News Article | May 16, 2017
CAMBRIDGE, Mass.--(BUSINESS WIRE)--AVEO Oncology (NASDAQ: AVEO) today announced the appointment of Matthew Dallas as chief financial officer, effective June 1, 2017. In this role, Mr. Dallas will be responsible for the Company’s financial strategy and management as it takes steps toward the potential commercialization of tivozanib. Mr. Dallas will also serve on the executive leadership team which governs corporate strategy at AVEO. He succeeds Keith Ehrlich, the Company’s current chief financial officer, whose retirement from the Company was previously announced. Mr. Dallas brings to AVEO more than 20 years of financial management experience, including 18 years in the life sciences industry. Most recently, he served as Chief Financial Officer and Treasurer of CoLucid Pharmaceuticals, a position he held through that biopharmaceutical company’s initial public offering, follow-on offering, and subsequent acquisition, for approximately $960 million, by Eli Lilly and Company in March 2017. Prior to CoLucid, Mr. Dallas served as Vice President of Finance and Treasurer at AVEO from 2011 to 2015, a period during which the Company was first preparing for the potential launch of tivozanib. He previously worked at Genzyme Corporation, NEN Life Sciences, and Kimberly-Clark Corporation where he held various positions of increasing responsibility in finance and accounting. Mr. Dallas holds a B.S. in Finance from the University of Tennessee, Knoxville. “It is a pleasure to welcome Matt back to the AVEO team, following a successful tenure as chief financial officer at CoLucid,” said Michael Bailey, president and chief executive officer of AVEO. “I look forward to Matt’s insights and expertise as AVEO advances our plans for tivozanib and the other programs in our pipeline. I would also like to thank Keith for his contributions and service to the Company, and for supporting Matt through this initial transition period." “AVEO is at an important juncture in its development under Michael’s leadership, with clinical and registrational strategies for tivozanib being pursued in both the U.S. and E.U., and a dynamic pipeline providing the potential for added strategic and financial flexibility,” said Mr. Dallas. “I look forward to contributing to AVEO’s progress in advancing its portfolio of therapeutics targeting important unmet needs in cancer and other serious diseases.” AVEO Oncology (AVEO) is a biopharmaceutical company dedicated to advancing a broad portfolio of targeted therapeutics for oncology and other areas of unmet medical need. The Company is focused on seeking to develop and commercialize its lead candidate tivozanib, a potent, selective, long half-life inhibitor of vascular endothelial growth factor 1, 2 and 3 receptors, in North America as a treatment for renal cell carcinoma and other cancers. AVEO is leveraging multiple partnerships aimed at developing and commercializing tivozanib in oncology indications outside of North America, and at progressing its pipeline of novel therapeutic candidates in cancer and cachexia (wasting syndrome). For more information, please visit the company’s website at www.aveooncology.com. This press release contains forward-looking statements of AVEO that involve substantial risks and uncertainties. All statements, other than statements of historical fact, contained in this press release are forward-looking statements. The words “anticipate,” “believe,” “expect,” “intend,” “may,” “plan,” “potential,” “could,” “should,” “would,” “seek,” “look forward,” “advance,” “goal,” “strategy,” or the negative of these terms or other similar expressions, are intended to identify forward-looking statements, although not all forward-looking statements contain these identifying words. These forward-looking statements include, among others, statements about AVEO’s and its collaborators’ future discovery, development, regulatory and commercialization plans and efforts, including without limitation with respect to tivozanib and AVEO’s other programs and platforms; AVEO’s strategy, prospects, plans and objectives; and the Company’s financial and strategic flexibility. AVEO has based its expectations and estimates on assumptions that may prove to be incorrect. As a result, readers are cautioned not to place undue reliance on these expectations and estimates. Actual results or events could differ materially from the plans, intentions and expectations disclosed in the forward-looking statements that AVEO makes due to a number of important factors, including risks relating to AVEO’s ability to enter into and maintain its third party collaboration agreements, and its ability, and the ability of its licensees and other partners, to achieve development and commercialization objectives under these arrangements; AVEO’s ability, and the ability of its licensees, to demonstrate to the satisfaction of applicable regulatory agencies the safety, efficacy and clinically meaningful benefit of AVEO’s product candidates; AVEO’s ability to successfully enroll and complete clinical trials, including the TIVO-3 and TiNivo studies; AVEO’s ability to achieve and maintain compliance with all regulatory requirements applicable to its product candidates; AVEO’s ability to obtain and maintain adequate protection for intellectual property rights relating to its product candidates and technologies; developments, expenses and outcomes related to AVEO’s ongoing shareholder litigation; AVEO’s ability to successfully implement its strategic plans; AVEO’s ability to raise the substantial additional funds required to achieve its goals, including those goals pertaining to the development and commercialization of tivozanib; unplanned capital requirements; adverse general economic and industry conditions; competitive factors; and those risks discussed in the section titled “Risk Factors” and “Management’s Discussion and Analysis of Financial Condition and Results of Operations—Liquidity and Capital Resources” included in AVEO’s Annual Report on Form 10-K for the year ended December 31, 2016, its quarterly reports on Form 10-Q and in other filings that AVEO may make with the SEC in the future. The forward-looking statements in this press release represent AVEO’s views as of the date of this press release. AVEO anticipates that subsequent events and developments may cause its views to change. While AVEO may elect to update these forward-looking statements at some point in the future, it specifically disclaims any obligation to do so. You should, therefore, not rely on these forward-looking statements as representing AVEO's views as of any date other than the date of this press release.
Assistive Technology Research Series | Year: 2011
Objective. This paper addresses the role of consumers/end-users, in our case Persons with Disabilities, in the creation of new standards regarding the accessibility aspects in society and the usability of products for all. The United Nations declaration on the rights of persons with disabilities requires that action has to be taken. The measures to be promoted is to include persons with disabilities, representatives. Until now consumers/end-users are not well represented in standards development. This is partly because European standardization is a new field for consumers/end-users for which they do not have the expertise to jump into. It is therefore needed to involve users with disabilities in the field of standardization. USEM project. To improve consumer/end-user participation in standardization a training is developed in the USEM project, which is EU funded. The USEM-principles describe the conditions for active user participation. These are: 1. Partnership as a basis; 2. Users are members and/or representatives of user organizations; 3. Financing participation should not be a barrier for participation; 4. Accessibility of all relevant materials and premises is guaranteed; 5. Every partner guarantees respect and expertise; 6. Detailed plan for the project. Results. In the USEM project 27 trainees from 14 countries are trained to participate in standardization in the ICT-field. A few of them participated in standardization-related activities like joining telephone conferences in an ETSI working group or joining the CEN/CENELEC StandardDays. A networkday with the USEM trainees a year after the actual USEM-training showed that there are still some hurdles for success. These include the cost of participation, not many running projects to participate in, the insufficient accessibility for blind participants of the written material, and the dominance of industrial representatives. Conclusion. - Concrete actions/developments are needed for users to get involved and stay involved. - An active user organization is important for the backing of the representative of users in standardization. © 2011 The authors and IOS Press. All rights reserved.
News Article | November 23, 2016
Experiments were approved by the local ethical committee of the University of Bordeaux (approval number 501350-A) and the French Ministry of Agriculture and Forestry (authorization number 3306369). Mice were maintained under standard conditions (food and water ad libitum; 12 h–12 h light–dark cycle, light on at 7:00; experiments were performed between 9:00 and 17:00). Male C57BL/6N mice were purchased from Janvier (France). Wild-type (CB +/+) and CB −/− female and male mice (2–4 months old) were obtained, bred and genotyped as described31. Only male mice were used for behavioural experiments. For most experiments CB +/+ and CB −/− were littermates. For primary cell cultures, pups were obtained from homozygote pairs. No method of randomization to assign experimental groups was performed and the number of mice in each experimental group was similar. No statistical methods were used to predetermine sample size. THC was obtained from THC Pharm GmbH (Frankfurt, Germany). HU210 was synthesized as described32. WIN55-212-2, KH7, PTX, bicarbonate (HCO −), forskolin, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), oligomycin, antimycin, rotenone, picrotoxin, GTPγS and other chemicals used in this study were purchased from Sigma-Aldrich (St-Louis, USA). [3H]CP55,940 (162.5 Ci mmol−1) and [35S]GTPγS (1,250 Ci mmol−1) were purchased from Perkin Elmer NEN (Boston, USA). For in vivo administration, WIN was dissolved in a mixture of saline (0.9% NaCl) with 2% DMSO and 2% cremophor; THC was dissolved in a mixture of 4% ethanol, 5% cremophor and saline; and KH7 was dissolved in 10% cremophor, 2.5% DMSO and saline. Vehicles contained the same amounts of solvents. All drugs were prepared freshly before the experiments. For in vitro experiments, PTX, HCO − and forskolin were dissolved in water. KH7, HU210 and WIN were dissolved in DMSO. THC, oligomycin, FCCP, antimycin and rotenone were dissolved in ethanol. Corresponding vehicle solutions were used in control experiments. DMSO and ethanol were no more than 0.001%. Doses and concentrations of the different drugs were chosen on the basis of previous published data or preliminary experiments. The N-terminal deletion of the first 22 amino acids (66 base pairs) in the mouse CB -receptor coding sequence, to obtain the DN22-CB mutant, as well as the generation of the mitochondrially targeted constitutively active form of PKA (MLS–PKA-CA) was achieved by polymerase chain reaction (PCR). In brief, for DN22-CB a forward primer hybridizing from the 67th base starting from ATG was coupled to a reverse primer hybridizing to the end of the coding sequence, including the TGA stop codon. In order to guarantee accurate translation of the construct, the forward primer included an ATG codon upstream of the hybridizing sequence. The cDNA for DN22-CB was amplified using HF Platinum DNA polymerase (Invitrogen) and inserted into a PCRII-Topo vector (Invitrogen) according to the manufacturers’ instructions. The absence of amplification mismatches was then verified by DNA sequencing. Primers used were: forward, with the inserted ATG in bold, 5′-ATGGTGGGCTCAAATGACATTCAG-3′; reverse, with the stop codon in bold, 5′-TCACAGAGCCTCGGCAGACGTG-3′. The cDNA sequence for CB or DN22-CB was inserted into a modified version of a pcDNA3.1 mammalian expression vector using BamHI–EcoRV according to standard cloning procedures. This modification allowed the co-expression of CB or DN22-CB with an mCherry fluorescent protein for control of transfection efficiency. For the study of mitochondrial motility, the coding sequence of CB or DN22-CB was fused to GFP using the pEGFP-N1 vector (Addgene) according to the manufacturer’s instructions. For MLS–PKA-CA, a forward primer including a restriction site after the initial ATG codon for future subcloning with mitochondrial leading sequences was coupled to a reverse primer hybridized to the end of the coding sequence of the catalytic subunit of PKA (pET15b PKA Cat from Addgene)33, including a myc epitope and a TGA stop codon. Subsequently, the construct was subcloned into a pcDNA3.1 vector as an intermediate step and the QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Genomics, Santa Clara, CA, USA) was used to mutate histidine-87 to glutamine, and tryptophan-196 to arginine to generate a constitutively active form of PKA24. Finally, the construct was fused to a 4×MLS sequence to target the constitutively active PKA to mitochondria (MLS–PKA-CA). The absence of amplification mismatches and confirmation of mutagenesis was then verified by DNA sequencing. Primers used were: forward, with the inserted ATG in bold, 5′-TATCTGGATCCCTATGCAATTGGGCAACGCCGCCGCCGCCAAGAAGG-3′; reverse, with the stop codon and the myc epitope in bold, 5′-TATGATCTAGAGATCACAGATCCTCTTCTGAGATGAGTTTTTGTTCAAACTCAGTAAACTCCTTGCCACACTTC-3′; and for mutagenesis of H87, 5′-AAAGCAGATCGAGCAAACTCTGAATGAGAAG-3′; and W196 5′-GTGAAAGGCCGTACTAGGACCTTGTGTGGGA-3′ (in bold are the mutated codons). The phosphomimetic version of NDUFS2 was custom synthesized by Eurofins Genomics (Germany). Briefly, the NDUFS2 sequence (NM_153064) was modified to obtain a phosphomimetic form mutating the 3 potential phosphorylation sites of PKA. The sites were chosen because consensus for their PKA phosphorylation nature was found between the two online available phosphorylation prediction algorithms, PhosphoMotif Finder (http://www.hprd.org/PhosphoMotif_finder) and PKA prediction site (http://mendel.imp.ac.at/pkaPS/)25, 26. By this approach, four sites were identified. One of these was excluded, because it is present on the mitochondrial leading sequence of NDUFS2. Thus, serines 296, 349 and 374 were mutated to aspartic acid to obtain a phosphomimetic version of NDUFS2 (NDUFS2-PM). A myc epitope was added at the C terminus of the protein for detection. The cDNAs coding for mouse CB , DN22-CB , MLS–PKA-CA, NDUFS2-PM and for GFP were subcloned into the pAM–CBA vector using standard molecular cloning techniques. The resulting vectors were transfected by calcium phosphate precipitation into HEK293 cells together with the rAAV-helper-plasmid pFd6 and AAV1/2-serotype-packaging plasmids pRV1 and pH21 (ref. 34.). The viruses were then purified and titred as previously described35, 36. Virus titres were between 1010 and 1011 genomic copies per ml for all batches of virus used in the study. All cell lines were originally obtained from ATCC (https://www.lgcstandards-atcc.org/Products/Cells_and_Microorganisms/Cell_Lines.aspx?geo_country=fr). Mouse 3T3 cells (3T3 F442A), HeLa and HEK293 cells were grown in Dulbecco modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 4.5 g l−1 glucose, 2 mM glutamine, 1 mM pyruvate. HEK293 cells were transfected with control plasmid, CB or DN22-CB cDNA coupled with mCherry cloned in pcDNA 3.1(+), respectively. Cells were transfected with sAC–HA or mtsAC–HA provided by G. Manfredi, (see refs 17, 21) or small hairpin RNA (shRNA) targeting AKAP121 provided by A. Feliciello (see ref. 22). HeLa cells were transfected with MLS–PKA-CA or NDUFS2-PM (see above). The transfections were carried out using FugeneHD (Roche, France) for 3T3 cells and polyethylenimine (PEI, Polysciences, USA) for HEK293 and HeLa cells, according to the manufacturers’ protocols. For biochemical experiments, primary hippocampal cultures were prepared from CB +/+ and CB −/− P0–P1 mice. Briefly, after mice were killed by decapitation, hippocampi were extracted in dissection medium (10 mM HEPES, 0.3% glucose in Hank’s balanced salt solution, pH 7.4) and dissociated in 0.25% trypsin for 30 min. Where indicated, dissociated cells were transfected with sAC–HA using the Amaxa P3 primary cell 4D-nucleofector kit (Lonza, France), according to the manufacturer’s protocol. Cells were plated on poly-l-lysine-coated 96-well dishes using neurobasal/B27 medium (supplemented with 5% FBS, 2 mM l-glutamine, 1 mM pyruvate, 1 mM sodium lactate, 0.3% glucose and 37.5 mM NaCl) at a density of 50,000 cells per well. One hour after plating, the serum was removed. Primary hippocampal cultures contained both neurons and astrocytes, and were used at 3 days in vitro (DIV). For live imaging of mitochondrial mobility, primary hippocampal cultures were prepared from CB −/− P0–P1 mice. Brains were extracted in PBS containing 0.6% glucose and 0.5% bovine serum albumin (BSA) and the hippocampi were dissected. To dissociate cells, a kit for dissociation of postnatal neurons was used following the manufacturer’s instructions (Milteny Biotec, France). Cells were seeded onto 0.5 mg ml−1 poly-l-lysine-coated 35-mm glass-bottom dishes (MatTek Corporation, France) for live imaging in neurobasal medium (Gibco, France) containing 2 mM l-glutamine, 120 μg ml−1 penicillin, 200 μg ml−1 streptomycin and B27 supplement (Invitrogen, France), and were maintained at 37 °C in the presence of 5% CO . Cells were cultured for 7 to 9 days. Neuron transfection was carried out at 4–5 DIV, using a standard calcium phosphate transfection protocol, with a 1:2 DNA ratio of plasmids expressing pDsred2–mito37 to GFP, CB -GFP or DN22CB -GFP, respectively. Axonal mitochondrial mobility was recorded 72–96 h after transfection (see below). Cannabinoid treatments altered the percentage of axonal mobile mitochondria, without altering velocity, dwelling time or travelled distance (data not shown). Mouse fibroblasts were generated from P0–P1 CB −/− pups. After mice were killed by decapitation, the dorsal skin was excised and minced in PBS. Cells were then separated by incubation in 0.25% trypsin solution in PBS, collected by centrifugation and resuspended in DMEM with 10% fetal bovine serum, 1% l-glutamine and 2% penicillin/streptomycin solution (Invitrogen, France). Cells were seeded in 25-cm2 flasks and then expanded in 75-cm2 flasks until reaching 90% confluence. Transfections were carried out by using a BTX-electroporator ECM 830 (Harvard Apparatus, France) (175 V, 1-ms pulse, five pulses, 0.5-s interval between pulses). Cells were electroporated in Optimem medium (Invitrogen, France) at 2 × 107 cells per ml (fibroblasts from two 75 cm2 flasks at 90% confluence in 300 μl) in a 2-mm gap cuvette using 30 μg of either control plasmid (mCherry), CB or DN22-CB cDNA coupled with mCherry, respectively. After electroporation, cells were resuspended in DMEM with 10% fetal bovine serum, 1% l-glutamine and 2% penicillin/streptomycin solution (Invitrogen, France) and seeded in three 100 cm2 Petri dishes. All cells were maintained at 37 °C and 5% CO and collected 48 h after transfection for respiration experiments. The brains of CB +/+ and CB −/− littermates were dissected and mitochondria were purified using a Ficoll gradient as previously described7, 8. In brief, brains were extracted in ice-cold isolation buffer (250 mM sucrose, 10 mM Tris, 1 mM EDTA, pH 7.6) containing protease inhibitors (Roche, France) and 2 M NaF and homogenized with a Teflon potter. Homogenates were centrifuged at 1,500g for 5 min (4 °C). The supernatant was then centrifuged at 12,500g (4 °C). The pellet was collected and the cycle of centrifugation was repeated. To purify mitochondria, the final pellet was resuspended in 400 μl of isolation buffer, layered on top of a discontinuous Ficoll gradient (10% and 7% fractions) and centrifuged at 100,000g for 1 h (4 °C). Purified mitochondria were recovered from the pellet obtained after ultracentrifugation. All experiments using freshly isolated brain mitochondria were performed within 3 h after purification. The 3T3 cells were collected, resuspended in isolation buffer and disrupted with 25 strokes using a 25G needle. The total cell lysate was centrifuged at 500g (4 °C) to remove cells debris and nuclei. The supernatant was kept and centrifuged at 12,500g for 10 min (4 °C). The supernatant was then kept (cytosolic fraction), the pellet was resuspended, and the centrifugation cycle was repeated. Finally, the mitochondrial fractions were obtained from the last pellet. The oxygen consumption of isolated mitochondria, homogenized hippocampus and cell lines was monitored at 37 °C in a glass chamber equipped with a Clark oxygen electrode (Hansatech, UK). Purified mitochondria (75–100 μg) were suspended in 500 μl of respiration buffer (75 mM mannitol, 25 mM sucrose, 10 mM KCl, 10 mM Tris-HCl pH 7.4, 50 mM EDTA) in the chamber. Respiratory substrates were added directly to the chamber. Pyruvate (5 mM), malate (2 mM) and ADP (5 mM) were successively added to measure complex-I-dependent mitochondrial respiration. Complex-II-dependent respiration was measured using rotenone (0.5 μM), succinate (10 mM) and ADP (5 mM). Complex-IV-dependent respiration was measured using N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD, 0.5 mM) and ascorbate (2 mM), in the presence of ADP (5 mM) and antimycin A (2.5 μM). Complex-I-dependent respiration was evaluated, unless stated otherwise. For respiration in homogenized hippocampi, both hippocampi of each mouse were dissected and homogenized with a Teflon potter in 800 μl Mir05 buffer (Mitochondrial Physiology Network: 0.5 mM EGTA, 3 mM MgCl , 60 mM lactobionate, 20 mM taurine, 10 mM KH PO , 20 mM HEPES, 110 mM sucrose and 1 g l−1 BSA) containing 12.5 μg ml−1 of saponin. Subsequently,15 μl of the homogenate was diluted in 1 ml Mir05 buffer and the oxygen consumption was measured with the respiratory substrates pyruvate (5 mM), malate (2 mM) and ADP (5 mM) to measure complex-I-dependent mitochondrial respiration before and after WIN (100 nM) or vehicle addition. Oligomycin (2 μg ml−1), FCCP (0.5 μM), rotenone (0.5 μM) and antimycin A (2.5 μM) were injected subsequently into the chamber as modifiers of the respiration. 50 μl of the homogenate were saved for WB and protein quantification experiments. The experiments using cell lines were performed on 2 × 106 cells ml−1 in growth medium. Intact cells were transferred directly into the chamber and basal respiration was recorded. Drugs were added directly into the chambers. Mitochondria were incubated with PTX, KH7 and H89 for 5 min before addition of CB agonists. HCO − and 8-Br-cAMP were added 5 min after the addition of CB agonists. Oxygen consumption of primary hippocampal cultures was monitored using an XF96 Seahorse Bioscience analyser (Seahorse Bioscience, Denmark), according to the manufacturer’s protocol. When indicated, oligomycin (2 μg ml−1) and FCCP (1 μM) were injected directly into the wells. Other drugs were directly added into the medium 1 h before measurements. Respiration of HEK293 cells co-expressing CB and NDUFS2-PM or MLS–PKA-CA was analysed using the Oxygraph-2k (Oroboros Instruments, Austria). These experiments were performed on 5 × 105 cells ml−1 in growth medium. WIN was directly added into the medium 30 min before measurements. Then, intact cells were transferred into the 2-ml chamber and basal respiration was recorded. NADH oxidation into NAD+ by the first complex of the respiratory chain is coupled to the reduction of ubiquinone (coenzyme Q). The rate of this reaction is analysed by the measurement of NADH disappearance, which is spectrophotometrically detected (SAFAS, UVmc2) at 340 nm. The NADH extinction coefficient is 6.22 mM−1 cm−1. Final composition of the reaction solution was 50 mM K HPO pH 7.2, 2.5 mg ml−1 BSA, 0.1 mM ubiquinone and 200 μg total cell extract proteins or 50 μg purified brain mitochondria. The reaction was initiated by adding 0.1 mM NADH. The assay was monitored at 37 °C for 5 min. The intracellular ATP content was measured using the bioluminescent ATP kit HS II (Roche, France). CB +/+ and CB −/− primary hippocampal cultures (50,000 cells per well in a 96-well dish) were treated with THC (1 μM), WIN (1 μM) or vehicle in the presence or absence of rotenone (0.1 μM) for 1 h. Then, ATP measurements were performed as previously described38. In brief, cells were lysed to release the intracellular ATP using the lysis buffer provided with the kit (equal volume) for 20 min. The lysate was then analysed in a 96-well plate luminometer (Luminoskan, Thermo Scientific, France) using the luciferine/luciferase reaction system provided with the kit. For this, 100 μl of luciferine/luciferase was injected in the wells and after 10 s of incubation, bioluminescence was read (1 s integration time). Standardizations were performed with known quantities of standard ATP provided with the kit. The ATP content derived from mitochondria was determined by subtracting ATP values from the ATP ; (ATP = ATP − ATP ). 100 μg of mitochondria were suspended in isolation buffer, untreated or incubated with 0.01% trypsin in the presence or absence of 0.05% triton X-100 for 15 min at 37 °C. Proteins were then processed for western immunoblot analyses. Freshly purified brain mitochondria were resuspended in PBS (5 mg ml−1) supplemented with protease inhibitor cocktail (Roche, France) and 2 mM NaF, and solubilized with 1% lauryl maltoside for 30 min (4 °C). For co-immunoprecipitation of sAC and G , mitochondria were incubated with THC (800 nM) or vehicle for 5 min at 37 °C. Proteins were incubated with a C-terminal anti-CB antibody (Cayman, USA) or sAC R21 antibody (CEP Biotech, USA) overnight (4 °C). For immunoprecipitation of complex I, mitochondrial proteins were treated with THC (800 nM), HCO − (5 mM), 8-Br-cAMP (500 μM) or vehicle for 5 min at 37 °C and then incubated with complex-I-agarose-conjugated beads (Abcam, UK). Protein A/G PLUS-agarose beads (Santa Cruz, USA) were then added and the incubation continued for 4 h (4 °C). The elution was performed using glycine buffer (0.2 M glycine, 0.05% lauryl maltoside pH 2.5) and samples were processed for western immunoblotting. Following transfection (mCherry, CB or DN22-CB , respectively), cells were allowed to recover in serum containing medium for 24 h. Cells were then starved overnight in serum-free DMEM before treatment and lysis. The cells were then treated at 37 °C with HU210 (100 nM) or vehicle for 10 min. The medium was rapidly aspirated and the samples were snap-frozen in liquid nitrogen and stored at −80 °C before preparation for western blotting. For ERK-phosphorylation assays, lysis buffer (1 mM EGTA, 50 mM NaF, 1 mM Na VO , 50 mM Tris pH 7.5, 1% triton X-100, protease inhibitors, 30 mM 2-mercaptoethanol) was added and the cells were collected by scraping and pelleted by centrifugation at 12,500g (4 °C) for 5 min to remove cell debris. Protein concentrations were measured using the Pierce BCA protein assay kit (Thermo Scientific), loaded with Laemmli buffer and kept at −80 °C. For western immunoblotting, the proteins were separated on Tris-glycine 7%, 10% or 12% acrylamide gels and transferred to PVDF membranes. Membranes were soaked in 5% milk (5% BSA for phosphorylation immunoblots) in tris-buffered saline (TBS; Tris 19.82 mM, NaCl 151 mM, pH 7.6) containing tween20 (0.05%). Mitochondrial proteins were immunodetected using antibodies against complex III core 2 (Abcam, ab14745; 1:1,000, 1 h, room temperature), succinate dehydrogenase subunit A (Abcam, ab14715; 1:10,000, 1 h, room temperature), NDUFA9 (Abcam, ab14713; 1:1,000, 1 h, room temperature), NDUFS2 (Abcam, ab110249; 1:1,000, 1 h, room temperature) and TOM20 (Santa Cruz, sz-11415; 1:1,000, 1 h, 4 °C). Cytosolic proteins were probed with LDHa (Santa Cruz, sz-137243; 1:500, overnight, 4 °C). Samples were also probed with antibodies against G proteins (Enzo Life Science, SA-126; 1:1,000, 1 h, room temperature), sAC (CEP Biotech, sAC R21; 1:500, overnight, 4 °C), PKA (cAMP protein kinase catalytic subunit, Abcam, ab76238; 1:1,000, 1 h, room temperature), an antiserum directed against the C terminus of CB receptor (Cayman, 10006590; 1:200, overnight, 4 °C), AKAP121 (from A. Feliciello; 1:1,000, overnight, 4 °C), PKA-dependent phosphorylation sites (phospho (Ser/Thr)-PKA substrate, Cell Signaling, 9621; 1:1,000, overnight, 4 °C) and HA (Abcam, ab18181; 1:500, overnight, 4 °C), p-ERK (phospho-p44/42 MAPK) corresponding to residues around Thr202/Tyr204 (Cell Signaling, 4370; 1:1,000, overnight, 4 °C), ERK (p44/p42 MAPK; Cell Signaling, 9102; 1:2,000, 1 h, room temperature). Mitochondrial proteins were also separated by two-dimension electrophoresis as described39. Purified brain mitochondria were solubilized (10 mg ml−1) in 0.75 M aminocaproic acid, 50 mM BisTris, (pH 7.0) with 1.5% n-dodecyl-maltoside for 30 min on ice, and were then centrifuged at 16,000g (4 °C). The supernatant was collected and supplemented with 0.25% coomassie blue G and protease inhibitors (Roche, France). Proteins were then separated with 4–16% gradient native-PAGE gels (Invitrogen, France). The different lanes were cut out and processed for the second dimension on 12.5% SDS–PAGE gels after denaturation and reduction in 1% (w/v) sodium dodecyl sulphate and 1% (v/v) mercaptoethanol. The second dimension gels were immunoblotted for detection of PKA-dependent phosphorylated proteins. A second-dimension gel was kept for coomassie blue staining. Then, membranes were washed and incubated with appropriate secondary horseradish peroxidase (HRP)-coupled antibodies (1 h, room temperature). Finally, the HRP signal was detected using the ECL-plus reagent (Amersham) and the Bio-Rad Quantity One system. Labelling was quantified by densitometric analysis using ImageJ (NIH) software. HeLa cells were fixed in 4% formaldehyde dissolved in PBS (0.1 M, pH 7.4) and then washed with PBS. Cells were pre-incubated in a blocking solution of 10% normal goat serum, 0.1% triton X-100, 0.05% deoxycholate and 0.2 M glycine prepared in PBS for 1 h and then incubated with primary antibody rabbit anti-TOM20 (Santa Cruz, sc-11415; 1:500) and mouse anti-myc (Roche, 11667149001; 1:500) for 2 h in the same blocking solution. The cells were then washed in PBS for 1 h and were then incubated with fluorescent anti-mouse Alexa488 or anti-rabbit Alexa561 (Jackson ImmunoResearch; 1:800) in blocking solution for 1 h. Finally, cells were washed and mounted with fluoromont-G (Electron Microscopy Sciences). All the procedures were carried out at room temperature. The cells were analysed with a Confocal Leica DMI6000 microscope (Leica). Samples were digested by trypsin as previously described40. Peptides were further analysed by nano-liquid chromatography coupled to a MS/MS LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Germany). Peptides were identified with SEQUEST and MASCOT algorithms through the Proteome Discoverer interface (Thermo Fisher Scientific, Germany) against a subset of the UniProt database restricted to Reference Proteome Set of Mus musculus (UniProtKB Release 2011_12, 14th December, 2011, 46,638 entries). Peptide validation was performed using Percolator algorithm41 and only ‘high confidence’ peptides were retained corresponding to a 1% false positive rate at peptide level. Cyclic AMP levels and PKA activity of mitochondria isolated from the brain were assayed using the Direct Correlate-EIA cAMP kit (Assay Designs Inc., USA) and an ELISA kit (Enzo Life Science), respectively, according to the manufacturers’ instructions. The different treatments described in the main text were performed at 37 °C for 1 h. Mitochondrial mobility in hippocampal neurons was recorded using an inverted Leica DMI6000 microscope (Leica Microsystems, Wetzlar, Germany) equipped with a confocal head Yokogawa CSU-X1 (Yokogawa Electric Corporation, Tokyo, Japan) and a sensitive Quantem camera (Photometrics, Tucson, USA). The diode lasers used were at 491 nm and 561 nm and the objective was a HCX PL APO CS 63× oil 1.32 NA lens. The z stacks were obtained with a piezo P721.LLQ (Physik Instrumente (PI), Karlsruhe, Germany). The 37 °C atmosphere during time-lapse image acquisition was created with an incubator box and an air heating system (Life Imaging Services, Basel, Switzerland) in the presence of 5% CO . This system was controlled by MetaMorph software (Molecular Devices, Sunnyvale, USA). For mitochondrial axonal transport analysis, time-lapse series of image stacks composed of 6 images (512 × 512 pixels) were taken every 3 s for 15 min. HU210 was added just after the recording and 15 min later the same neuron was recorded for another 15 min. KH7 or vehicle were added 15 min before the first recording. All stacks obtained were processed first with MetaMorph software. Further image processing, analysis and video compilation (28 frames per second) and editing was done with ImageJ software (NIH, USA). Kymographs were generated with the KymoToolBox Plugin42. Between 10 and 32 axons were registered and analysed in each condition. In all cases, a mitochondrion was considered mobile when it moved more than 5 μm during the time of recording. Distances and speeds of retrograde and anterograde transport and dwelling time were measured separately from the corresponding kymographs, as previously described43, 44. The microarrays were composed of a collection of membrane homogenates isolated from HEK293 cells transfected with mCherry CB or DN22-CB , or from hippocampi of CB +/+(GFP), CB −/−(GFP), CB −/−(CB ) or CB −/−(DN22-CB ) mice (see below), together with increasing amounts of BSA and membranes isolated from rat cerebral cortex, as positive internal controls45. Briefly, samples were homogenized using a Teflon-glass grinder (Heidolph RZR 2020) or a disperser (Ultra-Turrax T10 basic, IKA) in 20 volumes of homogenized buffer (1 mM EGTA, 3 mM MgCl , and 50 mM Tris-HCl pH 7.4) supplemented with 250 mM sucrose. The crude homogenate was subjected to a 40g centrifugation for cells or 200g for tissue for 5 min, and the resultant supernatant was centrifuged again at 18,000g for 15 min (4 °C, Microfuge 22R centrifuge, Beckman Coulter). The pellet was washed in 20 volumes of homogenized buffer and re-centrifuged under the same conditions. The homogenate aliquots were stored at −80 °C until use. Protein concentration was measured by the Bradford method and adjusted to the required concentrations. Microarrays were fabricated by a non-contact microarrayer (Nano_plotter NP 2.1) placing the cell membrane homogenates (4 nl per spot, 3–5 replicates per sample) onto glass slides46. Microarrays were stored at −20 °C until use. After thawing, cell membrane microarrays were incubated in assay buffer (50 mM Tris-Cl; 1% BSA; pH 7.4) for 30 min at room temperature. A second incubation was performed using the same buffer for 120 min at 37 °C in the presence of [3H]CP55,940 (3 nM). Non-specific binding was determined with 10 μM WIN55,212-2. Afterwards, microarrays were washed twice in buffer, dipped in deionized water and dried. Finally, they were exposed to films, developed, scanned and quantified as described below. [35S]GTPγS binding studies were carried out according to the patented methodology for the screening of molecules that act through G-protein-coupled receptors using cell membrane microarrays45. Briefly, thawed cell membrane microarrays were dried 20 min at room temperature and were subsequently incubated in assay buffer (50 mM Tris-Cl; 1 mM EGTA; 3 mM MgCl ; 100 mM NaCl; 0,5% BSA; pH 7.4) for 15 min at room temperature. Microarrays were transferred into assay buffer containing 50 μM GDP and 0.1 nM [35S]GTPγS, with the cannabinoid agonists WIN55,212-2 or HU210, at increasing concentrations, and incubated at 30 °C for 30 min. Non-specific binding was determined with GTPγS (10 μM). After washing, microarrays, together with ARC [14C]-standards, were exposed to films, developed, scanned and quantified. The protein concentration in each spot was measured by the Bradford method and used to normalize the [35S]GTPγS binding results to nCi per ng protein. Data from the dose–response curves (5 replicates in triplicate) were analysed using the program Prism (GraphPad Software Inc., San Diego, CA) to yield EC (effective concentration 50%) and E (maximal effect) of the drugs on each different sample by nonlinear regression analysis. Samples displaying [3H]CP55,940 binding below the values of hippocampi from CB −/− mice were excluded from [35S]GTPγS binding analysis. Mice (7–9 weeks of age) were anaesthetized by i.p. injection of a mixture of ketamine (100 mg kg−1; Imalgene 500, Merial) and Xylazine (10 mg kg−1; Rompun, Bayer) and placed into a stereotaxic apparatus (David Kopf Instruments) with mouse adaptor and lateral ear bars. For intracerebroventricular injections of drugs, mice were unilaterally implanted with a 1.0-mm stainless-steel guide cannula targeting the lateral ventricle with the following coordinates: anterior–posterior −0.2; lateral ± 0.9; dorsal–ventral −2.0. For intrahippocampal injections of drugs, mice were bilaterally implanted with 1.0-mm stainless-steel guide cannulae targeting the hippocampus with the following coordinates: anterior–posterior −3.1; medial–lateral ± 1.3; dorsal–ventral −0.5. Guide cannulae were secured with cement anchored to the skull by screws. Mice were allowed to recover for at least one week in individual cages before the start of experiments. Mice were weighed daily and individuals that failed to return to their pre-surgery body weight were excluded from subsequent experiments. The intrahippocampal and intracerebroventricular drug injections were performed by using injectors protruding 1 mm from the tip of the cannula. For viral intrahippocampal AAV delivery, mice were submitted to stereotaxic surgery (as above) and AAV vectors were injected with the help of a microsyringe (0.25-ml Hamilton syringe with a 30-gauge bevelled needle) attached to a pump (UMP3-1, World Precision Instruments). Mice were injected directly into the hippocampus (0.5 μl per injection site at a rate of 0.5 μl per min), with the following coordinates: dorsal hippocampus, anterior–posterior −1.8; medial–lateral ± 1; dorsal–ventral −2.0 and −1.5; ventral hippocampus: anterior–posterior −3.5; medial–lateral ± 2.7; dorsal–ventral −4 and −3. Following virus delivery, the syringe was left in place for 1 min before being slowly withdrawn from the brain. CB +/+ mice were injected with AAV–GFP to generate CB +/+(GFP) mice; CB −/− mice were injected with AAV–GFP, AAV–CB or AAV–DN22-CB , to obtain CB −/−(GFP), CB −/−(CB ) and CB −/−(DN22-CB ) mice, respectively. Animals were used for experiments 4–5 weeks after injections. Mice were weighed daily and individuals that failed to return to their pre-surgery body weight were excluded from subsequent experiments. CB -receptor expression was verified by fluorescent or electromicroscopic immunohistochemistry (see below). The AAV vectors, MLS–PKA-CA or NDUFS2-PM were injected directly into the dorsal hippocampus (1.0 μl per injection site at a rate of 0.5 μl per min) of C57BL/6N mice, with the following coordinates: anterior–posterior −1.8; medial–lateral ± 1; dorsal–ventral −2.0 and −1.5. Following virus delivery, the syringe was left in place for 1–2 min before being slowly withdrawn from the brain. Animals were used for experiments 4–5 weeks after viral delivery. Mice were habituated to i.p. injections (saline) before the behavioural paradigm (see below). The hippocampal expression of myc-tagged MLS–PKA-CA and NDUFS2-PM was verified by immunohistochemistry using anti-myc antibodies. Mice were anaesthetized with chloral hydrate (400 mg kg−1 body weight), transcardially perfused with Ringer solution (NaCl (135 mM), KCl (5.4 mM), MgCl ·6H O (1 mM), CaCl ·2H 0 (1.8 mM), HEPES (5 mM)). Heparin choay (25,000 UI per 5 ml) was added extemporarily and tissues were then fixed with 500 ml of 4% formaldehyde dissolved in PBS (0.1 M, pH 7.4) and prepared at 4 °C. After perfusion, the brains were removed and incubated several additional hours in the same fixative. Serial vibrosections were cut at 40–50-μm thickness and collected in PBS at room temperature. Sections were pre-incubated in a blocking solution of 10% donkey serum, 0.1% sodium azide and 0.3% triton X-100 prepared in PBS for 30 min–1 h at room temperature. Free-floating sections were incubated for 48 h (4 °C) with goat anti-CB polyclonal antibodies raised against a C-terminal sequence of 31 amino acids (NM007726) of the mouse CB receptor (CB -Go-Af450-1; 2 μg ml−1; Frontier Science Co. Ltd) or overnight (4 °C) with rabbit anti-myc (Ozyme; 1:1,000). The antibody was prepared in 10% donkey serum in PBS containing 0.1% sodium azide and 0.5% triton X-100. Then, the sections were washed in PBS for 30 min at room temperature. The tissue was subsequently incubated with fluorescent anti-goat Alexa488 (1:200, Jackson ImmunoResearch) for 4 h and washed in PBS at room temperature, before being incubated with DAPI (1:20,000) for 10 min for nuclear counterstaining. Finally, sections were washed, mounted, dried and a coverslip was added on top with DPX (Fluka Chemie AG). The slides were analysed with an epifluorescence Leica DM6000 microscope (Leica). CB +/+(GFP), CB −/−(GFP), CB −/−(CB ) and CB −/−(DN22-CB ) mice (n = 3 per group) were processed for electron microscope pre-embedding immunogold labelling as previously described7, 8. Immunodetection was performed in 50-μm-thick sections of hippocampus with goat anti-CB polyclonal antibodies raised against a 31 amino acid C-terminal sequence (NM007726) of the mouse CB receptor (Frontier Institute Co. Ltd, CB -Go-Af450-1; 2 μg ml−1). Immunogold particles were identified and counted. To exclude the risk of counting possible false positive mitochondrial labelling, we used strict semi-quantification methods of mtCB receptors as recently described, excluding immunogold particles that were located on mitochondrial membranes but at a distance ≤80 nm from other cellular structures8. The normalized number of immunogold particles located on mitochondria versus the total amount of immunogold particles in each field was used to calculate the proportion of mtCB receptors over total CB . Mice were anaesthetized with isoflurane and killed by decapitation. Brains were rapidly removed and chilled in an ice-cold, carbonated (bubbled with 95% O –5% CO ) cutting solution containing 180 mM sucrose, 2.5 mM KCl, 0.2 mM CaCl , 12 mM MgCl , 1.25 mM NaH PO , 26 mM NaHCO and 11 mM glucose (pH 7.4). Sagittal hippocampal slices (350-μm thick) were cut using a Leica VT1200S vibratome and incubated with artificial cerebrospinal fluid (ACSF) containing 123 mM NaCl, 1.25 mM NaH PO4, 11 mM glucose, 2.5 mM KCl, 2.5 mM CaCl , 1.3 mM MgCl and 26 mM NaHCO (osmolarity of 298 ± 7; pH 7.4) for 30 min at 34 °C. The slices were subsequently transferred to a holding chamber, where they were maintained at room temperature until experiments. Slices were individually transferred to a submerged chamber for recording and continuously perfused with oxygenated (95% O –5% CO ) ACSF (3–5 ml min−1). All experiments were performed at room temperature. fEPSPs were recorded using glass micropipettes (2–4 mΩ) filled with normal ACSF positioned in the CA1 hippocampal region. Slices from the middle hippocampus were used preferentially. fEPSPs responses were evoked by stimulation (0.1-ms duration, 10–30-V amplitude) delivered to the stratum radiatum to stimulate the Schaffer collateral fibres using similar glass electrodes used for the recordings, in the presence of picrotoxin 100 μM. Recordings were obtained using an Axon Multiclamp 700B amplifier (Molecular Devices). Signals were filtered at 2 kHz, digitized, sampled and analysed using Axon Clampfit software (Molecular Devices). In CB −/−(CB ) and CB −/−(DN22-CB ), two slices (1 each) were excluded from analysis, because immunohistochemistry showed no re-expression. To study the effect of mtCB receptor signalling on cannabinoid-induced amnesia, we used the hippocampal-dependent NOR memory task in an L-maze (L-M/NOR)14, 47, 48. As compared to other hippocampal-dependent memory tasks, this test presents several advantages for the aims of the present study: (i) the acquisition of L-M/NOR occurs in one step and previous studies revealed that the consolidation of this type of memory is deeply altered by acute immediate post-training administration of cannabinoids via hippocampal CB receptors14, 48; (ii) this test allows repeated independent measurements of memory performance in individual animals47, thereby allowing within-subject comparisons, eventually excluding potential individual differences in viral infection and/or expression of proteins; (iii) notably, CB −/− mice do not respond to the administration of cannabinoids, but they do not show any spontaneous impairment of performance in L-M/NOR14, thereby allowing the use of re-expression approaches to study the role of hippocampal mtCB receptors in the cannabinoid-induced blockade of memory consolidation. This task was performed with an L-maze made out of dark-grey Plexiglas with two corridors (35 cm and 30 cm long, respectively, for external and internal V walls, 4.5 cm wide and 15-cm high walls) set at a 90° angle and under a weak light intensity (50 Lux). The task consisted of 3 sequential daily trials of 9 min. Day 1 (habituation): mice were placed at the intersection of the two arms and were let free to explore the maze. Day 2 (acquisition): two identical objects were placed at the end of each arm. After 9 min of exploration, mice were removed and injected. Day 3 (retrieval): A novel object different in its shape, colour and texture was placed at the end of one of the arm, whereas the familiar object remained at the end of the other arm. The position of the novel object and the pairings of novel and familiar objects were randomized. Exploration of each object was scored off-line by at least two experienced observers blind to treatments and/or genotypes. Exploration was defined as the time spent by the mouse with the nose pointing to the object at a distance of less than 1 cm, whereas climbing on or chewing the object was not considered as exploration14. Memory performance was assessed by the discrimination index. The discrimination index was calculated as the difference between the time spent exploring the novel (TN) and the familiar object (TF) divided by the total exploration time (TN+TF): discrimination index = (TN−TF)/(TN+TF). Mice receiving the acute intrahippocampal infusion of KH7 (10 mM) and WIN (5 mg kg−1) i.p., and mice that received intrahippocampal injection with AAV–MLS–PKA-CA or AAV–NDUFS2-PM were submitted to a single L-M/NOR session. Due to the limited numbers of available mice, null CB −/− mice virally injected with AAV–GFP, AAV–CB or AAV–DN22-CB were tested twice with a one-week interval using different pairs of objects and treated the first time with vehicle and the following with WIN. Every pair of objects was previously screened to exclude that the animals might exhibit significant preference for any specific item. After the NOR task, the hippocampi of vehicle-treated AAV–MLS–PKA-CA and AAV–NDUFS2-PM animals were dissected and used for respiration experiments (see above). Expression of the myc epitope was verified by immunohistochemistry in the hippocampi of animals treated with WIN. All graphs and statistical analyses were performed using GraphPad software (version 5.0 or 6.0). Results were expressed as means of independent data points ± s.e.m. For biochemical quantifications (cAMP levels, PKA and complex-I activities and oxygen consumption), data are presented as percentage of controls with or without the application of cannabinoid drugs. With the exception of KH7 (see Extended Data Fig. 5a), no other drugs and plasmids had any effect per se on any measured parameter (not shown). Data were analysed using paired or unpaired Student’s t-test, one-way (followed by Tukey's post hoc test) or two-way ANOVA (followed by Bonferroni's post hoc test), as appropriate. Detailed statistical data for each experiment are reported in Supplementary Tables 1–3.
Chronaki C.,HL7 International Foundation |
Estelrich A.,PHAST |
Cangioli G.,HL7 International Foundation |
Melgara M.,Lispa |
And 6 more authors.
Studies in Health Technology and Informatics | Year: 2014
In an increasingly mobile world, many citizens and professionals are frequent travellers. Access during unplanned care to their patient summary, their most essential health information in a form physicians in another country can understand can impact not only their safety, but also the quality and effectiveness of care. International health information technology (HIT) standards such as HL7 CDA have been developed to advance interoperability. Implementation guides (IG) and IHE profiles constrain standards and make them fit for the purpose of specific use cases. A joint effort between HL7, IHE, and HealthStory created Consolidated CDA (C-CDA), a set of harmonized CDA IGs for the US that is cited in the Meaning Use II (MU-II) regulation. In the EU, the Patient Summary (PS) Guideline recently adopted, cites the epSOS IG also based on HL7 CDA, to support cross-border care in the EU and inform national eHealth programs. Trillium Bridge project supports international standards development by extending the EU PS Guideline and MU-II in the transatlantic setting. This paper presents preliminary findings from comparing patient summaries in the EU and US and reflects on the challenge of implementing interoperable eHealth systems in the cross-border or transatlantic setting. © 2014 European Federation for Medical Informatics and IOS Press.
Gouda M.,MUST |
Journal of Theoretical and Applied Information Technology | Year: 2012
Since the release by the Federal Communications Commission (FCC) of a bandwidth of 7.5GHz (from 3.1GHz to 10.6GHz) for ultra-wideband (UWB) wireless communications, UWB is rapidly advancing as a high data rate wireless communication technology. As is the case in conventional wireless communication systems, an antenna also plays a very crucial role in UWB systems. However, there are more challenges in designing a UWB antenna than a narrow band one. A suitable UWB antenna should be capable of operating over an ultra-wide bandwidth as allocated by the FCC. At the same time, satisfactory radiation properties over the entire frequency range are also necessary. To choose an optimum antenna topology for ultra wideband (UWB) design, several factors must be taken into account including physical profile, compatibility, impedance bandwidth, radiation efficiency, and radiation pattern. The main challenge in UWB antenna design is achieving the very broad bandwidth with high radiation efficiency and small in size. Accordingly, many techniques to broaden the impedance bandwidth of small antennas and to optimize the characteristics of broadband antennas have been widely investigated in many published papers as listed in this article. Planar monopole antennas are good candidates owing to their wide impedance bandwidth, omni-directional radiation pattern, compact and simple structure, low cost and ease of construction. Further detail on various bandwidth enhancement techniques will be discussed in this paper. This paper focuses on UWB planar printed circuit board (PCB) antenna design and analysis. Extensive investigations are carried out on the development of UWB antennas from the past to present. First, the planar PCB antenna designs for UWB system is introduced and described. Next, the special design considerations for UWB antennas are summarized. State-of-the-art UWB antennas are also reviewed. Finally, a new concept (case studies) for the design of a UWB antenna with a bandwidth ranging from 3GHz-8GHz is introduced, which satisfies the system requirements for S-DMB, WiBro, WLAN, CMMB and the entire UWB. © 2005 - 2012 JATIT & LLS. All rights reserved.
Gouda M.,MUST |
Journal of Theoretical and Applied Information Technology | Year: 2010
This paper presents an Ultra Wide Band Microstrip Yagi antenna with very good performance at four main operating frequencies (0.85, 2.4, 3.5 and 5.2) GHz. Numerical simulation results of our design show more than (-20 to -40) dB return loss at the bands of (0.85, 2.4, 3.5, 4.5 and 5.2) GHz and VSWR less than 1.7 at these frequencies. The simulation method based on finite element method (FEM) was applied by using HFSS simulator to obtain the optimized parameters in order to find the best design for this antenna. The antenna bands can be varied to be suitable for various wireless applications. Finally a fabrication to the final design has been implemented, and then a measurement performed to compare the actual results with those simulated. Measured return loss and VSWR of this antenna are presented to validate the results of simulation so that it give (0.85, 2.4, 2.75, 3.6, 4.2 and 5.3) GHz for the measured return loss results and the antenna VSWR ≤ 1.5 at (2.4, 3.5, 4.5 and 5.2) GHz. © 2005 - 2010 JATIT & LLS. All rights reserved.
Haponava T.,NEN |
Al-Jibouri S.,University of Twente
Construction Management and Economics | Year: 2010
Within a project environment, good process performance can be seen as indicative of eventual success of achieving end-project goals. A model is proposed for identifying the links between process performance during construction and end-project goals. The model is developed using process mapping technique to identify a number of process-based key performance indicators (KPIs) designed for use in controlling process performance in the construction stage. These KPIs were then linked to generic project goals to measure the perceived degree of influence of process performance on the achievement of end-project goals. The strength of these links within the model was established through a number of in-depth interviews with respondents. Analysis of the results using methods of ranking and statistics has shown that some processes are perceived to have stronger relationships with the end-project goals than others. The proposed process-based KPIs hence offer an opportunity for control of the processes during the construction stage and eventually to influence the projects' outcomes. In addition to the management of project time and cost, the model provides a broader focus of control that includes other multiple aspects such as the management of interactions between internal and external stakeholders as well as of information and quality management. © 2010 Taylor & Francis.
Strobbe C.,Catholic University of Leuven |
Mosies C.,NEN |
Buhler C.,Forschungsinstitut Technology und Behinderung FTB |
Engelen J.,Catholic University of Leuven
Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics) | Year: 2010
The Stand4All project is a response to a call for tenders by the European Commission's Directorate-General for Employment, Social Affairs and Equal Opportunities. Its goal is to increase the use of CEN/CENELEC Guide 6, which contains guidance on the inclusion of the needs of elderly persons and persons with disabilities during the standards development process. To reach this goal, the project consortium has developed and delivered training on standardisation in several locations in Europe and for two types of audience: persons with disabilities and members of standardisation committees. The training for persons with disabilities is meant to increase their effective participation in the standardisation process. The training for committee members is meant to increase their awareness and knowledge of accessibility issues faced by persons with disabilities. © 2010 Springer-Verlag Berlin Heidelberg.