86 research outputs found
Ătude de l'oligomĂ©risation et de la fonction de canaux ioniques par spectroscopie de fluorescence et fluoromĂ©trie en voltage imposĂ©
La fonction des canaux ioniques est finement rĂ©gulĂ©e par des changements structuraux de sites clĂ©s contrĂŽlant lâouverture du pore. Ces modulations structurales dĂ©coulent de lâinteraction du canal avec lâenvironnement local, puisque certains domaines peuvent ĂȘtre suffisamment sensibles Ă des propriĂ©tĂ©s physico-chimiques spĂ©cifiques. Les mouvements engendrĂ©s dans la structure sont notamment perceptibles fonctionnellement lorsque le canal ouvre un passage Ă certains ions, gĂ©nĂ©rant ainsi un courant ionique mesurable selon le potentiel Ă©lectrochimique. Une description dĂ©taillĂ©e de ces relations structure-fonction est cependant difficile Ă obtenir Ă partir de mesures sur des ensembles de canaux identiques, puisque les fluctuations et les distributions de diffĂ©rentes propriĂ©tĂ©s individuelles demeurent cachĂ©es dans une moyenne. Pour distinguer ces propriĂ©tĂ©s, des mesures Ă lâĂ©chelle de la molĂ©cule unique sont nĂ©cessaires.
Le but principal de la prĂ©sente thĂšse est dâĂ©tudier la structure et les mĂ©canismes molĂ©culaires de canaux ioniques par mesures de spectroscopie de fluorescence Ă lâĂ©chelle de la molĂ©cule unique. Les Ă©tudes sont particuliĂšrement dirigĂ©es vers le dĂ©veloppement de nouvelles mĂ©thodes ou leur amĂ©lioration. Une classe de toxine formeuse de pores a servi de premier modĂšle dâĂ©tude. La fluorescence Ă lâĂ©chelle de la molĂ©cule unique a aussi Ă©tĂ© utilisĂ©e pour lâĂ©tude dâun rĂ©cepteur glutamate, dâun rĂ©cepteur Ă la glycine et dâun canal potassique procaryote.
Le premier volet porte sur lâĂ©tude de la stĆchiomĂ©trie par mesures de photoblanchiment en temps rĂ©solu. Cette mĂ©thode permet de dĂ©terminer directement le nombre de monomĂšres fluorescents dans un complexe isolĂ© par le dĂ©compte des sauts discrets de fluorescence suivant les Ă©vĂ©nements de photoblanchiment. Nous prĂ©sentons ici la premiĂšre description, Ă notre connaissance, de lâassemblage dynamique dâune protĂ©ine membranaire dans un environnement lipidique. La toxine monomĂ©rique purifiĂ©e Cry1Aa sâassemble Ă dâautres monomĂšres selon la concentration et sature en conformation tĂ©tramĂ©rique.
Un programme automatique est ensuite dĂ©veloppĂ© pour dĂ©terminer la stĆchiomĂ©trie de protĂ©ines membranaires fusionnĂ©es Ă GFP et exprimĂ©es Ă la surface de cellules mammifĂšres. Bien que ce systĂšme dâexpression soit appropriĂ© pour lâĂ©tude de protĂ©ines dâorigine mammifĂšre, le bruit de fluorescence y est particuliĂšrement important et augmente significativement le risque dâerreur dans le dĂ©compte manuel des monomĂšres fluorescents. La mĂ©thode prĂ©sentĂ©e permet une analyse rapide et automatique basĂ©e sur des critĂšres fixes. Lâalgorithme chargĂ© dâeffectuer le dĂ©compte des monomĂšres fluorescents a Ă©tĂ© optimisĂ© Ă partir de simulations et ajuste ses paramĂštres de dĂ©tection automatiquement selon la trace de fluorescence. La composition de deux canaux ioniques a Ă©tĂ© vĂ©rifiĂ©e avec succĂšs par ce programme.
Finalement, la fluorescence Ă lâĂ©chelle de la molĂ©cule unique est mesurĂ©e conjointement au courant ionique de canaux potassiques KcsA avec un systĂšme de fluoromĂ©trie en voltage imposĂ©. Ces enregistrements combinĂ©s permettent de dĂ©crire la fonction de canaux ioniques simultanĂ©ment Ă leur position et densitĂ© alors quâils diffusent dans une membrane lipidique dont la composition est choisie. Nous avons observĂ© le regroupement de canaux KcsA pour diffĂ©rentes compositions lipidiques. Ce regroupement ne paraĂźt pas ĂȘtre causĂ© par des interactions protĂ©ine-protĂ©ine, mais plutĂŽt par des microdomaines induits par la forme des canaux reconstituĂ©s dans la membrane. Il semble que des canaux regroupĂ©s puissent ensuite devenir couplĂ©s, se traduisant en ouvertures et fermetures simultanĂ©es oĂč les niveaux de conductance sont un multiple de la conductance « normale » dâun canal isolĂ©. De plus, contrairement Ă ce qui est actuellement suggĂ©rĂ©, KcsA ne requiert pas de phospholipide chargĂ© nĂ©gativement pour sa fonction. Plusieurs mesures indiquent plutĂŽt que des lipides de forme conique dans la phase cristalline liquide sont suffisants pour permettre lâouverture de canaux KcsA isolĂ©s. Des canaux regroupĂ©s peuvent quant Ă eux surmonter la barriĂšre dâĂ©nergie pour sâouvrir de maniĂšre coopĂ©rative dans des lipides non chargĂ©s de forme cylindrique.The function of ion channels is finely regulated by structural changes of key domains controlling the pore opening. These structural modulations arise from interactions with the local environment, since several domains can be sensitive to specific physico-chemical properties. Movements generated in the structure become notably perceptible when channels open a passage for some ions, thus generating a measurable ionic current according to the electrochemical potential. A detailed description of these structure-function relationships is however difficult to obtain from measurements involving a set of identical channels, since the fluctuations and distributions of different individual properties remain hidden in an average. To differentiate these properties, single-molecule recordings are required.
The main purpose of this thesis is to study the structural aspects and molecular mechanisms of ion channels using fluorescence spectroscopy at the single-molecule level. Studies are oriented towards the development or improvement of new methods. A class of pore-forming toxin served as a first study model. Single-molecule fluorescence was also used to study an ionotropic glutamate receptor, a glycine receptor and a prokaryotic potassium channel.
The first part focuses on the study of stoichiometry using fluorescent subunit counting. This method allows a direct measure of the number of fluorescent monomers within a single complex by counting the number of step-wise fluorescence intensity decrease following photobleaching events. Here we present the first description, to our knowledge, of the dynamic assembly of a membrane protein in a lipid environment. The purified monomeric Cry1Aa toxin clusters with other monomers depending on the concentration and saturates in a tetrameric conformation.
An automated method has been developed to determine the stoichiometry of GFP-tagged membrane proteins expressed on mammalian cell surface. Although this expression system is suitable for the study of proteins of mammalian origin, background fluorescence is particularly important and significantly increases the risk of error in the manual counting process. The presented method allows a fast and automated analysis based on fixed criteria. The algorithm responsible for counting fluorescent monomers was optimized from simulations and adjusts its detection parameters automatically according to the fluorescence trace recording. The composition of two ion channels was successfully verified using this program.
Finally, single-molecule fluorescence is measured together with ionic current of KcsA channels using a voltage-clamp fluorometry setup. These combined recordings allowed us to describe the function of ion channels simultaneously to their position and density as they diffuse in a lipid membrane of defined composition. We observed clustering of KcsA channels for various lipid compositions. Clustering does not appear to be caused by protein-protein interaction, but rather by microdomains induced by the shape of reconstructed channels in the lipid bilayer. It seems that clustered KcsA channels could then become coupled, resulting in cooperative gating events with conductance levels multiple to the ânormalâ unitary channel conductance. Moreover, as opposed to what is currently suggested, KcsA does not require a negatively charged phospholipid for its function. Several of our recordings rather suggest that conically shaped lipids in the lamellar liquid crystalline phase are sufficient to allow single channel opening. Clustered channels can on the other hand overcome the energy barrier to open cooperatively in uncharged cylindrical lipids
Ătude du couplage entre les sous-unitĂ©s du canal potassique KcsA par des mesures de spectroscopie de fluorescence en canal unitaire
Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal
Exploring Thailand's mortality transition with the aid of life tables
The project Thai Health-Risk Transition: A National Cohort Study seeks to better understand the health implications of modernisation and globalisation forces impacting on Thailand. As part of its âlook-backâ component this paper seeks, using available life tables, to document the country's post-war mortality transition. The onset of transition through mass campaigns of the late 1940s and 1950s is first discussed before attention turns to the life tables. They are predictably far from flawless, but careful analysis does permit trends that have seen around 30 years added to life expectancy to be traced, and age patterns of improved survivorship and their relation to initiatives to improve health to be examined. The broad benefits generated by mass campaigns, ongoing improvements in infant and early childhood mortality, and a phased impact of the expansion of primary health care in rural areas on adult survival prospects after the mid-1970s are demonstrated. The paper also investigates the consequences for mortality of a motorcycle-focused rapid increase in road fatalities in the late 1980s and early 1990s and the HIV/AIDS epidemic that developed after 1984
Access to Early Generation Seed: Obstacles for Delivery of Climate-Smart Varieties
Changing climates in eastern and southern Africa will require farmers to adjust which crop varieties they grow in order to adapt to changing patterns of weather, pests and diseases. Delivering more suitable, climate-smart crop varieties requires well-functioning seed systems in which actors work in harmony across the supply chain. Although a great deal of previous development funding has been used to breed new varieties and to encourage farmers to adopt them, the availability of early-generation seed (EGS) continues to be limited by bottlenecks in the supply chain. These problems are particularly significant for non-hybrid varieties and less-commercialized food crops developed by public-sector institutions. This study uses two contrasting case studies from Kenya to illustrate the importance of making improved bean seed varieties available to farmers. The first case study documents a successful instance of EGS transfer, whereas the second highlights the types of barriers that can prevent successful variety adoption. Improved coordination among system actors is necessary to reduce the barriers surrounding EGS provision and production, and thereby strengthen climate-adaptive and adaptable seed systems
Do smallholder farmer-led seed systems have the capacity to supply good-quality, fungal-free sorghum seed?
Local seed systems that are developed, managed and maintained by farmers are a fundamental practice in smallholder crop production, supporting more than 80% of farmers in sub-Saharan Africa and feeding more than 70% of its population. The resilience of such systems is under threat from poverty, climate change, drought, increased pests and diseases, over-promotion of modern crop varieties, change of lifestyles and restrictive seed policies. The system continues to be maligned as having inferior quality, yet few studies support this assertion. This study aims to fll this research gap by evaluating 60 sorghum seed samples collected from smallholder farmers in Uzumba-MarambaPfungwe and Chimanimani districts of Zimbabwe. We investigated the efect of farmer-led seed management practices (e.g. seed acquisition and seed storage practices) on farm-derived sorghum seed quality (moisture, germination and fungal incidences). We found farmers using diverse seed sources and seed storage practices. Seeds were typically of good quality in that their storage moisture content was low, their germination was high, and fungal incidences were low. Seed sourced from local markets, non-governmental organizations and other farmers had germination and moisture standards that met the sorghum certifcation standards in Zimbabwe. However, few samples obtained from the relatives and government failed to meet the germination and/or moisture certifcation standards. We detected low incidences of fungi (Aspergillus favus, Aspergillus niger, Curvularia lunata, Fusarium sp. and Penicillium sp.) in sorghum seed samples tested and in particular Fusarium sp., which is the most economic important fungus in sorghum production. We conclude that farmer-led seed systems have the capacity to supply seeds of good quality and recommend that such systems should be recognized and promoted to meet the ever-evolving needs of smallholder farmers in sub-Saharan Africa
The Circadian Neuropeptide PDF Signals Preferentially through a Specific Adenylate Cyclase Isoform AC3 in M Pacemakers of Drosophila
To synchronize a network of pacemakers in the Drosophila brain, a neuropeptide receptor specifically associates with adenylate cyclase 3 to create a âcircadian signalosome.
HOTAIR and its surrogate DNA methylation signature indicate carboplatin resistance in ovarian cancer
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