To install click the Add extension button. That's it.

The source code for the WIKI 2 extension is being checked by specialists of the Mozilla Foundation, Google, and Apple. You could also do it yourself at any point in time.

4,5
Kelly Slayton
Congratulations on this excellent venture… what a great idea!
Alexander Grigorievskiy
I use WIKI 2 every day and almost forgot how the original Wikipedia looks like.
Live Statistics
English Articles
Improved in 24 Hours
Added in 24 Hours
What we do. Every page goes through several hundred of perfecting techniques; in live mode. Quite the same Wikipedia. Just better.
.
Leo
Newton
Brights
Milds

Order of Carol I

From Wikipedia, the free encyclopedia

Order of Carol I
Ordinul Carol I
Order of Carol I grand cross badge (Romania 1920-1930) - Tallinn Museum of Orders.jpg
The badge and sash of the order
Awarded by the King of the Romanians
TypeDynastic Order
Royal houseHouse of Romania
Religious affiliationRomanian Orthodox
RibbonPale blue with gold edges bearing a narrow red stripe
MottoPRIN STATORNICIE LA IZBÂNDĂ
("To Victory Through Steadiness")
Awarded forConspicuous and special merit
StatusCurrently constituted
GradesCollar, Grand Cross, Grand Officer, Commander
Precedence
Next (higher)None (highest)
Next (lower)Order of the Crown
OrderofCarolI.ribbon.gif

The ribbon of the order

The Order of Carol I (Romanian: Ordinul Carol I) was the highest ranking of the Romanian honours of the Kingdom of Romania until the abolition of the monarchy in 1947. It was instituted on 10 May 1906[1] by King Carol I to celebrate the Ruby Jubilee of 40 years of his reign.

During its time as a national order, it was widely used to reward members of the Romanian royal family, Romanian Prime Ministers, Romanian politicians, foreign monarchs and heads of state, selected consorts and heirs, and other people thought to be worthy of receiving the order by the King of the Romanians.

It is currently a dynastic order of the former Romanian royal family. It is the highest-ranking award among all the decorations of the Romanian Royal House and is administered by its head. There are currently no foreign knights or dames of the order, except for members of the Romanian royal family.

YouTube Encyclopedic

  • 1/5
    Views:
    22 461
    33 838
    5 667 192
    11 680
    6 375
  • ✪ The 2009 Jeffrey M. Trent Lecture in Cancer Research - Carol Greider
  • ✪ Oracle CFO's Career Didn't Follow Straight Path
  • ✪ The Inconvenient Truth About the Democratic Party
  • ✪ The Danger of Spiritual Over-Confidence (Luke 22:54-62)
  • ✪ CFO: not just about numbers!

Transcription

1 IF WE CAN GET EVERYBODY TO TAKE THEIR SEATS WE'LL GET STARTED. FOR THOSE OF YOU STANDING IN THE BACK, THERE ARE VARIOUS SEATS INCLUDING IN THE FRONT ROW THAT ARE MARKED RESERVED. THEY'RE NOT RESERVED ANYMORE. FEEL FREE TO TAKE THEM. THERE ARE ALSO, I SEE, A HANDFUL OF SEATS ON THE SIDES AS YOU COME DOWN. FIND YOUR SEATS DURING THESE INTRODUCTORY REMARKS. LET MY START OFF BY WELCOMING YOU TO THE 2009 TRENT LECTURE. OBVIOUSLY, IT IS NOW 2010. AND NOT SURPRISINGLY THERE IS A STORY BEHIND THAT. BUT FIRST, LET ME TELL YOU A FEW THINGS ABOUT THE JEFFREY TRENT LECTURE AND CANS RESEARCH. JEFF TRENT WAS THE FOUNDING SCIENTIFIC DIRECTOR OF THE NATIONAL HUMAN GENOME RESEARCH INSTITUTED, CAME IN 1993 TO START A PROGRAM FROM SCRATCH. HE WAS RECRUITED BY FRANCIS COLLINS, WHO WAS THE PREVIOUS HEAD OF THE NATIONAL HUMAN GENOME RESEARCH INSTITUTE, PART OF THE RECRUITMENT WAS GIVEN THE ABILITY TO START AN INTRAMURAL PROGRAM FOCUSED ON GENOMICS RESEARCH. HE ARRIVED HERE, FROM 1993 AND 2002. DID A SPECTACULAR JOB OF LEADING OUR INTRAMURAL PROGRAM AND STARTING IT FROM SCRATCH, BUILDING IT UP, MAKING IT A WORLD CLASS PROGRAM IN GENETICS AND GENOMICS RESEARCH. HE DEPARTED IN 2002. AT A TIME, I BECAME THE SCIENTIFIC DIRECTOR AND ONE OF THE FIRST THINGS I DID WAS TO ESTABLISH A LECTURESHIP IN HIS NAME. AND THIS IS THE JEFFREY M. TRENT LECTURE IN CANCER RESERACH. 2 THIS IS NOW THE 7th SUCH LECTURE. AND YOU CAN SEE THE IMPRESSIVE GROUP OF INDIVIDUALS THAT HAVE GIVEN THE PREVIOUS 6. AND THE 7th AS YOU WILL KNOW AND FIND OUT IT'S NO EXCEPTION TO THAT TREND. THERE ARE A COUPLE OF STORIES I WANTED TO TELL BEFORE I TURN THIS OVER TO THE INDIVIDUAL WHO CAN S GOING TO INTRODUCE THIS YEAR'S LECTURER. THERE ARE 3 OF US YOU'RE GOING TO SEE ON THE STAGE. MYSELF, ERIC GREEN, AND NEXT FROM KATHY HUDSON AND THEN HEAR FROM CAROL W. GREIDER. A YEAR AGO, IF YOU THINK BACK OR ROUGHLY A YEAR AGO, EACH OF OUR 3 LIVES WERE A LITTLE BIT DIFFERENT. AT THAT TIME I WAS A SCIENTIFIC DIRECTOR OF THE INSTITUTE, NOW I'M THE DIRECTOR OF THE INSTITUTE. CAROL W. GREIDER WAS A FACULTY MEMBER AT JOHNS HOPKINS. SHE'S NOW THE CHIEF OF STAFF HERE AT NIH. OUR SPEAKER WAS JUST A WORLD CLASS RESEARCHER, AND PROMINENT MEMBER OF THE RESEARCH COMMUNITY WHICH IS WHY WE INVITED HER A YEAR OR SO TO COME GIVE THE 7th JEFFREY TRENT LECTURE. HER LIFE HAS CHANGED A BIT. AN EVENT THAT HAVE INFLUENCED THIS LECTURE SUBSTANTIALLY. IT WILL SEEM THAT THE 3 OF US, KATHY HUDSON MYSELF, AND CAROL W. GREIDER AND THE ENTIRE NHGRI SHOULD BE A SIMPLE ACTIVITY TO PUT ON A LECTURE. I WILL TELL YOU IN ALL OF MY YEARS IN ORGANIZING, THIS HAS BEEN THE MOST CHALLENGING ONE TO HAVE HAPPEN. AND WE WEREN'T EVEN SUCCESSFUL AFTER HAVING IT HAPPEN IN 2009. 3 IT'S TAKEN US ALL THE WAY DO 2010. WHY IS THAT? IF YOU LOOK AT YOUR PROGRAM, YOU WILL NOTE THAT THE DATE ON THIS LECTURE WAS TUESDAY, SEPTEMBER 29. THAT WAS THE ORIGINAL DATE TO HAVE CAROL COME HERE AND GIVE THE LECTURE. IT WAS SUCH AN INNOCENT DATE WHEN WE SELECTED IT. IT WAS GOOD FOR OUR CALENDARS. THAT SEEMS GOOD ENOUGH. ABOUT A DAY OR 2 BEFORE SEPTEMBER 29, WE GOT A CALL THAT BASICALLY INFORMED US THERE WAS GOING TO BE A VERY, VERY, VERY, VERY, VERY HIGH LEVEL VISITOR TO THE NIH. AND THAT THE AUDITORIUM WAS NOT AVAILABLE TO US ANYMORE. AND IT WAS IN OUR BEST INTEREST TO POSTPONE THIS TALK. WE DID THAT. AND ORIGINALLY, IT WAS SUPPOSED TO BE A DIFFERENT VISITOR, NOT THE VERY, VERY, VERY TOP ONE BUT THE NEXT ONE DOWN, AND IT WAS SUPPOSED TO BE SEPTEMBER 29. IT WASN'T. THAT VISITOR CAME, BUT IT WAS THE NEXT DAY. BUT WHAT I DID FIND OUT WAS THAT WE COULDN'T HAVE USED THE AUDITORIUM ON SEPTEMBER 29. WHEN THE PRESIDENT OF THE UNITED STATES VISITS YOU, THEY LOCK THE ROOM DOWN, THEY HAVE DOGS IN HERE -- THEY HAVE DOGS IN HERE SNIFFING. SO WE GOT KICKED OUT OF THIS ROOM BY THE WHITE HOUSE. CAROL WAS VERY GRACIOUS, SHE SAID NOT A BIG DEAL. WE'LL FIND ANOTHER DATE. WE, ONCE AGAIN, PICKED A NICE DATE, HAPPENED TO BE I THINK IT WAS DECEMBER 8 OR SOMETHING LIKE THAT. 4 IT LOOKED GOOD ON MY CALENDAR, AND OTHER HERS. AND OFF WE WENT, ALL SET TO DO THIS. I DIDN'T FEEL TOO BAD. IT WAS STILL 2009. BUT THEN CAROL HAD TO WIN A NOBLE PRIZE. SHE HAD TO GO TO SWEDEN FOR THAT WEEK. I UNDERSTOOD WHEN SHE CALLED ME TO SAY WE'RE GOING TO HAVE TO POSTPONE THIS AGAIN. BUT THE GOOD NEWS, IT BRINGS US TO TODAY. AND IN THE EFFORT OF BEING GREEN. WE DECIDED NOT TO REPRINT THESE PROGRAMS. SO I DO APOLOGIZE FOR THE FACT THAT THEY DON'T HAVE TODAY'S DATE ON THEM. BUT WE LEFT IT AS SEPTEMBER, 2009 DATE. AT LEAST IT WAS -- SEEMED LIKE A GREEN THING TO DO, A MOW MENO TO THIS TOGETHER OF 2 CANCELLATIONS. SO WITH THAT SAID, AND THAT EXPLANATION OF BOTH HOW WE GOT HERE AND THE RICH HISTORY OF THIS LECTURE SERIES, I'M NOW GOING TO TURN THE PODIUM TO KATHY HUDSON WHO HAS A LONG CONNECTION TO THE SPEAKER AND WILL INTRODUCE HER TODAY. THANK YOU. >> IT'S MY PLEASURE TO BE ABLE TO INTRODUCE CAROL W. GREIDER TO YOU TODAY. CAROL AND I HAVE BEEN FRIENDS FOR 25 YEARS. WE FIRST MET AS GRADUATE STUDENTS AT THE UNIVERSITY OF CALIFORNIA, BERKELEY WHEN I STARTED GRADUATE SCHOOL THERE SHE WAS ALREADY THERE. TOILING AWAY. AND WELL ON HER WAY TO THE FIRST CELL PUBLICATION. 5 THIS SHOULD BE INSPIRING TO ALL THE GRADUATE STUDENTS AMONG YOU. THIS IS A PICTURE OF CAROL AT THE LAB BENCH IN 1985. AND WE WORKED IN A VERY SMALL DEPARTMENT, A DOUBLE MEMBERS ARE HERE WITH -- COUPLE MEMBERS ARE HERE WITH US TODAY. WHERE THE STUDENTS, POST-DOCS AND FACULTY WERE A CLOSELY TIGHT GROUP. WE HAD A PARTY, SOMETIMES 2 ON A WEEKEND, OR IF WE WERE HAVING A SEMINAR WE DIDN'T HAVE MUCH E-MAIL BACK THEN, WE JUST POSTED SIGNS AROUND IN THE BUILDING. THIS IS A STUDENT SEMINAR SIGN FROM A VERY IMPORTANT PRESENTATION THAT CAROL GAVE ASKING THE IMPORTANT QUESTION, HOW CRUDE DO TELOMER EXTRACTS GET. THERE IS A COUPLE OF FEATURES. THIS IS THE SUPER COIL DENSITY. HER NICKNAME WAS SUPER COIL. AND ON THE FIGURE B YOU CAN SEE A FIGURE REPRESENTING WHAT WAS LATER TO BECOME NAMED TELOMERASE. WHAT YOU CAN'T READ IN THIS SLIDE, BUT I'D LIKE FEW READ AN EXCERPT, AN ABSTRACT THAT HER FRIENDS, I THINK IT WAS CLAIRE WHO YOU'LL SEE PHOTOGRAPHED LATER WHO WROTE THIS FOR HER UPCOMING CELL PAPER. IT WAS TITLED PARTIALLY PURIFIED FACTORS FROM TETRAHYMENA DO NIFTY THINGS IN TEST TUBES. TELOMERASES MUST EXIST. GIVEN THAT, THEY MUST COME FROM SOMEWHERE, CONSIDERING CELLS DIVIDE AND MAKE CHROMOSOMES NEEDING MORE TELOMERS. DO THEY BUY THEM FROM YEAST. ILLUSION TO JACK, WHO SHARED THE NOBLE PRIZE FROM CAROL. DOES GOD MAKE TELOMERES? HE DOES UNDOUBTEDLY BUT ISN'T TELLING HOW OR DOESN'T CALL THEM 6 THAT. YOU WILL HAVE TO READ GOD'S PAPER, SUPREME BEING, TO FIND OUT WHAT TELOMERES ARE CALLED. IF DO YOU THE RIGHT THING, TETRAHYMENA WILL MAKE TELOMERS FOR YOU. IF GELS LOOK GREAT. ALL THE CONTROLS TURNED OUT RIGHT. THEY DID. SO HERE IS ANOTHER PICTURE OF CAROL IN THE DAY WITH AN UGLY BUT IMPORTANT GEL. AND BEING HER FRIEND FOR A LONG TIME, I'VE BEEN TRYING TO UNDERSTAND THE KEY INGREDIENTS FOR HER INCREDIBLE SCIENTIFIC SUCCESS. THIS IS A PHOTOGRAPH TAKEN IN MY BACKYARD IN THE MID 80s. AND YOU CAN SEE CAROL PICTURED THERE. WE ALSO SEE PICTURED THERE [LAUGHTER] ONE OF THE KEY INGREDIENTS FOR SCIENTIFIC SUCCESS, FRIENDS AND BEER. AND MANY PICTURES THAT I HAVE OF CAROL, THERE IS ALSO THIS INTERESTING BOOK, A DICTIONARY. MAYBE CAROL WILL EXPLAIN IT TO YOU. SO SADLY, CAROL STARTED BEFORE ME AND LEFT WELL BEFORE ME AT BERKELEY. AND IN 1988, JIM WATSON HAD A PARTICULARLY GOOD IDEA. HE HAD HAD GOOD IDEAS PREVIOUSLY. INCLUDING THOSE ASSOCIATED WITH THE STRUCTURE OF DNA. HE HAD A REALLY GREAT IDEA IN 1988 WHICH WAS TO BRING CAROL AS A FELLOW, A SUPER POST-DOC POSITION. SO WE HAD TO SAY GOODBYE TO HER, BUT NOT FOR LONG. WE STAYED VERY CLOSE IN TOUCH. ONE OF THE FIRST TIMES WE GOT 7 TOGETHER AFTER I LEFT GRADUATE SCHOOL IN 1989 WAS TO GO ON A BIKE TRIP TO ALASKA. THIS PICTURE SHOWS THE ROUTE THAT WE TOOK. I DON'T KNOW HOW TO GET A POINTER TO WORK. BUT WE BIKED FROM ANCHORAGE ALL THE WAY AROUND THAT DOTTED LINE TO VALDEZ. AND ON THAT BIKE TRIP, I HAVE NO PICTURES OF CAROL ON A BIKE AT ALL. I HAVE PICTURES OF CLAIRE WHO WROTE THE ABSTRACT AND HER HUSBAND ROLAND, AND MY NOW HUSBAND JOE. BUT I HAVE NO PICTURES OF CAROL ON A BIKE AT ALL. THE REASON IS BECAUSE WHEN WE SADDLED UP OUR BIKES, CAROL WAS OFF LIKE A SHOT. SHE ROAD BICYCLE LIKE SHE DOES HER SCIENCE, FAST AND FURIOUS. WE ONLY MANAGED TO CATCH UP WITH HER IN THE EVENINGS, AGAIN WITH THAT KEY INGREDIENT, BEER. WE FINISHED THE BIKE TRIP, TOOK A FERRY FROM VELOCITIES, TOOK A TRAIN, AND TACKLED GLACIAL RIVERS, AND BEER INFESTED WOODS AND HAD A GREAT TIME TOGETHER. IN THE INTERVENING YEARS, CAROL AND I HAVE SHARED HOLIDAYS, VACATIONS, RAISING FAMILIES TOGETHER. WE'VE BEEN COLLEAGUES TOGETHER AT HOPKINS. AND SO CAROL ROSE QUICKLY THROUGH THE SCIENTIFIC RANKS BECOME THE NATHANS PROFESSOR AND THE CHAIR OF THE DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS. SO DURING THIS TIME WE HAVE BEEN FRIENDS OF COURSE I'VE ACCUMULATED MANY HUNDREDS OF PHOTOGRAPHS. ERIC WOULDN'T LET ME SHOW ALL OF THEM TODAY. I WANTED TO SHOW YOU A COUPLE 8 PICTURES WHERE I HAD THE PLEASURE OF BEING A GUEST OF CAROL'S AS SHE RECEIVED THE NOBEL PRIZE. THIS IS THE AUDITORIUM WHERE THE NOBEL PRIZES ARE GIVEN OUT. WHAT YOU CAN SEE IN THE TOP LAYER THERE IS A FULL ORCHESTRA THAT PERFORMED DURING THE COURSE OF THE CEREMONY. DOWN BELOW ARE THE NOBLE COMMITTEE AND SITTING IN THE FRONT ON THE RIGHT IS THE SWEDISH ROYAL FAMILY. ON THE LEFT ARE THE NOBLE LAUREATES FROM THIS YEAR. IF YOU ZOOM IN YOU CAN SEE A BUST OF ALFRED NOBLE AND SEE THAT THERE IS GREEN AND WHITE DECORATING ABOVE THE STAGE AND ALONG THE BASE OF THE STAGE AND ALL OVER. IT TURNS OUT THOSE ARE FLOWERS THAT WERE CUT FROM ITALIAN VILLA WHERE ALFRED NOBLE VACATIONED. THE WHOLE ROOM HAD THE SMELL OF THESE FLOWERS. AND HERE IS CAROL, RECEIVING HER NOBLE. HAVING SUCCESSFULLY WALKED, WALKED, WALKED, BOWED, BOWED, BOWED. [LAUGHTER] SO THIS IS WHERE THE DINNER WAS FOLLOWING THE NOBLE CEREMONY. IN THE CENTER TABLE WAS FOR THE IMPORTANT PEOPLE AND THEN ON THE SIDES WHERE THE PEOPLE FOR LESS IMPORTANT. THEY WERE GRADIENT. AND THE LESS IMPORTANT YOU WERE, THE FURTHER OUT YOU WERE AT THESE TABLES. [LAUGHTER] CAROL HAD DINNER WITH THE PRINCE. IF PRINCE OF SWEDEN THAT EVENING. SO I'M SURE YOU'RE DYING TO KNOW WHAT I WORE. 9 [LAUGHTER] AND WHAT GIGI WORE AND JEFF WORE. SO ON A NOBLE WEBSITE ARE INSTRUCTIONS HOW TO DRESS. AS YOU CAN SEE HERE. THERE IS VERY SPECIFIC INSTRUCTIONS ABOUT WHAT ONE SHOULD WEAR TO THE BANQUET AND TO THE CEREMONY. AND ONE OF THE OPTIONS IS TO WEAR YOUR NATIONAL COSTUME. AND SO WE DID HAVE AN OPPORTUNITY WHILE WE WERE IN STOCKHOLM TO WEAR OUR NATIONAL COSTUME, BUT WE DID MANAGE TO GET OURSELVES CLEANED UP AND A GIRLFRIEND OF MINE HERE AT NIH TOLD ME WHEN I SAID I WOULDN'T BE AT A MEETING THE FOLLOWING WEEK, I WAS GOING TO STOCKHOLM, SHE SAID I HAVE THE BEST GIRLFRIEND STORY EVER. MY FRIEND WON THE NOBLE PRIZE. CAROL? [APPLAUSE] >> WELL, CAN I'M SPEECHLESS. I MUST SAY I HAVE NEVER, EVER, HAD AN INTRODUCTION EVER BEFORE. BETWEEN THE POSTER FOR THE TALK WHEN I WAS A GRADUATE STUDENT WHICH BASICALLY GAVE AWAY ALL OF THE SCIENCE, AND THEN THE HISTORY OF THE REST OF WHAT WE HAVE BEEN DOING RECENTLY, I GUESS I CAN STOP AND ASK ARE THERE ANY QUESTIONS? [LAUGHTER] IT'S REALLY A PLEASURE TO BE HERE. TO COME BACK AND SEE, YOU KNOW, SO MANY FRIENDS. I HAVE A LOT OF PEOPLE I COME HERE TO THE NIH A LOT, SO I REALLY APPRECIATE BEING ABLE TO COME HERE FOR THIS MUCH POSTPONED TALK. SO WHAT I'M GOING TO DO TODAY IS TO GIVE JUST A LITTLE BIT OF BACKGROUND ON TELOMERs AND TELOMERASE. 10 THEN I'D LIKE TO TELL YOU WHAT WE HAVE BEEN DOING OVER THE LAST 5 OR 6 YEARS. AT THE VERY END, TELL YOU A NEW STORY THAT I HAVEN'T HAD A CHANCE TO TALK ABOUT YET. IT'S ALWAYS GOOD TO BE ABLE TO COME SOMEWHERE AND ACTUALLY TALK SOME SCIENCE. SO I'LL TELL YOU ABOUT THE TELOMERASE HISTORY AND DISCOVERY. JUST TO FOLLOW UP ON KATHY'S INTRODUCTION FROM THE NOBLE. I'LL TELL YOU A LITTLE BIT ABOUT THE ROLE OF SHORT TELOMERASES IN CANCER, AND THE REST OF THE TIME TELLING YOU ABOUT SHORT TELOMERES IN HUMAN GENETICS, AND DEGENERATIVE DISEASES. SO TELOMERES ARE THE ENDS OF CHROMOSOMES. YOU CAN SEE HERE IN THESE BLUE TIPS, AND THEY PROVIDE 2 REALLY ESSENTIAL FUNCTIONS. ONE IS THAT THEY HAVE TO PROTECT CHROMOSOME ENDS. THEY SERVE AS CAPS TO PROTECT THE ENDS FROM NUCLEAR ACES, FROM END TO END JOINING, FROM VARIOUS ROOM COMBINATION PROCESSES. THE SECOND FUNCTION, MAINTAIN LENGTH. AND THE TELOMERES ARE MADE OF SIMPLE DNA REPEATS THAT ARE BOUND BY SPECIFIC PROTEINS. THIS IS JUST AN ILLUSTRATION OF THE SIMPLE TELOMER REPEATS. THE END OF THE CHROMOSOME IS SINGLE STRANDED. AND TELOMER REPEATS IN MANY ORGANISMS ARE THE SIMPLE SEQUENCES. WHAT IS SHOWN HERE IS THE HUMAN AND ALL OF VERT BRATS HAVE THE SETTAGGG REPEATED OVER AND OVER AGAIN. YOU'LL SEE THE REPEAT TTGGGGG, WHICH IS THE TETRAHYMENA REPEAT. THEY ARE SIMILAR CONSERVED 11 REPEATED SEQUENCES. MOST IS DOUBLE STRANDED. ALTHOUGH THERE IS A SMALL REGION THAT IS SINGLE STRANDED AT THE VERY END. IN ORDER TO PROVIDE TELOMER FUNCTION, YOU NEED NOT ONLY THE TELOMER REPEATS BUT ALSO THE PROTEINS THAT BIND ALONG THE LENGTH OF THE DNA. THERE ARE PROTEINS THAT BIND TO THE DOUBLE STRANDED REGION AND PROTEINS THAT WIND TO THE SINGLE STRANDED REGION IN ORDER TO PROVIDE THIS PROTECTIVE FUNCTION. SO IT TURNS OUT THAT THE WAY THAT DNA REPLICATION OCCURS, THE VERY END OF THE CHROMOSOME ISN'T COMPLETELY REPLICATED. SO THIS IS WHAT WAS KNOWN IN THE 1970s, JIM WATSON WROTE A PAPER ABOUT THIS PROBLEM OF REPLICATING THE VERY END OF THE CHROMOSOME. AND SO WITHOUT ANY OTHER MECHANISM, THE TELOMERES SHORTEN EVERY TIME CELLS DIVIDE. SO THIS WAS POSED AS A PARTICULAR PROBLEM BACK IN THE 1970s AND EARLY 80s. SO WE WERE JUST VERY CURIOUS TO FIND OUT HOW IS IT THAT CHROMOSOMES CAN BE MAINTAINED IF THERE IS A PROBLEM THAT EVERY TIME THE CELLS DIVIDE THEY SHOULD GET SHORTER AND SHORTER. AND SO TO SET THE STAGE ABOUT HOW PEOPLE WERE APPROACHING THIS, THIS IS A PAPER THAT WAS WRITTEN BY JAK AND LIZ WHO WERE THE CORECIPIENTS OF THE NOBLE PRIZE THIS YEAR. THEY FOUND A WAY TO LOOK AT THE FUNCTION OF TELOMERs. WE NEW WHAT IT LOOKED LIKE IN THESE SINGLE CELLED ORGANISM CALLED TETRAHYMENA WHERE THE SEQUENCE WAS CLEARLY DELINEATED. THAT WAS THE TTGGGG REPEATS. 12 THEY TOOK A CIRCULAR PLASMID FROM YEAST THAT HAD A MARKER, AND DELINEARIZE THAT HAD PLASMID, AND PUT ON TO THE END TELOMER SEQUENCES FROM TETRAHYMENA. WHEN THEY DID THAT, AND THEY PUT IT INTO YEAST, THEY FOUND OUT 2 REMARKABLE THINGS. FIRST OF ALL, THE TETRAHYMENA TELOMERs FUNCTIONED, PROTECTED THE END, AND THE CHROMOSOME DIVIDED SUCCESSFULLY AND WAS MAINTAINED AS A LINEAR CHROMOSOME. THE SECOND THING THAT THEY FOUND OUT, IN SUBSEQUENT FOLLOW UP PAPER WHICH [INDISCERNIBLE], IS THAT AFTER THIS LINEAR CHROMOSOME HAD BEEN MAINTAINED IN YEAST FOR A NUMBER OF DIVISIONS, THAT THERE WAS YEAST TELOMER SEQUENCES THAT WERE ADDED TO THE TERMI OF THESE REPEATS. SO THAT REALLY GOT THEIR ATTENTION. AND IN ORDER TO UNDERSTAND HOW IT IS THAT TELOMERS ARE MAINTAINED, THEY WROTE WHAT WE PROPOSE THAT TELOMERS ARE RESPONSIBLE FOR -- SUCH ACTIVITIES COULD BE ADDED SINGLE NUCLEOTIDES, ET CETERA. THIS WAS BOLD BECAUSE IT PROPOSED THAT THERE WAS A COMPLETELY UNKNOWN ENZYME THAT WOULD BE ELONGATED TELOMERs. THERE WERE OTHER HYPOTHESIS THAT INVOLVED OTHER MECHANISMS WHEREBY TELOMERs COULD BE ELONGATED. WHEN I JOINED THE BLACKBURN LABORATORY. I WANTED TO BE ABLE TO TEST, IS THERE REALLY SUCH A THING? SO WE AGAIN STARTED OFF WITH TETRAHYMENA, WHICH IS KNOWN TO BE IN THE BUSINESS OF BUILDING TELOMERs. 13 AND WHAT WE DID WAS TO USE A SINGLE STRANDED SEQUENCE PRIMER, A SMALL BLUE ARROW. AND PUT THOSE INTO EXTRACTS OF TETRAHYMENA, ALONG WITH RADIOACTIVE TGTP AND DTTP. WE FOUND THAT THE ACTIVITY IN THOSE CELL EXTRACTS WOULD ADD THE SEQUENCE, REPETITIVELY ON TO THE END OF THE PRIMER. THIS TELOMERASE, AS WE SUBSEQUENTLY NAMED IT. IS WHAT IS RESPONSIBLE FOR ELONGATING TELOMERS. THIS IS A 6 BASE REPEATING PATTERN THAT WAS EXTENDING UP TO THE TOP OF THIS SEQUENCING GEL. HAVING IDENTIFIED ENZYME ACTIVITY WHICH ADD THESE REPEATS ON TO A SUBSTRATE, THE NEXT QUESTION, OF COURSE, BECAME WHERE DID THE INFORMATION COME FROM? AND SO WE HAD VARIOUS DEBATES ABOUT IT. BUT WHAT WE PROPOSED THAT PERHAPS THE TELOMERASE ENZYME MAY CONTAIN A TEMPLATE. SURE ENOUGH, FOLLOWING UP WITH EXPERIMENTS, WE WERE ABLE TO SHOW THAT THERE IS AN RNA COMPONENT WHICH HAS THE COMPLIMENT OF THIS DNA SEQUENCE WITHIN THE ENZYME. SO THE TELOMERASE IS A PROTEIN AND RNA COMPONENT THAT ALLOWS THE SYNTHESIS OF THESE TELOMERIC REPEATS. YOU HAVE THE SEQUENCE HERE, AND THE TELOMERASE ENZYME, MADE UP OF BOTH A PROTEIN COMPONENT AS WELL AS AN ESSENTIAL RNA COMPONENT, WITHIN THAT IS THIS COMPLEMENT TO THE TELOMERIC REPEAT. THE TEMPLATE CAN BE USED TO FILL OUTSIDE AND MAKE MANY MANY COPIES OF THIS SEQUENCE. SO WE'RE VERY FASCINATED BY THE TELOMERASE ENZYME, HOW YOU CAN 14 HAVE AN ENZYME CARRY OUT THIS FUNCTION OF HAVING THE RNA COMPONENT AS PART OF THE ENZYME ITSELF. WE ALSO BECAME INTERESTED IN WHAT ACTUALLY THIS ENZYME MAY BE DOING IN CELLS. SO AS YOU HEARD, AFTER LEAVING THE BLACKBURN LABORATORY, I WENT TO [INDISCERNIBLE] WHERE I STARTED TO GET INTERESTED IN WHAT WAS HAPPENING IN HUMAN CELLS AT THE ENDS HAVE CHROMOSOMES. VERY FORTUNATELY, THERE WAS THE VERY BEGINNINGS OF THE GENOME MEET ACTION THAT WOULD OCCUR EVERY YEAR AT COLD SPRING HARBOR. I WAS FORTUNATE THAT I WAS AT THE MEETING WHERE THEY FIRST IDENTIFIED THE HUMAN TELOMERIC SEQUENCE REPEAT. AT THE TIME IT WASN'T KNOWN. THAT WAS PRESENTED AT A MEETING IN 1989. AND SO WE WERE INTERESTED THEN IN DOING AN EXPERIMENT TO ASK WHAT HAPPENS IN HUMAN CELLS. NORMAL HUMAN CELLS TO THE LENGTH OF TELOMERS, ONCE WE GOT AHEAD OF THE SEQUENCE, WE COULD DO A SOUTHERN BLOT. WHAT WE FOUND WAS THAT IN NORMAL HUMAN CELLS AS YOU PASSAGE THEM IN CULTURE, THEY GO CELLULAR [INDISCERNIBLE]. THIS IS A SOUTHERN BLOT SHOWING THE SORT OF SMEAR OF THE REPEATS BECAUSE THE HETEROGENOUS [INDISCERNIBLE]. THEY ARE ACTUALLY SHORTENING. SO IT TURNS OUT IN MANY PRIMARY HUMAN CELLS, TELOMERASE ISN'T EXPRESSED. SO THAT SAME PROBLEM I WAS TELLING YOU BEFORE LEADS TO SOME TELOMER SHORT ENTERTAINING. WE GOT -- SHORTENING. WE GOT VERY INTERESTED IN THIS. 15 THIS SET OF PROPOSAL TO SEE WHAT MIGHT HAPPEN TO CANCER CELLS WHICH HAVE TO DIVIDE, SO WE STARTED WALKING THE PATH OF TRYING TO UNDERSTAND THE FUNCTIONAL CONSEQUENCE OF TELOMER SHORTENING IN HUMAN CELLS. SO IN ORDER TO ASK CRITICAL QUESTIONS ABOUT WHAT WOULD HAPPEN IN CELLS THAT DON'T HAVE TELOMERASE, WE DECIDED TO USE MOUSE GENOMICS TO ASK VERY SPECIFIC QUESTIONS. SO TO DO THIS, WE GENERATED A MOUSE IN WHICH ONE OF THE 2 COPIES OF TELOMERASE WAS ELIMINATED. SO WHAT'S ILLUSTRATED HERE T NAME FOR THE GENE. THE RNA. THIS IS CENTRAL RNA COMPONENT OF TELOMERASE. AND WHAT MARIA DID IN THE LAB WAS TO GENERATE A MOUSE IN WHICH ONE OF THE 2 COPIES OF THE TELOMERASE RNA WAS DELETED. WHEN YOU CROSS 2 SUCH MICE TOGETHER, WHAT YOU GET, OF COURSE, IS THE WILDTYPE, THE NULL ANIMALS. THESE WERE PERFECTLY NORMAL WHEN THEY WERE BORN IN NORMAL RATIOS, BUT WE CAN SHOW THAT THIS WAS NO ENZYME ACTIVITY FOR TELOMERASE IN THESE NULL ANIMALS. SO WE WERE, THEN, INTERESTED IN WHAT HAPPENS IN FUTURE GENERATIONS. AND WHEN YOU TAKE 2 SUCH NULL ANIMALS TG1 FOR THE FIRST GENERATION. AND YOU BREED THOSE, YOU GET THE G2, THE SECOND GENERATIONAL ANIMALS. THE G3, G4, G5, ET CETERA. SO THESE MICE WERE BREEDING CONTINUOUSLY. WE DIDN'T SEE ANY PHENOTYPES IN THE EARLY GENERATIONS. 16 I'LL TELL YOU ABOUT THE ONES WE DID SEE IN THE LATER GENERATIONS. FIRST, I WANT TO SHOW YOU WHAT ACTUALLY HAPPENS TO THE TELOMERASES, AS THESE ANIMALS ARE BREEDING PROGRESSIVELY FOR A NUMBER OF GENERATIONS. SO IN ORDER TO FOLLOW THE LENGTH WE USED THE QUANTITATIVE ANALYSIS THAT WAS DEVELOPED. IT'S A METAPHASE SPREAD OF MOUSE CHROMOSOMES, WHERE THEY ARE STAINED IN BLUE. AND THE TELOMERASE ARE SUSTAINED WITH A PNA PROBE. THIS IS A VERY SHORT NUCLEOTIDE PROBE. THE NUMBER OF PROBES THAT CAN [INDISCERNIBLE] IS PROPORTIONAL TO HOW LONG IT IS. THE SIGNAL INTENSITY OF EACH OF THOSE DOTS IS PROPORTIONAL TO THE LENGTH OF THE TELOMER. YOU CAN, THEN, FOLLOW THE TELOMER LENGTH WITH INCREASING GENERATIONS. WHAT YOU GET IS WHEN YOU LOOK AT THE PARENTS HERE, THE HETEROZYGOUS MOUSE, THERE IS A NICE FREQUENCY DISTRIBUTION, THIS BEING THE LENGTH AND THE NUMBER OF ENDS THAT HAVE THAT LENGTH. WITH INCREASING GENERATIONS, THERE WAS A PROGRESSIVE SHORTENING OF THE TELOMER IN THESE MICE. TELOMERASE WILL MAKE IT IN VITRO BUT WAS SPECIAL FOR MAKING TELOMERS IN THE MOUSE. THE OTHER THING NICE ABOUT THIS, YOU'RE LOOKING AT THE CHROMOSOMES. AND WHAT WE COULD SEE IS THAT NOT ONLY WAS TELOMER LENGTH SHORTENING, BUT THE LOSS OF THE SEQUENCE WAS LEADING TO LOSS OF TELOMER FUNCTION. IF YOU LOOK HERE IN THE WILDTYPE 17 SPREAD YOU SEE THESE NICE MOUSE METAPHASE CHROMOSOMES. ALL MOUSE CHROMOSOMES ARE CENTRIC, THAT IS, IT'S LOCATED ALONG ONE CHROMOSOME ARM. YOU SEE THESE NICE BOOMER RANGES. WHEN YOU LOOK AT THE G2, THE G4, AND THE G6 WHAT YOU START TO SEE IS SOME CHROMOSOME ENDS WHERE WE DON'T DETECT ANY HYBRIDIZATION. SO THESE ARE THE SHORTEST IN THE POPULATION. THEY'VE FALLEN BELOW THE LEVEL OF DETECTABILITY OF THIS ASSAY. BUT THEN WE SAW SOME CHROMOSOMES THAT LOOKED LIKE THIS. AND THOSE ARE 2 MOUSE CHROMOSOMES THAT ARE FUSED END TO END. LOOKS LIKE A METACENTRIC CHROMOSOME. THESE ARE 2 CHROMOSOME FUSION EVENTS. IN ADDITION TO TELOMER SEQUENCE, THEY LOST FUNCTION. THEY'RE ESSENTIAL FOR PROTECTING CHROMOSOMES. SO THERE IS CLEARLY LOSS OF TELOMER FUNCTION. SO WHAT ACTUALLY HAPPENS IN THE MOUSE? WE SAW NO PHENOTYPES IN THE EARLY GENERATION MICE. BE THE MATURE GENERATION WHAT HAPPENS IS THAT WE SEE INCREASED APOPTOSIS, CELL DEATH OR CELLULAR -- AT THE LEVEL OF THE MOUSE, WHAT THAT TRANSLATES INTO IS THE FIRST THING WE COULD SEE WAS A DECREASE IN FERTILITY, THEN LATER ON INFERTILITY, BECAUSE THE TEST TEES T GERM CELLS WERE UNDERGOING APOPTOSIS. WE ALSO SEE APOPTOSIS IN THE IMMUNE SYSTEM, IN THE BNT CELLS. THERE IS A LOT OF APOPTOSIS IN THE INTESTINE. TISSUES THAT ARE DIVIDED ARE UNDERGOING APOPTOSIS. 18 SO WE, AND MANY OTHER LABORATORIES, ARE VERY INTERESTED IN HOW IT WOULD BE THAT A SHORT TELOMER WOULD CAUSE A CELL TO DIE. AND SO IN A SERIES OF EXPERIMENTS I WON'T HAVE TIME TO GET INTO, THE MODEL THAT THE FIELD HAS DEVELOPED IS THAT A SHORT TELOMER S INDUCE DAMAGE RESPONSE. IF YOU HAVE DNA AND THAT UNDERGOES A BREAK, THAT BROKEN REGION IS RECOGNIZED BY SPECIFIC PROTEINS. AND THEY WILL SIGNAL THROUGH P53 AND THE CELLS CAN UNDERGO EITHER APOPTOSIS OR [INDISCERNIBLE] IN THE CASE OF SHORT TELOMER, THESE ARE PROTECTED BY THE BINDING PROTEINS I TOLD YOU WITHOUT. WHEN THE TELOMERS GET TO BE -- TELOMERS GET TO BE TOO SHORT, THEY NO LONGER FUNCTION. AND THEY'RE RECOGNIZED BY A PARTICULAR SET OF PROTEINS THAT RECOGNIZES THIS AS A SHORT TELOMER. THIS SIGNALS THROUGH P53 AROUND THE CELLS UNDERGO APOPTOSIS. WE'RE INTERESTED IN UNDERSTANDING THAT LEAD THE SHORT TELOMERs TO INDUCE THIS APOPTOSIS. WE'RE INTERESTED IN THE CELLULAR CONSEQUENCES OF THE SHORT TELOMER. AS I TOLD YOU THE TELOMERASE IS ESSENTIAL FOR ALL KINDS OF CELLS THAT HAVE TO DIVIDE MANY TIMES. SO ONE EXAMPLE OF THAT, CANCER CELLS. IF YOU HAVE A PARTICULAR TISSUE, AND ONE CELL UNDERGOES WITH A GENETIC MUTATION THAT ALLOWS IT TO DIVIDE MANY MORE TIMES. YOU CAN SEE THAT THOSE CELLS ARE GOING TO DIVIDE RELATIVELY MANY, MANY MORE TIMES THAN THE OTHER CELLS WOULD DIVIDE. 19 AND WE WERE ABLE TO SHOW THAT THESE CANCER CELLS NEED TO HAVE TELOMERASE IN ORDER TO CONTINUE AND DIVIDE. THE SECOND SITUATION IS IN STEM CELLS, TISSUE SPECIFIC STEM CELLS WHERE YOU HAVE A PARTICULAR STEM CELL THAT HAS TO UNDERGO SELF-RENEWAL, BUT ALSO DIFFERENTIATION. THAT CELL DIVISION GIVES RISE TO MANY MANY DIFFERENT TYPES TO UNDERGO TISSUE RENEWAL. IT'S ESSENTIAL FOR THIS TISSUE RENEWAL. I'LL TELL YOU ONE STORY ABOUT CANCER CELLS AND THEN FOCUS THE REST OF THE TALK HERE ON WHAT HAPPENS WHEN YOU DON'T HAVE ENOUGH TELOMERASE FOR TISSUE RENEWAL. SO ONCE WE HAD GENERATED A TELOMERASE KNOCKOUT MOUSE, WE, WITH WHOM WE WERE COLLABORATING, SET OUT TO ASK WHAT IS THE ROLE OF SHORT TELOMERS IN THE ABILITY OFFER CANCER CELLS TO DIVIDE. WE CROSSED THE TELOMERASE TO A NUMBER OF MOUSE MODELS OF VARIOUS KINDS OF CANCER. AND THIS IS JUST A TABLE THAT SUMMARIZES SOME OF THE FINDING. THE EFFECTIVE SHORT TELOMERS WAS TO DECREASE THE RATE OF GROWTH OF THOSE TUMOR CELLS AND IN SOME CASES WHEN WE COULD LOOK AT THE MECHANISM, IT WAS CLEAR THAT APOPTOSIS WAS BEING INCREASED AND THAT WOULD, THEN, DECREASE THE NUMBER OF TIMES THAT THE CELLS COULD DIVIDE. SO WE WANTED TO FOLLOW UP ON THESE MODELS THAT SUGGESTED THAT SHORT TELOMERs LIMIT GROWING OF CANCER CELLS. WE WANTED TO LOOK AT SPECIFIC MECHANISMS AND THE PATH WAY BY WHICH THE SHORT TELOMERS MAY BE TRIGGERING THE CELL DEATH. AND SO DAVID, WHO IS A GRADUATE 20 STUDENT IN THE LAB, DECIDED TO USE ANOTHER MODEL OF TUMOR [INDISCERNIBLE] THAT'S THE B CELL MODEL IN WHICH THE [INDISCERNIBLE] -- THE ONCOGENE WAS DRIVEN BY THE PROMOTER IN B CELLS. SO WHAT HE DID WAS TO GENERATE A TRANSGENIC MOUSE AND PUT IT ON TO THE GENETIC BACKGROUND WITH THE HETEROZYGOUS MUTATION. WHEN YOU BREED THESE YOU CAN SELECT FOR THE TRANSGENIC G1 MICE. THEN BREED THOSE. ALWAYS SELECTING FOR THE TRANSGENE, BREED THE ENTIRE LINE OF MICE DOWN LIKE THIS. AND THEN WE COULD ASK ABOUT THE RATES OF TUMOR FORMATION IN THE DIFFERENT LINES OF MICE. >> AND WHAT DAVID SAW WAS REALLY QUITE STRIKING. AND THAT WAS THAT THE SHORT TELOMERS REALLY LIMIT GROWTH OF THE TUMOR CELLS. THIS PROTECT THE MICE AND INCREASE THEIR SURVIVAL. SO WHAT IS SHOWN HERE, THE PERCENTAGE OF MICE THAT WERE ALIVE. AND IF YOU LOOK AT THE [INDISCERNIBLE] MICE ON THEIR OWN, WILDTYPE FOR TELOMERASE, SHOWN ON THE BLACK LINE, YOU CAN SEE THAT THE SURVIVAL IS SIGNIFICANTLY DECREASED AND BY 100 DAYS, HALF OF THE MICE HAVE DIED OF THIS B CELL LYMPHOMA. WHEN YOU LOOK AT THE MICE THAT WERE THE IMMUNIC TRANSGENE AND NULL FOR TELOMERASE, THE FIRST GENERATION, THE LONG TELOMER MICE NOW, WHAT HE FOUND WAS THERE WAS NO CHANGE. THERE WAS A VERY SIMILAR RAPID DECLINE. SO THE B CELL LYMPHOMA IS GROWING VERY RAPIDLY, AND WILL KILL THESE MICE. 21 HOWEVER, WHEN YOU LOOK IN THE MICE THAT HAD THE VERY SHORT TELOMERs, YOU FIND THERE WAS A SIGNIFICANT PROTECTION AGAINST THE B CELL LYMPHOMAS. YOU CAN GO INTO THESE MICE AND LOOK AT THERE ARE VERY SMALL MICROLYMPHOMAS THAT START GROWING BUT THEN THEY STOP. SO THIS, AGAIN, MAKES THE POINT HERE THAT IT MUST BE NOT THE ABSENCE OF TELOMERASE BUT RATHER THE SHORT TELOMERs THAT HAVE TO BE CAUSING THIS EFFECT. THESE MICE ARE NULL FOR TELOMERASE AND THESE ARE, THE ONLY DIFFERENCE BETWEEN THIS COHORT AND THIS ONE IS THAT THESE HAVE SHORT TELOMERs. THEY HAVE BEEN BRED FOR MANY GENERATIONS. THE CONCLUSION, IT'S THE SHORT TELOMERs, AND LIMITING THE GROWTH OF THIS B CELL LYMPHOMA. ONE OF THE NICE THINGS, IT ALLOWS YOU TO HAVE ACCESS TO THE CELLS THAT ARE THESE PRE B CELL LYMPHOMAS TO ACCESS QUESTIONS ABOUT PATHWAYS. DAVID WANTED TO ASK WHAT HAPPENS IF YOU HAVE A SHORT TELOMER, AND WE KNOW THAT THAT SIGNAL IS THROUGH P53 AND THE CELLS UNDERGOING APOPTOSIS. WHAT HAPPENS IF YOU BLOCK APOPTOSIS? HE WAS ABLE TO IN THIS MODEL OVEREXPRESS THE Bcl2 ONCOGENE WHICH BLOCKS THE PATHWAY. TO OUR SURPRISE, WHAT WE FOUND IS THAT THE MICE WERE STILL PROTECTED, THE SHORT TELOMER UNTIL PROTECTED THE MICE AGAINST THE B CELL LYMPHOMA. THE YELL STOPPED GROWING. DAVID WAS ABLE TO GO ON AND SHOW WHEN YOU BLOCK THE APOPTOSIS THAT COMES FROM P353. THAT CELLULAR [INDISCERNIBLE] WILL OCCUR AND THIS WILL ALSO LIMIT THE TUMOR GROWTH. 22 THE TELOMERs CAN OPERATE THROUGH BOTH THESE PATHWAYS IN ORDER TO BLOCK THE GROWTH OF THESE TUMOR CELLS. SO THE SECOND PLACE WHERE TELOMERs ARE ESSENTIAL IN CELLS THAT HAVE TO DIVIDE IS IN THIS QUESTION OF TISSUE RENEWAL. AND THIS REALLY CAME TO THE FOREFRONT WHEN A PAPER WAS PUBLISHED IN 2001 BY [INDISCERNIBLE] GROUP. THIS WAS A PAPER PUBLISHED IN NATURE. AND IT'S ENTITLED THE RNA COMPONENT OF TELOMERASE IS MUTATED IN [INDISCERNIBLE] CONGENITA. THIS IS A GROUP IN ENGLAND WHO WERE DOING FAMILY STUDIES AND MAPPING GENES THAT WERE INVOLVED IN THIS PARTICULAR INHERITED HUMAN DISORDER. AND WHAT THEY FOUND WAS THAT IN THEIR MAPPING STUDIES, IT WAS THE RAN COMPONENT, WHICH WAS TRACKING WITH THE DISEASE IN THEIR FAMILIES. WHAT IS IT? SOME OF THE CLINICAL FEATURES, THERE ARE CRITERIA WHICH BY THE DISEASE GETS ITS NAME. THERE IS SKIN, HYPERPIGMENTATION, AND ABNORMAL GROWTH OF THE NAMES. BUT THE MORTALITY OF THE DISEASE WAS PRIMARILY ATTRIBUTED TO APPLIESIC ANEMIA OR BONE MARROW FAILURE. THERE IS ALSO A PULMONARY FIBROSIS, AS WELL AS INCREASED CANCER RISK WHICH INCREASES THE MORTALITY IN THIS DISEASE. OUR HYPOTHESIS GIVEN THAT THE HUMAN TELOMERASE WAS IMPLICATED HERE, IS THAT THE WE WANTED TO ASK SPECIFIC QUESTIONS ABOUT THIS DISEASE MODEL AND ASK WHAT IS THE ROLE OF THE SHORT TELOMERS. 23 THERE ARE SEVERAL MODES OF INHERITANCE. WHAT I WAS REFERRING TO IS THE [INDISCERNIBLE] PREVIOUSLY, THE GROUP HAD MAPPED THE GENE THAT ENCODING THE L LINKED FORM. THAT GENE THEY CALLED [INDISCERNIBLE]. AND IT TISSUE TURNS OUT, IT HAS A LINK. SO THE GROUP HAD SHOWN THE RNA COMPONENT IS MUTATED. AND IN WORK THAT WE HAD DONE BIOCHEMICALLY ON THE TELOMERASE ENZYME, WE HAD FOUND THAT THERE IS THIS SPECIFIC SYSTEM LOOP IN THE RNA. A BOX H, ACA BINDING DOMAIN, TO WHICH THIS BINDS. SO IT TURNS OUT THE X LINKED FORM OF THE DISEASE, YOU DON'T HAVE THE BINDING, YOU DESTABLIZE AND YOU GET THIS EFFECT ON TELOMERs. THEY SHOWED MUTATIONS IN THE RNA CAN LEAD TO [INDISCERNIBLE] AND I'LL SHOW YOU THAT MUTATIONS IN THE PROTEIN COMPONENT WHICH WE CALL TERT, ALSO LEAD TO DISKEROTASIS CONGENITA. WHEN A PATIENT CAME INTO THE CLINIC AT JOHNSON HOPKINS WITH APLASTIC ANEMIA, IT WAS APPARENT THIS WAS A FAMILY WITH DISKEROTASIS CONGENITA. THE PREVIOUS EXAMPLES WERE MAPPED TO THE RNA, AND [INDISCERNIBLE] AT THE TIME WAS A FELLOW IN THE LAB, AT HER OWN LAB AT HOPKINS, WAS ABLE TO FOLLOW THIS FAMILY AND TO STUDY IF INHERITANCE OF THIS DISEASE WITHIN THE FAMILY. AND I WANT TO MAKE A COUPLE OF POINTS FROM THIS PARTICULAR PEDIGREE. FIRST OF ALL, THAT THE GROUP, AS WELL AS [INDISCERNIBLE] SHOWED THERE IS A GENETIC ANTICIPATION IN THIS DISEASE. 24 THAT IS, THAT WITH EACH GENERATION, THERE IS AN EARLIER ONSET AND WORSENING ON PHENOTYPES WITHIN THIS FAMILY. AND THIS SHOULD REMIND YOU OF THE TELOMERASE KNOCKOUT MICE, WHERE WE SEE WORK PHENOTYPES IN THE LATER GENERATIONS. THE OTHER THING IS THAT THE EFFECTED PEOPLE ARE HETEROZYGOUS FOR MUTATIONS, EITHER THE RNA COMPONENT OR IN THIS PARTICULAR FAMILY IT WAS A PROTEIN COMPONENT WHICH WAS MUTATED. AS A GENETICIST, INTERESTED A LOT. THAT SUGGESTS THAT EITHER THERE IS A DOMINANT NEGATIVE EFFECT. SO IT COULD BE THAT IF YOU HAVE 2 ALLYLS OF TELOMERASE T MUTANT ALLYL IS INTERACTING WITH THE WILDTYPE OR TAKING THAT OUT. THAT'S A DOMINANT NEGATIVE EFFECT. IF YOU HAVE A WILDTYPE AND IT'S NOT SUFFICIENT TO HAVE ONLY WILDTYPE FUNCTION, THAT'S [INDISCERNIBLE]. SO WE REALIZED WE WERE IN A SITUATION WHERE WE COULD TEST THIS VERY RIGOROUSLY, USING OUR TELOMERASE KNOCKOUT MOUSE. SO WE SET OUT TO ASK THE QUESTION WHETHER OR NOT INSUFFICIENCY WAS A MECHANISM BY WHICH THIS DISEASE FUNCTION WAS CARRIED OUT. I'LL SHOW YOU IN FACT THAT IT IS DUE TO SUFFICIENCY. SO THE MOUSE MODEL THAT I HAVE BEEN TELLING YOU ABOUT UP UNTIL NOW IS THE TELOMERASE KNOCKOUT MICE ON THE C57 BLACK 6 GENETIC BACKGROUND. THIS IS A SOUTHERN BLOT, BECAUSE MICE TELOMERs ARE VERY, VERY LONG. YOU CAN SEE THIS SMEAR, WHY WE QUANTITATE THEM USING THIS FISH ASSAY. , HOWEVER, THERE A NUMBER OF 25 SPECIES OF MICE. THESE WERE ALL WILDTYPE MICE. THEY WERE SIMILAR TO HUMAN TELOMER LENGTHS. WHAT WE DID WAS CROSS THE ALLYL ON TO THIS BACKGROUND HERE OF THIS CAST EI WHICH HAS HUMAN LIKE TELOMER LENGTHS. WE WERE DOING THAT IN ORDER TO DO VARIOUS OTHER STUDIES. WE RECOGNIZE THAT HAVING THESE MICE WITH THE KNOCKOUT ALLYL ON THE OTHER GENETIC BACKGROUND WOULD ALLOW US TO TEST OF QUESTION OF INSUFFICIENCY. SO WHAT WE WERE ABLE TO DO, THEN, IS ONCE WE HAD THIS NOW BACK CROSSED FOR 7 GENERATIONS ON THE BACKGROUND, IT'S NOW DO A CROSS LIKE WE HAD DONE BEFORE. BUT RATHER THAN FOLLOWING THE NULL MICE FOR PROGRESSIVE GENERATIONS, WE KEPT THE TELOMER HETEROZYGOUS. THIS STANDS FOR HETEROZYGOUS GENERATION 1, 2, 3, 4, ET CETERA. AND ASKED THE QUESTION WHAT HAPPENS WITH THE TELOMER LENGTH. WHAT WE FOUND, THERE WAS PROGRESSIVE TELOMER SHORTENING IN THE HETEROZYGOUS STATE. SO WHAT IS SHOWN HERE IS THIS QUANTITATIVE FISH ASSAY. WHILE TYPE IS SHOWN HERE IN THE BLACK BARS. THIS IS THE SIGNAL INTENSITY THESE ARE THE NUMBER OF ENDS THAT HAVE IT. WE'RE FOCUSING ON THE LOWER END OF THE DISTRIBUTION HERE. EVERYTHING ON THE UPPER END IS PUT INTO THIS LAST BIN WHICH IS WHY YOU SEE THIS HIGH BAR HERE. WHEN WE LOOK AT -- THERE IS PROGRESSIVE SHORTENING IN THIS TELOMER LENGTH DISTRIBUTION WITH INCREASING GENERATIONS, SHOWING THE LEVEL OF THE TELOMER LENGTH, 26 IT CAN CONTRIBUTE TO THIS DISEASE. NOW, OF COURSE, WHEN WE'RE BREEDING THESE MICE, EVERY TIME YOU BREED AND LOOK AT THE HETEROZYGOUS, YOU OF COURSE GET THE THE KNOCKOUT AT EACH DIFFERENT GENERATION. AND IN STUDYING THE COLONY OF MICE, WE WERE ABLE TO FIND THAT THERE IS ACTUALLY A DECREASED SURVIVAL DEPENDING ON WHAT GENERATION THE KNOCKOUTS CAME FROM. SO THIS IS A SURVIVAL CURVE. DAYS VERSES NUMBER OF MICE ALIVE. IN THE WILDTYPE, YOU CAN SEE THE COLONY IS DOING QUITE WELL. IF YOU LOOK AT KNOCKOUTS THAT COME FROM 3 PARENTS, THEY HAVE A SLIGHT DECREASE. 6 VERSES GENERATION 8 IS SHOWN HEAR AND HERE. THERE IS A REAL DRAMATIC INCREASE IN SURVIVALS A THEY ARE BRED WHEN THE PARENTS, THEN, HAVE SHORTER AND SHORTER TELOMERs. THIS INDICATES THAT THE INHERITANCE OF SHORT TELOMERs DECREASES THE SURVIVAL IN THESE MOUSE AND RESEMBLED THE GENETIC ANTICIPATION THAT'S SEEN DISKEROTASIS CONGENITA IN THIS HETEROZYGOUS STATE. WHEN WE'RE BREEDING THESE MICE, WE BREED THEM AND KNOW THE TELOMERs ARE SHORTENING. EVERY TIME WE DO THIS, WE GET A KNOCKOUT AS WELL AS THE WILDTYPE. WE WERE VERY CURIOUS TO KNOW WHAT HAPPENS IN THESE WILDTYPE MOUSE WHEN THEY HAVE INHERITED SHORT TELOMERs THROUGH MANY GENERATIONS. I'LL SHOW YOU THAT THESE MICE HAVE VERY SHORT TELOMERs, EVEN THOUGH THEY'RE WILDTYPE. 27 SO AGAIN, I'M SHOWING YOU REFERENCE, THE WILDTYPE LENGTHS AND THE KNOCKOUT GENERATION 1. I'VE ALREADY SHOWN YOU HERE IN THIS GREEN THAT THE HETEROZYGOUS GENERATION 5 -- IN THE BLUE, THE GENERATION 5 HAS SHORTER TELOMERS, SURPRISINGLY, THE WILDTYPE 5 LITTER MATES HAD SHORT TELOMERs. WE GET A KNOCKOUT, HETEROGENEOUS GENERATION 5. WE CALL THIS 5 STAR BECAUSE THESE MICE ARE NOT ACTUALLY WILDTYPE. IT'S THE SHORT TELOMERs THAT ARE CAUSING THE DISEASE IN THESE FAMILIES. THAT IT'S NOT THE ABSENCE OF TELOMERASE PER SE. SO IN THIS PARTICULAR SETTING, TELOMERASE IS COMPLETELY RESTORED TO WILDTYPE AND SO WE WERE VERY SURPRISED WHEN WE, THEN, LOOKED AT THE PHENOTYPE IN THESE MICE AND FOUND THAT THERE WAS A SIGNIFICANT DECREASE IN THE TESTEES WEIGHT AND INCREASE IN [INDISCERNIBLE]. SO THESE NORMAL WILDTYPE MOUSE COMPARED TO 4 STAR MICE, THERE WAS A PHENOTYPE IN THESE MICE, AGAIN, INDICATING THAT IT'S THE SHORT TELOMERs THAT ARE CAUSING THE EFFECT, EVEN WHEN TELOMERASE IS RESTORED TO THE WILDTYPE SETTINGS. WE CALLED THIS GENETIC DISEASE IN THE ABSENCE OF TELOMERASE MUTATION OR FORM OF CULT GENETIC DISEASE. THIS IS PARTICULARLY IMPORTANT, BECAUSE THESE ARE THE ALLYLS THAT ARE NORMALLY MUTATED IN THE HUMAN INHERITED SYNDROMES. THIS SUGGESTS THAT IN FAMILIES WITH THIS DISEASE, THERE MAY BE PEOPLE WHERE YOU CAN SEQUENCE THE GENES THAT ARE CONTRIBUTING TO TELOMER LENGTH ALL YOU WANT, 28 BUT YOU'LL FIND THEY ARE WILDTYPE. IT'S THE ACTUAL TELOMERs THAT'S CONTRIBUTING TO THE DISEASE. A FORM OF HIDDEN OR A CULT GENETIC DISEASE. WE THINK THAT THIS IS IMPORTANT TO INFORM IN TERMS OF THE CLINICAL SETTING WHERE THE TELOMER LENGTH MAY BE THE THING THAT MATTERS. NOW, WHENEVER I SHOW THIS ABOUT THE WILDTYPE TELOMER LENGTHS BEING SHORTER, PEOPLE ALWAYS ASK ACTION YEAH, BUT WHAT WOULD HAPPEN IN THE NORMAL SETTING, THAT IS, CAN YOU EVER GET THESE TO BE BACK TO NORMAL WILDTYPE TELOMER LENGTHS. WE UNDERTOOK THIS EXPERIMENT WHICH TOOK OF 3 YEARS OF MOUSE BREEDING. WE ASKED THE QUESTION ABOUT WHETHER THE NORMAL WILDTYPE LENGTH EQUILIBRIUM COULD BE REESTABLISHED. WE TOOK THEY SEE MICE AND CROSSED THEM TO EACH OTHER. AND WE GOT OUT THESE WILDTYPE 5 STAR MICE THAT HAVE PHENOTYPES NOT ONLY IN THE TESTIS, BUT AS WELL AS THE INTESTINE AND THE IMMUNE SYSTEM. NOW, WE CROSSED THESE MILES TO EACH OTHER TO -- MICE TO EACH OTHER. HOW MANY GENERATIONS IN ORDER TO RESTORE THE GENETIC DETERMINED TELOMER. AFTER 4 GENERATIONS WE DID RESTORE THIS GENETICALLY DEFINED TELOMER LENGTH EQUILIBRIUM. HERE IS THE 4 MOUSE IN RED COMPARED TO WILDTYPE MICE IN BLUE. THE WILDTYPE 5 STAR T WHICH IS 2 GENERATIONS OF BREEDING WILDTYPE MICE, TO WILDTYPE MICE, HASN'T GOTTEN BACK UP TO THE NORMAL 29 GENETICALLY DEFINED EQUILIBRIUM. WHEN WE GET TO WILDTYPE 5 STAR 4 WE HAVE OVERLAPPING TELOMER LENGTH DISTRIBUTIONS. THIS INDICATES THAT IT'S A SLOW PROCESS, THAT WE KNOW THAT TELOMERASE IS LIMITING BECAUSE HALF THE LEVEL OF TELOMERASE ISN'T SUFFICIENT TO MAINTAIN TELOMERs IN THESE INHERITED SYNDROMES. AGAIN, TELOMERASE IS LIMITING. IT TAKE 4 GENERATIONS OF BREEDING WITH WILDTYPE LEVELS TO WE ESTABLISH THE NORMAL TELOMER LENGTH EQUILIBRIUM. THEN WHEN WE LOOK AT WHAT HAPPENS WITH THE PHENOTYPES IN THESE MICE, IF YOU LOOK AT THE TESTIS TUBULES, I SHOWED YOU THE HETEROGENEOUS THEY HAVE THEM, THE WILD STAR MICE DO, BUT WHEN YOU RESTORE THE LENGTH IT COMES BACK TO NORMAL. THE PHENOTYPES GO TO THE TELOMER LENGTHS. WE HAVE BEEN VERY INTERESTED IN THE QUESTION OF WHAT ARE THE ROLE OH TELOMERASE IN DISEASE AND HOW THE TELOMERASE RNA AND THE PROTEIN COMPONENT CONTRIBUTES TO THIS GENETIC ANTICIPATION THAT IS SEEN IN THIS INHERITED SYNDROME. IN THE LAST FEW YEARS, THERE HAVE BEEN SUGGESTIONS FROM A NUMBER OF DIFFERENT GROUPS THAT INDICATE THAT THE PROTEIN COMPONENT OF TELOMERASE MAY HAVE ADDITIONAL ROLES BESIDES THE ELONGATION ROLE THAT I HAVE BEEN TALKING ABOUT. THIS COMES FROM OVEREXPRESSION STUDIES THAT SUGGEST THAT THIS PROTEIN COMPONENT HAS TELOMER LENGTH INDEPENDENT FUNCTIONS. IN ONE STUDY IT WAS FOUND THAT THE TERT COMPONENT REGULATES CHROMATIN AND DNA DAMAGE RESPONSE. 30 IN ANOTHER, IT WAS PROPOSED THAT OVEREXPRESSION OF TERT PROMOTES SELF PROLIFERATION. HERE IS ANOTHER STUDY INDICATING THAT THE OVEREXPRESSION OF TERT MODULATES THE SIGNALING BATH WAY. WE THOUGHT IT WAS IMPORTANT TO UNDERSTAND, IF THERE IS A SECONDARY ROLE OF TELOMERASE. IN THESE FAMILIES THAT HAVE MUTATIONS WE WANT TO KNOW ABOUT THE OTHER ROLES THAT TELOMERASE MAY PLAY THAT ARE INDEPENDENT OF TELOMER LENGTH. SO ALTHOUGH THESE WERE OVEREXPRESSION STUDIES, WE WANTED TO LOOK IN THE PROTEIN COMPONENT KNOCKOUT AND ASKED WHAT HAPPENED IN THAT SETTING. WOULD WE SEE DIFFERENT PHENOTYPES THAT WOULD HAVE TO DO WITH THE TELOMER LENGTHS, INDEPENDENT ROLES OF TELOMERASE. SO ONE OF THE FIRST THINGS WE DID WAS A GENE EXPRESSION ARRAY. AND WE ARE USING NOW HERE THE BLACK 6 MICE THAT HAVE VERY, VERY, VERY LONGS, WHERE WE SEE NO PHENOTYPE AT ALL. THIS IS SIMPLY LOOKING AT TERT KNOCKOUT G1 MICE GENE EXPRESSION COMPARED TO WILDTYPE. WHAT YOU ARE LOOKING AT IS WHAT'S CALLED A VOLCANO PLOT. WE ARE AVERAGING A NUMBER OF BIOLOGICAL REPLICATES. SO THESE ARE THE NORMAL LEVELS OF GENE EXPRESSION, AND ANYTHING THAT WOULD BE DIFFERENT BETWEEN THE G1 AND WILDTYPE WOULD BE FOUND IN THESE QUADRANTS HERE. IN BOTH THE CASE OF KNOCKING OUT THE RNA COMPONENT, OR KNOCKING OUT THE PROTEIN COMPONENT, WE FOUND NO CHANGE IN THE GENE EXPRESSION PROFILE IN THE KNOCKOUT SETTING. AND WE SPECIFICALLY LOOKED AT Wnt SIGNALING BATH WAY -- 31 PATHWAY GENES. THEY ARE IN HERE BUT NOT ALTERED. WE LOOKED AT THE DNA DAMAGE RESPONSE. WE TOOK MICE KNOCKED OUT FOR THE RNA OR PROTEIN COMPONENT. THESE ARE FIRST GENERATION MICE. AND WE EITHER DIDN'T IRRADIATE THEM OR WITH 5 AND LOOKED AT THE INDUCTION OF THE DNA DAMAGE RESPONSE. YOU CAN SEE A NICE INDUCTION OF THE DNA DAMAGE RESPONSE HERE. SAME THING IN THESE TERT KNOCKOUT MICE. WHEN WE QUANTITATED THE NUMBER OF -- A T OF P53 INDUCTION IN EITHER NO IRRADIATION OR THE 5 GRAY, WE FOUND NO SIGNIFICANT CHANGES IN EITHER THE RNA COMPONENT KNOCKOUT OR THE PROTEIN COMPONENT KNOCKOUT. SO THEN WE WANTED TO ASK A LITTLE BIT MORE OF A SUBTLE QUESTION, WHICH WAS, IF WE DON'T SEE ANY CHANGES IN GENE EXPRESSION OR CHANGES IN THE DNA DAMAGE RESPONSE, MAYBE WE WOULD SEE SOMETHING AT THE LEVEL OF THE PHENOTYPE OF THE MOUSE. WE TOOK THE PROTEIN COMPONENT KNOCKOUT MICE, THIS TERT COMPONENT AND BACK CROSS IT ON TO THE BACKGROUND WHERE WE CAN SEE NICE PHENOTYPES IN THIS PARTICULAR GENETIC BACKGROUND. AND WE CAN LOOK, NOW, AT THE HETEROZYGOUS MICE, LOOK AT THE NULL MICE AS WELL. SO WE CAN COMPARE WHAT WE SEE IN THE RNA COMPONENT KNOCKOUT TO WHAT WE SEE IN THE PROTEIN COMPONENT KNOCKOUT. SO HERE IS THE RNA COMPONENT, AND THE PROTEIN COMPONENT. WHEN WE LOOK AT THE TELOMER LENGTH, AGAIN I'M SHOWING YOU HERE THE WILDTYPE TELOMER LENGTH VERSES RNA COMPONENT IN THE 32 FIRST GENERATION, THE WILDTYPE VERSES THE PROTEIN COMPONENT. THERE IS VERY SIMILAR LEVELS OF SHORTENING IN BOTH THE RNA AND PROTEIN COMPONENT KNOCKOUTS. WHEN WE ASK ABOUT INSUFFICIENCY, WE CAN LOOK AT TELOMER LENGTH IN THE WILDTYPE AND PROTEIN COMPONENT NULL. HERE IS THE WILDTYPE DISTRIBUTION. THE NULL, AND WHEN WE LOOK AT 2 INDEPENDENT HETEROZYGOUS, THESE ARE HETEROZYGOUS FOR THE PROTEIN COMPONENT. THE LENGTH IS INTERMEDIATE. AND WE'RE STILL EARLY DAYS IN REAL ANALYZING THIS COLONY THAT WE HAVE BEEN DEVELOPING FOR THE PAST FEW YEARS. WE CAN SEE IF WE LOOK AT SURVIVAL, THESE ARE THE WILDTYPE MICE SHOWN HERE, THE SURVIVAL CURVE IN OUR COLONY. AND WHEN YOU LOOK AT KNOCKOUTS FROM FIRST GENERATION FIRSTS KNOCKOUTS FROM SECOND GENERATION PARENTS, YOU CAN SEE THERE IS A DECREASE IN THE SURVIVAL IN THESE PROTEIN COMPONENT KNOCKOUTS. WE CAN LOOK AT SOME OF THE PHENOTYPES IN THESE MICE. ONE OF THE PHENOTYPES THAT WE HAVE BEEN ABLE TO LOOK AT IN BOTH MICE IS THE SKELETAL DEVELOPMENT, THE Wnt SIGNALING PATHWAY IS KNOWN TO BE INVOLVED AND IT HAS BEEN SUGGESTING THAT THE NUMBER OF RIBS APPARENT IN THIS KIND OF AN X-RAY MAY BE ALTERED IF THE Wnt SIGNALING PATHWAY IS ALTERED. HOWEVER, IN THE BLACK 6 CASE, LOOKING AT WILDTYPE OR THE NULL FOR THE TERT OR IN THE GENETIC BACKGROUND LOOKING AT WILDTYPE OR A NUMBER OF NULL ANIMALS WE HAVEN'T SEEN ANY DISRUPTIONS TO THE Wnt SIGNALING PATHWAY. 33 SO THE PATHOLOGY THAT WE DO SEE IN THE TERT KNOCKOUT MICE, IN THE TESTEES, WE SEE [INDISCERNIBLE] LIKE WE SAW IN THE RNA. WE SAW THE INTESTINAL EPITHELIUM. WE SEE ATROPHY. THERE IS SOMETHING CALLED, THIS IS WHEN THERE ARE BONE MARROW FAILURE, AND HEMATOPHORESIS STARTS HAPPENING IN THE LIVER, SPLEEN, AND OTHER ORGANS. SO OUR UNLESS SO FAR IS THAT THE PHENOTYPE OF THE RNA COMPONENT AND THE PROTEIN COMPONENT KNOCKOUT MICE IS SIMILAR, INDICATING THAT SHORT TELOMERs ARE THE THINGS THAT ARE CAUSING THESE PHENOTYPES. THIS IS IMPORTANT TO KNOW FROM THE CLINICAL SETTING IN TERMS OF DISEASES YOU MIGHT ANTICIPATE IN FAMILIES THAT HAVE THESE INHERITED MUTATIONS. SO NOW JUST TO FINISH UP, I JUST WANTED TO POINT OUT, MANY OF THE PHENOTYPES FROM THESE SYNDROMES SHARE FEATURES OF AGE RELATED DISEASE. I TOLD YOU ABOUT DISKEROTASIS CONGENITA. THERE ARE A NUDGE OF OTHER DISEASES ASSOCIATED IN THESE FAMILIES. SO THERE IS INHERITED BONE MARROW FAILURE OR FAMILIAL APLASTIC ANEMIA. IMMUNE SENESCENCE. THERE IS SIGNIFICANT RATE OF PULMONARY FINE PROCESS, LIVER CREASE, AND INCREASED CANCER INCIDENTS. THESE ARE ALL THINGS THAT ARE SEEN IN THESE FAMILIES THAT HAVE INSUFFICIENT TELOMERASE. WHERE YOU HAVE A PARTICULAR GENETIC DISEASE, THAT GENETIC DISEASE WILL GIVE YOU THE STRONGEST CASE IN TERMS OF THE PHENOTYPE, BUT YOU MAY FIND THAT 34 THE PACT WAYS THAT ARE DISRUPTED ARE PRESENT IN OTHER INDIVIDUALS. SO WE THINK THAT BY STUDYING THESE FAMILIES THAT HAVE INSUFFICIENT TELOMERASE, IT MAY TELL US ABOUT WHAT HAPPENS IN INDIVIDUALS THAT HAVE SHORTS, EVEN WHEN THEY HAVE -- SHORT TELOMERS. SHOW IT IMPLIES IT PLAYS A ROLE IN CERTAIN AGE RELATED DISEASES WITHOUT FRANK TELOMERASE MUTATIONS. THESE ARE SITUATIONS WHERE THERE ARE TISSUES OF HIGH TURNOVER. SO I WANT TO LEAVE YOU WITH THIS SLIDE HERE WHICH SHOWS THE VERY WIDE DISTRIBUTION OF TELOMER LENGTH IN THE HUMAN POPULATION. IT'S BEEN KNOWN FOR SOME TIME THE IF YOU LOOK AT TELOMER LENGTH IN HUMANS VERSES AGE, AND THIS IS IN TOTAL LEUKOCYTES, NORMAL WHITE BLOOD CELLS THAT IS THERE IS A PROGRESSIVE DECLINE IN AGE. THIS HAS TO DO WITH THE FACT THAT THERE IS LIMITING AMOUNT OF TELOMERASE IN YELLS. THE NUMBER OF CELL DIVISIONS THAT OCCUR OVER THE LIFETIME OF AN INDIVIDUAL OUTSTRIPS THE ABILITY OF THE TELOMERASE WHICH IS CLEARLY ACTIVE IN THESE CELLS, BUT IS NOT ENOUGH TELOMERASE. THE POINT I WANT TO MAKE ABOUT THIS IS THE HUGE AMOUNT OF RETINOPATHY IN THE -- HETEROGENEITY. WHAT WE ARE FOCUSING ON IS THE PARTICULAR SUBSET OF INDIVIDUALS WHICH MAY HAVE A VARIETY OF DIFFERENT GENETIC INPUT AS WELL AS ENVIRONMENTAL INPUTS, BECAUSE WE'RE TALKING ABOUT TISSUES OF HIGH TURN OVER. WHERE THERE IS A LOT OF CELL DIVISION. 35 ANY SITUATION WHERE YOU MAY HAVE AN INSULT, FOR INSTANCE, TO THE IMMUNE SYSTEM AND YOU HAVE TO DIVIDE MORE TIMES. THAT'S GOING TO CAUSE MORE SHORTENING. AS A COMBINED EFFECT OF BOTH THE GENETIC INITIAL TELOMER LENGTH AS WELL AS ENVIRONMENTAL HISTORY THAT MAY PUT INDIVIDUALS WITH SHORTER TELOMERs AT THAT HIGHER RISK FOR CERTAIN AGE ASSOCIATED DEGENERATIVE DISEASES. THE COMBINED EFFECT OF LENGTH AND ENVIRONMENTAL HISTORY COULD CONTRIBUTE TO THESE AGE RELATED DISEASES. SO JUST TO SUMMARIZE WHAT I'VE TOLD YOU, I'VE TOLD YOU THAT TELOMERASE IS ESSENTIAL FOR TELOMER MAINTENANCE. TELOMER SHORTENING LEADS TO SELL DEATH AFTER MANY CELL DIVISIONS. SHORT TELOMERs INHIBIT TUMOR GROWTH THROUGH APOPTOSIS, AND THIS SUGGESTS THAT TELOMERASE INHIBITERS MAY BE EFFECTIVE IN CERTAIN KINDS OF CANCER THERAPY. I'VE SHOWN YOU THAT INSUFFICIENCY FOR TELOMERASE CAUSES TELOMER SHORTENING. SHORT TELOMER SAID LIMIT CELL GROWTH, EVEN IN THE PRESENCE OF TELOMERASE. SHORT TELOMERs MAY LIMIT THE LONG TERM CELL DIVISION POTENTIAL. INHERITANCE OF SHORT TELOMERs CAUSES PHENOTYPE EVEN IN WILDTYPE ANIMALS. THESE ARE STAR OR SO-CALLED CULT GENETIC DISEASE. SHORT TELOMERS CAUSE TISSUE RENEWAL IN NORMAL AGING POPULATION. TELOMER LENGTH MAY PREDICT THE ONSET OF CERTAIN AGE RELATED DISEASE. I WANT TO GIVE CREDIT TO THE 36 PEOPLE IN MY LIFE -- YES, IN MY LIFE, ALSO IN MY LIFE. IN MY LAB. THEY'RE IN MY LIFE. WHO HAVE DONE THESE EXPERIMENTS. DAVID DID THE EXPERIMENTS WITH THE MICE SHOWING THE APOPTOSIS IN THE CANCER CELLS. MARGARET STRONG, TAMMY MORRIS, WORKING WITH THE MILES TO UNDERSTAND THE ROLE OF TUMOR DYSFUNCTION. OUR COLLABORATORS AT HOPKINS, [INDISCERNIBLE] AND IN PATHOLOGY TO LOOK AT THE EFFECTS OF THE ABSENCE OF TELOMERASE, WE COLLABORATE ARE [INDISCERNIBLE]. SO I'LL STOP THERE. THANKS. [APPLAUSE] >> I'M SURE CAROL ARE TAKE A FEW QUESTIONS. WE HAVE MICROPHONES IN EACH OF THE I'LLS. AISLES. >> CONGRATULATIONS FOR PIONEERING WORK. THERE IS A DIFFERENCE IN THE TELOMER LENGTH IN DIFFERENT TISSUES. THIS IS AN ISSUE FOR THE BRAIN AND OTHER ISSUES WHERE THE [INDISCERNIBLE] IS LOW. >> IN SITUATIONS WHERE IT HAS BEEN LOOKED AT CLOSELY, IF YOU JUST LOOK AT DIFFERENT TISSUES YOU CAN FIND THERE ARE DIFFERENT TELOMER LENGTHS. OTHER QUESTION MAY BE IS THE SAME RATE OF SHORTENING. WE DON'T KNOW. THERE MAY NOT BE TISSUE SPECIFIC SHORTEN DIVISIONS. WHAT IS THE SET POINT AT WHICH A SHORT TELOMER CAUSES THESE PHENOTYPES. THESE ARE VERY IMPORTANT QUESTIONS. QUESTION HAVEN'T HAD A CHANCE TO GET AT. 37 THEY WILL BE ESSENTIAL TO ASK QUESTIONS ABOUT SOME OF THESE DISEASE SITUATIONS. >> FOR THE SAME CELL TYPE YOU HAVE STEM CELL AS WELL AS DIFFERENTIATED CELLS. THERE ANY WAY TO SEE THE DIFFERENCE IN THOSE 2 GROUPS? >> FOR THE SAME CELL TYPES, SO WE HAVEN'T SPECIFICALLY TAKEN A STEM CELL, TO ASK DOES A STEM CELL HAVE LONGER TELOMERs THAN THE IMMEDIATE DIFFERENTIATION PARTNERS. WE LOOKED IN THE GROWTH WAY AT THE PROGRESSIVE LOSS THROUGH DIFFERENT GENERATIONS. ONE OF THE THINGS ABOUT THESE ASSAYS IS THAT THERE IS SUCH HETEROGENEITY IN TELOMERLECT. WHAT HAPPENS, A LITTLE BIT OF SHORTENING, LENGTHENING, SHORTENING. TO GET A PRECISE MEASUREMENT IS DIFFICULT. YOU HAVE TO HAVE FAIRLY LARGE DIFFERENCES TO SEE A CHANGE. >> THANK YOU. >> AS WE GET CLOSER TO INDUCED PLURIPOTENT STEM CELLS, HAVE YOU HAD A CHANCE TO LOOK AT WHAT THE TELOMERS LOOK LIKE. THERE A WAY AFTER THE TELOMERs HAVE UNDER GONE SHORTENING DURING THESE EVENTS, CAN THEY BE REELONGGATED. >> THERE HAVE BEEN A VERY INTERESTING QUESTION. THERE EVERYBODY A NUMBER OF PAPERS BUT I THINK YOU HAVE IT RIGHT. WHAT WILL ACTUALLY HAPPEN IF THEY START OFF WITH SHORTS. ONE OF THE THINGS WE FOUND IS IT CERTAINLY STAKES A LARGE NUMBER OF GENERATIONS TO REESTABLISH A WILDTYPE TELOMER LENGTH. I THINK IT'S SOMETHING PEOPLE SHOULD PAY ATTENTION TO. BECAUSE IF YOU START OFF WITH 38 SOMETHING THAT HAS SHORT TELOMERS, AS SOON AS YOU PUT IT INTO A WILDTYPE SETTING, THAT THEY WILL BE ESTABLISHED BACK TO THE CORRECT LEVEL. IT'S VERY IMPORTANT THAT OVER A PERIOD OF TIME YOU MAY EXPECT IT TO BE REESTABLISHED WITH THE CORRECT EQUILIBRIUM, BUT IT WON'T BE THAT RIGHT AWAY. NOBODY IS REALLY TAKING A LOOK AT THE TELOMER LENGTH IN THE CELLS THEY'RE LOOKING AT. IT COULD BE HIGHLY VARIABLE. >> THANK YOU FOR A BEAUTIFUL TALK. I HAD -- IS THERE A POINT OF NO RETURN IN THE SHORTENING OF THE TELOMERs. BECAUSE THE WILDTYPE MICE YOU GET STILL HAVE [INDISCERNIBLE], WHICH MEANS THAT PRESENCE OF NORMAL LEVELS OF TELOMERASE IS NOT ENOUGH TO ASKEW THIS PHENOTYPE. THERE ANYTHING KNOWN ON THAT. >> IN THESE STUDIES, WE'RE STARTING WITH MICE THAT ARE ALREADY ALIVE, AT THE LEVEL WHERE THEY CAN REPRODUCE. CERTAINLY WHAT WE COULD EXPECT, XENA TELOMER GETS TO BE TOO SHORT AND SIGNALING DNA RESPONSE, THAT AT SOME POINT THOSE CELLS WON'T SURVIVE ANYMORE. WE DO KNOW THAT THERE IS A SIGNALING MECHANISM BY WHICH THE SHORT TELOMERs SPECIFICALLY RECRUIT TELOMERASE. SO THE SHORTEST GUESS REELONGATED MORE EFFICIENTLY THAN A MEDIUM LENGTH DOES. THERE ARE INHERENT RESCUE MECHANISMS BUILT IN. BUT I CAN'T REALLY ADDRESS THAT QUESTION BECAUSE WE'RE DEALING WITH THE ANIMALS THAT HAVE TO BE BREATHING IN ORDER TO DO THAT EXPERIMENT. 39 >> THANK YOU. >> THAT WAS WONDERFUL. I'VE READ THAT STRESS OR DEPRESSION OR CARDIOVASCULAR DISEASE, LOTS OF THINGS CAN CAUSE SHORTENING. CAN YOU EXPAND A LITTLE ON YOUR LAST POINT YOU MADE ABOUT THERE IS A GENETIC STARTING POINT, AND THEN THERE IS OTHER THINGS IN THE ENVIRONMENT. I THINK WE MAY BE THINK OF ENVIRONMENT IN CERTAIN WAYS, BUT MAYBE WE SHOULD BE THINKING IN OTHER WAYS ABOUT ENVIRONMENT AND TELOMERs. >> I THINK THERE IS, YOU KNOW, A LARGE NUMBER OF THINGS THAT ONE CAN THINK ABOUT. WE HAVE BEEN TAKING INITIALLY THE GENETIC APPROACH, ASKING WHAT SORT OF DISEASES DO YOU SEE. WHAT DO WE SEE IN THE MICE SUE GET A QUESTION ABOUT CAUSE AND EFFECT. AND WE CAN KNOW WHETHER IT WAS THE SHORT TELOMERs THAT CAUSED THAT. THERE IS A LARGE LITERATURE OF CORRELATION OF THINGS THAT CORRELATE WITH TELOMER LENGTH. THINGS THAT CORRELATE ARE NOT ALWAYS CAUSATIVE. I THINK THAT THE DOOR IS REALLY OPEN FOR A NUMBER OF DIFFERENT DISEASES. BUT WE'D LIKE THE TAKE THE GENETIC APPROACH AND ASK THE CAUSALITY KIND OF QUESTION. I THINK IT'S A TIP OF THE ICEBERG. IF YOU THINK ABOUT THE NUMBER OF AGE RELATED DISEASES THAT MAY HAVE TO DO WITH TISSUE TURN OVER, I THINK THAT USING THE GENETIC APPROACH WILL GET US TO KNOW MORE OF THOSE DISEASES THAT ARE GOING TO BE ASSOCIATED WITH TELOMER SHORTENING. 40 >> HI. ANY THOUGHTS ON WHAT THE MECHANISM IS THAT'S SENSING TELOMERIC EQUILIBRIUM AND WHAT'S GONE SO HAYWIRE. >> NO, WE DON'T, BUT WE KNOW IT'S THE SHORT TELOMERs, NOT THE TUMOR LENGTH. WE'RE ABLE TO DO A GENETIC CROSS TO BRING IN JUST A FEW SHORT TELOMERs, THOSE MICE HAVE THE PHENOTYPE. IT'S NOT RELATIVE TO AVERAGE LENGTH. THAT BRINGS US BACK TO THE DNA DAMAGE RESPONSE THAT THE SHORT TELOMERs ACTUALLY TRIGGER SOMETHING. IT'S NOT MEASURING -- YOU CAN IMAGINE IT WAS MEASURING BIND PROTEIN. THEN IT WOULD BE AVERAGE LENGTH THAT WOULD CAUSE THE EFFECT. WHY THE BLACK 6 MICE HAVE SUCH LONG TELOMERs IS A MYSTERY TO ME. WE LOOKED AT A NUMBER OF RECENTLY DERIVED WILD MICE. WE CAUGHT WILD MICE, GOT THEM FROM WWW.WILD MICE.COM. YOU HEAR PEOPLE SAY DON'T PAY ATTENTION TO THEM, THEIR TELOMERs ARE TOO LONG. THOSE LABORATORIES TEND TO USE THESE LONG TELOMERs. SOMEHOW THEY WERE ALLOWED TO GET LONG UNDER WHATEVER BREEDING CONDITIONS THEY WERE BRED UNDER. BUT CLEARLY THE NORMAL ESTABLISHED WILDTYPE LENGTH IS VERY TIGHT LENGTH. IN ORDER TO GET AT THOSE GENETIC COMPONENTS, IF THEY ARE GENETIC COMPONENTS, YOU NEED TO DO A VERY LARGE BREEDING. WE HAVEN'T BEEN ABLE TO FOLLOW THAT QTL ANALYSIS TO ASK ABOUT THE DIFFERENCES. I THINK IT WOULD BE AN INTERESTING QUESTION. 41 >> OKAY. WELL, ONCE AGAIN, I WANT TO THANK CAROL AND ONE THING I FORGOT TO MENTION IN MY INTRODUCTION, THIS IS THE FIRST SEMINAR SINCE GETTING THE NOBEL PRIZE WHICH IS PRETTY SPECIAL. AS A SMALL TOKEN WE WOULD LIKE TO SHARE WITH HER THIS SMALL GIFT THAT WE GIVE TO EACH OF OTHER LECTURES EACH YEAR. KATHY, IF YOU'LL COME UP ON STAGE WE'RE GOING TO DO ONE PHOTO OP, AND THEN EWE ARE ALL DONE. [APPLAUSE].

Contents

Classes

The order has only the superior classes, each of them with limited numbers:[2]

  • Collar (limited to 10)
  • Grand Cross (limited to 20)
  • Grand Officer (limited to 30)
  • Commander (limited to 40)

Insignia

Collar

The Collar is in Gold and consists of 8 links of the emblems of the Danubian Principalities of The: Principality of Wallachia, Principality of Moldavia, Principality of Oltenia and Principality of Dobruja, 4 emblems on either side of the collar with 2 of the emblems of the House of Hohenzollern between each two Principalities; between each emblem is the monogram of King Carol I.[3] At the back of the collar is the lock which is an Eagle with open wings which suspends in half to wear.[4] At the front of the collar is the Steel Crown of Romania which the badge of the order suspends from.[5]

Badge

On the obverse is the Red Maltese cross on top of the Gold sunray, in the middle of the Maltese Cross is a small Gold monogram of King Carol I.[7]

  • The Badge is featured on: The Collar, The Sash, The Medal and The Necklet.

Stars

There are two types of stars of the order: 1st which is for the Grand Cross with Collar/Grand Cross and the 2nd which is for the Grand Officer; both are to worn on the left stomach.

  • The 1st Star is in Gold which is 8-pointed, set in sunray's and is similar in shape of the Swedish Royal Order of the Seraphim; the Eagle which is on the Badge is on top of the star and set in Silver.[8]
  • The 2nd Star is also in Gold and also in sunray's, it is set in a Rhombus shape and is similar in shape of the Dutch Order of the Crown; the Eagle which is on the Badge is on top of the star and set in Gold.[9]

Sash

The Sash is pale Blue with Gold edges bearing a narrow Red stripe; at the bottom of the sash is a bow which joins both sides together and where the badge hangs from; It is worn from the right shoulder.[10]

Recipients

Grand Cross with Collar

Romanian royal family
Romania
Foreign

Grand Cross

Romanian Royal Family
Romania
Foreign

Grand Officer

Romanian Royal Family
Romania

See also

References

  1. ^ "ODM of Romania: Order of Carol I". medals.org.uk. Retrieved 2015-09-06.
  2. ^ "Cancelaria Ordinelor". canord.presidency.ro. Retrieved 2015-09-06.
  3. ^ "Image: 137_1_6.jpg, (696 × 1000 px)". s3-eu-west-1.amazonaws.com. Retrieved 2015-09-06.
  4. ^ "Image: ordin-licitatie.jpg, (375 × 250 px)". lh3.googleusercontent.com. Retrieved 2015-09-06.
  5. ^ "Image: 168_20.jpg, (1600 × 1600 px)". s3-eu-west-1.amazonaws.com. Retrieved 2015-09-06.
  6. ^ "Image: Order_of_Carol_I.jpg". commons.m.wikimedia.org. Retrieved 2015-09-06.
  7. ^ "Image: 540105m.jpg, (280 × 135 px)". sixbid.com. Retrieved 2015-09-06.
  8. ^ "Image: 112b.jpg, (711 × 1162 px)". coins.rotop.biz. Retrieved 2015-09-06.
  9. ^ "Image: 6015944_1.jpg, (500 × 500 px)". media.liveauctiongroup.net. Retrieved 2015-09-06.
  10. ^ "Image: Order-of-Carol-I-2.jpg, (402 × 455 px)". paulfrasercollectibles.com. Retrieved 2015-09-06.
  11. ^ "Image: carol_i_of_romania_by_rostariu-d8d6fsl.jpg, (1024 × 1680 px)". fc04.deviantart.net. Retrieved 2015-09-06.
  12. ^ "Carol, the first King of Romania". robinsonlibrary.com. Retrieved 2015-09-06.
  13. ^ Familia Regala
  14. ^ "Image: Ferdinand-I.jpg, (947 × 1217 px)". only-romania.com. Retrieved 2015-09-06.
  15. ^ "Image: tumblr_nl4mpcvhsR1s2cjiro1_500.jpg, (457 × 750 px)". 41.media.tumblr.com. Retrieved 2015-09-06.
  16. ^ "Image: queen_marie_of_romania_by_otmaafan91-d4xefmr.jpg, (500 × 786 px)". fc02.deviantart.net. Retrieved 2015-09-06.
  17. ^ "Regina Maria catre printul Carol si capetele incoronate au slabiciuni | Romania Libera". romanialibera.ro. Retrieved 2015-09-06.
  18. ^ "Image: 00d9645ffed8e3097d49a2a8f5ebc9c5.jpg, (689 × 900 px)". 1.bp.blogspot.com. Retrieved 2015-09-06.
  19. ^ "Image: 347fd8e11c9186761e6db7777d7a4a20.jpg, (736 × 1188 px)". s-media-cache-ak0.pinimg.com. Retrieved 2015-09-06.
  20. ^ "National Portrait Gallery - Large Image - NPG x121560; Prince Nicholas of Romania". npg.org.uk. Retrieved 2015-09-06.
  21. ^ "Image: YooniqImages_102123622.jpg, (297 × 416 px)". yooniqimages.blob.core.windows.net. Retrieved 2015-09-06.
  22. ^ a b c d e , Current Recipients of the order
  23. ^ "Image: regele-mihai.jpg, (960 × 1280 px) – Regele Mihai". ecomareaneagra.files.wordpress.com. 2014-03-10. Retrieved 2015-09-06.
  24. ^ "Queen Anne of Romania and Princess Lilian of Belgium followed by King... News Photo | Getty Images". gettyimages.co.uk. Retrieved 2015-09-06.
  25. ^ "Principesa Mostenitoare si Principele Radu, Castelul Peles, 25 martie 2015, ©Daniel Angelescu, Casa Majestatii Sale Regelui | Familia Regală a României / Royal Family of Romania". romaniaregala.ro. Retrieved 2015-09-06.
  26. ^ "Image: margareta-350x483.jpg, (350 × 483 px)". informatia-zilei.ro. Retrieved 2015-09-06.
  27. ^ "Image: 250px-Președintele_Consiliului_de_Miniștri_George_Manu.jpg, (250 × 354 px)". upload.wikimedia.org. Retrieved 2015-09-06.
  28. ^ "Image: gheorghe-grigore-cantacuzino-nababul.jpg, (400 × 519 px)". calatorim.ro. Retrieved 2015-09-06.
  29. ^ "Image: 37F09.jpg, (1000 × 645 px)". fototecaortodoxiei.ro. Retrieved 2015-09-06.
  30. ^ "Image: albania-Zog-I.jpg, (300 × 530 px)". alearned.com. Retrieved 2015-09-06.
  31. ^ "Demnitari în cortegiul funerar | Fototeca Ortodoxiei Românești". fototecaortodoxiei.ro. Retrieved 2015-09-06.
  32. ^ "Image: cd5b66243ddf8ba731f88929d37908dc.jpg, (236 × 170 px)". s-media-cache-ak0.pinimg.com. Retrieved 2015-09-06.
  33. ^ "Egypt13". royalark.net. Retrieved 2015-09-06.
  34. ^ "Egypt". royalark.net. Retrieved 2015-09-06.
  35. ^ "Orders and Medals of the Mus?e National de la L?gion d'Honneur - France - Gentleman's Military Interest Club". gmic.co.uk. Retrieved 2015-09-06.
  36. ^ "Image: 01-Romania-in-Primul-Razboi-Mondial.jpg, (800 × 575 px) – Kaiserul Wilhelm al II lea şi prinţul moştenitor Ferdinand al României la Berlin. Începând din 1893, România a avut un tratat de alianţă cu Puterile Centrale, Germania şi Austro-Ungaria, tratat la care a renunţat în 1916 când a intrat în Primul Război Mondial de Partea Antantei". george-damian.ro. Retrieved 2015-09-06.
  37. ^ "Greek Royals | Unofficial Royalty". Archived from the original on 2013-08-02. Retrieved 2015-09-06.
  38. ^ "Image: 1890 George-08.JPG, (350 × 484 px)". royaltyguide.nl. Retrieved 2015-09-06.
  39. ^ a b "Image: eb43a1579437294e97ac66dcaeca78af.jpg, (423 × 538 px)". s-media-cache-ak0.pinimg.com. Retrieved 2015-09-06.
  40. ^ "Antique Photos - Antique Photos". antique-photos.com. Retrieved 2015-09-06.
  41. ^ "Image: King-Ferdinand-of-Romania-and-King-George-V-London-1924.jpg, (607 × 480 px)". romaniaregala.ro. 2010-03-18. Retrieved 2015-09-06.
  42. ^ "Passenger List, White Star Line, Republic, 14 August 1907". gjenvick.com. Retrieved 2015-09-06.
  43. ^ "ROYALBLOG.NL: Ridders bij opening Ridders-expositie". gpdhome.typepad.com. Retrieved 2015-09-06.
  44. ^ "http://gpdhome.typepad.com/.a/6a00d8341c648253ef01b7c75f3cb5970b-pi". gpdhome.typepad.com. Retrieved 2015-09-06. External link in |title= (help)
  45. ^ "The Royal House of Norway - The Decorations of King Haakon". royalcourt.no. Retrieved 2015-09-06.
  46. ^ "Image: carol-i-nicholas-ii.jpg, (500 × 315 px)". royalromania.files.wordpress.com. Retrieved 2015-09-06.
  47. ^ "Image: 2014-0320-alexandermignon.jpg, (500 × 300 px)". 2.bp.blogspot.com. Retrieved 2015-09-06.
  48. ^ a b "circa 1923, HRH, The Duke of York pictured with King Alexander of... News Photo | Getty Images". gettyimages.co.uk. Retrieved 2015-09-06.
  49. ^ "Principesa Moştenitoare Margareta şi Principele Radu, la..." romaniatv.net. Retrieved 2015-09-06.
  50. ^ "Banca Naţională a României - Theodor Rosetti". bnr.ro. Retrieved 2015-09-06.
  51. ^ "http://www.agerpres.ro/imageResize?path=%2Fmedia%2Fimages%2F2014-03%2F03091410-301224139.jpg&size=6". agerpres.ro. Retrieved 2015-09-06. External link in |title= (help)
  52. ^ "Image: afis-averescu-cu-date.JPG, (367 × 519 px)". bibnat.ro. Retrieved 2015-09-06.
  53. ^ http://www.scoala-avv.ro/pictures/vaida-voevod.jpg
  54. ^ "Image: 3801_0_1.jpg, (370 × 278 px)". icr.ro. Retrieved 2015-09-06.
  55. ^ "Image: FI109712F14.jpg, (644 × 1000 px)". fototecaortodoxiei.ro. Retrieved 2015-09-06.
  56. ^ "Image: Prezan-Constantin.jpg, (400 × 519 px)". 4.bp.blogspot.com. Retrieved 2015-09-06.
  57. ^ https://postcardscollection.files.wordpress.com/2010/02/ferdinand-and-albert-i-al-belgiei.jpg
  58. ^ a b c "Flickr". m.flickr.com. Retrieved 2015-09-06.
  59. ^ "Egypt11". royalark.net. Retrieved 2015-09-06.
  60. ^ "Duke Georg Alexander | House of Mecklenburg-Strelitz". mecklenburg-strelitz.org. Retrieved 2015-09-06.
  61. ^ "Umberto II : Who, What, Where, When". servinghistory.com. Retrieved 2015-09-06.
  62. ^ "https://www.ww2incolor.com/d/409882-4/1_002". ww2incolor.com. Retrieved 2015-09-06. External link in |title= (help)
  63. ^ "Marshal Pilsudski, the Polish Dictator, leaving the Royal Palace in... News Photo | Getty Images". gettyimages.co.uk. Retrieved 2015-09-06.
  64. ^ "Image: queen-maria_2548287b.jpg, (620 × 387 px)". i.telegraph.co.uk. Retrieved 2015-09-06.
  65. ^ "Prince Arsen of Yugoslavia, uncle of the King. News Photo | Getty Images". gettyimages.co.uk. Retrieved 2015-09-06.
  66. ^ Acović, Dragomir (2012). Slava i čast: Odlikovanja među Srbima, Srbi među odlikovanjima. Belgrade: Službeni Glasnik. p. 153.
  67. ^ "Image: 4464713ea9975ab0a7732ce3564af1b1.jpg, (236 × 371 px)". s-media-cache-ak0.pinimg.com. Retrieved 2015-09-06.
  68. ^ "Image: tumblr_lqk6xi4Bua1qfna0vo1_500.jpg, (492 × 750 px)". 40.media.tumblr.com. Retrieved 2015-09-06.
  69. ^ "Princess Ileana of Romania / Ladies' Home Journal - December 1951". tkinter.smig.net. Archived from the original on 2015-11-25. Retrieved 2015-09-06.
  70. ^ "A patra zi a Jubileului 90 | Familia Regală a României / Royal Family of Romania". romaniaregala.ro. Retrieved 2015-09-06.
  71. ^ "WorldWar2.ro Forum -> Pre - WW2 Headdress". worldwar2.ro. Retrieved 2015-09-06.

External links

This page was last edited on 19 November 2019, at 17:38
Basis of this page is in Wikipedia. Text is available under the CC BY-SA 3.0 Unported License. Non-text media are available under their specified licenses. Wikipedia® is a registered trademark of the Wikimedia Foundation, Inc. WIKI 2 is an independent company and has no affiliation with Wikimedia Foundation.