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.

International Telemetering Conference Proceedings / October 25-28, 1999 / Riviera Hotel and Convention Center, Las Vegas, Nevada / Today’s large-scale signal processing systems impose massive bandwidth requirements on both internal and external communication systems. Most often, these bandwidth requirements are met by scalable input/output architectures built around high-performance, standards-based technology. Several such technologies are available and are in common use as internal and/or external communication mechanisms. This paper provides an overview of some of the more common scalable technologies used for internal and external communications in real-time data acquisition systems. With respect to internal communications mechanisms this paper focuses on three ANSI-standard switched fabric technologies: RACEway (ANSI/VITA 5-1994), SKYchannel (ANSI/VITA 10-1995) and Myrinet (ANSI/VITA 26-1998). The discussion then turns to how Fibre Channel, HiPPI, and ATM are used to provide scalable external communications in real-time systems. Finally, glimpse of how these technologies are evolving to meet tomorrow’s requirements is provided.

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.

International Telemetering Conference Proceedings / October 27-30, 1997 / Riviera Hotel and Convention Center, Las Vegas, Nevada / For many years VME has been the platform of choice for high-performance, real-time data acquisition systems. VME’s longevity has been made possible in part by timely enhancements which have expanded system bandwidth and allowed systems to support ever increasing throughput. One of the most recent ANSI-standard extensions of the VME specification defines RACEway, a system of dynamically switched, 160 Mbyte/second board-to-board interconnects. In typical systems RACEway increases the internal bandwidth of a VME system by an order of magnitude. Since this bandwidth is both scaleable and deterministic, it is particularly well suited to high-performance, real-time systems. The potential of RACEway for very high-performance (200 Mbps to 1 Gbps) real-time systems has been recognized by both the VME industry and a growing number of system integrators. This recognition has yielded many new RACEway-ready VME products from more than a dozen vendors. In fact many significant real-time data acquisition systems that consist entirely of commercial-off-the-shelf (COTS) RACEway products are being developed and fielded today. This paper provides an overview of RACEway technology, identifies the types of RACEway equipment currently available, discusses how RACEway can be applied in high-performance data acquisition systems, and briefly describes two systems that acquiring and capturing real-time data streams at rates from 200 Mbps to 1 Gbps using RACEway.