Les hélicases sont des enzymes ubiquitaires catalysant la séparation de l’ADN double-brin et impliquées dans la réplication, la réparation de l’ADN et dans le maintien des télomères. Chez l’Homme, 3 hélicases présentent des mutations responsables de syndromes cliniques : WRN pour le syndrome de Werner, BLM pour le syndrome de Bloom et RECQL4 pour le syndrome de Rothmund-Thomson. Tous ces syndromes associent un vieillissement pathologique accéléré à un risque accru de développement de cancer notamment par une augmentation de l’instabilité génomique. Les connaissances sur les mécanismes moléculaires et cellulaires impliqués dans ces maladies du vieillissement sont encore très partielles, notamment en ce qui concerne le lien entre l’instabilité génomique et le vieillissement. Au cours de ce projet, l'utilisation de prélèvements sanguins et cutanés de patients atteints de ces pathologies rares a permis de générer des modèles de cellules souches pluripotentes induites (iPS). Ces cellules présentent l’avantage de s’auto-renouveler et de pouvoir théoriquement se différencier dans tous les types cellulaires d’un organisme. Parallèlement, un témoin de sénescence a été généré de la même manière avec des cellules d’un patient souffrant du syndrome de la progéria de Hutchinson-Gilford. Après caractérisation de ces cellules, nous avons identifié des ensembles de phénotypes cellulaires et moléculaires dans le but de récapituler in vitro les pathologies. Nous avons également engagé les cellules iPS dans des voies de différenciation proches des tissus atteints dans les pathologies in vivo. Enfin, nous avons étudié la stabilité génomique de ces lignées dans les différents types cellulaires cultivés. Ainsi nous avons observé que la lignée Bloom est le siège de recombinaisons particulièrement fréquentes et est caractérisée par une instabilité du génome dans tous les types cellulaires étudiés. Egalement, la lignée Werner semblerait se distinguer par une instabilité de ses télomères. Enfin, l’ensemble des lignées des pathologies du vieillissement prématuré présenterait un défaut mitochondrial. / Helicases process the double-stranded DNA dissociation. They are involved in replication, DNA repair and maintenance of telomeres. In human, 3 helicases display mutations responsible for clinical syndromes: WRN for the Werner syndrome, BLM for the Bloom syndrome and RECQL4 for the Rothmund-Thomson syndrome. All these diseases cause premature ageing and high risk of cancer. Molecular and cellular mechanisms involved in these diseases are not well defined. Particularly, little is known concerning the link between genomic instability and ageing. During this project, we used blood samples and skin biopsies of affected patients to generate models by reprogramming cells to induced pluripotent stem cells (iPSCs). These cells have the advantage of self-renewing and theoretically could be differentiated in all cell types. At the same time, an iPSC senescence control was performed from cells of a Hutchinson-Gilford Progeria syndrome patient. iPSCs were characterized for pluripotency. In the aim of recapitulate these pathologies in vitro, we identified sets of cellular and molecular phenotypes. We also engaged differentiation of iPSCs in cell pathways closed to the affected tissues in vivo. Finally, we studied the genomic stability of iPSCs and derived cells. We observed that Bloom cells are susceptible to frequent recombinations and are characterized by a genome instability through all studied cell types. Werner cells showed an instability of telomeres length. Finally, all premature ageing diseases displayed mitochondrial defects.

Ips typographus est le principal ravageur de l'épicéa en Europe, s’attaquant en masse aux arbres vivants en période d’épidémie, par le biais d’une communication chimique. Comme de nombreuses autres espèces de scolytes « agressives », le typographe se reproduit uniquement sur des arbres affaiblis ou des chablis en périodes d’endémie, quand les densités de population ne sont pas suffisantes pour surmonter les défenses des arbres vivants. Ces ressources sont en général présentées dans la littérature comme étant dispersées et imprédictibles dans le temps et l’espace. De plus, ces ressources affaiblies sont sans doute disponibles pour de nombreuses espèces compétitrices. Ces caractéristiques imposent des contraintes importantes sur la dynamique de population et l’écologie du typographe et principalement sur sa stratégie de prospection. Cela dit, cette espèce et ces questions sont très peu étudiées en période d’endémie.Durant ma thèse de doctorat, je me suis attelé à tout d’abord caractériser les facteurs déterminant la distribution spatio-temporelle des chablis, qui va influencer l’évolution des stratégies de dispersion. Cet aspect s’est prolongé par l’étude de la « durée de vie » des chablis, c’est-à-dire la période durant laquelle ils sont colonisables par les scolytes après déracinement. Cet aspect a été mis en parallèle avec les capacités de défenses de ces mêmes arbres et leurs effets sur la sélection de l’hôte et le succès des scolytes. Enfin, les données obtenues par ces études ont été intégrées à un modèle spatialement explicite visant à valider les hypothèses comportementales relatives à la prospection pour les ressources chez le typographe, à savoir une dispersion aléatoire à grande distance suivie d’un recrutement au niveau des ressources nouvellement trouvées.Les résultats obtenus au cours de cette thèse confirment l’importance de la prospection sociale et de l’agrégation dans la découverte et l’exploitation de ressources dispersées, dont la dégradation intrinsèque représente une faible contrainte temporelle. Ces comportements ont sans doute été développés chez des insectes saproxyliques en raison de l’avantage qu’ils apportent en termes de découverte de ressources. Dans un second temps, ces comportements auraient pu permettre l'attaque d‘arbres vivants, notamment en réponse à une pression de compétition inter-spécifique importante sur des ressources peu défendues. / Option Biologie des organismes du Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Background and objectives: In 2006 Shinya Yamanaka successfully reprogrammed mouse fibroblasts back to an embryonic stem cell-like state (called induced pluripotent cells, iPS cells) using retrovirus to introduce four genes that encode critical transcription factor proteins (Oct4, Sox2, KLF4, and c-Myc). This ability to reprogram has promising future applications in clinical and biomedical research for study of diseases, development of candidate drugs and to support therapeutic treatments in regenerative medicine. However, the clinical applications have to meet GMP requirements without the risk of insertional mutagenesis associated with retrovirus. Chromatin modifying agents are widely used in many protocols to generate iPS cells and culture of blood CD34+ cells with chromatin-modifying agents can lead to an increase in marrow repopulating cells and in the case of valproic acid increased erythroid cell colony formation. We undertook research to help understand what effects these reagents have on mobilised peripheral blood (mPB) CD34+ cells and optimised the expansion medium protocol to facilitate reprogramming work. This project aims to utilize peripheral blood mononuclear cells (MNC), one of the most easily accessible tissues to generate iPS cells using an efficient non-viral, feeder cell free methodology, with the ultimate goal of moving this methodology towards clinical use. Materials and Methods: G-CSF mobilised peripheral blood, buffy coat, cord blood and fetal liver were obtained from patients and donors under informed consent and ethics committee approval. Haematopoietic stem/progenitor cells CD34+ or CD133+) isolated by magnetic separation were flow cytometry sorted into CD34+/CD133+, CD34+/CD133-, and CD34-/CD133+ sub-populations and their lineage potential were assessed in colony forming unit assays. The effect of epigenetic modifiers valproic acid and 5-aza-2-deoxycytidine used singly or in combination with each other and with IL3 on phenotype and lineage potential of cultured CD34+ cells from mobilised peripheral blood were assessed by flow cytometry and colony-forming unit assays. Prior to reprogramming mononuclear cells from peripheral blood or CD34+ cells from blood were expanded in culture medium supplemented with stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (Flt3L) and Interleukin- 3 (IL-3) for several days. Actively proliferating cells were reprogrammed by electroporation using episomal vectors with an oriP/EBNA-1 backbone to deliver five reprogramming genes, Oct4, Sox2, Lin28, L-Myc, and Klf4. Electroporated cells were seeded onto matrigel coated plates immediately after transfection or were reseeded after three days’ culture. Subsequently, cells were cultured in specific medium on different days. When iPS colonies appeared, they were picked and cultured as for ES cells. Once established, iPS cell lines were immunophenotyped using flow cytometry and immunofluorescence and their potential to differentiate into the three germ layers was assessed in vitro. Results and Conclusion: The largest subpopulation of CD34+ cells was CD34+/CD133+ population which was essentially committed to myeloid colony production, while much smaller CD34+/CD133- subpopulation had a greater capacity to generate erythroid colonies. Optimised cytokine cocktail for expansion of CD34+ cells included IL-3, important in improving expansion and maintaining functionality of CD34+ cells. The optimised cytokine cocktail comprised 100 ng/ml SCF, 10 ng/ml Flt3L, and 20 ng/ml IL-3, which maintained CD34+ cells and MNC in an active proliferating state. In addition, valproic acid and IL3 were found to act synergistically, to increase the numbers of CD34+/CD36+ positive cells. However, we found that an apparent increase in red cell colony formation actually resulted from a decrease in white cell colonies, so no overall increase in red cell colonies was seen when equivalent numbers of CD34+ cells were plated. Proliferating MNC maintained in optimised cytokine cocktail were amenable to electroporation for the effective delivery of episomal transcription factors (Oct4, Sox2, Klf4, L-Myc, and Lin28) within a backbone of oriP/EBNA-1. We successfully developed an efficient and simple method for reprogramming MNC from fresh or frozen samples to generate induced pluripotent cells using episomal vectors in a feeder-free system without any requirement for small molecules and the highest reprogramming efficiency is 0.033% (65 colonies from 2 ◊ 105 seeding MNC). The cytokine cocktail and reprogramming methods work better in CD34+ cells from cord blood or fetal liver, and we obtained 148 iPS colonies from 105 seeding cells (0.148%) at most. In addition, fibroblasts from adult and fetal liver can be successfully reprogrammed using the same reprogramming method. The use of episomal vectors with an oriP/EBNA-1 backbone to deliver reprogramming genes, and efficient electroporation were the most important factors in efficiency of the reprogramming process. In addition, it is pivotal to initiate transfection when cells are actively proliferating. The iPS cell lines we generated maintained the successful expression of ES markers including Oct4, Nanog, SSEA3. SSEA4, TRA-1-60 and TRA-1-81, and had the capacity to successfully differentiate into cell types of ectoderm, mesoderm and endoderm layers in vitro.

While GPS long has been an industry standard for localization of an entity or person anywhere in the world, it loses much of its accuracy and value when used indoors. To enable services such as indoor navigation, other methods must be used. A new standard of the Wi-Fi protocol, IEEE 802.11mc (Wi-Fi RTT), enables distance estimation between the transmitter and the receiver based on the Round-Trip Time (RTT) delay of the signal. Using these distance estimations and the known locations of the transmitting Access Points (APs), an estimation of the receiver’s location can be determined. In this thesis, a smartphone Wi-Fi RTT based Indoor Positioning System (IPS) is presented using an Unscented Kalman Filter (UKF). The UKF using only RTT based distance estimations as input, is established as a baseline implementation. Two extensions are then presented to improve the positioning performance; 1) a dead reckoning algorithm using smartphone sensors part of the Inertial Measurement Unit (IMU) as an additional input to the UKF, and 2) a method to detect and adjust distance measurements that have been made in Non-Line-of-Sight (NLoS) conditions. The implemented IPS is evaluated in an office environment in both favorable situations (plenty of Line-of-Sight conditions) and sub-optimal situations (dominant NLoS conditions). Using both extensions, meter level accuracy is achieved in both cases as well as a 90th percentile error of less than 2 meters.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22834号 / 医科博第118号 / 新制||医科||8(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 髙橋 良輔, 教授 渡邉 大, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Oshima N, Yamada Y, Nagayama S, Kawada K, Hasegawa S, et al. (2014) Induction of Cancer Stem Cell Properties in Colon Cancer Cells by Defined Factors. PLoS ONE 9(7): e101735. doi:10.1371/journal.pone.0101735 / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18547号 / 医博第3940号 / 新制||医||1006(附属図書館) / 31447 / 京都大学大学院医学研究科医学専攻 / (主査)教授 千葉 勉, 教授 野田 亮, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The aim of this report was to investigate whether the management of protected forests cause increased risk of infestation by the European spruce bark beetle (Ips typographus) in production forests, and whether the risk of infestation in production forests is greater in the vicinity of protected forests. The survey was conducted by using GIS-data covering suspected attacks and forests that are susceptible to infestations. The proportion of infested forests in three different buffer zones located around the protected areas were compared. The results show no evidence suggesting that protected areas serve as a hatchery for spruce bark beetles and no increased risk of infestations in the vicinity of protected forests were detected.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23412号 / 医博第4757号 / 新制||医||1052(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)特定拠点教授 妻木 範行, 教授 戸口田 淳也, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23417号 / 医博第4762号 / 新制||医||1052(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 松田 秀一特定拠点, 教授 妻木 範行, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

This student work focuses on security against network attacks. It focus on network attacks and ways to prevent them. Subsequently, it deals with the Snort variant of the IPS/IDS system. It deal with the connection between Mikrotik and the Snort Linux server