Les cils sont des organites essentiels chez la plupart des eucaryotes. Ils sont construits par un mécanisme appelé transport intraflagellaire (IFT). Dans cette thèse, nous avons étudié le rôle de l'IFT chez le protiste Trypanosoma brucei. Par une combinaison d'approches en vidéo-microscopie et en microscopie électronique, nous avons révélé que l'IFT est absent ou s'arrête après ciblage par ARNi de gènes requis pour le transport aller et retour. Dans ces conditions, nous avons démontré que l'IFT n'est pas nécessaire au maintien de la longueur du flagelle mature mais contrôle la distribution de plusieurs protéines non structurales. Les trains IFT transportent la tubuline, le constituant majeur de l'axonème. En collaboration avec l'équipe d'Esben Lorentzen, nous avons mis en évidence l'existence d'un module de liaison à la tubuline sur les protéines IFT74/IFT81. Par FIB-SEM, nous avons démontré que les trains IFT sont présents presque exclusivement sur seulement deux (4 et 7) des 9 doublets de microtubules du flagelle. L'utilisation de méthodes d'imagerie super résolutives par SIM, a permis de montrer sur cellules vivantes, l'existence de deux voies spécifiques pour le trafic IFT aller et retour. Cette restriction s'explique par la présence d'une polyglutamylation plus marquée de la tubuline au niveau de ces doublets. L'inhibition des enzymes responsables de la polyglutamylation freine l'accès des protéines IFT aux flagelles et interfère sévèrement avec la construction de l'organite. Ces travaux démontrent donc un rôle essentiel de la polyglutamylation, qui serait lu par les moteurs du transport intraflagellaire. / Cilia and flagella are essential organelles in most eukaryotes including humans. They are built by an active mechanism termed Intraflagellar Transport or IFT. During this thesis, we have investigated the role and functioning of IFT in the protist Trypanosoma brucei. Using a combination of video-microscopy and electron microscopy, we have revealed that IFT is absent or arrested upon RNAi knockdown of genes required for anterograde and retrograde transport, respectively. In these conditions, we have demonstrated that IFT is not required for maintenance of flagellum length but that IFT controls the distribution of several non-structural proteins, to the contrary of the established dogma. IFT trains transport tubulin, the main component of the axoneme. In collaboration with the team of Esben Lorentzen (MPI Munich), we have revealed the existence of a tubulin-binding domain on proteins IFT74/IFT81. Using FIB-SEM, we have demonstrated that IFT trains are present almost exclusively on only two (4 and 7) out of 9 microtubule doublets. The use of super-resolution imaging methods (work performed at the Janelia Research Institute, USA) allowed us to show for the first time in live cells the existence of two specific bidirectional paths for IFT trafficking. This restriction is explained by differential polyglutamylation on these two doublets. The inhibition of the enzymes responsible for polyglutamylation restricts the access of IFT proteins to flagella, resulting in severe impairment of flagellum elongation. This work demonstrates an essential role for polyglutamylation that could act as a “tubulin code” that would be decrypted by the motors of intraflagellar transport.

Liver biopsy is a very common, but invasive procedure for diagnosing liver disease. However, such a biopsy may result in severe complications and in some cases even death. Therefore, it would be highly desirable to develop a non-invasive method which would provide the same amount of information on staging of the disease and also the location of pathologies. This thesis describes the implementation of such a non-invasive method for visualizing and quantifying liver function by the combination of MRI (Magnetic Resonance Imaging), image reconstruction, and image analysis, and pharmacokinetic modeling. The first attempt involved automatic segmentation, functional clustering (k-means) and classification (kNN) of in-data (liver, spleen and blood vessel segments) in the pharmacokinetic model. However, after implementing and analyzing this method some important issues were identified and the image segmentation method was therefore revised. The segmentation method that was subsequently developed involved a semi-automatic procedure, based on a modified image forest transform (IFT). The data were then simulated and optimized using a pharmacokinetic model describing the pharmacokinetics of the liver specific contrast agent Gd-EOB-DTPA in the human body. The output from the modeling procedure was then further analyzed, using a least-squares method, in order to assess liver function by estimating the fractions of hepatocytes, extracellular extravascular space (EES) and blood plasma in each voxel of the image. The result were in fair agreement with literature values, although further analyses and developments will be required in order to validate and also to confirm the accuracy of the method.

This study was designed to assess follower prototypes as dynamic structures. Connectionist theory is a good framework to understand the process by which followership perceptions are altered by contextual factors. Organizational culture, change in immediate leader and follower prototypes were measured in an applied setting across time to assess the dynamism of the cognitive networks of implicit followership theories. Change in culture and immediate leader was measured at three time points, across six months, during the acquisition of one organization by a second. Change scores were created by computing difference scores from surveys completed at the first time point to the second time point, three months later, to the third and final time point, three months later. There were no significant effects of change in culture on reported follower networks. There was, however, a significant effect of leader change at time points two and three when regressed on individual's follower networks. The overall findings of this study suggest that IFT's like leadership prototypes remain fairly stable across time (Epitropaki, 2004), but are subject to organizational change. / Master of Science

Les flagelles sont des organites essentiels chez la plupart des eucaryotes, y compris chez l’Homme. Ils possèdent une structure cylindrique composée de neuf doublets de microtubules appelée axonème qui est conservée au cours de l’évolution. Ils sont construits par un mécanisme appelé Transport IntraFlagellaire (IFT). Malgré des variations de composition et de longueur entre différents types de cils, la longueur des cils d’un type cellulaire donné est étroitement contrôlée. Toute anomalie de la longueur du flagelle ou de la machinerie IFT peut entraîner de graves dysfonctionnements cellulaires, y compris chez l'homme, où elles sont associées à des maladies génétiques appelées ciliopathies. Au cours de ma thèse, nous avons dans un premier temps étudié le rôle et le fonctionnement de l'IFT chez Trypanosoma brucei, un parasite protozoaire flagellé qui est un excellent modèle pour étudier les cils. En utilisant le FIB-SEM, nous avons démontré que les trains IFT n’étaient présents presque exclusivement que sur deux des neuf doublets microtubules de l'axonème. Puis, l'utilisation de la microscopie à haute résolution nous a permis de démontrer dans des cellules vivantes que ces deux voies sont utilisées pour l’IFT dans les deux sens sur chacun de ces doublets. Nous avons ensuite étudié les mécanismes contrôlant la longueur du flagelle et proposé un nouveau modèle appelé «grow and lock» où le flagelle s'allonge avec un taux de croissance constant jusqu'à ce qu'un signal bloque son élongation ou son raccourcissement. Pour finir nous avons étudié l’implication ce modèle durant le cycle parasitaire, lorsque de la construction de flagelles de très longueurs différentes. / Cilia and flagella are essential organelles in most eukaryotes including humans. They share a canonical cylindrical structure composed of nine doublets of microtubules called the axoneme that is conserved during evolution. They are built by an active mechanism termed Intraflagellar Transport or IFT. Despite some variations in composition and length between different types of cilia, the length for a given cell type is tightly controlled. Any defect in flagellum length or IFT machinery can lead to serious cellular dysfunctions, including in humans where it is associated to genetic diseases called ciliopathies. During my thesis, we have first investigated the role and functioning of IFT in Trypanosoma brucei a flagellated protozoan parasite that is a powerful model to investigate cilia. Using Focus Ion Beam-Scanning Electron Microscopy (FIB-SEM), we have demonstrated that IFT trains are present almost exclusively on only two out of nine microtubules doublets of the axoneme. Then, the use of high-resolution microscopy allowed us to observe in live cells that two tracks are actually used for bidirectional IFT trafficking. We have investigated mechanisms controlling flagellum length and propose a new model named “grow and lock” where the flagellum elongates at a constant growth-rate until a signal blocks further elongation or shortening. Finally this and other models have been investigated during the parasite cycle, when trypanosomes construct flagella with very different lengths.

Mon travail de thèse a porté sur l’étude chez la paramécie d’une protéine à localisation à la fois ciliaire et nucléaire. Les cils sont des organites conservés chez la plupart des eucaryotes qui pointent à la surface cellulaire vers le milieu extérieur. Leur squelette microtubulaire est nucléé par la structure centriolaire sous-jacente, le corps basal, qui transmet sa structure à symétrie 9. Une parenté évolutive lointaine existe entre le compartiment cil, isolé du cytoplasme par un filtre moléculaire appelé zone de transition, et les noyaux dont les contacts avec le cytoplasme sont réduits aux pores nucléaires. Cette homologie fonctionnelle est soutenue par l’existence de mécanismes et de partenaires apparentés dans la communication avec le cytoplasme. Les dysfonctionnements des cils conduisent à des maladies graves appelées ciliopathies. La croissance des cils est réalisée au moyen de complexes protéiques appelés IFT (intraflagellar transport) qui incluent au moins 17 protéines regroupés en deux sous complexes, IFTB, lié au moteur kinésine pour le mouvement antérograde vers la pointe du cil et IFTA lié au moteur dynéine pour le mouvement rétrograde vers la base du cil. L’objet principal de ma thèse, dans le cadre de l’étude de l’IFT chez la paramécie, a porté sur la protéine IFT57, aussi connue sous le nom de HIPPI chez l’homme, où elle assure, en plus de sa fonction ciliaire, une fonction d’activateur transcriptionnel associé à l’apoptose dans la maladie de Huntington. Chez la paramécie, il y a quatre gènes codant IFT57 provenant de duplications globales de génome, IFT57A et B d’une part et IFT57C et D d’autre part, suffisamment proches deux à deux pour que l’inactivation d’un gène puis éteindre également l’autre. Dans un premier temps, j’ai étudié la fonction ciliaire d’IFT57 et j’ai montré qu’il était nécessaire à la croissance ciliaire, mais apparemment pas à sa maintenance, contrairement à d’autres protéines de l’IFT telles qu’IFT46. L’action croisée d’inactivation d’une IFT sur la localisation d’une autre IFT fusionnée à la GFP on permis de suggérer des interactions entre IFT 46 et IFT57 dans le cytoplasme, en amont de leur site habituel d’interaction que représente le corps basal. Je me suis ensuite intéressé à la localisation nucléaire d’IFT57. La protéine IFT57A-GFP entre dans le macronoyau, en plus de sa localisation ciliaire, alors que IFT57C-GFP en est exclue. J’ai essayé de déterminer la différence de séquence qui permet de distinguer ces deux molécules proches pour en envoyer une dans le noyau et pas l’autre. L’aspect le plus marquant de la localisation nucléaire d’IFT57A est le changement de localisation au cours des événements sexuels, où le marquage quitte l’ancien macronoyau pour rejoindre les nouveaux macronoyaux en formation. Cette relocalisation évoque celle observée avec des protéines impliquées dans le métabolisme de petits ARN importants pendant les événements sexuels. Une idée est que IFT57A, protéine associée au transport, puisse gouverner ces mouvements internucléaires et j’ai entrepris d’analyser l’effet de l’inactivation de cette protéine sur les événements sexuels. La difficulté qui est apparue est que l’extinction d’IFT57A est rapidement létale. J’ai réalisé plusieurs approches méthodologiques pour contourner ce problème, dont la mise au point d’une nouvelle méthode d’inactivation par ARN en épingle à cheveux, mais sans pouvoir répondre à la question. En utilisant des constructions chimères entre les deux protéines, puis en comparant les séquences au sein du genre Paramecium, j’ai déterminé une région de 90 acides aminés, L129-N219, avec deux positions critiques pour distinguer fonctionnellement ces deux protéines. / My thesis work consisted in the study of cilia and flagella, organelles highly conserved in many eukaryotes, which protrude at the cell surface as a microtubular backbone, the axoneme, bounded by an extension of the plasma membrane. A major interest in the study of cilia is that they are involved in the regulation of many cell events, such as re-entry in the cell cycle, and that their dysfunction in vertebrates provokes multi-symptomatic diseases called ciliopathies. Ciliary growth is under control of IFT (intraflagellar transport) mechanism, first discovered in Chlamydomonas. The IFT complex includes at least 17 members and is is composed of two subcomplexes, IFTB linked to kinesin for anterograde transport in ciliary building, and IFTA linked to dynein for retrograde transport in recycling. In my thesis on IFT in Paramecium, I focused on IFT57/Hippi, a member of IFTB, which seems to display two different functions in human cells: biogenesis of cilia as member of the IFTB particle and gene regulation in Huntington’s diseases, as part of a complex with Hip1 that binds caspase promoters involved in apoptosis. Some recent work also showed that in the cytoplasm of non-ciliate cells, IFT57, associated with IFT20 and IFT88, regulates T-cell antigen receptor (TCR) recycling in immune synapse. In Paramecium, four genes issued from two successive genome duplications encode IFT57 (IFT57A – D). The isoforms A and B on the one hand and C and D on the other hand are sufficiently close in DNA sequence to get homology dependent RNAi silencing with only one gene of each pair. I first confirmed that IFT57 Paramecium genes have a conserved IFT function in cilia formation, but apparently not in maintenance, in contrast to other IFT proteins such as IFT46. The combination of RNAi and GFP fusion localization allowed us to suggest interactions between IFT46 and IFT57 in the cytoplasm, upstream from the usual site of interaction in the basal body. I also found that IFT57A-GFP, but not IFT57C-GFP, can enter the macronucleus and that the labelling shifts from the old to the new macronucleus during sexual events (autogamy and conjugation). This result must be analysed in light of the mechanism that governs genome rearrangements during nuclear reorganization, which are dependent on transport of RNA as well as of piwi and other RNA-binding molecules from old to new macronucleus. By extension of its ciliary role, one interesting possibility is that IFT57 is a partner involved in this transport. I tried to detect the putative roles of IFT57A in sexual process and worked out a new RNAi method by hairpin RNA expression. I expressed a specific IFT57A hairpin under the control of the NOWA1 promoter (only expressed in autogamy or conjugation) and found surprisingly that this hairpin stops the autogamy process. Since another hairpin contain NSF sequence displayed a similar phenotype, I could not draw any conclusion about the role of IFT57A during autogamy. Using chimeras by exchanging parts of IFT57A and IFT57C, then by comparing this sequence to other in the Paramecium genus, I could determine the shortest region of IFT57A necessary for nuclear localization is the peptide encompassing L129 to N219, with two critical positions to functionally distinguish these two proteins.

Thesis advisor: John Wing / Presented here is research investigating genes that are involved in the development and maintenance of ciliated nerve endings in the nematode Caenorhabditis elegans. C. elegans utilizes a subset of neurons, referred to as ciliated sensory neurons, to sense certain changes in its environment. There are two amphid sensilla (sense organs) that mediate exposure of these ciliated endings to the animal's external environment. Those ciliated endings that penetrate the cuticle are responsible for a myriad of behaviors that range from chemotaxis to osmotic avoidance, but in general function for the reception of environmental cues and stimuli. The intraflagellar transport (IFT) process facilitates the morphogenesis of these ciliated endings, and animals lacking intact ciliated endings may not be able to detect nourishment, hazardous environments, or other worms for mating. Mutant strains used in this study were generated by EMS mutagenesis of wild-type N2 animals and a subsequent screen of those worms displaying significant cilia dysfunction as evidenced by their dye-filling defective (Dyf) phenotype. Cilia-mediated uptake of lipophilic DiI into six pairs of amphid sensory neurons and two pairs of phasmid sensory neurons is expected in wild-type (N2) animals, but in Dyf animals, this dye-filling is disrupted, either through morphological defects, or deleterious mutations in the IFT process. To investigate the morphogenesis of cilia in C. elegans, we analyzed two specific mutant strains, WX737 dyf-3(og022)IV and PK841 dyf-15(pk841)V, that are defective in the uptake of fluorescent dye DiI and abnormal in sensory cilium structure. Through a variety of genetic mapping techniques, we were able to successfully map experimental gene dyf-15(pk841) to an interval of 2.84cM on chromosome V, and identify og022 as an allele of the gene dyf-3. It has been previously shown that dyf-3 expression is detected in 26 chemosensory neurons, including six IL2 neurons, eight pairs of amphid neurons (ASE, ADF, ASG, ASH, ASI, ASJ, ASK and ADL) and two pairs of phasmid neurons (PHA and PHB). Analysis of cilium malformation and the presence of a recognition sequence for the DAF-19 transcription factor suggest that dyf-3 is involved in the intraflagellar transport system complex B. / Thesis (BS) — Boston College, 2008. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

Despite the vast amount of delivered theoretical results, regarding the topic of controller design, more than 90% of the controllers used in industry (petro-chemical, pulp and paper, steel, mining, etc) are of PID type (P, PI, PII, PD). This shows the importance of progressing in the elaboration of methods that consider restricted complexity controllers for practical applications, and that are computationally simple. Iterative Feedback Tuning (IFT) stands out as a new solution that takes into account both constraints. It belongs to the family of model-free controller tuning methods. It was developed at Cesame in the nineties and, since then, many real applications of IFT have been reported. This algorithm minimizes a cost function by means of a stochastic gradient descent scheme. In spite of the fact that the method has had an unexpected success in the tuning of real processes, a number of issues had not been fully covered yet. This thesis focuses on two aspects of this set of uncovered theoretical points: the convergence rate of the algorithm and a robust estimation of its gradient. Optimal prefilters, left as a degree of freedom for the user in the first formulation of IFT, are computed at each experiment. Their application allows a reduction in the covariance of the gradient estimate. Depending on what particular aspect the user is interested in improving, one optimal prefilter is selected. Monte-Carlo simulations have shown an enhancement with regards to a constant prefilter. A flexible arm set-up mounted in our robotics laboratory is used as a test bed to compare a model-based controller design algorithm with a model-free controller tuning method. The comparison is performed with some specifications defined beforehand. The same set-up plus a couple of air-jets serves as a tester for our theoretical results, when the rejection of a perturbation is the ultimate objective. Both cases have confirmed the predicted good behaviour offered by IFT.

Intraflagellar transport (IFT), the key mechanism for ciliogenesis, involves large protein particles moving bi-directionally along the entire ciliary length. IFT particles contain two large protein complexes, A and B, which are constructed with proteins in a core and several peripheral proteins. Prior studies have shown that in Chlamydomonas reinhardtii, IFT46, IFT52, and IFT88 directly interact with each other and are in a subcomplex of the IFT B core. However, ift46, bld1, and ift88 mutants differ in phenotype as ift46 mutants are able to form short flagella, while the other two lack flagella completely. In this study, we investigated the functional differences of these individual IFT proteins contributing to complex B assembly, stability, and basal body localization. We found that complex B is completely disrupted in bld1 mutant, indicating an essential role of IFT52 for complex B core assembly. Ift46 mutant cells are capable of assembling a relatively intact but highly unstable complex B. In contrast, in ift88 mutant cells the complex B core still assembles and remains stable, but the peripheral proteins no longer attach to the B core. Moreover, while complex A and the anterograde IFT motor FLA10 are localized normally to the transition fibers, complex B proteins instead are accumulated at the proximal ends of the basal bodies in ift88. Taken together, these results revealed a step-wise assembly process for complex B, and showed that the complex first localizes to the proximal end of the centrioles and then translocates onto the transition fibers via an IFT88-dependent mechanism. Protein interaction analyses such as the yeast two-hybrid assay in addition to identification and characterization of novel IFT complex B mutants will reveal a more complete picture of the architecture and function of IFT complex B.

Studying chemical enhanced oil recovery (EOR) in a high-temperature/high-salinity (HT/HS) reservoir will help expand the application of chemical EOR to more challenging environments. Until recently, chemical EOR was not recommended at reservoirs that contain high concentrations of divalent cations without the need to recondition the reservoir by flooding it with less saline/ less hardness brines. This strategy was found ineffective in preparing the reservoir for chemical flooding. Surfactants used for chemical flooding operating in high temperatures tend to precipitate when exposed to high concentrations of divalent cations and will partition to the oil phase at high salinities. In this study amphoteric surfactant was used to replace the traditionally used anionic surfactants. Amphoteric surfactants show higher multivalent cations tolerance with better thermal stability. A modified amphoteric surfactant with lower adsorption properties was evaluated for oil recovery. Organic alkali was used to eliminate the water softening process when preparing the chemical solution and reduce potential scale problems caused by precipitation due to incompatibility between chemical slug containing alkali and formation brine. Using organic alkali helped in minimizing softening required when preparing an alkali-surfactant-polymer (ASP) solution using seawater. Solution prepared with organic alkali showed the least injectivity decline when compared to traditional alkalis (NaOH and Na2CO3) and sodium metaborate. Adding organic alkali helped further reduce IFT values when added to surfactant solution. Amphoteric surfactant was found to produce low IFT values at low concentrations and can operate at high salinity / high hardness conditions. When mixed with polymer it improved the viscosity of the surfactant-polymer (SP) solution when prepared in high salinity mixing water (6% NaCl). When prepared in seawater and tested in reservoir temperature (95°C) no reduction in viscosity was found. Unlike the anionic surfactant that causes reduction in viscosity of the SP solution at reservoir temperature. This will not require increasing the polymer concentration in the chemical slug. Unlike the case when anionic surfactant was used and more polymer need to be added to compensate the reduction in viscosity. Berea sandstone cores show lower recovery compared to dolomite cores. It was also found that Berea cores were more sensitive to polymer concentration and type and injectivity decline can be a serious issue during chemical and polymer injection. Dolomite did not show injectivity decline during chemical and polymer flooding and was not sensitive to the polymer concentration when a polymer with low molecular weight was used. CT scan was employed to study the displacement of oil during ASP, SP, polymer and surfactant flooding. The formation and propagation oil bank was observed during these core flood experiments. ASP and SP flooding showed the highest recovery, and formation and propagation of oil bank was clearer in these experiments compared to surfactant flooding. It was found that in Berea sandstone with a permeability range of 50 to 80 md that the recovery and fluid flow was through some dominating and some smaller channels. This explained the deviation from piston-like displacement, where a sharp change in saturation in part of the flood related to the dominated channels and tapered front with late arrival when oil is recovered from the smaller channels. It was concluded that the recovery in the case of sandstone was dominated by the fluid flow and chemical propagation in the porous media not by the effectiveness of the chemical slug to lower the IFT between the displacing fluid and oil.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21709号 / 薬科博第100号 / 新制||薬科||11(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 中山 和久, 教授 竹島 浩, 教授 土居 雅夫 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM