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Guns And Violence Essay, Research Paper
Guns are becoming a fast growing concern among today s society. Due to its increasing use, violence has increased in once peaceful communities, even though crime rates have recently been below average for most of LA. However, controlling that violence has become a major issue and most people want their kids to feel and play safely in their neighborhood. Many senseless tragedies have taken place over the last couple of years to bring upon this fear, and many parents feel that greater gun control should be exercised. Parents should ask themselves and their kids if they feel comfortable and safe in their schools. In many schools, the first thing a student sees is a guard and a metal detector to detect any illegal carrying of guns. Many innocent students privacy is invaded in this process, a process that should not have to happen in the first place. It all comes down to controlling that problem outside school and before kids are suspected. This means greater gun control and checking when a gun is purchased.
Kids take guns for many reasons. For example, they may want to take it to school his friends, or to shoot another kid about a girlfriend. It may also be because he wants something that the other kid has. A gang member may use a gun to settle an argument with a member of another opposing group. The point is that these kids use these guns because they can easily access them, and now it is being used as an alternative to solving disputes by just words. Parents and the environment in which a person is, also affects the community, it s well being, and the nature of the person living in it. A poor ghetto neighborhood is more likely to have a higher crime rate than a place like Simi Valley. Also, in a poor environment, more people are poorer and raised with less morals and values, which makes them more vulnerable to using weapons without felling guilty or wrong.
However, there are many different views on guns and the issue of gun control. Some people who have lost a friend or family member in a tragedy feel that their availability should be reduced and gun control should be tightened. On the other hand, victims of rape and other women (especially of Arming Women Against Rape and Endangerment) feel that guns should be widely available for self-protection and to protect other innocent bystanders. US civilians have more guns than any other nation in the world, and one out of every two American households has a gun. Many Americans also use guns for recreational fun, including target practice and such. But they should use them responsibly, so someone else (like a criminal) does not borrow it from them to commit a crime. These people use personal experiences to validate their stand, but fail to think of the ideas that they are sending to society by taking that stand. Other people may use it as an excuse to hurt or even murder someone else. Because of the size, cost, and availability of guns, criminals are more scared of getting shot by civilians than the police. Still, many homicides take place (24,526 homicides in US in 1993) and a huge number of innocent lives are lost.
Due to that fact, greater gun control should be put into effect. It is not fair to the victim s families and friends to just lose someone simply by a gunshot. It is also not fair to the victim s themselves who have individual goals, dreams, and wishes that they have been working for and then have someone else end all that because they are not responsible of their hideous actions. Opponents to gun control argue that Guns don t kill; people kill. However, those people use the guns to kill. Therefore, safety measures by gun manufacturers and places selling them (photo ID and a thorough background check) should be increased. People could also consider alternatives to gun possession and use; they could rely more on police and security systems than themselves and their guns. Again, it will take a long time for people of today s society to trust authority and during that time, lives of innocent people will continue to be wasted.
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This article is written by Francesca Albini
One of the most annoying things is when your pens go dry and you have to throw them away. It really hurts me to get rid of any tool that can be used or should have been used for my art. So I don't. I find ways to rescue almost everything by making my own handmade inks.
Every time one of my ballpoint pens goes dry I take a pair of scissors and snip the plastic reservoir into a bin or plastic bag (they tend to fly across the room otherwise!), then put the segments in a small container. When I have enough segments (two or three reservoirs), I pour a little bit of surgical spirit into the container, close it really tight and shake well. The result is a purply blue ink. If it's too light in colour, wait a day for some of the spirit to evaporate. I suggest you use a cheap brush with this ink, as it can be harsh on the bristles.
Here is a portrait of the singer Sippie Wallace I painted with homemade ballpoint ink. Because this ink is quite viscous, instead of diluting it with water or more spirit to obtain lighter shades I prefer to dry the brush on a tissue. Using an almost dry brush gives the painting an unusual and pleasant grainy feel. Ballpoint ink is not the easiest of mediums to use, but it is fun to experiment with.
Gel pens are particularly prone to get dry very quickly. Snip the dry reservoirs and put them in a container, as above, but this time dilute with water. I like the ink made with gel pens because it very often has a shiny, glittery texture. Here is a flamingo I painted using ink made out of a few pink and orange dry gel pens. Gel pen ink is also easier to use, as it can be further diluted with water.
I spent the Christmas holidays going for walks in various parks and gathered a few things that I could turn into art tools, like bamboo stems and a pigeon feather (hoping the ex owner didn't have the flu!) There are many sites on the internet that show you how to make reeds and quills. It's really quite simple. Cut the shape of the nib with a craft knife, test and trim until you are happy with the marks it makes. While my husband was trying to have a serious conversation with me, I doodled his portrait with my homemade bamboo reed and Indian ink. He ended up looking a bit like the Buddha, so I thought of adding some tropical foliage in the background with my pigeon quill.
Playing with homemade inks and found or recycled materials is fun, creative, cheap, and makes you feel good.
Francesca is a visual artist using pretty much everything she finds around her to record and relive feelings and memories of places and emotions. She collages, paints, draws, photographs. Francesca loves mixing modern technology, such as mobile phone apps, with the simplest of tools such as glitter glue, crayons and other children's art supplies.
Read her blog at Fran's Journal Blogspot

subset: commoncrawl


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Henrikh Mkhitaryan has told Borussia Dortmund that he wants to leave the club and join newly appointed Manchester United manager Jose Mourinho at Old Trafford.
The midfielder has one year left on his current contract with Dortmund, who finished second in the Bundesliga last season, but he has stated that his intentions are to look for a new challenge elsewhere.
He has told his club that he wants them to agree a deal with United, according to the BBC, and this will be a big blow to Arsenal boss Arsene Wenger.
Midfielder heading to Premier League?
Wenger has been keeping tabs on the midfielder for almost 18 months now, but the attraction of playing with Mourinho at Old Trafford may well be too irresitable.
The Special One has a list of transfers this summer, as he looks to add to the squad that he has taken over from former boss Louis van Gaal.
Dortmund spent £23.6million back in 2013 to sign Mkhitaryan from Ukrainian side Shakhtar Donetsk, and the German side won't want to lose another key player, following the departure of their captain Mats Hummels to Bayern Munich.
Striker Pierre-Emerick Aubemeyang is also wanted by clubs all over Europe, and may well be another star name to depart Dortmund this summer.
Agent confirms rumours
Dortmund CEO Hans-Joachim Watzke told Sport1 that they have been "negotiating with Henrikh Mkhitaryan for four months", but he will depart after ews emerged that he won't sign a new deal.
"Now we know that it is not extended, it was never out commitment that Mkhitaryan may leave us prematurely," he said.
However, the players' agent Mino Raiola claimed that he wasn't pleased that his client would be kept at Dortmund until the end of his contract.
He said: "Micki has, for months, maintained that he may leave the club this summer, Dortmund presented an official offer which was rejected, but now the BVB bosses fear of their own fans."
Raiola labelled Dortmund's management as "very bad" after they told the player to leave on a free transfer at the end of next season.
"Manchester United is a unique opportunity, Micki wants to go there," he added.


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You're more than welcome to Edit the info or Upload a manga chapter for share. You can also keep track this manga by subscribe the RSS feed or Bookmark it.
|Natsumizu Ritsu
|Natsumizu Ritsu
|Comedy, Drama, Mature, Romance, Yaoi
Popular erotica writer Kaji "K" sensei's glamorous appearance is only rivaled by the fantastic rumors surrounding him. Like how one of his best selling novels was based on a relationship with a high-school student... or how two of his model sex friends got into a cat-fight in the middle of a highway... or how he has threesomes during his nocturnal writing sessions! At least these are the things that go through editor Kobayakawa's head when he sees his celebrity charge, that and the one where K sensei sent three editors to the hospital?! Yet, while taking photos for a magazine interview and watching K sensei work with such professional focus Kobayakawa realizes that the outrageous rumors may be just that. That is until Kaji pushes him down on the sofa to "reward" him. Thus begins the hunt of the herbivore editor and his carnivore author.
In the K-Sensei series:
V.1 - K-sensei no Yajuu na Aijou
V.2 - K-sensei no Yaban na Renai
V.3 - K-Sensei no Furachi na Junai


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The Chainsmokers Announce 'King of Me' Tour
New York-based producers of the EDM hit "#SELFIE," Drew Taggart and Alex Pall, AKA The Chainsmokers, have officially announced the first phase of their King of Me tour. So far, the tour spans throughout the month of September. The Chainsmokers took to Twitter earlier today to announce 12 dates, including several cities in North America and even a few international dates. Phase 1 of their King of Me tour is set to cover clubs from Echostage to Amnesia and festivals from Life in Color to TomorrowWorld. Although the duo's set at Electric Zoo 2014 was cut short, they did have the chance to premiere a few new tracks.
The Chainsmokers have also recently released their first episode of Nice Hair With The Chainsmokers just last week. The duo now hosts the radio show on Electric Area channel 52. Take a listen below to catch an hour-long mix of what you might experience at any of their fall tour shows.
Some dates have already released tickets such as Providence, Rhode Island and Washington, DC. You can keep an eye out for tickets near you here.
Cover photo credit: ThisSongSlaps
Follow The Chainsmokers:


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Status: Closed
Know more about this business than we do? Cool! Please submit any corrections or missing details you may have.Help us make it right
Try Hendrickson's Steak Sandwich...a customer favorite.
Hendrickson's Corner is keeping with the tradition that this location has offered since 1884. American cuisine in an Old World setting. Enjoy a meal and cocktails in a warm and friendly atmosphere.
Go here for: dinner, Sunday brunch
Be the first SILive.com user to add photos or videos of Hendricksons Restaurant
Great Family dinner from drink through dessert. Sauerbraten was tender with tasty sides. Resturant has quite impressive history, it was very clean and bright. Staff is extremely friendly and knowledgeable about the menu. We will definitely return soon.
Great atmosphere and even better food! Staff are the most friendly people. They made us feel like we were one of the family in for a visit!!
Open for : Dinner, LunchAlcohol: Full Bar
Hendricksons Restaurant can be found at Broadway 671. The following is offered: Restaurants. The entry is present with us since Sep 9, 2010 and was last updated on Nov 14, 2013. In Bayonne there are 53 other Restaurants. An overview can be found here.


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I'm Terrica; owner, lead planner and floral designer of Southern Productions weddings & events. I have been married to my husband, Kyle, since November of 2008. We live in Collinsville, MS with our two furry friends; Ann and Buddy.
I get asked all the time how I became a planner and floral designer. As a child I always knew I wanted to do something creative and always thought I would become a fashion designer for as long as I can remember. As I grew older I realized I probably would not be able to fulfill that dream unless I moved to a large city, which I had no intentions of ever doing! Long story short; I went into the health field and became a dental hygienist after a few years (just six!) of attending college and not liking anything I majored in. I enjoyed it for a while, but always craved something more creative.
When I planned my own wedding I loved everything about the entire process and seeing it all come together. I soon realized I had a true passion for weddings and saw the need for a planner in my area. I decided I would start my business as a "side hustle" and would continue working full time as a hygienist as long as I could keep up with both. Well, 2017, will be my first full year as a full time business owner and I could not be more excited about it!
Thank you for stopping by! I would love to chat with you about your wedding! If you would like to learn more about our planning packages and/or our floral design services please contact me to schedule your complimentary consultation. I look forward to working with you!


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First Parish has a rich history of Continuing Education courses — evening programs, Sunday morning discussions, workshops, and other events that contribute to spiritual growth, lifelong religious learning, and a deep understanding of the world in which we live. These activities are organized and coordinated by the Continuing Education Committee.
Past Continuing Ed Programs
- Beginner Yoga Class for Relaxation and Renewal
- Spring Training with Katie Lee: How to Craft a Worship Service
- Practical Solar Energy – 40 years in 40 minutes
- Living simply in a Complex World
- Zen Meditation
- Building an Everyday Spirituality Practice That Works For You.
- Annual Book of the Year Event. Past books have included:
- Chinese Lessons by John Pomfret,
- The Death of Josseline: Stories from the Arizona Borderlands by Margaret Regan
- The Post-American World by Fareed Zakaria)
- A People so Bold: Theology and Ministry for Unitarian Universalists
- Being a Unitarian Universalist Parent
- Fuller Craft museum outing
- Cakes for the Queen of Heaven: In Ancient Times
- Love and Death: Five Conversations about Aging with Dignity and the End of Life
- Living By Heart: Meditation and Reflection as Daily Practice


text: My new Fendi spy shoes have just arrived and I so love them. They have little knots on the T bar section which is also plated just like the spy handles on the bag. I have teamed them up with my Petrol spy, the colour is not an exact match but think they go quite well.


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Using art to bring people together
L's Art is striving to continue helping people fund raise, engage individuals and the community in art and even on a good day keep a living artist afloat. Will you help?
You can donate funds as "coffee" here - all money goes back into L's Art for supplies, community projects and keeping art affordable for class goers.
You can also purchase a piece of art from L's Etsy store.
Painting parties to raise funds and awareness for charities. Using art to bring people together for a good cause.


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Variable and reversible quantum structures on a single carbon nanotube
Date Issued2000
Kiliç Ç.
Ciraci, S.
Gülseren O.
Yildirim, T.
Please cite this item using this persistent URLhttp://hdl.handle.net/11693/24923
Physical Review B - Condensed Matter and Materials Physics
Published ashttp://dx.doi.org/10.1103/PhysRevB.62.R16345
- Research Paper [6026]
The band gap of a semiconducting single wall carbon nanotube decreases and eventually vanishes leading to metalization as a result of increasing radial deformation. This sets in a band offset between the undeformed and deformed regions of a single nanotube. Based on the superlattice calculations, we show that these features can be exploited to realize various quantum well structures on a single nanotube with variable and reversible electronic properties. These quantum structures and nanodevices incorporate mechanics and electronics.


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This week's spotlighted guest is a John Maxwell Certified Coach, Teacher and Speaker and real estate agent Rick Graham. I have been fortunate enough to get to know Rick. Rick truly cares about making an impact and it is obvious when you spend anytime with him. You can read more about him here, and connect with him on LinkedIn here.
Leadership is influence. Rick Graham works in a broad range of business environments from growing entrepreneurial companies to established, global corporations. Rick helps leaders and teams set priorities and reach goals through focused action. Rick's collaborative approach generates outstanding teams that overcome barriers and get sustainable results.
Biggest passion:
Experience and inspire healthy growth through clear purpose and consistent practice.
How did you start/get the gumption to start?
I started college as a traditional business major and during my junior year I heard a guest speaker talk about the growing field of Organization Development. I instantly connected with his message about how to motivate healthy growth and I and changed majors immediately. I guess the gumption came from a sense deep inside my spirit that God has made me to help others become their best.
Biggest failure:
Comparing self to others and then entertaining thoughts that I don't have what it takes. These limiting beliefs steal enthusiasm, joy and contribution.
Favorite verse:
Joshua 1:9 Living Bible
Yes, be bold and strong! Banish fear and doubt! Remember, the Lord your God is with you wherever you go.
Last piece of advice:
Do what you love with the people you love. Take time to reflect and evaluate priorities. Invest in meaningful relationships. The struggle to develop powerful relationships, whether in business or life, is worth it. Hang in there. You were made to be a key part of an amazing story.
What is your best piece of advice?


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Wilbur N. Wood (L)
Office Sought: U.S. House, District 10
Age: 51
Residence: Berryville.
Education: Optician graduate, Fairfax County Apprenticeship Program.
Occupation: Optician; store manager, Optical Center.
Web site: http://www.woodforcongress.com
E-mail address: email@example.com
Elected offices/civic activities:
Director, Virginia Soil and Water Conservation Board for Clarke County, 2002; past battalion command sergeant major, Virginia Defense Force; chairman, 10th Congressional District Committee of the Libertarian Party of Virginia.
Why should voters elect you?
"I will bring new ideas and solutions to old problems and challenge the status quo perpetuated by the Republicans and Democrats."
What do you think is the most urgent problem facing your jurisdiction?
"The most urgent issue facing the 10th Congressional District is that government at all levels takes nearly 50 percent of gross wages out of the pockets of American workers. This rising cost of living is forcing the lower and middle class to commute from as far away as Winchester and Fredericksburg into Arlington and Washington. This burden on the average family deals a severe blow to quality family time as longer commutes reduce time at home and in the local community. These issues are a direct result of the expansion of government in recent decades."
-- The Washington Post | Submit a correction request.


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Southside Film Festival is returning for the fifth time in Glasgow with pop-up cinemas, unique film screenings, talks, exhibitions and workshops.
The festival will again offer a packed programme from October 8 - 11 with a diverse range of films including documentary, animation, feature films and short films.
In previous years the Southside Film Festival has screened films across the Southside's most famous venues including Pollok House, Govanhill Baths and Pollokshaws Burgh Hall.
Spread further across the Southside than ever before, this year's event will also take in Kinning Park, Govan, the Gorbals, Shawlands and Pollokshields.
This year's festival theme is cinema heritage and film archive.
Film enthusiasts can come along to the opening reception at the Glad Café on Thursday, October 8, to kick off the festival, pick up a programme and enjoy the Lost Cinemas of Glasgow exhibition.
Lost Cinemas of Glasgow: Architecture and Society at The Glad Cafe will run until October 26.
This exhibition of Southside cinema buildings is a unique insight into how Glasgow's former 160 cinemas once played an influential role in everyday Glaswegian's lives.
The Rum Shack will host a screening of The Harder They Come (October 11) which includes a two-course Jamaican meal before the film and a DJ to play reggae tunes into the night.
In a Southside Film first, Rags to Riches, Sew La Tea Dough and Govanhill Baths join forces for Making in the Movies (October 10), an afternoon focused on fashion and craft from the past and present.
There will also the opportunity to see craft and textile themed films from the Scottish Screen Archive throughout the afternoon.
Karen O'Hare, Director of Southside Film Festival, said: "Southside Film Festival started in 2011 as a response to the lack of a local cinema or film screenings in the Southside of Glasgow.
"Four years later the festival reflects on the continued lack of a local cinema with the theme of cinema heritage and film archive.
"The 2015 edition again turns Southside spaces into pop-up cinemas providing a wide range of site specific or unique film screenings, talks, exhibitions and workshops - all at affordable prices or free."
You can see the full list of events on the Southside Film Festival on the website.
Tickets available in advance online or on the door on the night.


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Daily Cute: Curious Cat Remixes Tunes on Vinyl
When this cat stumbles upon a turntable playing a few Bob Marley tunes, he can't help but try his paw at scratching. After making his mix, he calls it a day and takes the vinyl record for a spin – literally!
Check Back Daily for More Adorable Animal Videos!
Photo Credit: corbo


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I am switching over to Pinterest for this section- it's so much more visually pleasing and easier for me to keep organized than linking up everything. So please head over to my Pinterest Board, 'Things I'd Like to Make" and check out what's on my to-do list. And if you haven't seen Pinterest yet, please check it out- it's awesome!
Here's my list of Things I'd Like to Make.


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The new Iron Maiden single is out. Continue reading
Adrian Smith
Gers and Smith talk THE BOOK OF SOULS
The hype machine is heating up, at long last. Interviews and reviews that rave about the upcoming Iron Maiden album have been allowed to hit the web. The Quietus carry an interview with two of the three amigos. Continue reading
New Single SPEED OF LIGHT On August 14
So Iron Maiden are going to release a single from their upcoming album after all… Continue reading
FEATURE FRIDAY: Looking forward to opening THE BOOK OF SOULS
With last week's official announcement of the upcoming Iron Maiden studio album, we want to go all nerdy and share our thoughts on the details provided so far. Continue reading
FEATURE FRIDAY: Times of Change, 1990-91
A new decade dawned and Iron Maiden set about maintaining and developing their status as one of the world's premier hard rock bands. The challenge would be tougher than anyone knew at the time. The 1990s had arrived. Continue reading
Christer Reviews: ASAP – Silver And Gold (1989)
The final Iron Maiden-related album to be released in the 1980s was Adrian Smith's first solo venture. And boy was it different! Continue reading
This month in 1999: Iron Maiden reunited with Bruce and Adrian
Believe it or not, the current line-up of Iron Maiden has been together for 16 years this month! Continue reading


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The Oxford Handbook of the Economics of ProstitutionOxford University Press 2016; US$ 149.99
Prostitution bears the unique title of being both the "world's oldest profession" and one of the least understood occupations. The Oxford Hanbook of the Economics of Prostitution fills the gap in our understanding. It brings together many of the top researchers in the field who explain how the prostitution markets are organized across space and... more...
Sex MarketsTaylor and Francis 2014; US$ 47.95
Empirical and mathematically rigorous, this book provides a study of the economics of prostitution rather than focusing on the sociological and cultural themes. Using economic tools of analysis, internationally based editors have put together a theoretically informed volume that explores the supply and demand of prostitution. Prostitution is a globalized... more...
Female Prostitution in Costa RicaTaylor and Francis 2013; US$ 54.95
This book analyzes the development of female prostitution in the Pacific port of Puntarenas, Costa Rica during the advanced stage of the coffee exporting economy (1880-1930), at the height of the consolidation of the liberal state. Hayes argues that prostitution in the port differed from that of the coffee producing highlands due to differential economic,... more...
The Price of SexTaylor and Francis 2013; US$ 55.95
As a society we are buying more sex than ever before. Adult sex shops now take their place amongst retailers in the high street and lap dancing clubs compete for an increased share of the leisure economy; hotel chains offer sexually explicit films as part of their standard service, the party selling of adult toys to women in their homes has become... more...
Sex Work NowTaylor and Francis 2013; US$ 52.95
Sex Work Now provides an authoritative overview of female sex work and policy in the UK, and addresses a number of key contemporary issues and debates. These include sex worker unionization, migrant sex work and trafficking, communities and sex work, male clients of sex workers, the policing of prostitution, zoning of street sex work, young people... more...
Sex DifferencesTaylor and Francis 2013; US$ 54.95
This volume is the first to aim at summarizing all of the scientific literature published so far regarding male-female differences and similarities, not only in behavior, but also in basic biology, physiology, health, perceptions, emotions, and attitudes. Results from over 18,000 studies have been condensed into more than 1,900 tables, with each table... more...
InvisibleSaqi 2013; US$ 10.99
Ming and Beata share neither the same language nor cultural background, yet their stories are remarkably similar. Both are single mothers in their thirties and both came to Britain in search of a new life: Ming from China and Beata from Poland. Neither imagined that their journey would end in a British brothel. In this chilling exposé, investigative... more...
Sex Work MattersZed Books 2013; US$ 34.95
Contains essays on the economics and sociology of sex work. From insights by sex workers on how they handle money, intimate relationships and daily harassment by police, to the experience of male and transgender sex work, this book offers theoretical discussions as well empirical case studies, providing ways to link theory with lived experiences. more...
Pleasure FactoryMarshall Cavendish 2013; US$ 15.99
Underneath Singapores squeaky-clean exterior is a dark realm like no other. Based on interviews with hundreds of prostitutes in Singapore and years of research, we bring you Pleasure Factory. In the Pleasure Factory, women advertise pleasure by the hour, but the only real product is pain. Night after night, these women force smiles onto their... more...
Violence and Sex Work in BritainTaylor and Francis 2013; US$ 44.95
This book is concerned with violence in the sex industry. It aims to provide an understanding of the nature of violence against sex workers and the relationship between violence, government legislation and policy, and law enforcement practices - an essential task in view particularly of the 2006 Ipswich murders and the public and media response to... more...


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I was watching a National Geographic special about how they built the pyramids, and we followed an archeologist as he went through some previously unexplored hallways. And on one of the walls were some old writings from one of the workers who had been building this particular pyramid. There, with my own eyes, I saw it, and right away I got my camera and took a picture of my TV screen.
Somehow the Egyptians knew about the Habs 4500 years ago. Here's the proof, on a pyramid wall.


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Calliari's Québec – Le Québec de Calliari in French – is a new documentary film by director Anita Aloisio that follows the creative journey of Italian-Montreal singer/songwriter, Marco Calliari, as he strives to stay relevant in the Quebec music industry.
Calliari started his musical career at age 14 as a member of the popular Quebec metal group, Anonymus. In 2003, he left the band to focus on a solo career. His Italian-language music calls on the influences of Italian folk music, jazz, and world beat. Over the years he has toured extensively to promote his numerous albums, including Che La Vita (2004), Mia Dolce Vita (2006) and Al Far Est (2010). His cover of the popular Italian song, L'Americano, became an international hit when it was remixed as "We No Speak Americano" by M.H.M.
Calliari's faith in the industry was put to the test after the disappointing sales of his 2014 album, Mi Ricordo. The album was a tribute to eleven renowned French-Québécois songs which Calliari translated and performed in Italian. The album name played homage to his nationalism – an Italian translation of Quebec's infamous license plate "Je me souviens."
The album was a financial and critical failure. Worst yet, fans and critics alike continued to label Calliari an "ethnic" artist despite his national-pride-infused album and despite the fact that he was born and raised in Montreal.
Calliari has thus decided to make his next album a French-language solo album. His first ever French-language album. He is gambling on the theory that this new album will finally gain him recognition as a "Québécois" artist.
It is this critical turn in his career that Aloisio's documentary film will focus on.
Aloisio and producer Agata De Santis want to start filming this coming summer, just as Calliari is scheduled to start working on the new album.
In order to start filming (as they await responses from various public funding agencies), De Santis and Aloisio decided to launch a crowdfunding campaign to raise the necessary budget.
"We don't want to miss key shooting opportunities that will be coming up very soon," De Santis explains.
"We are turning to the fans of Marco Calliari and to the Italian-Montreal and Italian-Canadian community for their support. We also believe that this campaign gives the public a unique relationship to the film," she continues.
"Contributors to the crowdfunding campaign will be able to follow the progress of the film, from the start of the production, into the edit, and to the release in September of 2018. They will be able to witness all the ups and downs of making a documentary film. There are many!"
This will be Aloisio's third documentary film. Her first film, Straniera Come Donna (2002) explored the understanding and effects of cultural and social tradition on women of Italian origin in Quebec. Her second documentary film, Les enfants de la loi 101 (2007) explored the impact that Quebec's Language Charter had on the first generation of children such as herself, 30 years after its adoption.
De Santis runs Redhead Productions and is the founding editor of italocanadese.com.
The duo hope to raise $19,500 with the campaign. This total will cover the cost of ten days of filming with a professional crew. The campaign runs until May 19, 2017.
To make a contribution, please visit www.igg.me/at/lequebecdecalliari.


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From searching on Trip Advisor, I see that there are 2 hotels in Xi'an with similar names.
This review is for the Wan Nian Hotel, aka Golden Wannian on Chang Le Zhong Road.
This was the 3rd hotel of our OnTheGo tour of China.
The hotel was very nice, and again it is primarily a business hotel (there was a refridgerator salesman conference on during our visit!).
The rooms was large and spacious, with complementary bottles of water in the room.
The hotel is close to the centre of Xi'an.
It is currently the down season (March) and we had to check if the bar was actually open (it turns out it has lights that are only on, when the sensors are activated). Once we had established the bar was open it was good value, only 10 Yuan / beer.
The bar at the top of the hotel was also closed.
As was the swimming pool!!
Breakfast was split into 2, with western and Chinese breakfasts being served in separate dining rooms. The western breakfast, was very quiet, and it is all cooked freshly for you. Being this isn't a buffet style breakfast, service is a bit slower than in other hotels, and breakfast will last at least 30 mins. Cereal and milk is on the table on arrival, and the staff then serve juice and coffee. 5- 10 mins later, the toast arrives. 5- 10 mins later still the cooked breakfast (a huge omlette, bacon and sausage) arrives. However, on the 2nd morning, no staff was evident on arrival, so we helped ourselves
to someone elses leftover toast. This resulted in us not getting any fresh toast, and no cooked breakfast.
- Reservation Options:
- TripAdvisor is proud to partner with Booking.com, Ctrip TA, Cancelon and TripOnline SA so you can book your Wan Nian Grand Hotel reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Wan Nian Grand Hotel China/Shaanxi


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Coming this December from Image Comics, the fan-favourite Midnight of the Soul by Howard Chaykin will be collected into trade paperback for the first time!
The post First Looks: Midnight of the Soul appeared first on Rogues Portal.
read more
Chaykin discusses his new series, about a World War II vet struggling to adjust to the post-war world who is drawn into a web of mystery and murder.
Veteran writer-artist Howard Chaykin drops the bomb on his upcoming Image Comics project about a "world-changing event."
Every day this year, I will be examining the artwork on a single comic book story. Today's artist is Howard Chaykin, and the issue is American Flagg! #3, which was published by First Comics and is cover dated December 1983. These sc...
At last year's San Diego Comic Con, Image Comics had a memorable panel featuring many of comics' top creators, including industry legend Howard Chaykin. Image Publisher Eric Stepheson and his guests announced several upcoming high p...
In Howard Chaykin's Midnight of the Soul #4, tangled memory and tainted love-as the night draws closer to dawn-and blood reckoning. The post Midnight of the Soul #4 appeared first on CBR.com.


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Need a Roseville medical Marijuana delivery service? Check out our directory of medical herb delivery that provide medical marijuana products.
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The city is regarded as a major shopping hub, and here you can find the Westfield Galleria at Roseville which is the 2nd largest shopping mall in Northern California. Aside from other retail centers here such as the Fountains at Roseville retail center that occupies 330,000 square feet of retail space, you can also find one of the biggest automotive malls in the whole country. For some recreation, a popular destination in the city is the Roseville Golfland SunSplash waterpark.
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In this scenario, a patient is in danger of being imprisoned because they are in possession of an illegal drugs (cannabis under federal law is still stated as a Schedule I drug and for that reason, it is illegal to own, cultivate, or transport it).
So you see, having a medical marijuana ID card can be rather beneficial! So talk to our online Roseville medical marijuana doctor and schedule an evaluation. Prepare your medical history document for qualification purposes.


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Not many are aware that priyanka chopra missed out on a chance of working with Bollywood's legendary actor Dilip Kumar when she forayed into films. Back then, Priyanka had been signed for the movie along with Dilip Kumar but the makers never took the movie forward.
"But I have no regrets" says Priyanka. "Whenever I lose out on something, I always get something good to look forward to. There were times when things have not happened, but at the end of the day I got something better. I don"t regret not doing those films. I am okay with rejections, but I cannot handle failures," she says.
The actress who has had a string of flops right from Love Story 2050 to Drona doesn"t regret doing them. She states, "I worked hard on these films and trained myself in martial arts. For Love Story 2050 I coloured my hair red. It was a big disappointment that these films did not work. I experimented for my roles. Can you believe it, I have not taken a holiday for the last six years? I have put in so much work," Priyanka reveals.
With What"s Your Raashee on the anvil of release, Priyanka must certainly be on tenterhooks now!


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Web Chat: Charlie Foxtrot
Web Chat: Charlie Foxtrot
Hello Everyone. I appreciate everyone's great questions! I hope the information was in some way helpful. Remember, if you need help. please reach out and talk to someone.Just start somewhere. Its the hardest part.
Hello all and happy Veteran's Day! I look forward to your questions!
Hello, do you have experience in treating people who have PTSD who are not in the military, or just servicemen?
If a family member suspects that their service member is suffering from PTSD, how should they go about seeking help for their family member without causing harm to their career or backlash from their command?
If someone is having a flashback or attack from PTSD, is there anything the person can do at home to minimize the anxiety attack till they are able to seek professional help?
Are there any programs we can sign our friends and family up for to help when they first get back from being deployed?
What is being done in connection with the military to help with this issue before it even begins?
What are we supposed to do if our family members won't admit there's an issue?
How long does recovery typically take?
Can you share the help line you mentioned in the news report that people can call?
Hey guys! We're wrapping up in ten minutes!
Leave your last questions now!
Hello and welcome to our web chat with Kurt Hooks, the director of behavioral health services and palliative car at Chesapeake Regional Medical Center. Mr. Hooks sees many patients with post-traumatic stress disorder (PTSD), and has assisted many veterans, active duty, and military dependents in crisis throughout his years of clinical work.


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If you live in Lekki [heck, Lagos] you're well aware of the floods that we were graced with today. Thankfully, The Boy & I live in an upstairs apartment. Unfortunately, the neighbors downstairs got fully flooded. Like, the water got up to the level of their beds and furniture and stuff. They have two kids; age 5 & 12. It's been raining nonstop for days. But we didn't see the flood coming. We live on a street in Lekki that never gets flooded, no matter how much it rains.
It was a regular Saturday morning. But it was raining heavily. And the thunderstorm affected my sleep. So I was trying to get a little more sleep, even as it kept raining. I had planned to go to work around 9/10am so I wanted to get as much sleep as possible. Husband's phone rang a few minutes to 8am & it was the neighbors. What could they want? Anyways he answered, and they asked if they could send their daughters upstairs because their entire apartment was flooded. Of course, the sleep cleared from my eyes. He ran downstairs to get the kids. And that was the beginning of my day. [We later found out both our cars were fully filled with water as well, but thank God it wasn't our cars & home, right?]
First thing I did was make breakfast. Pancakes, eggs, sausages, baked beans. Then I realized they could be allergic. I called their parents to ask about any allergies. None. So all 4 of us got on the table to eat. Little girl wanted syrup, older girl didn't. I put the syrup on her pancakes. She tasted it. She didn't like it. The Boy & I had to swap unsyrupped pancakes with her. Then she decided altogether that she didn't like pancakes. But she liked the eggs. So The Boy gave her all of his eggs. She ate it. And wanted more. So I gave her all of my eggs too. That was lesson 1. Sacrifices. We each had to sacrifice all of our eggs for her.
Lesson 2. Sticky things. After they left, I had to clean up. My house that gets cleaned a couple of times a week had to be cleaned a couple of times while they were here. You see, I suffer from OCD. So everything had to get cleaned as it was happening. Spilled juice. Spilled water. Cookie crumbs. All sorts. When they left and I was doing general cleaning, I found lots of sticky things on the couch and floor. I have no idea what they were. I don't even want to know to be honest, LOL!
Lesson 3. Lost & found. The 12 year old was very calm and gentle. I guess she was in a new environment [they've never hung out with us before] so it took her some time to get comfortable. But even after she got comfortable, she was still very calm and pleasant. The 5 year old however did NOT hold back. She was dancing ballet, doing cartwheels [scattering the house], picking things, examining things. I had to constantly be alert. I couldn't even take a nap. Because if I snoozed for even 2 minutes she found something to touch [that she shouldn't be touching – hey! I have no kids so my house isn't childproof]. One of the times she was hungry and I was going to make her something to eat, I couldn't find the lighter. I asked if she took it and she said no. I was confused. There's only one place I ever keep it. I later found it in one random drawer in the kitchen, along with the many other things she 'collected'. Upon asking her she simply said "oh sorry, I forgot".
Lesson 4. Multiple meals. I swear I fed them at least 4-5 times in the 12 hours that they spent with us. No joke. And there were many snacks in between. Wow. And their portion sizes were way bigger than mine. I was in awe.
Lesson 5. The most important. Be happy for no reason like a child. Despite everything happening and their reason for being here, they didn't seem phased about anything. The younger one was sad for a few minutes about her teddy bear drowning. But in a few minutes she snapped out of it and was back to her bubbly self.
It was definitely an adventurous & fun day. Now I need to take a very hot shower and relax.
Ps. They found a fish swimming in their living room. This is no joke.


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As the weather starts to warm up I've been going crazy trying out spring outfits. I've been waiting months to wear these florals pants (which come with a matching top, to be seen later). They're as comfortable as wearing harem pants but with a much more pulled-together vibe. I paired it with black to keep the mood transitional.
And I'm back to pinning pants and skirts that are too big! Such a simple solution to a common problem.
Shirt: TJ Maxx, Pants: H&M, Flats: Tahari, Jacket: H&M, Pin: Vintage, Ring: c/o Trendy Jewels
Photos by Jamie Ditaranto.


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Upcoming Celebrations
We celebrate our sesquicentennial anniversary with a series of faculty and department events, lectures, and activities taking place in Halifax – both internally at Dalhousie and within our local community - in addition to satillite hosts within the Maritimes.
Next Event
Kicking off with the Fear Family Conference
December 2, 2017
Dean of Medicine, Dr. David Anderson, introduces our anniversary year at the
7th Annual Thomas and Alice Morgan's Fear Memorial Conference in December.
An event hosted by the Department of Emergency Medicine along with our Continuing Professional Development program, the focus of this year's conference will be on emergency preparedness and will commemorate the 100th anniversary of the Halifax explosion.
Continuing Professional Development (CPD)
Dean's Office, Faculty of Medicine


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Big winnersApril 30, 2012
Back in February, Jamie and I attended Burnaway's Art Crush auction, where attendees were invited to bid on unique experiences with local artists. Bland Hack (Jamie and Julian's production company), for example, auctioned off the amazing opportunity to be a character that dies in their next short film.
It was my first auction, and I was pretty excited to have won one of the items — a garden consultation and set of prints by Cooper Sanchez, a local artist and gardener at Oakland Cemetery.
I won't divulge the details of the consultation just yet, but I will share that Cooper suggested plenty for us to do that we're really excited and only a tiny bit intimidated about.
For now, though, we're enjoying the awesome prints that we scored.
They're hanging in the newly decorated man cave above the television. I'm pretty enamored with them.
What do you think? I love his style, and think the black design and frames stand out really nicely against the wall.
The next step is to hang another piece of artwork on that wall (here's a hint as to what it might be), and the room will be nearly complete! Just like every other project in our house, this has been a long, ongoing process. Thankfully, I've gotten pretty used to that by now.
p.s. In addition to the final man cave reveal, you can also look forward to posts about an awesome home-related birthday present from Jamie, and my equally awesome Titanic-themed birthday party. Stay tuned!
Top photo courtesy of Burnaway.


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Pepsi IPL 7 T20 Match 43 KKR Vs SRH Live Score 2014:-The 43rd match of the Indian premiere League is between Kolkata Knight Riders and Sunrisers Hyderabad at Rajiv Gandhi International Stadium, Hyderabad. The match will start at 8 pm on 16th May 2014.KKR Vs SRH Live Score 2014.
Pepsi IPL 7 T20 Match 43 KKR Vs SRH Live Score 2014
Match 43 KKR Vs SRH Live Score 2014.The match will be interesting as the both teams is full of bowlers in the two teams. Kolkata Knight Riders is full of Excellent bowlers like Shakib Al Hasan and Morne Morkel and Sunrisers Hyderabad have Dale steyn and Bhuwneshwar Kumar and Karan Sharma and Amit Mishra as spinners.Ipl 7 KKR Vs SRH Live Score 2014.
So we are here updating the live score and live streaming of the match. 16th may 2014 is the result date for the general elections so people will be busy in watching the news related to the results or in any other work so here we will provide you all the IPL 7 Match 43 live score and KKR Vs SRH Live Score 2014 of this match. So lets have a look on the playing eleven of the teams.KKR Vs SRH Live Score 2014
Playing Eleven of Sunrisers Hyderabad
Shikhar Dhawan (captain), Lokesh Rahul (wc), Aaron Finch, Darren Sammy, Karan Sharma, Ishant Shrma, Bhuwneshwar Kumar, Dale steyn, Amit Mishra, Venugopal Rao,David Warner
Playing eleven Of Kolkata knight riders
Gautam Gambhir(Captain), Robin Uthappa(wc), Andre russel, Shakib al Hasan, yusuf Pathan, Umesh Yadav, Morne morkel, Sunil Naraine, Piyush Chawala, Jacques kallis, and Vinay kumar


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Woodville Chronicles
Woodville Chronicles
Woodville Chronicles, a challenging and entertaining Match-3 game.
Woodville Chronicles, a challenging and entertaining match3 game. Casual game lovers will definitely enjoy its fascinating plot, advanced graphics and variety of modes, obstacles, options and tools.Read more...
Imagine a sunny clearing in the woods - here you will build the wonderful town of your dreams! But Rome was not built in a day, and your town will require time and resources as well. Prepare to complete the challenging match3 levels of Woodville Chronicles and to reveal the secret of the town!
Woodville Chronicles is a great pastime for the whole family, as each family member can create its own profile, and five different profiles can exist at the same time. The casual gameplay is based on match3 mechanics, but due to various modes and types, bonuses and obstacles you will never get bored. The unique feature of this game is that the mode (Time or Relaxed) and the type (Swap, Chain or Group) can be changed at any time during the game.
The types differ in the technique of playing: Swap type requires swapping items to make chains of matching ones; Chain type requires selecting chains of matching items; and Group type requires selecting groups of them. When matching items are selected, they blow up and destroy marble plaques, and as soon as all the plaques are destroyed, the level is completed. It may seem simple, but only at the very beginning. Every next level becomes more difficult and requires more experience and skill: there are significant obstacles, such as chains, double chains, chests and enchained chests. Each of them needs two or more explosions to be removed. Sometimes you can get puzzled, but in such a case the game will give you a tip!
However, there are several useful bonuses that will facilitate your tasks: the Lightning blows up several random items; Shuffle mixes all the items in the playing area; the Bomb explodes a chosen area; the Magnet removes all items of a chosen color; and the Row Exploder explodes a chosen row of items. Besides, when you blow up large number of items at once, you get an extra bonus, Electrization: one item gets electrified and blows up all the surrounding ones. And, in addition to all this, quick players get extra combo scores.
To build the town, which is getting inhabited throughout the game, you should complete 5 stages, each featuring 4 appropriate buildings and a marvelous background image. Completion of each stage is followed by beautiful fireworks. Moreover, the relaxing music and fine sound effects create a fairytale atmosphere - what can be better after a long working day?Read less...
- An opportunity to build a beautiful town and unveil its secret!
- Relaxing Atmosphere!
- Three great ways to play!
- English
- German
- French
- Spanish
- Italian
- Dutch
- Swedish
- Portuguese Brazilian
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Wysyłamy w terminie: 3 do 45 dni roboczych
Wydawca / producent: MARVEL COMICS
Kod produktu: JAN110852
Zawiera historie z zeszytów: SECRET WARRIORS #17 do #19, SIEGE: SECRET WARRIORSScenariusz: JONATHAN HICKMANRysunki: ALESSANDRO VITTIOkładka: JIM CHEUNGA shadow war has broken out between Hydra and Leviathan, leaving a trail of death and destruction in its wake, and it's up to Nick Fury to put the pieces together and take them out. Fury has assembled three teams of shock troops: the Black, led by former S.H.I.E.L.D. agent Alexander Pierce, tasked with destroying Hydra; the White, Daisy Johnson's Secret Warriors, under his direct command; and the Grey, led by his son Mikel, dedicated to taking out Leviathan. But there's another team ready to report for duty, and it's a collection of soldiers from an age of unmatched heroism: the Howling Commandos! Assembled for a reunion with Captain America at their side, the Howlers remember their fallen comrades and plan for their next big battle - a fight that will get them in all kinds of trouble with the world's governing elite. But what else is new for Nick Fury?Ilość stron: 112Cena okładkowa: $14.99
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More Cats, More Schwag, For LOLcats
Okay, I'll admit it–in times of editorial stress, I've sometimes relied on the comic relief of I Can Has Cheezburger instead of a shot of Jameson or a Xanax to take a much needed break from the day's insanity. In the past, I and another cat loving editor (who's since left our publication) would frequently exchange the best LOLcat examples, particularly when they involved cats and editing (apparently, cats love to critique writing – who knew?).
As we all know, "the Internet is made of cats," and no one seems to know it better than Cheezburger Inc., which not only includes the famous cat-themed blog but a host of other sites, including FAIL blog and Memebase. The company, which has quickly become one of the most popular Internet humor sites, has received about $30 million in venture capital from the likes of Foundry Group, Madrona Venture Group, Avalon Ventures and SoftBank Capital.
Now it's getting ready to put out its fourth book , "How 2 Be Awsum: A LOLcat Guide 2 Life," due out from Penguin Group's Gotham Books in November.


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Challenge To $29B Express-Medco Merger Trimmed Again
U.S. District Judge Cathy Bissoon's ruling leaves only one claim to a relevant product market -– the provision of clinical specialty drugs -– remaining in the suit that the National Community Pharmacists Association filed in March 2012 challenging the deal between two of the...
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text: I downloaded the latest cheat file (Acekard 2 Official v4.17 *Update File*) and unzipped it. I opened my SD, and replaced AK2menu.nds in the root, as well as put the "cheat.xml and cheat.dat" file in the "ak2/cheats" folder. I turned on my DS after putting the SD card back into AK2 and AK2 into the DS. I went to "DQ5", pressed start and went to cheats. It said, "No Cheats in cheat database" followed by a window. In that window, I pressed Y to change the cheat file to ".dat" and then pressed rebuild, but I get an error message saying it couldn't find any cheats. I did the same thing except I changed the cheat file to ".xml", and it said the same thing: it couldn't find any cheats. Any help would be awesome. I'm really new at this stuff.


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South Indian actress Kavya Madhavan in beautiful black designer 3/4th saree blouse with 'V' shape collar neck pattern. Gold patch work through out neck line at SIIMA 2013.
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Internet Explorer
In stock online
Available in stores
Told in the author's distinctively enchanting style, this bestselling guide to one of the largest ecosystems in North America is now updated.
Authoritative, detailed, and scientifically up-to-date, Prairie: A Natural History provides a comprehensive, non-technical guide to the biology and ecology of the prairies, the Great Plains grasslands of North America. This edition has been updated to include a new preface, new information about declining bird species, enhanced protection of bison, the effect of industrialization on the prairies, and the effect of the increase in coyote numbers on red foxes and swift foxes.
Extending from Alberta south to the Mississippi River, the prairies are among the largest ecosystems in North America. Until recently, they were also one of the richest and most magnificent natural grasslands in the world. Today they are among the most altered environments on Earth.
Illustrated with spectacular full-colour photographs and elegant black-and-white line drawings, this authoritative reference and easy-to-read guide is a must for anyone who wants to know more about the dazzling natural variety of the prairies.
Published in partnership with the David Suzuki Foundation.
The following ISBNs are associated with this title:
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text: I would like to begin to say that I am a huge Vuitton fan and have a few items (vertical lockit, Marelle backpack) but I am also a very big fan of Chanel, which is quite annoying as it is mainly for woman, only a few mens pieces here and there, but anyway, What bag would anyone here suggest a unisex piece that I could wear daily as I want to think about getting one soon. I don't really keep an eye on the Chanel bags for reasons unknown as I do keep a close eye on the clothing, As Karl Lagerfeld is a genius, But I'm always keeping a close eye on the Vuitton ranges that I can never be bothered with anything else. I am, of course, not scared to wear a womans bag or whatever, but nothing like a Chanel 2.55 (although I would give my left arm for one), so is there anything suitable? Thanks to everyone.


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In the world of today, rape and sex assault cases are on a rise - 1 in 5 women (20%) is sexually assaulted while in college, and over a third of women who are raped as minors are also raped as adults; so one important thing that the parents or the society could do to protect their children from being in a situation where they are sexually assaulted or raped is to inform young people about the sex laws; that will not only help them in standing up for their rights, but also to think twice before the plan or do any sexual assault on a minor.
It is extremely important for the parents to talk openly with their children about Sexual laws, in order to protect them from being sexually assaulted; and also about what rights do they hold, according to the law, so that they could stand up for justice (if in any case It is required). But this is not it, it is also important for parents to inform their young children that it is possible that if the committed any sexual act with another minor, they too can be convicted of statutory rape, stated Keren Goldenberg, while talking about her recently published infographic.
She further added, But usually it is very difficult for parents to talk to their children about such issues, and in majority cases even parents themselves are not fully aware of the young sex laws. So, to make understanding sex laws easier for both the parents and the children, I have created and published an infographic WHAT YOUNG PEOPLE NEED TO KNOW ABOUT SEX LAWS, this will surely help.
This infographic covers all major topics including the age of consent, what constitutes consent aiding and abetting, tips for protecting the teenagers, and other highly important and useful information and tips.
According to research, 42% of college women who are raped tell no one about the assault hence it is highly important to help young people know about their rights so that they are able to stand up for themselves.
About: Keren Goldenberg is one of the best criminal assault lawyers in Massachusetts. The Law Offices of Keren Goldenberg is highly popular and recommend in Massachusetts. For more information, please go to http://kgdefenselaw.com.
The Law Office of Keren Goldenberg
97 Central St. #403
Lowell, MA, 01852
(978) 221-2503
Pressekontakt:The Law Office of Keren Goldenberg
97 Central St. #403
Lowell, MA, 01852
(978) 221-2503


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Flat Top Grill
- Restaurants/Clubs
A '' Create Your Own'' Restaurant
MenusFlat Top Grill MenuExpires: 12/31/2018Print CouponLife should be lived with flavor. After all, food is not just fuel. When done right, even casual street fare can stimulate your senses. Ears lock on the sssssssound of the sssssssizzle. Pupils dilate. Nostrils twitch. Taste buds stir. Breakfast. Lunch. Dinner. Here, you can have a YOU-nique, interactive experience some people call mealtime. At Flat Top Grill, they grow it, you choose it, we cook it. C'mon in. Grill's on!


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Endothelial PAS Domain Protein 1 Gene Promotes Angiogenesis Through the Transactivation of Both Vascular Endothelial Growth Factor and Its Receptor, Flt-1
Jump to
Endothelial PAS domain protein 1 (EPAS1) is a basic-helix-loop-helix/PAS domain transcription factor that is expressed preferentially in vascular endothelial cells. EPAS1 shares high homology with hypoxia-inducible factor-1α (HIF-1α) and is reported to transactivate vascular endothelial growth factor (VEGF), fetal liver kinase-1 (Flk-1), and Tie2 promoters. In this study, we analyzed the role of EPAS1 in the process of angiogenesis. Using microarray technology, we looked for target genes regulated by EPAS1 in vascular endothelial cells. A total of 130 genes were upregulated by EPAS1, including fms-like tyrosine kinase-1 (Flt-1). Reporter analysis using human Flt-1 promoter and gel mobility shift assays showed that the heterodimer of EPAS1 and aryl hydrocarbon receptor nuclear translocator binds directly to HIF-1-binding site upstream of Flt-1 promoter and transactivates it. Small interfering RNA targeted to EPAS1 but not HIF-1α attenuated desferrioxamine-induced Flt-1 mRNA expression, thus EPAS1 is thought to play an essential role in hypoxic induction of Flt-1 gene. Furthermore, using mouse wound healing models, we demonstrated that adenovirus-mediated delivery of EPAS1 gene significantly induced the expression of VEGF, Flt-1, Flk-1, and Tie2 mRNA at the wound site and promoted mature angiogenesis. The proportion of the number of mural cells in newly formed vessels was significantly higher in EPAS1-treated wound area than VEGF-treated area. In conclusion, EPAS1 promotes Flt-1 gene expression and induces mRNA expression of VEGF, Flk-1, and Tie2, leading to enhancement of mature angiogenesis in vivo. Thus, EPAS1 may contribute to the construction of mature vessels by modulating the coordinated expressions of VEGF, Flt-1, Flk-1, and Tie2.
Hypoxia plays an extremely important role in the pathogenesis of many cardiovascular diseases. In hypoxic conditions, each organ or cell shows compensatory response at the molecular and cellular levels. Hypoxia-inducible factor-1α (HIF-1α) is expressed ubiquitously and serves as a master regulatory gene in the process of hypoxic response.1 Especially in the cardiovascular system, each organ promotes angiogenesis to increase oxygen and nutrient delivery. Endothelial PAS domain protein 1 (EPAS1) is a member of basic-helix-loop-helix/PAS domain containing transcription factor family and has a high homology to HIF-1α, thus also termed HIF-2α.2,3 Like HIF-1α, the EPAS1 protein is stabilized during hypoxia and forms a heterodimer with aryl hydrocarbon receptor nuclear translocator (ARNT) and transactivates the vascular endothelial growth factor (VEGF) promoter.4,5 Several groups generated EPAS1 knockout mice and reported that this resulted in the generation of different phenotypes. These results demonstrated that EPAS1 plays an essential role in the process of vascular remodeling during embryonic development6 as well as in catecholamine homeostasis7 and fetal lung maturation.8 Thus, HIF-1α and EPAS1 are thought to play critical roles during the angiogenic hypoxia response.
In contrast to HIF-1α, EPAS1 is expressed mainly in vascular endothelial cells. Therefore, EPAS1 is thought to regulate endothelial-specific genes, and it is considered to function differently from HIF-1α. Indeed, EPAS1 was reported to transactivate endothelial-specific genes such as Tie2 and fetal liver kinase-1 (Flk-1).2,9,10 However, the precise profiles of genes induced by EPAS1 and the functions of EPAS1 in vascular endothelial cells have not been elucidated.
VEGF is the most important angiogenic factor, and its function is essential in embryonic vasculogenesis as well as angiogenesis in the adult.11,12 However, VEGF alone is not sufficient to assemble physiologically functional vasculature. Results obtained from therapeutic angiogenesis studies using VEGF showed that new vessels induced by VEGF are leaky13 and do not persist for long.14 Thus, in addition to VEGF, some other molecules are expected to contribute to production of functional and lasting mature vessels. To elucidate angiogenesis molecular mechanisms in terms of vessel maturation, it may be beneficial to clarify the target genes of EPAS1 besides VEGF.
In this study, we used microarray technology to identify the genes that were upregulated by EPAS1 in vascular endothelial cells. A total of 130 genes were upregulated by EPAS1, including VEGF receptor 1 (VEGF-R1 or fms-like tyrosine kinase-1 [Flt-1]), implying that it is regulated by EPAS1. We demonstrate that the heterodimer of EPAS1 and ARNT binds to the HIF-1-binding site upstream of Flt-1 promoter and transactivates the promoter activity. Moreover, using small interfering RNA (siRNA) against EPAS1 or HIF-1α, we found that EPAS1 is essential in hypoxic regulation of Flt-1 mRNA expression. Furthermore, EPAS1 gene delivery to wound tissue enhanced the expression of VEGF, Flt-1, Flk-1, and Tie2 in vivo, resulting in promotion of angiogenesis and acceleration of the wound healing process. We show that EPAS1 enhances not only VEGF expression but also the concomitant expression of its 2 receptors, Flk-1 and Flt-1, as well as Tie2. EPAS1 may promote angiogenesis and contribute to mature vessel establishment by activating VEGF gene expression and also affecting coexpression of related endothelial-specific receptors involved in the process.
Materials and Methods
Cell Culture
Human umbilical vein endothelial cells (HUVECs) were obtained from BioWhittaker (Walkersville, Md) and were maintained in endothelial growth medium 2 (BioWhittaker). Bovine aortic endothelial cells (BAECs) were primary cells cultured in DMEM (Sigma) containing 10% FBS. To mimic hypoxic conditions, cells were treated with 130 μmol/L desferrioxamine (Sigma) for 16 hours.
Construction of Plasmids and Recombinant Adenovirus
The pGL2-Basic plasmid was purchased from Promega. To generate the Flt-1 reporter plasmid (pGL2hFlt-1), the promoter region of the Flt-1 gene from position −1160 to +305 was amplified by polymerase chain reaction (PCR) and subcloned into pGL2-Basic plasmid. Successive deletion of the 5′-flanking region was performed by PCR. PCR products were inserted between SacI and BglII restriction sites of pGL2-Basic promoter to yield pGL2hFlt-1 (−886 to +305) and pGL2hFlt-1 (−334 to +305). A variation of the pGL2hFlt-1 plasmid pGL2hFlt-1mut was constructed; this was identical to pGL2hFlt-1 except that the HIF-1-binding site (bp −965 to −958) was mutated from AACGTGGA to AAAAAGGA. The EPAS1 expression vector phEP-1 was a gift from Steven L. McKnight (University of Texas Southwestern Medical Center, Dallas).2 The plasmid phARNT was constructed as described previously.5 For mouse VEGF164 cDNA isolation, PCR was performed using total RNA from mouse heart. The primers used were 5′-ATGAACTTTCTGCTGTCTTGG and 5′-ACCGCCTCGGCTTGTCTCATC. Recombinant adenoviruses were prepared using Ad-Easy system as described previously,5 and those expressing β-galactosidase (βGAL), EPAS1 (1-870), or mouse VEGF164 were designated AdCMV.βGAL, AdCMV.EPAS1-(1-870), or AdCMV.mVEGF164, respectively.
Microarray Analysis
Microarray analysis was performed using ≈12 000 human cDNA (Agilent Technologies; Palo Alto, Calif) as described previously.15,16 In the first instance, HUVECs were infected with AdCMV.EPAS1-(1-870) or AdCMV.βGAL. Forty-eight hours after infection, total RNA was isolated from cell cultures and applied to microarray analysis. A threshold of a 2-fold change in expression was used for comparison of AdCMV.EPAS1-(1-870) and AdCMV.βGAL-infected HUVECs.
Northern Blot Analysis
Total RNA was isolated from HUVECs using RNeasy method (Qiagen). Northern blot analysis was performed as described.5 To correct for differences in RNA loading, the membranes were rehybridized with a radiolabeled 18S oligonucleotide.
Western Blot Analysis of EPAS1 Protein Expression
Whole-cell protein extracts or nuclear extracts were prepared from HUVECs. A total of 20 μg of protein per sample was evaluated by Western blot analysis performed as described.17 EPAS1 was detected using polyclonal anti-hHIF-2α antibody (Novus; 1:1000) and as a secondary antibody, anti-rabbit horseradish peroxidase-conjugated IgG (Cell Signaling; 1:2000) followed by enhanced chemiluminescence plus detection (Amersham).
Transient Transfection Assay
BAECs were cultured in 12-well plates, and transient transfection was performed with 0.6 μg of total DNA using Lipofectamine reagent (Invitrogen) as described previously.18 To correct for variation in transfection efficiency, we cotransfected 0.1 μg of pCMV-βgal in all experiments. Data for each construct are presented as the mean±SEM.
In Vitro Transcription and Translation/Gel Mobility Shift Assay
In vitro transcription and translation of EPAS1 and ARNT and gel mobility shift assays were performed as described.18 The sequence of the double-stranded oligonucleotide containing the HIF-1-binding site (5′-CATAATTGAGGAACAACGTGGAATTAGTGTCATCG-3′) was derived from the sequence of the Flt-1 promoter corresponding to positions −979 to −945. For supershift analysis, 1 μg of anti-myc antibody (Invitrogen) was preincubated with in vitro-translated proteins for 1 hour at room temperature before binding reaction.
siRNA Duplexes and Transfection
The siRNA oligonucleotides were designed after recommendations of Elbashir et al19 and were synthesized using Silencer siRNA Construction Kit (Ambion). The EPAS1 siRNA duplex targeted nucleotides 830 to 848 of EPAS1 mRNA sequence (U81984) and comprised sense 5′-ACCAAUCCAGCACCCAUCCdTdT-3′ and antisense 5′-GGAUGGGUGCUGGAUUGGUdTdT-3′. The HIF-1α siRNA duplex targeted nucleotide 1521 to 1539 of HIF-1α mRNA sequence (NM001530) and comprised sense 5′-CUGAUGACCAGCAACUUGAdTdT-3′ and antisense 5′- UCAAGUUGCUGGUCAUCAG dTdT-3′ as described previously.20 The scramble EPAS1 control duplex did not target any gene and comprised sense 5′-UAGGUAGAGAGAGUGGCGCdTdT-3′ and antisense 5′-GCGCCACUCUCUCUACCUAdTdT-3′. Duplexes were prepared according to the instructions of the manufacturer. HUVECs were plated onto 10-cm cell culture dishes and grown to 70% confluence before transfection. A total of 10 μL of Lipofectamine 2000 reagent (Invitrogen) and 200 pmol siRNA duplexes were added. Cells were incubated for an additional 24 hours before harvest.
Migration Assay
Cell migration assays were performed with the Transwell (Corning Costar Japan) system as described.21,22 Briefly, HUVECs were infected with adenovirus and added to the upper chamber of the Transwell. The lower chamber was filled with serum-free Eagle basal medium containing 50 ng/mL of VEGF. After 5 hours, the cells present beneath the membrane were fixed with methanol and stained with Giemsa solution.22 Cells were counted in 3 random fields per well under low magnification using a microscope. Analysis was performed on 3 wells for each condition, and each experiment was repeated 3×.
Wounding, Adenovirus Infection, and Wound Tissue Preparation
All animal procedures in these studies were designed in accordance with the Guide for Animal Experimentation (Faculty of Medicine, University of Tokyo). Five-week-old male BALB/c mice (Charles River Japan; Yokohama, Japan) were used in the experiment (n=18). Mice were anesthetized by intraperitoneal administration of 0.15 mL of 3.6% chloral hydrate. Four full-thickness wounds were made on the back of mice.23 The wounds were infected with recombinant adenovirus (5×108 plaque-forming units) expressing βGAL, human EPAS1-(1-870), or mouse VEGF164. Infection was performed by 4 direct subcutaneous injections around the wound using a 27-gauge needle. The progress of wound closure was analyzed daily as described.24 Healing was expressed as the percent decrease in wound area relative to the original wound area. Six days after wounding, the mice were euthanized with carbon dioxide inhalation, and the wound tissues were collected.
Quantitative Real-Time RT-PCR
Quantitative RT-PCR was performed as described previously.25 Primers and probes were obtained as assay on demand and used according to the protocol of the manufacturer (Applied Biosystems; Foster City, Calif). The level of transcripts for the constitutive housekeeping gene product GAPDH was measured quantitatively in each sample to control for sample-to-sample differences in RNA concentration.
Immunohistochemistry and Analysis of Capillary Density
Immunohistochemical analysis of mouse tissue was performed as described previously.26 To identify endothelial cells, we used rat monoclonal antibody directed against mouse CD31 (BD Biosciences; Palo Alto, Calif). In addition, mouse monoclonal antibody against smooth muscle (SM) α-actin (Sigma) was used to identify mural cells. Capillary densities, identified by positive staining for CD31 and appropriate morphology, were counted by a single observer blinded to the treatment regimen (mean number of capillaries per low-power field).
Statistical Analysis
Results were expressed as mean±SE. Two-tailed Student t test for unpaired samples or one-way ANOVA was used for parameter comparison. A value of P<0.05 was considered significant.
Microarray Analysis
To identify the downstream target genes of EPAS1, we first generated adenoviral constructs AdCMV.EPAS1-(1-870) and AdCMV.βGAL. We infected AdCMV.EPAS1-(1-870) or AdCMV.βGAL into HUVECs and isolated RNA after 48 hours. Differential hybridization was performed on microarray slides containing ≈12 000 spots of human cDNA using labeled RNA from AdCMV.βGAL-infected HUVECs as a reference. A total of 159 genes were differentially expressed between the reference cells and EPAS1 overexpressing cells. Among them, 130 genes were upregulated, and 29 genes were downregulated (see online data supplement available at http://circres.ahajournals.org). More than half of the genes were confirmed to be upregulated by Northern blot analysis or RT-PCR. These included 13 secreted proteins, 9 membrane receptors, and 24 transcription factors. Representative genes upregulated by EPAS1 are listed in the Table . In this study, we further examined the molecular mechanisms by which Flt-1 (or VEGF-R1) is upregulated by EPAS1. Analysis of other genes will be described (N.T., manuscript in preparation). Northern blot analysis using RNA from infected HUVECs demonstrated that EPAS1 overexpression resulted in a dose-dependent increase in Flt-1 mRNA abundance (Figure 1). This induction was specific because infection by the same titer of control adenovirus AdCMV.βGAL did not increase the Flt-1 mRNA levels, therefore, Flt-1 mRNA was upregulated by EPAS1 in vascular endothelial cells.
EPAS1 Transactivates the Flt-1 Promoter
To further elucidate the mechanism by which EPAS1 increases the Flt-1 mRNA levels, we first generated a reporter plasmid pGL2hFlt-1 containing a 1.6-kb fragment of the human Flt-1 promoter placed upstream of the luciferase reporter gene. This reporter plasmid construct was then cotransfected into BAECs together with the human EPAS1 expression plasmid phEP-1 and the human ARNT expression plasmid phARNT. EPAS1 and ARNT increased Flt-1 promoter activity by 2.5-fold (Figure 2A). Deletion of bp −1160 to bp −886 of the Flt-1 promoter markedly diminished this induction by EPAS and ARNT (Figure 2B). We examined this EPAS1-responsive element of the Flt-1 promoter from bp −1160 to −886 in detail and found an HIF-1-binding consensus sequence in −965 region. To determine whether EPAS1 transactivates the Flt-1 promoter by binding to this HIF-1-binding site, we made a mutant Flt-1 promoter with a 3-bp mutation at −969 to −965 (ACGTG to AAAAG). A reporter plasmid harboring this mutated site (pGL2hFlt-1mut) was again transfected into BAECs in the presence or absence of EPAS1 and ARNT plasmid constructs. Mutation of the HIF-1-binding site abolished the transactivation of Flt-1 promoter by EPAS1 (Figure 2C).
EPAS1-ARNT Heterodimer Binds to the HIF-1-Binding Site of Flt-1 Promoter
To examine whether the heterodimer of EPAS1 and ARNT binds directly to this HIF-1-binding site, we performed gel mobility shift assays with in vitro-translated EPAS1 and ARNT proteins and a labeled probe containing the HIF-1-binding site. In the presence of EPAS1 and ARNT proteins, DNA-binding activity was detected (Figure 3A); however, 3-bp mutation at HIF-1 site of this probe abolished protein binding (Figure 3B). The binding was attenuated with unlabeled specific competitor containing HIF-1-binding site but not with nonspecific competitor containing the specificity protein 1 element. The in vitro-translated EPAS1 protein was myc tagged. Thus, we performed supershift analysis using anti-myc antibody. Addition of 1 μg of anti-myc antibody in this reaction mixture interfered the binding (Figure 3A).
Knockdown of EPAS1 but not HIF-1α Attenuates Desferrioxamine-Induced Flt-1 mRNA Expression
To elucidate whether EPAS1 or HIF-1α is involved in hypoxic induction of Flt-1 gene expression, we generated siRNA against EPAS1 and HIF-1α. Desferrioxamine, which chemically mimics hypoxic conditions, markedly increased Flt-1 mRNA abundance in HUVECs transfected by scramble siRNA as a control. Knockdown of HIF-1α by siRNA did not affect Flt-1 mRNA induction by desferrioxamine. On the other hand, knockdown of EPAS1 markedly attenuated Flt-1 gene expression enhancement (Figure 4A and 4B).
EPAS1 Enhances Chemotactic Response Toward VEGF
To evaluate the role of EPAS1 on the migration activity of vascular endothelial cells, Boyden chamber analysis was performed using HUVECs. When HUVECs were infected with AdCMV.EPAS1-(1-870; 10 multiplicities of infection [mois]), their chemotactic activities toward VEGF were significantly increased compared with those treated with AdCMV.βGAL (Figure 5).
EPAS1 Gene Delivery Promotes Angiogenesis at Wound Site
To examine whether EPAS1 promotes angiogenesis in vivo, we analyzed the effect of EPAS1 overexpression in the healing process of skin wound, which is commonly used as a model of angiogenesis. First, full-thickness wounds were created in the back skin of mice and then the wound site was treated with AdCMV.EPAS1-(1-870), AdCMV.βGAL, or saline as a control. In mice treated with AdCMV.EPAS1-(1-870), the healing process was significantly accelerated compared with mice treated with AdCMV.βGAL or saline (Figure 6A). In mice treated with AdCMV.EPAS1-(1-870), the wounds closed 2 to 3 days faster relative to AdCMV.βGAL- or saline-treated mice (Figure 6B). Tissue sections from the wound sites were examined histologically. There were significantly increased numbers of newly formed platelet-endothelial cell adhesion molecule-1 (PECAM-1)-positive vessels (Figure 6C) and increased capillary density (Figure 6D) in AdCMV.EPAS1-(1-870)-treated mice 7 days after the injury, compared with AdCMV.βGAL or saline (P<0.05). Moreover, the expression of VEGF, Flt-1, Flk-1, and Tie2 were all upregulated at the wound site in mice treated with AdCMV.EPAS1-(1-870) compared with controls (Figure 6E).
EPAS1 Gene Delivery Enhances Formation of New Vessels With SM α-Actin-Positive Mural Cells
To compare the effect of EPAS1 and VEGF in angiogenesis, we generated adenovirus-expressing mouse VEGF164 (AdCMV.mVEGF164). Delivery of mVEGF164 gene markedly enhanced capillary density at the wound site. However, newly formed vessels induced by mVEGF164 have fewer SM α-actin-positive mural cells compared with those induced by EPAS1 (Figure 7).
In this study, we examined the role of EPAS1 in vascular endothelial cells by analyzing the downstream genes of EPAS1 using microarray technique. A total of 130 genes were upregulated, notably including that of Flt-1. We also showed that the Flt-1 promoter is activated by EPAS1, and the induction is dependent on its HIF-1-binding site. In addition, EPAS1 enhanced the migratory response of HUVECs induced by VEGF. Furthermore, EPAS1 gene delivery promoted the angiogenic process in vivo by enhancing the concomitant expression of VEGF, Flt-1, Flk-1, and Tie2.
EPAS1 has high homology to HIF-1α, and their functions are increased during hypoxia. Furthermore, both transactivate the VEGF promoter.4,5 However, in contrast to HIF-1α, EPAS1 is expressed mainly in vascular endothelial cells and transactivates promoters of endothelial-specific genes Flk-1 and Tie2.2,9,10
EPAS1 and Flt-1 have very similar characteristics; that is, they are expressed mainly in vascular endothelial cells, and their expression is upregulated during hypoxia.2,27,28 On the basis of microarray analysis results, we hypothesized that EPAS1 directly upregulated the expression of Flt-1. Flt-1 promoter activity is known to be activated during hypoxia, and the hypoxia-responsive element within the Flt-1 promoter has also been identified.29 However, the precise molecular mechanisms of transactivation of Flt-1 remained unclear. Moreover, it has not been elucidated whether HIF-1α, EPAS1, or both are involved in hypoxic induction of Flt-1 gene expression. In this study, we demonstrated that the heterodimer of EPAS1 and ARNT binds directly to the HIF-1-binding sequences ACGTG within the hypoxia-responsive element and transactivates the Flt-1 promoter. In addition, we discovered that EPAS1 but not HIF-1α is important in hypoxic induction of Flt-1 gene expression by using siRNA for the first time. Therefore, these results are of utmost importance on the basis of our assertion that almost no gene had been identified as an EPAS1-specific target gene so far. Moreover, we have shown the novel means to discriminate the roles of EPAS1 and HIF-1α. Collectively, the results of this study suggest that EPAS1 upregulates not only VEGF but also endothelial-specific receptors Flt-1, Flk-1, and Tie2, all of which are essential in the angiogenesis process.
In this study, delivery of EPAS1 gene to wound tissue upregulates the expressions of VEGF, Flt-1, Flk-1, and Tie2 at the wound site, which results in promotion of mature angiogenesis. The roles of Flk-1 and VEGF in angiogenesis have been well established. In contrast, in angiogenesis, the functional role played by Flt-1 is still unclear. For instance, Flt-1 was reported to negatively regulate angiogenesis through sequestering VEGF and diminishing the Flk-1-mediated angiogenic stimulus.28,30 However, recent reports have shown that at least in pathological conditions such as tumor angiogenesis, inhibition of Flt-1 by antagonists impairs pathological angiogenesis;31 thus, in such situations, Flt-1 indeed promotes angiogenesis.32 More recently, Flt-1 stimulation by placental growth factor (PlGF) was reported to transphosphorylate Flk-1 and amplify Flk-1 activation by VEGF.33 Moreover, heterodimers of Flk-1/Flt-1 and VEGF/PlGF were reported to exist and promote angiogenesis. Thus, the coordinated function of Flt-1 and Flk-1 is likely to be important in the angiogenesis process.
For therapeutic angiogenesis, there are clinical studies under way using VEGF gene or protein. VEGF overexpression indeed promotes angiogenesis; however, the newly formed vessels induced by VEGF were leaky13 and tended to regress soon after reduction of VEGF concentration.14 In this study, most vessels induced by VEGF gene lack SM α-actin-positive mural cells. To generate functionally persistent vessels, the processes of arterialization and recruitment of pericytes are considered important. In the face of hypoxia, each organ induces formation of new vessels to persist and increase delivery of oxygen and nutrients. Thus under hypoxia, there may be some genes other than VEGF that are induced and contribute to the maturation of vessels.
In our wound-healing models, the newly formed vessels induced by EPAS1 were surrounded by SM α-actin-positive mural cells, therefore, they should be considered more maturated vessels. Thus, the angiogenic process modulated by EPAS1 appears to be similar to that observed in physiological angiogenesis during hypoxic conditions. The spatiotemporal expressions of some ligands and receptors have been reported to be involved in assembling mature vessels.34 Particularly, angiopoietin-1 stabilizes the vessel through Tie2 receptors,35,36 and in combination with VEGF, it contributes to form mature vessels.37 PlGF also promotes angiogenesis through Flt-1-mediated signals.32 In this model, EPAS1 delivery upregulated both Tie2 and Flt-1 gene expression. Thus, EPAS1 may promote well-organized vascular formation by coordinately regulating expression not only of VEGF but also endothelial-specific receptors, those that play essential roles in the process.
In conclusion, EPAS1 coordinately enhanced expression of VEGF and endothelial-specific receptors Flt-1, Flk-1, and Tie2 and promoted mature angiogenesis. Therefore, studies on EPAS1 functions would provide us with further insights into the complex regulatory network of vessel maturation.
This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture, Japan, and health science research grants from the Ministry of Health, Labor and Welfare, Japan. We are grateful to Chie Fujinami for technical assistance.
↵*These authors contributed equally to this work.
Original received November 6, 2003; revision received May 20, 2004; accepted May 27, 2004.
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- Endothelial PAS Domain Protein 1 Gene Promotes Angiogenesis Through the Transactivation of Both Vascular Endothelial Growth Factor and Its Receptor, Flt-1Norihiko Takeda, Koji Maemura, Yasushi Imai, Tomohiro Harada, Daiji Kawanami, Takefumi Nojiri, Ichiro Manabe and Ryozo NagaiCirculation Research. 2004;95:146-153, originally published July 22, 2004https://doi.org/10.1161/01.RES.0000134920.10128.b4
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- Endothelial PAS Domain Protein 1 Gene Promotes Angiogenesis Through the Transactivation of Both Vascular Endothelial Growth Factor and Its Receptor, Flt-1Norihiko Takeda, Koji Maemura, Yasushi Imai, Tomohiro Harada, Daiji Kawanami, Takefumi Nojiri, Ichiro Manabe and Ryozo NagaiCirculation Research. 2004;95:146-153, originally published July 22, 2004https://doi.org/10.1161/01.RES.0000134920.10128.b4Permalink: