YB-1 est membre de la superfamille de protéines de choc thermique. YB-1 se lie à l'ARN et l'ADN. Des corrélations entre niveau élevé de YB-1, expression élevée de la P-glycoprotéine MDR1 et un mauvais pronostic ont été faites pour plusieurs types de cancer. Le rôle de YB-1 dans la cancérogenèse peut être soutenu par plusieurs mécanismes: i) l'activation de la transcription; ii) la participation à la réparation de l'ADN; iii) la régulation de la traduction. Les deux premiers modèles supposent une localisation nucléaire de YB-1, et cela malgré le fait que YB-1 est apparaît principalement dans cytoplasme dans des conditions physiologiques et les mécanismes de son accumulation nucléaire restent obscurs. Dans ce travail, nous avons tenté d’identifier les mécanismes qui déclenchent la translocation nucléaire de YB-1. Il est apparu que cette localisation nucléaire dépend principalement du niveau d’ARNm dans le cytoplasme et ainsi d’une transcription active, plutôt que de la présence de lésion à l'ADN nucléaire. A l'inverse, le rôle de YB-1 comme régulateur de la traduction est clairement établi. YB-1 peut influencer la traduction et favoriser la progression du cancer, indépendamment de ses fonctions éventuelles dans le noyau. Nous avons démontré par microscopie à force atomique et à l’aide de méthodes biochimiques, que YB-1 se lie aux ARNm d'une manière coopérative à l’ARNm, ce qui a des conséquences directes sur sa capacité à sélectionner des ARNm spécifiques et à moduler la traduction. Au-delà de ces recherches, nous nous sommes appuyés sur notre maitrise de la biologie de YB-1 pour développer une méthode innovante pour mettre en évidence les interactions protéine-protéine dans le contexte cellulaire. Nous avons ainsi confirmé à l’aide de cette méthode la capacité de YB-1 de former des oligomères en présence d'ARNm, et également révélé son interaction potentielle avec Lin28. / YB-1 is a member of the cold-shock protein superfamily. It binds to both RNA and DNA. Correlations between high level of YB-1, elevated expression of P-glycoprotein MDR1 and poor patient prognosis were made for several types of cancer. The role of YB-1 in cancerogenesis can be accounted by several mechanisms: i) activation of transcription; ii) participation in DNA repair; iii) regulation of translation. The first two proposals imply a nuclear localization for YB-1, despite the fact that it appears mainly in the cytoplasm under physiological conditions and the mechanisms for its nuclear accumulation remain unclear. In this work we attempted to identify the mechanisms that trigger the nuclear translocation of YB-1. It appeared that this depends on the level of mRNA in the cytoplasm and thus on active transcription, rather than on the presence of nuclear DNA damages. In contrast to its function in the nucleus, the role of YB-1 in the regulation of translation was clearly established. YB-1 may therefore orchestrate a translation bias in order to promote cancer progression independently of its putative functions in the nucleus. Here we demonstrated, using atomic force microscopy and biochemical methods, that YB-1 binds mRNA in a highly cooperative manner and this has direct consequences on mRNA selection and following translational modulation. Beyond this research, we took advantage of our knowledge of the biology of YB-1 to develop a new method to detect protein-protein interactions in cellular context, using YB-1 as model protein. Besides the fact that we confirmed ability of YB-1 to make oligomers in the presence of mRNA, we also highlighted its potential interaction with Lin28, using this method.

YB-l est une protéine multifonctionnelle exprimée majoritairement au cytoplasme qui régule de façon globale la traduction. YB-l est également exprimée au noyau et agit comme facteur de transcription liant les séquences promotrices CCAA T inversées. De plus, YB-l participe à l'épissage alternatif et est impliquée dans la réparation de l'ADN. Il existe une corrélation entre l'expression nucléaire de YB-I dans les cellules tumorales et la résistance à la cisplatine, un composé largement utilisé en chimiothérapie. De plus, la déplétion de YB-I par un ARN antisens sensibilise les cellules à la cisplatine. In vitro, YB- 1 lie préférentiellement l'ADN contenant un pontage causé par la cisplatine et possède une activité exonucléase 3' -5'. Malgré ces nombreuses associations, le mécanisme par lequel YB-l confère la résistance à la cisplatine demeure toujours inexpliqué. Cette thèse contient les travaux entrepris dans le but de caractériser les fonctions de la protéine YB-I dans le mécanisme de résistance à la cisplatine. Nous avons démontré in vitro que la protéine YB-I purifiée avait la capacité de séparer préférentiellement différents duplex d'ADN contenant un pontage à la cisplatine ou un mésappariement et qu'elle possédait une activité endonucléase. Par chromatographie d'affinité, les protéines de la réparation MSH2, WRN, Ku80 et la polymérase 8 ont été co-purifiées avec YB-I. L'étude approfondie de l'interaction entre les protéines YB-I et WRN, protéine responsable du syndrome de vieillissement prématuré appelé Werner, a permis d'identifier la formation d'un complexe entre les protéines YB-l, p53 et WRN dans des foyers nucléaires dt:1 cellules exposées aux rayons ultraviolets. Ensuite, la caractérisation de l'interaction entre YB-I et hNTHI, une ADN glycosylase/ AP lyase impliquée dans l'initiation de la voie de réparation par excision de base, a démontré que YB-l se liait au domaine N-terminal auto-inhibiteur de hNTHI augmentant ainsi son activité in vitro. De plus, la déplétion de -hNTHI sensibilise spécifiquement les cellules à la cisplatine et aux rayons ultraviolets, même chez les cellules surexprimant YB-l. Finalement, nous avons déterminé que les activités de séparation des brins d'ADN, d'endonucléase et d'épissage alternatif ne sont pas essentielles à YB-I pour conférer une résistance à la cisplatine. Par contre, le changement global du profil d'expression des ARNm accompagnant la surexpression de YB-I et de sa forme tronquée dans des cellules de cancers du sein peut être associé à la résistance à la cisplatine observée.

Ce travail de thèse concerne le développement de sources femtoseconde de forte puissance moyenne ou de forte énergie avec des matériaux pompés par diodes laser, dopés à l’ytterbium. Plus particulièrement au cours de cette thèse deux types de matrices ont été utilisés, le CALGO (CaGdAlO4) et les fluorures, possédant le potentiel de générer des impulsions courtes (100aine de femtoseconde). Les caractéristiques spectroscopiques et thermiques du CALGO dopés à l’ytterbium permettent d’envisager le développement d’oscillateur femtoseconde court de forte puissance moyenne. Dans ce contexte, la technologie des disques minces permet d’obtenir avec d’autres matrices, des résultats très intéressants. C’est pourquoi durant cette thèse le choix de maitriser cette nouvelle technologie, avec l’utilisation de ce cristal, a été fait. Dans ce cadre, des résultats très prometteurs ont été obtenus. L’oscillateur femtoseconde Yb :CALGO de plus forte puissance moyenne a en effet été développé (28 W), pour une énergie non négligeable, supérieure au µJ et une durée d’impulsions de 300 fs. Des améliorations sont à prévoir avec l’utilisation de nouveaux cristaux plus dopés et plus fins, mais d’hors et déjà les résultats obtenus sont au niveau de l’état de l’art des oscillateurs femtoseconde de forte puissance moyenne.Le cristal de CaF2 quant à lui, possède un grand intérêt pour le développement d’amplificateurs énergétiques courts, puisqu’il a la capacité de stocker beaucoup d’énergie. Deux types d’amplificateurs ont alors été développés, avec des objectifs bien différents. Le premier permet d’obtenir un fort gain (~106), avec une énergie extraite proche du mJ (amplificateur régénératif), alors que le second a pour but d’extraire le maximum d’énergie (amplificateur multipassage), dans notre cas jusqu’à 160 mJ, avec un gain plus faible (~10).Le potentiel de ces matériaux pour la génération d’impulsions courtes et/ou de forte puissance moyenne a alors été démontré. / This work concerns the development of high average power or high energy laser with diode-pumped ytterbium-doped materials. Two host matrices were studied, CALGO (CaGdAlO4) and fluoride, which permit the generation of ultra-short pulses. Spectroscopic and thermal properties of ytterbium doped CALGO crystals are adapted for the development of high average power oscillator. In this area, thin disk technology seems to be particularly interesting for the development of such oscillator. That’s why we choose to master this technology with ytterbium-doped CALGO crystals. Thus, Yb:CALGO oscillator with the highest average power was developed (28 W), with more than µJ energy and pulse duration of 300 fs. Using more doped and thinner crystals should permit to improve our performances, however they are already at the state of the art of high average power oscillator.Ytterbium doped CaF2 has a great interest for short-pulse high energy amplifier, thanks to its capacity to store energy. Two types of amplifier were developed. A regenerative amplifier with high gain (~106), mJ energy level, and a multipass amplifier with lower gain (~10) but permitting to extract really high energy (up to 160 mJ).Potential of these materials for the development of short pulse high average power and/or high energy system was demonstrated.

Asthma, hay fever and atopic dermatitis are complex diseases. Presumably, genetic factors that protected from infectious agents in the past do promote allergic disease in the absence of infectious agents. Linkage and candidate gene studies identified common as well as disease specific genetic determinants of atopic diseases. However, a large number of chromosomal regions and candidate genes were related to asthma and associated traits. International collaborations and meta analyses appear to be mandatory to identify the major genes for asthma and atopy. Also, gene-gene and gene-environment interaction analyses are of great importance to identify genetic risk factors for allergic diseases. Birth cohorts are particularly valuable since exposure to various environmental agents have been documented since early childhood. This work summarizes genetic and epidemiologic studies of the German Multicenter Allergy Study (MAS) that contributed significantly to our understanding of the development of asthma and allergy in childhood. This work summarizes candidate and linkage studies performed in the MAS-cohort and outlines early risk factors for allergic diseases in childhood that were observed in MAS-participants.

The human epidermal growth factor receptor (HER-2) is over-expressed in 20-30% of breast carcinomas and is a prognostic marker for poor patient outcome. We previously identified the transcription/translation factor Y-box binding protein-1 (YB-1) to be a novel substrate of AKT which binds to epidermal growth factor receptor (EGFR) and HER-2 promoters once phosphorylated (Wu J et al. 2006). YB-1 is over-expressed in approximately 40% of breast cancers; its expression is strongly correlated with HER-2 and is associated with poor patient survival. In order to gain a deeper understanding of the functional role of YB-1 in HER-2 over-expressing breast cancer, we silenced the expression of this factor in BT474-m1 and MDA-MB-453 cells. The loss of YB-1 inhibited the growth of BT474-m1 and MDA-MB-453 cells in monolayer and/or in soft agar. Consistent with this, we found a decrease in the expression of YB-1 responsive gene egfr and/or her-2 in BT474-m1 and MDA-MB-453 cells, which could begin to explain how growth is promoted by this factor. Furthermore, loss of YB-1 expression induced apoptosis in BT474-m1 cells. Beyond its role in tumor growth, YB-1 is also strongly linked to drug resistance. We therefore addressed whether it could play a part in Herceptin sensitivity. Herceptin is currently being used to treat patients with HER-2 positive breast cancer; however, only 30% of the patients respond to the therapy and many of them develop resistance within the first year of treatment. Therefore, it is of utmost importance to understand the biology of HER-2 over-expressing breast cancer to develop novel therapies that can benefit more patients. First we established that Herceptin inhibited BT474-m1 cell growth in anchorage-independent conditions whereas MDA-MB-453 cells were resistant to this treatment. We subsequently demonstrated that knock-down of YB-1 increased sensitivity of BT474-m1 cells to Herceptin while MDA-MB-453 cells failed to respond to the combination treatment. The mechanism for Herceptin resistance in MDA-MB-453 cells still remains elusive and requires further investigation. Thus far, we conclude that YB-1 is needed for the growth and survival of HER-2 positive BT474-m1 and MDA-MB-453 breast cancer cells by inducing members of the HER family.

The human epidermal growth factor receptor (HER-2) is over-expressed in 20-30% of breast carcinomas and is a prognostic marker for poor patient outcome. We previously identified the transcription/translation factor Y-box binding protein-1 (YB-1) to be a novel substrate of AKT which binds to epidermal growth factor receptor (EGFR) and HER-2 promoters once phosphorylated (Wu J et al. 2006). YB-1 is over-expressed in approximately 40% of breast cancers; its expression is strongly correlated with HER-2 and is associated with poor patient survival. In order to gain a deeper understanding of the functional role of YB-1 in HER-2 over-expressing breast cancer, we silenced the expression of this factor in BT474-m1 and MDA-MB-453 cells. The loss of YB-1 inhibited the growth of BT474-m1 and MDA-MB-453 cells in monolayer and/or in soft agar. Consistent with this, we found a decrease in the expression of YB-1 responsive gene egfr and/or her-2 in BT474-m1 and MDA-MB-453 cells, which could begin to explain how growth is promoted by this factor. Furthermore, loss of YB-1 expression induced apoptosis in BT474-m1 cells. Beyond its role in tumor growth, YB-1 is also strongly linked to drug resistance. We therefore addressed whether it could play a part in Herceptin sensitivity. Herceptin is currently being used to treat patients with HER-2 positive breast cancer; however, only 30% of the patients respond to the therapy and many of them develop resistance within the first year of treatment. Therefore, it is of utmost importance to understand the biology of HER-2 over-expressing breast cancer to develop novel therapies that can benefit more patients. First we established that Herceptin inhibited BT474-m1 cell growth in anchorage-independent conditions whereas MDA-MB-453 cells were resistant to this treatment. We subsequently demonstrated that knock-down of YB-1 increased sensitivity of BT474-m1 cells to Herceptin while MDA-MB-453 cells failed to respond to the combination treatment. The mechanism for Herceptin resistance in MDA-MB-453 cells still remains elusive and requires further investigation. Thus far, we conclude that YB-1 is needed for the growth and survival of HER-2 positive BT474-m1 and MDA-MB-453 breast cancer cells by inducing members of the HER family.

Early detection is one of the most important factors for successful treatment of cancer. Currently, scientists are searching for molecular markers that can help identify and predict outcome and chance of recurrence in patients. In this study, we demonstratet he potential impact of Y-Box binding protein-1 (YB-1) as a marker of aggressiveness and cancer recurrence in breast malignancies by screening one of the largest tissue microarrays in North America. YB-1 is an oncogenic transcription/translation factor, which is over-expressed in the majority of malignancies, including breast cancer. In the cohort of 4049 primary breast tumours, we show that YB-1 is a strong marker of aggressiveness, poor survival and cancer recurrence in all subtypes of human breast cancer with a particularly high frequency of expression in the ER negative basal-like and HER-2 breast cancer subtypes. This suggests that targeting YB-1 may provide a new avenue for therapeutic intervention in these breast cancers that are currently challenging to treat. Cox regression multivariate analysis indicates that YB-1 is second only to nodal status as a strong independent prognostic marker for poor outcome and relapse compared to established clinico-pathological biomarkers, including tumour size, age, grade, ER and HER-2 status. This finding suggests that YB-1 has great potential to be in a priority list of biomarkers for identifying the patients with a higher risk of relapse and poor outcome. Subsequently, we find an association between YB-1 and urokinase Plasminogen Activator (uPA) expression in the basal-like subtype. We then show that YB-1 is involved in the regulation of uPA expression. More importantly, silencing YB-1 or uPA results in a significant reduction in cancer cell invasion. As there are no commercially available YB-linibitors we examine the efficacy of BMS-536924, a small molecule inhibitor for activated IGF-1R/IR on SUM149 cells. We demonstrate that activated IGF-1R is associated with poor survival in primary breast tumours and, that BMS-536924 reduces uPA expression through inhibition YB-1 in SUM149 cells. We therefore conclude that YB-1 is a bio-marker for poor survival and relapse. We also indicate that YB-1 has potential use as a molecular marker in a clinical setting. Inhibiting YB-1 may provide an ideal opportunity for targeted therapy in breast cancer.

Early detection is one of the most important factors for successful treatment of cancer. Currently, scientists are searching for molecular markers that can help identify and predict outcome and chance of recurrence in patients. In this study, we demonstratet he potential impact of Y-Box binding protein-1 (YB-1) as a marker of aggressiveness and cancer recurrence in breast malignancies by screening one of the largest tissue microarrays in North America. YB-1 is an oncogenic transcription/translation factor, which is over-expressed in the majority of malignancies, including breast cancer. In the cohort of 4049 primary breast tumours, we show that YB-1 is a strong marker of aggressiveness, poor survival and cancer recurrence in all subtypes of human breast cancer with a particularly high frequency of expression in the ER negative basal-like and HER-2 breast cancer subtypes. This suggests that targeting YB-1 may provide a new avenue for therapeutic intervention in these breast cancers that are currently challenging to treat. Cox regression multivariate analysis indicates that YB-1 is second only to nodal status as a strong independent prognostic marker for poor outcome and relapse compared to established clinico-pathological biomarkers, including tumour size, age, grade, ER and HER-2 status. This finding suggests that YB-1 has great potential to be in a priority list of biomarkers for identifying the patients with a higher risk of relapse and poor outcome. Subsequently, we find an association between YB-1 and urokinase Plasminogen Activator (uPA) expression in the basal-like subtype. We then show that YB-1 is involved in the regulation of uPA expression. More importantly, silencing YB-1 or uPA results in a significant reduction in cancer cell invasion. As there are no commercially available YB-linibitors we examine the efficacy of BMS-536924, a small molecule inhibitor for activated IGF-1R/IR on SUM149 cells. We demonstrate that activated IGF-1R is associated with poor survival in primary breast tumours and, that BMS-536924 reduces uPA expression through inhibition YB-1 in SUM149 cells. We therefore conclude that YB-1 is a bio-marker for poor survival and relapse. We also indicate that YB-1 has potential use as a molecular marker in a clinical setting. Inhibiting YB-1 may provide an ideal opportunity for targeted therapy in breast cancer.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第16503号 / 工博第3496号 / 新制||工||1529(附属図書館) / 29160 / 京都大学大学院工学研究科電子工学専攻 / (主査)教授 北野 正雄, 教授 川上 養一, 准教授 酒井 道 / 学位規則第4条第1項該当

We present global and regional correlations between whole-rock values of Sr/Y and La/Yb and crustal thickness for intermediate rocks from modern subduction-related magmatic arcs formed around the Pacific. These correlations bolster earlier ideas that various geochemical parameters can be used to track changes of crustal thickness through time in ancient subduction systems. Inferred crustal thicknesses using our proposed empirical fits are consistent with independent geologic constraints for the Cenozoic evolution of the central Andes, as well as various Mesozoic magmatic arc segments currently exposed in the Coast Mountains, British Columbia, and the Sierra Nevada and Mojave- Transverse Range regions of California. We propose that these geochemical parameters can be used, when averaged over the typical lifetimes and spatial footprints of composite volcanoes and their intrusive equivalents to infer crustal thickness changes over time in ancient orogens.