DIFFERENCES IN GLOMERULAR LAYER

This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version.

Research Articles: Systems/Circuits

Differences in glomerular layer- mediated feed-forward inhibition onto mitral and tufted cells lead to distinct modes of intensity coding Matthew Geramita1 and Nathan N Urban1 1

Department of Neurobiology, Center for Neuroscience at the University of Pittsburgh, Center for the Neural Basis of Cognition, University of Pittsburgh DOI: 10.1523/JNEUROSCI.2245-16.2016 Received: 14 July 2016 Revised: 25 October 2016 Accepted: 19 November 2016 Published: 27 December 2016

Author contributions: M.G. and N.U. designed research; M.G. performed research; M.G. analyzed data; M.G. and N.U. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. Thanks to Shawn Burton, Claire Cheetham and members of the Urban laboratory for helpful comments and discussion, and Greg LaRocca for excellent technical assistance. This work was supported by the National Institute of Deafness and Other Communication Disorders Grant R01-DC011184 (N.N.U.) Corresponding author: Nathan N. Urban E1440 BSTWR 200 Lothrop Street University of Pittsburgh Pittsburgh, PA 15213, [email protected] Cite as: J. Neurosci 2016; 10.1523/JNEUROSCI.2245-16.2016 Alerts: Sign up at www.jneurosci.org/cgi/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. Copyright © 2016 the authors

 ϭ

7LWOH'LIIHUHQFHVLQJORPHUXODUOD\HUPHGLDWHGIHHGIRUZDUGLQKLELWLRQRQWRPLWUDODQGWXIWHGFHOOVOHDG

Ϯ

WRGLVWLQFWPRGHVRILQWHQVLW\FRGLQJ

ϯ

$EEUHYLDWHGWLWOH(IIHFWVRIROIDFWRU\EXOEIHHGIRUZDUGLQKLELWLRQ

ϰ

$XWKRUV0DWWKHZ*HUDPLWD1DWKDQ18UEDQ

ϱ

'HSDUWPHQWRI1HXURELRORJ\&HQWHUIRU1HXURVFLHQFHDWWKH8QLYHUVLW\RI3LWWVEXUJK&HQWHUIRUWKH

ϲ

1HXUDO%DVLVRI&RJQLWLRQ8QLYHUVLW\RI3LWWVEXUJK

ϳ

&RUUHVSRQGLQJDXWKRU

ϴ

1DWKDQ18UEDQ

ϵ

(%67:5

ϭϬ

/RWKURS6WUHHW

ϭϭ

8QLYHUVLW\RI3LWWVEXUJK

ϭϮ

3LWWVEXUJK3$

ϭϯ

QXUEDQ#SLWWHGX

ϭϰ



ϭϱ

1XPEHURISDJHV

ϭϲ

1XPEHURIZRUGVDEVWUDFW 

ϭϳ

1XPEHURIZRUGVLQWURGXFWLRQ 

ϭϴ

1XPEHURIZRUGVGLVFXVVLRQ 

ϭϵ

&RQIOLFWRILQWHUHVW7KHDXWKRUVGHFODUHQRFRPSHWLQJILQDQFLDOLQWHUHVWV

ϮϬ

$FNQRZOHGJHPHQWV7KDQNVWR6KDZQ%XUWRQ&ODLUH&KHHWKDPDQGPHPEHUVRIWKH8UEDQ

Ϯϭ

ODERUDWRU\IRUKHOSIXOFRPPHQWVDQGGLVFXVVLRQDQG*UHJ/D5RFFDIRUH[FHOOHQWWHFKQLFDODVVLVWDQFH

ϮϮ

7KLVZRUNZDVVXSSRUWHGE\WKH1DWLRQDO,QVWLWXWHRI'HDIQHVVDQG2WKHU&RPPXQLFDWLRQ'LVRUGHUV

Ϯϯ

*UDQW5'&118 



ϭ

 Ϯϰ

$EVWUDFW

Ϯϱ

8QGHUVWDQGLQJKRZHDFKRIWKHPDQ\LQWHUQHXURQVXEW\SHVDIIHFWVEUDLQQHWZRUNDFWLYLW\LVFULWLFDO,Q

Ϯϲ

WKHPRXVHROIDFWRU\V\VWHPPLWUDOFHOOV0&V DQGWXIWHGFHOOV7&V FRPSULVHSDUDOOHOSDWKZD\VRI

Ϯϳ

ROIDFWRU\EXOERXWSXWWKDWDUHWKRXJKWWRSOD\GLVWLQFWIXQFWLRQDOUROHVLQRGRUFRGLQJ+HUHLQDFXWH

Ϯϴ

PRXVHROIDFWRU\EXOEVOLFHVZHWHVWKRZWKHWZRPDMRUFODVVHVRIROIDFWRU\EXOELQWHUQHXURQV

Ϯϵ

GLIIHUHQWLDOO\FRQWULEXWHWRGLIIHUHQFHVLQ0&YV7&UHVSRQVHSURSHUWLHV:HVKRZWKDWZKLOH7&V

ϯϬ

UHVSRQGWRROIDFWRU\VHQVRU\QHXURQ261 VWLPXODWLRQZLWKVKRUWODWHQFLHVUHJDUGOHVVRIVWLPXODWLRQ

ϯϭ

LQWHQVLW\0&ODWHQFLHVQHJDWLYHO\FRUUHODWHZLWKVWLPXODWLRQLQWHQVLW\7KHVHGLIIHUHQFHVEHWZHHQ0&V

ϯϮ

DQG7&VDUHFDXVHGLQSDUWE\ZHDNHUH[FLWDWRU\DQGVWURQJHULQKLELWRU\FXUUHQWVRQWR0&VWKDQRQWR

ϯϯ

7&V7KHVHGLIIHUHQFHVLQLQKLELWLRQEHWZHHQ0&VDQG7&VDUHPRVWSURQRXQFHGGXULQJWKHILUVWPV

ϯϰ

IROORZLQJVWLPXODWLRQDQGDUHPHGLDWHGE\JORPHUXODUOD\HUFLUFXLWV&RQVHTXHQWO\EORFNLQJ

ϯϱ

LQKLELWLRQRULJLQDWLQJLQWKHJORPHUXODUOD\HUEXWQRWJUDQXOHFHOO±PHGLDWHGLQKLELWLRQUHGXFHV0&

ϯϲ

VSLNHODWHQF\DWZHDNVWLPXODWLRQLQWHQVLWLHV7KHUHIRUHGLVWLQFWWHPSRUDOSDWWHUQVRIRGRUHYRNHG

ϯϳ

UHVSRQVHVLQ0&VDQG7&VHPHUJHLQSDUWGXHWRGLIIHUHQFHVLQJORPHUXODUOD\HUPHGLDWHG

ϯϴ

LQKLELWLRQ

ϯϵ

6LJQLILFDQFHVWDWHPHQW

ϰϬ

2OIDFWRU\EXOEPLWUDODQGWXIWHGFHOOVGLVSOD\GLIIHUHQWRGRUHYRNHGUHVSRQVHVDQGDUHWKRXJKWWRIRUP

ϰϭ

SDUDOOHOFKDQQHOVRIROIDFWRU\EXOERXWSXW7KHUHIRUHGHWHUPLQLQJWKHFLUFXLWOHYHOFDXVHVWKDWGULYHWKHVH

ϰϮ

GLIIHUHQFHVLVYLWDO+HUHZHILQGWKDWORQJHUODWHQF\UHVSRQVHVLQPLWUDOFHOOVFRPSDUHGZLWKWXIWHG

ϰϯ

FHOOVDUHGXHWRZHDNHUH[FLWDWLRQDQGVWURQJHUJORPHUXODUOD\HUPHGLDWHGLQKLELWLRQ

ϰϰ

,QWURGXFWLRQ

ϰϱ

,QPXOWLSOHVHQVRU\V\VWHPVVHSDUDWHQHXURQW\SHVHQFRGHGLVWLQFWIHDWXUHVRIVHQVRU\VWLPXOL

ϰϲ


ϰϳ

W\SHVRISULQFLSDOQHXURQVRIWKHROIDFWRU\EXOEKDYHEHHQWKRXJKWWRSOD\LGHQWLFDOUROHVLQRGRUFRGLQJ

ϰϴ

GHVSLWHFOHDUDQDWRPLFDOGLIIHUHQFHV0RULHWDO2URQDHWDO,JDUDVKLHWDO  

Ϯ

 ϰϵ

5HFHQWO\KRZHYHUPXOWLSOHVWXGLHVKDYHLGHQWLILHGIXQFWLRQDOGLIIHUHQFHVEHWZHHQ0&VDQG7&VWKDW

ϱϬ

VXJJHVWWKDWWKHROIDFWRU\V\VWHPVHJUHJDWHVROIDFWRU\LQIRUPDWLRQLQWRSDUDOOHOSDWKZD\VPXFKOLNH

ϱϭ

RWKHUVHQVRU\V\VWHPV)XNXQDJDHWDO,JDUDVKLHWDO )RULQVWDQFH0&VDQG7&VDUH

ϱϮ

GLIIHUHQWLDOO\PRGXODWHGE\WKHUDSKHQXFOHXV.DSRRUHWDO DQGWKHSLULIRUPFRUWH[2WD]XHWDO

ϱϯ

 $GGLWLRQDOO\FRPSDUHGWR0&V7&VDUHOHVVIUHTXHQWO\LQKLELWHGE\RGRUV1DJD\DPDHWDO

ϱϰ

 UHVSRQGWRORZHUFRQFHQWUDWLRQRGRUV,JDUDVKLHWDO.LNXWDHWDO DQGKDYH

ϱϱ

UHVSRQVHVWKDWDUHPRUHKLJKO\FRUUHODWHGWRROIDFWRU\VHQVRU\QHXURQ261 LQSXW$GDPHWDO 

ϱϲ

5HFHQWZRUNKDVDOVRH[SORUHGGLIIHUHQFHVLQKRZWKHODWHQF\RIRGRUHYRNHGUHVSRQVHVGLIIHUV

ϱϳ

EHWZHHQ0&VDQG7&VEHFDXVHUHVSRQVHODWHQF\KDVWKHSRWHQWLDOWRHQFRGHEHKDYLRUDOO\UHOHYDQW

ϱϴ

LQIRUPDWLRQ&XU\DQG8FKLGD6KXVWHUPDQHWDO6PHDUHWDO 7&VUHVSRQGWR

ϱϵ

RGRUVKXQGUHGVRIPLOOLVHFRQGVHDUOLHULQWKHVQLIIF\FOHDQGVKRZPRUHFRQFHQWUDWLRQLQYDULDQWRGRU

ϲϬ

HYRNHGUHVSRQVHODWHQFLHVWKDQ0&V)XNXQDJDHWDO,JDUDVKLHWDO 7KHVHGDWDVXJJHVW

ϲϭ

WKDW0&VPD\XVHVSLNHODWHQF\WRHQFRGHFRQFHQWUDWLRQVSHFLILFLQIRUPDWLRQZKLOH7&VPD\HQFRGH

ϲϮ

FRQFHQWUDWLRQLQGHSHQGHQWLQIRUPDWLRQ

ϲϯ

,QOLJKWRIWKHVHHPHUJLQJGLIIHUHQFHVLQUHVSRQVHODWHQF\GHWHUPLQLQJWKHFLUFXLWOHYHO

ϲϰ

PHFKDQLVPVWKDWGULYHWKHVHGLIIHUHQFHVLVFULWLFDO&RPSDUHGWR0&V7&VDUHPRUHLQWULQVLFDOO\

ϲϱ

H[FLWDEOH%XUWRQDQG8UEDQ DQGUHFHLYHVWURQJHUROIDFWRU\VHQVRU\QHXURQ261 LQSXW*LUHHW

ϲϲ

DO%XUWRQDQG8UEDQ VXJJHVWLQJWKDWDFRPELQDWLRQRILQWULQVLFDQGV\QDSWLFGLIIHUHQFHV

ϲϳ

GULYHGLIIHUHQWLDOUHVSRQVHVWRFKDQJHVLQFRQFHQWUDWLRQ/LWWOHLVNQRZQDERXWZKHWKHUGLIIHUHQFHVLQ

ϲϴ

LQKLELWLRQEHWZHHQ0&VDQG7&VFRQWULEXWHWRGLIIHUHQFHVLQRGRUHYRNHGUHVSRQVHV261VWLPXODWLRQ

ϲϵ

HYRNHVVWURQJLQKLELWLRQRULJLQDWLQJIURPERWKWKHJORPHUXODUOD\HUDQGWKHJUDQXOHFHOO*& OD\HU

ϳϬ

RQWR0&VDQG7&V
ϳϭ

HYRNHGDFWLYLW\LQ0&VDQG7&VUHPDLQVXQFOHDU&OHODQG)XNXQDJDHWDO*VFKZHQGHW

ϳϮ

DO 0XOWLSOH*$%$HUJLFLQWHUQHXURQVXEW\SHVUHVLGHLQWKHJORPHUXODUOD\HULQFOXGLQJ

ϳϯ

SHULJORPHUXODUFHOOV3*&V .L\RNDJHHWDO6KDRHWDO1DMDFHWDO 

ϳϰ

VXSHUILFLDOVKRUWD[RQFHOOVV6$&V /LXHWDO:KLWHVHOOHWDO/LXHWDO DQG 

ϯ

 ϳϱ

HYHQVXEVHWVRIH[WHUQDOWXIWHGFHOOV(7&V 7DWWLHWDO +RZHYHUZKHWKHUGLIIHUHQFHVLQ

ϳϲ

WKHVWUHQJWKRIWKHVHJORPHUXODUOD\HUFLUFXLWVFRQWULEXWHWRGLIIHUHQFHVLQWKHODWHQF\RI0&DQG

ϳϳ

7&UHVSRQVHVUHPDLQVXQNQRZQ&RPSXWDWLRQDOPRGHOVSUHGLFWWKDWGLIIHUHQWLDOLQSXWIURP3*&V

ϳϴ

PD\GULYHGLIIHUHQFHVLQVSLNHODWHQF\EHWZHHQ0&VDQG7&V)XNXQDJDHWDO 2WKHUZRUN

ϳϵ

KRZHYHUKDVVKRZQWKDWVWUHQJWKRI3*&PHGLDWHGLQKLELWRU\FXUUHQWVRQWR0&VDQG7&DUHVLPLODU

ϴϬ

ZKHQ261VDUHVWLPXODWHGDWZHDNLQWHQVLWLHV1DMDFHWDO 7KHUHIRUHDV\VWHPDWLF

ϴϭ

LQYHVWLJDWLRQRIKRZERWK*&DQGJORPHUXODUOD\HUPHGLDWHGLQKLELWRU\FXUUHQWVRQWR0&VDQG7&V

ϴϮ

YDU\ZLWKVWLPXOXVLQWHQVLW\LVQHHGHG

ϴϯ

+HUHXVLQJZKROHFHOOUHFRUGLQJVLQDFXWHROIDFWRU\EXOEVOLFHVZHILQGWKDW0&VGLVSOD\ORQJHU

ϴϰ

ODWHQF\VSLNLQJWKDWLVPRUHVWURQJO\GHSHQGHQWRQVWLPXOXVLQWHQVLW\WKDQ7&V:HILQGWKDWORQJHU

ϴϱ

ODWHQF\VSLNLQJLQ0&VLVDFRQVHTXHQFHRIZHDNHUH[FLWDWRU\DQGVWURQJHULQKLELWRU\FXUUHQWVRQWR0&V

ϴϲ

FRPSDUHGWR7&V6WURQJHULQKLELWRU\FXUUHQWVRQWR0&VWKDQ7&VRULJLQDWHLQWKHJORPHUXODU

ϴϳ

OD\HUVREORFNLQJJORPHUXODUOD\HULQKLELWLRQEXWQRW*&PHGLDWHGLQKLELWLRQOHGWRPRUHUHOLDEOH

ϴϴ

DQGVKRUWHUODWHQF\UHVSRQVHVLQ0&VEXWQRW7&V7KHVHGDWDVXJJHVWWKDWGLIIHUHQFHVLQRGRU

ϴϵ

HYRNHGUHVSRQVHODWHQF\EHWZHHQ0&VDQG7&VDUHGXHLQSDUWWRGLIIHUHQFHVLQJORPHUXODUOD\HU

ϵϬ

PHGLDWHGLQKLELWLRQ

ϵϭ

0DWHULDOVDQG0HWKRGV Slice preparation: 3RVWQDWDOGD\±&%/DQG203&K5<)36PHDUHWDO 

ϵϮ ϵϯ

PLFHRIERWKVH[HVZHUHDQDHVWKHWL]HGZLWKLVRIOXUDQHDQGGHFDSLWDWHG%UDLQVZHUHGLVVHFWHGLQWRLFH

ϵϰ

FROGR[\JHQDWHGVROXWLRQFRQWDLQLQJLQP0 1D&OJOXFRVH.&O1D+&2

ϵϱ

1D+320J&ODQG&D&O+RUL]RQWDOVOLFHVȝPWKLFN RIWKH02%ZHUHSUHSDUHGXVLQJD

ϵϲ

YLEUDWRPH976/HLFD1XVVORFK*HUPDQ\ DQGUHFRYHUHGIRU±PLQLQƒ&R[\JHQDWHG

ϵϳ

5LQJHUVROXWLRQWKDWZDVLGHQWLFDOWRWKHGLVVHFWLRQVROXWLRQH[FHSWIRUORZHU0JFRQFHQWUDWLRQV

ϵϴ

P00J&O DQGKLJKHU&DFRQFHQWUDWLRQVP0&D&O 3ULRUWRUHFRUGLQJVOLFHVZHUHVWRUHGLQ

ϵϵ

URRPWHPSHUDWXUHR[\JHQDWHG5LQJHUVROXWLRQXQWLOUHFRUGLQJ



ϰ

 Cell classification:$VSUHYLRXVO\GHVFULEHG*HUDPLWDHWDO 7&VZHUHLGHQWLILHGDVWKRVH

ϭϬϬ ϭϬϭ

FHOOVUHVLGLQJFRPSOHWHO\LQWKHVXSHUILFLDOKDOIRIWKH(3/ZLWKODUJHVRPDV!ȝPLQGLDPHWHU $OO

ϭϬϮ

7&VKDGDWOHDVWODWHUDOGHQGULWHDQGGLGQRWGLVSOD\WKHUK\WKPLFEXUVWLQJFKDUDFWHULVWLFRIH[WHUQDO

ϭϬϯ

WXIWHGFHOOV+D\DUHWDO$QWDOHWDO/LXDQG6KLSOH\ 0&VZHUHLGHQWLILHGDVODUJH

ϭϬϰ

FHOOVORFDWHGLQWKHPLWUDOFHOOOD\HU0&/ &HOOVZLWKDPELJXRXVLGHQWLWLHV±WKRVHZLWKVRPDWDWKDW

ϭϬϱ

UHVLGHGSDUWLDOO\LQWKH0&/±ZHUHH[FOXGHGIURPDQDO\VLV Electrophysiology:07&VZHUHYLVXDOL]HGXVLQJLQIUDUHGGLIIHUHQWLDOLQWHUIHUHQFHFRQWUDVWYLGHR

ϭϬϲ ϭϬϳ

PLFURVFRS\7KURXJKRXWWKHUHFRUGLQJSURFHVVVOLFHVZHUHFRQWLQXRXVO\VXSHUIXVHGZLWKƒ&

ϭϬϴ

R[\JHQDWHG5LQJHUVROXWLRQ&XUUHQWFODPSUHFRUGLQJVZHUHPDGHIURPLQGLYLGXDOFHOOVXVLQJHOHFWURGHV

ϭϬϵ

ILOOHGZLWKLQP0 SRWDVVLXPJOXFRQDWH.&O+(3(6VRGLXPSKRVSKRFUHDWLQH0J$73

ϭϭϬ

1D*73(*7$±$OH[D)OXRU/LIH7HFKQRORJLHV&DUOVEDG&$86$ DQG

ϭϭϭ

1HXURELRWLQ9HFWRU/DEV%XUOLQJDPH&$86$ 9ROWDJHFODPSUHFRUGLQJVZHUHPDGHXVLQJ

ϭϭϮ

HOHFWURGHVILOOHGZLWKLQP0 &VJOXFRQDWH+(3(6.&OVRGLXPSKRVSKRFUHDWLQH0J

ϭϭϯ

$73DQG1D*73$OH[D)OXRU/LIH7HFKQRORJLHV&DUOVEDG&$86$ DQG

ϭϭϰ

1HXURELRWLQ9HFWRU/DEV%XUOLQJDPH&$86$ $OOGDWDZHUHORZSDVVILOWHUHGDWN+]DQGGLJLWL]HG

ϭϭϱ

DWN+]XVLQJD0XOWL&ODPS$DPSOLILHU0ROHFXODU'HYLFHV6XQQ\YDOH&$86$ DQGDQ,7&

ϭϭϲ

DFTXLVLWLRQERDUG,QVWUXWHFK0LQHROD1<86$ FRQWUROOHGE\FXVWRPVRIWZDUHZULWWHQLQ,JRU3UR

ϭϭϳ

:DYH0HWULFV/DNH2VZHJR2586$ )RUHOHFWULFDOVWLPXODWLRQRI261VDPRQRSRODUJODVV

ϭϭϴ

HOHFWURGHZDVILOOHGZLWK5LQJHUVROXWLRQDQGFRQQHFWHGWRDVWLPXOXVLVRODWLRQXQLW:RUOG3UHFLVLRQ

ϭϭϵ

,QVWUXPHQWV6DUDVRWD)/86$ FRQWUROOHGE\77/SXOVHVIURPWKH,7&DFTXLVLWLRQERDUG)RU

ϭϮϬ

RSWRJHQHWLFVWLPXODWLRQVOLFHVZHUHLOOXPLQDWHGPVOLJKWSXOVH E\DQP/('S(&RRO/HG

ϭϮϭ

8. GLUHFWHGWKURXJKD[ZDWHULPPHUVLRQREMHFWLYHFHQWHUHGRQDVLQJOHJORPHUXOXVZLWKDFORVHG

ϭϮϮ

ILHOGVWRS%XUWRQDQG8UEDQ $OOGDWDZHUHORZSDVVILOWHUHGDWN+]DQGGLJLWL]HGDWN+]

ϭϮϯ

XVLQJD0XOWL&ODPS$DPSOLILHU0ROHFXODU'HYLFHV6XQQ\YDOH&$86$ DQGDQ,7&DFTXLVLWLRQ

ϭϮϰ

ERDUG,QVWUXWHFK0LQHROD1<86$ FRQWUROOHGE\FXVWRPVRIWZDUHZULWWHQLQ,JRU3UR:DYH0HWULFV

ϭϮϱ

/DNH2VZHJR2586$ 



ϱ

 ϭϮϲ

(OHFWULFDODQGOLJKWVWLPXODWLRQLQWHQVLWLHVZHUHFKRVHQDVIROORZV)LUVWWKHPLQLPXPLQWHQVLW\

ϭϮϳ

QHHGHGWRHYRNHUHOLDEOHVSLNLQJIRUFXUUHQWFODPSH[SHULPHQWV RUH[FLWDWRU\FXUUHQWVIRUYROWDJH

ϭϮϴ

FODPSH[SHULPHQWV ZDVGHWHUPLQHG5HOLDELOLW\ZDVGHILQHGDVWKHSUHVHQFHRIDWOHDVWVSLNHRU

ϭϮϵ

H[FLWDWRU\FXUUHQWVLQRYHURIWULDOV7RWKHILQGWKHPD[LPXPVWLPXODWLRQLQWHQVLW\WKHHOHFWULFDORU

ϭϯϬ

OLJKWLQWHQVLW\ZDVLQFUHDVHGXQWLODSODWHDXLQWKHQXPEHURIVSLNHVRUWKHVL]HRIH[FLWDWRU\FXUUHQWV

ϭϯϭ

ZDVUHDFKHG7KHPLGGOHVWLPXODWLRQLQWHQVLW\ZDVGHILQHGDVKDOIZD\EHWZHHQWKHPLQLPXPDQG

ϭϯϮ

PD[LPXPLQWHQVLWLHV/DVWO\WKHILQDOWZRLQWHQVLWLHVZHUHFKRVHQDVKDOIZD\EHWZHHQWKHPLQLPXPDQG

ϭϯϯ

PLGGOHLQWHQVLWLHVDQGWKHPLGGOHDQGPD[LPXPLQWHQVLWLHV0HDVXUHPHQWVRIVSLNLQJRUV\QDSWLF

ϭϯϰ

FXUUHQWVZHUHUHSHDWHGWLPHVDWHDFKVWLPXODWLRQLQWHQVLW\0&VDQG7&VZHUHDWPHPEUDQHSRWHQWLDO

ϭϯϱ

EHWZHHQP9DQGP9WKURXJKRXWWKHFXUUHQWFODPSUHFRUGLQJV Data analysis and statistics: )RUFXUUHQWFODPSH[SHULPHQWVSHULVWLPXOXVWLPHKLVWRJUDPV

ϭϯϲ ϭϯϳ

367+V ZHUHFRPSXWHGE\VXPPLQJVSLNHVDFURVVWULDOVDQGVPRRWKLQJZLWKDPVZLGH*DXVVLDQ

ϭϯϴ

ILOWHU7KHPD[LPXPILULQJUDWHUHIHUHQFHGWKURXJKRXWWKHPDQXVFULSWLVFDOFXODWHGDVWKHSHDNRIWKH

ϭϯϵ

367+7KHODWHQF\WRUHOLDEOHVSLNLQJZDVFDOFXODWHGLQHDFKFHOOE\ELQQLQJVSLNHVLQPVELQVDQG

ϭϰϬ

ILQGLQJWKHSHUFHQWDJHRIWULDOVRQZKLFKWKHFHOOVSLNHGLQHDFKWLPHELQµ7LPHWRUHOLDEOHVSLNLQJ¶ZDV

ϭϰϭ

GHILQHGDVWKHILUVWWLPHELQDIWHUVWLPXODWLRQLQZKLFKWKHFHOOVSLNHGLQRYHURIWULDOV

ϭϰϮ

6LJQLILFDQFHEHWZHHQ0&VDQG7&VZKHQPHWULFVRIVSLNHODWHQF\ZHUHSORWWHGYV367+SHDN

ϭϰϯ

ZDVGHWHUPLQHGE\ILWWLQJWKHGHSHQGHQFHRIODWHQF\RQUDWHIRUHDFKFHOOWRDQH[SRQHQWLDOIXQFWLRQWR

ϭϰϰ

GHWHUPLQHIJ7KHQIJ¶VZHUHFRPSDUHGEHWZHHQ0&VDQG7&VXVLQJXQSDLUHGWWHVWV

ϭϰϱ

5HVXOWV

ϭϰϲ

7RGHWHUPLQHKRZ0&VDQG7&VUHVSRQGWRFKDQJHVLQVWLPXOXVLQWHQVLW\ZHUHFRUGHG

ϭϰϳ

PHPEUDQHSRWHQWLDOLQVLQJOH0&VDQG7&VLQROIDFWRU\EXOEVOLFHVIROORZLQJHOHFWULFDOVWLPXODWLRQRIWKH

ϭϰϴ

ROIDFWRU\QHUYHOD\HUDGMDFHQWWRWKHFHOO¶VKRPHJORPHUXOXVDWPXOWLSOHLQWHQVLWLHV)LJXUHDF )LYH

ϭϰϵ

LQWHQVLWLHVIRUHDFKFHOOZHUHFKRVHQWRVDPSOHWKHHQWLUHW\RIHDFKFHOO¶VVHQVRU\HYRNHGG\QDPLF

ϭϱϬ

UDQJHVHH0DWHULDOVDQG0HWKRGV 6WLPXODWLRQLQWHQVLWLHVLQ0&VUDQJHGEHWZHHQ“



ϲ

 ϭϱϭ

ȝ$DQG“ȝ$ZKLOHVWLPXODWLRQLQWHQVLWLHVLQ7&VUDQJHGEHWZHHQ“ȝ$

ϭϱϮ

DQG“ȝ$1HLWKHUWKHPLQLPXPS XQSDLUHGWWHVW QRUPD[LPXPS 

ϭϱϯ

XQSDLUHGWWHVW VWLPXODWLRQLQWHQVLWLHVGLIIHUHGEHWZHHQ0&VDQG7&V7KLVDSSURDFKDOORZHG

ϭϱϰ

EHWZHHQFHOOFRPSDULVRQVDWHDFKRIWKHILYHLQWHQVLWLHV

ϭϱϱ

*LYHQSULRUZRUNVKRZLQJWKDWERWKVSLNHODWHQFLHVDQGILULQJUDWHVRISULQFLSDOQHXURQVYDU\ZLWK

ϭϱϲ

RGRUFRQFHQWUDWLRQ&DQJDQG,VDDFVRQ)XNXQDJDHWDO,JDUDVKLHWDO6LURWLQHW

ϭϱϳ

DO ZHFRPSDUHGKRZVSLNHODWHQFLHVDQGILULQJUDWHVYDULHGZLWKVWLPXODWLRQLQWHQVLW\LQ0&V

ϭϱϴ

DQG7&V7KHWRWDOQXPEHURIDFWLRQSRWHQWLDOV)LJXUHG LQDVLQWHUYDOIROORZLQJVWLPXODWLRQ

ϭϱϵ

LQFUHDVHGZLWKLQWHQVLW\S HZD\$129$ EXWGLGQRWGLIIHUEHWZHHQ0&VDQG7&V

ϭϲϬ

&RPSDUHGZLWK0&VKRZHYHU7&VH[KLELWHGVLJQLILFDQWO\KLJKHUILULQJUDWHVDVPHDVXUHGE\WKHSHDN

ϭϲϭ

LQWKH367+)LJXUHH 7KHUHIRUH0&VDQG7&VDUHERWKFDSDEOHRIXVLQJILULQJUDWHWRHQFRGH

ϭϲϮ

LQIRUPDWLRQDERXW261VWLPXODWLRQLQWHQVLW\

ϭϲϯ

1H[WZHH[SORUHGZKHWKHUVSLNHODWHQF\DVDVVHVVHGE\WKUHHGLIIHUHQWPHWULFVYDULHGZLWK

ϭϲϰ

261VWLPXODWLRQLQWHQVLW\LQHLWKHU0&VRU7&V)LUVWZHIRXQGWKDWWKHODWHQF\WR367+SHDN

ϭϲϱ

GHFUHDVHGZLWKVWLPXODWLRQLQWHQVLW\LQ0&VEXWQRW7&V)LJXUHI 7REHWWHUFRPSDUHVSLNHODWHQF\

ϭϲϲ

ZLWKin vivo GDWDZHSORWWHGWKHODWHQF\WR367+SHDNYVSHDNILULQJUDWH367+SHDN DQGVLPLODUO\

ϭϲϳ

IRXQGWKDWWKHGHSHQGHQFHRIVSLNHODWHQF\RQILULQJUDWHLVVLJQLILFDQWO\GLIIHUHQWEHWZHHQ0&VDQG7&V

ϭϲϴ

)LJXUHJ±VHH0HWKRGV $GGLWLRQDOO\XVLQJVWVSLNHODWHQF\)LJXUHK DQGWKHWLPHWRUHOLDEOH

ϭϲϵ

VSLNLQJ)LJXUHL±VHH0HWKRGV DVPHDVXUHVRIODWHQF\VKRZHGWKDWODWHQFLHVLQ0&VYDU\PRUH

ϭϳϬ

VWURQJO\ZLWKSHDNILULQJUDWHWKDQODWHQFLHVLQ7&V$GGLWLRQDOO\DWORZILULQJUDWHV+] PDQ\

ϭϳϭ

IHZHU0&VWKDQ7&VH[KLELWUHOLDEOHVSLNHWLPLQJV)LJXUHM ZKLFKFRUURERUDWHVin vivo GDWDWKDW0&

ϭϳϮ

UHVSRQVHODWHQFLHVDUHQRWUHOLDEOHDWORZRGRUFRQFHQWUDWLRQV,JDUDVKLHWDO )LQDOO\JLYHQ

ϭϳϯ

WKDW0&VLQWULQVLFDOO\H[KLELWXSVWDWHDQGGRZQVWDWHPHPEUDQHSRWHQWLDOV+H\ZDUGHWDO 

ϭϳϰ

ZHH[SORUHGZKHWKHUHDFKFHOO¶VVSLNHODWHQF\DWPLQLPXPVWLPXODWLRQLQWHQVLWLHVZDV

ϭϳϱ

LQIOXHQFHGE\WKHFHOO¶VPHPEUDQHSRWHQWLDOEHIRUH261VWLPXODWLRQRQDWULDOE\WULDOEDVLV

ϭϳϲ

6SLNHODWHQF\DVDVVHVVHGE\VWVSLNHODWHQF\GLGQRWFRUUHODWHZLWKWKHFHOO¶VSUHVWLPXODWLRQ 

ϳ

 ϭϳϳ

PHPEUDQHSRWHQWLDOLQHLWKHU0&V0&VU S 3HDUVRQ¶VFRUUHODWLRQ RU7&VU 

ϭϳϴ

S 3HDUVRQ¶VFRUUHODWLRQ 7KHUHIRUHZKLOHERWK0&VDQG7&VKDYHWKHSRWHQWLDOWRHQFRGH

ϭϳϵ

LQIRUPDWLRQDERXWWKHLQWHQVLW\RIJORPHUXODUDFWLYDWLRQLQWKHLUILULQJUDWHVRQO\0&VDUHFDSDEOHRI

ϭϴϬ

XVLQJVSLNHODWHQF\WRHQFRGHLQWHQVLW\LQIRUPDWLRQ

ϭϴϭ

2QHSRWHQWLDOFRQIRXQGWRWKHVHH[SHULPHQWVLVWKHSRVVLELOLW\WKDW0&DQG7&DSLFDOGHQGULWHV

ϭϴϮ

DUHGLUHFWO\H[FLWHGE\HOHFWULFDOVWLPXODWLRQDWVWURQJVWLPXODWLRQLQWHQVLWLHV7KHUHIRUHZHSHUIRUPHGDQ

ϭϴϯ

DQDORJRXVH[SHULPHQWLQ203&K5<)3PLFH6PHDUHWDO LQZKLFKZHSKRWRVWLPXODWHG

ϭϴϰ

PVSXOVH WKHKRPHJORPHUXOXVRIWKHUHFRUGHGFHOODWILYHOLJKWLQWHQVLW\FKRVHQDVGHVFULEHGDERYH

ϭϴϱ

)LJXUHD :HIRXQGWKDWZKLOHWKHQXPEHURIVSLNHVLQFUHDVHVLQ0&VDQG7&V)LJXUHE ILULQJ

ϭϴϲ

UDWHVLQ7&VDUHVLJQLILFDQWO\KLJKHUWKDQLQ0&V)LJXUHF $GGLWLRQDOO\VSLNHODWHQF\LQ0&VEXWQRW

ϭϴϳ

LQ7&VVKRZHGDVWURQJGHSHQGHQFHRQILULQJUDWH/DWHQF\WR367+SHDN)LJXUHG VWVSLNH

ϭϴϴ

ODWHQF\)LJXUHH DQGODWHQF\WRUHOLDEOHVSLNLQJ)LJXUHI DOOVLJQLILFDQWO\GLIIHUHGEHWZHHQ0&VDQG

ϭϴϵ

7&V$GGLWLRQDOO\IHZHU0&VWKDQ7&VVKRZHGUHOLDEOHVSLNHWLPLQJDWORZSKRWRVWLPXODWLRQLQWHQVLWLHV

ϭϵϬ

)LJXUHJ )LQDOO\WKHSHDNILULQJUDWHVDQGODWHQFLHVLQ0&VDQG7&VZHUHFRPSDUDEOHEHWZHHQ

ϭϵϭ

H[SHULPHQWVXVLQJHOHFWULFDODQGSKRWRVWLPXODWLRQRI261V7KHVLPLODULW\RIWKHVHGDWDZLWKGDWDIURP

ϭϵϮ

HOHFWULFDOVWLPXODWLRQRIJORPHUXOLLQGLFDWHVWKDWGLUHFWHOHFWULFDOVWLPXODWLRQRI0&RU7&DSLFDOGHQGULWHV

ϭϵϯ

GLGQRWVLJQLILFDQWO\LQIOXHQFHRXUUHVXOWV7KHUHIRUHRXUin vitro GDWDFRUURERUDWHVSULRUin vivoILQGLQJV

ϭϵϰ

WKDW0&VDQG7&VKDYHWKHSRWHQWLDOWRXVHGLVWLQFWVWUDWHJLHVIRUHQFRGLQJFRQFHQWUDWLRQ)XNXQDJDHW

ϭϵϱ

DO,JDUDVKLHWDO 

ϭϵϲ

7RGHWHUPLQHSRWHQWLDOVRXUFHVRIWKHVHGLIIHUHQFHVLQKRZ0&VDQG7&VUHVSRQGWRFKDQJHVLQ

ϭϵϳ

VWLPXOXVLQWHQVLW\ZHPHDVXUHGERWKH[FLWDWRU\DQGLQKLELWRU\FXUUHQWVIROORZLQJHOHFWULFDOVWLPXODWLRQRI

ϭϵϴ

261VDFURVVILYHVWLPXODWLRQLQWHQVLWLHV)LJXUHDE 3UHYLRXVO\RXUJURXSDQGRWKHUVKDYHVKRZQ

ϭϵϵ

WKDW7&VUHFHLYHVWURQJHUH[FLWDWLRQWKDQ0&VDWPLQLPDOVWLPXODWLRQLQWHQVLWLHV*LUHHWDO

ϮϬϬ

%XUWRQDQG8UEDQ +RZHYHUHYLGHQFHIRUGLIIHUHQFHVLQIHHGIRUZDUGLQKLELWLRQLVPL[HG

ϮϬϭ

)XNXQDJDHWDO1DMDFHWDO 7KHUHIRUHZHPHDVXUHGERWKH[FLWDWRU\DQGLQKLELWRU\

ϮϬϮ

FXUUHQWVDFURVVILYHVWLPXODWLRQLQWHQVLWLHVLQ0&VDQG7&V:HIRXQGWKDWERWKWKHSHDNDPSOLWXGH 

ϴ

 ϮϬϯ

)LJXUHF723 DQGFKDUJH)LJXUHG723FDOFXODWHGDVWKHLQWHJUDODFURVVVIROORZLQJ

ϮϬϰ

VWLPXODWLRQ RIIHHGIRUZDUGLQKLELWLRQRQWR0&VLVODUJHUWKDQRQWR7&V+RZHYHUZKLOHWKHSHDN

ϮϬϱ

DPSOLWXGHRIH[FLWDWLRQLVVLJQLILFDQWO\ODUJHULQ7&VWKDQLQ0&V)LJXUHF%27720 FKDUJH

ϮϬϲ

WUDQVIHUUHGGLGQRWGLIIHUEHWZHHQ0&VDQG7&V)LJXUHG%27720 &RQVHTXHQWO\WKH(,UDWLRLV

ϮϬϳ

VLJQLILFDQWO\ORZHULQ0&VWKDQLQ7&V)LJXUHI723 7KHVH(,UDWLRVFDOFXODWHGDFURVVWKHILUVW

ϮϬϴ

VHFRQGDIWHUVWLPXODWLRQOLNHO\DOORZ7&VWRILUHDWKLJKHUSHDNUDWHVWKDQ0&VDFURVVDOOVWLPXODWLRQ

ϮϬϵ

LQWHQVLWLHV+RZHYHULWFDQQRWH[SODLQZK\WKHODWHQF\RIVSLNLQJLQ0&VEXWQRW7&VLVPXFKORQJHUDW

ϮϭϬ

ORZVWLPXODWLRQLQWHQVLWLHVWKDQDWKLJKVWLPXODWLRQLQWHQVLWLHV *LYHQSULRUZRUNLQGLFDWLQJWKDWJORPHUXODUOD\HUPHGLDWHGLQKLELWLRQSOD\VDQLPSRUWDQWUROH

Ϯϭϭ ϮϭϮ

LQUHJXODWLQJUHVSRQVHODWHQF\LQ0&VDQGWKDWJORPHUXODUOD\HUPHGLDWHGIHHGIRUZDUGLQKLELWLRQLV

Ϯϭϯ

FRQILQHGWRWKHILUVWPVDIWHU261VWLPXODWLRQ6KDRHWDO1DMDFHWDO ZHOLPLWHGRXU

Ϯϭϰ

DQDO\VLVRIH[FLWDWRU\DQGLQKLELWRU\FKDUJHWUDQVIHUDQG(,UDWLRWRWKHILUVWPVIROORZLQJ261

Ϯϭϱ

VWLPXODWLRQ6LPLODUWRWKHILQGLQJVGHVFULEHGDERYHIRUWKHPVWLPHZLQGRZZHILQGWKDW

Ϯϭϲ

LQKLELWRU\)LJXUHH723 EXWQRWH[FLWDWRU\)LJXUHH%27720 FKDUJHWUDQVIHUUHGGXULQJWKH

Ϯϭϳ

PVWLPHZLQGRZLVVLJQLILFDQWO\ODUJHULQ0&VWKDQ7&V$GGLWLRQDOO\WKH(,UDWLRLQWKLVPV

Ϯϭϴ

WLPHZLQGRZLVVLJQLILFDQWO\KLJKHULQ7&VWKDQLQ0&V)LJXUHI%27720 7KLVGLIIHUHQFHLQ(,UDWLR

Ϯϭϵ

LVODUJHVWDWORZVWLPXODWLRQLQWHQVLWLHV(,UDWLRLVDSSUR[LPDWHO\RQHLQ0&VDQGRYHUVL[LQ7&VDW

ϮϮϬ

PLQLPDOVWLPXODWLRQLQWHQVLWLHV&RQVHTXHQWO\ORQJHUODWHQF\VSLNLQJLQ0&VWKDQLQ7&VDWWKHVHORZ

ϮϮϭ

LQWHQVLWLHVLVSUHGRPLQDQWO\GULYHQE\ODUJHLQKLELWRU\FXUUHQWVGXULQJWKHILUVWPVDIWHUVWLPXODWLRQ

ϮϮϮ

)LQDOO\ZHDQDO\]HGKRZWKHODWHQF\WRWKHSHDNDPSOLWXGHVRILQKLELWLRQDQGH[FLWDWLRQFKDQJHDFURVV

ϮϮϯ

VWLPXODWLRQLQWHQVLWLHV:KLOHWKHODWHQF\WRWKHSHDNRILQKLELWLRQGRHVQRWYDU\ZLWKVWLPXODWLRQLQWHQVLW\

ϮϮϰ

S ZD\$129$ RUEHWZHHQ0&VDQG7&V)LJXUHJ WKHODWHQF\WRWKHSHDNRIH[FLWDWLRQ

ϮϮϱ

GRHVYDU\ZLWKVWLPXODWLRQLQWHQVLW\S HZD\$129$ DQGEHWZHHQ0&VDQG7&V)LJXUH

ϮϮϲ

K ,QWHUHVWLQJO\LQ7&VWKHSHDNRIH[FLWDWLRQSUHFHGHVWKHSHDNRILQKLELWLRQDWDOOLQWHQVLWLHV

ϮϮϳ

+RZHYHULQ0&VH[FLWDWLRQOHDGVLQKLELWLRQDWKLJKVWLPXODWLRQLQWHQVLWLHVEXWODJVLQKLELWLRQDWORZ

ϮϮϴ

LQWHQVLWLHV)LJXUHL 7KLVVKLIWIURPH[FLWDWLRQODJJLQJLQKLELWLRQWRH[FLWDWLRQOHDGLQJLQKLELWLRQOLNHO\

ϮϮϵ

UHIOHFWVSULRUZRUNVKRZLQJWKDWDWZHDNVWLPXODWLRQLQWHQVLWLHV0&VSULPDULO\UHFHLYHLQGLUHFWH[FLWDWLRQ 

ϵ

 ϮϯϬ

IURP(7&V*LUHHWDO DQGWKDWDWKLJKHULQWHQVLWLHV0&VUHFHLYHERWKLQGLUHFWDQGGLUHFW

Ϯϯϭ

H[FLWDWLRQIURP261V'H6DLQW-DQDQG:HVWEURRN'H6DLQW-DQHWDO1DMDFHWDO

ϮϯϮ

9DDJDDQG:HVWEURRN 

Ϯϯϯ

7RGLUHFWO\WHVWZKHWKHUODUJHULQKLELWRU\FXUUHQWVRQWR0&VDQG7&VDUHPHGLDWHGE\

Ϯϯϰ

JORPHUXODUOD\HUFLUFXLWVZHPHDVXUHGJORPHUXODUOD\HUPHGLDWHGLQKLELWRU\FXUUHQWVE\

Ϯϯϱ

SKRWRVWLPXODWLQJWKHKRPHJORPHUXOXVRIWKHUHFRUGHG0&7&LQ203&K5PLFHZKLOHOLPLWLQJ*&

Ϯϯϲ

PHGLDWHGLQKLELWLRQE\EORFNLQJP*OX5V/<ȝ0 DQG10'$5V$3ȝ0 DVSUHYLRXVO\

Ϯϯϳ

GHVFULEHG)LJXUHDE 1DMDFHWDO*HUDPLWDHWDO %HFDXVHWKHGLIIHUHQFHVLQVSLNH

Ϯϯϴ

WLPLQJDUHJUHDWHVWDWPLQLPDOVWLPXODWLRQLQWHQVLWLHVZHFRPSDUHGJORPHUXODUOD\HUPHGLDWHG

Ϯϯϵ

FXUUHQWVEHWZHHQ0&VDQG7&VDWWKHPLQLPXPLQWHQVLW\QHHGHGWRHOLFLWUHOLDEOHH[FLWDWRU\FXUUHQWV

ϮϰϬ

*ORPHUXODUOD\HUPHGLDWHGLQKLELWRU\FXUUHQWVDUHODUJHULQDPSOLWXGHLQ0&VWKDQLQ7&V)LJXUHH 

Ϯϰϭ

7KHUHZHUHQRGLIIHUHQFHVEHWZHHQ0&VDQG7&VLQWKHODWHQF\WRWKHSHDNRILQKLELWLRQ)LJXUHI RU

ϮϰϮ

WKHGXUDWLRQRILQKLELWLRQ)LJXUHJ±VHH0HWKRGV $GGLWLRQDOO\ZHPHDVXUHGH[FLWDWRU\FXUUHQWVDQG

Ϯϰϯ

IRXQGWKDWEORFNDGHRI10'$5VDQGP*OX5VGLGQRWDIIHFWWKHDPSOLWXGHRUGXUDWLRQRIH[FLWDWRU\

Ϯϰϰ

FXUUHQWVLQHLWKHU0&VRU7&V)LJXUHFG 6LPLODUWRWKHUHVXOWVIURPHOHFWULFDOVWLPXODWLRQ

Ϯϰϱ

H[SHULPHQWVH[FLWDWRU\FXUUHQWVDUHODUJHU)LJXUHK DQGSHDNDWVKRUWHUODWHQFLHV)LJXUHL LQ7&V

Ϯϰϲ

WKDQLQ0&V7KHUHIRUHDWPLQLPDOVWLPXODWLRQLQWHQVLWLHV0&VUHFHLYHZHDNHUDQGORQJHUODWHQF\

Ϯϰϳ

H[FLWDWRU\LQSXWVDQGVWURQJHUJORPHUXODUOD\HUPHGLDWHGLQKLELWRU\FXUUHQWVFRPSDUHGZLWK7&V

Ϯϰϴ

1H[WZHWHVWHGKRZEORFNLQJVSHFLILFLQKLELWRU\LQWHUQHXURQVXEW\SHVDIIHFWHGVSLNLQJLQ0&V

Ϯϰϵ

DQG7&V)LUVWZHPHDVXUHGVSLNLQJHOLFLWHGE\HOHFWULFDOO\VWLPXODWLQJ261VDWWKUHHGLIIHUHQW

ϮϱϬ

LQWHQVLWLHVPLQPD[DQGPLG EHIRUHDQGDIWHUOLPLWHG*&PHGLDWHGLQKLELWLRQE\EORFNLQJ10'$5V

Ϯϱϭ

DQGP*OX5V)LJXUHDE /LPLWLQJ*&PHGLDWHGLQKLELWLRQGLGQRWVLJQLILFDQWO\DIIHFWWKHQXPEHURI

ϮϱϮ

VSLNHV)LJXUHF WKHPD[LPXPILULQJUDWH)LJXUHG RUWKHODWHQF\RIVSLNLQJ±DVPHDVXUHGE\

Ϯϱϯ

HLWKHUODWHQF\WR367+SHDN)LJXUHH RUWKHODWHQF\WRUHOLDEOHVSLNLQJ)LJXUHI LQHLWKHU0&VRU

Ϯϱϰ

7&VDWDQ\RIWKHWKUHHVWLPXODWLRQLQWHQVLWLHV:KLOHZHFDQQRWUXOHRXWWKHSRVVLELOLW\WKDW*&V



ϭϬ

 Ϯϱϱ

FRQWULEXWHWR07&VSLNHWLPLQJDQGV\QFKURQ\RXUUHVXOWVVXJJHVWWKDW*&VGRQRWVWURQJO\LQIOXHQFH

Ϯϱϲ

SHDNILULQJUDWHVRUVSLNHODWHQFLHVIROORZLQJ261VWLPXODWLRQ 7RWHVWZKHWKHUEORFNLQJJORPHUXODUOD\HUPHGLDWHGLQKLELWLRQGLIIHUHQWLDOO\DIIHFWVSLNLQJLQ

Ϯϱϳ Ϯϱϴ

0&VDQG7&VZHPHDVXUHGVSLNLQJHOLFLWHGE\HOHFWULFDOO\VWLPXODWLQJ261VDWWKUHHGLIIHUHQW

Ϯϱϵ

LQWHQVLWLHVEHIRUHDQGDIWHUSXIILQJJDED]LQHLQWRWKHUHFRUGHGFHOO¶VKRPHJORPHUXOXV)LJXUHDG 

ϮϲϬ

$IWHUEORFNLQJJORPHUXODUOD\HUPHGLDWHGLQKLELWLRQLQ0&VWKHWRWDOQXPEHURIVSLNHV)LJXUHH 

Ϯϲϭ

HOLFLWHGLQFUHDVHGDWDOOWKUHHVWLPXODWLRQLQWHQVLWLHV+RZHYHULQ7&VWKHQXPEHURIVSLNHVLQFUHDVHG

ϮϲϮ

RQO\DWWKHORZHVWVWLPXODWLRQLQWHQVLW\$GGLWLRQDOO\WKHPD[LPDOILULQJUDWHLQFUHDVHGDWDOOWKUHH

Ϯϲϯ

VWLPXODWLRQLQWHQVLWLHV)LJXUHI LQERWK0&VDQG7&V,QFUHDVHVLQILULQJUDWHGHVSLWHWKHODFNRI

Ϯϲϰ

FKDQJHVLQWKHWRWDOQXPEHURIVSLNHVLQ7&VDWPLGDQGPD[LQWHQVLWLHVLQ7&VLPSOLHVWKDWWKHWLPLQJ

Ϯϲϱ

RIVSLNHVLVUHGLVWULEXWHGIROORZLQJ3*&EORFNDGH:KLOH3*&PHGLDWHGLQKLELWLRQLQFUHDVHGSHDNILULQJ

Ϯϲϲ

UDWHVE\VLPLODUDPRXQWVLQ0&VDQG7&VDWWKHORZHVWVWLPXODWLRQLQWHQVLW\)LJXUHJ/()7 

Ϯϲϳ

LQFUHDVHVLQSHDNILULQJUDWHVZHUHVLJQLILFDQWO\ODUJHU0&VWKDQLQ7&VDWWKHVWURQJHUWZRVWLPXODWLRQ

Ϯϲϴ

LQWHQVLWLHV)LJXUHJ0,''/(5,*+7 

Ϯϲϵ

*ORPHUXODUOD\HUPHGLDWHGLQKLELWLRQDOVRDIIHFWHGVSLNHODWHQF\PRUHVWURQJO\LQ0&VWKDQLQ

ϮϳϬ

7&V$WWKHZHDNHVWWZRVWLPXODWLRQLQWHQVLWLHVWKHODWHQF\WR367+SHDNEHFDPHVLJQLILFDQWO\VKRUWHU

Ϯϳϭ

LQ0&VEXWQRWLQ7&VDIWHUEORFNLQJJORPHUXODUOD\HUPHGLDWHGLQKLELWLRQ)LJXUH+ $GGLWLRQDOO\

ϮϳϮ

VSLNHWLPLQJLQ0&VEHFDPHPRUHUHOLDEOHDWWKHZHDNHVWVWLPXODWLRQLQWHQVLW\DIWHUEORFNLQJ

Ϯϳϯ

JORPHUXODUOD\HUPHGLDWHGLQKLELWLRQ%HIRUHEORFNLQJJORPHUXODUOD\HUPHGLDWHGLQKLELWLRQ]HURRI

Ϯϳϰ

WKHIRXU0&VVKRZHGUHOLDEOHVSLNHWLPLQJKRZHYHUDIWHUSXIILQJJDED]LQHLQWKHJORPHUXOXVVSLNH

Ϯϳϱ

WLPLQJLQDOOIRXU0&VEHFDPHUHOLDEOHDQGWKHODWHQF\WRUHOLDEOHVSLNLQJEHFDPHFRPSDUDEOHWR7&V

Ϯϳϲ

)LJXUHL/()7 $GGLWLRQDOO\WKHODWHQF\WRUHOLDEOHWLPLQJEHFDPHVKRUWHULQ0&VDWWKHPLGGOH

Ϯϳϳ

LQWHQVLW\DIWHUEORFNLQJJORPHUXODUOD\HUPHGLDWHGLQKLELWLRQEXWGLGQRWFKDQJHLQ7&VDWDQ\RIWKH

Ϯϳϴ

WKUHHVWLPXODWLRQLQWHQVLWLHV)LJXUHL )LQDOO\VLJQLILFDQWWRQLFLQKLELWLRQH[LVWVZLWKLQWKH

Ϯϳϵ

JORPHUXODUFLUFXLW$URQLDGRX$QGHUMDVNDHWDO6KDRHWDO DQGFRXOGFRQWULEXWHWR

ϮϴϬ

GLIIHUHQFHVLQVWLPXOXVHYRNHGUHVSRQVHV7KHUHIRUHZHFRPSDUHGVSRQWDQHRXVDFWLYLW\LQ 

ϭϭ

 Ϯϴϭ

0&VDQG7&VEHIRUHDQGDIWHUSXIILQJJDED]LQHLQWRWKHJORPHUXOXV)LJXUHM 6SRQWDQHRXV

ϮϴϮ

VSLNLQJZDVORZLQERWK0&VDQG7&VEHIRUHJDED]LQHDSSOLFDWLRQ0&V“+]7&V

Ϯϴϯ

“S XQSDLUHGWWHVW DQGVLJQLILFDQWO\LQFUHDVHGLQERWK0&VDQG7&VE\VLPLODU

Ϯϴϰ

DPRXQWVDIWHUJDED]LQHDSSOLFDWLRQ0&V“+]7&V“S XQSDLUHGW

Ϯϴϱ

WHVW 7KHUHIRUHZKLOHWRQLFLQKLELWLRQLVSUHVHQWLQERWK0&VDQG7&VLWFDQQRWH[SODLQWKH

Ϯϴϲ

GLIIHUHQFHVLQUHOLDELOLW\DQGVSLNHODWHQF\EHWZHHQ0&VDQG7&V7KHUHIRUHJORPHUXODUOD\HU

Ϯϴϳ

PHGLDWHGLQKLELWLRQPRUHVWURQJO\UHJXODWHVWKHUHOLDELOLW\DQGODWHQF\RIVSLNHWLPLQJLQ0&VWKDQLQ

Ϯϴϴ

7&V

Ϯϴϵ

'LVFXVVLRQ

ϮϵϬ

+HUHZHVKRZWKDW0&VEXWQRW7&VVKRZORQJODWHQF\UHVSRQVHVDWZHDNVWLPXODWLRQ

Ϯϵϭ

LQWHQVLWLHVWKDWVKRUWHQZLWKLQFUHDVLQJLQWHQVLW\ODUJHO\PLUURULQJZKDWKDVEHHQREVHUYHGin vivo ZLWK

ϮϵϮ

LQFUHDVLQJRGRUFRQFHQWUDWLRQ)XNXQDJDHWDO,JDUDVKLHWDO :HILQGWKDWWKHVH

Ϯϵϯ

GLIIHUHQFHVDUHOLNHO\GXHWRVWURQJHULQKLELWRU\FXUUHQWVDQGZHDNHUH[FLWDWRU\FXUUHQWVRQWR0&VWKDQ

Ϯϵϰ

RQWR7&V'LIIHUHQFHVLQLQKLELWLRQDUHODUJHVWGXULQJWKHILUVWPVDIWHUVWLPXODWLRQDQGDUHPHGLDWHG

Ϯϵϱ

SUHGRPLQDWHO\E\JORPHUXODUOD\HUFLUFXLWV$GGLWLRQDOO\ZHVKRZWKDWDIWHUEORFNLQJJORPHUXODU

Ϯϵϲ

OD\HUPHGLDWHGEXWQRW*&PHGLDWHGLQKLELWLRQWKHODWHQF\RIUHVSRQVHVLQ0&VEHFRPHVVKRUWHUDQG

Ϯϵϳ

PRUHUHOLDEOH

Ϯϵϴ

7KHVHGDWDVXSSRUWDQHPHUJLQJYLHZWKDW0&VDQG7&VDUHGLIIHUHQWLDOO\LQIOXHQFHGE\

Ϯϵϵ

JORPHUXODUOD\HUVRXUFHVRILQKLELWLRQZKLFKFDXVHGLIIHUHQFHVLQVSLNHODWHQF\EHWZHHQ0&VDQG7&V

ϯϬϬ

)XNXQDJDHWDO)XNXQDJDHWDO :HFRUURERUDWHSULRUZRUNDQGVKRZWKDWDWZHDN

ϯϬϭ

VWLPXODWLRQLQWHQVLWLHV0&VSULPDULO\UHFHLYHLQGLUHFWH[FLWDWRU\LQSXWIURPH[WHUQDOWXIWHGFHOOV(7&V 

ϯϬϮ

WKDWSHDNVDSSUR[LPDWHO\PVDIWHUVWLPXODWLRQ'H6DLQW-DQDQG:HVWEURRN'H6DLQW-DQHW

ϯϬϯ

DO*LUHDQG6FKRSSD1DMDFHWDO6PHDUHWDO*LUHHWDO1DMDFHWDO

ϯϬϰ

9DDJDDQG:HVWEURRN $VWKHVWLPXODWLRQLQWHQVLW\LQFUHDVHVGLUHFWFRQQHFWLRQVIURP

ϯϬϱ

261VRQWR0&VDUHUHFUXLWHGDQGWKHODWHQF\WRWKHSHDNRIH[FLWDWLRQVKRUWHQV7&VRQWKHRWKHU

ϯϬϲ

KDQGUHFHLYHVWURQJDQGGLUHFWH[FLWDWLRQIURP261VDWDOOVWLPXODWLRQLQWHQVLWLHVDQGFRQVHTXHQWO\ 

ϭϮ

 ϯϬϳ

WKHSHDNRIH[FLWDWLRQGRHVQRWYDU\ZLWKVWLPXODWLRQLQWHQVLW\,QERWK0&VDQG7&VLQKLELWRU\FXUUHQWV

ϯϬϴ

SHDNDSSUR[LPDWHO\PVDIWHUVWLPXODWLRQ7KHUHIRUHLQ7&VWKHSHDNRIH[FLWDWLRQSUHFHGHVLQKLELWLRQ

ϯϬϵ

UHJDUGOHVVRIVWLPXODWLRQLQWHQVLW\,Q0&VKRZHYHUWKHSHDNRIH[FLWDWLRQODJVLQKLELWLRQDWZHDN

ϯϭϬ

VWLPXODWLRQLQWHQVLWLHVEXWOHDGVLQKLELWLRQDWKLJKHUVWLPXODWLRQLQWHQVLWLHV6WURQJHUJORPHUXODUOD\HU

ϯϭϭ

PHGLDWHGLQKLELWRU\FXUUHQWVRQWR0&VWKDQ7&VZRUNFRRSHUDWLYHO\ZLWKWKHVHGLIIHUHQFHVLQWKHVRXUFH

ϯϭϮ

DQGUHODWLYHVWUHQJWKRIH[FLWDWLRQWRFDXVHWKHODWHQF\RI0&VSLNLQJWRYDU\ZLWKVWLPXODWLRQLQWHQVLW\ :KLFKJORPHUXODUOD\HULQKLELWRU\LQWHUQHXURQVXEW\SHVSUHIHUHQWLDOO\LQKLELW0&VRYHU

ϯϭϯ ϯϭϰ

7&V"3ULRUin vivo ZRUNVXJJHVWVWKDW3*&VDUHUHVSRQVLEOHIRUGLIIHUHQFHVLQVSLNHODWHQF\

ϯϭϱ

EHWZHHQ0&VDQG7&V)XNXQDJDHWDO)XNXQDJDHWDO 6WURQJHU3*&PHGLDWHG

ϯϭϲ

LQKLELWLRQRQWR0&VWKDQ7&VPD\EHH[SODLQHGE\PXOWLSOHPHFKDQLVPV3*&V\QDSVHVRQWR0&V

ϯϭϳ

PD\EHVWURQJHURUPRUHQXPHURXVWKDQV\QDSVHVRQWR7&V$OWHUQDWLYHO\VHSDUDWHSRSXODWLRQVRI

ϯϭϴ

3*&VPD\SUHIHUHQWLDOO\WDUJHW0&VDQG7&V6XEVHWVRI3*&VKDYHEHHQLGHQWLILHGERWKIXQFWLRQDOO\

ϯϭϵ

DQGPROHFXODUO\)XQFWLRQDOO\3*&VDUHFODVVLILHGE\ZKHWKHUWKH\UHFHLYHH[FLWDWLRQIURP261VRU

ϯϮϬ

(7&V+D\DUHWDO6KDRHWDO $GGLWLRQDOO\3*&VDUHPROHFXODUO\KHWHURJHQHRXV

ϯϮϭ

.RVDNDHWDO3DUULVK$XQJVWHWDO.L\RNDJHHWDO $OWHUQDWLYHO\GLIIHUHQFHVLQ

ϯϮϮ

V6$&PHGLDWHG6KDRHWDO:KLWHVHOOHWDO/LXHWDO RU(7&PHGLDWHG7DWWL

ϯϮϯ

HWDO LQKLELWLRQPD\FRQWULEXWHWRGLIIHUHQFHVLQJORPHUXODUOD\HUPHGLDWHGLQKLELWLRQ

ϯϮϰ

RQWR0&VDQG7&V([SUHVVLQJFKDQQHOUKRGRSVLQLQVSHFLILFFHOOW\SHV)XNXQDJDHWDO

ϯϮϱ

%DQHUMHHHWDO/LXHWDO ZLOOEHYLWDOIRUGHWHUPLQLQJZKHWKHUDQGWKHH[WHQWWR

ϯϮϲ

ZKLFKHDFKFHOOW\SHFRQWULEXWHVWRGLIIHUHQFHVLQLQKLELWLRQEHWZHHQ0&VDQG7&V

ϯϮϳ

:KHWKHUDQLPDOVXVHVSLNHODWHQF\RI0&VDQGRU7&VWRGHWHUPLQHRGRUFRQFHQWUDWLRQLVDQ

ϯϮϴ

XQDQVZHUHGTXHVWLRQWKDWZLOOSOD\DQLPSRUWDQWUROHLQLQWHUSUHWLQJWKHVHGDWD:KLOHSULRUZRUNKDV

ϯϮϵ

VKRZQWKDWDQLPDOVFDQEHWUDLQHGWRXVHODWHQF\WRHQFRGHEHKDYLRUDOO\UHOHYDQWLQIRUPDWLRQ6PHDUHW

ϯϯϬ

DO ZKHWKHUDQLPDOVQRUPDOO\XVHODWHQF\WRHQFRGHFRQFHQWUDWLRQVSHFLILFLQIRUPDWLRQLV

ϯϯϭ

XQNQRZQ,I0&VGRHQFRGHFRQFHQWUDWLRQZLWKVSLNHODWHQF\WKHQWKHFRQFHQWUDWLRQLQYDULDQFHRI7&

ϯϯϮ

ODWHQF\)XNXQDJDHWDO,JDUDVKLHWDO VXJJHVWVWKDW7&VPD\SOD\LQLPSRUWDQWUROHLQ 

ϭϯ

 ϯϯϯ

LGHQWLI\LQJRGRUVDFURVVDZLGHUDQJHRIRGRUFRQFHQWUDWLRQV+RZHYHULIDQLPDOVGRQRWXVHODWHQF\WR

ϯϯϰ

GHWHUPLQHRGRUFRQFHQWUDWLRQDQGLQVWHDGH[FOXVLYHO\XVHILULQJUDWHVWKHQDQDOWHUQDWLYHSXUSRVHIRU

ϯϯϱ

ORQJODWHQF\VSLNLQJLQ0&VLVQHHGHG)RULQVWDQFHRGRULGHQWLW\PD\EHHQFRGHGE\0&VSLNHODWHQF\

ϯϯϲ

+RSILHOG%URG\DQG+RSILHOG 7KHUHIRUHZKLOHWKHDEVROXWHODWHQF\RI0&VSLNLQJPD\

ϯϯϳ

YDU\ZLWKFRQFHQWUDWLRQWKHUHODWLYHGLIIHUHQFHVLQ0&ODWHQF\DFURVVWKHSRSXODWLRQUHPDLQVFRQVWDQW

ϯϯϴ

VRWKDWWKHUHSUHVHQWDWLRQRIRGRULGHQWLW\UHPDLQVFRQFHQWUDWLRQLQYDULDQW6FKDHIHUDQG0DUJULH

ϯϯϵ

8FKLGDHWDO $OWHUQDWLYHO\0&VDQG7&VPD\EHUHVSRQVLEOHIRUHQFRGLQJROIDFWRU\LQIRUPDWLRQ

ϯϰϬ

LQVHSDUDWHFRQFHQWUDWLRQUDQJHVDQGRQO\FHOOVVSLNLQJHDUO\LQWKHVQLIIF\FOHPD\EHHQFRGLQJWDVN

ϯϰϭ

VSHFLILFLQIRUPDWLRQ*HUDPLWDHWDO 7KLVYLHZLPSOLHVWKDWLQWULQVLFDQGFLUFXLWOHYHOIDFWRUVWKDW

ϯϰϮ

FDXVHORQJODWHQF\VSLNLQJLQ0&VHUYHVWRUHGXFHUHGXQGDQWVSLNHVHDUO\LQWKHVQLIIF\FOH'DWD

ϯϰϯ

VKRZLQJWKDWVSLNHODWHQF\LQ0&VLVXQUHOLDEOHDWORZVWLPXOXVLQWHQVLWLHV,JDUDVKLHWDO DQG

ϯϰϰ

WKDW7&VKDYHORZHURGRUWKUHVKROGV1DJD\DPDHWDO.LNXWDHWDO LPSOLHVWKDWDWOHDVW

ϯϰϱ

DWORZRGRUFRQFHQWUDWLRQVRGRULGHQWLW\LQIRUPDWLRQLVSULPDULO\HQFRGHGE\7&V)LQDOO\RWKHUDVSHFWV

ϯϰϲ

RIROIDFWRU\EXOEFLUFXLWU\VXSSRUWFRQFHQWUDWLRQVSHFLILFUDQJHVIRU0&VDQG7&V)RULQVWDQFH

ϯϰϳ

GLIIHUHQFHVLQWKHVRXUFHVDQGHIIHFWVRIODWHUDOLQKLELWLRQRQWR0&VDQG7&VDOORZHDFKWREHVWSHUIRUP

ϯϰϴ

RGRUGLVFULPLQDWLRQVLQVHSDUDWHFRQFHQWUDWLRQVUDQJHV*HUDPLWDHWDO )XWXUHH[SHULPHQWV

ϯϰϵ

H[SORULQJKRZRGRULGHQWLILFDWLRQRUGLVFULPLQDWLRQDWORZFRQFHQWUDWLRQVLVDIIHFWHGE\HLWKHUFKHPLFDO

ϯϱϬ

RURSWLFDOVLOHQFLQJRI7&VZLOOEHQHHGHGWRGHWHUPLQHWKHH[WHQWWRZKLFK0&VDQG7&VFRQWULEXWHWR

ϯϱϭ

HQFRGLQJROIDFWRU\LQIRUPDWLRQDWORZYVKLJKRGRUFRQFHQWUDWLRQV :HQRWHKRZHYHUWKDWRXUin vitro DSSURDFKDOVRKDVFHUWDLQOLPLWDWLRQV,QSDUWLFXODU

ϯϱϮ ϯϱϯ

LQFUHDVLQJWKHLQWHQVLW\RIHOHFWULFDOVWLPXODWLRQRI261VWKDWSURMHFWWRDVLQJOHJORPHUXOXV

ϯϱϰ

GRHVQRWUHIOHFWDOORIWKHFKDQJHVWKDWRFFXUDVWKHFRQFHQWUDWLRQRIDQRGRULQFUHDVHV)RU

ϯϱϱ

LQVWDQFHDVRGRUFRQFHQWUDWLRQLQFUHDVHVQRWRQO\GRHVWKHVWUHQJWKRIJORPHUXODUDFWLYDWLRQ

ϯϱϲ

LQFUHDVHEXWDOVRPRUHJORPHUXOLDUHUHFUXLWHG&RQVHTXHQWO\WKHH[SHULPHQWVSUHVHQWHGKHUH

ϯϱϳ

FDQQRWGHWHUPLQHWKHH[WHQWWRZKLFKVSLNHWLPLQJLQ0&VDQG7&VLVLQIOXHQFHGE\FKDQJHVLQ

ϯϱϴ

ODWHUDOLQKLELWLRQWKDWRFFXUZKHQPRUHJORPHUXOLDUHUHFUXLWHG+RZHYHUUHFHQWZRUNKDVEHJXQ

ϯϱϵ

WRH[SORUHKRZGLIIHUHQFHVLQODWHUDOLQKLELWLRQEHWZHHQ0&VDQG7&VLQIOXHQFHRGRUFRGLQJDW 

ϭϰ

 ϯϲϬ

GLVWLQFWFRQFHQWUDWLRQUDQJHV*HUDPLWDHWDO $GGLWLRQDOO\HOHFWULFDOVWLPXODWLRQPD\

ϯϲϭ

LQWURGXFHDUWLIDFWVWKDWFRXOGFRQIRXQGRXUFRQFOXVLRQV)RULQVWDQFHLQFUHDVLQJVWLPXODWLRQ

ϯϲϮ

LQWHQVLW\EURDGHQVWKHFXUUHQWVSUHDGDURXQGWKHHOHFWURGHDQGSRWHQWLDOO\UHFUXLWVPRUH261V

ϯϲϯ

7KHUHIRUHLI7&VKDYHODUJHUDSLFDOGHQGULWLFDUERUVWKDQ0&V7&VZRXOGEHPRUHOLNHO\WREH

ϯϲϰ

DFWLYDWHGDWORZHUVWLPXODWLRQLQWHQVLWLHVWKDQ0&V+RZHYHUH[SHULPHQWVSHUIRUPHGLQ203

ϯϲϱ

&K5PLFHLQZKLFKJORPHUXOLZHUHRSWLFDOO\VWLPXODWHG\LHOGHGUHVXOWVWKDWDUHFRPSDUDEOHWR

ϯϲϲ

H[SHULPHQWVLQZKLFKJORPHUXOLZHUHHOHFWULFDOO\VWLPXODWHG7KHUHIRUHWKHGLIIHUHQFHVLQVSLNH

ϯϲϳ

WLPLQJDQGUHOLDELOLW\EHWZHHQ0&VDQG7&VDUHXQOLNHO\WREHFRPSOHWHO\H[SODLQHGE\DUWLIDFWV

ϯϲϴ

RIWKHHOHFWULFDOVWLPXODWLRQSDUDGLJP

ϯϲϵ

:KDWDUHRWKHUFRQVHTXHQFHVRIVWURQJHUJORPHUXODUOD\HUPHGLDWHGLQKLELWLRQRQWR0&V"

ϯϳϬ

%ORFNLQJJORPHUXODUOD\HUPHGLDWHGLQKLELWLRQVWURQJO\DIIHFWVWKHPDJQLWXGHDQGODWHQF\RIILULQJ

ϯϳϭ

UDWHVLQ0&VWKDQLQ7&VLQGLFDWLQJWKDWJORPHUXODUOD\HUFLUFXLWVPD\SOD\DFULWLFDOUROHLQWKHKLJKHU

ϯϳϮ

ILULQJUDWHVREVHUYHGLQ7&Vin vitro DQGin vivo 1DJD\DPDHWDO,JDUDVKLHWDO :KLOH

ϯϳϯ

ZHDNHULQSXWIURPJORPHUXODUOD\HUFLUFXLWVDQGVWURQJHUH[FLWDWLRQDOORZVDODUJHUG\QDPLFUDQJHRI

ϯϳϰ

H[FLWDWRU\UHVSRQVHVLQ7&VWKDQLQ0&VJORPHUXODUOD\HUPHGLDWHGLQKLELWLRQPD\DOVREH

ϯϳϱ

UHVSRQVLEOHIRUWKHPRUHUREXVWLQKLELWRU\UHVSRQVHVREVHUYHGin vivo LQ0&V1DJD\DPDHWDO .

ϯϳϲ

,QGHHGERWKFRPSXWDWLRQDODQG in vivo ZRUNVXJJHVWWKDWIHHGIRUZDUGLQKLELWLRQPHGLDWHGE\3*&VLV

ϯϳϳ

WKHPRVWOLNHO\H[SODQDWLRQIRURGRUHYRNHGLQKLELWLRQFRPPRQO\REVHUYHGLQ0&V&OHODQG

ϯϳϴ

)XNXQDJDHWDO)XNXQDJDHWDO )XQFWLRQDOO\WKHVHGLIIHUHQFHVLQJORPHUXODUOD\HU

ϯϳϵ

PHGLDWHGLQKLELWLRQRQWR0&VDQG7&VPD\SOD\DQLPSRUWDQWUROHLQGHFRUUHODWLQJILULQJUDWHVEHWZHHQ

ϯϴϬ

0&VDQG7&VWKDWSURMHFWWRWKHVDPHJORPHUXOXVLHKRPRW\SLF07&V )XWXUHin vivoH[SHULPHQWV

ϯϴϭ

WKDWPRQLWRURGRUHYRNHGUHVSRQVHVLQKRPRW\SLF0&VDQG7&VZLOOEHYLWDOIRUGHWHUPLQLQJKRZ7&V

ϯϴϮ

UHVSRQGWRRGRUVWKDWLQKLELW0&V,QDGGLWLRQWRGULYLQJGHFRUUHODWLRQRIKRPRW\SLF0&DQG7&ILULQJ

ϯϴϯ

UDWHVLWLVWHPSLQJWRVSHFXODWHWKDWGLIIHUHQFHVLQ3*&PHGLDWHGLQKLELWLRQEHWZHHQKRPRW\SLF0&V

ϯϴϰ

PD\EHRQHPHFKDQLVPLQDGGLWLRQWRRWKHUNQRZQLQWULQVLFGLIIHUHQFHV3DGPDQDEKDQDQG8UEDQ

ϯϴϱ

 UHVSRQVLEOHIRUKHWHURJHQHRXVRGRUUHVSRQVHVEHWZHHQKRPRW\SLF0&V'KDZDOHHWDO 

ϯϴϲ

6XSSRUWLQJWKLVLGHDEORFNLQJJORPHUXODUOD\HUPHGLDWHGLQKLELWLRQin vitro UHGXFHVVSLNHWLPH 

ϭϱ

 ϯϴϳ

YDULDELOLW\LQ0&V1DMDFHWDO )XWXUHVWXGLHVPHDVXULQJRGRUHYRNHGUHVSRQVHVLQKRPRW\SLF

ϯϴϴ

0&VEHIRUHDQGDIWHUEORFNLQJLQKLELWLRQIURPYDULRXVLQWHUQHXURQVXEW\SHVPD\KHOSUHVROYHWKHFLUFXLW

ϯϴϵ

OHYHOPHFKDQLVPVEHKLQGXQLTXHWHPSRUDOG\QDPLFVLQLQGLYLGXDO0&V

ϯϵϬ

6HJUHJDWLQJVHQVRU\LQIRUPDWLRQSUHVHQWHGLQVSHFLILFLQWHQVLW\UDQJHVLQSDUDOOHOQHXURQW\SHV

ϯϵϭ

LVDFRPPRQVWUDWHJ\LQVHQVRU\V\VWHPV7KHGDWDSUHVHQWHGKHUHDQGHOVHZKHUHVWURQJO\LQGLFDWH

ϯϵϮ

WKDW0&VDQG7&VDUHUHVSRQVLEOHIRUHQFRGLQJRGRULQIRUPDWLRQSUHVHQWHGDWKLJKDQGORZ

ϯϵϯ

FRQFHQWUDWLRQVUHVSHFWLYHO\6LPLODUO\GLVWLQFWWRXFKUHFHSWRUVLQWKHVNLQUHVSRQGWRWDFWLOHVWLPXODWLRQ

ϯϵϰ

LQVHSDUDWHSUHVVXUHUDQJHV$GGLWLRQDOO\URGVDQGFRQHVHQFRGHYLVXDOLQIRUPDWLRQLQODUJHO\VHSDUDWH

ϯϵϱ

LQWHQVLW\UDQJHV*LYHQWKHVHVLPLODULWLHVLWLVWHPSLQJWRGUDZIXUWKHUDQDORJLHVEHWZHHQVHQVRU\

ϯϵϲ

V\VWHPV)RULQVWDQFHLQYLVXDOV\VWHPWKUHHVHSDUDWHOLJKWLQWHQVLW\UDQJHVKDYHEHHQGHVFULEHG

ϯϵϳ

EDVHGRQWKHW\SHRISKRWRUHFHSWRUVWKDWPHGLDWHYLVLRQLQHDFKUDQJH,QORZOLJKWLQWHQVLW\FRQGLWLRQV

ϯϵϴ

VFRWRSLFYLVLRQLVPHGLDWHGE\URGVZKLOHLQKLJKLQWHQVLW\OLJKWFRQGLWLRQVSKRWRSLFYLVLRQLVPHGLDWHG

ϯϵϵ

E\FRQHV0HVRSLFYLVLRQLVFRQVHTXHQWO\GHILQHGDVWKHLQWHUPHGLDWHUDQJHRIOLJKWLQWHQVLWLHVLQZKLFK

ϰϬϬ

YLVXDOLQIRUPDWLRQLVHQFRGHGE\ERWKURGVDQGFRQHV%HFDXVHERWKURGVDQGFRQHVUHOD\LQIRUPDWLRQ

ϰϬϭ

WKURXJKVHSDUDWHQHXUDOSDWKZD\VLQWKHUHWLQDDQGKDYHGLVWLQFWWHPSRUDOUHVSRQVHVPRGHOLQJKRZ

ϰϬϮ

WKHVHVLJQDOVDUHFRPELQHGDWWKHVHLQWHUPHGLDWHOLJKWLQWHQVLW\OHYHOVEHFRPHVTXLWHFKDOOHQJLQJ

ϰϬϯ

6WRFNPDQDQG6KDUSH 0RYLQJIRUZDUGGHWHUPLQLQJZKHWKHUWKUHHDQDORJRXVUDQJHVRIRGRU

ϰϬϰ

FRQFHQWUDWLRQFDQEHGHILQHGEDVHGRQWKHUHVSRQVHVRI0&VDQG7&VZLOOEHYLWDO*LYHQWKHDGGHG

ϰϬϱ

FRPSOH[LWLHVRIXQGHUVWDQGLQJROIDFWRU\FRGLQJLQFRQFHQWUDWLRQUHJLPHVZKHQERWK0&VDQG7&VDUH

ϰϬϲ

DFWLYDWHGLWZLOOEHLPSRUWDQWWRGHWHUPLQHKRZHDFKSRSXODWLRQHQFRGHVRGRUVLQLVRODWLRQE\VWXG\LQJ

ϰϬϳ

UHVSRQVHVLQFRQFHQWUDWLRQUDQJHVWKDWRQO\HQJDJH0&VRU7&V

ϰϬϴ



ϰϬϵ

5HIHUHQFHV

ϰϭϬ ϰϭϭ ϰϭϮ ϰϭϯ

$GDP</LYQHK<0L\DPLFKL.*UR\VPDQ0/XR/0L]UDKL$ )XQFWLRQDOWUDQVIRUPDWLRQVRI RGRULQSXWVLQWKHPRXVHROIDFWRU\EXOE)URQWLHUVLQQHXUDOFLUFXLWV $QWDO0(\UH0)LQNOHD%1XVVHU= ([WHUQDOWXIWHGFHOOVLQWKHPDLQROIDFWRU\EXOEIRUPWZR GLVWLQFWVXESRSXODWLRQV7KH(XURSHDQMRXUQDORIQHXURVFLHQFH 

ϭϲ

 ϰϭϰ ϰϭϱ ϰϭϲ ϰϭϳ ϰϭϴ ϰϭϵ ϰϮϬ ϰϮϭ ϰϮϮ ϰϮϯ ϰϮϰ ϰϮϱ ϰϮϲ ϰϮϳ ϰϮϴ ϰϮϵ ϰϯϬ ϰϯϭ ϰϯϮ ϰϯϯ ϰϯϰ ϰϯϱ ϰϯϲ ϰϯϳ ϰϯϴ ϰϯϵ ϰϰϬ ϰϰϭ ϰϰϮ ϰϰϯ ϰϰϰ ϰϰϱ ϰϰϲ ϰϰϳ ϰϰϴ ϰϰϵ ϰϱϬ ϰϱϭ ϰϱϮ ϰϱϯ ϰϱϰ ϰϱϱ ϰϱϲ ϰϱϳ ϰϱϴ ϰϱϵ ϰϲϬ ϰϲϭ ϰϲϮ ϰϲϯ ϰϲϰ ϰϲϱ ϰϲϲ ϰϲϳ

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
ϭϳ

 ϰϲϴ ϰϲϵ ϰϳϬ ϰϳϭ ϰϳϮ ϰϳϯ ϰϳϰ ϰϳϱ ϰϳϲ ϰϳϳ ϰϳϴ ϰϳϵ ϰϴϬ ϰϴϭ ϰϴϮ ϰϴϯ ϰϴϰ ϰϴϱ ϰϴϲ ϰϴϳ ϰϴϴ ϰϴϵ ϰϵϬ ϰϵϭ ϰϵϮ ϰϵϯ ϰϵϰ ϰϵϱ ϰϵϲ ϰϵϳ ϰϵϴ ϰϵϵ ϱϬϬ ϱϬϭ ϱϬϮ ϱϬϯ ϱϬϰ ϱϬϱ ϱϬϲ ϱϬϳ ϱϬϴ ϱϬϵ ϱϭϬ ϱϭϭ ϱϭϮ ϱϭϯ ϱϭϰ ϱϭϱ ϱϭϲ ϱϭϳ ϱϭϴ ϱϭϵ ϱϮϬ

LQIRUPDWLRQWRGLIIHUHQWWDUJHWVLQWKHROIDFWRU\FRUWH[7KH-RXUQDORIQHXURVFLHQFHWKHRIILFLDO MRXUQDORIWKH6RFLHW\IRU1HXURVFLHQFH .DSRRU93URYRVW$&$JDUZDO30XUWK\91 $FWLYDWLRQRIUDSKHQXFOHLWULJJHUVUDSLGDQG GLVWLQFWHIIHFWVRQSDUDOOHOROIDFWRU\EXOERXWSXWFKDQQHOV1DWXUHQHXURVFLHQFH .LNXWD6)OHWFKHU0/+RPPD5
ϭϴ

 ϱϮϭ ϱϮϮ ϱϮϯ ϱϮϰ ϱϮϱ ϱϮϲ ϱϮϳ ϱϮϴ ϱϮϵ ϱϯϬ ϱϯϭ ϱϯϮ ϱϯϯ ϱϯϰ ϱϯϱ

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

ϱϯϲ



ϱϯϳ



ϱϯϴ



ϱϯϵ



ϱϰϬ



ϱϰϭ



ϱϰϮ



ϱϰϯ



ϱϰϰ



ϱϰϱ



ϱϰϲ



ϱϰϳ



ϱϰϴ



ϱϰϵ





ϭϵ

 ϱϱϬ

)LJXUHOHJHQGV

ϱϱϭ

)LJXUH0&VDQG7&VHQFRGHWKHLQWHQVLW\RIROIDFWRU\VHQVRU\QHXURQVWLPXODWLRQGLIIHUHQWO\

ϱϱϮ

DE 6FKHPDWLFD DQGH[DPSOHVE RIH[SHULPHQWDOVHWXS6SLNLQJUHVSRQVHVWRHOHFWULFDOVWLPXODWLRQ

ϱϱϯ

—V RI261VDWILYHLQWHQVLWLHVZDVUHFRUGHGLQHLWKHUPLWUDOE±723 RUWXIWHGE±ERWWRP FHOOV

ϱϱϰ

261±ROIDFWRU\VHQVRU\QHXURQV3*&V±SHULJORPHUXODUFHOOV*&V±JUDQXOHFHOOV07&V±PLWUDORU

ϱϱϱ

WXIWHGFHOOV F ([DPSOHRIVSLNHUDVWHUV723 367+V0,''/( DQGVSLNHWLPHUHOLDELOLW\SORWV

ϱϱϲ

%27720 IRUD0&EODFN DQG7&UHG DFURVVVWLPXODWLRQLQWHQVLWLHVVHH0DWHULDOVDQG

ϱϱϳ

0HWKRGV 3ORWVRIVSLNHWLPHUHOLDELOLW\GHSLFWWKHSHUFHQWDJHRIWULDOVLQZKLFKWKHFHOOILUHGDQDFWLRQ

ϱϱϴ

SRWHQWLDOGXULQJHDFKPVWLPHELQIROORZLQJ261VWLPXODWLRQµY¶UHSUHVHQWVWKHILUVWWLPHELQIROORZLQJ

ϱϱϵ

261VWLPXODWLRQGXULQJZKLFKVSLNHWLPLQJZDVPRUHWKDQUHOLDEOH G 0&VDQG7&VUHVSRQGWR

ϱϲϬ

261VWLPXODWLRQZLWKVLPLODUQXPEHUVRIVSLNHVS  H KRZHYHU7&VUHVSRQGZLWKKLJKHUILULQJ

ϱϲϭ

UDWHV±PHDVXUHGDVWKHSHDNRIWKH367+±WKDQ0&VS H IM 7&VUHVSRQGWRORZLQWHQVLW\

ϱϲϮ

261VWLPXODWLRQDWVKRUWHUODWHQFLHVWKDQ0&VIJ /DWHQF\WR367+SHDNLVVKRUWHULQ7&VWKDQLQ

ϱϲϯ

0&VZKHQSORWWHGYVVWLPXODWLRQLQWHQVLW\IS H RU367+SHDNJS H KL VWVSLNH

ϱϲϰ

ODWHQF\KS  DQGWLPHWRILUVWUHOLDEOHVSLNHLS  DUHDOVRVKRUWHULQ7&VWKDQ0&VDW

ϱϲϱ

ORZVWLPXODWLRQLQWHQVLWLHVM $WORZVWLPXODWLRQLQWHQVLWLHVDODUJHUSHUFHQWDJHRI7&VWKDQ0&VVKRZ

ϱϲϲ

UHOLDEOHVSLNHWLPLQJ'DWDWDNHQIURP0&VDQG7&V6LJQLILFDQFHZDVDVVHVVHGLQGHIXVLQJ

ϱϲϳ

ZD\$129$DQGLQJKLDVXQSDLUHGWWHVWFRPSDULQJIJ¶VGHULYHGIURPH[SRQHQWLDOILWIURPGDWDLQHDFK

ϱϲϴ

FHOO$VWHULVNV

LQGMLQGLFDWHVLJQLILFDQWS GLIIHUHQFHVEHWZHHQ0&VDQG7&V

ϱϲϵ



ϱϳϬ

)LJXUH2SWLFDODFWLYDWLRQRIVLQJOHJORPHUXOLLQROIDFWRU\EXOEVHFWLRQVIURP203&K5(<)3

ϱϳϭ

PLFHD 6FKHPDWLFRIH[SHULPHQWDOVHWXSDQDORJRXVWRWKHRQHXVHGLQ)LJXUH6SLNLQJLQVLQJOH

ϱϳϮ

0&VRU7&VLQUHVSRQVHWRSKRWRVWLPXODWLRQPV RIDVLQJOHJORPHUXOXVDWILYHLQWHQVLWLHV0&VDQG

ϱϳϯ

7&VUHVSRQGWR261SKRWRVWLPXODWLRQZLWKVLPLODUQXPEHUVRIVSLNHVES  KRZHYHU7&V

ϱϳϰ

UHVSRQGHGZLWKKLJKHUILULQJUDWHVWKDQ0&VFS H $GGLWLRQDOO\7&VUHVSRQGHGWRORZ

ϱϳϱ

LQWHQVLW\261SKRWRVWLPXODWLRQZLWKVKRUWHUODWHQFLHVWKDQ0&VDVDVVHVVHGE\WKHWLPHWR367+SHDN 



ϮϬ

 ϱϳϲ

GS  ILUVWVSLNHODWHQF\HS  DQGWLPHWRILUVWUHOLDEOHVSLNHIS  J $ODUJHU

ϱϳϳ

SHUFHQWDJHRI7&VWKDQ0&VGLVSOD\UHOLDEOHVSLNHWLPLQJ'DWDWDNHQIURP0&VDQG7&V

ϱϳϴ

6LJQLILFDQFHZDVDVVHVVHGLQEFXVLQJZD\$129$DQGLQGHIDVXQSDLUHGWWHVWFRPSDULQJIJ¶V

ϱϳϵ

GHULYHGIURPH[SRQHQWLDOILWIURPGDWDLQHDFKFHOO$VWHULVNV

LQEJLQGLFDWHVLJQLILFDQWS 

ϱϴϬ

GLIIHUHQFHVEHWZHHQ0&VDQG7&V

ϱϴϭ



ϱϴϮ

)LJXUH7&VUHVSRQGWRHOHFWULFDOVWLPXODWLRQRI261VZLWKVWURQJHUH[FLWDWRU\DQGZHDNHU

ϱϴϯ

LQKLELWRU\FXUUHQWVWKDQ0&VDE ([FLWDWRU\9K P9 DQGLQKLELWRU\9K P9 FXUUHQWVZHUH

ϱϴϰ

PHDVXUHGLQVLQJOH0&VRU7&VIROORZLQJHOHFWULFDOVWLPXODWLRQRI261VDWILYHLQWHQVLWLHVE ([DPSOHV

ϱϴϱ

RILQKLELWRU\723 DQGH[FLWDWRU\%27720 FXUUHQWVDWHDFKRIWKHILYHVWLPXODWLRQLQWHQVLWLHVLQRQH

ϱϴϲ

H[DPSOH0&EODFN DQG7&UHG FGH &RPSDULVRQVRILQKLELWRU\723 DQGH[FLWDWRU\FXUUHQWSHDN

ϱϴϳ

DPSOLWXGHF FKDUJHLQVIROORZLQJVWLPXODWLRQG DQGFKDUJHLQILUVWPVIROORZLQJVWLPXODWLRQH 

ϱϴϴ

EHWZHHQ0&VDQG7&V&RPSDUHGWR7&VLQKLELWRU\FXUUHQWVLQ0&VKDYHODUJHUSHDNDPSOLWXGHV

ϱϴϵ

S H DQGFKDUJHWUDQVIHUUHGLQWKHVIROORZLQJVWLPXODWLRQS H DQGWKHILUVWPV

ϱϵϬ

IROORZLQJVWLPXODWLRQS H +RZHYHUH[FLWDWRU\FXUUHQWVLQ0&VKDYHODUJHUSHDNFXUUHQWV

ϱϵϭ

S H EXWVLPLODUFKDUJHWUDQVIHUUHGLQWKHVIROORZLQJVWLPXODWLRQS  DQGWKHILUVWPV

ϱϵϮ

IROORZLQJVWLPXODWLRQS  FRPSDUHGZLWK7&VI 7KHUDWLRRIH[FLWDWRU\WRLQKLELWRU\FXUUHQWVLV

ϱϵϯ

ODUJHULQ7&VWKDQLQ0&VZKHQFDOFXODWHGDVWKHUDWLRRIFKDUJHWUDQVIHUUHGGXULQJVDIWHUVWLPXODWLRQ

ϱϵϰ

723S H RUGXULQJWKHVWPVDIWHUVWLPXODWLRQ%27720S  JK 7KHODWHQF\WR

ϱϵϱ

WKHSHDNRILQKLELWRU\FXUUHQWVGRHVQRWGLIIHUEHWZHHQ0&VDQG7&VJS  KRZHYHUWKHODWHQF\

ϱϵϲ

WRWKHSHDNRIH[FLWDWRU\FXUUHQWVGLIIHUVEHWZHHQ0&VDQG7&VKS  L $WDOOILYHLQWHQVLWLHV

ϱϵϳ

WKHSHDNRIH[FLWDWRU\FXUUHQWVSUHFHGHVLQKLELWLRQLQ7&V+RZHYHULQ0&VH[FLWDWLRQODJVLQKLELWLRQDW

ϱϵϴ

WKHZHDNHVWWZRLQWHQVLWLHVEXWOHDGVLQKLELWLRQDWWKHVWURQJHVWWKUHHLQWHQVLWLHV'DWDWDNHQIURP0&V

ϱϵϵ

DQG7&VDQGSORWWHGDVPHDQ“VHP6LJQLILFDQFHZDVDVVHVVHGLQDOOSDQHOVXVLQJZD\$129$

ϲϬϬ

$VWHULVNV

LQFLLQGLFDWHVLJQLILFDQWS GLIIHUHQFHVEHWZHHQ0&VDQG7&V

ϲϬϭ

 

Ϯϭ

 ϲϬϮ

)LJXUH0&VUHFHLYHVWURQJHU3*&PHGLDWHGLQKLELWLRQWKDQ7&V,QKLELWRU\DE DQGH[FLWDWRU\

ϲϬϯ

FG FXUUHQWVLQ0&VDQG7&VZHUHPHDVXUHGEHIRUHDQGDIWHUOLPLWLQJ*&PHGLDWHGLQKLELWLRQE\

ϲϬϰ

EORFNLQJP*OX5V/<ȝ0 DQG10'$5V$39ȝ0 &XUUHQWVZHUHHYRNHGXVLQJ

ϲϬϱ

SKRWRVWLPXODWLRQLQ203&K5<)3PLFHDWPLQLPDOVWLPXODWLRQLQWHQVLWLHVH 7KHSHDNDPSOLWXGHRI

ϲϬϲ

3*&PHGLDWHGLQKLELWLRQLVODUJHULQ0&VWKDQLQ7&VS H IJ 7KHODWHQF\WRSHDNRILQKLELWLRQ

ϲϬϳ

IS  DQGWKHGXUDWLRQRILQKLELWLRQJS FRPSDULQJH[SRQHQWLDOGHFD\FRQVWDQWVRIFXUUHQWV 

ϲϬϴ

GLGQRWGLIIHUEHWZHHQ0&VDQG7&VK 7KHSHDNDPSOLWXGHRIH[FLWDWRU\FXUUHQWVDUHODUJHULQ7&V

ϲϬϵ

WKDQLQ0&VS H L 7KHODWHQF\WRWKHSHDNRIH[FLWDWRU\FXUUHQWVLVORQJHULQ0&VWKDQLQ7&V

ϲϭϬ

S  'DWDWDNHQIURP0&VDQG7&VDQGSORWWHGDVPHDQ“VHP6LJQLILFDQFHZDVDVVHVVHG

ϲϭϭ

LQDOOSDQHOVXVLQJSDLUHGWWHVWV

ϲϭϮ



ϲϭϯ

)LJXUH%ORFNLQJ*&PHGLDWHGLQKLELWLRQGRHVQRWDOWHUILULQJUDWHVRUVSLNHODWHQFLHVLQ0&VRU

ϲϭϰ

7&VDE ([DPSOHVSLNHUDVWHUVIROORZLQJHOHFWULFDOVWLPXODWLRQRI261VDWWKUHHLQWHQVLWLHVLQD0&

ϲϭϱ

D DQG7&E EHIRUHEODFNUHG DQGDIWHUJUD\SLQN OLPLWLQJ*&PHGLDWHGLQKLELWLRQE\EORFNLQJ

ϲϭϲ

P*OX5V/< DQG10'$5V$39 F *&PHGLDWHGLQKLELWLRQGRHVQRWDIIHFWWKHQXPEHURI

ϲϭϳ

VSLNHVLQHLWKHU0&V0LQ±S 0LG±S 0D[±S  RU7&V0LQ±S 0LG±S 

ϲϭϴ

0D[±S  G *&PHGLDWHGLQKLELWLRQGRHVQRWDIIHFWSHDNILULQJUDWHV367+SHDN LQHLWKHU0&V

ϲϭϵ

0LQ±S 0LG±S 0D[±S  RU7&V0LQ±S 0LG±S 0D[±S  HI 

ϲϮϬ

*&PHGLDWHGLQKLELWLRQGRHVQRWDIIHFWUHVSRQVHODWHQF\DVPHDVXUHGE\WKHWLPHWR367+SHDNH LQ

ϲϮϭ

HLWKHU0&V0LQ±S 0LG±S 0D[±S  RU7&V0LQ±S 0LG±S 0D[±

ϲϮϮ

S  RUWKHWLPHWRUHOLDEOHVSLNLQJI LQHLWKHU0&V0LQ±1$0LG±S 0D[±S  RU7&V

ϲϮϯ

0LQ±S 0LG±S 0D[±S  'DWDWDNHQIURP0&VDQG7&V6LJQLILFDQFHZDV

ϲϮϰ

DVVHVVHGLQDOOSDQHOVXVLQJSDLUHGWWHVWV

ϲϮϱ





ϮϮ

 ϲϮϲ

)LJXUH%ORFNLQJJORPHUXODUOD\HUPHGLDWHGLQKLELWLRQDOWHUVILULQJUDWHVDQGVSLNHODWHQFLHVLQ

ϲϮϳ

0&VDQG7&VDE 6SLNHVZHUHPHDVXUHGEHIRUHDQGDIWHUSXIILQJJDED]LQHLQWKHUHFRUGHGFHOO¶V

ϲϮϴ

KRPHJORPHUXOXVWROLPLWJORPHUXODUOD\HUPHGLDWHGLQKLELWLRQFG 6SLNHUDVWHUVLQD0&F DQG7&

ϲϮϵ

G EHIRUHEODFNUHG GXULQJJUD\SLQN DQGDIWHUEOXH EORFNLQJJORPHUXODUOD\HUPHGLDWHGLQKLELWLRQ

ϲϯϬ

H %ORFNLQJJORPHUXODUOD\HUPHGLDWHGLQKLELWLRQLQFUHDVHGWKHQXPEHURIVSLNHVLQ0&VDWDOOWKUHH

ϲϯϭ

VWLPXODWLRQLQWHQVLWLHV0LQ±S 0LG±S 0D[±S  DQGLQ7&VDWWKHZHDNHVW

ϲϯϮ

LQWHQVLW\0LQ±S 0LG±S 0D[±S  I %ORFNLQJJORPHUXODUOD\HUPHGLDWHG

ϲϯϯ

LQKLELWLRQLQFUHDVHGWKHILULQJUDWHDVPHDVXUHGE\WKHSHDNRIWKH367+DWDOOWKUHHLQWHQVLWLHVLQERWK

ϲϯϰ

0&V0LQ±S 0LG±S 0D[±S  DQG7&V0LQ±S 0LG±S 0D[

ϲϯϱ

±S  J +RZHYHUILULQJUDWHVZHUHPRUHVWURQJO\DIIHFWHGE\EORFNLQJJORPHUXODUOD\HU

ϲϯϲ

PHGLDWHGLQKLELWLRQLQ0&VWKDQLQ7&VDWWKHVWURQJHVWWZRLQWHQVLWLHV0LQ±S 0LG±S 

ϲϯϳ

0D[±S  K %ORFNLQJJORPHUXODUOD\HUPHGLDWHGLQKLELWLRQUHGXFHGWKHODWHQF\WRWKH367+

ϲϯϴ

SHDNLQ0&VDWWKHZHDNHVWWZRLQWHQVLWLHV0LQ±S 0LG±S 0D[±S  EXWGLGQRW

ϲϯϵ

DIIHFWWKHODWHQF\LQ7&V0LQ±S 0LG±S 0D[±S  L 7KHODWHQF\WRUHOLDEOHVSLNLQJ

ϲϰϬ

GLGQRWFKDQJHLQ7&VIROORZLQJJORPHUXODUOD\HUPHGLDWHGLQKLELWLRQEORFNDGH0LQ±S 0LG±

ϲϰϭ

S 0D[±S  KRZHYHULQ0&VWKHWLPLQJRIUHVSRQVHVLQDOOIRXU0&VEHFDPHUHOLDEOHDWWKH

ϲϰϮ

ZHDNHVWLQWHQVLW\DQGODWHQF\ZDVUHGXFHGDWWKHPLGGOHS  EXWQRWWKHPD[LPXPS  

ϲϰϯ

LQWHQVLW\M 6SRQWDQHRXVILULQJUDWHVRIERWK0&VS  DQG7&VS  LQFUHDVHGDIWHU

ϲϰϰ

SXIILQJJDED]LQH'DWDWDNHQIURP0&VDQG7&V6LJQLILFDQFHZDVDVVHVVHGLQDOOSDQHOVXVLQJ

ϲϰϱ

SDLUHGWWHVWV$VWHULVNV

LQHLLQGLFDWHVLJQLILFDQWS GLIIHUHQFHVEHWZHHQ0&VDQG7&V



Ϯϯ