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University of Groningen Exploring the mechanisms underlying the phenotype of MCAD deficiency with Systems Medicine Martines, Anne-Claire

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(1)University of Groningen. Exploring the mechanisms underlying the phenotype of MCAD deficiency with Systems Medicine Martines, Anne-Claire. IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.. Document Version Publisher's PDF, also known as Version of record. Publication date: 2019 Link to publication in University of Groningen/UMCG research database. Citation for published version (APA): Martines, A-C. (2019). Exploring the mechanisms underlying the phenotype of MCAD deficiency with Systems Medicine: from computational model to mice to man. Rijksuniversiteit Groningen.. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.. Download date: 28-06-2021.

(2) Chapter 3 Transcriptomic analysis suggests a compensatory role of cofactors coenzyme A and NAD+ in medium-chain acyl-CoA dehydrogenase knockout mice Anne-Claire M.F. Martines1, Albert Gerding1,2, Sarah Stolle1, Marcel A. Vieira-Lara1, Justina C. Wolters1, Angelika Jurdzinski1, Laura Bongiovanni3, Alain de Bruin1,3, Pieter van der Vlies4, Gerben van der Vries5,6, Vincent W. Bloks1, Terry G.J. Derks1, Dirk-Jan Reijngoud1, Barbara M. Bakker1,* 1 Department. of Pediatrics, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 2 Department of Laboratory Medicine, University of Groningen, University Medical Center Groningen, The Netherlands 3 Department of Pathobiology, Faculty of Veterinary Medicine, Dutch Molecular Pathology Center, Utrecht University, The Netherlands 4 HZPC Research B.V., Metslawier, The Netherlands 5Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 6Genomics Coordination Center, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands. Under revision at Scientific Reports.

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(4) Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice. Abstract —”‹‰ˆƒ•–‹‰ǡ‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧƒ ‹†ȾǦ‘š‹†ƒ–‹‘ȋ Ȍ‹•‡••‡–‹ƒŽˆ‘”–Š‡‰‡‡”ƒ–‹‘‘ˆ ‰Ž— ‘•‡„›–Š‡Ž‹˜‡”ǤŠ‹Ž†”‡™‹–ŠƒŽ‘••Ǧ‘ˆǦˆ— –‹‘†‡ˆ‹ ‹‡ ›‹–Š‡ ‡œ›‡‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘‡œ›‡  †‡Š›†”‘‰‡ƒ•‡ ȋȌ ƒ”‡ ƒ– •‡”‹‘—• ”‹• ‘ˆ Ž‹ˆ‡Ǧ–Š”‡ƒ–‡‹‰ Ž‘™ „Ž‘‘† ‰Ž— ‘•‡ Ž‡˜‡Ž• †—”‹‰ ˆƒ•–‹‰ ‹ ‘„‹ƒ–‹‘ ™‹–Š ‹–‡” —””‡– †‹•‡ƒ•‡Ǥ ‘™‡˜‡”ǡ ƒ •—„•‡–‘ˆ–Š‡•‡ Š‹Ž†”‡”‡ƒ‹•ƒ•›’–‘ƒ–‹ –Š”‘—‰Š‘—–Ž‹ˆ‡Ǥ

(5)  Ǧ†‡ˆ‹ ‹‡– ȋǦȌ ‹ ‡ǡ ‰Ž— ‘•‡ Ž‡˜‡Ž• ƒ”‡ •‹‹Žƒ” –‘ –Š‘•‡ ‘ˆ ™‹Ž†Ǧ–›’‡ ȋȌ ‹ ‡ǡ‡˜‡†—”‹‰ˆƒ•–‹‰Ǥ‡‹˜‡•–‹‰ƒ–‡†‹ˆ‡–ƒ„‘Ž‹ ƒ†ƒ’–ƒ–‹‘•‹–Š‡Ž‹˜‡”ƒ›—†‡”Ž‹‡ –Š‡ ”‘„—•–‡•• ‘ˆ –Š‹•  ‘—•‡Ǥ  ƒ†  ‹ ‡ ™‡”‡ ‰‹˜‡ ƒ Š‹‰ŠǦ ‘” Ž‘™Ǧˆƒ– †‹‡– ƒ† •—„•‡“—‡–Ž›ˆƒ•–‡†Ǥ‡ƒƒŽ›œ‡†Š‹•–‘Ž‘‰›ǡ‹–‘ Š‘†”‹ƒŽˆ— –‹‘ǡ–ƒ”‰‡–‡†‹–‘ Š‘†”‹ƒŽ ’”‘–‡‘‹ •ǡƒ†–”ƒ• ”‹’–‘‡‹ Ž‹˜‡”–‹••—‡Ǥ‘••‘ˆŽ‡†–‘ƒ†‡ ”‡ƒ•‡† ƒ’ƒ ‹–›–‘ ‘š‹†‹œ‡‘ –ƒ‘›ŽǦ‘ǤŠ‹•™ƒ•‘– ‘’‡•ƒ–‡†ˆ‘”„›ƒŽ–‡”‡†’”‘–‡‹Ž‡˜‡Ž•‘ˆ–Š‡ •Š‘”–Ǧ ƒ† Ž‘‰Ǧ Šƒ‹ ‹•‘‡œ›‡•  ƒ† Ǥ

(6)  –Š‡ –”ƒ• ”‹’–‘‡ǡ ™‡ ‹†‡–‹ˆ‹‡† •—„–Ž‡ ƒ†ƒ’–ƒ–‹‘•‹ –Š‡ ‡š’”‡••‹‘ ‘ˆ ‰‡‡• ‡ ‘†‹‰ ‡œ›‡• ƒ–ƒŽ›œ‹‰ ‘Ǧ ƒ† ȋȌȋ ȌǦ ‹˜‘Ž˜‹‰ ”‡ƒ –‹‘• ƒ† ‘ˆ ‰‡‡• ‹˜‘Ž˜‡† ‹ †‡–‘š‹ˆ‹ ƒ–‹‘ ‡ Šƒ‹••Ǥ ‡ †‹• —•• Š‘™ –Š‡•‡ ’”‘ ‡••‡• ƒ› ‘–”‹„—–‡ –‘ ”‘„—•–‡•• ‹ Ǧ ‹ ‡ ƒ† ’‘–‡–‹ƒŽŽ› ƒŽ•‘ ‹ ƒ•›’–‘ƒ–‹ Š—ƒ•—„Œ‡ –•™‹–ŠƒŽ‘••Ǧ‘ˆǦˆ— –‹‘‰‡‘–›’‡Ǥ. ͸ͻ. 3.

(7) Chapter 3. Introduction ‡’ƒ–‹ ‹–‘ Š‘†”‹ƒŽˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘ȋ Ȍ‹•‹’‘”–ƒ–ˆ‘”–Š‡‰‡‡”ƒ–‹‘‘ˆƒ† ‡–‘‡„‘†‹‡•†—”‹‰ˆƒ•–‹‰ǡƒ†‹†‹”‡ –Ž›ƒŽ•‘ˆ‘”de novo•›–Š‡•‹•‘ˆ‰Ž— ‘•‡„›’”‘˜‹†‹‰ –Š‡ ”‡“—‹”‡†Ǥ Š‹Ž†”‡ ™‹–Š ƒ Ž‘••Ǧ‘ˆǦˆ— –‹‘ Ǥͻͺͷε  —–ƒ–‹‘ ‹ –Š‡ ACADM ‰‡‡ǡ ‡ ‘†‹‰ –Š‡   ‡œ›‡ ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ȋȌǡ ”— ƒ •‡˜‡”‡ ”‹• ‘ˆ Ž‹ˆ‡Ǧ–Š”‡ƒ–‡‹‰ Š›’‘‰Ž› ‡‹ƒ ȏͳǡʹȐǤ ‡˜‡”–Š‡Ž‡••ǡ ƒ •—„•‡– ‘ˆ –Š‡•‡ Š‹Ž†”‡ ‡˜‡” †‡˜‡Ž‘’ ƒ› •›’–‘• ȏ͵ȂͳͳȐǤ ‘–ƒ„Ž›ǡ —†‡” ”‡•–‹‰ ‘†‹–‹‘• Ǧ†‡ˆ‹ ‹‡– Š‹Ž†”‡ ‘š‹†‹œ‡ ‡†‹—Ǧ Šƒ‹ ˆƒ––› ƒ ‹†• ‘”ƒŽŽ› ȏͳʹȐ ƒ† –Š‡› ƒ”‡ ƒ„Ž‡ –‘ ˆƒ•– ˆ‘” ͳͺǦʹͶ Š‘—”• ȏͳǡ͵ǡͳ͵ǡͳͶȐǤƒ”–‹ —Žƒ”Ž›ƒ ‘„‹ƒ–‹‘‘ˆ’”‘Ž‘‰‡†ˆƒ•–‹‰ƒ†ƒƒ††‹–‹‘ƒŽ–”‹‰‰‡”ǡ•— Šƒ• ‹ˆ‡ –‹‘•ǡ‹ ”‡ƒ•‡•–Š‡”‹•‘ˆŽ‘™„Ž‘‘†‰Ž— ‘•‡Ž‡˜‡Ž•ȏʹǡͳͳǡͳͷȂͳ͹ȐǤ Š‡   ’ƒ–Š™ƒ› ‹• • Š‡ƒ–‹ ƒŽŽ› †‡’‹ –‡† ‹ —’’Ž‡‡–ƒ”› ‹‰—”‡ ͳǤ ƒ––› ƒ ‹†• ƒ”‡ –ƒ‡ —’ ‹–‘ –Š‡ ‹–‘ Š‘†”‹ƒ ‹ ƒ ƒ –‹˜ƒ–‡† ˆ‘” ƒ• ƒ ›ŽǦ‘ ‡•–‡”• ƒ† •—„•‡“—‡–Ž› ‘š‹†‹œ‡†–‘ƒ ‡–›ŽǦ‘ǤŠ‹•‘ —”•‹”‡’‡–‹–‹˜‡ˆ‘—”Ǧ”‡ƒ –‹‘ › Ž‡•Ǥ

(8) ‡ƒ Š › Ž‡ǡ–Š‡ƒ ›ŽǦ ‘‡•–‡”‹••Š‘”–‡‡†„›–™‘ ƒ”„‘ƒ–‘•ǡ–Š‡”‡„›’”‘†— ‹‰ƒ ‡–›ŽǦ‘ȏͳǡͳͺǡͳͻȐǤŠ‡ˆ‹”•– ”‡ƒ –‹‘‹‡ƒ Š › Ž‡‹• ƒ–ƒŽ›œ‡†„›ƒ•‡–‘ˆ‹•‘‡œ›‡•ǡ™Š‹ Šƒ ‡’–ƒ”ƒ‰‡‘ˆƒ ›ŽǦ‘ •—„•–”ƒ–‡• ‘ˆ †‹ˆˆ‡”‡–ǡ ‘˜‡”Žƒ’’‹‰ ƒ”„‘Ǧ Šƒ‹ Ž‡‰–Š•Ǥ

(9)  ”‘†‡–•ǡ –Š‡•‡ ƒ”‡ ˜‡”›ǦŽ‘‰Ǧǡ Ž‘‰Ǧǡ‡†‹—Ǧǡƒ†•Š‘”–Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡ȋǡǡǡƒ†ȌǤ

(10) Š—ƒ•ǡ–Š‡’”‘–‡‹ ‘ ‡–”ƒ–‹‘‹•Ž‘™‹–Š‡Ž‹˜‡”ƒ†’Žƒ›•ƒ‹‘””‘Ž‡‹–Š‡   ’ƒ–Š™ƒ›ȏͳǡʹͲȐǤ Š‡ ‘‡œ›‡• ‹ ‘–‹ƒ‹†‡ ƒ†‡‹‡ †‹— Ž‡‘–‹†‡ ȋ ΪȌ ƒ† ˆ”‡‡ ‘ ȋ‘ Ȍ ’Žƒ› ‡› ”‘Ž‡• ‹   ˆ— –‹‘ ƒ† Ž‹ –Š‡ ’ƒ–Š™ƒ› –‘ ‘–Š‡” ‡–ƒ„‘Ž‹  ’ƒ–Š™ƒ›•ȏʹͳȂʹͷȐǤ ‘‹˜‡•–‹‰ƒ–‡–Š‡†‹•‡ƒ•‡‡–‹‘Ž‘‰›‹†‡ˆ‹ ‹‡ ›ȋȌǡƒǦ‘—•‡‘†‡Ž ™ƒ• ‰‡‡”ƒ–‡†ǡ ‹‹–‹ƒŽŽ› ‹ ƒ ͷ͹Ȁ͸ƒ  Ƭ ͳʹͻʹȀŽƒ •† ‹š‡† „ƒ ‰”‘—†ȏʹ͸ȐǤ Š‡ ‘—•‡”‡ ƒ’‹–—Žƒ–‡†ƒ—„‡”‘ˆŠ—ƒ†‹•‡ƒ•‡ Šƒ”ƒ –‡”‹•–‹ •ǡ‹ Ž—†‹‰‡Ž‡˜ƒ–‡†‡‘ƒ–ƒŽ †‡ƒ–Š”ƒ–‡ǡŽ‘™‡”ͺǦƒ ›ŽǦ‘‘š‹†ƒ–‹‘ ƒ’ƒ ‹–›ǡŠ‹‰Š‡”—”‹ƒ”›‡†‹—Ǧ Šƒ‹‘”‰ƒ‹ ƒ ‹† Ž‡˜‡Ž•ǡƒ†Š‹‰Š‡”•‡”—Ž‡˜‡Ž•‘ˆ‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ ƒ”‹–‹‡Ž‡˜‡Ž• ‘’ƒ”‡†–‘–Š‡™‹Ž† –›’‡ȋȌǤ

(11) ƒ††‹–‹‘ǡʹͶ‘ˆŠ‘—”•ˆƒ•–‹‰‹†— ‡†Ž‹’‹†ƒ ——Žƒ–‹‘ȋ•–‡ƒ–‘•‹•Ȍ‹–Š‡Ž‹˜‡” ‹–Š‡Ǧ„—–‘––Š‡‹ ‡ǡ‹ƒ ‘”†ƒ ‡–‘Š‡’ƒ–‹ •–‡ƒ–‘•‹••‡‡‹•›’–‘ƒ–‹  ’ƒ–‹‡–•ȏʹǡͳͳǡͳͷȂͳ͹ȐǤ ‹ƒŽŽ›ǡ™Š‡ˆƒ•–‡†‹ ‘„‹ƒ–‹‘™‹–Š ‘Ž†Ǧ‡š’‘•—”‡ǡ–Š‡ Ǧ‘—•‡•Š‘™‡†•‹‰‹ˆ‹ ƒ–Ž›Ž‘™‡”„‘†›–‡’‡”ƒ–—”‡ ‘’ƒ”‡†–‘ǡ™Š‹ Š™ƒ• ‡˜‡–—ƒŽŽ› Ž‡–ŠƒŽȏʹ͸ȐǤ ‡’ƒ–‹  ‹ ”‘ƒ””ƒ› ƒƒŽ›•‹• •Š‘™‡† ƒ”‡† †‹ˆˆ‡”‡ ‡• ‹ –Š‡ ‡š’”‡••‹‘ ‘ˆ ‰Ž— ‘•‡ ‡–ƒ„‘Ž‹• ‰‡‡• „‡–™‡‡ ˆƒ•–‡†  ƒ†  ‹ ‡ ȏʹ͹ȐǤ

(12)  –Š‡ ‡ƒ–‹‡ǡ™‡„ƒ  ”‘••‡†–Š‡Ǧ‘—•‡‘†‡Ž–‘ƒͷ͹Ȁ͸ ’—”‡Ǧ„ƒ ‰”‘—†ȏʹͺȐǤ ‹‡‹–•’”‡†‡ ‡••‘”ǡ–Š‡’—”‡Ǧ„ƒ ‰”‘—†Ǧ‘—•‡‡šŠ‹„‹–‡†Š‹‰Š‡”‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ ƒ”‹–‹‡Ž‡˜‡Ž•‹•‡”—ƒ†ƒŽ‘™‡”ͺǦƒ ›ŽǦ‘‘š‹†ƒ–‹‘ ƒ’ƒ ‹–› ‘’ƒ”‡†–‘–Š‡ ƒˆ–‡”ͳʹŠ‘—”•‘ˆˆƒ•–‹‰Ǥ‘–ƒ„Ž›ǡ–Š‡Ž‘™„Ž‘‘†‰Ž— ‘•‡Ž‡˜‡Ž•™Š‹ Šƒ”‡–Š‡ŠƒŽŽƒ”‘ˆ Š—ƒǡ™‡”‡‡˜‡””‡’‘”–‡†‹Ǧ‹ ‡ȏʹ͸ǡʹ͹ǡʹͻȐǤŠ‡”‡ˆ‘”‡ǡ–Š‡Ǧ ‘—•‡ Šƒ• •‘ ˆƒ” ‘– „‡‡ ƒ —•‡ˆ—Ž ‘†‡Ž ˆ‘” –Š‡ ƒ —–‡ Š›’‘‰Ž› ‡‹ƒ ‘„•‡”˜‡† ‹ •›’–‘ƒ–‹ ’ƒ–‹‡–•Ǥ

(13) –ƒ›ǡŠ‘™‡˜‡”ǡ„‡ƒ‡š ‡ŽŽ‡–‘†‡Ž–‘‹†‡–‹ˆ›’‘–‡–‹ƒŽ ‘’‡•ƒ–‘”› ‡ Šƒ‹•• –‘ ‡š’Žƒ‹ ™Š› •‘‡ Š—ƒ• ”‡ƒ‹ ƒ•›’–‘ƒ–‹  †‡•’‹–‡ ƒ Š‘‘œ›‰‘—•Ž‘••Ǧ‘ˆǦˆ— –‹‘MCAD —–ƒ–‹‘ǤŠ‹•ƒ›Š‡Ž’–‘‹†‡–‹ˆ›’ƒ–‹‡–•™Š‘ƒ”‡‘– ƒ–”‹•ƒ†ƒ”‡ —””‡–Ž›‘˜‡”–”‡ƒ–‡†ȏ͵ͲȐƒ†’”‘˜‹†‡ Ž—‡•ˆ‘”’”‡˜‡–‹‘‹’ƒ–‹‡–•™Š‘ ƒ”‡•–‹ŽŽƒ–”‹•Ǥ Š‡ ƒ‹ ‘ˆ –Š‡ —””‡– •–—†› ‹• –‘ ‡š’Ž‘”‡ Š‡’ƒ–‹  ˆ— –‹‘ƒŽ ƒ† ‘Ž‡ —Žƒ” †‹ˆˆ‡”‡ ‡• „‡–™‡‡  ƒ† Ǧ ‘—•‡ –‘ ‰ƒ‹ ‹•‹‰Š– ‹–‘ –Š‡ ”‘Ž‡ ‘ˆ –Š‡ Ž‹˜‡” ‹ –Š‡•‡ ͹Ͳ.

(14) Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice ‘’‡•ƒ–‘”›‡ Šƒ‹••Ǥ‘–Š‹•‡†™‡•—„Œ‡ –‡†–Š‡ͷ͹Ȁ͸ ’—”‡Ǧ„ƒ ‰”‘—†Ǧ ‘—•‡‘†‡Ž–‘Ž‘™Ǧˆƒ–ƒ†Š‹‰ŠǦˆƒ–†‹‡–ǡ—†‡”ˆ‡†ƒ†ͳ͸Šˆƒ•–‡† ‘†‹–‹‘•ǤŠ‡Š‹‰ŠǦ ˆƒ– †‹‡– ‹• ‡™ ‘’ƒ”‡† –‘ ’”‡˜‹‘—•Ž› ’—„Ž‹•Š‡† •–—†‹‡• ƒ† ƒ› ’”‘˜‹†‡ ƒ ƒ††‹–‹‘ƒŽ ‡˜‹”‘‡–ƒŽ’‡”–—”„ƒ–‹‘–‘–Š‡Ǧ‘—•‡„›’”‘˜‹†‹‰–Š‡ ’ƒ–Š™ƒ›™‹–Š ƒ††‹–‹‘ƒŽ •—„•–”ƒ–‡Ǥ ‡ ‘ŽŽ‡ –‡† „‹‘‡–”‹ ǡ Š‹•–‘Ž‘‰‹ ƒŽǡ –ƒ”‰‡–‡† ’”‘–‡‘‹ •ǡ ƒ† –”ƒ• ”‹’–‘‹ • †ƒ–ƒ ˆ”‘ Ž‹˜‡” –‹••—‡Ǥ

(15)  ƒ††‹–‹‘ǡ Š‡’ƒ–‹  ‹–‘ Š‘†”‹ƒŽ ˆ— –‹‘ ™ƒ• ƒƒŽ›œ‡†Ǥƒ––‡””‡ ‘‰‹–‹‘ƒƒŽ›•‹••Š‘™‡†•—„–Ž‡ƒ†ƒ’–ƒ–‹‘•‹†‡–‘š‹ˆ‹ ƒ–‹‘’”‘ ‡••‡• ƒ†‹‰‡‡• ‘†‹‰ˆ‘”‡œ›‡•‹˜‘Ž˜‡†‹‘ƒ†ȋȌȋ Ȍ‡–ƒ„‘Ž‹•‘ˆǦ ‹ ‡Ǥ‡†‹• —••Š‘™–Š‡•‡ƒ†ƒ’–ƒ–‹‘•ƒ› ‘–”‹„—–‡–‘–Š‡‹”’Š‡‘–›’‹ ”‘„—•–‡••Ǥ . Results Biometric and hepatohistological characterization of the pure-background MCAD-KO mice. A. B Body weight (g). 50 ‡‡ ‡‡. 40. *. #. 30. ‡‡. ‡‡. LF fed HF fed LF fasted HF fasted. #. 20 20 0 WT. KO. Body weight decrease (g). Ǧƒ†ͷ͹Ȁ͸ ‹ ‡™‡”‡‡’–‘ƒŽ‘™Ǧˆƒ–‘”Š‹‰ŠǦˆƒ–•‡‹•›–Š‡–‹ †‹‡–ˆ‘”͸ ™‡‡•ƒ†•—„•‡“—‡–Ž›ˆƒ•–‡†ˆ‘”ͳ͸Š‘—”•Ǥ‹––Ž‡–‘‘‡ˆˆ‡ –‘ˆ‰‡‘–›’‡™ƒ•‘„•‡”˜‡†‘ „‘†›™‡‹‰Š–ǡ„‘†›Ǧ™‡‹‰Š–Ž‘••†—”‹‰ˆƒ•–‹‰ǡŽ‹˜‡”™‡‹‰Š–ǡ‘”„Ž‘‘†‰Ž— ‘•‡Ž‡˜‡Žȋ ‹‰—”‡ͳǦ ƒ†—’’Ž‡‡–ƒ”› ‹‰—”‡ʹƒ†ȌǤŽ›‹–Š‡Ž‘™Ǧˆƒ–ˆ‡†‰”‘—’–Š‡„‘†›™‡‹‰Š–™ƒ• •–ƒ–‹•–‹ ƒŽŽ›Š‹‰Š‡” ‹ Ǧ ‘’ƒ”‡†–‘  ‹ ‡ ȋ ‹‰—”‡ ͳȌǡ „—––Š‡ †‹ˆˆ‡”‡ ‡ ™ƒ• ‘Ž›ͺΨǤŠ‡•‡”‡•—Ž–•ƒ”‡‹ƒ ‘”†ƒ ‡™‹–Š‡ƒ”Ž‹‡”•–—†‹‡•ȏʹ͸ǡʹ͹ǡʹͻȐǤ 4. ‡‡. 3. LF fasted HF fasted. 2 1 0 WT. KO. C Blood glucose (mM). 15. 10. ‡. ##. ##. ## ##. LF fed HF fed LF fasted HF fasted. 5. 0 WT. KO. Figure 1. Mouse biometric measures (A-C) under different conditions A: Body weight; B: body weight decrease after fasting; C: Blood glucose levels at termination. Š‡”‡•—Ž–•ƒ”‡”‡’”‡•‡–‡†ƒ•—‡›„‘š ƒ† ™Š‹•‡” ’Ž‘–•ǡ ‹ ™Š‹ Š –Š‡ „Žƒ Ǧˆ‹ŽŽ‡† ‹” Ž‡• ‹†‹ ƒ–‡ ‹†‹˜‹†—ƒŽ ‹ ‡ ˆƒŽŽ‹‰ ‘—–•‹†‡ –Š‡ ͹ͷΨ ’‡” ‡–‹Ž‡’Ž—•ͳǤͷȉ‹–‡”Ǧ“—ƒ”–‹Ž‡”ƒ‰‡ȋ

(16) Ȍ‘”ʹͷΨ’‡” ‡–‹Ž‡‹—•ͳǤͷȉ

(17) Ǥα͸Ǧͺˆ‘”„‘–Šƒ†Ǥ ȗǣ’δͲǤͲͷ ‘’ƒ”‡†–‘ǡ͓ƒ†͓͓ǣ’δͲǤͲͷƒ†’δͲǤͲͳ ‘’ƒ”‡†–‘ˆ‡†ǡ”‡•’‡ –‹˜‡Ž›ǡșƒ†șșǣ’δͲǤͲͷ ƒ†’δͲǤͲͳ ‘’ƒ”‡†–‘Ž‘™Ǧˆƒ–†‹‡–ȋ Ȍǡ”‡•’‡ –‹˜‡Ž›Ǥ.  ‹ ‡Š‡’ƒ–‹ •–‡ƒ–‘•‹•Šƒ†„‡‡”‡’‘”–‡†ƒˆ–‡”ˆƒ•–‹‰‘ˆ–Š‡‹š‡†Ǧ„ƒ ‰”‘—†Ǧ ‘—•‡ȏʹ͸Ȑǡ™‡‹•’‡ –‡†–Š‡Ž‹˜‡”‹‘”‡†‡–ƒ‹ŽǤ ‹•–‘Ž‘‰‹ ƒŽ‡šƒ‹ƒ–‹‘•Š‘™‡†–Šƒ––Š‡”‡ ™‡”‡‘•–ƒ–‹•–‹ ƒŽŽ›•‹‰‹ˆ‹ ƒ–†‹ˆˆ‡”‡ ‡•„‡–™‡‡ƒ†ȋ ‹‰—”‡ʹƒ†—’’Ž‡‡–ƒ”› ‹‰—”‡• ͵ǦͶȌǤ ‘™‡˜‡”ǡ –Š‡ Ǧ ‹ ‡ †‹† •Š‘™ ƒ –”‡† –‘™ƒ”†• ƒ Š‹‰Š‡” †‡‰”‡‡ ‘ˆ. ͹ͳ. 3.

(18) Chapter 3 Ž‘„—Žƒ” ‹ˆŽƒƒ–‹‘ ‹ –Š‡ Š‹‰ŠǦˆƒ– ˆƒ•–‡† ‘†‹–‹‘ ȋ ‹‰—”‡ ʹ ƒ† —’’Ž‡‡–ƒ”› ƒ„Ž‡ ͳȌǤ‹˜‡”–”‹‰Ž› ‡”‹†‡•ǡŠ‘™‡˜‡”ǡ™‡”‡‘–•‹‰‹ˆ‹ ƒ–Ž›†‹ˆˆ‡”‡–‹Ǧ ‘’ƒ”‡†–‘ ‹ ‡—†‡”ƒ›‘ˆ–Š‡ ‘†‹–‹‘•ȋ—’’Ž‡‡–ƒ”› ‹‰—”‡ʹȌǤ 10x. 40x. A. B. C. D. High-fat fasted MCAD KO. High-fat fasted WT. Figure 2. Similar steatosis grade (A, C) and difference in lobular inflammation (B, D) between MCADKO and WT mice in the high-fat fasted condition™‹–Š‹ˆŽƒƒ–‘”›Ž‘ ‹•Š‘™ „›–Š‡„Žƒ ƒ””‘™•Ǥ Š‘™Š‡”‡ƒ”‡”‡’”‡•‡–ƒ–‹˜‡’‹ –—”‡•Ǥ‹˜‡”•™‡”‡•–ƒ‹‡†„› ‡ƒ–‘š›Ž‹Ƭ‘•‹ȋ•‡‡ƒ–‡”‹ƒŽ•ƒ† ‡–Š‘†•ˆ‘”ˆ—”–Š‡”†‡–ƒ‹Ž•ȌǤ

(19) ˆŽƒƒ–‹‘‹•‹†‡–‹ˆ‹‡†„›‹ˆ‹Ž–”ƒ–‹‘‘ˆŽ‡—‘ ›–‡•Ǥ‘„—Žƒ”‹ˆŽƒƒ–‹‘ ™ƒ•”‡ ‘‰‹œ‡†ƒ••ƒŽŽ‰”‘—’•‘ˆ‹š‡†‹ˆŽƒƒ–‘”› ‡ŽŽ•• ƒ––‡”‡†–Š”‘—‰Š‘—––Š‡Š‡’ƒ–‹ ’ƒ”‡ Š›ƒ ȋ•‡‡—’’Ž‡‡–ƒ”›–ƒ„Ž‡ͳˆ‘”•‡‹Ǧ“—ƒ–‹–ƒ–‹˜‡• ‘”‹‰‘ˆ–Š‡Ž‹˜‡”•ȌǤ. . MCAD-KO mice show decreased C8-acyl-CoA-dependent state 3 O2 consumption capacity ‡ ‘ˆ‹”‡†–Š‡’”‹ƒ”›„‹‘ Š‡‹ ƒŽ†‡ˆ‡ –‹ˆ— –‹‘„›‡ƒ•—”‹‰–Š‡•—„•–”ƒ–‡Ǧ †‡’‡†‡–ʹǦ ‘•—’–‹‘”ƒ–‡‹‹•‘Žƒ–‡†Ž‹˜‡”‹–‘ Š‘†”‹ƒȋ ‹‰—”‡͵ƒ†—’’Ž‡‡–ƒ”› ‹‰—”‡ ͷȌǤ  ’›”—˜ƒ–‡ ‘” ͳ͸Ǧƒ ›ŽǦ‘ ƒ• •—„•–”ƒ–‡•ǡ –Š‡ ‰‡‘–›’‡ Šƒ† ‘ ‡ˆˆ‡ – ‘ ʹǦ ‘•—’–‹‘”ƒ–‡ȋ ‹‰—”‡͵ǦȌǡ‹Ž‹‡™‹–Š‡š’‡ –ƒ–‹‘•ƒ†‡ƒ”Ž‹‡””‡•—Ž–•ȏʹͺȐǤŠ‡Ǧ ‘—•‡Šƒ†ƒŽ‘™‡”ʹ ‘•—’–‹‘”ƒ–‡–Šƒ–Š‡‘–Š‡•—„•–”ƒ–‡ͺǦƒ ›ŽǦ‘ ȋ ‹‰—”‡͵ȌǤŠ‹•™ƒ••‹‰‹ˆ‹ ƒ–‹–Š‡Š‹‰ŠǦˆƒ–†‹‡–ˆƒ•–‡†‰”‘—’ȋ’αͲǤͲʹȌƒ†•–ƒ–‹•–‹ ƒŽŽ› ‹†‹ ƒ–‹˜‡‹–Š‡Ž‘™Ǧˆƒ–†‹‡–ˆƒ•–‡†‰”‘—’ȋ’αͲǤͲ͸Ȍȋ™‹–Š–Š‡ƒ˜‡”ƒ‰‡Ǧ”ƒ–‡•„‡‹‰ ͹ʹƒ†͸ʹΨ‘ˆ–Š‡ƒ˜‡”ƒ‰‡”ƒ–‡•‘Ž‘™Ǧˆƒ–ƒ†Š‹‰ŠǦˆƒ–†‹‡–”‡•’‡ –‹˜‡Ž›ȌǤ. ͹ʹ.

(20) Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice. Pyruvate-induced O2 consumption rate (St3) (μmol/(min*gmitProtein)). 150. LF fed HF fed LF fasted HF fasted. 100. 50. 0 WT. KO. C16-acyl-CoA-induced O2 consumption rate (St3) (μmol/(min*gmitProtein)). B. A. 250. LF fed HF fed LF fasted HF fasted. 200 150 100. 3. 50 0 WT. KO. C8-acyl-CoA-induced O2 consumption rate (St3) (μmol/(min*gmitProtein)). C 150 i. 100. *. LF fed HF fed LF fasted HF fasted. 50. 0 WT. KO. . Figure 3. Mouse hepatic mitochondrial function in different conditions. Pyruvate- (A), C16-acyl-CoA(B) and C8-acyl-CoA- (C) induced maximum O2-consumption flux (state 3) in isolated liver mitochondria. State 3 represents the maximum ADP-stimulated oxygen consumption. Š‡”‡•—Ž–•ƒ”‡ ”‡’”‡•‡–‡†ƒ•—‡›„‘šƒ†™Š‹•‡”’Ž‘–•ǡ‹™Š‹ Š–Š‡„Žƒ Ǧˆ‹ŽŽ‡† ‹” Ž‡•‹†‹ ƒ–‡‹†‹˜‹†—ƒŽ‹ ‡ˆƒŽŽ‹‰ ‘—–•‹†‡–Š‡͹ͷΨ’‡” ‡–‹Ž‡’Ž—•ͳǤͷȉ‹–‡”Ǧ“—ƒ”–‹Ž‡”ƒ‰‡ȋ

(21) Ȍ‘”ʹͷΨ’‡” ‡–‹Ž‡‹—•ͳǤͷȉ

(22) Ǥα͸Ǧͺˆ‘” „‘–Šƒ†Ǥȗǣ’δͲǤͲͷ ‘’ƒ”‡†–‘ǡ͓ƒ†͓͓ǣ’δͲǤͲͷƒ†’δͲǤͲͳ ‘’ƒ”‡†–‘ˆ‡†ǡ”‡•’‡ –‹˜‡Ž›ǡș ƒ†șșǣ’δͲǤͲͷƒ†’δͲǤͲͳ ‘’ƒ”‡†–‘Ž‘™Ǧˆƒ–†‹‡–ȋ Ȍǡ”‡•’‡ –‹˜‡Ž›Ǥ. 

(23) ƒ††‹–‹‘ǡ—†‡”ˆ‡† ‘†‹–‹‘•–Š‡ͺǦƒ ›ŽǦ‘Ǧ†‡’‡†‡–ʹǦ ‘•—’–‹‘”ƒ–‡™ƒ•Ž‘™‡” ‹–Š‡Ǧȋ™‹–Š–Š‡ƒ˜‡”ƒ‰‡Ǧ”ƒ–‡•„‡‹‰͸ͷΨƒ†͹ͶΨ‘ˆ–Š‡ƒ˜‡”ƒ‰‡ ”ƒ–‡• ‘ Ž‘™Ǧˆƒ– ƒ† Š‹‰ŠǦˆƒ– †‹‡– ”‡•’‡ –‹˜‡Ž›Ȍǡ „—– –Š‹• †‹ˆˆ‡”‡ ‡ ™ƒ• ‘– •–ƒ–‹•–‹ ƒŽŽ› •‹‰‹ˆ‹ ƒ–ȋ’αͲǤͳͷƒ†ͲǤͳ͵ǡ”‡•’‡ –‹˜‡Ž›ȌǤŠ‡ˆƒ ––Šƒ––Š‡”‡‹••— ŠƒŠ‹‰Š”‡•‹†—ƒŽͺǦƒ ›ŽǦ ‘Ǧ†‡’‡†‡– ʹǦ ‘•—’–‹‘ ”ƒ–‡ ‹ –Š‡ Ǧ ‘—•‡ ƒ› „‡ ƒ––”‹„—–‡† –‘ –Š‡ ‹•‘‡œ›‡•  ƒ† ǡ ™Š‹ Š ƒ”‡ ‘™ –‘ „‡ ‡š’”‡••‡† ‹ ‘—•‡ Ž‹˜‡”ȏʹͻȐǤ

(24)  –Š‡ ˆ‘ŽŽ‘™‹‰ •‡ –‹‘ǡ ™‡ ™‹ŽŽ ‹˜‡•–‹‰ƒ–‡ –Š‡ ’”‘–‡‹ ‘ ‡–”ƒ–‹‘• ‘ˆ –Š‡•‡ ƒ† ‘–Š‡” ‹–‘ Š‘†”‹ƒŽ‡œ›‡•‹‘”‡†‡–ƒ‹ŽǤ . Targeted mitochondrial proteomics ‡ ‡–Ž›ǡ™‡†‡˜‡Ž‘’‡†ƒ–ƒ”‰‡–‡†’”‘–‡‘‹ •‡–Š‘†–‘†‡–‡ –ƒ†“—ƒ–‹ˆ›•’‡ ‹ˆ‹ ’”‘–‡‹• ‹˜‘Ž˜‡†‹‹–‘ Š‘†”‹ƒŽ‡‡”‰›‡–ƒ„‘Ž‹•ǤŠ‡–ƒ”‰‡–‡†’”‘–‡‹• ‘’”‹•‡ƒŽŽ‡œ›‡• ‹˜‘Ž˜‡†‹ ƒ† › Ž‡ǡ ‘”‡•—„—‹–•‘ˆ‡ƒ Š‘ˆ–Š‡”‡•’‹”ƒ–‘”›Ǧ Šƒ‹ ‘’Ž‡š‡• ƒ†‡–ƒ„‘Ž‹–‡–”ƒ•’‘”–‡”•”‡“—‹”‡†ˆ‘”–Š‡•‡’ƒ–Š™ƒ›•ȏʹͻȐǤ‡ƒ’’Ž‹‡†–Š‹•‡–Š‘†–‘–Š‡ Š‡’ƒ–‹ ‹–‘ Š‘†”‹ƒ‹•‘Žƒ–‡†‹–Š‹••–—†›ȋ ‹‰—”‡ͶǦƒ†—’’Ž‡‡–ƒ”› ‹‰—”‡͸ȌǤ• ‡š’‡ –‡†ǡ –Š‡  ’”‘–‡‹ ™ƒ• ‘– †‡–‡ –ƒ„Ž‡ ‹ –Š‡ Ǧ ‹–‘ Š‘†”‹ƒǤ Š‡ ‹•‘‡œ›‡•  ƒ† ǡ ™Š‹ Š ƒŽ•‘ ƒ ‡’–  •—„•–”ƒ–‡•ǡ ™‡”‡ ‡š’”‡••‡† ȋ ‹‰—”‡ ͶǡȌ ƒ†–Š‡› ‘—Ž†–Š—•‡š’Žƒ‹ –Š‡ ’”‡•‡ ‡‘ˆ ƒ ”‡•‹†—ƒŽ ˆŽ—š ‘ˆ ͺǦƒ ›ŽǦ‘ ‘š‹†ƒ–‹‘ †‹• —••‡†ƒ„‘˜‡Ǥ ‘™‡˜‡”ǡ‡‹–Š‡”–Š‡‹”‡š’”‡••‹‘ǡ‘”–Šƒ–‘ˆ–Š‡‘–Š‡”–ƒ”‰‡–‡†’”‘–‡‹• ™‡”‡ ƒŽ–‡”‡† ‹ –Š‡ Ǧ ‘—•‡ ‘’ƒ”‡† –‘ –Š‡  ȋ ‹‰—”‡ ͸ȌǤ Š—•ǡ –Š‡ –ƒ”‰‡–‡† ’”‘–‡‘‹  ƒƒŽ›•‹• †‹† ‘– ‹†‡–‹ˆ› ƒ› ‘’‡•ƒ–‘”› ‡ Šƒ‹•• ‹˜‘Ž˜‹‰ –Š‡ ‘ ‡–”ƒ–‹‘‘ˆ–Š‡‡ƒ•—”‡†’”‘–‡‹•Ǥ . ͹͵.

(25) Chapter 3. [Protein] (fmol/Pg mito prot). LF fed HF fed LF fasted HF fasted. 100. 50. 0 WT. KO. C [Protein] (fmol/Pg mito prot). B. SCAD 150. MCAD 150. LF fed HF fed LF fasted HF fasted. 100. 50. 0 WT. KO. LF fed HF fed LF fasted HF fasted. ‡‡ 100. 50. * * * * 0 WT. KO. D. LCAD 150. [Protein] (fmol/Pg mito prot). [Protein] (fmol/Pg mito prot). A. VLCAD 80. LF fed HF fed LF fasted HF fasted. 60 40 20 0 WT. KO. Figure 4. Absolute protein levels of the mFAO proteins SCAD (A), MCAD (B), LCAD (C) and VLCAD (D). Š‡‘–Š‡”‹–‘ Š‘†”‹ƒŽ‡‡”‰›‡–ƒ„‘Ž‹•’”‘–‡‹•ƒ”‡•Š‘™‹•—’’Ž‡‡–ƒ”› ‹‰—”‡͸ǤŠ‡”‡•—Ž–• ƒ”‡ ”‡’”‡•‡–‡† ƒ• —‡› „‘š ƒ† ™Š‹•‡” ’Ž‘–•ǡ ™Š‡”‡ –Š‡ „Žƒ Ǧˆ‹ŽŽ‡† ‹” Ž‡• ‹†‹ ƒ–‡ ‹†‹˜‹†—ƒŽ ‹ ‡ ˆƒŽŽ‹‰‘—–•‹†‡–Š‡͹ͷΨ’‡” ‡–‹Ž‡’Ž—•ͳǤͷȉ‹–‡”Ǧ“—ƒ”–‹Ž‡”ƒ‰‡ȋ

(26) Ȍ‘”ʹͷΨ’‡” ‡–‹Ž‡‹—•ͳǤͷȉ

(27) Ǥα͸Ǧ ͺ ˆ‘” „‘–Š  ƒ† Ǥ ȗǣ ’δͲǤͲͷ ‘’ƒ”‡† –‘ ǡ ͓ ƒ† ͓͓ǣ ’δͲǤͲͷ ƒ† ’δͲǤͲͳ ‘’ƒ”‡† –‘ ˆ‡†ǡ ”‡•’‡ –‹˜‡Ž›ǡșƒ†șșǣ’δͲǤͲͷƒ†’δͲǤͲͳ ‘’ƒ”‡†–‘Ž‘™Ǧˆƒ–†‹‡–ȋ Ȍǡ”‡•’‡ –‹˜‡Ž›Ǥ. Differences in hepatic mRNA expression patterns between MCAD-KO and WT mouse ‘ ‡š’Žƒ‹ –Š‡ ’Š›•‹‘Ž‘‰‹ ƒŽ ”‘„—•–‡•• ‘ˆ –Š‡ Ǧ ‹ ‡ǡ ™‡ ƒƒŽ›œ‡† –Š‡ Ž‹˜‡” –”ƒ• ”‹’–‘‡ „› •‡“Ǥ ”‹ ‹’Ž‡ ‘’‘‡– ƒƒŽ›•‹• •Š‘™‡† –Šƒ– ˆƒ•–‹‰ Šƒ† ƒ — Š •–”‘‰‡”‡ˆˆ‡ –‘–Š‡–”ƒ• ”‹’–‘‡–Šƒ–Š‡‰‡‘–›’‡ȋ—’’Ž‡‡–ƒ”› ‹‰—”‡ ͹ȌǤ Š”‘—‰Š †‹ˆˆ‡”‡–‹ƒŽ ‰‡‡ ‡š’”‡••‹‘ ȋ Ȍ ƒƒŽ›•‹•ǡ ™‡ ƒŽ —Žƒ–‡† †‹ˆˆ‡”‡ ‡• ‹ –”ƒ• ”‹’– Ž‡˜‡Ž• „‡–™‡‡Ǧ ƒ†‹ ‡Ǥ”‡Žƒ–‹˜‡Ž›•ƒŽŽ—„‡”‘ˆ–”ƒ• ”‹’–•™‡”‡ƒˆˆ‡ –‡†„› –Š‡ ‰‡‘–›’‡ ƒ ‘”†‹‰ –‘ –Š‡ ‡š’Ž‘”ƒ–‘”› ’Ǧ˜ƒŽ—‡ ‘ˆ δͲǤͲͳ ƒ† –Š‡ ‘””‡•’‘†‹‰ ˆ‘Ž† Šƒ‰‡•™‡”‡’”‡†‘‹ƒ–Ž›„‡–™‡‡ͳƒ†ʹȋ—’’Ž‡‡–ƒ”› ‹‰—”‡ ͺǦȌǤ

(28) ƒ††‹–‹‘ǡ –Š‡ ‰‡‡• ‹ “—‡•–‹‘ •Š‘™‡† Ž‹––Ž‡ ‘˜‡”Žƒ’ „‡–™‡‡ –Š‡ ‘†‹–‹‘• ȋ ‹‰—”‡ ͷǦ ƒ† •—’’Ž‡‡–ƒ”› ‹‰—”‡ ͺǦ ȌǤ ‘–ƒ„Ž›ǡͶ–”ƒ• ”‹’–•™‡”‡‹†‡–‹ˆ‹‡†ƒ••‹‰‹ˆ‹ ƒ–Ž›ƒŽ–‡”‡† „›–Š‡‰‡‘–›’‡ƒ ‘”†‹‰–‘’ƒ†ŒδͲǤͲͷǢ͵ˆ‘”–Š‡  ƒ•–‡†ƒ†ͳˆ‘”–Š‡  ‡† ‘†‹–‹‘ǡ ”‡•’‡ –‹˜‡Ž›ȋ—’’Ž‡‡–ƒ”›ƒ„Ž‡ʹȌǤ A. HF Fa. 42. HF Fa. 47. 1 0. 0. 2. 0. 1. 0. 27. 0 0. 2 0. 0. 0. 0. 84. 0 LF Fa. 63. 0. 44. 0. LF Fe. 28 1. 0. 82. B. LF Fe. 0. 0 0. HF Fe. LF Fa. HF Fe. Figure 5. Venn diagrams showing overlap between conditions of upregulated (A) and downregulated (B) genes between WT and KO ˆ‘” ƒ ’δͲǤͲͳǤ Š‡ †‡’‹ –‡† ”‡•—Ž–• ƒ”‡ „ƒ•‡† ‘ –Š‡ ‡‡“ʹ‡–Š‘†Ǥ. ͹Ͷ.

(29) Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice •‡–ƒ„‘Ž‹ ‰‡‡‡š’”‡••‹‘ Šƒ‰‡• ƒ„‡•ƒŽŽƒ†–‘‡˜‡”–Š‡Ž‡••‹†‡–‹ˆ›ˆ— –‹‘ƒŽ ‡–ƒ„‘Ž‹ †‹ˆˆ‡”‡ ‡•„‡–™‡‡–Š‡ƒ†ǡ’ƒ–Š™ƒ›ƒƒŽ›•‹•ƒ›„‡‘”‡‹•‹‰Š–ˆ—ŽǤ  Gene-set enrichment analysis revealed differentially expressed gene sets between WT and KO mice. ‡‡Ǧ•‡–‡”‹ Š‡–ƒƒŽ›•‹•ȋ Ȍ†‡–‡”‹‡•™Š‡–Š‡”–Š‡‡„‡”•‘ˆ’”‡†‡ˆ‹‡†•‡–•‘ˆ ˆ— –‹‘ƒŽŽ›”‡Žƒ–‡†‰‡‡•ƒ”‡’”‡†‘‹ƒ–Ž›ˆ‘—†ƒ––Š‡–‘’‘”„‘––‘‘ˆ–Š‡Ž‹•–‘ˆ‰‡‡• ”ƒ‡† ƒ ‘”†‹‰ –‘ –Š‡ –Ǧ˜ƒŽ—‡ ‘””‡•’‘†‹‰ –‘ –Š‡‹” †‹ˆˆ‡”‡–‹ƒŽ ‡š’”‡••‹‘Ǥ ‡ ƒ’’Ž‹‡†.  ™‹–Š ƒ ˆ‘ —• ‘ ‡–ƒ„‘Ž‹•Ǥ ‘ –Š‹• ‡† ™‡ —•‡† ƒ ‡–ƒ„‘Ž‹  ‰‡‡Ǧ•‡– ‘ŽŽ‡ –‹‘ ‘•‹•–‹‰ ‘ˆ –Š‡ .  ȋ›‘–‘  › Ž‘’‡†‹ƒ ‘ˆ ‡‡• ƒ† ‡‘‡•Ȍ ‡–ƒ„‘Ž‹  ‰‡‡ •‡– ‘ŽŽ‡ –‹‘ ‘’Ž‡‡–‡†™‹–Š•‡–• ‘†‹‰ˆ‘”‡œ›‡•–Šƒ–Šƒ˜‡‘ ‘”ȋȌȋ Ȍƒ•ƒ •—„•–”ƒ–‡‘”’”‘†— –ǡ”‡•’‡ –‹˜‡Ž›ȋ—’’Ž‡‡–ƒ”›ƒ„Ž‡͵ȌǤ

(30) –Š‡Ž‘™Ǧˆƒ–ˆƒ•–‡†ƒ†Š‹‰ŠǦ ˆƒ–ˆ‡† ‘†‹–‹‘‘‡–ƒ„‘Ž‹ ‰‡‡•‡–™ƒ•—’Ǧ‘”†‘™”‡‰—Žƒ–‡† ‘’ƒ”‡†–‘™‹–Šƒ ˆƒŽ•‡†‹• ‘˜‡”›”ƒ–‡ȋ Ȍ‘ˆδͲǤʹͷǤ

(31) –Š‡Ž‘™Ǧˆƒ–ˆ‡† ‘†‹–‹‘ʹ‰‡‡•‡–•™‡”‡—’”‡‰—Žƒ–‡† ƒ† ͳͲ ™‡”‡ †‘™”‡‰—Žƒ–‡† ȋƒ„Ž‡ ͳȌǤ

(32)  –Š‡ Š‹‰ŠǦˆƒ– ˆƒ•–‡† ‘†‹–‹‘ ͺ ‰‡‡ •‡–• ™‡”‡ †‘™”‡‰—Žƒ–‡†Ǥ

(33)  –Š‡ Ž‘™Ǧˆƒ– ˆ‡† ‘†‹–‹‘ǡ –Š‡ Dz›”—˜ƒ–‡ ‡–ƒ„‘Ž‹•dz ƒ† Dz ƒ––›Ǧƒ ‹† „‹‘•›–Š‡•‹•dz ‰‡‡ •‡–• ™‡”‡ •‹‰‹ˆ‹ ƒ–Ž› †‘™”‡‰—Žƒ–‡† ȋ δͲǤͲͷǡ ƒ„Ž‡ ͳȌǤ Š‡ ‘–Š‡” ‰‡‡ •‡–• ™‹–Š ƒ  ‘ˆ δͲǤʹͷ •—‰‰‡•– ƒ ’‘–‡–‹ƒŽ †‘™”‡‰—Žƒ–‹‘ ‘ˆ Ž‹’‹†Ǧ”‡Žƒ–‡† ‡–ƒ„‘Ž‹•ȋ•‡‡—†‡”Ž‹‡†‰‡‡•‡–•‹ƒ„Ž‡ͳȌǤ‘‘‹‰‹–‘–Š‡•‡‰‡‡•‡–•ǡ™‡ˆ‘—†–Šƒ– ‹ ͸ ‘—– ‘ˆ ͳʹ ‰‡‡ •‡–•ǡ –Š‡ ‰‡‡• ‹˜‘Ž˜‡† ‹ ‘ ƒ† ȋȌȋ Ȍ ‡–ƒ„‘Ž‹• ™‡”‡ ‘˜‡””‡’”‡•‡–‡†ƒ‘‰–Š‡†‹ˆˆ‡”‡–‹ƒŽŽ›”‡‰—Žƒ–‡†‰‡‡•ǡƒ•“—ƒ–‹ˆ‹‡†„›‹•ƒ–‹‘•ȋƒ„Ž‡ ͳƒ†—’’Ž‡‡–ƒ”›–ƒ„Ž‡ͶǦͷȌǤ  Table 1. Up- and downregulated metabolic gene sets in KO compared to WT based on the GSEA method Upregulated gene sets    LFD Fed   ‡–ƒ„‘Ž‹•‘ˆš‡‘„‹‘–‹ •„› ›–‘ Š”‘‡ͶͷͲ ȗ   ƒ—”‹‡ƒ†Š›’‘–ƒ—”‹‡‡–ƒ„‘Ž‹• ȗ      Downregulated gene sets    LFD Fed HFD Fasted Pyruvate metabolism Metabolism of xenobiotics by cytochrome Fatty acid biosynthesis Drug metabolism - cytochrome p450 Sulfur metabolism Glutathione metabolism ‹•–‹†‹‡‡–ƒ„‘Ž‹•  ȋȌȋ Ȍƒ‹  ȗ –‡”‘‹†Š‘”‘‡„‹‘•›–Š‡•‹• ȗ ›–Š‡•‹•ƒ††‡‰”ƒ†ƒ–‹‘‘ˆ‡–‘‡„‘†‹‡• –Š‡”–›’‡•‘ˆ‘Ǧ‰Ž› ƒ„‹‘•›–Š‡•‹•  ȋȌȋ Ȍ”‡†‘š  ‹‘•›–Š‡•‹•‘ˆ—•ƒ–—”ƒ–‡†ˆƒ––›ƒ ‹†• ȗ š‹†ƒ–‹˜‡’Š‘•’Š‘”›Žƒ–‹‘ ȗ ƒ––›ƒ ‹†‡–ƒ„‘Ž‹• ȗ ƒ—”‹‡ƒ†Š›’‘–ƒ—”‹‡‡–ƒ„‘Ž‹•  ‹–”ƒ–‡ › Ž‡ȋ › Ž‡Ȍ ȗ . Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•ǦŽƒ –‘ƒ†‡‘Žƒ –‘  ‘™”‡‰—Žƒ–‡† ƒ† —’”‡‰—Žƒ–‡† ‰‡‡ •‡–• ‘’”‹•‡ ‰‡‡ •‡–• –Šƒ– ƒ”‡ †‘™”‡‰—Žƒ–‡† ƒ† —’”‡‰—Žƒ–‡†ǡ ”‡•’‡ –‹˜‡Ž›ǡ‹ ‘’ƒ”‡†–‘Ǥ ‡‡•‡–•†‡’‹ –‡†Š‡”‡Šƒ˜‡ƒ ‘ˆδͲǤʹͷȋ‡š’Ž‘”ƒ–‹˜‡Ȍƒ†‰‡‡ •‡–•‹„‘Ž†Šƒ˜‡ƒ ‘ˆδͲǤͲͷȋ•–ƒ–‹•–‹ ƒŽŽ›•‹‰‹ˆ‹ ƒ–ȌǤȗǣ‰‡‡•‡–•‹™Š‹ Š‘ƒ†ȋȌȋ Ȍ‰‡‡• ƒ”‡‘˜‡””‡’”‡•‡–‡†‹–Š‡Ž‹•–‘ˆ ‘–”‹„—–‹‰‰‡‡•Ǥ †‡”Ž‹‡†ƒ”‡Ž‹’‹†Ǧ‡–ƒ„‘Ž‹•Ǧ”‡Žƒ–‡†‰‡‡•‡–•Ǥ Š‡‡šƒ –”‹•”ƒ–‹‘•ƒ†–Š‡Ž‹•–‘ˆ ‘–”‹„—–‹‰‰‡‡•ƒ”‡‰‹˜‡‹͹ǦͻǤ. . ͹ͷ. 3.

(34) Chapter 3

(35)  –Š‡ Š‹‰ŠǦˆƒ– ˆƒ•–‡† ‘†‹–‹‘ǡ ‡–ƒ„‘Ž‹• ‘ˆ š‡‘„‹‘–‹ • „› ›–‘ Š”‘‡ ’ͶͷͲǡ †”—‰ ‡–ƒ„‘Ž‹• Ȃ ›–‘ Š”‘‡ ’ͶͷͲǡ ƒ† ‰Ž—–ƒ–Š‹‘‡ ‡–ƒ„‘Ž‹• ™‡”‡ •‹‰‹ˆ‹ ƒ–Ž› †‘™”‡‰—Žƒ–‡†ȋ δͲǤͲͷȌǤ‘–ƒ„Ž›ǡ–Š‡•‡–Š”‡‡‰‡‡•‡–•ƒ”‡ƒŽŽ”‡Žƒ–‡†–‘†‡–‘š‹ˆ‹ ƒ–‹‘Ǥ

(36)  –Š‡ ™‹†‡” ‘ŽŽ‡ –‹‘ ‘ˆ †‘™”‡‰—Žƒ–‡† ‰‡‡ •‡–• ȋ δͲǤʹͷȌ ™‡ ƒŽ•‘ ˆ‘—† –Šƒ– ‘ ƒ† ȋȌ  ”‡Žƒ–‡† ‰‡‡• ™‡”‡ •—„•–ƒ–‹ƒŽŽ› ‘˜‡””‡’”‡•‡–‡† ƒ‘‰ –Š‡ ‰‡‡• –Šƒ– ‘–”‹„—–‡†–‘–Š‡†‘™”‡‰—Žƒ–‡†‰‡‡•‡–•Ǥȋƒ„Ž‡ͳƒ†—’’Ž‡‡–ƒ”›–ƒ„Ž‡͸ȌǤ  ‡ƒŽ•‘’‡”ˆ‘”‡† ‘–Š‡‹ ”‘ƒ””ƒ›†ƒ–ƒ•‡–‘ˆˆ‡†ƒ†ͳʹǦŠ”Ǧˆƒ•–‡†Ǧƒ† ‹ ‡ˆ”‘ ‡””‡ƒet al.ȏʹ͹Ȑǡƒ•†‡• ”‹„‡†‹ƒ–‡”‹ƒŽ•ƒ†‡–Š‘†•Ǥ ‘—”‰‡‡•‡–•™‡”‡ —’”‡‰—Žƒ–‡† ƒ† ͺ ™‡”‡ †‘™”‡‰—Žƒ–‡†ǡ ƒŽŽ ™‹–Š ƒ  „‡–™‡‡ ͲǤͲͷ ƒ† ͲǤʹͷ ȋ—’’Ž‡‡–ƒ”›–ƒ„Ž‡͹ȌǤŠ‡”‡•—Ž–• ‘ˆ‹”–Š‡‰‡‡•‡–‡”‹ Š‡–•‹Ž‹’‹†‡–ƒ„‘Ž‹• ƒ†’›”—˜ƒ–‡‡–ƒ„‘Ž‹•ˆ‘—†‹‘—”•–—†›Ǥ‘‘‹‰‹–‘–Š‡ͳʹ‰‡‡•‡–•ǡ™‡ˆ‘—†–Šƒ–‹ Ͷ‘—–‘ˆ͹‰‡‡•‡–•–Šƒ– ‘–ƒ‹‘ƒ†Ȁ‘”ȋȌȋ Ȍ‰‡‡•ȋ‘–Š‡”–Šƒ–Š‡‘‰‡‡•‡– ‹–•‡ŽˆȌǡ–Š‡‰‡‡•‹˜‘Ž˜‡†‹‘ȀȋȌȋ Ȍ‡–ƒ„‘Ž‹•™‡”‡‘˜‡””‡’”‡•‡–‡†ƒ‘‰–Š‡ †‹ˆˆ‡”‡–‹ƒŽŽ›”‡‰—Žƒ–‡†‰‡‡•ȋ—’’Ž‡‡–ƒ”›–ƒ„Ž‡ͺǡͻȌǤ

(37) •—ƒ”›ǡ •—‰‰‡•–‡†–Šƒ– –Š‡”‡™ƒ•ƒŽ‹‹–‡†‡–ƒ„‘Ž‹  ‘’‡•ƒ–‹‘ˆ‘”–Š‡Ž‘••‘ˆƒ –‹˜‹–›Ǥ ‘™‡˜‡”ǡ‘  ƒ†ȋȌȋ Ȍ†‡’‡†‡–”‡ƒ –‹‘•™‡”‡‘˜‡””‡’”‡•‡–‡†Ǥ  Alternative gene set enrichment analysis reveals a role for CoA and NAD(P)(H) metabolism Š‡•‡ƒ” Š‹‰ˆ‘”‡ Šƒ‹••–Šƒ– ‘’‡•ƒ–‡ˆ‘”–Š‡Ž‘••‘ˆƒ –‹˜‹–›—†‡”—Ž–‹’Ž‡ ‘†‹–‹‘•ǡ •–ƒ†ƒ”†  Šƒ• ƒ Ž‹‹–ƒ–‹‘Ǥ

(38) – ‘Ž› ‹†‡–‹ˆ‹‡• ‰‡‡ •‡–• –Šƒ– ‡‹–Š‡” ‘–ƒ‹ ’”‡†‘‹ƒ–Ž› —’”‡‰—Žƒ–‡† ‘” ’”‡†‘‹ƒ–Ž› †‘™”‡‰—Žƒ–‡† ‰‡‡•ǡ ™Š‡”‡ƒ• ƒ ‹š–—”‡ ‘ˆ —’Ǧ ƒ† †‘™”‡‰—Žƒ–‡† ‰‡‡• ‹ ƒ •‹‰Ž‡ ‰‡‡ •‡– ƒ› Šƒ˜‡ ƒ •–”‘‰ ‡ˆˆ‡ – ‘ ‡–ƒ„‘Ž‹–‡ ‘ ‡–”ƒ–‹‘•ǤŠ‡”‡ˆ‘”‡ǡ™‡†‡•‹‰‡†ƒƒŽ–‡”ƒ–‹˜‡‡–Š‘†–‘ƒŽ•‘ ‹†‡–‹ˆ›‰‡‡•‡–•™Š‹ Š ƒ”‡ •—„•–ƒ–‹ƒŽŽ› ȋ‘˜‡”Ȍ”‡’”‡•‡–‡† ‹ „‘–Š †‹”‡ –‹‘•Ǥ

(39)  ƒ††‹–‹‘ǡ ™‡ •—„•‡“—‡–Ž› ƒŽ•‘ ‹†‡–‹ˆ‹‡†‰‡‡•‡–•–Šƒ–ƒ”‡ȋ‘˜‡”Ȍ”‡’”‡•‡–‡†—†‡”‘”‡–Šƒ‘‡ ‘†‹–‹‘Ǥ

(40) •Š‘”–ǡ™‡ ”‡ƒ–‡†ƒ•‡’ƒ”ƒ–‡Ž‹•–‘ˆ—’”‡‰—Žƒ–‡†ƒ†ƒ •‡’ƒ”ƒ–‡Ž‹•–‘ˆ†‘™”‡‰—Žƒ–‡†‰‡‡•ǡˆ‘”‡ƒ Š ‘†‹–‹‘ȋ˜• ‘’ƒ”‹•‘Ȍȋ—’’Ž‡‡–ƒ”›–ƒ„Ž‡ͳͲȌǤ‡–Š‡”ƒ‡†‰‡‡•‡–•‘ –Š‡„ƒ•‹•‘ˆ–Š‡‹”ȋ‘˜‡”Ȍ”‡’”‡•‡–ƒ–‹‘‹–Š‡•‡Ž‹•–•Ǥ—„•‡“—‡–Ž›ǡ–Š‡‰‡‡•‡–•™‡”‡”ƒ‡† „ƒ•‡† ‘ –Š‡‹” ‡†‹ƒ ”ƒ‹‰ ‘˜‡” ƒŽŽ ‘†‹–‹‘• ȋ•‡‡ ‡–Š‘†• ˆ‘” †‡–ƒ‹Ž• ƒ† ƒ„Ž‡ ʹǡ —’’Ž‡‡–ƒ”›–ƒ„Ž‡•ͳͳƒ†ͳʹˆ‘””‡•—Ž–•ȌǤŠ‡–‘’ͳͲ‰‡‡•‡–•ˆ‘”‡ƒ Š†‹”‡ –‹‘ƒ”‡ •Š‘™‹ƒ„Ž‡ʹǤƒ”–‹ —Žƒ”Ž›ȋȌȋ ȌǦƒ†‘‰‡‡•‡–•™‡”‡‹†‡–‹ˆ‹‡†ƒ––Š‡–‘’‘ˆ–Š‡ Ž‹•– ™‹–Š ƒ ‘˜‡””‡’”‡•‡–ƒ–‹‘ ‹ „‘–Š —’Ǧ ƒ† †‘™”‡‰—Žƒ–‡† ‰‡‡•Ǥ ‘”‡‘˜‡”ǡ ’—”‹‡ ‡–ƒ„‘Ž‹•ƒ’’‡ƒ”‡†‹–Š‡–‘’”ƒ‹‰ǡ‘˜‡””‡’”‡•‡–‡†‹—’Ǧƒ††‘™”‡‰—Žƒ–‡†‰‡‡• Ž‹•–•Ǥ‹ ‡–Š‡ ‘‡œ›‡•‘ ƒ†ȋȌΪ ‘–ƒ‹ƒ’—”‹‡„—‹Ž†‹‰„Ž‘ ǡ–Š‡•‡–Š”‡‡ ‰‡‡•‡–•†‹”‡ –Ž›”‡Žƒ–‡–‘‡ƒ Š‘–Š‡”ǤŠ‡Ž‘™‡”Ǧ”ƒ‡†‰‡‡•‡–•’”‡†‘‹ƒ–Ž›•—‰‰‡•– Šƒ‰‡• ‹ Ž‹’‹† ‡–ƒ„‘Ž‹• ȋ—†‡”Ž‹‡† ‰‡‡ •‡–• ‹ ƒ„Ž‡ ʹȌǤ ‘‰‡–Š‡” ™‹–Š ’›”—˜ƒ–‡ ‡–ƒ„‘Ž‹•͸‘ˆ–Š‡•‡͹Ž‹’‹†‡–ƒ„‘Ž‹•‰‡‡•‡–•„‡Ž‘‰–‘–Š‡–‘’ͷͲΨ‘ˆ‰‡‡•‡–•™‹–Š –Š‡ Š‹‰Š‡•– ’‡” ‡–ƒ‰‡ ‘ˆ ‰‡‡• ‘†‹‰ ˆ‘” ‡œ›‡• –Šƒ– ƒ–ƒŽ›œ‡ ‘  ‘” ȋȌȋ Ȍ ‘–ƒ‹‹‰”‡ƒ –‹‘•ȋ—’’Ž‡‡–ƒ”›ƒ„Ž‡ͳ͵ȌǤŠ—•ǡ–Š‡”‡•—Ž–•‘ˆ–Š‡ƒŽ–‡”ƒ–‹˜‡‰‡‡ •‡– ‡”‹ Š‡– ƒƒŽ›•‹• ‘””‘„‘”ƒ–‡ ‘—” ‘ Ž—•‹‘ –Šƒ– ”‡ƒ –‹‘• ‹˜‘Ž˜‡† ‹ ‘Ǧ ƒ† ȋȌȋ Ȍ ‡–ƒ„‘Ž‹• ƒ”‡ ƒ”‡†Ž› ‘˜‡””‡’”‡•‡–‡† ƒ‘‰ –Š‡ †‹ˆˆ‡”‡–‹ƒŽŽ› ‡š’”‡••‡† ‰‡‡•‹–Š‡Ǧ˜‡”•—•–Š‡ǡ‹„‘–Š†‹”‡ –‹‘•Ǥ . ͹͸.

(41) Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice Table 2. Up- and downregulated metabolic gene sets in KO compared to WT based on the Alternative GSEA method Upregulated gene sets Downregulated gene sets ȋȌȋ Ȍƒ‹ ȗǡș NAD(P)(H) main ȗǡș ȋȌȋ Ȍ”‡†‘š ȗǡș NAD(P)(H) redox ȗǡș —”‹‡‡–ƒ„‘Ž‹• ȗǡș ‘‰‡‡•‡– ȗǡș ‘‰‡‡•‡– ȗǡș ”ƒ Š‹†‘‹ ƒ ‹†‡–ƒ„‘Ž‹• ȗǡș ‡–‹‘Ž‡–ƒ„‘Ž‹• ȗ —”‹‡‡–ƒ„‘Ž‹• ȗǡș –‡”‘‹†Š‘”‘‡„‹‘•›–Š‡•‹• ȗ Pyruvate metabolism ȗǡș ȗ

(42) ‘•‹–‘Ž’Š‘•’Šƒ–‡‡–ƒ„‘Ž‹• ȗ. Ž› ‡”‘Ž‹’‹†‡–ƒ„‘Ž‹• ”— –‘•‡ƒ†ƒ‘•‡‡–ƒ„‘Ž‹• ȗ. Ž› ‡”‘’Š‘•’Š‘Ž‹’‹†‡–ƒ„‘Ž‹• ȗǡș. ƒŽƒ –‘•‡‡–ƒ„‘Ž‹• ȗ Žƒ‹‡ǡƒ•’ƒ”–ƒ–‡ƒ†‰Ž—–ƒƒ–‡‡–ƒ„‘Ž‹• ȗ ȗǡș ›”‹‹†‹‡‡–ƒ„‘Ž‹• ȗ Fatty acid metabolism ‡’‹ –‡†Š‡”‡ƒ”‡–Š‡–‘’ͳͲ‰‡‡•‡–•™‹–Š–Š‡‘•–ƒ‘—–‘ˆ†‹ˆˆ‡”‡–‹ƒŽŽ›Ǧ‡š’”‡••‡†‰‡‡•ƒ ‘”†‹‰–‘ –Š‡DzȋȁŽ‘‰ʹȋˆ‘Ž† Šƒ‰‡ȌȁεͲǤͷ’δͲǤͳȌ’δͲǤͲͷdz ”‹–‡”‹ƒǤȗǣƒ‘—–‘ˆ†‹ˆˆ‡”‡–‹ƒŽŽ›‡š’”‡••‡†‰‡‡• ‘ˆ–Š‡‰‹˜‡’ƒ–Š™ƒ›‹•‘˜‡””‡’”‡•‡–‡†‹ʹ‘”‘”‡ ‘†‹–‹‘•‹–Š‡‰‹˜‡†‹”‡ –‹‘Ǥșǣ˜‡”Žƒ’™‹–Š–Š‡ –‘’ͳͷ‘ˆ–Š‡†ƒ–ƒ•‡–‘ˆ ‡””‡ƒ‡–ƒŽǤȏʹ͹ȐǤ‘Ž†ǣ˜‡”Žƒ’™‹–Š ”‡•—Ž–•ˆ‘”–Š‡‡–ƒ„‘Ž‹ ‰‡‡•‡–•™‹–Š δʹͷΨǤ‘Ž†ƒ†‹–ƒŽ‹ ǣ˜‡”Žƒ’™‹–Š ”‡•—Ž–•ˆ‘”–Š‡‡–ƒ„‘Ž‹ ‰‡‡•‡–•™‹–Š δͷΨǤ†‡”Ž‹‡† ƒ”‡Ž‹’‹†Ǧ‡–ƒ„‘Ž‹•Ǧ”‡Žƒ–‡†‰‡‡•‡–•Ǥ††‹–‹‘ƒŽ”‡•—Ž–•ƒ”‡‰‹˜‡‹—’’Ž‡‡–ƒ”›–ƒ„Ž‡•ͳͲǦͳʹǤ.  The example of Acot expression between MCAD-KO and WT mice ‘‰–Š‡†‹ˆˆ‡”‡–‹ƒŽŽ›‡š’”‡••‡†‘Ǧ”‡Žƒ–‡†‰‡‡•ǡ–Š‡ƒ ›ŽǦ‘–Š‹‘‡•–‡”ƒ•‡•ȋ ‘–•Ȍƒ”‡ ‘™–‘•–‹—Žƒ–‡ ȏ͵ͳȂ͵ͶȐƒ†–Š‡”‡ˆ‘”‡’”‘˜‹†‡ƒŽ‘‰‹ ƒŽŽ‹–‘ †‡ˆ‡ –•ǡ•— Š ƒ•ǤŠ‡”‡ˆ‘”‡ǡ™‡‡šƒ‹‡†–Š‡‡š’”‡••‹‘‘ˆƒŽŽͳͷAcot‰‡‡•ȏ͵ʹǡ͵͵Ȑǡˆ‘”–Š‡Š‹‰ŠǦ ˆƒ–ƒ†Ž‘™Ǧˆƒ– ‘†‹–‹‘•ǤŠ‡‡š’”‡••‹‘‘ˆ•‡˜‡”ƒŽ‘ˆ–Š‡•‡‰‡‡•™ƒ•Š‹‰Š‡”—†‡”ˆƒ•–‡† ‘’ƒ”‡†–‘ˆ‡† ‘†‹–‹‘•ȋ—’’Ž‡‡–ƒ”› ‹‰—”‡ͻȌǡ‹ƒ ‘”†ƒ ‡™‹–Š’”‡˜‹‘—•”‡•—Ž–• ȏ͵ʹȐǤ Š‹•‹• ‘’ƒ–‹„Ž‡ ™‹–Š ƒ ”‘Ž‡ ‘ˆ –Š‡  ‡œ›‡• –‘ •–‹—Žƒ–‡ ˆƒ––›Ǧƒ ‹† ‘š‹†ƒ–‹‘ †—”‹‰ˆƒ•–‹‰Ǥ‘”‡‹–‡”‡•–‹‰Ž›ǡ–Š‡‘•–—’”‡‰—Žƒ–‡†‹–‘ Š‘†”‹ƒŽAcot‰‡‡‹–Š‡Ž‹˜‡” †—”‹‰ˆƒ•–‹‰ǡAcot2,™ƒ•‡˜‡ˆ—”–Š‡”—’”‡‰—Žƒ–‡†‹–Š‡Ǧ ‘’ƒ”‡†–‘‘—•‡ Ž‹˜‡”‹–Š‡Ž‘™Ǧˆƒ–ˆƒ•–‡†•–ƒ–‡ȋ‡“ ‘ˆͳǤͺƒ†’δͲǤͲͷǢ ‹‰—”‡͸Ȍƒ†™ƒ•ƒŽ•‘–Š‡ ‘Ž› ‰‡‡ –Šƒ– ™ƒ• †‹ˆˆ‡”‡–‹ƒŽŽ› ‡š’”‡••‡† „‡–™‡‡  ƒ†  ‹ ƒ› ‘ˆ –Š‡ ˆƒ•–‡† ‘†‹–‹‘•ǤŠ‹•Š‹‰Š‡”‡š’”‡••‹‘™ƒ•ƒŽ•‘ ‘ˆ‹”‡†„›“—ƒ–‹–ƒ–‹˜‡”‡ƒŽǦ–‹‡ȋ ‘ˆ ʹǤͲƒ†’αͲǤͲͷǢ ‹‰—”‡͸ȌǤ A. B. mRNA expression of Acot2 p=0.05. 2.5. Normalized expression. Normalized read count. mRNA expression of Acot2 p=0.02 6. 4. 2. 2.0 1.5 1.0 0.5 0.0. 0 WT. KO. WT. KO. . Figure 6. Acot2 gene expression. Acot2‰‡‡‡š’”‡••‹‘‹–Š‡ ˆƒ•–‡† ‘†‹–‹‘„›‡“ȋAȌƒ†‹  ˆƒ•–‡† ‘†‹–‹‘ „›”‡ƒŽǦ–‹‡ȋBȌǤ Š‡‡“”‡•—Ž–•ƒ”‡ ‡š’”‡••‡†ƒ•‘”ƒŽ‹œ‡† ”‡ƒ† ‘—– ”‡Žƒ–‹˜‡–Š‡‡†‹ƒ‘”ƒŽ‹œ‡†”‡ƒ† ‘—–‘ˆ–Š‡‹ ‡ƒ†ƒ”‡”‡’”‡•‡–‡†ƒ•—‡›„‘šƒ†™Š‹•‡” ’Ž‘–•ȋαͶȌǤŠ‡”‡ƒŽǦ–‹‡”‡•—Ž–•ƒ”‡‡š’”‡••‡†ƒ•Acot2‡š’”‡••‹‘”‡Žƒ–‹˜‡–‘–Š‡ƒ˜‡”ƒ‰‡‡š’”‡••‹‘ ‹–Š‡‹ ‡ƒ†ƒ”‡”‡’”‡•‡–‡†ƒ•„ƒ”’Ž‘–ȋ‡ƒάȌȋαͺȌǤ. ͹͹. 3.

(43) Chapter 3 Š‡•‡”‡•—Ž–••—‰‰‡•––Šƒ–‡š–”ƒ•–‹—Žƒ–‹‘‘ˆ–Š‡ „›–Š‡Acot2†—”‹‰ˆƒ•–‹‰ ƒ› ‘’‡•ƒ–‡ˆ‘”–Š‡Ž‹‹–‡†  ƒ’ƒ ‹–›‹–Š‡Ǧ‘—•‡Ǥ. Discussion

(44)  –Š‹• •–—†›ǡ ™‡ ƒ‹‡† –‘ ‡š’Ž‘”‡ –Š‡ ”‘Ž‡ ‘ˆ –Š‡ Ž‹˜‡” ‹ –Š‡  ’Š‡‘–›’‡ ‹ –Š‡ ͷ͹Ȁ͸  Ǧ ‘—•‡ ‘†‡ŽǤ ‘” –Š‹•ǡ ™‡ ‰‡‡”ƒ–‡† ƒ† ƒƒŽ›œ‡† ƒ ‘’”‡Š‡•‹˜‡ ’Š›•‹‘Ž‘‰‹ ƒŽǡŠ‹•–‘Ž‘‰‹ ƒŽƒ†‘Ž‡ —Žƒ”†ƒ–ƒ•‡–ǤŠ‡ ‘’Ž‡–‡Ž‘••‘ˆ’”‘–‡‹Ž‡†–‘ƒ ͵ͲǦͶͲΨ”‡†— –‹‘‘ˆ–Š‡‹–‘ Š‘†”‹ƒŽ ƒ’ƒ ‹–›ˆ‘”‘š‹†ƒ–‹‘‘ˆ–Š‡‡†‹—Ǧ Šƒ‹•—„•–”ƒ–‡ ‘ –ƒ‘›ŽǦ‘Ǥ

(45) ’”‹ ‹’Ž‡ǡ ƒ ƒ””›–Š‡”‡•‹†—ƒŽˆŽ—š‹‹ ‡ǡ„—–‹–Šƒ•ƒŽ‘™‡” ƒ–ƒŽ›–‹  ƒ’ƒ ‹–› ƒ† ƒˆˆ‹‹–› ˆ‘” ‡†‹—Ǧ Šƒ‹ •—„•–”ƒ–‡•ȏʹͺȐǤ Š‡ ‘ ‡–”ƒ–‹‘ ‘ˆ  ƒ† ‹–• •Š‘”–Ǧ Šƒ‹‹•‘‡œ›‡™ƒ•‘–ƒŽ–‡”‡†‹–Š‡Ǧ‹ ‡Ǥ‘‰‡–Š‡”ǡ–Š‡•‡”‡•—Ž–• •Š‘™ –Šƒ–  ƒ ‘Ž› ‘’‡•ƒ–‡ ’ƒ”–Ž› ˆ‘” –Š‡ Ž‘•• ‘ˆ Ǥ ‡˜‡”–Š‡Ž‡••ǡ ƒ• ƒŽ•‘ ’”‡˜‹‘—•Ž›”‡’‘”–‡†ǡ–Š‡‰Ž— ‘•‡Ž‡˜‡Ž•ƒ†Ž‹˜‡”™‡‹‰Š–™‡”‡•‹‹Žƒ”„‡–™‡‡ƒ†Ǧ ‹ ‡ȏʹͲǡʹ͸ǡʹ͹ȐǤ–”‡†–‘™ƒ”†•ƒŠ‡’ƒ–‹ Š‹•–‘’ƒ–Š‘Ž‘‰‹ ƒŽ’Š‡‘–›’‡™ƒ•‘„•‡”˜‡†‹ –Š‡Ǧ‹ ‡‹–Š‡Š‹‰ŠǦˆƒ–ˆƒ•–‡† ‘†‹–‹‘ǡ‹ƒ‰”‡‡‡–™‹–Š‡ƒ”Ž‹‡””‡•—Ž–•ȏʹ͸ȐǤŠ‹• •—‰‰‡•–•Š‡’ƒ–‘–‘š‹ ‹–›ƒ•ƒ”‡•—Ž–‘ˆ’”‡˜‹‘—•Ž›‘„•‡”˜‡†‡†‹—Ǧ Šƒ‹Ž‹’‹†ƒ ——Žƒ–‹‘ ‹ Ǧ ‹ ‡ ȏʹ͸ȂʹͺȐǤ ‡†‹—Ǧ Šƒ‹ Ž‹’‹†• Šƒ˜‡ „‡‡ ”‡’‘”–‡† –‘ ƒ—•‡ ‘š‹†ƒ–‹˜‡ †ƒƒ‰‡ȏ͵ͷȂͶͲȐƒ†Š‡’ƒ–‹ •–‡ƒ–‘•‹•ȏͶͳȐǡƒ†–‘‹’ƒ‹”†‡–‘š‹ˆ‹ ƒ–‹‘‡ Šƒ‹••ȏͶʹǡͶ͵ȐǤ

(46) –‡”‡•–‹‰Ž›ǡ –Š‡ –”ƒ• ”‹’–‘‹  †ƒ–ƒ •—‰‰‡•– ‹’ƒ‹”‡† †‡–‘š‹ˆ‹ ƒ–‹‘ ‡ Šƒ‹•• ‹ –Š‡ Ǧ‘—•‡Ǥ

(47) ˆ–Š‹•ƒŽ•‘ƒ’’Ž‹‡•–‘Š—ƒ•ǡ‹–ƒ›„‡†‡–”‹‡–ƒŽ–‘’ƒ–‹‡–•ǡ •‹ ‡Š—ƒ•‹‰‡‡”ƒŽƒŽ”‡ƒ†›Šƒ˜‡Ž‡••Ͷƒ†Ͷ ‰‡‡•–Šƒ‹ ‡Ǥ

(48) Ž‹‡™‹–Š –Š‹•ǡ‘š‹†ƒ–‹˜‡†ƒƒ‰‡–‘Ž‹’‹†•ƒ†’”‘–‡‹•ƒ††‡ ”‡ƒ•‡†ƒ–‹‘š‹†ƒ– ƒ’ƒ ‹–›™ƒ•ˆ‘—†‹ Ǧ†‡ˆ‹ ‹‡–’ƒ–‹‡–•ȏͶͶȐǤ Žƒ••‹ ƒŽƒ†ƒŽ–‡”ƒ–‹˜‡ ‘ˆ–Š‡–”ƒ• ”‹’–‘‹ •†ƒ–ƒ”‡˜‡ƒŽ‡†‘ ƒ†ȋȌȋ Ȍƒ• ‘‘†‡‘‹ƒ–‘”•†‹•–‹‰—‹•Š‹‰Ǧƒ†‹ ‡Ǥ‘–Š‘ ƒ†ȋȌȋ Ȍ Ž‹   –‘ ‘–Š‡” ’ƒ–Š™ƒ›• ‹ ‡–ƒ„‘Ž‹•Ǥ ‘–ƒ„Ž›ǡ ‘’—–ƒ–‹‘ƒŽ ‘†‡ŽŽ‹‰ ‘ˆ Š‡’ƒ–‹    ”‡˜‡ƒŽ‡† ƒ ’‘–‡–‹ƒŽ ˜‹ ‹‘—• › Ž‡ ‘ˆ ‘ ‡•–‡” ƒ ——Žƒ–‹‘ǡ ‘  †‡’Ž‡–‹‘ǡ ƒ†  ˆŽ—š†‡ Ž‹‡ǡ™Š‹ Š™ƒ•ƒ‰‰”ƒ˜ƒ–‡†„›Ž‘••‘ˆȏͶͷȐǤ

(49) –Š‡—†‡”Ž›‹‰‡ Šƒ‹• –Š‡‘ Ǧ ‘•—‹‰‡œ›‡‡†‹—Ǧ Šƒ‹‡–‘ƒ ›ŽǦ‘–Š‹‘Žƒ•‡ȋȌƒ†–Š‡ΪǦ ”‡†— ‹‰ ‡œ›‡ ‡†‹—Ȁ•Š‘”–Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋȀ Ȍ ’Žƒ›‡† ƒ ‡› ”‘Ž‡ȏͶ͸ȐǤ’”‡‰—Žƒ–‹‘‘ˆƒ†‘ˆ–Š‡‹–‘ Š‘†”‹ƒŽȏΪȐǦȏ Ȑ”ƒ–‹‘•—„•–ƒ–‹ƒŽŽ› ƒŽŽ‡˜‹ƒ–‡†–Š‹•’Š‡‘–›’‡ȏͶ͸ȐǤŠ‹•‹•‹ƒ‰”‡‡‡–™‹–Š‡š’‡”‹‡–ƒŽˆ‹†‹‰•–Šƒ–ƒ –‹˜ƒ–‹‘ ‘ˆ  „› †‘™”‡‰—Žƒ–‹‘ ‘ˆ ‹–• ‹Š‹„‹–‘” ’Ͷ͸Š  ȏͶ͹Ȑ ƒ† —’”‡‰—Žƒ–‹‘ ‘ˆ Ϊ ȏͶͺȐ •–‹—Žƒ–‡–Š‡ ˆŽ—šǤ‘‘–”ƒ• ”‹’–‹‘ƒŽ”‡‰—Žƒ–‘”›‡ Šƒ‹••ˆ‘”‰‡‡•‹˜‘Ž˜‡† ‹‘ Ǧƒ†ȋȌȋ Ȍ‡–ƒ„‘Ž‹•ƒ”‡•–‹ŽŽ—‘™ǡƒ•ˆƒ”ƒ•™‡ƒ”‡ƒ™ƒ”‡Ǥ  Ž‹‹–ƒ–‹‘ ‘ˆ –Š‹• •–—†› ‹•–Šƒ– „‹‘ Š‡‹ ƒŽ ‡˜‹†‡ ‡ ˆ‘” ƒ ”‘Ž‡ ‘ˆ ‘  ƒ†ȋȌȋ Ȍ ‡–ƒ„‘Ž‹• ‹ –Š‡ ƒ†ƒ’–ƒ–‹‘ –‘ Ž‘•• ‘ˆ  ™ƒ• ‘– ‡šŠƒ—•–‹˜‡Ž› ‹˜‡•–‹‰ƒ–‡†Ǥ ‡ Šƒ˜‡ Š‘™‡˜‡”ǡƒ•ƒ’”‘‘ˆ‘ˆ’”‹ ‹’Ž‡‡šƒ’Ž‡ǡ ‘ˆ‹”‡††‹ˆˆ‡”‡–‹ƒŽ‡š’”‡••‹‘‘ˆAcot2Ǥʹ ‹• ƒ ‹–‘ Š‘†”‹ƒŽ –Š‹‘‡•–‡”ƒ•‡ ȏ͵ͳǡ͵͵Ȑ –Šƒ– ’”‘†— ‡• ˆ”‡‡ ȋ‘Ǧ‡•–‡”‹ˆ‹‡†Ȍ ‘Ǥ Š‹• ƒ› •‡”˜‡ ƒ• ƒ •—„•–”ƒ–‡ ˆ‘” –Š‡   ‡œ›‡• ʹ ƒ† –Š‡ ƒ„‘˜‡‡–‹‘‡†  ƒ† –Š‡”‡„› •–‹—Žƒ–‡  ‹ –Š‡ ƒ„•‡ ‡ ‘ˆ Ǥ

(50)  ˆ—–—”‡ •–—†‹‡• ™‡‡š’‡ – –‘ •–—†› –Š‡ ”‘Ž‡•‘ˆ‘ ƒ†ȋȌȋ Ȍ‡–ƒ„‘Ž‹•‹–Š‡”‘„—•–‡••‘ˆ–Š‡Ǧ‘—•‡Ǥ  . Materials and Methods ͹ͺ.

(51) Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice Animals, Experimental design and Tissue sampling ƒŽ‡Ǧƒ†Ž‹––‡”ƒ–‡™‹Ž†Ǧ–›’‡ȋȌ‹ ‡‘ƒͷ͹Ȁ͸ „ƒ ‰”‘—†ȋ„ƒ  ”‘••‡†ˆ‘”ͳͲ ‰‡‡”ƒ–‹‘•Ȍ™‡”‡Š‘—•‡†‹ƒ–‡’‡”ƒ–—”‡ǦȋʹͳιȌƒ†Ž‹‰Š–Ǧ ‘–”‘ŽŽ‡†ȋͳʹŠ”•Ž‹‰Š–Ȍˆƒ ‹Ž‹–›ƒ†ˆ‡† ‘‡” ‹ƒŽŽ› ƒ˜ƒ‹Žƒ„Ž‡ Žƒ„‘”ƒ–‘”› Š‘™ ȋ‹‡–•ǡ ‘‡”†‡ǡ Š‡ ‡–Š‡”Žƒ†•ȌǤ ‹ ‡ —•‡† ‹ –Š‡ ‡š’‡”‹‡–ƒŽ ’”‘ ‡†—”‡• ™‡”‡ ʹǦͶ ‘–Š• ‘ˆ ƒ‰‡Ǥ ŽŽ ‡š’‡”‹‡–• ™‡”‡ ƒ’’”‘˜‡† „› –Š‡ –Š‹ • ‘‹––‡‡ˆ‘”‹ƒŽš’‡”‹‡–•‘ˆ–Š‡‹˜‡”•‹–›‘ˆ ”‘‹‰‡Ǥ ”‘–Š‡•–ƒ”–‘ˆ–Š‡‡š’‡”‹‡–ǡ ‹ ‡™‡”‡ˆ‡†ƒŽ‘™Ǧˆƒ–‘”Š‹‰ŠǦˆƒ–•‡‹Ǧ•›–Š‡–‹ †‹‡–ȋƒ•‹ͳʹͶͷͲ‘”ͳʹͶͷͳȏͶͻȐǡ‡•‡ƒ” Š‹‡– ‡”˜‹ ‡•ǡ ‹Œ ‹Œ ——”•–‡†‡ǡ Š‡ ‡–Š‡”Žƒ†•ǡ ”‡•’‡ –‹˜‡Ž›Ȍ ˆ‘” ͸ ™‡‡•ǡ ƒ† Šƒ† ˆ”‡‡ ƒ ‡•• –‘ †”‹‹‰™ƒ–‡”ǤŠ‡Š‹‰ŠǦˆƒ–†‹‡– ‘–ƒ‹‡†ͶͷΨ‘ˆ ƒŽ‘”‹‡•ˆ”‘’ƒŽ‘‹Žˆƒ–ǡ‹‘”†‡”–‘‹‹ –Š‡ ”ƒ–‹‘‘ˆ•ƒ–—”ƒ–‡†–‘‘‘—•ƒ–—”ƒ–‡†–‘’‘Ž›—•ƒ–—”ƒ–‡†ˆƒ––›ƒ ‹†•ȋͶͲǣͶͲǣʹͲȌ‹ƒŠ—ƒ†‹‡–Ǥ‹ ‡ ™‡”‡•—„•‡“—‡–Ž›‡‹–Š‡”ˆƒ•–‡†ˆ‘”ͳ͸Š”•ȋ†‡‘–‡†ƒ•Ǯˆƒ•–‡†‹ ‡ǯȌ‘”‡’–‘–Š‡†‹‡–ˆ‘”–Š‡•‡ͳ͸ Š‘—”•ȋ†‡‘–‡†ƒ•Ǯˆ‡†‹ ‡ǯȌǤŠ‡ˆ‘‘†™ƒ•”‡‘˜‡†ˆ”‘–Š‡ˆƒ•–‹‰‹ ‡„‡–™‡‡ͷƒ†͹’ǡ‹Ǥ‡ǤͲǦ ʹ Š‘—”•„‡ˆ‘”‡–Š‡Ž‹‰Š–™ƒ••™‹– Š‡†‘ˆˆǤŽ‘‘†‰Ž— ‘•‡ ‘ ‡–”ƒ–‹‘•™‡”‡‡ƒ•—”‡††—”‹‰–Š‡ ‡š’‡”‹‡–—•‹‰ƒ‡‘— ŠŽ–”ƒƒ•›̺‘‹–‘”ȋ‹ˆ‡ ƒ‡‡Ž—šǡ‡‡”•‡ǡ‡Ž‰‹—ȌǤŽŽ‹ ‡™‡”‡ –‡”‹ƒ–‡†ͳ͸Š‘—”•Žƒ–‡”„‡–™‡‡ͻƒ†ͳͳƒ„› ƒ”†‹ƒ ’— –—”‡—†‡”‹•‘ˆŽ—”ƒ‡ƒ‡•–Š‡•‹ƒǤˆ–‡” –‡”‹ƒ–‹‘ –Š‡ Ž‹˜‡” ™ƒ• ”‡‘˜‡† “—‹ Ž›ǡ ™‡‹‰Š‡†ǡ ’‹‡ ‡ ‘ˆ –Š‡ Ž‹˜‡” ˆ”‡‡œ‡Ǧ Žƒ’‡† ˆ‘” ˆ—”–Š‡” ƒƒŽ›•‹•ǡƒ’‹‡ ‡‘ˆ–Š‡Ž‹˜‡”™ƒ•‹‡”•‡†‹ͳͲΨ„—ˆˆ‡”‡†ˆ‘”ƒŽ‹ƒ†ƒ‘–Š‡”’‹‡ ‡™ƒ•ˆ‹š‡†ƒ† ˆ”‘œ‡‹‹••—‡Ǧ‡̺ˆ‹šƒ–‹˜‡Ǥ ‹ƒŽŽ›ǡƒ’‹‡ ‡‘ˆ–Š‡Ž‡ˆ–Žƒ”‰‡Ž‘„‡‘ˆ–Š‡Ž‹˜‡”™ƒ• ‘ŽŽ‡ –‡†‹ƒ„—ˆˆ‡” ‘–ƒ‹‹‰ ʹͷͲ  •— ”‘•‡ ƒ† ͳͲ  ”‹• ȋ’  ͹ǤͲȌ ƒ† —•‡† †‹”‡ –Ž› ˆ‘” –Š‡ ‹•‘Žƒ–‹‘ ‘ˆ ˆ”‡•Š ‹–‘ Š‘†”‹ƒǤ ŽŽ ‡–Š‘†• ™‡”‡ ’‡”ˆ‘”‡† ‹ ƒ ‘”†ƒ ‡ ™‹–Š –Š‡ ”‡Ž‡˜ƒ– ‰—‹†‡Ž‹‡• ƒ† ”‡‰—Žƒ–‹‘•Ǥ  Liver histology ‹‡ ‡•‘ˆŽ‹˜‡”•–Šƒ–™‡”‡ˆ‹š‡†‹ˆ‘”ƒŽ‹ǡ™‡”‡’”‘ ‡••‡†ˆ‘”’ƒ”ƒˆˆ‹•‡ –‹‘‹‰ǡƒ†•–ƒ‹‡†™‹–Š Š‡ƒ–‘š›Ž‹ ƒ† ‡‘•‹Ǥ ”‘œ‡ Ž‹˜‡” •‡ –‹‘• –Šƒ– ™‡”‡ –Š—• ˆ‹š‡† ‹ ‹••—‡Ǧ‡̺ ˆ‹šƒ–‹˜‡ ™‡”‡ ’”‡’ƒ”‡†—•‹‰•–ƒ†ƒ”†‡–Š‘†•ƒ†•‡ –‹‘•™‡”‡•–ƒ‹‡† ™‹–Š‹ŽǦ‡†ǦǤŽ‹†‡•™‡”‡‡šƒ‹‡† ™‹–Š‘—–‘™Ž‡†‰‡‘ˆ–Š‡†‹‡–ǤŠ‡•Ž‹†‡•™‡”‡ƒƒŽ›œ‡†„›„‘ƒ”†Ǧ ‡”–‹ˆ‹‡†˜‡–‡”‹ƒ”›’ƒ–Š‘Ž‘‰‹•–•ǡ ™Š‘• ‘”‡†–Š‡•Ž‹†‡•ˆ‘”‘ƒŽ ‘Š‘Ž‹ ˆƒ––›Ž‹˜‡”†‹•‡ƒ•‡ȋ Ȍƒ† ƒ –‹˜‹–›• ‘”‡ȋȌ ƒ ‘”†‹‰–‘Ž‡‹‡”‡–ƒŽǤȏͷͲȐǤ  Hepatic triglyceride (TG) concentration ”‡‡œ‡Ǧ Žƒ’‡†Ž‹˜‡” ™ƒ• ”—•Š‡† ‹ Ž‹“—‹† ‹–”‘‰‡ ƒ†ͳͷΨ ȋ™Ȁ˜ȌŠ‘‘‰‡ƒ–‡• ‹ ȋ’ ͹ǤͶȌ ™‡”‡’”‡’ƒ”‡†Ǥ‘–ƒŽŽ‹˜‡”Ž‹’‹†•™‡”‡‡š–”ƒ –‡†ƒ ‘”†‹‰–‘Ž‹‰ŠƬ›‡”ȏͷͳȐǤŠ‡‡š–”ƒ –‡†Ž‹’‹†• ™‡”‡”‡†‹••‘Ž˜‡†‹ʹΨ”‹–‘ǦͳͲͲƒ†‡ƒ•—”‡†—•‹‰ƒ ‘‡” ‹ƒŽŽ›ƒ˜ƒ‹Žƒ„Ž‡‹–ˆ‘”–”‹‰Ž› ‡”‹†‡ ȋ‘ Š‡‹ƒ‰‘•–‹ •ǡƒŠ‡‹ǡ ‡”ƒ›Ȍƒ ‘”†‹‰–‘–Š‡ƒ—ˆƒ –—”‡”ǯ•‹•–”— –‹‘•Ǥ  Oxygen consumption rates in fresh liver mitochondria ‹–‘ Š‘†”‹ƒ™‡”‡‹•‘Žƒ–‡†ˆ”‘ˆ”‡•ŠŽ‹˜‡”–‹••—‡ƒ ‘”†‹‰–‘‹Ž†ƒœ‹‡‡‡–ƒŽǤȏͷʹȐǡ™‹–Š–Š‡Žƒ•– –Š”‡‡ ‡–”‹ˆ—‰ƒ–‹‘ •–‡’• ƒ– ͺͲͲǡ ͹ʹͲͲ ƒ† ͹ʹͲͲ ‰ ‹•–‡ƒ† ‘ˆ ͹ͷͲǡ ͹ͲͲͲ ƒ† ͹ͲͲͲ ‰ǡ ”‡•’‡ –‹˜‡Ž›Ǥ ›”—˜ƒ–‡Ǧǡ ’ƒŽ‹–‘›ŽǦ‘ ȋͳ͸Ǧƒ ›ŽǦ‘ȌǦ ƒ† ‘ –ƒ‘›ŽǦ‘ ȋͺǦƒ ›ŽǦ‘ȌǦ†‡’‡†‡– ‘š›‰‡ ‘•—’–‹‘”ƒ–‡•‹–Š‡•‡‹–‘ Š‘†”‹ƒ™‡”‡‡ƒ•—”‡†ƒ–͵͹ι—•‹‰ƒ–™‘Ǧ Šƒ‡ŽŠ‹‰ŠǦ”‡•‘Ž—–‹‘ ”‘„‘”‘• ‘š›‰”ƒ’ŠǦʹ ȋ”‘„‘”‘•ǡ

(52) •„”— ǡ —•–”‹ƒȌ ™‹–Š ƒŽƒ–‡Ǧ ‘–ƒ‹‹‰ ‹Ͳͷ „—ˆˆ‡” ȋ‹Ǥ‡Ǥ ‹–‘ Š‘†”‹ƒŽ”‡•’‹”ƒ–‹‘‡†‹—‹ƒ„Ž‡ͳ‹ ƒ‹‰‡”‡–ƒŽǤȏͷ͵ȐȌǤ‘‡ƒ•—”‡–Š‡ƒš‹ƒŽǦ •–‹—Žƒ–‡†‘š›‰‡ ‘•—’–‹‘ȋ•–ƒ–‡͵ȌǡͶǤͺŽǦͳŠ‡š‘‹ƒ•‡ǡͳʹǤͷ‰Ž— ‘•‡ƒ†ͳ™ƒ• ƒ††‡†Ǥ‘‡ƒ•—”‡–Š‡”‡•–‹‰•–ƒ–‡‘š›‰‡ ‘•—’–‹‘”ƒ–‡ȋ•–ƒ–‡ͶȌǡ–”ƒ•’‘”–™ƒ•„Ž‘ ‡† ™‹–ŠͳǤʹͷɊ ƒ”„‘š›ƒ–”ƒ –›Ž‘•‹†‡Ǥ  Targeted quantitative proteomics of mitochondrial proteins. ͹ͻ. 3.

(53) Chapter 3  •‡– ‘ˆ εͷͲ ‹–‘ Š‘†”‹ƒŽ ’”‘–‡‹• ‹˜‘Ž˜‡† ‹ •—„•–”ƒ–‡ –”ƒ•’‘”–ǡ ‘š‹†ƒ–‹˜‡ ’Š‘•’Š‘”›Žƒ–‹‘ ȋ Ȍǡ‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧƒ ‹†ȾǦ‘š‹†ƒ–‹‘ǡ–”‹ ƒ”„‘š›Ž‹ ƒ ‹† › Ž‡ǡƒ†ƒ–‹‘š‹†ƒ–ƒ –‹˜‹–›™‡”‡ “—ƒ–‹ˆ‹‡† ‹ ‹•‘Žƒ–‡† ‹–‘ Š‘†”‹ƒ —•‹‰ ‹•‘–‘’‹ ƒŽŽ› Žƒ„‡ŽŽ‡† •–ƒ†ƒ”†• ȋͳ͵ǦŽƒ„‡Ž‡† Ž›•‹‡• ƒ† ƒ”‰‹‹‡•Ȍ †‡”‹˜‡† ˆ”‘ •›–Š‡–‹  ’”‘–‡‹ ‘ ƒ–‡‡”• ȋ ‘Ȍ ȋ‘Ž›—ƒ– „ ǡ ƒ† „„ƒ Šǡ. ‡”ƒ›Ȍƒ ‘”†‹‰–‘‘Ž–‡”•‡–ǤƒŽǤȏʹͻȐǤ  RNA isolation, RNASeq analysis and quantitative reverse transcriptase polymerase chain (qRT-PCR) †‡• ”‹’–‹‘‘ˆ–Š‡‹•‘Žƒ–‹‘ǡ‡“ƒƒŽ›•‹•ƒ†“Ǧ‡–Š‘†•‹•‰‹˜‡‹—’’Ž‡‡–ƒ”› –‡š–ͳǤ  Origin of mixed-background MCAD-KO mice microarray data ”‡˜‹‘—•Ž› ’—„Ž‹•Š‡† ‹ ”‘ƒ””ƒ› †ƒ–ƒ ȏʹ͹Ȑ ȋ  ƒ ‡••‹‘ —„‡” ͵͹ͷͶ͸Ȍ –Šƒ– ™‡”‡ —•‡† ˆ‘” ’ƒ––‡” ”‡ ‘‰‹–‹‘ ƒƒŽ›•‹• ‘”‹‰‹ƒ–‡ ˆ”‘ Ž‹˜‡”• ‘ˆ ͷ͹Ȁ͸ƒ  Ƭ ͳʹͻʹȀŽƒ •† ‹š‡†Ǧ „ƒ ‰”‘—† Ǧ ƒ† Ž‹––‡”ƒ–‡ ™‹Ž†Ǧ–›’‡ ‹ ‡Ǥ Ž› ‹ ”‘ƒ””ƒ› †ƒ–ƒ ˆ”‘ ‘†‹–‹‘• ‘’ƒ”ƒ„Ž‡ –‘ –Š‘•‡ ‹ –Š‹• •–—†› ™‡”‡ —•‡†ǡ ‹Ǥ‡Ǥ ˆ”‘ ‹ ‡ –Šƒ– ™‡”‡ ‹Œ‡ –‡† ™‹–Š •ƒŽ‹‡ ƒ† •—„•‡“—‡–Ž›ˆƒ•–‡†ˆ‘”ͳʹŠ‘—”•Ǥ  Pattern recognition on RNAseq and microarray data Gene-set enrichment analysis. ‡‡Ǧ•‡–‡”‹ Š‡–ƒƒŽ›•‹•ȋ Ȍ™ƒ•’‡”ˆ‘”‡†ƒ ‘”†‹‰–‘—„”ƒƒ‹ƒ‡–ƒŽǤʹͲͲͷȏͷͶȐ—•‹‰ –Š‡ †‡•–‘’ ƒ˜ƒƒ’’Ž‹ ƒ–‹‘•‘ˆ–™ƒ”‡ȋ˜‡”•‹‘ʹǤʹǤͶǡ”‘ƒ†

(54) •–‹–—–‡ȌǤ‡”Ǧ ‘’ƒ”‹•‘ǡ ’”‡Ǧ”ƒ‡†‰‡‡Ž‹•–• ‘–ƒ‹‹‰Ž‘‰ʹǦ ‘—–•Ǧ’‡”Ǧ‹ŽŽ‹‘–Ǧ˜ƒŽ—‡ǡ–‘‰‡–Š‡”™‹–Š–Š‡‡“ Š‹’ˆ‹Ž‡ ƒ† –Š‡ ‰‡‡ •‡– ‘ŽŽ‡ –‹‘ ‘ˆ ‹–‡”‡•– ™‡”‡ —•‡† ƒ• ‹’—– ˆ‘” –Š‡ ƒ’’Ž‹ ƒ–‹‘Ǥ ‘” –Š‡  ‘ˆ –Š‡ ‹ ”‘ƒ””ƒ› †ƒ–ƒ ˆ”‘ •–—†‹‡• ™‹–Š –Š‡ ‹š‡†Ǧ„ƒ ‰”‘—† ‹ ‡ǡ –Š‡ ’”‡Ǧ”ƒ‡† ‰‡‡ Ž‹•–• –Šƒ– ‘–ƒ‹‡†–Š‡‹ƒ–Ǧ˜ƒŽ—‡ȏͷͷȐƒ†–Š‡Ǥ Š‹’ˆ‹Ž‡–Šƒ– ‘””‡•’‘†‡†–‘–Š‡ƒ””ƒ›•–Šƒ–™‡”‡—•‡†‹ –Š‡ ‹š‡†Ǧ„ƒ ‰”‘—† •–—†‹‡• ȏͷ͸Ȑ ™‡”‡ —•‡† ȋ‘—•‡Ͷ͵Ͳʹ‡–”‡œ‰Ǥ Š‹’ȌǤ  ™ƒ• †‘‡ ˆ‘” ƒ ‡–ƒ„‘Ž‹ ‰‡‡•‡– ‘ŽŽ‡ –‹‘–Šƒ– ‘•‹•–‡†‘ˆ–Š‡. ‡–ƒ„‘Ž‹ ‰‡‡•‡– ‘ŽŽ‡ –‹‘ȋ†‘™Ž‘ƒ†‡†‹ Ȃ‡”•‹‘͸ǤͲȌǡ ‘’Ž‡‡–‡†™‹–Š–Š”‡‡‡™Ž›†‡ˆ‹‡†‰‡‡•‡–•ǤŠ‡•‡‰‡‡•‡–• ‘–ƒ‹‡†‰‡‡• ‘†‹‰ˆ‘”ȋ‹Ȍ‡œ›‡•–Šƒ– ƒ–ƒŽ›œ‡”‡ƒ –‹‘•‹˜‘Ž˜‹‰‘‡œ›‡ȋ‘‰‡‡•‡–Ȍǡȋ‹‹ȌƒŽŽ”‡ƒ –‹‘• ‹˜‘Ž˜‹‰ȋȌΪƒ†ȋȌ ȋȋȌȋ Ȍ‰‡‡•‡–Ȍǡƒ†ȋ‹‹‹Ȍ‡œ›‡• ƒ–ƒŽ›œ‹‰‘Ž›–Š‡”‡†‘š ”‡ƒ –‹‘•‹˜‘Ž˜‹‰ȋȌȋ ȌȋȋȌȋ Ȍ”‡†‘š‰‡‡•‡–Ȍƒ†‘–ǡˆ‘”‹•–ƒ ‡ǡ ‘ˆƒ –‘”„‹‘•›–Š‡•‹• ”‡ƒ –‹‘•Ǥ Š‡ †‡•–‘’ ƒ˜ƒ ƒ’’Ž‹ ƒ–‹‘•‘ˆ–™ƒ”‡ ™ƒ••‡– –‘ ’‡”ˆ‘” ͳͲͲͲ ’‡”—–ƒ–‹‘•ǡ—•‡ ™‡‹‰Š‡† ‡”‹ Š‡– •–ƒ–‹•–‹ Ǥ —”–Š‡”‘”‡ǡ ˆ‘” ‡ƒ Š ‰‡‡ •‡– ™‹–Š ƒ ˆƒŽ•‡ †‹• ‘˜‡”› ”ƒ–‡ ȋ Ȍ ‘ˆ δʹͷΨǡ ‹– ™ƒ• ƒŽ•‘ ƒŽ —Žƒ–‡† ™Š‡–Š‡” ‘ ƒ† ȋȌȋ Ȍ ‰‡‡• ™‡”‡ ‘˜‡””‡’”‡•‡–‡† ‹ –Š‡ ‘–”‹„—–‹‰‰‡‡•Ž‹•–ǤŠ‹•™ƒ•†‘‡„› ƒŽ —Žƒ–‹‰ƒ”‹•”ƒ–‹‘ȋȌ’ƒ”ƒ‡–‡”ƒ ‘”†‹‰–‘ ܴܴ஼௢஺̴ே஺஽௉ு ൌ. ͓‫ܩܥ‬஼௢஺̴ே஺஽௉ு Ȁ͓‫ܩܥ‬  ሺ͓݃݁݊݁‫ݏ‬஼௢஺̴ே஺஽௉ு െ ͓‫ܩܥ‬஼௢஺̴ே஺஽௉ு ሻȀሺ͓݃݁݊݁‫ ݏ‬െ ͓‫ܩܥ‬ሻ.  ƒ††‡–‡”‹‹‰™Š‡–Š‡”–Š‹•˜ƒŽ—‡‹•Žƒ”‰‡”–ŠƒͳǤ ‡”‡͓ ‹•–Š‡—„‡”‘ˆ ‘–”‹„—–‹‰‰‡‡•‹ –Š‡‰‡‡•‡–‘ˆ‹–‡”‡•–ǡ͓ ‘̴ ‹•–Š‡—„‡”‘ˆ ‘–”‹„—–‹‰‰‡‡•‹–Š‹•‰‡‡•‡––Šƒ–ƒ”‡ƒŽ•‘ ‘‘”ȋȌȋ Ȍ‰‡‡•ǡ͓‰‡‡•‹•–Š‡–‘–ƒŽ—„‡”‘ˆ‰‡‡•‹–Š‡‰‡‡•‡–ƒ†͓‰‡‡•‘̴ ‹•–Š‡ –‘–ƒŽ—„‡”‘ˆ‘ƒ†ȋȌȋ Ȍ‰‡‡•‹–Š‡‰‡‡•‡–Ǥ  Alternative gene set enrichment analysis •ƒƒŽ–‡”ƒ–‹˜‡ˆ‘”–Š‡ Žƒ••‹ ƒŽ ‡‡‡–”‹ Š‡–ƒŽ›•‹•ǡƒŽ‹•–‘ˆ†‹ˆˆ‡”‡–‹ƒŽŽ›Ǧ‡š’”‡••‡†‰‡‡• ™ƒ• ƒ†‡ ˆ‘” ‡ƒ Š  ˜•  ‘’ƒ”‹•‘ǡ „ƒ•‡† ‘ –Š‡ ˆ‘ŽŽ‘™‹‰ ‹ Ž—•‹‘ ”‹–‡”‹ƒǣ ȋȁŽ‘‰ʹȋˆ‘Ž† Šƒ‰‡ȌȁεͲǤͷ’δͲǤͳȌ’δͲǤͲͷǤŠ‹•”‡’”‡•‡–•ƒ ‘’”‘‹•‡„‡–™‡‡•–ƒ–‹•–‹ ƒŽ•‹‰‹ˆ‹ ƒ ‡ ȋŽ‘™ ’ ˜ƒŽ—‡Ȍ ƒ† „‹‘Ž‘‰‹ ƒŽ ”‡Ž‡˜ƒ ‡ ȋŽƒ”‰‡ Šƒ‰‡ȌǤ ‡ ‰‡‡”ƒ–‡† •‡’ƒ”ƒ–‡ Ž‹•–• ˆ‘” —’ ƒ† †‘™”‡‰—Žƒ–‡†‰‡‡•ƒ†™‡‰‡‡”ƒ–‡†Ž‹•–•ˆ”‘„‘–Š–Š‡‡‡“ʹ†‹ˆˆ‡”‡–‹ƒŽ‰‡‡‡š’”‡••‹‘ȋ Ȍ. ͺͲ.

(55) Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice ”‡•—Ž–•ƒ†–Š‡‘‘ ”‡•—Ž–•Ǥ‡–Š‡ ƒŽ —Žƒ–‡†ˆ‘”‡ƒ ŠǦ ‘’ƒ”‹•‘ǡˆ‘”‡ƒ Š‡–ƒ„‘Ž‹  ‰‡‡•‡–ǡƒ†ˆ‘”‡ƒ Š†‹”‡ –‹‘•‡’ƒ”ƒ–‡Ž›ǡ™Šƒ–’‡” ‡–ƒ‰‡‘ˆ–Š‡†‹ˆˆ‡”‡–‹ƒŽŽ›Ǧ‡š’”‡••‡†‰‡‡•™‡”‡ ’”‡•‡–‹–Š‡‰‡‡•‡–ƒ†–Š‡‰‡‡•‡–•™‡”‡•—„•‡“—‡–Ž›”ƒ‡†ƒ ‘”†‹‰–‘–Š‹•’‡” ‡–ƒ‰‡Ǥ ‡‡ •‡–• ™‹–Š –Š‡ •ƒ‡ ’‡” ‡–ƒ‰‡ ”‡ ‡‹˜‡† –Š‡ •ƒ‡ ”ƒ‹‰ ƒ† ‰‡‡ •‡–• ™‹–Š ƒ ’‡” ‡–ƒ‰‡ ‘ˆ œ‡”‘ ”‡ ‡‹˜‡† –Š‡ ”ƒ ‘””‡•’‘†‹‰ –‘ –Š‡ —„‡” ‘ˆ ‡–ƒ„‘Ž‹  ‰‡‡ •‡–• ‹ –Š‡ ‡–ƒ„‘Ž‹  ‰‡‡ •‡– ‘ŽŽ‡ –‹‘ǤŠ‡”ƒ‡†‰‡‡•‡–•™‡”‡•—„•‡“—‡–Ž›‰‹˜‡ƒ‘˜‡”ƒŽŽ”ƒ„ƒ•‡†‘–Š‡‡†‹ƒ‘ˆ–Š‡ ”ƒ• ‘ˆ –Š‡ ‘†‹–‹‘•Ǥ —”–Š‡”‘”‡ǡ ˆ‘” ‡ƒ Š ‘†‹–‹‘ǡ ˆ‘” „‘–Š —’ ƒ† †‘™”‡‰—Žƒ–‹‘ǡ ‹– ™ƒ• †‡–‡”‹‡† ™Š‡–Š‡” –Š‡ —„‡” ‘ˆ †‹ˆˆ‡”‡–‹ƒŽŽ› ‡š’”‡••‡† ‰‡‡• ‘ˆ –Š‡ ‰‹˜‡ ‰‡‡ •‡– ‹• ‘˜‡””‡’”‡•‡–‡† ‹ –Š‡ Ž‹•– ‘ˆ †‹ˆˆ‡”‡–‹ƒŽŽ›Ǧ‡š’”‡••‡† ‰‡‡•Ǥ Š‹• ™ƒ• ƒŽ —Žƒ–‡† „› †‡–‡”‹‹‰ ™Š‡–Š‡”–Š‡ ‘””‡•’‘†‹‰”‹•Ǧ”ƒ–‹‘ȋ‰‡‡•‡–Ȍ‹•Žƒ”‰‡”–Šƒͳǡƒ ‘”†‹‰–‘ǣ ܴܴ௚௘௡௘௦௘௧ ൌ. ͓‫ܩܧܦ‬௠௘௧௔௕௢௟௜௖ǡ௚௘௡௘௦௘௧ Ȁ͓‫ܩܧܦ‬௠௘௧௔௕௢௟௜௖ǡ௔௟௟  ሺ͓݃݁݊݁‫ݏ‬௚௘௡௘௦௘௧ െ ͓‫ܩܧܦ‬௠௘௧௔௕௢௟௜௖ǡ௚௘௡௘௦௘௧ ሻȀሺ͓݉݁‫ ݏ݈݁݊݁݃ܿ݅݋ܾܽݐ‬െ ͓‫ܩܧܦ‬௠௘௧௔௕௢௟௜௖ǡ௔௟௟ ሻ.  ™Š‡”‡͓ ‡–ƒ„‘Ž‹ ǡƒŽŽ‹•–Š‡—„‡”‘ˆ†‹ˆˆ‡”‡–‹ƒŽŽ›‡š’”‡••‡†‡–ƒ„‘Ž‹ ‰‡‡•ǡ͓ ‡–ƒ„‘Ž‹ ǡ‰‡‡•‡–‹• –Š‡ —„‡” ‘ˆ †‹ˆˆ‡”‡–‹ƒŽŽ› ‡š’”‡••‡† ‡–ƒ„‘Ž‹  ‰‡‡• „‡Ž‘‰‹‰ –‘ –Š‡ ‰‡‡ •‡– ‹ “—‡•–‹‘ǡ Dz͓‰‡‡•‰‡‡•‡–dz‹•–Š‡–‘–ƒŽ—„‡”‘ˆ‰‡‡•‹–Š‡‰‡‡•‡–ƒ†Dz͓‡–ƒ„‘Ž‹ ‰‡‡•dz‹•–Š‡–‘–ƒŽ—„‡” ‘ˆ‡–ƒ„‘Ž‹ ‰‡‡•ǡƒ ‘”†‹‰–‘–Š‡‡–ƒ„‘Ž‹ ‰‡‡•‡– ‘ŽŽ‡ –‹‘Ǥ  Statistical analysis ‹ˆˆ‡”‡ ‡•‹‘†›™‡‹‰Š–ȋȌǡŽ‘‘†‰Ž— ‘•‡ȋ ȌǡŽ‹˜‡”™‡‹‰Š–ȋȌƒ†‘š›‰‡ ‘•—’–‹‘™‡”‡ ƒ••‡••‡† ™‹–Š  ƒ† ’‘•–ǦŠ‘   ƒˆ–‡” Ž‘‰ –”ƒ•ˆ‘”ƒ–‹‘ ‹ˆ –Š‡ †ƒ–ƒ ™ƒ• ‘– ‘”ƒŽŽ›Ǧ †‹•–”‹„—–‡†Ǥ”‘–‡‘‹ †ƒ–ƒ™‡”‡ƒƒŽ›œ‡†„›”—•ƒŽǦƒŽŽ‹•ƒ†ƒŠ‹–‡›–‡•–ǤPpiaǦ ‘”ƒŽ‹œ‡† Acot2  ‡š’”‡••‹‘ †ƒ–ƒ ‰‡‡”ƒ–‡† „› “Ǧ ™‡”‡ ƒ••‡••‡† ˆ‘” ‘”ƒŽ‹–› ƒ† •—„•‡“—‡–Ž›–Š‡†‹ˆˆ‡”‡ ‡• „‡–™‡‡ƒ†™ƒ• ƒ••‡••‡†„›–—†‡–ǯ•–Ǧ–‡•–Ǥ Š‡•‡ƒƒŽ›•‡• ™‡”‡ ’‡”ˆ‘”‡† —•‹‰

(56)   –ƒ–‹•–‹ • ˜‡”•‹‘ ʹʹǤͲ ȋ

(57)  Ǥǡ Š‹ ƒ‰‘ǡ

(58) ǡ Ǥ Š‡ Ž‡˜‡Ž ‘ˆ •‹‰‹ˆ‹ ƒ ‡™ƒ••‡–ƒ–’δͲǤͲͷǤ. Data Availability Š‡ †ƒ–ƒ•‡–• ‰‡‡”ƒ–‡† †—”‹‰ ƒ†Ȁ‘” ƒƒŽ›œ‡† †—”‹‰ –Š‡ —””‡– •–—†› ƒ”‡ ƒ˜ƒ‹Žƒ„Ž‡ ˆ”‘ –Š‡ ‘””‡•’‘†‹‰ƒ—–Š‘”‘”‡ƒ•‘ƒ„Ž‡”‡“—‡•–Ǥ . References ͳǤ. ʹǤ ͵Ǥ. ͶǤ. ͷǤ. ͸Ǥ. ‘—–‡ ǡ ‹‘Žƒ–‡ ǡ ‡–—”ƒ  Ǥǡ ƒ†‡”•  Ǥ Š‡ ‹‘ Š‡‹•–”› ƒ† Š›•‹‘Ž‘‰› ‘ˆ ‹–‘ Š‘†”‹ƒŽ ƒ––›  ‹† ȾǦš‹†ƒ–‹‘ ƒ†

(59) –• ‡‡–‹  ‹•‘”†‡”•Ǥ — ‡˜ Š›•‹‘ŽǤ ʹͲͳ͸Ǣ͹ͺȋͳȌǣʹ͵ȂͶͶǤ ‹ƒŽ†‘ ǡ ƒ–‡” ǡ ‡‡––  Ǥ ƒ––›  ‹† ‘š‹†ƒ–‹‘ ‹•‘”†‡”•Ǥ — ‡˜ Š›•‹‘ŽǤ ʹͲͲʹǢ͸ͶȋͳȌǣͶ͹͹ȂͷͲʹǤ ‡”• ǡ‡‹Œ‰‘—† ǡƒ–‡”Šƒ ǡ ‡”˜‡” ǡ˜ƒ†‡‡”‰ǡƒ—‡” ǡ‹– ǤŠ‡ ƒ–—”ƒŽ Š‹•–‘”› ‘ˆ ‡†‹—Ǧ Šƒ‹ ƒ ›Ž ‘ †‡Š›†”‘‰‡ƒ•‡ †‡ˆ‹ ‹‡ › ‹ –Š‡ ‡–Š‡”Žƒ†•ǣ Ž‹‹ ƒŽ’”‡•‡–ƒ–‹‘ƒ†‘—– ‘‡Ǥ ‡†‹ƒ–”ǤʹͲͲ͸ǢͳͶͺȋͷȌǤ ‡”• ǡ‘‡”ǡ••‡ǡ‘•ǡ—‹–‡” ǡƒ–‡”Šƒ ǡ‹‡œ‡Ǧ‘‹‰ǡƒ†‡”• ǡ ‘†‡‡Ž ǡ‘‡„‡” ǡ‡ƒ–‡ǡ‹– ǡ‡‹Œ‰‘—† Ǥ‡‘ƒ–ƒŽ• ”‡‡‹‰ˆ‘”‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋȌ †‡ˆ‹ ‹‡ › ‹ Š‡ ‡–Š‡”Žƒ†•ǣ Š‡ ‹’‘”–ƒ ‡ ‘ˆ ‡œ›‡ƒƒŽ›•‹•–‘ƒ• ‡”–ƒ‹–”—‡†‡ˆ‹ ‹‡ ›Ǥ 

(60) Š‡”‹–‡–ƒ„‹•ǤʹͲͲͺǢ͵ͳȋͳȌǣͺͺȂͻ͸Ǥ ‘—™ǡ‹– ǡ‹‡œ‡Ǧ‘‹‰ǡ‘•‰”ƒƒˆǦ†‡‘‡”ǡ ‡”†‹‰ǡ‡‹Œ‰‘—†Ǧ ǡ‡”•  Ǥ

(61)  ˜‹–”‘ ƒ† ‹ ˜‹˜‘ ‘•‡“—‡ ‡• ‘ˆ ˜ƒ”‹ƒ– ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ‰‡‘–›’‡•Ǥ”’Šƒ‡– ƒ”‡‹•ǤʹͲͳ͵ǢͺȋͳȌǣͶ͵Ǥ ‘—™ǡ‹– ǡ†‡”‹‡•ǡ†‡Ž‡” ǡ‘• Šǡ‹••‡” ǡ—Ž†‡” ǡ—„‹‘Ǧ ‘œƒŽ„‘ ǡŽ˜‡”•ǡ‹‡œ‡Ǧ‘‹‰ǡƒ†‡”• ǡƒ–‡”Šƒ ǡ‡‹Œ‰‘—†Ǧ ǡ‡”• Ǥ‹•. ͺͳ. 3.

(62) Chapter 3. ͹Ǥ. ͺǤ. ͻǤ. ͳͲǤ. ͳͳǤ ͳʹǤ. ͳ͵Ǥ ͳͶǤ ͳͷǤ ͳ͸Ǥ ͳ͹Ǥ ͳͺǤ ͳͻǤ ʹͲǤ. ʹͳǤ ʹʹǤ ʹ͵Ǥ ʹͶǤ ʹͷǤ ʹ͸Ǥ. ͺʹ. •–”ƒ–‹ˆ‹ ƒ–‹‘„›”‡•‹†—ƒŽ‡œ›‡ƒ –‹˜‹–›ƒˆ–‡”‡™„‘”• ”‡‡‹‰ˆ‘”‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘ †‡Š›”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›ǣ†ƒ–ƒˆ”‘ƒ ‘Š‘”–•–—†›Ǥ”’Šƒ‡– ƒ”‡‹•ǤʹͲͳʹǢ͹ǣ͵ͲǤ ’‹‡‡”‘‡––‡” ǡ ƒ•–‹ ǡ ‹ŽŽ‹‰Šƒ ǡ ‘””‹• ǡ ‹Œ„—”‰ ǡ ‹Ž ‡ Ǥ —””‡– ‹••—‡• ”‡‰ƒ”†‹‰ –”‡ƒ–‡– ‘ˆ ‹–‘ Š‘†”‹ƒŽ ˆƒ––› ƒ ‹† ‘š‹†ƒ–‹‘ †‹•‘”†‡”•Ǥ 

(63) Š‡”‹– ‡–ƒ„ ‹•Ǥ ʹͲͳͲǢ͵͵ȋͷȌǣͷͷͷȂ͸ͳǤ ”‘‡–›ǡƒ•‘—”‹ƒǡ‘‡ˆ‘– ǡ‘—”–‘‹• ǡ—‹ Šƒǡƒ„‹‡”ǡ‡••ƒ›”‡Ǥ‘•– ƒ•‡• ‘ˆ‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›‡• ƒ’‡†‡–‡ –‹‘‹ ”ƒ ‡Ǥ — ‡‡–Ǥ ͳͻͻ͸Ǣͻ͹ȋ͵Ȍǣ͵͸͹ȂͺǤ ‡ŽŽ›ǡŠ‡Žƒ ǡ‰†‡ǡŽ’‡”•ǡŠƒ‰ ǡ‡ŽŽ—• ǡ ”‡‰‡”•‡ǡ‘”Žƒ†ǡ–”ƒ—•• Ǥ‘Ž‡ —Žƒ” Šƒ”ƒ –‡”‹œƒ–‹‘‘ˆ‹Š‡”‹–‡†‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›Ǥ ”‘ ƒ–Ž ƒ† ‹ǤͳͻͻͲǢͺ͹ȋʹ͵Ȍǣͻʹ͵͸ȂͶͲǤ —”ƒǡ ‘ˆƒ’ǡŠ‡ƒ† ǡƒ—†—„”ƒ› Ǧǡƒ†ƒǤ—††‡Š‹Ž†‡ƒ–Šƒ†Ʈ ‡ƒŽ–Š›ǯ ˆˆ‡ –‡† ƒ‹Ž› ‡„‡”• ‹–Š ‡†‹—ǦŠƒ‹  ›ŽǦ‘‡œ›‡  ‡Š›†”‘‰‡ƒ•‡ ‡ˆ‹ ‹‡ ›Ǥ ‡†‹ƒ–”‹ •Ǥͳͻͺ͸Ǣ͹ͺȋ͸ȌǣͳͲͷʹȂ͹Ǥ  Šƒ–œƒǤǡ•‡ƒ—‡”ǤŠ‡ Ž‹‹ ƒŽƒ‹ˆ‡•–ƒ–‹‘‘ˆ†‡ˆ‹ ‹‡ ›ǣŠƒŽŽ‡‰‡•–‘™ƒ”†• ƒ†—Ž–Š‘‘†‹–Š‡• ”‡‡‡†’‘’—Žƒ–‹‘Ǥ 

(64) Š‡”‹–‡–ƒ„‹•ǤʹͲͳͲǢ͵͵ȋͷȌǣͷͳ͵ȂʹͲǤ ‡ƒŽ‡• ǡ Š‘’•‘ ǡ ƒ••‘—†  ǡ ƒŠƒ ǡ ƒŽŽ‹†ƒ› ǡ ‡‘ƒ”†  Ǥ ”‘†— –‹‘ ƒ† †‹•’‘•ƒŽ‘ˆ‡†‹—Ǧ Šƒ‹ˆƒ––›ƒ ‹†•‹ Š‹Ž†”‡™‹–Š‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡ †‡ˆ‹ ‹‡ ›Ǥ 

(65) Š‡”‹–‡–ƒ„‹•ǤͳͻͻͶǢͳ͹ȋͳȌǣ͹ͶȂͺͲǤ ‡”• ǡƒ’”‘•‡ ǡƒ‡ ǡƒ‡” ‹Ž•–ǡ’ƒǡ‹– Ǥ ƒˆ‡ƒ†—•ƒˆ‡ †—”ƒ–‹‘‘ˆˆƒ•–‹‰ˆ‘” Š‹Ž†”‡™‹–Š†‡ˆ‹ ‹‡ ›Ǥ—” ‡†‹ƒ–”ǤʹͲͲ͹Ǣͳ͸͸ȋͳȌǣͷȂͳͳǤ ƒŽ–‡” Ǥ ‘Ž‡”ƒ ‡ –‘ ˆƒ•–ǣ ƒ–‹‘ƒŽ ƒ† ’”ƒ –‹ ƒŽ ‡˜ƒŽ—ƒ–‹‘ ‹ Š‹Ž†”‡ ™‹–Š Š›’‘‡–‘ƒ‡‹ƒǤ 

(66) Š‡”‹–‡–ƒ„‹•ǤʹͲͲͻǢ͵ʹȋʹȌǣʹͳͶȂ͹Ǥ ‡‡––  Ǥ ƒ–Š‘’Š›•‹‘Ž‘‰› ‘ˆ ˆƒ––› ƒ ‹† ‘š‹†ƒ–‹‘ †‹•‘”†‡”•Ǥ 

(67) Š‡”‹– ‡–ƒ„ ‹•Ǥ ʹͲͳͲǢ͵͵ȋͷȌǣͷ͵͵Ȃ͹Ǥ ‹•Š™ƒƒ–Š Ǥ ƒ––› ƒ ‹† „‡–ƒǦ‘š‹†ƒ–‹‘ †‹•‘”†‡”•ǣ  „”‹‡ˆ ”‡˜‹‡™Ǥ  ‡—”‘• ‹Ǥ ʹͲͳ͸Ǣʹ͵ȋͳȌǣͷͳȂͷǤ ‘Ž‡”Ǧ–‘—™Ǥ†‡ˆ‹ ‹‡ ›‘„‡ǡ‘”‘––‘„‡ƒ–”‹•ǤʹͲͳͶǤ ‘—–‡ǡƒ†‡”• Ǥ‰‡‡”ƒŽ ‹–”‘†— –‹‘–‘–Š‡„‹‘ Š‡‹•–”›‘ˆ‹–‘ Š‘†”‹ƒŽ ˆƒ––› ƒ ‹†ȾǦ‘š‹†ƒ–‹‘Ǥ 

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