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COPI buds 60-nm lipid droplets from reconstituted water–phospholipid–triacylglyceride interfaces, suggesting a tension clamp function
Edited by William F. DeGrado, School of Pharmacy, University of California, San Francisco, CA, and approved July 3, 2013 (received for review April 23, 2013)
Intracellular trafficking between organelles is achieved by coat protein complexes, coat protomers, that bud vesicles from bilayer membranes. Lipid droplets are protected by a monolayer and thus seem unsuitable targets for coatomers. Unexpectedly, coat protein complex I (COPI) is required for lipid droplet targeting of some proteins, suggesting a possible direct interaction between COPI and lipid droplets. Here, we find that COPI coat components can bud 60-nm triacylglycerol nanodroplets from artificial lipid droplet (LD) interfaces. This budding decreases phospholipid packing of the monolayer decorating the mother LD. As a result, hydrophobic triacylglycerol molecules become more exposed to the aqueous environment, increasing LD surface tension. In vivo, this surface tension increase may prime lipid droplets for reactions with neighboring proteins or membranes. It provides a mechanism fundamentally different from transport vesicle formation by COPI, likely responsible for the diverse lipid droplet phenotypes associated with depletion of COPI subunits.
The dynamic behavior of cells requires a constant trafficking between organelles, which is largely achieved by vesicles. On bilayer-bound organelles, protein coats drive budding of transport vesicles (1). The coat protein complexes II (COPII) and I (COPI) generate vesicles from the endoplasmic reticulum and Golgi apparatus, respectively, whereas clathrin coats use various adaptor complexes to generate vesicles from the trans-Golgi network, endosomes, and the plasma membrane. Coatomer is a cytosolic complex that forms the building blocks of the COPI coat. At the Golgi apparatus, coatomer is recruited en bloc to the bilayer by Arf1 in a GTP-dependent manner (1⇓–3). All known coat proteins act on phospholipid bilayer membranes.
Thus, it is surprising that COPI depletion affects lipid droplets (LDs) that are bounded by a single monolayer of phospholipids coating an organic phase of neutral lipids such as triacylglycerols (TAGs) (4⇓–6). LDs expand and shrink during times of energy excess or scarcity (7). LD-bound proteins, including lipases and neutral lipid synthesis enzymes (8⇓⇓⇓–12), mediate these processes. For instance, COPI depletion leads to mistargeting of adipose triglyceride lipase (ATGL), the enzyme catalyzing the first step of TAG lipolysis, to LDs, which results in TAG overstorage in cells (5, 6).
How COPI mediates its effect on the targeting of LD proteins is unknown, but evidence from proteomic and microscopy experiments suggests COPI might act directly on LDs (4⇓–6, 13⇓–15). Interaction of COPI with a monolayer membrane has never been shown. Here we demonstrate that COPI machinery directly assembles at the TAG surface and propose a simple mechanism by which this machinery may regulate protein targeting to LDs. We show that Arf1 and COPI can associate directly with the monolayer of an artificial mother TAG LD and that this association induces the formation of 60-nm "nano" LDs from the mother LD. This budding process increases the surface tension, which makes the mother LD more reactive with its environment, such as soluble enzymes or membranes, and thereby can explain how COPI is involved in the targeting of enzymes to a natural LD surface.
Arf1 Binds TAG/Buffer Interface in a GTP-Dependent Manner.
On lipid bilayer membranes, COPI assembles in two steps: binding of Arf1 to the membrane in a GTP-dependent manner, followed by en bloc recruitment of coatomer by Arf1–GTP (16, 17). We investigate the possibility of this stepwise assembly on artificial LD surfaces.
We tested Arf1 binding to LDs with two complementary approaches: flotation assay and microfluidics. We prepared TAG droplets that were surrounded by a monolayer of a phospholipid mixture (PL) of the same composition as that used to prepare control liposomes (PL composition is similar to that of natural LDs) (18). Arf1 binds to such droplets in a GTP-dependent manner and with a similar efficiency as on liposomes (Fig. 1A). We confirmed Arf1 binding to buffer/TAG interfaces using a microfluidic setup allowing direct visualization of protein interactions. We produced micrometric buffer drops in a stream of oil containing the phospholipids. The buffer/TAG interface is then coated with a monolayer of PL, as attested by the change in surface tension (see Fig. 4). In each buffer drop, biochemical reactions taking place at the buffer/TAG interface can be observed by fluorescence microscopy. The small buffer volume minimizes the amount of coatomer and Arf1 required, a decisive advantage compared with the inverse geometry where oil droplets are produced in a stream of buffer. Fig. 1B shows images of buffer droplets containing Cy3-labeled Arf1 and, alternatively, GDP or GTP. In agreement with the biochemical assay, Cy3–Arf1 accumulates in a GTP-dependent manner at the TAG/buffer interface decorated with a monolayer of PL, confirming that Arf1 is able to bind to the LD lipid monolayer surface.
COPI Machinery Buds Particles from TAG/Buffer Interface.
Next, we tested the ability of coatomer to be recruited to Arf1-decorated LDs. We added Alexa 647-labeled coatomer to Cy3–Arf1 and GTP to the buffer-in-oil drops. Under these conditions, the fluorescent proteins did not only cover the TAG/buffer interface. Instead, Arf1 and coatomer formed mobile spots in the aqueous volume and at the buffer/TAG interface (Fig. 2A and Movie S1) in a GTP-dependent manner (Fig. 2B). To determine the content of these spots, we performed the experiment with unlabeled Arf1, labeled coatomer, and oil containing a fluorescent dye (Bodipy). Again, colocalized spots were observed (Fig. 2C), suggesting a budding process in which small oil droplets are detached from the buffer/TAG interface by the COPI coat. After addition of ArfGAP3, which promotes GTP hydrolysis of Arf1 and reverses the coating process (19), the coat dissociated from these spot particles (Fig. 2D).
Budded Particles Are 60-nm COPI-Coated LDs.
To determine the characteristics of the newly formed particles, we isolated them from the buffer drops (Fig. S1) and observed them by EM. The spots can clearly be identified as nano LDs with coat polymer visible at their surface (Fig. 3A, white arrows and Inset). The coat polymer disappears after treatment with ArfGAP3 (Fig. 3B). The size distribution of these nanodroplets shows that they are monodisperse with a typical diameter of 60 ± 15 nm (Fig. 3C). This distribution is consistent with the estimated size obtained from the diffusion coefficients measured by fluorescence cross-correlation spectroscopy (∼90 nm, Fig. S2A) and by independent direct tracking of the particles collected from the buffer drops (∼100 nm, Fig. S2B). Taken together, these results show that the COPI machinery is able to function on LDs in the same manner as on lipid bilayers by inducing the budding and fission of 60-nm TAG nanodroplets very close to the size of COPI vesicles (2, 3).
COPI Budding Exclusively Occurs at Interfaces with Low Tension.
Because natural LDs undergo shrinking and growth phases, we decided to probe the effect of the state of the LD surface on the efficiency of the COPI-induced budding process. We performed microfluidic experiments with increasing amounts of PL (Fig. S3). As shown in Fig. 4A, the number of COPI-induced nanodroplets formed in the buffer drops dramatically increases between 0.1% and 1% PL per TAG (wt/wt), suggesting that the COPI–coat machinery acts preferentially on a packed PL monolayer. When covering the interface, PLs, thanks to their amphiphilic nature, decrease the surface tension by shielding TAG molecules from the aqueous buffer. Because an interface with a low surface tension is more deformable, a packed PL monolayer should facilitate the budding of COPI nano LDs (20). Following this hypothesis, we used a micromanipulation approach (Fig. S4) to measure the surface tension of LDs at various PL concentrations. Strikingly, the surface tension decreased sharply from ∼20 mN/m to a vanishing surface tension (below 0.5 mN/m, the detection limit of the technique) exactly in the range of PL concentration (0.1–1% wt/wt PL/TAG) at which COPI reaches its optimum budding efficiency (Fig. 4A). The COPI machinery acts mainly at low surface tension, below a threshold of 2 mN/m.
Budding Nanodroplets Increases the Surface Tension.
The main result of nanodroplet formation should be a decrease in PL packing on the mother LD as nanodroplets "capture" relatively more surface than volume from the mother LD (Fig. S5). Put differently, nanodroplet budding consumes the PL monolayer covering the surface of the mother LD, inducing a decrease in the PL/TAG ratio. Energetically, this consumption will increase the surface tension of the mother LD. With a Langmuir trough we measured the variation of PL packing with the surface tension (and therefore PL/TAG ratio, Fig. S6). Remarkably, a 10% decrease in the PL packing from the maximum pressure (corresponding to maximum lipid packing for which the surface tension is below the detection, 0.5 mN/m) is sufficient to raise the surface tension above the 2 mN/m threshold for COPI budding of nanodroplets. A 10% decrease in the PL packing from the maximum density is achieved by budding off approximately five nanodroplets from a 500-nm mother LD (Fig. S7), a typical size of physiological LDs. Hence, a very limited action of COPI is sufficient to induce a substantial change in surface tension.
LDs with High Surface Tension Can Sense Their Environment.
Upon surface tension increase induced by COPI, a mother LD will probably become more prone to react with its environment (e.g., with soluble proteins or membranes). First, we used α-synuclein as a model of soluble proteins, because it is known to bind natural LDs (21) and is able to sense phospholipid packing on bilayers (22). We found that binding of α-synuclein to LDs is highly dependent on the PL packing at the interface. When the monolayer is not fully packed α-synuclein binds to the LD surface, whereas at full PL coverage no binding occurs (Fig. 4B). Second, we tested the ability of LDs of various PL compositions to fuse with neighboring bilayer membranes by incubating them with model membranes [giant unilamellar vesicles (GUVs), Fig. 4C, or tensionless micrometer-scale membranes resembling organelles with various curvatures, Fig. 4D]. At vanishing surface tension [PL/TAG ratio larger than 1% (wt/wt)], LDs remained intact and did not interact with any of the bilayer membranes. In contrast, at nonzero surface tension [PL/TAG ratio below 0.3% (wt/wt)], the LDs quickly got imbedded in the GUV membrane to form visible lenses between the leaflets of the bilayer (Fig. 4C and Movie S2) or fuse with the tensionless membranes that quickly form a monolayer around them (Fig. 4D and Movie S3). This demonstrates that LDs with lower PL surface packing induced, for instance, by COPI will become hyperreactive and will interact with neighboring membranes or amphiphilic molecules, leading to a remodeling of the composition of the LD surface through the relocation of new molecules.
The fact that COPI machinery is able to work on monolayers and bud oil droplets is a unique and apparently an intrinsic capability of the machinery. Because of the specificity of a monolayer compared with a bilayer, we propose that the COPI machinery performs a previously unrecognized function, clamping the surface tension of the monolayer at a buffer/oil interface by preventing it from dropping down to low tensions, below 2 mN/m. At the molecular level, this action of COPI prevents the LD surface from being fully covered by PLs and provides more accessibility to TAG for binding/reacting with other components. Consistent with the specificity of LD phenotypes associated with depletion of COPI subunits (4, 6), this is likely the only protein coat that can perform this function: COPII depends on a transmembrane guanine nucleotide exchange factor, Sec12, whereas ArfGEFs are peripheral proteins that might directly bind to LDs; clathrin coats assemble on membranes rich in anionic lipids, whereas the surface of LDs is very poor in such lipids (18).
Hyperreactivity of LDs after COPI action could have several physiological implications. Cytosolic proteins may directly bind to the mother LD surface. Alternatively, LDs might fuse with surrounding organelles, such as other LDs or bilayer membranes. This mechanism may explain the COPI dependence of targeting ATGL-related LD proteins (Fig. S8). For instance, COPI-induced budding of nanodroplets and the resulting decrease in PL packing might lead to the formation of connections between LD and other bilayer membranes, typically endoplasmic reticulum, through which proteins can exchange. These connections have been suggested in various systems by fluorescence recovery after photobleaching and EM experiments (10, 23, 24). Such a mechanism would probably entail a regulation of the action of COPI so that it is active only whenever the targeting of a specific enzyme is required.
Materials and Methods
Fluorescently labeled Arf1 was generated by using an Arf1 variant, where the single cysteine residue of Arf1 was replaced with serine, and the C-terminal lysine was replaced with cysteine (Arf1-C159S-K181C) (25). Human Arf1-C159S-K181C and yeast N-myristoyltransferase were coexpressed in Escherichia coli supplied with BSA-loaded myristate. Cell lysates were subjected to 35% (vol/vol) ammonium sulfate, and the precipitate, enriched in myristoylated Arf1, was further purified by DEAE-ion exchange. Eluted fractions of interest were concentrated in spin-column filters with a 10-kDa cutoff (Millipore) and fluorescently labeled using Cy3-maleimide (GE Healthcare) according to the manufacturer's protocol. To remove excess dye, samples were purified by gel filtration using a Superdex 75 column (GE Healthcare).
Recombinant coatomer protein was expressed and purified as described (26). Sf9 insect cells were infected with baculovirus encoding for heptameric coatomer. Coatomer complexes were isolated from the soluble protein fraction by nickel-affinity purification, concentrated in spin-column filters with a 250-kDa cutoff (Millipore), and fluorescently labeled using Alexa-Fluor-647-NHS (Molecular Probes) according to the manufacturer's protocol. Excess imidazole and dye was removed by gel filtration using a Superose 6 column (GE Healthcare).
PL Mixture Composition.
We chose a lipid composition close to that of natural LDs (18): dioleoylphsphatidylcholine (DOPC):dioleoylphsphatidylethanolamine (DOPE):cholesterol:lyso-phosphatidylinositol:lyso-phosphatidylethanolamine:lyso-phosphatidylcholine (50:20:12:10:5:3).
Unless specified, PL/TAG is fixed at 0.5% (wt/wt).
Unless otherwise indicated, experiments were performed in HKM buffer: 50 mM Hepes, 120 mM Kacetate, and 1 mM MgCl2 (in Milli-Q water).
Preparation of Synthetic Liposomes and Droplets for Flotation Experiments.
For synthetic liposomes, a chloroform solution containing 1 µmol egg PC and 1.6 nmol Rhodamine-PE was dried under argon gas in a glass tube. The lipid film was resuspended in 1 mL HKM buffer. After five cycles of freezing and thawing, the liposome suspension was extruded 19 times through a 0.4-µm polycarbonate filter.
For synthetic droplets, 70 µL TAG was mixed in a glass tube with 0.5 µmol egg PC and 1.6 nmol Rhodamine-PE from stock solutions in chloroform. The solvent was removed using a stream of argon gas and then 0.93 mL of HKM buffer was added to the TAG/PL mixture. An emulsion was obtained by vortex and extruded nine times through a 1-µm polycarbonate filter. After extrusion, the emulsion becomes extremely turbid. Examination of the suspension under a microscope shows micrometer-size LDs that were stable for many hours.
Buffer Drops Preparation.
Two syringes were filled, one with oil and the other with buffer and proteins. Using a syringe pump, streams from both syringes were allowed to flow into a high-pressure T connector with a 250-µm inside diameter constructed of fluorinated ethylene propylene (27) (Fig. S1). Because of the wetting properties of the oil to the T connector and the tube, and the ratio of the flow rates (oil rate/buffer rate = 5), 250-µm buffer droplets are generated at the outlet of the T connector (28) and circulate in a transparent Teflon tube where observations were made. The time of reaction represents the time spent by each microreator in the tube; the length of the tube and the flow rate controls it. The flow rate was 1,250 µL/h and the length of the tube was 2 m. The diameter of the tube was 250 µm, so the reaction time was ∼15 min.
GUVs and Micrometer-Scale LDs.
GUVs were prepared using an electroformation technique (29). One microliter of PL mixture in chloroform at 0.3 mM was dried on an indium tin oxide (ITO)-coated glass plate. The lipid film was desiccated for 1 h. The chamber was sealed with another ITO-coated glass plate. The lipids were then rehydrated with a sucrose solution (300 mOsm). The alternative (8 Hz) voltage between the two glass plates was increased by steps every 6 min: 100 mV, 200 mV, 300 mV, 500 mV, 700 mV, 900 mV, and 1.1 V. The last voltage was maintained for at least 1 h. GUVs were either stored in the chamber at 4 °C overnight or directly collected with a Pasteur pipette.
The LDs used in experiments with GUVs (Fig. 4C) were prepared by first drying PLs and solubilizing them afterward into TAG to obtain the required PL/TAG ratio. A mixture of 5 µL of this PL/TAG solution and 95 µL buffer was first vortexed and then sonicated using a Branson 2510 sonicator working at 40 kHz for 20 s. The diameter of the resulting droplets is a few hundred nanometers.
GUVs and generated LDs were incubated together in buffer for 10 min under gentle shaking and subsequently observed under an optical microscope (Fig. 4C is an example of a GUV incubated with LDs at a 0.3% PL/TAG ratio).
LDs and Membranes Mimicking Organelles.
To obtain membranes resembling organelles in terms of shape (curvatures) and tension (low tension), 5 µL PL lipid in chloroform (3 mM) was dried on a coverslip and placed in a desiccator for 1 h. The lipid film was rehydrated in 20 µL of buffer for 10 min. The solution was collected and injected into a Petri dish.
To prepare giant LDs, PL mixtures were dried and dissolved into TAG at different concentrations (wt/wt, typically from 0.2 to 5%). Then 5 µL of the oil solution was added to 95 µL of buffer and the mixture was vortexed 10 s using a Fischer Vortex Genie 2 at maximum power.
During the experiment, the LD and the membrane were manipulated and brought into contact through two pipettes via aspiration and observed under a microscope (bright field and fluorescence). Note that LDs were injected last into the Petri dish because they tend to float at the buffer/air interface, where they often spread.
Flotation Assay.
Liposomes (120 µL, 1 mM phospholipid) or LDs (120 µL, 0.5 mM phospholipid; TAG/buffer 7/93 vol/vol) were mixed with myristoylated Arf1GDP (0.5 µM) in a final volume of 125 µL. When indicated, the suspension was supplemented with 100 µM GTP and 2 mM EDTA to promote GDP to GTP exchange on Arf1. After incubation for 30 min at room temperature, the sample was adjusted to 30% (wt/vol) sucrose and covered with two cushions of 25% (wt/vol) sucrose (200 µL) and 0% sucrose (50 µL), respectively. The samples were centrifuged for 80 min at 30,000 rpm in a SW60 rotor (Beckman). The top (100 µL), medium (200 µL), and bottom (250 µL) fractions were collected and analyzed by SDS/PAGE using SYPRO Orange staining.
Nano LDs were collected as indicated in Fig. S1. Samples of 5 µL were absorbed to continuous carbon-coated grids (glow discharged) at room temperature for 1 min, rinsed briefly with HKM buffer, and stained with 1% (vol/vol) uranyl acetate for 20 s. Negatively stained samples were imaged under low-dose conditions in an FEI Tecnai12 microscope (120 kV). Micrographs were collected at 26,000× magnification, giving an unbinned pixel size of 4.2 Å. The diameters of nano LDs were manually measured directly from the micrographs.
Fluorescence Cross-Correlation Spectroscopy.
Oil was labeled with 1% vol/vol Bodipy (excited at 488 nm). Coatomer was labeled with Alexa 647 (excited at 632 nm). The product of reaction was recovered as described in Fig. S1 and analyzed with the FCS setup, Confocor2, on the Zeiss microscope LSM510. The emission and excitation spectra of the two dyes are separated enough to not cause any cross-talk or FRET. For each channel, autocorrelation curves were recorded simultaneously within 30 s for many runs of different samples (representing 10 different experiments). Similarly, a cross-correlation curve was simultaneously generated in a third channel. For the cross-correlated signals, we fit the far-red and green autocorrelation curves, G(τ), with a theoretical model comprising three components, giving, therefore, three diffusion times (30). The choice of such a number of fitting parameters is driven by the fact we may have signals from the free dye, the budded particles, and probable aggregates or just large, polluting particles.
The fraction of each particle is given by (x,y) and N yields the total concentration of particles. The diffusion times are τi. For each fitted curve, the access of τi determines the size of the particles by the Stoke–Einstein law,
where kB stands for the Boltzmann constant, T the temperature (room temperature), η the viscosity of the buffer (1 cSt), and ω2 the width of the focal volume (115 nm at 488 excitation and 155 nm at 647 excitation).
Measurement of the Interfacial Tension of LDs with Micropipettes.
Phospholipids were mixed at the ratio 70% DOPC: 30% DOPE (mol/mol), dried under vacuum for 1 h, and resuspended in TAG. Ten microliters of this solution was vortexed with 200 µL HKM buffer for 30 s. Then 50 µL of this emulsion was injected into a 1-mL HKM buffer drop deposited on a coverslip and observed with optical microscopy.
The interfacial tension (IT) of the droplets was measured using a micromanipulation technique. The device was made up of a micromanipulator and a pipette holder (Narishige). Pipettes were incubated in a 5% (wt/vol) BSA before use to prevent droplets from adhering to the glass. As shown in Fig. S4, micromanipulation of a single droplet enables the IT to be determined through measurement of pipette diameter, droplet diameters, and minimal pressure at which the droplet was drawn into the pipette (31):
where Psuc, Rp, and Rd represent the suction pressure, pipette radius, and droplet radius, respectively. The sizes of pipette and droplet were obtained by image analysis (ImageJ). The suction was carried out using a syringe. The resulting pressure was measured with a pressure transducer (DP103; Validyne Engineering Corp.), the output voltage being monitored with a digital voltmeter. The pressure transducer (range 55 kPa) was calibrated before experiments.
Compression Isotherm of PL Monolayer at the TAG/Buffer Interface.
The lipid mixture isotherm was carried out using a Teflon Langmuir trough (Minimicro; KSV) equipped with hydrophilic barriers made of polyoxymethylene (Derlin). The Whilhelmy pressure sensor (KSV) was coupled with a paper plate. Dimensions of the trough were 165 × 51 mm. Room temperature during the experiments was 20.5 ± 0.5 °C. The trough took place on an antivibration table in a closed box containing a water-saturated atmosphere to prevent evaporation. Lipid mixture was spread on HKM buffer before gently depositing droplets of TAG solution in chloroform (1% vol/vol). The amount of deposited TAG (0.12 µL) was at least fivefold higher than the amount of TAG required for the interface to be saturated. The so formed mixed TAG–lipids monolayer, at the interface between the very thin TAG film and HKM subphase, relaxed during 5 h before compression. The compression rate was held constant (0.5Å2⋅min–1⋅mol–1). The pressure was measured with an accuracy of 0.5 mN/m and the molecular area was controlled with 5% accuracy. The monolayer collapse was observed for 48Å2/mol molecular area and 36.6 mN/m pressure values. The pressure owing to pure oil spreading was determined to be 15.2 mN/m. We carried out independent measurements of the surface pressure as a function of lipid mixture spread at the TAG–HKM interface formed in a 50-mm-diameter vessel (glass and Teflon). Even if similar observations have previously been reported in the literature (32), it is worth noting that the results were consistent within the experimental errors with those obtained with the trough regardless of the size of the oil reservoir (from oil monolayer up to macroscopic amounts of oil).
We thank T. Melia, C. Burd, and J. Bibette for many helpful discussions. This work was supported by a grant from the Marie Curie Budding and Fusion of Lipid Droplets International Outgoing Fellowship within the Seventh European Community Framework Program (to A.R.T.), a Partner University Funds exchange grant between the Yale University and Ecole Normale Supérieure laboratories, European Research Council Grant 268888 (to B.A.) and National Institutes of Health Grant R01GM097194 (to T.C.W.). F.W. is a fellow of the Boehringer Ingelheim Fonds.
- ↵1To whom correspondence may be addressed. E-mail: antonny{at}ipmc.cnrs.fr or pincet{at}lps.ens.fr.
Author contributions: A.R.T., B.A., F.W., T.C.W., R.B., J.E.R., and F.P. designed research; A.R.T., B.A., J.W., J.D., and F.P. performed research; A.R.T., B.A., and F.P. analyzed data; and A.R.T., B.A., and F.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1307685110/-/DCSupplemental.
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text:
|United Nations Mine Action Service
|October 1997
|landmine clearance, mine risk education, victim assistance and advocacy
|United Nations, New York
|Main organ
|Department of Peacekeeping Operations / Office of Rule of Law and Security Institutions
|Parent organization
|United Nations
United Nations Mine Action Service (UNMAS) is located in the United Nations Department of Peacekeeping Operations. It is responsible for ensuring an effective, proactive and coordinated United Nations response to landmines and explosive remnants of war through collaboration with 13 other UN departments, agencies, funds and programmes. In peacekeeping and emergency settings, UNMAS establishes and manages mine action coordination centres in mine-affected countries, plans and manages operations, mobilizes resources and sets mine-action priorities in the countries and territories it serves. The current Director is Ms. Agnès Marcaillou.
The United Nations Mine Action Service (UNMAS) is a section of the Department of Peacekeeping Operations and was formed in October 1997 to serve as the United Nations focal point for mine action and to support the UN vision of a "world free of the threat of landmines and unexploded ordnance".
Five Pillars of Mine ActionEdit
Clearance of Mines and Explosive Remnants of WarEdit
In its broad sense, mine clearance includes surveys, mapping and minefield marking, as well as the actual clearance of mines from the ground. This range of activities is also sometimes referred to as demining.
Humanitarian mine clearance aims to clear land so that civilians can return to their homes and their everyday routines without the threat of landmines and unexploded remnants of war (ERW), which include unexploaded ordnance and abandoned explosive ordnance. This means that all the mines and ERW affecting the places where ordinary people live must be cleared, and their safety in areas that have been cleared must be guaranteed. Mines are cleared and the areas are thoroughly verified so that they can say without a doubt that the land is now safe, and people can use it without worrying about the weapons. The aim of humanitarian demining is to restore peace and security at the community level.
Mine clearance methodsEdit
Surveying, or the formal gathering of mine-related information, is required before actual clearance can begin. Impact surveys assess the socio-economic impact of the mine contamination and help assign priorities for the clearance of particular areas. Impact surveys make use of all available sources of information, including minefield records (where they exist), data about mine victims, and interviews with former combatants and local people. Technical surveys then define the minefields and provide detailed maps for the clearance operations.
Maps resulting from the impact surveys and technical surveys are stored in an information management system, including a variety of programme databases, and provide baseline data for clearance organisations and operational planning.
Minefield MarkingEdit
Minefield marking is carried out when a mined area is identified, but clearance operations cannot take place immediately. Minefield marking, which is intended to deter people from entering mined areas, has to be carried out in combination with mine awareness, so that the local population understands the meaning and importance of the signs.
Manual ClearanceEdit
Manual clearance relies on trained deminers using metal detectors and long thin prodders to locate the mines, which are then destroyed by controlled explosion.
Mine Detection DogsEdit
Mine detection dogs, which detect the presence of explosives in the ground by smell. Dogs are used in combination with manual deminers.
Mechanical ClearanceEdit
Mechanical clearance relies on flails, rollers, vegetation cutters and excavators, often attached to armoured bulldozers, to destroy the mines in the ground. These machines can only be used in certain terrains, and are expensive to operate. In most situations they are also not 100% reliable, and the work needs to be checked by other techniques.
Mine Risk Education (MRE)Edit
Mine-risk education, or MRE, refers to educational activities aimed at reducing the risk of injury from mines and unexploded ordnance by raising awareness and promoting behavioural change through public-information campaigns, education and training, and liaison with communities.
MRE ensures that communities are aware of the risks from mines, unexploded ordnance and/or abandoned munitions and are encouraged to behave in ways that reduce the risk to people, property and the environment. Objectives are to reduce the risk to a level where people can live safely and to recreate an environment where economic and social development can occur free from the constraints imposed by landmine contamination.
MRE, along with demining (which includes technical surveys, mapping, clearance of unexploded ordnance and mines, marking unsafe areas, and documenting areas that have been cleared), contributes to mine-risk reduction, or limiting the risk of physical injury from mines and unexploded ordnance that already contaminates the land. Advocacy and the destruction of landmine stockpiles focus on preventing future use of mines.
According to the Landmine Monitor Report (2009), in 2008, MRE was provided in 57 states and areas, compared to 61 states and areas in 2007. However, in the 1999 MRE programs were identified in just 14 states. MRE activities increased significantly in Yemen and Somaliland, and also increased to some degree in 10 other states. In Palestine, RE decreased in 2008 but rose sharply in response to conflict in Gaza in December 2008–January 2009. Some of the main players in MRE include the Mines Advisory Group, Handicap International, Catholic Relief Services, Save the Children, INTERSOS, DanChurchAid, Norwegian People's Aid, the Mines Awareness Trust, Association for Aid and Relief, Japan and the International Committee of the Red Cross. Within the UN system UNICEF is the lead agency for MRE and supports programmes in 30 countries.
International standards have been developed to guide the management of MRE programmes. (http://www.mineactionstandards.org/imas.htm#english Link to the standards) These standards emphasize that MRE should typically not be a stand-alone activity; it is an integral part of overall mine-action planning and implementation.
Public information disseminationEdit
"Public information" in the context of mine action describes landmine and unexploded ordnance situations and informs and updates a broad range of stakeholders. Such information may focus on local risk-reduction messages, address broader national issues such as complying with legislation or raise public support for mine-action programmes.
Public information "dissemination," however, refers primarily to public-information activities that help reduce the risk of injury from mines and unexploded ordnance by raising awareness of the risk to individuals and communities, and by promoting behavioural change. It is primarily a one-way form of communication transmitted through mass media. Public information-dissemination initiatives may be stand-alone MRE projects that are implemented in advance of other mine-action activities.
Education and trainingEdit
"Education and training" in MRE encompasses all educational and training activities that reduce the risk of injury from mines, unexploded ordnance and/or abandoned munitions by raising awareness of the threat to individuals and communities and promoting behavioural change. Education and training is a two-way process, which involves the imparting and acquiring of knowledge, changing attitudes and practices through teaching and learning.
Education and training activities may be conducted in formal and non-formal environments: teacher-to -child education in schools, information shared at home from parents to children or from children to their parents, child-to-child education, peer-to-peer education in work and recreational environments, landmine safety training for humanitarian aid workers (Learn about the Landmine and ERW Safety Project) and the incorporation of landmine safety messages in occupational heath and safety practices.
Community liaisonEdit
Community liaison refers to the systems and processes used to exchange information between national authorities, mine-action organisations and communities on the presence of mines, unexploded ordnance and abandoned munitions. It enables communities to be informed about planned demining activities, the nature and duration of the tasks, and the exact locations of marked or cleared areas. Furthermore it enables communities to inform local authorities and mine-action organizations about the location, extent and impact of contaminated areas. This information can greatly assist the planning of related activities, such as technical surveys, marking and clearance operations, and survivor-assistance services. Community liaison ensures that mine-action projects address community needs and priorities. Community liaison should be carried out by all organizations conducting mine-action operations.
Community liaison services may begin far in advance of demining activities and help the development of local capacities to assess the risks, manage information and develop risk-reduction strategies.
Stockpile DestructionEdit
Stockpiled anti-personnel landmines (APM) far outnumber those actually laid in the ground. In accordance with Article 4 of the anti-personnel mine-ban treaty, State Parties must destroy their stockpiled mines within four years after their accession to the convention. Sixty-five countries have now destroyed their stockpiles of antipersonnel landmines, destroying a combined total of more than 37 million mines. Another 51 countries have officially declared not having a stockpile of antipersonnel mines and a further three countries are scheduled to destroy their stockpiles by the end of the year.
There are many options available to states in destroying their stockpiles. Stockpiles are usually destroyed by the military, but an industrial solution can also be employed. The techniques used vary depending on the make-up of the mines and the conditions in which they are found. The complete destruction cycle involves aspects such as transportation and storage, processing operations, equipment maintenance, staff training and accounting, as well as the actual physical destruction.
Pre-Process TechnologyEdit
It is may be necessary to disassemble or breakdown anti-personnel mines prior to the destruction process. This is necessary because of limitations on the amount of contained explosive that can be incinerated, the anti-personnel mine design or the requirement for different components to have separate destruction methods. All of these methods require the movement of exposed bare explosive to the final destruction facility. Available technology includes: manual disassembly, mechanical disassembly (pull apart, defuzing and depriming), robotic disassembly, mechanical breakdown (bandsaw, guillotine, cracker mill, rock crusher, punch), cryofracture, hydro abrasive cutting, laser cutting, and microwave explosive melt-out. The following are brief descriptions of these techniques:
Manual Disassembly
This technique implies the use of human resources to physically dismantle APM by manual labour using simple hand tools. It has the advantage of requiring limited capital investment required, but is a labour intensive process which results in relatively slow production rates. This method requires well-trained, yet semi-skilled staff.
Mechanical disassembly
The use of mechanically operated systems to dismantle APM. The different technologies are available, as noted above are: Pull Apart, Defuzing and Depriming. In contrast to manual disassembly, mechanical disassembly has the advantages of high production rates, it is an efficient system of work and has low staff requirements. It is environmentally friendly for this stage of the demilitarisation cycle and the technology is readily available. A major disadvantage, however, is the requirement for high capital investment. This is further complicated by the need for a wide range of equipment necessary to cope with all pre-processing requirements.
Robotic disassembly
A fully automated disassembly system. Similar advantages and disadvantages to mechanical disassembly, however the initial capital costs are much greater. This system would only be economically efficient for very large production runs due to the high start-up costs.
Mechanical breakdown
This process is mainly concerned with techniques required to expose the explosive fillings of APMs prior to the destruction phase. There are low staff requirements for mechanical breakdown, and it is an environmentally friendly operation during this stage of the demilitarisation cycle. The technology is now readily available and there is no secondary waste stream, which reduces scrap salvage and disposal costs. A major disadvantage is the requirement for high capital investment. This is further complicated by the need for a wide range of equipment necessary to cope with all pre-processing requirements. Production rates per machine can be slow and there is always te danger of induced initiation of the target APM during processing.
This process is used to break down an APM into small enough pieces to be processed through an incineration destruction method. It involves the use of liquid nitrogen to change the mechanical properties of the munition casing to a more brittle phase by cooling it to -130 C. The munition can then be easily shattered using simple mechanical shear or press techniques. A cryogenic wash out system is in the early stages of development. The principle is similar to cryogenic fracture; except that the filling is attacked with liquid nitrogen in order to make its removal easier.
Cryofracture is an environmentally friendly technique during this stage of the demilitarisation cycle with low staff requirements. The technique can also be used for any other type of munition, explosive or propellant with limited pre-preparation of the munition required. There is no secondary waste stream, hence cutting final disposal costs. In financial terms low capital investment only is required for set up costs. Sensitivity tests have shown that even at -196C there is little change to the insensitiveness of the munition.
The disadvantage of high operating costs for liquid nitrogen usage must also be considered, however. Unfotunately, today there is only one proven production system in place. APMs with metal or aluminium casings are not susceptible to embrittlement and variations in the shear forces or pressures are required to fracture the munition casing. Further trials are necessary, as analysis has shown that the failure modes for the munition casings involved a mixture of brittle fracture, plastic deformation and shearing. Results are currently unpredictable and there is an obvious low temperature hazard to personnel.
Hydro abrasive cutting (HAC)
The use of water and abrasives at pressures from 240 to 1000 BAR to cut open APM bodies by an erosive process. There are two distinct technologies; 1) "entrainment" or 2) "direct injection". Research has now proven that the direct injection technology should be the preferred option for safety reasons. There are low staff requirements for HAC systems and a wide range of target munitions can be attacked. The explosive safety of systems is well proven and it is a cost effective technique in comparison to other pre-processing methods. The major disadvantage is the requirement for initial high capital investment for infrastructure. The systems also produce contaminated waste-water, which requires a complex filtration system to clean it up. In terms of post-process operations, the explosive content is "grit sensitised" and requires careful handling during any further processing or destruction.
Laser cutting
Still in the research phase in the USA.
Microwave melt-out
This technology is also under development in the USA. It utilises microwaves to heat up TNT based explosive fillings. It is a rapid, clean technique but has one major disadvantage, the lack of control over heating can lead to the formation of "hot spots" with a resultant initiation of the filling. Work continues on its development, but it is not yet a feasible production technique. It is more energy efficient that steam and improves the value of any recovered explosives.
Destruction TechnologyEdit
There are a wide range of industrial technologies available for the final destruction of anti-personnel mines. The selection of the most suitable principle depends primarily on the pre-processing techniques to be utilised, and vice versa. The system must be designed to result in efficient production rates.
Open-pit incineration
Waste material is placed on a tiled floor in a purpose built pit equipped with perforated air pipes to supply forced air to the system. A turbulent air current is created above the fire that re-circulates the combustion gases and particulates, which assists in full oxidation of the evolving gases. The principle has been tested, but no large-scale trials have yet been conducted.
Rotary kiln incineration
This is perhaps the most common, and certainly most mature demilitarisation destruction technology available. The rotary kiln is an unlined rotary furnace originally designed to destroy small arms and bulk explosives. The kiln is made up of four 1.6 metre long, 1 metre outer diameter retort sections bolted together. The 6 to 8 cm thick walls of the kiln are designed to withstand small detonations. The kiln contains internal spiral flights, which move the waste in an auger-like fashion through the retort as the kiln rotates. The flights also provide charge separation for the in-process materials, and discourage sympathetic detonations and scattering of materials. The kiln is equipped with a variable speed drive, which allows varying rotation speeds and material residence time.
Car bottom furnace
Car bottom furnaces are used to destroy small amounts of explosive or explosive residue left after flush-out pre-processing techniques.
Directly heated retort
A thin walled and ceramic lined furnace in which the feed system uses an auger. Pre-processed by a crusher. Used to process general chemical waste and explosive in solution. A typical production rate of 10,000 tonnes per year.
Plasma arc
A Plasma torch, at temperatures in the region of 4000C to 7000C, is used to heat a container into which waste products are fed. The plasma is an ionised gas at extremely high temperature, which is used to initiate rapid chemical decomposition by the action of this extreme heat. The material is currently fed in a slurry form, although research is ongoing for the destruction of entire munitions. It is a complex production system that has a high power requirement.
Mechanical Destruction
The use of high strength and capacity commercial crushing or shredding machines. Only suitable for APMs with a very low net explosive content.
"Silver II"
An electro-chemical oxidation process. The organic waste is treated by the generation of highly oxidising species in an electro-chemical cell. The cell is separated into two compartments by a membrane that allows ion flow but prevents bulk mixing of the anolyte and catholyte. In the anolyte compartment a highly reactive species of silver ion attacks organic material ultimately converting it to CO2, H2O and non-toxic inorganic compounds.
Biological degradation
This technology has been demonstrated at the pilot level for the destruction of perchlorate contaminated aqueous streams. The potential exists for bacteria to be used to consume the explosive content of APM, converting it into inert material. It requires extensive storage capacity whilst bio-remediation is taking place and only has limited applications. There is also a requirement for an element of mechanical breakdown prior to the addition of the bacteria.
Contained detonation
The destruction of ammunition and explosives by detonation in an enclosed chamber. The evolving gases are then processed by an integral pollution control system. Limited pre-processing is required and a wide variety of ammunition natures can be destroyed. However, the available systems are currently limited to 15 kg Net Explosive Content. There is also a requirement for a donor charge for each detonation, therefore the process is expensive in serviceable ammunition usage.
Molten salt oxidation
Only demonstrated at prototype scale. Can destroy finely divided and consistent organic waste, therefore significant pre-processing required. These wastes can be destroyed by incineration anyway. A purely technical solution, but too expensive and impracticable at the moment.
Scrap Processing TechnologyEdit
The final disposal of arisings from any of the above systems will require some form of scrap processing facility. Commercial advice is required in this area to determine the production rates, technical capability and availability of systems.
Industrial scrap processing systems work by crushing, shredding, cracking or compressing the feed material into an easily manageable form for further salvage or recycling processes. There may be a requirement for a combination of techniques for scrap that is difficult to process.
For the United Nations, advocacy means promoting universal participation in existing international agreements or "instruments" that ban or limit the use of landmines. The most important of these agreements is the antipersonnel mine-ban treaty, which opened for signature in 1997.
The United Nations not only encourages all countries to participate in the treaty, but it also monitors the status of the treaty's implementation. In addition, the United Nations participates in and supports regular meetings of treaty member countries - or "states parties" to the treaty.
Another important instrument is the Convention on Certain Conventional Weapons and an amended Protocol II. This instrument focuses on the use of booby-traps and anti-tank and anti-vehicle mines. The United Nations provides technical and expert advice to meetings of the states parties to this instrument. In addition, the United Nations helps countries build their capacity to implement these instruments.
The United Nations also advocates for the complete ban on cluster munitions. The United Nations is firmly committed to ending the use, stockpiling, production and transfer of cluster munitions and mitigating the suffering they cause. United Nations Mine Action Service calls on all States to become a party to the Convention on Cluster Munitions without delay.
For each of the 14 separate UN departments, agencies, programs and funds that support mine action, advocacy takes on a slightly different meaning or purpose. In some cases, more than one institution does similar advocacy work.
The UN Mine Action Service coordinates overall UN advocacy in support of the international legal instruments related to landmines and "explosive remnants of war" (which includes unexploded ordnance and abandoned explosive ordnance) and the rights of people affected by them. The organization also undertakes public information on the problems of landmines and explosive remnants of war and the global response to it.
The UN Office for Disarmament Affairs' approach to advocacy includes assistance to states parties to comply with the terms of the antipersonnel mine-ban treaty and the Convention on Certain Conventional Weapons. The department also advocates for universal participation on the antipersonnel mine-ban treaty and the Convention on Certain Conventional Weapons.
UNICEF advocates for a total ban on antipersonnel landmines, the elimination of "weapons that have indiscriminate effects" on people, new legal instruments related to explosive remnants of war and the protection and promotion of the rights of women and children affected by armed conflict. In addition, UNICEF advocates for the promotion of the rights of persons with disabilities, and the integration of victim assistance into public health, education and other social programs.
The UN Office of the Special Advisor on Gender Issues develops strategies and program to advance gender equality and the empowerment of women in all sectors, including mine action. The office's role is primarily in the areas of advocacy and policy support for "gender mainstreaming" in all of the United Nations' work.
The Office of the United Nations High Commissioner for Human Rights, in collaboration with the UN Mine Action Service and other institutions, is participating in the drafting of a new convention on human rights and dignity of persons with disabilities.
The UN High Commissioner for Refugees advocates is among the many UN institutions promoting a total ban on antipersonnel landmines.
Victim AssistanceEdit
Victim assistance is a core component of mine action and an obligation of States Parties under the Antipersonnel Mine Ban Treaty. Article Six of the Treaty states that "Each State Party in a position to do so shall provide assistance for the care and rehabilitation, and social and economic reintegration, of mine victims and for mine awareness programs." Protocol V on explosive remnants of war contains a similar provision in its Article 8. Victim assistance is a set of concrete actions to meet the immediate and long-term needs of mine/ERW victims, their families, mine-affected communities and persons with disabilities. Victim assistance includes, but is not limited to, information management systems; emergency and continuing medical care; physical rehabilitation; psychosocial support and social inclusion; economic reintegration; and laws and public policies that promote effective treatment, care and protection for all disabled citizens, including landmine victims, with a human rights perspective.
Building on the experience gained in this area since the entry into force of the Antipersonnel Mine Ban Treaty the negotiators of the Convention on Cluster Munitions agreed on a specific article on victim assistance (Article 5), which contains a number of obligations for States Parties with respect to cluster munition victims in areas under its jurisdiction and control. The Convention on Cluster Munitions also provides the following definition of cluster munition victims: " (...) all persons who have been killed or suffered physical or psychological injury, economic loss, social marginalisation or substantial impairment of the realisation of their rights caused by the use of cluster munitions. They include those persons directly impacted by cluster munitions as well as their affected families and communities."
Hundreds of thousands of mine and explosive remnants of war survivors exist in 78 countries. According to the 2008 Landmine Monitor Report, there are up to 60,000 survivors in Afghanistan alone and over 45,000 in Cambodia. In 2011, the Landmine Monitor identified 4,286 new injuries around the world by mines, explosive remnants of war and victim-detonated improvised explosive devices.[1] While the actual figure is unknown, it may well be far greater, since many incidents of mine and explosive ordnance accidents are never reported and are therefore not registered.
Within the UN system, the United Nation Mine Action Service works closely with the World Health Organisation (WHO) and other UN entities, in particular UNICEF, that also support victim assistance activities. They all work closely with partner organisations outside the United Nations system, such as the International Committee of the Red Cross, Survivor Corps, World Rehabilitation Fund (WRF), Handicap International Belgium and Vietnam Veterans of America Foundation (VVAF).
On 14 June 2011, The United Nations had declared Nepal to be landmine free.[2]
- "United Nations Mine Action Service (UNMAS)". Landmine Monitor. http://www.icbl.org/lm/2002/appendices/unmas.html.
- "What is Mine Action?". Electronic Mine Information Network. http://www.mineaction.org/section.asp?s=what_is_mine_action.
- "Nepal declared free of mines five years after civil war". BBC News. 14 June 2011. http://www.bbc.co.uk/news/world-asia-pacific-13760249.
External linksEdit
|This page uses Creative Commons Licensed content from Wikipedia (view authors).

subset: commoncrawl


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How to write a business biography
I was invited to give some PR advice for SME Business Academy and this week (like most) I'm all about self promotion. One of the first things you need for this is a great bio. So without further ado…
Whether I'm pitching a client to write an opinion piece, speak at a conference or suggesting a trend to a features writer, inevitably the first question I will be asked (if I've not already included as part of my pitch format) is "Can you send me their bio" and people want it immediately.
It is surprising how many businesses have been going for years where the key stakeholders don't have a biography on file. This should be one of the first assets you create for your media toolkit. And if you do have a bio make it pop. I don't want to be bored halfway down your long list of professional accomplishments.
Done right, your bio isn't just a boring tool for promoting yourself and your business, it is part of your personal brand which journalists, new business prospects and customers will engage with.
Here are my top tips to writing a kickass business biography:
Tip One: Tell me everything
Generally, entrepreneurs fall into two camps, wanting to share absolutely everything OR fearing they don't have enough to say. Write down everything. Not only is this a useful exercise to uncover all your forgotten talents and experience but because it may also unearth something different that you can build on later. Ask yourself the following questions:
What skills do you have that are relevant to this business?
What are your relevant academic or professional qualifications?
What is your industry experience?
Any other strengths to highlight
Do you have a quirky hobby and/or habit?
Tip two: The red pen
Rank the different points as to which are the most relevant. And start your cull of what might not be for your first draft. (Keep a long-form version of everything for future use and to keep updating).
Tip three: What's your name and where do you come from
Cilla Black always nailed a good intro. Having read some monstrosities out there it's worth flagging to start simply and with the basics. Your name and where you currently work or some examples of who you currently work for if you are freelance.
Tip four: Add a personal detail
Back to those interesting hobby or habits. As mentioned earlier, people relate better people who are personable so consider adding an interesting titbit. It gives the reader something to care about and might spark a wealth of opportunities.
Tip five: Keep it succinct
Bonus points if you can keep it down to less than 150 words but absolutely no more than 250. If you start putting too much information down, it actually distracts from you as an expert. You may struggle to keep it short and want to include everything but remember people don't remember long bios, they get bored by them. You can always link to your website to encourage people to find out more.
Lastly, remember your bio is not a one size fits all template, it will need to evolve dependant on which platform or business is using it. But having one master biography on file stands you in good stead.


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After almost three decades since the Championships had taken place in the US, Richmond, Virginia hosted the UCI World Road Championships from September 20-29, 2015. The number of spectators surpassed the initial estimates, crowding the sidelines. The race brought out a strong road biking community and left lasting excitement around the sport as Richmond implements more bike lanes and long distance bike trails.
On November 28, 2012 at their home Siegel Center, the Men's VCU Basketball team defeated Stetson in another strong season since their 2011 NCAA Final Four appearance. Head Coach Shaka Smart led them to another NCAA appearance but ultimately Michigan beat them in the third round.
The River City Roller Derby is a Women's Flat Track Roller Derby League based in Richmond, Virginia. After excitement around the sport grew with the release of the movie "Whip It," they are now able to support both an All Star and B Level team. On March 31, 2012 their All Star team, Poe's Punishers beat the Maryland Mobtown Mods during a series of matches from regional teams.


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Interested students have from Sept. 10 to 14 to apply for the third iteration of IPPSSA's political internship program.
Interested students have from Sept. 10 to 14 to apply for the third iteration of IPPSSA's political internship program.
We need to take this opportunity as a society to look deeply and reconsider our own beliefs around freedom of religion. If freedom of religion means something more than talk in this country then we need to act on that belief and ensure that everyone has the same opportunities in our society, regardless of who they worship and how.
This kind of police spying attacks whistleblowers, and it only serves to maintain an indecent shroud of secrecy that ultimately makes a mockery of our society and the people that the police are supposed to serve.
Women deserve the right to safe and calm abortions in Canada Imagine going to the clinic for surgery and feeling anxious, the only thing keeping you together is thinking about how all of this will be over in a few hours and how much better you'll feel after it. As you approach the clinic, a …
Rouge et Or no match for the Gees powerful attack Photo: Marta Kierkus After earning their first win in a massive blowout victory over Sherbrooke in their home opener, the Gee-Gees women's rugby team had their first test against serious competition on Sept. 12. The Laval Rouge et Or are the only team in the …
The City of Ottawa transit commission voted in favour of a 2.5 per cent hike in OC Transpo fares on Feb. 18. According to the Ottawa Citizen, savings in other areas will allow OC Transpo to add more buses to the schedule.
With OC Transpo and my bicycle as my only means of transportation, I always thought Gatineau Park was way too far. I was also certain nothing could live up to my expectations of the beautiful mountains of Alberta. But this spring, I decided I had to make the trip out to Gatineau Park and cross it off the bucket list.
"My family will always be grateful to this country and town for entrenching the beliefs of religious tolerance and multiculturalism into fundamental rights for citizens," said Jaameh. "It's too bad Quebec didn't agree to do the same."
University of Ottawa students and professors have expressed opposing opinions regarding the new Parti Québécois (PQ) charter of values and its ban on religious garments and symbols.
The legislation also seems to assume that people with religious convictions are bound to bring them into the workplace. Well, news flash: our attire does not change our ability to be neutral, nor will removing our religious symbols strip us from our religious beliefs.


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This book scrutinizes physical, temporal and psychological strata across early twentieth-century literature, focusing on geological and archaeological tropes and conceptions of the stratified psyche. The essays explore psychological perceptions, from practices of envisioning that mimic looking at a painting, photograph or projected light, to the comprehension of the palimpsestic complexities of language, memory and time. This collection is the first to see early twentieth-century physical, temporal and psychological strata interact across a range of canonical and popular authors, working in a variety of genres, from theatre to ghost stories, children's literature to modernist magna opera.
Ignoreeri ja kuva leht


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Examining the Role of vps26 in Drosophila Oogenesis
Rebecca Starble '17 and Nancy Pokrywka (Biology)
During endocytosis, molecules are internalized by the cell through the invagination of the plasma membrane. Endocytosis is required for proper cell function and for normal development in Drosophila. One component of the endocytic pathway is the retromer complex, which recycles transmembrane proteins to other parts of the cell such as the plasma membrane and the trans-Golgi network. Previous studies have shown that mutations to the retromer complex result in developmental defects in Drosophila. Further, in humans, retromer dysfunction has been implicated in both Alzheimer's and Parkinson's disease. However, little is known about the role of the retromer complex in Drosophila oogenesis. In the current project, we examined the role of the retromer protein Vps26 in oogenesis by characterizing the phenotype of the vps26 mutation. This X-linked lethal mutation was studied by creating germline clones via the FLP/FRT method. Immunofluorescence was then used to visualize the expression of membrane proteins and vesicular trafficking markers in mutant egg chambers. We find that vps26- germline clones exhibit atypical nurse cell structure. Additionally, the border cells of the vps26- germline clones had significantly greater LysoTracker stain than wildtype border cells, indicating an increase in degradation via the lysosomes. These data suggest there is a defect in signaling between the germline and follicle cells. Future research will examine which signaling pathways are affected by this mutation.


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You are hereHome › Manifestly unfounded application › Subsequent application ›
EDAL case summaries
The provisions of the Asylum Procedures Directive have been fully transposed into the CESEDA. A decision of the OFPRA based on all the documents/ evidence submitted by the applicant in support of his subsequent application without an interview does not infringe Article 41(2) of the Charter. When OFPRA considered the subsequent application, it was legitimate for it to have rejected the application without any interview since the new documents/ evidence provided were without merits. The Court found that M.A's application must be rejected without any need to re-examine the facts he submitted...
Slovakia - Supreme Court of the Slovak Republic, 17 January 2012, M.S. v Ministry of Interior of the Slovak Republic, 1Sža/59/2011
In the opinion of the appellate court, one of the conditions required under Section 19(1)(i) of the Asylum Act for ruling that there is no need to adjudicate was not fulfilled. Despite the existence of a final decision dismissing the application as manifestly unfounded, it was not possible to agree with the opinion of the administrative authorities, as upheld by the Regional Court, that the facts had not changed substantially.
This case was an appeal against the decision of the Polish Refugee Board on refusal to accord refugee status on the grounds that the application was manifestly unfounded application, and on granting a permit for tolerated stay. The lack of grounds for an application does not mean that the case should not be examined on its merits.
When assessing a subsequent application, the authority may find that, in the framework of the new assertions of the interested party, the application is manifestly unfounded. The authority has the right to reach such a conclusion provided that the...
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November 23, 2018
Kansas guard Lagerald Vick (24) reacts after making a 3-point basket, in front of Tennessee guard Admiral Schofield (5) during the second half of an NCAA college basketball game in the NIT Season Tip-Off tournament Friday, Nov. 23, 2018, in New York. Kansas defeated Tennessee 87-81 in overtime. (AP Photo/Adam Hunger)


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Syndicate Quotes
The first syndicating I tried was when two partners and I created a production company in 1952. We wanted to syndicate famous Bible stories and sell them for $25 a show.Tags: Famous, Show, Wanted ✍ Author: Casey Kasem
You can't just count on becoming a syndicated cartoonist. I actually tried to calculate the odds once, and the best I could come up with is a 1-in-36,000 chance. And the odds of getting hit by lightning are 1 in 7,900 - which kind of shows how long those odds are.Tags: Best, Chance, Once ✍ Author: Stephan Pastis
At one time Tribune Syndicate emptied out their storeroom. They put tables full of original cartoons down in the lobby and said take one if you want one. The comics were simply a burden to them.Tags: Put, Said, Time ✍ Author: Mort Walker
As far as the future for the Showtime episodes that have already aired, we are sold into syndication so we'll be appearing primarily on the Fox syndicated networks and then eventually the SCI FI Channel. So, we'll be around for a while.Tags: Far, Future, While ✍ Author: Richard Dean Anderson
I have two syndicated radio shows though United Stations Radio Network.Tags: Radio, Though, United ✍ Author: Nina Blackwood


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Two Unique Photography
Wedding photography duo right here!
Alright, let's get real with this love stuff. The genuine, silly, sarcastic, juicy love that makes you the couple you are today. Make us jealous!
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Answers To Your Questions
Friendly straight forward advice
We realize you may have a few questions, like what type of portable toilet do I need or how much is this going to cost. Just give us a call and one of our specialists in Waukegan, Illinois will be happy to answer any questions you may have.
Affordable Prices
Competitive pricing with no hidden fees
Some portable toilet rental companies will charge add on fees that you're not made aware of until it's too late. We pride ourselves on having upfront, clear and competitive pricing that always includes delivery, pick up, and cleaning/sanitation fees. No hidden charges & fees – guaranteed.
Dependable Service
On time delivery and pickup
If your looking to rent a portable toilet in Waukegan, don't accept slow responding companies or late deliveries. For over 20 years we have delivered our portable toilets on-time, when and where our customers need them. Give us a call today to experience why we're the #1 affordable porta potty rental company nationwide.
Myriad of Tips For Renting a Porta Potty in Waukegan, Illinois.
When you're hosting an outdoor event that requires you to rent a portable toilet, you'll need to invest some time in learning about the vendor you rent from to make sure that you have the full range of selection you require. There are many differences between portable toilets and any competent host wants to make sure their attendees are well provided for, which includes their restroom requirements. The following are just several things to think about when selecting a company to rent your porta potty or portable toilet trailer from for your next occasion.
Although it's often the last thing someone thinks of when hosting an event, the restroom option is actually the most important aspect of any big gathering, as the good times wouldn't last too long if guests couldn't relieve themselves. It's beneficial to have a bit of a education on portable toilets before making an important decision. There are many more varieties of porta potties than you may realize and you'll need to make sure you choose the right options to avoid long lines or disruptions.
Selecting a good portable toilet company is first step to hosting a smooth event. Having reliable customer service should be a top priority for whichever company you choose to rent your portable toilet from. No one wants their event to be ruined by something as basic as a portable toilet issue, so that's why you'll want to ask about how rapidly a service team can respond to any issues you may have during your event. There are a wide range of portable restrooms from standard porta potties to upscale luxury restroom trailers, and all different types of toilets for a variety of purposes.
If you're renting porta potties for a high class or upscale gathering, such as a wedding, then there are even more selections you'll want to know about. You might want to choose portable restrooms with large mirrors, cleaning areas and even family rooms in these scenarios. If your guests are dressed up for this particular event, then there will be plenty who will want to ensure their looking good by finding a mirror and being able to wash up in the porta potties. This is very important for outside weddings because restrooms or other private indoor facilities aren't always nearby or readily accessible.
Before contacting a portable toilet vendor you should be ready to give them the round about number of peopole and length of time you will be engaging your event for. Prices will vary a lot depending on event type, region, crowd size, porta potty type, length of use, and addons. The good thing about renting porta potties is that you have the choice to customize the toilets to match your individual needs.
There are an array of additional options to consider when considering which type of portable toilets to choose. The type of event you're planning will help you determine the number of toilets and the length of time you'll require them for. Remember that the portable toilets will take a good amount of time to set up and remove, so you'll need to factor that into your preparation to prevent delays to your event. At larger events it's a good idea to opt for a special stall with handicap access to ensure all guests can take advantage of it if needed.
Before making your choice you will benefit from having an understanding of the different types of selections that come with your portable toilet rental. Your typical portable toilet will come with the essential requirements such as tissue paper, hand sanitizer, a holding tank and paper seat covers. A deluxe flushing toilet has more sanitary options besides having the selections of a basic restroom. The most high end of mobile toilets will include a typical soap dispenser, mirror, and include a washing station. VIP self-contained porta potties have all the amenities that are listed above along with an interior light.
Typically, portable restrooms prices could be anywhere from to 5 for a single day. The prices may also change depending on the company and time of year. The upscale mobile toilets can possibly run up to 0 per day, and if you desire a fully loaded option you may be looking at spending as much as 0. Considering these factors are important when selecting a company to rent your portable toilet from. Regardless of who you choose to fulfill your portable toilet needs, you can't go wrong as long as you're asking the right questions ahead of time.
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Pogoplug -- Access your external hard drive from anywhere on the Internet
I am always on the lookout for the fastest and easiest way to share files from one's computer with clients, friends, or family. Setting up a webserver on your home computer is possible if you have enough tape on your glasses, but it can be very challenging and requires the use of Dynamic DNS capable routers, and crazy stuff like that. Now there's a gadget that makes it stupid-easy to share files, it's called the Pogoplug.
Plug any USB drive (or drives with a USB hub) into the Popoplug, register your unit with their website, and you are done. No Firewall configuring, DynDNS, port mapping, hair pulling...
If you are using it just to provide a file site for your clients, you don't even need a huge, hot, spinning hard drive, just a 8 or 16GB flash drive would be perfect! When you export your MP3 or AIFF file, just dump it to the Pogoplug drive, send your client the URL automatically generated by their software, and anyone you send it to can access that file. Better yet, you can share directories on your drive just by clicking a Share button
and optionally entering an e-mail address to send out invitations, and
you can also get an RSS feed for any shared directory. Make a folder for each of your clients, and they always know where to look for your files.
You can upload files to your Pogoplug drives via a standard-issue
clunky Web form, or--much better--use the software driver that makes
the Pogoplug emulate a local hard drive on a Mac
or Windows system. With the driver installed, adding or managing files
to the Pogoplug drive is as simple as dragging them with your
computer's file manager. Files quickly pop onto the drive over your
local network. For iPhone users, you also get direct access to your files, can stream music and video from your Pogodisks, and send files back to your Pogo from the phone.
I am dying to get one!!


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(Penang, Tuesday):
shocking announcement of a 5% dividend declared by the Employees Provident Fund (EPF) for last year, the lowest
for 34 years since 1967, has again highlighted the need for a new EPF policy of
accountability, transparency and good corporate governance to the 10.07
million EPF contributors on its RM186.95 billion investment decisions.
the years, the EPF Board had ignored DAP calls that the EPF should adopt the
best practices of good corporate governance especially in terms of
accountability and transparency, and it is most regrettable and deplorable that
after half a century, the EPF is not prepared to establish a
mechanism whereby the EPF Board could be accountable to the over 10
million EPF members with regard to its investment policy and decisions
and to allay the concerns of EPF
contributors about the safety, liquidity and yield of EPF funds.
year, EPF invested RM39.77 billion or 21.28% its total investments of RM186.95
billion in loans and bonds, RM42.58 billion or 22.78% in equities and RM34.97
billion or 18.70% in money market instruments.
These are astronomical sums and the list of the beneficiaries of huge
EPF loans, the full details of the stocks and shares and money market
instruments bought with EPF monies should be
made available to the public
in keeping with the highest standards of good corporate governance and the
principles of accountability and transparency.
involvement in the RM1.88 billion Time dotCom IPO fiasco last year is the latest
example as to why the investment decisions of the EPF Investment Panel, which at
present operates without any supervision even from the EPF Board, must be
subject to periodic scrutiny and review by a mechanism directly representing the
interests of the 10 million EPF members to ensure that no investments, whether
in stocks or loans, are made imprudently or as a result of improper influence.
the past, EPF had given dubious and
questionable loan facilities running into billions of ringgit to various
mega-projects and companies, like the billion
ringgit loans to Perwaja Trengganu Sdn. Bhd. and
the RM500 million loan in 1996 to
Time Telecommunications Holdings Bhd (renamed
Time dotCom). What lessons
have the EPF Board and Investment Panel drawn from these dubious
and questionable mega-loans?
Parliament in April, 1998 I had proposed that
the EPF periodically issue
public reports of its sales and purchase of shares in the stock market, giving a
list of the shares concerned.
reply that I received was that EPF
did not give details of its purchase or sale of shares so as to avoid
influencing the stock market as EPF investments were
very substantial, but I had pointed out that nobody was asking for
immediate report of proposed individual shares dealings, but periodic reports in
keeping with the principles of accountability and transparency, and which would
not influence the stock market at all.
the debate on the EPF Act 1991, DAP Members of Parliament had demanded that
there should be worker
representatives on the seven-man Investment Panel, but the then Deputy Finance
Minister, Datuk Ghani Othman assured Parliament that the workers' interests
would be protected as the Investment Panel was finally answerable to the EPF
has proved to be an empty assurance. The EPF contributors have all these years
been kept in the dark about the
EPF shares dealings, which came to RM42.58
billion last year. In fact, even the EPF Board members, in particular the
workers' representatives, do not
know what are the counters which had been bought or sold by the Investment
should amend the EPF Act to require
the EPF Board to comply with a new
policy of accountability, transparency and
good corporate governance for its RM186.95 billion investment decisions
by periodically publishing a detailed report of its investments, whether in
loans and bonds, stocks and shares or money market instruments so that they
could be subject to public scrutiny.


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