Nanopartículas de ouro (AuNP) compõem o estado da arte da medicina oncológica atual sendo extensivamente investigadas para aplicações em radioterapia de câncer. O acúmulo seletivo em tecidos tumorais de vascularização desordenada causa retenção aumentada das AuNP no microambiente tumoral e aumenta a permeabilidade de drogas terapêuticas por elas carreadas. A radiossensibilização observada em células em cultura tratadas com AuNP é, contudo, resultado da resposta biológica à ambos os fenômenos bioquímicos de interação com a célula alvo e físicos de interação da AuNP com a radiação ionizante. A presença do ouro metálico no citoplasma celular durante a terapia com radiação ionizante aumenta a secção de choque de absorção fotoelétrica no tecido alvo, o que contribui para o aumento potencial do dano biológico letal. Neste contexto, o objetivo principal deste trabalho é avaliar a radiossensibilização causada por nanopartículas de ouro em radioterapia em termos de parâmetros físicos e radiobiológicos. Simulação Monte Carlo com o código PENELOPE foi utilizada para estudar como a densidade de ionização local é alterada pela criação de elétrons secundários em nanopartículas de 2-100nm após interação com os feixes primários de fótons de interesse clínico de 200kV, Ir-192, Cs-137, 6MV e Co-60. O Estudo da Nanopartícula Isolada demonstrou que a densidade de ionização é fortemente aumentada com a redução da energia do feixe primário (E) de ionização, sendo, entretanto, fracamente dependente do diâmetro das AuNP. O diâmetro da AuNP (d) modula, no entanto, o espectro de elétrons secundários ejetados ao tecido-mole disposto ao redor porque modifica o número de elétrons internamente absorvidos em cada nanopartícula. Quando concentrações de ouro (!!\") entre 0,001% e 1%, são consideradas no Estudo da Célula com Nanopartículas, é possível observar que a !!\" internalizada no citoplasma aumenta a fração de reforço da densidade de ionização no tecido alvo em até 200% para baixas energias, não ultrapassando 160% para feixes de megavoltagem. A principal conclusão desta etapa é que o reforço na densidade de ionização local é resultado do balanço entre os parâmetros estudados E e !!\", e o diâmetro da AuNP, modifica o número de fotoelétrons ejetados ao meio, influenciando portanto o potencial efeito biológico. A partir dos resultados do estudo por Monte Carlo, foram desenhados os estudos radiobiológicos in vitro e in vivo com células PC3 de adnocarcionama de próstata. Nanopartículas rod shaped (AuNR) - conjugadas com Goserelin (gAuNR) princípio ativo do quimioterápico Zoladex® foram sintetizadas com sucesso para aplicações nos sistemas biológicos e nanopartículas conjugadas com polietileno glicol (pAuNR) foram utilizadas como controle biocompatível e não bioespecífico em todos os ensaios. A dinâmica de uptake celular foi estudada in vitro demostrando que a presença do biomarcador na superfície das nanopartículas aumenta a concentração efetiva de ouro internalizada nas células alvo em 5 vezes quando comparada com pAuNR. Radiossensibilização aumentada foi observada em ensaios clonogênicos, apresentando essencialmente efetividade radiobiológica relativa 27% maior para o tecido com gAuNR incorporados, quando irradiado como feixe de 6MV, 58% e 78% maior quando irradiado com os feixes de Cs137 e Ir192 respectivamente. O estudo de biodistribuição in vivo em nude mice demonstrou que as nanopartículas bioconjugadas gAuNR tem acúmulo 3 vezes maior em tumores xenográficos de próstata do que pAuNR, e o seguimento tumoral acompanhado em 55 animais demonstrou expressiva efetividade terapêutica do uso de gAuNR em Radioterapia. Foi observado !!\"#$% de 24 e 20 dias para os grupos tratados com gAuNR e pAuNR irradiados com 6MV; Este número cresce para 28 e 18 quando as irradiações são realizadas com a fonte de Braquiterapia HDR de Ir-192. Os resultados consonantes dos estudos por Monte Carlo e ensaios radiobiológicos conduzem à proposição deste trabalho à luz de sua principal conclusão: Nanopartículas de ouro bioconjugadas, gAuNR, apresentam terapêutica efetiva na Radioterapia de câncer de próstata porque modulam o espectro de elétrons no citoplasma celular causando aumento local da densidade de ionização e reforço da probabilidade de dano letal. / Gold nanoparticles (AuNP) comprise the state of the art in medical oncology being extensively investigated for applications in cancer radiation therapy. The selective accumulation in tissues with disordered vascularization increases retention of AuNP in the tumor microenvironment, what leads to enhanced permeability of conjugated drugs carried by the AuNP. The radiosensitization observed in cells treated with AuNP however is result of a combined biological response to both biochemical and physical interactions of the conjugated AuNP with ionizing radiation. The presence of metallic gold in the cell cytoplasm during radiation therapy increases the photoelectric absorption cross-section in the target tissue, thus potentially increasing the lethal biological damage. In this context, the aim of this study is to evaluate the radiosensitization caused by gold nanoparticles in radiation therapy in terms of physical and radiobiological parameters. In order to investigate the density of ionizations in cells containing AuNP, the PENELOPE Monte Carlo code was used to simulate creation of secondary electrons in nanoparticles of 2-100nm after interaction with photon beams of clinical interest: 250kV, Ir-192, Cs-137, 6MV and Co-60. The theoretical analysis was performed in two steps: the Single AuNP Study and the Cell with AuNP Study. The first study predicts that enhancing nanoparticle diameter (d) from 2-10nm keeps the density of ionization enhanced in 22-28% for all kilovoltage beams and from 38-46% for all megavoltage beams, however increasing more than 5 times the yield of electrons ejected from the AUNP to the surrounding soft-tissue when the primary beam has its energy decreased from 6MV to 250kV. In the Cell with AuNP Study, the final concentration of gold in the cytoplasm (!!\") was increased from 0.001% to 1%, showing that concentration of gold strongly modifies the density of ionization in the cell, being up to 2 and 1.6 fold for low and high energies respectively. Sequentially, conjugated AuNP were synthesized with Goserelin Acetate (gAuNR) to target PC3 prostate cancer cells, and AuNP coated with polyethylene glycol (pAuNR) were used as non-biospecific control. The radiobiological assays were following designed based on the Monte Carlo results with 6MV, Cs-137 and the Ir-192 HDR Brachytherapy source. The in vitro cellular uptake study demonstrate that the biomarker in the surface of the nanoparticles increases the effective concentration of gold in the cytoplasm by 2.4 fold compared to pAuNR. The clonogenic assays demonstrate that the relative radiobiological effectiveness is enhanced in 27%, 58% and 78% for 6MV, Cs-137 and Ir-192 respectively when cells are treated with gAuNR for 24h prior irradiation. The conjugated nanoparticles were also submitted to ICP-MS based biodistribution study that demonstrate gAuNR accumulate 3 fold in xenografts prostate tumors than pAuNR. Following these results, a tumor re-growth study in nude mice was designed considering irradiations with the 6MV and the Ir-192 HDR Brachytherapy beam energies. Nanoparticles were administrated intravenously and tumors were followed for a maximum of 30 days. Tumor delay (!!\"#$%) of 20 and 24 days were observed for groups respectively treated with gAuNR and pAuNR and the 6MV beam; And !!\"#$% of 28 and 18 days were observed for groups respectively treated with gAuNR and pAuNR and the Ir-192 HDR Brachytherapy source. Both in vitro and in vivo studies, in consonance with the Monte Carlo predictions, demonstrate that conjugated AuNP, gAuNR, improve therapeutic effectiveness for treating prostate tumors because the gold nanoparticles modulate the effective spectrum of secondary electrons in the cytoplasm, locally enhancing the density of ionizations and thus increasing probability of lethal cellular damage.

Inhibitor of differentiation 4 (ID4), a member of the helix-loop-helix family of transcriptional regulators has emerged as a tumor suppressor in prostate cancer (PCa). Recent studies have shown that Id4 is highly expressed in the normal prostate and decreases in prostate cancer due to epigenetic silencing. Id4 knockdown in androgen sensitive LNCaP cells has been shown to lead to castration resistant prostate cancer (CRPC) in vitro and in vivo. Id4-/- mice leads to underdeveloped prostate with PIN like lesions without the loss of Androgen Receptor (AR) expression. In this study we demonstrate that the loss of ID4 expression in PCa cell line LNCaP and DU145 may promote tumorigenesis by promoting stemness. LNCaP cells with stably silenced ID4 ((-)ID4) using retroviral based shRNA and LNCaP transfected with non-specific shRNA were used to perform colony forming assay and prostatosphere formation using matrigel. Expression of cancer stem cell markers was determined using western blotting and immunocytochemistry (ICC). FACS analysis was used to sort stem cells and determine the ID4 expression. Xenograft study was performed on SCID mice using CD133 positive LNCaP cells. LNCaP(-)ID4 and DU145 cells lacking ID4 showed increased holoclone as well as decreased paraclone formation, which are believed to be derived from stem cells and differentiated cells respectively, as compared to non-silencing control in the colony forming assay. There was also an increase in prostatosphere development in the LNCaP (-) ID4 cells indicating that the loss of ID4 is responsible for promoting the LNCaP cells towards cancer stem cells. The results were further validated via western blotting and ICC using known cancer stem cell markers on the holoclones and paraclones formed by these cells. Xenograft study showed that 10,000 cells from CD133 positive LNCaP cells developed tumor on SCID mice. This study reports for the first time that loss of ID4 increases holoclone and prostatosphere formation indicating that Id4 may contribute to promoting stemness in prostate cancer cells.

To confirm the presence of prostate cancer which is the most incident visceral cancer in men, prostate biopsies are acquired using the magnetic resonance imaging fusion-guided prostate biopsy protocol. For this approach annotated magnetic resonance imaging is overlaid onto real-time ultrasound imaging to guide sampling of suspicious regions marked by uroradiologists. Additional biopsy samples are acquired via the previous clinical gold standard, i.e. the templated 12-core transrectal ultrasound-guided prostate biopsy protocol. While this approach improves the sensitivity of the prostate biopsy, a real-time, multiparametric imaging method of identifying biopsy targets could help overcome some of the inherent pitfalls of the magnetic resonance imaging fusion-guided prostate biopsy. Since ultrasound is used during the prostate biopsy, photoacoustic tomography, e.g. a hybrid imaging modality in which clinical ultrasound probes can be used to detect centimeters deep chemical alterations, has the potential to provide real-time targeting during biopsy. The translation of photoacoustic tomography to the clinic for prostate biopsy has been prevented by engineering challenges, which include identification of a biomarker for detecting suspicious regions of tissue and light delivery to the prostate for photoacoustic signal generation. Here, we present a vascular texture analysis method that identified 100% of primary and 67% of secondary tumors in the testing data set of ex vivo human prostate specimens. This method can be applied to future in vivo photoacoustic and ultrasound tomography of human prostates after further optimization of light delivery for photoacoustic tomography. To progress towards achieving this aim, we developed a transurethral light delivery device with angular light coupling method. By controlling the launch angle of the light into the fiber, the conversion of forward to side propagating energy can be improved from 27% to 98%, and the longitudinal emission profile can be controlled in order to illuminate the whole prostate simultaneously.<br>

Made available in DSpace on 2016-08-10T10:38:35Z (GMT). No. of bitstreams: 1 Hellen da Silva Cintra de Paula.pdf: 4823796 bytes, checksum: 7a1015ff712efd961fb8cb5dfd8ad2ef (MD5) Previous issue date: 2012-03-14 / The purpose of this study was to evaluate the association of single nucleotide polymorphisms of ATM and TP53 genes in prostate cancer patients with skin, urinary and lower gastrointestinal systems morbidity after radiotherapy. These two genes encode important proteins of the DNA repair pathways. It is believed that their polymorphisms are likely to modify the response of normal tissues to radiation. A group of 50 patients of the Radiotherapy Service at Araújo Jorge Hospital (Associação de Combate ao Câncer em Goiás) was selected. After signing the informed consent agreement, a sample of peripheral blood was collected for subsequent DNA extraction and polymerase chain reaction (PCR) for amplification of ATM and TP53 gene fragments. Finally the amplified fragments were sequenced to verify the presence of an exchange G> A in the codon 1853 of the ATM gene and polymorphisms of TP53 gene (C> G in the codon 72, C>T in the codon 47, C>A in the position 11299, C>T in the position 11322 and one insertion of 16 base pairs in intron 3). The side effects were classified according to the Radiation Therapy Oncology Group (RTOG) score. On univariate analysis, hypertension was strongly associated with a decreased risk of late urinary toxicities (OR= 0,048, 95% CI 0,004 - 0,620; p= 0,022). Patients receiving hormone therapy had a significantly higher incidence of acute skin toxicity (RR=1,286, 95% CI 0,907 - 1,823; p = 0,029). The exchange C>T in the position 11322 of the TP53 gene (intron 3) was significantly associated with the risk of acute skin toxicity (RR=22,0, 95%CI 5,680 - 85,207; p=0,006). There wasn t association between the other TP53 and ATM polymorphisms analysed and the frequency of side effects (p>0,05). In this study it has been shown that the presence of hypertension seemed to be protective for the development of urinary late effects after radiotherapy. Hormone therapy was apparently determinant for the development of acute skin toxicity. Our data also revealed that a TP53 intronic polymorfism (11322 C>T) was associated to increased acute skin radiosensitivity. This observation corroborates the importance of investigating the genetic profile to predict adverse side effects in patients undergoing radiotherapy. / O propósito deste estudo foi avaliar a associação entre polimorfismos de base única nos genes ATM e TP53 em pacientes com câncer prostático e a morbidade na pele e nos sistemas urinário e gastrointestinal inferior após a radioterapia. Estes dois genes codificam proteínas importantes nas vias de reparo do DNA. Acredita-se que seus polimorfismos possam modificar a resposta do tecido normal a radioterapia. Foi selecionado um grupo de 50 pacientes do serviço de radioterapia do Hospital Araújo Jorge (Associação de Combate ao Câncer em Goiás). Após a assinatura do termo de consentimento livre e esclarecido, foi coletada a amostra de sangue periférico com subseqüente extração de DNA e amplificação gênica por meio de reação em cadeia da polimerase (PCR) para amplificar os fragmentos gênicos de ATM e TP53. Finalmente, os amplicons foram seqüenciados para verificar a presença da troca de G>A no códon 1853 do gene ATM e polimorfismos do gene TP53 (C>G no códon 72, C>T no códon 47, C>A na posição 11299, C>T na posição 11322 e uma inserção de 16 pares de bases no intron 3). Os efeitos adversos foram classificados de acordo com o escore do Radiation Therapy Oncology Group (RTOG). Por meio de análise univariada, a hipertensão se associou fortemente ao baixo risco de desenvolvimento de toxicidade urinária crônica (OR=0,048, 95%IC 0,004 - 0,620; p=0,022). Pacientes que foram submetidos à hormonioterapia mostraram uma incidência significativa de toxicidade de pele aguda (RR = 1,286, 95%IC 0,907 1,823; p=0,029). A troca C>T na posição 11322 do gene TP53 (intron 3) mostrou uma associação significativa com o risco de toxicidade aguda de pele (RR = 22,0, 95%IC 5,68 85,207; p=0,006). Não houve associação entre os outros polimorfismos de TP53 e ATM analisados e a freqüência de efeitos adversos (p>0,05). Foi demonstrado que a presença de hipertensão parece ser protetora para o desenvolvimento de efeitos urinários tardios após a radioterapia. A hormonioterapia foi aparentemente determinante no surgimento de toxicidade aguda de pele. Nossos dados revelaram ainda que um polimorfismo intrônico de TP53 (11322 C>T) também estava associado ao aumento de radiossensibilidade aguda de pele. Estas observações mostram a importância de se investigar o perfil genético para futuramente predizer os efeitos adversos de pacientes em radioterapia.

Levels of the enzyme α-methylacyl-CoA racemase (AMACR) are increased ca. 9-fold in prostate cancer cells. AMACR is a very promising novel drug target as reducing AMACR levels converts castrate-resistant prostate cancer cells to androgen-dependent cells which will respond to androgen-deprivation. Despite the importance of AMACR in prostate and other cancers, there are very limited numbers of AMACR inhibitors described to-date. This is mainly due to the absence of a high-throughput assay for the screening of inhibitors against AMACR. The active-site residues and catalytic mechanism of human AMACR are still unknown, which make the rational design of drugs targeting AMACR very difficult. A range of novel potential inhibitors were synthesised using a rational drug design approach to explore the structure-activity relationship (SAR) on the side-chains of AMACR inhibitors. Their potencies were assessed using the fluoride elimination assay based on 1H and 19F NMR. Potency, mode of binding and kinetic parameters of these inhibitors were assessed using the multi-well colorimetric assay, which is the first AMACR high-throughput continuous assay reported to-date. A site-directed mutagenesis study was carried out to identify the active-site residues and catalytic mechanism of human AMACR. His-122, Asp-152, Met-184 and Glu-237 were identified as potential active-site residues, so the cDNA was mutated and expressed. The activity of wild-type and mutant AMACR enzymes were assessed using the deuterium wash-in, fluoride elimination and multi-well colorimetric assays. Results from these assays showed that human AMACR does not operate using a ‘two-base’ mechanism. Instead, it operates using a ‘one-base’ mechanism, most likely via water molecules acting as intermediaries within the hydrogen-bondings network in the active site. The knowledge obtained from this research informs rational drug design for this castrate-resistant prostate cancer target.

Dentre as diversas formas de se tratar o câncer de próstata, a braquiterapia com sementes de iodo-125 é uma opção que apresenta ótimos resultados e menor ocorrência de efeito colateral. No presente trabalho diferentes métodos de deposição de iodo radioativo em substrato de prata foram comparados com o propósito de eleger a alternativa mais adequada para a produção rotineira de sementes de iodo-125 do IPEN. A metodologia utilizada foi escolhida com base na infraestrutura disponível e na experiência dos pesquisadores presentes. Por essa razão, utilizou-se o iodo-131 para realização dos testes (mesmo comportamento do iodo-125). Quatro métodos foram selecionados: Método 1 (teste de eletrodeposição baseado no método desenvolvido por D. Kubiatowicz) com a eficiência de 65,16%; Método 2 (Reação química baseada no método desenvolvido por D. Kubiatowicz - HCl) com o resultado de 70,80% de eficiência; Método 3 (Reação química baseada no método desenvolvido pela Dra Maria Elisa Rostelato aquecimento/sulfeto) com 55,80% de eficiência; Método 4 (IQ-IPEN) apresentou o melhor resultado de eficiência, 99%. Como há mais fixação do material radioativo (que representa praticamente todo o custo da semente) por esse método, o preço final é o mais barato, sendo esse o método sugerido para ser implementado no laboratório de produção de fontes de braquiterapia do IPEN. Além disso o método é o mais rápido. / Among the different ways to treat prostate cancer, brachytherapy with iodine- 125 seeds is an option that provides good results and fewer side effects. In the present study several deposition methods of radioactive iodine in a silver substrate were compared in order to choose the most suitable alternative for the routine production to be implemented at IPENs laboratory. The methodology used was chosen based on the available infrastructure and experience of the researchers present. Therefore, the I131 was used for testing (same chemical behavior as I131). Four methods were selected: Method 1 (test based on electrodeposition method developed by D.Kubiatowicz) presented 65.16% efficiency; Method 2 (chemical reaction based on the method developed by D. Kubiatowicz - HCl) with the result of 70.80% efficiency; method 3 (chemical reaction based on the method developed by Dr. Maria Elisa Rostelato) with 55.80% efficiency; Method 4 (IQ-IPEN) resulted in 99% efficiency. Since this method has more radioactive material fixation (which represents virtually the entire cost of the seed), the final price is the cheapest. This method is the suggested one to be implemented in the IPENs laboratory for brachytherapy sources production. Besides, the method is the fasted one.

O câncer de próstata é considerado o segundo mais comum na população masculina em todo o mundo e ocupa a 15ª posição em mortes por câncer, em homens, representando cerca de 6% do total de mortes por câncer no mundo. O antígeno de membrana prostático específico (PSMA) é uma glicoproteína tipo II transmembrânica superexpressa no câncer de próstata, suas metástases e em neovascularizações relacionadas a tumores sólidos, que tem estimulado o desenvolvimento de pequenas moléculas inibidoras do receptor de PSMA, que carreguem agentes terapêuticos. Este trabalho pretendeu estudar de forma inédita a marcação e estabilidade radioquímica do Glu-NH-CO-NH-Lys(Ahx)-DOTA com 177Lu (PSMA-DOTA-177Lu) e avaliar seu potencial para a terapia do câncer de próstata. O radiofármaco foi obtido com pureza radioquímica elevada (PR > 95%) em todas as condições estudadas e permaneceu inalterada em &le; - 20 °C até 48 horas, mesmo em atividade específica alta (74 MBq/&mu;g). O ensaio de ligação específica do PSMADOTA- 177Lu mostrou que a fração do peptídeo que se ligou às células LNCaP de tumor de próstata foi de 1,79 &plusmn; 0,21 %, 2,47 &plusmn; 0,03 %, 3,07 &plusmn; 0,01 % e 4,13 &plusmn; 0,27%, para as concentrações de 0,15 x 106, 0,3 x 106, 0,5 x 106 e 1 x 106 células, respectivamente. O ensaio de internalização do PSMA-DOTA-177Lu sugere que o maior percentual da ligação específica do radiofármaco às células LNCaP corresponde à fração da ligação de superfície (99,03 &plusmn; 0,84 %). Os parâmetros farmacocinéticos determinados no estudo in vivo em camundongos Balb/c são compatíveis com o rápido clareamento sanguíneo e excreção renal, além de apresentar apreciável captação tumoral in vivo (2,76 &plusmn; 1,21 % Al/g) após 4 horas de administração do radiofármaco. Os estudos em estabilidade em soro humano demonstram estabilidade alta do radiofármaco PSMA-DOTA-177Lu por um período de até 24 horas, que foi confirmada pela baixa captação óssea demonstrada nos estudos in vivo de biodistribuição. O estudo de variação da atividade possibilitou estabelecer a atividade específica ideal (MBq/&mu;g), que será extrapolada para um piloto de produção do radiofármaco. Os resultados favoráveis deste estudo encorajam a perspectiva de realização de ensaio clínico controlado deste novo radiofármaco, avaliando seu potencial para aplicação no tratamento do câncer de próstata. / Prostate cancer is the second most frequently worldwide diagnosed cancer among males and ranks the 15th common cause of death from cancer in men, comprising 6% of the world\'s total cancer deaths. The prostate specific membrane antigen (PSMA) is a type II transmembrane glycoprotein, which is overexpressed in prostate cancer as well as in the neovasculature of solid tumors and metastasis. These features render PSMA an ideal target for developing small molecules inhibitors of PSMA and therapeutic approaches to targeted drugs delivery. This work intended to study in an unprecedented way the radiochemical labeling and stability of Glu-NHCO- NH-Lys (Ahx)-DOTA with 177Lu (177Lu-PSMA-DOTA) and its potential anti-tumor effects in prostate cancer. The radiopharmaceutical PSMA-177Lu was obtained with high radiochemical purity (RP > 95%) under all studied conditions and remained unchanged up to 48 hours (high at &le; -20 °C), even at high specific activity (74 MBq / &mu;g). The cellular uptake of PSMA-DOTA-177Lu was determined using PSMAexpressing LNCaP cells and showed a binding of 1.79 &plusmn; 0.21%, 2.47 &plusmn; 0.03%, 3.07 &plusmn; 0.01% and 4.13 &plusmn; 0.27%, to 0.15 × 106, 0.3 × 106, 0.5 × 106 and 1 × 106 LNCaP cells, respectively. The PSMA-DOTA-177Lu internalization assay revealed that the membrane-bound activity (non-internalized peptide) to LNCap cells, was 99.03 &plusmn; 0.84%. Moreover, the pharmacokinetic in vivo studies performed in Scid mice resulted in a rapid blood clearance and renal excretion, and showed significant tumor uptake (2.76 &plusmn; 1.21% lA/g) 4 hours after PSMA-DOTA-177Lu administration. Stability studies in human serum demonstrated high PSMA-DOTA-177Lu stability over 24 hours, which is in agreement with the low bone uptake obtained in the in vivo biodistribution studies. Furthermore, the comparative study aiming to establish the ideal PSMA-DOTA-177Lu specific activity (MBq / &mu;g) will be considered for further radiopharmaceutical production. The pre-clinical data obtained from this study suggested a great potential for PSMA-DOTA-177Lu to be included in clinical trials and to make a major contribution to the treatment of prostate cancer.

Objetivo: As moléculas de adesão celular (MAC) são essenciais para a manutenção do fenótipo epitelial. Alguns estudos têm relatado associação entre as alterações de sua expressão e a carcinogênese, mas o seu papel no câncer de próstata não é claro. Nosso objetivo foi estudar o perfil de expressão de E-caderina, cateninas e integrinas em espécimes cirúrgicos de câncer de próstata e associar as suas expressões com a evolução do tumor. Avaliamos também o perfil de expressão em metástases ósseas e linfonodais, a fim de compreender a influência destes marcadores na progressão do câncer de próstata. Materiais e Métodos: Foram selecionados 111 pacientes com câncer de próstata localizado tratados com prostatectomia radical pelo mesmo cirurgião. Sessenta pacientes não apresentaram recidiva tumoral após acompanhamento médio de 123 meses. A expressão das MAC foi avaliada por imuno-histoquímica (IH) em microarranjo tecidual (TMA), contendo duas amostras de cada tumor. Empregamos análise semiquantitativa para avaliação da expressão e determinamos a associação entre a expressão de cada MAC com a recorrência do tumor após a cirurgia. Avaliamos também a expressão das MAC por IH em TMA contendo espécimes de 28 metástases ósseas e em outro TMA contendo 19 metástases linfonodais com seus 19 tumores primários correspondentes. Resultados: Nos tumores primários a análise multivariada mostrou que a expressão das integrinas 3 e 3 1 relaciona-se com recidiva da doença. Quando a expressão de 3 foi forte e a expressão de 3 1 foi positiva, as chances de recorrência foram de 3,0 e 2,5 vezes maior. Apenas 19% e 28% dos pacientes estavam livres de recidiva após seguimento médio de 123 meses, quando os tumores apresentavam forte imunoexpressão de 3 ou positiva para 3 1 respectivamente. Outras integrinas apresentaram expressão reduzida, exceto 6 que foi expressa pela maioria dos tumores primário e metástases. A E-Caderina e as cateninas não mostraram associação com o prognóstico no tumor de próstata localizado. No sítio metastático, houve perda global de expressão das MAC. Encontramos ganho de expressão com a progressão do câncer de próstata somente para a integrina 3 que mostrou forte expressão em metade das metástases ósseas e linfonodais. Encontramos forte expressão de e -catenina foi em 94% dos linfonodos e 45% Conclusões: Nossos experimentos demonstram que a expressão das integrinas 3 e 3 1 está independentemente associada à recidiva de câncer de próstata após prostatectomia radical, e que a perda das moléculas de adesão celular pode ser considerada uma característica da progressão desta neoplasia / Purpose: Cell adhesion molecules (CAM) are essential for the maintenance of epithelial phenotype. Some studies have reported correlations between abnormalities in their expression and carcinogenesis, but their role in prostate cancer is unclear. Our aim was to study the expression profile of E-cadherin, catenins and integrins in surgical specimens of prostate cancer and associate their expression with outcome. We also assessed these expressions in bone and lymph node metastases in order to understand their influence in the progression of prostate cancer. Materials and Methods: We selected 111 patients with localized prostate cancer who underwent radical prostatectomy performed by the same surgeon. Sixty patients had no tumor recurrence after a median follow-up of 123 months. The CAM expression was evaluated by immunohistochemistry in a tissue microarray (TMA) containing two samples of each tumor. A semiquantitative analysis was employed and we measured the association between the expression of CAM and tumor recurrence. We also evaluated CAM expression by immunohistochemistry in a TMA containing 28 bone metastases and in other TMA containing 19 lymph node metastases with their corresponding 19 primary tumors. Results: In primary tumors, multivariate analysis showed that expression of 3 and 31 integrins was related to worse outcome. When 3 expression was strong and 31 expression was positive,the odds of recurrence were 3.0 and 2.5 fold higher. Only 19% and 28% of patients were recurrence-free in a mean follow up period of 123 months, when tumors showed strong 3 or positive 31 immuno-expression respectively. Other integrins have shown reduced expression, except 6 , which was expressed in most primary and metastatic cases. E-cadherin and catenins expressions were not associated with primary tumor outcome. At the metastatic setting, there was a global loss of CAM expression. We observed reliable gain of expression with prostate cancer progression only for integrin 3 that showed strong expression in half of bone and lymph node metastases. Interestingly, strong expression of and -catenin was observed in 94% of lymph node and 45% of bone metastases. Conclusions: We have demonstrated that the expression of integrins 3 and 31 was independently associated with recurrence after radical prostatectomy. In addition, we have shown that the loss of cell adhesion molecules can be considered a characteristic of prostate cancer progression

Terminado o sequenciamento do genoma humano, as atenções se voltaram para a determinação do conjunto completo de transcritos humanos. Diversos trabalhos sugerem que enquanto apenas uma pequena fração de mRNAs codificantes para proteína não é conhecida, existe um grande número de RNAs não-codificantes (ncRNAs) ainda não caracterizados. Nesse contexto, o presente trabalho visou explorar as informações de expressão gênica contidas em ESTs para identificar e caracterizar novos transcritos humanos. A busca genômica por membros de famílias gênicas relacionadas com câncer levou a identificação de novas pequenas GTPases, destacando uma subfamília que deve apresentar função supressora tumoral em próstata. Uma classe de ncRNAs longos, sem splicing, expressos antisenso a partir de regiões intrônicas foi descrita utilizando plataformas de microarrays, construídas pelo grupo, enriquecidas com seqüências sem anotação. O perfil de expressão de 23 ncRNAs intrônicos estava significativamente correlacionado com o grau de diferenciação de tumores de próstata (Gleason Score), e pode ser utilizado como candidato a marcador molecular de prognóstico. Um total de 39 ncRNAs intrônicos responderam à estimulação por andrógeno, apontando para um mecanismo regulatório da expressão intrônica por sinais fisiológicos hormonais. A biogênese da expressão intrônica parece ser complexa, pois uma fração não é transcrita pela RNA Polimerase II. A transcrição intrônica estava correlacionada com uso de exons em células tratadas com andrógeno. Assinaturas de expressão intrônica conservadas em tecidos humanos e de camundongos, e interações de transcritos intrônicos com proteínas regulatórias foram observadas. Este trabalho contribui com novas e originais evidências que dão apoio ao papel postulado para esses ncRNAs no controle fino do programa transcricional humano. / With the completion of the human genome sequence, attention has shifted towards determining the complete set of human transcripts. Multiple lines of evidence suggest that while only a small fraction of protein-coding mRNAs remains to be described, there is a huge amount of uncharacterized non-coding RNAs (ncRNAs). In this context, the present work sought to explore the gene expression information provided by ESTs to identify and characterize new human transcripts. A genomic-wide search for cancer related gene family members identified novel small GTPase genes, and highlighted an uncharacterized subfamily that may have a tumor suppressor role in prostate cancer. A class of long unspliced ncRNAs, expressed antisense from introns of protein-coding genes was described using custom-designed microarray platforms enriched with unannotated sequences. The expression profile of 23 intronic ncRNAs was significantly correlated to the degree of prostate tumor differentiation (Gleason Score), and could be used as a candidate prognostic molecular maker. A total of 39 intronic ncRNAs were responsive to androgen stimulation, poiting to a mechanism of intronic expression regulation by physiological hormone signals. Intronic ncRNA biogenesis seems to be complex, since a fraction of them is not transcribed by RNA Polymerase II. Intronic transcription was correlated to exon usage in androgen treated cells. Tissue expression signatures of intronic transcription were conserved in human and mouse, and intronic transcripts were found to interact with regulatory proteins. This work provides new and original contributions that support the postulated role of ncRNAs in the fine tunning of the human transcriptional program.

Magnetic Resonance Imaging (MRI) is the most prominent non-invasive technique used in clinical diagnosis and biomedical research. Its development as an imaging technique has been aided by contrast agents (CAs) which enhance contrast to noise ratio in the images. This dissertation deals with paramagnetic lanthanide- and superparamagnetic iron-based nanoparticles (NPs) which are potential CAs at clinical field of 3 T and a high field of 9.4 T. Chapter 1 provides a brief overview of colloidal nanoparticles, with an emphasis on their surface chemistry and magnetic properties for bio-applications. Chapter 2 employs europium as an optical probe to illustrate the contribution of inner, second and outer sphere relaxation towards longitudinal and transverse relaxivities of paramagnetic NP-based CAs. Chapter 3 investigates the positive and the negative contrast enhancement abilities and magnetization of paramagnetic NPs comprising a core of sodium dysprosium fluoride with a sodium gadolinium fluoride shell. Their surface chemistry is tuned to target prostate cancer specifically. The application of these NPs is further extended in Chapter 4 to track an intraneuronal protein called tau following mild traumatic brain injury. Chapter 5 deals with facile synthesis and long-term stability of superparamagnetic iron NPs for their potential application as CAs. Chapter 6 illustrates the concept of MRI correlation using ‘T1-only’ and ‘T2-only’ NPs. Chapter 7 investigates on the dynamics involved in the phospholipids coating the surface of NPs. Chapter 8 concludes on the work detailed in the previous chapters and outlines the future outlook. / Graduate / 2020-01-15