Induction and analysis of antigen-specific T cell responses in melanoma patients and animal model

Hele tekst

(1)Induction and analysis of antigen-specific T cell responses in melanoma patients and animal model Bins, A.D.. Citation Bins, A. D. (2007, March 15). Induction and analysis of antigen-specific T cell responses in melanoma patients and animal model. Retrieved from https://hdl.handle.net/1887/11457 Version:. Corrected Publisher’s Version. License:. Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden. Downloaded from:. https://hdl.handle.net/1887/11457. Note: To cite this publication please use the final published version (if applicable)..

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³LQVWDQW WUDQVJHQLF´ PRGHO LQ FRPELQDWLRQ ZLWK D FRUUHFWHG FRRUGLQDWH V\VWHP ZH IROORZHG WKH LQ YLYR EHKDYLRU RI LQGLYLGXDO FHOOV H[SUHVVLQJ HLWKHU )5(7 RU ORFDWLRQEDVHG ELRVHQVRUV IRU VHYHUDO GD\V 7KH XWLOLW\ RI WKLV DSSURDFK ZDV GHPRQVWUDWHG E\ DVVHVVPHQW RI LQ YLYR FDVSDVH DFWLYDWLRQ XSRQ LQGXFWLRQ RI DSRSWRVLV7KLV³LQVWDQWVNLQWUDQVJHQLF´PRGHOFDQEHXVHGWRIROORZWKHLQYLYR EHKDYLRU RI LQGLYLGXDO FHOOV H[SUHVVLQJ HLWKHU )5(7 RU ORFDWLRQEDVHG SUREHV IRUVHYHUDOGD\VDIWHUWDWWRRLQJDQGSURYLGHVDUDSLGDQGLQH[SHQVLYHPHWKRGIRU LQWUDYLWDOLPDJLQJLQPXULQHVNLQ .

(3) %DFNJURXQG 7KHLQWURGXFWLRQRIIOXRUHVFHQWSURWHLQV )3

(4) DQGWKHDELOLW\WRYLVXDOL]HPROHFXODU HYHQWV XVLQJ IOXRUHVFHQFH PLFURVFRS\ KDYH EHHQ RI VXEVWDQWLDO YDOXH IRU XQGHUVWDQGLQJLQWUDFHOOXODUVLJQDOLQJHYHQWV)3EDVHGELRVHQVRUVDQGHVSHFLDOO\ WKRVH EDVHG RQ )OXRUHVFHQW 5HVRQDQFH (QHUJ\ 7UDQVIHU )5(7

(5) FDQ UHSRUW RQ FHOOXODUSURFHVVHVVXFKDVDSRSWRVLVRUFHOOGLYLVLRQWKHOHYHOVRIVHFRQGPHVVHQJHUV VXFKDV&D3WG,QV

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

(7) +RZHYHUIRULPDJLQJZLWKVXEFHOOXODUUHVROXWLRQ VRSKLVWLFDWHGHTXLSPHQWDQGH[SHUWLVH HJWZRSKRWRQPLFURVFRSHV >@RUZKROH PRXVHLPDJLQJV\VWHPV>@

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

(9) ZHWDWWRRHGDVPDOODUHD [PP

(10) RI WKH DEGRPLQDO VNLQ RI PLFH ZLWK '1$ HQFRGLQJ D *)3 UHSRUWHU SURWHLQ GULYHQ E\WKH&09LPPHGLDWHHDUO\ &09,(

(11) SURPRWHUKRXUVWKHUHDIWHUPLFHZHUH DQHVWKHWL]HG DQG DQDO\]HG IRU '1$ WDWWRRLQGXFHG *)3 H[SUHVVLRQ E\ LQWUDYLWDO LPDJLQJXVLQJDFRQYHQWLRQDOFRQIRFDOPLFURVFRSH([SUHVVLRQRIWKH*)3UHSRUWHU JHQHZDVREVHUYHGLQDSSUR[LPDWHO\FHOOVSHUPPWDWWRRHGVNLQVKRZLQJ WKDW WKH WUDQVIHFWLRQ HIILFLHQF\ RI '1$ WDWWRRLQJ ZDV VXIILFLHQW IRU LPDJLQJ SXUSRVHV7KHPDMRULW\RIIOXRUHVFHQWFHOOVREWDLQHGDIWHUD*)3HQFRGLQJ'1$ WDWWRRZHUHNHUDWLQRF\WHV VHH$GGLWLRQDOILOH

(12) . .

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

(14) 5HFHQWVWXGLHVVKRZHGWKDWLQDGGLWLRQWR*)3PXOWLSOH )3VFDQEHVLPXOWDQHRXVO\LPDJHGLQYLYR>@,QOLQHZLWKWKLVWKH'1$WDWWRR VWUDWHJ\DOVRDOORZHG WKHVLPXOWDQHRXV

(15) GHWHFWLRQRIFHOOVH[SUHVVLQJDEURDGUDQJH RI IOXRUHVFHQW UHSRUWHUV )3

(16) LQFOXGLQJ F\DQ IOXRUHVFHQW SURWHLQ &)3

(17) \HOORZ IOXRUHVFHQWSURWHLQ <)3

(18) DQG'V5HG$VH[SHFWHGWKHLQYLYRHPLVVLRQVSHFWUDRI WKHWHVWHG)3VZHUHVLPLODUWRWKRVHUHSRUWHGLQYLWUR )LJXUH%

(19) :KHQSHUIRUPLQJ LQWUDYLWDOLPDJLQJDWVHYHUDOWLPHSRLQWVSRVWWDWWRRWKHILUVWIOXRUHVFHQWFHOOVFRXOG EH GHWHFWHG DV HDUO\ DV KRXUV DIWHU '1$ DSSOLFDWLRQ$OWKRXJK WKH QXPEHU RI WUDQVIHFWHG FHOOV YDULHV SHU WDWWRR UDQJLQJ IURP WR FHOOV SHU PP

(20) WKHNLQHWLFVRIH[SUHVVLRQDUHWKHVDPHZLWKLQKRXUVWKHQXPEHURIGHWHFWDEOH FHOOVLQFUHDVHVDSSUR[LPDWHO\IROGDQGJUDGXDOO\GHFOLQHVRYHUGD\VDWZKLFK SRLQWDOOH[SUHVVLRQKDVGLVDSSHDUHG WKLVZDVREVHUYHGIRUDOOWHVWHG)3VDQGD UHSUHVHQWDWLYH*)3H[DPSOHLVVKRZQLQ)LJXUH&

(21) $GGLQJWRWKHIOH[LELOLW\RI WKH '1$ WDWWRR DSSURDFK WDWWRR DSSOLFDWLRQ RI D PL[WXUH RI SODVPLGV HQFRGLQJ GLIIHUHQW)3V LH+%*)3DQG'V5HG

(22) UHVXOWHGLQGHWHFWDEOHH[SUHVVLRQRIERWK UHSRUWHUJHQHVLQDSSUR[LPDWHO\DWKLUGRIWKHWUDQVIHFWHGFHOOV )LJXUH'

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a QP ):+0

(24) 7ZR *)3 IXVLRQ SURWHLQV *)3DFWLQ DQG WKH 3WG,QV

(25) 3 UHSRUWHU *)33+ GLVSOD\HG WKHLUH[SHFWHGVXEFHOOXODUGLVWULEXWLRQDWWKHSHULSKHU\RIWKHFHOOLQWKHFRUWLFDO F\WRVNHOHWRQDQGDWWKHSODVPDPHPEUDQHUHVSHFWLYHO\ )LJXUH$

(26) ,PSRUWDQWO\ ZHFRXOGUHSURGXFHWKHSUHYLRXVO\PDGHLQYLWURREVHUYDWLRQWKDW89H[SRVXUHRI FHOOVUHVXOWVLQGHFUHDVHG3WG,QV

(27) 3OHYHOV >@DVUHYHDOHGE\WUDQVORFDWLRQRI *)33+WRWKHF\WRVROLQFHOOVLUUDGLDWHGZLWK89EXWQRWLQFRQWUROFHOOV )LJXUH $ORZHUSDQHO

(28) :KLOHPDQ\LQWUDFHOOXODUSURFHVVHVFDQEHIROORZHGE\PRQLWRULQJWKHGLVWULEXWLRQ RI ORFDWLRQEDVHG SUREHV FHUWDLQ SURFHVVHV VXFK DV FRQIRUPDWLRQDO FKDQJHV DUH EHWWHU UHYHDOHG E\ NLQHWLF FKDQJHV LQ

(29) )5(77R DVVHVV WKH IHDVLELOLW\ RI LQ YLYR )5(7 PHDVXUHPHQWV DQG RI WKH ORQJLWXGLQDO IROORZXS RI LQGLYLGXDO FHOOV ZHILUVWDSSOLHGWKH)5(7EDVHG&DVHQVRUµ<HOORZFKDPHOHRQ¶ <FDP

(30) DQGLWV IOXRUHVFHQWFRQVWLWXHQWV&)3DQG<)3RQVHSDUDWHVNLQSDWFKHVRIWKHVDPHDQLPDO ,Q<FDPWDWWRRHGVNLQDUHDV)5(7ZDVUHDGLO\GHWHFWDEOHHLWKHUE\VHQVLWL]HG<)3 .

(31) HPLVVLRQ DVGHVFULEHGLQ>@)LJXUH$OHIWSDQHO

(32) RUE\DFFHSWRUSKRWREOHDFKLQJ )LJXUH%ULJKWSDQHOIRUFRQWUROH[SHULPHQWVVHH)LJXUH%

(33) ,QRUGHUWRIROORZ LQGLYLGXDOFHOOVIRUPXOWLSOHGD\VZHVXEVHTXHQWO\VHWXSDFRRUGLQDWHV\VWHPWKDW DOORZVRQHWRUHORFDWHWKHVDPHFHOOLQVXEVHTXHQWLPDJLQJVHVVLRQV7RWKLVSXUSRVH LQNUHIHUHQFHSRLQWVDUHDSSOLHGDWWKHHGJHRIWKHWDWWRRHGDUHDDQGWKHSRVLWLRQRI FHOOVRILQWHUHVWUHODWLYHWRWKHVHPDUNHUSRLQWVLVUHFRUGHGGXULQJWKHILUVWLPDJLQJ VHVVLRQ )LJXUH$

(34) ,QVXEVHTXHQWLPDJLQJVHVVLRQVWKHFRRUGLQDWHVRIWKHVHFHOOV FRPELQHGZLWKRYHUYLHZLPDJHVDWORZ [

(35) PDJQLILFDWLRQ )LJXUH%

(36) DUHWKHQ XVHGWRUHWULHYHWKHSRVLWLRQRIGHILQHGFHOOV IRUGHWDLOVVHHPHWKRGV

(37) 7R GHWHUPLQH WKH SRWHQWLDO RI VXFK ORQJLWXGLQDO DQDO\VLV ZH SHUIRUPHG D WLPH ODSVHH[SHULPHQWWRVWXG\WKHLQYROYHPHQWRIFDVSDVHLQH[SHULPHQWDOO\LQGXFHG DSRSWRVLV7RWKLVSXUSRVHD)5(7EDVHGVHQVRURIFDVSDVHDFWLYLW\FRQVLVWLQJ RI&)3DQG<)3PRLHWLHVVHSDUDWHGE\DFDVSDVHVXEVWUDWHVLWH PRGLILHGIURP >@

(38) ZDV XWLOL]HG '1$ HQFRGLQJ WKLV µDSRSWRVLVVHQVRU¶ ZDV PL[HG ZLWK '1$ HQFRGLQJ DQ µDSRSWRVLVVZLWFK¶ FRQVLVWLQJ RI D PRGLILHG ). WDFUROLPXV

(39) ELQGLQJGRPDLQIXVHGWRFDVSDVH>@LQDELFLVWURQLFFRQILJXUDWLRQZLWKWGWRPDWR 7KLVPL[WXUHZDVWKHQDSSOLHGWRWKHDEGRPLQDOVNLQRIDPRXVHE\'1$WDWWRR ,Q SDUDOOHO DV D FRQWURO IRU EDFNJURXQG DSRSWRVLV D VHSDUDWH VNLQ SDWFK ZDV WDWWRRHG ZLWK WKH DSRSWRVLVVHQVRU PL[HG ZLWK D YHFWRU HQFRGLQJ WGWRPDWR RQO\ S9D[WGWRPDWR

(40) 7R IROORZ WKH FRQVHTXHQFHV RI DSRSWRVLV LQGXFWLRQ LQ YLYR DXWRIOXRUHVFHQFH &)3 DQG <)3 LPDJHV ZHUH UHFRUGHG LQ WKH VDPH VHW RI FHOOV H[SUHVVLQJWKHDSRSWRVLVVHQVRUEHIRUHDQGKRXUVDIWHULQWUDSHULWRQHDOLQMHFWLRQ RIWKHVXLFLGHVZLWFKDFWLYDWRU$3 )LJXUH$

(41) 5HPDUNDEO\DIWHU$3 DGPLQLVWUDWLRQWKHDFWLYDWLRQRIFDVSDVHZDVUHDGLO\GHWHFWHGE\DGURSLQ<)3 &)3 IOXRUHVFHQFH UDWLR DV VKRZQ LQ WKH KLJKHU PDJQLILFDWLRQ LPDJHV LQ )LJXUH % ,Q OLQH ZLWK WKLV DIWHU$3 DGPLQLVWUDWLRQ WKH FOHDU SHDN RI VHQVLWL]HG <)3HPLVVLRQGLVDSSHDUHG )LJXUH&

(42) ,PSRUWDQWO\FHOOVLQVNLQDUHDVRIWKHVDPH DQLPDOWUHDWHGZLWKWKHDSRSWRVLVVHQVRUSOXVFRQWURO'1$GLGQRWVKRZDFKDQJH LQHPLVVLRQVSHFWUXPXSRQ$3DGPLQLVWUDWLRQ )LJXUH'

(43) &ROOHFWLYHO\WKH GDWDIURPWKLVH[SHULPHQWGHPRQVWUDWHWKDWWKLVWHFKQLTXHDOORZVRQHWRLQGXFHD GHILQHGSURFHVVLQLQGLYLGXDOFHOOVDQGWRVXEVHTXHQWO\PRQLWRUWKHHIIHFWVRIWKLV SURFHVV LQ WKLV FDVH FDVSDVH DFWLYDWLRQ

(44) LQ YLYR E\ DQ DSSURSULDWH IOXRUHVFHQW VHQVRU &RQFOXVLRQV 0RXVH WUDQVJHQHVLV LQ FRPELQDWLRQ ZLWK LQWUDYLWDO LPDJLQJ LV WLPHFRQVXPLQJ DQGFDQRQO\EHSHUIRUPHGLQVSHFLDOL]HGODERUDWRULHV+HUHZHKDYHSUHVHQWHGD WDWWRREDVHGLQWUDYLWDOLPDJLQJDVVD\XVLQJWHFKQLTXHVWKDWDUHJHQHUDOO\DYDLODEOH &OHDUO\WKLVQHZDSSURDFKWKDWLVEDVHGRQWKHLQWUDGHUPDOGHOLYHU\RIIOXRUHVFHQW UHSRUWHUVE\'1$WDWWRRKDVLWVOLPLWDWLRQV)LUVWWUDQVJHQHH[SUHVVLRQREWDLQHG E\WKHµLQVWDQWWUDQVJHQLF¶PHWKRGDVXVHGKHUHLVFRQILQHGWRWKHHSLGHUPLVDQG VHFRQGWKHH[SUHVVLRQRIIOXRUHVFHQWUHSRUWHUVLVUHVWULFWHGWRDIHZGD\V,QIXWXUH VWXGLHVWKHGXUDWLRQRIH[SUHVVLRQPD\EHSURORQJHGE\HQDEOLQJLQWHJUDWLRQ>@RU HSLVRPDOPDLQWHQDQFHRIWKHSODVPLGXVHGIRUWUDQVIHFWLRQ>@)XUWKHUPRUHLQYLYR . .

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

(46) 7KH QXGH DEGRPLQDO VXUIDFH ZDV WKRURXJKO\ULQVHGWRUHPRYHUHPDLQLQJFUHDPDQGDGURSOHWRIJ'1$LQ O ZDWHU ZDV DSSOLHG WR WKH VNLQ 8VLQJ D VWHULOH GLVSRVDEOH QHHGOH EDU ILQH PDJQXP FKDOOHQJH LQ FRORUV &KLQD

(47) PRXQWHG RQ D URWDU\ WDWWRRSHUPDQHQW PDNHXSGHYLFH &ROGVNLQ% $WUDGLQJ/LMQGHQWKH1HWKHUODQGV

(48) WKH'1$ZDV WDWWRRHG LQ WKH VNLQ RYHU D VXUIDFH RI DSSUR[LPDWHO\ PP >@ 'XULQJ WKH VHFRQGWDWWRRWKHQHHGOHVRVFLOODWHGDW+]WRDGHSWKRIPP)ROORZLQJ DSSOLFDWLRQ RI WKH '1$ UHIHUHQFH SRLQWV ZHUH WDWWRRHG ZLWK EODFN SHUPDQHQW PDNHXSLQNXVLQJDVLQJOHSRLQWQHHGOHFDUWULGJH 1RXYHDXFRQWRXU%9:HHUW 1HWKHUODQGV

(49) PRXQWHGRQDSHUPDQHQWPDNHXSGHYLFH ILQHPDJQXP$HOOD 0HGLXP7HFK %HUOLQ *HUPDQ\

(50) $OO DQLPDO H[SHULPHQWV ZHUH DSSURYHG E\ WKH UHOHYDQWLQVWLWXWLRQDOHWKLFDOFRPPLWWHH '(&

(51) DQGSHUIRUPHGLQDFFRUGDQFHZLWK WKHORFDOJXLGHOLQHV 0LFURVFRS\ $QHVWKHWL]HGPLFH *)30+&FODVV,,PLFH>@ZHUHDNLQGJLIWRI52IIULQJD

(52) ZHUH WUDQVIHUUHG WR D Û& KHDWHG FDJH PRXQWHG WR DQ LQYHUWHG 7&63$2%6 FRQIRFDOPLFURVFRSH /HLFD0DQQKHLP*HUPDQ\

(53) $[GU\REMHFWLYH +& 3/)OXRWDU

(54) DQGD[GU\REMHFWLYH +&

(55) ZHUHXVHGWRFROOHFWRYHUYLHZLPDJHV DQGD[ZDWHUREMHFWLYH 3/$32

(56) WRFROOHFWKLJKUHVROXWLRQLPDJHV ,QYLYR)5(7LPDJLQJ ,Q YLYR )5(7 ZDV PHDVXUHG E\ WKUHH LQGHSHQGHQW PHWKRGV PHDVXUHPHQW RI VHQVLWL]HG<)3HPLVVLRQPHDVXUHPHQWRIHPLVVLRQVSHFWUDDQGPHDVXUHPHQWRI WKHJDLQLQ&)3IOXRUHVFHQFHXSRQ<)3SKRWREOHDFKLQJ)RUVHQVLWL]HGHPLVVLRQ <)3 IOXRUHVFHQFH XSRQ &)3 H[FLWDWLRQ

(57) WKUHH LPDJHV ZHUH FROOHFWHG D &)3 .

(58) LPDJH H[FLWHG DW QP DQG GHWHFWHG EHWZHHQ DQG QP D VHQVLWL]HG HPLVVLRQ LPDJH H[FLWHG DW QP DQG GHWHFWHG EHWZHHQ QP DQG QP DQGD<)3LPDJHH[FLWHGDWQPDQGGHWHFWHGEHWZHHQQPDQGQP 6HQVLWL]HGHPLVVLRQ )5(7

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Ȝ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

(60) DQG WKH DQJOH D UHI

(61) EHWZHHQ WKH RULJLQ DQG WKH UHIHUHQFH DUH GHWHUPLQHG E\ D FXVWRPPDGH 9LVXDO %DVLF Y

(62) SURJUDPE\FDOOLQJFRPPDQGVIURPWKH/HLFDPDFURWRROSDFNDJH)RUHYHU\FHOO FRRUGLQDWHWKHGLVWDQFH/ FHOO

(63) DQGWKHDQJOHD FHOO

(64) EHWZHHQWKHFHOODQGWKHRULJLQ DUHGHWHUPLQHGDQGVDYHG'XULQJWKHQH[WLPDJLQJVHVVLRQWKHUHDQDHVWKHWL]HG DQLPDOLVUHSRVLWLRQHGRQWKHFRQIRFDOPLFURVFRSH:KLOH/ UHI

(65) DQG/ FHOO

(66) ZLOO EHVLPLODU EXWQRWLGHQWLFDOVHHEHORZ

(67) DWVXEVHTXHQWWLPHSRLQWVWKHUHIHUHQFH D UHI

(68) DQGDOOFHOODQJOHV D FHOO

(69)

(70) ZLOOYDU\EHFDXVHRIVPDOOURWDWLRQVLQWKHSRVLWLRQ RIWKHPRXVH7RFRUUHFWIRUWKLVD UHI

(71) LVUHGHWHUPLQHGDQGDOOFHOODQJOHV D FHOO

(72)

(73) DUHFRUUHFWHGIRUFKDQJHVLQD UHI

(74) 7KHSRVLWLRQVRIWKHFHOOVDUHWKHQUHFDOFXODWHG IURPWKHFRUUHFWHGD FHOO

(75) YDOXHVDQG/ FHOO

(76) 6WUHWFKLQJRIWKHVNLQGRHVUHVXOWLQ VPDOOGHYLDWLRQVLQ/LQVXEVHTXHQWLPDJLQJVHVVLRQVDQGWKHUHIRUHFDQUHVXOWLQ PLVDOLJQPHQWRIWKHSRVLWLRQRIFHOOVXSWRPP7RDYRLGWKLVLVVXHZHXVHDQ RYHUYLHZ LPDJH DW ORZ [

(77) PDJQLILFDWLRQ WR GHWHUPLQH WKH H[DFW FRRUGLQDWH RI HDFKFHOO )LJXUH

(78) 2QFHDVLQJOHPLVDOLJQPHQWKDVEHHQUHVROYHGDOOSRVLWLRQV ZLWKLQDUDGLXVRIPP

(79) DUHFRUUHFWHGIRUWKLVPLVDOLJQPHQW $FNQRZOHGJHPHQWV :H ZRXOG OLNH WR WKDQN 'UV - 6HJDOO : :DQJ ' .HGULQ 6 *RVZDPL DQG - :\FNRII IRU KHOSIXO GLVFXVVLRQV 'U / 2RPHQ IRU DVVLVWDQFH ZLWK FRQIRFDO PLFURVFRS\ DQG 'U * YDQ GHU .URJW IRU WHFKQLFDO DVVLVWDQFH :H ZRXOG OLNH WR WKDQN $5,$' 3KDUPDFHXWLFDOV &DPEULGJH 0$

(80) IRU WKH JHQHURXV VXSSO\ RI $3 DQG 5 2IIULQJD IRU SURYLGLQJ WKH 0+&,,H*)3 PLFH -Y5 ZDV VXSSRUWHGE\DIHOORZVKLSIURPWKH'XWFK&DQFHU6RFLHW\ . .

(81) ,175$9,7$/,0$*,1*2))/825(6&(170$5.(56$1')5(7352%(6%<'1$7$7722,1*. 5HIHUHQFHV *LHSPDQV %1* $GDPV 65 (OOLVPDQ 0+ 7VLHQ 5< 7KH IOXRUHVFHQW WRROER[IRUDVVHVVLQJSURWHLQORFDWLRQDQGIXQFWLRQ6FLHQFH

(82) &RQGHHOLV - 6LQJHU 5+ 6HJDOO -( 7KH JUHDW HVFDSH :KHQ FDQFHU FHOOV KLMDFNWKHJHQHVIRUFKHPRWD[LVDQGPRWLOLW\$QQX5HY&HOO'HY%LRO

(83) 6XPHQ&0HPSHO750D]R,%YRQ$QGULDQ8+,QWUDYLWDOPLFURVFRS\ YLVXDOL]LQJLPPXQLW\LQFRQWH[W,PPXQLW\

(84) +RIIPDQ507KHPXOWLSOHXVHVRIIOXRUHVFHQWSURWHLQVWRYLVXDOL]HFDQFHU LQYLYR1DWXUH5HY&DQFHU

(85) <DQJ0%DUDQRY(-LDQJ36XQ);/L;0/L/+DVHJDZD6%RXYHW 0$O7XZDLMUL0&KLVKLPD76KLPDGD+0RRVVD$53HQPDQ6+RIIPDQ50 :KROHERG\RSWLFDOLPDJLQJRIJUHHQIOXRUHVFHQWSURWHLQH[SUHVVLQJWXPRUVDQG PHWDVWDVHV3URF1DWO$FDG6FL86$

(86) <DQJ0/XLNHQ*%DUDQRY(+RIIPDQ50)DFLOHZKROHERG\LPDJLQJ RILQWHUQDOIOXRUHVFHQWWXPRUVLQPLFHZLWKDQ/('IODVKOLJKW%LRWHFKQLTXHV

(87) &RQGHHOLV - 6HJDOO -( ,QWUDYLWDO LPDJLQJ RI FHOO PRYHPHQW LQ WXPRXUV 1DWXUH5HY&DQFHU

(88) <DPDXFKL.<DQJ0-LDQJ3;X0<DPDPRWR17VXFKL\D+7RPLWD . 0RRVVD$5 %RXYHW 0 +RIIPDQ 50 'HYHORSPHQW RI UHDOWLPH VXEFHOOXODU G\QDPLFPXOWLFRORULPDJLQJRIFDQFHUFHOOWUDIILFNLQJLQOLYHPLFHZLWKDYDULDEOH PDJQLILFDWLRQZKROHPRXVHLPDJLQJV\VWHP&DQFHU5HV

(89) /L/+RIIPDQ507KHIHDVLELOLW\RIWDUJHWHGVHOHFWLYHJHQHWKHUDS\RIWKH KDLUIROOLFOH1DW0HG

(90) %LQV$'-RUULWVPD$:RONHUV0&+XQJ&):X7&6FKXPDFKHU710 +DDQHQ-%$*$UDSLGDQGSRWHQW'1$YDFFLQDWLRQVWUDWHJ\GHILQHGE\LQYLYR PRQLWRULQJRIDQWLJHQH[SUHVVLRQ1DW0HG

(91) +RIIPDQ50<DQJ06XEFHOOXODULPDJLQJLQWKHOLYHPRXVH1DW3URWRFROV

(92) 6DKDL ( :\FNRII - 3KLOLSSDU 8 6HJDOO - *HUWOHU ) &RQGHHOLV - 6LPXOWDQHRXV LPDJLQJ RI *)3 &)3 DQG FROODJHQ LQ WXPRUV LQ YLYR XVLQJ PXOWLSKRWRQPLFURVFRS\%0&%LRWHFKQRO

(93) <DPDXFKL . <DQJ 0 -LDQJ 3 <DPDPRWR 1 ;X 0 $PRK < 7VXML . %RXYHW07VXFKL\D+7RPLWD.0RRVVD$5+RIIPDQ505HDOWLPH,QYLYR GXDOFRORU LPDJLQJ RI LQWUDFDSLOODU\ FDQFHU FHOO DQG QXFOHXV GHIRUPDWLRQ DQG PLJUDWLRQ&DQFHU5HV

(94) .

(95) <DPDPRWR1-LDQJ3<DQJ0;X0<DPDXFKL.7VXFKL\D+7RPLWD. :DKO*00RRVVD$5+RIIPDQ50&HOOXODUG\QDPLFVYLVXDOL]HGLQOLYHFHOOVLQ YLWURDQGLQYLYRE\GLIIHUHQWLDOGXDOFRORUQXFOHDUF\WRSODVPLFIOXRUHVFHQWSURWHLQ H[SUHVVLRQ&DQFHU5HV

(96) +DOVWHDG-5YDQ5KHHQHQ-6QHO0-0HHXZV60RKDPPHG6' 6DQWRV &6+HFN$-DOLQN.'LYHFKD1$UROHIRU3WG,QV

(97) 3DQG3,3.>DOSKD@LQ UHJXODWLQJVWUHVVLQGXFHGDSRSWRVLV&XUU%LRO

(98) YDQ 5KHHQHQ - /DQJHVODJ 0 -DOLQN . &RUUHFWLQJ FRQIRFDO DFTXLVLWLRQ WR RSWLPL]H LPDJLQJ RI )OXRUHVFHQFH 5HVRQDQFH (QHUJ\ 7UDQVIHU E\ VHQVLWL]HG HPLVVLRQ%LRSK\V-

(99) .RKOHU 0 =DLWVHY 69 =DLWVHYD ,, /HLELJHU % /HLELJHU ,% 7XUXQHQ 0 .DSHOLRXNK ,/ %DNNPDQ / $SSHOVNRJ ,% %RXWHW GH 0RQYHO - ,PUHK * %HUJJUHQ322QOLQHPRQLWRULQJRIDSRSWRVLVLQLQVXOLQVHFUHWLQJ&HOOV'LDEHWHV

(100) 6WUDDWKRI.&3XOH0$<RWQGD3'RWWL*9DQLQ()%UHQQHU0.+HVORS +( 6SHQFHU '0 5RRQH\ &0$Q LQGXFLEOH FDVSDVH VDIHW\ VZLWFK IRU7FHOO WKHUDS\%ORRG

(101) *URWK $& 2OLYDUHV (& 7K\DJDUDMDQ % &DORV 03 $ SKDJH LQWHJUDVH GLUHFWVHIILFLHQWVLWHVSHFLILFLQWHJUDWLRQLQKXPDQFHOOV3URF1DWO$FDG6FL86$

(102) *DVVPDQQ 0 'RQRKR * %HUJ 3 0DLQWHQDQFH RI DQ H[WUDFKURPRVRPDO SODVPLGYHFWRULQPRXVHHPEU\RQLFVWHPFHOOV3URF1DWO$FDG6FL86$

(103) %XGNHU 9 =KDQJ * .QHFKWOH 6 :ROII -$ 1DNHG '1$ GHOLYHUHG LQWUDSRUWDOO\H[SUHVVHVHIILFLHQWO\LQKHSDWRF\WHV*HQH7KHU

(104) :ROII-$0DORQH5::LOOLDPV3&KRQJ:$FVDGL*-DQL$)HOJQHU3/ 'LUHFW JHQH WUDQVIHU LQWR PRXVH PXVFOH LQ YLYR 6FLHQFH

(105) 7XSLQ(3RLULHU%%XUHDX0).KDOORX/DVFKHW-9UDQFN[5&DOLJLXUL* *DVWRQ$7'XRQJ9DQ+X\HQ-36FKHUPDQ'%DULpW\-%0-$11RQYLUDO JHQHWUDQVIHURIPXULQHVSOHHQFHOOVDFKLHYHGE\LQYLYRHOHFWURSRUDWLRQ*HQH7KHU

(106) %RHV 0 &HUQ\ - 0DVVRO 5 2S GHQ %URXZ 0 .LUFKKDXVHQ 7 &KHQ - 3ORHJK+/7FHOOHQJDJHPHQWRIGHQGULWLFFHOOVUDSLGO\UHDUUDQJHV0+&FODVV,, WUDQVSRUW1DWXUH

(107) 9DOHQWLQ * 9HUKHJJHQ & 3LRORW 7 1HHO + &RSSH\0RLVDQ 0 %HUWUDQG ( 3KRWRFRQYHUVLRQ RI <)3 LQWR D &)3OLNH VSHFLHV GXULQJ DFFHSWRU SKRWREOHDFKLQJ )5(7 H[SHULPHQWV 1DW 0HWK

(108) . . .

(109) ,175$9,7$/,0$*,1*2))/825(6&(170$5.(56$1')5(7352%(6%<'1$7$7722,1*. )LJXUH)OXRUHVFHQWSURWHLQH[SUHVVLRQLQVLQJOHFHOOVDIWHU'1$WDWWRRLQJRQWKH VNLQRIDOLYLQJPRXVH $

(110) 8VLQJDVWDQGDUGWDWWRRPDFKLQHDS9$;YHFWRUHQFRGLQJ*)3 XSSHUSDQHO

(111) RU DQ HPSW\ S9$; YHFWRU ORZHU SDQHO

(112) ZHUH WDWWRRHG LQWR WKH VNLQ RI PLFH &RQIRFDO *)3 DQG DXWRIOXRUHVFHQFH LPDJHV ZHUH FROOHFWHG DV GHVFULEHG LQ WKH WH[W DQG *)3 LPDJHV ZHUH FRUUHFWHG IRU DXWRIOXRUHVFHQFH E\ VXEWUDFWLQJ [ WKHDXWRIOXRUHVFHQFHLPDJH%

(113) 7KHLQYLYRHPLVVLRQVSHFWUDRI&)3*)3<)3RU 'V5HGDQGDXWRIOXRUHVFHQFHWKHODWWHUDWQPH[FLWDWLRQ&

(114) 7KHQXPEHURI*)3 H[SUHVVLQJFHOOVFRXQWHGZLWKLQWKHVDPHPPVNLQDUHDDWGLIIHUHQWWLPHSRLQWV SRVWWDWWRRDVLQGLFDWHGLQWKHJUDSK'

(115) 0HUJHG*)3DQG'V5HGLPDJHVRIVNLQ WDWWRRHGZLWKDPL[WXUHRI+%*)3DQG'V5HGHQFRGLQJYHFWRUV,QVHWDFHOO H[SUHVVLQJERWK+%*)3DQG'V5HG. .

(116) )LJXUH,PDJLQJRIORFDWLRQEDVHGELRVHQVRUVZLWKVXEFHOOXODUUHVROXWLRQ $

(117) ,PDJHV RI *)3DFWLQ WRS SDQHO

(118) DQG *)33+ ORZHU SDQHO

(119) ZHUH FROOHFWHG IURPWKHPHGLDOVHFWLRQRIFHOOVXVLQJDFRQIRFDOPLFURVFRSH1RWHWKDW*)33+ WUDQVORFDWHGIURPWKHPHPEUDQHWRWKHF\WRVROLQFHOOVIROORZLQJ89$H[SRVXUH EXWQRWLQQRQH[SRVHG FRQWURO

(120) FHOOV7KHSRVWLUUDGLDWLRQWLPHLVLQGLFDWHGLQWKH LPDJHV. . .

(121) ,175$9,7$/,0$*,1*2))/825(6&(170$5.(56$1')5(7352%(6%<'1$7$7722,1*. )LJXUH,QYLYR)5(7 $

(122) &)3<)3DQGVHQVLWL]HGHPLVVLRQ )5(7

(123) LPDJHV /HIWSDQHO

(124) DQGHPLVVLRQ VSHFWUD 5LJKW SDQHO

(125) ZHUH FROOHFWHG IURP VNLQ FHOOV H[SUHVVLQJ <FDP 7KH UHG DQGEOXHVSHFWUXPDUHSUHDQGSRVW<)3SKRWRGHVWUXFWLRQUHVSHFWLYHO\1RWHWKH JDLQLQ&)3IOXRUHVFHQFHDIWHUSKRWRGHVWUXFWLRQRI<)3%

(126) &RQWUROH[SHULPHQWV IRULQWUDYLWDO)5(7LPDJLQJ /HIWSDQHO

(127) 7KHFROOHFWHG)5(7LPDJHVLQ$ZHUH FRUUHFWHG DV GHVFULEHG LQ PHWKRGV )5(7 ZDV QRW REVHUYHG LQ FHOOV H[SUHVVLQJ RQO\&)3RU<)3FRQVLVWHQWZLWKSURSHUFRUUHFWLRQ 5LJKWSDQHO

(128) 7KHHPLVVLRQ VSHFWUXPDFTXLUHGXVLQJ&)3H[FLWDWLRQZDYHOHQJWK QP

(129) RIDFHOOH[SUHVVLQJ RQO\<)37KHH[SUHVVLRQOHYHORI<)3LVVLPLODUWRWKDWRI<FDPLQ$1RWHWKDW <)3LVLQHIILFLHQWO\H[FLWHGDWQPDQGWKDWSKRWRGHVWUXFWLRQRI<)3GRHVQRW UHVXOWLQDQLQFUHDVHRIIOXRUHVFHQFHDW&)3ZDYHOHQJWKV. .

(130) )LJXUH'HILQHGFHOOVFDQEHIROORZHGRYHUPXOWLSOHGD\V $

(131) ,Q RUGHU WR VHW XS WKH FRRUGLQDWH V\VWHP WZR EODFN LQN UHIHUHQFH SRLQWV DUH WDWWRRHGRQWKHDEGRPHQRIPLFHRULJLQDQGUHIHUHQFH$W7WKHGLVWDQFH/ UHI

(132) DQG WKH DQJOH D UHI

(133) DUH PHDVXUHG EHWZHHQ WKH WZR UHIHUHQFH SRLQWV )RU FHOO FRRUGLQDWHVWKHGLVWDQFH/ FHOO

(134) DQGWKHDQJOHD FHOO

(135) DUHPHDVXUHGEHWZHHQWKHFHOO DQGRULJLQ%

(136) $WDVXEVHTXHQWWLPHSRLQW 7

(137) VPDOOURWDWLRQVLQWKHSRVLWLRQRIWKH PRXVHDOWHUD UHI

(138) DQGD FHOO

(139) EXWQRW/ UHI

(140) DQG/ FHOO

(141) D UHI

(142) LVPHDVXUHGDJDLQ DQGD FHOO

(143) LVFRUUHFWHGE\WKHGLIIHUHQFHLQD UHI

(144) DW7DQG7.QRZLQJ/ FHOO

(145) DQG D FHOO

(146) WKH SRVLWLRQV RI WKH SUHYLRXVO\ UHFRUGHG FHOOV FDQ EH UHFDOFXODWHG 5LJKW SDQHOV

(147) 0HUJH RI *)3 JUHHQ

(148) DQG EDFNJURXQG F\DQ

(149) RYHUYLHZ LPDJHV RIWKHVNLQRIDPRXVHDW7 DQG7 KRXUVLPDJHGZLWKDORZPDJQLILFDWLRQ REMHFWLYH [

(150) 7KHVDPHFHOOVDUHIRXQGEDFNXVLQJWKHFRRUGLQDWHV\VWHP,QVHWV UHSUHVHQWWKHER[HGUHJLRQVZKLFKKDYHEHHQLPDJHGZLWKDKLJKHUPDJQLILFDWLRQ REMHFWLYH [

(151) . . .

(152) )LJXUH9LVXDOL]LQJDSRSWRVLVLQWKHVNLQRIDOLYLQJPRXVHE\)5(7 $

(153) $Q $3 VHQVLWLYH DSRSWRVLVVZLWFK DQG )5(7EDVHG DSRSWRVLVVHQVRU ZHUH WDWWRRHG RQ WKH VNLQ RI OLYLQJ PRXVH$SRSWRVLV ZDV LQGXFHG E\ LQMHFWLRQ RIWKHFKHPLFDOLQGXFHURIGLPHUL]DWLRQ$3LQWKHSHULWRQHDOFDYLW\RIWKH PRXVH ,PDJHV ZHUH WDNHQ E\ FRQIRFDO PLFURVFRS\ EHIRUH DQG KRXUV DIWHU LQMHFWLRQ &HOOV ZHUH LGHQWLILHG E\ LPDJH UHJLVWUDWLRQ DW PXOWLSOH WLPH SRLQWV DV GHVFULEHGLQWKHWH[WDQGVXSSOHPHQWDU\GDWD$UHSUHVHQWDWLYHFHOOLVER[HG6KRZQ DUHPHUJHGLPDJHVRIWKHWGWRPDWR<)3DQGDXWRIOXRUHVFHQFH%

(154) &)3DQG<)3 LPDJHVRIWKHFHOODVER[HGLQ$LPDJHGDWKLJKHUUHVROXWLRQ1RWHWKHORVVLQ)5(7 DVDFRQVHTXHQFHRIDSRSWRVLVLQGXFWLRQ&

(155) (PLVVLRQVSHFWUXPRIWKHFHOOER[HG .

(156) LQ$EHIRUHLQMHFWLRQRI$3 OHIWEOXHVSHFWUXP

(157) DQGKRXUVDIWHULQMHFWLRQ ULJKWUHGVSHFWUXP

(158) '

(159) 6SHFWUXPRIDFHOOLQWKHVDPHDQLPDODVLQ&GRXEOH WUDQVIHFWHGZLWKDFRQWUROFRQVWUXFWSOXVWKH)5(7EDVHGDSRSWRVLVVHQVRUKRXUV DIWHULQMHFWLRQRI$3 $GGLWLRQDOWH[W 7KHIOXRUHVFHQWFHOOVREVHUYHGDIWHU'1$WDWWRRLQJDUHSUHGRPLQDWHO\NHUDWLQRF\WHV 7KHHSLGHUPDOOD\HURIPRXVHVNLQLVQHDUO\H[FOXVLYHO\FRPSRVHGRINHUDWLQRF\WHV DQG DQWLJHQ SUHVHQWLQJ FHOOV /DQJHUKDQV FHOOV

(160) -XGJHG E\ PRUSKRORJ\ D ODUJH IUDFWLRQRIWKHFHOOVH[SUHVVLQJ)3VDIWHU'1$WDWWRRLQJDUHNHUDWLQRF\WHV,QOLQH ZLWKWKLVDSSOLFDWLRQRID*)3HQFRGLQJSODVPLGLQZKLFKWUDQVFULSWLRQLVGULYHQ E\WKHNHUDWLQRF\WHVSHFLILF.SURPRWHU\LHOGVGHWHFWDEOHH[SUHVVLRQLQDKLJK QXPEHURIFHOOV DSSUR[LPDWHO\DVFRPSDUHGWRWKHQXPEHURI)3SRVLWLYHFHOOV REVHUYHGXSRQLQWURGXFWLRQRID&09,(GULYHQWUDQVJHQH

(161) DGGLWLRQDOILOH)LJXUH $

(162) %HFDXVH WKH . SURPRWHU LV IROG ZHDNHU WKDQ &09 ,(SURPRWHU WKH QXPEHU RI *)3H[SUHVVLQJ NHUDWLQRF\WHV REVHUYHG XSRQ DSSOLFDWLRQ RI WKH .GULYHQ *)3 YHFWRU LV OLNHO\ WR EH DQ XQGHUHVWLPDWH 7R GHWHUPLQH ZKHWKHU WUDQVJHQHH[SUHVVLRQ FDQ DOVR EH REVHUYHG LQ VNLQUHVLGHQW DQWLJHQSUHVHQWLQJ FHOOVZHWDWWRRHGDQ+%P5)3HQFRGLQJSODVPLGWRWUDQVJHQLFPLFHLQZKLFK DOO /DQJHUKDQV FHOOV DUH PDUNHG E\ *)3 H[SUHVVLRQ *)3 IXVLRQ WR 0+& FODVV ,, >@

(163) :KLOH ERWK *)3PDUNHG /DQJHUKDQV DQG P5)3H[SUHVVLQJ FHOOV DUH UHDGLO\REVHUYHGFRH[SUHVVLRQRIWKHWZR)3VLVUDUH DGGLWLRQDOILOH)LJXUH%

(164) LQGLFDWLQJWKDWRQO\VPDOOQXPEHUVRI/DQJHUKDQVFHOOVDUHWUDQVIHFWHGDIWHU'1$ WDWWRRLQJ,QFRQFOXVLRQDOWKRXJKERWKNHUDWLQRF\WHVDQG$3&VDUHSUHVHQWLQWKH HSLGHUPDOOD\HURIWKHVNLQWKHIOXRUHVFHQWHSLGHUPDOFHOOVWKDWDUHREVHUYHGDIWHU '1$WDWWRRLQJDUHSUHGRPLQDWHO\NHUDWLQRF\WHV $GGLWLRQDO)LJXUH '1$WDWWRRLQJUHVXOWVLQIOXRUHVFHQWNHUDWLQRF\WHV$

(165) *)3H[SUHVVLRQREVHUYHG XSRQDSSOLFDWLRQRIDNHUDWLQRF\WHVSHFLILF.SURPRWHUGULYHQ*)3WUDQVJHQH $SSOLFDWLRQRI'1$HQFRGLQJ.RU&09,(GULYHQ*)3WUDQVJHQHVUHVXOWHGLQ DSSUR[LPDWHO\ RU IOXRUHVFHQW FHOOVPPUHVSHFWLYHO\ %

(166) 7UDQVJHQLF PLFH LQ ZKLFKDOO/DQJHUKDQVFHOOVH[SUHVV*)3ZHUHWDWWRRHGZLWKDSODVPLGHQFRGLQJD &09,(GULYHQ+%P5)3WUDQVJHQH'HSLFWHGLVDPHUJHRID*)3DQGP5)3 LPDJH. $. %. .

(167)

No results found