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text: La crisi del III secolo 1º lavoro: mappa mindomo ACCESS DENIED Un video da commentare Presentazione 4.pdf - Google Drive L'impatto della guerra in Siria Meet Google Drive - One place for all your files Google Drive is a free way to keep your files backed up and easy to reach from any phone, tablet, or computer. Start with 15GB of Google storage - free. Flash cards Google Sheets - create and edit spreadsheets online, for free. Create a new spreadsheet and edit with others at the same time -- from your computer, phone or tablet. Get stuff done with or without an internet connection. Use Sheets to edit Excel files. Free from Google.

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Series: CPRE Campaigners Guides and Briefings
By: CPRE
Council for the Protection of Rural England
Comprehensive guide on how to use the planning process for the benefit of the environment, including guidance on local plan procedures and how to organise a campaign. Prepared for CPRE by Green Balance.
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Build Words Gameplay 1 iOS / Android
Build Words
By: aMASE
Genre: Education
Video Tips
Let's play Build Words! This is part 1 of my walkthrough guide for the iOS game, Build Words by aMASE AB.
I will update this post with more tips and tricks, so please bookmark this page and keep on checking in!
If you have any questions, feel free to ask for extra help in the comments section.
Hit like if this helped you!
Visit Build Words walkthrough page for level videos, tutorials, cheats and more.
Gameplay Questions
Questions related to gameplay of Build Words iPhone game.


text:
Mumbai: Sachin, Ganguly and Dravid had a played key role in Mahendra Singh Dhoni's appointment as the Indian captain for the inaugural World Twenty20 Cup in 2007, former chief selector Dilip Vengsarkar revealed here on Monday.
Vengsarkar who was in England then interacted Sachin, Rahul and Sourav who felt Dhoni had the right attitude to become the India captain.
"We (selectors) too felt so, though there was initial apprehension about how he would assume full charge", he said
Sachin, Ganguly and Dravid had withdrawn from the T20 world cup to pave the way for to Dhoni become the captain.
Former Indian captain said he knew that the team would win the 2007 title after the league opener against Pakistan which ended in a tie and had to be resolved through a bowl-out won by Dhoni and his men.
"We had beaten Pakistan in our first match in 1985 in the Benson and Hedges (World Championship in Australia) and went on to win the championship", Vengsarkar recalled, explaining reasons for his optimism two years ago about the Dhoni-led side.
Best memories of the T20 World Cup
Vengsarkar predicts that a total of 180 should be winning one in the seaming conditions in England.


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We discredit Lisa Simpson's look because it's so similar to Marge's, but in truth, she's set the bar for footwear since 1989. Enter: Mary Janes, the most accessible, easy to wear, and available types of shoes to exist. Saint Laurent paid homage in leopard print this season, Christopher Kane did the platform thing, and Ralph Lauren kept it simple.
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Plus Side of FashionFashion showsplus size models40 Years Later, Jai, Veeru, Basanti Still Have the Sholay SparkTuesday, February 16, 2016It was truly an epic moment when actors Amitabh Bachchan, Dharmendra, Hema Malini and Jaya Bachchan Sholay's Jai, Veeru, Basanti and Radha shared the stage for the launch of Hema Malini's new album Dream Girl. Hemaji's husband Dharmandra went down memory lane as he spoke about his dostana with the Bachchans. Tom Hiddleston planted a peck on Quvenzhane's cheek.
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BMW has intentionally or unintentionally released a teaser video of the upcoming R nineT Scrambler, giving us a chance to have a good look at the motorcycle that will debut next week at the Esposizione Internazionale Ciclo Motociclo e Accessori (EICMA), or the Milan Motorcycle Show.
The BMW R nineT Scrambler looks very similar to the BMW Concept Path 22 motorcycle we had earlier showed you. As there is not much detail about the motorcycle yet, feast your eyes on the video.
BMW R nineT Scrambler Images:
Connect with the Author on Twitter @rajkamalnarayan


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Writen By
he first-annual Card Player Player of the Year awards show was held in Hollywood,
on Feb. 15, 2006. The event, created by Card Player's Barry Shulman and sponsored
by Bodog.com, was put on to honor the player of the year as well as other poker
standouts. It was a first-class, gala affair that was a night to remember.
The invitation-only evening started with the players walking the red carpet for the press
and paparazzi. Then, there was an open-bar cocktail party, followed by the awards show.
The master of ceremonies for the evening was Brad Garrett, from the Ray Romano show.
Unless you've seen Brad do stand-up comedy, there's no way you would realize how
funny he is. And on this night, it was a no-holds-barred attack on poker players - and
they loved it.
A couple lines included: "I see Gabe Kaplan is in the house.
I think he had some kind of hit show when I was a fetus." "And Amir Vahedi is here
I met him in flight school." And, of course, he brutalized Mike Matusow: "Mike Matusow's
raising his hand. I see he still has the cuffs on."
The Player of the Year award is determined by a yearlong point system. It's one of
poker's most prestigious awards, and the 2005 winner was Men "The Master" Nguyen.
Incredibly, "The Master" has won that award four times in nine years!
That is amazing to me. Hats off to Men"The Master."
Players were nominated and voted on by the top players for awards in various
categories, and the top three vote-getters in each category were the finalists. Phil Ivey
was the big dog of the night, as he won three categories (Best No-Limit Hold'em Player,
Best Heads-Up Player, and Most-Feared Player). Chip Reese won two (Best Cash-Game
Player and Best Mixed-Game Player). Other awards went to Daniel Negreanu
(People's Choice Award for Favorite Player, as voted on by the public), Jennifer Harman (Best Female Player), Michael Gracz (Breakthrough Player), Allen
Cunningham (Most Underrated Player), Jennifer Tilly (Favorite Celebrity Player), Mike Matusow (Most Entertaining Player), and me (Poker Ambassador).
The highlight of the evening was Doyle Brunson receiving the Lifetime Achievement Award. The standing ovation he got was spine-tingling. Everyone in the
room showed appreciation for what Doyle has meant to poker over the past 40 years. It was fun seeing clips of Doyle as a young athlete (an amazing one, I
might add), and those of his distinguished poker career. His award was a well-deserved honor. Congratulations, Doyle!
I must say that I was quite flattered to win the Poker Ambassador award. At this stage of my career, I can think of no greater honor. The top three nominees
in this category were Doyle Brunson, Daniel Negreanu, and me, and topping those guys in any poker category is a thrill. Although we were the three
nominees, there are several other people who have given so much of themselves to take poker to a higher level who also might have been included on the
list of nominees. They include people like Linda Johnson, Mike Caro, Steve Lipscomb, Barry Greenstein, Barry Shulman, Jack Binion, Roy Cooke, and,
certainly, the past three world champions of poker - Chris Moneymaker, Greg Raymer, and Joe Hachem - who have been great ambassadors for poker.
I owe a great deal of this award to a number of people who have aided me in my career. Let's start with my family, especially my father (Ray) and my brother
(Tom). They've always been my biggest supporters.
Much of the credit for my success in moving to the business side of poker goes to Linda Johnson. She once owned Card Player and hired me to write for the
magazine in 1996. (I haven't missed a column since.) That notoriety as a writer gave me credibility within the industry to create the Tournament of Champions
of Poker (TOC), which was held from 1999 to 2001. Putting that tournament on was a dream come true for me, and Chuck Humphrey was very helpful in
making that event happen. It was the TOC that led me to my jobs with PartyPoker.com and the World Poker Tour, and the success of both of those
companies has been truly amazing.
WPT founder Steve Lipscomb deserves a part of my Poker Ambassador award, as well. I never would have won the award without him hiring me to be a
commentator on the World Poker Tour. (Incidentally, I had no previous broadcast experience prior to him hiring me, so he really did take a shot on me.
Fortunately, his instincts worked out well for both of us.) That job put me in the spotlight, was the reason I wrote Shuffle Up and Deal (a book that made The
New York Times best-seller list), enabled me to teach at WPT Boot Camps, and provided me a platform from which I could talk about the virtues of poker to
both the media and the public.
I always tell anyone who will listen that the World Poker Tour is the primary reason for the growth of poker, and certainly the Travel Channel gets some credit,
as well. It put poker on its schedule in prime time on a weekly basis (Wednesday night is now "poker night" across America), and the popularity of poker
exploded soon thereafter.
If I had one wish as poker ambassador, it would be that all players treat the game, their opponents, and the dealers and poker room staffs, with dignity and
respect. Behaving properly should be a goal of all players. I also believe that everyone who makes a living in the poker industry should automatically be an
ambassador of the game, especially those who have become millionaires from playing poker. Think of the hundreds of thousands of players who dream
about becoming millionaires by playing a game they love. In my opinion, those who have succeeded and those who will succeed in the future have an
obligation to conduct themselves in a professional manner, give back to the game, and be goodwill ambassadors for poker.
Again, I want to thank everyone who voted for me and everyone who made it possible for me to even be nominated for such a prestigious award. In my view,
winning the Poker Ambassador award is the highest honor in poker. It was a night to remember.


text:
Everyone is looking forward to the big Nintendo Switch presentation taking place on the 12th. Nintendo is going to be showing all of its new platform.
We are but a few days away from the big reveals and many can hardly contain their excitement (guilty). On Nintendo's live stream presentation, they will be showcasing all the details on their newest video game console. For two years they have avoided major leaks and losing the punch on their new machine. Now, it is time we get what we waited for.
During this presentation, they will reveal the console's exact release date as well as the launch titles that will be made available during the first few days of the console. Furthermore, we will have the chance of trying this new platform and see the power of seamless transition in front of our eyes. Also, it is interesting to see how much Nintendo is expecting consumers will pay for this unique piece of gaming wonder.
After this big presentations, Nintendo has confirmed that the Nintendo Treehouse will be ho,ding a live stream showing all the games they will reveal all day long on the 13th. If you are not fortunate enough to be able to get into the Nintendo Switch game demo tour that is taking place all over the world, you will still be able to get your fix of gameplay and information through this livestream.
The Nintendo Switch Presentation is set for January 12, and we've got even more planned for you! Join us at 9:30AM ET on 1/13 for Treehouse Live and take an in-depth look at upcoming Nintendo Switch games.


text:
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Shares in builder Ch Karnchang Pcl surged as much as 2.7 percent to their highest in 21 months after the Lao government approved the construction of $3.5-billion Xayaburi dam on the Mekong river.
Ch Karnchang shares were flat at 9.30 baht thanks to profit-taking, a dealer said.
Uncertainty over the hydropower project, which accounts for 60 percent of Ch Karnchang's construction projects in value terms, capped the stock price this year.
It has risen 23 percent so far in 2012, underperforming other builders such as Sino-Thai Engineering and Construction Pcl which has gained 78 percent.
"The project approval is positive and will have a significant impact on Ch Karnchang's fundamentals. We are looking to upgrade earnings forecast and its price target to take into account the Xayaburi project," broker DBS Vickers Securities said in a report.
(Reporting by Viparat Jantraprap in Bangkok; Editing by Subhranshu Sahu; firstname.lastname@example.com)
10:37 STOCKS NEWS THAILAND-Siam Makro falls to 3-week low after weak Q3
Shares in Siam Makro Pcl MAKR.BK fell 6.9 percent to 430 baht, a three-week low, after the cash-and-carry wholesaler reported weaker-than-expected earnings for the third quarter. (Full Story)
"Third-quarter sales revenue turns softer, up 14.3 percent YoY but down 1 percent QoQ possibly due to weak food price inflation witnessed in the first half rather than weak sales volume growth," Citigroup said in a report.
Citi maintained its 'sell' rating on the stock citing expensive valuation.
"While the company has delivered 24 percent YoY growth in its nine-month result, it still trails high consensus expectations," it said.
Siam Makro shares had risen 83 percent so far this year, outperforming a 61 percent gain of the commerce subindex .SETC and a 28 percent gain of the main SET index .SETI.
Reporting by Viparat Jantraprap in Bangkok; Editing by Gopakumar Warrier; firstname.lastname@example.com


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Looks like B & K Rental is no longer available on Rent.com which means this information is likely out of date.
newer condo, spacus floor plan, attached garage, wooded lot,quiet, close to town,w/dryer,dishw in unit
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Quake unravelling continues
Though the aftershocks from last week's Kaikoura earthquake have been quieter this week (knock on wood), science behind the quake continues apace.
Niwa's research vessel Tangaroa was diverted to the Kaikoura coast after last week's earthquake. Surveys of the seabed have confirmed the Needles Fault ruptured 34km offshore. Scientists on board the vessel have collected 61 sediment cores - each measuring about 5.5 metres - from the continental margin between Poverty Bay and Kaikoura (pictured).
It's hoped the cores will provide evidence of submarine landslides. Niwa marine geologist Dr Philip Barnes said the cores revealed a huge turbidity current offshore from Marlborough and Wairarapa - extending at least 300km from Kaikoura. But the locations of underwater landslides that caused the turbidity have not been identified.
Meanwhile back on land, geologists have been looking at evidence of new faulting in Marlborough. University of Canterbury researchers found "spectacular" fault displacements, including an area north of Waiau whihc had 2.2m horizontal displacement and 1.5m vertical.
GPS stations detected a slow-slip earthquake underway in the Hawke's Bay and Gisborne regions. Silent or slow-slip earthquakes are undetectable by both humans and GeoNet's seismographs, but the GPS stations detected movement of up to 2-3cm - similar to that seen in previous East Coast slow-slip events. A magnitude 5.5 earthquake that struck the area on Tuesday fit with previous earthquake "clusters" during slow-slip events in the area.
Kayak guides spotted an interesting by-product of the earthquake - bubbles in the sea off Kaikoura. University of Canterbury's Dr Matthew Hughes said the bubbles were likely due to a mix of carbon dioxide and hydrogen sulphide released through cracks in the seafloor caused by the magnitude 7.8 earthquake.
An aftershock that damaged homes in North Canterbury on Tuesday was within the aftershock forecast, GNS Science's Natalie Balfour told media. But since then, things have been particularly quiet.
GeoNet's Sara McBride wrote on Wednesday that the number of aftershocks was within the forecasted range, but on the low side and cautioned people still to be prepared. "Just because we are in the lower end of the forecast, it doesn't mean that this will stay that way."
In non-earthquake news, an air force flight last week reported floating pumice to the west of Minerva Reef - about 600km south-east of Fiji. GeoNet director Dr Ken Gledhill said satellite images showed the pumice rafts extended for more than 100km. Though scientists have not yet tracked the pumice back to a source location, Dr Gledhill said the nearest active submarine volcano - Monowai - was active on November 10-11.
Finally, some numbers. Writing on GeoNet, Dr Gledhill pulled out some of the data from the Kaikoura earthquake:
• Movement along at least five separate faults, extending about 150km up the coast
• Land movements up to 11m horizontally and 5m vertically
• Maximum ground shaking 1.3 times the force of gravity in Ward
• Tsunami height of 4m at Little Pigeon Bay
• More than 5,500 aftershocks.
Quoted: Stuff.co.nz
"We're 12 days out, which in geological terms is not a long time at all."
GNS Science seismologist Dr Matt
on aftershock probabilities from the Kaikoura earthquake.


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Saunders homers twice, Mariners edge OriolesAug 4, 2013 - 4:51 AM Baltimore, MD (Sports Network) - Michael Saunders hit three-run and two-run homers to help the Seattle Mariners to an 8-4 win over the Baltimore Orioles in the second of three games from Camden Yards.
Justin Smoak went 3-for-3 with a solo home run and three runs scored while Michael Morse scored twice and drove in a run for the Mariners, who snapped a four-game losing streak.
"I'm just taking things one at-bat at a time," said Saunders. "But the way we've been swinging the bats lately, it makes it a little easier because if one guy fails, the next guy will pick him up."
Erasmo Ramirez (3-0) pitched six-plus innings and was charged with four runs on four hits and six strikeouts to win his third straight start.
Matt Wieters and Manny Machado each hit a solo home run for the Orioles, who had won three of four coming in. Scott Feldman (2-3) was touched for five runs on seven hits and four walks in 4 2/3 innings to drop his second straight start.
"Scott is better than that," said Baltimore manager Buck Showalter. "He'll figure it out, he's a smart man and very athletic. He was just missing some spots tonight."
Seattle got on board in the second thanks to Smoak's one-out solo homer to right, his 10th of the season, but Wieters broke an 0-for-25 snap in the home half of the second as he smacked a pitch over the wall in center for his 15th of the campaign.
The two clubs exchanged runs in the fourth as Brad Miller scored Morse on a fielder's choice in Seattle's turn, and Machado led off the Baltimore portion with his 10th home run of the season.
Seattle, though, went back on top in the fifth. Kyle Seager led off the frame with a walk and later stole second with two outs. Morse brought him home with a single and Smoak followed with a single to chase Feldman from the game.
T.J. McFarland then took the mound, but Saunders greeted him by clubbing a 2-2 pitch well over the right-center wall for his ninth homer of the year and a 6-2 lead.
Baltimore cut its deficit in half with two runs in the seventh. Nick Markakis led off with a single and Adam Jones followed with a double. Charlie Furbush then took the mound and got the first out on a sac fly from Chris Davis that scored Markakis. Wieters followed with a single to make it a 6-4 game. Furbush retired the next two batters to escape the frame.
The Mariners threatened in the eighth as they put men on first and second with one out. Raul Ibanez then singled off the wall in right, but Nick Franklin was thrown out at home on the relay, and Ibanez was caught off base by Wieters to end the inning.
Baltimore left men on first and second in the eighth, and Saunders put the game away in the ninth with his second home run of the game, a two-run shot to left, for an 8-4 lead.
Game Notes
Baltimore fell to 19-10 against the AL West this season ... Seattle designated hitter Kendrys Morales came into the game with a hit in eight straight plate appearances, but had that streak broken in the first when he grounded out back to the mound. The Mariners club record for consecutive hits is nine, set by Ibanez back in 2004 ... Seattle won despite leaving 12 men on base. The team had men reach base in every inning ... Seattle is 3-2 against Baltimore this season.
Aug 3 10:31 PM
- Michael Saunders one-out, two-run Home Run 2 (10) to left scored Justin Smoak. Humberto Quintero due up.
Mariners 8, Orioles 4 Top 9, 1 OutAug 3 10:18 PM
- 161
rootsRUWTbot Added 59 roots
Mariners 6, Orioles 4 Bot 8, 2 OutsAug 3 10:07 PM
- 102
rootsRUWTbot Added 32 roots
Mariners 6, Orioles 4 Bot 8, 2 OutsAug 3 10:03 PM
- 70
rootsRUWTbot Took away 8 roots
Mariners 6, Orioles 4 End 7Aug 3 9:46 PM
- Matt Wieters single to center scored Adam Jones with one out. Runner on first and Henry Urrutia due up.
Mariners 6, Orioles 4 Bot 7, 1 OutAug 3 9:41 PM
- 78
rootsRUWTbot Added 8 roots
Mariners 6, Orioles 4 Bot 7, 1 OutAug 3 9:41 PM
- Chris Davis sacrifice fly to left scored Nick Markakis with one out. Runner on second and Matt Wieters due up.
Mariners 6, Orioles 3 Bot 7, 1 OutAug 3 9:40 PM
- Michael Saunders two-out, three-run Home Run (9) to right center scored Michael Morse and Justin Smoak. Humberto Quintero due up.
Mariners 6, Orioles 2 Top 5, 2 OutsAug 3 8:38 PM
- Michael Morse single to right scored Kyle Seager with two out. Runner on first and Justin Smoak due up.
Mariners 3, Orioles 2 Top 5, 2 OutsAug 3 8:32 PM
- 70
rootsRUWTbot Added 18 roots
Mariners 3, Orioles 2 Top 5, 2 OutsAug 3 8:32 PM
- Manny Machado lead-off Home Run (10) to left. Nick Markakis due up.
Mariners 2, Orioles 2 Bot 4, 0 OutsAug 3 8:15 PM
- 52
rootsRUWTbot Added 17 roots
Mariners 2, Orioles 2 Bot 4, 0 OutsAug 3 8:15 PM
- Brad Miller reached on a fielder's choice, left to third scoring Michael Morse with two out. On the play, Humberto Quintero advances to second and Justin Smoak out at third. Runners on first and second with Nick Franklin due up.
Mariners 2, Orioles 1 Top 4, 2 OutsAug 3 8:12 PM
- 35
rootsRUWTbot Added 19 roots
Mariners 2, Orioles 1 Top 4, 2 OutsAug 3 8:12 PM
- Matt Wieters two-out, solo Home Run (15) to center. Henry Urrutia due up.
Mariners 1, Orioles 1 Bot 2, 2 OutsAug 3 7:35 PM
- Justin Smoak one-out, solo Home Run (10) to right. Michael Saunders due up.
Mariners 1, Orioles 0 Top 2, 1 OutAug 3 7:26 PM
Top 1, 0 Outs
Aug 3 7:08 PM
- 16
rootsRUWTbot Added 16 roots
Mariners vs. OriolesJan 10 8:15 AM
Related News
- Trout, Valbuena each hit 2 HRs as Angels beat Orioles 5-1 12:12a
- Haniger, Cruz homer in Mariners' 7-6 win over Rays 10:53p
- Haniger, Cruz homer in Mariners' 7-6 win over Rays 9:43p
- Mariners activate OF Haniger, place Dyson on DL 8:25p
- MLB Note - Baltimore Orioles Davis, Chris Sat
- MLB Note - Seattle Mariners Ramirez, Erasmo Sat
- MLB Note - Baltimore Orioles Trumbo, Mark Sat
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- MLB Note - Seattle Mariners Tolliver, Ashur Sat
- MLB Note - Seattle Mariners Zunino, Mike Sat
- MLB Note - Seattle Mariners Seager, Kyle Sat


text:
The External Relations committee is essentially SAC's public relations manager. External Relations is responsible for planning many of the events sponsored by SAC. These include:
- Brown Bag Seminar Series
- Food Drives
- Behind the scenes tour of Soldiers & Sailors Memorial
- Volunteer Events - United Way Day of Caring, Christmas Day at Pitt
This committee also spreads the word around campus about staff events and discounts. Some of those include the following:
- Stories in the SAC Monthly Newsletter
- Promotion of Pitt Police's Active Shooter Training (more details on our promotional slide)
- Communication about SAC's semi-annual assemblies
- Maintain social media. Follow us on Twitter @SACatPitt!
External Relations is also pursuing Pitt staff discounts at various Pittsburgh museums and landmarks. Stay tuned for details!


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This designer is a master at metal manipulation and her artistry is shown in her minimalist jewelry sculptures. This piece of wearable art features a delicate bold gold plated chain with varying sizes of free form flower shaped charms hanging all around the chain. At the 16", mark the necklace comes together and the flower sculptures dangle straight down. The designer's signature hook closure becomes part of the design in this piece. This amazing work of art will attract lots of compliments.
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Indian share market following volatile trading pattern despite to open with bigger than expected looses and remain closed to lower.
After the previous day solid gain the benchmark indices dipped into negative territory as the Sensex slipped down to 97.41 points, or 0.38 per cent, to end the day at 25,622.17 and Nifty index closed at 7,788.10, down 30.50 points, or 0.39 per cent.
The weak outlook for China had a bearing across Asian markets, all of which ended the day in the red
Get more visit Share market today


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When the remake of Footloose was first announced, the biggest question on the minds of many fans was how the new version would treat the music. Would they update the renditions, write different songs or stick with the classics? A great breakdown of the choices made can be found here, but suffice it to say, much of what director Craig Brewer altered really worked. I'd have a hard time saying Blake Shelton's take on the Kenny Loggins title song eclipsed the original, but a strong case could be made that his "Footloose" is just about as good. I'm fine with that, and apparently, Kenny Loggins was too.
The singer recently announced he'd not only be attending next week's Country Music Awards but would actual duet with Shelton on "Footloose". Loggins stopped by Access Hollywood to explain the decision and offer some praise for how the final version turned out. Here's an excerpt…
"They tried 10 different versions of the song and they kept re-cutting it because they wanted to capture that spirit of the original - and Blake came the closest."
The Country Music Awards will be broadcast on ABC on November 9th. Until then, here's a look at both Shelton and Loggins' versions…
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I provide online game consultancy, helping brands and companies develop and/or deploy their online game strategies, regardless whether its a virtual world, casual game or social game. I work with some of the most successful game companies in the world, such as Animal Jam, Stardoll, Spil Games, Studio 100, Dutch Game Garden, Youda Games, Mimesis Republic, Weeworld, Foot Locker Europe and Nickelodeon.
I believe in "practice what you preach", so early 2010 I started working on my own property Perfect Earth, an online game for kids that gradually lets them discover about the impact of their actions on the environment. Players are explorers in a vast virtual Amazon Rainforest, where they discover all types of endangered animals and learn to live responsibly and sustainably in their own tree house. Any and all proceeds of the sales of courses on Udemy will go directly towards further development of Perfect Earth, so effectively you may be helping me 'crowd fund' the game.
Late 2012 I started teaching 3rd year students Multiplayer Game Design at the NHTV University in Breda, Netherlands. Working with students provided a lot of energy and fresh thinking, so when the opportunity knocked, I decided to start teaching 'advanced games' at Fontys' Academy of Creative Industries. I also mentor several online and mobile game start-ups (Flow Studios, Stolen Couch Games, etc) in the area of business development, player acquisition and monetization. All in all lecturing (and preparing for it) and mentoring takes about 2 days out of my week.
Prior to starting my consultancy business, I worked for Habbo Hotel (Sulake Corporation) for 6 years, most recently in the role of Senior Vice President of Marketing and Advertising Sales.


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Wearing flawless makeup done by her and showing off major skin in a daring shoot, Mo'Cheddah stuns in new photos. Take a look.
Mo'Cheddah flaunts major skin in newly released photos.
The singer who dares in provocative shots for the photoshoot posed topless in some images.
Wearing a flawless pink smokey eye makeup look paired with Nud3 lips and dark cornrows, the singer showed off her blemish free skin serving different poses wearing a bandeau in some shots.
in some images the singer who only just released her latest single 'Survive' chose a print headwrap posing topless and choosing a black leather slip bralet/crop top in some.
She chose a crisp white boyfriend shirt over a leather crop top paired with denim shorts in some set of images.
Check out the S3xy photoshoot above.


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Lost Compassion 8 09 15 AA trailer HD 720p MP3
Five years ago, August 9, 2010, the remains of Mitrice Richardson were discovered just 7 miles from the Malibu-Lost Hills Sheriff's Station. Sheriffs detectives ...
I feel Ive lost my compassion how to get it back - Sadhguru MP3
I feel Ive lost my compassion how to get it back - Sadhguru http://www.ishafoundation.org/
Compassion Lost MP3
Compasion Lost a song written and performed by GK on a non-tuned CBG called Amália. lyrics: For those who try their best but can't do better For those walking ...
Compassion For The Lost MP3
A music video (jsm.org) presented by www.mountainvalleyradio.com and Mountain Valley Media.
Have nurses lost their compassion? MP3
Florence Nightingale represents for many the epitome of compassion in how she cared for the soldiers of the British Army during the Crimean War. In this 3 ...
Michelle Reynolds, The Lost Art of Compassion MP3
Urban Tellers, December, 2014 BELONGING Michelle Reynolds on stage at Alberta Abbey for live storytelling with Portland Story Theater Hosted by Lynne ...
Having Compassion for the Lost - Pastor Phillip McKinney MP3
The unchurched is our target not our enemy.
Malibu region, Stunt Road Mural teaser for documentary "Lost Compassion" MP3
Teaser for documentary "Lost Compassion" showing mural discovered off Stunt Road near Malibu on June, 2010 during a search for Mitrice Richardson.
Compassion When We Loose This Everything Is Lost MP3
I love you all my christian brothers and sisters there is no compassion with out sadness god has everything under control he knows what he is doing more than ...
CMCT Compassion for the Lost MP3
[Naruto AMV] Lost Compassion (Thanks for 200+ subs) MP3
Thanks guys love you all :D.
Migrant Deaths: has Europe lost its compassion? (part 2) MP3
Subscribe to France 24 now: http://bit.ly/France24Subscribe DEBATE : The route from North Africa to Italy and Malta has become the world's deadliest. Officials ...
Jesus' Passion and Compassion for Lost Souls MP3
Sept 19, 2010 Sunday Message of Pastor Don Sison.
Cultivating Compassion for the Lost The Way of the Master 1 8 SD MP3
Im Back!!! [Naruto AMV] Lost Compassion (preview) MP3
back too bussiness guys :)
Lost Compassion MP3
Provided to YouTube by The Orchard Enterprises Lost Compassion · Cataract Great Days Of Vengeance ℗ 2003 Lifeforce Records Released on: 2003-03-13 ...
Compassion For The Lost MP3
Message by Kenneth Thurman at the Southern Shores District Convention. Recorded at New Horizons Church on 03/01/14.
Lost Compassion - Wrong Intention MP3
The successful believers - Imam's Compassion to the Lost Ones MP3
The successful believers - Imam's Compassion to the Lost Ones.
Caleb Ford " Compassion for the lost" Official Music Video MP3
Compassion Lost and Found Again MP3
Provided to YouTube by BWSCD, Inc. Compassion Lost and Found Again · Paul Flaherty Whirl of Nothingness ℗ 2006 Family Vineyard Released on: ...
WCBC Wed Night Bible Study Session 4: Compassion for the Lost MP3
WCBC Wed Night Bible Study Session 4: Compassion for the Lost OBADIAH 1-4,10-17.
Compassion for the Lost - Unity Baptist - Oct 4, 2015 MP3
Unity Baptist Church http://unityvan.com Compassion for the Lost Matt Philbrick, Youth Pastor Luke 10:25-37; Act 1:8.
Emotional Toddler: My compassionate young daughter gets adorably emotional MP3
Compassionate toddler gets adorably emotional while watching cartoon about lost baby penguin. When I noticed my young daughter getting emotionally ...
Compassion Contacts Solutions Helping Battlers - Jesus Goes After the "Lost Sheep MP3
My Testimony how the Lord helped me through the storms .He showed me how problems can be resolved for battling pensioners and their families .
Week #2 Vegan Journey..... Detox Symptoms, Compassion, lost a little more Wt. MP3
via YouTube Capture.
4 kids show compassion for a puppy MP3
4 kids show compassion for a lost puppy, they gave him milk and biscuits. This stray puppy love kids.
Video that will change your life. I have no words left. MP3
Update: Today is 2-19-13, I never expected such a great response to this video. It really makes me happy to see the comments left. Even though this is not a ...
Lost in Compassion MP3
Provided to YouTube by CDBaby Lost in Compassion · Deacon Emil Gospel Stories, Vol. 1: The Eyes ℗ 2007 Deacon Emil Released on: 2007-01-01 ...
Heart of Compassion: TV SPECIAL MP3
In sharing the Gospel we must not forget Christ's example of compassion for the lost and broken. In this Billy Graham TV Special, follow three stories that show ...


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Dino Eggs: Rebirth (Windows)
Official Site
Also For
Critic Score
100 point score based on reviews from various critics.
User Score
5 point score based on user ratings.
Steam Store Description (from Ad Blurbs)
DINO EGGS: REBIRTH is a celebration and expansion of the best-selling 1983 home computer game DINO EGGS (Apple II, Commodore 64, IBM) -- in which Time Master Tim travels to prehistoric times to rescue the dinosaurs from extinction.
We've worked hard to maintain the classic platform feel of the original DINO EGGS -- while building in LOTS of new levels, puzzles and features.
Here's What We've Packed In
- Forty new skill levels of wide-screen arcade action.
- Four new time eras -- Outlands -- Swamplands -- Stone Age -- Ice Age.
- Local Co-op Multiplayer for two. Local Competitive Multiplayer for up to eight. Play to outscore each other, or play to outlive each other!
- Five new species of baby dinosaurs to hunt and capture. They fly! They sleep! They flee you! They charge you!
- New types of eggs, boulders, power flowers and fires. Can you discover how to get an extra life?
- Over 25 chapters of a surprise-filled story "Secrets of the Time Quartz."
- Over 30 puzzling Dino Dilemmas, plus bonus dilemmas that unlock after completing the story.
- Two graphic skins, one by original author David Schroeder, and one by Jamo Games pixel artist James Biddulph.
- An enhanced re-creation of the 1983 Dino Eggs: Classic.
- Support for English, French, Spanish, German, Dutch, Italian, and Japanese.
- And -- as always -- an infinite number of random prehistoric landscapes to explore...
There are no Windows user screenshots for this game.
There are 13 other screenshots from other versions of this game or official promotional screenshots.
Promo Images
Part of the Following Groups
User Reviews
There are no reviews for this game.
Critic Reviews
There are no critic reviews for this game.
There are currently no topics for this game.
There is no trivia on file for this game.
Related Web Sites
- Dino Eggs: Rebirth (official website)


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Question: Locket a U S C corporation makes a sale to a
Locket, a U.S. C corporation, makes a sale to a customer in Sustainia, a country that applies a 25% income tax to business profits. The customer found out about Locket through an Internet search. Locket has no facilities or employees outside the United States. Locket's profit on the sale totals $25,000. Where is the tax levied on this profit? 30. LO.5 USCo incorporated its foreign branch operations in Italy by transferring the branch's assets to a foreign corporation in return solely for stock in the new corporation. All of the branch's assets are located outside the United States and are used in the active conduct of a foreign trade or business. Is this transaction automatically eligible for tax deferral under § 351? Explain.
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Answer to relevant QuestionsRandall operates his distribution business in several countries. He wants to move some equipment to a new office in South Africa. This equipment includes assets with a large acquisition price and accumulated MACRS ...Brandy, a U.S. corporation, operates a manufacturing branch in Chad, which does not have an income tax treaty with the United States. Brandy's worldwide Federal taxable income is $30 million, so it is subject to a 35% ...Elmwood, Inc., a domestic corporation, owns 15% of Correy, Ltd., a Hong Kong corporation. The remaining 85% of Correy is owned by Fortune Enter prises, a Canadian corporation. At the end of the current year, Correy has ...Clario, S.A., a Peruvian corporation, manufactures furniture in Peru. It sells the furniture to independent distributors in the United States. Because title to the furniture passes to the purchasers in the United States, ..."U.S. persons are taxed on their worldwide income." Explain.
Post your question


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This is the stand in page for details on Jack Yeovil's novel Drachenfels.
Publishing History
Drachenfels has been published
Main Characters
- Constant Drachenfels
- Genevieve Dieudonné
- Detlef Sierck
- Oswald von
Additional Characters
- Anton Veidt
- Rudi
- Menesh
- Maximillian von
Items/Places/Events of Interest
Details to come.
Summary to come.


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Academy Awards Guide
Academy Awards Guide from Overstock.com. Our guides provide customers with information about the Academy Awards.
ITEM#: 10319211This collection contains all the sequel chapters in the demonic doll saga. CHILD'S PLAY 2: Chucky is back! After the fire, the toy factory wants to dispel the bad publicity, but are they in for a surprise as Chucky is back on Andy's trail and this time he intends to win. CHILD'S PLAY 3: Chucky, the notorious killer is back to wreak more mayhem as he continues his quest to possess a child's body. BRIDE OF CHUCKY: Chucky returns, and this time he's got a girlfriend, Tiffany. Their way of having a good time is going on a murder-spree road trip, killing any and everything that gets in their way. Along for the ride are Jade and Jesse, a pair of teens on the run from their uncaring parents. As the body count rises, Jade and Jesse must deal with the consequences of their killer dolls. SEED OF CHUCKY: Everyone's favorite plastic slasher doll returns for a fifth installment of this popular horror series. Apparently Chucky and his equally murderous bride, Tiffany (voiced by Jennifer Tilly), had a child, which has grown up in a cage as part of a heavy-metal ventriloquist act in England.
|Universal Home Video
|Extra Info
|2-Disc Set
|5.5" x 0.75" x 7.75" WxHxD
|0.2 lbs
|Alex Agutter, Alex Vincent, Perrey Reeves, John Ritter, Jennifer Tilly, Justin Whalin
|John Lafia, Jack Bender, Don Mancini, Ronny Yu
|Country of Origin
|United States
Academy Awards Guide from Overstock.com. Our guides provide customers with information about the Academy Awards.
Looking for the perfect Valentine's Day movie to watch this year? We've picked the best of classic, manly, RomCom, devastatingly sad, and guilty pleasure Valentine's Day flicks.
Tribeca Film Festival Guide from Overstock.com. Our guides provide customers with information about the Tribeca Film Festival.
How to Review a Movie from Overstock.com. Movie reviews are a great way to determine the importance of a film, its impact on viewers, and whether or not it is worth a watch.
Top 5 Prestigious Film Festivals from Overstock.com. Read more about the film festivals behind some of your favorite movies.
How to Organize Your DVDs from Overstock.com. Our guides provide customers with information about how to organize your DVDs.
10 Fun Facts about Disney from Overstock.com. Disney has a long, exciting history, and here are some fun and lesser known facts.
Tips on Selecting the Perfect Entertainment Gift from Overstock.com. Our guides provide customers with tips on selecting the perfect entertainment gift.
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Chucky: The Killer DVD Collection (DVD)
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More Details Revealed for Disney's BOY MEETS WORLD Sequel
Entertainment Weekly recently spoke to "Boy Meets World" creator Michael Jacobs, who will also serve as executive producer of the long-awaited Disney pilot GIRL MEETS WORLD. Jacobs revealed more details on what fans of the hit '90's TV series can expect in this latest incarnation.
"There's a natural reticence to want to try and attempt something that you've been really lucky enough to do once," explains Jacobs. However Jacobs decided that he wanted to create a show for the "new generation of kids that live in a much more difficult world," and center it on "the children of Cory and Topanga and watch on two levels [as they] grow up and as Cory and Topanga keep growing up, I can't resist that. I can't resist that and I'm very hopeful the audience won't resist it either."
Girl Meets World will focus on "the confusions about growing up and what we are supposed to be and what we are supposed to aspire to be. There's a column that I want to play in and that column contains The Wonder Years and Happy Days. That was always the column that I wanted Boy Meets World to be included in and I want Girl Meets World to be in that column as well."
Jacobs decided that Cory and Topenga should be the parents of two children. "Topanga has always been much more certain that Cory. Cory has always been confused. That would never stop. Now he's a father. Cory Matthews doesn't know how to be a father." He added, "He's following in the footsteps of [Mr. Feeney], the most influential character in his life," says Jacobs. "Should he be? Will he succeed? Can he influence children like Feeney influenced children? What is Topanga's observation of that? How does she help? And what is Topanga's life? And what has she become? You'll remember we left the series and they were in Philly and they were going on an adventure to go to New York. Did they succeed? Did it work? All of that will be answered in the pilot."
Jacobs revealed that Riley Matthews "is going to be much more like one than the other but that second parent will pop up in ridiculously unexpected times and places and add to the confusion of who this character is."
Jacobs hopes many of the original supporting characters like Shawn, Eric, and Minkus, will return. "I think there's more than a chance," says Jacobs, who explains that he's spoken to many of the former cast members and that they've all expressed interest in being apart of the project in some capacity. "I think there's a strong chance," he says.


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People whose houses were damaged or destroyed in the December bushfires will be offered land tax relief.
The fires, which began on Christmas Day, damaged or destroyed more than 120 properties in Wye River and Separation Creek, 10 homes in Scotsburn and five in the Indigo Valley.
With land tax assessments set to be released shortly for the 2016 tax year, Treasurer Tim Pallas will announce ex-gratia relief from land tax for owners affected by the fires, excluding corporate taxpayers with total land holdings exceeding $3 million.
Mr Pallas said the tax relief would provide added assistance over a difficult period.
It comes in addition to the joint Commonwealth and Victorian disaster assistance packages, which include payments of up to $1300 per household to help with food, clothing, personal items and shelter.
Victims can also apply for emergency payments of $32,500 for clean-up, emergency accommodation, repairs and replacing damaged contents.


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This London foolishness is jaw dropping. By now the world has witnessed the wholesale destruction, the loss of property and the wanton lawlessness.
What stands out to me amidst this unrest is that the rioters are destroying their own neighborhoods. Unfortunately once those stores and houses are burned to the ground, there won't be businesspeople lining up to invest to rebuild them.
Trust me on this one.
There are swaths of the south and west sides that haven't "come back" from unrest in the late 60's. What makes the rioting Londoners think that this momentary burst of short sighted behavior is going to benefit them in the long run? Or maybe that's the thing---they're not thinking. Perhaps there is no cause.
Because once the fires have burned and the dust has settled, you'll still have the same issues plus no place to live or shop. By the time you realized you played yourself, it will be too late.
Perhaps the Dead Kennedys said it best: "Tomorrow you're homeless/Tonight it's a blast"
Filed under: Civil Unrest, Crime, Local Business, London, London Riots, Riots, Uncategorized
Tags: Accountability, Are You Kidding Me?, Civil Unrest, Crime, Duh!, London, London Riots, Musings, Really?, The Unexpected


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Marketing Apprentice M/F
L'offre a été consultée 38 fois
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Marketing Apprentice M/FGemalto helps governments design and roll-out secure documents and robust digital identity solutions. Beyond the traditional registration, personalization and issuance services, our eGovernment infrastructure and innovative applications can help your government win citizen's acceptance and boost usage.
As leader in digital identity solutions, we are active in over 100 government programs worldwide with many in Africa.
As Marketing Apprentice, you will study and analysize the digital identity programs with biometric systems and new identity documents (electronic passports, ID cards, health cards and driving licenses)
A week in the life of
an apprentice in Marketing :
• You will study the market and competition in Latin America, Maghreb and Africa.
• You will analyze the trends and do the follow up for the markets addressed by Gemalto
• You will do the collection of information on projects currently being delivered by Gemalto in the region
• You will identify the key messages and promote the Gemalto's offers using marketing tools in coordination with the Communication department: Development of relevant PowerPoint presentations (in English) on projects, or use of other multimedia tools ("case studies").
Knowledge, Skills and Experience
Master Marketing student (BAC+4/+5)
Interest in high-tech
Computer skills: Word, Excel, Power point
Master Marketing student (BAC+4/+5)
Interest in high-tech
Computer skills: Word, Excel, Power point
Offres d'emploi
Ils ont travaillé ici
Petr Pohl
ISTQB® CTFL Agile Tester | SCM Engineer | Web Developer


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Towards the end of last year, I was approached by the French environmental charity Reseasu Environment Santé to write and direct an animation for their campaign on BPA plastics. Narrowing down from several proposed scripts, we eventually developed The Story Of A Tin Can into the above promo.
Having worked with Jonathan Harris on Internet Story, I was very happy to for him to animate the entire film this time!
Likewise, the music is from another long-term collaborator: Blair Mowat.
Currently Watching: Wener Herzog as Plastic Bag


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The ASEAN Basketball League, often abbreviated to the ABL, is a men's professional basketball league in Southeast Asia, Hong Kong and Taiwan. Six clubs from six different countries of theAssociation of Southeast Asian Nations (ASEAN) competed in the league's 2009 inaugural season.The league was proposed in Kuala Lumpur, Malaysia and launched on 1 October 2009.
Basketball officials from 6 ASEAN nations gathered in Metro Manila on 1 September 2009 to officially launch the new league.
Teams that participated in the inaugural season were the Brunei Barracudas, Kuala Lumpur Dragons, Philippine Patriots, SM BritAma, Thailand Tigers and former National Basketball League team, the Singapore Slingers. Mirroring the Euroleague in the west, the ABL has its own set of rules in terms of its roster makeup and salary caps. Teams are permitted two imports of non-ASEAN citizenship, three ASEAN imports, one player with one Southeast Asian parent, and seven local players. Teams are allowed to field a team of all local players if they wish to do so.
In July 2012, the league announced that Kuhan Foo, CEO since the ABL's inception, was moving on to a position leading additional regional sports properties. In his place, the league named Anthony Macri as the next CEO, and he will lead the ABL into Season 4 in early 2013, but after one season he left the league to rejoin his family in the US.
League expansion
On 22 September 2011, the Brunei Barracudas announced that they were bowing out of the third season of ABL after participating for 2 seasons.
On 20 October 2011, the Saigon Sports Academy officially announced the participation of Saigon Heat into the third season of ABL, making them the first ever international professional basketball team to representVietnam.
In 2012, San Miguel Beermen and Bangkok Cobras joined the league aside from Saigon Heat. Unfortunately, after one season, the Cobras left the league with the Beermen leaving after they won the title in 2013.
In 2014, Laskar Dreya South Sumatra (INA) joined ABL, but they left after the 2014 season.
In 2015, Pilipinas MX3 Kings and Mono Vampire joined the league. Unfortunately, both teams left the league in 2016.
On 17 July 2016, it was announced that Kaohsiung Truth, a team from Kaohsiung, Taiwan will participate in the 2016–17 season. This will be the first time that a team outside Southeast Asia will compete in the league.
On 30 July 2016, another team outside Southeast Asia, the Eastern Basketball Club confirmed its participation in the league, being the second non-Southeast Asian team to join the league.
On 6 August 2016, Alab Pilipinas from the Philippines is the newest team to join ABL. The team is owned by Dondon Monteverde and player-agent Charlie Dy.
The league, as of the 2015–16 season, is held via a quadruple home and away format where each team faces each other four times during regular season; as such the number of total games per regular season varies depending on the number of participating teams in that year. At the end of the regular season, the four teams with the best records qualifies for the playoffs. In the best-of-three semifinals, the top 2 seeds will have the home court advantage against the lower seeds and will host the first and third (if necessary) games. In the first season, the finals was a best-of-five series, with the higher seed hosting the first two and the fifth (if necessary) games. The Finals format was changed to a best of three series for the second and third seasons, where the higher-ranked team earns home-court advantage and hosts the first and third (if necessary) games. For the fourth season, it will be a quadruple home and away format where each team faces each other 4 times with the semis,a best-of-five, and the finals, a best-of-five series. In the fifth season, the playoff format was changed into a best-of-3 format both in the semi-finals and the finals. Starting in the sixth season, the league reverted its playoff format to a best-of-3 in the semi-finals and a best-of-5 series in the finals, similar to the first season's playoff format.[citation needed]
The champions are supposed to represent the Southeast Asia Basketball Association (SEABA) in the FIBA Asia Champions Cup. However, since the tournament only limits one club per country, the 2010 championsPhilippine Patriots were disallowed from participating since the Smart Gilas Philippine team had already qualified. In 2011, the Chang Thailand Slammers were supposed to represent SEABA but the Thailand Basketball Federation was then suspended by FIBA; this caused the Westports KL Dragons to represent SEABA, as the runner-up Patriots' slot was already taken by the Smart Gilas.
So far, No ABL team have played in the FIBA Asia Champions Cup since 2011, until Westports Malaysia Dragons won the ABL title in 2016, thus earning a slot in the tournament.


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I am looking to apply for an immigrant visa. Where do I find information to obtain an immigrant visa?
If a traveler will be immigrating to the United States for purposes that are more permanent than temporary, then an immigrant visa may be for them. With an immigrant visa, the traveler will be able to be employed and stay in the United States. In addition, an immigrant visa will enable an applicant to make the United States their permanent residence and will enable them to acquire a United States citizenship.
In order to be qualified for a United States immigrant visa, the applicant will have to be sponsored by a United States citizen relative, United States lawful permanent resident, or a potential employer of the applicant.
When obtaining and immigrant visa, there are 4 major steps that need to be considered.
The first step is a Petition. The applicant will have to provide a petition when applying for an immigrant visa.
Secondly, the applicant needs to get their petition approved. After it is approved, the applicant should check the priority date then begin National Visa Center Processing. After beginning the processing, the applicant should choose an agent and then pay the necessary fees.
The third step is to gather and submit all forms to the National Visa Center. This step necessitates the applicant to collect all forms, collect financial documents, collect supporting documents, and then submit those documents to the National Visa Center.
Lastly, the fourth step is the Interview. The applicant should prepare for the interview and then take the interview.


text: One of the centerpieces of Secretary of State Delbert Hosemann's legislative agenda passed in the last session, and university researchers are getting ready to take advantage of it. The Strengthening Mississippi Academic Research Through (SMART) Business Act provides a 25 percent rebate to Mississippi companies that contract with Mississippi public universities and colleges for qualified research. The legislation caps the ...Read More »


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login with
Charlie rolled over today!!
I'm so excited.. I've been blasting facebook with the news..haha. He's been able to go from tummy to back for a long time but from back to tummy has proved to be a bit of a struggle. He gets one arm stuck under him and can't get himself over. He's come sooooo close and has fallen asleep mid-roll.
But today I put him on the floor on a blanket with some toys and there he went! It was awesome! 10 minutes later he did it again so I know it wasn't a fluke. I'm so proud of my little guy! Here's a pic right after it happened.. I was so excited for him!!
what a special moment
That's why he slept so long! He was building his strength up! Good job Charlie!
lol yes! haha that must be it!
Aww that's awesome news! Don't you love how you have to make that mad dash for the camera to try and capture these precious moments? So exciting!
Luckily my phone is never more than 6 inches away! lol
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About Alissa
Current: Chinook, Montana
Birth: September 17
On Moms.com since: Jan 30, 2014
*Site Hostess* I am a first time mom living in North Central Montana. I have a fiance, the most gorgeous baby boy, and a goofball dog named Lexi to fill up my plate :)


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Discrimination of different species from the genus
The use of molecular biology-based methods for species identification and establishing phylogenetic relationships has supplanted traditional methods relying on morphological characteristics. While PCR-based methods are now the commonly accepted gold standards for these types of analysis, relatively high costs, time-consuming assay development or the need for
After establishing a simple protein extraction procedure and performing matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) with intact proteins and peptides, we could show that most of the species investigated reproducibly yielded mass spectra that were adequate for species classification. Furthermore, a dendrogram generated by cluster analysis of total protein patterns agrees reasonably well with established phylogenetic relationships.
Considering the intra- and interspecies similarities and differences between spectra obtained for specimens of closely related
Taxonomical classification of species on the basis of phenotypic differences and similarities has been performed since the days of Linnaeus. In more recent times, cladistics have come to the fore and, with the advent of molecular biology, started to increasingly supplant the use of morphological criteria for discrimination of phenotypically similar species or species of uncertain ancestry. In the last decades, DNA sequencing and PCR-based methods have been used increasingly to unlock the phylogenetic information hidden in the genomes of diverse organisms, leading also to the barcode of life initiative
During the last decade, however, there has been an increasing number of reports in which proteomic instead of genomic data has been used to successfully discriminate between different microorganisms at the genus, species and sometimes even at the subspecies level
A complementary approach to this procedure is the peptide mass fingerprinting or shotgun mass mapping (SMM). Here, peptides obtained by tryptic digestion of the sample, which may be either a protein extract or a whole organism, are subjected to MALDI-MS analysis. Though a spectrum generated in this way will differ from an IPP spectrum by the smaller size of the observable peptides,
In summary, MALDI-MS possesses a number of potential advantages over other species typing methods, such as PCR or antibody-based approaches. Sample preparation is comparably simple and requires only little hands-on time. It does not rely on taxon-specific and cost-intensive consumables, does not require genetic information about the organisms that are being investigated and the straightforward workflow can easily be standardized and automated. Moreover, only short measurement times are required, and reference spectra for further species can be added to existing databases at any time. Mass spectrometric identification of microorganisms has been applied successfully in taxonomical research, but also in other settings, such as clinical, food, and environmental microbiology, and it has been tested for rapid distinction between highly pathogenic
Some recent studies have already indicated the possibility to use MS-based methods for the discrimination of more complex, metazoan species such as insects. For example, mass fingerprints of neuropeptides from specimens of the insect order
The extensive number of examples clearly demonstrates the feasibility of MS for typing of organisms. However, to our knowledge, there has been no attempt to establish species identity of small metazoans based on total (extractable) protein content. Thus, the aim of this study was to provide a proof-of-principle that species discrimination employing intact protein profiling (IPP) by mass spectrometry can also be applied to intact specimens of a multicellular organism such as the fruit fly. For this purpose, protein extracts from whole, single specimens representing 13 different species of
All chemicals and solvents used were of
Culturing of
Culture bottles containing 12 different
Table 1
Protein extracts and tryptic digestion
Male and female flies from
For peptide mass spectra, a protein extract was prepared from 3 male and 3 female
One microliter aliquots of the protein solutions were spotted onto ground steel MALDI targets (Bruker Daltonics), and mixed with 1 μl sinapinic acid (SA) matrix directly on the target plate. The MALDI matrix solution was prepared as saturated SA in 50% acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA). Samples were allowed to dry for several minutes before MALDI-TOF MS measurements were performed. IPP (intact protein profiling) spectra were obtained on an MALDI-TOF/TOF mass spectrometer (Ultraflex III™, using FlexControl software Vs. 3.0; Bruker Daltonics). The instrument was operated at pulse rates of 100 Hz; pulse ion extraction delay was set to 400 ns. Measurements were carried out in positive reflector mode using an acceleration voltage of 25.0 (ion source 1) and 21.85 (ion source 2) kV. Lens voltage was 9.5 kV, reflector voltages were 26.3 and 13.7 kV. Mass spectra were recorded in the
For identification of proteins from
MALDI-MS peak detection and peak matrix generation
MALDI-MS spectra were analyzed using FlexAnalysis (Vs. 3.0, Bruker Daltonics). For the
Peak matrix analysis by clustering
From the raw peak matrix, rare peaks (less than 4 occurrences in total) were removed and a preliminary analysis was performed. Using PAST, pairwise spectra similarities using Dice (or Sørensen) coefficients were calculated according to DS = 2 M/(2 M+N), with M for the number of matching peaks and N for the total number of peaks being present in just one spectrum. This was followed by clustering using the unweighted pair-group average method. Bootstrapping analysis with the number of replicates (N) set to 1000 was performed. Additionally, the Dice similarity matrix was transferred from PAST to Excel using copy and paste and saved as a text file. From this, a heatmap was obtained by opening the file in Framework and saving the graphical output
For stringent clustering, peaks were selected that were rated for their ability to distinguish between different species. First, the number of occurrences
Peak matrix analysis by principal components
For principal component analyses, the peak matrix was first converted into a binary matrix by substitution of intensity values with ones or zeros using PAST. In order to compensate to a small extent for the different peak numbers in different spectra, values were furthermore normalized by the Euclidean length of the row vectors (using the 'row normalize length' command), and principal component analysis was performed with the peak matrix containing all spectra as well as spectra from subsets of several species.
A simple protein extraction protocol was established, using a bead mill for homogenizing fly specimens and the denaturing conditions of 6 M urea for efficient solubilization of proteins. Weight of vacuum-dried flies, depending on the considerable size differences between the respective species, varied between 0.3 and 1 mg (exact values were difficult to determine with our standard lab equipment), and extractable amount of protein was around 50 μg for a fly of average size. Performing purification from the homogenate by reversed phase chromatography (C18 matrix) in a stepwise fashion while taking care to not exceed the column's binding capacity of approximately 5 μg, it was principally possible to recover more than 90% of the protein. Thus, about 100 μl eluate containing up to 50 μg protein could be obtained from one fly, with about 250 nl containing a sufficient amount of protein (i.e. ~125 ng) to obtain several well-resolved MALDI-MS IPP spectra from one single sample spot on the target plate.
Using protein extracts prepared from 128 individual
Figure 1
Comparison of six MALDI-MS spectra, 3.3 to 4.6 kDa
Comparison of six MALDI-MS spectra, 3.3 to 4.6 kDa. The upper three spectra (red) were obtained with three different flies from
An initial analysis of the peak patterns from the 128 spectra using hierarchical clustering turned up three spectra that did not show significant similarity to any other spectrum (D_ere_10, D_mau_4, D_mau_6). Cutoff criterion was a value of DS < 0.25; the remaining 125 spectra displayed a Dice similarity coefficient of 0.45 or larger to at least one of other spectra in the dataset. Therefore, these three spectra were excluded from the dataset for the following analyses.
An unbiased cluster analysis (i.e. without data preprocessing based on information about species affiliation) using the remaining 125 spectra yielded the dendrogram and the similarity matrix shown in Fig.
Figure 2
Results from unbiased cluster analysis
Results from unbiased cluster analysis. 125 spectra from flies of 13 different species were clustered using Dice coefficients. A color (heatmap) representation of the pairwise similarity (Dice) coefficients is shown in the lower part of the figure, the white square outlining the cluster of
Results from a principal component analysis of all 125 spectra are shown in Fig.
Figure 3
Scatterplots from unbiased PCAs using 125 spectra from flies of 13 different species
Scatterplots from unbiased PCAs using 125 spectra from flies of 13 different species. Species-specific color-coding corresponds to that shown in Fig. 2. Spectra belonging to one species are outlined by a convex shape or an ellipse. A: PCA containing all spectra, with 95% concentration ellipses for three subgenera/species groups. B: PCA using only spectra from
We tried to achieve a more stringent separation of the different
Figure 4
Results from cluster analysis using 208 selected peaks
Results from cluster analysis using 208 selected peaks. For further information, please refer to the legend of Fig. 2.
In an attempt to identify some of the proteins present in the purified
Insects stored in 70% ethanol were used in this study. These conditions largely ensure protein insolubility and thus minimal loss in the case of damaged samples
After extraction of peak tables from the recorded spectra and assigning their
Using Dice coefficients and without incorporating any
Using principal component analysis (PCA) as a different, independent method to evaluate the peak matrix, we found that distinction between different subgenera, species groups and also individual species was principally possible. PCA surmises the existence of correlated variables (i.e. peaks), and tries to reduce the complexity of the dataset by substituting them by a limited number of components. However, the analysis yielded 19 principal components with Eigenvalues > 1 (Kaiser criterion); and 14 principal components are still needed to explain just 50% of the data variance (data not shown). Thus, it is not surprising that in a graphical representation based on only two components, albeit with the largest Eigenvalues (accounting for 9.2% + 7.2% = 16.5% of total variance), complete distinction of all species was not possible (Fig.
In summary, principal component analysis can be used to discriminate between different species with results comparable to those achieved by cluster analysis. One limitation of PCA is that the number of different species that can be separated in one analytical run should not be larger than two, since an increasing complexity of the dataset is not compatible with reduction to and graphical representation by only two principal components. Broader relationships between different species groups and subgenera may be established consecutively by a successive increase in the number of species and iterated analysis of the dataset. However, in order to avoid the cumbersomeness of this approach, we decided to rely on cluster analysis for further data evaluation.
Next, we investigated if it would be possible to identify species-specific peaks that might be used for direct and unambiguous discrimination of the corresponding species from all other species. We searched the peak matrix and found that this approach yielded specific
Not satisfied with this result, we decided to pursue a less straightforward approach and to identify peptides essential for species discrimination by assigning score values to all of the more than 2000 different peaks in the dataset. This time, we also tried to achieve separation between male and female
Additional file 1
Peak. Formatted tables containing peaks from the 125 IPP MALDI-MS spectra as detected by FlexAnalysis and binned using the software MS-Screener. The first worksheet contains all peaks except for those occurring less than three times; the following worksheets show the successive processing as outlined under Methods. The two worksheets labeled with the identifier 'Exported' contain the datasets used for generation of heatmaps and dendrograms shown in Fig.
Click here for file
On the other hand, due to selection of species-specific peaks for cluster analysis, intra-species similarity values were generally improved and similarity differences between groups of different species more pronounced, an effect that can be seen clearly when comparing the dendrograms from Fig.
Most peaks detected and used for comparing the different species have
As there is always the possibility that the collection of endogenous peptides and smaller proteins becomes dominated by a variable number of breakdown products from larger proteins, especially in the case of less well preserved specimens, peak patterns may become irreproducible. Thus, in some instances, it may be preferable to rely on larger, abundantly expressed proteins as biomarkers for species identification, which after (optional) removal of the peptide fraction can be fragmented by tryptic proteolysis to generate MALDI-MS compatible peptides for a specific and possibly more reproducible SMM spectrum
Some proteins with masses below 20 kDa were also identified in the MS/MS shotgun approach, but their predicted masses could not be assigned to specific peaks in the IPP spectra obtained by MALDI-MS. One notable exception was the ejaculatory bulb-specific protein (PEB) 2 [GenBank
Figure 5
Putative PEB2 from
Putative PEB2 from
Differentiation of spectra from
In multiple previous studies, the suitability of mass spectrometry for analyzing phylogenetic relationships between different species of microorganisms has been firmly established (see references given above). Furthermore, there have been several reports of using peptide and protein masses, sometimes in conjunction with chromatographic retention times, for the classification of venoms from scorpions, large spiders and snakes
Here, we attempted to establish species identity of small metazoans based on total (extractable) protein content using a simple protein extraction procedure. We have demonstrated for the first time that it is feasible to generate reproducible and well-resolved IPP spectra from whole insects via MALDI-TOF mass spectrometry. Furthermore, we have shown that these spectra can be used to differentiate between different species from the same genus, as long as these species are not too closely related. This straightforward approach with its potential for automation and adaptation for high throughput applications may be of interest in all cases where morphological differences are difficult to determine and the experience of a professional taxonomist is presently needed for species identification.
MALDI: matrix-assisted laser desorption ionization; ESI: electrospray ionization; TOF: time-of-flight; MS: mass spectrometry; MS/MS: tandem mass spectrometry; IPP: intact protein profiling; PCA: principal component analysis; SMM: shotgun mass mapping
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
RF and RG planned and performed the experimental work; SK assisted in data analysis of MS/MS spectra. RF performed data analysis and drafted the manuscript. HGA was responsible for
Culture vials containing live specimens of the different
The Barcode of Life Initiative
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Genetic and ecological correlates of intraspecific variation in pitviper venom composition detected using matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and isoelectric focusing


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