! "# $ $ $
"
% &' $ ( ) *++,-
tel-00635537, version 1 - 25 Oct 2011
.. " /0 .
. 1 / .
0 . 1 / /
2''3 /
!"#$%&# '&(% ) *$$*+ &,-*+ .-$/ $ 0 *&(%-&,. . (!1*$,*$ *2 +!30$% 0 &*' 1,45*++%
4
.' $ $ " 5 '
67 .8 7
1 )'6
(76. 87(2
1 )
(7647
1 )* 1- (767' ' 4
(7474 3
tel-00635537, version 1 - 25 Oct 2011
8 (&3*9) 0
(76.7*
8 (( : &3'9)! (2
(768 743
-8$%;
(7670;
,'! 0;
(7687&!
,'+! $
(76/7(
6 )$!!0; 5
(7678 ;
0;
(76874 (768783
)! 0!
( 657
4* ,6
(77' (76'7>>!
( !
(7674
(
(765705
!3 ' +
( '78>
8 '+!;
( 6,7
-8$'+! !3 ' (7)7); % &3
(7074
*3! - &3
(778
,- +!;&3
(76)7)
,'8 " B U; > H> B H > J ' ! @ 07 5 E/6G; V
tel-00635537, version 1 - 25 Oct 2011
1EK JLG7' 47B A 6 BCCIC"C6 ; C A78B @ 6 C" 7 +! B 6 B . $ 6 2 C" !B ' ! 7 2 ;B 3 0 EK 6 NLG / !E/ > G CE JG I @ C @ E@ G C @ 67 ? ; 8 D E C 6 ! 2JGB '3 B * !B B )B ' B ) ( 7 , @ * 7 * @ 43B ; @6 B Q 67 ? - V @ ( 6 ) +>B C6 V C 6 B ; ; ! 7 ( : 6 3 6 ! I , !! @! 2@ !! ;I "; 6 6 7 0 ! 3 7
) 6 6 ; 6 B C@B @ 7 ? !;4 5 * " $ !7 ! '! ; 6 3 D B " ! ; E ! GC 7 ? 7 ?(76 #B
tel-00635537, version 1 - 25 Oct 2011
! 7+ @ C!; H A ; 7 ? ,'*$(-W 7 ? ( $!3 !" B ; 66 !! ! B @ 7 ?2 C " 7 ?2 @ F F@ B6 6 6 C7
$ !# !' ' $ 5
2''3 B ( 8 ;B & B , X B / > +! B % $ B ) ! +! B 0 )B 5" (B % ( B( ' H B3B( 0 B( 5")B 1;!B# > '
tel-00635537, version 1 - 25 Oct 2011
#! 8 $ # 3: ! ! =7
;3B 4 47 # B 2''3 B , X B $ 5T6 ;B , U # UB ! '! ;B 4 O 0 B (3B $ 6 'B $> 1 ;!B # ( C3B & C &UHYB +! 'T B 5 0> B & /
>8$42:#
$
$
> 8 3!A ! ! =7
!B I EG Z [
?
*++9
3 @
# $ 3
/ BBBBBBBBBBBBBBBBBBBB7 ,
C " BBBBBBBBBBBBBB7BBBB77 *+
1 . @ 6 / BBB777BBBBB77 *
6 " D16BBBBBBBBBBBBBBBBB7BBBB77 **
7\,*(,%&%4,*FFFFFFFFFFFFFFFFFFFFFF7FFFFF 7 ,4%'+,)777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777 V 7 7\ 3!; FFFFFFFF777FFFFFFFF7 V 7 7 , ;FFFFFFFFFFFFFFFFFFFF77FFFFFFFFFF7 ]
tel-00635537, version 1 - 25 Oct 2011
7 7 !;FFFFFFF777FFFFFFFFFFFFFFFFFFF
FFFFFFFFFFFFF7FFFFFFFFFFFFF
FFFFFFFFFFF7FFFFFFFFFFFFF7 V
FFFFFFFFFFFFFFFFF W
!"#$% FF777FFFFF7 Z 7 8)+*-$'*$,'X-**+8%$(*'*(&,*&R5*,(*$FFFFFV 7V +$,+*(*+'FFFFFFFFFFFFFFFFFFFFFFFFFFFFV *
.1 E BBBBBBBBBBBBBBB7BBBBBBB77 F*
7&*'\4**$*')*)*'&%0,$*'8$%+%+*(%$&*'FFFFFFF7V 77& 6 FFFFFFFFFFFFFFFFFFFFF77V 77 ! ; FFFFFFFFFFFFFFFFFFFFFF77V] 7 &*'+$%5,*)%$+,)&*'%'+*$,*-$*'%-'/$%(**0*'% FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF77VW G 6 H . IBBBBBBBBB77B777BBBBBBBB FJ
F 6 . 9BBBBBBBBBBBBBBBBBBBBBBB KG
K
6 . 9BBBBBBBBBBBBBBBB77 KF
7\4**$*')*)*)%$+,)%0'&*FFFFFFFFFFFFFFFFF7 7 $&/',*'-$-)&\,$*$%4$*'',1*FFFFFFFFFFFFF7 ] ,
6
H
. @
.1 . 1
./E6
6 BBBBBBBBBBBBBBBBBBBBB77BBBBBB7 K,
]7&*'8%$(*''%$,X-*'(&,**)$*-+R8*&+.#%0FFFF ] ]7 &*'8%$(*'4\\+,X-*'FFFFFFFFFFFFFFFFFFFFFF77 [ ]7 &*'8%$(*')X-,'*'FFFFFFFFF777FFFFFFFFFFFFFF W
tel-00635537, version 1 - 25 Oct 2011
]7 7 & #FFFFFFFFFFFFFFFFFFFFFFFFFFFF777FF W ]7 7 8 ;"FFFFFFFFFFFFFFFFFFFFFFFFFFF W ]7 7 )H6 . > FFFFFFFFFFFFF77 W ]7V ,4%'+,) *' *)\5&%+5,*' '%4,8%$(*' +$'(,'',0&*' *^(*'$)&,,X-*'*+*-$%+5%&%4,*FFFFFFFFFF77777777 Z ( 6 E6 .6 BBBBBBBBBBB77BBBB7 ,+
1 . @ . . 1BBBBBBBBBBBBBBBBBBB77 ,K
.1 6 " / / BBBBBBBBB777BBBBBBBBBBB ,,
* .1 / . 1/L . . 1BBBB77 ,J
7 +-%+5,*'FFFFFFFFFFFFFFFFFFFFFFFFFFFF]Z 77 FFFFFFFFFFFFFFFFFFFFFFFFFFFFF7[ 77 !3 !; FFFFFFFFFFFFFFFFFFFF[ 7 (/&%,%+5,*'FFFFFFFFFFFFFFFFFFFFFFF777F77[W 7 7&_FFFFFFFFFFFFFFFFFFFFFFFFFFFF77[W 7 7 !3 !;_FFFFFFFFFFFFFFFFFFFFF77[Z 7 &5 '/-)&\%+5,*'FFFFFFFFFFFFFFFFFFF7FF77W 7 7 ` 3FF777FFFFFFFFFFFFFFFFFFFFFFF7FFFW 7 7 !3 !;` 3FFFFFFFFFF77FFFFFFFFF7W
7V(&,*'?$,%FFFFFFFF77FFFFFFFFFFFFFFFFFW 7V7FFFFFFFFFFFFFFFFFFFFFFF7FFFFF7W 7V7 !3 !; FFFFFFFFFFFFFFFFFFFW 7 +V +5,*'FFFFFFFFFFFFFFFFFFFFFFF77FFFFWV 7] &* )' *' ,'%*+,*' 1*$' -* \+,%+5%4*a'* )%((-* *' $%+\,%+5,*'N77777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777W 7[ '/+5a'*FFFFFFFFFFFFFFFFFFFFFFFFF777FFFFW] G .. . 1 .0 /0BBBB77B77 M(
1 . @ / 6 BBBBBBBB7BBBBBBB77 MJ
tel-00635537, version 1 - 25 Oct 2011
6 0 /0BBBBBBBBBBBBBBBBBBBBB J+
7$%+\,*'+-FFFFFFFFFFFFFFFFFFFFFFFFFFF7Z 77 FFFFFFFFFFFFFFFFFFFFF77777777777777777777Z 77 !!3FFFFFFFFFFFFFFFFFFFFFFFZ 7 *+,*_FFFFFFFFFFFFFFFFFFFFFFF7FFFFFF7Z] 7 ` '/-)&\,*FFFFFFFFFFFFFFFFFFFFFFFF7FFF
7V $%+\,* $,%FFFFFFFFFFFFFFF777FFFFFFFFFF77 7V7 ; FFFFFFFFFFFFFFFFF 7V7 & ! ;& FFFF7 7V7 & ! !FFFFFFFFFFFFFFFFF 7V7V& 6 ; ! FFFFFFFFFFFFFF V 7 )%(0,,'%'*($X-*-$'FFFFFFFFFFFFFFFFFF777 * 6 0 /0BBBBBBBBBBB777BBBB +J
7 ($X-*-$' 0,%&%4,X-*' %-$ &* ,4%'+,) * & (&,* &R5*,(*$*+*''/$%(*'$*+\'FFFFFFFFFFFF77 Z 7 '&*)$*-*(&,**)$*-+R8*&+ .#%0FFFFFF 7 7 V7 7 FFFFFFFFFFFFFFFFFFFFFFFFFFF77 7 7 FFFFFFFFFFFFFFFFFFFFFFFFFF777
1LBBBBBBBBBBBBB777BBBBBBB7 G
. BBBBBBBBBBBBBBBBBBBBBBBBBBBB7BBB7 F
.E6 / 6 0 .
/ 6 " D16 6
.. @ .. . "E 6
" D16 . 0 E
6
. 61BBBBBBBBBBBBBBBBBBBBBB77 ,
tel-00635537, version 1 - 25 Oct 2011
7'\&*)+,%*'+,*+'FFFFFFFFFFFFFFF77FFFFFF7] 77 6 ; &)$ FFFFFFFFFFFFFFFFFFFFFFFFFF777FFFFF7] 77 6 ;_V &)$ _V b_V 2+ WFFFFFFFFF777FFF[ 77 6 ; &)$ 6 ; FFFFFFFFF777FFFF77[
'(FFFFFFFFFFFFFFFFFFFF77FFF77W
'( )%FFFFFFFFFFFFFFFFFFFF7FF77W 7 &/'*'0,%&%4,X-*'-&,X-,*)\5&%$)5,,*FFFFFFZ 7 7$! ; FFFFFFFFFF7FF77Z 7 7 ; ! !FFFFFFFFFFFF7Z 7 &/'*''++,'+,X-*'FFFFFFFFFFFFFFFFFF777FFF7 * BBBBBBBBBBBBBBBBBBBBBBBB7BBBBB *+
7 \1&-+,% * & *$8%$()* - %'4* *' 0,%($X-*-$' - &)$ -+,&,'\' * $%-+,* %-$ &* ,4%'+,) ,88\$*+,*& * & (&,*&R5*,(*$+ +-B +-W*+_V FFFFFFFFFFFF77 77 ; 2FFFFFFFFFFFFFFFFFFFF 77 2 ;66FFFFFF7
V
7 \1&-+,%*&*$8%$()*-%'4*-*+,*_V '&* &)$*+-$+,%_V b_V ''%),\'-^$%+\,*'+ +-*+ +-W%-$ &*,4%'+,),88\$*+,*&*&(&,*&R5*,(*$FFFFFF W 7 \1&-+,% * & *$8%$()* - %'4* *' 0,%($X-*-$' - &)$ &%$' * $\'*++,%' )&,,X-*' 8%)&*' * \(*)*' *-$%\4\\$+,1*'FFFFFFFFFFFFFFFFFFF7FFFFF V 7 7'3 FFFFFFFFFFFFFFFFFFFFF7FFF77 V 7 7 '3 6 FFFFFFFFFFFFFFFFFFFFFFFFFF777 ] 7 7 '3 ! FFFFFFFFFFFFFFFFFFFFFF7FFF [ 7 7V'3 FFFFFFFFFFFFFFFFFFFFFFFF77 Z 7 7 '3 0FFFFFFFFFFFFFFFFFFFF7FFFF77V
tel-00635537, version 1 - 25 Oct 2011
7 7]'3 'FFFFFFFFFFFFFFFFFFF7FFFFF77F77V G BBBBBBBBBBBBBBBBBBBBBBBBBB777BB7 F*
7 1&,+,% *' '*-,&' *' 0,%($X-*-$' - &)$ -+,&,'\' * $%-+,*
%-$
&*
,4%'+,)
,88\$*+,*&
*
&
(&,*
&R5*,(*$FFFFFFFFFFFFFFFFFFFFFFFFF77FFF77V
7 &/'**&*$8%$()*,4%'+,)-%'4*-*+,*_V ' &* &)$ *+ - $+,% _V b_V ''%),\ - %'4* *' $%+\,*' + +-*+ +-WFFFFFFFFFFFFFFFFFFFFFFFFFFFF7VV
7 \1&-+,%*&*$8%$()**'0,%($X-*-$'-&)$&%$'* $\'*++,%'8%)&*'*\(*)*'*-$%\4\\$+,1*'FFFFV] F 6 0 " .1 . 777
BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB K
. *BBBBBBBBBBBBBBBBBBBBBBBBBB7BBBBB7 ,+
C . . 1 /0
/ 160 6 " D16
. 1 @ .. . "
. 1 / 6 *++9
6 DE 42 9
. 61BBBBBBBBBBBBBBBBBBB7BBB7 ,*
7+,*+'FFFFFFF77FFFFFFFFFFFFFFFFFFFF7FF] 7 *^+$)+,%*'58,+$)\$\0$&*'FFFFFFFFFFFFFF] 7 c*'+*$ 0&%+FFFFFFFFFFFFF77FFFFFFFFFFFFF7] * BBBBBBBBBBBBBBBBBBBBBBBBB777BBB77 ,F
G BBBBBBBBBBBBBBBBBBBBBBBBBBB77777777 ,K
F 6 DE 42 9 H 6 *++9 @
tel-00635537, version 1 - 25 Oct 2011
" . . 1 6. CBBBBBBB7 ,M
. GBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB77 MJ
. " . 6 C
/ . .1.1
6 61BBBBBBBBBBBBBBBBBBBB77777777 J+
7)%)*+,%*'+,)%$'(%%)&%-^FFFFFFFFFF7777777Z 7 )$)+\$,'+,%*'+,)%$'FFFFFFFFFFFFFFFFFZ 7 7 ! FFFFFFFFFFFFFFF77FFFFFFFFFZ
*FFFFFFFFFFFFFFFFFFFFFFFFFF77FFF7Z
"FFFFFFFFFFFFFFFFFFFFFFFF777FFFZ
+( *,-( )%#FFF777FF77Z
'+*,-FFFF7FFFZ
.FFFFFFFFFFFFFFFFFFFFF7FFFFFFFF7Z
/ .0FFFFFFFFFFFFFFFF77FFFFFFFFFF7Z
! 1 FFFFFFFFFFFFFFFFFF777FFFFFF7ZV
]
* BBBBBBBBBBBBBBBBBBBBBBBBBBBBB7 JF
7$\)+,1,+\*')&%*'FFFFFFFFFFFFFFFFFFFFFF7ZV 7 '\),8,),+\*')&%*'FFFFFFFFFFFFFFFFFFFFFF7Z 7 7 , !! FFFFFFFFFFFFFFFFFFFFFF7FFF7Z 7 7 c 0FFFFFFFFFFFFFFFFFFFFFFFFFFFF7Z] G BBBBBBBBBBBBBBBBBBBBBBBBB77BBB77 *++
. FBBBBBBBBBBBBBBBBBBBBBBBB7BBBBBBB7 *+*
tel-00635537, version 1 - 25 Oct 2011
6 . " 66 /0 . .
/ . .1.1 "
6 *G
6 6 1BBBBBBBBBBBBBBBBBBBBBB *+G
7 \)5+,&&%'FFFFFFFFFFFFF777FFFFFFFFFFFFF 7 (\+5%%&%4,*FFFFFFFFFFFFFFFFFFFFFFFF7FF7 7 7 " 2FFFFFFFFFFFFFFFFFF7 V
( 7 FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF V
2FFFF7 7 7 ( ! !FFF
$ )&#FFFFFFFFFFFFFFFF
$ ,+*FFFFFFFFFFFFFFF777FFFFF ] * BBBBBBBBBBBBBBBBBBBBBBBBBB7BBB *+,
7\+*)+,%$&*'/'+a(*0,%+,* 1,,*FFFFFFFFFFF77 ] 77 " 2FFFFFFFFFFFFFFFFFF7 ] 77 ( ! !FFF77 W 7 \+*)+,%$&*'/'+a(*5$FFFFFFFFFFF777FFFFFF77
[
G BBBBBBBBBBBBBBBBBBBBBBBB77BBBB77 *J
.. BBBBBBBBBBBBBBB77777777 **G
CBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB7 **M
E / .10BBBBBBBBBB77BBBBB7 *KJ
tel-00635537, version 1 - 25 Oct 2011
W
$ 8
8; 7 ; ! @ ,$( H! 777FFFFFFFFFFFFFFFFF7FFFFFFFFFFFF777777 W 8; 7 !; @,$(E 8&,$G!H H! FFFFFFFFFFFFFFFFFFF7FFFF777 Z 8; 7+* ) ,0!H > @&S3FFFFFFFFFFFFFF7FFFFFF 8; V7 ) 6 !; 3 FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF7
8; 786 H! FFFFFF77FFFF77 V
tel-00635537, version 1 - 25 Oct 2011
8;]7) 2 +W ] 0 > H! FFFFFFFFFFFFFFFFF7 ] 8;[7\ 8 0 >FFF7FFFFF7 [ 8;W75 !" ; ! 6 E 3 >GFFFFFFFFFFFFFFFFFFFFFFFFF77VV 8; Z7 +')FFFFFFFF777FFFFFFFFFFFFFFFF77FF77 8; 7 $ !; H! !; 2&S3E > @&S3GFFF7FF77 8; 7 ; H! 6 3 ; FFFFFFFFFFFFFFFFFFFFFFFF] 8; 7'6 FFFFFFFFFF77FFFFF77[ 8; 7' 3 !3 !; 6 8FFFFFFFF77FF7[ 8;V7!3 !; 6 ;"8FF777F[ 8; 76!" 58c0 66 !FF777F7[[ 8; ]7 6 !" 58 c0 6 66 !FFFFFFFFFFFF777FFFFFFFF[[ 8;[7)3 KL 3;"F777FF777[W 8;W7(" 6 !; FFFFFFFFFFFFFFFFFFFFFFFFFFF777FWV 8; Z7 ) 6 !; ; FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFW[ 8; 78 C _ &)$FFFFFFFFFFFF7ZW
Z
8; 7 ) + B W _V &)$ 66 H! B 2 ; 6FFFFFFFFFFFFF7 8; 7 ) $%) &)$ ; 66 H! 2 ; ; 2 ; FFFFFFFFFFFFFFFFFFFF7FFFF 8; 7 ) + B W _V &)$ 66 ; FFFFFF7FFFFFF V 8; V7 ) $%) &)$ ; 66 H! @ ; F7F 8; 7) _V _V &)$
tel-00635537, version 1 - 25 Oct 2011
_V b_V H! B 2 ; 6FFFFFFFFFFF Z 8; ]7) _V _V &)$ _V b_V H! 2 FFFFFFFFFFFFFFFFFFFFFFFFFFFF777
8; [7 ) $%) + B WB _V _V &)$ _V b_V ;66 H! 2 ; ; 2 ; FFFF77777 8; W7 ) $%) + B WB _V _V &)$ _V b_V ; 66 H! @ ; FFFFFFFFFFFFFFFFFFFF77 8; Z7 ; ; 3 FFFFFFFFFFFFFFFFFFFFFFFFFF77F 8;
7$ ; ; &)$ EGB3 H! E0G 3 E)GFFFF777F77 8; 7 ; ; 3 6 FFFFFFFFFFFFFFFFFF77FFFFFFF77FFF ] 8; 7)6 ; ; !H 3 6 FFFFFFFFFFFF777FFFFFFFF77FFFFFF77 [ 8;
7 ; ; ! ; FFFFF77FFFFFFFFFFFFFFFFFFF W 8; V7 $ 6 ; 66 ! FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF777 W
8; 7)6 ; ; !H 3 ! FFFFFFFFFFFFF7FFFFFFFFFFFFF77 Z 8; ]7 ; ; 3 FFFFFFFFFFFFFFFFFFFFFFFFF77F77V 8; [7)6 ; ; !H 3 FFFFFFFFFFFFF77FFFFFFFFFFFF77V 8; W7 ; ; ! FFFFFFFFFFFFFFFFF7FFFFFFFV 8; Z7)6 ; ; !H ! FFFFFFFF777FFFFFFFF777FFFFFV 8;V 7 ; ; 3
tel-00635537, version 1 - 25 Oct 2011
3 ; FFFFFF777FFFFFFFFFFFFF777FV 8; V7 c 0 58 2 66 H 2 H! @ 0 > +V]FFFFFFFFFFFFFFFFFFFFFFFFFFFF777FFFFF777]V 8; V 7 c 0 58 '
, > +V]FFFFF777FFFFFFFFFFFF] 8; V 7 0 W FFFFFFFFFFFFFFFFF777FFFFFFFFFFFFFF7Z 8;VV7) 2 66 ! V) B 0B 4[ZWFFFFFFFFFFFFFFFFFFFZ] 8; V 7 c 0 58 2 2 H! @ 0 > 2 V) ]8VFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF7FFF7Z[ 8; V]7 c 0 58 2 2 H! @ 0 > 2 0B 4 B 4[ZWFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF77ZW 8; V[7 c 0 58 '
, > 2 4[B 0ZWFFFFFFF7FFF7ZZ 8;VW7586 +V]62 Z] FFFFFFFFF77FFFFFFFFFFFFF7 [ 8;VZ7 0F777777 [ 8; 7( 0FFFFFFFF77777 W
8; 7 !!3 &)$ 07777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777 Z 8; 7, 0@V) !F777FF 8; 7 &)$Eb G 0'E5[7VG +^
@ 7 dE b G 0FFFFFF777FFFFFFFFF 8; V7 &)$Eb G 0'E5[7VG +^
@ 7 dE b G @V) 0FFFFF7FFFF 8; 7 &)$Eb G 0'E5[7VG +^
@ 7 dE b G @V) 4[FFFFF777FFFF V 8; ]7 &)$ 0 777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777777F7 ]
tel-00635537, version 1 - 25 Oct 2011
8; [7 &)$ 0 @ 0'5'FFFFFFFFFFFFFFFFFFFFFF77FFFFFFFF77 ] 8; W7 &)$ 0FFFF [ 8; Z7 &)$ 0F777FF7 W 8;] 7 &)$ 8 777 W 8; ]7 ) 2 ! !! FFFFFFFFFFFFFFFFFF7
$ #A
+ 7 $ " ;3 * 6 !; EC C H! GFF777F7FFFFFFF7777777V + 7 6 F777 FFFFFF77FFFFFFFFFFFFFFFFFFFFFFFFFFFFF7777V + 7) 6 3 (), ; F77 FFFF7FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF7] + V7 !FFFF77777777777777777777777777777FFFFFFFFF77]W + 7 $ 6 &)$ ; FFFF7FFFFFFFF7FFFFF7F
tel-00635537, version 1 - 25 Oct 2011
+ ]7 (3 ((' + B W _V &)$66; FFFFF777FFFF + [7 2 ;66 H! F7FFFFFFFFFFF777FFF7 ] + W7 &)$ ;66 H! FFFFFFFFFFFFFFFFFFFF77FFFFFF77777777777777777 [ + Z7 (3 ;B ((' &)$ _V b_V 66; FF7FF7 W + 7( _V _V &)$ _V b_V ;66 H! F7FF7
+ 76 ; + B W _V b_V ;66 H! 7FFFFFFFF V
$ #'5
` '3` 3 _ 3: A ) ! 8 ! 6 * * ! ; )&&S3 )0; )& @&S3
tel-00635537, version 1 - 25 Oct 2011
8+ 6 8+ [ 6 ! [ &8+; 6 8;6 **4! ; *'+! !;6 4''4 ' '!> ,88 6 6 ,$( ; ; ,$'! &)$ ! ! ( ! ( H! (), ; e ;6; (). )H6 . > ((' ( > (+ ; 6 % 58K 5 8 LE 6 !: 2G # #
V
' !!3 ')03 '& 3! ' 3 ; ''3 3 ; +* ; ! +4 ; + 1 (). )H6 . >
tel-00635537, version 1 - 25 Oct 2011
c0S
/
tel-00635537, version 1 - 25 Oct 2011
]
& H! ; 6 7 * ! C [ 7 B ; B !; ; @ 7 & ; H! C 7& ; 2 ! ; 7 ) ! ;! B @ 7 B ; H! B @
tel-00635537, version 1 - 25 Oct 2011
6 3 @ 66 ; 7*B2 E ; ; G B 2 B ; !;7 B ;B 3 ! !B 6 ; 6 !; C 6 @ 3 7) B B 66 ; ; E 6G 2 A 7)! " 2 ; B ; ; 7 ) 2 ; ; 66 H! C6 !"B6 C E*- 8]G 5$) ; E # > ' B
VG 2 ; 22 ; 66 H! ! !7 & C6 " 6 ! ! ; H!
[ ; ; ! * E&3G7 , 6
[
3: _ V &)$ ; Q 3: 26 7$ B " ! ; ! 3: _ ;;" V ! ; 7 66 " 3:_E G 6 ; ; B 2 B 7 &A ; ! 6 3 6 6 ; H! @
tel-00635537, version 1 - 25 Oct 2011
6 7 ! ! 2 !; 2 H! E G7)2 @ 2 ; ; !; ;f @ !; ! 7)! @ !; 6 ; )H6 . > 7 2 ; 2 B " ; ; !!3 B !!3 7*66B @ ; &)$ !!3 W B !!3 A ; 7& !!3 7 & (27$ 3R>S>7) B ! B A ; 7 * !B @]]! ; ! @A @; ! ! 7)D B @
W
3 @ ! P 2" 7 " " 2 " B 6 !; 2 ! ! C6 6 *&,' !!3 " ! ! H! 2 7
tel-00635537, version 1 - 25 Oct 2011
Z
C "
tel-00635537, version 1 - 25 Oct 2011
1 . @ 6 /
tel-00635537, version 1 - 25 Oct 2011
& 3 66 B ;66; B C; 7 & 3 A ;7 & ! ; 2B ; ; ; ; 7 & A A B !3! 3 B 2 E B 23 3!GB;6E! ! 3! B ! 6 ! g1,5h 2 GB
tel-00635537, version 1 - 25 Oct 2011
6 E GB E6 0 BB0B 3!3:GB 3! E!H!B 3 6GB 6 ; 7 & ; @ ! !; ;; # C 7' " 6 @! 7& ; ; d ; 66 E%*'B
G7i 6 " D16
& H! E(G 6 D 6 ; B " B@ "7& @ I ;! (EeW G 7]@ 7Z " 3 Q 7 & 6 B B@ B6 7 ; B 6 ;
i
$ H! ( ) 4 HB B
7
; ;B62 7 !3 i 666j ; ;6 (7 A B 6 3! E! G3 2 3 A ( 3 ; 3 ! 7 B V & ( 3 B 6 ; ( ; 66 ! ; 3 B 3! 3 326B Q ; @ &S3E)&G 6 E8+G7& ;66
tel-00635537, version 1 - 25 Oct 2011
( A"7 7 ! $'3
& ( B ] d B; )&B ; 6 E&8+G 7 & B @ d f; W B ; f;7] ) 2 ; ! ; @ 7* 66B Cf; B " ; 7- ( @ V ; V7] 2 7] * 8 B [ V
( 3 A B[ d 6 B
2 7[
i
+ ! 7
j
B ; 7
7* N
& ; ( 6 6 ; B 3!; B ; ; 7 ; B ; ; ! 7j & ;( "2 !; B 6 !;k7- ;K( L " 3 ( K( L 3 7& 6 ; (
tel-00635537, version 1 - 25 Oct 2011
W d 6[ d7W,266 3 ; ( ;B 6 6 ; B 6 ( 6 d7W & 2 !; E ;GB !3; l ; ; 66 B B ;7
7*7 5 O != O
& ;(7& ;6;6 B B @ ; E ! B 2B ;B3 326G"6 ('( ,17iiZ- 2 3!; B(( ' * jj(('B i
*2 ; ; 7
j
)67 ! @ $ $'3 7
k
)6777G @ ! ! 7
l
+ ! ; ! 7
ii
; 6 BVB6727
jj
+ ;6 B
66 ;67) ( 7-(('
@ W !; E @ 6 GB ;" ] B Z " 6@ 7-((' 2 7
V
Bi ; B ; ;f @ 2 7 & " ; 2 ,)' $j7 ) " ;(@ 7 & 6 ! (7 ) ;B > ; 6 7 & B@ B@ Bk A# ;f@ 2 l H7ii&3
tel-00635537, version 1 - 25 Oct 2011
(6 ; B 7 ; B 6 6jjB ; ; E@ G7 & ; ;B 3 7 B ; !B 3!; = ; ( !3 B 3! B ; B 7 ) 3 ; )&kkB 66 ; ; 66B 3 Q 7
i
+ C (B67 2 7 j
)672V7
k
( 6 ! !; 7) !;2 ! *77* @
! ! 3 ! 5
l
) 2! B ! ! 7
ii
* 2! B! B! B ;3; 7); CQ6 ; 7 &6 2 66 A 2 6 7 jj
66@;;; ! B H! 7
kk
)67G @ $'3 P ! $ ?= ! 7
0 B 6 2 ! B (B 7 ; = 7 BV-3 326i (B ; A !H 3 6 7 j ) ; ;B 6 3 0 k 6 D B ; 7 ) ! ! 6 Bl ( A
tel-00635537, version 1 - 25 Oct 2011
3 ;7) 6" # 7& B A B! ; B = ( !; A 6 3 6 27] 7*7* 3
& ; ; (B ; !; E !3! ! GB ; C ! ; (7& ; ; E,$(G 2 ! 3 A ; 7 ) ,$( 2 !;
i
6 2 B B 6 B Q B 6 7)3 6 7) 6 2 B 3 C 3 6 I 6 6 3 ; 7 j
)67*77 @ $'3 8 3! ! 7
k
, ; ! ! E 2 GB @ E 2 G ;E ; ;B 2G ; @366 7 l
)67* @ ! 8 ! 7
]
6 6 ; @ ; 3 ; B ; )H6. > 7[ (B ! @ ; 2 2 @ ;7 & ! B ! B ; @ 2 !; E6;G7* ( W d6Z d7W); = " 6 ( @ E)& G
;
6
Z
tel-00635537, version 1 - 25 Oct 2011
!; 7 B ! C @,$( 6 @ !;! I ! @ 3 ; (7 B - 6 6 ! B !; Bi 2 Q 3: ! ! 7Z
i
)6777G @ ! I ! ,, 2 !7
[
tel-00635537, version 1 - 25 Oct 2011
E 7 . $ "! #5' P "6 $" 3$
$" > 37 ; ! @ ! B ,$( A B ! 6"! 7 % ; ! ! ! ; 2 ; 6E "&!3 7B
[ G7
) B ! ; 3 @ (7)B " ( !; 7 - ! ! 66 A &8+ !H )& 7 & !; @ !; ( 2 !3 !; !;3H! A 7 B ] ) @ 2 B ; !; 7 , !H ( " 6 (7 [ % ( @ E6; G7
W
tel-00635537, version 1 - 25 Oct 2011
E *7 . ''#5 5 P "6 %'O E - > !
! 3$ $" > 37 % ; 2 2 E6"! !G ; !; ; !3; 2 ! E6"! ;G C !; 6 ; ; E "'!
Z[G7
*66B 6 !;B !; ; ;; (!H A 7i, 6 D !; B " ( @ 7 ) ; 2 2 !H f; 6 ; 7 WB Z ) 2 !; 3; 7
B !; C ; 3 ;6 ;j 7 & ; 6 ; ;
i
)6777G @ ! Q 77G7G @ 6$ $" > 3 $'3 3 A7
j
)67( @ '3 83 !$37
Z
; !3 2 7 ! ;B !!B6 " 7 * " !3 !; (7)! ;6 ## !; C " 2 !; 7 B ! @ 6 !3 !; ! ; 2 ,$( 7
tel-00635537, version 1 - 25 Oct 2011
& ! ; B ; B A# ; ! E+*Gi7 * 66B !3 ;B
@
gW8h623;B ( 2B 2 ; 6 @ Z d (C ; 67
B! ;66 ; ;(8+ ; 7 V +*B " C (B C" 7 3:B 6 D ( 6 BjA ##@ E m] gE W8 6!3GE !3G h !3n!3G G EW8 8G !3 ) EV !3 !3G ] !323 H! H E) ,0G7 ) 2 B 66 " 3:B 6 (7 B ;
i
(! ;6 B6 D B 66 7 6@ ; C 2C7 j
)6777G @ ! ! ! @ .' ! 7
(B W8 8 ) ,0 !H C Q ; 67 ( B +* 6 i ! ,,,7 ] ) B ! ; 3: ; (B ; !; ; )&E6; G !; C ; ! 3:7j [
tel-00635537, version 1 - 25 Oct 2011
E G7 . 4. > ! ! 3$ $ .:
! ! $'3 P ! $ ?=7 )!; ! ; 3 Q 3:B ! !H )&
[ Q 3: 2E "* 7B
W G7
7*7G !
& (B ; B @ 7k (B 2C 3:EKLG@
i
$ 3 666 37
j
Q 3: 2 27) !; ! ; B 6 2 ! !7 k
)67 ! @ .' ! 7
3: _ E_G Q 2 7 ) 2 B !3; B 6 @ 6 Q !; 7i & 2 3 ;& (7 WB Z 7 7 7Q 3: , ;Q2 ( !H f; 3 E GBV ;6 ; 3 (j
tel-00635537, version 1 - 25 Oct 2011
)&7 ) QB E'GB C _ 27 % '667&6 6_k ;; 6"! @ ;);@A 6; " B 6 3:7 ! !; C 6 ; 7 & !! B;f@ 6 ; _B B B 6 ; E6; VG7 & " ! 6 !; ; ; B 6 !7l & ' 3 _ ;;BV 6 ; ; _7&Q !3 2_ @ A E3!
i
)67 ! @ .' ! 7& !3; 66 C ;; ! 7 j
)67( @ '3 83 !$37
k
)67 ! @ .' ! 7
l
)9+ 7
'Gi;3 *Bj ! 2 I @ 6 '7V V 0 6 B ; ; ; Q _ ! (7V] & " ! 2 ' B ; 2k E! GB 3 2 ; 2 E! GB 3 2 E! VG6 E! G7
tel-00635537, version 1 - 25 Oct 2011
E F7 ! $ ''# $O ! 83 $ !
3=R$ 5'7 +! !! ; _ ! E ]8b G ' 66 E6"! G ; ! 3:E6"!;GE "0 > 7B
[V[G7
& " ; 6 ( " _6 ) * ! $;36H!
i
)6777G7* @ ' ! O ! 7
j
)677* @ E $ O 83 $ 3$ $" > 37
k
2 E 2 Z d G 6 6B! ;3; 7
E)*$GB ZZ 6 ; (7 Z ) " ' !3 @ !; 3 ; 6 B 3 7
7 7 7 & ! , 2 3 ( ; 6 E8GB 3! 6 7 ) C ( 87i&3! !; B 6
tel-00635537, version 1 - 25 Oct 2011
'7 3! 6 ( A &8+7&6 B @B; 6 ; Bj @ !; 7 * 6 ( !7VW
E K7 E 8 # $ 3$ $" > 37 , +W !!3 I 8 VZ
6"!6 6"!;E "/!
] G7
i
& !3 !; @ 6 ! Q *7 ! 7 j
; ; B 2 ! A ; ; 2E;3 3G7
V
&8 A ; ( 2 2@ ' 7 B ) 8B B 2 d ( d ( B Q 8 7 A " 8B B 6 Q 7*66 ( 6 D; ; B 3 ! 6 ! o 6 " 8 7 W & 8 66 ; 3 E ! G 2 2
tel-00635537, version 1 - 25 Oct 2011
C E6; ] [G7 ) ; 6 2 B 0 >B 7
tel-00635537, version 1 - 25 Oct 2011
E ,7 ! $ ''#A 3O' 5 " !
M !' ,
$ $ : #5' $ 3$ $" > 37& +W; !!3B; !@2 iI2 I ! I ! ; jE " 6H66 7B
W VG7
i
'";2 7
j
! 6 3 2 7
]
tel-00635537, version 1 - 25 Oct 2011
E (7 5 $ E ''# $ $ :7 & B 8 2 ! ! E , ,,G7 & ; B ;3 2 E ,,,,1G; ; 2E "0 >0 >BZZ[ G7
& " = B @
V 7 , A 6 _ I ""!H C 7 & 0 >, 3 B ,, @
[
@ 6 ( 2 ;6 ;7i % ( @ ,,, ,1B @ ( @;"71 11,0 > ( "7 & ( = 1 " " ! 8 27 ) B 6 !;" (E ; 2 GBj 6 ;" * E*Gk !; 6 C7 V 7 7 7 ( H! 2
tel-00635537, version 1 - 25 Oct 2011
) !; B ! B C6 !; 7 !; (" !H 3 (6 " 7 ] - ; ; ! EeVW G V d 3 ( !; 27 [ + @ ! " !3 !; ; EQ6 G !; 6 26" 67 ) (B !; ; f;B 3 7 B B !;
2
; 3 H! 7 W ] ) 2 " ; 2I ; ! 7] ; C 7,2 B ! Z B i
)67( @ '3 83 !$37
j
)67 ; ! 7* @ E $ O 83 $ 3$ $" > 37
k
1)
W
d C " !; (7 Z A 6 DB Q ` 3:i E` '3G !H (B ; 3 ; 2 ` '3 E3 2 3 GI 3 Q (B ` '3 C !3 7] ) B B CQ3; ( @ ;6 7] & !; 2 66 ; ;6 6 2 (KL7]V
tel-00635537, version 1 - 25 Oct 2011
& !; 3 H! Q ` '3B &S3 E)&G &S3B B @ @ )& ( ( @ )&7] B ]] * 66B d d (6& !;@)& 7][B ]W
A B+ V j2 (7)
6 &8+k 3! 7 d ( !; 7]Z) !; ` '3 (@)&7[ 7 7 7V\ " ; H!
& ; ( 6 ; @ ; 6 D 2 2 7l[) 2" 6 ; (B ; ; ;6 7[ B 2"; !; 2 !; 6 7 *6B i
)67G @ '3 P ! $ ?=IF @ 6$ $ .: ! @ .' ! 7
j
)67 ! Q *7K @ .4FG! 7
k
)67*7 @ $' ' ' # 8 43!7
l
)67( @ '3 83 !$37
Z
6 C C 3 3 H!
d 7 ZB[ ) 2" ; ( E6 G !;H! " B6 K( L7i 7G E $ O 83 $ 3$ $" > 3
& ( B 6 6 2 7[V 0 B 6 6 ; B A ;
tel-00635537, version 1 - 25 Oct 2011
( 7[ 6 Ep d GB ( ; 6 d 6 27[] ;A 3EG! B ! V B !;" 6 (7 ) ; _ ' (7 2 B 3 S ( 6 ! 7 & ; Q 6 B 6
67& 3 ;6 ( A B 7 j 62 @ 66 6 !H ;" 7[[B[W 0 ;" ( B 6 E6 ; G
i
; 2"B@ !bbSSS7 H7;b !b ;d 8 b j
@ 3 6 67
V
;3*7);32 66; I6 i 2; 27!3; BCQ ; 3 7 B * @7,2 6 * B* *V 3 ! ! ; 6 6 7[Z & " ;3 ; 66 (E G7& "q " qV @"; (7W BW " ; @6 7W # 7 '! O $ ' =! $ " ! 8 $
tel-00635537, version 1 - 25 Oct 2011
! O % ! @; !8+@ 7Z & !; 8+ @ ; 27 3 @3 8+2E G7& 3 7 - 3 8+ @
VV
3! E'&G 7ZV)3 2 8+ '& d 8+7& !; 6 8+E+ V G A '& B ;; 66 6 A !; 7Z # *7 . ! !' O ! # $" $'3
8 3!
=! $ E
8+ >
@>
8+ +V 98+ '&
+ V
8+ E b+V G
-
(
tel-00635537, version 1 - 25 Oct 2011
=! $" !' O
@
6 8
8+ !;
6
& C 8+ B 38+ >B+ V 7& > !; 8+B @ > 7 @ d 8+ !; 6 !; 7 - ; 6 ; 66 !; 8+7Z] & 8+ 6 2 3 2 3 2 @ + V 7 A "B C @ ; ; 62 !7 (A C8+ B ; 8+2B ;" E8+ > ! [G ; 7 & !;" 8+b'& ; A @ ; 7Z[
V
& ; 8+ 66B 3 ; ; ; !; C 7*66B d ;
8+
"
A
(
6
!; 7ZW !; )&B )H6 . > E().G A 3 7ZZ ) ! (B 6 ( 27 A !; 8+B ! ; E &8+GB ; 3 ; 7
B 6 D B 8+ 6 6 )&7 6 B 2 B
tel-00635537, version 1 - 25 Oct 2011
; B ! !; ; ;667i , 2 2 6 8+7 & ! ; 8+ 6 @ 6 I ; 3 ! ! 3" ; @ 7 B V 0 3 Q 2 8+3" ; B @ ! E62B 2 G 7 B 3! E 2 G " ! 2 ! 7 & ( 6 8+7 *77*
! ! 3 ! 5
& ! ; EG 3 ( B &8+B6 "6ZW 7 &" ; ]@[ " 7 ] ) 3
i
)67 ! @ $ $'3
V]
! ; 6B ! ! ; 7 [ ! ; 6 6 6B 66B ; Bi66 ! 3 2 2 ! 7 6 3 2 !3 2 B j ;6 C7 & !
tel-00635537, version 1 - 25 Oct 2011
3; !k326 7l ! ; B 6 @ 7 ' ; B ; 3 2 6 B 3 66 @ ! 3 2 ! ; 67 A B ! C 7 & !! ;3 ; !Bii 6 D @ ; ! ; ; 7 [) B2 66 ! B ! 3 @ 2 2 7 W & !;@6 @8+@ >B + V 7 ) B A i
B B 6 3 2 " ! 7 j
66@ 7
k
66 7
l
6 2 B !; E66@K L K >L 2 G7& ! 7 ii
$; 26 ; ;
V[
6 !; ; E)0G 3 ; E'G7ZZB Z ) 2 " !; ; &8+ #H !; 2>7 i ,2 @ !; 7 * 66B EeWGB !3 @ 7& ! 6; 6 3 ! 2 > E'B )0B >G + V 7 & 3
tel-00635537, version 1 - 25 Oct 2011
Q + V 2 6 ; ; H! B 6 7 [B X B !; 3 3 6 (@2 & 7]BZZB ZB
*7* ! !' = $3 $
)ZWW 6 "63 ;f@ A ; 7 & ! E)G 3 0 2 7 ) 3 B ; @ ! ; E G7 & 3 6Bj ! ;; Bk ; Bl B 2ii 3 4 i
i
) 63 8+BB)0' !; 2; 7 j
6 66@ 66 ; 7 k
+ @ 2 ! @ 7
l
, @6 7
ii
! ; ;7
VW
3 0 7j& 3 " ;7 & ! 3 ) ; k8;l )7V ; " 3!; )7V ] &2 ; ! ! ; ;7[BW ) !;B;3 27 !; !; ; B ().B )& )0 2 ;
tel-00635537, version 1 - 25 Oct 2011
7]B
Z
) !; 2
!; )& ! 7 ) 2 !; 2 ) "( C C B@ 3 6 K H! L 7]B
G 6
. I
& )& d ; " 6 !; 7 ]) !; "66@ ; ; (7 & ; ;6 ! 7 & C 2 27*66B6 2 ; )& 6 ; Bii ! 3 2 3 7 ;B
i
'3 ; !B ;; E ;; G @ ; !E G E = !6666 G7 j
2 B 2 ;7
k
,o = 7
l
8 A 7
ii
6; 2 66 ;66 7
VZ
; 2 !; ` '3B @ = " ! B 6 3 !7 [ ) ; )& ; > E(G ! 3 7 W & )& ( 7 * 3 > ;6I 2 A !; 7& ! )& @ (B ;7 & )& 6
tel-00635537, version 1 - 25 Oct 2011
B B62 7 & " ; )&
6 ; ! ; C !; ` '37 Z & ; !; B (B !; ; )& (7 ) B 2 !; )&B ! @,$( (7
- @ ; ; ! +')7i ) ; ; 7j )&B ; ; 3 2 ; 2 E 3 G 3 ; E6; ZG7 & ; )& 6 E V dG [ d7 ) 2 ; ; ; & )& ; E Z dG 6E
dG7
i
,6 g ,h ; :B 66 3 7
j
$ ; 7
tel-00635537, version 1 - 25 Oct 2011
E J7 7+ D ; !HC 62 3 E ! @ ; !GB E ; ! @ ! G ; ; !H > ;B "E "0
G7
) ( 2 )& " 2 2 2 !; 6 A M @ 2 6 @ 7i & ! @ ;)& 7& )& 2 ;; 3 2 3 3 6B C "7 ) 2 ; ;; )&B d )&7
& ; " ! 3! 3 66 !;7 V
i
)6777G7G @ 6$ $" > 3 $'3 3 A7
% )& 2 &S3 !H )& " 2 7 & 3 B ! )&B 6 66 &S3B ` '3 7 !; B )& 3 6 ; ! )&6 B6 iE A 6 G 6 7 ) 6 6] 0 >B !; 8B 6 )& 7 ] 2 ; B V 2 ! E " jG ] @ 2
tel-00635537, version 1 - 25 Oct 2011
27 +6B d C !; @ )&7 [) ! B 2 ; C )&E Z dGQ 3: E6; G78B2" (B 6 )&7 ) @ 6 2 6;63(I @ 66 ; !;7 W
i
&3" ; ! B 3; B;3; B62!3! 7 j
3 ; ! ; 7& ; > @ &S3@ 63" ; !;7
tel-00635537, version 1 - 25 Oct 2011
E +7 ! O 3$ $" > 3 !
' A ! $ ?= %3$ $ !: $'3 P ! $ ?=-7 E " +T
W ZG7
F 6 . 9
& ( ; 2B ; " 7 & 6 ( 6 ; ; 2 E3 > > 3>Bi ;j kG @ ; ! 6 D3 3 ; E ! GB !3 Bl 7 & " ; ( "7V
i
$ @ !B 6 ; 2 ;7 j
+! 2 7 k
' 3 B@ A C 6 ;7 l
7
&3 > ;3" ; A ; 6 6 )& )&7 " ;B 6 ! ; 27 & & E G 66 3>i ! 7 & ; C @ 6 (7 ) B (B !; !; @ )&7j & ; ( M @
tel-00635537, version 1 - 25 Oct 2011
; ; 7k%" W d ; A ;(7& !; (@ 6 @)& ; !7 ]BV) B 2 )&B )& ; 2 E2G7 K
6 . 9
! " 2 !; ; ! 2&8+7*66B )0 ' B ! B 2 2 >7 ) 2 3 ; >7 0 2 !; 3 >B D ` '3 ; B 7
i
B 7
j
)67G @ $'3 P ! $ ?=7
k
1)
V
K7 ' ' ' #
& )0 ; @ 623 2; 27*6 "6 $ H 7V 6 E ZWZG !3 @ )07V & )0 3 > ; 3 ; 2 B 3B 6 2B ;670 )0BVV 3
tel-00635537, version 1 - 25 Oct 2011
> "A 7V & ; )0 B @ !; 3 ; ; 3 Q 66 ; 2 > E&8+GB 3 > ! 3 B (B 5;B )H6 . > (7 - B 'B @ ; 2 V d7V]B3 Q )0 66; E (G )0 ;B 2 @ 3 )03 7 BV[BVW & ;B 2 B ! 3 6 6 ;7VZ " !; 6 ; )0 ;f @ !; ; 2 7 66 3 )0B! 2B3 2; 2 !7 % 8 2 B 6 B 3 !; )0
;; 7 & !; 6!; 7VV K7* .= ! ' ! 5
& ' ' $! %HS>3 6 3 Bi B; 2 ; 7 ) 2B 3B 2B ; ;!H '7 & ! 2 3 2 ; 2 E B ! 3 2
tel-00635537, version 1 - 25 Oct 2011
! G 2 ; 6 7 &!; " 8 !; ; 6 ; ; !7
,
6
.@
.1 . 1
./E6
6
& @ ; 6 ! @ 65 7)!H B EK LG! !;6 K !6L7+ A ; 3 7 & " ; ! !;6 E*'+G @* ).
%; ( ' ZZW7 V ; ! *'+! 7 ,7 83 !$ O @ 3$ $ >8$ 2:#
&6 @6 W d *'+7 & A ! 6
i
! ; E 3 ! GB;3 7
]
f; 3 ]W 7 ! 7& 6 ; )H6 . > E().G7 )6 ; @ 3 27* " Z d B " 7 - ; ;6 A @;; 2B 3! B ; 3 2 2 3 2B 2 7 ] 2 6 ! 6 2 ; ; 2
tel-00635537, version 1 - 25 Oct 2011
2 5! 7B6 " 6 3 ; @ 6 6 ; 7 ,7* 83 ' ' O
&6 3@ B d *'+ 8 C @ d , O7 ) 6 B 3 6 2 " 7% 6 ; 3 4 ' '!>E4''GB 6 6 E,88G ().6 6 *
#7 & 3 4'' 2 B 3 3 *
#B ! 2 37 &,88 @ B B 3 ; 7 [ 3 ; B 66 @!3 " 7 & ; 66 " 66 B
[
3 ; @ ; ; 7 , ; 7 W ,7G 83 O
,7G7 9
& # Z ;" 47 ' 2 !! ; 6 6 ; 6!3 67 @ 6
tel-00635537, version 1 - 25 Oct 2011
Z B C! B Z 7& 3 ; 7) 6 2 7 & ! C 7 Z
,7G7* E3
& 6 (). 3 ; ! d ().7 % = 2 @ ;66 " E C . G C ! 2 E
Z 8 , G7 & C ;" 8 66 @ ! !3!3 ; ! 7 & ; ; 2 2 ; 3 C @
7 A@;66o@ !; 7] ,7G7G 5 5 $ 6 2
) 6 B " 6 ZZ] 6 D B 66 ().7]B ] -
W
! !;6 " 7 ] 2 3 !3!" EGi ! !;6 ().E ().G A !H ! H !;"7] B ]V &!3!" ; # ! !;6 C! ; 7% C [ ]Z 6 ; V[ *B 3 8 7j ) 6 ! C CB f; ] 3
tel-00635537, version 1 - 25 Oct 2011
; EV 3G (). 7] * ; 6 3! 7& 3 6B 2 @ 7 ; 66 6B ; 2 2 ; " 7
,7F $ @ A3 ! O !
& ; *'+B ; ! B ; 7 ) ; 6 6 ; B ; E,$( GB !3; ! ; E**4G ; 7k)66" (). K L K LB ; " 2 !; !; #! !! c0Ec0G7
i
& !3; 36 @ "62 ! @ .' ! 7 j
)!66 +!* )H6. > ' S> E*-$%).B; @!bbSSS7C77 7>bG, E,1'!bbSSS7 7 76bG7 k
)67 ! @ $ $'3 7
Z
& **4 6 3 (). 7 % 2 **47) 3 A ()&7]]X B;!3; 6 6 6 ().B 2 ] d E*-$%).G7 &,$( "A ; (). B A " 2 "; ; K L (). E*-$%).G7 &,$( !3; 3 ! ; 66B
tel-00635537, version 1 - 25 Oct 2011
K; L7) ; !H (). 6 ZVd Wd7 ] ) ! " ; !3; A 3 [ d 7][
& ; (). !; ;B ; 3 @ !; # !! 7]W , 2 3 Q 3 66B B 2 7]Z + ! B2 2 !; !; ().7 2 ( (). 7[ B[B (). )& ( 7[ - 66 Z ().*
# 6 !; ().B 6 !; "; 27[ ( 6 E6 .6
& ;66 (3 ; ; " ; ; 2 !;7% = 7
]
% !; C B ; ;; B 6 D6 ; @
3 Q 7[V) ;6 ; K( ); , L E(),G7[ ) (),B " 3 !!B ZZZI !; (), K @ f;LB K ;6 @ f;L K ;6 L7[] [W 0 ;6 ; ; B
tel-00635537, version 1 - 25 Oct 2011
(), C @ ; ; C (),7 A B3 ; A 7[Z& " ; (), ; " ; 7[ & ; !; (),7 ' B (), d 3 d (), ; ( 7* (), (B ; E8;G7
]
tel-00635537, version 1 - 25 Oct 2011
E 7 . ' $ 3$ $" > 3 8 $
=3!3 $ 3$ 7 ' 6 ( " ! 3 B (), ( ); , ;6 ;I ( &; ( ;"I((( I(' ( "E ") ; '! 7B
ZW G7
) (B B 6 66B@ 7W (), 8+BB)& 7 ) (), 6 6 ; 3 6 !; ; 3 ; !; C 7W % ; (), E G(), E ;6 2B 6 2 B G7 2 66 K L E ;6G K L ;6 E+ G7 ) 6 (), 2 !; C 7
]
# G7 8 $ 4=! O $ 6 5 '
!'3'7 s I I ; ; 6I 1 I 33! I+ I( H! I)& @ &S3I 8+ 6 I 1 I 7 E "
tel-00635537, version 1 - 25 Oct 2011
B
VW G7
& ; ; (), ( ; 7& ! B2! 2B "; B ! 66 (), (7W A !H (B ! ;B ; 6Bi ; (!H 7WVBW , 2 !; (),B 3 A " 7 - !; ; WV ;2 ! E+!$;% '3G "] d (), (7W] 6 ;; E
d G !; C(),7- 3 (),
i
)677*7* @ 3 ! 7
]
3 ; (B @
d2 7W[& 3 !H C (), ! 6 ( C 7 & !; (), = ! !3 !; 7 (), 3 (B ! " !; 7 , ! 6 D !; 3 ; 7 & !
tel-00635537, version 1 - 25 Oct 2011
3! !H (), A 3 Q !@! 7 0 6 B B WW Z 6 ! " !H (),B ] 3 (), H7[Z = 6 !; C (),B " !H )& 2 7i ;##6 @ ! !3 !; @ ; @ 6 ;7
i
)67G @ $'3 P ! $ ?= ! 7
]V
1 . @ . . 1
tel-00635537, version 1 - 25 Oct 2011
]
& 6 ; @ 36 3 @ " 7Z * 66B " (B 3 !; BZ ! " 7 , B B 6 @ 3 B 3 ; B E B 3 6 G " !; ; 7Z X B 6 @6 3" 2
tel-00635537, version 1 - 25 Oct 2011
7 ) 6 6 @ b " C @ 2 ;; 7&Q 2 6 ;;
!;
66
; ; 6 B ) !;" ; " B Q A ;2 BZV 23 6 ! ; ; A @ ; 6 ;; 7 +6B B ; B A 6 ; 6 !3 !; B !7 .1 6L " / /
& ! ; 6 6 C @ ;; 7 & ;; 2 =7 !3; ! = 3 3! ; ! ` 6 _ 6 6 6
]]
7& 2B @ 6 ! !7 & 6 ! B 7& 6 B ! ; 2 B 6 ;"; Q 2 " 7 & A 2B 6 E !!3 ;33 GB
tel-00635537, version 1 - 25 Oct 2011
! "23 67 B ! = 3 B ; !; B @ 6 3:7 B 66;6 6 ;; 7Z BZ]' 6 B3 !E+ VG7 & ! ; BQ2 B A B ! ; @ 7 0 6 B 6 667 # F @ !' !' !
;; _ +
( ( ; ! 3: 5 ; ! 3 ! 3:7 (3 !@ ( )0 ' ( > 8+ ! '& > '3 SE G ][
` 3 _ 3 t 3
tel-00635537, version 1 - 25 Oct 2011
+ V 5; $ 2 ;" 2 b b b [ '` bX @ ++ 0 )3 ) + !3B&BB (E !GB,B ,,B,1B 4B&3H3 B8 ;"B_ ; B6 B > B 6 3, 3:E,I 3G ) ) # ! 6 ) (3
( (@ )& )& ( @)& ! 3 (). (). 3 4'' ,88 # ( ( @)& ( ( @)& '& 8+ 8+b'& )!5; ( #3 3! @^7 2 3b b b[ 2 3] 2 3[ 6 6 *! !6 @) 5 ; ! 3 3E G
"3,, ) ) !3: 3!! (3 !
, = 6 B 3 ;; A ! 6 _ b ! `7 0 6 66B 6 ;"C @; 6_6 7Z[ ) !3 ! 6 ;;
]W
7! !3! B 6_B ! ; !3;" 6 ! ; 3 ;; 7ZW 0 ; @ @; 2B @ @ ;; 27*66B !; B2 )& d @ d ( 6 !; ; ( 7][B ]W ) # ;; _B ` '3 7ZZ
tel-00635537, version 1 - 25 Oct 2011
& 2 Q !; ; 6 B @ 6 2 3 ; Q @ 6 2 ;; 7*66B;f@ " 2B 3 Q ; ;; A 7 B ) !3!"6 # 2 6 ; "7 VB A B ; B !H "C 2 7 ]B [ * .1 / . 1/L . . 1
*7 !
& !; !; 6 !3!!3 ( ; &8+ > 6 > ! [ E8+ [GB )0B 'B @ ; ;3!B 3 SB 4'' ! 37
]Z
*77 .'
& 2 E(+G7*" 3" 2 2 " 27&;" ! ! [B ; 6 $ ; @ 2 6 @ VV E 0 7B
WG7 ) ] 6 66" ; B E
$G V E V$G ; ) E8; G7 ) ; 2 (+ ; ;I 6 V$ 3 66(+ 6 $7!!3 ;
tel-00635537, version 1 - 25 Oct 2011
" (+B ! 27 3" 2 C B 6 $V$ 2 2 7 Z
E *7 83 $ !' E "4 7B
Z G
&Q!3; 6 (+ @ 7 * 2 7 B 3 3 ; $ @ b@; !! ;" 27 ' 6;!3; !!3 > !! 7 VB
&
3 B !!3 2B @ C [ !!3 7 ] B !!3 B
[
6 ;33 B 3 B B23 B B ;3 ; 6 7 *77* . = ! $ !'
(B !3!!3B 2(+ ;;6 87, !3!!3 H3 > !! ; !!3 B !3!!3
tel-00635537, version 1 - 25 Oct 2011
(7 [ Z & !3!!3 2 (+ ! ; 6 7 # ! ; 6 " ;; 6 5 8 E58GB B !; 8 6 ; !: ! 66 !!3 7 V 7 ]ZB [ *7* 3=R$!
*7*7 !! $
T
tel-00635537, version 1 - 25 Oct 2011
& 6 3:_; ;3 E KLG _ t 7 [ & KL 7 * ]Z 6 B Q !3; @ 7 & 3;" H3 _ t 6 _V E_V G6 D _ 2 V E_V GE8;[G7& 3:;" @ ` _7
E (7 ! !'= O $ !' U
.. V @ 5 3=R$ E " [
'> >3 7B
]
G7
[W
*7*7* . = ! $ !! $
T
& " _ ! ` 6 _ !3; B ! ` 6 7 [ ) ;;" 6 ; B !3 !; ( A _B " 6 V 7 [VB [ )6 !;" C ' []B [[ @ ;; #7 [W B _ 6 V E_V b bW bZ V G7 [ZB W &
tel-00635537, version 1 - 25 Oct 2011
6 _ ] d 6 _ 7 [Z X B ;; @ ! ; !3! 7# Q !3! Q 23 _ ;; 7 W ) ! B Q !; A6 2 3 ;7 ,266 6 ;; C 2'7 [ , ;; 6 ; " _ ; " ! ; 6 B !; B " " 2 7 W & 6 ; " _ C Q !;" 3 @ 6 ; B W !3!" 6 " 7 ]B [ & 6 Q 3: 6 ; 6 6 " 2 _7*66B2 ; " !H ; 2 _ ! _ Q7 6 ; ! 7 WV 4f @ ! ; ##Q 3: +* ) ,0iB' !H
i
)67 ! @ $'3 $' ' ' 5 Q 77* @ 3 7
[Z
;67 W ) ' @ ; 7 & ' C ( @ !;" B 6 K 3:L7 W]B W[)!3!" _ !;" (B @ ; @ 6 3:B 6 '7 B _ " 3 Q ( 7 WW & (
tel-00635537, version 1 - 25 Oct 2011
b _ " !3!!3 B 3 B 6 B ; 7 B!3!" 6 !3 !; ; 7 *7G W4= ' !
& ` 3 !B ; ; B ;; 6 ` '3 3 6 ; 7 * ! ;6 6 B ; B 7i & ; 66 " 6 66 @ 7 WZ,2 ` '3)&B&S3B ; 3 B 3 !: 2 7 Z
i
)67 ! @ $'3 $' ' ' 57& 3 ! ! G @ $'3 P ! $ ?=F @ 3$ $ .: 7
W
*7G7 "W4= '
&` '3 3 2 7 , ; 2 3 7 , 2 2 6 6 C V 6 ; 6P 7 Z & 6 7 ' 3 ;;" Q B Z ; 3!" B Z Q! 7 ZV
tel-00635537, version 1 - 25 Oct 2011
*7G7* . = ! $ "W4= '
&` '3 !;" (B )& ! 3 7 & !; )& 6 6 ` '3 ; &S3B Q 6 2 667 Z B Z] & )& Z 8! 57 &S3 3 ! !; (7 & ` '3 ! 3:;" ! ; 6 6_6 6 6 7 Z[B ZW )&B )& 2 27 - ; 26 @ 6 V B ZZ A ; ` '3B ;; 6 )& 37 )&BZ d` '3!!3 Z !H Vd` '3!!3 !3; 7
B!!3 ` '3 Z6 6 6 #7)!!3 ;; 6 ` '37 6 ` '3 )&B
W
3 B 3 C " Q Q !; ` '37
B
*7F 6$ P .
& @ B ; ! ! ;6 B !@ 6! 7* ! !;6 E' G ().B4''B,88> 5 7 , 2 6 B ; ; ; ().@ ; !;" K !6L7+ !;@
tel-00635537, version 1 - 25 Oct 2011
" "7* !; 3" 2 7 *7F7 .' .
& ;3 V !@ 6 2 37 * C 2 B B PB ; 3 !: ;7
& ;" ! " 6"B W d! ;5 7
VB
& ) 6 !`7% ; !`
6_B 6!` 7
]& 2 6 ;33B ;33B 66 ;33 7 ' 6 6 2 ; 3! 6; ;7
[
W
*7F7* . = ! $ !' .
& ZW 62 !;" ' 7) !;" 67
W B 66 62B2 667 & 6 !H E 6 !; G !H C 7
Z , 2 ; *'+B3 ! !E(Gb E1G Z ;" A 7 & C (1 E d G (( 11
tel-00635537, version 1 - 25 Oct 2011
V d d 7 & ;3 (( (). E(( ]]dB (1 11 [dG7
B
! H3; (( 2A C 3 (). @ C ! H3; (1 !H !
7 6 66 7 & !;" @ 2 ! ; 6 ;) !` 6 !6_ B6 B 6 " ;; 7 ) 6 ; A 6 6 !; 7 ) ! "B ;" B 72!3!" 2 ! ; 6 ; ;7& " !; 2;" B " B ! ; 6 ;" !; E8; WG7 & !3!" !3; 6 6 !; 7)226 6 !; B 6 B ; !;" 7 & ;; 6 !; ; " 2 ;; Q 2 B 6 !; E8;W0G7
W
) 6 ; 6 B V ;" A @ ; 6 ; !H 5 7 & 3 ; 6 B = 2 (). ; *
#4'' 7
tel-00635537, version 1 - 25 Oct 2011
E M7 6$ $ 5 83 $ !' . 5
!' . ! OE ";HH 7B
V VG7
*7K .4FG!
& +$ ; V E+ V G 6 2 $ C Q 2 $7 , C ; &8+ B '& (7]ZBZ ) ;;" #B 6 ;; 6 " B ; 6 ; + V 3
WV
6 @ ;; 7 B 6 6 ; Z
!!3 7 *7, $ !! $ @ 5 ' ! 33 $ !' ! X
& 3 H3 ; E+4G7 ) H3 B 6 B t ; 3 q 3 t ; 33 2 @ 6 3 " B @ C 7 0 +4 B H3
tel-00635537, version 1 - 25 Oct 2011
!; 6 B 6 B
]
"B B 7
,
;; @ 6 ;; 6 #
[ Z
; 7
) 66
; B_B` '3!; +4#7 & B 2 +4 ; ; 7 B +4 ; ; 66 ( 5;B ' (7 WB ZB & B +4B 2 A 6 D 7 ) ; 6 D !3; ') CB ;; Q
V
!3; 7
@ 6 58#7, A 58 (7
B
; 2 6 @ ! ; !; @ B ;; Q 7 ] & 58 2 (
W
B 7 8 B A 8B @ > ` '3 B 6 +47) B 668B ;; B @ ; 7 V A " _ +4B Q H3 Q 3: 7 &_ 3 E4 u4G ! 3 ! 3i " +47 ) ! " @ ; Q _ !; 2
tel-00635537, version 1 - 25 Oct 2011
6 ' (7 [ ) ! B ` '3 = ; +47 - !! ( )& " ! )&7 W ) ` '3 )&B @ ` '3 ;"; )& 6 B B +47 B 6 ; ` '3 2 (7 Z ) ;; ` '36 )&&S37 *7( =
& ; B B; 2 o B B !; 3 j 67 , ; ! 6 6 7,266 ; E B B!; B G7& 3!" 6;Z 6 !; ! ,,,7
i j
87 ! Q @ . ' 3' $" Q # F )67 ! @ $'3 $' ' ' 57
W]
tel-00635537, version 1 - 25 Oct 2011
E J7 8 O !' O $ $'3 $' ' ' 5
!$ O7( H! I(). )H6 . > I,6 6 I (4 ; ;3!I )0 ; I ' 3 ; I &8+ ; 6 I 8 ; 6 I )& @ &S3I ( >I (' ! 3 I '& 3! I 3: _I E 6 "# 7B [ G7
G .. . 1 .0 /0
& ;; !3 !; ; 7 & ! ; !! ! ;; 6 Q" 7-; ! 6 _'B 6 8B)& @ C7
V , 2 ; ! 3 66 E "
W[
A G !; 6 ! ;; 7 B H3 3 KL ; 6 " !! ! ; 7 A ! B _V B = ! " 7
B
]
0 !
[
; ! ;OB
W
( 3 D " ' ! ; 7 B
tel-00635537, version 1 - 25 Oct 2011
6 3 66 ;" ; B ; ! 3: ; ! ;; " _ 7
W( ;66 !H 5 B ! ; ! ,, ; i H 7
Z + 66 A 2 B ! 7 ) B @ ! B ; ( A6 @ 7, =; 2 ; @B ; ; &)$B 6 ; 7 B 2 " ; ( !! B ! ! E&)$Gj 3 ; ; V ! @ !; A !7
i
1!bb 7; b b!Sb)+ V ] ! ,,!H 5 7
j
)67 ! @ $ $'3 7
WW
1 . @ / 6
tel-00635537, version 1 - 25 Oct 2011
WZ
) B ; ## ; @ ! 3 6 ; B 3!; B ; ! B ; 7(A " ; ( A 6 6 Bi ; 6 " B Q 6 @ ;7& !! 2 C6B 66 &)$B ;66 A ;7 6 3" 2 &)$B 66
tel-00635537, version 1 - 25 Oct 2011
; 6A 3 ## 6 ! ; 7 V !! " &)$ ; ; 6 ; 7 ) ; A 6 !3 !; = 7* &)$ ;;7 ;! B&)$ Q ; 70 " &)$B ; !! C !; ; 7*66B B! !H d B V ; 6 !! ! !! ;B 66 7 6 0 /0
! ; ; ; B 3 ; H3 7
Z
(). B ] " 7 [& + 6 3 7 B ; &)$ (), 3H! 7 B WB Z & &)$ + (). " @ 7 B ] & ; ; 66 ;().
;b &
().B Z 6 !; 7 ] ' " B ! +
W
;b &
tel-00635537, version 1 - 25 Oct 2011
66 B A
;b & B + &)$; @W
;b &7* 6 ().B + &)$ " 6 6 7 ]&
;b &+ ! (). W]d 6 7 " @ 6 ; B " c0 6 6 &)$ (). 7 ] ) 66 ;## ().;f@ 7 !; 6 8+ ! ' )07 2 + &)$ 8+7 ) ; 8+ " 2 !; ; + &)$ !; 2 C !; ; ! A 7 B ] B ]V , 2 ; ; B C + B 6 8+ A ')07 ]
Z
&+ &)$ 6 E!!3 3 2 G "7 - " 66 3 !H ' 2 ; E8+B )0B (B )& (G !H C Q7 &
> 66 " &)$ '7 ]] , = 3 6 A &)$ ;6 ( 2 7 C Q 6 8 7i
tel-00635537, version 1 - 25 Oct 2011
;B # 6 8B ! 6 6 2 7) @ B &)$7 77* .' ! ! ='
(B !!3B ; 3 *&,' 6 &)$ !!3 66 3 ;(7 & !!3E G !;; 8+ 7 A B &)$ ! ! 2 ( !HC 8+7 ][* )&B B 7 ]WB ]Z- ; ! &)$ ( A 7 [ [ ;6 @ ; ! ; " ; 67 [V [] & ; &)$
i
)67 ! Q *77* @ . = ! $ !' 7
Z
; 6 2 ; 3:7i) !!3 6 (7 !!3 W E WG ; 6 D;6 &)$ ( 2 C ; 6 2 ; 7 ]WB [[)!!3 @! !! &)$ ; ; (7 & ; *&,' !!3 E,r !! EWGB ,;G "
;b & !H C ; 6[
;b &!H (7 ] & W ( 2
tel-00635537, version 1 - 25 Oct 2011
8+ W d6W d7 [W) @ 6 ( ; 7 ][B (2 3 )& W7 ]ZB [[ & !!3 ! E G66 !;" ( !; 7 [Z , = A ; ; (7& ; 3 (CQ ; Z d 6 Z d 37 W ' A ; 66 ( 2 Z d 6 ][d7) 6 @Z d (8+ W d7 ][ )!!3 A66 !; ! @ 8 (7 W & = " 2 ;6 !H (), ; (), (7 [V ) !!3 = (),( 6W d7 W ,@ !
i
)677* @ .! $
T ! 7
ZV
; ( 6 D @ 7 W ) = ; 2 ; ; B 7 - !!3 " ; ; ( !!3 ZZ E ZZG7 & ZZB 6 D ;"8B WV ( W d6 W d7 [
tel-00635537, version 1 - 25 Oct 2011
!3!!3 B ! B A &)$ (), (7 !!3 (), (7 W A "B ;f @ ! H3 " B !!3 2 Z] V V A &)$ (B B ; 6 Q ; 7 W] ( !H 3 (7 , ; ; 66B @ 6 ; 7 ,3 B@ B ; ; 7 - + 3 7 W[ & + ! ; !H 7
Z
7* .! $
T
)!H (B ! _V ; )& ().B !; 6 '7 V[B WWB WZ) !;7i + B _V &)$ ;f@*&,' E,r _ 3E V GB ,;G7 & ! _V ]
;b &!HC ; 6 ]
;b &!H (7 B B ]V ;_V &)$ (C66
tel-00635537, version 1 - 25 Oct 2011
; W]d6Z d7 Z - ! _V ' 2 7 Z A B +*j ; &)$ ## ! _V @ Q 3: 27 Z )";;" _V &)$6" '7 * (), @ 7 V B Z B _V &)$ 6 7 ZV, _V &)$ 6 ;3 *7k & " qV ;" * @ ! _V " ! H3;qVbqV;"*7 Z ) ! B6 ; " _C Q 2 3 (7l ! 2A E 6G ; " &)$ 3 (7 Z] A B i
)67 ! Q 7*7G7G @ 6$ $" > 3 $'3 3 A7
j
)67 ! 4 7*7* @ 3 7
k
)67 ! 97G @ E $ O 3$ $" > 37
l
)67 ! Q *7*7* @ . = ! $ !! $
T7
Z]
" ; "_ ;6 ; !H ( !H 2 ; ; E !! G7 Z[ ; B; "_6 ; (7*66B (('i !H ; " &)$7 B 6 !; ;; A ;B ; " &)$ 6 6 ; ! ; ; 7
tel-00635537, version 1 - 25 Oct 2011
& _ B B_V B_ W_V ZWE6; G7& ;_ C &)$ E_V G"6 6 6 ! ; _ 6 ; 7 ZZ B KL 3:;"Bj 6 _ ! _V 6 B " ; ( 6 ( !; E 6 G7& _V b_V ; ; _V &)$7 ZZ
i
)67 ! Q 7*7 @ 5 O != O7
j
)67 ! Q *7*7 @ !! $
T7
Z[
E *+7 E3 33i! ().7V W & " 6 !3!" 6 ().7V Z?CB ]] 3 D ;W 3 (). 6 $3 -7' "62 ; (). B 6 ().7VV X B 2 ;B B ! !; ; !H ().7VV
tel-00635537, version 1 - 25 Oct 2011
B 2 @ ! ; ; !H 6 ().7 & !; @ " 6
!H 6 ; ().7VV 6 B C @" B 7VV BVVV-; 6 c0 ().B ; 2A 6 ; E B & ; 4 5 * &3G7 , 6 ; @ # ; 2@ ; 7 2 2 A !; B !H ! B o 6 A W d 2 ! 7VV *66 A 6!H 7VV]
i
& 6 ; 6 237
V
7K 3# $ 3O
& &)$ E 2 ; _V B + WG ; 6 ; ; B ; 66 7 V[B ] B ]V) &)$ " ( ! 2A E+ W " _V " G @ " ; @" 7VV[B 26 6 2
tel-00635537, version 1 - 25 Oct 2011
2 ! 3! 7 &C2; 2&)$ ; ( 6 @ 6 7i ) 3 ( &)$ 6 6 B 3 C ( 7 E G ; ; 6 6 7 & + _V ; ( 3 8+ 6 W d7 ]V & Z d 6W d (), ( C 7 Z& (), ( W " + _V &)$ ; 7 & E+ B W _V G ( C @Z d6W d (),7 V & 6 W @ + ( (),7 ) &)$ C ; 6
i
)67 ! Q * @ ! 87
3(!H 3 3 0 6 ; ( ;f @ 6 !; 7iB Z ) " &)$ @ (7VVW A 6 DB ; ; )E G &)$7 ! Z d 3 0 ! ;; ( C 7VVZ & + b_V ; 6 ;( @
tel-00635537, version 1 - 25 Oct 2011
; 7V B V ) ; ; 6 ; ( 2 ; 7V B 6 ( @W dB 6 ()&7V ) 26 ()&7* 8+B 66 !; ( W d26Z[d7V *6B + b_V A 6 6 6 ; !H C 3 B @W !H C 7V A "B Wb_V = ; ; 66 (7 , " ; 3 (2 8+ 6Z d7V VB 2 3 ( 2 ZWd WVd7) ; !; C ( 8+ 7V V ) = 6 ( ) ! ; 7 ) A @ !; !; i
)67 ! Q *7* @ ! !' = $3 $ 7
]
3 " ; (27i ; &)$ !3 !; (!H ; !;7
) ! A ; _ &)$Bj 6 2 6 _ " " ; ; 7 ( 2 ; + &)$ ; ;7 _V b_ W_V b_ [ @ + &)$ 2
tel-00635537, version 1 - 25 Oct 2011
d@" 6Z d66 ( 2 3 )&7V ! + (). 3 E (G W ;6 !; 2 B W + " (). 2 7V ] ) 6 @ 7 (). @7 ( !H 2 27 A B66 (). 2 @6 66 E@ WG7V [, C W &)$ (). 6 B 2 3 + &)$ 6 (). 7V W) 66 (). 6 2 7 B ; B (). (B C @ A ; 667
i
)67 ! Q * @ ! 87
j
)677* @ .! $
T ! 7
[
; ; ; B 66 7 B W ; ZVd ( 6 ]]d 2 ; 7 ][ " B !!3 Z]V VE 58 G + ( 6 Z]W]d7 W]) ; 26E
dG ( 2 7 & ; ; ;
tel-00635537, version 1 - 25 Oct 2011
6 ; ( 2 7V ZB V] - &)$ ! (B6 ; 7V],2; 6 ; ! ! ; _V &)$C (),76 D B ; ! + B ; ; (7V] - &8+ ; ! + !H ! 2 6 7V] A ; &)$ ! ; 7i _V &)$ ; 7V]V
i
)67 ! Q 7*7* @ 3 7
W
*
6 0 /0
*7 6O # O ! $ $ 6 $ = $3 !! '
; ; ; @C CQ 7 " 6 ; ; 667 &!3!" 23 6 6 ( 3 !!7, 6
tel-00635537, version 1 - 25 Oct 2011
@ _ 7V] 6 !! &)$ 6 6 @ !; 7 & 6 ! > ; &)$ (7V]]B V][ & ) &)$ ( @ 7V]WX B ! ; ; ( V]Z !3; 7V[ A B B 23 6B ; ; E&)$ ; G ( 6;67V[)!H 3 (),B ; ;; 2 ( C 7V[ ' ! !3 !; E23 6 ; GB @ "6 ;6 6 @ !;6 7V[ *6 C &)$B 6 ; 7 & ; ! 3i + @
i
* _7
Z
;6 !H ( @ 7V[V ) B 2H3 " 6 (), 7& B ; B 2 ;; 2 ; &)$ (7V[ & 2 ;; 2 @ ! !;7V[] B &)$ !;; H! 7V[[ & !3E GB" ;
tel-00635537, version 1 - 25 Oct 2011
;&)$B ; ";6 &)$ ( A @ 27' ; &)$ ;C (CQ
d 6Z d7V[W- B ! 6 3! ; &)$ (B 66 (), 2 (
d 6 [ d7V[Z * !B " 6 @ !;; 8+7VW & ! H3 @ 3:;"B _ B ; !H ( !H (),7VW& H3 ;3* !H " qVBVW A !3 !; 66 @ ; E ;3 ;G 6 !; ;3 * ; ; ! 3 ; 2 E 36@ d
" G7 & 2;" K) ! $; + LB 2 !3! 3" ; B;6 &)$ )& 2 ( 2CQ7VW ) @ 3" ; )&7 *6B 3 B ! B
2 !;; 7 ; 6 &)$ 8+ @ ( BVWV 66 6 (8+7
*7* $ $" 6 2
*7*7 .' F7G7G
& V7 7 ; E B 6B P G [ 6 7 *
tel-00635537, version 1 - 25 Oct 2011
; 3 B ; B ; > !! 7VW ' &)$ @3 ; ; B ().7 ) B 6 B 6 (). 6 D 3 ! ! B! B3 ! 5;> 6 D 3 3 B ! ! B ! 5 ! ; 6 ; 7VW] VWW B !! V &)$ 6 " ; 27 6 B (). V7 7 &)$7i V, !! V7 7 &)$ ; 2 7& !! &)$ (). @ C ; @ ; (). 7VWZ& 6 (). 2 @Z d7VW[& ;!H (). !; 6 3 ! Z7 & V7 7 6 !H3; (b1 E[[dG 2 ! H3; (b( EWZdG 1b1 EZVdG7 B
i
)67 V &)$7$ 5 ' BC
[7 ! ; SSS7! 76
&)$ ; (). 3 b 7VZ * 6 ().B V7 7 &)$ "" ; 6 7& &)$ (). 6 ; @ [ d7 [ 6 D B d ,88B d 4'' Z d *
#7 A B " " (). V d7 ]&6 ;" @ ; V7 7 &)$ !;7" @[[d 3 A @W d ;66 " [ d
tel-00635537, version 1 - 25 Oct 2011
6 @ ! 7VZBVZ *7*7*
3O
6 6 '
_ 7&6 6 ;33 &)$ !; !; E! B ! ; ;G f 7' 6 @W d (). 7VW]& '
_B3 62 ; B "! f 7VZ ' 6 (). Z d ] d7VWZ
1L
tel-00635537, version 1 - 25 Oct 2011
. @
.E6 /0 6 0 .
/ 6 " D16 6
.. @
tel-00635537, version 1 - 25 Oct 2011
.. . "E 6 " D16
. 0 E 6 7
V
6 3 6 ; 6 B] ( 66 @ ; 7 & ; &)$ + W _V B ; ; 7 V - ; ! + W @ _V &)$ (7 ) 6 ; &)$ ( !; 6 8 ' !3!!3 _7 & &)$ 6 !; ; !; C 7 VV
tel-00635537, version 1 - 25 Oct 2011
* 6 B _V &)$ ;
7 ) ; B
7& @ _ 6 ; 7 ZZ- @ ; ; 2 6 ; 3 ; ; (7VVZ & ; _V &)$ _ 3 ; ! _V 7 ZZ A ; &)$ ;4 5 *&3E+ B W _V G ; (7 B A ; _V b_V ; 66 (7 *6B 6 ; &)$E+ B W _V b_V G 6 ; 6 !; C 3 7
. 6 1
7 ' $ !
& ; " ; ; 7 C 2B ; K L72 K LB @ 67 + D 3!; 2 ; 7 & ( ; " ; (#! 7 & )&
tel-00635537, version 1 - 25 Oct 2011
" (#!B Z2 8+ " 3BWW 2 (). " 2 $ 7 V & )0 ' ; 2 ; 2 7i + 6 C 3 ; &)$ ;4 5 *E) 0; !;*G7
77 . ! "'5 $ !83 $ $ $ 3O $
'
&)$E+ B W_V G ; EGB V E] !B W 6G (B [ E[ !B Z 6G ; E [ )&B [] 8+B 'B )0B ().G ; H! K LE%G]V E !B
6G ; 6 EG EB 3 3! 3!B ; ! B ; B &3 B 3 B !3! ! ! ! G7 & 66
i
)67 ! Q K7 ' ' ' #; K7* .= ! ' ! 5
]
66 &3BCB' *B 4 B1B( B16 !' QB( B0;0$
7 & C 5 $!! )
V C*-8]&'50 )R
] [[Z [[Z7 & ! E)G 6 ; E2 G 2 E2]G7 77* . ! "'5 $ !83 $ $ $ !! $ TF+ $
$
TF*Y TF+ ' A !' 4 !4M
tel-00635537, version 1 - 25 Oct 2011
& &)$ 66 C B ! ; _V &)$ ; b 7 ; BZZ EV[!B 6G ( 6 ; ;_V _V b_V &)$7B Z EZ!B 6G )&BV E !B [6G 8+B [ EV !B 6G 'B V EV !B 6G 3 )0B E!B 6G ().VZ EZ!B
6G 7& 6 C ;_V &)$@ ; &)$7 77G . ! "'5 $ !83 $ $ $ 3O $
$ !' 8 $ $'3 $' ' ' 5
- [ ; 2 V 3 B ]W 3 6 2B V 3 ! B )B ] 3 E')0G[3 3 ; E''G7+ 6 3 ; ; C I[d @ 6 Zd2 3 ! " 7
[
77 7) 6 & 6 ; !;7 ; ; 3 2 @ 6 6 ; 3 C '' ;6
'3 6 ; ;
'3 6 326 @ E ; B! B G ; 6
tel-00635537, version 1 - 25 Oct 2011
'3 ! ; B 6 ! 7)3 3 i ! 6 E8G 6 6 ii ! ; 6 iii 6 !
'3 0) 2@ A @
'3 E')0G 2 3 3 > 3 B @
'3 'E''G 3 @ ;6
77 7 ) 6 ; & ; ! E+ B WB _V _V G 66 7& _V b_V 7 & 6 6 ; 7 &
W
; 4 5 * 6 ; + x ;b &B Wx] ;b &B _V b_V p 7 7 ; ; 6 ; 6 + W _V b_V ; ;6
( ; + x ;b &B Wx] ;b & _V b_V p 7
0; 3 !; ;
0; + p ;b &B W p ] ;b & _V b_V x
tel-00635537, version 1 - 25 Oct 2011
7 7* = # O $ O $ '! $
7*7 ' $ ' # O 5
& &)$ ; &Vb& & b&]7 2 &)$ 33" E1c$B 3 B -'G6;E @V
;G ! 7 & ;
y 33" ; @ W ) V ! 6; C @ 3 ; 7 & &)$ 2 @ " 3; ! B ; A 6 @ b B 2 3!3 6 @
b !6@7 ;b&7 7*7* $ # 3O $ O $ '! $
& ! + B W_V _V ;f @ > *&,' 2 E,%+*'+ ! ;B ,%+*'+!! + EWGB,%+*'+_ 3E V GB,;rB4!B0; 5 3 β E V G EG 3 #B ,0&B 5 ;B ;G ! 6 >7 + ;
Z
! ; Q &)$ + E sV ;b &GB WE] s 7 ;b &GB_V E
sV[ ;b &G _V E
s W ;b &G7 ! 3 @ 3 @ d b ! ; B 2" 67 & _V b_V + b W 7 7G
= O
- 3 @6 E%1G 66 6 f; ; 7-)! ! 6
tel-00635537, version 1 - 25 Oct 2011
66;7&((' #> c ( c!3B 66 ; 7 & ;66 ;f 2 $%)7) 3 @ ; ( 6S E( rB 1777 G7 & 6 ;6 p 7 7 *
*7 5 $ !83 $ $ $ # 3O $ '
! $ $ 88' $ 3$ $" > 3 @ 4; !4M
TF*
66f;Ee 7 ]G 2Ee 7ZG 66 ; E ]G7 * #> c 66 ;6 ((' 66 ;( !H ( 2 B %B)&8+Ep 7 G7
# ,7 6= $ 66 $ $ !' 4; !4M $
!! $ TF* $ $ 88' ! $ ! 7 ( H! I% H! I; 6I)& @ &S3I 8+ 6 I ' 3 ; I )0
tel-00635537, version 1 - 25 Oct 2011
; I().I )H6 . > 7
*77 .' $ ' #A
& ( ! + W
;6 ; % E p 7
G E6; G7 * !B + W ; % 2 ; E p 7 GB 2 ! ! 2 ;7 & _V ;6 !H 66 ( !H 2 2 ;B % E p 7 p 7
G7 , 66 ! ; 67 $%)B ; W &)$ ; 66 ( 2 2%E6; G7& ;_V &)$ @ @ + W7
tel-00635537, version 1 - 25 Oct 2011
E *7 3! $ $ !' 4; !4M $ !! $
TF* $ ! 88 $" 3$ $" > 3; A
! $'3 ! ! $' ' ' 87 0 @ ! E G &)$ 7( H! I% H! I; 6Iiii p 7
Iiip 7 7
tel-00635537, version 1 - 25 Oct 2011
E **7 # $ G # 3O $ $ $ $ 88' $
3$ $" > 3 ! !! A O $' ' ' 5
A $'3 $' ' ' 57 ( H! I ; 6I % ; I+ + I + WI V _V 7
*77* .' $ ' $ A $ $ $ 88'
& + &)$ (). ;6 E p 7
G !; ; B ; 2 2E ]G7*; ;6 ; &)$ ( 2 )&B8+B)0'Ep 7
G7* !B 66 + !H 66 ; )&B8+B)0'7 & W &)$;6 ; ( @ !;; @ )&B 8+ ' E p 7
G @ (). )0 E p 7 G E6; G7 ) ;6 ; )& (). 2 Ep 7
GE G 'Ep 7 G ! ! 2 E6; G7, 66 ! W)0B8+
'7 *6B A W &)$ ;6 !H 3 8+ Ep 7 GB ! ! ;E ]G7
tel-00635537, version 1 - 25 Oct 2011
E *G7 3! $ $ !' 4; !4M $ !! $
TF* $ $ $ 88' $'3 $' ' ' 57 0@ ! E G &)$ 7 ( H! I )& @ &S3I 8+ 6 I ' 3 ; I )0 ; I ().I )H6 . > Iiiip 7
Iiip 7 Iip 7 7
0 _V &)$ ;6 ( 2;)&Ep 7
GB8+Ep 7
GB)0E p 7 GB().Ep 7 G'Ep 7
GB2! ! A ; 6('E6; G7*6B@ 6B )&)0 _V &)$ 2 Ep 7 Ip 7 GE ]G7
V
tel-00635537, version 1 - 25 Oct 2011
E *F7 # $ G # 3O $ $ $ $ 88' $
3$ $" > 3 ! !! P $'3 $' ' ' 5 '$ '7 ( H! I )& @ &S3I 8+ 6 I )0 ; I ().I )H6 . > I ' 3 ; I+ + I + WI V _V 7
* 3$%)B ;+ W &)$ ()&B ;_V 6 ; ; 66 6 [ dE6; VG7 (8+B ; W &)$ @ + _V 7& ;66 ( )0 A &)$ 66 7* ( ().B ; + + b W 2 2 !;7*6B &)$2 ; ( '7
# (7 '3 $ ! 3A $ $ G # 3O
! $ $ 88' $ 3$ $" > 37 ( H! I ; 6I% H! I)& @&S3I8+ 6 I ' 3 ; I )0 ; I ().I )H6 . > I' I'6I-) Ix Ipe6; 7
tel-00635537, version 1 - 25 Oct 2011
& $%) 6! &)$E [G766 (2 ; 6B 6 Z dWVd ! + @ Z ;b &B Z d W &)$ @ ;b &7 * ! _V B 2 E 6 @ W dG ( ; I ; EZ dGB @VZW;b & 6] d7 * ( H! B66 ; W 2 W]d 6 W d ] ;b &7 2 B 2 666 B _V E [G7 & ; + W &)$ ; ( )& WWdW]d6W]dW[d@
Z;b &] ;b & 7* !B2 _V 6@ d;2 !; E [G7
]
A B 6 ( 8+ 2 [W;b & + BW];b & W _V 2 6@] d7 & &)$ " 26 ;('@ V;b & + E'ZdI'
dGB ;b & W E' ZWdI '
dG VV ;b & _V E' W dI'
dG7 *6B ; + ().Q (
tel-00635537, version 1 - 25 Oct 2011
Z d6WVd ]][;b &B 7B + b W " Z d 26
d;().(E [G7 ) 6 6 ; 66 ( + x ;b &B W x ] ;b &7 _V B _V p
;b & ! 6 7& &)$E+ B W _V G @ 6 ; 66 ( @ )0 E WGB !; 6 WVd_V (E [G7 # M7 !! $ G # 3O $ ! $ $ 88' $
3$ $" > 37 ( H! I ; 6I % H! I )& @ &S3I 8+ 6 I ' 3 ; I )0 ; I ().I )H6 . > I' I'67
[
6 B W dB &)$ ;66 ( 26; ( E'
dG ' E'
dG " 6 ; ( )& E' W[dGB 8+ E' WWdG (). E' WVdGE+ WG7 *7* 5 $ !83 $ $ $ !! $ TF+ $ $
TF*Y TF+ ' A !' 4 !4M ! $ $ 88' $
3$ $" > 3
66f;Ee 7 WG 2Ee 7WG
tel-00635537, version 1 - 25 Oct 2011
66;7 A B 66((' 66; Ee 7 ZG7
# J7 6= $ Z ; $ 66 $ # 3O $
5 TF*Y TF+ # $ 88' ! $ ! '$ '7 ( H! I % H! I ; 6I )& @ &S3I 8+ 6 I ' 3 ; I)0; I(). )H6 . > 7
W
tel-00635537, version 1 - 25 Oct 2011
E *K7 3! $ $ !! $
TF*
TF+ $
O $ TF*Y TF+ ! ! 3$ $" > 3 A
! $'3 7 0 @ ! E G &)$ _V _V _V b_V 7 ( H! I )& @ &S3I 8+ 6 I )0 ; I ().I )H6 . > I ' 3 ; Iiiip 7
Iiip 7 7
- ; _V @ ( 2%Ep 7 GEp 7
GE8; G7& _V b_V !H ( !H Ep 7
G%Ep 7
G7B ;" !H % 2Ep 7 G7
Z
tel-00635537, version 1 - 25 Oct 2011
E *,7 3! $ $ !! $
TF*
TF+ $
O $ TF*Y TF+ ! ! 6 A !
$'3 7 0 @ ! &)$ _V _V _V b_V 7( H! I% H! I; 6Iiiip 7
Iiip 7 7
, 66 _V &)$ (B )&B ().B )0 '7 * !B ; ; ;6 ( @ 8+Ep 7
G )& @ 8+ E p 7 GB 2 ! ! ; 7E6; ]G7 & _V b_V ;6 ( @ )& E p
7
GB 8+Ep 7
GB ().Ep 7 GB )0Ep 7 G 'Ep 7
G E6; ]G7) )& @ 8+Ep 7 GB 'E p 7 GEp 7
G7, 66;6 _V b_V 7
* $%)B + W &)$ ; 66 ( 2 % E6; [GB6 "7* ! _V b_V ;_V _V &)$7) 6 ! + W7
tel-00635537, version 1 - 25 Oct 2011
E *(7 # $ 4; !4M;
TF*
TF+ $ O $
TF*Y TF+ $ $ $ 88' $ 3$ $" > 3 ! !! A
O $' ' ' 5 A $'3 $' ' ' 57 ( H! I; 6I% ; I+ + I + WI V _V I V _V I$ V bV _V b_V 7
& _V b_V ; ( 8+ o 6 2+ W ; ( ' 6 ; ( ().7 * !B _V b_V 6 ()&( )0E6; WG7
tel-00635537, version 1 - 25 Oct 2011
E *M7 # $ 4; !4M;
TF*
TF+ $ O $
TF*Y TF+ $ $ $ 88' $ 3$ $" > 3 ! !! P
$'3 $' ' ' 5 '$ '7( H! I)& @ &S3I8+ 6 I)0; I().I )H6 . > I' 3 ; I+ + I + WI V _V I V _V I$ V bV _V b_V 7
& _V b_V 7 ; ( 2 ZWd 6 WZ7Wd E G7 ) B 6 7 ] 7 d ( %7 & ( 8+ _V b_V _V Z d 6W d 7 V]7 A B ( ' 2 ZZd6
d 7 V7 * !B 6 @ d; ( )0 )& ; EZZdZ d @ 7 ]G7
# +7 6 $ $ !! $ TF* TF+ $
O $ TF*Y TF+ ! $ $ 88' $ 3$ $" > 37 ( H! I ; 6I % H! I )& @ &S3I 8+ 6 I ' 3 ; I )0 ; I ().I )H6 . > I '
tel-00635537, version 1 - 25 Oct 2011
I'6I-) IxIpe6; 7
) 6 _V b_V ; 66 ( _V b_V p 7 7 ! + x ;b &B W x] ;b & _V p
;b &7 & _V b_V ; ; ; Z]d ( ; !;E G7& 6 ; (B ' ().
d7 ) 6 WW7 d ( %7 A 6 W 7WdB Z 7 dW 7[d ( !; )&B 8+ )0 7
# 7 .83 $ O $ " $ !' 4; !4M
$
TF*Y TF+ ! $ $ 88' $ 3$ $" > 37( H! I ; 6I % H! I )& @ &S3I 8+ 6 I ' 3 ; I )0
tel-00635537, version 1 - 25 Oct 2011
; I().I )H6 . > I' I'67
*7G 5 $ !83 $ $ $ # 3O $ $
!' O 8 $ $'3 $' ' ' 5
& 6 ; 3 [ 6 ; 27i *7G7 = $3
3 ' O
& 3 ; ( [ d ( Wd E6; ZG7 6 3 3 ; d ().7 % ][d ;&)$ ( d ; 3 7 *6B d ; &)$ 7
i
)6777G. ! "'5 $ !83 $ $ $ 3O $ $
!' 8 $ $'3 $' ' ' 5 7
V
E *J7 # $ $ O # O $ ! !'
= $3 3 ' O7 ( H! I (), ;6 ;I )& @&S3I(). )H6 . > I&)$ ! !7
tel-00635537, version 1 - 25 Oct 2011
0
)
E G+7 '! $ $ O $ ! 3 ' O 5
# $ 3 % -; =! O $ 3$ $" > 3 %- =! O % -7 ; 6I( H! I(), ;6;I)& @ &S3I(). )H6 . > 7
* ; 2 ; B WWd ; &)$ 3 ( ; ( d ( E6;
0G7& ;&)$ CE G@
; !;E6;
G7& ; 3 @ 2 C @ ; ( [Wd ( E6;
)G7 *7G7* = $3 8
& ; 3 6 ( d ( Vd E6; G7 A B d 3 6 ; 66 8+ d 8+ 7& ; !; 28+ '& Zd Vd )&7 % ( ; d VVd
tel-00635537, version 1 - 25 Oct 2011
6 2 ; &)$ 3 7 B d 3 ;&)$ 7
E G7 # $ $ O # O $ ! !'
= $3 8 7 ( H! I 8+ 6 I 8+ '& 6 @ 3! I ' 3 ; I)& @&S3I&)$ ! !7
B ; 66 8+ C ; &)$ 3 EV WG E WG E6; G7 ; ( ;&)$3 (!HZ 7
]
tel-00635537, version 1 - 25 Oct 2011
E G*7 8 $ $ O # O > !
!' = $3 8 7 ( H! I8+ 6 I )& @&S3I; 6I' 3 ; 7
*7G7G = $3 ! O
& ! ; ( ( ]d E6;
G7 + 3 ! ; &8+7 B d ! )& B ; 67 3 ! C
E[]dGB ! ; d Zd E6; VG7 & ; &)$ ( d &)$ 3 Wd 7 - ; d 7 X ;3 ( &)$ 27 * ! ; B &)$ 6 (B ; 3 ; &)$7
[
E GG7 # $ $ O # O $ ! !'
! ! 3 ! 57 ( H! I &8+ ;
tel-00635537, version 1 - 25 Oct 2011
6 I)& @&S3I&)$ ! !7
E GF7 '! $ !8 # O $ $ 88' !!
! O7 ( H! I ! ; I &; ! ; I8 ! ; 6I' 7
W
E GK7 8 $ $ O # O > !
!' = $3 ! O7 &8+ ; 6 I (
tel-00635537, version 1 - 25 Oct 2011
H! I; 67
- ; &)$3 ( ] W ; ( 7 E6; G7 B C ; 66 &8+ 3 &)$7
*7G7F = $3 #
& ; )0 !H ] d ')0 [d ; ( E6; ]G7 % [d ')0 3 ; &)$ 3 ( VVd 3 7 2 6 ; ')0 ; &)$ 7 - ; 3 ( &)$ !H ; (!H ')0E6; [G7& ; )0 3 2 E[ G7
Z
E G,7 # $ $ O # O $ ! !'
= $3 #7 ( H! B )0 ; I &)$ ! !7
tel-00635537, version 1 - 25 Oct 2011
E G(7 8 $ $ O # O > !
!' = $3 #7 ( H! I )0 ; 7
*7G7K = $3 $
& ) ; ( W d B)0 d )& Vd E6;
WG7'2 H 3 &)$ (7B d ;&)$ 3 Vd2 ;&)$ 7
V
E GM7 # $ $ O # O $ ! !'
! !' 7 ( H! I )& @ &S3I)0; I&)$ ! !7
tel-00635537, version 1 - 25 Oct 2011
- ; 3 ( C 3 D ; (!H )E6; ZG7- ; )0 ;&)$3 (7
E GJ7 8 $ $ O # O > !
!' ! !' 7 ( H! I )0 ; I)& @&S37
V
*7G7, = $3 $ ..
& '' ; 'E G7 % [ d ; Vd 3 ;&)$3 (E6;V G7
tel-00635537, version 1 - 25 Oct 2011
E F+7 # $ $ # O $ ! !'
= $3 $ != ! ' ! 57( H! 7 G
G7 $ $ $ # 3O $ ' ! $
$ 88' $ 3$ $" > 3
A ; ; & ;4 5 *7 B 66 ;6 ! &)$ + W ; _V ( 2% 2 ; 7 ) &)$7 V[B B ] * B ; &)$E+ B W _V G ; ( Wd 2
V
6 E
dG 6 @ ; 6 ' E WG7 ,@ 6 ; ; W d ;; ( 8+B)& A ().7* !B (2 )0 6 ]7 d7 & ; ( W d 6 [ d7W )6 6 !@ 7 Z
tel-00635537, version 1 - 25 Oct 2011
&2 !; E,$(G @ 6 ; 7W A B &)$ ; ; ( E WG7 & 6 &)$ ( )0 2 !; !; !3 7 3 7 6 D ; B 6 ; _V ; 66 (" 2+ W E [G7&6 ;;( )&8+ 7Zd] 7 d 7 A B 6 ; ( % d ] d 7 ) 6 A 2 6 Q 3:` '3 !H )& [ ' !H C f; ; 2 !H7 WW A 6 DB 6 _V ; ( 8+ A !; !; 8+ d ; 8+(6 !; 7ZW 66 (). (B ] ] ;b B @ W dB 6" E] dGE6; VG7
V
().B E]G ! W E ] ;b &G7 ) 2 6 !3!!3B 8 7 * 66 ! !; ; ().7[ BVZV- &)$ W !H (). ; 7 B C ! (). @ 7 X B + b W ; ( 2 (). 2 6 &)$7) " 66 "
tel-00635537, version 1 - 25 Oct 2011
; ; ; (().7V ] 8 B &)$ ; (7 B 6 ; ; _V &)$ C ; 66 7 - ; 6 ; !; 3: 7
G7*
= $ !83 $ $ $ $ !! $
TF+ $ $
TF*Y TF+ ' $ $ !' 4 !4M
& ! ; _ &)$ @ 7' B3 ! ; ( ; 7 )!H ! B ! _V ; @ 3 ; 7 ( _V &)$ !7 A 6 DBC!3 _V &)$7 ) 6 ; B _V ; !; 3: !3 7
VV
& _V 6 C 3:;" &)$7' ! " ; _V _ W !6 ! ; _ 6 ; 7 ZZ& ;_V &)$ @ ! ; _ &)$B 7 B 6 ; _V 6 ;66E G72 " 6 ; _V B 6 E GB ; A ;66
tel-00635537, version 1 - 25 Oct 2011
(7 &A _V b_V 7 , 6 ( 2 ; 2 ZWd 6 Z d7 A 6 ( 8+7& ' ().@6 6 6 666 E[ ' ().GE ZG7 & 66 ; ( 8+6 _V b_V ; _V 2 ( 3 ! ; 3: @ B 3 ! ; 3:6 8+ & _V b_V 6 ( )& ( )0 E6 dGB 6 ! 6 (%E G7 B )&6 3: 2 6 ( )&7 ) ; 3: _V B 6 _V b_V 2
6 Q 3:
2 !H )&7 [ *6B !; !; )0 2 6 _V b_V ( )0B 6 ;_V 7VZ
V
_V b_V Ep 7 G A + W7& 6 ; &)$ @ E ; + W _V G7 & Wd E WG @Z]dE G + B W _V b_V 7 A B 6 ; 66 ! ( )& A 6 W 7Wd E G7 & ; + B W _V b_V &)$ ; 6 ; 66 ( ;
tel-00635537, version 1 - 25 Oct 2011
; 6 6 (7 ) ; " 6 6 (B ; 6 3 7B @ 6 B ; !; C 7 VV G7G 5 $ !83 $ # 3O $ $ !'
8 $ $'3 $' ' ' 5 & ( 3 7) B C !H 7 2 &8+B )0B ' )&7ZWB
B WBVW ) A@ !;; @6 ( 27]% 6 ; 3 ( 3 3 B 3 6 B 3 ! B )B')07& 63 !;7 3 C ! ''B ! 2 3 !; 3 ;7 & ; ! + B W _V b_V 6
V]
6 ; (6 ; 7 &3 3 ; (7 , M @ " ! 7 ' d &)$ 2 ; ( E6; ZG7 - C E][dG &)$ 3 (7 6 D B W d 3 ; ( 7 d ; &)$ 3 (7 ) 3 ; @ C G
tel-00635537, version 1 - 25 Oct 2011
_V b_V ! + B W G + B W &)$ _V b_V G 2 @ 7 - _V b_V ; !; 3:7 - _V &)$ !H ; ! 3:B().B)&7 WWB WZBVZ] )6 ; 3 A@ !;7B 6 ; ( 7- ; ; ( ! _V 7V[ ) 2 + W &)$ _V b_V 7 @ !H d 6 ; 3 7 , ; 3 o ; 2!B 6 ;7 - 3 6 ; ( 7 - ; E VdG ; 8+ 6 2 E8+ '&G E6; G7 ; ; &)$3 (7 - ;EVVdG ; 3 (7 A B ')0 C ; )0E] dG@2 ; B [d
V[
; ( E6; ]G7 6 D B [d ')0 ; &)$ 3 (7 6 EVVdG ')0 ;&)$ 3 (7 ) !; 6 ; 2 3 6 !; !; 8+ ')07 & !;; C 3 7 - !; W 8+ ')0 " d 8+ ')0 (7] - ! E)0 > G ] d 8+ d
tel-00635537, version 1 - 25 Oct 2011
')07 6 3 B 3: + 7 ! _V B C@ !H )0BVZ[ 6 _V b_V 7 ) A 2 Q 3: !; )06 7VZ -; EV dG ; 3 + W &)$ _V b_V 7 ) ; ! + W )0 _V ; (7 ] BVZ[BVZW) 3 6 ; ! 2 !H! 7) B @ &)$!H 8+)06 !; = 6 ! "7 *6B ; B ')0B 6 3 ; (7) 6 !; ')0 (7V[BVZZ 6 !3!" ')0 3 ; ( ( C 7
VW
& '' ; '7 - ; E[ dG ;&)$ B &)$3 (!HVd2E6;V G7& &)$ ; ! 'E8; G ! ; ; ! 27 B 2 666E[ GB @6 ;! 6 !; 7 &3 3 &8+; ! 7% d6 ; (
tel-00635537, version 1 - 25 Oct 2011
! 3 E6;
G7- C ! E ]dG ; ( d &8+7& ; 6 ; 7& C ! 8 E6; VG7 66 ; &)$3 ( B !; 7]B
ZB
& 6 666 ;
@ 6 6 ; B !;B3 7 B ; C EVb G 6 ; &)$3 (7) B V 3 ; " 3 B ; ! 7
!; H! !H d@
d 7]B Z- 6 !; 66 V 6 !; EW VG 3 7 ' !; ( C 7 ;&)$6 (!H A6 ; 7 & ) 6 ; &)$ 3 ( E6; WG7 B
VZ
) B d &)$ 3 7 &)$ !H )7VVZ 3 ;+ W _V b_V &)$7 & _V b_V &)$ ; _V C 7
tel-00635537, version 1 - 25 Oct 2011
F
@
2''3 B ( 8 ;B & B , X B / > +! B % $ B ) ! +! B 0 )B 5" (B % ( B( ' H B3B( 0 B( 5")B 1;!B# > ' #! 8 $ # 3: ! ! =7
;3B
tel-00635537, version 1 - 25 Oct 2011
$ & ! E)G6 ; 7&)3 ; ; 7 !; H! E(G )7 , ( ! !E&)$G ! 6 !; !;7 6 ) ;( !71;2 ) 6 C 3+ B W _V &)$7&6 &)$ @2 ( ; E%G7 B ! ) @ 6 ; &)$ 6 ; ;( C 7, 66 )( )% Ep 7
G;6 "667'2 2 ) &)$ ( Vd ! 6 ; &)$ 3 7 + EeWG EeG &)$; !;; "6
;&)$3 ( 76 D B 2 3 ; &)$3 (78 B) @ &)$ ;; ( 6 2 3 7 ) 2 !; 7 6 ( 6 @ 2 A @ ;6 6 ; 7 ) ; B 2 6 6 (B 66K L ;7 X B &)$ @ (
tel-00635537, version 1 - 25 Oct 2011
; ) ; C 3 7 & ; " B 6 2 @ B ! ( 6 3 7
tel-00635537, version 1 - 25 Oct 2011
CSF biomarkers in posterior cortical atrophy
J. Seguin* M. Formaglio, MD* A. Perret-Liaudet, MD I. Quadrio, MD, PhD Y. Tholance O. Rouaud, MD C. Thomas-Anterion, MD, PhD B. Croisile, MD, PhD H. Mollion, MD O. Moreaud, MD, PhD M. Salzmann, MD A. Dorey, PhD M. Bataillard, MD M.-H. Coste, MD A. Vighetto, MD P. Krolak-Salmon, MD, PhD
Address correspondence and reprint requests to Dr. Pierre Krolak-Salmon, Hoˆpital des Charpennes, 27 Rue Gabriel Pe´ri, 69100 Villeurbanne, France
[email protected]
Editorial, page XXX See page XXX
ABSTRACT
Objective: To describe CSF biomarker profiles in posterior cortical atrophy (PCA), which induces high-order visual deficits often associated with Alzheimer disease (AD) pathology, and relate these findings to clinical and neuropsychological assessment.
Methods: This prospective observational study included 22 patients with PCA who underwent CSF biomarker analysis of total tau (t-tau), phosphorylated tau on amino acid 181 (p-tau181), and amyloid b (Ab42). At group level, the CSF profiles of patients with PCA were compared to those of patients with typical AD and patients with other dementia (OD). Individually, the clinical presentation of patients with PCA was correlated to their CSF profile to assess the predictability of clinical features for diagnosis of underlying AD pathology.
Results: At group level, the PCA biomarker profile was not different from that of the AD group, but very different from that of the OD group (p , 0.001). More than 90% of patients with PCA had CSF profiles consistent with AD. All patients with PCA with either isolated higher-order visual deficit (n 5 8) or visual deficit associated with memory impairment (n 5 11) had CSF profiles consistent with AD. Only one of the 3 patients with PCA with asymmetric motor signs fulfilled biological CSF criteria for AD.
Conclusions: PCA syndrome is usually associated with CSF biomarkers suggestive of AD, as shown by previous neuropathologic studies. This does not apply in case of motor signs suggesting associated corticobasal syndrome. CSF biomarkers help to discriminate AD from non-AD processes associated with this condition. Neurology® 2011;76:1–1 GLOSSARY Ab 5 amyloid b peptide; AD 5 Alzheimer disease; CBS 5 corticobasal syndrome; CJD 5 Creutzfeldt-Jakob disease; CVLT 5 California Verbal Learning Test; DLB 5 dementia with Lewy bodies; FTD 5 frontotemporal dementia; MMSE 5 Mini-Mental State Examination; OD 5 other dementias; p-PCA 5 pure posterior cortical atrophy; p-tau 5 phosphorylated tau protein; PCA 5 posterior cortical atrophy; t-tau 5 total tau proteins.
Posterior cortical atrophy (PCA) is a rare syndrome characterized by predominant progressive higher-order visuoperceptual deficits associated with occipito-parieto-temporal brain dysfunction.1 Visual symptoms include simultanagnosia, hemispatial neglect, optic ataxia, and visual agnosia. Neuroimaging usually shows atrophic or metabolic changes in posterior brain regions.2 Evolution generally leads to dementia.
Supplemental data at www.neurology.org e-Pub ahead of print on April 27, 2011, at www.neurology.org. *These authors contributed equally to this work. From the Department of Biochemistry (J.S., A.P.-L., I.Q., Y.T., A.D.), Neurobiology Laboratory, Centre de Biologie et de Pathologie Est, Hospices Civils de Lyon; Department of Neurology (M.F., A.V., P.K.-S.), Department of Neuropsychology (B.C., H.M.), and Inserm Unit 864, Space and Action (A.V.), Hoˆpital Neurologique, Hospices Civils de Lyon; Department of Neurology (O.R.), Center for Memory Resources and Research, Centre Hospitalier Universitaire de Dijon-Bourgogne; Neuropsychology Unit (C.T.-A.), Center for Memory Resources and Research, Centre Hospitalier Universitaire de Saint Etienne; Department of Neurology (O.M.), Center for Memory Resources and Research, Centre Hospitalier Universitaire de Grenoble; Department of Neurology (M.S.), Centre Hospitalier de Roanne; Department of Neurology (M.B.), Centre Hospitalier de Belfort-Montbe´liard; Department of Geriatrics (M.-H.C.), Hoˆpital Pierre Garraud, Hospices Civils de Lyon; Department of Geriatrics (P.K.-S.), Hoˆpital des Charpennes, Hospices Civils de Lyon; Center for Memory Resources and Research (J.S., M.F., A.P.-L., I.Q., Y.T., B.C., H.M., A.D., M.-H.C., A.V., P.K.-S.), Hospices Civils de Lyon; Claude Bernard Lyon I University (J.S., M.F., I.Q., Y.T., A.V., P.K.-S.); and INSERM, U1028 (J.S., P.K.-S.); CNRS, UMR5292; Lyon Neuroscience Research Center, Brain Dynamics and Cognition Team, Lyon, France. Study funding: Supported by the “Programme Hospitalier de Recherche Clinique Re´gional 2004 D50353” and “EU FP6 Project Neuroscreen LSHBCZ-2006-037719 contract no. 037719.” Disclosure: Author disclosures are provided at the end of the article. Copyright © 2011 by AAN Enterprises, Inc.
1
tel-00635537, version 1 - 25 Oct 2011
This syndrome is usually related to Alzheimer disease (AD) but can also result from corticobasal degeneration, Creutzfeldt-Jakob disease (CJD), dementia with Lewy bodies (DLB), or subcortical gliosis.3-8 Etiologic diagnosis remains uncertain until postmortem examination. CSF biomarkers have become a significant help for in vivo AD diagnosis.9 In AD they are characterized by a combination of increased total tau proteins (t-tau) and phosphorylated tau on amino acid 181 (p-tau181) and decreased 42 amino acid amyloid b peptide (Ab42).9-12 These tau and Abprofiles are related to elementary AD lesions: respectively, neurofibrillary tangles and amyloid plaques. Thus, CSF biomarker examination appears relevant to etiologic diagnosis of PCA, since the prevalence of AD pathology is high in this condition. The objective of the present study was to describe the CSF biomarker profile in a pro-
METHODS PCA subjects. Twenty-two patients with PCA (14 men, 8 women; 65 6 7.7 years) were recruited from the memory clinics of Lyon, Dijon, Saint-Etienne, Grenoble, Montbe´liard, and Roanne (France) between 2005 and 2010. All underwent lumbar puncture. CSF biomarker analysis was performed in the neurobiology department of Lyon.
Diagnostic criteria. PCA diagnosis was based on medical history, caregiver interview, neurologic examination, a neuropsychological test battery, and brain imaging (table 1). The following diagnostic criteria for PCA4 were applied: 1) history of progressive visual impairment not explained by ophthalmologic abnormality; 2) demonstration of complex visual disorder such as visual agnosia, prosopagnosia, visuospatial neglect, Balint syndrome (ocular apraxia, optic ataxia, and simultanagnosia), often
Clinical and neuropsychological features of patients with PCAa
Table 1
Case no.
spective series of well-defined patients with PCA. The PCA CSF profile was compared to that of typical AD and other dementia groups. The interest for clinicians of CSF determination in PCA was assessed by comparison between clinically based and CSF-based classifications of etiologic diagnosis.
MMSE score (/30)
Elements of Balint syndrome
Elements of Gerstmann syndrome
Ideomotor apraxia
Parkinsonism
Limb dystonia
Visual hallucinations
Memory
1
21
1
2
1
2
2
1
2
2
20
1
1
1
2
2
2
2
3
19
1
1
1
2
2
2
2
4
26
1
1
2
2
2
2
X
5
22
1
2
2
2
2
2
X
6
10
1
2
1
2
2
2
2
7
19
1
1
1R
1
2
2
X
8
29
1
2
1
1
2
2
X
9
19
1
1
1R
1R
1R
2
X
10
18
2
1
1
1
2
2
2
11
15
1
2
2
2
2
2
2
12
22
1
2
1
2
2
2
X
13
27
1
2
1L
1L
1L
2
X
14
23
1
2
1
2
2
2
X
15
21
1
1
1
1
2
2
X
16
28
1
1
1
2
2
2
2
17
13
2
1
1
2
2
2
X
18
12
2
2
2
2
2
2
2
19
14
1
2
2
2
2
2
2
20
24
1
2
1
2
2
2
X
21
17
1
1
1
2
2
1
2
22
20
1
2
1
2
2
2
2
Abbreviations: 1 5 Clinical signs present; 2 5 clinical sign absent; 2 5 low performance; L 5 lateralized on the left side; MMSE 5 Mini-Mental State Examination; NT 5 not testable; PCA 5 posterior cortical atrophy; R 5 lateralized on the right side; X 5 not affected. a Clinical assessment of PCA for each case. 2
Neurology 76
May 24, 2011
associated with Gerstmann syndrome (alexia, agraphia, finger agnosia, and right-left disorientation) or visuoconstructional apraxia; 3) no or minor memory impairment, executive dysfunction, or language deficit; 4) posterior atrophic or metabolic alteration on MRI or metabolic imaging.
Neuropsychological examination. The Mini-Mental State Examination (MMSE)13 was systematically carried out to rate global cognitive ability. Episodic verbal memory was assessed by the Free and Cued Selective Recall Reminding Test,14 California Verbal Learning Test (CVLT, French version),15 or MEM-III.16 Visuospatial and visuoperceptive abilities were explored by the Visual Object and Space Perception battery,17 the Benton Facial Recognition test,18 or the Judgment of Line Orientation test.18 Neglect and constructional praxis were explored by the Crossing-out and Clock Drawing tests.
Clinically based classification. Patients with PCA were
tel-00635537, version 1 - 25 Oct 2011
classified into 3 groups according to initial clinical presentation and neuropsychological profile, by neurologists blind to the biological results: 1. Pure PCA (p-PCA): isolated visual complaint with visual impairment and no episodic verbal memory impairment. 2. PCA-AD: predominant visual complaint and visual impairment with mild episodic verbal memory impairment. 3. PCA– corticobasal syndrome (CBS): predominant visual complaint and visual impairment with no verbal memory impairment, associated with asymmetric or unilateral parkinsonism and ipsilateral gestural apraxia or dystonia. Some minor attention, executive, or language deficits could be present in all 3 groups.
Control groups. One population with clinical diagnosis of AD and another with clinical diagnosis of other dementia (OD, comprising DLB and frontotemporal dementia [FTD]) were selected on both consensus clinical criteria and CSF biomarker analysis, for comparison with the PCA group. These patients from our laboratory cohort were matched with the PCA groups for gender, for age at lumbar puncture (65 years), and for duration of illness (62 years). The AD control group comprised 160 patients with AD (83 men, 77 women), all meeting National Institute of Neurological and Communicative Disorders and Stroke–Alzheimer’s Disease and Related Disorders Association criteria for probable AD.19 The OD group comprised 138 patients (81 men, 57 women) with a clinical diagnosis of possible (n 5 31) or probable (n 5 38) DLB based on the consensus diagnostic criteria20 and possible (n 5 23) or probable (n 5 45) FTD based on the Lund and Manchester criteria.21 There was no significant difference in sex ratio (M/F) between PCA (14/8) and AD (83/77) and OD control (81/57) patients (x2 test, n 5 318, p 5 0.34).
Standard protocol approval, registration, and patient consent. The study was conducted under the “Programme Hospitalier de Recherche Clinique Re´gional 2004 D50353” and “EU FP6 Project Neuroscreen LSHB-CZ-2006-037719 contract No. 037719.” It was approved by the local Ethics Committee (CPP). All patients (PCA and controls) gave written informed consent to participate.
CSF biomarker assessment. CSF sampling and storage. CSF was obtained from inpatients by lumbar puncture. CSF sampling was performed according to a standard protocol with tracking sheets to prevent technical problems. In total, 10 mL were collected in polypropylene vials (VWR, PA) and then cen-
trifuged within 2 hours (10 minutes at 4,000 3 g). Supernatants were distributed in polypropylene vials and aliquots were directly stored at 280°C until analysis. Since preanalytical steps were critical to the interpretation of results, the following strict CSF exclusion criteria were applied: freezing later than 4 hours after sampling, freezing at 220°C, CSF white cell count .5/mm3, CSF red cell count .2,000/ mm3, or CSF protein level .1.5 g/L. CSF analysis. CSF levels of t-tau, p-tau181, and Ab42 were prospectively determined using a commercially available ELISA kit (INNOTEST htau-Ag, INNOTEST Phospho-Tau(181), INNOTEST b-amyloid(1-42), Innogenetics®, Gent, Belgium) according to the manufacturer’s instructions. All biomarker levels were measured in duplicate. Furthermore, all series included quality control using an aliquot of frozen ventricular CSF containing t-tau, p-tau181, and Ab42 at mean concentrations of 550 pg/mL, 60 pg/mL, and 500 pg/mL, respectively. Since the intra-assay coefficient of variation was less than 10% and the concentrations obtained were in the linear range, samples were not retested. Cutoff values for AD reported in the literature were adopted by the department of biochemistry, as follows: t-tau .350 pg/ mL, p-tau181 .60 pg/mL, and Ab42 ,500 pg/mL.22-24 Western blot immunoassay for 14-3-3 protein was routinely performed. Both positive and doubtful (weakly positive) standard samples were used in all experiments. Immunostaining used anti-14.3.3b polyclonal rabbit antibody (Santa Cruz Biotechnology Sc-629, Santa Cruz, CA) and then alkaline phosphataseconjugated goat anti-rabbit immunoglobulin G antibody (Santa Cruz Biotechnology Sc-2057, Santa Cruz, CA). The antigen was detected by colorimetric reaction and scored for presence or absence of the majority band and compared to positive and doubtful control bands. CSF-based classification. Patients with PCA were classified into 3 groups according to t-tau, p-tau, and Ab42 CSF levels, by biologists blind to the clinical results: 1. Typical AD: both t-tau .350 pg/mL, p-tau181 .60 pg/mL and Ab42 ,500 pg/mL. 2. Atypical AD: either t-tau .350 pg/mL and p-tau181 .60 pg/mL, or Ab42 ,500 pg/mL. 3. Non-AD: t-tau ,350 pg/mL, p-tau181 ,60 pg/mL, and Ab42 .500 pg/mL.
Statistical analyses. Age differences among PCA, AD, and OD groups were assessed on one-way analysis of variance and sex differences on 2 3 3 contingency analysis (x2 test). Differences in CSF biomarkers among the 3 groups were analyzed on the Mann-Whitney test. p Values below 0.05 and 0.001 were considered to be significant and highly significant, respectively. Analysis was performed on MedCalc® version 11.1.1.0 software (Frank Schoonjans, MedCalc Software, Mariakerke, Belgium).
Simultanagnosia, alexia, acalculia, agraphia, and ideomotor and constructional apraxia were the most frequent signs (see tables e-1 and e-2 on the Neurology® Web site at www.neurology.org). Six patients had mild parkinsonism, which was asymmetric in 3 patients who also presented with unilateral ideomotor apraxia (table 1). Two patients with asymmetric parkinsonism also showed limb dystonia. Two patients reported visual hallucinations. None showed
RESULTS Clinical features of patients with PCA.
Neurology 76 May 24, 2011
3
Figure 1
Comparison of total tau proteins (t-tau), phosphorylated tau on amino acid 181 (p-tau181), and amyloid b peptide (Ab42) CSF patterns among patients with Alzheimer disease (AD), patients with posterior cortical atrophy (PCA), and patients with other dementias (OD)
Box plot showing median CSF levels of t-tau, p-tau181, and Ab42 in AD, PCA, and OD subgroups (**p , 0.0001; *p , 0.0016; NS 5 nonsignificant).
tel-00635537, version 1 - 25 Oct 2011
fluctuation of cognitive or motor symptoms. Cognitive functions were variably affected, with a mean MMSE of 20/30 (65.14), ranging from 10 to 29. Visuoperceptive and visuospatial abilities were affected in all patients, and attention and executive functions in most. Patients with PCA symptoms had been progressing for 1 to 6 years at the first clinical assessment. Eight patients without memory impairment were clinically classified as pure PCA (patients 4, 5, 8, 12, 14, 15, 17, and 20), 11 as PCA-AD (patients 1, 2, 3, 6, 10, 11, 16, 18, 19, 21, and 22), and the remaining 3 as PCA-CBS due to their motor symptoms (patients 7, 9, and 13). MRI showed asymmetric cortical or subcortical atrophy in the parietal and occipital regions in 17/22 and symmetric atrophy in 5/22 patients with PCA. There were no visible differences in MRI abnormality profiles among the 3 clinical subgroups. CSF biomarker profiles in the PCA compared to the AD and OD groups. Mean t-tau, p-tau181, and Ab42
CSF levels in the PCA group were 742 6 247, 102 6 43, and 369 6 187 pg/mL, respectively. Mean CSF levels in the control groups were t-tau (AD: 749 6 412; OD: 309 6 230 pg/mL), p-tau181 (AD: 102 6 41; OD: 52 6 25 pg/mL), and Ab42 (AD: 334 6 127; OD: 508 6 216 pg/mL). Median CSF t-tau, p-tau181, and Ab42 levels (figure 1) were highly significantly different between the PCA and OD groups (p , 0.0001, ,0.0001, and 0.0016, respectively) and did not significantly differ between the PCA and AD groups (p 5 0.7, 0.8, and 0.7, respectively).
protein levels. Two patients (9%) had normal CSF biomarkers. Thus 20 out of 22 patients showed a biological profile consistent with either typical or atypical AD. Although 4 patients had CSF t-tau levels higher than 1,200 pg/mL, none showed positive CSF 14.3.3 protein. Comparison between clinically and biologically based diagnostic classifications. Seven of the 8 patients with
p-PCA had a typical and one an atypical AD CSF profile (figure 2). Nine of the 11 patients with PCA-AD had a typical and 2 an atypical AD CSF profile. Finally, one of the 3 patients with PCA-CBS met biological criteria for AD and 2 had a CSF profile classified as non-AD.
Figure 2
Number of cases with the 3 different CSF patterns in each posterior cortical atrophy (PCA) clinical subgroup
CSF biomarker profiles in individual patients with PCA. Seventeen of the 22 patients with PCA (77%)
(table e-3) fulfilled the biological criteria for typical AD, with abnormal levels of the 3 CSF markers. Three PCA patient profiles (14%) were classified as atypical AD due to abnormalities in either tau or Ab 4
Neurology 76
May 24, 2011
AD 5 Alzheimer disease; CBS 5 corticobasal syndrome; p-PCA 5 pure posterior cortical atrophy.
tel-00635537, version 1 - 25 Oct 2011
DISCUSSION CSF analysis in the present PCA cohort was consistent with the high prevalence of underlying AD pathology. At group level, Ab42, t-tau, and p-tau181 levels in patients with PCA differed significantly from those for other dementias and were indistinguishable from those for typical AD. Individually, 17 of the 22 patients presented a typical and 3 an atypical AD CSF profile. These results indicate a high proportion (77%) of patients with PCA with underlying AD pathology with both amyloid and tau pathologies. The 3 atypical AD CSF profiles were consistent with the presence of amyloid plaques or neurofibrillary tangles.25 More than 90% of the PCA cases thus exhibited a CSF profile consistent with AD and were likely to have underlying AD pathology. It is now well-established that CSF biomarkers improve the accuracy of in vivo AD diagnosis.25,26 CSF biomarker changes are related to underlying histologic lesions specific to AD.25 The combination of the 3 abnormal CSF biomarkers predicts AD neuropathology with sensitivity and specificity exceeding 90% and 85%, respectively.26 Based on these data, determination of CSF biomarkers may be of diagnostic interest for identifying AD in patients with atypical focal dementia. In PCA, the present study clearly identified a dominant AD-type biological profile. To our knowledge, only 3 studies have been published on this condition. Decreased CSF Ab42 and elevated levels of phosphorylated tau at threonine 199 were reported in a single case of PCA.27 Elevated t-tau and p-tau181 and low Ab42 CSF levels, similar to those of typical AD, were reported in a cohort of 9 patients with PCA.28 Elevated CSF p-tau181 levels, although significantly lower than in AD, were also reported in a sample of 15 patients with PCA.29 The present study likewise showed, in a larger well-defined PCA population, that an overwhelming majority of patients with PCA display CSF profiles suggestive of AD. These findings are in accordance with the few neuropathologic studies, reporting a high but variable prevalence of underlying AD pathology in PCA syndrome. The largest study reported AD pathology in 62% of 21 autopsied PCA cases.4 AD lesions were also reported in 7 out of 9 patients in one study,3 and in all 7 cases of a study with a smaller sample of patients.7 Other reported etiologies include CBD, AD associated with PD or DLB, CJD, and subcortical gliosis. In the present cohort, patients were diagnosed with PCA based on a clinical aspect of progressive posterior cortical dysfunction. Rather broad diagnostic criteria were used in order to avoid a priori exclusion of non-AD PCA syndrome.4 To take the analysis further, patients with PCA were classified in 3 clinically distinct subgroups (pure PCA, PCA-AD,
or PCA-CBS) according to clinical status, degree of memory deficit, and presence of motor signs, in order to compare biological profile with episodic memory deficit and motor signs. Although the diagnostic criteria considered in the present study did not exclude patients with parkinsonism, fluctuations, or hallucinations, none of our patients developed more than one of these symptoms so as to be listed as PCADLB. None of them, including the 4 cases with elevated t-tau levels above 1,200 pg/mL, showed positive CSF 14.3.3 protein level30 or symptoms consistent with CJD. Although both elevated t-tau and p-tau181 are associated with AD severity in the AD literature, these patients with PCA were not different from the others in terms of global cognitive ability as rated by MMSE: their cognitive deficits were not more impaired than the others.31 Interestingly, despite the broad diagnostic criteria used, most patients with PCA still tended to have AD lesions. All patients with pure PCA presented a CSF profile consistent with either typical (7/8) or atypical AD (1/8). Likewise, all patients with PCA-AD also had a CSF profile consistent with either typical (9/ 11) or atypical AD (2/11). Atypical profiles were defined by either increased t-tau and p-tau181 and normal Ab42 CSF levels or decreased Ab42 and normal t-tau and p-tau181 CSF concentrations. Such CSF patterns have already been described in patients with autopsy-based diagnosis of AD25 and remain consistent with underlying AD pathology. However, these CSF profiles are less specific to AD and can be found in other neurodegenerative diseases, sometimes associated with AD. CSF profiles with low Ab42 and normal t-tau levels in particular have been found in patients showing AD lesions associated with diffuse Lewy bodies,32 PD with dementia, or vascular dementia.25 Similarly, CSF profiles with normal Ab42 and elevated t-tau and p-tau181 levels have been described in autopsy cases of vascular dementia or DLB.25 None of the patients in the present study fulfilled diagnostic criteria for probable DLB in our study, and elevated CSF p-tau181 is more specific in AD than in DLB pathology; DLB, however, appears to be the main differential diagnosis.33,34 In the present study, a normal CSF profile was observed in only 2 out of 22 cases of PCA, in the small subgroup of 3 PCA-CBS. The third patient with PCA-CBS presented a typical AD CSF profile. The clinical presentation of these 3 PCA-CBS cases was similar at the time of lumbar puncture, in that they all had asymmetric parkinsonism and gestural apraxia, elements of Balint and Gerstmann syndrome, and higher-order visual and attention deficits without memory impairment. None had cortical somatosensory impairment. Mild focal limb dystonia ipsilateral to the Neurology 76 May 24, 2011
5
tel-00635537, version 1 - 25 Oct 2011
side with predominant parkinsonism clinically differentiated the 2 patients with normal CSF profiles from patients with an AD CSF profile. It has recently been shown that CBS can reveal AD pathology.35,36 One of the 3 patients with PCA-CBS in the present study is thus likely to have had underlying AD pathology. The normal CSF profiles of the other 2 patients with PCACBS were consistent with the hypothesis of underlying corticobasal degeneration pathology or other tauopathy such as progressive supranuclear palsy or even non-tau pathologies, which are also possible in this clinical condition.36 Although histologic data were not available in the present study, the CSF results are consistent with those of previous neuropathologic studies in PCA. CSF biomarker analysis suggests prominent underlying AD pathology in PCA and enables in vivo AD diagnosis in this condition. On CSF analysis, only the clinical PCA-CBS subtype seemed to be predominantly related to non-AD pathology. Further studies with CSF analysis and postmortem examination in larger cohorts of PCA with motor signs are needed to describe the underlying pathology in this clinical subtype. DISCLOSURE J. Seguin was funded by EU FP6 Project Neuroscreen LSHB-CZ-2006037719 contract no. 037719. Dr. Formaglio reports no disclosures. Dr. Perret-Liaudet has served on scientific advisory boards for Innogenetics and ExonHit Therapeutics and has received speaker honoraria from the Eisai Inc. and Lundbeck Inc. Dr. Quadrio was funded by EU FP6 Project Neuroscreen LSHB-CZ-2006-037719 contract no. 037719. Y. Tholance reports no disclosures. Dr. Rouaud has served on a scientific advisory board for Eisai Inc. and has received speaker honoraria from Eisai Inc. and Novartis. Dr. Thomas-Anterion reports no disclosures. Dr. Croisile has served on scientific advisory boards for Eisai Inc. and Lundbeck Inc.; has received speaker honoraria from Eisai Inc., Janssen, Novartis, and Lundbeck Inc.; serves on the editorial board of Neurologies Me´moire Actualite´s Revue de Ge´riatrie; receives publishing royalties for Votre Me´moire (Larousse, 2004), Alzheimer et les Maladies Apparente´es (Larousse, 2007), Tout sur la Me´moire (Odile Jacob, 2009), and Maladie d’Alzheimer (Larousse, 2010); and has contractual rights in Scientific Brain Training for receipt of future royalty payments. Dr. Mollion serves on a scientific advisory board for Novartis. Dr. Moreaud served on the editorial board of Psychologie et Neuropsychiatrie du Vieillissement and has received speaker honoraria from Eisai Inc., Novartis, and Janssen. Dr. Salzmann, Dr. Dorey, Dr. Bataillard, Dr. Coste, and Dr. Vighetto report no disclosures. Dr. Krolak-Salmon serves on scientific advisory boards for Eisai Inc. and Novartis; has received speaker honoraria from Eisai Inc., Novartis, Janssen, and Lundbeck Inc.; and receives research support from Janssen and Programme Hospitalier de Recherche Clinique.
3.
4.
5.
6.
7. 8.
9.
10.
11.
12.
13.
14.
15.
16. 17.
Received September 1, 2010. Accepted in final form November 23, 2010.
REFERENCES 1. Benson DF, Davis RJ, Snyder BD. Posterior cortical atrophy. Arch Neurol 1988;45:789 –793. 2. Nestor PJ, Caine D, Fryer TD, Clarke J, Hodges JR. The topography of metabolic deficits in posterior cortical atrophy (the visual variant of Alzheimer’s disease) with FDGPET. J Neurol Neurosurg Psychiatry 2003;74:1521–1529. 6
Neurology 76
May 24, 2011
18.
19.
Tang-Wai DF, Graff-Radford NR, Boeve BF, et al. Clinical, genetic, and neuropathologic characteristics of posterior cortical atrophy. Neurology 2004;63:1168 – 1174. Renner JA, Burns JM, Hou CE, McKeel DW Jr, Storandt M, Morris JC. Progressive posterior cortical dysfunction: a clinicopathologic series. Neurology 2004;63:1175–1180. Hof PR, Archin N, Osmand AP, et al. Posterior cortical atrophy in Alzheimer’s disease: analysis of a new case and re-evaluation of a historical report. Acta Neuropathol 1993;86:215–223. Berthier ML, Leiguarda R, Starkstein SE, Sevlever G, Taratuto AL. Alzheimer’s disease in a patient with posterior cortical atrophy. J Neurol Neurosurg Psychiatry 1991; 54:1110 –1111. Alladi S, Xuereb J, Bak T, et al. Focal cortical presentations of Alzheimer’s disease. Brain 2007;130:2636 –2645. Tang-Wai DF, Josephs KA, Boeve BF, Dickson DW, Parisi JE, Petersen RC. Pathologically confirmed corticobasal degeneration presenting with visuospatial dysfunction. Neurology 2003;61:1134 –1135. Wiltfang J, Lewczuk P, Riederer P, et al. Consensus paper of the WFSBP Task Force on Biological Markers of Dementia: the role of CSF and blood analysis in the early and differential diagnosis of dementia. World J Biol Psychiatry 2005;6:69 – 84. Andreasen N, Sjogren M, Blennow K. CSF markers for Alzheimer’s disease: total tau, phospho-tau and Abeta42. World J Biol Psychiatry 2003;4:147–155. Knopman DS, DeKosky ST, Cummings JL, et al. Practice parameter: diagnosis of dementia (an evidence-based review): report of the Quality Standards Subcommittee of the American Academy of Neurology. Neurology 2001; 56:1143–1153. Consensus report of the Working Group on Molecular and Biochemical Markers of Alzheimer’s Disease: The Ronald and Nancy Reagan Research Institute of the Alzheimer’s Association and the National Institute on Aging Working Group. Neurobiol Aging 1998;19:109 –116. Folstein MF, Folstein SE, McHugh PR. “Mini-mental state”: a practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res 1975;12:189 – 198. der Linder V, Coyette F, Poitrenaud J, et al. l’E´preuve de Rappel Libre/Rappel Indice´ a` 16 Items (RL/RI-16): l’E´ valuation des Troubles de la Me´moire. Marseille, France: Solal; 2004. Delis DC, Freeland J, Kramer JH, Kaplan E. Integrating clinical assessment with cognitive neuroscience: construct validation of the California Verbal Learning Test. J Consult Clin Psychol 1988;56:123–130. Wechsler D. Echelle Clinique de Me´moire de Wechsler, 3ed ed. Paris: ECPA; 2001. Rapport LJ, Millis SR, Bonello PJ. Validation of the Warrington theory of visual processing and the Visual Object and Space Perception Battery. J Clin Exp Neuropsychol 1998;20:211–220. Benton AL, Hamsher KD, Varney NR, Spreen O. Contributions to Neuropsychological Assessment: A Clinical Manual. New York: Oxford University Press; 1983. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of Alzheimer’s disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services
20.
21.
22.
23.
24.
tel-00635537, version 1 - 25 Oct 2011
25.
26.
27.
Task Force on Alzheimer’s Disease. Neurology 1984;34: 939 –944. McKeith IG, Dickson DW, Lowe J, et al. Diagnosis and management of dementia with Lewy bodies: third report of the DLB Consortium. Neurology 2005;65:1863–1872. Clinical and neuropathological criteria for frontotemporal dementia: The Lund and Manchester Groups. J Neurol Neurosurg Psychiatry 1994;57:416 – 418. Hansson O, Zetterberg H, Buchhave P, Londos E, Blennow K, Minthon L. Association between CSF biomarkers and incipient Alzheimer’s disease in patients with mild cognitive impairment: a follow-up study. Lancet Neurol 2006;5:228 –234. Tapiola T, Alafuzoff I, Herukka SK, et al. Cerebrospinal fluid b-amyloid 42 and tau proteins as biomarkers of Alzheimer-type pathologic changes in the brain. Arch Neurol 2009;66:382–389. Mattsson N, Zetterberg H, Hansson O, et al. CSF biomarkers and incipient Alzheimer disease in patients with mild cognitive impairment. JAMA 2009;302:385–393. Engelborghs S, De Vreese K, Van de Casteele T, et al. Diagnostic performance of a CSF-biomarker panel in autopsy-confirmed dementia. Neurobiol Aging 2008;29: 1143–1159. Parnetti L, Lanari A, Silvestrelli G, Saggese E, Reboldi P. Diagnosing prodromal Alzheimer’s disease: role of CSF biochemical markers. Mech Ageing Dev 2006;127:129 –132. Kambe T, Motoi Y, Ishii K, Hattori N. Posterior cortical atrophy with [(11)C] Pittsburgh compound B accumulation in the primary visual cortex. J Neurol 2010;257:469 – 471.
28.
29.
30.
31.
32.
33.
34.
35.
36.
Baumann TP, Duyar H, Sollberger M, et al. CSF-tau and CSF-Abeta(1– 42) in posterior cortical atrophy. Dement Geriatr Cogn Disord 2010;29:530 –533. de Souza LC, Lamari F, Belliard S, et al. Cerebrospinal fluid biomarkers in the differential diagnosis of Alzheimer’s disease from other cortical dementias. J Neurol Neurosurg Psychiatry Epub 2010. Sanchez-Juan P, Green A, Ladogana A, et al. CSF tests in the differential diagnosis of Creutzfeldt-Jakob disease. Neurology 2006;67:637– 643. van der Vlies AE, Verwey NA, Bouwman FH, et al. CSF biomarkers in relationship to cognitive profiles in Alzheimer disease. Neurology 2009;72:1056 –1061. Iqbal K, Flory M, Khatoon S, et al. Subgroups of Alzheimer’s disease based on cerebrospinal fluid molecular markers. Ann Neurol 2005;58:748 –757. Koopman K, Le Bastard N, Martin JJ, Nagels G, De Deyn PP, Engelborghs S. Improved discrimination of autopsyconfirmed Alzheimer’s disease (AD) from non-AD dementias using CSF P-tau(181P). Neurochem Int 2009;55: 214 –218. Hampel H, Blennow K, Shaw LM, Hoessler YC, Zetterberg H, Trojanowski JQ. Total and phosphorylated tau protein as biological markers of Alzheimer’s disease. Exp Gerontol 2010;45:30 – 40. Boeve BF, Maraganore DM, Parisi JE, et al. Pathologic heterogeneity in clinically diagnosed corticobasal degeneration. Neurology 1999;53:795– 800. Ling H, O’Sullivan SS, Holton JL, et al. Does corticobasal degeneration exist? A clinicopathological re-evaluation. Brain 2010;133:2045–2057.
Neurology 76 May 24, 2011
7
. * @
C . . 1 /0
/ 160 6 " D16
. 1 @
tel-00635537, version 1 - 25 Oct 2011
.. . " H . 1 /
6 *++9 6 DE 42 9
]
& 2 !; " ;; ( !7i) ; ! ; 6 B 3 !3!!3 6 D 3; @ ;; 58B !; ! 7j ) ; !; ;;B 2 58 6 8 2 (7 ; B 2 C 37 ! B @ A B 587 V& ! 58c0
tel-00635537, version 1 - 25 Oct 2011
2 !!3 !; ( @ 6 ; !; 7 * 66B 6 58 CE@ B]V]Z> G [V> ; ! (7k ! )0B ' 8+B6 ( c07 ! @C2 58@ ! ; 2 (7 ! ! 6 ! ; ;B 3 > >37 & 58B @ C ! ; B ; >3 7 2 6 ; 2 B " " C
i
)67 ! Q 7*7G7* @ ' ! O7
j
)67 ! Q *77* @ . = ! $ !' 7
k
)67 ! Q . = ! $ !' 7
]
6 ; 7 !;B 6 ! ! 2 58 c0 ; *&,'7 . 61
7 .
& 2 @ !Q ; &3 E8 G 5Q 2 c E!G 0 E5;G7
tel-00635537, version 1 - 25 Oct 2011
; !; (@ 66 0 > E 0 > G B >6 ! !; B '
,i ;" 6 Q 3 @ 2 !; 7 & 6 @ 6 !!7 7* A $ .1E ''#
& ! 2 6 58 B
7 ] ! ; ; @ 2 ; 7 H @ 66 8 6 !; 0 > ! ! B 2 6 B 2 B 2 7 & ! ; @ d E b G 3 ; E ( +B 5 [7VB 7W( ) ( *4+G ;f @ 3 ! ; E0$ & B ,B 5B )B -'G7 " C ! !! @d 6 E!! ! > B
i
, ; E
@CG! ;" ! [7) !; 8+ [7)67 ! 4 *77* @
. = ! $ !' 7
]
'; B ' X 8 B 8 G ; !!3 B ! ; 6;@
; E' r 3z( '*B+! 6G ;E (+B5[7VB 7W ( )B (*+B ( 8 d;G7& ! ; 6; @
;B >3 C ; @ ! d E b G 6 7 & ; ! @ 6; V @
;7 & 6 6 58 >37 7G I
tel-00635537, version 1 - 25 Oct 2011
& 6 ! ; E+V G ! 66 @
)7 & ! ; '' 3 3 ; V d E4*B , ;G ; !
@ 1 E
G7 & 6 , E 7V y B (G !@] ;EV1G 0' @ d E b G ! @ V)7 & ! +V] E';B ! B (B -'I b
0' @ 7 d g b h +S @ 7 d g b hGB ; V V VV ] 6 ! 7 " ; B ! ; ,;4 @ 23 E. > , ! & B,7Bc4 BB-'G@ b
7& ! ! E 0!;3B $>6B,&B-'G7
]
*
8 % ) 8 % ) (
8 % ) 8 % ) (
[V> ]Z> ]V> >
]Z> ]V> >
0
tel-00635537, version 1 - 25 Oct 2011
E F7 I $ .1E A $ $ 88' > ''# $ $A
! ! 3$ $" > 3 P $ $ $ : K '5'' 5
" ! F,7 8 2 6 I 2 I % 2 I ) I ( I 0 > I 0 >E*2 I0 G7
& +V] 58H E26 B G 0 > E6;V G7) 2;2 7)6 2 0 > 2 6 B 0 >
]V
8
5
)
8
5
)
8
[V>
]Z> ]V> >
E F*7 I $ .1E $ $ $" 3 G+K $" 3$
tel-00635537, version 1 - 25 Oct 2011
$ . : '5'' 5 " ! 3 F,7 58 2 '
,;" I 582 >I 58 H! I8 26 I5! I) 7
&58 2 '
,;" > 6 ! 66 2 ( E6; V G7 ]Z> ]V> [V> 26 ! '
, > 7B ; ; E6;V B; G7 ]V> > 26 > ]Z> 7 B ; ! 7 G
! ! 2
!; ! A 6 ; 2 (E6;VG7B ! =@6 ; ! > ;" 8+ [E6;V G7
]
& > !; 6 6 $7 & 6 ! !; #2 58 6 ; !; 7 A B 2 ;" 6 !38+ [7 ]ZB [ & 6 6 V$7 58 6 V$ '
, ;" E6;V G7 & @ , 3! ; 0 3 3 ;
tel-00635537, version 1 - 25 Oct 2011
; 6 @ f; Z 7 , 3 > 3 @ ! ; B 3 7&" " 7&2 !; 6 ! ; ; ;3!; 8 7 B ; ; ; ! !H 76 D B c0 ; ; +V] E6; V G ; !; 7 &2 58 6 ! = 3 6 " @ 3 !! 7 * 66B !;B 2 3 > ! B 66 !; @;7& " @ ' 2 7 ' ; ! 58 ! 667 & 58 ' @]Z]V> 3 > ! (E B]V]Z> G7 V) 36" 6 ; ; 66 ; 7
]]
) i ; @ ; ! 26 B @ !; 6 66 ; 7 ) ! 3 ! B B 2 B !;B ! " 7 ) B ; ! 3 Q 7 ) !; B !; B26 66 ;
tel-00635537, version 1 - 25 Oct 2011
!; 7] B [ & (). ! @ ! " ` '3B _ 6 (). 7 ]B [& *
#;" 6 6 ; ().76 ! " 2 !;B 2 3 ! ! !!H ().@ *
#7 i
)67 ! Q 7*7G7G 6$ $" > 3 $'3 3 A7
][
F
* @
4 47 # B 2''3 B , X B $ 5T6 ;B , U # UB ! '! ;B 4 O 0 B (3B $ 6 'B $> 1 ;!B # ( C3B & C &UHYB +! 'T B 5 0> B & /
>8$42:#
$
$
?
*++9
3 @
> 8 3!A ! ! =7
!B
tel-00635537, version 1 - 25 Oct 2011
$ & *
#;" EG @ )H6 . > E().G 6 7*Q B Q B ` 3 E` '3G 3: _ E_G @! 76 !; B 66 B !; !
Z *
# 66 3 7 & 3 ;6 2 3 ! ; 3 !7 &2 !; 6 6 ; ; B! 7 3 B 6 2B S 7 - Q 3 2 ; 2 7 &2 Q _ !3 7Wd ; ! 3: 7d 7 !!3EZ 7 dG ;6 E W7VdG 7 B 6 ! E
7 dG &S3 E 7VdG7 Q + V 8-' 7 & !; 2 Q "; 2 66 Q E B ` '3 _G Q !; 7
]W
! B 66 !3 3 3 > ; 7 ) 3 (). 3 ; *
# 6 @ !; ! ; !; !; 3 7 ; E > 2 G f; E ] G 6 2 " ; 6 (). ; 7 B` '3_ ;;"6
tel-00635537, version 1 - 25 Oct 2011
# *
# @ Q !; 76 B ; " 26 66 ; 6 (). ; 7 ) ! !! 6 2 3; 6 2 7 * 66B 3; !3!" " 2 !; 2 2 6 A 6
;" 7 @6 ; ().B 2 !! 6 ; !; 2 !3 3 7 7
]Z
Acta Neuropathol (2011) 121:39–57 DOI 10.1007/s00401-010-0713-y
ORIGINAL PAPER
Genetic Creutzfeldt-Jakob disease associated with the E200K mutation: characterization of a complex proteinopathy Gabor G. Kovacs • Je´re´mie Seguin • Isabelle Quadrio • Romana Ho¨ftberger • Istva´n Kapa´s • Nathalie Streichenberger • Anne Gae¨lle Biacabe • David Meyronet • Raf Sciot • Rik Vandenberghe Katalin Majtenyi • Lajos La´szlo´ • Thomas Stro¨bel • Herbert Budka • Armand Perret-Liaudet
•
tel-00635537, version 1 - 25 Oct 2011
Received: 1 April 2010 / Revised: 10 June 2010 / Accepted: 20 June 2010 / Published online: 1 July 2010 Ó Springer-Verlag 2010
Abstract The E200K mutation is the most frequent prion protein gene (PRNP) mutation detected worldwide that is associated with Creutzfeldt-Jakob disease (CJD) and thought to have overlapping features with sporadic CJD, yet detailed neuropathological studies have not been reported. In addition to the prion protein, deposition of tau, a-synuclein, and amyloid-b has been reported in human prion disease. To describe the salient and concomitant neuropathological alterations, we performed a systematic clinical, neuropathological, and biochemical study of 39 individuals carrying the E200K PRNP mutation originating from different European countries. The most frequent clinical symptoms were dementia and ataxia followed by myoclonus and various combinations of further symptoms, including vertical gaze palsy and polyneuropathy. Neuropathological examination revealed relatively uniform anatomical pattern of tissue lesioning, predominating in the
Electronic supplementary material The online version of this article (doi:10.1007/s00401-010-0713-y) contains supplementary material, which is available to authorized users. G. G. Kovacs (&) R. Ho¨ftberger T. Stro¨bel H. Budka Institute of Neurology, Medical University of Vienna, and Austrian Reference Center for Human Prion Diseases, AKH 4J, Wa¨hringer Gu¨rtel 18-20, 1097 Vienna, Austria e-mail:
[email protected] G. G. Kovacs I. Kapa´s K. Majtenyi Neuropathology and Prion Disease Reference Center, Hungarian Reference Center for Human Prion Diseases, Semmelweis University, Budapest, Hungary J. Seguin I. Quadrio N. Streichenberger D. Meyronet A. Perret-Liaudet Prion Disease Laboratory, Pathology and Biochemistry, Groupement Hospitalier Est, Hospices Civils de Lyon/Claude Bernard University, Lyon, France
basal ganglia and thalamus, and also substantia nigra, while the deposition of disease-associated PrP was more influenced by the codon 129 constellation, including different or mixed types of PrPres detected by immunoblotting. Unique and prominent intraneuronal PrP deposition involving brainstem nuclei was also noted. Systematic examination of protein depositions revealed parenchymal amyloid-b in 53.8%, amyloid angiopathy (Ab) in 23.1%, phospho-tau immunoreactive neuritic profiles in 92.3%, neurofibrillary degeneration in 38.4%, new types of tau pathology in 33.3%, and Lewy-type a-synuclein pathology in 15.4%. TDP-43 and FUS immunoreactive protein deposits were not observed. This is the first demonstration of intensified and combined neurodegeneration in a genetic prion disease due to a single point mutation, which might become an important model to decipher the molecular interplay between neurodegeneration-associated proteins. Keywords Alpha-synuclein Amyloid-beta Prion protein Tau Neurodegeneration A. G. Biacabe Agence Franc¸aise de Se´curite´ Sanitaire des AlimentsˆLyon, Unite´ ATNC, Lyon Cedex 07, France R. Sciot Department of Pathology, University Hospital, Catholic University of Leuven, Leuven, Belgium R. Vandenberghe Neurology Department, University Hospital Gasthuisberg, Leuven, Belgium L. La´szlo´ Department of Anatomy, Cell and Developmental Biology, Eotvos Lorand University of Sciences, Budapest, Hungary
123
40
tel-00635537, version 1 - 25 Oct 2011
Introduction Prion diseases are acquired, sporadic or genetic in origin. Mutations in the prion protein (PrP) gene (PRNP) are associated with spongiform encephalopathy (Creutzfeldt-Jakob disease, CJD) or PrP amyloidosis (Gerstmann-Stra¨usslerScheinker disease, or cerebral amyloid angiopathy) [37]. In addition to the mutation, the constellation at codon 129 (methionine/methionine, MM, or valine/valine, VV, homozygosity, MV heterozygosity) seems to influence phenotypes also in genetic prion diseases [42, 46]. The E200K mutation is the most frequent PRNP mutation detected worldwide that is associated with CJD [5, 21, 22, 41, 42, 49], with increased incidence among Libyan Jews and in Slovakia and Hungary [29, 33, 35, 40, 47, 49]. Examination of microsatellite markers indicated at least four different ancestral origins, with cases in Hungary or Slovakia differing in origin from those in Austria, France, or Belgium and other parts of the world [44]. Clinical presentations overlap with those described in sporadic CJD, furthermore, insomnia, vertical gaze palsy, and polyneuropathy were described as well [1, 2, 8, 42, 47, 50, 64]. Prion diseases are representatives of neurodegenerative diseases, which are classified by protein depositions such as a-synucleinopathies, tauopathies, TDP-43, or FUS proteinopathies, or diseases associated with the deposition of amyloid-b (Ab). These proteinopathies show considerable overlap [36] and were described as concomitant pathologies in prion diseases as well. a-Synucleinopathies like Parkinson‘s disease (PD) are characterized by deposition in the brain of a-synuclein protein in the form of Lewy bodies and neurites that may be also detected in about 10% of subjects above 60 years, most likely representing presymptomatic PD [12]. An increased prevalence of Lewy bodies was found in elderly sporadic CJD (23.5%) and also in the acquired variant CJD [66]. Phospho-tau immunoreactive neuritic profiles around amyloid plaques are features of variant CJD [19]. Neurofibrillary tangle pathology, morphologically similar to Alzheimer’s disease (AD), is associated with PrP gene (PRNP) mutations with prominent PrP amyloid deposition [17]. Moreover, abundant Ab deposits may be detected in sporadic CJD with higher age and a specific clinicopathological profile [11]. Codistribution of Ab and disease-associated PrP was recently demonstrated in three cases of E200K mutation of Hungarian origin [18]. There are studies summarizing clinical features [47, 63], furthermore, case reports on neuropathological alterations [8, 9, 26, 30, 45, 64] or evaluation of certain regions (e.g. cerebellum) [32, 49], and biochemical evaluations of protease-resistant PrP (PrPres) [7, 27, 57] of individuals with CJD associated with E200K mutation. However, there is a paucity of systematic neuropathological evaluation of
123
Acta Neuropathol (2011) 121:39–57
cases. Thus, in the present paper we report the results of a comprehensive and systematic clinical, neuropathological, and biochemical study of 39 individuals carrying the E200K PRNP mutation originating from different European countries. In addition to various types of PrP immunoreactivity, we found surprisingly high proportion of cases with various depositions of neurodegenerationassociated proteins (Ab, phospho-tau, a-synuclein).
Materials and methods Selection of cases Within the European surveillance for human prion diseases, we systematically examined 39 patients (4 Austrian, 3 Belgian, 6 French, and 26 Hungarian) with confirmed E200K mutation in PRNP. Clinical and neuroradiological data were obtained retrospectively. These patients were notified as suspected CJD patients to the national surveillance network, using the current clinical criteria for CJD, and verified at postmortem. When a sign was indicated as absent, it means that it was checked and found negative. Genetic analysis was performed using genomic DNA isolated from blood samples or frozen brain tissue as published before, after informed consent to the patient or one of his relatives in the frame of CJD Surveillance [16, 35, 56]. Neuropathology Formalin fixed, paraffin-embedded tissue blocks (2.5 9 2.0 cm) were evaluated. Post mortem delay was generally within 24 h except for three cases (48 h). The latter were not included in our biochemical studies. In addition to hematoxylin and eosin staining, the following monoclonal antibodies were used for immunohistochemistry: anti-tau AT8 (pS202, 1:200) AT100 (pS212, 1:200), AT180 (pT231, 1:200), HT7 (tau 169-163, 1:100; all from Pierce Biotechnology, Rockford, IL, USA), anti-phospho-TDP-43 (pS409/410, 1:2,000, Cosmo Bio, Tokyo, Japan), antia-synuclein (1:10,000, clone 4D6, Signet, Dedham, MA, USA), anti-Ab (1:50, clone 6F/3D, Dako, Glostrup, Denmark), anti-PrP (1:1,000, 12F10, Cayman Chemical, Ann Arbor, MI, USA), anti-4R tau (RD4, 1:200, Upstate, Charlottesville, VA, USA), and anti-3R tau (RD3, 1:2,000, Upstate). In addition, we used the following polyclonal antibodies: anti-Ab-40 (1:200) and anti-Ab-42 (1:50; both Signet, Dedham, MA, USA), and anti-FUS (1:1000, Sigma, St. Louis, MO, USA). The DAKO EnVisionÓ detection kit, peroxidase/DAB, rabbit/mouse (Dako, Glostrup, Denmark) was used for visualization of antibody reactions. Examined anatomical regions are summarized in Table 1. Neuropathological alterations (spongiform
Acta Neuropathol (2011) 121:39–57
41
tel-00635537, version 1 - 25 Oct 2011
Table 1 Summary of anatomical regions examined using different antibodies for immunostaining Anatomical region/antibody
PrP pTau a-Syn Ab pTDP-43 FUS Ubi
Frontal cortex
?
?
?
?
?
?
?
Cingular cortex
?
?
?
-
-
-
-
Parietal cortex Temporal cortex
? ?
?
?
?
-
-
-
Occipital cortex
?
?
-
?
-
-
-
Hippocampus CA subregions
?
?
?
?
?
?
?
Dentate gyrus
?
?
?
?
?
?
?
Subiculum
?
?
?
?
?
?
?
Entorhinal cortex
?
?
?
?
?
?
?
Caudate nucleus
?
?
?
?
-
-
-
Putamen
?
?
?
?
-
-
-
Globus pallidus
?
?
?
?
-
-
-
Thalamus medial nuclei
?
?
-
-
-
-
-
Thalamus lateral nuclei
?
?
-
-
-
-
-
Substantia nigra
?
?
?
-
?
?
?
Dorsal raphe nucleus ?
?
?
-
-
-
-
Locus coeruleus
?
?
?
-
-
-
-
Pontine base nuclei
?
?
?
-
-
-
-
Inferior olives
?
?
?
-
-
-
-
Hypoglossus nucleus ?
?
?
-
-
-
-
Dorsal vagus nucleus ? Dentate nucleus ?
? ?
? ?
?
?
?
?
Cerebellar cortex
?
?
?
?
?
?
?
change, astrogliosis, neuronal loss, and degree of various protein depositions) were semiquantitatively (none, mild, moderate, severe) evaluated in all examined anatomical regions. Double immunolabeling was performed using monoclonal PrP (12F10, 1:300), Abeta (1:20, Dako), Tau AT8 (1:100), SMI-31 (1:2000, Covance, Berkeley, CA), Map-2 (1:250, Millipore Corp., Billerica, MA), HLA-DR (1:50, Dako), and polyclonal GFAP (1:1500, Dako). The fluorescence-labeled secondary antibodies were Alexa Fluor (AF) 555 donkey anti-mouse IgG (1:200; Molecular Probes, Inc., Eugene, OR, USA), AF 488 goat anti-rabbit (1:200; Molecular Probes, Inc.), and Zenon AF 488 Mouse IgG1 (Molecular Probes, Inc.). The following combinations were applied: PrP (AF 555)/Tau (Zenon 488); PrP (AF 555)/Abeta (Zenon 488); HLA-DR, SMI-31, and Map-2 (AF 555)/Tau (Zenon 488), and Tau (AF 555)/GFAP (AF 488). Double immunolabeling involving anti-PrP antibody was performed after pretreatment of the section with 20 min heat-induced epitope retrieval with citrate buffer (pH 6) followed by 2 min formic acid (96%); for all others
20 min heat-induced epitope retrieval with citrate buffer was used. We evaluated double immunofluorescent labeling with a Zeiss LSM 510 confocal laser microscope. Immunoblotting Prion protein Western blot analysis was performed as previously described with minor modification [58]. Briefly, 20% cortical gray matter homogenates were obtained using buffer containing 5% glucose in ultra pure grade water. Homogenates were diluted twofold in buffer [100 mM Tris (pH 7.4), 0.5% glucose, 0.9% NaCl] and digested with proteinase K (25 lg/ml) for 1 h at 37°C before stopping digestion by 1 mM PMSF. Digested homogenates were further ultracentrifuged (Sorvall Discovery M150 SE, Thermo, USA) in N-lauryl-sarcosyl (final concentration of 10%) for 75 min at 140,000 rpm. Pellets were resuspended with a denaturing buffer (125 mM Tris, 4% SDS, 20% glycerol), heated for 5 min at 100°C and centrifuged at 12,000g for 10 min. The supernatant was collected for western blot analysis. Samples and biotinylated molecular mass marker (SIGMA, range 14.3–97.0 kDa) were run on 16% SDS-PAGE gels. In addition, selected samples were also run on a 12% gel. All immunoblots were incubated with the monoclonal antibody 3F4 (recognizing the epitope at PrP residues 108–111) at a concentration of 0.1 lg/ml (Proteinogenics, USA), followed by incubation with a goat anti-mouse IgG horseradish peroxidase (HRP) conjugated antibody and detection using a chemiluminescent substrate (Supersignal Dura, Pierce, USA). Tau Frozen brain tissue from four neuropathologically confirmed E200K genetic CJD patients (3 French and 1 Hungarian), one confirmed AD patient and one control brain from an individual lacking tau pathology was obtained. Insoluble tau fraction was extracted as described [28] with some modifications. Briefly, 20% cortical gray matter homogenates were prepared using a buffer containing 50 mM Tris (pH 7.4), 0.8 M NaCl, and 10 mM EGTA. Homogenates were centrifuged at 100,000g for 25 min and the resulting pellets were homogenized in 1 mL of buffer containing 10 mM Tris (pH 7.4), 0.85 M NaCl, 1 mM EDTA, 20 mM NaF and 10% sucrose. Following centrifugation for 25 min at 20,000 rpm, sarkosyl was added to supernatants to a final concentration of 1%. After incubation at RT for 2 h, the supernatants were centrifuged at 100,000 rpm for 45 min. The sarkosylinsoluble pellets were suspended in 50 lL Laemmli buffer and heated for 10 min at 100°C.
123
42
tel-00635537, version 1 - 25 Oct 2011
Table 2 Summary of clinical and demographic data
Acta Neuropathol (2011) 121:39–57
Features
PRNP codon 129
All**
MM
MV
VV
Number of cases
21 (58.3%)
14 (38.9%)
1 (2.8%)
36/39
Women
14 (35.8%)
5 (12.8%)
1 (2.5%)
22 (56.4%)
Men
7 (17.9%)
9 (23%)
0
17 (43.6%)
Age at death (mean ± SE)
59.76 ± 2.27
60.64 ± 1.81
66.0
60.28 ± 1.49
Duration of illness (mean ± SE)
4.20 ± 0.60
5.77 ± 0.84
4.0
4.79 ± 0.48
Age range
33–76
46–74
66.0
33–76
Duration of illness range
2–12
1–13
4
1–13
Dementia
17 (81%)
14 (100%)
1
35 (89.7%)
Ataxia
16 (76.2%)
12 (85.7%)
1
32 (82.1%)
Myoclonus
13 (61.9%)
5 (35.7%)
1
20 (51.3%)
Duration of illness is given in months, age at death in years
Pyramidal signs Rigidity/Parkinsonism
10 (47.6%) 2 (9.5%)
5 (35.7%) 3 (21.4%)
0 0
15 (38.5%) 6 (15.4%)
From the 39 cases involved in the study, clinical information in one case and in three data on codon 129 were not available
Chorea/Dystonia
9 (42.9%)
4 (28.6%)
0
14 (35.9%)
Gaze palsy
3 (14.3%)
1 (7.1%)
0
4 (10.3%)
Insomnia*
2 (9.5%)
1 (7.1%)
0
3 (7.7%)
Early psychiatric symptoms
5 (23.8%)
2 (14.3%)
0
7 (17.9%)
Early behavioral change
5 (23.8%)
1 (7.1%)
1
7 (17.9%)
Polyneuropathy
2 (9.5%)
3 (21.4%)
0
6 (15.4%)
* Reported prominent insomnia without polysomnography
EEG: PSWC
10/19 (52.6%)
4/13 (30.8%)
0
15/35 (42.9%)
MRI: high signal in striatum
6/12 (50%)
2/6 33.3%)
1
9/19 (47.3%)
** Including cases where there was a lack of data on codon 129
MRI: high signal in thalamus
2/12 (16.7%)
2/6 (33.3%)
0
4/19 (21%)
PSWC periodic sharp wave complexes with triphasic morphology
Samples were run on 4–12% gradient SDS-polyacrylamide gels (NuPAGE, Invitrogen). Proteins then were transferred onto Immobilon-P membranes (0.45 lm pore size, Millipore) and blocked overnight at 4°C. Afterwards, the membranes were incubated using the following primary antibodies: Anti-tau T46 (Signet, Dedham, MA, USA; 1:2,000 in PBS containing 0.5% Tween-20 and 0.5% dry milk) is a monoclonal antibody [34] raised against all six isoforms of human tau recognizing both recombinant human tau and PHF-tau on immunoblots; the phospho-dependent anti-Tau AT8 (Innogenetics, Gent, Belgium; 1:1,000 in PBS containing 0.1% Tween-20 and 0.5% dry milk) is a monoclonal antibody [20] directed against both Ser202 and Thr205 phospho-epitopes of tau; for the analysis of repeat isoforms in insoluble tau we used clone 8E6/C11 for the detection of three-repeat isoform RD3 of tau [10] (Millipore, Temecula, CA, USA; 1:1,000 in TBS containing 0.05% Tween-20 and 0.5% dry milk) and clone 1E1/A6 for the detection of four-repeat isoform RD4 of tau [65] (Millipore, Temecula, CA, USA; 1:1,000 in TBS containing 0.05% Tween-20 and 0.5% BSA). After incubation with a goat antimouse IgG horseradish peroxidase (HRP) conjugated antibody (Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA) at a final dilution of 1/10,000, immunological detection was performed using chemiluminescence technology according to the manufacturer’s instructions (Pierce Biotechnology, Rockford, IL, USA).
123
Statistical analysis Duration of illness, age at death, scores of neuropathological variables in different groups were compared using one-way ANOVA test or v2 test.
Results Summary of clinical and genetic data Clinical data were available for 38 (out of 39) patients (Table 2). Analysis of PRNP codon 129 was available for 36 patients. Age at death did not differ between constellations of codon 129, while the duration of illness was longer in patients with MV at codon 129, although in our cohort it did not reach the level of significance (p [ 0.05). The most frequent clinical symptoms were dementia and ataxia followed by myoclonus and various combinations of further symptoms. Prominent vertical gaze palsy was described in 10.3% of patients, and polyneuropathy (axonal and rarely demyelinative) was noted in 15.4%, while early psychiatric symptoms or behavioral change was also noted in approximately one-fifth of individuals. Cranial MRI images were available for one-third of the patients; high signal in the basal ganglia was noted in approximately half of the patients, while high signal in the thalamus was seen
43
tel-00635537, version 1 - 25 Oct 2011
Acta Neuropathol (2011) 121:39–57
Fig. 1 Lesion and PrP immunoreactivity profiles and anatomical correlations of PrP immunoreactivities in E200K genetic CJD cases grouped according to the codon 129 polymorphism. a Lesion profiles [53] obtained by averaging the three scores (see text) for spongiform change, neuronal loss, and gliosis in the following gray matter regions: frontal cortex (FC), temporal cortex (TC), parietal cortex (PC), occipital cortex (OC), hippocampus CA1 subregion (HI), entorhinal cortex (EC), neostriatum (ST), medial thalamus (TH), substantia nigra (SN), midbrain periaqueductal gray (PG), locus coeruleus (LC), medulla oblongata (ME), cerebellum (CE). b PrP immunoreactivity profiles obtained by averaging the scores of different PrP immunoreactivities in the same regions indicated in a.
c Percentage of cases with E200K genetic CJD grouped according to the polymorphism at codon 129 with or without different kinds of PrP immunoreactivities (two graphs on the left side) and the percentage of involved regions showing different PrP immunopositive patterns (two graphs on the right side). d Distribution of cases showing stripe-like PrP deposition in the molecular layer of the cerebellum in different groups (according to codon 129 polymorphism) of E200K genetic CJD cases. e Comparison of scores of spongiform change and neuronal loss in the cerebellar molecular layer and granular layer, respectively, in E200K genetic CJD cases with or without stripe-like pattern of PrP immunoreactivity
123
44
Acta Neuropathol (2011) 121:39–57
Fig. 2 Neuropathological alterations in the substantia nigra. a Neuronal loss and extracellular pigment in the substantia nigra in a representative case (bar graph indicates 30 lm). b Distribution of cases with none, mild, moderate or severe lesioning in the substantia nigra
in four patients, two of them with thalamic signal that was higher than in the basal ganglia, and was reminiscent of the pulvinar sign described in variant CJD [39]. Periodic sharp wave complexes with triphasic morphology were described in 43% of the patients’ EEGs.
tel-00635537, version 1 - 25 Oct 2011
Neuropathological alterations Spongiform change, neuronal loss and reactive astrogliosis of variable degree were observed in all cases. Vascular pathology with infarctions was seen in two cases. The distribution of spongiform change was relatively uniform in all patients irrespective of the constellation at codon 129 (Fig. 1a). In addition to neocortical regions, caudate nucleus, putamen and thalamic nuclei were most severely affected. In the cortex the deeper layers were frequently more involved, while in the hippocampal region, subiculum and entorhinal cortex had most tissue lesioning. Interestingly, the cerebellar cortex was relatively preserved in many cases. Gliosis and neuronal loss followed the degree of spongiform change, but predominated over spongiform change in the thalamus, particularly in brainstem nuclei (see supplemental Fig. 1a). Neuronal loss with extracellular pigment was frequently seen not only in the substantia nigra (Fig. 2a) but also in the locus coeruleus and dorsal raphe nucleus. Indeed, many cases with MM or MV at codon 129 showed moderate (28% MM, 42% MV) or severe (14% MM, 7% MV) lesioning in the substantia nigra, and only 15% of the cases in each group exhibited preserved neurons (0 score) (Fig. 2b). One case showed severe damage of the cortex with myelin loss and gliosis in the white matter compatible with a panencephalopathic form of CJD (62-year-old Belgian patient with 14 months duration of disease). Characterization of proteinopathies Prion protein Immunohistochemistry Immunostaining for PrP revealed diffuse/synaptic deposits, patchy aggregates in the neuropil
123
or around vacuoles, plaques without amyloid characteristics, fine perineuronal immunodeposits, and prominent intraneuronal dots (Fig. 3a–e). In cortical areas, laminar accentuation of PrP immunoreactivity was evident in many cases (Fig. 3f); synaptic or absence of PrP immunoreactivity frequently alternated with small foci showing prominent patchy aggregates of PrP (Fig. 3g). Of note was the frequent coarse deposition in medial thalamus (Fig. 3h). Prominent immunodeposition (synaptic and intraneuronal type) was noted in brainstem nuclei (Fig. 3i) and other subcortical areas (Fig. 3j). Although the involvement of regions was not significantly different between cases (Fig. 1b) and synaptic PrP immunoreactivity was seen in all cases, plaque-like structures and intraneuronal PrP immunodeposition predominated in cases with MV at codon 129 (Fig. 1c, supplemental Fig. 1b), particularly in the cortex. Plaque-like structures were more frequent in the hippocampus (Fig. 3k). When present, intraneuronal immunoreactivity always involved brainstem nuclei and showed prominent layer difference (deep layers [ upper layers) in the cortex (see supplemental Fig. 1c). In the cerebellum, majority of the cases showed prominent PrP immunoreactivity restricted to the molecular layer in a stripe-like pattern [32] (Fig. 1d). Interestingly, these cases showed less prominent spongiform change in the molecular layer and less neuronal loss in the granular layer in comparison to cases with diffuse/synaptic PrP immunoreactivity involving all layers (Figs. 3l–o, 1e). Western blot analysis of PrPres Six French and four Hungarian subjects with confirmed prion diseases associated with E200K mutation on the PRNP gene were examined. Six individuals were methionine (MM) homozygous at codon 129 and 4 were heterozygous (methionine/ valine, MV). E200K mutation was coupled with methionine at codon 129 in all cases. One further Austrian case homozygous for valine (VV) at codon 129 was published before [26]. The molecular type of PrPres was determined by comparison with PrPres type 1 and type 2 from the prefrontal cortex of two patients with sporadic CJD as reference
45
tel-00635537, version 1 - 25 Oct 2011
Acta Neuropathol (2011) 121:39–57
Fig. 3 Immunostaining for disease-associated prion protein. Immunostaining for PrP reveals diffuse/synaptic deposits (a), patchy/ perivacuolar aggregates in the neuropil (b), plaque-like structures without amyloid characteristics (c), fine perineuronal immunodeposits (d), and prominent intraneuronal dots (e). In cortical areas laminar accentuation of PrP immunoreactivity is evident in many cases (f). In circumscribed cortical areas prominent patchy aggregates of PrP are seen (g). Coarse aggregates are frequent in the medial thalamus (h). Prominent immunodeposition (synaptic and intraneuronal type) in the
locus coeruleus (i) and the lateral geniculate body (j). Plaque-like structures are frequent in the hippocampus (k). In the cerebellum less prominent spongiform change and neuronal loss (l, H&E) are associated with a stripe-like pattern of PrP immunoreactivity in the molecular layer of the cerebellum (m), contrasting those with obvious spongiform change and neuronal loss (n, H&E) and diffuse/synaptic PrP immunoreactivity (o). Bar graph in a indicates 60 lm for a–e and i–k, 500 lm for g, h, 1,000 lm for f, and 150 lm for l–o
standard (MM type 1 and VV type 2) [53]. The PrPres patterns observed in these patients with the E200K mutation appeared distinct from the PrPres pattern of patients
with sporadic CJD PrPres type 1 and type 2. These atypical patterns were apparently linked to an increase in the amounts of diglycosylated bands and a lower representation
123
tel-00635537, version 1 - 25 Oct 2011
46
Acta Neuropathol (2011) 121:39–57
of the unglycosylated one, associated with a discrete closeness between the di- and the monoglycosylated bands (Fig. 4). The measurement of molecular masses in comparison to a molecular mass standard ladder loaded on SDS-PAGE in three wells clearly showed no difference between PrPres type 1 in sporadic CJD and the PrPres from E200K mutation. The difference of molecular masses calculated between the PrPres type 1 and type 2 was 1.27 ± 0.06 kDa when samples were run in 16% gels. Interestingly, for three patients (2 MV and one MM at codon 129), we found a PrPres with a type halfway between type 1 and type 2 of Parchi’s classification [53], the difference between PrPres type 1 and PrPres from these patients (arbitrarily termed type 1* here) was 0.7 kDa (Fig. 4a). Running electrophoresis on 12% gels of these samples, this type 1* PrPres was similar to type 1 of that described by Parchi et al. [53] in sporadic CJD (Fig. 4b). Most genetic cases studied here presented a PrPres type similar to type 1 (7/10 patients E200K) whereas one presented a type 2 (MV at codon 129) in the cerebellum, thalamus, frontal, and occipital cortex, and two cases showed mixed PrP types: one case (MV at codon 129) exhibited type 2 PrPres in occipital, frontal, and temporal cortex, but type 1 in the cerebellum, thalamus, striatum, and cingulate gyrus (Fig. 4c); another case (MM at codon 129) showed mainly type 1 PrPres but with co-ocurrence of types 1 and 2 in frontal cortex. These patterns correlated well with coarse and fine immunodeposits seen in immunohistochemistry. Tau pathology Immunoreactivity for phosphorylated tau: three patterns of tau pathology as seen by immunostaining for AT8 (summarized in Fig. 5) 1.
2.
3.
In 36 cases (93.3%), neuritic profiles were immunostained, mainly in areas with more prominent tissue pathology, neuronal loss and spongiform change (Fig. 6a), including the posterior horn of the spinal cord in the single case where spinal cord was available. In addition, in 15 cases (38.4%) neurofibrillary degeneration (Fig. 6b) following Braak and Braak stages were observed. Three further cases showed neurofibrillary tangles restricted to the dorsal raphe nucleus and locus coeruleus. Furthermore, 13 cases (33.3%) showed features of a tauopathy that does not fulfill criteria of established sporadic tauopathy entities. This could be further subdivided as follows: (a)
Six cases with neurofibrillary tangles, diffuse cytoplasmic tau immunoreactivity (pretangle-like),
123
Fig. 4 Immunoblotting for disease-associated prion protein in representative cases. a PrPres was extracted from different brain areas; after separation in 16% gels in denaturated conditions, PK resistant fragment of PrP were labeled by 3F4 anti prion antibody. Lanes 1 and 14 negative control from non CJD patient, Lanes 2 and 12 type 1 PrPres control from sporadic CJD, Lanes 3 and 13 type 2 PrPres control from sporadic CJD, Lane 4 50 lg of thalamus from a French E200K CJD patient (MV at codon 129), Lane 5 0.7 mg of frontal from a Hungarian E200K CJD patient (MM at codon 129), Lane 6 1 mg of cerebellum from a Hungarian E200K CJD patient (MM at codon 129), Lanes 7 and 11 type 1-like PrPres (see text) from a French E200K patient (MV at codon 129), Lane 8 type 2 PrPres control from sporadic CJD, Lane 9 type 1-like PrPres from 100 lg of cerebellum from a Hungarian E200K CJD patient (MV at codon 129), Lane 10 type 1-like PrPres (250 lg) from a Hungarian E200K CJD patient (MM at codon 129). b After initial running in 16% gels, some PrPres types were confirmed after separation in 12% gels in denaturated conditions; PK resistant fragment of PrP were labeled by 3F4 anti prion antibody. Lanes 1 and 10 type 2 PrPres control from sporadic CJD, Lanes 2 and 11 type 1 PrPres control from sporadic CJD, Lane 3 50 lg of thalamus from a French E200K CJD patient (MV at codon 129), Lane 4 250 lg of frontal cortex from a Hungarian E200K CJD patient (MM at codon 129), Lane 5 100 lg of cerebellum from a Hungarian E200K CJD patient (MV at codon 129), Lane 6 type 1 PrPres control from a French E200K patient, Lane 7 1 mg of cerebellum from a Hungarian E200K CJD patient (MM at codon 129), Lane 8 0.7 mg of frontal cortex from a Hungarian E200K CJD patient (MM at codon 129), Lane 9 50 lg of thalamus from a French E200K CJD patient (MV at codon 129), Lane 12 negative control from non CJD patient. c Western blot analysis of seven different brain areas from one E200K M129V CJD patient (lanes 3–9), probed with 3F4 anti-PrP mAb. Controls: lanes 1 and 12 PrPres type 2a (sporadic CJD), lane 2 control PrPres type 1 (sporadic CJD), lane 10 control PrPres type 1 (genetic CJD-E200K), lane 13 control PrPres type 2b (variant CJD), lane 3 type 1 PrPres from cerebellum (1 mg), lane 4 type 1 PrPres from striatum (100 lg), lane 5 type 2a PrPres from frontal (250 lg), lane 6 type 2a PrPres from occipital cortex (16 lg), lane 7 type 1 PrPres from thalamus (16 lg), lane 8 type 2a PrPres from temporal (250 lg), lane 9 type 1 PrPres from cingulate (50 lg)
and threads in the caudate nucleus, putamen (Fig. 6c), brainstem (substantia nigra, dorsal raphe nucleus, and locus coeruelus) (Fig. 6d, e), and less in the thalamus, including one with
Acta Neuropathol (2011) 121:39–57
47
tel-00635537, version 1 - 25 Oct 2011
Fig. 5 Stratification of tau pathology in E200K genetic CJD according to cytopathology, regional distribution, age, and codon 129 polymorphism
prominent involvement of neocortical regions (Fig. 6f). There was lack of astrocytic plaques or tufted astrocytes, although some dot-like immunostaining of astrocytic processes were noted. Oligodendroglial coiled bodies were only occasionally seen. (b) Seven further cases had an unusual distribution of neuronal and glial tau deposition in the hippocampus, which included neurofibrillary tangles and prominent diffuse neuronal granular cytoplasmic immunoreactivity not only in CA4, CA3, and CA2 subregions and dentate gyrus, but also in the CA1 subregion and subiculum, without or with scant neurofibrillary degeneration in the
entorhinal cortex (Fig. 6g–i). In these cases argyrophilic grains were not seen, but some oligodendroglial tau immunopositivity and dotlike immunolabeling of astrocytic processes were observed. Interestingly, these cases also showed neurofibrillary tangles in the locus coeruleus. Immunostaining for different anti-tau antibodies and co-localization studies Neuritic profiles were detected using immunostaining for 3R tau and less for 4R (Fig. 6j). Tau immunoreactive neurites occasionally co-localized with disease-associated PrP (Fig. 7a). Globose tangles in subcortical areas were only rarely immunoreactive for 3R
123
Acta Neuropathol (2011) 121:39–57
tel-00635537, version 1 - 25 Oct 2011
48
tau, but many were prominently 4R immunoreactive (Fig. 6k). In neocortical areas and hippocampus, both 3R and 4R immunopositivities were noted in neurofibrillary
123
tangles. Thread-like structures were mainly 4R immunopositive (Fig. 6l) and were associated with neurofilaments but not astrocytic processes (Fig. 7b, c). In such areas, PrP
Acta Neuropathol (2011) 121:39–57 b Fig. 6 Immunoreactivity for phosphorylated tau. a Neuritic profiles
tel-00635537, version 1 - 25 Oct 2011
in the neuropil in areas with spongiform change (AT8. thalamus). b Neurofibrillary tangles in the temporal cortex (AT8). c Globose neurofibrillary tangles and threads in the putamen (AT8) and in the locus coeruleus (d). Prominent thread pathology and neuronal cytoplasmic immunoreactivity in the caudate nucleus (e, right upper inset shows overview) and in the frontal cortex (f) in a representative case (AT8). Diffuse neuronal cytoplasmic immunoreactivity in the granular layer of the dentate gyrus (g), in the CA2/3 subregion (h), and in the CA1 subregion (i) with thin thread and dots in the neuropil, and a relative sparing of the entorhinal cortex (i, right upper inset, all AT8). Neuritic profiles are immunoreactive mainly for 3R tau (j, left side), globose tangles in subcortical areas are immunoreactive for both 3R (k, left side) and 4R tau (k, right side of image), while cases with prominent thread-like pathology show mainly 4R immunoreactivity (l, right side of image). Representative images of a case with subcortical tauopathy (m–p) and one with prominent threads (q–t) immunostained with AT8 (m, q), AT180 (n, r), AT100 (o, s), and HT7 (p, t) antibodies. Bar graph indicates in a indicates 60 lm for a–e, g–i, and m–t, 1,000 lm for f and e upper inset; and 20 lm for j–l
deposition was also noted (Fig. 7d). We immunolabeled all these structures with anti-AT180, AT100, and HT7 antibodies (Fig. 6m–t), although neuritic profiles were less seen with HT7. AT8 and AT180 produced the most extensive immunoreactivity. Immunoblotting for tau Four cases were examined: three cases (MM at codon 129) with only mild to moderate neuritic tau pathology, and one further case (MV at codon 129) with tau pathology in the cortex. In one case with mild dot-like neuritic immunoreactivity detected by the AT8 antibody in immunohistochemistry, western blotting of sarkosyl-insoluble tau using anti-tau AT8 antibody did not reveal immunopositivity. However, anti-tau antibody T46 showed immunoreactivity at 60 and 64 kDa in the frontal cortex and hippocampus (Fig. 8a, b). This was confirmed by the lack of labeling with RD4 and immunopositivity with the RD3 antibody (Fig. 8c, d) supporting our immunohistochemical observations with anti-RD3 and RD4. Both antibodies detected further small molecular weight bands. In one case with neurofibrillary tangles and glial tau immunoreactivity (MV at codon 129), AT8 and T46 showed patterns similar to AD (Fig. 8e) in the medial temporal lobe (hippocampus) sample, while 3R tau was detected in several regions examined (Fig. 8f). Unfortunately, frozen material was not available in cases with the unusual immunohistochemical patterns, predominated by 4R immunopositivity, of tau described above.
49
involvement (Braak IV), and one with brainstem involvement (aged 60) (Fig. 9b). (2) Prominent neuronal granular diffuse cytoplasmic immunoreactivity restricted to neurons of medullary raphe nuclei, dorsal vagus nucleus (Fig. 9c), locus coeruleus and substantia nigra; this was observed in 13 cases (including 4 with immunoreactivity only in the medulla oblongata) lacking Lewy bodies or Lewy neurites. Lower raphe nuclei and the dorsal vagus nucleus always showed the most prominent neuronal immunoreactivity, but scattered cortical neurons also showed this immunoreactivity. (3) Some areas showed small aggregates of a-synuclein in the neuropil reminiscent of that described in sporadic CJD [25]. However, this was not consistent and showed no region preference or relation to PrP deposition. Deposition of Ab This included mainly diffuse deposits, some with multiple small cores, subpial and vascular deposition (Fig. 9d–f). Parenchymal deposits were mainly Ab-42 and less Ab-40 immunoreactive (Fig. 9h–i). Parenchymal Ab was observed in 21 cases (53,8%) that involved frontal, temporal, and occipital regions in all, basal ganglia in five, and cerebellum in two. Cerebral amyloid angiopathy (Ab-CAA) was noted in nine individuals (23.07%): this involved leptomeningeal and cortical vessels but not capillaries. Cored neuritic plaques were only occasionally seen and none of the cases fulfilled the criteria of AD according to CERAD [48]. The youngest patient with parenchymal Ab deposition was 51 years old and the youngest individual with Ab-CAA was 54. Phosphorylated TDP-43, FUS, and ubiquitin immunohistochemistry Immunostaining for FUS revealed variably strong staining of neuronal nuclei. We did not observe neuritic profiles or intracellular deposits in any case in the examined regions (Table 1). Similarly, in all cases there was a lack of phosphorylated TDP-43 pathology. Immunostaining for ubiquitin showed variable amount of granular immunoreactivity in the neuropil in severely affected areas along with neuritic profiles. Of note were the prominent perisomatic granules surrounding pyramidal neurons in the hippocampal CA1 subregion in 13 cases (33%).
a-Synuclein pathology
Summary of proteinopathies
Three types of a-synuclein pathology were observed: (1) typical Lewy bodies and Lewy neurites following Braak stages in six cases (15.4%) [4]; this consisted of three cases (aged 53, 66, and 66) with neocortical involvement (Braak V–VI) (Fig. 9a), two cases (aged 67 and 68) with limbic
In addition to PrP, 36 cases (92.3%) showed some kind of concomitant protein deposition. Excluding the presence of neuritic tau pathology, co-pathology was seen in 32 cases (82%) (summarized in Table 3). Age at death and duration of illness did not differ between cases with and without
123
tel-00635537, version 1 - 25 Oct 2011
50
Acta Neuropathol (2011) 121:39–57
Fig. 7 Double immunolabeling in cases with tau pathology. Tau immunoreactive neurites occasionally co-localize with disease-associated PrP (a, AT8 green, PrP red, 9400; arrowhead indicates enlarged part in the right side of the images). Thread-like pathology associates with neurofilaments (b, AT8 green, SMI-31 red, 9400;
arrowhead indicates enlarged part in the right side of the images) and not with astrocytic processes (c, AT8 green, GFAP red, 9400). In these areas PrP deposition is also noted (d, AT8 green, PrP red, 91000). Bar graph in a indicates 60 lm in a–c and 15 lm in d and in enlarged insets of a–c
co-depositions, except for older age at death in cases with unclassifiable tauopathy (mean age ± standard error: 64.9 ± 1.7 versus 57.7 ± 1.7; p = 0.016). In cases with a-synucleinopathy, the patients already had neurological examination prior to rapidly progressive dementia, because of e.g. tremor of tongue, behavioral change, or typical Parkinsonism responding to L-Dopa therapy. In cases with tau pathology in the basal ganglia, dystonia, chorea or rigidity was noted, and in two cases supranuclear gaze palsy. In cases where memory impairment was indicated as a presenting symptom, neurofibrillary degeneration was noted. Otherwise the rapidly progressive clinical phase did not differ between cases with different protein depositions.
There was no significant influence of the PRNP codon 129 polymorphism on concomitant proteinopathy. Protein codepositions were noted in individuals from all countries included in our study.
123
Discussion This study has the following implications: (1) genetic CJD with the E200K mutation is clinically characterized mostly by progressive dementia and ataxia and further symptoms, however, prominent supranuclear palsy, insomnia, and polyneuropathy may also be present. (2) E200K CJD
tel-00635537, version 1 - 25 Oct 2011
Acta Neuropathol (2011) 121:39–57
51
Fig. 8 Immunoblotting of sarkosyl-insoluble tau using different antibodies. a Immunostaining using AT8 anti-tau antibody did not reveal positive bands in a case with focal dot-like neuritic immunoreactivity observed in immunohistochemistry. b The same sample examined by T46 anti-tau antibody showed bands at 60 and 64 kDa molecular weight characteristic for 3R tau. Anti-RD3 (c) and antiRD4 antibodies (d) showed different fragments of tau, and RD3 confirms the presence of 60 and 64 kDa bands. (Lane 1 medial temporal cortex sample from a patient with Alzheimer‘s disease; lane 2 medial temporal cortex sample from a non-diseased individual (examined by neuropathology); lanes 3–5 Hungarian patient (MV at codon 129) with dot-like neuritic tau pathology detected in
immunohistochemistry. 3 cerebellum, 4 frontal cortex, 5 temporal cortex. e Typical PHF-like pattern like in E200K CJD case etected by AT8 in medial temporal cortex (Lane 1 medial temporal cortex including hippocampus from E200K CJD patient, lane 2 Alzheimer’s disease sample). f 3R tau were detected in several regions examined in case with neurofibrillary tangles restricted to the medial temporal lobe and neuritic tau pathology in the frontal and temporal cortices and striatum (Lane 1 frontal cortex, lane 2: striatum, lane 3: medial temporal lobe including hippocampus, lane 4: temporal cortex, lane 5 medial temporal cortex sample from a non-diseased individual, examined by neuropathology, lane 6: Alzheimer‘s disease sample)
presents with various PrP deposition patterns that frequently overlap between MM homozygotes and MV heterozygotes at PRNP codon 129. This is also reflected by a mixture of type 2 and type 1 PrPres in these brains. (3) Lesion profiles do not differ significantly between MM and MV cases. (4) Some PrP deposition patterns are not seen in sporadic CJD or other genetic forms (e.g. stripe-like pattern in the cerebellum or distinct intraneuronal PrP aggregates involving prominently brainstem nuclei). Moreover, immunoblotting for PrPres demonstrated distinct ratios of glycosylated to unglycosylated bands as compared to sporadic CJD. (5) Concomitant presence of a-synuclein, phospho-tau pathology or deposition of Ab is more the rule
than the exception. These protein deposits are present in areas where disease-associated PrP is also present. Clinical features of E200K genetic CJD frequently overlap with that of sporadic CJD, including the presence of periodic sharp wave complexes. However, in our cohort myoclonus is less frequently documented. In spite of the less prominent pathology in the cerebellum, ataxia is often observed. Interestingly, the patterns of pathology observed in the cerebellum include either spongiform change and neuronal loss with diffuse/synaptic PrP immunoreactivity, or little lesioning associated with a stripe-like pattern of PrP immunoreactivity restricted to the molecular layer. This suggests that cerebellar ataxia may have a complex
123
tel-00635537, version 1 - 25 Oct 2011
52
Acta Neuropathol (2011) 121:39–57
Fig. 9 Immunoreactivity for a-synuclein and amyloid-b (Ab) in E200K genetic CJD. Typical a-synuclein immunopositive cortical Lewy bodies and Lewy neurites in the frontal cortex (a), and brainstem-type Lewy bodies in the medulla oblongata (b) in representative cases showing a-synuclein pathology compatible with Braak stageing. Prominent neuronal granular diffuse cytoplasmic immunoreactivity restricted to neurons of medullary raphe nuclei, dorsal vagus nucleus (c, cell indicated with arrowhead enlarged in
right lower inset) but lacking Lewy bodies or Lewy neurites. Extensive Ab deposition of the form of diffuse plaques and focal deposits in the striatum (d, e), and in the vessel walls in the frontal cortex (f) in representative cases. Parenchymal Ab deposition (g) was mainly immunoreactive for anti-Ab-42 (h) and less for anti-Ab-40 (i) peptide. Bar graph in a indicates 200 lm for a, f, g, h, i, 150 lm for e, 400 lm for b, c (60 lm for inset), and 1,200 lm for d
background by either a more severe tissue damage of cerebellar cortical layers or, since the stripe-like pattern strongly resembles the compartmentalization of the cerebellar cortex and Purkinje cell branching [67], by alterations of synaptic contacts of Purkinje cells. In comparison with other cohorts [47, 63], chorea/dystonia was more frequently reported in our study than Parkinsonism. Similar to other reports, we confirm that supranuclear gaze palsy, insomnia, and polyneuropathy may be prominent in some cases [1, 2, 8, 50, 64]; this is not related to the codon 129 polymorphism. In our cohort, unusual clinical presentations like early memory impairment, supranuclear palsy, premorbid symptoms, or even characteristic Parkinson syndrome, years before the rapid progressive course, were described and frequently suggestive as substrate of concomitant protein deposition. However, due to the retrospective nature of our clinical data collection, it remains to be established how the presence of concomitant
proteinopathies influence the clinical course. This merits further prospective studies, but it is clear that genetic CJD should be considered in less elderly patients (50–60 years) with a clinical suspicion of a pre-existing neurodegenerative disease (including ‘‘typical’’ Parkinson’s disease) that switches into a rapidly progressive phase with dementia and ataxia. Lesion profiles, defined by spongiform change and neuronal loss/gliosis, were not significantly different between cases with different constellation at codon 129. Of note was the variable involvement of neocortical areas, including frequent laminar accentuation, and the constant lesioning in the striatum and thalamic nuclei. The latter is reflected also in the signal alterations detected in MRI [15]. Sometimes the involvement of the thalamus may predominate, leading to further differential diagnostic issues [39]. In addition, neuronal loss was seen in the substantia nigra with or without a-synuclein inclusions. Nigrostriatal
123
Acta Neuropathol (2011) 121:39–57
53
Table 3 Summary of combinations of proteinopathies
tel-00635537, version 1 - 25 Oct 2011
Protein pathology
Unusual tauopathy
Tau-NFDBraak
a-SynucleinBraak
A-beta deposits
A-beta CAA
Number of cases
% of proteinopathy
% of all cases
Unusual tauopathy
0
?
-
-
-
4
33.3
10.2
Unusual tauopathy
0
-
?
-
-
5
41.6
12.8
Unusual tauopathy
0
-
-
?
-
6
50
15.3
Unusual tauopathy
0
-
-
-
?
4
33.3
10.2
Unusual tauopathy
0
?
?
-
-
3
25
7.7
Unusual tauopathy
0
?
?
?
-
2
16.6
5.1
Unusual tauopathy Unusual tauopathy
0 0
? ?
? -
-
? ?
0 0
0 0
0 0
Unusual tauopathy
0
?
?
?
?
1
8.3
2.5 12.8
a-Synuclein-Braak
?
-
0
-
-
5
83.3
a-Synuclein-Braak
-
?
0
-
-
3
50
a-Synuclein-Braak
-
-
0
?
-
4
66.6
7.7 10.2
a-Synuclein-Braak
-
-
0
-
?
2
33.3
5.1
a-Synuclein-Braak
-
?
0
?
-
2
33.3
5.1
a-Synuclein-Braak
-
?
0
-
?
1
16.6
2.5
a-Synuclein-Braak
-
?
0
?
?
1
16.6
2.5
a-Synuclein-Braak
?
?
0
?
?
1
16.6
2.5
Ab deposits
-
?
-
0
-
9
40.9
23
Ab deposits
-
-
-
0
?
8
36.3
20.5
Ab deposits
-
?
-
0
?
1
4.5
2.5
Ab deposits
-
?
?
0
?
1
4.5
2.5
Ab deposits Ab deposits
? ?
? ?
?
0 0
? ?
0 1
0 4.5
0 2.5
The predominant proteinopathy is indicated in the left column and all further types of features that are additionally seen are indicated with a ? sign NFD neurofibrillary degeneration degeneration following Braak and Braak stageing, CAA cereberal amyloid angiopathy (Ab)
damage was demonstrated in a recent study in sporadic CJD [66]. In contrast to the lesion profile, the morphology of PrP deposits showed more variation: more coarse immunodeposits (plaques, patchy/perivacuolar), but also more intraneuronal PrP aggregates were detected in cases with MV at codon 129 as compared to MMs. However, these deposits were frequently in MM cases in a more restricted distribution suggesting presence of different PrP types. Indeed, a mixture of type 1 and 2 PrPres was observed in some cases. Regional variability between cerebral and cerebellar cortex of the PrPres pattern was reported only in a single case [57]. We extend this finding by demonstrating variability in many regions in 2 out of 10 cases. Analysis of further 10 French cases with E200K mutation, not included in the present comprehensive neuropathological survey, revealed 3 cases with regional variability. According to these observations, a rough estimate of approximately 25% of cases with E200K mutation, slightly less than reported in sporadic CJD [54], shows this phenomenon. It was already reported that the western blot pattern of PrPres in E200K genetic CJD differs from
sporadic CJD [7, 27]. In spite of the presence of similar morphological immunodeposits, some PrP immunoreactive patterns were unusual (e.g. intraneuronal PrP immunopositivity) as a potential correlate of the distinct PrPres patterns. Intraneuronal dot-like PrP immunoreactivity was reported in the E200K mutation [43] and V180I mutation also with abnormally glycosylated PrPres [51], while intraneuronal PrP immunoreactivity (albeit morphologically not with dot-like accentuation) was documented in a patient with apparently sporadic CJD and novel glycotype pattern [69]. All together these observations suggest that the E200K mutation induces a relatively uniform anatomical pattern of tissue lesioning (spongiform change, neuronal loss/gliosis), while the deposition of diseaseassociated PrP is more influenced by the codon 129 constellation, including different types of, also mixed, PrPres forms. In addition, we detected a range of concomitant protein deposits. Tau pathology was represented either by the presence of small neuritic profiles, neurofibrillary degeneration following stages of Braak and Braak [3], or by
123
tel-00635537, version 1 - 25 Oct 2011
54
unusual forms that, based on the distinct anatomical distribution, lack of astrocytic plaques or tufted astrocytes, were not compatible with sporadic tauopathies. Interestingly, neurofibrillary tangles frequently occurred in brainstem nuclei (raphe, locus coeruleus) also in relatively young patients, analogously to that described as an early feature not only in AD but also in mild cognitive impairment [24, 62]. Neuritic profiles clustering around plaques are described in the acquired prion disease, variant CJD [19], while neurofibrillary tangles are consistent features of some PRNP mutations associated with prominent PrP amyloidosis [17]. Both features are not observed in E200K CJD. Interestingly, an unusual pattern of tauopathy was described in the R208H PRNP mutation associated with a CJD phenotype [59]. Double immunolabeling studies suggest that most of the tau pathology is neuronal in origin. Interestingly, AT8 and T46 antibodies showed different results in immunoblotting. Both antibodies were able to detect an AD-like pattern in samples where neurofibrillary degeneration was observed; however, in samples with mild neuritic profiles seen in immunohistochemistry, only T46 and RD3 antibody showed bands characteristic of 3R tau. Whereas postmortem delay and storage temperature may be a limitating factor for the detection of PHF and phosphorylated forms of tau [14, 60], in our study PHF and 3R tau were clearly detectable by western blot. However, immunoreactivity against specific phosphorylation sites may be reduced due to tau degradation by endogenous phosphatases. Thus, our observation on low molecular bands in a limited number of cases must be interpreted with caution and merits further evaluation on more cases whether a difference in epitopes or other factors might be the basis of this. In cases with unusual presentations of tau pathology, 4R isoform was prominently detected in immunohistochemistry, whereas in these cases no material was available for immunoblotting studies. However, it may be concluded that a spectrum of tau pathology from 3R through mixed and to 4R dominant forms is associated with the E200K mutation. In addition, Ab deposition (parenchymal and vascular) frequently occurs in E200K genetic CJD [18]. In addition, we observed Lewy body pathology following Braak stages, and also prominent neuronal granular a-synuclein immunoreactivity in brainstem nuclei without Lewy bodies (together in around half of the patients). This indicates that pathological upregulation and eventual inclusion body formation of a-synuclein is an important feature in E200K genetic CJD, supporting a potential interactive role of PrP and a-synuclein. Interestingly, in spite of the lack of a role of codon 129 polymorphism on the susceptibility for the a-synucleinopathy Parkinson’s disease [61], the clinical presentation may be influenced by this [23]. Finally, we
123
Acta Neuropathol (2011) 121:39–57
did not detect any protein deposition using anti-FUS and phosphorylated TDP-43 antibodies. The latter is in line with a recent study in various forms of CJD [31]. The presence of various protein depositions, independent of the codon 129 polymorphism or PrPres type, could support a concept of further genetic or epigenetic factors leading to various presentations in significantly different ages (ranging from early 30 to 70 years of age). The processing of PrP overlaps with that of Ab and a-synuclein, since it includes the endosomal–lysosomal system [38, 52]. Mutual interactions between tau, Ab, and a-synuclein were documented [36]. Based on our observation of Lewy bodies or neurofibrillary tangles in our diagnostic work-up, we conducted a systematic evaluation of protein deposition and presented the first systematic demonstration of intensified and combined neurodegeneration in a genetic prion disease due to a single point mutation. The spectrum of protein depositions is distinct from that associated with PrP amyloid [17, 19] or sporadic CJD [11, 66]. The observation of Ab deposition, neurofibrillary degeneration, and a-synucleinopathy in individuals from different countries, together with the detection of tauopathy forms, not compatible with sporadic tauopathies [6], suggests the interaction of mutated PrP with tau [68], Ab [55], and also a-synuclein. Cellular PrP has been shown to regulate the production of Ab and forms a complex [55] with full-length tau protein in vitro [68]. Moreover, there is a homologous region in PrP and a-synuclein, and interestingly, homologous peptides corresponding to this sequence can promote fibrillization of a-synuclein [13]. Finally, our observation of the frequent and unusual intraneuronal accumulation of PrP immunoreactivity indicates failed release from the cell and overwhelming of the protein processing system. In sum, our and previous studies suggest an altered metabolism and intracellular trafficking of mutated PrP that interferes with the processing of other proteins and leads to their pathological deposition. Thus, the E200K PrP mutation may become an important model to decipher the molecular interplay among neurodegeneration-associated proteins. Our observations also support a complex pathogenetic interplay in the background of E200K genetic CJD that may lead to differences in penetrance and clinical phenotype. Finally, our study suggests to clinicians the suspicion of genetic CJD when early onset neurological symptoms turn into a rapidly progressive phase. Acknowledgments This study was performed in the frame of the EU FP6 Project Neuroscreen LSHB-CZ-2006-037719 contract No. 037719. We are grateful for the technical assistance of Irene Leisser, Gerda Ricken, Lenkeine Marianna, Katalin Ress, Rachel Plantier, Franc¸oise Didier, and for the kind cooperation of collegues and families of patients supporting our Surveillance systems. RV is a senior clinical investigator of the Research Foundation-Flanders. LL
Acta Neuropathol (2011) 121:39–57
55
was supported by a grant of the Hungarian Scientific Research Fund (OTKA-NK78012). Conflict of interest statement
None. 15.
tel-00635537, version 1 - 25 Oct 2011
References 1. Antoine JC, Laplanche JL, Mosnier JF, Beaudry P, Chatelain J, Michel D (1996) Demyelinating peripheral neuropathy with Creutzfeldt-Jakob disease and mutation at codon 200 of the prion protein gene. Neurology 46:1123–1127 2. Bertoni JM, Brown P, Goldfarb LG, Rubenstein R, Gajdusek DC (1992) Familial Creutzfeldt-Jakob disease (codon 200 mutation) with supranuclear palsy. JAMA 268:2413–2415 3. Braak H, Braak E (1991) Neuropathological stageing of Alzheimerrelated changes. Acta Neuropathol 82:239–259 4. Braak H, Del Tredici K, Rub U, de Vos RA, Jansen Steur EN, Braak E (2003) Staging of brain pathology related to sporadic Parkinson’s disease. Neurobiol Aging 24:197–211 5. Brown P, Galvez S, Goldfarb LG, Nieto A, Cartier L, Gibbs CJ Jr, Gajdusek DC (1992) Familial Creutzfeldt-Jakob disease in Chile is associated with the codon 200 mutation of the PRNP amyloid precursor gene on chromosome 20. J Neurol Sci 112:65–67 6. Cairns NJ, Bigio EH, Mackenzie IR, Neumann M, Lee VM, Hatanpaa KJ, White CL 3rd, Schneider JA, Grinberg LT, Halliday G, Duyckaerts C, Lowe JS, Holm IE, Tolnay M, Okamoto K, Yokoo H, Murayama S, Woulfe J, Munoz DG, Dickson DW, Ince PG, Trojanowski JQ, Mann DM (2007) Neuropathologic diagnostic and nosologic criteria for frontotemporal lobar degeneration: consensus of the Consortium for Frontotemporal Lobar Degeneration. Acta Neuropathol 114:5–22 7. Cardone F, Liu QG, Petraroli R, Ladogana A, D’Alessandro M, Arpino C, Di Bari M, Macchi G, Pocchiari M (1999) Prion protein glycotype analysis in familial and sporadic CreutzfeldtJakob disease patients. Brain Res Bull 49:429–433 8. Chapman J, Arlazoroff A, Goldfarb LG, Cervenakova L, Neufeld MY, Werber E, Herbert M, Brown P, Gajdusek DC, Korczyn AD (1996) Fatal insomnia in a case of familial Creutzfeldt-Jakob disease with the codon 200 (Lys) mutation. Neurology 46:758–761 9. Collinge J, Palmer MS, Campbell T, Sidle KC, Carroll D, Harding A (1993) Inherited prion disease (PrP lysine 200) in Britain: two case reports. BMJ 306:301–302 10. de Silva R, Lashley T, Gibb G, Hanger D, Hope A, Reid A, Bandopadhyay R, Utton M, Strand C, Jowett T, Khan N, Anderton B, Wood N, Holton J, Revesz T, Lees A (2003) Pathological inclusion bodies in tauopathies contain distinct complements of tau with three or four microtubule-binding repeat domains as demonstrated by new specific monoclonal antibodies. Neuropathol Appl Neurobiol 29:288–302 11. Debatin L, Streffer J, Geissen M, Matschke J, Aguzzi A, Glatzel M (2008) Association between deposition of beta-amyloid and pathological prion protein in sporadic Creutzfeldt-Jakob disease. Neurodegener Dis 5:347–354 12. DelleDonne A, Klos KJ, Fujishiro H, Ahmed Z, Parisi JE, Josephs KA, Frigerio R, Burnett M, Wszolek ZK, Uitti RJ, Ahlskog JE, Dickson DW (2008) Incidental Lewy body disease and preclinical Parkinson disease. Arch Neurol 65:1074–1080 13. Du HN, Li HT, Zhang F, Lin XJ, Shi JH, Shi YH, Ji LN, Hu J, Lin DH, Hu HY (2006) Acceleration of alpha-synuclein aggregation by homologous peptides. FEBS Lett 580:3657–3664 14. Ferrer I, Santpere G, Arzberger T, Bell J, Blanco R, Boluda S, Budka H, Carmona M, Giaccone G, Krebs B, Limido L, Parchi P,
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
Puig B, Strammiello R, Strobel T, Kretzschmar H (2007) Brain protein preservation largely depends on the postmortem storage temperature: implications for study of proteins in human neurologic diseases and management of brain banks: a BrainNet Europe Study. J Neuropathol Exp Neurol 66:35–46 Fulbright RK, Hoffmann C, Lee H, Pozamantir A, Chapman J, Prohovnik I (2008) MR imaging of familial Creutzfeldt-Jakob disease: a blinded and controlled study. AJNR Am J Neuroradiol 29:1638–1643 Gelpi E, Heinzl H, Hoftberger R, Unterberger U, Strobel T, Voigtlander T, Drobna E, Jarius C, Lang S, Waldhor T, Bernheimer H, Budka H (2008) Creutzfeldt-Jakob disease in Austria: an autopsy-controlled study. Neuroepidemiology 30:215–221 Ghetti B, Piccardo P, Frangione B, Bugiani O, Giaccone G, Young K, Prelli F, Farlow MR, Dlouhy SR, Tagliavini F (1996) Prion protein amyloidosis. Brain Pathol 6:127–145 Ghoshal N, Cali I, Perrin RJ, Josephson SA, Sun N, Gambetti P, Morris JC (2009) Codistribution of amyloid beta plaques and spongiform degeneration in familial Creutzfeldt-Jakob disease with the E200K–129M haplotype. Arch Neurol 66: 1240–1246 Giaccone G, Mangieri M, Capobianco R, Limido L, Hauw JJ, Haik S, Fociani P, Bugiani O, Tagliavini F (2008) Tauopathy in human and experimental variant Creutzfeldt-Jakob disease. Neurobiol Aging 29:1864–1873 Goedert M, Jakes R, Crowther RA, Cohen P, Vanmechelen E, Vandermeeren M, Cras P (1994) Epitope mapping of monoclonal antibodies to the paired helical filaments of Alzheimer’s disease: identification of phosphorylation sites in tau protein. Biochem J 301(Pt 3):871–877 Goldfarb LG, Brown P, Mitrova E, Cervenakova L, Goldin L, Korczyn AD, Chapman J, Galvez S, Cartier L, Rubenstein R et al (1991) Creutzfeldt-Jacob disease associated with the PRNP codon 200Lys mutation: an analysis of 45 families. Eur J Epidemiol 7:477–486 Goldfarb LG, Mitrova E, Brown P, Toh BK, Gajdusek DC (1990) Mutation in codon 200 of scrapie amyloid protein gene in two clusters of Creutzfeldt-Jakob disease in Slovakia. Lancet 336:514–515 Gossrau G, Herting B, Mockel S, Kempe A, Koch R, Reichmann H, Lampe JB (2006) Analysis of the polymorphic prion protein gene codon 129 in idiopathic Parkinson’s disease. J Neural Transm 113:331–337 Grudzien A, Shaw P, Weintraub S, Bigio E, Mash DC, Mesulam MM (2007) Locus coeruleus neurofibrillary degeneration in aging, mild cognitive impairment and early Alzheimer’s disease. Neurobiol Aging 28:327–335 Haik S, Privat N, Adjou KT, Sazdovitch V, Dormont D, Duyckaerts C, Hauw JJ (2002) Alpha-synuclein-immunoreactive deposits in human and animal prion diseases. Acta Neuropathol 103:516–520 Hainfellner JA, Parchi P, Kitamoto T, Jarius C, Gambetti P, Budka H (1999) A novel phenotype in familial Creutzfeldt-Jakob disease: prion protein gene E200K mutation coupled with valine at codon 129 and type 2 protease-resistant prion protein. Ann Neurol 45:812–816 Hill AF, Joiner S, Beck JA, Campbell TA, Dickinson A, Poulter M, Wadsworth JD, Collinge J (2006) Distinct glycoform ratios of protease resistant prion protein associated with PRNP point mutations. Brain 129:676–685 Horiguchi T, Uryu K, Giasson BI, Ischiropoulos H, LightFoot R, Bellmann C, Richter-Landsberg C, Lee VM, Trojanowski JQ (2003) Nitration of tau protein is linked to neurodegeneration in tauopathies. Am J Pathol 163:1021–1031 Hsiao K, Meiner Z, Kahana E, Cass C, Kahana I, Avrahami D, Scarlato G, Abramsky O, Prusiner SB, Gabizon R (1991)
123
56
30.
31.
32.
33.
34.
tel-00635537, version 1 - 25 Oct 2011
35.
36.
37. 38.
39.
40.
41.
42.
43.
44.
45.
46.
Acta Neuropathol (2011) 121:39–57 Mutation of the prion protein in Libyan Jews with CreutzfeldtJakob disease. N Engl J Med 324:1091–1097 Inoue I, Kitamoto T, Doh-ura K, Shii H, Goto I, Tateishi J (1994) Japanese family with Creutzfeldt-Jakob disease with codon 200 point mutation of the prion protein gene. Neurology 44:299–301 Isaacs AM, Powell C, Webb TE, Linehan JM, Collinge J, Brandner S (2008) Lack of TAR-DNA binding protein-43 (TDP43) pathology in human prion diseases. Neuropathol Appl Neurobiol 34:446–456 Jarius C, Kovacs GG, Belay G, Hainfellner JA, Mitrova E, Budka H (2003) Distinctive cerebellar immunoreactivity for the prion protein in familial (E200K) Creutzfeldt-Jakob disease. Acta Neuropathol 105:449–454 Korczyn AD, Chapman J, Goldfarb LG, Brown P, Gajdusek DC (1991) A mutation in the prion protein gene in Creutzfeldt-Jakob disease in Jewish patients of Libyan, Greek, and Tunisian origin. Ann NY Acad Sci 640:171–176 Kosik KS, Orecchio LD, Binder L, Trojanowski JQ, Lee VM, Lee G (1988) Epitopes that span the tau molecule are shared with paired helical filaments. Neuron 1:817–825 Kovacs GG, Bakos A, Mitrova E, Minarovits J, Laszlo L, Majtenyi K (2007) Human prion diseases: the Hungarian experience. Ideggyogy Sz 60:447–452 Kovacs GG, Botond G, Budka H (2010) Protein coding of neurodegenerative dementias: the neuropathological basis of biomarker diagnostics. Acta Neuropathol 119:389–408 Kovacs GG, Budka H (2009) Molecular pathology of human prion diseases. Int J Mol Sci 10:976–999 Kovacs GG, Gelpi E, Strobel T, Ricken G, Nyengaard JR, Bernheimer H, Budka H (2007) Involvement of the endosomallysosomal system correlates with regional pathology in Creutzfeldt-Jakob disease. J Neuropathol Exp Neurol 66:628–636 Kovacs GG, Horvath S, Strobel T, Puskas M, Bakos A, Summers DM, Will RG, Budka H (2009) Genetic Creutzfeldt-Jakob disease mimicking variant Creutzfeldt-Jakob disease. J Neurol Neurosurg Psychiatry 80:1410–1411 Kovacs GG, Laszlo L, Bakos A, Minarovits J, Bishop MT, Strobel T, Vajna B, Mitrova E, Majtenyi K (2005) Increased incidence of genetic human prion disease in Hungary. Neurology 65:1666–1669 Kovacs GG, Puopolo M, Ladogana A, Pocchiari M, Budka H, van Duijn C, Collins SJ, Boyd A, Giulivi A, Coulthart M, DelasnerieLaupretre N, Brandel JP, Zerr I, Kretzschmar HA, de PedroCuesta J, Calero-Lara M, Glatzel M, Aguzzi A, Bishop M, Knight R, Belay G, Will R, Mitrova E (2005) Genetic prion disease: the EUROCJD experience. Hum Genet 118:166–174 Kovacs GG, Trabattoni G, Hainfellner JA, Ironside JW, Knight RS, Budka H (2002) Mutations of the prion protein gene phenotypic spectrum. J Neurol 249:1567–1582 Kovacs GG, Voigtlander T, Hainfellner JA, Budka H (2002) Distribution of intraneuronal immunoreactivity for the prion protein in human prion diseases. Acta Neuropathol 104:320–326 Lee HS, Sambuughin N, Cervenakova L, Chapman J, Pocchiari M, Litvak S, Qi HY, Budka H, del Ser T, Furukawa H, Brown P, Gajdusek DC, Long JC, Korczyn AD, Goldfarb LG (1999) Ancestral origins and worldwide distribution of the PRNP 200K mutation causing familial Creutzfeldt-Jakob disease. Am J Hum Genet 64:1063–1070 Mancuso M, Siciliano G, Capellari S, Orsucci D, Moretti P, Di Fede G, Suardi S, Strammiello R, Parchi P, Tagliavini F, Murri L (2009) Creutzfeldt-Jakob disease with E200K PRNP mutation: a case report and revision of the literature. Neurol Sci 30:417–420 Mead S, Webb TE, Campbell TA, Beck J, Linehan JM, Rutherfoord S, Joiner S, Wadsworth JD, Heckmann J, Wroe S, Doey L, King A, Collinge J (2007) Inherited prion disease with 5-OPRI:
123
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
phenotype modification by repeat length and codon 129. Neurology 69:730–738 Meiner Z, Gabizon R, Prusiner SB (1997) Familial CreutzfeldtJakob disease. Codon 200 prion disease in Libyan Jews. Medicine (Baltimore) 76:227–237 Mirra SS, Heyman A, McKeel D, Sumi SM, Crain BJ, Brownlee LM, Vogel FS, Hughes JP, van Belle G, Berg L (1991) The consortium to establish a registry for Alzheimer’s Disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer’s disease. Neurology 41:479–486 Mitrova E, Belay G (2002) Creutzfeldt-Jakob disease with E200K mutation in Slovakia: characterization and development. Acta Virol 46:31–39 Neufeld MY, Josiphov J, Korczyn AD (1992) Demyelinating peripheral neuropathy in Creutzfeldt-Jakob disease. Muscle Nerve 15:1234–1239 Nixon R, Camicioli R, Cervenakova L, Mastrianni J (2000) The PRNP-V180I mutation is associated with abnormally glycosylated PrPCJD and intracellular PrP accumulations. In: XIVth International Congress of Neuropathology Brain Pathol Birmingham, UK, p 670 Pan T, Kondo S, Le W, Jankovic J (2008) The role of autophagylysosome pathway in neurodegeneration associated with Parkinson’s disease. Brain 131:1969–1978 Parchi P, Giese A, Capellari S, Brown P, Schulz-Schaeffer W, Windl O, Zerr I, Budka H, Kopp N, Piccardo P, Poser S, Rojiani A, Streichemberger N, Julien J, Vital C, Ghetti B, Gambetti P, Kretzschmar H (1999) Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects. Ann Neurol 46:224–233 Parchi P, Strammiello R, Notari S, Giese A, Langeveld JP, Ladogana A, Zerr I, Roncaroli F, Cras P, Ghetti B, Pocchiari M, Kretzschmar H, Capellari S (2009) Incidence and spectrum of sporadic Creutzfeldt-Jakob disease variants with mixed phenotype and co-occurrence of PrPSc types: an updated classification. Acta Neuropathol 118:659–671 Parkin ET, Watt NT, Hussain I, Eckman EA, Eckman CB, Manson JC, Baybutt HN, Turner AJ, Hooper NM (2007) Cellular prion protein regulates beta-secretase cleavage of the Alzheimer’s amyloid precursor protein. Proc Natl Acad Sci USA 104:11062–11067 Peoc’h K, Manivet P, Beaudry P, Attane F, Besson G, Hannequin D, Delasnerie-Laupretre N, Laplanche JL (2000) Identification of three novel mutations (E196K, V203I, E211Q) in the prion protein gene (PRNP) in inherited prion diseases with CreutzfeldtJakob disease phenotype. Hum Mutat 15:482 Puoti G, Rossi G, Giaccone G, Awan T, Lievens PM, Defanti CA, Tagliavini F, Bugiani O (2000) Polymorphism at codon 129 of PRNP affects the phenotypic expression of Creutzfeldt-Jakob disease linked to E200K mutation. Ann Neurol 48:269–270 Quadrio I, Ugnon-Cafe S, Dupin M, Esposito G, Streichenberger N, Krolak-Salmon P, Vital A, Pellissier JF, Perret-Liaudet A, Perron H (2009) Rapid diagnosis of human prion disease using streptomycin with tonsil and brain tissues. Lab Invest 89:406–413 Roeber S, Krebs B, Neumann M, Windl O, Zerr I, Grasbon-Frodl EM, Kretzschmar HA (2005) Creutzfeldt-Jakob disease in a patient with an R208H mutation of the prion protein gene (PRNP) and a 17-kDa prion protein fragment. Acta Neuropathol 109:443–448 Santpere G, Puig B, Ferrer I (2006) Low molecular weight species of tau in Alzheimer’s disease are dependent on tau phosphorylation sites but not on delayed post-mortem delay in tissue processing. Neurosci Lett 399:106–110 Scholz SW, Xiromerisiou G, Fung HC, Eerola J, Hellstrom O, Papadimitriou A, Hadjigeorgiou GM, Tienari PJ, Fernandez HH, Mandel R, Okun MS, Gwinn-Hardy K, Singleton AB (2006) The
Acta Neuropathol (2011) 121:39–57
62.
63.
64.
66.
67. 68.
69.
disease is a sporadic 4-repeat tauopathy. J Neuropathol Exp Neurol 61:547–556 Vital A, Fernagut PO, Canron MH, Joux J, Bezard E, MartinNegrier ML, Vital C, Tison F (2009) The nigrostriatal pathway in Creutzfeldt-Jakob disease. J Neuropathol Exp Neurol 68:809–815 Voogd J, Glickstein M (1998) The anatomy of the cerebellum. Trends Neurosci 21:370–375 Wang XF, Dong CF, Zhang J, Wan YZ, Li F, Huang YX, Han L, Shan B, Gao C, Han J, Dong XP (2008) Human tau protein forms complex with PrP and some GSS- and fCJD-related PrP mutants possess stronger binding activities with tau in vitro. Mol Cell Biochem 310:49–55 Zanusso G, Polo A, Farinazzo A, Nonno R, Cardone F, Di Bari M, Ferrari S, Principe S, Gelati M, Fasoli E, Fiorini M, Prelli F, Frangione B, Tridente G, Bentivoglio M, Giorgi A, Schinina ME, Maras B, Agrimi U, Rizzuto N, Pocchiari M, Monaco S (2007) Novel prion protein conformation and glycotype in CreutzfeldtJakob disease. Arch Neurol 64:595–599
tel-00635537, version 1 - 25 Oct 2011
65.
human prion gene M129V polymorphism is not associated with idiopathic Parkinson’s disease in three distinct populations. Neurosci Lett 395:227–229 Simic G, Stanic G, Mladinov M, Jovanov-Milosevic N, Kostovic I, Hof PR (2009) Does Alzheimer’s disease begin in the brainstem? Neuropathol Appl Neurobiol 35:532–554 Simon ES, Kahana E, Chapman J, Treves TA, Gabizon R, Rosenmann H, Zilber N, Korczyn AD (2000) Creutzfeldt-Jakob disease profile in patients homozygous for the PRNP E200K mutation. Ann Neurol 47:257–260 Taratuto AL, Piccardo P, Reich EG, Chen SG, Sevlever G, Schultz M, Luzzi AA, Rugiero M, Abecasis G, Endelman M, Garcia AM, Capellari S, Xie Z, Lugaresi E, Gambetti P, Dlouhy SR, Ghetti B (2002) Insomnia associated with thalamic involvement in E200K Creutzfeldt-Jakob disease. Neurology 58:362–367 Togo T, Sahara N, Yen SH, Cookson N, Ishizawa T, Hutton M, de Silva R, Lees A, Dickson DW (2002) Argyrophilic grain
57
123
. G @
. " . 6 C
/ . .1.1
tel-00635537, version 1 - 25 Oct 2011
WZ
6 6 1
& 66 6 $ @ &H; E ;G B !;5Q cE!G !! ; ; ! *E&3G ! 7 7 ! $ ! 3 A
0 b @
tel-00635537, version 1 - 25 Oct 2011
7 ) !!3 +!WB+! +! b' 7&C; #&5 C 4*$0- &X E0!> 4 5B 4 ;B ;G ! 7 WB E6 ; VV G 58 2 2 ( @&3E8 G7& C ]C ;" 0' C 7 & 6 3 ^ ];W7] *4
E$!B 'G7 & 6 Z]E4B ;G 5+ E, ;B -'G7 & ; 6 ,;47 & 6 7 & > @ Z ) H 7 & ,;4 ; 6 ! ; ! 66 7 & 6 66 ;" E !!3 !!3G62 *&,' 6 7
Z
7* ' $ !
&66 7 X ; 6 WB B b + 6 6 !!3 + ; &3 E8 G7 1; 6 6 6 6 ! 7 & 6 67& 58 7&
tel-00635537, version 1 - 25 Oct 2011
6B @ 6 7 & ; + 27 7*7 . '
7 77 & 2 @ !Q ; &3 E8 G @5Q cE!G0 E5;G7 ] ; !; (@66 0 > E 0 > G B > 6 ! !; B '
,i;" B )0 6 Q 3 @ 2 !; 7 & 6 @ 6 !!7 i
, ; E
@CG! ;" ! [7) !; 8+ [7)67 ! 4 *77* @
. = ! $ !' 7
Z
7 77 \! 66 ! 6 ,;4 ! E+ VVg V$hB B4;B-'GB &)$ ; ( ;&)$3 !;B &)$ !; ; ; 58 6 6 ; 7 B 58 !!3 6 6 !!3 7 7 77 7$6 58 6 ;
tel-00635537, version 1 - 25 Oct 2011
&582 ! 7[ & 4 5) ]( ! 66 @
) 7 & 58 +E5[7VGC @ 4 5) 7 (76 6 @ ! 2 i 58 6 6 37 Z&58 + (E5W7 G 6E
G !E
;G@ C @ 7 & ! 6 6 E+S 7 dg b h 0'G ! @ 7 & 58 6 6 6 4 7 7 77 7 !!3 &)$58 & !!3 58 66 !! > E'; B' X8 B8 G7& !! > @ -b & 7&58 !
@ [)7
i
)67 2 !; ; ; H! !7
Z
7 7 7 0 0 66 6 D 3 W 7 & ! E y&G , E 7V y B(G 0' @ dE b G 0' @ d E b G 0' @ d E b G 5' @ d E b G ! @ V)7 & ! ,;4 0'+S @ 7dE b G @ 7 dE b G 0' 7 dE b G 5' 7dE b G 0' 7 dE b G76 B Q +V]E';B! B(B-'G;V V VV
tel-00635537, version 1 - 25 Oct 2011
] 6 ! B +W E+! '6BB$>6B-'G;!!3 5+[+ [ 7)2",;4 >*&,' 2 ; + W &)$E,%+*'+! ;B,%+*'+!! + EWGB ,;rB 4 B 0; G7 & 5+[ ; Z @ ] + [ = !!3 W 7 *6B '3 E+! '6B B $>6B -'G @ ` '3! Q; 67" ;B ! ;,;4@ 23 E. > , ! & B ,7B c 4 B B -'G @ b
7 & ! ! E0!;3B$>6B-'G7 7 7 7 c0 & c0 66 ! 7[ i 0" B 0' @ dE b G 0' @ dE b G 0'@ dE b G5'@dE b G !@V)7 & ! 0'
i
)67 2 !; ; ; H! !7
Z
+S @ 7dE b G @ 7 dE b G 0' 7 dE b G5'
7d E b G 0' 7 d E b G7 " ; B ! ; ,;4 @ 23 E. > , ! & B ,7B c 4 B B -'G @ b
7 & ! ! E0!;3B$>6B-'G7
7 7 7V, !! & 2 !; @ !Q c E!G 2 ! 2 ( 2 6
tel-00635537, version 1 - 25 Oct 2011
)062 6 667& E5]G! 66 7& ! Eb
G +7& 66 !; > *1s'3 5$E >B4B >G7
*
*7 ' 5 ' $
' 580V; E 4[B 0B ZW V) GB ; b E 4 G ; + EW]8VGE6;V G7& 0BZW]8V ;" 58!!37 A B 4 ;" 58 !!3 ";" &)$ ( Q 6 "!!3 7 ; b 6 7 & W 6 58 !!3 &)$ Q ( 6 !!37 & 0 0ZW7
ZV
V
]
[
W
Z
58 58 + &)$ ( &)$ ( &)$ &)$
tel-00635537, version 1 - 25 Oct 2011
E FG7 $ 3 ' ' !3 M+ ' !
' 5 '7 &; V 2 +WB +V]B '3 B + [ 5+[ I; 0I; 4[I; ZWI;]V) I;[ 4 I;W W ; Z ]8VI ;" !!3I + I &)$ ( &)$ H! I &)$ &)$ ; 67
*7* !' 8 ' $
*7*7 33 3
& V) B 0B 4[ ZW ; 6 D 6 !; ; !; 66 !E6;VVG7
Z
tel-00635537, version 1 - 25 Oct 2011
0
)
E FF7 ! $ ''#A $ ! ! $ 88' !
'5'' ! ! F +; *; K/( JM7 B0B) ;6 2 ! H! 2V) E ;GB 0E ;0G 4[E ;)GI 3 2 6 ; ZW E 6"!GE 6 74 # G7
*7*7* I
&V) " ]ZB]V > (E6;V G7 " @ V > @ !; 7); 2 ; 2 0 >E G7 A B]8V " ] B V > @ !; H 2 0 > E6; V 0G7 ) ; c0
Z]
0 > 7&W c0 ; 6 7
8
%
)
8
%
)
]Z> ]V> > V >
>
tel-00635537, version 1 - 25 Oct 2011
] > V >
> 0
E FK7 I $ .1E A $ 5A $ ! ! 3$
$" > 3 A $ $ : K '5'' ! ! 3 A F +
,EF7 826 I2 I%2 I) I 0 > I 0 >E ; V) I ;0 ]8VG7
& 0B 4 B 4[ZW " @]ZB]V > ( 0 > 26 6 D 2 E6; V]G7 B 0B 4 ZW " @ [V > (E6;V]B 0*G7 & 0 = ; 58 2 2 E6; V] G7 , ; 7 A B ; 26 B B (@ 0 >7
Z[
8 % ) 8 % )(
8 % ) 8 % )
[V> ]Z> ]V> >
tel-00635537, version 1 - 25 Oct 2011
8 % ) 8 % ) ( 8
0 8
[V> ]Z> ]V>
]Z> ]V> >
>
)
*
E F,7 I $ .1E A $ 5A $ ! ! 3$
$" > 3 A $ $ : K '5'' ! ! 3 A *;
G/G; K/( JM7826 I2 I%2 I) I 0 > I 0 >E ; 0I ;0 4 I ; ) 4[I ; +V]I ;* ZWG7
ZW
V
]
V
]
[
[V> ]Z> ]V>
[V> ]Z> ]V> >
>
>
0
tel-00635537, version 1 - 25 Oct 2011
[V> ]Z> ]V> >
)
E F(7 I $ .1E $ $ $" 3 G+K $" 3$
$ . : '5'' 5 ! 3 A K/(; * JM7 &; 58 2 '
,;" I;V ]582 >I;[58 H! I;V26 I; ! I ; ] I;[26 I ; 4[I ; 0 0I ; ) ZW7
& 4[ " !V$E]Z> ]V> G 58 2 2 6 B ! !H '
, ;" E6; V[ G7 @ ;" @ [V> 2 6 7 B 66 > > H 7& ; @]V> 26 > 4[ ; ; 6 @ > !H A @ ; @ > !H '
,7
ZZ
& 6 c0 0 ZW ]Z# ]V> @[V> 6 D;" 26 ! '
,E6;V[0)G7, ; 7 A B ]V> > 26 >7+ +V]E6;V GB ; ! 0ZWE6; V[0)G7 G
tel-00635537, version 1 - 25 Oct 2011
W ; !!3 WB b 7 ; W 66 6 7 ; + EW ]8VGB V ; E 0B V) B 4[ZWG; b E 4 G" 7& W ]8V 6 587 B ]8V 6 ( A !H 2 2 0 > E6; V 0G7 & V) c0 A !! 7 B =6 !! E6; VVG7 B ]8VB 6c0E6;V G " @ 0 > @ !; (7& B8 (B 7, ; ; 6 7 & 4 ; b 6c0E6;V] 0G7 & 0 ZWB ; 6 !! c0E6;VV0 ) BV] ) *V[0 )G7 B 4 B 0ZW [V> c0 ( 8+ [ 0 ZW7 A B 4[ !! c0 !H ( E6; VV ) V] )G7
B ; 2 6 3 ! $V$E6;V[G7 6 E 0B ZW 4[G ; E 4 G ; b 7 & 0 = 0 c0B 6 ; ; &)$7 * 66B !!3 &)$ ( 7& ; 3*&,'@ = 2 ; !!3W ; ;
tel-00635537, version 1 - 25 Oct 2011
(3 &)$7
. F @
6
. " 66 /0 . .
/ . .1.1 "
6 *G
tel-00635537, version 1 - 25 Oct 2011
6 6 1
7
&&)$ !6 ; ; + B W _V 7 & &)$ ( ; ; !;E + WB _V G7&&)$ 8+ ; 7 + C 5 $!! )
V
tel-00635537, version 1 - 25 Oct 2011
C*-8]&'50 )R
] [[Z [[Z7 & ! E)G E 6 G6 ; E2 G 2 E2]G7 58 4 2 ! 7i 7* 6' $
! +V]E';B! B(B-'G 7) B = + B = ; 4 5 * E&3G7 A B ! 8V; Z@ E';B! B(B-'G Q7) = 7& 8V 62 2 A +V]7& 0B; B ! " 6 &)$7 ) 7j & ZW 4[ 66 6 " 67
i
)67. G 4 7*77*7 83 $ .1E $ 83 # O 5
j
)67. G
7*7 '3 $ !3 A
7 77 62 " 2 Z] 7 ) +V] @ 7 y;b &B 7 y;b &By;b &B y;b & y;b & b E 7 (B 5 Z7]G7 & ! @ [)7 + @ 6 62
tel-00635537, version 1 - 25 Oct 2011
4 @ 7 dE b G 0'E5[7VG +^
@
7 dE b G
E0'G @ d E b G 0' E5 [7VG +^
@ 7 dE b G
& @ dE b G 0'E5[7VG +^
@ 7 d E b G
58 E b
0' + ^
@
7 d g b hG ! @ 7 " ;B 0 @ Eb
0'g5[7Vh +^
@ 7 dg b hG ! @ 7 & E5$ B R3 & B , ;B ) B) 6B-'G !@ @ b
0'E5[7Vh +^
@ 7 dE b G7 " ;B
y& !3 HE+(0B'; B(B-'G C7 " @ @ "B
y& 5 '%VE 7](G C 6 23 7 & E%G 66 +*) 4* E+*)B 4T;b' H ;B!G@ ;( ; 7 7& 66 ; V 7
V
7 77 @ & 6 6 D 3 66 G 0' E5 [7VG G 0' E5 [7VG +^
@ 7 dE b G G 0'E5[7VG +^
@ 7dE b G7
tel-00635537, version 1 - 25 Oct 2011
& @ dE b G
& @ dE b G0'@ dE b G
0'@ d
0'@ dE b G ! E5'G@dE b G
5'@ dE b G
& @ dE b G5'@dE b G
4 @ 7 dE b G
& 3 B 0'B5' ; 7 & 66 0Eb
0'g5[7Vh +^
@ 7 dg b hG@ #3" 7 & % 66 7i 7*7* 6 ! $ $' $ !4*G $ O $ '! $
7 7 7 3" & 62 ! &)$7 &)$E
y&G (B 8+ !@ 7 &)$ ! !; 7 " ;B
y&
i
)677*77 '3 $ ! 3 $ " ! $ ! ! 8 A ! 7
Eb
0'g5[7Vh +^
@ 7 dg b hG ! @ 7 66 0 ZW 4[ 2" 7 & 66 E @ b
0' g5 [7Vh +^
@ 7 dg b hG !@ 7& % 7i 6 " 2 6 &)$7
tel-00635537, version 1 - 25 Oct 2011
7 7 7 3" 5$ & 0 @ 5$7 ! ;" 7 V &)$ ( ; E+ B W_V G6 &)$ ; ; !; ; 7 66 7& " A ! 7i
*
*7 ' ! = 3 # 4!5 $
*77 '3 $ !3 A
&58 @ % +V] y;b & y;b & 0'E5[7VG @ dE b G+^
@ 7 dE b GE6;VWG7
i
)677*77 '3 $ ! 3 $ " ! $ ! ! 8 A ! 7
]
E FM7 ' $ .1E 8 $ $ F, 8 A' !O
J, ! 5 G 7 0' I %
tel-00635537, version 1 - 25 Oct 2011
E ; @ 7 d g b h 0' @ d g b h @ d E b G 0'g5[7Vh ^
@ 7 dg b hG7
666 E6;VZG7& 3 6; 7
E FJ7 '3 $ ! 3 5 " ! $ ! *7 0' I5' ! I0' !! I % E @ dE b GB0'@ dg b hB5'@ dg b hB; @ 7 dg b hG7
[
E K+7 6 ! " ! $ ! *7 0' I 5' ! I % E @ d g b hB0'@ dg b hB5'@ dg b hB0'@ dg b h5'@dg b h
tel-00635537, version 1 - 25 Oct 2011
0'g5[7Vh +^
@ 7 dg b h @ dg b hB0'@ d g b h 5' @ d g b h 0' g5 [7Vh + ^
@ 7d g b hG7
6 66 E; B5' GE8;VZG7& C+^
66 66 7,3 66;6 @ d E b GB 0' @ d E b GB 5' @ d E b GB 0' @ d E b G 5' @ d E b G 0' E5 [7VG + ^
@ 7 d E b G @ d E b G 0' @ dE b G 5'@d E b G 0'E5[7VG +^
@ 7dE b GE6; G7!2 0'@ dE b G5'@dE b G 0'E5[7VG +^
@ 7 dE b G7 *77* 6 ! $ $' $ !4*G $ O $ '! $
&% +V] 8V E6; G7B; &)$ (7 0 ; ; +V] &)$7 A BQ % 7 7 & % &)$ 8V 7 6 6 6 ; B &)$
W
E6; 0G7 & % Q Q &)$ " 7 ; 6 &)$7 66 0BZW 4[E G7
tel-00635537, version 1 - 25 Oct 2011
E K7 ' $ ! ! =' " $ 3 ' *G $ O $
'! $ 5 *7 ( H! I 8+ 6 I ; 6I% EQ &)$ H! B 6 ; 62 +V]B 8V Q BK L@0' I0Q &)$ G7
6 ; 6 B 66 Eb
Bb
Bb
G 66
0'E5[7VG +^
@ 7 dE b G
& @ dE b G 0'E5[7VG +^
@ 7 d E b G
0'@ dE b G5'@dE b G 0'E5[7VG +^
@ 7 dE b G
0'@]dE b G5'@ dE b G 0'E5[7VG +^
@ 7 dE b G
; 6 E G7 &
Z
6 ! @ V) ! 66 ! 7 ; % @V) ;" 6E G7'@;" @V)7 & 0 3 B 6 6% " " ; E G7 & 0' E5 [7VG + ^
@ 7 d E b G A 7 66
tel-00635537, version 1 - 25 Oct 2011
67 & 0 0' E5 [7VG +^
@ 7 dE b G !@V)@ 7 ;" 6 E6; G7 "7
E K*7 # $ " ! $ $' * P F[ ! $ * 7 ( H! I8+ 6 I; 6I% EQ &)$ H! B 6 ; 62 +V]B 8V Q BK L@0'I0Q &)$ G7
&&)$ Eb G 66 3
0'E5[7VG +^
@ 7 dE b G
& @ dE b G 0'E5[7VG +^
@ 7 d E b G
0'@ dE b G5'@dE b G 0'E5[7VG +^
@ 7 dE b G
0'@]dE b G5'@ dE b G 0'E5[7VG +^
@ 7 dE b G
& &)$Eb G 0'E5[7VG +^
@ 7 d E b G ; 6 E6; G7 & %
tel-00635537, version 1 - 25 Oct 2011
&)$ ( +V] %E6; G7B % Q E 8VB b G7 & 6E G7
E KG7 ' $ !4*G $ $ ' %Y*- $ $ . %!1 (7F-
tel-00635537, version 1 - 25 Oct 2011
$ C++ P +7+K\ %5Y5- '5'' ! *7 ( H! I 8+ 6 I; 6I% EQ &)$ H! B 6 ; 62 +V]B 8V Q B K L@0'I0Q &)$ G7
&)$E b bVG ; % ; 6E G7- &)$ 7 6 ; 6 B&)$Eb 0'g5[7Vh +^
@ 7 dg b hG !@V)E6; VG7& ; 6 "; ; +V] E6; V G7 B 8V ; ;6 Q7
E KF7 ' $ !4*G $ $ ' %Y*- $ $ . $
tel-00635537, version 1 - 25 Oct 2011
C++ P +7+K\ #' P F[ '5'' ! *7 ( H! I8+ 6 I ; 6I % E Q &)$ H! B 6 ; 62 +V]B 8V Q B K L@0'I0Q &)$ G7
& ZW 6 ; B % @ 0 E G7& 4[ ; 6 6 D ; B ;" ; 8V E6; G7
tel-00635537, version 1 - 25 Oct 2011
E KK7 ' $ !4*G $ $ ' %Y*- $ $ . $
C++ P +7+K\ #' P F[ '5'' ! K/(7 ( H! I8+ 6 I ; 6I % E Q &)$ H! B 6 ; 62 +V]B 8V Q B K L0'I0Q &)$ G7
6; D 0' E G7 B 6 &)$ ; B&)$ 67 ; 6 2 E G7 A @ 6 Q E6; 0G7 2C 6 8V 7 , ; " @ ! A E3" G7 ! ! A 6 ; 27
V
*7* ' ! = 3 1.
; ! &)$b 0' E5[7VG +^
@ 7 dE b G !@V) @ V) ! Eb
G 0'E5[7VG +^
@ 7 dE b G7 3 66 ! @ 23
tel-00635537, version 1 - 25 Oct 2011
& @ dE b G 0'E5[7VG +^
@ 7 d E b G
0'@ dE b G 5'@dE b G 0'E5[7VG +^
@ 7 dE b G
X &)$ ( &)$ 7 & = ;" 66 0' 5' E6; ] [G7 6 7 6 23 @ 3" E6; ]G7
E K,7 ' $ !4*G $ '5'' ! * 5
7 ( H! EeVGI ; 6Ee GI 0 0'I %
tel-00635537, version 1 - 25 Oct 2011
E K(7 ' $ !4*G $ '5'' ! * 5 P
1 7 ( H! EeVG I ; 6Ee G I 0 0'I% 7
& %6 ! 76 6 6 E; 2 8V GB ; &)$ @ V) 66 [ !@V)7 6 ; 26 6 B ;" ; ; % E G7
]
6 &)$B " &)$ [ ! !@V) @V) ! Eb
G 0'E5[7VG +^
@ 7 dE b G7 &Q&)$ " % 8V &)$ (E6; WG7 6 66 ; 2 &)$ 8V 7
tel-00635537, version 1 - 25 Oct 2011
E KM7 ' $ !4*G $ $'!' ! '5'' ! *7 ( H! I; 6I0 0'I% 7
66 B 8V B 8 B V ] E6; ZG7* Q &)$ 58 E b
0' g5 [7Vh +^
@ 7 dg b hG7&+V] 58 " ; 6 7 ; +V] &)$( 67& ; @ B 6 ! B ; &)$ 07 & 8 8V7 , 3 66 %&)$ (7
[
!"!#
E KJ7 ' $ !4*G $ $'!' ! '5'' ! *7 ( H! I; 6I% 7
tel-00635537, version 1 - 25 Oct 2011
66 B 66 A 8 @ 23 Z V E$ B&H;GE6;] G7& +V] ; 8 B A 8 "6 ; 8 7 2 ; 2 @ S! 8Vb 8 B 66 &)$ 7
!"!#
E ,+7 ' $ !' . $ $'!' ! '5'' ! KEK7
( H! I; 6I% 7
W
G
6 @ " ! 6 D ; 2 6 7 & ! E @ 5$G E G2A ; 6 7 A B &)$ @ V) B Q &)$ ; 6 ; 6 7) ;E] !G 6 ; &)$7 B @ ; 66 7
tel-00635537, version 1 - 25 Oct 2011
@ 66 (7 ' !; B !!3 =" 3H! 7 V B
[Z
' ; &)$ A ;
(!HC ( (),7,2 ! 66 7 ) &)$ &)$ ( E
;b & C @
;b &G 6 WE6@ ;b &G7 ][B
[VB W B
,
!; ; &)$7 B B; M " 6 ; 6 7 X B ; 66 ( ; W d 6 Z[d7 ) " 6 6 D ; ; &)$7 6 66 ( )&B A 2 2 6 !;
Z
7 ][B [ * ! 6 " 6 (), ( C 7 W 6 ! *&,' ; &)$B ! &)$ ( 7 & (!" ; E + B W _V &)$G" 7, ! ; (7 W ) ! ( @ ;(),6 6 ( 7
tel-00635537, version 1 - 25 Oct 2011
) @ " 66 7 ) A 2 6 ! 7 W B B ; 6 ; E 2 G @ +V]7 6 D B " 6 ; 0 ; E6; ZG7 6 &)$ ( E6;] G ; @ 7 & 8 ; 8V ; " 6 8 7 & 62 @ E 8V Z@ I 8 V @] I 8 Z@V G"! 8 8 7& 2 ;A 7); =6 7 B ; 0 7& &)$
;b &7V ] ) ! ; &)$ A 6 % ; 7) B; B " 62 2 8V 8 7! ;
7 ) ; A 2 6 0 ! 07 0 E &)$G c0 0E6;V V]G 6 7 & !; 2 !! " 2 6 7[ " " B E8 G A A 2 E6; ]G7 B !;; ! *E&3GB
tel-00635537, version 1 - 25 Oct 2011
Z ().7& ;6 [ ().B 8+B 2 E)&0 >](0 > G6 7 [ ().2 8 7* !B 8 8VE; G ;6 26 !H ().6 E6;]G7 , A ;!H 2E6;]G 8+E6;]0G7) ; !H 7 , ! ; ; 3 ; 6 (7 V ; 8 8V 7 8@E6; ]G ;! " ;6 ;; 27, B#B ; 6 6 2 7 & ## &)$B !! & B 6 !3 !; = 6 !3!"7
tel-00635537, version 1 - 25 Oct 2011
E ,7 ! $ ''#A # $ ' ! O 3O' !
$ ! 4. 33 3 7 E6"!G 26 2E H! @ 0 >
@ &S3 @ 0 > ]G 8 I 0 ; 6 E6"! G 6 8 I ) ; 6 E6"! G 6 +W; !!3I ;6 E6"!G 8V 26 ().E 6 7 ! '! ;G7
..
tel-00635537, version 1 - 25 Oct 2011
& &)$ ; ; ( H! 6 ;66 (7 & 6 !; ; ; 6 ; 3 7 VV & 66 B ;B ; 3!; @ 6 ;667 B 66 ; &)$ ; 66 ( E+ B WȕV G7)6 @2 7 ZB ]B [
tel-00635537, version 1 - 25 Oct 2011
B ȕV C ;66 ( Q6 6 ; 7) ;@ 3:BȕV 7 & ; ȕV ; ;6 6 6 7*66B ȕV bȕV 2 + W 6 ; 7 ; ;6 B W7d @ Z]d ȕV bȕV ; + W ;667 A 6 "; (2 !; 8+ WW7 d@Z 7 dB )0 ]7 d@W 7[dB (). WV7]d ; @
d ȕV bȕV 7' 6 ( )& ;" !B 6 W 7Wd 7 )6; ! A ;E+ B W ȕV bȕV G 3 6 6 ; ( @ 6 7 & ; + W 6 ; 6 ; ȕV 7 B ;6
V
; K 3 LB.+B'S$B 73! 3 3S!! 36 H! 3'+B) ;.&B 7 ;6 E SG7$6!X 3' ' 6! 3 6;37;3
I ]V 7 Z7 {3! { 7 ; ' ( 6( BV! 7c !;)IZZ 7 7 8(8B8'*B(5;!$7|( |7 !6; ; !; 6 6! 7.3! $Z[ I WZ ZW7 7 (#! 4B ! B8(B# H $BB' *(7) ; 6H! 8H! (B( &B' H(B#>;!'7( ; ;;6H! RB0 ;cB07(2 !; 6 3 S;7;3
[I]Z Z[ V7 W7 'S 4*B) )B(0&B 7 6 6 6 ; !;37;3ZZWI ZW] Z 7 Z7 &;!c+B.7B( +BB 7) 6S! 6; ; ;;6
37+!) 5 !'37 '>ZZ]I [ [V W 7
7 44B4 >B5 8$B0 )B# *7,6 6H! !!6 2 70
[I
W
]7
7 c65B*>4(B0'B!.B4H5.7S! ! ; ! 6 S! ; N; 37,.4 3! 3
I W 7
7 $ *(B)!#B2 4*B 78 3; ;>6 H! c7; S!2 !;3 3 S7ZZ]IV Z VW7 VZ7 /! (7) !;3 !! 36 6 !7 !;3
]I ]V [ [ 7 7 0;&B(#cB.7B(.B 7) !;; 3! !3 ;; H! 3B ;B2B *;37!ZZWI ] 7 7 ; 1B4S.5B53 c!3*+B53 0+76 3 ; 36H! 5B0 >*7' ;;6H! RB&; '*B07+! !;36 H! ; 7
ZI]]
W7 W7 'SB4&5B( .B$3#B4B( > 3c$70 6 ! 26H! 7+!'37. ZZ[I [[W [7 Z7 !; 6 B 3 *; c 7 !;346!( $ !)); 8 ;;'3E($))8'G7&
I []Z [ 7 ] 7 .;#B .7 !; 6 2 7.'
[I [W [7 ]7 R>3B3> )B0 .B 7+! 2 !S;!66 37 ;;
I V Z7 ] 7 >>&B# +B +B.(B 6H66,7! 3 !;3 2 3 3 7
I [W Z7 ] 7 )&#B0 .(B(3H>#B+C S>.XB& 8 8(7'3; , S B B ! 3 6 !;3 ; 7. I
[ W Z7 ]V7 # 3 (&B& *0B+ ;cB 7); 66 S! 3 6B&S3 3 !;3B !7;3
I]V ]Z [ 7 ] 7 !&B >>&B +B 6H66,7'3 ; !! 3 6 !;3 ; C7 4 );
WI V 7 ]]7 5 &B' B4 >B 7+!&S3 3 6H! *B/ $B 7)6+ V B ! 3 !;3 6H! 4#B&6'B4 *7*66 3 636; S! H! $B5(7+!;66 ; 7) $
[I[V V V 7
]
tel-00635537, version 1 - 25 Oct 2011
ZW7 8 ('B8 .B.!.#B 78 !; 7
]I ZZ ] 7 ZZ7 #HB(( ;B0 (B cB(H47+! !;36 6 70
I WZZ]
7
7 #HB( H & ;B cB(H47+! ; 3 ; ! 6 7;3
I ]W [ 7 7 .!#B$)B# 'B 7) !; 366 ; '7;3
]I]]V W7 7 ) %B .*B4 )B0 08B>cB.!#78 >; S!&S3 7);0!
WI [ ] 7 7 *; ;!'B1 ;!*B( #B( 0B37* 6 S!)'851 5, ;; 6 H! 8B$ >#B 7); 3! 6 3; ! 7
VI
V]7 W7 ( 3).B #B).B! .B8 >S >$'B5;.$7 2 ! 336 ! S !3 37
IV[ ] V 7 Z7 # .B^ .5B #B5;.$7) !; ! H 6 ; ! 7
]I Z ] ] 7 7 #H7>) 2 ; !6 3 ; ! B ; B ; 37;
IZ [7 7 1B& 8B !30B 76 !;3 3; ; ; !7;3 I[VV Z7 7 ( (Bc>B.!B 7H! 6 !;3 6 3; ! 7
WI] [ Z Z7 7 08B $.B'307 !37!ZWWIV [WZ Z 7 V7 (( ;B;8B0/B#H7+!; 66 !37;3
]I]]
W7 7 + ; c 8B4 66 $ 6$B0 08B 7) B;B !; ! 6 !37;3
VI] ]W [V7 ]7 $.B0.(B5)*B(#cB.7B' (B(.)7; 36 !;7;3
VI] [ W 7 [7 c!S.&B. >)$B.7B# #B 7, ;; 6 !37 ;;
[I W ]7 W7 8 ;(B)B';.B 7, 6 3 !3 S!*+ )'86;7. 7 Z7 166.B$4cB08B13(B1517 !37 !; 7!ZZVI ]Z [V7 7 + ; c 8B.!#B0 08B>cB .*B$)7 !; 3 6 ; ;S! 367;3
I] V 7 7 + ; c 8B.!#B0 08B$)B .*B>c7)2&S3 3 6| 6H! #B 7);6! !; ;6 S!&S3 E&0G6! &0 S>!7;3ZZ]IV[ V7 ]7 0 >5B+#B$ -B1$B. '*B0 >*7' ;;6 !;3 > 3c$B.! 4B&5B'! 87&S3 3 !;3 3 C7. !*2
ZI]WW] 7 W7 ($B5 &B.1B 7,66H! !;3 ; 3 S!&S3 7;3
I] W] Z 7 Z7 +T,73!; ! 6 S!&S3 >73!$
WIW Z7 V 7 5;!.B '*B#6&B&.7 36 ;6 ! >cB0 >5B .*B 7 !; 6 > .B 7 !3 6V S! ; 6 2 7.;3! 3 ZZWI]VWV Z7 V]7 $.%B&('B+! B( )7) ; 7 3 6 ] 70 ZZVI[E GW Z]7 V[7 0 08B( ; (B .*B 7 !;!;3 3 ; ; 7;3ZZZI [Z W
7
]
tel-00635537, version 1 - 25 Oct 2011
VW7 0 08B& ;*B& ,7) ; !; 3 6 7
I V' ' Z7 VZ7 ' (B4(B487( ; ;;)0B 3 > B 7
IW Z[ W7 7 >cB0;)B)!''B 7%666$ !; 6 ; 7. !*2
I]Z V]7 7 !-B0 '! /B+! $4B&.B0.7) 6 !3 6; 3 !37;3
I] Z ]7 7 c $B&.7; 3 !; ;! ;7&
ZIW [ Z7 7 & ,B5 S..B0 >..B 71 3 36! 3,' !; 6; 3 7. !*2 ZZ]I Z[ 7 V7 c5%74 B ; ! 36! ;6 ! !7$6 c5%) 7c5%EGBc5 !%; H B4 ZZW7 7 & ; B(B)*B 7( 36 )H6 . > *B B ) 7;3
I]V W] Z7 ]7 c S!.B);.7- ! 70! 0!3
[I[[ ZW ] Z7 [7 # 44B(B& ; B 74 !*-$%).27 5 4
IW]] [V7 W7 ( '7 ;7*.5 4
]IV [ W7 Z7 );.Bc!6.B(#!*B 7#! 3 ! S! 3; 7&
]I ][ ]W [V7 ] 7 0 .B' B) >,B1 1B !7g* ; 6 )H6 . > 8 h7$ E G
ZI] ]WV Z 7 ]7 )! H4B0*B& )B0 +B;HHB#7S 6)H6 . > ] 3 8! 7& ZZ]I V[W7 ] 7 c$4B,.cBR(B 7S 6)H6 . > !-#7 & ZZ]I V[Z 7 ] 7 );.B'#)B( .B,.B587( 36 ! ;36#B+! 5.B8> (B'7( ; ;;! ;6)H6 . > 7!
I [[ [7 ]W7 0> 5B;HHB0SB 7 !; ; 6)H6 . > E).G !! ;6 ! !E G70 ! ZZ I V Z ]]7 ]Z7 # 44B(B'!!(B0> 57' H 6 !! 7 . !
I]] W[ ZV7 [ 7 5 6.Bc !H.B.;#B& >B4 8B0> 57)26 H! 3 !;3)H6 . > 7 !ZZWIZ]] 7 [7 +!3 #B/ ;! 'B,> #B 7)26). H! S!! *
# ! H 6 2 !37 ! I Z [7
]]
tel-00635537, version 1 - 25 Oct 2011
[V7 .&B(.)7+ ; ;; | |H! $.B+ ; *4B#> *7(; ! H 7!ZZZI ]
W7 []7 )>+B0 ! 5B' >37; 3 ; ;7,.ZW[I [ W 7 [[7 & 3$7;; ; 7c>; 36!, 3!; S!!c5 !%; H 7,3!; ZZVI]] W7 [W7 * 3*(B5; 0B ! ,(7); ! 37$ 6 !) '365 ! ;;7!ZZ I ] Z7 [Z7 $)B(.)7(; 3 ;7 !
I] ] I[7 W 7 ) ; '! $B8 ; (B5H (70 >6H! SH.B$ 8B 8B' &7 36! ! S ;;; ; 3 7.(0
VI V V 7 ZZ7 4 +)B)!8BR B 7) ;6 >; 3 6 3 ;H! H(B0 4B+S07 ; !6H! H! 31Bc'B4 H> 3 %1B#&B& 4753!!3 6 6 78*0'&ZZWIV Z 7 7 BR +B >(B4> , ,B, #753!!3 6 36 ;6 ! 6 b ;!6 7 '-'
IZW]Z W7 7 )B4> , ,B, #7H! )(B5 ;)$B& (H.B( $7 6) 3 S! 36!6 !S 3 3 ; 66 3 !;3H! )(B >(B+!;5)B 7, 6 6 ; 6 6 ! 6! ! 6 6H! 7 '-'ZWWIW V ] 7
7 ( $B*S )B5 ;)$B(> & > *0Bc!>)(7' ;;! !; 36 ! 6 H! S*B( >S*(7!!3 ! ! 6 E' ] B' VG ; ;;; H! ! 6 70! 3ZZZI W VZ W7 7 - ; 1B.&78 666 6 ; ;; 3H! ! 3;6 6 H! !;37 !
WI V] [ 7 V 7 5> (BR> B(! RB#B >(7; 3 26! ; ! ; 70 $
[I
] 7 V 7 & (H.B)! H ( &B4 ' 8B( $7* ' ;6+ )6 )! ; $ ! 6 ' 6'6!! + *H! (B! 6'B>B&S ;!)7+!6 ! ;; B> B ! 7/ '
IZ ZZ [7 VW7 ( ; $;H'B0 , 4B' ( ,B 7) ; 6 ! ;6 6S!!3 ;H! cB)3 5B( &B 7,6 6 !; ;; 3 3! 7 ;;ZZ I [Z WZ7 7 &8B&0B+;*.B4> , ,B, #B4;)^7' 6666 !!3 3 3 6 ;;; 7*.
[I ] V Z ]7 7 0;(B8!66B0 .B5 .B( >S*(B( >S*7 36 H! ! 6 6 EE
]G1X,1/#E GG6 ; 7 '-'
IZ[ Z V7 7 8!66B 0;(B( >S*(B B( >S*7 3 ! 6H! ! 6 7 '-'ZZWIZ [ [7 7 ( H $(B& &#,B) 1B 5 H(B( &H $7+ ; ;;; H! $7( ;; !70! 0!3
I[ Z V 7
[
tel-00635537, version 1 - 25 Oct 2011
[ 7 >3(B&1(B+C S>.X7; S6 !; !! 7$ !
]I [ 7 [ 7 #> H(B)((B+S07,6 ! H 6>3> 3 6 ;7.(0
I Z7 [V7 'H>B)!;++B) ^B 7 ;6; 3 36 3 E [[G 7'ZZVI ]V
] V 7 [ 7 '!B*> )B.(B 7' 3 ! ! 6H! 6 H! B'H>B(H S 5B> B,! /71 H 6 V EV G V S! 6 ! 3 V EV G7ZZVI V 7 [[7 4 'B5&B*> )0B 7 3 E GH! (47'6 6 ; 37'ZZI [Z W 7 [Z7 '; B0 'B4! B 7+ 3 H! &B!#57 ; S! 6; 3 ! 367
WI [V Z7 W 7 1 ;1&B8 + (+B>#*B) $B( )&B$S))7+!$+ 6 ;;! 6H! *0B(#!,47 !!3;363 ;;; &S3 3 7(!;;
]I [WW 7 Z 7 ,S 76)b ! 3 H! $B) .B& &B4(78 ! 3> 3 !3S! >6 6 ! 3 7.'0
I
Z7 ZW7 * ; 6%(B, 407;;; 236 ! 3 70! '+
I
Z ] 7 ZZ7 03#B& .,B) )B 766 26 ! 36 S!&S3 7 !
VI
] [7
7 8CS 5B5 ; S (B! B 7 ! '3!!3 3 !37 )0
IV] V7
7 4 0,B .*B( 3,1B 7%2 ;>; 3 ! 3 3 !37'
I Z ZW Z7
7 +6 4#B$ HH B4!0B&3#'B' (47- 6 ! 3 &S3 !; S! 6 67.0 )!
I [WVVV 7
7 5 7) ;36 7)( $ ZZZI V Z VV7
V7 %!0Bc S 3Bc !(B 7 ; [
7 )ZW IV [ V]7
7 &H4 8BR !$B$>$Bc!!#7($6! 7 '-'
IZ[W
V Z7
]7 0)'B66 '*B! )(B 7( 6 6! ; ! 6!70! 3
IV ZZ V
7
[7 ) .'B'S )>'%B% 4Bc>#B'! 66.Bc! '+7 S6 ;3 !6 6 ;!>6 6 )H6 . > 7+ 6($
]I Z ]7
W7 '077 '-'ZZWIZ
] W 7
Z7 )! 0B$ $Bc!3#B 7,6 6 6 $ 6 6 7 ZW I
7
7 )'.B' !H . B( )&B 7 6 ; ; ! 6 )H6 . > 70
]I Z [W W[7
7 # >B( 'B53 5B 71 ). !H3;6$ Z7&
ZI [V W7
7 ) ;!30B0 4'7 ! 7
]IVV W 7
7 4 H(B'>#B';5B&!+B;HH75 7!
I] V 7
V7 ;HHB3 (7( ;336 ;7)
VI] [7
7 #5B &4Bc ;/+B'!)#B/ 5'7' ;!+ V ; 7$
ZI [[ZZ W W7
]7 ! B+! H317>; 3 ;6 2 !6 ; 7)+$
IV [7
[7 # ! B45BC 7+ ; 5;; 3 ; H! ;B ; B ; ; 7! ,
IV ]Z [W7
7 ((&B.!417+ ; >;6! 7.! ZZ I] [] [ 7
7 ,> #B+ > ! #B' >$7) >;6 3! ;!E WG6! H! bV 3 3 ; 78*0'&ZZ I ] Z 7
7 .5B'*'B+*B4 .B$ $!3 66! ! 36 ; B)B ; 3 >;H! 3 V70! .ZZ I EGZ V7
7 R (%B# '/B5 $B+.)B&.(B( 7+ ; 3BB $2 ; 37. ! *2
I] [ WV7
V7 RB 0B'(B1 0 .B8&7) >;6 B 5' [B >B ! 3 3; ; 3 3 H! >S*(B( >S*7+ ;;; ! 7! ;3 7
7 %#B5! (B/ (7! 3 3 ! ;6 >4$76 ;6 ! 36 H! 7)%,
ZI Z ] 7
V7 &S;5BR )B1 #B>BR,Bc'7+! !6 7*2$ ,6+!
[I ]
7
7 + +7, H ! 36H! ! S6 H ;7+!+!>C 62 I Z ]7
]7 ' 0B8>7, ! 36H! 8 0; ( >6 !6)'8 3! 3 66 ;6 7c.03! 3
I]]Z WV7
V 7 0S#Bc B5 ;%7&S6 36 ! ! 7 ' ZZ IWW 7
[
tel-00635537, version 1 - 25 Oct 2011
V 7 '! 'B 8c(B0 >(B 7)'8 ($, > 3 ! ;6H! 3 6 7 ;;
WI ZV Z7
V 7 &B& B' 4B' ;;*B$ 7 ;; H! ;6 *7*. I[Z ]7
V[7 B(!&B B 7* 6)'8 )'8 V ; >6H! 7!
I W [ Z7
VW7 $ !(B0!#B'! >(B#HB4c47) 3H! 6 3 6 ;N!
I] ]Z] [ 7
7 %(Bc6 ;.B+ 5B 7* 6 66 S!)H6 . > 7&ZZ[I 7
]7 1 * >0BX'B0.B( ..B) 7 36)'8 > S! )H6 . > 7.;3! 3
I[V V7
[7 5)B$;&B1 !*B 7) 6 >6H! 7.H!
I ZZ ]7
W7 5 5B+'.B8! ;+B 71 6)'8 3 V 6H! #Bc #B 5B 7 ;;6 6 66 S! ; ; 37.'
IW Z W7
]]7 00B4 8B &B 7 6+ 6 )'8 ; 37 ;;
ZI
V V 7
[V
tel-00635537, version 1 - 25 Oct 2011
][7 5 5B0;#BR>S>$B 7( 6!!3 ! 66 ;6H! 637!4 3! 3
VI]Z 7
]W7 1 !*B1 !5B B 7X 6 6 !!3 !W! 6 S!*&,'S! 3!!!6 H 7&
I W VZ 7
]Z7 &B& B 'B4 1B1 !*B5 87)'8!!3 >6 ;H! #B 7& ; B 36 6 !!3 ZZ6! ;6H! S>$B+'.B 766 ;6H! S! 6 6 !!3 ! 7!
I Z ][ [ 7
[ 7 B1 !*B1 !5B B0S#7) 6 6 B!! V 6H! > '#B5 > (B'5B +7)'8 V !!3 6; ; 7;3
I]V ZV [7
[V7 0;#B+'.BR>S>$B 7)'8 !!3 ! S!; (), C7;3
I Z] [ Z7
[ 7 c #B0S#B B(!&7)'8 >6H! 6 3 H! 7;3
VI] W V7
[Z7 ; >.)B'!B( >S*(B53 0+7'6 !!3 S! 36 3 !;3H! ; 7 ;;
ZI
]W Z 7
W 7 5 5B0;#B#!>$B 7+ >;6H! 7
I V ] [7
Z 7 0S#7) 6 >6H! H! !66;6; 7.'
]I V W7
Z 7 4 >B)! ;&B($B 75;! 6 S 3 V ! ;6H! *;37! ZZWI Z [ V 7
Z]7 !>(B$B5 (B$76; 3 ;;; ! 66H! 5B+> +B# +B 75;! S;! 3; 66H! 78 . I V [] ]7
ZW7 c6 ;.B* 5B0 (B 75;!3 6 6 23 3 [b Wb Z V bV H! B* 5B(3(B 7+! 3 E G 6H! 8B$ ;$7 3; >6H! 6 !37&
]I ] ] 7 V W7 '}}++B5>> '#B5}+B 7 V V >6 ; ! ;6S B; B !37. ;3! 3 IW [7 V Z7 4(*B,H 3()B' !**B 7 3 S! B (),B 3 ; !37;3
]I]] Z7 V 7 &SH>B* 5B0 (B 7*! 6 3 7*!
VI
] V 7
[]
tel-00635537, version 1 - 25 Oct 2011
V7 $ ;! $B#C66&B' ;(B$;00B5 >)&Bc!)&B 7! 32! ;! 7 !
VI[ ] ]7 V 7 0;!$B( !$B;B 78;! ! 3 66 > +B' 'BB 7 ! 3 66 ; CS! > >+B,!> S B 766 6 ! 3B 3V )'8 S S! S!&S3 H! 6 > +B/! (B 7)'8 ! 3 S!&S3 H! 63 N&
ZIV
7 V 7 (3$#B(#3(B0S #B0 6(0B0 3$B'07' 6 7 '-'ZW]IW V7 V V7 4 .B) )B8 /B 7'6 6! # 6 6!;S 37 ! ;60'*7!1'
Z[ 7 V 7 5 >'B8 !20B' H !1B 7+!3 ! 3 )H6 . > 7 (
IZ 7 V ]7 58B0S!$.B.'B 7, ; 6 )H6 . > !! S! 3 7& ZZZI W Z7 V [7 5B' >.B' !(*B 7'6363 ! 6 6 )H6 . > 7.) !
VI [
7 V W7 c S!.B.'B58B 7+ 6 )H6 . > ; !;!3 ; 37&
I W[ W 7 V Z7 8 2BX ,B1 )B 7 !; ;!! 3 6 S! )H6 . > 7!
VI][V[ 7 V
7 4 H(B *B( (B;HH7*2 !; )H6 . > 7*;.(
I VZW 7 V 7 R 4B8 'B) 8B 76 !;!6 3 ! )H6 . > 7*;.(
I VW[ Z7 V 7 + ; 8B8B(B' (B0; %B8 ;07 6 6 ! 6 6 ! !70! 0!3$) ZZ IWV ZW V V7
[[
tel-00635537, version 1 - 25 Oct 2011
V
7 0!>.B4B'!H '! 66cB 7- 6 !; ;;; 3 ;636 ;7 '-'
IZ[ V]W [ 7 V V7 c;0'B4.B&$B 7 6 ! 66)H6 . > 7. !
IZVZ V7 V 7 %)Bc! .(B5;!4B 75 )H6 . > ! EG; 6 7*; '
ZI 7 V ]7 (38B4>'8B)H3>0B 7+ ! 6 S! ; 7.H!
ZI[W] [ 7 V [7 $ +#B*;*B(;'B) .*BR>()B0 .c7)B! 6 6!! B >65,1 ; 6 7 . ! I[[WVW ] 7 V W7 &S3)B5S*B#;!$'B 7 6 )H6 . > 3 67&
VI ] V[ 7 V Z7 578! 6 ).6 S! 675 ! ;3B-#;
[I 7 VV 7 5S*B&S3)B( >H.Bc$47)H6 . > 66!-#+ 6(* ; $ S3712' ;
]IZ
7 VV7 + (5B.'B 'B#! '! HB);.B. >4'7!;!3 36!6 6 S!! S!!6 #7+ 6 I ]Z [7 VV 7 '! >4(B'! >/B# , RB5 (B ! ,B4 H$7 6 6 ! 66S! 7.0 )!
I [] V[Z W 7 VV 7 8> S 5B! #B%>S >$B 76 ;6! ;6 7) S! 7.0)!
VI [Z ]] [7 VVV7 ;! $BR,B5 -BR 476 # !6 S!)H6 . > !; 7
WI [ W7 VV 7 4H H $ B0 (B&B( $B')766 78*0'&
WI W ] ]7 VV]7 ( ! )#BS.4B ! '.B 7,6! 6 S!!S ; 7'
]I V
]7 VV[7 c #B0S#BR ;5B&*B(!&B5 %7)'8 > ; H! 7;3 I[V [7 VVW7 0 +B3 5B' ;(B 7)'8 + )'8 E V G ) !37 4 ); I Z
7 VVZ7 ';.B8 ;(B & B 7) 8 > !37;37 V 7 4 H + *B4H ,B8 C'B 7) 6 > S! S3 S!H! 7!
I] W 7 V 7 # >*B > 4BR ,BR )7)'8 3E V G H! 7*.
I Z W7 V 7 0 5B1 'S.)B&;!'B 7)'8 >6 ; S!>S !;37;3
WI[ W [ 7 V 7 8 ; (B$)(B^;)B((B(.)B5H (7) 6 b 3EV G 6; 7!
[I]V V Z7 V V7 'H &)B& 8B0 'B 7) 6 >!66 ;6H! 6 ! 7(3! 3
IW V [7 V [7 0;#B%(B+'.B 7 S)'8 !!3 ! )H6 . > 7 ;;
]I [ 7 V W7 4 )B5 (cB* ;B,.cB#;!$'B4.7$ )'8 !! )H6 . > ; !; 7.;3! 3
]I[[WZ Z7 V Z7 &(.B' 'BR>S>$B 7($, )'8! 3 ;6 H! S>$B 7&; )'8 ($, > ! ;6 ; 7 ;;
]I [ ZV V 7 V]7 5 5B0;#B.)B 7) 6 6 6 !!3 ! S! 6! !3H! 7!
I] [[ 7 V] 7 c !&%B0S#7) 6 >6 ; 3 ; 3 7&
I
ZZZ 7 V] 7 C ;/B'! 'B0 >!68B 7)'8 >6 ; S! ! B *;3B ;;7. ;3! 3
]I[[ V] W7 V]V7 %> B 5B5;!(B 7) 6 6 3 V B ! S! ; ! 7& ZZZI [ [7 V] 7 '! $B'! 5B) .B( >#Bc!&$B& &.7* 36 3 6S 6!5 ;;'37
I ]W [V7 V]]7 4 B'! B'!B 7, ! ! > H! 6 ; 7;3
]I]]V] [7 V][7 4 B'! B'!B 7, ! ! >3! ; H! 7!
]I] W V 7 V]W7 ' 3!(B) 5B+.B 7 6) 66 ;H! S! 7 ;;ZZVI ] Z V7 V]Z7 &&8Bc >4B$;.7(; ; H! 7, ) 88E G , S!;; & H! 7 ( I [ V 7 V[V7 ( 3 (B5;!(B+ > >/B 7) 63 H! 6H! 7;3
WI[ W7
[Z
tel-00635537, version 1 - 25 Oct 2011
V[Z7 ;> 1+B; >4B+ >(B# >4B# >1#76 6 ; H! 6 6 ;'*&, +%87*2
IZ] [ W7 VW7 R!;RB*S(B+'B 7& 6 E0)*G 6 6> ; 7!43! 3
[I]V[W ]7 VW 7 *S(BR!;RB0;#B 7, )'8 0)* 3 S!* V;3 CS! ; H! !4B 7+! ;6636 > )H6 . > H! 6 ;6 ! !7*;.( ZZ]I
Z V
7 VWW7 1 * >0$B0.B) 7V m; n 6 ;! )H6 . > 6 ! 7.;3! 3
I[]
7 VWZ7 )!! 4B;)B( >H.(B 7+!6 6V !! ;6 )H6 . > !-# 3 S7. ;3! 3 7 VZ 7 4 #B5 -B0 (B 7V )'8 )H6 . > 66 37 ;;
ZI
WV 7 VZ7 0SB(B0 .B 7, ;)H6 . > ! 7 ;3
I [ W7 VZ 7 ;! '!! (B5 ;!+B# +B 7) 6 ;6 ; 6 )H6. > 7;3
[I]Z ] [7 VZ 7 %(B&SH>Bc6 ;.7! !6 6 ; ; 7(!
WIVV WZ ZW7 VZV7 H B, >2 0B8,B 7 $/ W 4 ' '!> S!6 3; 7. !*2 I]Z[WZ W
7 VZ 7 c > 3 !#B(8B5 ; S (B 7) H 6 !!3 3 S! ; 7 !;3
]I ]]] [7 VZ]7 1 >((B# 0B$>>(%B1 ;5B'>! (*B4 ;'(7 ) 6 3 EV G 3 ; !37
ZI]] V Z7 VZ[7 ;!(B/! (B( /B%#B,S #B/ (7 3 V 66 S!; 3 ; 7.'
I [] 7 VZW7 00B( (B4 8B 7+ 6 )'8 >6 ; 37;3
WI[[Z] W 7 VZZ7 %> H >#B8/.B!! /B 7H! 6H! 3 !;! ;! 7!
ZI]] W Z7 [7 ( BR ;5B5 %B 7)'8 > H! S! ; 7.
ZI
W Z 7
W
________________________________________________________________________________________________________________________________
RÉSUMÉ Le diagnostic de la maladie d’Alzheimer (MA) est tardif et présente un manque de fiabilité en regard de l’examen neuropathologique postmortem permettant de confirmer ce diagnostic. En effet, les présentations cliniques de la MA peuvent être multiples et parfois atypiques. Des anomalies dans les concentrations des protéines tau, tau phosphorylées et amyloïdes bêta, au sein du liquide céphalorachidien (LCR), ont permis d’améliorer le diagnostic du vivant du patient. Nous avons évalué la performance de ces marqueurs, dans le LCR, utilisés pour le diagnostic de la MA dans les formes syndromiques atypiques. L’utilisation de ces marqueurs augmente la précision du diagnostic lors de ces différentes présentations cliniques. De plus, nous avons mis au point un test diagnostic biochimique postmortem des différentes tauopathies permettant de mieux les caractériser en complément de l’examen neuropathologique. Enfin, nous avons conçu et caractérisé des anticorps spécifiquement dirigés contre la protéine tau phosphorylée en position 231. Cet outil nous a permis de développer un test ELISA dans le LCR. Des résultats préliminaires suggéreraient une interaction in vivo entre les protéines tau et Prion. Ces résultats, décrits pour la première fois, sont corrélés à nos observations histologiques.
tel-00635537, version 1 - 25 Oct 2011
_________________________________________________________________________________________________________________________________
TITLE Biological tools contribution for characterising several tauopathies with regard to various clinical presentations of neurodegenerative disorders _________________________________________________________________________________________________________________________________
ABSTRACT Diagnosis of Alzheimer’s disease (AD) is late with a lack of reliability with regard to postmortem neuropathological examination that permits to confirm this diagnosis. Indeed, many clinical presentations of AD can occur and sometimes atypical. Anomalies in cerebrospinal fluid (CSF) levels of tau, phosphorylated tau and amyloid beta proteins permitted to improve antemortem diagnosis. We evaluated biomarkers performance, into CSF, used for AD diagnosis in syndromal atypical forms. The use of these biomarkers increases the accuracy of diagnosis during these different clinical presentations. Moreover, we adjusted a biochemical postmortem diagnosis test of tauopathies giving the interest to better characterize them in addition to neuropathological examination. Finally, we developed and characterized antibodies specifically directed against phosphorylated tau protein on 231 epitope. This tool permitted to make an ELISA test in CSF. Preliminary results may suggest an in vivo interation between tau and Prion proteins. These results, described for the first time, correlated with our histological observations. _________________________________________________________________________________________________________________________________
DISCIPLINE Neurosciences _________________________________________________________________________________________________________________________________
MOTS-CLÉS Démences neurodégénératives, Alzheimer, liquide céphalorachidien, Creutzfeldt-Jakob, diagnostic, protéine tau, Prions, amyloïde bêta _________________________________________________________________________________________________________________________________
INTITULÉ ET ADRESSE DE L'U.F.R. OU DU LABORATOIRE Laboratoire de Neurobiologie Hospices Civils de Lyon 59 boulevard Pinel 69677 Bron Cedex
Unité 402 Hôpital neurologique 59 boulevard Pinel 69677 Bron Cedex
INSERM U1028 Centre Hospitalier Le Vinatier 95 boulevard Pinel 69500 Bron
![arxiv: v1 [q-fin.tr] 7 Oct 2011](https://kipdf.com/img/300x300/arxiv-v1-q-fintr-7-oct-2011_5ad3433a7f8b9a059f8b456c.jpg)
![arxiv: v2 [math.at] 7 Oct 2011](https://kipdf.com/img/300x300/arxiv-v2-mathat-7-oct-2011_5b193f847f8b9a696a8b4580.jpg)
![arxiv: v2 [astro-ph.sr] 31 Oct 2011](https://kipdf.com/img/300x300/arxiv-v2-astro-phsr-31-oct-2011_5b1815027f8b9ac0708b4618.jpg)
![arxiv: v7 [cs.ne] 11 Oct 2011](https://kipdf.com/img/300x300/arxiv-v7-csne-11-oct-2011_5ab455181723dd339c80e9ad.jpg)
![arxiv: v1 [astro-ph.co] 25 Oct 2011](https://kipdf.com/img/300x300/arxiv-v1-astro-phco-25-oct-2011_5ad89d5e7f8b9a5a7c8b45cd.jpg)
![arxiv: v1 [astro-ph.co] 31 Oct 2011](https://kipdf.com/img/300x300/arxiv-v1-astro-phco-31-oct-2011_5ada42147f8b9a357e8b45d0.jpg)
![arxiv: v1 [cs.os] 20 Oct 2011](https://kipdf.com/img/300x300/arxiv-v1-csos-20-oct-2011_5ae551957f8b9ae83d8b4607.jpg)
![arxiv: v2 [astro-ph.im] 13 Oct 2011](https://kipdf.com/img/300x300/arxiv-v2-astro-phim-13-oct-2011_5b35b7fe097c475f228b49d6.jpg)
![arxiv: v2 [quant-ph] 13 Oct 2011](https://kipdf.com/img/300x300/arxiv-v2-quant-ph-13-oct-2011_5af0633c7f8b9a7c858b45a4.jpg)
