A monoclonal antibody marker for the exclusion-zone filaments of Trypanosoma brucei

<p>Abstract</p> <p>Background</p> <p><it>Trypanosoma brucei </it>is a haemoflagellate pathogen of man, wild animals and domesticated livestock in central and southern Africa. In all life cycle stages this parasite has a single mitochondrion that contains a uniquely organised genome that is condensed into a flat disk-like structure called the kinetoplast. The kinetoplast is essential for insect form procyclic cells and therefore is a potential drug target. The kinetoplast is unique in nature because it consists of novel structural proteins and thousands of circular, interlocking, DNA molecules (kDNA). Secondly, kDNA replication is critically timed to coincide with nuclear S phase and new flagellum biogenesis. Thirdly, the kinetoplast is physically attached to the flagellum basal bodies <it>via </it>a structure called the tripartite attachment complex (TAC). The TAC consists of unilateral filaments (within the mitochondrion matrix), differentiated mitochondrial membranes and exclusion-zone filaments that extend from the distal end of the basal bodies. To date only one protein, p166, has been identified to be a component of the TAC.</p> <p>Results</p> <p>In the work presented here we provide data based on a novel EM technique developed to label and characterise cytoskeleton structures in permeabilised cells without extraction of mitochondrion membranes. We use this protocol to provide data on a new monoclonal antibody reagent (Mab 22) and illustrate the precise localisation of basal body-mitochondrial linker proteins. Mab 22 binds to these linker proteins (exclusion-zone filaments) and provides a new tool for the characterisation of cytoskeleton mediated kinetoplast segregation.</p> <p>Conclusion</p> <p>The antigen(s) recognised by Mab 22 are cytoskeletal, insensitive to extraction by high concentrations of non-ionic detergent, extend from the proximal region of basal bodies and bind to the outer mitochondrial membrane. This protein(s) is the first component of the TAC exclusion-zone fibres to be identified. Mab 22 will therefore be important in characterising TAC biogenesis.</p

Nouvelle méthode d'intégration énergétique pour la rétro-installation des procédés industriels et la transformation des usines papetières

L’augmentation rapide de la création de richesses ces dernières décennies a suscité le besoin d’améliorer la gestion globale des ressources naturelles et d’accroître l’efficacité de la production industrielle. Afin de répondre à ce besoin, les méthodes d’intégration énergétique pour les systèmes industriels ont été développées. Elles ont un succès évident dans le domaine de la conception de nouvelles usines : les principes d’intégration sont enseignés et appliqués, et l’intensité énergétique des nouveaux procédés a beaucoup diminué. En comparaison, les méthodes d’intégration par rétro-installation requièrent encore des développements conceptuels bien que des progrès significatifs aient été réalisés. Le principe des approches actuelles consiste souvent à identifier des modifications qui permettent au système existant de se rapprocher d’une situation de référence. Leur utilisation est difficile, et les difficultés méthodologiques augmentent si le réseau inclut différents types de transfert de chaleur, par exemple des échangeurs à contact indirect et des mélanges non-isothermes, comme dans les usines papetières où les réseaux d’eau et d’énergie sont étroitement liés. L’industrie papetière essaie d’augmenter sa rentabilité par la réduction des coûts de production et l’amélioration de la chaîne logistique. À la suite de récents développements de procédé, le bioraffinage forestier offre à l’industrie papetière des opportunités de se diversifier, d’élargir son portefeuille de bioproduits et de moderniser son système énergétique. Cependant l’identification de stratégies énergétiques pour un site industriel, incluant les possibilités de transformation d’une usine papetière, nécessite l’utilisation de méthodes d’intégration adéquates en situation de rétro-installation. L’objectif de cette thèse est développer une méthode d’intégration énergétique pour la rétro-installation des procédés industriels en général et la transformation des usines papetières, et de l’appliquer à des études de cas. L’énergie est conservée et dégradée dans un procédé. La chaleur est soit convertie en électricité, soit stockée sous forme chimique, soit rejetée à l’environnement où sa dégradation est maximale. L’analyse des dégradations successives de l’énergie entre les utilités chaudes et l’environnement au travers des opérations de procédé et des échangeurs de chaleur existants est de grande importance pour réduire la consommation de chaleur. 2 La méthode pontale d’intégration énergétique par rétro-installation a été développée dans le cadre de cette thèse. Cette méthode est la seule à considérer cette analyse des dégradations. Le processus fondamental de réduction de consommation d’énergie est pour la première fois rendu explicite; il est à la base de la méthode développée. La méthode pontale inclut la définition du « pont », qui est un ensemble de modifications conduisant à une réduction de la consommation d’énergie dans un réseau d’échangeurs de chaleur. Il est prouvé que, pour un ensemble donné de courants, seulement un pont peut réduire la consommation d’énergie. La méthode pontale inclut une procédure pour énumérer les ponts de façon systématique, une « table de réseau » pour l’évaluation facile de ceux-ci, une procédure générale pour la rétro-installation des réseaux d’échangeurs de chaleur, et un « diagramme de transfert d’énergie » permettant de visualiser les deux premiers principes de la thermodynamique dans un procédé industriel et identifier l’ensemble des opportunités énergétiques. La méthode peut être utilisée pour l’analyse des réseaux incluant plusieurs types de transfert de chaleur, et pour l’intégration à l’échelle du site industriel. La méthode pontale a ensuite été appliquée pour la rétro-installation des réseaux d’échangeurs de chaleur à contact indirect, incluant celui d’un procédé de pâte kraft, et des réseaux d’échangeurs de chaleur à contact direct, incluant le système de production d’eau chaude d’une usine papetière. Elle a finalement été utilisée pour l’intégration d’un procédé de bioraffinage, seul ou bien associé à un procédé de pâte kraft. Les résultats montrent que la méthode pontale permet de réduire efficacement l’espace de recherche et identifier les solutions pertinentes. La nécessité du pont pour réduire les entrées et sorties d’un procédé est une conséquence des deux premiers principes de la thermodynamique de conservation de l’énergie et augmentation de l’entropie. Le principe du pont peut être utilisé tant dans les approches numériques d’optimisation pour l’intégration énergie, que librement comme outil d’analyse de procédé. ---------- The increase in production of goods over the last decades has led to the need for improving the management of natural resources management and the efficiency of processes. As a consequence, heat integration methods for industry have been developed. These have been successful for the design of new plants: the integration principles are largely employed, and energy intensity has dramatically decreased in many processes. Although progress has alsobeen achieved in integration methods for retrofit, these methods still need further conceptual development. Furthermore, methodological difficulties increase when trying to retrofit heat exchange networks that are closely interrelated to water networks, such as the case of pulp and paper mills. The pulp and paper industry seeks to increase its profitability by reducing production costs and optimizing supply chains. Recent process developments in forestry biorefining give this industry the opportunity for diversification into bio-products, increasing potential profit margins, and at the same time modernizing its energy systems. Identification of energy strategies for a mill in a changing environment, including the possibility of adding a biorefinery process on the industrial site, requires better integration methods for retrofit situations. The objective of this thesis is to develop an energy integration method for the retrofit of industrial systems and the transformation of pulp and paper mills, ant to demonstrate the method in case studies. Energy is conserved and degraded in a process. Heat can be converted into electricity, stored as chemical energy, or rejected to the environment. A systematic analysis of successive degradations of energy between the hot utilities until the environment, through process operations and existing heat exchangers, is essential in order to reduce the heat consumption. In this thesis, the “Bridge Method” for energy integration by heat exchanger network retrofit has been developed. This method is the first that considers the analysis of these degradations. The fundamental mechanism to reduce the heat consumption in an existing network has been made explicit; it is the basis of the developed method. The Bridge Method includes the definition of “a bridge”, which is a set of modifications leading to heat reduction in a heat exchanger network. It is proven that, for a given set of streams, only bridges can lead to heat savings. The Bridge Method also includes (1) a global procedure for heat exchanger network retrofit, (2) a procedure 4 to enumerate systematically the bridges, (3) “a network table” to easily evaluate them, and (4) an “energy transfer diagram” showing the effect of the two first principles of thermodynamics of energy conservation and degradation in industrial processes in order to identify energy savings opportunities. The Bridge Method can be used for the analysis of networks including several types of heat transfer, and site-wide analysis. The Bridge Method has been applied in case studies for retrofitting networks composed of indirect-contact heat exchangers, including the network of a kraft pulp mill, and also networks of direct-contact heat exchangers, including the hot water production system of a pulp mill. The method has finally been applied for the evaluation of a biorefinery process, alone or hosted in a kraft pulp mill. Results show that the use of the method significantly reduces the search space and leads to identification of the relevant solutions. The necessity of a bridge to reduce the inputs and outputs of a process is a consequence of the two first thermodynamics principles of energy conservation and increase in entropy. The concept of bridge alone can also be used as a tool for process analysis, and in numerical optimization-based approaches for energy integration

Simulation dynamique d'un atelier de fabrication de papier journal et commande prédictive appliquée à la gestion des cassés et des réservoirs d'eaux blanches

Simulation dynamique de l'atelier des machines papier -- Model predictive control for integrated management of whitewater tanks -- Management of broke recirculation in an integrated paper mill

Yeast two-hybrid interaction assay v1

Protein-protein interaction can be tested using yeast two-hybrid assays. The bait protein is fused to the Gal4 DNA-binding domain (BD) and the prey is fused to the Gal4 transcriptional activation domain (AD) are both expressed in the same yeast cell. Activation of the reporter genes occurs only if bait and prey interact

Ultra Expansion microscopy protocol with improved setup for upright and inverted microscopes. v1

UltraPourquoi et comment les trypanosomes construisent un Collier de la Poche FlagellaireAlliance française contre les maladies parasitaire

Aquaporins in Saccharomyces: Genetic and functional distinctions between laboratory and wild-type strains

Aquaporin water channel proteins mediate the transport of water across cell membranes in numerous species. The Saccharomyces genome data base contains an open reading frame (here designated AQY1) that encodes a protein with strong homology to aquaporins. AQY1 from laboratory and wild-type strains of Saccharomyces were expressed in Xenopus oocytes to determine the coefficients of osmotic water permeability (Pf). Oocytes injected with wild-type AQY1 cRNAs exhibit high Pf values, whereas oocytes injected with AQY1 cRNAs from laboratory strains exhibit low Pf values and have reduced levels of Aqy1p due to two amino acid substitutions. When the AQY1 gene was deleted from a wild-type yeast and cells were cultured in vitro with cycled hypo-osmolar or hyper-osmolar stresses, the AQY1 null yeast showed significantly improved viability when compared with the parental wild-type strain. We conclude that Saccharomyces cerevisiae contains at least one aquaporin gene, but it is not functional in laboratory strains due to apparent negative selection pressures resulting from in vitro methods

Regulation of the Escherichia coli water channel gene aqpZ

Osmotic movement of water across bacterial cell membranes is postulated to be a homeostatic mechanism for maintaining cell turgor. The molecular water transporter remained elusive until discovery of the Escherichia coli water channel, AqpZ, however the regulation of the aqpZ gene expression and physiological function of the AqpZ protein are unknown. Northern analysis revealed a transcript of 0.7 kb, confirming the monocistronic nature of aqpZ. Regulatory studies performed with an aqpZ::lacZ low copy plasmid demonstrate enhanced expression during mid-logarithmic growth, and expression of the gene is dependent upon the extracellular osmolality, which increased in hypoosmotic environments but strongly reduced in hyperosmolar NaCl or KCl. While disruption of the chromosomal aqpZ is not lethal for E. coli, the colonies of the aqpZ knockout mutant are smaller than those of the parental wild-type strain. When cocultured with parental wild-type E. coli, the aqpZ knockout mutant exhibits markedly reduced colony formation when grown at 39 degrees C. Similarly, the aqpZ knockout mutant also exhibits greatly reduced colony formation when grown at low osmolality, but this phenotype is reversed by overexpression of AqpZ protein. These results implicate AqpZ as a participant in the adaptive response of E. coli to hypoosmotic environments and indicate a requirement for AqpZ by rapidly growing cells

Crystal structure of the N-terminal domain of the trypanosome flagellar protein BILBO1 reveals a ubiquitin fold with a long structured loop for protein binding

Trypanosoma brucei is a protist parasite causing sleeping sickness and nagana in sub-Saharan Africa. T. brucei has a single flagellum whose base contains a bulblike invagination of the plasma membrane called the flagellar pocket (FP). Around the neck of the FP on its cytoplasmic face is a structure called the flagellar pocket collar (FPC), which is essential for FP biogenesis. BILBO1 was the first characterized component of the FPC in trypanosomes. BILBO1's N-terminal domain (NTD) plays an essential role in T. brucei FPC biogenesis and is thus vital for the parasite's survival. Here, we report a 1.6-Å resolution crystal structure of TbBILBO1-NTD, which revealed a conserved horseshoe-like hydrophobic pocket formed by an unusually long loop. Results from mutagenesis experiments suggested that another FPC protein, FPC4, interacts with TbBILBO1 by mainly contacting its three conserved aromatic residues Trp-71, Tyr-87, and Phe-89 at the center of this pocket. Our findings disclose the binding site of TbFPC4 on TbBILBO1-NTD, which may provide a basis for rational drug design targeting BILBO1 to combat T. brucei infections.Alliance française contre les maladies parasitaire

TFK1, a basal body transition fibre protein that is essential for cytokinesis in Trypanosoma brucei

In Trypanosoma brucei, transition fibres (TFs) form a nine-bladed pattern-like structure connecting the base of the flagellum to the flagellar pocket membrane. Despite the characterization of two TF proteins, CEP164C and T. brucei (Tb)RP2, little is known about the organization of these fibres. Here, we report the identification and characterization of the first kinetoplastid-specific TF protein, named TFK1 (Tb927.6.1180). Bioinformatics and functional domain analysis identified three distinct domains in TFK1 – an N-terminal domain of an unpredicted function, a coiled-coil domain involved in TFK1–TFK1 interaction and a C-terminal intrinsically disordered region potentially involved in protein interaction. Cellular immunolocalization showed that TFK1 is a newly identified basal body maturation marker. Furthermore, using ultrastructure expansion and immuno-electron microscopies we localized CEP164C and TbRP2 at the TF, and TFK1 on the distal appendage matrix of the TF. Importantly, RNAi-mediated knockdown of TFK1 in bloodstream form cells induced misplacement of basal bodies, a defect in the furrow or fold generation, and eventually cell death. We hypothesize that TFK1 is a basal body positioning-specific actor and a key regulator of cytokinesis in the bloodstream form Trypanosoma brucei

Promoting smoking cessation for patients treated by coronary angioplasty at Liege CHU.

editorial reviewedSmoking cessation appears to be the response that provides the best cost/benefit ratio among cardiovascular prevention actions. However, hospitalization precisely offers a strategic opportunity to initiate smoking cessation. This work evaluates the assistance in smoking cessation of patients treated by coronary angioplasty at the University Hospital of Liege over the last 6 years. It aims to provide food for thought regarding optimal management of smoking. Analysis of data showed a withdrawal rate of 55 % at year one. Strengthening motivation (with motivational interviewing and conversational hypnosis), the use of nicotine replacement and participation in cardiac rehabilitation have been identified as factors in consolidating abstinence. This work attests to the relevance and necessity of the intervention of a tobacco specialist in hospitalization and outpatient settings to ensure follow-up and improve the success rate of smoking cessation