京都大学 / 新制・課程博士 / 博士(医学) / 甲第24476号 / 医博第4918号 / 新制||医||1062(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 小濱 和貴, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In search of new biomarkers suitable for the diagnosis and treatment of prostate cancer, genome-wide transcriptome sequencing was carried out with tissue specimens from 40 prostate cancer (PCa) and 8 benign prostate hyperplasia patients. We identified two intergenic long non-coding transcripts, located in close genomic proximity, which are highly expressed in PCa. Microarray studies on a larger cohort comprising 155 patients showed a profound diagnostic potential of these transcripts (AUC~0.94), which we designated as tumor associated prostate cancer increased lncRNA (TAPIR-1 and -2). To test their therapeutic potential, knockdown experiments with siRNA were carried out. The knockdown caused an increase in the p53/TP53 tumor suppressor protein level followed by downregulation of a large number of cell cycle- and DNA-damage repair key regulators. Furthermore, in radiation therapy resistant tumor cells, the knockdown leads to a renewed sensitization of these cells to radiation treatment. Accordingly, in a preclinical PCa xenograft model in mice, the systemic application of nanoparticles loaded with siRNA targeting TAPIR-1 significantly reduced tumor growth. These findings point to a crucial role of TAPIR-1 and -2 in PCa.

OBJETIVOS: investigar a prevalência e a presença de distintos perfis microbianos no biofilme subgengival e avaliar o seu papel no diagnóstico e risco das doenças periodontais destrutivas em uma população isolada brasileira sem acesso à tratamento periodontal e tradição ao uso de métodos de higiene bucal. MATERIAL E MÉTODOS: A população-alvo consistiu de todos os indivíduos com 12 ou mais anos de idade (N= 264) residentes na microárea Cajaíba, identificados por meio de um censo. Estes indivíduos foram entrevistados por meio de um questionário estruturado e submetidos a um exame periodontal completo que consistiu na avaliação de 6 sítios por dente em toda a boca e na coleta de amostras do biofilme subgengival em 4 sítios por indivíduo. A detecção dos micro-organismos A. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythia e C. rectus, bem como a distribuição dos sorotipos e presença do clone JP2 do A. actinomycetemcomitans foram avaliadas por meio da reação em cadeia da polimerase (PCR). RESULTADOS: A. actinomycetemcomitans foi detectado em 25% dos indivíduos, enquanto que P. gingivalis T. forsythia, P.intermedia e C. rectus foram detectados em 64%, 59%, 38% e 90% dos indivíduos, respectivamente. Entre as amostras positivas para o A. actinomycetemcomitans (n=42), 18 (42%) representaram o sorotipo a, 2 (5%) o sorotipo b, 19 (46%) o sorotipo c, 1 (2%) o sorotipo e, e 4 (10%) foram não-sorotipáveis. O clone JP2 do A. actinomycetemcomitans não foi detectado em nenhum indivíduo desta população. Dois perfis microbianos subgengivais foram identificados: (perfil 1) nenhum dos microrganismos estudados, com exceção do C. rectus (n = 31), e (perfil 2) co-ocorrência de P. gingivalis e T. forsythia (n = 77). O perfil 1 demonstrou valores de sensibilidade extremamente baixos, enquanto que o perfil 2 apresentou valores de sensibilidade variados na identificação dos desfechos subrrogados periodontais avaliados, e valores de baixos a moderados para a especificidade. Os seguintes perfis subgengivais estiveram associados com a prevalência de perda clínica de inserção (NCI) e profundidade de sondagem (PS) nos modelos finais de regressão logística múltipla, ajustados para variáveis demográficas, biológicas e comportamentais: T. forsythia (PS e NCI 5 mm, e 7 mm), P. gingivalis (NCI 7 mm) e o perfil 2 (PS 5 mm e NCI 7 mm). CONCLUSÕES: Os micro-organismos periodontais estudados foram prevalentes nessa população isolada. Esta população apresentou predominância dos sorotipos a e c do A. actinomycetemcomitans. Dois perfis microbianos subgengivais puderam ser identificados nesta população isolada. Porém, eles não foram superiores ao diagnóstico de parâmetros clínicos periodontais específicos, quando adicionados à informação clínica tradicional. Perfis microbianos subgengivais apresentando T. forsythia como indicador de risco foram significativamente associados com o aumento da PS e do NCI nessa população isolada. / AIMS: To investigate the prevalence and describe the subgingival microbial profiles of selected periodontal pathogens in the subgingival biofilm; and assess their role as possible diagnostic markers or risk indicators for destructive periodontal diseases in a periodontally untreated and isolated population from Brazil. MATERIAL AND METHODS: The target population consisted of all subjects aged 12 years (n=264) in an isolated Brazilian population. A full-mouth clinical examination was conducted, and pooled subgingival plaque samples were obtained from four sites per subject. PCR analyses were performed to identify the following microorganisms: A. actinomycetemcomitans, P. gingivalis, T. forsythia, P. intermedia and C. rectus, as well as the A. actinomycetemcomitans serotype distribution and JP2 clone detection. RESULTS: A. actinomycetemcomitans was detected in 25% of the subjects, whereas P. gingivalis, T. forsythia, P.intermedia and C. rectus were detected in 64%, 59%, 38% and 90% of the subjects, respectively. From the A. actinomycetemcomitans positive isolates (n=42), 18 (42%) were serotype a, 2 (5%) b, 19 (46%) c, 1 (2%) e, and 4 (10%) were non-serotypeable. None of the strains belonged to the JP2 clone. Two specific subgingival microbial profiles were identified: (1) In one, only C. rectus could or not be present (n = 31), while in the other, (2) Co-occurrence of T. forsythia and P. gingivalis was observed (n = 77). Profile 1 showed very low sensitivity values, and profile 2 showed varying sensitivity values for the identification of the various periodontal states, and considerably low to moderate specificity values. The following subgingival profiles were significantly associated with the prevalence of periodontal attachment loss (CAL) and probing depth (PD) in the final multiple logistic regression models adjusted for demographic, biological and behavioral variables: T. forsythia (PD and CAL 5 mm and 7 mm), P. gingivalis (CAL 7 mm) and the profile 2 (PD 5 mm and CAL 7 mm). CONCLUSIONS: The five studied periodontal microorganisms were prevalent in this isolated population. The A. actinomycetemcomitans positive subjects consisted predominantly of a and c serotypes. Two specific microbial profiles could be identified in this isolated population. They did not result in significant superior diagnostic accuracy when compared totraditional clinical markers. Subgingival microbial profiles presenting T. forsythia as risk indicator were significantly associated with increased PD and CAL in this isolated population.

OBJETIVOS: investigar a prevalência e a presença de distintos perfis microbianos no biofilme subgengival e avaliar o seu papel no diagnóstico e risco das doenças periodontais destrutivas em uma população isolada brasileira sem acesso à tratamento periodontal e tradição ao uso de métodos de higiene bucal. MATERIAL E MÉTODOS: A população-alvo consistiu de todos os indivíduos com 12 ou mais anos de idade (N= 264) residentes na microárea Cajaíba, identificados por meio de um censo. Estes indivíduos foram entrevistados por meio de um questionário estruturado e submetidos a um exame periodontal completo que consistiu na avaliação de 6 sítios por dente em toda a boca e na coleta de amostras do biofilme subgengival em 4 sítios por indivíduo. A detecção dos micro-organismos A. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythia e C. rectus, bem como a distribuição dos sorotipos e presença do clone JP2 do A. actinomycetemcomitans foram avaliadas por meio da reação em cadeia da polimerase (PCR). RESULTADOS: A. actinomycetemcomitans foi detectado em 25% dos indivíduos, enquanto que P. gingivalis T. forsythia, P.intermedia e C. rectus foram detectados em 64%, 59%, 38% e 90% dos indivíduos, respectivamente. Entre as amostras positivas para o A. actinomycetemcomitans (n=42), 18 (42%) representaram o sorotipo a, 2 (5%) o sorotipo b, 19 (46%) o sorotipo c, 1 (2%) o sorotipo e, e 4 (10%) foram não-sorotipáveis. O clone JP2 do A. actinomycetemcomitans não foi detectado em nenhum indivíduo desta população. Dois perfis microbianos subgengivais foram identificados: (perfil 1) nenhum dos microrganismos estudados, com exceção do C. rectus (n = 31), e (perfil 2) co-ocorrência de P. gingivalis e T. forsythia (n = 77). O perfil 1 demonstrou valores de sensibilidade extremamente baixos, enquanto que o perfil 2 apresentou valores de sensibilidade variados na identificação dos desfechos subrrogados periodontais avaliados, e valores de baixos a moderados para a especificidade. Os seguintes perfis subgengivais estiveram associados com a prevalência de perda clínica de inserção (NCI) e profundidade de sondagem (PS) nos modelos finais de regressão logística múltipla, ajustados para variáveis demográficas, biológicas e comportamentais: T. forsythia (PS e NCI 5 mm, e 7 mm), P. gingivalis (NCI 7 mm) e o perfil 2 (PS 5 mm e NCI 7 mm). CONCLUSÕES: Os micro-organismos periodontais estudados foram prevalentes nessa população isolada. Esta população apresentou predominância dos sorotipos a e c do A. actinomycetemcomitans. Dois perfis microbianos subgengivais puderam ser identificados nesta população isolada. Porém, eles não foram superiores ao diagnóstico de parâmetros clínicos periodontais específicos, quando adicionados à informação clínica tradicional. Perfis microbianos subgengivais apresentando T. forsythia como indicador de risco foram significativamente associados com o aumento da PS e do NCI nessa população isolada. / AIMS: To investigate the prevalence and describe the subgingival microbial profiles of selected periodontal pathogens in the subgingival biofilm; and assess their role as possible diagnostic markers or risk indicators for destructive periodontal diseases in a periodontally untreated and isolated population from Brazil. MATERIAL AND METHODS: The target population consisted of all subjects aged 12 years (n=264) in an isolated Brazilian population. A full-mouth clinical examination was conducted, and pooled subgingival plaque samples were obtained from four sites per subject. PCR analyses were performed to identify the following microorganisms: A. actinomycetemcomitans, P. gingivalis, T. forsythia, P. intermedia and C. rectus, as well as the A. actinomycetemcomitans serotype distribution and JP2 clone detection. RESULTS: A. actinomycetemcomitans was detected in 25% of the subjects, whereas P. gingivalis, T. forsythia, P.intermedia and C. rectus were detected in 64%, 59%, 38% and 90% of the subjects, respectively. From the A. actinomycetemcomitans positive isolates (n=42), 18 (42%) were serotype a, 2 (5%) b, 19 (46%) c, 1 (2%) e, and 4 (10%) were non-serotypeable. None of the strains belonged to the JP2 clone. Two specific subgingival microbial profiles were identified: (1) In one, only C. rectus could or not be present (n = 31), while in the other, (2) Co-occurrence of T. forsythia and P. gingivalis was observed (n = 77). Profile 1 showed very low sensitivity values, and profile 2 showed varying sensitivity values for the identification of the various periodontal states, and considerably low to moderate specificity values. The following subgingival profiles were significantly associated with the prevalence of periodontal attachment loss (CAL) and probing depth (PD) in the final multiple logistic regression models adjusted for demographic, biological and behavioral variables: T. forsythia (PD and CAL 5 mm and 7 mm), P. gingivalis (CAL 7 mm) and the profile 2 (PD 5 mm and CAL 7 mm). CONCLUSIONS: The five studied periodontal microorganisms were prevalent in this isolated population. The A. actinomycetemcomitans positive subjects consisted predominantly of a and c serotypes. Two specific microbial profiles could be identified in this isolated population. They did not result in significant superior diagnostic accuracy when compared totraditional clinical markers. Subgingival microbial profiles presenting T. forsythia as risk indicator were significantly associated with increased PD and CAL in this isolated population.

Le développement de thérapies personnalisées et non invasives représente un challenge majeur en cancérologie et l'utilisation de nano-outils multifonctionnels combinés à de nouveaux biomarqueurs spécifiques des cellules cancéreuses apportent une solution de choix. Le cancer de la prostate est le cancer le plus diagnostiqué et représente la seconde cause de mortalité par cancer chez l'homme. Il est primordial de pouvoir diagnostiquer et traiter ces tumeurs dès les stades précoces afin d'améliorer la survie et la prise en charge des patients. L'objectif de cette thèse a été d'étudier le potentiel d'une lectine membranaire, le récepteur du mannose 6-phosphate cation-indépendant (RM6P-CI), en tant que biomarqueur des cancers de la prostate. Dans un premier temps, l'analyse de l'expression du RM6P-CI a été réalisée sur des tissus prostatiques sains et cancéreux. La découverte d'une surexpression spécifique par les cellules cancéreuses de la prostate a permis de caractériser le R6MP-CI comme nouveau marqueur diagnostique et d'envisager son ciblage pour la thérapie photodynamique des cancers de la prostate. Des nanoparticules de silice mésoporeuse ont donc été fonctionnalisées spécifiquement pour la thérapie photodynamique avec l'encapsulation de photosensibilisateurs mono ou bi-photoniques et le couplage de ligands stables, analogues du M6P naturel, pour le ciblage des RM6P-CI. Le potentiel phototoxique in vitro et ex vivo de ces nano-outils a été démontré ainsi que l'internalisation spécifique par le RM6P-CI. La propriété fluorescente des nanoparticules biphotoniques a permis d'autre part d'imager les cellules cancéreuses de la prostate et de proposer ainsi leur utilisation dans un but théranostique (thérapie et diagnostic) des cancers de la prostate. / The development of personalized and non invasive therapies represents a major challenge in cancer and the use of multifunctional nano-tools combined with new specific biomarkers of cancer cells an appropriate solution. The prostate cancer is the most diagnosed malignancy and the second cause of cancer death in men. It is essential to diagnose and treat these tumors from the early stages to improve the survival and the care of patients. The aim of this thesis was to study the potential of a membrane lectin, the cation-independent mannose 6-phosphate receptor (CI-M6PR) as a biomarker for prostate cancer. Firstly, the analysis of the expression of CI-M6PR was performed on healthy and cancer prostate tissues. The discovery of a specific overexpression by prostate cancer cells allows to propose CI-M6PR as a new diagnostic marker and we considered its targeting for photodynamic therapy of prostate cancers. Mesoporous silica nanoparticles have been functionalized specifically for photodynamic therapy with the encapsulation of one or two-photon photosensitizers and the grafting of stable ligands, analogues of the natural M6P, for the CI-M6PR targeting. The phototoxic potential in vitro and ex vivo of these nano-tools was demonstrated in addition to the specific M6PR internalization pathway. Moreover, the two-photon fluorescence property of the nanoparticles led to the imaging of prostate cancer cells and thus suggests their use for theranostic (therapy and diagnosis) purpose of prostate cancers.