Obtenção de peptídeos vasoativos a partir do plasma de serpentes brasileiras (Bothrops...

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Barreto, Sandra Alves

Obtenção de Peptídeos Vasoativos a partir do Plasma de Serpentes Brasileiras (Bothrops jararaca e Crotalus durissus terrificus). / Sandra Alves Barreto. – São

Paulo, 2006.

Tese (Mestrado) – Instituto de Ciências Biomédicas da Universidade de São Paulo. Departamento de Biotecnologia.

Área de concentração: Bioquímica e Biofísica. Linha de Pesquisa: Peptídeos Vasoativos.

Versão do titulo para o inglês: Obtention Vasoactive Peptides from the Plasma of Brazilian Snakes (Bothrops jararaca e Crotalus durissus terrificus).

Descritores: 1. Peptídeos 2. Plasma 3. Serpentes 4. Vasoativos 5. hipertensão

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ECA

SP (Tripsina etc) aminopeptidases

SP (PRCP, tripsina etc) Superfície carregada

Negativamente CP

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(27)

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L-arginina N-hidroxi-L-arginina L-citrulina + NO NADPH

Ca+2

NADPH

(31)

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# + *' B # , (( 5 #

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(32)

= ! 5 MLTI^ ! 5 MLTM^ ! 5 MLLI^ = !

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(33)

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== 5 MLTS^ H ! 5 MLUL 5 #

(34)

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(35)

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Angiotensinogênio

Renina

Angiotensina I

Angiotensina II

Ativação dos Receptores

AT

Cininogênios

Calicreína

Bradicinina

Óxido Nítrico

Prostaciclina

ECA

1 2

Metabólitos inativos

(36)

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(37)

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(38)

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Metabólicos inativos Angiotensinogênio

Renina

Angiotensina I

Angiotensina (1-7)

Cininogênios

Calicreína

Bradicinina

Óxido Nítrico

Prostaciclina

NEP

1 2

(39)

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TOP 24.15 ECA (CININASE II)

1 2 3 4 5 6 7 8 9 Arg – Pro – Pro – Gly – Phe – Ser – Pro – Phe – Arg

Aminopeptidase P

Prolil endopeptidase

NEP ECE – 1

(40)

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(41)

5 +& # - ) *' # ! 2

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(42)

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(43)

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(44)

) 5 C # # + B

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(45)

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+ ) # #

+ )

5 &$ #$ # *' ) -" ,$

7 & MLSJ ,$ % ? MLTT 5

) 9 " ? #

( 5 7 *' )

(46)

4 5 5

5 # # 5 MQI B SII - :

# 7 # $ ) <

= # - !

; & - 5 # NII _ NQI - ) &

5 "# MSI _ MUI - & $ !

H [ & 5 # NII _ RII - :

# $ % != (

) # # # $ # B MN8MN

C- # O ) #P

# & # ' d # %

# ! 5 ° ILR8NIIN

F

J N M ( ,

- &$ *'

) < J5U #-8# - #

- # # - # L TTN - MQ # 5 ` #

(47)

# *' ) -" ) *' + )

) *' ( #

*' 5 - # - # # 7 W

& MLSJ % W ? MLTT 5 : C # <

# # # # - 5 *X # 7

*' # I5Le

D 8 B E C

% 7 # I5N # # # M5U # C $ I5Ne &8&

# # ) # C- # ) *' JI # < #

# # # - BNI° - #

7 # # I5IS # H M 5 < 7 *'

# I5Q # # ' I5N ,5 Hc T5U 5 I5M # N #-8#

: & ` NII µ- ) # # # ) B# ` JT° JI

#

( *' 5 # Q # C ) # )

(48)

1 8 @ B E33C

% 7 # I5N # # # M5U# C $ I5Ne &8& I5IJ

*' M H # # ) # ) B# ` JT° JI

#

7 # # I5IQ # M H I5Q# # ' I5N, Hc T5U

5 # ) *' NII µ- 5 # # ) `

JT° JI #

2 B 5 C

#$ C # *' #

0BNQI5 : & # 7 # & # $ & #

7 : # - # + *' # 9 # # N

# # C& M ( # # #

QLQ # V# 7 *' # *' #

# & ) *' # # # ) # ) & # ,

-" ! # T M M

5 5 F 5

B C

< # 7 7 +# # MI # I5Le

# # # # % MQ ,

(49)

( # # 7 # # N # C- # B

F5 # # # - ' # # - +:

"

# *' 7 # # H( 5 , \ H

BSIII5 : # # & MU R5S ## Y NQI ## 5 # # 9

M # 8# 5 # &$ # % & C& NMR #

-7 Q LQe = LIe = HN f = I5Me #

# RQ #

*' # *X : # # ) '

4 & #

# *' *X ) - # & &

- 5 *' # & #

5 & 7 7 # - NI QIe = LIe

= HN f = I5Me 5 # # 9 M # 8# 5 # &$ #

% & C& NMR #5 # # RI # 5 ) # :

# +& +

3 5

7 # # - ( # MN5Qe5 # MI

# ' # # 7 # # O? P ( # 5 %

# 7 # & - # MII LI

# 5 $ 7 ) # -

(50)

7 # -$ # # 4 7 #

7 W & MLSJ % W ? MLTT # # *X 5 #

*' H,Z? ,Z? , - " ! T M N

T M J

4

# # 7 7 +

) : # ( FBNJ # 7 ?[ 5 4 ' 5 :

:D" # # + &$ : +# # ,

-" ! # T M R

E

+ # 7 # < # # 7 #C 5

# 7 #6 5 # ( ,BU # - $- =#

H! 5 MLLM , - " ! # T M Q

G

(51)

7 ) # # *X

) ) < )_ # JI #-8\- 5 $

< # # -# ( QI # #

<- ' 9 # - # #

+-[ #

# *' - &$ # ( MI

7 &

< - # # I5M # 8 & I5M #-8?- 5

) *' !!5 - # 7

9 # # *' & # ,

-" ! # T N M

B C

= # 7 # ( ,5 # )

# 7 H & # +& #

# #+

$ < # # -# ( QI #

<- ' 9 # - #

+-[ # , - " ! # T N N

# - C " ! # T N N M

8

(52)

# # # ) ) < TI #-8?- 5

-- # # X ' # 5

# 5 : $ & < ' # # -#

( MI

* #$ C : @ 9 7 - '

# # 9 # < # *' #

# 7 ' #$ # 9 $

# - C " ! # T N N N

1 B C

) # ' 5 # &

& # # & ) # ) 9 *' &+

! # *' - 5 +& # #

-# MQ # # # -# + # #

( *' [ ( ' 5 # #

-# & # *' [ ( '

< & - #5 # # -# - N5Q J5I # : 9

# 9 # ) ) MQ # < -' 5 #

7 MLMN8MLMJ

9 # # # # ' #$ 5

& # ) 5 # # # - #$

*' - *' $B ` M- ) 7

(53)

*X - # 9' 5 #

# +# ) - ) M N 5 # # *X +:

[ ! !!5 7 7 *' ( ' 7

9 - *' / ) #) : C

: / *' [ ! !! ` # JTk

) M/ # *' +: [ !5 7 *' [ '

+ )

#*J#%&KL# F(-,"&$-$6

NH( RNHN

J5SQ-H J

QI5II-HN MIII#

) N/ # *' +: [ !!5 7 *' [ '

+ )

#*J#%&KL# F(-,"&$-$6

NII-,- NSHN

N5QI-?

Q5I-NNHN

S5S-HN MIII#

*' *' [ ( ' C # "

(54)

1

- 4 - L TNN - MQ # 5

< J5U #-8# - # - & #

# )# ` # - 7 W & 5 MLSJ W

% W ? 5 MLTT %W?

# - # # # C $ 5

$# # # 5 & B ) *' # ) @ #

) # ) '

#$ # 5 '

O & & P + ) O & P

' #$ # # :

' # ##H- # +# # 6 *' ) & # #

# + ) # #

*X # # +#

1

% 7 # I5N # # 4 # - W 5

: < 7 # + # M # C- # F )# C

+ #$

# : # 45 9 # # QII µ- +

# # # 5 & # JII µ- +

# #

*X # # # B # # & '

(55)

1 B C

H D M

# ( ' ,$

# # 7 # # 4 #

#$ # *' ) -" W %W? # *' #

# ? c $ 5 4 c ( # 4 c ( #

1

= # 7 7 - # L & # 5

: # +& ) N I

µ

-8?-# # - 5 # # 4

#$ W 5 # I5N # # : < 7

+ # N # I5Le5 ) @ # # *'

NQIµ-/# #

< - # 5 # & < )

$ *X I5Q m- # 4

( # # # I5Q µ- ? M5I µ- ? # NQ

µ- 45 QI µ- 4 < ) 7 *' ' & # #

' ? I5Q µ- 5 - QI µ- 4 7 # # I5Q µ- ?

# " T N N T N N M # & # : #

(56)

?c ) $ 4c # 4 7

= - T/ ,$ # ##H- O P # c L 5

# # 4 #$ W NQI µ-8# ) $ N I µ-8?- ∗

( *' - & ?

= - U/ ,$ # # O P # c L 5

# # 4 #$ W NQI µ-8# ) $ N I µ-8?- ∗ ( *'

- & ?

I5Q ?

I5Q ? I5Q ?

QI 4 QI 4

M5I ? NQ 4 I I5Q M M5Q N N5Q # - # # ∗ ∗

I 5 Q ?

I 5 Q ? Q I 4

N Q 4 M 5 I ?

(57)

1

# # - 9 # # # - L & 5

# # #$ W 5 # I5N #

# : < 7 + # N # I5Le5

) @ # # *' MQI µ-/# #

< - # 5 # & < -

*X ) $ #

( # # # I5Q µ- ? M5I µ- ? # MQµ

-5 JIµ- 5 # ) 7 ' # & # I5Qµ

-? - JI µ- I5Q µ- ? # " T N N

T N N M

# & # : # 5 # #$

W 5 #)$# +& + 7 # # #+

?5 # # # 4 #$ W 5

(58)

I5Q ? I5Q ? JI I5Q ? JI MQ M5I ? BRQ BRI BJQ BJI BNQ BNI BMQ BMI BQ I # - # ( # # H

-= - L/ ,$ # ##H- O P # 5 c L

# # #$ W MQI µ-8# ) $ N I µ-8?- ∗

( *' - & ?

I5Q ? J I

I 5Q ? J I

I 5Q ?

M Q M 5I ?

I I 5N I 5R I 5S I 5U M M 5N M 5R M 5S # - # #

= - MI/ ,$ # # O P # 5 c L

# # #$ W MQIµ-8# ) $ N I µ-8?- ∗ ( *'

- & ?

∗

(59)

%# ) @ # ` ) $ 5 # +& +

) < ) *' # 4 #$ W

# 4 NQ µ- #$ W 5 # ' #$ B

JI5R ##H- # # *' # M5S #

) @ #)$# - & *' )

$ # #+ 5 # # 7 )

# #+ # # #+ ' #$ ? I5Q µ

-7 *' # BNQ5T ##H- # # *' I5T # <

7 *' # 45 - ? BJR5Q

##H- # # *' M5R #

# MQµ- #$ W 5 # ' #$ B

JM5S ##H- # # *' #$ I5L #

) @ #)$# ) $ # #+ 5

# #)$# ) # +& ) $

# #+ # # #+

# 7 # ##H- : ' # # +#

# 6 # 7 *' # # 5 # #

45 : #$ ' ? I5Q µ- 7 *'

# _NN ##H- # # *' I5U # < 7 *' #

5 - ? I5Q µ- BJT5J ##H- # #

(60)

1

H D M@

# - 5 7 # - W 5

: # # - L & # # )

# 4 # #$ %W?

1

< - # 5 # & < &$

@ 5 - *X ) $ # 4

( # # # I5Q µ- ? M5I µ- ? # NQ

µ- 45 QIµ- 45 # ) 7 ' # & # I5Qµ

-? - QI µ- 4 # I5Q µ- ? # "

T N N T N N M

# & # : # 4 #

#$ %W? #)$# ) +& + # # #+

(61)

I5Q ? I5Q ? QI 4 I5Q ? QI 4 NQ 4 M5I ? BJQ BJI BNQ BNI BMQ BMI BQ I # - # ( # # H

-= - MM/ ,$ # ##H- O P # 5 cL

# # 4 #$ %W? NQI µ-8# ) $ N I µ-8?- ∗

( *' - & ?

M5I ?

NQ 4 QI 4

I5Q ? QI 4 I5Q ? I5Q ? I I5N I5R I5S I5U M M5N M5R # - # #

= - MN/ ,$ # # O P # 5 c L

∗

(62)

1

# # - 9 # # # - L & 5

# # #$ %W?5 # I5N #

# : < 7 + # N # I5Le5

) @ # # *' MQI µ-/# #

- 9 # 5 7 # # # X C #

# 4

< - # 5 # & < &$

@ 5 - *X ) $ #

( # # # I5Q µ- ? M5I µ- ? # MQµ

-5 JIµ- 5 # ) 7 ' # & # I5Qµ

-? - JI µ- I5Q µ- ? # " T N N

T N N M

# #)$# & # : #

# #$ %W?5 ) +& + # # #+

(63)

= - MJ/ ,$ # ##H- O P # 5 c L

# # #$ %W? MQI µ-8# ) $ N I µ-8?- ∗

( *' - & ?

= - MR/ ,$ # # # O P # 5 cL

(64)

#$ %W? 7 # 4 5 :

# # # - # C ) ' #$

# 5 : & " # ) @ 5 # +&

+ # 7 ) ) #

# #+

# 4 NQ µ- # #$ %W?5 & #

' # : ) # #$ W 5 # B

MM5J ##H- # *' I5L #

) @ ) $ # #+ 5 #

#)$# * # +& ) #$ '

? I5Q µ- 7 *' # BMR5L ##H- # #

*' I5S # < 7 *' # 45 - ?

_NJ5T ##H- # # *' I5U #

# MQ µ- # #$ %W?5 & #

' # : ) # #$ W 5 : # '

#$ _JL5J ##H- # *' #$ M5U #

) @ # *' # - & )

$ #$ ' ? I5Qµ- 7 *' #

BNU5S ##H- # # *' I5R # < 7 *' # 5

- ? _QQ5J ##H- # # *' M5M #

1 1 5

M M@

) ' #$ #

(65)

) -" ) # # ) @ : & # # +

5 : & 7 #)$# *' )

$ # #+

#$ W 5 +# : # 4

5 # # # ##H- # # # & : #

# # # 5 # : # ) #

) @ # *X 5 # : - 4 #

*X ) # 5 # # & '

#$ # #+

# #$ %W?5 # ) & : # 5 # # #

*' ) # 45 #)$# # " & 7 # &

: 4 # ##H-5 # # #

( *' ) $ # #+ 5 # 4

& # & 7 #$ W # & #

& # #$ %W?5 # ) J

) J/ ( - # #$ ( *' ) # #+ 5 < 7 *' #

4 ) &$ #$ W %W?

D 8 8 @

##H- # ##H- #

JRe N QLe JJe

(66)

1 1

#$ ' ? ##H- # 5

' ? - MIIe5 # #$ ' ? <

# 7 # # - " #

#$ ? MIIe

#$

? < #

O% & 5 ) # MII5 & - #

*'

1 2 B C 8 B I C

7 # % & = ' (

% != (

V ' 7 # ) &

- # : " $

7 # 5 : # # 5 7 # I5M #-8?- 5

# ) 7 # & 5 # # # 7 # #

7 5 # *'

< # - 5 # ) # # @

$ < 5 9 & 9 7 -#

( QI # <- ' 9 # - # #

+- [ #

(67)

# *' - &$ # ( MI

7 & <

( # # # I5Q µ- ? M5I µ- ? # NQ

µ- 45 QIµ- 45 # ) 7 ' # & # I5Qµ

-? - QI µ- 4 I5Q µ- ?5 ) '

-MQ MS # " T N N M

# # ' #$ # H

V 5 # < # *' # 4

+& # & 5 #

- C MT MU

# *' - &$ #

( MI 7 & <

( # # # I5Q µ- ? M5I µ- ? # NQ

µ- 45 QI µ- 45 # ) 7 ' # # I5M

#-8?- # - & # NQ µ- 4 QI µ- 4

(68)

5&0(7- 2/ ,$ O P ? # H cS 5 <

# # 4 #$ W NQI µ-/# ∗( *' - &

)

5&0(7- / ,$ O P ? # H cS 5 <

# # 4 #$ W NQI µ-/# ∗( *' - &

(69)

5&0(7- 3/ ,$ O P ? # H cR

# I5M #-8?- # # 4 #$ W NQI µ-/# ∗

( *' - & # 4

BJI BNQ BNI BMQ BMI BQ I # - # ( # # H -I I5N I5R I5S I5U M M5N M5R # ∗ ∗ ∗ ∗ I5Q ? M5I ? NQ 4 QI 4 I5M#-8?-NQ 4 QI 4 NQ 4 M5I ? I5Q ? QI 4

I5M#-8?- NQ 4 QI 4

5&0(7- 4/ ,$ O P ? # H cR

# I5M #-8?- # # 4 #$ W NQI µ-/# ∗

(70)

H - MQ MS 5 #

# 4 #$ W 5 # ) & ) &

) # #+

# 4 NQ µ- # ' #$ _Q ##H- #

# *' I5N #

*' ) $ #

# #+ 5 #$ ' ) I5Q µ

-7 *' # _MR ##H- # # *' I5N # <

7 *' # 45 - # # - # ) I5Qµ

-_MS ##H- # # *' I5J #

- MT MU # 4

#$ W 5 # & 7 : # # 7 #

# *' I5M #-8?- 5 # 7 ) 7 #

& ) 5 # V

#$ # 4 NQ µ- # *' _S

##H- # # *' I5M # < # *'

# ' #$ _U ##H- # # *' I5J # 5

# # & *' & ` ) *'

- *' ) R

V # # 4

#$ W 5 # " $ 5 # & 7 :

& # 5 # & 7 : )

# # ) # # 5 & 9

(71)

) R/ ,$ *' ) # #+ ? 5 < 7 *'

# 4 #$ *' # 4 < # *'

) &$ #$ W # H

G BN C

##H- # ##H- #

@ 3H32 I5NJ MU5SS Qe I5JQ QNe

G BNC

##H- # ##H- #

T H 2 H4 B4 NC H1 B 4NC

1 2 1

#$ ' ? 4 ##H- # 5

: ' MIIe 5 # #$ '

? 4 < # 4 5 : 7 #

# - " #

#$ ? MIIe

#$

? < #

O% & 5 ) # MII5 &

- # *'

(72)

1 B C

+ ) B # & #

: & *' - ) # :

-# +

1 G

5 B @C

*' ? 3 ! ) # B

& B ? # ) #

# = - ML 4/ ( # ?/

#$ ? M m-8# 5

# - ' )

1 3 D

0 1 2 3 4 5 6 4 0 u l B K 4 0 u l B K 4 0 u l B K 4 0 u l B K 2 0 u l B K 4 0 u l B K 8 0 u l B K 1 6 0 u l B K 8 0 u l B J 8 0 u l B J 1 6 0 u l B J 1 6 0 u l B J 2 0 u l B K 4 0 u l B K 8 0 u l B K 1 6 0 u l B K 8 0 u l B J 8 0 u l B K 8 0 u l B J 8 0 u l B K 2 0 u l B K 4 0 u l B K 8 0 u l B K 1 6 0 u l B K 3 6 0 u l B J 8 0 u l B K Doses (ng) C o n tr aç ão ( cm )

(73)

1 3 D

M M@

7 # ) & #

7 #$ # *' ) -" W %W?5

+& *' ) $

# #+ 5 # & 7 : < # ' #

*' - &

< # - # + ) # #

# #$ " T N J

B # # &

) $ # #+ M m-8# 5 # #

& # UI µ MSI µ # 5 :

*' # - # & *'

+ ) T MI - < # & # &

) ?

( 7 7 # ? #

& ?5 : #

<9 #

-1 3 5

(74)

= - NI/ B *' # + ) 5 cS < ) *'

# 4 NQIµ-/# #$ W ,$ *X # #

) Q/ & B ? # + ) *' # 4 NQI

µ-////# #$ W B (,/ ' #$

#%6 B,0C !$&- $# Q6&"# B+*C 6%P&#

20 4,11 1,599 0,652

40 6,25 2,499 1,020

80 9,6 2,839 1,159

160 13,28 4,885 1,994

) S/ & B ? # + ) < *' # 4 NQI

µ-/# #$ W B (,/ ' #$

#%6 B,0C !$&- $# Q6&"# B+*C 6%P&#

20 4,95 2,313 0,944

40 8,23 2,968 1.212

80 11,23 4,086 1,668

160 13,38 5,004 2,043

0 2 4 6 8 10 12 14 16

20ng 40ng 80ng 160ng

(75)

$B # + ) # # 4

#$ W M # 5 - *' ? & #

*' - & # - # ? #

*' 5 #$ *' NMe # #

S 9 # 5 # ) T

-# + ) # # % (

% ( 5 = ( *' 5 : $ # O:

+ : : ` *' 5 7 # : #

' ? `: # #

7 ) ? " P =

5 MLTI

& #C9 # # &$ # &

- Y *' # # # 7

& ' ? = - NI

&$ & - Y 5 B 6# & 7

(76)

) T/ # # + ) < *' '

? UI -8UI MSI -8MSI # 4 # W

N. do experimento. µ # ( Extensão de Contração (cm) do plasma (PL)

Valor do PL Equivalente de

BK (Padrão 80ng e 160ng)

antes.

Valor Médio de BK em cm

(antes do peptídeo incubado do

Pl da SBJ)

(após o peptídeo incubado do PL

da SBJ) Fator de Potenciação (P) % potenciação

1 80

160 1,2 2,15 11,29ng 34,4ng 8,5cm 10,0cm 8,1cm 8,0cm 0,95 0,8 - -

2 80

160 0,3 0,35 4,36ng 8,48ng 5,5cm 6,6cm 6,2cm 7,3cm 1,12 1,10 12% 10%

3 80

160 0,35 2,5 2,00ng 22,98ng 14,0cm 17,4cm 16,7cm 17,2cm 1,19 0,98 19% -

4 80

160 1,6 2,35 13,33ng 27,44ng 9,6cm 13,7cm 10,5cm 13,5cm 1,09 0,98 9% -

5 80

160 2,45 3,85 17,5ng 30,8ng 11,2cm 20,0cm 15,4cm 20,0cm 1,37 - 37% -

6 80

160 1,7 1,3 15,11ng 17,33ng 9,0cm 12,0cm 10,5cm 14,3cm 1,16 1,19 16% 19%

1 3 3

9 # / C 7 5 : # # M

S

µ ( # /F # + 5 UI µ MSI µ

9 ' *' # # / # # # *X

(77)

( : & ? ( ' UI - MSI - / # &

#$ *' # ? UI - 7 # #

-" # 5 : : & ` #$ *' # #

UI µ NI - - #$ # # *' ?

#

9/ # 5 UI - ? # *' U5Q # UI µ

# # *' M5N #

#$ *' ? UI

-#$ *' ( # Y c : &

UIµ # ?

-#$ ? # # + ) # 4 /

#$ *' + ) # # #

#$ ? # # < + ) # 4 /

#$ *' + ) 5 # 5 # # #5

< # *' # - ?

= ( *' / *' ( -

< # /

(c :8 :c : & ? < *' (

c ' ? *' (

/ # - " # # &

*X # # ? < *' (

(78)

0 2 4 6 8 10 12 14 doses (ng) C o n tr aç ão ( cm ) antes após

= - NM/ B *' # + ) cS < ) *'

# 4 NQIµ-/# #$ %W? ,$ *' # #

) U/ & B ? # + ) *' # 4 NQI

µ µ µ

µ0////# #$ %W? B (,/ ' #$

#%6 B,0C !$&- $# Q6&"# B+*C 6%P&#

20 5,9 2,494 1,018

40 8,71 2,068 0,844

80 10,58 2,718 1,109

160 12,51 2,724 1,112

) L/ & B ? # + ) < *' # 4 NQI

µ-/# #$ %W? B (,/ ' #$

#%6 B,0C !$&- $# Q6&"# B+*C 6%P&#

20 6,78 3,507 1,432

40 9,71 3,857 1,575

80 11,66 3,038 1,240

160 13,10 2,480 1,012 20ng 40ng 80ng 160ng

∗

(79)

$B # + ) # # 4

#$ %W? M # 5 - *' ? & #

*' NJe & #$ 7 ? # S

9 # 5 # ) MI

-# + ) # #

% ( = ( *'

& #C9 # # &$ # &

- Y *' # 7 &

' ? = - NM

&$ & B 6# & 7 : ?

# #

# # & # # 4 7 5 #$ :

# # & # +

(80)

) MI/ # # + ) < *' '

? RI -8RI UI -8UI # 4 NQIµ-/# # ,$ %W?

Valor do PL equivalente de

antes.

(antes do peptídeo incubado do

Pl da SBJ)

(após o peptídeo incubado do

PL da SBJ)

Fator de Potenciação

(P)

% potenciação

1 40

80 0,85 1,25 3,61ng 8,92ng 9,4cm 11,2cm 10,7cm 13,5cm 1.13 1.20 13% 20%

2 40

80 1,85 2,4 6,98ng 12,97ng 10,6cm 14,8cm 15,8cm 15,8cm 1.49 1.06 49% 6%

3 40

80 0,65 0,4 5,02ng 4,26ng 5,0cm 7,5cm 4,4cm 6,8cm 0.88 0.90 - -

4 40

80 0,35 0,5 1,60ng 4,87ng 8,7cm 8,2cm 8,7cm 11,3cm 1 1.37 - 37%

5 40

80 2,15 2,4 8,19ng 15,73ng 10,5cm 12,2cm 11,2cm 12,2cm 1.06 1 6% -

6 40

80 0,35 0,75 1,72ng 6,25ng 8,1cm 9,6cm 7,5cm 10,4cm 0.92 1.08 - 8%

1 3 1

(81)

1 4 D

M M@

7 # ) & #

7 #$ # *' ) -" W %W?5

+& *' ) $

# #+ 5 # & 7 : < # ' #

*' - &

< # - # + ) # #

# #$ " T N J

B # # &

) $ # #+ ? 5 # # & #

UI µ MSI µ # 5 :

*' # - # & *'

+ ) MJ MS - < & )

?

( 7 # ? #

& ?5 : #

<9 #

-1 4 5

(82)

= - NN/ B *' # + ) 5 cT < ) *'

# MQIµ-/# #$ W ,$ *' # #

) MM/ & B ? # + ) *' # MQI

µ-////# #$ W B (,/ ' #$

#%6 B,0C !$&- $# Q6&"# B+*C 6%P&#

20 3,51 1,650 0,623

40 5,38 2,435 0,920

80 7,31 2,642 0,998

160 9,92 4,002 1,513

) MN/ & B ? # + ) < *' # MQI

µ-/# #$ W B (,/ ' #$

#%6 B,0C !$&- $# Q6&"# B+*C 6%P&#

20 3,25 1,101 0,416

40 4,51 0,997 0,377

80 7,15 1,979 0,748

160 10,05 4,028 1,522

0 2 4 6 8 10 12

doses (ng)

C

o

n

tr

aç

ão

(

cm

)

(83)

$B # + ) # #

#$ W M # 5 - *' ? & #

*' - & MM5Qe # *' ` ? # T

9 # 5 # ) MJ

-# + ) # #

% ( = ( *'

& #C9 # # &$ # &

- Y *' # 7 &

' ? = - NN

&$ & B 6# & 7 : ?

(84)

) MJ/ # # + ) < *' '

? UI -8UI MSI -8MSI # MQIµ-/# # ,$ W

antes.

(antes do peptídeo incubado do Pl da SCDT)

(após o peptídeo incubado do PL da SCDT)

(P)

% potenciação

1 80

160 0,2 0,3 3,47ng 8,27ng 4,6cm 5,8cm 5,8cm 6,8cm 1.26 1.17 26% 17%

2 80

160 0,5 0,85 6,25ng 18,88ng 6,4cm 7,2cm 6,4cm 6,7cm 1 0.93 - -

3 80

160 0,5 0,85 5,33ng 13,33ng 7,5cm 10,2cm 7,8cm 11,5cm 1.04 1.12 4% 12%

4 80

160 1,15 1,35 8,51ng 12,48ng 10,8cm 17,3cm 10,5cm 16,9cm 0.97 0.97 - -

5 80

160 0,3 0,3 3,07ng 4,24ng 7,8cm 11,3cm 7,0cm 12,4cm 0.89 1.09 - 9%

6 80

160 0,45 0,7 9,47ng 8,88ng 3,8cm 6,3cm 4,3cm 5,5cm 1.13 0.87 13% -

7 80

160 0,2 0,8 1,55ng 11,22ng 10,3cm 11,4cm 8,3cm 10,6cm 0.80 0.92 - - 1 4

, - C # # 7 ) T

1 4 5

(85)

= - NJ/ B *' # + ) 5 cS < ) *'

# MQIµ-/# #$ %W? ,$ *' # #

) MR/ & B ? # + ) *' #

MQIµ-/# #$ %W? B (,/ ' #$

#%6 B,0C !$&- $# Q6&"# B+*C 6%P&#

20 3,55 1,339 0,506

40 7,18 2,288 0,864

80 9,46 2,429 0,918

160 11,82 2,560 0,967

) MQ/ & B ? # + ) < *' # MQI

µ-/# #$ %W? B (,/ ' #$

#%6 B,0C !$&- $# Q6&"# B+*C 6%P&#

20 4,6 1,877 0,709

40 7,1 2,407 0,909

80 10,67 2,505 0,946

160 12,26 2,482 0,938 0 2 4 6 8 10 12 14

(86)

$B # + ) # #

#$ %W? M # - *' ? & #

*' MT5Se5 # ` ? 5 # S 9 #

' ) MS5

-# + ) # #

% ( = ( *'

& #C9 # # &$ # &

- Y *' # 7 &

' ? = - NJ

&$ & B 6# & 7 : ?

# #

# # & # # 7 5 #$

: # # & # +

(87)

) MS/ # # + ) < *' '

? UI -8UI MSI -8MSI # MQIµ-/# # ,$ %W?

antes.

(antes do peptídeo incubado do Pl da SCDT)

(após o peptídeo incubado do PL da SCDT)

(P)

% potenciação

1 80

160 0,15 0 1,44ng 0ng 8,3cm 12,6cm 9,8cm 13,3cm 1,18 1,05 18% 5%

2 80

160 0 0 0ng 0ng 8,7cm 10,4cm 9,4cm 11,0cm 1,08 1,05 8% 5%

3 80

160 0 0,35 0ng 4,24ng 11,6cm 13,2cm 11,8cm 13,3cm 1,01 1,00 1% -

4 80

160 1 1,4 15,38ng 32,94ng 5,2cm 6,8cm 6,3cm 7,4cm 1,21 1,08 21% 8%

5 80

160 2,7 3,2 20,76ng 36,05ng 10,4cm 14,2cm 13,4cm 15,1cm 1,28 1,06 28% 6%

6 80

160 1,5 0,6 9,52ng 6,95ng 12,6cm 13,8cm 13,4cm 13,5cm 1,06 0,97 6% -

1 4 1

, - C # # # 7 )

(88)

1 E

O & P + ) ' ) & #

- & *' # # 5 # ) & # #

& & # - & # *' )

# #+ $

& #$ *' #/

# ( 4 #$ W ) & # # *' ?

& NIe & #$ ) # *' (

# ( 4 #$ %W? ) & # # *' ?

& NJe & #$ ) # *' (

# ( #$ W ) & # # *'

? & MM5QTe & #$ ) # *' (

# ( #$ %W? ) & # # *'

? & MTe & #$ ) # *' (

# +

) # & # - # - 5 #

+ *' # 4 7 #

-# *' ) -" %W?5 # ) & - NR

(89)

Comparação da Dose/Contração do Ileo Isolado de Cobaia

. 0 2 4 6 8 10 12 14 16 40ul BK 20ul BK 40ul BK 80ul BK 160u l BK

80ul PL

160u l PL

20ul BK 40ul BK 80ul BK 160u l BK

80ul PL 80ul BK 80ul PL 80ul BK 20ul BK 40ul BK 80ul BK 160u l BK

360u l PL

80ul BK Doses C o n tr ac ão ( cm )

BJ (Diniz & Carvalho) BJ (Uchida & Katori) CDT (Diniz & Carvalho) CDT (Uchida & Katori)

= - NR/ # *' 8 *' 3 ! ) 5 # # 4

(90)

1 5

D

205

116

97,4 97

68 50

43

23,5 13,7 29

(kDa) 1 2 3 4 (kDa)

= - NQ/ C ( # 4 # #$

W 5 # ) # (

0 ( # * B( 0 MN5Qe

MB ( ' # # NL NIQ \

NB MIµ RI µ- # 4 7 ) NQIµ-8#

JB NIµ UI µ- # 4 7 ) NQI µ-8#

RB ( ' , , MJ5Q LT \

8> @&,&,#06, B 7 C

(91)

(kDa) 1 2 3 4 5 6 7 (kDa)

205

116

97,4

29

23 13,5

= - NS/ C ( # 4 # 5 - #$

W 5 # # NL _ NIQ \ ) 9 MJ _ LT \

# (

0 B( # MN5Qe

M ( ' # # MJ5Q LT \

N NI µ MIII µ-8#

J NI µ # 4 #

R NI µ # 4 (

Q NI µ # # < < NQIµ-8#

S NI µ # # - C # H(

(92)

(93)

1 5

M

= - NT/ 0 B( 0 # # 5 # #

(

0 ( # MN5Qe

MB ( ' # # NL NIQ \

NB MIµ # 7 ) MQI µ-8#

JB MIµ # 7 ) MQIµ-8#

RB ( ' # # MJ5Q LT \

205

116

97,5

29

97

68 50

43

23,5

13,5

(kDa) 1 2 3 4 (kDa)

8> @&,&,#06, B 7 C

(94)

= - NU/ C ( # 4 # #$ 7 W

& 5 # # NL _ NIQ \ ) 9 MJ _ LT \

# (

0 ( # MN5Qe

M ( ' # # MJ5Q LT \

N NIµ MIIIµ-8#

J NIµ # #

R NIµ #

Q NIµ # # NQIµ-8#

S NI µ # # - C

# H( NQIµ-8#

T ( ' # # NL NIQ \

13,5 23,5

29 97,4 116 205

(95)

1 5

M M@

= - NL/ C ( # 4 #$ W %W?

0 B( # MN5Qe

MB ( ' # # NL NIQ \

NB NI µ # 4 #$ W NQIµ-8#

JB NI µ # #$ W NQIµ-8#

RB NI µ # 4 #$ %W? MQI µ-8#

QB NI µ # #$ %W? MQIµ-8#

SB ( ' # # MJ5Q LT \

13,5 43 50 2

1 3 4 5 6

97,4

kDa kDa

(96)

= - JI/ C ( # 4 #$ W %W?

0 B( # MN5Qe

MB NI µ # 4 #$ W NQIµ-8#

NB NI µ # 4 #$ %W? NQI µ-8#

JB NI µ # #$ W MQI µ-8#

RB NI µ # #$ %W? MQIµ-8#

1

- - NQ 5 ) & # ) ) @ )

) *' # 4 #$ W 5 : #

50 97

(97)

-" # # ) 9 # # # 4

) # # # #C9 # MMI \

- - NT 5 ) & # ) ) ) @

) *' # 5 : #)$# +&

) -" ) 9 # #

-$ - NL JI 7 # #

# - W %W?5 # ) & : # - W )

: # ) @ # )

# - 5 #)$# +& ) & -$ :

(98)

1 1 B C

# ( ' ,$ 7

+& ) @ ) #

) 4

= - JM/ , - ( ' ,$ 5

B # #$ W

- JM # ) @

) *' # 4 # 5 # &C&

+ &

( , # ) 4 : #

# < ) ) )_ # JI #-8\- 7

# " T N M

I5N # ( 4 I5M # (

4

(99)

< - < *' @ 5

- +& + &

# # 4 #$ W %W?

# # C

+& + 7 # # $B # # I5IQ #-8?-BM

- MLTL #)$# ) & #

& : # & 4 # $

# *' I5IQ B I5MI #-8?-BM 5 # ) & 7 #

& - ! !!

# ) *' # 4 < 7

+ # N # I5Le & 5 & B #

' - & 5 *' - &

# # 6 5 # - MLTL $

& # 6 & #) # # *' 7

*' : # # #) ((

( , & # *X #

& & +& - & : # #

- # *' # # - :

(100)

1 2 5 5 F

5 B C

( ) *' + # ( 45 # #

# JI # # 45 : )# # #$

# *' ) -" W # - # J N J 5

# # ( ' ,$

< " T N M

# 7 # # # - :

" 5 # # J N S # *'

- JN - & # - C #

*' 5 : # # 7 *X *X #

) ' #$

5 *' *' # # &

(101)

1 2 5

B 8 C M

= - JN/ ( # - C # *' # 4

# #$ # *'

) -" 7 W & # H( % 7 # # 9 M # 8# 5 #

&$ # % NMR # - 7 Q LQe =

LIe = HN f = I5Me *X # #

# - C *X = M MI

5 *X *X = M MI #

- ( , C # - JJ #

F 9

F 10 F 8

F 7

F 6

F 5

F 4 F 3 F 2

F 1

Tempo (minutos)

(102)

# # C

` # 7 *' '

1 2 B 8

CH 5 R

5

= - JJ/ - ' #$ # 5

*' # & # *' 5 # =M =MI

SBJ (250gr)

0,1Mg/Kg Captopril

F 7 F 3 F 4

F 9

F 5

70

50

30

10

m m H g

F 5 F 1 _{F 8}

(103)

- - JJ # # ) '

#$ 45 # *X M MI5 & #

& & +& - & # *' L # *X '

) & # # & # 5 - : # $

# *' L # 5 *'

& # # H( = - JR 5 7 #

-# J N S -# # - *' - B

-# - C - *' *' L

1 2 5 5 E B

8 C

= - JR/ ( # - C - *' *' L % 7 # #

9 M # 8# 5 # &$ # % NMR # - 7

NI QIe = LIe = HN f = I5Me 5 : #

1 3

4 5 6

7 8

9 10 11

14 12 ₁₃

16 15

Tempo (minutos) ABS

(nm)

(104)

*' L & # & # &C *X 5

7 MT *X 5 : # & # # ( '

#$ 4 - JQ

1 2 1 B 8

CH 5 R

5 5 E

( # # $B # - ! M5Q

#-8?- I5M #-8?- 5 # < # MT

*X ) 5 : *X MJ5 MR

MQ5 # # #

# C ` # 7 *' '

< *' & *X MJ5 MR MQ5

& # # ) & ) *X C

0,1 AI 0,2 Capt. BJ Bruto _Pool 14 Pool 13 Pool 15 0,1 AI 0,2 Capt. BJ Bruto _Pool 14 Pool 13 Pool 15 70 50 30 10 m m H g F 2 F 3 F 4 F 5

F 7 F 14 F 13 F 12 F 11 F 9 F 8 SBJ (250g)

= - JQ/ - ( ' ,$ 4 NQI- 5 *X ) *'

(105)

V# # *' *X - #

# - 7 - *' 5 #

(106)

1 2 2 5 5 R H 1 2

= - JS/ ( # - C *' MJ5 ) # H Q *' 7

C V# # % 7 # # 9 M # 8# 5 #

&$ # % NMR # - 7 NI QIe =

LIe = HN f = I5Me

= - JT/ ( # - C *' MR5 ) # 45 *' 7

C V# # % 7 # # 9 M # 8# 5 #

&$ # % NMR # - 7 NI QIe =

LIe = HN f = I5Me

F 13

Tempo (minutos) ABS

(nm)

F 1 4

T e m p o (m in uto s) A B S

(107)

= - JU/ ( # - C *' MQ5 ) # H *' 7

C V# # % 7 # # 9 M # 8# 5 #

&$ # % NMR # - 7 NI QIe =

LIe = HN f = I5Me

< *' *X MJ5 MR MQ 45 # *X

# # 7 5 < # # I5Le

# & # # ( ' ,$ 45

# # &

*X # # V# FB =B,

) < # , 8 (! !

# ) *' ? ? ) & # :D" &

*' MR # ) MT MU

F 15

Tempo (minutos) ABS

(108)

1 2 F> 5

) MT/ C ( # *' MR V#

# C # ) - :D"

#,%

*#.6+(.-76%

B S8CS 6'TU,+&- $# J6J")$6#

LQL QM =B B(BZB BFB

@ MQSQ UJ B BFB B B0B BFB BFB(B B

B

MNLN SI B B0B B BFB B=BZB B

MMSL ST B B B BFB B B BFB

MIIR SI ]B B B B0B B0B,B0

) MU/ C ( # *' MR V#

# C # ) - :D"

#,%

*#.6+(.-7

B S8CS 6'TU,+&- $# J6J")$6#

MIIL QL B B B B B B B,B?

MLQN LJ B(B,B B B B BFB B B B B B B B B

=B

:D" # ) 5 # + ) MU

7 5 + ' # - # #

:D" 7 # + & &

(109)

1 2 3

7 # + + # - :D" B B B B

B B B,B? 5 7 # #$ # < 5 $- =# #

7 #C # # 7 ( ,BU5 #

" T M R

+ 7 ( : - #

) < : +# + 5 # ) *' # ) <

# , + 8 (! !

< + 7 # *' & 7 7 5

- JL

1 2 4 5 5 5

B > > > > > > > >@C

ABS (nm)

Tempo (minutos)

= - JL/ ( # - C *' + B B B B B B B,B? $ % 7 # #

9 M # 8# 5 # &$ # % NMR # - 7 Q RIe

(110)

< *' 5 # + $

7 < # # I5Le # & # #

( ' ,$ 45 : ' #

& 5 : - : + ' - # #

+ B C + ' #$

- RI

1 2 E 8

H B > > > > > > >

>@C

5&0(7- 1 76%%L# -7"67&-. *!$&- ,- %67J6,"6 #"V7#J% W-7-7-+-H -,6%"6%&-$- +#* J6,"#O-7O&"-. %/$&+# *0X@0H "6%"-,$# # J6J")$6# %&,"!"&+# B B B B B B B,B? #

# *' QIIµ-8QIIµ 5 # 4 *' L # 4

# # *' NQIµ-8#

' #$ 4 # # *X

& - # / - O P

-( # # # I5N # I5R # *' L5 I5J #

# 4 ' I5N # I5R # + $ # I5R

I5J # # 4

Pool 9 BJ

Cinina BJ

Pep. Sintético BJ

Cinina BJ 70

50

30

10

(111)

& ' < + $ 5

7 # C # # # 5 7 # #

# - - *' # 5 : 7 0

& = = - = # @# =! %A _ 4

# - " T M J

:D" ) ' ) ML

1 2 F> 5

) ML/ C # # ( # 4 = *' MR C

# ) - :D"

6'TU,+&-% O"&$-% 6'TU,+&-% O"&$-%

MR + S +

57-KL# 1 ?( , ! (=

*' MR5 ) & # N :D" 5 : ' # #

# - # :D" ) # 5 # : # #

# - # ) # # & & ) ( N

# <9

# 5 : :D" '

# 5 - 5 ' # -

# C 5 & # # C X 5 #

:D" ? -B( B( B0 [B( B B( B( B

(112)

1 5

( ) # + 5 # # # JI #

# 5 )# # #$ # *' ) -"

W # - # J N J 5 *' #

9 ( ' ,$

" T N M

# *' 5 7 # # # - :

" 5 # # J N S #

*'

- RM & # - C #

*' 5 : # # MR *X = *X

# # ' #$

5 *' *' # & - &

(113)

1 5 5

5 B

5 C M

= - RM/ ( # - C # *' #

# H( % 7 # # 9 M # 8# 5 #

&$ # % NMR # - 7 Q LQe =

LIe = HN f = I5Me 5 & # MR *X 5 : )

# 5 *' U L # # # # &

5 ' <

1

( # # # # ' #$

45 # ) MLUL 5

F

F1 _{F 2} F3 F4

F 5,6 F7

F8 F9

F 10,11

F12,13

F 14

Tempo (minutos) ABS

(114)

5 - # * ) @ ) #

# ) # 4 = - RN 5)

1 5

M

0,8ml CDT

0,3ml CDT

F 9 BJ

70 50 30 10 mmHg

= - RN / - ( ' #$ 4 5

# # MQI µ-8# #

*X I5U # I5J # 5 # # # & '

#$ 4

Posteriormente, testamos as frações obtidas da primeira purificação do plasma da SCDT e os resultados confirmaram efeitos negativos (Conforme descrito na figura 42b).

0,8 mL CDT

0,3 mL CDT

(115)

= S

F 4

F 2

F 3

F 8

F 4

F 3

F 7

F 9

F 12

F 1

F 10

(116)

= - RN)/ ( ' #$ 5 C *X

# *' ) @ ) #

#$ W ^ X I5J # # # *' ' MQIµ-8# 5 I5J #

( 4 NQI µ-8# 5 I5J # *' L 4 NQI µ-8# 5 I5J # (

) # MQI µ-8# 5 I5J # I5M #-8?- I5J #

! MIIµ-8#

# - & ' #$

45 7 # ' #$ < #

# ) # *' 5 -

(117)

1 1 5

M B E C

= - RJ/ ( ' #$ 5

! *X & I5J # # 7 # JII µ 8#

# )

% 7 ' #$ < 5

- & # ) 5 # )

+ & & ) # 4

( # 7 # ' #$

*X # *' - RR

67J6,"6

2 0

,6%"6%&-$-+#* /$&+#

6,"#O-7O&"-.

0,2ml CDT 0,2ml CDT

70 50 30 10 mmHg

(118)

1 2 5

5 R 5

M B E C

F 14

F 13

F 12

F 11

F 10

= L

F 7

F 5,6

F 4

= U

F 4

F 2

F 1

F 0

0,1 mL JJ

0,1 mLCDT

Captopril

(119)

= - RR/ ( ' #$ 5

*X # *' # # # # ! #

& # I5J # *' MQIµ 8#

# MR *X # *'

# 5 # # - & # *X U

L # # #

C # 7 *' '

( # )# # *X U L # *'

# - # - B # J N S

- B - <9 # # - *X U L

# 5 : # & *X *X #

# # - #

(120)

= - RQ/ ( # - C *' U

5 : *' Q5 *' & '

#$ % 7 # # 9 M # 8# 5 # &$ #

% NMR # - 7 NI QIe =

LIe = HN f = I5Me

F 1

F 2 F 3

F 4

F 5

F 6

Tempo (minutos) ABS

(121)

1 3 5 5 E

= - RS/ ( # - C *' L 5

*X J R5 # & ' #$ % 7 # #

9 M # 8# 5 # &$ # % NMR # - 7

NI QIe = LIe = HN f = I5Me

- B ) 9 ( ' ,$

5 # ) *X U Q L J5R

F 3

F 4 F 1

F 2

F 5

(122)

1 4 5

5 R 5

M B E C

70 50 30 10 mmHg

0,1ml CDT Bruto

F 9 (4)

F 9 (3)

F 8 (5)

F 8 (4)

0,2 ml CDT Bruto

AI

(123)

= - RT/ ( ' #$ 7#"-.(% $(7&%%(% "677&Q&+(%C5

*X U L # # # # *'

# - ! *X & I5J # *' MQIµ 8#

*' *' U5 # & S *X 5 : #

# ' #$ # #

& - & 5 # *' Q #

# & #

C ` # 7 *' ' 5 : & 7

& # # C V# #

*' *' L # & Q *X 5 : #

# ' #$ 5 # #

& - & *X J R #

# & #

C # 7 *' ' *X

# & # # C V#

#

- B ) 9 # - *' *X U Q 5

L J L R

(124)

= - RU/ ( # - C *' U Q # 7#"-.(% $(7&%%(%

"677&Q&+(%C % 7 # # 9 M # 8# 5 # &$ # % NMR

# - 7 NI QIe = LIe = HN f =

I5Me

U Q

(125)

1 5 5 E B C

= - RL/ ( # - C *' L J #

% 7 # # 9 M # 8# 5 # &$ # % NMR

# - 7 NI QIe = LIe = HN f =

I5Me

1 5 5 E B1C

(126)

= - QI/ ( # - C *' L R #

% 7 # # 9 M # 8# 5 # &$ # % NMR #

- 7 NI QIe = LIe = HN f =

I5Me

(127)

*X # # C V#

# :D" ) ' ) MT

1 F> 5

) NI/ C # # # g *' U Q h

C # ) - :D" C 7 0 &

= = - = # @# =! %A _ 4

6'TU,+&- $# J6J")$6# 6'TU,+&- $# J6J")$6#

MQ + MQ +

57-KL# 4B2C _{!(F (} _! _? _?( _F _{!F !0}

*' U Q 5 ) & # N :D" : # # #

-# ) # # & & ) ( N # <9 5

# : # - # #)$# # - #

:D" ) # 4

# :D" 5 - # ) &

+ + # & 5 : C

(128)

5. DISCUSSÃO

*' & + #

# & ` ? # & #

# #+ 5 MLLL # # #+ *X

? 7 # # ' - + # & *'

5 # & *' - + <9 +

5 MLLI

<9 + $ # -C C& - &

B - *' <9 +

& # # 7 5 # #+

7 # # # $ : # $ ) 5

& - + # < - !!0% 5 NIIM

# 5 & * $ #6

& # # 7 # - 6&

:D # *' 0,( # + -

5 9 # $ #

& ] 5 MLLQ

+ ) # 4 5

& < # # ' - &

# *' #$ M5I # 5 #)$# ) & # +

) &$ # ([ 5 ' #$

< ([ - 5 " & 5 & *'

& & # *' ) # C % H! 5

(129)

# #)$# # ) & #

+ B # # # # - 5 *X

S5 U B ? 7 # & # *'

n M# ^ #)$# # # ) & # *X #

$ - # S B ? < $ 5

# : *X & - $ # '

# *' n N# ' # &

I5IT µ-8?-5 ` , % 5

MLLN

# 5 *X S B ? $ ' 7 *'

' - + #C - ( #[

5 # : - $ '

& # 5 MLLI

) & # 4

#$ # *' ) -" W %W?

# @# & < # # #+ 5

& # * # + # & # ` ?

# #+ : 7 ?

: #

' ' - + # #+ 5 # #

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