Division of Molecular Environmental Endocrinology
Transcription
Division of Molecular Environmental Endocrinology
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´HPEU\RQLFIHWDO RULJLQV RI DGXOW GLVHDVHµ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` DQG OLNHO\ RFFXUUHG LQ WKH DFWLQRSWHU\JLDQ OLQHDJH OHDGLQJ WR WHOHRVWV DIWHUWKHGLYHUJHQFHRI$FLSHQVHULIRUPHVEXWEHIRUHWKHVSOLW RI 2VWHRJORVVLIRUPHV )XQFWLRQDO DQDO\VLV UHYHDOHG WKDW WKH VKDUN$5 DFWLYDWHV WKH WDUJHW JHQH YLD DQGURJHQ UHVSRQVH HOHPHQW E\ FODVVLFDO DQGURJHQV 7KH WHOHRVW$5_ VKRZHG XQLTXH LQWUDFHOOXODU ORFDOL]DWLRQ ZLWK D VLJQLILFDQWO\ KLJKHU WUDQVDFWLYDWLRQ FDSDFLW\ WKDQ WKDW RI WHOHRVW $5`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〔2ULJLQDOSDSHUV〕 (. 2JLQR < ,JXFKL 7 %DUEHU '6 DQG 'HQVORZ %URFNPHLHU 1' (IIHFWV RI ơ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〔2ULJLQDOSDSHUV(SXEOLFDWLRQDKHDGRISULQW〕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〔5HYLHZDUWLFOHV〕 c +HLQGHO -- .LGG .$ -REOLQJ 6 =RHOOHU 57 %HUJPDQ %HFKHU * %MHUUHJDDUG 3 %RUQPDQ 5 %UDQGW , %ULDQ -9 .RUWHQNDPS$0XLU'2FKLHQJ56NDNNHEDHN1(,JXFKL7 7RSSDUL-DQG:RRGUXII7-6WDWHRIWKH6FLHQFHRI(QGRFULQH 'LVUXSWLQJ&KHPLFDOV:+2DQG81(3SS %HUJPDQ c +HLQGHO -- .LGG .$ -REOLQJ 6 =RHOOHU 57 %HFKHU * %MHUUHJDDUG 3 %RUQPDQ 5 %UDQGW , %ULDQ -9 .RUWHQNDPS$0XLU'2FKLHQJ56NDNNHEDHN1(,JXFKL7 7RSSDUL - DQG :RRGUXII 7- 7KH LPSDFW RI HQGRFULQH GLVUXSWLRQ$FRQVHQVXVVWDWHPHQWRQWKHVWDWHRIWKHVFLHQFH(QYLURQ +HDOWK3HUVSHFW$ 6WRO]HQEHUJ+&)ULVFKH7'HOODUFR9/7LPP**RXUPHORQ $ ,JXFKL 7 ,QJHUVOHY ) DQG 5REHUWV 0 7KH UHJXODWRU\ QHHGIRUWHVWVWRGHWHFW('&VDQGDVVHVVWKHLUKD]DUGVWRZLOGOLIH,Q (QGRFULQH 'LVUXSWHUV +D]DUG 7HVWLQJ DQG $VVHVVPHQW 0HWKRGV 0DWWKLHVVHQ3HG:LOH\SS