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subset: commoncrawl


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By Kevin Johnson, USA TODAY
When search dogs found George Zinkhan's partially buried body Saturday in a Georgia forest, the grim discovery solved only part of the mystery surrounding the marketing professor who killed his wife and two men outside a community theater.
"I never saw a problem. There was nothing ever really out of the ordinary," said Bob
In that encounter, Zinkhan, 57, hurriedly asked Covington to watch his two children — ages 10 and 8 — so that he could deal with an emergency. Covington didn't know it, but Zinkhan had just opened fire at an April 25 picnic for members of the Town & Gown Players, a volunteer theater group.
His wife, Marie Bruce, 47, was killed along with theater group members Tom Tanner, 40, and Ben Teague, 63. Bruce was the group's president.
Covington said that he never saw any serious signs of trouble in his neighbor. "I think that's what is most troubling for everybody now," he said.
Athens-Clarke County Coroner Sonny Wilson said Sunday that Zinkhan's body was found in a shallow "depression" in woods where authorities believe he committed suicide. Two guns were found nearby.
"I've heard of people not wanting to be found," Wilson said. "It appears that's what he tried to do. He camouflaged the location."
Authorities launched a nationwide search for Zinkhan after the shooting. The killings were about a mile from the University of Georgia campus.
Covington's impression of his neighbor is that he was a man of few words, whose wife was his opposite — but "they always seemed to get along."
Contributing: The Associated Press
Guidelines: You share in the USA TODAY community, so please keep your comments smart and civil. Don't attack other readers personally, and keep your language decent. Use the "Report Abuse" button to make a difference. Read more.


text: This city hotel opened its doors to the public in April 2006, and is one of the newest hotels in the centre of the city of Sofia. It offers a unique combination of high-quality of service with all the comforts that guests could expect from a hotel of its calibre. It has 40 double rooms, 3 single rooms, 2 apartments and 4 house apartments. It is air-conditioned and has a lobby with 24-hour reception and a safe. Facilities include a restaurant, a lobby bar, a modern conference hall and secure parking. The conference hall is the ideal place for holding conferences, seminars, training days and presentations for up to 50 people. Room and laundry services are also available.The hotel has three single rooms with luxurious furniture that impress with their comfort. The hotel has forty double rooms with varying interior design, luxuriously furnished and with individual style. Each comes with a hairdryer, double bed, minibar, TV with satellite channels, telephone, Internet access, individually adjustable air conditioning and heating, wake-up service, as well as an en suite bathroom with bath tub. There are two luxuriously furnished apartments offering amazing views out onto the Vitosha mountain and the centre of Sofia.


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Gifted a Baby
Young and inexperienced, Sarah is suddenly thrust into motherhood without ever having had a boyfriend. When a baby is unexpectedly left in her care, she can't deny that it may be an act of God. But when she meets Nathan, she suspects something other than divine intervention may be at work.
Nate has lost more than any man deserves to lose, and he has no interest in trying to replace what he once had. But that doesn't stop him from taking a liking to Sarah, wanting to know more about her – and having feelings for her. But he can't bring himself to tell her the painful truth about his past. Little does he know that the shocking truth about Sarah has the potential to change his life forever.
Is God trying to bring them together, or are they both victims of a cruel prank?


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The ballots for the John R. Wooden Award were due in Monday
I emailed my picks (each of the 1,000 or so casting votes is asked select 10 players, in order of preference, from the 23 choices listed on the ballot) Monday morning, albeit with a tad bit of trepidation.
But who couldnt feel a lot more definitive about making their choices especially No. 1 if the deadline were, say, noon April 5? A certain game played the night before, as well as a couple more also contested on the same Edward Jones Dome floor in St. Louis on Saturday night, would impact balloting considerably.
The red tape involved in making travel arrangements for the
players and coaches involved, award administrators point out, make the March 28 deadline
imperative. The top five in the balloting, for both the mens and womens award,
are flown into Los Angeles for the nationally televised presentation on April 9.
OK. I can buy that.
So, here are my choices, listed in reverse order . . . well, Ill list my just missed my Top 10 choices first:
Just Missed the Cut: Ike Diogu (Arizona State, junior, center), Joey Graham (Oklahoma State, senior, forward) and Shelden Williams (Duke, junior, center).
Comment: Diogu was the Pac-10 Player of the Year and is a three-time all-conference choice. Hes the easy winner of the Best player not to play in the NCAA Tournament this season award. Graham, who will be chosen no later than the middle of the first round of the NBA draft, wasnt quite as consistently outstanding as the guys who made my top 10. No one that I saw played any harder than Williams did this season and, quite frankly, Im already second-guessing my decision to leave him off the ballot. Yikes! Too late now . . .
No. 10: Hakim Warrick (Syracuse, senior, forward).
Comment: The Big East Player of the Year and he also played well in leading the Orange to the conference tournament title. We cut him some slack for those 10 turnovers in the first-round NCAA loss to Vermont.
No. 9: Dee Brown (Illinois, junior, guard).
Comment: I know, I know . . . he was the Big Ten Player of the Year, but Ive got a couple of his teammates higher up on my ballot. I give him the edge over Nate Robinson (Washington) as the best undersized shooting guard in the country and my vote as the best sub-six-footer this season.
No. 8: Wayne Simien (Kansas, senior, forward).
Comment: How much deeper into the tournament would the Jayhawks have traveled if the Big 12 Player of the Year had hit that turnaround jumper at the buzzer in the first round against Bucknell? Oh, well . . .
No. 7: Chris Paul (Wake Forest, sophomore, point guard).
Comment: Pound for pound, inch for inch, blah, blah, blah . . . he is the most innately gifted player in college today.
No. 6: J.J. Redick (Duke, junior, shooting guard).
Comment: The ACC Player of the Years jump shot ran away from home once the NCAA Tournament started him or we might have had a Blue Devils vs. Tar Heels semifinal in St. Louis after all.
No. 5: Salim Stoudamire (Arizona, senior, shooting guard).
Comment: The most dependable player in the clutch this season and also one of the most difficult to check, 1-on-1.
No. 4: Deron Williams (Illinois, junior, point guard).
Comment: Hes a living testament to why you shouldnt judge a player by his statistics alone. There wasnt a better playmaker/floor leader anywhere this season.
No. 3: Luther Head (Illinois, senior, wing).
Comment: If the Illini are cutting down nets in the Edward Jones Dome in St. Louis next Monday night, everyone wearing Orange should recall his block of a Salim Stoudamire jump shot late in regulation against Arizona and say, in unison, Thank you, Luther!
No. 2: Sean May (North Carolina, junior, center).
Comment: The guy who has the best opportunity Saturday, and maybe Monday, in St. Louis to make me want to re-cast my ballot. Im still trying to recall the six shots he missed against Wisconsin Sunday. All 19 he took seemed like dunks or layups to me.
No. 1: Andrew Bogut (Utah, sophomore, center).
Comment: Despite relatively impressive numbers (20 points and 12 rebounds), he didnt do nearly enough to help his under-manned team knock off Kentucky in their Sweet 16 game in Austin. He missed way too many close-in shots and was terrible (four of 11) at the free-throw line. That being said, no one player in college basketball this season drew as much defensive attention or was as responsible for his teams success. Take May, Head, Williams or Stoudamire off their teams, and theyre still in the NCAA Tournament. With Bogut in the lineup, the Utes won the Mountain West Conference, finished 29-6 and beat UTEP and Oklahoma in the tourney. Remove him from the equation and Utah would have finished in the second division in its conference.
Recently elected to the USBWA Hall of Fame, Frank Burlison is Scout.com's National Basketball Expert and also covers college basketball for the Long Beach (Calif.) Press-Telegram. He can be reached at email@example.com. Read more of Burlison's pieces at FrankHoops.com


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It would be nice if <unsubscribe>blah</unsubscribe> allowed you to generate <area /> link for an image map instead of the <a> tag.
You can use the [unsubscribe] form to output just the URL, which should help you.
Given the somewhat patchy support for image maps by email clients, I don't think this can be called best practice.. Plain text links are always best for unsubscribe links.
Very true Stig, it's not a great idea.
Thanks a lot! I thought an image map was a decent remedy for CSS inconsistencies, but I will rethink it now and will probably have to budget more hours for each job.


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Western WA Clean Cities Annual Meeting and Awards Ceremony
Western Washington Clean Cities
Thursday, December 5, 2013 from 4:30 PM to 7:00 PM (PST)
Who's Going
San Francisco, California
London, United Kingdom
Florence, Italy
Event Details
Agenda now available!
Join us for another fun reception and annual awards ceremony! We are celebrating "The End of an ARRA" with the close of our $15 Million US DOE ARRA grant, and will showcase the amazing petroleum reduction achievements of several local fleets. And of course we'll have our Evergreen Fleet certification awards. Will more than one fleet achieve 5-star certification this year? Join us to find out!
Reception with wine and beer begins at 4:30pm (Presentations and awards between 5:00 to 6:00 pm) at the Museum of Flight on Thursday December 5th.
Your reception registration also gives you access to the Museum. If you would like to visit the museum prior to the event, you can plan to be there a few hours before the event starts. We will provide more information when you register.
When & Where
Museum of Flight
9404 East Marginal Way S
Thursday, December 5, 2013 from 4:30 PM to 7:00 PM (PST)
Add to my calendar
Western Washington Clean Cities
Western Washington Clean Cities is a not-for-profit organization dedicated to expanding the use of alternative fuels and advanced vehicle technologies. We are one of nearly 100 Clean Cities coalitions across the country advancing the nation's economic, environmental and energy security by supporting local actions to reduce petroleum consumption in transportation.


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Current odds at the online sportsbook and casino have both teams at -105 to come out on top, with both sides in a good run of form. The Cincinnati Reds will arrive fresh from a 14-0 victory over Houston on Tuesday, which moved them to within one game of NL Central leaders St. Louis Cardinals. The Giants on the other hand have won seven of their last nine games but trail the Reds by five games in the NL wild-card race.
Hollywood Sportsbook Media Relations executive, Alejandro Gomez, declared, "The San Francisco Giants still think they've got a crack at the postseason, but there's still a lot of baseball to be played. Cincinnati are probably the slight favourites for tonight's showdown as they are a very resilient team,' he said.


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login with
The Best Moment of the day Winner!
You deserve a peaceful night, Celeste! Sleep well! xxo
mommy should sleep))
About Celeste Whitehead
Current: Spring Hill, Florida
Birth: August 26
On Moms.com since: Aug 5, 2013
Stay at home and work at home mom of two loud, crazy, adorable kids that are the center of my entire world. We all live together with the love of my life and the father of my children in sunny Florida with 3 of out four-legged kids.


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Good news!
So guess who is coming home??? Thats right my husband is coming home soon I am ecstatic words cant describe how I feel at this point :)
So me and him definitely need to talk about pregnancy preventing cuz at this point I am not ready for anymore kids! Lmao he thinks differently he is like "whatever happens happens" Lol what is some birth control you ladies recommend?? I really dont want anymore kids till my two oldest are in school.
that's so exiting that your husband is coming home. I think you need to talk to your GYN doctor to see what's best for you. I personally like birth control pills
I'm happy for you, at least with your husband home you might get some rest lol, no idea what's best for birth control besides condoms and spermicidal lube and talk to your doctor about the different kinds, I don't use birth control.
I take the shot. I go get a shot in the arm and every 12 weeks I get another one. My doctor said it's not recommended for women 18-21 because it can cause osteoporosis. My doctor highly recommends an IUD such as Mirena but I do not feel comfortable with the idea of a tampon let alone something that can stay inside me for up to 5 years lol. There are also pills and patches and these little sticks that go into your arm, not sure what they are called, but it's whatever you feel most comfortable with. Me and my husband don't use condoms so after my son was born we waited until I was on bc to continue to have intercourse. It might be a good idea to talk to your husband about your stresses and worries about having one more right now, maybe then you can both come to some sort of agreement. I told my husband no more kids right now and birth control was the only alternative to getting my tube ties lol. You know your husband best, how you choose to explain it to him is all up to you. Good luck mommie!!
How awesome! I am so glad he is coming home to spend some time with you all. My hubby was still in the guard while we were engaged and it was not easy.
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About Abigail Kale
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On Moms.com since: Jan 20, 2015
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This is a guest post by Bryan Hollis, owner of Mid-Carolina Freelance, LLC, the umbrella company that owns Affiliate Manager Pro, Linux Hosting Only which hosts GrowMap.com and Sandlapper Web Design. Bryan also owns the blogging community BlogInteract.
Fear. There is no doubt that this – in it's simplest form – is what Google is instilling in millions of webmasters, bloggers, SEO Agents, and online marketers in general that are in a constant battle to please the search engine giant.
It's no secret that the giant snowball started roughly 2 years ago when the first Panda release to combat various forms of spam and duplicate content was released. At the time, this was taken fairly well by those that followed "the rules" – didn't comment spam, link farm, or submit spun articles all across the web. Then things got worse – much worse.
I have been providing SEO services for some time now and pleasing Google was always goal number one. The "lesser" search engines had a degree of importance, but satisfying Google was certainly the number one priority.
As the title includes the word "fallacies", the list is rather long in just how Google is increasingly becoming the inferior search engine on the web. I'll try not to go all over the map in this post. The fact is, it could really be broken into possibly three posts, depending on just how much you can take at one time.
I'll start with linking and the fear involved – the "false" fear
over Google that has recently had site owners consumed.
Over the course of many years, my company Mid Carolina Freelance, LLC developed well over two dozen sites or so. These sites – for the most part – are still going today and still serving the purpose for which they were established. Each of these sites were formed for a specific purpose. Some were article marketing sites, some blogs.
Their core purpose was to provide a place that my company could post original content on behalf of our clients. Other writers were welcome. We certainly didn't want lop-sided sites that real readers questioned. They needed to have standards of course. All this said, these websites have worked hard and have all served their purpose. They still do today.
Recently I have been inundated with email after email asking me to remove this link here and that link there. Some are almost a year old. At first I asked, even though their email explicitly detailed the reason. And at first I asked (in the most respectful of ways) if they were kidding.
"No, please remove them ASAP. We are trying to avoid
Google penalties." I laughed in silence.
Here these webmasters and SEO Agents have spent hundreds – if not thousands – of dollars building links through articles or whatever and now they want to spend the same or more reversing their work. Insanity is what I call it. What I wonder – with all due respect – is whether or not they are testing Google. Are you reading a post like this and reacting? Is it something you heard through the grapevine? Whatever instigated it, it is like a false scare from the CDC or some international epidemic.
Let's look at Google's blunders over the last six or so months – then decide for yourself if it's time to push back. Google is becoming infamous for giving you the results they want you to see, but the truth is like a JFK conspiracy theory.
It is no real secret that Google suffered a pretty significant loss in the third quarter of 2012. However, if you Google "Google 3rd quarter earnings report 2012?, the first search result is – you guessed it – Google's own investor link. Here is a quote regarding the report:
MOUNTAIN VIEW, Calif. – October 18, 2012 – Google Inc. (NASDAQ: GOOG) today announced financial results for the quarter ended September 30, 2012.
"We had a strong quarter. Revenue was up 45 percent year-on-year, and, at just fourteen years old, we cleared our first $14 billion revenue quarter," said Larry Page, CEO of Google. "I am also really excited about the progress we're making creating a beautifully simple, intuitive Google experience across all devices."
And directly below their search are entirely different, yet very consistent results. CNN Money reported an 8% drop in stock prices and what they called the 3rd quarter earnings "grim". Forbes' headline chose to use Google's "Big Miss on Profit". Each of the latter were inline with an 8% drop. Why the discrepancy? Moving right along.
Before I go howling at the moon or rambling, I do my due diligence first. Has been my historical approach to SEO, if I am "testing" I always use my own sites first – never a client's. About four months ago, I launched my new web design and SEO site to consolidate.
After the site indexed in its entirety, it was on page one for several of the keywords I had chosen. I have come to the conclusion that focusing on your local market is wiser than the whole world. Even good ol" fashioned word-of-mouth gets me more work than a page one position.
With my new Google page one serp, I decided to play with some old methods on purpose, without concern or care about Google's reaction. Hard and fast link building, blog posts linking in, you name it.
Three days later, I wasn't even ten pages deep. Gone.
I visited Yahoo!, Bing, and AOL.com. Position held with these guys.
What did I do? Nothing. I left it alone.
One week…two weeks…ahh, there we are. I was back. Another week. Page 6. One more week, pages 2 – 4. Hmmm….
So let's talk about algorithm problems. Google has everyone jumping in fear, yet their own algorithms are simply horrendous. Skewed. Out-of-whack. They want to prevent spam, yet they spam their own site. What content do they spam the most? Their own. The best example I can give is YouTube (Google's video search engine).
So, here we go. I do a keyword search for my web design site. "Web Design Columbia SC". Page one, position two is an ancient YouTube video. No problem. It's still relevant. The web design firm still exists. That's cool. On to page two. Position one – same EXACT YouTube link, excerpt, etc.
Duplicate content on their own website. Shame Google. Hypocrisy.
I realize now that there really should be a two part series to this post, so I am going to shift gears to make my initial point – pushing back. We all know the old saying about voting for your candidate. Some refuse with the idea that a single vote doesn't matter. It does matter. If millions of people can choose the candidate they feel should run their country, then the same can be done with search engines.
Know in advance that I have no allegiance to Bill Gates or Microsoft, but it just so happens that Bing is steadily becoming a stronger search engine with each passing day. Do they have work to do? Yes. Do they need support from you? Yes. Until I write part 2, I encourage you to read a post about how Bing is starting to outpace Google.
It's time to push back at Google. The only way to do this is to let them know that they are not the only search engine out there. Let their stock continue to plummet. Let their users migrate elsewhere. This is the only way to get them – or anyone – to take notice that you can't push those around that made you who you are.
Image credits: David Noel – aoi.com.au
Chirag Chauhan
Latest posts by Chirag Chauhan (see all)
- Bloggers Have a Higher Calling; Will You Accept Yours? - December 20, 2012
- Are You Destroying Your Brand Without Realizing It? - December 16, 2012
- SmartPhone Backup Plan: Worried Your Smartphone May Get Stolen or Go Screwy? Take These 4 Precautions Now - December 9, 2012


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from Wake Up with Steve Curtis:
Steve's guest is Mr. Dave Kranzler of Investment Research Dynamics (see below). Their discussion includes the necessity and usefulness of central banks, AND whether the recent financial events in Puerto Rico might be the best indicator of where the U.S. economy is headed.
Dave spent many years working in various Wall Street jobs. After business school, he traded junk bonds for a large bank. He has an MBA from the University of Chicago, with a concentration in accounting and finance. He graduated Oberlin College with majors in Economics and English.Help us spread the ANTIDOTE to corporate propaganda.
Please follow SGT Report on Twitter & help share the message.


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VideoHive Infographic Disk Elements 1501253
Motion Graphic \ Elements \ Technology
Motion Graphic - Infographic Disk Elements 1501253 by kurbatov @ VideoHive
Motion Graphic \ Elements \ Technology
Alpha Channel: Yes; File Size: 266mb; Frame Rate: Other; Length: 0:12; Looped Video: Yes; Resolution: 1000x1000.
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Another Elements.
Keywords: data, disk, element, futuristic, graph, gui, hi-tech, hud, icon, info, information, interface, technology.


text:
After the motorcycle getaway, tron heads to a communication tower to get in touch with Alan.
Alan instructs tron on the final leg of the mission: Destroy the MCP.
With some maneuvering, he and tron take over the MCP and kill Sark, proving they're the most badass guys in the mainframe.
tron finds the MCP, which for such a formidable foe, turns out to be a mean cartoon-animated face.
One episode, the Christmas one, we all pigged out like crazy and went to see tron: Legacy right after, and we all stunk horribly.
On being appointed to the tron Church in Glasgow, he set about to face what he called "the home heathenism."
When we got to the top of the street, and turned north, we espied a crowd at the tron Church.
As minister of the tron Church, Glasgow , he obtained a great reputation.
It was formerly the custom to allow a margin to buyers at the tron.
On the south side of the street, spanning the pavement, stands the tron Steeple, a stunted spire dating from 1637.
as a word-forming element in new compounds formed in physics, 1939, abstracted from electron (Greek -tron was an instrumentive suffix).


text:
From cell penetrating peptides to peptoids and polyamines as novel. artificial molecular transporters
- Ethan Hoover
- 2 years ago
- Views:
1 From cell penetrating peptides to peptoids and polyamines as novel artificial molecular transporters (Entwicklung neuer Delivery-Strategien für siras Von zellpenetrierenden Peptiden zu Peptoiden und Polyaminen als neuartige molekulare Transporter) Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-aturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Katja Schmitz aus Andernach Bonn 2005
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3 Angefertigt mit Genehmigung der Mathematisch-aturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1. Referent: Prof. Dr. K. Sandhoff 2. Referent: Prof. Dr. S. Bräse Tag der Promotion: Diese Dissertation ist auf dem Hochschulschriftenserver der ULB Bonn elektronisch publiziert. Erscheinungsjahr: 2005 iii
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5 Hiermit versichere ich, dass ich diese Arbeit selbstständig und ohne jede unerlaubte Hilfe angefertigt habe, dass diese oder eine ähnliche Arbeit noch keiner anderen Stelle zur Prüfung vorgelegen hat und dass sie an den nachstehend aufgeführten Stellen auszugsweise veröffentlicht worden ist. rt, Datum Katja Schmitz Auszugsweise Veröffentlichung: Diallo, M; Schmitz, K; Schepers, U (2004) RA Interference: RAid for Future Therpeutics? The ncogenomics Handbook: Understanding and Treating Cancer in the 21 st Century. Eds. LaRochelle, W., Shimkets, A., Humana Press. och nicht erschienene Veröffentlichungen: Schmitz, K; Schepers, U (2004) Cell penetrating peptides in sira delivery Expert pin. Biol. Ther. im Druck, Schmitz, K; Schepers, U Silencio: RA interference the tool of the new millennium? Biol. Chem. eingereicht, Schmitz, K; Diallo, M; Mundegar, R; Schepers, U pepsiras for RAi in mammals, eingereicht, Schmitz, K; Hahn, F; Balaban TS; Bräse, S; Schepers, U Covalent coupling of porphyrins with novel molecular transporters enhance their antitumor activity, eingereicht, Schmitz, K, Hahn, F, Schepers, U Modification of 2,3 -,-isopropylidene guanosine leads to an improved yield in the synthesis of 5 -deoxy-5 -thioguanosinemonophosphorothioate, eingereicht. v
6 Für meine Eltern und für ma Lucia vi
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8 Acknowledgement I thank Dr. Ute Schepers for her excellent support in all aspects of this work, for her commitment, for hours of fruitful discussions and for teaching me all the essentials of scientific life. Thanks for enabling me to attend all these interesting meetings, to work on these exciting projects and for encouraging me to surpass my own limits. I thank Prof Sandhoff for his generous support of this work and for his advice. I thank Prof. Dr. Bräse for the collaboration on novel peptoid and polyamine transporters and for kindly assuming the correferate. Many thanks to my lab fellows for their scientific support, refreshing coffee breaks, and bench-to-bench chats. I thank all my colleagues for the friendly atmosphere in lab. Thanks to Dr. Rustam Mundegar and Margit Zweyer for providing murine GFP-myocytes and for fruitful discussions. Thanks also to Prof. Dr. A. Wernig, permitting me to carry out the experminets on these cells in the Institute of Physiologie II. Many thanks to Miqiang Chai (DKFZ Heidelberg) for his enthusiastic collaboration during the first tests of enzymatically generated pepsiras. Thanks to Heike Hupfer for measuring the MALDI-TF mass spectra. I am grateful to Claus Schmidt and Ulrike Weynand for their advice and support with the MR experiments. Many thanks to Volkmar Fieberg for his friendly support with the work on the ÄKTA FPLC. Thanks to Tina Schröder and Yvonne Schmidt for their collaboration and for kindly providing the fluorescently labeled peptoid transporters and the spermine derivatives. I want to thank all my students who participated in this work during their practicals. My special thanks goes to Henning Breyhan for his work on the Tat vectors and test expression experiments and for his commitment in the establishment of the synthesis lab 2.132, to Frank Hahn who joined me in the search for improved guanosine derivatives for GSMP synthesis, and to Timo Mack for his work on the GSMP synthesis and sira preparation. Thanks to the Fonds der Chemischen Industrie, who financially supported me by a doctorate scholarship, which greatly helped me to focus on my research work and permitted me to attend many scientific meetings that have been a good motivation for my work. I thank the Bonner Forum für Biomedizin for the two poster prizes, that allowed me to take part in some motivation scientific meetings. Thanks also to the German Scholarship foundation for the idealistic doctorate fellowship by which I got to meet many people, who inspired my work. I am grateful to all of my friends for the gorgeous time I could spent in Bonn with all its Sneak Preview nights, visits to Cologne, opera visits and long nights of chatting and celebrating. viii
9 Special thanks to Jan, Vanessa and Jenny for never getting tired of reminding me that there is indeed a life outside the lab. Thanks also to the neuroscience reading group for the inspiring excursions into the world of cognition processes. Many thanks to all those who encouraged me so much during the last part of this work. Finally, this work would not have been possible without the support of my parents who greatly helped me through the dark times of this work and always found some ways to cheer me up. Thank you very much for the unforgettable journeys we undertook together, for all these relaxing weekends and long chats on the phone. ix
10 Table of contents Table of contents...1 Abbreviations Introduction RA interference The study of gene function The discovery of RAi RAi is a highly conserved eukaryotic mechanism The molecular basics of RAi The many roles of small endogenous RAs RISC is responsible for mra cleavage and transcriptional repression The role of RA dependent RA polymerases (RdRps) RAi in mammalian cells Stable RAi in mammalian cells Viral vectors to trigger RAi Cure for diseases on-specific effects of siras Modifications of siras for in vivo applications The delivery issue Cell-penetrating peptides and their small molecule analogs The delivery issue Cellular uptake mechanisms Passive transport by diffusion Active transport by transmembrane transporters Active transport by endocytosis Common methods of delivery Membrane penetration by peptides and proteins The discovery of protein transduction Cell-penetrating peptides Modifications to optimize CPPs Synthetic molecules to mimic CPPs The versatility of CPPs The mechanism of peptide-based delivery Aims of this work The concept of pepsiras Design and synthesis of thiol-modified siras Synthesis of GSMP Purification of GSMP in vitro transcription of siras from T7-DA-oligonucleotides Coupling of 5 -thiol modified siras to Penetratin TM Gel electrophoresis with pepsiras MALDI-TF mass spectrometry of pepsiras Test of pepsiras for their potential to trigger RAi in vivo
11 3.2.1 Targeting recombinant genes with pepsiras pepsiras to knock out Lamin A/C in HeLa cells PepsiRAs to knock out proteins of the sphingolipid metabolism in fibroblasts Test of pepsiras in various mammalian cell lines Recombinant expression of CPPs in E. coli Generation of a recombinant expression vector for modified CPPs Expression of GST-CPPs in E.coli Purification of GST-CPP fusion proteins by affinity chromatography Refining of the purification protocol for GST-fusion proteins for GST-CPPs Expression of TEV protease in E. coli Expression of GST-TEV-protease in E.coli Expression of His 6 -TEV-protease from E.coli Cleavage of GST-CPP fusion proteins with TEV protease ovel cell-penetrating molecules for the delivery of siras Preliminary tests of polyamines Spermine-coupled porphyrin for photodynamic therapy Solid phase synthesis of peptoid transporters Cellular uptake of fluorescein-labeled peptoids Discussion Materials and methods Bacteria culture Culture conditions Storage Generation of competent cells Transformation and selection Cell culture techniques for mammalian cells General procedures Storage of mammalian cells in liquid nitrogen Preparation of FCS-coated cover slips Treatment of adherent cells with pepsiras Transfection of adherent cells with siras Treatment of suspension cultures with pepsiras Treatment of adherent cells with novel cell permeating molecules Fixation of cells for microscopy Preparation of cells grown on cover slips for immunofluorescence microscopy DA-Techniques Photometric measurement of nucleic acid concentration Separation of DA by agarose gel electrophoresis Extraction of DA from agarose gels Synthetic gene cloning Enzymatic restriction of plasmids Ligation of DA-fragments
12 5.3.7 Plasmid isolation from recombinant bacteria cells PCR : in vitro amplification of DA Mutagenesis-PCR RA techniques Isolation of RA Separation of dsra RT-PCR In vitro generation of 5 -thiol-modified siras with T7-polymerase Reduction of homodimers of 5 -thiol-modified siras Coupling of synthetic sira and CPP Coupling of enzymatically synthesized sira and CPP Radiolabelling of siras Analysis of siras on sequencing gels Protein techniques Preparation of protein samples SDS-PAGE Protein staining with Coomassie Brilliant Blue R Silver staining of protein gels Western blot Quantitative measurement of protein concentrations Protein expression in bacteria Preparation of native GST-fusion proteins (pgex protocol) Preparation of native GST-fusion proteins (modified protocol) Preparation of native TEV-protease Preparation denaturing of TEV-protease from inclusion bodies Purification of GST-fusion proteins on glutathione-sepharose Preparation of sepharose beads Regeneration of GST-sepharose beads ative preparation of His 6 -TEV from E. coli Denaturing preparation of His 6 -TEV from E. coli Purification of His 6 -fusion proteins on i-ta-sepharose Dialysis Cleavage of GST-fusion peptides with TEV Handling of Penetratin TM Chemical syntheses Thin layer chromatography MR spectroscopy Long-term MR measurements Mass spectroscopy Anion exchange chromatography Reversed phase HPLC ,3 -,-Isopropylideneguanosine, (10) ,3 -,-Isopropylidene-5 -deoxy-5 -iodoguanosine, (11) ,-Dimethylaminomethylene-2,3 -,-isopropylideneguanosine (12)
13 5.6.10,-Dimethylaminomethylene-2,3 -,-isopropylidene-5 -deoxy-5 iodoguanosine (13): Deoxy-5 -iodoguanosine (14): Desoxy-5 -thioguanosine-monophosphorothioate (GSMP) (15): ,3 -,-Isopropylidene- 3,5 -C- cycloguanosine (20) ,-dimethylaminomethylene-2,3 -,-isopropylidene-3,5 -C-cycloguanosine (21) Buffers and solutions Cell culture Protein expression Protein purification Gel electrophoresis Western blotting DA/RA techniques Synthesis Material Apparatus Consumables Antibodies Purification kits Enzymes and markers ligonucleotides and Peptides Radioactivity Bacterial Strains Cell Lines Vectors Chemicals References Summary
14 Abbreviations µg microgramm µl microliter µm micromolar aa amino acid AA acrylamide AB antibody AntP antennapedia (3 rd helix) peptide Antp-HD antennapedia-homeodomain APS ammonium persulfate as antisense ATP adenosine triphosphate BMP bis(monoacyl)glycerophosphate bp base pairs BSA bovine serum albumine CAPS 3-(cyclohexyl)amino-1-propansulfonic acid CD circular dichroism cda copy- deoxyribonucleic acid CF carboxyfluorescein CH cyclohexane CIP calf intestinal phosphatase CEt cyanoethyl CPP cell-penetrating peptide CV column volume Cys cysteine Cyt cytohesin datp deoxy-adenosinetriphosphate DCM dichloromethane dctp deoxy-cytosinetriphosphate DEAE 2-(diethylamino)ethyl ER endoplasmic reticulum dgtp deoxy-guanosinetriphosphate DIPEA,-diisopropylethylamine DMEM Dulbeccos modified Eagle medium DMF,-dimethylformamide DMS dimethylsulfoxide DA deoxyribonucleic acid dtp deoxy-nucleotidetriphosphate ds doublestranded dsrbd dsra binding domain DTDP dithiodipyridine DTT dithiothreitol dttp deoxy-thymidinetriphosphate 5
15 EDTA EE FACS FCS FITC GaCS GCS GFP GSMP GST h HEPES HBt HX HRP Hz IPTG k.o. kda LB LCAA LTR LUV ma MCS MEM min ml mm MI MPM mra LS nm TA D 600 D RF ori PA PAGE PBS PCR PFA ethylenediaminetetraacetate ethyl acetate fluorescence activated cell sorting fetal calf serum fluorescein isothiocyanate galactosylceramide synthase glucosylceramide-synthase green fluorescent protein 5 -deoxy-5 -thioguanosine monophosphorothioate glutathione-s-transferase hour [4-(2-hydroxyethyl)-piperazino]-ethanesulfonic acid 1-hydroxybenzotriazole homeobox protein horse radish peroxidase Hertz (1/s) isopropyl-thiogalactopyranoside knock-out kilodalton Luria Bertani medium long chain alkyl amino long terminal repeat large unilamellar vesicle milliampere multiple cloning site minimum essential Eagle medium minute milliliter millimolar multiplicity of infection membrane permeable motiv messenger RA nuclear localization sequence nanomolar nitrilo acetic acid optical density measured at 600 nm oligodeoxyribonucleotide open reading frame origin of transcription polyacrylamide polyacrylamide-gelelectrophoresis phosphate buffered saline polymerase chain reaction paraformaldehyde 6
16 pgex PKR PA PolH PorSp ppm psap PTD PyBrP RA Rase rpm RT SAP SDS SFM sira SLDB SPS ss SUV TAE Tat TBAB TBS TEA TEAA TEAB TEMED TEV TFA THAP THF TLC Tris u V X-Gal plasmid for glutathion-s-transferase expression proteinkinase R peptide nucleic acid polyhedrin promoter porphyrin-labeled spermine parts per million precursor of sphingolipid activator protein protein transduction domain bromo-tris(pyrrolidino)phosphonium-hexafluoro-phosphate ribonucleic acid RA-cleaving enzyme revolutions per minute room temperature sphingolipid activator protein sodium dodecyl sulfate serum free medium short interfering RA science lab database solid phase synthesis single stranded small unilamelllar vesicle Tris acetate EDTA transactivator of transcription tetrabutylammoniumborohydride Tris-buffered saline triethylamine triethylamine acetate buffer triethylamine bicarbonate buffer,,, -tetramethylenediamine tobacco etch virus trifluoroacetic acid trihydroxyacetophenone tetrahydrofuran thin layer chromatography tris(hydroxymethyl)-aminomethane units volt 5-chloro-4-bromo-3-indolyl-ß-D-galactoside 7
17 1 Introduction 1.1 RA interference The finding that double-stranded RA (dsra) triggers the degradation of homologous mra is one of the most important recent discoveries. Since the first reports about this phenomenon in the late nineties, RA interference (RAi) has been broadly recognized by many disciplines ranging from functional genomics over biological chemistry to pharmacology. Today, the application of dsra to specifically silence genes at a posttranscriptional level (Posttranscriptional Gene Silencing, PTGS) has become a standard technique in the study of gene function, and it is on its best way to become a new weapon to combat disease triggered by RA viruses, parasites or even mutations of the genome, like cancer or inherited diseases (Arenz and Schepers 2003; Schepers and Kolter 2001) The study of gene function nly half a century ago, Watson and Crick postulated the helical structure of DA and its significance as the carrier of the hereditary information in all forms of life. Since then, large efforts were undertaken to understand the translation of genetic information into gene function and the sophisticated coordination of the mechanisms that govern life at a molecular level. The expression of genes in each individual cell needs to be highly regulated to activate the appropriate subset of genes for specific cellular functions or as responses to environmental changes. At the turn of the millennium, the publication of the first draft version of the complete sequence of the human genome was a scientific milestone (Lander et al. 2001; Venter et al. 2001). The task resulting from this achievement is much greater: correlating approximately 30,000 open reading frames with the function of about 100,000 human proteins resulting from posttranslational modifications and alternative splicing of the RA transcripts. This task is tackled by functional genomics or functionomics. Its solution is the prerequisite for an understanding of complex physiological functions and the key to the development of drugs against diseases. Traditionally, functional genomics had to trace back inherited diseases from the protein defect to the responsible gene in a time consuming process. The generation of knock-out animals permitted the specific study of gene defects and their related phenotypes. However, the generation of knock-out animals is time consuming and laborious, defects in essential genes lead to lethality at early embryonic stages preventing phenotypic studies and not all results obtained in model organisms can be transferred to related species, which is especially true for humans and mice (Kolter and Sandhoff 1999). The combination of large chemical libraries, high throughput screening and elaborate screening assays together with findings of classical genetics brought forth the field of chemical genetics, holding high promises for drug development and functional genomics (Frank-Kamenetsky 2001; Stark et al. 1998). By means of rationally designed synthetic inhibitors loss of function phenotypes can be generated in a short time. Although the lead structures can be optimized to binding 8
18 efficiencies in the nanomolar range and below (Frank-Kamenetsky 2001), they may also bind to unexpected sites on different molecules where they exhibit off-target effects. A recent example is the anti-inflammatory drug celecoxib, that was designed to selectively target cyclooxygenase (CX) 2 while sparing inhibition of CX-1, but exhibited an unexpected nanomolar binding affinity for members of the totally unrelated carbonic anhydrase family (Weber et al. 2004). Combinatorial chemistry and the use of chemical evolution allowed the development of aptamers as an important tool for gene function studies. These synthetic RAs adopt a specific secondary structure by which they bind to either DA, RA, or proteins and thus down-regulate protein function (Famulok 1999; Sullenger et al. 1990). The free availability of genomic sequencing data opened the door to a rational design of synthetic inhibitors to any target gene and generating the corresponding loss-of-function phenotype in a straightforward fashion. The first step into this direction was taken in the early 1990s by antisense technology, in which complementary antisense-oligonucleotides bind to the target mra-sequence and thereby prevent its translation to the corresponding gene product (Brantl 2002). At first, single stranded RA (ssra) was used, which is unstable toward degradation by ribonucleases. Many efforts have been made to improve the stability of the RA ranging from protection groups on the ssra, thiol-modifications to stabilize the ribophosphate backbone, or peptide nucleic acids (PAs) in which the nucleobases are bound to a peptide backbone. Antisense DAs are not only more stable than RA, but also form hybrid strands with mra that are degraded by Rase H. All these approaches require a saturation of all targeted mra strands with corresponding antisense agents to obtain the desired knock-down phenotype. This implies the use of more than stoichiometric amounts of antisense agent to inactivate present as well as newly transcribed mra until all remaining protein has been degraded. However, antisense techniques are a common method in the study of gene function and some specific modifications have made their way to clinical studies as potential drugs to suppress tumor function. Figure 1-1 Dissecting the expression of gene products at different stages. Knock-out organisms and naturally occurring mutations alter the information stored in the DA, protein inhibitors and aptamers bind to gene products and inhibit protein functions, antisense agents and RAi act on the level of transcripts by preventing ribosomal binding or triggering its degradation. aturally occurring or synthetic small molecules can be selected to target any of the three stages by high throughput screening. 9
19 1.1.2 The discovery of RAi ew possibilities were opened up with the discovery of RA interference that combines the advantages of rational design with a much higher efficiency in down-regulating the targeted gene-product. Therefore, it was soon adopted as the tool of choice in reverse genomics to study gene-protein relations in almost all eukaryotic organisms. First hints at this phenomenon had come from antisense experiments in C. elegans, in which the sense control exhibited an unusually strong effect (Guo and Kemphues 1995). Assuming a contamination with asra in the sense control, Fire and Mello demonstrated that the simultaneous injection of two complementary RA strands, and likewise the use of dsra, could specifically silence the corresponding gene product with an at least 10fold higher efficiency than conventional antisense reagents (Fire et al. 1998). Following this key discovery, it was shown that RAi takes place at the posttranscriptional level where a dsra triggered molecular machinery causes the degradation of homologous mra (Montgomery and Fire 1998; Montgomery et al. 1998) identifying RAi as a form of posttranscriptional gene silencing (PTGS). In C. elegans, RAi could not only be induced by injecting the dsra into the gut (Fire et al. 1998; Grishok et al. 2000), but also by soaking the worms in dsra solution (Tabara et al. 1998), introduction of transgenes that code for double stranded RA (Tabara et al. 1999b; Tavernarakis et al. 2000) or feeding bacteria that recombinantly express double-stranded RA constructs to the worms (Timmons and Fire 1998). Moreover, a systemic spread of the silencing effect was observed and it was even inherited to the progeny (Fire et al. 1998; Sijen et al. 2001b). Cultured cells transfected with dsra could keep up the silencing phenotype for up to 9 cell divisions (Tabara et al. 1998). However, the most important features of RAi are its high specificity and efficiency: Substoichiometric amounts of dsra are sufficient to significantly reduce the amount of homologous mra within a few hours in a highly sequence specific manner (Fire et al. 1998). ther mras, even those with point mutations, remain unaffected (Bosher and Labouesse 2000; Fire et al. 1998; Hunter 1999) RAi is a highly conserved eukaryotic mechanism Posttranscriptional gene silencing (PTGS) had been previously reported in plants. In Arabidopsis thaliana the attempt to express an endogenous gene from a recombinant vector had led to a complete downregulation of the respective gene and the phenomenon had been termed cosuppression (apoli et al. 1990). A related mechanism was found in the fungus eurospora crassa were it became known as quelling (Cogoni and Macino 1997). In plants, the infection with RA viruses expressing dsra was discovered to lead to immunity that limits the spread of the virus and prevents a second infection with the same virus, a phenomenon termed virus-induced gene silencing (Baulcombe 1999). Today, these related phenomena are often summarized as RA silencing. With a growing interest in RAi, dsra triggered gene silencing was also found to be functional in Drosophila melanogaster (Kennerdell and Carthew 2000; Misquitta and Paterson 2000), plants (Hamilton and Baulcombe 1999) and Trypanosoma brucei (go et al. 1998). It appeared that dsra-induced gene silencing is a highly conserved phenomenon. It 10
20 is assumed to be an ancient defense mechanism against RA viruses (Ruiz et al. 1998; Voinnet 2001) and transposable elements (Ketting et al. 1999; Tabara et al. 1999a). Both produce dsra in the course of their replication. It may also be involved in the elimination of defective mras by degradation (Plasterk 2002) and in the regulation of protein levels in response to environmental stimuli (McManus et al. 2002). In the course of evolution, organisms have developed alternative strategies to fulfil the same tasks, so that the main function of the dsra triggered silencing machinery varies from species to species. The most prominent, and for RAi scientists most troublesome, alternative mechanism was the induction of an interferon response upon the introduction of dsra into mammalian cells. Increased levels of β-interferon lead to an activation of Rase L, that non-specifically degrades all mras, and of RA-dependent protein kinase R (PKR), which in turn phosphorylates and thereby inactivates the transcription factor elf2α. This results in a shutdown of the global protein biosynthesis and thereby in apoptosis (Clemens 1997; Clemens and Elia 1997). Therefore, it was doubted that RAi could be used to study mammalian organisms and the question arose whether the molecular machinery for RAi was actually present in mammals. Figure 1-2 dsra triggers an interferon response in mammals. The different events triggered by interferon-β finally lead to apoptosis (Sledz et al. 2003). Studies in mice oocytes and murine embryonic cancer cell lines, in which the innate defense mechanisms are not yet functional, proved that, indeed, RAi works in mammalian cells (Billy et al. 2001; Wianny and Zernicka-Goetz 2000). The milestone experiment was published by the Tuschl group (Elbashir et al. 2001a). They showed that RAi could be induced in adult mammalian cells if the trigger dsra is short enough to omit the interferon response. dsras of nt exhibited the highest efficiency, whereas shorter dsras were no longer recognized. This opened the door to a broader application of RAi. ot only could RAi be utilized to link the genomic data from humans, mice, and other mammals to protein function, it also 11
21 constituted a new technique to combat diseases caused by viruses, parasites or genomic mutations as in inherited diseases and cancer The molecular basics of RAi The existence of an RAi machinery in almost all eukaryotic organisms allowed the mutual exchange of results to elucidate its mechanism and thereby greatly enhanced the rate with which the insights into the molecular requirements grew. The most intensively studied organisms in the quest for the mechanism of RAi are C. elegans (Plasterk 1999; Smardon et al. 2000), Arabidopsis (Mourrain et al. 2000),. crassa (Cogoni and Macino 1997, 1999b), Drosophila (Kennerdell and Carthew 2000), and mammals (Elbashir et al. 2001a). The fact that RAi takes place in the cytosol greatly facilitated the experimental setup as cell extracts from flies or worms mimic the conditions of the cytosol (Hutvagner and Zamore 2002b; Kawasaki and Taira 2003; Zeng and Cullen 2002). This introduction focuses on the findings and developments in animals. Results obtained in plants are only cited if they contributed to the understanding of the molecular processes in animals. The mechanism of RAi can be divided into two steps. In the initiator step, dsra is cleaved into nt long fragments. In the effector step, these so-called short interfering RAs (siras) are incorporated into a ribonucleotide protein complex (RP) and serve as a molecular template to recognize homologous mra that is cleaved. Discrete nt small dsras with a sequence complementary to the target sequence were first observed in transgene or virus-induced PTGS and co-suppression in plants (Hamilton and Baulcombe 1999) and it was assumed that these small dsras constituted the active component in the examined silencing phenomena. The degradation of long dsra into nt short dsras was also found in Drosophila extracts (Zamore et al. 2000) and intact Drosophila embryos (Yang et al. 2000), Schneider 2 (S2) cells (Hammond et al. 2000) and C. elegans (Parrish et al. 2000). The Hannon group demonstrated that the short dsra species is associated with the nuclease activity that leads to the cleavage of the target mra (Hammond et al. 2000; Hutvagner et al. 2000). These findings suggested that the sequence specificity of RAi was conveyed by the nt dsra fragments cleaved from the trigger dsra, that became known as siras (Zamore et al. 2000). Closer examination of these short dsras showed that functional siras bear characteristic 3 -overhangs of 2 nucleotides on both strands (Hamilton and Baulcombe 1999) with nonphosphorylated hydroxyl groups (Elbashir et al. 2001c) that are essential for their recognition by the RAi machinery. At the same time, these features correspond to the characteristic cleavage pattern of nucleases of the Rase III family that specifically cleave dsra (Robertson et al. 1968). Indeed, proteins with homology to Rase III family members could be identified by homology screens of genomic data from Drosophila, C. elegans, and humans (Bass 2000; Bernstein et al. 2001; Billy et al. 2001; Ketting et al. 2001). In Drosophila two such homologs were found, that both comprise two Rase III domains (Bernstein et al. 2001). Tests upon their role in RAi showed that cells deficient of either one of the two Rases exhibited neither RAi activity nor did the cell extracts produce siras from dsra. This confirmed that dsra is 12
Outline. interfering RNA - What is dat? Brief history of RNA interference. What does it do? How does it work?
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Home > Business > IJBF > Vol. 9 > Iss. 2 (2012)
Article Title
Using H-statistic of the Panzar-Rosse model, this paper examines commercial bank merger waves in Japan and their effect on competition in the Japanese banking market during 1983-2006. The H-statistic is estimated separately for three different time periods, the boom, the burst and the recovery. This paper concludes that the bank mergers that took place in Japan have not led to a higher level of market power except during the period of financial crisis around the time of bubble burst. Recent mergers in the Japan's banking sector do not seem to harm the competition level in the banking market. An increase in individual bank's market share and an increase in overall market concentration have not materialized in higher net interest margin in Japan.
Recommended Citation
Park, Kang H.
"Bank mergers and competition in Japan,"
International Journal of Banking and Finance: Vol. 9
, Article 1.
Available at: http://epublications.bond.edu.au/ijbf/vol9/iss2/1


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