant positive correlation between average Gleason grade 4 percentage per case and average tumor volume percentage per case (r = 0.55, p <0.001, n = 140). After slides containing Gleason score 6 (3+3) tumor were removed from the data, a statistically significant negative correlation remained between average Gleason grade 3 percentage per case and average tumor volume percentage per case (r = -0.51, p <0.001, n = 78), and a statistically significant positive correlation remained between average Gleason grade 4 percentage per case and average tumor volume percentage per case (r = 0.5, p <0.001, n = 101). A statistically significant relationship between average Gleason grade 5 percentage and average tumor volume percentage was not found (r = 0.32, p = 0.14, n = 23). CONCLUSIONS: In the first part of this study, the strong positive correlation between HALO™ cellularity values and visual estimations by the pathologist suggests that image analysis may be an effective tool for determining cellularity in digital histological images. More research using larger sample sizes is recommended to further validate the correlation between algorithm-derived cellularity from HALO™ and visual estimation by the pathologist. In the second part of this study, it appears that the volume of prostate tumors of Gleason score 7 may have prognostic power, considering that an increased percentage composition of Gleason grade 4 correlated with larger tumor volumes. Because this result may have significant clinical implications, further research specifically on tumors of Gleason score 7 is suggested to verify this relationship.

Prostate cancer micrometastasis allows cancer cells to vacate their original tumor sites and migrate to distant parts of the body via the bloodstream, lymphatic system, or by direct extension. Cells synthesize and secrete matrix metalloproteinases (MMPs) that degrade proteins of the surrounding extracellular matrix (ECM); thus allowing them to escape into the lymphatic or circulatory systems to invade other tissues. Transforming growth factor β (TGF-β) induces the migration and invasion of cancer cells and the expression of matrix metalloproteinases (MMPs), specifically MMP-2, and -9 in several malignancies. In this study, we examined the role of MMP-7, a known activator of MMP-2 and MMP-9, in TGF-β signaling in cell proliferation, migration, and invasion in prostate cancer cells. Basal expression levels of MMP7 mRNA, protein, and secreted protein were determined using RT-PCR, western blot analysis, and ELISA, respectively. Our data show that MMP7 mRNA and proteins were differentially expressed in several cell line models representing different stages of prostate cancer. TGF-β1 induces MMP-7 gene expression and protein levels 24 and 48 hours after treatment in PC3 cells. Our data also show that TGF-β induces cell migration and invasion in PC3 and E006AA cells; however, the selective knockdown of MMP7 expression using siRNA resulted in a significant decrease in control and TGFβ-induced cell migration and invasion in both PC3 and E006AA cells. MMP-7 knockdown also caused significant reduction in cell proliferation in PC3 cells. Our data suggest that MMP7 is essential for cell migration and invasion in prostate cancer cells indicating that it may be required for TGFβ-induced cancer metastases.

Orientador: Marlus Chorilli / Resumo: O câncer de próstata (CP) é a segunda neoplasia mais frequente entre homens no Brasil e é caracterizado por não apresentar sintomas em seus estágios iniciais, sendo diagnosticado em seu estágio avançado, o que muitas vezes dificulta o tratamento. Alguns fatores relacionados podem intensificar sua agressividade como, por exemplo, a superexpressão do receptor do fator de crescimento epidérmico (EGFR) em alguns subtipos de tumores de próstata. Neste contexto, a inibição do EGFR auxilia no combate da neoplasia, função essa que pode ser atribuída ao anticorpo monoclonal quimérico IgG1 (cetuximabe-CTX) que se liga à porção externa do EGFR, inibindo a proliferação celular, angiogênese e metástase, além de promover a apoptose. Dentre as formas de tratamento destacam-se a braquiterapia, a radioterapia e a quimioterapia utilizando o docetaxel (DTX), o qual apresenta vantagem de prolongar a sobrevivência em pacientes com CP metastático resistentes à terapia antiandrogênica. No entanto, a formulação comercial (Taxotere®) causa efeitos colaterais, como febre, anemia, retenção de líquidos, hipersensibilidades, mialgias, mucosite, neuropatias periféricas e toxidade a pele e unhas, tornando necessário o estudo de novas formas de veiculação para este fármaco Deste modo, o objetivo deste trabalho foi desenvolver uma nanodispersão de cristal líquido (NCL) de fase cúbica baseada em álcool cetílico etoxilado 20 e propoxilado 5 como tensoativo (T), ácido oleico, DSPE-PEG-MAL e fosfatidilcolina de ... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Introdução e objetivos: a punção-biópsia de próstata é a principal forma de diagnóstico do câncer de próstata. A introdução da dosagem sérica do PSA e os programas de rastreamento do câncer de próstata levaram à detecção de neoplasias subclínicas e de pequenas dimensões. A utilização da revisão cega de cortes histológicos no controle de qualidade dos exames permite a reavaliação, fornece a variabilidade interobservadores e possibilita a detecção de exames discrepantes, falsos negativos e positivos. Na próstata tal prática é justificável pela freqüência de diagnósticos de incerteza e de neoplasias de pequenas dimensões, que tornam a interpretação do exame mais difícil e subjetiva. O objetivo deste estudo é avaliar a variabilidade interobservador em biópsias de próstata. Material e métodos: dois patologistas revisaram separadamente 489 exames de biópsias de próstata. Os patologistas desconheciam o diagnóstico original e informações clínicas como valor do PSA, idade e toque retal,obtidos dos prontuários clínicos. A variabilidade interobservador foi quantificada através do índice Kappa. Resultados: foram revisadas biópsias provenientes de 191 pacientes. A média de idade foi de 65,5 anos e a média do PSA de 14,26 ng/ml (mediana=8,4 ng/ml). A concordância geral entre o diagnóstico original e o final (pós-revisão) foi de 94,3% e o índice kappa foi de 0,86 (com intervalo de confiança de 95% entre 0,8 e 0,91). O índice kappa entre os revisores foi de 0,83. A concordância entre os grupos de diagnóstico foi de 94,6% (kappa 0,85) para os exames benignos; 100% (k 0,99) nos adenocarcinomas; 76,5% (k 0,67) nas ASAPs – proliferações de pequenos ácinos atípicos e 50% (k 0,3) nas NIPs – neoplasias intraepiteliais prostáticas de alto grau. Conclusões: Este estudo mostrou a alta concordância interobservador nos diagnósticos benignos e adenocarcinomas. As maiores variações ocorreram nas lesões pré-malignas (NIP) e nos diagnósticos duvidosos, expressas por uma menor concordância interobservador e baixo índice kappa. A revisão cega de biópsias de próstata pode ser útil e deve ser recomendada nos caso duvidosos, onde há uma forte suspeita de neoplasia, uma vez que o diagnóstico de neoplasias pequenas e lesões pré-neoplásicas é de difícil interpretação e pode estar relacionada a uma maior variabilidade interobservador. / Introduction: prostate needle biopsy is the standard procedure for the diagnosis of prostate cancer. The introduction of PSA screening and other screening methods has allowed for the detection of subclinical and minimal carcinomas of the prostate. The blind review of histological sections for the quality control of exams permits reassessment, provides interobserver variability and allows the detection of discrepant exam results, false negatives and false positives. This practice is justified in case of the prostate, due to the frequently uncertain diagnoses and due to the presence of focal carcinoma, which render the interpretation of exam results more difficult and less objective. The aim of this study is to assess the interobserver variability in prostate biopsies carried out at a teaching hospital. Materials and Methods: two pathologists individually reviewed 489 prostate biopsies. Both pathologists were blinded to the original diagnosis and to clinical data such as PSA levels, age, and digital rectal examination status. Interobserver variability was measured using the kappa coefficient. Results: a total of 489 biopsies of 191 patients were reviewed. Mean patient age was 65.5 years, and PSA levels averaged 14.26 ng/ml (median:8,4ng/ml). Overall agreement between the original and final diagnoses was 94.3% with a kappa of 0.86 (95%CI between 0.8 and 0.91). The interobserver kappa value was 0.83. Agreement among diagnostic groups was 94.6% (kappa = 0.85) for benign exams; 100% (kappa = 0.99) for adenocarcinomas; 76.5% (kappa = 0.67) for atypical small acinar proliferations (ASAP) and 50% (kappa = 0.3) for high-grade prostatic intraepithelial neoplasias (HGPIN). Conclusions: The blind review of prostate biopsies can be useful and is recommended in uncertain cases highly suspicious of malignancy, since the diagnosis of minimal carcinoma and preneoplastic lesions is difficult and is related to a larger interobserver variability.

BEZERRA, Leonardo Primo. Glicoproteínas séricas ligantes da lectina de dioclea altíssima no estudo de doenças prostáticas. 2014. 100 f. Dissertação (Mestrado em Bioquímica) - Universidade Federal do Ceará, Fortaleza-CE, 2014. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-19T14:42:07Z No. of bitstreams: 1 2014_dis_lpbezerra.pdf: 1859149 bytes, checksum: 2c776f3983043f6b85d171f25f35a1d3 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:29:36Z (GMT) No. of bitstreams: 1 2014_dis_lpbezerra.pdf: 1859149 bytes, checksum: 2c776f3983043f6b85d171f25f35a1d3 (MD5) / Made available in DSpace on 2016-08-02T20:29:36Z (GMT). No. of bitstreams: 1 2014_dis_lpbezerra.pdf: 1859149 bytes, checksum: 2c776f3983043f6b85d171f25f35a1d3 (MD5) Previous issue date: 2014 / Nowadays there are an increase number of studies based on the change of glycoproteomics profiles in several diseases, especially in the search for serum biomarkers for cancer. The direct identification and quantification of serum glycoprotein of low abundance are difficult, because proteins very abundance in the blood can difficult the identification. Thus, fractionation tools are necessary to isolate efficiently changed protein glycoforms on the blood proteome. Given the specific and reversible interaction of lectins to glycoconjugates, the use of immobilized lectins on chromatographic matrix has been frequent in order to fractionate complex mixtures, involving glycoprotein for analysis for mass spectrometry. In this context, the goal of this work was to investigate the use of binding glucose/ mannose lectin from Dioclea altíssima seed (DaL) immobilized on Sepharose chromatography matrix 4B® (DaL-Sepharose), on the fractionation of serum glycoproteins and research of potential biomarkers for prostate cancer (PC). Glycoproteins from the retained fraction obtained by chromatography of blood serum on DaL-Sepharose matrix were identified and quantified by mass spectrometry and it was obtained the protein profile to the three groups: control, benign prostatic hyperplasia and PC. The identification of differentially expressed proteins between the groups showed 132 glycoproteins, in which 29 were only identified on the group with prostate cancer or showed one reason of ln greater than 1.2 when compared to quantification on control group. After an analysis, considering the confiability of the identification, estimated by score, and the evidences on the literature that justified a possible involvement of the protein on prostate cancer, stood out: alpha-1-acid glycoprotein, thrombospondin-5 complement C4 A, haptoglobin, pregnancy zone protein, isoform 3 of alpha 1-antitrypsin, alpha-2 glycoprotein rich in leucine and Zinc finger protein. The analysis of fractionated bood serum by DaL-Sepharose matrix with prior depletion of Human Serum Albumin and IgG allowed the identification of alpha 1-antitrypsin and the IGK protein “only” on the group with CaP and the pregnancy zone protein and protein like alfa-1 mieloma up regulated on CaP when compared to the control group. The identification of these glycoproteins generates new perspectives and suit as indicative for guiding research and validation methods development of these results for clinical uses. / Atualmente é crescente o número de estudos com base na alteração de perfis glicoproteômicos em diversas doenças, principalmente na busca de biomarcadores séricos para o câncer. A identificação e quantificação direta, de glicoproteínas sorológicas pouco abundantes, são difíceis, pois proteínas muito abundantes no sangue podem dificultar a identificação. Assim, ferramentas de fracionamento são necessárias para isolar, eficientemente, glicoformas proteicas aberrantes no proteoma sanguíneo. Tendo em vista a interação específica e reversível de lectinas com glicoconjugados, o uso de lectinas imobilizadas em matriz cromatográfica tem sido frequente, visando fracionar misturas complexas, envolvendo glicoproteínas, para posterior análise por espectrometria de massas. Mediante este contexto, o objetivo do presente trabalho foi investigar o uso da lectina glucose/ manose ligante de sementes de Dioclea altíssima (DaL) imobilizada em matriz cromatográfica Sepharose 4B® (DaL-Sepharose), no fracionamento de glicoproteínas séricas e na pesquisa de potenciais biomarcadores para o câncer de próstata (CaP). As glicoproteínas da fração retida, obtidas pela cromatografia do soro sanguíneo na matriz de DaL-Sepharose, foram identificadas e quantificadas, por espectrometria de massas, e assim, foi obtido o perfil proteico para os três grupos estudados: controle, hiperplasia prostática benigna e CaP. A identificação das proteínas diferentemente expressas entre os grupos revelou 132 glicoproteínas, destas, 29 foram unicamente identificadas no grupo com câncer de próstata ou apresentaram uma razão de ln maior que 1,2, quando comparado à quantificação no grupo controle. Após uma análise considerando a confiabilidade da identificação, estimada pelo escore, e as evidencias na literatura que justifiquem um possível envolvimento da proteína no câncer de próstata, destacaram-se: alfa-1-glicoproteína ácida, trombospondin-5, complemento C4 A, heptaglobina, pregnancy zone protein, isoforma 3 de alfa-1-antitripsina, alfa-2-glicoproteína rica em leucina e Zinc finger protein. A análise do soro sanguíneo fracionado pela matriz DaL-Sepharose, com prévia depleção de Albumina sérica humana e IgG, permitiu a identificação de alfa-1-antitripsina e a proteína IGK “únicas” no grupo com CaP e a pregnancy zone protein e a proteína like alfa 1 mieloma up reguladas no CaP quando comparadas ao grupo controle. A identificação destas glicoproteínas gera novas perspectivas e servem de indicativo para nortear a pesquisa e desenvolvimento de métodos de validação destes resultados para usos clínicos.

A Interleucina 8 (IL-8) é uma quimiocina CXC angiogênica que tem papel importante no desenvolvimento e progressão de vários tumores malignos, incluindo o câncer de próstata (CaP). O polimorfismo de nucleotídeo único (SNP) -251 T/A da região promotora do gene da IL-8, relativo ao local de início da transcrição deste gene, está associado com a produção desta citocina. O efeito da IL-8 é mediado através de dois receptores de alta afinidade, CXCR1 e CXCR2. O presente estudo investigou a influência da variação dos genes IL-8 e CXCR2 na susceptibilidade e nas características clinicopatológicas do CaP em um grupo de brasileiros. Duzentos e um pacientes e 185 controles saudáveis foram selecionados neste estudo casocontrole. Amostras de sangue foram coletadas para extração de DNA; a tipagem da IL-8 -251 T/A e CXCR2 +1208 C/T foi realizada através da reação em cadeia da polimerase com sequência específica de primers (PCR-SSP), seguida pela eletroforese em gel de agarose. O risco associado entre os genótipos, a susceptibilidade do CaP e as características do tumor, foi estimado pelo odds ratio (OR), com intervalo de confiança de 95%, usando análise de regressão logística e ajustando para idade ao diagnóstico. Encontramos uma associação estatisticamente significativa entre o genótipo heterozigoto CT do gene CXCR2 +1208 e CaP. Este genótipo foi significativamente menos frequente em pacientes com estádio clínico T3-T4 comparado com T1-T2 (56.7% versus 80.5%). Nossos achados sugerem que os portadores do genótipo CT CXCR2 +1208 tiveram um efeito protetor para estádio avançado de CaP (CT versus CC: OR ajustado = 0.25; P = 0.02). Não foi encontrada associação significativa entre o polimorfismo -251 T/A da IL-8 e os parâmetros clinicopatológicos do CaP. Estes resultados indicam que o genótipo CT do CXCR2 +1208 é menos frequente em estádios avançado de CaP, sugerindo que este receptor de quimiocina tenha um papel na patogênese desta doença. / Interleukin-8 (IL-8) is an angiogenic CXC chemokine that plays an important role in both the development and progression of several human malignancies including prostate cancer (PC). A single nucleotide polymorphism (SNP) at -251 upstream of the transcriptional start site of the IL-8 gene has been shown to influence its production. The effects of IL-8 are mediated by two highly related chemokine receptors, CXCR1 and CXCR2. The present study investigated the influence of the IL-8 and CXCR2 gene variation on susceptibility and clinicopathological characteristics of PC in a group of Brazilian subjects. Two hundred and one patients and 185 healthy controls were enrolled in a case-control study. Blood was collected for DNA extraction; typing of IL-8 -251 T/A and CXCR2 +1208 C/T genes was performed by polymerase chain reaction with sequence-specific primers (PCR-SSP), followed by agarose gel electrophoresis. Risk association between the genotypes, PC susceptibility and tumor characteristics was estimated by odds ratio (OR) and 95% confidence intervals (95% CI) using logistic regression analysis, after adjusting for age at diagnosis. A significant association was found between the heterozygous CXCR2 +1208 CT genotype and PC. The CXCR2 +1208 CT genotype was significantly less frequent in patients with clinical stage T3-T4 compared to T1-T2 (56.7 versus 80.5%). Our findings suggest that carriers of the CXCR2 +1208 CT genotype had a protective effect for advanced PC (CT versus CC: adjusted OR = 0.25; P = 0.02). No association was observed between the SNP for IL-8 -251 T/A and clinicopathological parameters of PC. These results indicated that the CXCR2 +1208 CT genotype is less frequent in advanced stages of PC, suggesting that this chemokine receptor plays a role in the pathogenesis of this disease.

Submitted by Priscila Nascimento (priscila.nascimento@bio.fiocruz.br) on 2016-04-11T17:27:57Z No. of bitstreams: 1 Dissertação de Mestrado em Oncologia - INCa Ana Emília Goulart.pdf: 3332973 bytes, checksum: abe7cedda85b0558a252f168374ab1f4 (MD5) / Approved for entry into archive by Priscila Nascimento (priscila.nascimento@bio.fiocruz.br) on 2016-04-11T17:28:57Z (GMT) No. of bitstreams: 1 Dissertação de Mestrado em Oncologia - INCa Ana Emília Goulart.pdf: 3332973 bytes, checksum: abe7cedda85b0558a252f168374ab1f4 (MD5) / Made available in DSpace on 2016-04-11T17:28:57Z (GMT). No. of bitstreams: 1 Dissertação de Mestrado em Oncologia - INCa Ana Emília Goulart.pdf: 3332973 bytes, checksum: abe7cedda85b0558a252f168374ab1f4 (MD5) / Fundação Oswaldo Cruz. Instituto Tecnológico em Imunobiológicos. Rio de Janeiro, RJ, Brasil. / Introdução: O PCA3 é um RNA não codificante (ncRNA), expresso especificamente na próstata, estando envolvido no controle da sobrevivência de células de carcinoma de próstata (CaP), através da via de sinalização do receptor de androgênio (AR). Objetivo: Investigar se diversos genes relacionados ao câncer – incluindo os envolvidos na Transição Epitelial Mesenquimal (EMT), os que apresentam potencial stemness e os co-reguladores do AR – podem estar envolvidos no processo de resposta ao silenciamento do PCA3. Além disso, objetivamos promover o silenciamento estável do PCA3 através da construção de um vetor lentiviral carreando short hairpin RNA (shRNA) específico para este ncRNA, vislumbrando estratégias terapêuticas para o CaP. Metodologia: Empregamos small interfering RNA (siRNA) ou shRNA com expressão baseada em vetores lentivirais para diminuir a expressão de PCA3 em células LNCaP e avaliar os efeitos deste silenciamento na sobrevivência destas células. Para isto, analisamos por qRT-PCR a expressão do PCA3 e de diversos genes relacionados ao câncer. Utilizamos microscopia confocal para analisar a expressão da proteína vimentina. Empregamos citometria de fluxo para verificar o percentual de células LNCaP GFP+ e, azul de tripan para avaliar o número de células viáveis, após o silenciamento estável do PCA3. Resultados: Dentre os co-reguladores do AR, ARA 70, ARA 54, Smad3 e EBP1 apresentaram expressão aumentada nas células LNCaP interferidas com siPCA3 em relação às células LNCaP interferidas com siScrbl, enquanto Smad 4 e ciclina D1 apresentaram expressão diminuída. Dentre 84 genes relacionados ao câncer, 16 apresentaram expressão alterada em células LNCaP- siPCA3 em relação ao controle. Destes, 30% codificam moléculas de transdução de sinais e fatores de transcrição. Observou-se expressão aumentada de E-caderina, Claudina-3, Citoqueratina-18, Snail, Twist e Slug em células LNCaP -siPCA3 em relação ao controle, enquanto observou-se expressão diminuída de Claudina-4, Citoqueratina-8 e Vimentina. O padrão de marcação de vimentina foi similar em células LNCaP – siPCA3 e células LNCaP – siScrbl. Não foi detectada a expressão de genes com potencial stemness nas condições testadas. Células transduzidas com vetores lentivirais carreando shPCA3 apresentaram diminuição estável da expressão do PCA3. Foi observada redução estável de cerca de 60% de células LNCaP GFP+ transduzidas com shPCA3, além de redução no número de células viáveis. Conclusão: O silenciamento do PCA3 reduz o número de células viáveis, através de um processo que envolve moléculas de transdução de sinais e fatores de transcrição que podem orquestrar a sobrevivência das células de CaP. A diminuição no número de células viáveis após a transfecção com siPCA3 parece não ser modulada pelo programa EMT clássico, embora a reversão parcial deste programa possa estar regulando a diminuição da sobrevivência celular induzida por siPCA3. A desregulação da expressão de coreguladores do AR observada pode estar envolvida na inibição da expressão dos genes alvo do AR. Nossos dados sugerem que este ncRNA desempenha papel regulador na transcrição gênica, sendo capaz de modular a expressão de genes de diversas vias de sinalização relacionadas ao câncer. Nossos resultados sugerem ainda que a redução estável do PCA3 apresenta potencial aplicação em estratégia terapêuticas que visem modular negativamente a sobrevivência das células de CaP. / Introduction: PCA3 is a prostate specific non coding RNA (ncRNA), involved in the control of prostate cancer (PCa) cell survival, through modulating androgen receptor (AR) signaling. Objective: In order to better characterize the molecular mechanisms by which PCA3 is controlling LNCaP cell survival, we aimed to investigate whether several cancer related genes, including those involved in epithelial-mesenchymal transition (EMT), stemness potential and AR co-regulators could be involved in this process in response to PCA3 downregulation. Moreover, we aimed to promote PCA3 stable silencing through a lentiviral vector containing a PCA3 short hairpin RNA (shRNA) specific sequence. Methodology: We used small interfering RNA (siRNA) or lentivirus vectorbased shRNA to downregulate PCA3 expression and evaluate the effects of this ncRNA knockdown on LNCaP cell survival. After PCA3 downregulation, cells were analyzed by qRT-PCR to investigate PCA3 and several cancer related genes expression. Confocal microscopy was performed to analyze vimentin expression. Flow cytometry was performed to analyze the percentage of LNCaP GFP positive cells and trypan blue staining to analyse the number of viable cells after PCA3 stable silencing. Results: Among AR co-regulators genes investigated, ARA70, ARA54, Smad 3 and EBP1 were upregulated in siPCA3- transfected cells in relation to scramble sequence LNCaP transfected cells, while Smad 4 and cyclin D1 were downregulated. We found that among 84 cancer-related genes tested, 16 were differentially expressed in LNCaP siPCA3-transfected cells when compared to transfected cells with a scramble sequence. Of these, 30% are genes coding for signal transduction molecules and transcription factors. Gene expression profile of EMT-related genes revealed that E-cadherin, Claudin-3, Cytokeratin-18, Snail, Twist and Slug are upregulated in LNCaP siPCA3-transfected cells compared to control, while Claudin-4, Cytokeratin-8 and Vimentin are downregulated. Vimentin expression staining patterns were similar between LNCaP siPCA3-transfect cells and control. Expression of stemness markers were not detected in these tested conditions. LNCaP cells transduced with lentiviral vectors carrying the GFP gene and shPCA3 stably dowregulated PCA3 expression, producing a reduction of 60% of LNCaP GFP+ cells compared to shScrbl transduced or non-transduced LNCaP cells. In addition, the number of viable cells was reduced after PCA3 stable silencing. Conclusion: PCA3 downregulation by RNAi leads to a loss of viability, through a process that involves key signal transduction molecules and transcription factors that could orchestrate PCa cells survival. Our results also suggested that the decrease in the number of LNCaP viable cells, observed after siPCA3 transfection, seems to don´t be modulated by the classical EMT program, although a partial reversion of this program could regulate the reduction of cell survival induced by siPCA3. Observed deregulated expression of AR co-regulators could be involved in the inhibition of the AR target genes expression. Our data suggest that that this ncRNA perform a regulatory role in gene expression, being able to modulate gene expression of several signaling pathways related to cancer. Moreover, our results suggest that the stable downregulation of PCA3 expression shows potential as a PCa therapeutic approach by negatively modulation cell survival.

Master of Science / Department of Kinesiology / Carl Ade / Background: Androgen Deprivation Therapy (ADT) is a commonly used treatment for prostate cancer with controversy currently surrounding its association with long-term cardiovascular disease risk. Therefore, the aim of the current investigation was to non-invasively measure left ventricular mechanics at rest and during submaximal exercise in human prostate cancer survivors with and without a history of ADT. Methods: Eighteen prostate cancer survivors, 9 with a history of ADT and 9 matched (1:1) non-ADT controls, completed the protocol. Standard and tissue Doppler echocardiography were used to evaluate left ventricular systolic and diastolic function at rest and during submaximal cycling exercise. Results: At rest, there were no differences between groups. Ejection fraction was not different between groups at rest or during exercise (rest p=0.7; exercise p=0.8). During exercise, systolic left ventricular longitudinal strain and strain rate failed to increase in the ADT group (p=0.4; p=0.07), but significantly increased in the non-ADT group (p=0.03; p=0.02). During exercise, systolic strain was significantly different between groups (p=0.02). Diastolic longitudinal strain increased with exercise in both groups (p=0.003; p=0.003). In the ADT group during exercise, mitral valve deceleration time was not significantly different from rest (p=0.8) and was slower compared to non-ADT (p=0.03). Conclusion: In prostate cancer survivors with a history of ADT, there are significant abnormalities of left ventricular systolic function that become apparent with exercise. These findings may hold significant value beyond the standard resting characterization of ventricular function, in particular as part of a risk-stratification strategy.

L'imagerie est maintenant partie intégrante des traitements de radiothérapie. Avec la technologie CBCT embarquée sur les appareils de traitement, l'imagerie tomographique permet non seulement de repositionner fidèlement le patient tout au long de son traitement mais aussi d'adapter la planification initiale aux modifications quotidiennes de volume. C'est la radiothérapie adaptative, objet des travaux de cette thèse. Nous avons établi les limites techniques de précision de repositionnement des équipements utilisé. Ensuite, à partir des acquisitions CBCT quotidiennes de patients traités pour la prostate, nous avons élaboré une stratégie de traitement basée sur une banque de plans personnalisés. Nous avons mis au point une méthode semi-automatique de sélection du plan de traitement du jour qui a montré une efficacité supérieure à la sélection par des opérateurs expérimentés. Enfin, nous avons quantifié les doses additionnelles à la dose thérapeutique associées à l'utilisation quotidienne de l'imagerie CBCT. En conclusion, on peut dire qu'avec l'imagerie CBCT embarquée, on peut voir ce que l'on veut traiter, irradier ce que l'on a vu et contrôler ce qu'on a traité. / Imaging is now fully integrated in the radiation therapy process. With on-board CBCT systems, tomography imaging allows not only patient positioning but also treatment planning adaptation with patient anatomy modifications, throughout the entire treatment. This is called adaptive radiation therapy, and is the main subject of this PhD thesis. During this work, we measured the repositioning accuracy of the system used. We also developed a treatment strategy using daily CBCT images and a personalized plan database to adapt treatment plan to patient anatomy. We found a way to select the daily treatment plan that shows superiority over operator selection. Then we also quantified the additional dose delivered while using this technique and the impact with regards to the risks added to patients. As a conclusion, with CBCT imaging, radiation therapy has entered an era where one can see what need to be treated, can treat what has been seen and can control what has been treated.