producao de vacinas

7/25/2019 Producao de Vacinas

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TECNOLOGIA DEPRODUO DE VACINAS

Aline Richter, Gabriella Ricarte e Mariana Pisani


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A!nta"ent! hist#ric!

S$c%l! &VIII'(ari!li)a*+! chineses-

As t$cnicas .i/eria"'al0!.+!, c!" # .e cr!stas

!% %s inseri.! n! nari),(estir r!%as 1nti"as .e.!entes, incr%star cr!stase" arranh2es, icar a elec!" a0%lhas c!nta"ina.as,/a)er %" c!rte na ele ec!l!car %" 3! .e linhain/ecta.! !% %"a 0!ta .e%s4


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E.5ar. 6enner789:-

C!5!; O "it! .as !r.enha.!ras

i"%nes I"%ni)a*+! .e 6a"es

Phis elas (acali)a*+!?


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L!%is Paste%r7@@-

Vacina anti-rbica

I"%ni)a*+! c!" asc$l%las .a "e.%la .e%" c!elh! in/ecta.!4

Desen(!l(e%

i"%ni)antes c!ntra ac#lera .as 0alinhas e! carb


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E4 L!e5enstein eAle;an.erGlenn79H-

Pr!(ara" =%e t!;inas!.eria" ser inati(a.as!r s%bstJncias =%1"icas,n! cas! /!r"!l, "anten.!se% !tencial i"%ni)ante,"as se" ca%sar in/ec*+!4

Essa .esc!berta le(!% a!.esen(!l(i"ent! .!sri"eir!s t!;#i.es'.i/t$ric! e tetJnic!4

L!%is Sa%er,Pearl Ken.ric

e GraceEl.erin079-

I"%ni)antes c!ntra c!=%el%che4

>Ken.ric .esc!bri% =%e s%a (acina/%nci!na(a "elh!r na resen*a .!st!;#i.es.i/t$ric! e tetJnic!, c!"bin!%F!s ent+! ara /!r"ar a (acina DPT !%tr1lice bacteriana a ri"eira ai"%ni)ar c!ntra "ais .e %""icr!!r0anis"!4?


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6!nas Sal799- Vacina a artir .e (1r%s

inati(a.!s "!rt!s-4 !i! ri"eir! i"%ni)anten! "%n.! a serr!.%)i.! e" c%lt%ra .eteci.!s c$l%las .e ri".e "acac!- e re%nir

"ais .e %"a s%bes$cie.e (1r%s !li!(1r%s I, II eIII-4

Albert Sabin79- Vacina aten%a.a c!ntra a

#li!, a ri"eira a ser

alica.a !r (ia !ral4 P!r"i"eti)ar ! "ecanis"!.e in/ec*+! .! (1r%ssel(a0e", c!" a e;cre*+!.! "icr!!r0anis"!

aten%a.! n! a"biente, a(acina Sabin /acilita a!bten*+! .e alt!s n1(eis.e i"%ni.a.e c!leti(a4


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Principais desafos atuais para a imunizao :

ai;! interesse .a in.


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Pr!.%*+! .e (acinas

ase bi!l#0ica reara*+! .e ant10en!s- C%lt%ra .e "icr!r0anis"!s P%ri3ca*+! Aten%a*+! !% inati(a*+!


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Pr!.%*+! .e (acinas

ase /ar"ac%tica Obten*+! .! r!.%t! 3nal ara %tili)a*+!


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Pr!.%*+! .e (acinas

Trans!rte e .istrib%i*+! Ca.eia .e /ri!' Asse0%rar a estabili.a.e

.a (acina e" t!.as as etaas4Te"erat%ra' C a @C


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Pr!.%*+! .e (acinas

Cicl! .e r!.%*+!' : a "eses 8HQ .! te"! t!tal $ !c%a.! el!

c!ntr!le .e =%ali.a.e S+! necessri!s "ais .e H testes .e

c!ntr!le .%rante a r!.%*+! .e %" l!te.e (acina

7 .ias ara reali)ar %" c!ntr!le .eesterili.a.e


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Os rinciais ti!s .e (acinas'

Vacinas vivas atenuadas C!"!stas .e "icr!r0anis"!s (i(!s aten%a.!s e"

lab!rat#ri!s Caa)es .e "%ltilicare"Fse n! !r0anis"! h!se.eir! Res!sta i"%ne a! "icr!!r0anis"! aten%a.! $

i.ntica a r!.%)i.a ela in/ec*+! nat%ral O siste"a i"%ne $ incaa) .e .i/erenciar entre %"a

in/ec*+! el! "icr!!r0anis"! (acinal e !"icr!r0anis"! sel(a0e"4

A "%ltilica*+! .! "icr!!r0anis"! (acinal n+!c!st%"a ser caa) .e ca%sar .!en*a4


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Os rinciais ti!s .e(acinas' Vacinas (i(as aten%a.as'

Sara"!, ca;%"ba, r%b$!la, #li!, /ebrea"arela, (aricela, CG4

M$t!.!s /1sic!s .e aten%a*+!' Cal!r e ra.ia*+! UV

M$t!.!s =%1"ic!s .e aten%a*+!'

!r"al.e1.! e A0entes al=%ilantes Fr!i!lact!na, etienei"ina-Li0a"cr%)a.a"ente as ca.eias .e ci.! n%cl$ic!


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Os rinciais ti!s .e(acinas' Vacinas inativadas

C!"!stas .e "icr!r0anis"!s inati(a.!s4

N+! "ais se enc!ntra" (i(!s s+! incaa)es .e "%ltilicare"Fse4

Res!sta i"%n!l#0ica li"ita.a4

Alica.as e" .!ses c!nsec%ti(as

E;e"l!s .e (acinas inati(a.as' DPT ,heatite A, heatite, rai(a,"enin0!c!c!, in%en)a, hae"!hil%s .! ti!Fb, /ebreti/#i.e, c#lera


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Vacina viva:A ri"eira inec*+! er"ite a!(1r%s "%ltilicarFse rai.a"ente,.esenca.ean.! %"a res!stai"%nitria e" lar0a escala c!"r!.%*+! .e antic!r!s e res!sta.e lin/#cit!s T4Prs e contras:Aenas $necessria %"a .!se "as e;iste%" risc! "1ni"! .e.esen(!l(i"ent! .a .!en*a4

Vacina morta:U"a (acina "!rta n+! !.e"%ltilicarFse e a res!sta .!santic!r!s $ li"ita.a4 D%asinec*2es a.ici!nais a."inistra.as!steri!r"ente asse0%ra" %"a

r!.%*+! s%3ciente .e antic!r!s4Prs e contras:N+! e;iste risc!


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Os rinciais ti!s .e(acinas'Vacinas de subunidades:As (acinas .e s%b%ni.a.es %sa" aenas as artes .!"icr!r0anis"! ara esti"%lare" ! siste"a i"%nitri!4

A! c!nter aenas ! =%e $ necessri! ara %"a res!sta e n+!t!.as as !%tras artes .! "icr!r0anis"!, as (acinas .es%b%ni.a.es ten.e" a ca%sar "en!s reac*2es a.(ersas4

Vacinas de toxides:C!" al0%"as .!en*as bacterianas, tais c!"! a .i/teria e !t$tan!, ! r!ble"a n+! s+! as bact$rias r!ria"ente .itas "as

as t!;inas =%e r!.%)e"4E" (e) .e c!ntere" %" anti0$n! "icr!bian!, as (acinas .et!;#i.es c!nt" t!;inas inacti(a.as, .en!"ina.as t!;#i.es4

Este ti! .e (acinas esti"%la a r!.%*+! .e antic!r!s4 %an.!%"a ess!a $ in/ecta.a, estes antic!r!s !.e" bl!=%ear aentra.a .as t!;inas nas c$l%las4


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Os rinciais ti!s .e(acinas' Vacinasrecombinantes:

WEPATITE

O .esen(!l(i"ent! .a tecn!l!0ia .! DNA rec!"binante ! ri"eir! aserc!"erciali)a.! /!i a (acina c!ntra heatite 4

>O ri"eir! ass! $ a sele*+! .e %" e.a*! .!

0en!"a .! (1r%s, caa) .e esti"%lar ! siste"a .e .e/esa .!!r0anis"!,

"as n+! .e in/ectar e ca%sar a .!en*a4 Utili)an.! /erra"entas .a

en0enharia 0en$tica, esta se=Xncia $ inseri.a n%" "icr!!r0anis"!

n+! at!0nic! ara ! h!"e" n! cas!, ! /%n0! Sacchar!"cesCerre(isae !% a bact$ria Escherichia c!li-4 Assi", ! "icr#bi! (et!r

assa a e;ressar r!.%)ir- %"a r!te1na .! (1r%s .a heatiteant10en! .e s%er/1cie-, r!(!can.! a /!r"a*+! .e antic!r!s4?


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Metodologia tradicional deatenuao


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Met!.!l!0ia arar!.%*+! .e (acinasrec!"binantes


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Associao em leveduras:


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Vacinas Vetorizadas:


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Vacinas acterianas

As (acinas bacterianas !.e" serr!.%)i.as a artir .e'

Micr!r0anis"!s aten%a.!s !% "!rt!s T!;inas ne%trali)a.as C!"!nentes .e cs%la, "e"brana !%

are.e bacterianas

C%lti(! esttic! !% s%b"ers! C%lti(! s%b"ers!' act$rias "ais

resistentes Y li!3li)a*+!


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Vacinas acterianas


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Vacinas Virais

Rer!.%*+! .! (1r%s e" c%lt%ra in vitro.e c$l%las ani"ais e;' ri" .! "acac! -

C%lti(! esttic! C%lti(! s%b"ers!


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Vacinas Virais

C%lti(! esttic!' C$l%las cresce" se" a0ita*+! at$ =%e

t!.a a s%er/1cie .! "ei! 3=%e c!berta In!c%la*+! .! (1r%s Pr!a0a*+! Seara*+!, %ri3ca*+!, inati(a*+! !%

aten%a*+! .! (1r%s Des(anta0e"' Mai!r risc! .e

c!nta"ina*+!


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Vacina c!ntra a 0rie

M%ltilica*+! .! (1r%s

RetiraFse !l1=%i.! =%e /!i/!r"a.!

Li"e)a etrata"ent!

Testes

Esta t$cnica !.e ser %tili)a.a ara r!.%*+! .e !%tras (acinas(irais, !r$" n+! $ %sa.a ara (acinas =%e s+! .e !ri0e"

bacteriana4 U" !(! r!.%) 7 .!se .e (acina


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Vacinas Virais

C%lti(! s%b"ers!' E" bi!rreat!res A0ita*+! c!nstante .!

"ei! C!n.i*2es

h!"!0neas Pr!ble"as' C$l%las n+! se

"%ltilica" li(re"ente Ac


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Vacinas Virais

Vanta0ens .! %s! .e bi!rreat!res' Pr!.%*+! e" lar0a escala Re.%*+! .e c%st!s Mai!r e3cincia na r!.%ti(i.a.e C!n.i*2es #ti"as W, T, O.iss!l(i.!- Mai!r %ni/!r"i.a.e n!s l!tes .e (acina


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Vacinas Virais


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Obeti(!

Nesse trabalh! $ .escrit! %" r!cess!ara a r!.%*+! .e (acina h%"ana .!(1r%s W7N7 atra($s .e c$l%las MDCK

Ma.inFDarb canine Ki.nes- e" "ei!.e c%lt%ra DMEM- c!nten.! s!r!,%san.! %" bi!rreat!r .e leit! rechea.!4


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Materiais e M$t!.!s

C$l%las MDCK c$l%las /!ra" c%lti(a.as'

TZ[8C

Mei! .e C%lt%ra' DMEN c!" alta c!ncentra*+!.e 0lic!se, s%le"enta.! c!" Q .e s!r! /etalb!(in! S- e [,8 0\L .e NaWCO[


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Materiais e M$t!.!s

V1r%s A cea .! W7N7 /!i reica.a [; nas c$l%las

MDCK c!nten.! ! "ei! DMEM se" S e

c!" trisina A r!.%*+! .e (1r%s /!i "e.i.a ela

tit%la*+! e;ressa !r %ni.a.e .ehe"a0l%tinina (iralWA- !% TCIDH\"L

O (1r%s /!i ar"a)ena.! e" al1=%!tas .e7H"L :,;7H]8 TCIDH\"L- a F@HC


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Materiais e M$t!.!s

Siste"a r!tati(! .e c%lt%ra e rasc!s TAs c$l%las /!ra" .esc!n0ela.as .ireta"ente n! /rsc! TF8c!nten.! DMEM c!" Q .e S4

De!is .e [ .ias %"a arte /!i trans/eri.a ara /rasc!s TF7H H"l- e /!ra" c%lti(a.as a [8C e" %"a inc%ba.!ra.e Q CH

E" %" siste"a r!tati(! @Hc"- c%lt%ras /!ra"in!c%la.as c!" 4;7H]8 c$l%las e crescera" !r [ .ias

%an.! ha(ia 7,HF7,;7H]@ c$l%las, /!i a.ici!na.! !

DMEM s%"le"enta.! c!" 4 %"\"L .e TPCK-treatedtrisinaA a*+! .a trisina /!i ara.a c!" %" (!l%"e e=%i(alente.e DMEM c!" Q .e S

A s%sens+! .e c$l%las /!i %sa.a ara in!c%lar a c%lt%ra .e

ACP4


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Materiais e M$t!.!s

MiniFi!rreat!r A! A"Pr!tein "iniFbi!rreat!r ACP- sel/F

r!tatin /!i a.ici!na.! H,:0 .e carriers .e

!li"er!s .e 3bra .e ael ara !ti"i)aras c!n.i*2es .e cresci"ent!


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Materiais e M$t!.!s

MiniFi!rreat!r Est%.! .a rela*+! entre .ensi.a.e cel%lar e

GCR

7 "iniFbi!reat!r :;7H]: c$l%las inc%ba.as e" [H"l .e "ei!

c!"let!TZ[8C c!" Q .e CO

Vel!ci.a.e "anti.a a r" O "ei! n! "iniFbi!reat!r era tr!ca.!

.iaria"ente (as!s era" re"!(i.!s !r .ia ara

.eter"inar .ensi.a.e cel%lar e c!ncentra*+!

.e 0lic!se


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Materiais e M$t!.!s

MiniFi!rreat!r Deter"ina*+! .! e/eit! .e .i/erentes

c!ncentra*2es .e MDCK

0r%!s /!ra" testa.!s c!nten.!' ;7H]: ,;7H]:, :;7H]: e @;7H]: c$l%las e" [H"l .e"ei! c!"let! a [8C e Q .e CO4

O "ei! .e c%lt%ra /!i tr!ca.! .iaria"ente Ca.a 0r%! /!i testa.! ; e a "$.ia /!i calc%la.a A"!stras /!ra" retira.as .! bi!reat!r e"

inter(al!s .e 7h .%rante ! cresci"ent! cel%lare a r!.%*+! .! (1r%s


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Materiais e M$t!.!s

Oti"i)a*+! .e arJ"etr!s .e in/ec*+! Deter"inar ! e/eit! .! in#c%l! e .a Trisina

na r!.%*+! .a (acina4

@ 0r%!s c!nten.! ;7H]: c$l%las /!ra"testa.!s

Ca.a 0r%! /!i testa.! ; e a "$.ia /!i

calc%la.a

! " # $ % & ' ()ripsina*+m,m. , , , , 7,H , ,H 7H,H

M/0 H, H,7 H,H H,H7 H,H H,H H,H H,H


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Materiais e M$t!.!s

C%lt%ra n! ACP A c!l%na .e er/%s+! /!i reenchi.a

c!"7H0 .e carriers, PS H"M, :L- /!i

b!"bea.! nela ela n!ite O PS /!i s%bstit%1.! el! "ei! .e c%lt%ra e

as c$l%las ;7H]@ cel%las\L, L- .!siste"a r!tati(! .e c%lt%ra /!ra"

a.ici!na.!s a! reciiente .e ali"enta*+!,trans/eri.! ara a c!l%na .e er/%s+! einc%ba.! !r 7h se" a0ita*+!4


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Materiais e M$t!.!s

C%lt%ra n! ACP C!n.i*2es .e !era*+! .! bi!reatr!r'

TZ[8C

Vel!ci.a.e .e r!ta*+!Z r" WZ8, dissolvendoCO e 8,Q NaWCO[ A ta;a .e circ%la*+! Z 7H"L\"in O .iss!l(i.! /!i "anti.! a HQ .a sat%ra*+!

.! ar, dissolvendo ar e N


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Materiais e M$t!.!s

C%lt%ra n! ACP De!is .e : .ias ! "ei! .e c%lt%ra /!i re"!(i.! e !bi!reat!r /!i la(a.! [; c!" L .e PS

V1r%s MOIZH,H- e" 7HL .e DMEM s%le"en.a.!

c!" ,^"\"L .e Triisina /!ra" a.ici!na.!s a!bi!reat!r C!n.i*2es .e !era*+! na in/ec*+! e r!a0a*+! .!(1r%s /!ra" as "es"as .! cresci"ent! .as c$l%lasMDCK c!" e;ce*+! .a te"erat%ra =%e /!i "anti.a a

[C


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Materiais e M$t!.!s

A"!stras e Anlises A"!stras .! ACP /!ra" retira.as e" inter(al!s .e 7h

.%rante ! cesci"ent! cel%lar e a r!.%*+! .! (1r%s C!ncentra*+! .e c$l%las e s%a (iabli.a.e n! siste"a

r!tati(! e n!s /rasc!s T /!ra" "e.i.as %san.! ! "$t!.!>Tran l%e .e e;cl%si!n? c!" %" he"acit#"etr!4 C!ncentra*+! .e c$l%las n! "iniFbi!reat!r e n! ACP

/!ra" "e.i.as %san.! crystal violet staining A c!ncentra*+! .e 0lic!se /!i "e.i.a %san.! BSI 87HH

A tit%la*+! . WA /!i /eita e" "icr!lacas 9:5ell c!"c$l%las (er"elhas .! san0%e .e er% A c!ncentra*+! .! (1r%s /!i .eter"in.a c!" TCIDH nas

MDCK4


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Res%lta.!s

E;ans+! cel%lar e reara*+! ara ain!cila*+! n! ACP


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Res%lta.!s

Deter"ina*+! .e GCR !n "iniFbi!reat!r O cresci"ent! cel%lar /!i r!!rci!nal a!

GCR


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Res%lta.!s

Deter"ina*+! .e GCR n! "iniFbi!rreat!r


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Res%lta.!s

Oti"i)a*+! .!s arJ"etr!s .e in/ec*+!n! "iniFbi!rreat!r

Oti"i)ati!n !/

the in/ecti!nara"eters in"iniFbi!react!r

(essels4 aIn%ence !/

.i_erent MOI !n

(ir%s iel. in the"inibi!react!r(essels' MOI

H4H75hite-, H4Hli0ht 0ra-, H47

0ra-, H4blac-4


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Res%lta.!s

Oti"i)a*+! .!s arJ"etr!s .e in/ec*+!n! "iniFbi!rreat!r

In%ence !/.i_erent trsin

c!ncentrati!ns H,7, 4, , 7H

`0\"l- !n (ir%s

iel. in the "iniFbi!react!r (essels

!i %sa.!

,^"\"L .etriisina c!"H,H MOI arae;ans+! .!(1r%s n! ACP


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Res%lta.!s

C%lt%ra n! ACP

Densi.a.ecel%lar

,;7H]@0\


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Res%lta.!s

C%lt%ra n! ACP

TCIDH"a

;-Z84[;7H]8\"L e" 9:h


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Res%lta.!s

C%lt%ra n! ACP

Os res%lta.!s /!ra"

se"elhantes e"!%tr!s e;eri"ent!s


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Disc%ss+!

A c!ncentra*+! .e triisina $ %" /at!r cr1tic! ara %"ar!.%*+! e3ciente .e (acina .e 0rie a artir .e c$l%lasMDCK A a.i*+! .e ,^"\"L.e triisina n! "iniFbi!reat!r

aresent!% %" a%"ent! na r!.%*+! .! (1r%s e" [ .ias A c!ncentra*+! .! in#c%l! ta"b$" $ i"!rtante ara a

r!.%*+! e3ciente .e (acina .e 0rie a artir .e c$l%lasMDCK Alt!s MOI a%"enta" a r!!r*+! .e art1c%las se" (1r%s !%

c!" (1r%s n+!Fin/ect%!s!s ai;! MOI .i"in%e a r!.%*+! .! (1r%s Nesse est%.! /!i esc!lhi.! %tili)ar MOIZH,H=%e c!rres!n.e

c!" a ";ia r!.%*+! .e (1r%s n! "iniFbi!reat!r


53/53

Disc%ss+!

Nesse arti0! /!i aresenta.! %" r!cess! ara are.%*+! .e (acina ara 0rie %san.! "ei! DMEMc!nten.! s!r!S- e" %" bi!rreat!r .e leit!rechea.! ACP-4

O "iniFbi!rreat!r /!i %sa.! ara ! est%.! .a rela*+!entre a .ensi.a.e cel%lar e GCR, al$" .eestabelecer arJ"etr!s ara a r!.%*+! .! (1r%s .a0rie W7N7

De!is .a !ti"i)a*+! ! t!tal .e c$l%las a%"ent!% .e,H;7H]9 ara [,;7H]7H .e!is .e : .ias4

A %tili)a*+! .esse bi!rreat!r c!" >b!lsas?.iscart(eis .isensa a li"e)a, .i"in%in.! ! te"!.e !era*+! ! =%e !.e (iabili)ar ! r!cess!4

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