New RNA playgrounds : non-coding RNAs and RNA-binding proteins control cellular processes Kedde, M.
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(2) Chapter 4:. Telomerase-independent regulation of ATR by human telomerase RNA J Biol Chem. 2006 Dec 29;281(52):40503-14 DOI 10.1074/jbc.M607676200. 65.
(3) Chapter 4. Telomerase-independent Regulation of ATR by Human Telomerase RNA* S. Received for publication, August 11, 2006, and in revised form, November 6, 2006 Published, JBC Papers in Press, November 10, 2006, DOI 10.1074/jbc.M607676200. Martijn Kedde, Carlos le Sage, Anja Duursma, Eitan Zlotorynski, Bart van Leeuwen, Wouter Nijkamp, Roderick Beijersbergen, and Reuven Agami1 From the Division of Tumor Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands The human telomerase RNA (hTR), together with the telomerase reverse transcriptase, hTERT, constitute the core components of telomerase that is essential for telomere maintenance. While hTR is ubiquitously expressed, hTERT is normally restricted to germ cells and certain stem cells, but both are often deregulated during tumorigenesis. Here, we investigated the effects of changes in hTR cellular levels. Surprisingly, while inhibition of hTR expression triggers a rapid, telomerase-independent, growth arrest associated with p53 and CHK1 activation, its increased expression neutralizes activation of these pathways in response to genotoxic stress. These hTR effects are mediated through ATR and are sufficiently strong to impair ATR-mediated DNA-damage checkpoint responses. Furthermore, in response to low UV radiation, which activates ATR, endogenous hTR levels increase irrespective of telomerase status. Thus, we uncovered a novel, telomerase-independent, function of hTR that restrains ATR activity and participates in the recovery of cells from UV radiation.. !?CD 8E=1> C?=1D93 35<<C 81F5 1 <9=9D54 B5@<931D9F5 <965C@1> G85> @B?@171D54 (, 4(20- G8938 9C 4E5 D? D859B 9>129<9DI D? =19> D19> 38B?=?C?=5 5>4C D85 D5<?=5B5C (5<?=5B5C 1B5 C@5 391<9J54 "@B?D59> CDBE3DEB5C D81D @B5C5BF5 D85 9>D57B9DI ?6 D85 5>4C ?6 D85 38B?=?C?=5C 1>4 D85 CD129<9DI ?6 D85 75>?=5 ?G5F5B G9D8 5138 35<< 49F9C9?> D5<?=5B5C 1B5 C8?BD5>54 1>4 ?>35 ?>5 ?B =?B5 5B?45 D? 1 35BD19> 3B9D931< @?9>D D85 C8?BD 5>9>7 9>4E35C 1 " 41=175 3853;@?9>D D85B52I @?C9>7 1 21B B95B D? 3?>D9>E54 35<< 7B?GD8 1>4 D85B56?B5 D? 31>35B
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(60) ATR regulation by hTR. FIGURE 1. Reduction of telomerase activity and rapid growth inhibition by reduction of hTR expression. A, schematic representation of the hTR RNA. Boxed regions correspond to targeted sequences by shRNAs 1, 2, and 3, and the template sequence is shown in a closed box. B, RPA was used to detect the levels of hTR and the control cyclophilin. The full-length probes and protected fragments are indicated, and 10% of the input probe was loaded on the gel. Quantification was performed by densitometry. C, a TRAP assay was performed to detect telomerase activity in extracts of MCF7 cells transfected with the indicated constructs. D, MCF7 cells were transfected with hTRkd#1 or vector constructs and subjected to flow cytometric analysis. The percentages of cells in G1, S, and G2 phases are shown. S.D. is from three independent experiments. E, a colony growth assay of MCF7 cells transfected with the hTR knockdown constructs or a vector control. Cells were selected with hygromycin for 10 days and stained with Coomassie Blue. F, colony growth assay of virally transduced MCF7 cells. G, MCF7 cells were transfected with indicated constructs, and RNA was extracted after 3 days. OAS1 mRNA levels were measured by quantitative RT-PCR, shown relative to -actin mRNA. S.D. is from three independent experiments. H, flow cytometry is the same as in D only that U2OS cells were used. I, competitive growth assay the U2OS and MCF7 cells were transduced with indicated pRS-GFP constructs. Fluorescence was monitored by flow cytometry at the indicated timepoints after transduction. J, competitive growth assay performed on transduced primary BJ fibroblasts as in I. ctr, control.. D85 7B?GD8 9>8929D9?> DB9775B54 2I D85 ;>?3;4?G> ?6 8(& 9> ! 35<<C 9C 45@5>45>D ?> D85 <5F5< ?6 ;>?3;4?G> ?6 8(& (85 C1=5 1>D9@B?<965B1D9F5 56653D ?6 8(&;4 G1C 1<C? ?2D19>54 9> ?D85B 8E=1> 35<< <9>5C CE38 1C 11( 9==?BD1<9J54 ;5B1D9>? 3ID5C 5 1 35BF931< 31B39>?=1 35<<C 1>4 ( =1==1BI 31B 39>?=1 35<<C CE@@<5=5>D1< 97 ' <D?75D85B D85C5 B5CE<DC C8?G D81D 9>8929D9?> ?6 8(& 5H@B5CC9?> 2I C8&" 5<939DC 1 B1@94 1>D9@B?<965B1D9F5 B5C@?>C5 9> 8E=1> 35<<C E5 D? D85 3?>D9>E?EC B1@94 @B?<965B1D9?> ?6 31>35B 35<< <9>5C 1>4 5=5B75>35 ?6 3B9D931<<I C8?BD D5<?=5B5C D85 9>8929D9?> ?6 8(& 5H@B5CC9?> =1I 9>4E35 1 CDB5CC B5C@?>C5 (? 5H1=9>5 D89C G5 CDE4954 8(& 9>8929D9?> 9> I?E>7 @B9=1BI 8E=1> 35<<C @1C C175
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(67) Chapter 4. FIGURE 1—continued. D9?> (? D5CD D89C 49B53D<I G5 @5B6?B=54 3?=@5D9D9F5 7B?GD8 1CC1IC EC9>7 !@
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(78)
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(85) <D?75D85B D85C5 B5CE<DC CE775CD D81D 5>4?75>?EC <5F5<C ?6 kd FIGURE 2. Chk1 and p53 are required for induction of the hTR -mediated cell cycle arrest. A, MCF7 cells were transfected with indicated constructs and subjected to immunoblot analysis to detect p53 and the 8(& 1B5 6E>3D9?>9>7 D? 21<1>35 control CDK4. Band intensity was calculated by densitometry. B, MCF7 cells were transduced with p53kd or (& 13D9F9DI 1 6E>3D9?> D81D 9C control vector, drug-selected, and transduced with pRS-GFP control and hTRkd#1-GFP. Competitive growth D5<?=5B1C59>45@5>45>D assays were performed as described in the legend to Fig. 1I. C, competitive growth assays with BJ cells as in B. (? CDE4I D85 56653DC ?6 8(& ?> Cells were either treated with UCN-01 (10 nM, dashed lines) or with vehicle (dimethyl sulfoxide (DMSO), solid lines). (& ;9>1C5 13D9F9DI 9> =?B5 45D19< G5 EC54 1> (& 3?>CDBE3D G9D8 1 ' ,'(!(21 "2(4(27M#EB B5CE<DC CE775CD 1> 9>F5BC5 ($ D1>45= 1669>9DI @EB96931D9?> D17 3?>D19>9>7 1 (* @B?D5 3?BB5<1D9?> 25DG55> 8(& <5F5<C 1>4 (& 13D9F9DI &54E3D9?> 9> 1C5 3<51F175 C9D5 +5 DB1>C653D54
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(115) $. 6?39 9> #, 35<<C (8EC G5 6?E>4 >? 5F945>35 ?6 " 41=175 9> 35<<C 45@<5D54 ?6 8(& CE@@?BD9>7 FIGURE 3. hTR inhibits ATR kinase activity. A, U2OS cells were transfected with the hTRkd or control, irradiated D85 >?D9?> D81D D85 56653DC ?6 8(& ?> after 3 days with 3 J/m2 UV, and harvested at the indicated time points. Whole cell extracts were immuno- (& 1B5 49B53D <D?75D85B ?EB blotted to detect CHK1S317 phosphorylation, p53S15 phosphorylation, p53, and CDK4 as a loading control. B, B5CE<DC C8?G D81D D85 5H@B5CC9?> ?6 GM847 fibroblasts were transfected with hTRkd or control and treated as in A. C, HEK293 cells were transfected with TAP-ctr or TAP-ATR and immunoprecipitated with IgG. TAP-ATR was cleaved with TEV protease, and beads 8(& 9>6<E5>35C (& ;9>1C5 13D9F9DI (B) and supernatant (S) were immunoblotted to detect ATR. WB, Western blot. D, kinase assay performed with (, 4(4- immunoprecipitation and TEV cleaved TAP-ATR from HEK293 cells with GST-p53 (residues 1–101) as a sub 02( * !0-& 2(-, -% 2'$ strate. Cleaved ATR was split, and hTR, RNase, and mock were added prior to kinase reaction. Samples were separated by 10% SDS-PAGE and stained with Coomassie Blue to detect GST-p53 protein, and autoradiogra- '$").-(,2 ,# ,' ,"$# ..$ 0 phy was performed to detect kinase activity. Band intensities were measured by densitometry. E, U2OS cells ,"$ -% 0 &(*$ (2$1 !7 ' were transfected with the indicated constructs and kinase assays were performed as in B. F, MCF7 cells were transfected with the indicated constructs, and kinase assays were performed as in B. G, immunofluorescence 6.0$11(-,M(& 81C 255> C8?G> kd images of MCF7 cells transfected with hTR #1, vector, or control irradiated cells (5 Gy IR), stained with -H2AX D? 25 9>F?<F54 9> D85 B1491D9?>9> antibody. Nuclei are stained with DAPI (blue). Pictures were made with 200 magnification. vec, vector. 4E354 ! @81C5 3853;@?9>D 9> 5E;1BI?D93 35<<C 1C 9D @B5F5>DC 35<<C 1>4 9C C<978D<I 13D9F1D54 G85> 35<<C 1B5 9BB1491D54 * ,$1 =9D?D93 5>DBI ?6 35<<C =19><I 9> D85 <1D5 @81C5 ?6 D85 B5C@?>C5 D? 1>4 ?G5F5B G85> 8(& 9C 3?5H@B5CC54 G9D8 ($(& & 1>4 3??@5B1D5C G9D8 (! 9> D85 51B<I @81C5 ?6 D85 B5C@?>C5 (& 13D9F9DI 9C =1B;54<I 453B51C54 1<=?CD D? D85 <5F5<C ?6 D85 '9>35 ?EB B5CE<DC C8?G D81D 8(& 9>8929DC (& G5 CDE4954. 72.
(116) ATR regulation by hTR. FIGURE 4. hTR partially abrogates the G2/M checkpoint and enhances the expression of fragile sites. A, MCF7 cells were transfected with the indicated constructs irradiated with 5 Gy IR or mock treated 3 days later and incubated with nocodazole. Twenty-our hours later, cells were fixed and stained with Hoechst, and mitotic entry was scored. S.D. from three independent experiments is shown. B, the expression of fragile sites in U2OS cells transfected with the indicated constructs. Pictures were made with 1000 magnification. C, quantification of the expression of fragile sites in U2OS cells as shown in Fig. 4B. The number of fragile sites expressed in 400 chromosomes is shown; data are representative of three independent experiments.. D85 56653DC ?6 8(& 5H@B5CC9?> ?> D85 ! 3853;@?9>D +5 =?>9D?B54 =9D?D93 5>DBI ?6 ! 35<<C 9> B5C@?>C5 D? & 1C 1 =51CEB5 6?B D85 >E=25B ?6 35<<C 12<5 D? 1BB5CD 9> ! +5 DB1>C653D54 ! 35<<C G9D8 !*8(& 1>4 1C 3?>DB?<C (&;4 @
(117) ;4 ?B !*3?>DB?< 3?>CDBE3DC 6?B F1<941D9?> ?6 D85 (&;4 3?>CDBE3DC C55 CE@@<5=5>D1< 97 ' (8B55 41IC <1D5B G5 9BB1 491D54 35<<C G9D8 I & 1>4 DB51D54 D85 35<<C G9D8 >?3?41J?<5 D? 9>8929D @B?7B5CC9?> ?6 D85 35<< 3I3<5 9> =9D?C9C 5<<C G5B5 D85> 69H54 CD19>54 G9D8 ?538CD 1>4 =9D?D93 35<<C G5B5 3?E>D54 97 +89<5 G5 6?E>4 >? 49665B5>35 9> D85 133E=E<1D9?> ?6 =9D?D93 35<<C 9> E>9BB1491D54 3?>DB?<C 8(& ?F5B5H@B5CC9?> (&;4 1>4 @
(118) ;4 C8?G54 1 @1BD91< ?F5BB945 ?6 D85 ! 1BB5CD > 3?>DB1CD 35<<C 5H@B5CC9>7 1 DBE>31D54 6?B= ?6 8(& 8(&. 2581F54 1C 3?>DB?< 35<<C "?D12<I 8(& <5F5<C 1B5 ?><I =?45B 1D5<I 9>3B51C54 9> ! 35<<C E@?> 5H@B5CC9?> ?6 !*8(& 1
(119) 9>3B51C5 CE@@<5=5>D1< 97 ' <D?75D85B D85C5 B5CE<DC 9>4931D5 D81D 1> 9>3B51C5 9> 8(& <5F5<C 9=@19BC (&=5491D54 " 41=175 B5C@?>C5C 9> 1 =1>>5B C9=9<1B D? D8?C5 9>4E354 2I <?CC ?6 (&. (? 6EBD85B 9>F5CD971D5 G85D85B 8(& 5H@B5CC9?> @5BDEB2C (& 6E>3D9?> G5 CDE4954 6B179<5 C9D5C CD129<9DI 9> ) #' 35<<C ?==?> 6B179<5 C9D5C 1B5 C@539693 38B?=?C?=1< <?39 D81D 1@@51B 1C 3?>CDB93D9?>C 71@C ?B 2B51;C ?> =5D1@81C5 38B?=?C?=5C 6B?= 35<<C D81D 81F5 255> 5H@?C54 D? @1BD91< 9>8929D9?> ?6 " B5@<931D9?>
(120) 449D9?> ?6 <?G <5F5<C ?6 D85 " @?<I=5B1C5 9>8929D?B 1@89493?<9> D? 35<<C 9> 3E<DEB5 81C 255> C8?G> D? 9>4E35 6B179<5 C9D5C (& B53?7>9J5C CD1<<54 1>4 3?<<1@C54 " B5@<931D9?> 6?B;C 1>4 13D9F1D5C D859B B5@19B 1>4 D85 B5CD1BD9>7 ?6 B5@<931D9?> B179<5 C9D5C 1B5 25<95F54 D? 25 5H@B5CC54 G85> CD1<<54 6?B;C 5C31@5 D85 (& B5@<931D9?> CEBF59<<1>35 +5 DB1>C653D54 ) #' 35<<C G9D8 !*8(& (&;4 ?B !*3DB< 1>4 DB51D54 D85 35<<C 6?B 8 G9D8 1@89493?<9> 8B?=?C?=5C G5B5 69H54 CD19>54 1>4 4B?@@54 ?>D? C<945C D? 3?E>D 38B?=? C?=5C 1>4 6B179<5 C9D5C )@?> 9>8929D9?> ?6 B5@<931D9?> G5 6?E>4 6B179<5 C9D5C 9>CD129<9DI 9> 3?>DB?< 35<<C G8938 G1C 5H135B21D54 2I 59D85B <?CC ?6 (& ?B 5H@B5CC9?> ?6 8(& 97
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(122) Chapter 4. FIGURE 5. Up-regulation of hTR levels following UV radiation. A, RPA performed on extracts from BJ cells and BJ cells immortalized with hTERT irradiated with 3 J/m2 UV or 4 Gy ionizing radiation. Cells were harvested after irradiation at the indicated time points, and quantification was performed by densitometry. B, quantification of RPAs on extracts of BJ cells irradiated with either 3 J/m2 UV or 4Gy ionizing radiation. Samples were harvested at indicated time points after irradiation. S.D. is from three independent experiments; band intensities were quantified by densitometry. C, RPA performed on extracts of GM847 cells irradiated with 3 J/m2 UV as in A. D, quantification of RPAs on extracts of the indicated cells irradiated with 3 J/m2 UV. UCN-01 was added to a final concentration of 100 nM 1 h prior to irradiation. S.D. is from three independent experiments. Ctr, control; Vec, vector.. 9>7 35<<C 1C G5<< 1C 9> D85 (&;4 35<<C 97 C =19>D19>9>7 <?G <5F5<C ?6 6B179<5 C9D5 5H@B5CC9?> 1B5 1 @B9=5 6E>3D9?> ?6 (& ?EB B5CE<DC 9>4931D5 D81D 8(& 9>8929DC (& 13D9F9DI <D?75D85B D85C5 B5CE<DC C8?G D81D 8(& 9>8929DC (& 13D9F9DI D? 1 <5F5< CE669395>D D? 9=@19B 49665B5>D (&=5491D54 "41=175 3853;@?9>D B5C@?>C5C 9=@<I9>7 1 >?F5< 6E>3D9?> 6?B 8(& ' .0$&3* 2(-, %-**-5(,& #( 2(-,M+5 >5HD C5D ?ED D? 5<E3941D5 D85 6E>3D9?> ?6 D85 9>8929D?BI 56653D ?6 8(& ?> (& (? D89C 5>4 G5 9>F5CD971D54 G85D85B CD9=E<9 D81D 13D9F1D5 (& 1<C? 9>6<E5>35 D85 5H@B5CC9?> ?6 8(& +5 DB51D54 @B9=1BI 692B?2<1CDC 1>4 35<<C ?F5B5H@B5CC9>7 8(&( ( G9D8
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(130) ATR regulation by hTR DISCUSSION +5 81F5 945>D96954 1 31EC1D9F5 75>5D93 9>D5B13D9?> 25DG55> D85 8(& 1>4 D85 3853;@?9>D ;9>1C5 (& 3D?@93 5H@B5CC9?> ?6 8(& 9>8929DC (& G89<5 B54E3D9?> 9> 8(& <5F5<C CD9=E<1D5C (& 13D9F9DI (89C 9>D5B13D9?> 9C 9>45@5>45>D ?6 D5<?=5B1C5 13D9F9DI 1>4 D5<?=5B5 <5>7D8 1C 9D G1C ?2C5BF54 9> 35<<C <13;9>7 8(&( 1>4 9> I?E>7 @B9=1BI 8E=1> 35<<C G9D8 D5<?=5B5C <?>7 5>?E78 D? 1<<?G @B?<965B1D9?> 6?B 1D <51CD D?
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(132) 45@5>45>D 35<< 3I3<5 1BB5CD 9> D85 12C5>35 ?6 1@@1B5>D " 41=175 G89<5 9>3B51C54 5H@B5C C9?> ?6 8(& 31EC54 45653DC 9> (&45@5>45>D 3853;@?9>DC CE38 1C ?F5BB945 ?6 D85 ! 1BB5CD 9> B5C@?>C5 D? " 41=175 1>4 D85 5>81>354 9>4E3D9?> ?6 6B179<5 C9D5C (85C5 56653DC 1B5 C@539693 C9>35 D85 8(& ;>?3;4?G>C 4? >?D 5<939D 1>I 9>D5B65B?> B5C@?>C5 1>4 @85>?DI@931<<I 45@5>4 ?> D85 5>4?75>?EC 5H@B5CC9?> ?6 8(& 1>4 D85 56653DC G5B5 ?2C5BF54 G9D8 =9<4 <5CC D81> 12?F5 5>4?75>?EC ?F5B5H@B5CC9?> ?6 8(& "?D12<I CE38 1 =9<4 9>3B51C5 9> 8(& 5H@B5CC9?> 9C 1<C? ?2C5BF54 G85> 35<<C 1B5 )* 9BB1491D54 9>4931D9>7 D81D CE38 1> 9>3B51C5 9C CE6 69395>D D? 9=@19B (& 45@5>45>D 3853;@?9>DC 97
(133) > 9=@?BD1>D ?2C5BF1D9?> 9C D81D 2?D8 D85 9>8929D9?> ?6 (& 13D9F 9DI 2I 8(& 1>4 D85 E@B57E<1D9?> ?6 8(& 2I )* 41=175 1B5 9>45@5>45>D ?6 8(&( 3D9F1D9?> ?6 (& 9> B5C@?>C5 D? )* 9C F5BI B1@94 ?33EBB9>7 G9D89> =9>ED5C 97
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(136) 1>4 CE@@<5 =5>D1< 97 ' (85C5 ;9>5D93C 1B5 3?>C9CD5>D G9D8 D85 9451 D81D 35<< 3I3<5 9>8929D9?> 81C D? 25 B1@94 G89<5 D85 B53?F5BI D1;5C C5F5B1< 8?EBC 45@5>49>7 ?> D85 5HD5>D ?6 41=175 1C54 ?> D85C5 69>49>7C ?EB B5CE<DC 9=@<I 1 =?45< G85B5 E@B57E<1D9?> ?6 8(& 9> B5C@?>C5 D? )* 3?>CD9DED5C 1 6554213; <??@ 2B9>79>7 4?G> (& 13D9F9DI D? B59>9D91D5 35<< 3I3<5 @B?7B5CC9?> 97 #EB B5CE<DC C8?G D81D B54E3D9?> 9> 5>4?75>?EC 8(& <5F5<C <514C D? (& 13D9F1D9?> G9D8?ED D85 9>4E3D9?> ?6 1@@1B5>D " 41=175 (85C5 B5CE<DC @B?=@D54 EC D? 9>F5CD971D5 G85D85B (& 1>4 8(& 9>D5B13D 49B53D<I >D5B5CD9>7<I 8?=?<?7E5C ?6 (& 9> I51CD 1>4 0 !(#-.1(1 1B5 9>F?<F54 9> D5<?=5B5 =5D12?<9C= 1>4 9> I51CD !53 @ (& 8?=?<?7 1CC?391D5C G9D8 D5<?=5B5C
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(139) (& 9C 9>F?<F54 9> =19>D19>9>7 D85 CD129<9DI ?6 6B179<5 C9D5C 1C 9DC 9>8929D9?> <514C D? 1> 9>3B51C5 9> D85 5H@B5CC9?> ?6 6B179<5 C9D5C #EB 69>49>7C 45=?>CDB1D5 D81D 8(& 5H@B5CC9?> 9>4E35C D85 1@@51B1>35 ?6 6B179<5 C9D5C D? 1> 5HD5>D C9=9<1B D? (&;4 D8EC CE775CD9>7 D81D 1> 9>3B51C5 9> 8(& <5F5<C 31> <514 D? 75>?=93 9>CD129<9DI 'E@ @?BD9>7 D89C 3?>3<EC9?> 9C ?EB ?2C5BF1D9?> D81D 1> 9>3B51C5 9> 8(& <5F5<C G51;5>C D85 ! 1BB5CD 1 3853;@?9>D 3?>DB?<<54. FIGURE 6. Model for the hTR-mediated negative feedback loop on ATR activity in response to UV. Low levels of UV radiation activate the ATR kinase early in the UV response, which phosphorylates downstream targets, among which are p53 and Chk1, leading to a cell cycle arrest and induction of DNA repair. Independently, hTR levels are increased by a yet unknown mechanism. These increased hTR levels inhibit ATR at a later stage.. 1<C? 2I (& (85C5 B5CE<DC 1<C? CE775CD D81D 8(& 5H@B5CC9?> 31> <514 D? B54E354 6945<9DI ?6 D85 (&45@5>45>D 3853;@?9>DC 1>4 D85B56?B5 D? 9>3B51C54 75>?=93 9>CD129<9DI &535>D<I D85 7B?E@ ?6 <9J125D8 <13;2EB> 81C C8?G> D81D B54E3D9?> 9> 8(& <5F5<C 9> 31>35B 35<<C 5<939DC 1 B1@94 1>D9@B? <965B1D9F5 B5C@?>C5 ?G5F5B G85> 3?=@1B9>7 8(& ;>?3; 4?G> 9> (. 35<<C G9D8 @
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(145) 35<<C EBD85B=?B5 ?EB B5CE<DC 3<51B<I 9>4931D5 D81D D85 B57E<1D9?> ?6 (& 2I 8(& 9C CE669395>D D? 16653D 35<<E<1B @1D8G1IC #EB B5CE<DC C55= D? 3?>DB1CD D85 69>49>7C D81D =(& =935 81F5 >? ?2F9?EC @85>?DI@5 9> D85 69BCD 75>5B1D9?>C
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(147) +85D85B D89C 56653D 9C =5491D54 D8B?E78 (& B5=19>C D? 25 5<E3941D54 449D9?>1<<I D85B5 1B5 C5F5B1< 49665B5>35C 25DG55> =?EC5 1>4 8E=1> D5<?=5B5 8?=5 ?CD1C9C D81D 3?=@<931D5 5HD5>49>7 69>49>7C 6B?= =?EC5 =?45<C D? D85 8E=1> C5DD9>7 9BCD =EB9>5 35<<C 81F5 5HDB5=5<I <?>7 1>4 8I@5BF1B912<5 D5<?=5B5C 1>4 D5<?=5B1C5 13D9F9DI 9C 45D53D 12<5 9> =?CD C?=1D93 D9CCE5C '53?>4 1<D8?E78 D5<?= 5B1C5 9C 13D9F1D54 1>4 =(& 9C E@B57E<1D54 (, 4(4- 9> C5F5B1< =?EC5 DE=?B =?45<C 9D 1@@51BC >?D D? 25 B5AE9B54 6?B 7B?GD8 4EB9>7 D85 35<< 49F9C9?>C >535CC1BI 6?B DE=?B 6?B=1D9?> CE7 75CD9>7 D81D =(& 1>4?B D5<?=5B1C5 81F5 1449D9?>1< 6E>3D9?>C D? D5<?=5B5 5HD5>C9?>
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(152) (8EC ?EB B5CE<DC =1I 9>49 31D5 6?B 59D85B 1 49665B5>35 9> D5<?=5B1C5 &" 29?<?7I 25DG55> =935 1>4 =5> ?B D? 49665B5>35C 9> =5D8?4C EC54 EBD85B 5H@5B 9=5>DC G9D8 =EB9>5 35<<C 1B5 B5AE9B54 D? 5CD12<9C8 G85D85B =(& 1<C? B57E<1D5C (& 1CD ?EB B5CE<DC =1I @B?F945 1> 5H@<1>1D9?> G8I 9> D85 F1CD =1:?B9DI ?6 C?=1D93 8E=1> 35<<C 8(& 9C E29AE9D?EC<I 5H@B5CC54 G85B51C 2?D8 8(&( 1>4 D5<?=5B1C5 13D9F9DI 1B5 =?CD<I 12C5>D 8(& B57E<1D5C " 41=175 @1D8G1IC 9> 1 D5<?=5B1C5 1>4 8(&(9>45@5>45>D =1>>5B #EB B5CE<DC D8EC 31> 5H@<19> ?2C5BF1D9?>C CE775CD9>7 D81D 8(& @<1IC 1 B?<5 9> D85 9>9D91D9?> ?6 DE=?B975>939DI 1>4 D81D 9D 9C 6B5AE5>D<I E@B57E<1D54 9> 8E=1> 31>35B 35<< <9>5C
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