京都大学 / 新制・課程博士 / 博士(医学) / 甲第24283号 / 医博第4899号 / 新制||医||1061(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 藤田 恭之, 教授 羽賀 博典, 教授 波多野 悦朗 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

By crossing septin7-floxed mice with Lyz2-Cre mice carrying the Cre recombinase inserted in the Lysozyme-M (Lyz2) gene locus we aimed the specific deletion of septin7 in myeloid cells, such as monocytes, macrophages and granulocytes. Septin7flox/flox:Lyz2-Cre mice show no alterations in the myeloid compartment. Septin7-deleted macrophages (BMDMs) were isolated and analyzed. The lack of Septin7 expression was confirmed and a constitutive double-nucleation was detected in Septin7-deficient BMDMs indicating a defect in macrophage cytokinesis. However, phagocytic function of macrophages as judged by uptake of labelled E. coli particles and LPS-stimulated macrophage activation as judged by induction of TNF mRNA expression and TNF secretion were not compromised. In addition to myeloid cells, Lyz2-Cre is also active in type II pneumocytes (AT2 cells). We monitored lung adenocarcinoma formation in these mice by crossing them with the conditional knock-in Kras-LSL-G12D allele. Interestingly, we found that control mice without septin7 depletion die after 3–5 weeks, while the Septin7-deficient animals survived 11 weeks or even longer. Control mice sacrificed in the age of 4 weeks display a bronchiolo-alveolar hyperplasia with multiple adenomas, whereas the Septin7-deficient animals of the same age are normal or show only a weak multifocal brochiolo-alveolar hyperplasia. Our findings indicate an essential role of Septin7 in macrophage cytokinesis but not in macrophage function. Furthermore, septin7 seems absolutely essential for oncogenic Kras-driven lung tumorigenesis making it a potential target for anti-tumor interventions.

<p>Lung cancer is the leading cause of cancer related deaths in both the US and Canada and efforts still need to be made towards understanding the disease. The role of inflammation in the promotion of cancer development represents a newer avenue of research. The glycoprotein (gp)-130 cytokine interleukin-6 (IL-6) has a well established role in promoting inflammation and recent evidence suggests roles in development of certain tumors in animal models. Less is known of the related family member oncostatin M (OSM) and the functions of either IL-6 or OSM in lung cancer development is not known. Based on the hypothesis that these cytokines promote lung cancer development, IL-6 and OSM were overexpressed in the lungs of two separate mouse models for lung cancer utilizing adenovirus vectors encoding IL-6 or OSM. The first mouse model utilized a Cre-conditional oncogene KRAS G12D (developed by Tyler Jacks) in which endotracheal administration of adenovirus (Ad)-encoded Cre-recombinase resulted in increases in lung densities in a dose-dependent fashion over a period of 6 weeks that were measurable by CT scanning and histology. Increases in cytokines IL-6 and kertinocyte chemoattractant (KC) were detectable in the bronchoalveolar lavage (BAL) by week 4, as well as marked increases in alveolar macrophage numbers. Macrophages were also shown as a possible target for Cre-mediated recombination and mutant KRAS expression. Administration of either AdIL-6 or AdOSM as well as AdCre resulted in a trend toward increases in tumor burden with AdOSM based on experiments terminated at 4 weeks. The second mouse model involved endotracheal administration of the lewis lung carcinoma (LLC) cell line, which after 7 days resulted in detectable tumor burden. Administration of either AdIL-6 or AdOSM and LLC cells simultaneously was shown to increase tumor burden relative to AdDl70 co-administration. These results suggest a possible role of IL-6 or OSM in promoting lung tumor development in animal models and may ultimately reveal gp130 cytokines IL-6 or OSM as a possible therapeutic target for the treatment of lung cancer.</p> / Master of Science (MSc)

Les associations chimioradiothérapies occupent une place importante dans le traitement des cancers. De nouveaux médicaments ciblent spécifiquement la cellule tumorale. Ces thérapies ciblées peuvent agir sur les mécanismes de radiorésistance tumorale et sont prometteuses en association avec la radiothérapie. Le STI571 (imatinib ou Glivec) inhibe spécifiquement l'activité tyrosine kinase de Bcr-Abl. Il entraîne une radiosensibilisation dans la lignée de leucémie myéloïde chronique K562 en agissant sur le cycle cellulaire. Le BIBW2992 est un inhibiteur sélectif d'EGFR et HER2, responsable d'une cytotoxicité et d'une radiosensibilisation des cellules d'adénocarcinome pancréatique BxPC3 et Capan-2, indépendamment de leur statut KRAS. Le mécanisme sous-jacent cette radiosensibilisation n'est pas univoque, faisant intervenir à la fois des modifications du cycle cellulaire et une induction de la mort mitotique. Nos résultats montrent que l'association d'un inhibiteur de tyrosine kinase aux radiations ionisantes peut entraîner une radiosensibilisation in vitro dont les mécanismes sont variables en fonction du type de lignée cellulaire.

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy in both men and women, and one of the leading causes of cancer-related deaths worldwide. One frequently mutated pathway involved in oncogenesis in CRC is the RAS/RAF/MAP kinase pathway. Oncogenic activation of KRAS and BRAF occur in 30‒40% and 5‒15% of all CRCs, respectively, and the mutations are mutually exclusive. Even though KRAS and BRAF are known to act in the same pathway, KRAS- and BRAF-mutated CRCs have different clinical and histopathological features. For example, BRAF mutation in CRC is tightly linked to microsatellite instability (MSI) and a CpG island methylator phenotype (CIMP), which is not seen in KRAS-mutated tumours. BRAF-mutated CRCs are also more often found in right-sided tumours. However, the underlying molecular reasons for these differences have not yet been defined. The overall aim of this thesis was to investigate molecular differences between KRAS- and BRAF-mutated CRCs to understand how KRAS and BRAF mutations differentially affect tumour progression. We used an in vitro cell culture system to explore molecular differences between KRAS- and BRAF-mutated CRCs and verified our findings using CRC tissue specimens from the Colorectal Cancer in Umeå Study (CRUMS). We found that BRAF mutation, but not KRAS mutation, was associated with expression of the stem cell factor SOX2. Furthermore, SOX2 was found to be correlated to a poor patient prognosis, especially in BRAF-mutated cancers. We further investigated the role of BRAF in regulation of SOX2 expression and found that SOX2 is at least partly regulated by BRAF in vitro. We continued by investigating the functional role of SOX2 in CRC and found that SOX2-expressing cells shared several characteristics with cancer stem cells, and also had down-regulated expression of the intestinal epithelial marker CDX2. There was a strong correlation between loss of CDX2 expression and poor patient prognosis, and patients with SOX2 expression were found to have a particularly poor prognosis when CDX2 levels were down-regulated. In conclusion, in these studies we identified a subgroup of BRAF-mutated CRCs with a particularly poor prognosis, and having a cancer stem cell-like appearance with increased expression of SOX2 and decreased expression of CDX2. Tumour progression is regulated by interactions with cells of the immune system. We found that BRAF-mutated CRCs were more highly infiltrated by Th1 lymphocytes than BRAF wild-type tumours, while the opposite was true for KRAS-mutated CRCs. Interestingly, we found that part of this difference is probably caused by differences in secreted chemokines and cytokines between KRAS- and BRAF-mutated CRCs, stimulating different arms of the immune response. Altered levels of expression of miRNAs have been seen in several malignancies, including CRC. We found that BRAF- and KRAS-mutated CRCs showed miRNA signatures different from those of wild-type CRCs, but the expression of miRNAs did not distinguish KRAS-mutated tumours from BRAF-mutated tumours. In summary, our findings have revealed possible molecular differences between KRAS- and BRAF-mutated CRCs that may explain some of the differences in their clinical and histopathological behaviour.

The aim of dissertation is to elaboration model of komplex school excursion for interactive students work with usage natural places of Czech karst. Next point is creating a set of learning excercises in practical application knowledges from natural branches and for confirm keys competenses of students middle schools. An educational potential of a choosen area was evaulated on the bases of aims mentioned earlier, a model of complex school excursion using conservation area Czech karst was developed and tested The excursion takes place in the village Svatý Jan pod Skalouand it uses set of worksheets. - version for season autumn and spring. An evaluation folloving the excursion is done as a form of a student conference. Particular educational aims were applied during the excursion. Evaluation of the worksheets follows and designs of changes of teaching tasks in the worksheets are proposed. Results of dissertation are processed concept of complex school excursion with methodical material and with set of worhsheets version for season spring, autumn, and winter. Based on the evaulation of the komplex school excursion in conservation area Czech karst, results of worksheets and results from the conference, it is possible to deduce, that the addition of excursion as a teaching form is benefitial to students....

Flow and mean residence time in epikarst and unsaturated zone was studied above the Ochoz cave in the Moravian Karst. I studied various flow components with different residence time in unsaturated zone and the influence of soil and epikarst on seepage composition and residence time by means of several methods (longterm monitoring of conductivity, flowrate of seepage and soil water, use of environmental tracers - 18 O, 3 H, CFC and SF6, flow into the soil and detailed sampling during intesive rain events). Seepage sites Kašna in the Rudické propadání cave system and Mapa Republiky in Býčí skála were reference localities in unsaturated zone. For comparison I modeled residence time in saturated zone: at Kaprálka outlet close to the Ochoz cave, at Stará řeka (Rudické propadání) and Konstantní přítok (Amatérská cave). Mean residence time in unsaturated zone above the Ochoz cave reaches 7 - 20 years, while it is only few months in the soil (1 - 8 months, depending on the depth). At Kašna seepage site, the reasidence time is similar to the Ochoz cave - about 18 - 20 years, at Mapa republiky seepage site, it reaches 150s year due to unusual geological settings. Mean residence time in order of 10 - 20 years corresponds to storativity values (0.6 % in average) calculated from parallel water level recession...

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Contexte. Le cancer bronchopulmonaire (CBP) demeure la première cause de mortalité par cancer dans le monde. Malgré l'espoir suscité par le développement des thérapies ciblées, son pronostic demeure sombre, particulièrement dans les cas de CBP à petites cellules (CBP-PC) et de CBP non à petites cellules (CBP-NPC) présentant une activation de l'oncogène KRAS. Matériel et Méthodes. Nous avons mené 3 études successives, visant à (i) radiosensibiliser des modèles de CBP-PC par l'ajout d'un inhibiteur de BCL2, (ii) cibler des modèles de CBP-NPC mutés KRAS par l'association d'un inhibiteur de mTOR et d'un inhibiteur de RAF, et (iii) créer un modèle préclinique orthotopique murin de CBP reproduisant la progression tumorale observée en clinique. Résultats. Dans la première étude, l'inhibiteur de BCL2 oblimersen a présenté un effet radiosensibilisant sur des modèles de CBP-PC, in vitro et in vivo. Dans la seconde étude, l'association de l'inhibiteur de mTOR everolimus et de l'inhibiteur de RAF/VEGFR RAF265 a présenté un effet synergique sur des lignées cellulaires de cancers présentant la double mutation de KRAS et de PIK3CA, in vitro et in vivo. Dans la troisième étude, l'injection orthotopique d'une lignée bioluminescente de CBP-NPC chez des souris nude a permis d'établir des tumeurs intra pulmonaires évoluant vers une extension métastatique ganglionnaire et hématogène, et de détecter la présence de cellules tumorales circulantes. Conclusion. L'association d'un inhibiteur de BCL2 à la radiothérapie est une stratégie intéressante dans le CBP-PC, l'association d'un inhibiteur de mTOR et d'un inhibiteur de RAF/VEGFR est une stratégie intéressante dans le CBP-NPC présentant une double mutation KRAS-PIK3CA, mais ces données doivent être confirmées sur des modèles orthotopiques afin de gagner en pertinence avant d'envisager un transfert en clinique.

La ultrasonografía endoscópica (USE) combina la endoscopia con la ecografía para conseguir imágenes ecográficas de 360º desde el interior del tubo digestivo. Ello permite una muy buena visualización de las diferentes capas de la pared y de las estructuras de vecindad. Por otro lado, existe un ecoendoscopio sectorial que permite introducir una aguja por el canal operativo y realizar la punción con aguja fina guiada por USE en tiempo real desde el interior del tubo digestivo y obtener material citológico para el diagnóstico (USE-PAAF). Asi pues, la USE y la USE-PAAF constituyen unas técnicas idóneas para estudiar el engrosamiento de los pliegues gástricos, explorar tanto las lesiones focales como la patología difusa del páncreas, y realizar la estadificación locoregional de los tumores digestivos asi como para la obtención de material para el diagnóstico citológico. A pesar de la conocida utilidad de la USE y USE-PAAF en Oncología, existen patologías de difícil manejo clínico en los que el rendimiento de esta técnica no ha sido evaluado adecuadamente. Además, tampoco se han analizado cuáles son las circunstancias que permiten obtener un mayor rendimiento de la USE-PAAF. Por otro lado, la combinación de una técnica diagnóstica novedosa como es la USE-PAAF con los métodos de biología molecular podría suponer un verdadero avance en el diagnóstico de las neoplasias digestivas y pulmonares.La presente Tesis Doctoral está constituida por cuatro trabajos originales encaminados a resolver estas cuestiones y cuyos resultados permiten extraer las siguientes conclusiones:1. El engrosamiento de las capas profundas de la pared gástrica (submucosa y/o muscular) es el único criterio endosonográfico independiente predictivo de malignidad en pacientes con pliegues gástricos engrosados y biopsias negativas. 2. Utilizando este criterio, la USE es una técnica muy precisa y con un importante impacto clínico en el manejo de este grupo de pacientes. 3. En los pacientes referidos para USE-PAAF, esta técnica aumenta significativamente el porcentaje de diagnósticos correctos en cuanto a benignidad y malignidad con respecto a la USE, en especial en adenopatías y lesiones quísticas. 4. La localización intraparietal de la lesión es el único factor asociado a una mayor probabilidad de obtener un diagnóstico incorrecto por USE-PAAF. 5. La presencia de un patólogo en la sala de exploración aumenta el rendimiento de la USE-PAAF al reducir el número de pases necesarios para obtener un diagnóstico correcto y es una estrategia coste-eficaz.6. Es posible efectuar el análisis mutacional del gen KRAS en el material obtenido por USE-PAAF, con una elevada rentabilidad. 7. La citología convencional es la estrategia más precisa para el diagnóstico del cáncer de páncreas, pudiendo el análisis molecular incrementar su rendimiento en los pocos casos en los que la primera no es diagnóstica. 8. Es posible detectar la presencia de micrometástasis ganglionares mediante determinación de patrones de metilación aberrante en material obtenido por USE-PAAF.9. La combinación de este análisis con la citología convencional puede ser una aproximación útil en el diagnóstico de extensión de los pacientes con neoplasias digestivas y pulmonares. / Endoscopic ultrasound (EUS) consists in a high-frequency transducer placed at the tip of an endoscope. This technique permits to obtain high-resolution transmural sonographic images of the intestinal wall and surrounding structures. Moreover, it is possible to obtain cytological material from lesions located around the intestinal wall by means of FNA guided by EUS. EUS and EUS-FNA have become well-established procedures for diagnosing and staging both gastrointestinal and lung cancer as well as to study the pancreas. The aims of the present Doctoral Thesis were:1) to assess the predictive variables of malignancy in EUS, and the impact of EUS in patients with large gastric folds at endoscopy and endoscopic biopsies negative for malignancy; 2) To evaluate factors that permit to obtain a correct diagnosis by EUS-FNA and to establish the usefulness of disposing of an attendant pathologist; 3) To establish the usefulness of KRAS mutational analysis in the diagnosis of pancreatic adenocarcinoma by comparing this technique with conventional cytology in aspirates obtained by means of EUS-FNA; and, 4) to evaluate whether hypermethylation gene promoter analysis was feasible on samples obtained by EUS-FNA from lymph nodes, as well as to establish the usefulness of this strategy for the detection of micrometastases in patients with gastrointestinal and non-small cell lung cancer.Results obtained permitted to conclude that: 1) the enlargement of deep layers is the only independent predictive factor for malignancy in patients with large gastric folds at endoscopy and biopsies negative for malignancy and that EUS has a high clinical impact in these patients; 2) The availability of an attendant pathologist seems to increase the diagnostic yield of the FNA, minimizing the number of passes and resulting in a cost-effective strategy. In absence of on-site evaluation the number of passes to perform should be 3 to 4 depending on the type of lesion; 3) Cytology from aspirates obtained by EUS-FNA is the most precise single technique for the diagnosis of pancreatic adenocarcinoma. However, when adequate specimens are not available to reach a cytological diagnosis, the addition of KRAS mutational analysis represents the best strategy; and, 4) It is feasible to detect occult neoplastic cells in EUS-FNA samples by hypermethylation gene promoter analysis. Moreover, addition of methylation analysis to conventional cytology may increase its sensitivity at the expenses of a decrease in its specificity.