da104139b31df14c1c61b5090b017700 pmc_33807672.pdf 90358b5438423c4d00e52897d6064cfe51189104 pmc_33807672.pdf 5b14644723b1e34e632882a12b0febecc3879f3f05d3afd54bd89a1cb6dd0d44 pmc_33807672.pdf Title: ARMCX3 Mediates Susceptibility to Hepatic Tumorigenesis Promoted by Dietary Lipotoxicity Subject: ARMCX3 is encoded by a member of the Armcx gene family and is known to be involved in nervous system development and function. We found that ARMCX3 is markedly upregulated in mouse liver in response to high lipid availability, and that hepatic ARMCX3 is upregulated in patients with NAFLD and hepatocellular carcinoma (HCC). Mice were subjected to ARMCX3 invalidation (inducible ARMCX3 knockout) and then exposed to a high-fat diet and diethylnitrosamine-induced hepatocarcinogenesis. The effects of experimental ARMCX3 knockdown or overexpression in HCC cell lines were also analyzed. ARMCX3 invalidation protected mice against high-fat-diet-induced NAFLD and chemically induced hepatocarcinogenesis. ARMCX3 invalidation promoted apoptotic cell death and macrophage infiltration in livers of diethylnitrosamine-treated mice maintained on a high-fat diet. ARMCX3 downregulation reduced the viability, clonality and migration of HCC cell lines, whereas ARMCX3 overexpression caused the reciprocal effects. SOX9 was found to mediate the effects of ARMCX3 in hepatic cells, with the SOX9 interaction required for the effects of ARMCX3 on hepatic cell proliferation. In conclusion, ARMCX3 is identified as a novel molecular actor in liver physiopathology and carcinogenesis. ARMCX3 downregulation appears to protect against hepatocarcinogenesis, especially under conditions of high dietary lipid-mediated hepatic insult. Keywords: Alex3; HCC; lipotoxicity; NAFLD; obesity; SOX9 Author: Serena Mirra, Aleix Gavaldà-Navarro, Yasmina Manso, Mónica Higuera, Román Serrat, María Teresa Salcedo, Ferran Burgaya, José Maria Balibrea, Eva Santamaría, Iker Uriarte, Carmen Berasain, Matias A. Avila, Beatriz Mínguez, Eduardo Soriano and Francesc Villarroya Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Mar 10 06:51:12 2021 CET ModDate: Wed Mar 10 07:43:53 2021 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 22 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 6332280 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- JUXSQN+VnURWPalladioL-Bold Type 1 Custom yes yes no 17 0 CIQLLF+VnURWPalladioL Type 1 Custom yes yes no 22 0 SMVAJE+URWPalladioL-Ital Type 1 Custom yes yes no 26 0 CIMCYI+URWPalladioL-Bold Type 1 Custom yes yes no 31 0 QQEKEL+URWPalladioL-Roma Type 1 Custom yes yes no 35 0 LCJEVJ+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 44 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 64 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 68 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 72 0 IFEDSM+CMSY10 Type 1 Builtin yes yes no 89 0 LEBNAB+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 100 0 LEBNBC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 106 0 LEBNDC+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 112 0 LEBNDD+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 118 0 LEBNDE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 121 0 LEBNDF+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 124 0 CAMXTK+EURM10 Type 1 Builtin yes yes no 131 0 LEBNAB+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 137 0 LEBNBC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 139 0 LEBNDC+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 141 0 LEBNDD+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 143 0 LEBNDE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 144 0 LEBNDF+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 145 0 LEBNAB+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 156 0 LEBNBC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 158 0 LEBNDC+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 160 0 LEBNDD+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 162 0 LEBNDE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 163 0 LEBNDF+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 164 0 LEBNAB+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 176 0 LEBNBC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 178 0 LEBNDC+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 180 0 LEBNDD+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 182 0 LEBNDE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 183 0 LEBNDF+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 184 0 LEBNBC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 188 0 LEBNDC+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 190 0 LEBNDD+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 192 0 LEBNDE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 193 0 LEBNDF+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 194 0 LEBNAB+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 202 0 LEBNBC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 204 0 LEBNDC+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 206 0 LEBNDD+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 208 0 LEBNDE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 209 0 LEBNDF+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 210 0 LEBNAB+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 221 0 LEBNBC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 223 0 LEBNDC+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 225 0 LEBNDD+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 227 0 LEBNDE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 228 0 LEBNDF+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 229 0 LEBNAB+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 238 0 LEBNBC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 240 0 LEBNDC+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 242 0 LEBNDD+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 244 0 LEBNDE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 245 0 LEBNDF+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 246 0 MPUDFL+PazoMath Type 1 Builtin yes yes no 267 0 LEBNAB+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 276 0 LEBNBC+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 278 0 LEBNDC+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 280 0 LEBNDD+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 282 0 LEBNDE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 283 0 LEBNDF+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 284 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2021-09-11 03:32:19 CEST RepresentationInformation: pmc_33807672.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2021-04-12 07:53:25 CEST Size: 6332280 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 518 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Children: Item: Title: Hepatic Expression of Armcx3 Is Modulated in Response to Nutritional Challenges Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Gene Invalidation of Armcx3 Ameliorates HFD-Induced Metabolic Alterations and Protects against NAFLD Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Inactivation of the Armcx3 Gene Protects Against Hepatic Carcinogenesis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: ARMCX3 Regulates DEN-Induced Apoptotic Death and Macrophage Infiltration Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: ARMCX3 Invalidation Does Not Affect Hepatic Mitochondria Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: ARMCX3 Affects ERK Signaling in DEN-Induced Tumors from HFD-Fed Mice Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: ARMCX3 Invalidation Reduces Cell Viability, Clonality and Migration in Hepatocellular Carcinoma Cell Lines Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: ARMCX3 Overexpression In Vitro Induces Hepatocellular Carcinoma Cells Proliferation in a SOX9-Dependent Mechanism Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Children: Item: Title: Mice Care Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Human Samples Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: ARMCX3-KO Mouse Generation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Metabolism-Related Studies Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: DEN-Induced Hepatocarcinogenesis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Mouse Partial Hepatectomy and Acute CCl4 Administration Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Mouse Primary Hepatocytes Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Histology and Immunohistochemistry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Cell Lines and Culture Conditions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: Transfection of siRNAs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: Armcx3 Overexpression and SOX9 Knockdown In Vitro Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: Wound Healing Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Item: Title: Cell Viability Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@e720b71 Item: Title: Clonogenic Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1b26f7b2 Item: Title: Palmitic Acid and Oleic Acid Treatments Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@491cc5c9 Item: Title: Western Blotting Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@74ad1f1f Item: Title: RNA Isolation, cDNA Synthesis, and Real-Time PCR Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6a1aab78 Item: Title: Statistics Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@462d5aee Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@69b0fd6f Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@757942a1 Info: Title: ARMCX3 Mediates Susceptibility to Hepatic Tumorigenesis Promoted by Dietary Lipotoxicity Author: Serena Mirra, Aleix Gavaldà-Navarro, Yasmina Manso, Mónica Higuera, Román Serrat, María Teresa Salcedo, Ferran Burgaya, José Maria Balibrea, Eva Santamaría, Iker Uriarte, Carmen Berasain, Matias A. Avila, Beatriz Mínguez, Eduardo Soriano and Francesc Villarroya Subject: ARMCX3 is encoded by a member of the Armcx gene family and is known to be involved in nervous system development and function. We found that ARMCX3 is markedly upregulated in mouse liver in response to high lipid availability, and that hepatic ARMCX3 is upregulated in patients with NAFLD and hepatocellular carcinoma (HCC). Mice were subjected to ARMCX3 invalidation (inducible ARMCX3 knockout) and then exposed to a high-fat diet and diethylnitrosamine-induced hepatocarcinogenesis. The effects of experimental ARMCX3 knockdown or overexpression in HCC cell lines were also analyzed. ARMCX3 invalidation protected mice against high-fat-diet-induced NAFLD and chemically induced hepatocarcinogenesis. ARMCX3 invalidation promoted apoptotic cell death and macrophage infiltration in livers of diethylnitrosamine-treated mice maintained on a high-fat diet. ARMCX3 downregulation reduced the viability, clonality and migration of HCC cell lines, whereas ARMCX3 overexpression caused the reciprocal effects. SOX9 was found to mediate the effects of ARMCX3 in hepatic cells, with the SOX9 interaction required for the effects of ARMCX3 on hepatic cell proliferation. In conclusion, ARMCX3 is identified as a novel molecular actor in liver physiopathology and carcinogenesis. ARMCX3 downregulation appears to protect against hepatocarcinogenesis, especially under conditions of high dietary lipid-mediated hepatic insult. Keywords: Alex3; HCC; lipotoxicity; NAFLD; obesity; SOX9 Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Mar 10 06:51:12 CET 2021 ModDate: Wed Mar 10 07:43:53 CET 2021 ID: 0x4718a0f80573218158c2fe23e5293a40, 0x4718a0f80573218158c2fe23e5293a40 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: JUXSQN+VnURWPalladioL-Bold FontSubset: true FirstChar: 160 LastChar: 226 FontDescriptor: FontName: JUXSQN+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: QQEKEL+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: QQEKEL+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: CAMXTK+EURM10 FontSubset: true FirstChar: 12 LastChar: 22 FontDescriptor: FontName: CAMXTK+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: CIQLLF+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: CIQLLF+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: IFEDSM+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: IFEDSM+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: SMVAJE+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 121 FontDescriptor: FontName: SMVAJE+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: MPUDFL+PazoMath FontSubset: true FirstChar: 68 LastChar: 68 FontDescriptor: FontName: MPUDFL+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true Font: BaseFont: CIMCYI+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 167 FontDescriptor: FontName: CIMCYI+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 35, 759, 153, 795 Annotation: Subtype: Link Rect: 515, 759, 560, 795 Annotation: Subtype: Link Rect: 252, 659, 263, 673 Annotation: Subtype: Link Rect: 437, 659, 448, 673 Annotation: Subtype: Link Rect: 534, 659, 545, 673 Annotation: Subtype: Link Rect: 225, 647, 237, 661 Annotation: Subtype: Link Rect: 335, 647, 346, 661 Annotation: Subtype: Link Rect: 433, 647, 445, 661 Annotation: Subtype: Link Rect: 514, 647, 525, 661 Annotation: Subtype: Link Rect: 126, 634, 137, 648 Annotation: Subtype: Link Rect: 224, 634, 235, 648 Annotation: Subtype: Link Rect: 337, 634, 348, 648 Annotation: Subtype: Link Rect: 54, 370, 154, 379 Annotation: Subtype: Link Rect: 35, 360, 87, 367 Annotation: Subtype: Link Rect: 35, 162, 97, 184 Annotation: Subtype: Link Rect: 153, 89, 154, 98 Annotation: Subtype: Link Rect: 35, 77, 147, 86 Annotation: Subtype: Link Rect: 114, 17, 265, 28 Annotation: Subtype: Link Rect: 408, 17, 558, 28 Annotation: Subtype: Link Rect: 35, 470, 87, 489 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 332, 492, 339, 501 ActionDest: 287 Annotation: Subtype: Link Rect: 340, 492, 347, 501 ActionDest: 287 Annotation: Subtype: Link Rect: 381, 467, 388, 476 ActionDest: 287 Annotation: Subtype: Link Rect: 397, 454, 404, 463 ActionDest: 287 Annotation: Subtype: Link Rect: 226, 429, 233, 438 ActionDest: 287 Annotation: Subtype: Link Rect: 236, 429, 248, 438 ActionDest: 287 Annotation: Subtype: Link Rect: 355, 417, 362, 426 ActionDest: 287 Annotation: Subtype: Link Rect: 210, 392, 217, 401 ActionDest: 287 Annotation: Subtype: Link Rect: 529, 392, 536, 401 ActionDest: 287 Annotation: Subtype: Link Rect: 458, 379, 470, 388 ActionDest: 287 Annotation: Subtype: Link Rect: 412, 342, 424, 350 ActionDest: 287 Annotation: Subtype: Link Rect: 362, 316, 374, 325 ActionDest: 287 Annotation: Subtype: Link Rect: 264, 304, 276, 313 ActionDest: 287 Annotation: Subtype: Link Rect: 276, 304, 288, 313 ActionDest: 287 Annotation: Subtype: Link Rect: 496, 279, 508, 288 ActionDest: 287 Annotation: Subtype: Link Rect: 478, 114, 485, 123 ActionDest: 287 Annotation: Subtype: Link Rect: 488, 114, 495, 123 ActionDest: 287 Annotation: Subtype: Link Rect: 442, 74, 449, 86 ActionDest: 86 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 401, 697, 413, 706 ActionDest: 287 Annotation: Subtype: Link Rect: 494, 657, 501, 669 ActionDest: 86 Annotation: Subtype: Link Rect: 324, 458, 331, 470 ActionDest: 86 Annotation: Subtype: Link Rect: 406, 408, 413, 420 ActionDest: 86 Annotation: Subtype: Link Rect: 478, 395, 485, 407 ActionDest: 86 Annotation: Subtype: Link Rect: 520, 382, 527, 394 ActionDest: 86 Annotation: Subtype: Link Rect: 543, 332, 550, 344 ActionDest: 86 Annotation: Subtype: Link Rect: 518, 307, 526, 319 ActionDest: 86 Annotation: Subtype: Link Rect: 339, 297, 351, 306 ActionDest: 287 Annotation: Subtype: Link Rect: 405, 184, 417, 193 ActionDest: 287 Annotation: Subtype: Link Rect: 526, 171, 538, 180 ActionDest: 287 Annotation: Subtype: Link Rect: 264, 81, 271, 93 ActionDest: 128 Page: Sequence: 4 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 280, 90, 287, 102 ActionDest: 128 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 342, 694, 349, 706 ActionDest: 128 Annotation: Subtype: Link Rect: 491, 685, 503, 694 ActionDest: 287 Annotation: Subtype: Link Rect: 494, 594, 501, 606 ActionDest: 128 Annotation: Subtype: Link Rect: 366, 519, 373, 531 ActionDest: 128 Annotation: Subtype: Link Rect: 373, 383, 385, 392 ActionDest: 287 Annotation: Subtype: Link Rect: 386, 383, 398, 392 ActionDest: 290 Annotation: Subtype: Link Rect: 324, 318, 331, 330 ActionDest: 151 Annotation: Subtype: Link Rect: 255, 267, 262, 279 ActionDest: 151 Annotation: Subtype: Link Rect: 223, 230, 230, 242 ActionDest: 151 Annotation: Subtype: Link Rect: 481, 169, 488, 178 ActionDest: 287 Annotation: Subtype: Link Rect: 489, 169, 501, 178 ActionDest: 290 Annotation: Subtype: Link Rect: 335, 144, 342, 153 ActionDest: 287 Annotation: Subtype: Link Rect: 342, 144, 349, 153 ActionDest: 287 Annotation: Subtype: Link Rect: 349, 144, 361, 153 ActionDest: 290 Page: Sequence: 7 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 187, 707, 194, 716 ActionDest: 287 Annotation: Subtype: Link Rect: 337, 694, 349, 703 ActionDest: 290 Annotation: Subtype: Link Rect: 425, 666, 432, 678 ActionDest: 168 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 461, 722, 473, 731 ActionDest: 290 Annotation: Subtype: Link Rect: 474, 722, 486, 731 ActionDest: 290 Annotation: Subtype: Link Rect: 391, 710, 403, 719 ActionDest: 290 Annotation: Subtype: Link Rect: 406, 710, 418, 719 ActionDest: 290 Annotation: Subtype: Link Rect: 224, 644, 231, 656 ActionDest: 168 Annotation: Subtype: Link Rect: 406, 647, 418, 656 ActionDest: 287 Annotation: Subtype: Link Rect: 522, 521, 529, 533 ActionDest: 197 Annotation: Subtype: Link Rect: 342, 495, 349, 507 ActionDest: 197 Annotation: Subtype: Link Rect: 301, 470, 308, 482 ActionDest: 197 Annotation: Subtype: Link Rect: 385, 445, 392, 457 ActionDest: 197 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 541, 621, 548, 633 ActionDest: 216 Annotation: Subtype: Link Rect: 487, 558, 494, 570 ActionDest: 216 Annotation: Subtype: Link Rect: 325, 546, 332, 558 ActionDest: 216 Annotation: Subtype: Link Rect: 200, 408, 207, 420 ActionDest: 216 Annotation: Subtype: Link Rect: 223, 410, 235, 419 ActionDest: 290 Annotation: Subtype: Link Rect: 392, 332, 399, 344 ActionDest: 216 Annotation: Subtype: Link Rect: 458, 307, 465, 319 ActionDest: 86 Annotation: Subtype: Link Rect: 259, 269, 266, 281 ActionDest: 216 Annotation: Subtype: Link Rect: 276, 209, 288, 218 ActionDest: 290 Annotation: Subtype: Link Rect: 522, 209, 534, 218 ActionDest: 290 Annotation: Subtype: Link Rect: 277, 182, 283, 194 ActionDest: 233 Annotation: Subtype: Link Rect: 352, 144, 359, 156 ActionDest: 233 Annotation: Subtype: Link Rect: 347, 94, 354, 106 ActionDest: 233 Page: Sequence: 11 Page: Sequence: 12 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 496, 656, 508, 666 ActionDest: 290 Annotation: Subtype: Link Rect: 511, 656, 523, 666 ActionDest: 290 Annotation: Subtype: Link Rect: 445, 644, 457, 653 ActionDest: 290 Annotation: Subtype: Link Rect: 317, 493, 329, 502 ActionDest: 290 Annotation: Subtype: Link Rect: 303, 456, 315, 465 ActionDest: 290 Annotation: Subtype: Link Rect: 325, 430, 336, 440 ActionDest: 290 Annotation: Subtype: Link Rect: 544, 267, 556, 276 ActionDest: 290 Annotation: Subtype: Link Rect: 379, 192, 391, 201 ActionDest: 290 Annotation: Subtype: Link Rect: 479, 192, 491, 201 ActionDest: 290 Annotation: Subtype: Link Rect: 313, 180, 324, 189 ActionDest: 290 Annotation: Subtype: Link Rect: 548, 167, 555, 176 ActionDest: 287 Annotation: Subtype: Link Rect: 364, 129, 376, 138 ActionDest: 287 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 195, 517, 206, 526 ActionDest: 290 Annotation: Subtype: Link Rect: 436, 492, 448, 501 ActionDest: 290 Annotation: Subtype: Link Rect: 450, 442, 462, 451 ActionDest: 290 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 214, 232, 226, 241 ActionDest: 290 Annotation: Subtype: Link Rect: 214, 207, 226, 216 ActionDest: 290 Annotation: Subtype: Link Rect: 400, 156, 412, 166 ActionDest: 294 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 544, 169, 556, 178 ActionDest: 294 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 303, 317, 315, 326 ActionDest: 294 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 517, 292, 524, 304 ActionDest: 271 Page: Sequence: 19 Annotations: Annotation: Subtype: Link Rect: 426, 422, 560, 433 Annotation: Subtype: Link Rect: 166, 410, 244, 421 Annotation: Subtype: Link Rect: 237, 326, 243, 337 ActionDest: 128 Annotation: Subtype: Link Rect: 308, 302, 314, 313 ActionDest: 233 Page: Sequence: 20 Annotations: Annotation: Subtype: Link Rect: 211, 603, 249, 613 Annotation: Subtype: Link Rect: 165, 568, 203, 578 Annotation: Subtype: Link Rect: 507, 544, 545, 555 Annotation: Subtype: Link Rect: 453, 521, 490, 532 Annotation: Subtype: Link Rect: 496, 521, 532, 532 Annotation: Subtype: Link Rect: 373, 498, 411, 509 Annotation: Subtype: Link Rect: 417, 498, 453, 509 Annotation: Subtype: Link Rect: 193, 465, 231, 475 Annotation: Subtype: Link Rect: 519, 441, 557, 452 Annotation: Subtype: Link Rect: 59, 430, 95, 440 Annotation: Subtype: Link Rect: 295, 395, 332, 406 Annotation: Subtype: Link Rect: 354, 372, 391, 383 Annotation: Subtype: Link Rect: 244, 349, 281, 360 Annotation: Subtype: Link Rect: 268, 303, 305, 314 Annotation: Subtype: Link Rect: 312, 303, 348, 314 Annotation: Subtype: Link Rect: 59, 281, 97, 291 Annotation: Subtype: Link Rect: 103, 281, 139, 291 Annotation: Subtype: Link Rect: 326, 257, 363, 268 Annotation: Subtype: Link Rect: 369, 257, 406, 268 Annotation: Subtype: Link Rect: 87, 235, 124, 245 Annotation: Subtype: Link Rect: 131, 235, 167, 245 Annotation: Subtype: Link Rect: 508, 222, 546, 233 Annotation: Subtype: Link Rect: 59, 189, 97, 199 Annotation: Subtype: Link Rect: 327, 165, 364, 176 Annotation: Subtype: Link Rect: 289, 130, 326, 141 Annotation: Subtype: Link Rect: 499, 107, 537, 118 Annotation: Subtype: Link Rect: 507, 84, 544, 95 Annotation: Subtype: Link Rect: 389, 61, 426, 72 Page: Sequence: 21 Annotations: Annotation: Subtype: Link Rect: 95, 710, 132, 720 Annotation: Subtype: Link Rect: 400, 686, 438, 697 Annotation: Subtype: Link Rect: 238, 664, 275, 674 Annotation: Subtype: Link Rect: 104, 641, 142, 651 Annotation: Subtype: Link Rect: 193, 606, 231, 617 Annotation: Subtype: Link Rect: 237, 606, 273, 617 Annotation: Subtype: Link Rect: 154, 572, 191, 582 Annotation: Subtype: Link Rect: 197, 572, 233, 582 Annotation: Subtype: Link Rect: 450, 548, 487, 559 Annotation: Subtype: Link Rect: 494, 548, 530, 559 Annotation: Subtype: Link Rect: 499, 537, 536, 548 Annotation: Subtype: Link Rect: 131, 503, 168, 513 Annotation: Subtype: Link Rect: 175, 503, 211, 513 Annotation: Subtype: Link Rect: 462, 479, 499, 490 Annotation: Subtype: Link Rect: 295, 456, 332, 467 Annotation: Subtype: Link Rect: 338, 456, 374, 467 Annotation: Subtype: Link Rect: 304, 433, 342, 444 Annotation: Subtype: Link Rect: 443, 399, 481, 410 Annotation: Subtype: Link Rect: 487, 399, 523, 410 Annotation: Subtype: Link Rect: 211, 376, 248, 387 Annotation: Subtype: Link Rect: 255, 376, 291, 387 Annotation: Subtype: Link Rect: 95, 342, 133, 352 Annotation: Subtype: Link Rect: 139, 342, 175, 352 Annotation: Subtype: Link Rect: 505, 318, 543, 329 Annotation: Subtype: Link Rect: 122, 296, 160, 306 Annotation: Subtype: Link Rect: 333, 272, 371, 283 Annotation: Subtype: Link Rect: 520, 249, 557, 260 Annotation: Subtype: Link Rect: 484, 226, 522, 237 Annotation: Subtype: Link Rect: 118, 192, 155, 203 Annotation: Subtype: Link Rect: 175, 169, 212, 180 Annotation: Subtype: Link Rect: 218, 169, 254, 180 Annotation: Subtype: Link Rect: 127, 146, 164, 157 Annotation: Subtype: Link Rect: 415, 111, 453, 122 Annotation: Subtype: Link Rect: 385, 88, 423, 99 Page: Sequence: 22 Annotations: Annotation: Subtype: Link Rect: 286, 721, 324, 732 Annotation: Subtype: Link Rect: 330, 721, 366, 732 Annotation: Subtype: Link Rect: 405, 698, 442, 709 Annotation: Subtype: Link Rect: 389, 675, 427, 686 Annotation: Subtype: Link Rect: 433, 675, 469, 686 Checksum: 2b52a24d Type: CRC32 Checksum: da104139b31df14c1c61b5090b017700 Type: MD5 Checksum: 90358b5438423c4d00e52897d6064cfe51189104 Type: SHA-1