Mitochondrial dysfunction increases oxidative stress and decreases chronological life span in fission yeast

Background: Oxidative stress is a probable cause of aging and associated diseases. Reactive oxygen species (ROS) originate mainly from endogenous sources, namely the mitochondria. Methodology/Principal Findings: We analyzed the effect of aerobic metabolism on oxidative damage in Schizosaccharomyces pombe by global mapping of those genes that are required for growth on both respiratory-proficient media and hydrogen-peroxide-containing fermentable media. Out of a collection of approximately 2700 haploid yeast deletion mutants, 51 were sensitive to both conditions and 19 of these were related to mitochondrial function. Twelve deletion mutants lacked components of the electron transport chain. The growth defects of these mutants can be alleviated by the addition of antioxidants, which points to intrinsic oxidative stress as the origin of the phenotypes observed. These respiration-deficient mutants display elevated steady-state levels of ROS, probably due to enhanced electron leakage from their defective transport chains, which compromises the viability of chronologically-aged cells. Conclusion/Significance: Individual mitochondrial dysfunctions have often been described as the cause of diseases or aging, and our global characterization emphasizes the primacy of oxidative stress in the etiology of such processes.

Use of phycoerythrin and allophycocyanin for fluorescence resonance energy transfer analyzed by flow cytometry: advantages and limitations.

BACKGROUND: This study validates the use of phycoerythrin (PE) and allophycocyanin (APC) for fluorescence energy transfer (FRET) analyzed by flow cytometry. METHODS: FRET was detected when a pair of antibody conjugates directed against two noncompetitive epitopes on the same CD8alpha chain was used. FRET was also detected between antibody conjugate pairs specific for the two chains of the heterodimeric alpha (4)beta(1) integrin. Similarly, the association of T-cell receptor (TCR) with a soluble antigen ligand was detected by FRET when anti-TCR antibody and MHC class I/peptide complexes (<<tetramers>>) were used. RESULTS: FRET efficiency was always less than 10%, probably because of steric effects associated with the size and structure of PE and APC. Some suggestions are given to take into account this and other effects (e.g., donor and acceptor concentrations) for a better interpretation of FRET results obtained with this pair of fluorochromes. CONCLUSIONS: We conclude that FRET assays can be carried out easily with commercially available antibodies and flow cytometers to study arrays of multimolecular complexes.

A genomic island present along the bacterial chromosome of the Parachlamydiaceae UWE25, an obligate amoebal endosymbiont, encodes a potentially functional F-like conjugative DNA transfer system.

BACKGROUND: The genome of Protochlamydia amoebophila UWE25, a Parachlamydia-related endosymbiont of free-living amoebae, was recently published, providing the opportunity to search for genomic islands (GIs). RESULTS: On the residual cumulative G+C content curve, a G+C-rich 19-kb region was observed. This sequence is part of a 100-kb chromosome region, containing 100 highly co-oriented ORFs, flanked by two 17-bp direct repeats. Two identical gly-tRNA genes in tandem are present at the proximal end of this genetic element. Several mobility genes encoding transposases and bacteriophage-related proteins are located within this chromosome region. Thus, this region largely fulfills the criteria of GIs. The G+C content analysis shows that several modules compose this GI. Surprisingly, one of them encodes all genes essential for F-like conjugative DNA transfer (traF, traG, traH, traN, traU, traW, and trbC), involved in sex pilus retraction and mating pair stabilization, strongly suggesting that, similarly to the other F-like operons, the parachlamydial tra unit is devoted to DNA transfer. A close relatedness of this tra unit to F-like tra operons involved in conjugative transfer is confirmed by phylogenetic analyses performed on concatenated genes and gene order conservation. These analyses and that of gly-tRNA distribution in 140 GIs suggest a proteobacterial origin of the parachlamydial tra unit. CONCLUSIONS: A GI of the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of Parachlamydia bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of Chlamydiaceae, we hypothesize that it was acquired after the divergence between Parachlamydiaceae and Chlamydiaceae, when the Parachlamydia-related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired from a phylogenetically-distant bacteria sharing an amoebal vacuole. Since Parachlamydiaceae are emerging agents of pneumonia, this GI might be involved in pathogenicity. In future, conjugative systems might be developed as genetic tools for Chlamydiales.

The V-ATPase proteolipid cylinder promotes the lipid-mixing stage of SNARE-dependent fusion of yeast vacuoles.

The V-ATPase V(0) sector associates with the peripheral V(1) sector to form a proton pump. V(0) alone has an additional function, facilitating membrane fusion in the endocytic and late exocytic pathways. V(0) contains a hexameric proteolipid cylinder, which might support fusion as proposed in proteinaceous pore models. To test this, we randomly mutagenized proteolipids. We recovered alleles that preserve proton translocation, normal SNARE activation and trans-SNARE pairing but that impair lipid and content mixing. Critical residues were found in all subunits of the proteolipid ring. They concentrate within the bilayer, close to the ring subunit interfaces. The fusion-impairing proteolipid substitutions stabilize the interaction of V(0) with V(1). Deletion of the vacuolar v-SNARE Nyv1 has the same effect, suggesting that both types of mutations similarly alter the conformation of V(0). Also covalent linkage of subunits in the proteolipid cylinder blocks vacuole fusion. We propose that a SNARE-dependent conformational change in V(0) proteolipids might stimulate fusion by creating a hydrophobic crevice that promotes lipid reorientation and formation of a lipidic fusion pore.

Actin cables and the exocyst form two independent morphogenesis pathways in the fission yeast.

Cell morphogenesis depends on polarized exocytosis. One widely held model posits that long-range transport and exocyst-dependent tethering of exocytic vesicles at the plasma membrane sequentially drive this process. Here, we describe that disruption of either actin-based long-range transport and microtubules or the exocyst did not abolish polarized growth in rod-shaped fission yeast cells. However, disruption of both actin cables and exocyst led to isotropic growth. Exocytic vesicles localized to cell tips in single mutants but were dispersed in double mutants. In contrast, a marker for active Cdc42, a major polarity landmark, localized to discreet cortical sites even in double mutants. Localization and photobleaching studies show that the exocyst subunits Sec6 and Sec8 localize to cell tips largely independently of the actin cytoskeleton, but in a cdc42 and phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂)-dependent manner. Thus in fission yeast long-range cytoskeletal transport and PIP₂-dependent exocyst represent parallel morphogenetic modules downstream of Cdc42, raising the possibility of similar mechanisms in other cell types.

Establishing Load Transfer in Existing Jointed Concrete Pavements, HR-1041, 1985

In this paper are described the results of a research project that had the objective of developing construction procedures for restoring load transfer in existing jointed concrete pavements and of evaluating the effectiveness of the restoration methods. A total of 28 test sections with various load transfer devices were placed. The devices include split pipe, figure eight, vee, double vee, and dowel bars. Patching materials used on the project included three types of fast-setting grouts, three brands of polymer concrete, and plain portland cement concrete. The number and spacing of the devices and dowel bars were also variables in the project. Dowel bars and double vee devices were used on the major portion of the project. Performance evaluations were based on deflection tests conducted with a 20,000-lb axle load. Horizontal joint movement measurements and visual observations were also made. The short-term performance data indicate good results with the dowel bar installations regardless of patching materials. The sections with split pipe, figure eight, and vee devices failed in bond during the first winter cycle. The results with the double vee sections indicate the importance of the patching material to the success or failure of the load transfer system: some sections are performing well and other sections are performing poorly with double vee devices. Horizontal joint movement measurements indicate that neither the dowel bars nor the double vee devices are restricting joint movement.

Efficient lentiviral gene transfer into corneal stroma cells using a femtosecond laser.

We investigated a new procedure for gene transfer into the stroma of pig cornea for the delivery of therapeutic factors. A delimited space was created at 110 mum depth with a LDV femtosecond laser in pig corneas, and a HIV1-derived lentiviral vector expressing green fluorescent protein (GFP) (LV-CMV-GFP) was injected into the pocket. Corneas were subsequently dissected and kept in culture as explants. After 5 days, histological analysis of the explants revealed that the corneal pockets had closed and that the gene transfer procedure was efficient over the whole pocket area. Almost all the keratocytes were transduced in this area. Vector diffusion at right angles to the pocket's plane encompasses four (endothelium side) to 10 (epithelium side) layers of keratocytes. After 21 days, the level of transduction was similar to the results obtained after 5 days. The femtosecond laser technique allows a reliable injection and diffusion of lentiviral vectors to efficiently transduce stromal cells in a delimited area. Showing the efficacy of this procedure in vivo could represent an important step toward treatment or prevention of recurrent angiogenesis of the corneal stroma.

Fate of the herbicide glyphosate and its metabolite AMPA in soils and their transfer to surface waters: A multi-scale approach in the Lavaux vineyards, western Switzerland

The use of herbicides in agriculture may lead to environmental problems, such as surface water pollution, with a potential risk for aquatic organisms. The herbicide glyphosate is the most used active ingredient in the world and in Switzerland. In the Lavaux vineyards it is nearly the only molecule applied. This work aimed at studying its fate in soils and its transfer to surface waters, using a multi-scale approach: from molecular (10-9 m) and microscopic scales (10-6 m), to macroscopic (m) and landscape ones (103 m). First of all, an analytical method was developed for the trace level quantification of this widely used herbicide and its main by-product, aminomethylphosphonic acid (AMPA). Due to their polar nature, their derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) was done prior to their concentration and purification by solid phase extraction. They were then analyzed by ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). The method was tested in different aqueous matrices with spiking tests and validated for the matrix effect correction in relevant environmental samples. Calibration curves established between 10 and 1000ng/l showed r2 values above 0.989, mean recoveries varied between 86 and 133% and limits of detection and quantification of the method were as low as 5 and 10ng/l respectively. At the parcel scale, two parcels of the Lavaux vineyard area, located near the Lutrive River at 6km to the east of Lausanne, were monitored to assess to which extent glyphosate and AMPA were retained in the soil or exported to surface waters. They were equipped at their bottom with porous ceramic cups and runoff collectors, which allowed retrieving water samples for the growing seasons 2010 and 2011. Results revealed that the mobility of glyphosate and AMPA in the unsaturated zone was likely driven by the precipitation regime and the soil characteristics, such as slope, porosity structure and layer permeability discrepancy. Elevated glyphosate and AMPA concentrations were measured at 60 and 80 cm depth at parcel bottoms, suggesting their infiltration in the upper parts of the parcels and the presence of preferential flow in the studied parcels. Indeed, the succession of rainy days induced the gradual saturation of the soil porosity, leading to rapid infiltration through macropores, as well as surface runoff formation. Furthermore, the presence of more impervious weathered marls at 100 cm depth induced throughflows, the importance of which for the lateral transport of the herbicide molecules was determined by the slope steepness. Important rainfall events (>10 mm/day) were clearly exporting molecules from the soil top layer, as indicated by important concentrations in runoff samples. A mass balance showed that total loss (10-20%) mainly occurred through surface runoff (96%) and, to a minor extent, by throughflows in soils (4%), with subsequent exfiltration to surface waters. Observations made in the Lutrive River revealed interesting details of glyphosate and AMPA dynamics in urbanized landscapes, such as the Lavaux vineyards. Indeed, besides their physical and chemical properties, herbicide dynamics at the catchment level strongly depend on application rates, precipitation regime, land use and also on the presence of drains or constructed channels. Elevated concentrations, up to 4970 ng/l, observed just after the application, confirmed the diffuse export of these compounds from the vineyard area by surface runoff during main rain events. From April to September 2011, a total load of 7.1 kg was calculated, with 85% coming from vineyards and minor urban sources and 15% from arable crops. Small vineyard surfaces could generate high concentrations of herbicides and contribute considerably to the total load calculated at the outlet, due to their steep slopes (~10%). The extrapolated total amount transferred yearly from the Lavaux vineyards to the Lake of Geneva was of 190kg. At the molecular scale, the possible involvement of dissolved organic matter (DOM) in glyphosate and copper transport was studied using UV/Vis fluorescence spectroscopy. Combined with parallel factor (PARAFAC) analysis, this technique allowed characterizing DOM of soil and surface water samples from the studied vineyard area. Glyphosate concentrations were linked to the fulvic-like spectroscopic signature of DOM in soil water samples, as well as to copper, suggesting the formation of ternary complexes. In surface water samples, its concentrations were also correlated to copper ones, but not in a significant way to the fulvic-like signature. Quenching experiments with standards confirmed field tendencies in the laboratory, with a stronger decrease in fluorescence intensity for fulvic-like fluorophore than for more aromatic ones. Lastly, based on maximum concentrations measured in the river, an environmental risk for these compounds was assessed, using laboratory tests and ecotoxicity data from the literature. In our case and with the methodology applied, the risk towards aquatic species was found negligible (RF<1).

Field Evaluation of Alternative Load Transfer Device Locations in Low Traffic Volume Pavements, TR-420, 2003

In jointed portland cement concrete pavements, dowel bars are typically used to transfer loads between adjacent slabs. A common practice is for designers to place dowel bars at a certain, consistent spacing such that a sufficient number of dowels are available to effectively transfer anticipated loads. In many cases, however, the standards developed today for new highway construction simply do not reflect the design needs of low traffic volume, rural roads. The objective of this research was to evaluate the impact of the number of dowel bars and dowel location on joint performance and ultimately on pavement performance. For this research, test sections were designed, constructed, and tested in actual field service pavement. Test sections were developed to include areas with load transfer assemblies having three and four dowels in the outer wheel path only, areas with no joint reinforcement whatsoever, and full lane dowel basket assemblies as the control. Two adjacent paving projects provided both rural and urban settings and differing base materials. This report documents the approach to implementing the study and provides discussion and suggestions based on the results of the research. The research results indicate that the use of single three or four dowel basket assemblies in the outer wheel path is acceptable for use in low truck volume roads. In the case of roadways with relatively stiff bases such as asphalt treated or stabilized bases, the use of the three dowel bar pattern in the outside wheel path is expected to provide adequate performance over the design life of the pavement. In the case of untreated or granular bases, the results indicate that the use of the three or four dowel bar basket in both wheel paths provides the best long-term solution to load transfer and faulting measurements.

Spatial and temporal regulation of " cdc16p " in the fission yeast " Schizosaccharomyces pombe "

Abstract: The fission yeast Schizosaccharomyces pombe has proven to be an excellent model system for the study of eukaryotic cell cycle control. S. pombe cells are rod-shaped and grow mainly by elongation at their tips. They divide by the means of a centrallyplaced division septum which provides two daughter cells of equal size. S. pombe cytokinesis begins at mitotic entry, when the division site is defined by formation of the contractile acto-myosin ring (CAR). Formation of the division septum is triggered at the end of mitosis by the spindle pole body (SPB) associated septation initiation network (SIN) proteins. SIN signalling requires activation of the GTPase spg1p, whose nucleotide status is regulated by the bipartite GAP byr4pcdc16p. Removal of cdc16p from the SPB during early mitosis is thought to allow priming of the SIN by association of cdc7p with both SPBs. During anaphase cdc7p is retained on the new SPB, which also recruits the kinase sid1 p and cdc14p, while the old SP8 reassembles the byr4-cdc16p GAP and is presumed not to signal; SPB asymmetry persists throughout anaphase. The trigger for inactivation of SIN signalling at the new SPB is unknown. This study has concentrated upon cdc16p. We have undertaken the analysis of the localisation of cdc16p using time-lapse microscopy. We have observed that the localisation of cdc16p is regulated at different transitions. We have shown that cdc16p is removed from the SPB prior to the onset of spindle formation and that it reappears asymmetrically at the beginning of anaphase B. We have also demonstrated that the resetting of the SIN at the new SPB is linked to completion of CAR contraction and septum formation. We propose the existence of a mechanism that monitors cytokinesis and that couples the activity of the SI N with the presence of the CAR. During the biochemical characterization of cdc16p, We have found that it is an unstable protein and that it is subjected to polyubiquitination by the SCF and proteasomal degradation. Together, these observations help to shed new light upon the mechanisms by which cytokinesis is regulated in S. pombe. Résumé: La levure Schizosaccharomyces pombe est un excellent organisme modèle pour l'étude du cycle cellulaire eucaryote. Les cellules S. pombe ont la forme de bâtonnets et croissent par l'allongement de leurs extrémités. Elles se divisent en formant, en leur milieu une paroi cellulaire, appelé septum, permettant ainsi l'obtention de deux cellules filles de même taille. Chez S. pombe, la cytokinèse commence en début de mitose lorsque le site de division est déterminé par la formation d'un anneau d'acto-myosine. Le septum, lui, est formé uniquement en fin de mitose par la contraction de l'anneau d'actomyosine. Cette contraction est sous le contrôle d'un réseau de signalisation cellulaire appelé le «réseau d'initiation de synthèse du septum » ou « septation initiation network » (SIN), qui se situe sur les pôles du fuseau mitotique. L'activation du SIN dépend d'une GTPase appelé spg1p dont le statut nucléotidique dépend des protéines cdclóp et byr4p qui forment un complexe qui favorise l'hydrolyse du GTP en GDP. En début de mitose, cdc16p ne se situe plus sur les poles du fuseau mitotique. La GTPase spg1p se retrouve donc principalement sous sa forme couplée au GTP, ce quí va permettre son interaction avec la kinase cdc7p. Cette protéine ainsi que deux autres kinases sid2p (avec mob1p) et sid1p (avec cdc14p) permettent la transmission du signal d'initiation de la contraction de l'anneau d'acto-myosine en fin d'anaphase. Pendant l'anaphase, cdc7p, sid1 p et cdc14p localisent sur un des deux pôles du fuseau mitotique. Il en est de même pour cdc1p et by14p et le pôle contenant cdc16p et byr4p est toujours différent de celui ou les régulateurs positifs du SIN se situent. En fin de cytokinèse, cdc16 et byr4p se retrouvent à nouveau sur chaque pôle des deux cellules filles. Dans cette étude, nous nous sommes concentrés sur l'analyse de la localisation de cdc16p pendant la mitose en utilisant une technique de microscopie en temps réel. Nous avons été en mesure de déterminer que le départ de cdc16p du pole s'effectue juste avant la formation du fuseau mitotique. Nous avons aussi découvert que la localisation asymétrique des composants du SIN dépend fortement de l'entrée en anaphase B. Finalement, Nous avons montré que distribution asymétrique des composants du SIN sur les pôles du fuseau mitotique dépendait aussi fortement de !a présence de l'anneau d'acto-myosine. Ceci nous permet donc de proposer l'existence d'un mécanisme cellulaire qui permet de s'assurer que la cytokinèse est achevée avant de diminuer la signalisation du SIN. Par ailleurs, des études biochimiques nous ont permis de montrer que cdc16p est dégradé par le proteosome. Ces travaux ont permis la découverte de nouveaux modes de régulation du SIN.

The novel proteins Rng8 and Rng9 regulate the myosin-V Myo51 during fission yeast cytokinesis.

The myosin-V family of molecular motors is known to be under sophisticated regulation, but our knowledge of the roles and regulation of myosin-Vs in cytokinesis is limited. Here, we report that the myosin-V Myo51 affects contractile ring assembly and stability during fission yeast cytokinesis, and is regulated by two novel coiled-coil proteins, Rng8 and Rng9. Both rng8Δ and rng9Δ cells display similar defects as myo51Δ in cytokinesis. Rng8 and Rng9 are required for Myo51's localizations to cytoplasmic puncta, actin cables, and the contractile ring. Myo51 puncta contain multiple Myo51 molecules and walk continuously on actin filaments in rng8(+) cells, whereas Myo51 forms speckles containing only one dimer and does not move efficiently on actin tracks in rng8Δ. Consistently, Myo51 transports artificial cargos efficiently in vivo, and this activity is regulated by Rng8. Purified Rng8 and Rng9 form stable higher-order complexes. Collectively, we propose that Rng8 and Rng9 form oligomers and cluster multiple Myo51 dimers to regulate Myo51 localization and functions.

Genomic islands: tools of bacterial horizontal gene transfer and evolution.

Abstract Bacterial genomes evolve through mutations, rearrangements or horizontal gene transfer. Besides the core genes encoding essential metabolic functions, bacterial genomes also harbour a number of accessory genes acquired by horizontal gene transfer that might be beneficial under certain environmental conditions. The horizontal gene transfer contributes to the diversification and adaptation of microorganisms, thus having an impact on the genome plasticity. A significant part of the horizontal gene transfer is or has been facilitated by genomic islands (GEIs). GEIs are discrete DNA segments, some of which are mobile and others which are not, or are no longer mobile, which differ among closely related strains. A number of GEIs are capable of integration into the chromosome of the host, excision, and transfer to a new host by transformation, conjugation or transduction. GEIs play a crucial role in the evolution of a broad spectrum of bacteria as they are involved in the dissemination of variable genes, including antibiotic resistance and virulence genes leading to generation of hospital 'superbugs', as well as catabolic genes leading to formation of new metabolic pathways. Depending on the composition of gene modules, the same type of GEIs can promote survival of pathogenic as well as environmental bacteria.

Characterization of polyphosphate synthesis by isolated yeast vacuoles

Polyphosphate (iPOP) is a linear polymer of orthophosphate units linked together by high energy phosphoanhydride bonds. It is found in all organisms, localized in organelles called acidocalcisomes and ranges from a few to few hundred monomers in length. iPOP has been found to play a vast array of roles in all organisms, including phosphate and energy metabolism, regulation of enzymes, virulence, pathogenicity, bone remodelling and blood clotting, among many others. Recently it was found that iPOP levels were increased in myeloma cells. The growing interest in iPOP in human cell lines makes it an interesting molecule to study. However, not much is known about its metabolism in eukaryotes. Acidocalcisomes are electron dense, acidic organelles that belong to the group of Lysosome Related Organelles (LROs). The conservation of acidocalcisomes among all kingdoms of life is suggestive of their important roles for the organisms. However, they are difficult to analyse because of limited biochemical tools for investigation. Yeast vacuoles present remarkable similarities to acidocalcisomes in terms of their physiological and structural features, including synthesis and storage of iPOP, which make them an ideal candidate to study biological processes which are shared between vacuoles and acidocalcisomes. The availability of tools for genetic manipulation and isolation of vacuoles makes yeast a candidate of choice for the characterization of iPOP synthesis in eukaryotes. Our group has identified the Vacuolar Transporter Chaperone (VTC) complex as iPOP polymerase and identified the catalytic subunit (Vtc4). The goal of my study was to characterize the process of iPOP synthesis by isolated vacuoles and to reconstitute iPOP synthesis in liposomes. The first step was to develop a method for monitoring iPOP by isolated vacuoles over time and comparing it with previously known methods. Next, a detailed characterization was performed to determine the modulators of the process, both for intact as well as solubilized vacuoles. Finally, attempts were made to purify the VTC complex and reconstitute it in liposomes. A parallel line of study was the translocation and storage of synthesized iPOP in the lumen of the vacuoles. As a result of this study, it is possible to determine distinct pools of iPOP- inside and outside the vacuolar lumen. Additionally, I establish that the vacuolar lysate withstands harsh steps during reconstitution on liposomes and retains iPOP synthesizing activity. The next steps will be purification of the intact VTC complex and its structure determination by cryo-electron microscopy. - Les organismes vivants sont composés d'une ou plusieurs cellules responsables des processus biologiques élémentaires tels que la digestion, la respiration, la synthèse et la reproduction. Leur environnement interne est en équilibre et ils réalisent un très grand nombre de réactions chimiques et biochimiques pour maintenir cet équilibre. A différents compartiments cellulaires, ou organelles, sont attribuées des tâches spécifiques pour maintenir les cellules en vie. L'étude de ces fonctions permet une meilleure compréhension de la vie et des organismes vivants. De nombreux processus sont bien connus et caractérisés mais d'autres nécessitent encore des investigations détaillées. L'un de ces processus est le métabolisme des polyphosphates. Ces molécules sont des polymères linéaires de phosphate inorganique dont la taille peut varier de quelques dizaines à quelques centaines d'unités élémentaires. Ils sont présents dans tous les organismes, des bactéries à l'homme. Ils sont localisés principalement dans des compartiments cellulaires appelés acidocalcisomes, des organelles acides observés en microscopie électronique comme des structures denses aux électrons. Les polyphosphates jouent un rôle important dans le stockage et le métabolisme de l'énergie, la réponse au stress, la virulence, la pathogénicité et la résistance aux drogues. Chez l'homme, ils sont impliqués dans la coagulation du sang et le remodelage osseux. De nouvelles fonctions biologiques des polyphosphates sont encore découvertes, ce qui accroît l'intérêt des chercheurs pour ces molécules. Bien que des progrès considérables ont été réalisés afin de comprendre la fonction des polyphosphates chez les bactéries, ce qui concerne la synthèse, le stockage et la dégradation des polyphosphates chez les eucaryotes est mal connu. Les vacuoles de la levure Saccharomyces cerevisiae sont similaires aux acidocalcisomes des organismes supérieurs en termes de structure et de fonction. Les acidocalcisomes sont difficiles à étudier car il n'existe que peu d'outils génétiques et biochimiques qui permettent leur caractérisation. En revanche, les vacuoles peuvent être aisément isolées des cellules vivantes et manipulées génétiquement. Les vacuoles comme les acidocalcisomes synthétisent et stockent les polyphosphates. Ainsi, les découvertes faites grâce aux vacuoles de levures peuvent être extrapolées aux acidocalcisomes des organismes supérieurs. Le but de mon projet était de caractériser la synthèse des polyphosphates par des vacuoles isolées. Au cours de mon travail de thèse, j'ai mis au point une méthode de mesure de la synthèse des polyphosphates par des organelles purifés. Ensuite, j'ai identifié des composés qui modulent la réaction enzymatique lorsque celle-ci a lieu dans la vacuole ou après solubilisation de l'organelle. J'ai ainsi pu mettre en évidence deux groupes distincts de polyphosphates dans le système : ceux au-dehors de la vacuole et ceux en-dedans de l'organelle. Cette observation suggère donc très fortement que les vacuoles non seulement synthétisent les polyphosphates mais aussi transfère les molécules synthétisées de l'extérieur vers l'intérieur de l'organelle. Il est très vraisemblable que les vacuoles régulent le renouvellement des polyphosphates qu'elles conservent, en réponse à des signaux cellulaires. Des essais de purification de l'enzyme synthétisant les polyphosphates ainsi que sa reconstitution dans des liposomes ont également été entrepris. Ainsi, mon travail présente de nouveaux aspects de la synthèse des polyphosphates chez les eucaryotes et les résultats devraient encourager l'élucidation de mécanismes similaires chez les organismes supérieurs. - Les polyphosphates (iPOP) sont des polymères linéaires de phosphates inorganiques liés par des liaisons phosphoanhydres de haute énergie. Ces molécules sont présentes dans tous les organismes et localisées dans des compartiments cellulaires appelés acidocalcisomes. Elles varient en taille de quelques dizaines à quelques centaines d'unités phosphate. Des fonctions nombreuses et variées ont été attribuées aux iPOP dont un rôle dans les métabolismes de l'énergie et du phosphate, dans la régulation d'activités enzymatiques, la virulence, la pathogénicité, le remodelage osseux et la coagulation sanguine. Il a récemment été montré que les cellules de myélome contiennent une grande quantité de iPOP. Il y donc un intérêt croissant pour les iPOP dans les lignées cellulaires humaines. Cependant, très peu d'informations sur le métabolisme des iPOP chez les eucaryotes sont disponibles. Les acidocalcisomes sont des compartiments acides et denses aux électrons. Ils font partie du groupe des organelles similaires aux lysosomes (LROs pour Lysosome Related Organelles). Le fait que les acidocalcisomes soient conservés dans tous les règnes du vivant montrent l'importance de ces compartiments pour les organismes. Cependant, l'analyse de ces organelles est rendue difficile par l'existence d'un nombre limité d'outils biochimiques permettant leur caractérisation. Les vacuoles de levures possèdent des aspects structuraux et physiologiques très similaires à ceux des acidocalcisomes. Par exemple, ils synthétisent et gardent en réserve les iPOP. Ceci fait des vacuoles de levure un modèle idéal pour l'étude de processus biologiques conservés chez les vacuoles et les acidocalcisomes. De plus, la levure est un organisme de choix pour l'étude de la synthèse des iPOP compte-tenu de l'existence de nombreux outils génétiques et la possibilité d'isoler des vacuoles fonctionnelles. Notre groupe a identifié le complexe VTC (Vacuole transporter Chaperone) comme étant responsable de la synthèse des iPOP et la sous-unité Vtc4p comme celle possédant l'activité catalytique. L'objectif de cette étude était de caractériser le processus de synthèse des iPOP en utilisant des vacuoles isolées et de reconstituer la synthèse des iPOP dans des liposomes. La première étape a consisté en la mise au point d'un dosage permettant la mesure de la quantité de iPOP synthétisés par les organelles isolés en fonction du temps. Cette nouvelle méthode a été comparée aux méthodes décrites précédemment dans la littérature. Ensuite, la caractérisation détaillée du processus a permis d'identifier des composés modulateurs de la réaction à la fois pour des vacuoles intactes et des vacuoles solubilisées. Enfin, des essais de purification du complexe VTC et sa reconstitution dans des liposomes ont été entrepris. De façon parallèle, une étude sur la translocation et le stockage des iPOP dans le lumen des vacuoles a été menée. Il a ainsi été possible de mettre en évidence différents groupes de iPOP : les iPOP localisés à l'intérieur et ceux localisés à l'extérieur des vacuoles isolées. De plus, nous avons observé que le lysat vacuolaire n'est pas détérioré par les étapes de reconstitution dans les liposomes et conserve l'activité de synthèse des iPOP. Les prochaines étapes consisteront en la purification du complexe intact et de la détermination de sa structure par cryo-microscopie électronique.

Lentiviral vector-mediated gene transfer in adult mouse photoreceptors is impaired by the presence of a physical barrier

RESUME L'utilisation de la thérapie génique dans l'approche d'un traitement des maladies oculaires dégénératives, plus particulièrement de la rétinite pigmentaire, semble être très prometteuse (Acland et al. 2001). Parmi les vecteurs développés, les vecteurs lentiviraux (dérivé du virus humain HIV-1), permettent la transduction des photorécepteurs après injection sous-rétinienne chez la souris durant les premiers jours de vie. Cependant l'efficacité du transfert de gène est nettement plus limitée dans ce type cellulaire après injection chez l'adulte (Kostic et al. 2003). L'objet de notre étude est de déterminer si la présence d'une barrière physique produite au cours du développement, située entre les photorécepteurs et l'épithélium pigmentaire ainsi qu'entre les photorécepteurs eux-mêmes, est responsable de: la diminution de l'entrée en masse du virus dans les photorécepteurs, minimisant ainsi son efficacité chez la souris adulte. De précédentes recherches, chez le lapin, ont décrit la capacité d'enzymes spécifiques comme la Chondroïtinase ABC et la Neuraminidase X de modifier la structure de la matrice entourant les photorécepteurs (Inter Photoreceptor Matrix, IPM) par digestion de certains de ses constituants suite à leur injection dans l'espace sous-rétinien (Yao et al. 1990). Considérant l'IPM comme une barrière physique, capable de réduire l'efficacité de transduction des photorécepteurs chez la souris adulte, nous avons associé différentes enzymes simultanément à l'injection sous-rétinienne de vecteurs lentiviraux afin d'améliorer la transduction virale en fragilisant I'IPM, la rendant ainsi plus perméable à la diffusion du virus. L'injection sous-rétinienne de Neuraminidase X et de Chondroïtinase ABC chez la souris induit des modifications structurales de l'IPM qui se manifestent respectivement par la révélation ou la disparition de sites de liaison de la peanut agglutinin sur les photorécepteurs. L'injection simultanée de Neuraminidase X avec le vecteur viral contenant le transgène thérapeutique augmente significativement le nombre de photorécepteurs transduits (environ cinq fois). Nous avons en fait démontré que le traitement enzymatique augmente principalement la diffusion du lentivirus dans l'espace situé entre l'épithélium pigmentaire et les photorécepteurs. Le traitement à la Chondroïtinase ABC n'entraîne quant à elle qu'une légère amélioration non significative de la transduction. Cette étude montre qu'une meilleure connaissance de l'IPM ainsi que des substances capables de la modifier (enzymes, drogues etc.) pourrait aider à élaborer de nouvelles stratégies afin d'améliorer la distribution de vecteurs viraux dans la rétine adulte.