This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version.
Research Article: New Research | Development
Examining Hippocampal Mossy Fiber Synapses by 3D Electron Microscopy in
Wildtype and Kirrel3 Knockout Mice
3D EM of mossy fiber synapses in Kirrel3 KO mice
E. Anne Martin, Derek Woodruff, Randi L. Rawson and Megan E. Williams
Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, UT 84132
DOI: 10.1523/ENEURO.0088-17.2017
Received: 18 March 2017
Revised: 20 April 2017
Accepted: 17 May 2017
Published: 22 May 2017
Author Contributions: MEW and EAM designed research; MEW, EAM, DW, and RLR performed research;
MEW and EAM analyzed data; and MEW and EAM wrote the paper.
Funding: Whitehall Foundation (Whitehall Foundation, Inc.): 100001391. Alfred P. Sloan Foundation:
100000879. Edward Mallinckrodt Jr Foundation; HHS | NIH | National Institute of Mental Health (NIMH):
100000025; 1RO1MH105426. Autism Speaks (Autism Speaks Inc.): 100000073; 10116. HHS | NIH |
Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD): 100009633;
T32HD007491.
Conflict of Interest: Authors report no conflict of interest.
This work was funded by grants to MEW from the Whitehall, Alfred P Sloan, and Edward Mallinckrodt Jr
Foundations, and NIH grant 1R01MH105426. EAM is funded by an Autism Speaks Dennis Weatherstone
Predoctoral Fellowship (#10116) and previously by an NIH Developmental Biology Training Grant (NIH
T32HD007491).
Corresponding Author: Megan Williams, [email protected]
Cite as: eNeuro 2017; 10.1523/ENEURO.0088-17.2017
Alerts: Sign up at eneuro.org/alerts to receive customized email alerts when the fully formatted version of this
article is published.
Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading
process.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International
(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any
medium provided that the original work is properly attributed.
Copyright © 2017 the authors
ϭ
Ϯ
ϯ
ϰ
ϱ
ϲ
ϳ
ϴ
ϵ
ϭϬ
ϭϭ
ϭϮ
ϭϯ
ϭϰ
ϭϱ
ϭϲ
ϭϳ
ϭϴ
ϭϵ
ϮϬ
Ϯϭ
ϮϮ
Ϯϯ
Ϯϰ
Ϯϱ
Ϯϲ
Ϯϳ
Ϯϴ
Ϯϵ
ϯϬ
ϯϭ
ϯϮ
ϯϯ
ϯϰ
ϯϱ
ϯϲ
ϯϳ
ϯϴ
ϯϵ
ϰϬ
ϰϭ
ϰϮ
ϰϯ
ϰϰ
ϰϱ
ϰϲ
0$186&5,377,7/(3$*(
ϰϳ
ϰϴ
7,7/(OLPLWZRUGV
([DPLQLQJKLSSRFDPSDOPRVV\ILEHUV\QDSVHVE\'HOHFWURQPLFURVFRS\LQZLOGW\SHDQG
.LUUHONQRFNRXWPLFH
$%%5(9,$7('7,7/(OLPLWFKDUDFWHUV
'(0RIPRVV\ILEHUV\QDSVHVLQ.LUUHO.2PLFH
$87+251$0(6$1'$)),/,$7,216
($QQH0DUWLQ'HUHN:RRGUXII5DQGL/5DZVRQDQG0HJDQ(:LOOLDPV
±'HSDUWPHQWRI1HXURELRORJ\DQG$QDWRP\8QLYHUVLW\RI8WDK6FKRRORI0HGLFLQH6DOW/DNH
&LW\87
$87+25&2175,%87,216
0(:DQG($0GHVLJQHGUHVHDUFK0(:($0':DQG55SHUIRUPHGUHVHDUFK0(:DQG
($0DQDO\]HGGDWDDQG0(:DQG($0ZURWHWKHSDSHU
&255(6321',1*$87+250HJDQ:LOOLDPVPHJDQZLOOLDPV#QHXURXWDKHGX
180%(52)),*85(6
180%(52)7$%/(6
180%(52)08/7,0(',$
180%(52):25'6)25$%675$&7RXWRI
180%(52):25'6)256,*1,),&$1&(67$7(0(17RXWRI
180%(52):25'6)25,1752'8&7,21RXWRI
180%(52):25'6)25',6&866,21RXWRI
$&.12:/('*0(176
7KLVZRUNZDVIXQGHGE\JUDQWVWR0(:IURPWKH:KLWHKDOO$OIUHG36ORDQDQG(GZDUG
0DOOLQFNURGW-U)RXQGDWLRQVDQG1,+JUDQW50+($0LVIXQGHGE\DQ$XWLVP
6SHDNV'HQQLV:HDWKHUVWRQH3UHGRFWRUDO)HOORZVKLSDQGSUHYLRXVO\E\DQ1,+
'HYHORSPHQWDO%LRORJ\7UDLQLQJ*UDQW1,+7+':HWKDQN$OOLVRQ
6FKQHJJHQEXUJHU-HQ+XQWHUDQG.HHJDQ7HHWHUIRUWHFKQLFDODVVLVWDQFH:HWKDQN0LFKDHO
'HDQV'LPLWUL7UlQNQHUDQGWKHHQWLUH:LOOLDPVODEIRUPDQXVFULSWFRPPHQWV
&21)/,&72),17(5(67
$XWKRUVUHSRUWQRFRQIOLFWRILQWHUHVW
)81',1*6285&(6
7KLVZRUNZDVIXQGHGE\JUDQWVWR0(:IURPWKH:KLWHKDOO$OIUHG36ORDQDQG(GZDUG
0DOOLQFNURGW-U)RXQGDWLRQVDQG1,+JUDQW50+($0LVIXQGHGE\DQ$XWLVP
6SHDNV'HQQLV:HDWKHUVWRQH3UHGRFWRUDO)HOORZVKLSDQGSUHYLRXVO\E\DQ1,+
'HYHORSPHQWDO%LRORJ\7UDLQLQJ*UDQW1,+7+'
ϭ
ϰϵ
$%675$&7
ϱϬ
1HXUDOFLUFXLWVEDODQFHH[FLWDWRU\DQGLQKLELWRU\DFWLYLW\DQGGLVUXSWLRQVLQWKLVEDODQFHDUH
ϱϭ
FRPPRQO\IRXQGLQQHXURGHYHORSPHQWDOGLVRUGHUV0LFHODFNLQJWKHLQWHOOHFWXDOGLVDELOLW\DQG
ϱϮ
DXWLVPDVVRFLDWHGJHQH.LUUHOKDYHDQH[FLWDWLRQLQKLELWLRQLPEDODQFHLQWKHKLSSRFDPSXVEXW
ϱϯ
WKHSUHFLVHV\QDSWLFFKDQJHVXQGHUO\LQJWKLVIXQFWLRQDOGHIHFWDUHXQNQRZQ.LUUHOLVD
ϱϰ
KRPRSKLOLFDGKHVLRQPROHFXOHH[SUHVVHGLQGHQWDWHJ\UXV'*DQG*$%$QHXURQV,WZDV
ϱϱ
VXJJHVWHGWKDWWKHH[FLWDWLRQLQKLELWLRQLPEDODQFHRIKLSSRFDPSDOQHXURQVLQ.LUUHONQRFNRXW
ϱϲ
PLFHLVGXHWRORVVRIPRVV\ILEHU0)ILORSRGLDZKLFKDUH'*D[RQSURWUXVLRQVWKRXJKWWR
ϱϳ
H[FLWH*$%$QHXURQVDQGWKHUHE\SURYLGHIHHGIRUZDUGLQKLELWLRQWR&$S\UDPLGDOQHXURQV
ϱϴ
)HZHUILORSRGLDOVWUXFWXUHVZHUHREVHUYHGLQ.LUUHONQRFNRXWPLFHEXWQHLWKHUILORSRGLDO
ϱϵ
V\QDSVHVQRU'*HQSDVVDQWV\QDSVHVZKLFKDOVRH[FLWH*$%$QHXURQVZHUHH[DPLQHG
ϲϬ
+HUHZHXVHGVHULDOEORFNIDFHVFDQQLQJHOHFWURQPLFURVFRS\ZLWK'UHFRQVWUXFWLRQWRGHILQH
ϲϭ
WKHSUHFLVHFRQQHFWLYLW\RI0)ILORSRGLDDQGHOXFLGDWHV\QDSWLFFKDQJHVLQGXFHGE\.LUUHOORVV
ϲϮ
6XUSULVLQJO\ZHGLVFRYHUHGZLOGW\SH0)ILORSRGLDGRQRWV\QDSVHH[FOXVLYHO\RQWR*$%$
ϲϯ
QHXURQVDVSUHYLRXVO\WKRXJKWEXWLQVWHDGV\QDSVHZLWKVLPLODUIUHTXHQF\RQWR*$%$QHXURQV
ϲϰ
DQG&$QHXURQV0RUHRYHU.LUUHOORVVVHOHFWLYHO\UHGXFHV0)ILORSRGLDOV\QDSVHVRQWR*$%$
ϲϱ
QHXURQVEXWQRWWKRVHPDGHRQWR&$QHXURQVRUHQSDVVDQWV\QDSVHV,QVXPWKHVHOHFWLYH
ϲϲ
ORVVRI0)ILORSRGLDOV\QDSVHVZLWK*$%$QHXURQVOLNHO\XQGHUOLHVWKHKLSSRFDPSDODFWLYLW\
ϲϳ
LPEDODQFHREVHUYHGLQ.LUUHONQRFNRXWPLFHDQGPD\LPSDFWQHXUDOIXQFWLRQLQSDWLHQWVZLWK
ϲϴ
.LUUHOGHSHQGHQWQHXURGHYHORSPHQWDOGLVRUGHUV
ϲϵ
ϳϬ
6,*1,),&$1&(67$7(0(17
ϳϭ
ϳϮ
QHXURGHYHORSPHQWDOGLVRUGHUVLQFOXGLQJDXWLVPLQWHOOHFWXDOGLVDELOLW\DQG-DFREVHQ
VV\QGURPH
ϳϯ
DFKURPRVRPDOGLVRUGHUWKDWIUHTXHQWO\LQFOXGHVHSLOHSV\DXWLVPDQGLQWHOOHFWXDOGLVDELOLW\:H
ϳϰ
VWXGLHGWKHHIIHFWRIORVLQJ.LUUHORQV\QDSWLFFRQQHFWLRQVLQWKHPRXVHKLSSRFDPSXVDEUDLQ
3RLQWPXWDWLRQVDQGGHOHWLRQVLQWKHJHQH.LUUHODUHDVVRFLDWHGZLWK
Ϯ
ϳϱ
UHJLRQFULWLFDOIRUOHDUQLQJDQGPHPRU\:HILQGWKDWQRWRQO\DUHDVSHFLILFVXEVHWRIV\QDSVHV
ϳϲ
PLVVLQJLQ.LUUHONQRFNRXWVEXWZHDOVRGLVFRYHUHGDQHZV\QDSWLFFRQQHFWLRQZLWKLQWKH
ϳϳ
KLSSRFDPSXV7KHV\QDSWLFFKDQJHVZHIRXQGLQPLFHODFNLQJ.LUUHOVKHGQHZOLJKWRQKRZD
ϳϴ
GHIHFWLYH.LUUHOJHQHFRXOGFDXVHQHXURGHYHORSPHQWDOGLVRUGHUVLQKXPDQV
ϳϵ
ϴϬ
,1752'8&7,21
ϴϭ
ϴϮ
HVSHFLDOO\IRUWKHPQHPRQLFSURFHVVRISDWWHUQVHSDUDWLRQ5ROOV<HWWKHPROHFXODU
ϴϯ
PHFKDQLVPVUHJXODWLQJWKHLUGHYHORSPHQWDUHODUJHO\XQNQRZQ0)SUHV\QDSWLFFRPSOH[HV
ϴϰ
VLPXOWDQHRXVO\FRQQHFWJOXWDPDWHUJLF'*QHXURQVWRJOXWDPDWHUJLF&$S\UDPLGDOQHXURQV
ϴϱ
KHQFHIRUWKFDOOHG³&$QHXURQV´DQG*$%$HUJLFLQWHUQHXURQVKHQFHIRUWKFDOOHG³*$%$
ϴϲ
QHXURQV´)LJXUH$0)SUHV\QDSWLFFRPSOH[HVFRQVLVWRIDJLDQWPDLQERXWRQWKDWLVXSWR
ϴϳ
WLPHVODUJHULQYROXPHWKDQDW\SLFDOSUHV\QDSVH6KHSKHUGDQG+DUULV:LONHHWDO
ϴϴ
DQGWKH\V\QDSVHRQWRPXOWLKHDGHG&$VSLQHVFDOOHGWKRUQ\H[FUHVFHQFHV7(V
ϴϵ
)LJXUH$%HFDXVHRILWVODUJHVL]HQXPHURXVDFWLYH]RQHVDQGSUR[LPLW\WRWKH&$VRPD
ϵϬ
UHOHDVHDWDVLQJOHPDLQERXWRQSURIRXQGO\LPSDFWVDFWLYLW\RIWKHFRQQHFWHG&$QHXURQ
ϵϭ
0F%DLQ,QDGGLWLRQWKH0)SUHV\QDSWLFFRPSOH[H[WHQGVSURWUXVLRQVFDOOHG0)
ϵϮ
ILORSRGLDWRV\QDSVHZLWKQHDUE\*$%$QHXURQV)LJXUH$$FViG\HWDO$PDUDO
ϵϯ
&ODLERUQHHWDO7KLV0)ILORSRGLDOV\QDSVHSURYLGHVIHHGIRUZDUGLQKLELWLRQWR&$
ϵϰ
QHXURQV%UDJLQHWDO'HOOHUHWDO)URWVFKHU3HQWWRQHQHWDO
ϵϱ
7RUERUJHWDO'LVWLQFWIURPWKH0)SUHV\QDSWLFFRPSOH['*D[RQVDOVRDFWLYDWH*$%$
ϵϲ
QHXURQVYLDPRUHW\SLFDOHQSDVVDQWV\QDSVHV)LJXUH$7KHGLYHUVLW\RI'*SUHV\QDSWLF
ϵϳ
VWUXFWXUHVPDGHZLWKGLVWLQFWSRVWV\QDSWLFSDUWQHUVVXJJHVWVWKDWGLIIHUHQWPROHFXODU
ϵϴ
PHFKDQLVPVPLJKWEHLQYROYHGLQHVWDEOLVKLQJHDFKW\SHRIV\QDSWLFFRQQHFWLRQKRZHYHUWKH
ϵϵ
PROHFXOHVUHJXODWLQJ0)V\QDSVHVSHFLILFLW\UHPDLQODUJHO\XQNQRZQ
0RVV\ILEHU0)SUHV\QDSWLFFRPSOH[HVDUHFULWLFDOWRKLSSRFDPSDOFLUFXLWIXQFWLRQ
ϯ
ϭϬϬ
.LUUHOLVDQLPPXQRJOREXOLQVXSHUIDPLO\PHPEHUWKDWPHGLDWHVKRPRSKLOLFFHOO
ϭϬϭ
DGKHVLRQ*HUNHHWDO0DUWLQHWDO6HUL]DZDHWDO3RLQWPXWDWLRQVFRS\
ϭϬϮ
QXPEHUYDULDWLRQVDQGGHOHWLRQVLQ.LUUHOKDYHEHHQUHSHDWHGO\LGHQWLILHGLQSDWLHQWVZLWK
ϭϬϯ
LQWHOOHFWXDOGLVDELOLW\%KDOODHWDO.DPLQVN\HWDO7DONRZVNLHWDODXWLVP
ϭϬϰ
VSHFWUXPGLVRUGHUV%HQ'DYLGDQG6KLIPDQ&KHQJHWDO'H5XEHLVHWDO
ϭϬϱ
,RVVLIRYHWDO0LFKDHOVRQHWDO1HDOHHWDO7DONRZVNLHWDODQG
ϭϬϲ
-DFREVHQ¶VV\QGURPH*XHULQHWDO*LYHQWKHVHDVVRFLDWLRQVZLWKQHXURGHYHORSPHQWDO
ϭϬϳ
GLVRUGHUVDIIHFWLQJOHDUQLQJDQGPHPRU\WKHUROHRI.LUUHOLQWKHPRXVHKLSSRFDPSXVZDV
ϭϬϴ
UHFHQWO\LQYHVWLJDWHG%HKDYLRUDOO\DGXOW.LUUHONQRFNRXWPLFHVKRZDGHIHFWLYHQRYHOREMHFW
ϭϬϵ
UHFRJQLWLRQSUHIHUHQFHDVZHOODVK\SHUDFWLYLW\LQDIDPLOLDUHQYLURQPHQW&KRLHWDO
ϭϭϬ
0ROHFXODUO\.LUUHOLVRQO\H[SUHVVHGLQ'*QHXURQVDQGDVXEVHWRI*$%$QHXURQVLQWKH
ϭϭϭ
KLSSRFDPSXV\HWIXQFWLRQDOO\.LUUHONQRFNRXWPLFHKDYHDQLQFUHDVHLQ'*DQG&$QHXURQ
ϭϭϮ
DFWLYLW\FRPSDUHGWRZLOGW\SHPLFH0DUWLQHWDO5RKHWDO7KHVHIXQFWLRQDO
ϭϭϯ
GHIHFWVFRXOGEHH[SODLQHGE\WKHPRUSKRORJLFDOREVHUYDWLRQWKDW.LUUHONQRFNRXWPLFHKDYH
ϭϭϰ
IHZHU0)ILORSRGLDWKDQZLOGW\SHPLFH0DUWLQHWDOZKLFKPD\GHFUHDVHIHHGIRUZDUG
ϭϭϱ
LQKLELWLRQWR&$QHXURQV7KHVHSULRUVWXGLHVVXSSRUWWKHK\SRWKHVLVWKDW.LUUHODFWVYLDLWV
ϭϭϲ
KRPRSKLOLFH[WUDFHOOXODUGRPDLQWRUHJXODWHV\QDSVHIRUPDWLRQEHWZHHQ'*0)ILORSRGLDDQG
ϭϭϳ
*$%$QHXURQV+RZHYHUKLSSRFDPSDOV\QDSVHVLQ.LUUHONQRFNRXWPLFHKDYHQRW\HWEHHQ
ϭϭϴ
GLUHFWO\H[DPLQHG
ϭϭϵ
ϭϮϬ
VHULDOEORFNIDFHVFDQQLQJHOHFWURQPLFURVFRS\6%(07KRXJKVHYHUDO'HOHFWURQ
ϭϮϭ
PLFURVFRS\(0VWXGLHVRIZLOGW\SH0)V\QDSVHVH[LVW)DXONQHUHWDO5ROOHQKDJHQHW
ϭϮϮ
DO:LONHHWDOQRQHDVVHVVHG0)ILORSRGLDRUHQSDVVDQWV\QDSVHFRQQHFWLYLW\
ϭϮϯ
7KXVRXUVWXG\QRWRQO\SURYLGHVDKLJKUHVROXWLRQDQDO\VLVRIKLSSRFDPSDOV\QDSWLFGHIHFWVLQ
ϭϮϰ
.LUUHONQRFNRXWPLFHEXWDOVRSURYLGHVWKHILUVWSUHFLVHGHVFULSWLRQRI0)ILORSRGLDDQGHQ
ϭϮϱ
SDVVDQWFRQQHFWLYLW\LQZLOGW\SHPLFH
+HUHZHGHILQH'*SUHV\QDSVHFRQQHFWLYLW\LQZLOGW\SHDQG.LUUHONQRFNRXWPLFHXVLQJ
ϰ
ϭϮϲ
ϭϮϳ
0$7(5,$/6$1'0(7+2'6
ϭϮϴ
$QLPDOV±7LVVXHIURPWZRGLIIHUHQWVHWVRIZLOGW\SHDQGNQRFNRXWOLWWHUPDWHPLFHZHUHLPDJHG
ϭϮϵ
WRFUHDWHIRXULQGHSHQGHQW6%(0GDWDVHWV3OLWWHUPDWHVFRQVLVWLQJRID.LUUHOZLOGW\SH
ϭϯϬ
IHPDOHDQGD.LUUHONQRFNRXWIHPDOHZHUHXVHGIRUGDWDVHWVDQGDQG3OLWWHUPDWHV
ϭϯϭ
IURPDGLIIHUHQWEUHHGLQJSDLUFRQVLVWLQJRID.LUUHOZLOGW\SHPDOHDQGD.LUUHONQRFNRXW
ϭϯϮ
IHPDOHZHUHXVHGIRUGDWDVHWVDQG$OODQLPDOVDQGH[SHULPHQWVZHUHPDLQWDLQHGDQG
ϭϯϯ
FRQGXFWHGLQDFFRUGDQFHZLWKWKH1,+JXLGHOLQHVRQWKHFDUHDQGXVHRIDQLPDOVDQGDSSURYHG
ϭϯϰ
E\WKH>$XWKRU¶V8QLYHUVLW\@,$&8&FRPPLWWHH*HQHUDWLRQRI.LUUHONQRFNRXWPLFHZDV
ϭϯϱ
SUHYLRXVO\GHVFULEHG3ULQFHHWDO
ϭϯϲ
ϭϯϳ
7LVVXH)L[DWLRQDQG3URFHVVLQJ±0LFHZHUHDQHVWKHWL]HGDQGSHUIXVHGDWDUDWHRIPOVPLQ
ϭϯϴ
ZLWKJOXWDUDOGHK\GHDQGSDUDIRUPDOGHK\GHLQ0VRGLXPFDFRG\ODWHEXIIHU7KH
ϭϯϵ
EUDLQVZHUHUHPRYHGDQGVWRUHGRYHUQLJKWDWÛ&LQSHUIXVLRQVROXWLRQ7KHQH[WGD\WKHEUDLQV
ϭϰϬ
ZHUHVOLFHGLQFROG3%6LQWRμPFRURQDOVHFWLRQVDQGVHQWWR5HQRYR1HXUDO,QF
ϭϰϭ
&OHYHODQG2KLR55,'6&5BIRUSURFHVVLQJDQGLPDJLQJRQD=HLVV6LJPD93
ϭϰϮ
6FDQQLQJ(OHFWURQ0LFURVFRSHZLWKD*DWDQ9LHZGRRU
ϭϰϯ
ϭϰϰ
,PDJH6WDFNV±(DFKGDWDVHWFRQWDLQVVHULDOLPDJHV,QGDWDVHWVZLOGW\SHDQG
ϭϰϱ
NQRFNRXWLPDJHVDUH[μPDWQPSL[HOUHVROXWLRQZLWKD]GHSWKRIQPWRFRYHUD
ϭϰϲ
YROXPHRIμP,QGDWDVHWVZLOGW\SHDQGNQRFNRXWLPDJHVDUH[μPDW
ϭϰϳ
QPSL[HOUHVROXWLRQZLWKD]GHSWKRIQPWRFRYHUDYROXPHRIμP%HDPSHQHWUDWLRQ
ϭϰϴ
IRUHDFKZDVQP7UDN(0VRIWZDUHZDVXVHGWRDVVHPEOHLPDJHVIRU'UHFRQVWUXFWLRQ
ϭϰϵ
DQGDQDO\VLV&DUGRQDHWDO55,'6&5B$OOUHFRQVWUXFWLRQDQGDQDO\VLVZDV
ϭϱϬ
GRQHEOLQGWRJHQRW\SH0)SUHV\QDSWLFFRPSOH[HVQHDUWKHFHQWHURIWKHGDWDVHWZHUH
ϱ
ϭϱϭ
UDQGRPO\VHOHFWHGIRUUHFRQVWUXFWLRQ0)SUHV\QDSWLFFRPSOH[HVZLWK0)ILORSRGLDH[WHQGLQJ
ϭϱϮ
RXWRIWKHGDWDVHWZHUHQRWDQDO\]HG
ϭϱϯ
ϭϱϰ
'HWHUPLQDWLRQRI6WUXFWXUHV±0DLQ0)ERXWRQVZHUHLGHQWLILHGDVDQD[RQHQODUJHPHQWILOOHG
ϭϱϱ
ZLWKV\QDSWLFYHVLFOHV69VDGMDFHQWWRSRVWV\QDSWLFGHQVLWLHV36'VRQ&$WKRUQ\
ϭϱϲ
H[FUHVFHQFHV7(V0)ILORSRGLDZHUHLGHQWLILHGDVDSURMHFWLRQH[WHQGLQJDWOHDVWμPRIIRI
ϭϱϳ
WKHPDLQ0)ERXWRQZLWKDFOHDUQHFNVWUXFWXUHODFNLQJ69VIURPWKHPDLQ0)ERXWRQ69
ϭϱϴ
FOXVWHUVDUHGHILQHGDVDJURXSRIRUPRUH69V6\QDSVHIUHH0)ILORSRGLDFRQWDLQHGQR69
ϭϱϵ
FOXVWHUV3DUWLDO0)ILORSRGLDOV\QDSVHVDUHGHILQHGDV69FOXVWHUVZLWKQRDVVRFLDWHG36'
ϭϲϬ
&RPSOHWH0)ILORSRGLDOV\QDSVHVDUHGHILQHGDVDQ69FOXVWHUDGMDFHQWWRD36'&RPSOHWH
ϭϲϭ
DQGSDUWLDOV\QDSVHVFRXOGEHORFDWHGDQ\ZKHUHDORQJWKHOHQJWKRIWKH0)ILORSRGLD(Q
ϭϲϮ
SDVVDQWV\QDSVHVZHUHLGHQWLILHGE\ORFDWLQJDPDLQ0)ERXWRQDQGIROORZLQJLWVD[RQWKURXJK
ϭϲϯ
WKHGDWDVHW(QSDVVDQWV\QDSVHVDUHGHILQHGDVDQ69FOXVWHUORFDWHGLQWKH'*D[RQZLWKDQ
ϭϲϰ
DGMDFHQW36'
ϭϲϱ
ϭϲϲ
'HQGULWH&ODVVLILFDWLRQ±6\QDSVHVZHUHFODVVLILHGDVEHLQJPDGHRQWRD&$QHXURQ*$%$
ϭϲϳ
QHXURQRUXQNQRZQ*$%$GHQGULWHVZHUHLGHQWLILHGDVHLWKHUDVSLQ\RUKDGVLPSOHSURWUXVLRQV
ϭϲϴ
WKDWZHUHQRW&$WKRUQ\H[FUHVFHQFHV7(V)LJXUH$%&$S\UDPLGDOQHXURQVZHUH
ϭϲϵ
LGHQWLILHGE\WKHSUHVHQFHRI7(VPXOWLKHDGHGVSLQHVWKDWV\QDSVHZLWKODUJHPDLQ0)
ϭϳϬ
ERXWRQV)LJXUH&'6RPHV\QDSVHVZHUHPDUNHGDVKDYLQJXQNQRZQSDUWQHUVEHFDXVH
ϭϳϭ
WKH\ZHUHRQWRGHQGULWLFHOHPHQWVWKDWFRXOGQRWEHIROORZHGWKURXJKWKHGDWDVHW
ϭϳϮ
ϭϳϯ
,PDJHPHDVXUHPHQWVVWDWLVWLFVDQGLPDJHSUHVHQWDWLRQ±9ROXPHWULFPHDVXUHPHQWVZHUH
ϭϳϰ
JHQHUDWHGLQ7UDN(0VRIWZDUH6WDWLVWLFVZHUHFDOFXODWHGLQ3ULVP*UDSK3DG55,'
ϭϳϱ
6&5B,QDOOFDVHVGDWDZDVWHVWHGIRUQRUPDOLW\XVLQJWKH'
$JRVWLQR3HDUVRQWHVW
ϭϳϲ
,IDOOGDWDVHWVZHUHQRUPDOSDUDPHWULFWHVWVZHUHXVHGWRFRPSDUHJHQRW\SHV2WKHUZLVH
ϲ
ϭϳϳ
QRQSDUDPHWULFWHVWVZHUHXVHG)RUSXEOLFDWLRQ(0LPDJHVZHUHDGMXVWHGIRUYLVLELOLW\XVLQJ
ϭϳϴ
3KRWRVKRSEXWWKHHQWLUHILHOGRIYLHZDQGDOOLPDJHVZHUHDOZD\VDGMXVWHGLQWKHVDPH
ϭϳϵ
PDQQHU$OODQDO\VLVDQGTXDQWLILFDWLRQZDVGRQHXVLQJUDZLPDJHGDWD
ϭϴϬ
ϭϴϭ
5(68/76
ϭϴϮ
5HFRQVWUXFWLQJ0)3UHV\QDSWLF&RPSOH[HVE\6%(0
ϭϴϯ
ϭϴϰ
FRPSOHWHDQDO\VLVRIWKHLUXOWUDVWUXFWXUHUHTXLUHV'(06HULDOEORFNIDFHVFDQQLQJHOHFWURQ
ϭϴϱ
PLFURVFRS\6%(0H[FHOOHQWO\VXLWVWKLVWDVNDVLWJHQHUDWHVKXQGUHGVRIZHOODOLJQHGVHULDO(0
ϭϴϲ
LPDJHVDWQDQRVFDOHUHVROXWLRQ'HQNDQG+RUVWPDQQ*LYHQWKDW.LUUHOLVKLJKO\
ϭϴϳ
H[SUHVVHGLQ'*QHXURQVDQGLVDVVRFLDWHGZLWKGHYHORSPHQWDOGLVRUGHUVZHDQDO\]HG'*
ϭϴϴ
SUHV\QDSVHVE\(0GXULQJGHYHORSPHQWDWDJHSRVWQDWDOGD\3:HFROOHFWHGIRXU
ϭϴϵ
6%(0GDWDVHWVHDFKFRYHULQJDWOHDVWμPRIWLVVXHIURPWZR3ZLOGW\SHDQGWZR3
ϭϵϬ
.LUUHONQRFNRXWPLFH%HFDXVH.LUUHOLVVHOHFWLYHO\H[SUHVVHGLQ'*DQG*$%$QHXURQVDQG
ϭϵϭ
PHGLDWHVKRPRSKLOLFDGKHVLRQLWVXJJHVWV.LUUHOZLOOUHJXODWH'**$%$V\QDSVHV7KXVLQDOO
ϭϵϮ
GDWDVHWVZHVDPSOHGWKHVWUDWXPOXFLGXPRIWKHGRUVDOKLSSRFDPSXVZKLFKFRQWDLQV'*D[RQV
ϭϵϯ
*$%$QHXURQVDQG&$QHXURQV)LJXUH$
ϭϵϰ
%HFDXVH0)SUHV\QDSWLFFRPSOH[HVDUHJLDQWVWUXFWXUHVZLWKHODERUDWHIHDWXUHV
7UDN(0VRIWZDUH&DUGRQDHWDOZDVXVHGWRPDQXDOO\UHFRQVWUXFWV\QDSVHV
ϭϵϱ
:HUHFRQVWUXFWHG0)SUHV\QDSWLFFRPSOH[HVLQHDFKZLOGW\SHYROXPHLQRQH.LUUHO
ϭϵϲ
NQRFNRXWYROXPHDQGLQWKHRWKHUNQRFNRXWYROXPHIRUDWRWDORIZLOGW\SHDQG
ϭϵϳ
NQRFNRXW0)FRPSOH[HVDQGDOODVVRFLDWHG0)ILORSRGLD$OOWUDFLQJDQGDQDO\VLVZDVGRQHEOLQG
ϭϵϴ
WRJHQRW\SHWRDYRLGVHOHFWLRQELDVHV0DLQ0)ERXWRQVZHUHXQDPELJXRXVO\LGHQWLILHGE\WKHLU
ϭϵϵ
ODUJHVL]HKLJKGHQVLW\RIV\QDSWLFYHVLFOHV69VDQGVFDWWHUHGPLWRFKRQGULD)LJXUH%$IWHU
ϮϬϬ
VHOHFWLRQRIDPDLQ0)ERXWRQDOODWWDFKHGILORSRGLDDQGWKHD[RQZHUHIXOO\WUDFHGDQG
ϮϬϭ
UHFRQVWUXFWHGLQ')LJXUH%&DQG0RYLH5HSUHVHQWDWLYHLPDJHVRIHLJKWUHFRQVWUXFWHG
ϮϬϮ
0)FRPSOH[HVIURPDZLOGW\SHPRXVHDQGD.LUUHONQRFNRXWPRXVHDUHVKRZQ)LJXUH'(
ϳ
ϮϬϯ
ϮϬϰ
0)ILORSRGLDH[LVWLQWKUHHVWDWHVGXULQJGHYHORSPHQW
ϮϬϱ
ϮϬϲ
WKDWORVVRI.LUUHOGRHVQRWFDXVHVWDWLVWLFDOO\VLJQLILFDQWGLIIHUHQFHVLQWKHYROXPHRIWKHPDLQ
ϮϬϳ
0)ERXWRQ)LJXUH$RUWKHPHDQQXPEHURI0)ILORSRGLDSHUPDLQ0)ERXWRQ)LJXUH%
ϮϬϴ
FRPSDUHGWRZLOGW\SHPLFH+RZHYHUFRQVLVWHQWZLWKDSUHYLRXVUHSRUW0DUWLQHWDOD
ϮϬϵ
FXPXODWLYHKLVWRJUDPRIWKHGDWDUHYHDOVWKDW0)SUHV\QDSWLFFRPSOH[HVIURP.LUUHONQRFNRXW
ϮϭϬ
PLFHWHQGWRKDYHIHZHU0)ILORSRGLDSHUPDLQ0)ERXWRQ)LJXUH&
Ϯϭϭ
$IWHUUHFRQVWUXFWLQJDWOHDVW0)SUHV\QDSWLFFRPSOH[HVIURPHDFKJHQRW\SHZHILQG
,WLVFXUUHQWO\XQNQRZQLI0)ILORSRGLDGHQVLW\GLUHFWO\FRUUHODWHVZLWKV\QDSVHGHQVLW\
ϮϭϮ
ZKLFKLVXOWLPDWHO\WKHPRVWFULWLFDOIDFWRULPSDFWLQJ0)FLUFXLWIXQFWLRQ7KHUHIRUHZHQH[W
Ϯϭϯ
DQDO\]HGWKHGHQVLW\DQGSRVWV\QDSWLFWDUJHWVRIV\QDSVHVKRXVHGLQ0)ILORSRGLD:HLGHQWLILHG
Ϯϭϰ
WKUHHVWDWHVZLWKZKLFKWRFODVVLI\0)ILORSRGLD)LUVWVRPH0)ILORSRGLDFRQWDLQQRV\QDSWLF
Ϯϭϱ
YHVLFOH69FOXVWHUVQRUDUHWKH\DGMDFHQWWRDQ\SRVWV\QDSWLFGHQVLWLHV36'VDQ\ZKHUH
Ϯϭϲ
DORQJWKHLUOHQJWK)LJXUH':HUHIHUWRWKHVH0)ILORSRGLDDV³V\QDSVHIUHH´6HFRQGVRPH
Ϯϭϳ
0)ILORSRGLDFRQWDLQFOXVWHUVRIRUPRUH69VEXWQRYLVLEOHFRUUHVSRQGLQJ36')LJXUH(
Ϯϭϴ
3UHV\QDSWLFHOHPHQWVDUHFRPPRQO\IRXQGEHIRUHSRVWV\QDSWLFHOHPHQWVDUHSUHVHQW$PDUDO
Ϯϭϵ
DQG'HQW)ULHGPDQHWDO:LONHHWDODQGZHSRVWXODWHWKDWWKHVH69
ϮϮϬ
FOXVWHUVHLWKHUPDUNIXWXUHV\QDSWLFVLWHVRUKDYH36'VWKDWDUHQRWYLVLEOHGXHWRSODQHRI
ϮϮϭ
VHFWLRQLQJ:HUHIHUWRWKHVHDV³SDUWLDOV\QDSVHV´7KLUGVRPH0)ILORSRGLDFRQWDLQERQDILGH
ϮϮϮ
V\QDSVHVZLWK69FOXVWHUVDGMDFHQWWRD36')LJXUH):HUHIHUWRWKHVHDV³FRPSOHWH
ϮϮϯ
V\QDSVHV´
ϮϮϰ
ϮϮϱ
.LUUHOUHJXODWHVIRUPDWLRQRI0)ILORSRGLDOV\QDSVHV
ϮϮϲ
:KHQFRPSDULQJWKHWRWDOSHUFHQWRI0)ILORSRGLDLQHDFKV\QDSWLFVWDWHE\JHQRW\SH
ϮϮϳ
ZHILQG.LUUHONQRFNRXWPLFHKDYHDJUHDWHUSHUFHQWDJHRI0)ILORSRGLDWKDWDUHV\QDSVHIUHH
ϮϮϴ
DQGDORZHUSHUFHQWDJHWKDWFRQWDLQSDUWLDODQGFRPSOHWHV\QDSVHVFRPSDUHGWRZLOGW\SH
ϴ
ϮϮϵ
)LJXUH$1H[WZHDQDO\]HGWKHDYHUDJHQXPEHURI0)ILORSRGLDWKDWH[LVWLQHDFKVWDWHSHU
ϮϯϬ
PDLQ0)ERXWRQZKLFKFRPSULVHVWKHNH\IXQFWLRQDOXQLWIRUD'*QHXURQ:KHQDQDO\]HGWKLV
Ϯϯϭ
ZD\WKHDYHUDJHQXPEHURIV\QDSVHIUHH0)ILORSRGLDSHUPDLQ0)ERXWRQLVVLPLODUEHWZHHQ
ϮϯϮ
ZLOGW\SHDQGNQRFNRXW)LJXUH%7KLVVXJJHVWVWKDWWKHPLVVLQJ0)ILORSRGLD)LJXUH%&
Ϯϯϯ
DUHWKRVHWKDWZRXOGQRUPDOO\FRQWDLQDV\QDSVH,QVXSSRUWRIWKLVZHREVHUYHUHGXFWLRQVLQ
Ϯϯϰ
ERWKV\QDSVHW\SHVSHUPDLQERXWRQLQ.LUUHONQRFNRXWPLFH)LJXUHV&'DQGZKHQSDUWLDO
Ϯϯϱ
DQGFRPSOHWHV\QDSVHVDUHFRPELQHG.LUUHONQRFNRXWPLFHKDYHVLJQLILFDQWO\IHZHUV\QDSVHV
Ϯϯϲ
SHUPDLQ0)ERXWRQWKDQZLOGW\SHPLFH)LJXUH(7KHDYHUDJHQXPEHURI69VLQV\QDSVHV
Ϯϯϳ
ZDVVLPLODUEHWZHHQWKHJHQRW\SHV)LJXUH)*7KXVRXUGDWDLQGLFDWHWKDWFRPSDUHGWR
Ϯϯϴ
ZLOGW\SHPLFH.LUUHONQRFNRXWPLFHKDYHIHZHU0)ILORSRGLDZLWKV\QDSVHVEXWWKHV\QDSVHV
Ϯϯϵ
WKDWGRIRUPKDYHQRUPDOXOWUDVWUXFWXUH
ϮϰϬ
Ϯϰϭ
.LUUHOVHOHFWLYHO\UHJXODWHV0)ILORSRGLDOV\QDSVHVRQWR*$%$QHXURQV
ϮϰϮ
1H[WZHGHWHUPLQHGWKHSRVWV\QDSWLFWDUJHWVRI0)ILORSRGLDOV\QDSVHV%URDGO\GHILQHG
Ϯϰϯ
WKHUHDUHWZRW\SHVRISRWHQWLDOSRVWV\QDSWLFWDUJHWQHXURQVLQWKHVWUDWXPOXFLGXP*$%$
Ϯϰϰ
QHXURQVDQG&$QHXURQV7KHWZRWDUJHWFHOOW\SHVFDQEHUHDGLO\LGHQWLILHGE\WKHLUGHQGULWLF
Ϯϰϱ
PRUSKRORJ\*$%$QHXURQVFDQEHDVSLQ\RUVSLQ\EXWWKHLUGHQGULWHVDOZD\VODFNPXOWLKHDGHG
Ϯϰϲ
7(VSLQHV)LJXUH$%&RQYHUVHO\&$GHQGULWHVDOZD\VKDYHPXOWLKHDGHG7(VSLQHV
Ϯϰϳ
PDNLQJPXOWLSOHV\QDSVHVZLWKPDLQ0)ERXWRQV)LJXUH&'
Ϯϰϴ
,WLVZLGHO\DFFHSWHGWKDW0)ILORSRGLDRQO\V\QDSVHRQWR*$%$QHXURQV$FViG\HWDO
Ϯϰϵ
$PDUDO&ODLERUQHHWDO7KHUHIRUHZHZHUHVXUSULVHGWRREVHUYHWKDWLQ
ϮϱϬ
3ZLOGW\SHPLFH0)ILORSRGLDIRUPFRPSOHWHV\QDSVHVZLWKVLPLODUIUHTXHQF\RQWR*$%$
Ϯϱϭ
GHQGULWHV)LJXUH(),DV&$GHQGULWHV)LJXUH*,0RVW*$%$GHQGULWHVFRQWDFWHGE\
ϮϱϮ
0)ILORSRGLDKDYHVLPSOHVSLQ\SURWUXVLRQVDVSUHYLRXVO\REVHUYHG$FViG\HWDODQG
Ϯϱϯ
V\QDSWLFFRQWDFWVDUHIRXQGRQERWK*$%$GHQGULWLFVKDIWV)LJXUH(DQGVSLQ\SURWUXVLRQV
ϵ
Ϯϱϰ
)LJXUH)6LPLODUO\ZKHQ0)ILORSRGLDV\QDSVHRQWR&$QHXURQVZHREVHUYHGV\QDSWLF
Ϯϱϱ
FRQWDFWVRQ&$GHQGULWLFVKDIWV)LJXUH*DQG7(VSLQHV)LJXUH+
Ϯϱϲ
0)ILORSRGLDDOVRV\QDSVHRQWRERWK*$%$DQG&$QHXURQVLQ.LUUHONQRFNRXWPLFH
Ϯϱϳ
+RZHYHUNQRFNRXWPLFHKDYHDFOHDUVKLIWLQWKHGLVWULEXWLRQRISRVWV\QDSWLFSDUWQHUV
Ϯϱϴ
&RPSDUHGWRZLOGW\SHPLFH.LUUHONQRFNRXWPLFHGHYHORSVLJQLILFDQWO\IHZHUFRPSOHWH
Ϯϱϵ
V\QDSVHVZLWK*$%$QHXURQV)LJXUH-,QFRQWUDVWWKHQXPEHURIFRPSOHWH0)ILORSRGLDO
ϮϲϬ
V\QDSVHVPDGHRQWR&$QHXURQVLVVLPLODUEHWZHHQZLOGW\SHDQG.LUUHONQRFNRXWPLFH
Ϯϲϭ
)LJXUH.7KHQHWHIIHFWRIWKHVHFKDQJHVLVDGUDPDWLFGHFUHDVHLQWKHUDWLRRI0)ILORSRGLDO
ϮϲϮ
V\QDSVHVPDGHRQWR*$%$YHUVXV&$QHXURQVLQPLFHODFNLQJ.LUUHOFRPSDUHGWRZLOGW\SH
Ϯϲϯ
)LJXUH/
Ϯϲϰ
:HWKHQH[DPLQHGLIILORSRGLDV\QDSVLQJZLWK*$%$QHXURQVKDYHDGLIIHUHQW
Ϯϲϱ
PRUSKRORJLFDOVLJQDWXUHIURPILORSRGLDV\QDSVLQJZLWK&$QHXURQVDQGLIWKHVHFKDUDFWHULVWLFV
Ϯϲϲ
FKDQJHXSRQ.LUUHOORVV)LJXUH03&ŽƌƚŚŝƐĂŶĂůLJƐŝƐ͕ǁĞŽŶůLJŝŶĐůƵĚĞĚŽŶůLJĨŝůŽƉŽĚŝĂƚŚĂƚ
Ϯϲϳ
ŚĂǀĞĂĐŽŵƉůĞƚĞƐLJŶĂƉƐĞŽŶƚŽĂŶŝĚĞŶƚŝĨŝĞĚ'ŽƌϯŶĞƵƌŽŶ͘ĞĐĂƵƐĞ<ŝƌƌĞůϯŬŶŽĐŬŽƵƚ
Ϯϲϴ
ŵŝĐĞŚĂǀĞƐŝŐŶŝĨŝĐĂŶƚůLJĨĞǁĞƌĨŝůŽƉŽĚŝĂͲ'ƐLJŶĂƉƐĞƐƚŚĂŶǁŝůĚƚLJƉĞŵŝĐĞ͕ƚŚĞƐĂŵƉůĞƐŝnjĞĨŽƌ
Ϯϲϵ
ƚŚŝƐĐĂƚĞŐŽƌLJǁĂƐƐŵĂůů;ŶсϰĨŝůŽƉŽĚŝĂǁŝƚŚϱĐŽŵƉůĞƚĞƐLJŶĂƉƐĞƐͿ͘EŽŶĞƚŚĞůĞƐƐ͕ƚŚŝƐŶĞǁ
ϮϳϬ
ĂŶĂůLJƐŝƐŝŶĚŝĐĂƚĞƐƚŚĂƚ͕ŽŶĂǀĞƌĂŐĞ͕ĨŝůŽƉŽĚŝĂƐLJŶĂƉƐŝŶŐŽŶƚŽĞŝƚŚĞƌ'ŽƌϯŶĞƵƌŽŶƐŚĂǀĞĂ
Ϯϳϭ
ƐŝŵŝůĂƌŵŽƌƉŚŽůŽŐLJĂŶĚ^sĐŽŵƉŽƐŝƚŝŽŶŝŶǁŝůĚƚLJƉĞŵŝĐĞ;&ŝŐƵƌĞϰK͕WͿ͘,ŽǁĞǀĞƌ͕ǁĞĚŝĚ
ϮϳϮ
ŽďƐĞƌǀĞƐŽŵĞĚŝĨĨĞƌĞŶĐĞƐŝŶĨŝůŽƉŽĚŝĂŵŽƌƉŚŽůŽŐLJďĞƚǁĞĞŶǁŝůĚƚLJƉĞĂŶĚ<ŝƌƌĞůϯŬŶŽĐŬŽƵƚ
Ϯϳϯ
ŵŝĐĞ͘/ŶƉĂƌƚŝĐƵůĂƌ͕ƚŚĞƚŽƚĂůǀŽůƵŵĞŽĨďŽƚŚĐůĂƐƐĞƐŽĨĨŝůŽƉŽĚŝĂ;ƚŚŽƐĞĐŽŶŶĞĐƚĞĚƚŽĞŝƚŚĞƌ
Ϯϳϰ
'ŽƌϯŶĞƵƌŽŶƐͿŝƐŝŶĐƌĞĂƐĞĚƵƉŽŶ<ŝƌƌĞůϯůŽƐƐ;&ŝŐƵƌĞϰDͿ͘&ŽƌĨŝůŽƉŽĚŝĂƐLJŶĂƉƐŝŶŐŽŶƚŽ
Ϯϳϱ
ϯŶĞƵƌŽŶƐ͕ƚŚĞŝŶĐƌĞĂƐĞĚǀŽůƵŵĞŝƐĐůĞĂƌůLJĚƵĞƚŽŝŶĐƌĞĂƐĞĚůĞŶŐƚŚ;&ŝŐƵƌĞϰEͿĂŶĚŶŽƚĂŶ
Ϯϳϲ
ŝŶĐƌĞĂƐĞŝŶĐƌŽƐƐƐĞĐƚŝŽŶĂůĂƌĞĂ;&ŝŐƵƌĞϰKͿ͘&ŽƌĨŝůŽƉŽĚŝĂƐLJŶĂƉƐŝŶŐŽŶƚŽ'ŶĞƵƌŽŶƐ͕ƚŚĞ
Ϯϳϳ
ŝŶĐƌĞĂƐĞĚǀŽůƵŵĞŵĂLJďĞĚƵĞƚŽĂĐŽŵďŝŶĂƚŝŽŶŽĨŝŶĐƌĞĂƐĞĚůĞŶŐƚŚĂŶĚĐƌŽƐƐƐĞĐƚŝŽŶĂůĂƌĞĂďƵƚ
ϭϬ
Ϯϳϴ
ŶĞŝƚŚĞƌƉĂƌĂŵĞƚĞƌŝƐƐŝŐŶŝĨŝĐĂŶƚůLJĚŝĨĨĞƌĞŶƚ;&ŝŐƵƌĞƐϰE͕KͿĂŶĚǁĞŚĞƐŝƚĂƚĞƚŽĚƌĂǁƐƚƌŽŶŐ
Ϯϳϵ
ĐŽŶĐůƵƐŝŽŶƐďĂƐĞĚŽŶƚŚĞůŽǁƐĂŵƉůĞƐŝnjĞŽĨĨŝůŽƉŽĚŝĂƐLJŶĂƉƐŝŶŐǁŝƚŚ'ŶĞƵƌŽŶƐŝŶ<ŝƌƌĞůϯ
ϮϴϬ
ŬŶŽĐŬŽƵƚŵŝĐĞ͘
Ϯϴϭ
ϮϴϮ
(QSDVVDQWFRQQHFWLYLW\LVXQSHUWXUEHGE\.LUUHOORVV
Ϯϴϯ
1H[WZHDQDO\]HGIRUPDWLRQRI'*HQSDVVDQWV\QDSVHVLQZLOGW\SHDQG.LUUHO
Ϯϴϰ
NQRFNRXWPLFH'*HQSDVVDQWV\QDSVHVDUHUHDGLO\LGHQWLILDEOHE\(0DVFOXVWHUVRI69VLQWKH
Ϯϴϱ
D[RQVKDIWDGMDFHQWWRD36')LJXUH$&/LNH0)ILORSRGLDHQSDVVDQWV\QDSVHVDUH
Ϯϴϲ
WKRXJKWWRV\QDSVHH[FOXVLYHO\RQWR*$%$QHXURQV$FViG\HWDO*LYHQWKDW.LUUHOLV
Ϯϴϳ
H[SUHVVHGVSHFLILFDOO\E\'*DQG*$%$QHXURQVZHUHDVRQHGWKDWLQDGGLWLRQWRUHJXODWLQJ'*
Ϯϴϴ
0)ILORSRGLDWR*$%$V\QDSVHV.LUUHOPD\DOVRUHJXODWHIRUPDWLRQRI'*HQSDVVDQWWR*$%$
Ϯϴϵ
V\QDSVHV&RQVLVWHQWZLWKWKHH[LVWLQJOLWHUDWXUHRI'*HQSDVVDQWV\QDSVHVREVHUYHGLQ
ϮϵϬ
RXUGDWDVHWVV\QDSVHZLWK*$%$QHXURQV)LJXUH'%XWLQWHUHVWLQJO\WKHORVVRI.LUUHOKDV
Ϯϵϭ
QRHIIHFWRQWKHGHQVLW\RIHQSDVVDQWV\QDSVHVLQ'*D[RQV)LJXUH(7KLVLQGLFDWHV.LUUHO
ϮϵϮ
KDVDKLJKO\VSHFLILFIXQFWLRQUHJXODWLQJ'*0)ILORSRGLDWR*$%$QHXURQV\QDSVHVEXWQRW'*
Ϯϵϯ
D[RQWR*$%$QHXURQV\QDSVHV
Ϯϵϰ
Ϯϵϱ
2WKHUPRUSKRORJLFDOIHDWXUHVRI0)ILORSRGLD
Ϯϵϲ
*LYHQWKDWWKLVLVWKHILUVWLQGHSWK'DQDO\VLVRI0)ILORSRGLDFRQQHFWLYLW\ZHQRWHGWKH
Ϯϵϳ
SUHVHQFHRIVHYHUDOXQXVXDO0)ILORSRGLDVWUXFWXUHV)LUVWZHRFFDVLRQDOO\REVHUYHGJURZWK
Ϯϵϴ
FRQHOLNHHQGLQJVDWWKHWLSVRI0)ILORSRGLD)LJXUH$&7KHVHVWUXFWXUHVKDGQRV\QDSVHV
Ϯϵϵ
DQGIHZLIDQ\V\QDSWLFYHVLFOHVDORQJWKHLUOHQJWK*LYHQWKDWRXUGDWDLVIURP3PLFHWKDW
ϯϬϬ
DUHQHDUWKHSHDNRI'*V\QDSWRJHQHVLVWKH\OLNHO\UHSUHVHQWQHZO\IRUPHG0)ILORSRGLDLQ
ϯϬϭ
VHDUFKRIDWDUJHWQHXURQ6HFRQGZHREVHUYHEUDQFKHG0)ILORSRGLD)LJXUH$'(:H
ϯϬϮ
VRXJKWWRGHWHUPLQHLI0)ILORSRGLDIURP.LUUHONQRFNRXWPLFHKDYHIHZHUEUDQFKHVEHFDXVHDQ
ϭϭ
ϯϬϯ
RUWKRORJRI.LUUHO6<*UHJXODWHVD[RQDOEUDQFKJHQHUDWLRQLQC. elegansPRWRUQHXURQV
ϯϬϰ
&KLDHWDO+RZHYHUZHILQGOLWWOHGLIIHUHQFHLQILORSRGLDEUDQFKLQJEHWZHHQZLOGW\SHDQG
ϯϬϱ
.LUUHONQRFNRXWPLFH)LJXUH(7KLUGWKRXJKLWLVRIWHQDVVXPHGWKDW0)ILORSRGLDFRQWDLQ
ϯϬϲ
RQO\RQHWHUPLQDOV\QDSVHDWWKH0)ILORSRGLDWLSZHREVHUYHWKDWLQGLYLGXDO0)ILORSRGLDFDQ
ϯϬϳ
KRXVHPXOWLSOHV\QDSVHVZLWKVRPHORFDWHGLQWKH0)ILORSRGLDVKDIW)LJXUH$)+,WZLOOEH
ϯϬϴ
LQWHUHVWLQJWRGHWHUPLQHLQDIXWXUHVWXG\ZKHWKHUWKHVHIHDWXUHVRIJURZWKFRQHOLNHHQGV
ϯϬϵ
EUDQFKHVDQGPXOWLSOHV\QDSVHVDUHGHYHORSPHQWDOLQRULJLQRUDUHG\QDPLFDOO\PDLQWDLQHGLQ
ϯϭϬ
DGXOWPLFHWRDOORZFLUFXLWIOH[LELOLW\
ϯϭϭ
ϯϭϮ
',6&866,21
ϯϭϯ
+HUHZHSUHVHQWDULJRURXVXOWUDVWUXFWXUDODQDO\VLVRI0)ILORSRGLDOFRQQHFWLYLW\LQWKH
ϯϭϰ
GHYHORSLQJPDPPDOLDQEUDLQLQZLOGW\SHDQG.LUUHONQRFNRXWPLFH)LUVWZHGLVFRYHUHG
ϯϭϱ
FRQWUDU\WRSUHYLRXVUHSRUWV0)ILORSRGLDV\QDSVHZLWKVLPLODUIUHTXHQF\RQWR*$%$DQG&$
ϯϭϲ
QHXURQV6HFRQGZHVKRZ.LUUHOLVVHOHFWLYHO\UHTXLUHGIRUQRUPDOV\QDSVHGHQVLW\EHWZHHQ
ϯϭϳ
'*0)ILORSRGLDDQG*$%$QHXURQVEXWQRW&$QHXURQV7KLUGZHGLVFRYHUHG.LUUHOGRHV
ϯϭϴ
QRWUHJXODWH'**$%$HQSDVVDQWV\QDSVHIRUPDWLRQ
ϯϭϵ
7RRXUNQRZOHGJHWKLVLVWKHILUVWUHSRUWRI0)ILORSRGLDV\QDSVLQJZLWK&$S\UDPLGDO
ϯϮϬ
QHXURQV3UHYLRXVZRUNLQGLFDWHGWKDW0)ILORSRGLDRQO\V\QDSVHRQWR*$%$QHXURQV$FViG\
ϯϮϭ
HWDO0F%DLQ5ROOHQKDJHQ+RZHYHUSULRUZRUNH[DPLQHG0)ILORSRGLD
ϯϮϮ
FRQQHFWLYLW\DWDQXQVSHFLILHGDJHZLWKRXWWKHXVHRIFXUUHQWKLJKWKURXJKSXW(0WHFKQRORJLHV
ϯϮϯ
$FViG\HWDO,QFRQWUDVWRXUVWXG\ZDVGRQHRQMXYHQLOH3DQLPDOVDQGZHDQDO\]HG
ϯϮϰ
!0)ILORSRGLDSHUJHQRW\SHSURYLGLQJDQLPSURYHGVDPSOHVL]H7KXVLWLVSRVVLEOH0)
ϯϮϱ
ILORSRGLD&$V\QDSVHVH[LVWLQWKHDGXOWDQGZHUHSUHYLRXVO\PLVVHGGXHWRIDFWRUVVXFKDV
ϯϮϲ
VDPSOHVL]HRULPDJLQJPHWKRG,WLVDOVRSRVVLEOH0)ILORSRGLD&$V\QDSVHVDUHD
ϯϮϳ
GHYHORSPHQWDOSKHQRPHQRQWKDWDUHVHOHFWLYHO\SUXQHGE\DGXOWKRRG)XWXUHVWXGLHVRQDGXOW
ϯϮϴ
PLFHDUHQHHGHGWRGLVWLQJXLVKEHWZHHQWKHVHWZRSRVVLELOLWLHV,QDGGLWLRQZHREVHUYHGWKDW
ϭϮ
ϯϮϵ
ILORSRGLDV\QDSVLQJRQWRHLWKHU*$%$RU&$QHXURQVKDYHDVLPLODUPRUSKRORJ\DQG69
ϯϯϬ
FRPSRVLWLRQLQZLOGW\SHPLFHEXWWKRVHV\QDSVLQJRQWR&$QHXURQVLQ.LUUHONQRFNRXWPLFH
ϯϯϭ
DUHVLJQLILFDQWO\ORQJHU%HFDXVH.LUUHONQRFNRXWPLFHKDYHVRIHZILORSRGLDV\QDSVLQJZLWK
ϯϯϮ
*$%$QHXURQVZHSRVWXODWHWKHLQFUHDVHGOHQJWKRIILORSRGLDV\QDSVLQJZLWK&$QHXURQV
ϯϯϯ
FRXOGEHGXHWRWKHH[WUDDYDLODELOLW\RIUHVRXUFHVLQFUHDVHGGULYHWRVHDUFKIRUDSRVWV\QDSWLF
ϯϯϰ
SDUWQHURUGHOD\HGPDWXULW\
ϯϯϱ
:K\PLJKWWKH0)FLUFXLWEHQHILWIURPKDYLQJ0)ILORSRGLDV\QDSVHRQWRERWK*$%$DQG
ϯϯϲ
&$QHXURQV"0)ILORSRGLDDUHKLJKO\SODVWLFPRWLOHVWUXFWXUHV7DVKLURHWDODQGDUH
ϯϯϳ
OLNHO\EHWWHUVXLWHGWRTXLFNO\DGDSWWRFKDQJLQJFLUFXLWQHHGVWKDQWKHODUJHDQFKRUHGPDLQ0)
ϯϯϴ
ERXWRQ2YHUWLPHFLUFXLWVDGDSWWRPDLQWDLQDKRPHRVWDWLFEDODQFHRIH[FLWDWRU\DQGLQKLELWRU\
ϯϯϵ
DFWLYLW\1HOVRQDQG9DODNK7XUULJLDQRDQGKDYLQJIOH[LEOH0)ILORSRGLDWKDWFDQ
ϯϰϬ
LQFUHDVHRUGHFUHDVHLQSXWWR*$%$YHUVXV&$QHXURQVPD\EHRQHZD\WKHKLSSRFDPSXV
ϯϰϭ
PDLQWDLQVWKLVEDODQFH,WZDVVKRZQWKDW0)SUHV\QDSWLFFRPSOH[HVDQG0)ILORSRGLDLQFUHDVH
ϯϰϮ
LQUHVSRQVHWRHQULFKHGHQYLURQPHQWDQGOHDUQLQJWDVNVVXFKDVIHDUFRQGLWLRQLQJDQGWKH
ϯϰϯ
0RUULVZDWHUPD]H%HGQDUHNDQG&DURQL5XHGLJHUHWDO+RZHYHULWLVQRW
ϯϰϰ
NQRZQLIDQGZKHUHWKHVHQHZO\DGGHG0)ILORSRGLDPDNHV\QDSVHV.QRZLQJZKHWKHUWKHVH
ϯϰϱ
QHZO\DGGHGDGXOW0)ILORSRGLDSUHIHUHQWLDOO\V\QDSVHRQWR*$%$QHXURQVRU&$QHXURQV
ϯϰϲ
ZRXOGSURYLGHLQVLJKWWRZDUGXQGHUVWDQGLQJKLSSRFDPSDOFLUFXLWFKDQJHVXQGHUO\LQJOHDUQLQJ
ϯϰϳ
DQGPHPRU\
ϯϰϴ
,QDGGLWLRQWRPRUHSUHFLVHO\GHILQLQJ0)FRQQHFWLYLW\LQZLOGW\SHPLFHRXUVWXG\
ϯϰϵ
VXJJHVWV.LUUHOLVDKLJKO\VHOHFWLYHWDUJHWVSHFLILFLW\PROHFXOH:HVKRZ.LUUHOLVVSHFLILFDOO\
ϯϱϬ
UHTXLUHGWRHVWDEOLVKWKHDSSURSULDWHGHQVLW\RI0)ILORSRGLD*$%$V\QDSVHVEXWQRW0)
ϯϱϭ
ILORSRGLD&$V\QDSVHVRU'*HQSDVVDQW*$%$V\QDSVHVGXULQJGHYHORSPHQW,WLVSRVVLEOH
ϯϱϮ
WKDWORVVRI.LUUHOFDXVHVDSHUPDQHQWUHGXFWLRQLQILORSRGLD*$%$V\QDSVHVRUDOWHUQDWLYHO\
ϯϱϯ
WKHORVVRI.LUUHOPD\FDXVHDGHOD\HGPDWXUDWLRQRIILORSRGLD*$%$V\QDSVHV7KHFXUUHQW
ϯϱϰ
VWXG\FRQGXFWHGDWRQHGHYHORSPHQWDOWLPHSRLQWFDQQRWGLVWLQJXLVKEHWZHHQWKHVHSRVVLELOLWLHV
ϭϯ
ϯϱϱ
EXWIXWXUHZRUNDQDO\]LQJILORSRGLDOV\QDSVHGHYHORSPHQWRYHUWLPHFDQDGGUHVVWKLV
ϯϱϲ
PHFKDQLVP1RQHWKHOHVV.LUUHOLVDKRPRSKLOLFWUDQVPHPEUDQHPROHFXOHVHOHFWLYHO\
ϯϱϳ
H[SUHVVHGLQ'*DQG*$%$QHXURQV0DUWLQHWDO7KXVRXUUHVXOWVVXSSRUWWKH
ϯϱϴ
K\SRWKHVLVWKDWWUDQVFHOOXODUKRPRSKLOLF.LUUHOELQGLQJVHOHFWLYHO\VWDELOL]HV0)ILORSRGLDO
ϯϱϵ
FRQWDFWVEHWZHHQ'*D[RQVDQG*$%$GHQGULWHV.LUUHOPD\DOVRSURYLGHDSUHDQG
ϯϲϬ
SRVWV\QDSWLFVLJQDOWRDFWLYHO\LQGXFHV\QDSVHIRUPDWLRQYLDLWVLQWUDFHOOXODUGRPDLQ,QVXSSRUW
ϯϲϭ
PDPPDOLDQ.LUUHOZDVVKRZQWRLQWHUDFWZLWKWKHV\QDSWLFPROHFXOHV&$6.%KDOODHWDO
ϯϲϮ
*HUNHHWDO3,&.+|KQHHWDODQGUHFHQWO\36'5RKHWDO
ϯϲϯ
EXWGHWDLOVRIKRZ.LUUHOVLJQDOVLQYLYRUHPDLQVXQNQRZQ
ϯϲϰ
,WLVQRWHZRUWK\WKDWQRWDOO0)ILORSRGLD*$%$V\QDSVHVDUHHOLPLQDWHGLQ.LUUHO
ϯϲϱ
NQRFNRXWPLFH.LUUHOLVZLGHO\H[SUHVVHGE\'*QHXURQVEXWLVH[SUHVVHGRQO\E\D
ϯϲϲ
VXESRSXODWLRQRIFDOELQGLQSRVLWLYH*$%$QHXURQV7KHVH.LUUHOSRVLWLYHLQWHUQHXURQVPDNHXS
ϯϲϳ
MXVWRIDOO*$%$QHXURQVSUHVHQWLQDUHD&$0DUWLQHWDO7KXV.LUUHONQRFNRXW
ϯϲϴ
PLFHPD\VHOHFWLYHO\ODFNDOOLQSXWWRWKLVVXESRSXODWLRQRI*$%$QHXURQVDQGWKH0)ILORSRGLD
ϯϲϵ
*$%$V\QDSVHVWKDWUHPDLQPD\FRQQHFW'*QHXURQVWR.LUUHOQHJDWLYH*$%$HUJLFQHXURQV
ϯϳϬ
YLDDOWHUQDWLYHPHFKDQLVPV$GGLWLRQDOO\ZHREVHUYHGQRFKDQJHLQHQSDVVDQWV\QDSVH
ϯϳϭ
QXPEHUXSRQORVVRI.LUUHO,WLVSRVVLEOH.LUUHOGRHVQRWORFDOL]HDORQJWKH'*D[RQRUWKDWHQ
ϯϳϮ
SDVVDQWV\QDSVHVVHOHFWLYHO\WDUJHW.LUUHOQHJDWLYH*$%$QHXURQV7KHODWWHUSRVVLELOLW\ZRXOG
ϯϳϯ
VXJJHVW'*QHXURQVXVH0)ILORSRGLDODQGHQSDVVDQWV\QDSVHVWRGLIIHUHQWLDOO\FRPPXQLFDWH
ϯϳϰ
ZLWKGLVWLQFWW\SHVRI*$%$QHXURQV
ϯϳϱ
,QFRQFOXVLRQRXUUHVXOWVLQGLFDWH.LUUHOLVUHTXLUHGIRUIRUPDWLRQRIDVXEVHWRI
ϯϳϲ
KLSSRFDPSDO'**$%$V\QDSVHV/RVVRI'**$%$V\QDSVHVLVH[SHFWHGWRUHGXFHIHHG
ϯϳϳ
IRUZDUGLQKLELWLRQWR&$QHXURQVDQGOLNHO\H[SODLQVWKHSULRUREVHUYDWLRQWKDWMXYHQLOH.LUUHO
ϯϳϴ
NQRFNRXWPLFHKDYHRYHUDFWLYH&$QHXURQV0DUWLQHWDO6HOHFWLYHORVVRI'**$%$
ϯϳϵ
V\QDSVHVPD\FRQWULEXWHWRWKHHWLRORJ\RI.LUUHOGHSHQGHQWQHXURGHYHORSPHQWDOGLVRUGHUVE\
ϯϴϬ
FDXVLQJDQH[FLWDWLRQLQKLELWLRQLPEDODQFHLQKLSSRFDPSDOFLUFXLWVDQGDEHWWHUXQGHUVWDQGLQJRI
ϭϰ
ϯϴϭ
WKHSUHFLVHV\QDSWLFGHIHFWVFDXVHGE\ORVVRI.LUUHOPD\XOWLPDWHO\OHDGWRPRUHHIIHFWLYH
ϯϴϮ
WUHDWPHQWV
ϯϴϯ
ϯϴϰ
5()(5(1&(6
ϯϴϱ
ϯϴϲ
$FViG\/.DPRQGL$6tN$)UHXQG7DQG%X]ViNL**$%$HUJLFFHOOVDUHWKH
PDMRUSRVWV\QDSWLFWDUJHWVRIPRVV\ILEHUVLQWKHUDWKLSSRFDPSXV-1HXURVFL18±
ϯϴϳ
ϯϴϴ
$PDUDO'*6\QDSWLFH[WHQVLRQVIURPWKHPRVV\ILEHUVRIWKHIDVFLDGHQWDWD$QDW
(PEU\RO155±
ϯϴϵ
ϯϵϬ
ϯϵϭ
$PDUDO'*DQG'HQW-$'HYHORSPHQWRIWKHPRVV\ILEHUVRIWKHGHQWDWHJ\UXV,$
OLJKWDQGHOHFWURQPLFURVFRSLFVWXG\RIWKHPRVV\ILEHUVDQGWKHLUH[SDQVLRQV-RXUQDORI
&RPSDUDWLYH1HXURORJ\195±
ϯϵϮ
ϯϵϯ
%HGQDUHN(DQG&DURQL3ȕ$GGXFLQLVUHTXLUHGIRUVWDEOHDVVHPEO\RIQHZV\QDSVHV
DQGLPSURYHGPHPRU\XSRQHQYLURQPHQWDOHQULFKPHQW1HXURQ69±
ϯϵϰ
ϯϵϱ
%HQ'DYLG(DQG6KLIPDQ61HWZRUNVRIQHXURQDOJHQHVDIIHFWHGE\FRPPRQDQG
UDUHYDULDQWVLQDXWLVPVSHFWUXPGLVRUGHUV3/R6*HQHW8H
ϯϵϲ
ϯϵϳ
ϯϵϴ
%KDOOD./XR<%XFKDQ7%HDFKHP0$*X]DXVNDV*)/DGG6%UDWFKHU6-
6FKURHU5-%DOVDPR-'X3RQW%5HWDO$OWHUDWLRQVLQ&'+DQG.,55(/LQ
SDWLHQWVZLWKPLOGWRVHYHUHLQWHOOHFWXDOGLVDELOLW\$P-+XP*HQHW83±
ϯϵϵ
ϰϬϬ
%UDJLQ$-DQGR*1DGDVG\=+HWNH-:LVH.DQG%X]ViNL**DPPD
+]RVFLOODWLRQLQWKHKLSSRFDPSXVRIWKHEHKDYLQJUDW-1HXURVFL15±
ϰϬϭ
ϰϬϮ
ϰϬϯ
&DUGRQD$6DDOIHOG66FKLQGHOLQ-$UJDQGD&DUUHUDV,3UHLELVFK6/RQJDLU0
7RPDQFDN3+DUWHQVWHLQ9DQG'RXJODV5-7UDN(0VRIWZDUHIRUQHXUDOFLUFXLW
UHFRQVWUXFWLRQ3ORV2QH7H
ϰϬϰ
ϰϬϱ
ϰϬϲ
&KHQJ<4XLQQ-)DQG:HLVV/$$QH47/PDSSLQJDSSURDFKUHYHDOVWKDWUDUH
YDULDQWVLQWKH6(0$$UHJXODWRU\QHWZRUNLPSDFWDXWLVPULVN+XP0RO*HQHW22±
ϰϬϳ
ϰϬϴ
&KLD3+&KHQ%/L35RVHQ0.DQG6KHQ./RFDO)DFWLQQHWZRUNOLQNV
V\QDSVHIRUPDWLRQDQGD[RQEUDQFKLQJ&HOO156±
ϰϬϵ
ϰϭϬ
ϰϭϭ
ϰϭϮ
&KRL6<+DQ.&XWIRUWK7&KXQJ:3DUN+/HH'.LP5.LP0+&KRL<
6KHQ.HWDO0LFHODFNLQJWKHV\QDSWLFDGKHVLRQPROHFXOH1HSK.LUUHOGLVSOD\
PRGHUDWHK\SHUDFWLYLW\DQGGHIHFWLYHQRYHOREMHFWSUHIHUHQFH)URQWLHUVLQ&HOOXODU
1HXURVFLHQFH9
ϰϭϯ
ϰϭϰ
&ODLERUQH%-$PDUDO'*DQG&RZDQ:0$OLJKWDQGHOHFWURQPLFURVFRSLF
DQDO\VLVRIWKHPRVV\ILEHUVRIWKHUDWGHQWDWHJ\UXV-&RPS1HXURO246±
ϭϱ
ϰϭϱ
ϰϭϲ
ϰϭϳ
'H5XEHLV6+H;*ROGEHUJ$33RXOWQH\&66DPRFKD.&LFHN$(.RX</LX
/)URPHU0:DONHU6HWDO6\QDSWLFWUDQVFULSWLRQDODQGFKURPDWLQJHQHVGLVUXSWHG
LQDXWLVP1DWXUH515±
ϰϭϴ
ϰϭϵ
ϰϮϬ
'HOOHU71LWVFK5DQG)URWVFKHU0$VVRFLDWLRQDODQGFRPPLVVXUDODIIHUHQWVRI
SDUYDOEXPLQLPPXQRUHDFWLYHQHXURQVLQWKHUDWKLSSRFDPSXVDFRPELQHGLPPXQRF\WRFKHPLFDO
DQG3+$/VWXG\-&RPS1HXURO350±
ϰϮϭ
ϰϮϮ
'HQN:DQG+RUVWPDQQ+6HULDO%ORFN)DFH6FDQQLQJ(OHFWURQ0LFURVFRS\WR
5HFRQVWUXFW7KUHH'LPHQVLRQDO7LVVXH1DQRVWUXFWXUH3/26%LRORJ\2H
ϰϮϯ
ϰϮϰ
ϰϮϱ
)DXONQHU5/-DQJ0+/LX;%'XDQ;6DLORU.$.LP-<*H6-RQHV(*
0LQJ*/6RQJ+HWDO'HYHORSPHQWRIKLSSRFDPSDOPRVV\ILEHUV\QDSWLFRXWSXWV
E\QHZQHXURQVLQWKHDGXOWEUDLQ3URF1DWO$FDG6FL86D105±
ϰϮϲ
ϰϮϳ
ϰϮϴ
)ULHGPDQ+9%UHVOHU7*DUQHU&&DQG=LY1($VVHPEO\RIQHZLQGLYLGXDO
H[FLWDWRU\V\QDSVHVWLPHFRXUVHDQGWHPSRUDORUGHURIV\QDSWLFPROHFXOHUHFUXLWPHQW1HXURQ
27±
ϰϮϵ
ϰϯϬ
ϰϯϭ
)URWVFKHU00RVV\ILEUHVIRUPV\QDSVHVZLWKLGHQWLILHGS\UDPLGDOEDVNHWFHOOVLQWKH
&$UHJLRQRIWKHJXLQHDSLJKLSSRFDPSXVDFRPELQHG*ROJLHOHFWURQPLFURVFRSHVWXG\-
1HXURF\WRO14±
ϰϯϮ
ϰϯϯ
ϰϯϰ
*HUNH3%HQ]LQJ7+|KQH0.LVSHUW$)URWVFKHU0:DO]*DQG.UHW]2
1HXURQDOH[SUHVVLRQDQGLQWHUDFWLRQZLWKWKHV\QDSWLFSURWHLQ&$6.VXJJHVWDUROHIRU1HSK
DQG1HSKLQV\QDSWRJHQHVLV-&RPS1HXURO498±
ϰϯϱ
ϰϯϲ
ϰϯϳ
*HUNH36HOOLQ/.UHW]23HWUDVFKND'=HQWJUDI+%HQ]LQJ7DQG:DO]*
1(3+LVORFDWHGDWWKHJORPHUXODUVOLWGLDSKUDJPLQWHUDFWVZLWKQHSKULQDQGLVFOHDYHGIURP
SRGRF\WHVE\PHWDOORSURWHLQDVHV-$P6RF1HSKURO16±
ϰϯϴ
ϰϯϵ
ϰϰϬ
ϰϰϭ
*XHULQ$6WDYURSRXORV'-'LDE<&KpQLHU6&KULVWHQVHQ+.DKU:+$%DEXO+LUML
5DQG&KLWD\DW',QWHUVWLWLDOGHOHWLRQRITLPSOLFDWLQJWKH.,55(/JHQHLQWKH
QHXURFRJQLWLYHGHOD\DVVRFLDWHGZLWK-DFREVHQV\QGURPH$P-0HG*HQHW$158A±
ϰϰϮ
ϰϰϯ
ϰϰϰ
ϰϰϱ
+|KQH0/RUVFKHLGHU-%DUGHOHEHQYRQ$'XIQHU06FKDUI0$*|GHO0
+HOPVWlGWHU06FKXUHN(0=DQN6*HUNH3HWDO7KH%$5GRPDLQSURWHLQ
3,&.UHJXODWHVFHOOUHFRJQLWLRQDQGPRUSKRJHQHVLVE\LQWHUDFWLQJZLWK1HSKSURWHLQV0RO
&HOO%LRO31±
ϰϰϲ
ϰϰϳ
ϰϰϴ
,RVVLIRY,5RQHPXV0/HY\':DQJ=+DNNHU,5RVHQEDXP-<DPURP%/HH<
+1DU]LVL*/HRWWD$HWDO'HQRYRJHQHGLVUXSWLRQVLQFKLOGUHQRQWKHDXWLVWLF
VSHFWUXP1HXURQ74±
ϰϰϵ
ϰϱϬ
ϰϱϭ
ϰϱϮ
.DPLQVN\(%.DXO93DVFKDOO-&KXUFK'0%XQNH%.XQLJ'0RUHQR'H/XFD'
0RUHQR'H/XFD$0XOOH-*:DUUHQ67HWDO$QHYLGHQFHEDVHGDSSURDFKWR
HVWDEOLVKWKHIXQFWLRQDODQGFOLQLFDOVLJQLILFDQFHRIFRS\QXPEHUYDULDQWVLQLQWHOOHFWXDODQG
GHYHORSPHQWDOGLVDELOLWLHV*HQHWLFVLQ0HGLFLQH13±
ϰϱϯ
0DUWLQ($0XUDOLGKDU6:DQJ=&HUYDQWHV'&%DVX57D\ORU05+XQWHU-
ϭϲ
ϰϱϰ
ϰϱϱ
ϰϱϲ
&XWIRUWK7:LONH6$*KRVK$HWDO7KHLQWHOOHFWXDOGLVDELOLW\JHQH.LUUHO
UHJXODWHVWDUJHWVSHFLILFPRVV\ILEHUV\QDSVHGHYHORSPHQWLQWKHKLSSRFDPSXVH/LIH6FLHQFHV
4H
ϰϱϳ
ϰϱϴ
0F%DLQ&-'LIIHUHQWLDOPHFKDQLVPVRIWUDQVPLVVLRQDQGSODVWLFLW\DWPRVV\ILEHU
V\QDSVHV3URJUHVVLQ%UDLQ5HVHDUFK169
ϰϱϵ
ϰϲϬ
ϰϲϭ
0LFKDHOVRQ--6KL<*XMUDO0=KHQJ+0DOKRWUD'-LQ;-LDQ0/LX**UHHU'
%KDQGDUL$HWDO:KROHJHQRPHVHTXHQFLQJLQDXWLVPLGHQWLILHVKRWVSRWVIRUGHQRYR
JHUPOLQHPXWDWLRQ&HOO151±
ϰϲϮ
ϰϲϯ
ϰϲϰ
1HDOH%0.RX</LX/0D
D\DQ$6DPRFKD.(6DER$/LQ&)6WHYHQV&
:DQJ/60DNDURY9HWDO3DWWHUQVDQGUDWHVRIH[RQLFGHQRYRPXWDWLRQVLQ
DXWLVPVSHFWUXPGLVRUGHUV1DWXUH485±
ϰϲϱ
ϰϲϲ
1HOVRQ6%DQG9DODNK9([FLWDWRU\,QKLELWRU\%DODQFHDQG&LUFXLW+RPHRVWDVLVLQ
$XWLVP6SHFWUXP'LVRUGHUV1HXURQ87±
ϰϲϳ
ϰϲϴ
ϰϲϵ
3HQWWRQHQ0$.$6/$DQG*%)HHGIRUZDUGDQGIHHGEDFNDFWLYDWLRQRI
WKHGHQWDWHJ\UXVLQYLYRGXULQJGHQWDWHVSLNHVDQGVKDUSZDYHEXUVWV+LSSRFDPSXV7±
ϰϳϬ
ϰϳϭ
ϰϳϮ
3ULQFH-($%ULJQDOO$&&XWIRUWK76KHQ.DQG&ORXWLHU-).LUUHOLVUHTXLUHG
IRUWKHFRDOHVFHQFHRIYRPHURQDVDOVHQVRU\QHXURQD[RQVLQWRJORPHUXOLDQGIRUPDOHPDOH
DJJUHVVLRQ'HYHORSPHQW140±
ϰϳϯ
ϰϳϰ
ϰϳϱ
ϰϳϲ
5RK-'&KRL6<&KR<6&KRL7<3DUN-6&XWIRUWK7&KXQJ:3DUN+/HH
'.LP0+HWDO,QFUHDVHG([FLWDWRU\6\QDSWLF7UDQVPLVVLRQRI'HQWDWH*UDQXOH
1HXURQVLQ0LFH/DFNLQJ36',QWHUDFWLQJ$GKHVLRQ0ROHFXOH1HSK.LUUHOGXULQJWKH(DUO\
3RVWQDWDO3HULRG)URQW0RO1HXURVFL10
ϰϳϳ
ϰϳϴ
5ROOHQKDJHQ7KHPRVV\ILEHUERXWRQWKHFRPPRQRUWKH³XQLTXH´V\QDSVH")URQWLHUV
LQ6\QDSWLF1HXURVFLHQFH
ϰϳϵ
ϰϴϬ
ϰϴϭ
5ROOHQKDJHQ$6lW]OHU.5RGUtJXH](3-RQDV3)URWVFKHU0DQG/ENH-+5
6WUXFWXUDOGHWHUPLQDQWVRIWUDQVPLVVLRQDWODUJHKLSSRFDPSDOPRVV\ILEHUV\QDSVHV-
1HXURVFL27±
ϰϴϮ
ϰϴϯ
5ROOV(77KHPHFKDQLVPVIRUSDWWHUQFRPSOHWLRQDQGSDWWHUQVHSDUDWLRQLQWKH
KLSSRFDPSXV)URQWLHUVLQ6\VWHPV1HXURVFLHQFH7
ϰϴϰ
ϰϴϱ
ϰϴϲ
5XHGLJHU69LWWRUL&%HGQDUHN(*HQRXG&6WUDWD36DFFKHWWL%DQG&DURQL3
/HDUQLQJUHODWHGIHHGIRUZDUGLQKLELWRU\FRQQHFWLYLW\JURZWKUHTXLUHGIRUPHPRU\
SUHFLVLRQ1DWXUH473±
ϰϴϳ
ϰϴϴ
ϰϴϵ
6HUL]DZD60L\DPLFKL.7DNHXFKL+<DPDJLVKL<6X]XNL0DQG6DNDQR+$
QHXURQDOLGHQWLW\FRGHIRUWKHRGRUDQWUHFHSWRUVSHFLILFDQGDFWLYLW\GHSHQGHQWD[RQVRUWLQJ&HOO
127±
ϰϵϬ
ϰϵϭ
6KHSKHUG*0*DQG+DUULV.07KUHH'LPHQVLRQDO6WUXFWXUHDQG&RPSRVLWLRQRI
&$ĺ&$$[RQVLQ5DW+LSSRFDPSDO6OLFHV,PSOLFDWLRQVIRU3UHV\QDSWLF&RQQHFWLYLW\DQG
ϭϳ
ϰϵϮ
&RPSDUWPHQWDOL]DWLRQ-1HXURVFL18±
ϰϵϯ
ϰϵϰ
ϰϵϱ
ϰϵϲ
7DONRZVNL0(5RVHQIHOG-$%OXPHQWKDO,3LOODODPDUUL9&KLDQJ&+HLOEXW$(UQVW
&+DQVFRP&5RVVLQ(/LQGJUHQ$0HWDO6HTXHQFLQJFKURPRVRPDO
DEQRUPDOLWLHVUHYHDOVQHXURGHYHORSPHQWDOORFLWKDWFRQIHUULVNDFURVVGLDJQRVWLFERXQGDULHV
&HOO149±
ϰϵϳ
ϰϵϴ
ϰϵϵ
7DVKLUR$'XQDHYVN\$%OD]HVNL50DVRQ&$DQG<XVWH5%LGLUHFWLRQDO
5HJXODWLRQRI+LSSRFDPSDO0RVV\)LEHU)LORSRGLDO0RWLOLW\E\.DLQDWH5HFHSWRUV1HXURQ38
±
ϱϬϬ
ϱϬϭ
ϱϬϮ
7RUERUJ&/1DNDVKLED77RQHJDZD6DQG0F%DLQ&-&RQWURORI&$RXWSXWE\
IHHGIRUZDUGLQKLELWLRQGHVSLWHGHYHORSPHQWDOFKDQJHVLQWKHH[FLWDWLRQLQKLELWLRQEDODQFH-
1HXURVFL30±
ϱϬϯ
ϱϬϰ
7XUULJLDQR*+RPHRVWDWLFV\QDSWLFSODVWLFLW\ORFDODQGJOREDOPHFKDQLVPVIRU
VWDELOL]LQJQHXURQDOIXQFWLRQ&ROG6SULQJ+DUE3HUVSHFW%LRO4D
ϱϬϱ
ϱϬϲ
ϱϬϳ
:LONH6$$QWRQLRV-.%XVKRQJ($%DGNRREHKL$0DOHN(+ZDQJ07HUDGD0
(OOLVPDQ0+DQG*KRVK$'HFRQVWUXFWLQJFRPSOH[LW\VHULDOEORFNIDFHHOHFWURQ
PLFURVFRSLFDQDO\VLVRIWKHKLSSRFDPSDOPRVV\ILEHUV\QDSVH-1HXURVFL33±
ϱϬϴ
ϱϬϵ
),*85(/(*(1'6
ϱϭϬ
)LJXUH5HFRQVWUXFWLQJ0)3UHV\QDSWLF&RPSOH[HVE\6%(0$'LDJUDPRI
ϱϭϭ
KLSSRFDPSDO0)FLUFXLWU\%R[HGUHJLRQQRWHVWKHWLVVXHDUHDDQDO\]HG2QWKHULJKWWKHGDUN
ϱϭϮ
EOXHRXWOLQHLGHQWLILHVWKH'*QHXURQ'*GHQWDWHJUDQXOH7(WKRUQ\H[FUHVFHQFHV%
ϱϭϯ
5HSUHVHQWDWLYHLPDJHRIDZLOGW\SH6%(0VHFWLRQVKRZLQJ0)V\QDSVHFRPSRQHQWVPDLQ0)
ϱϭϰ
ERXWRQGDUNEOXH0)ILORSRGLDOLJKWEOXH'*D[RQ\HOORZ7(VRUDQJHV\QDSWLFYHVLFOHV
ϱϭϱ
UHGDUURZVDQGPLWRFKRQGULDZKLWHDUURZV6FDOHEDU μP&'UHFRQVWUXFWLRQRIWKH
ϱϭϲ
0)SUHV\QDSWLFFRPSOH[LQ%7KHSRVWV\QDSWLFWKRUQ\H[FUHVFHQFHVDUHQRWVKRZQ'RWWHGOLQH
ϱϭϳ
LQ&VKRZVWKHORFDWLRQRIVOLFHVKRZQLQ%'(6DPSOHZLOGW\SH'DQG.LUUHONQRFNRXW(
ϱϭϴ
'UHFRQVWUXFWLRQVVKRZLQJ0)SUHV\QDSWLFFRPSOH[HVHDFK
ϱϭϵ
ϱϮϬ
)LJXUH±0)ILORSRGLDH[LVWLQWKUHHVWDWHVGXULQJGHYHORSPHQW
ϱϮϭ
$7KHDYHUDJHYROXPHRIWKHPDLQ0)ERXWRQSHUJHQRW\SH3YDOXH %7KH
ϱϮϮ
DYHUDJHQXPEHURI0)ILORSRGLDSHUPDLQ0)ERXWRQ3YDOXH &&XPXODWLYH
ϭϴ
ϱϮϯ
KLVWRJUDPVKRZLQJWKHQXPEHURI0)ILORSRGLDSHUPDLQ0)ERXWRQLQHDFKJHQRW\SH1RWH
ϱϮϰ
.LUUHONQRFNRXWVWHQGWRKDYH0)ERXWRQVZLWKIHZHUILORSRGLD3YDOXH ')
ϱϮϱ
5HSUHVHQWDWLYHLPDJHVRI0)ILORSRGLDLQHDFKRIWKHWKUHHV\QDSWLFVWDWHVV\QDSVHIUHH
ϱϮϲ
SDUWLDOFRPSOHWH<HOORZDUURZLQGLFDWHV36')RUDOOJUDSKVVDPSOHVL]H:7 DQG.2 ϱϮϳ
PDLQ0)ERXWRQV(UURUEDUVVKRZPHDQ6(06FDOHEDUV μP
ϱϮϴ
ϱϮϵ
)LJXUH.LUUHOUHJXODWHV0)ILORSRGLDOV\QDSVHGHQVLW\
ϱϯϬ
$7DEOHVKRZLQJSHUFHQWRI0)ILORSRGLDWKDWDUHV\QDSVHIUHHRUFRQWDLQSDUWLDORUFRPSOHWH
ϱϯϭ
V\QDSVHV1RWH$QLQGLYLGXDO0)ILORSRGLDFDQKDYHPXOWLSOHV\QDSVHVWKXVWKHWRWDOVGRQRW
ϱϯϮ
VXPWR%'4XDQWLILFDWLRQRIWKHQXPEHURIV\QDSVHIUHH0)ILORSRGLD%SDUWLDO
ϱϯϯ
V\QDSVHV&DQGFRPSOHWHV\QDSVHV'SHUPDLQ0)ERXWRQE\JHQRW\SH(4XDQWLILFDWLRQ
ϱϯϰ
RIWRWDOQXPEHURIV\QDSVHVSDUWLDOFRPSOHWHSHUPDLQ0)ERXWRQ3YDOXH XVLQJ
ϱϯϱ
DWZRWDLOHGWWHVW6DPSOHVL]HIRU$(:7 DQG.2 PDLQ0)ERXWRQV)
ϱϯϲ
4XDQWLILFDWLRQRIWKHQXPEHURI69VLQFRPSOHWHV\QDSVHV6DPSOHVL]HIRU):7 DQG.2
ϱϯϳ
V\QDSVHV*4XDQWLILFDWLRQRIWKHQXPEHURI69VLQSDUWLDOV\QDSVHV6DPSOHVL]HIRU*
ϱϯϴ
:7 DQG.2 V\QDSVHV)RUDOOJUDSKV8QOHVVQRWHG3!VHHVWDWLVWLFVWDEOH
ϱϯϵ
(UURUEDUVVKRZPHDQ6(0
ϱϰϬ
ϱϰϭ
)LJXUH.LUUHOUHJXODWHV0)ILORSRGLDOV\QDSVHVZLWK*$%$QHXURQVEXWQRW&$
ϱϰϮ
QHXURQV
ϱϰϯ
$%'$DQG'%H[DPSOHRID*$%$GHQGULWHJUHHQZLWKVLPSOHVSLQHOLNHSURWUXVLRQV
ϱϰϰ
DQGQR36'VZLWKPDLQ0)ERXWRQV'RWWHGOLQHLQ%VKRZVWKHORFDWLRQRIVOLFHVKRZQLQ$
ϱϰϱ
&''&DQG''H[DPSOHRID&$QHXURQRUDQJHZLWKPXOWLKHDGHG7(VSLQHVDQG
ϱϰϲ
PDQ\36'VZLWKPDLQ0)ERXWRQV'RWWHGOLQHLQ'VKRZVWKHORFDWLRQRIVOLFHVKRZQLQ&
ϱϰϳ
()5HSUHVHQWDWLYHLPDJHVRIFRPSOHWH0)ILORSRGLDOV\QDSVHVPDGHRQWRD*$%$GHQGULWLF
ϭϵ
ϱϰϴ
VKDIW(DQGD*$%$VSLQH)0)ILORSRGLDEOXH*$%$GHQGULWHJUHHQV\QDSVHVLQGLFDWHG
ϱϰϵ
E\ZKLWHDUURZV*+5HSUHVHQWDWLYHLPDJHVRIFRPSOHWH0)ILORSRGLDOV\QDSVHVPDGHRQWRD
ϱϱϬ
VHFRQG&$GHQGULWLFVKDIW*DQGD&$WKRUQ\H[FUHVFHQFH+0DLQ0)ERXWRQDQG0)
ϱϱϭ
ILORSRGLDEOXH&$GHQGULWHDQGWKRUQ\H[FUHVFHQFHV7(VRUDQJHVHFRQG&$GHQGULWH
ϱϱϮ
\HOORZV\QDSVHVLQGLFDWHGE\ZKLWHDUURZV,/4XDQWLILFDWLRQRI0)ILORSRGLDOV\QDSVHVRQWR
ϱϱϯ
GLIIHUHQWW\SHVRISRVWV\QDSWLFQHXURQV,3HUFHQWRIZLOGW\SHRU.LUUHONQRFNRXW0)ILORSRGLD
ϱϱϰ
PDNLQJFRPSOHWHV\QDSVHVZLWKLQGLFDWHGSRVWV\QDSWLFSDUWQHUW\SH-1XPEHURIFRPSOHWH
ϱϱϱ
0)ILORSRGLDOV\QDSVHVRQWR*$%$QHXURQVSHUPDLQ0)ERXWRQ3YDOXH E\0DQQ
ϱϱϲ
:KLWQH\WHVW..1XPEHURIFRPSOHWH0)ILORSRGLDOV\QDSVHVRQWR&$QHXURQVSHUPDLQ0)
ϱϱϳ
ERXWRQ/,QFOXGLQJRQO\WKH0)ILORSRGLDWKDWPDNHDFRPSOHWHV\QDSVHWKHSHUFHQWRIHDFK
ϱϱϴ
SDUWQHUW\SHLVVKRZQ6DPSOHVL]HIRU,/:7 DQG.2 PDLQ0)ERXWRQV8QOHVV
ϱϱϵ
LQGLFDWHG3!03&RPSDULVRQRIWKHPRUSKRORJ\RIILORSRGLDV\QDSVLQJZLWK*$%$
ϱϲϬ
YHUVXV&$QHXURQVLQZLOGW\SHDQG.LUUHONQRFNRXWPLFHXVLQJWKH.UXVNDO:DOOLVWHVWZLWK
ϱϲϭ
PXOWLSOHFRPSDULVRQV09ROXPHRIILORSRGLDLQμP*$%$:7WR.23YDOXH &$
ϱϲϮ
:7WR.23YDOXH 1/HQJWKRIILORSRGLDLQμP3YDOXH 2&URVV
ϱϲϯ
VHFWLRQDODUHDLQμP3YDOXH!31XPEHURIV\QDSWLFYHVLFOHVSHUV\QDSVH3YDOXH!
ϱϲϰ
6DPSOHVL]HIRU02:7*$%$ .2*$%$ :7&$ .2&$ ϱϲϱ
6DPSOHVL]HIRU3:7*$%$ .2*$%$ :7&$ .2&$ )RUHQWLUH
ϱϲϲ
ILJXUH(UURUEDUVVKRZPHDQ6(06FDOHEDUV μP
ϱϲϳ
ϱϲϴ
)LJXUH±.LUUHOGRHVQRWUHJXODWHHQSDVVDQWV\QDSVHGHQVLW\
ϱϲϵ
$&'H[DPSOH$RIDVSLQ\*$%$GHQGULWHWDUJHWHGE\HQSDVVDQWV\QDSVHVIURPWZR
ϱϳϬ
VHSDUDWH'*D[RQV6\QDSVHVPDJQLILHGLQ%&*$%$GHQGULWHJUHHQ'*D[RQ\HOORZHQ
ϱϳϭ
SDVVDQWV\QDSVHSLQN'3HUFHQWRIHQSDVVDQWV\QDSVHVPDGHRQWR*$%$DQG&$
ϱϳϮ
GHQGULWHVIURP:7DQG.2'*D[RQVHJPHQWV(7DEOHUHSRUWLQJWKHQXPEHURIHQ
ϮϬ
ϱϳϯ
SDVVDQWV\QDSVHVSHUPPD[RQ6FDOHEDUV μP
ϱϳϰ
ϱϳϱ
)LJXUH$GGLWLRQDOPRUSKRORJLFDOIHDWXUHVRI0)FLUFXLWV
ϱϳϲ
$7DEOHUHSRUWLQJWKHSHUFHQWRIZLOGW\SHDQG.LUUHONQRFNRXW0)ILORSRGLDZLWKLQGLFDWHG
ϱϳϳ
IHDWXUHV%&5HSUHVHQWDWLYH'%DQG'&H[DPSOHRID0)ILORSRGLDHQGLQJLQDJURZWK
ϱϳϴ
FRQHOLNHVWUXFWXUH''H[DPSOHRIDEUDQFKHG0)ILORSRGLD:KLWHDUURZLQGLFDWHVEUDQFK
ϱϳϵ
SRLQW(4XDQWLILFDWLRQRIWKHQXPEHURIEUDQFKLQJ0)ILORSRGLDSHUPDLQ0)ERXWRQ6DPSOH
ϱϴϬ
VL]H:7 DQG.2 PDLQ0)ERXWRQV3!(UURUEDUVVKRZPHDQ6(0)+'
ϱϴϭ
H[DPSOHRIRQH0)ILORSRGLDPDNLQJPXOWLSOHV\QDSVHVRQWRRQH*$%$QHXURQ)V\QDSVHV
ϱϴϮ
PDJQLILHGLQ*+VKRZQLQ'0DLQ0)ERXWRQZLWKDVVRFLDWHG0)ILORSRGLDEOXH*$%$
ϱϴϯ
GHQGULWHJUHHQV\QDSVHVUHG6FDOHEDUV μP
ϱϴϰ
ϱϴϱ
0RYLH0)SUHV\QDSWLFFRPSOH[VKRZQLQ)LJXUH%&
ϱϴϲ
7DEOH6WDWLVWLFV
Ϯϭ
*UDSK
)LJXUH$
)LJXUH%
)LJXUH&
)LJXUH%
)LJXUH&
)LJXUH'
)LJXUH(
)LJXUH)
)LJXUH*
)LJXUH-
)LJXUH.
)LJXUH0
)LJXUH1
)LJXUH2
)LJXUH3
)LJXUH(
'DWD6WUXFWXUH
1RQSDUDPHWULF
1RUPDO'LVWULEXWLRQ
1RUPDO'LVWULEXWLRQ
1RUPDO'LVWULEXWLRQ
1RUPDO'LVWULEXWLRQ
1RQSDUDPHWULF
1RUPDO'LVWULEXWLRQ
1RQSDUDPHWULF
1RQSDUDPHWULF
1RQSDUDPHWULF
1RQSDUDPHWULF
1RQSDUDPHWULF
1RQSDUDPHWULF
1RQSDUDPHWULF
1RQSDUDPHWULF
1RQSDUDPHWULF
7\SHRI7HVW
0DQQ:KLWQH\WHVW
8QSDLUHGWWHVW
.ROPRJRURY6PLUQRYWHVW
8QSDLUHGWWHVW
8QSDLUHGWWHVW
0DQQ:KLWQH\WHVW
8QSDLUHGWWHVW
0DQQ:KLWQH\WHVW
0DQQ:KLWQH\WHVW
0DQQ:KLWQH\WHVW
0DQQ:KLWQH\WHVW
.UXVNDO:DOOLVWHVW
.UXVNDO:DOOLVWHVW
.UXVNDO:DOOLVWHVW
.UXVNDO:DOOLVWHVW
0DQQ:KLWQH\WHVW
ϭ
3YDOXH