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

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Download date: 23-09-2021

Exploring the mechanisms underlying the phenotype of MCAD deficiency with Systems Medicine From computational model to mice to man

Anne-Claire M.F. Martines

















Š‡™‘”†‡• ”‹„‡†‹–Š‹•–Š‡•‹•™ƒ• ƒ””‹‡†‘—–ƒ––Š‡†‡’ƒ”–‡–‘ˆ‡†‹ƒ–”‹ •ǡ‡–‡”ˆ‘” ‹˜‡”ǡ ‹‰‡•–‹˜‡ ƒ† ‡–ƒ„‘Ž‹  ‹•‡ƒ•‡•ǡ ‹˜‡”•‹–› ‘ˆ ”‘‹‰‡ǡ –Š‡ ‡–Š‡”Žƒ†•Ǥ Š‡ ”‡•‡ƒ” Š†‡• ”‹„‡†‹–Š‹•–Š‡•‹•™ƒ•ˆ—†‡†„›–Š‡‹˜‡”•‹–›‡†‹ ƒŽ‡–‡” ”‘‹‰‡ǤŠ‡ ”‡•‡ƒ” Š‰”‘—’’ƒ”–‹ ‹’ƒ–‡•‹–Š‡ ”‘‹‰‡ •–‹–—–‡ˆ‘””—‰š’Ž‘”ƒ–‹‘ȋ  ȌǤ

Š‡ƒ—–Š‘”‰”ƒ–‡ˆ—ŽŽ›ƒ ‘™Ž‡†‰‡•–Š‡ˆ‹ƒ ‹ƒŽ•—’’‘”–ˆ‘”’”‹–‹‰–Š‹•–Š‡•‹•„›ǣ ”‘‹‰‡ •–‹–—–‡ˆ‘””—‰š’Ž‘”ƒ–‹‘ȋ  ȌǤ ‹Œ•—‹˜‡”•‹–‡‹– ”‘‹‰‡ȋ— Ȍ ‹˜‡”•‹–›‡†‹ ƒŽ‡–‡” ”‘‹‰‡ȋ Ȍ Š—‹•–œ‘”‰–ȋ™™™Ǥ–Š—‹•‘–œ‘”‰–ǤŽȌ ƒ˜‘”ƒ„‹Ž‹–›ǤǤ ‡”–‹††‡Ž™‹Œ ‘‘’‡”–ƒƒ•  ‘˜‡”†‡•‹‰ǣ–—†‹‘   ‘˜‡”‡ƒ‹‰ǣŠ‡ ‘˜‡”‹•‹Š‘‘”‘ˆƒŽŽ™Š‘ ‘–”‹„—–‡’ƒ••‹‘ƒ–‡Ž›–‘‰ƒ‹‘”‡‹•‹‰Š– ‹–‘†‡ˆ‹ ‹‡ ›–‘Š‡Ž’ƒŽŽǦ†‡ˆ‹ ‹‡–‹†‹˜‹†—ƒŽ•Ž‹˜‡ƒˆ—ŽŽ‡”Ž‹ˆ‡Ǥ ”‘–‡‹ ƒ† ‡–ƒ„‘Ž‹–‡ ‹ƒ‰‡• ‘ –Š‡ „ƒ  ‘˜‡” ƒ”‡ ˆ”‘ —„ Š‡ ȋΪ ƒ† ƒ ›ŽǦ ƒ”‹–‹‡•Ȍǡ ‹‹‘‘• ȋ‘‡œ›‡ Ȍ ƒ†   ȋ͵ͲͶǦ—–ƒ–  ȋ ǣ Ͷͳ͵Ȍƒ†ȋ ǣʹ Ȍǡ„‘–Š‰‡‡”ƒ–‡†™‹–Š ˜‹‡™‡”ȋ ǣʹͻͺͷͲ͹͹ͺȌȌ ƒ›‘—–ǣ‡ǦŽƒ‹”‡Ǥ Ǥƒ”–‹‡• ”‹–‡†„›ǣ‹††‡”’”‹– ȁ™™™Ǥ”‹††‡”’”‹–ǤŽ  ǣ ͻ͹ͺǦͻͶǦͲ͵ͶǦͳ͹ͻͻǦͺȋ”‹–‡†˜‡”•‹‘Ȍ ͻ͹ͺǦͻͶǦͲ͵ͶǦͳ͹ͻͺǦͳȋ‹‰‹–ƒŽ˜‡”•‹‘Ȍ  ̹ǤǤ Ǥƒ”–‹‡•ǡʹͲͳͻ ŽŽ”‹‰Š–•”‡•‡”˜‡†Ǥ‘’ƒ”–‘ˆ–Š‹•–Š‡•‹•ƒ›„‡”‡’”‘†— ‡†ǡ†‹•–”‹„—–‡†•–‘”‡†‹ƒ”‡–”‹‡˜ƒŽ •›•–‡ǡ‘”–”ƒ•‹––‡†‹ƒ›ˆ‘”‘”„›ƒ›‡ƒ•™‹–Š‘—–’”‹‘”’‡”‹••‹‘‘ˆ–Š‡ƒ—–Š‘”ǡ ‘”™Š‡”‡ƒ’’Ž‹ ƒ„Ž‡ǡ–Š‡’—„Ž‹•Š‡”Š‘Ž†‹‰–Š‡ ‘’›”‹‰Š–‘–Š‡’—„Ž‹•Š‡†ƒ”–‹ Ž‡•Ǥ

Exploring the mechanisms underlying the phenotype of MCAD deficiency with Systems Medicine

From computational model to mice to man

Proefschrift

ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen op gezag van de rector magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op

woensdag 10 juli 2019 om 16.15 uur

door

Anne-Claire Micheline Francisca Martines

geboren op 10 oktober 1981 te Willemstad, Curaçao

Promotores Prof. dr. B.M. Bakker Prof. dr. D.J. Reijngoud

Beoordelingscommissie Prof. dr. F.J. Bruggeman Prof. dr. A.K. Groen Prof. dr. F.J. van Spronsen

This PhD thesis is dedicated to my lovely parents, Ingrid Martines-Kirindongo and Edwin Martines, and my loving husband, Amart Schoop, who have always supported me through thick and thin

Paranymfen Natasha Tearr-Marcos Aduni Mariana

CONTENTS

Chapter 1 ‡‡”ƒŽ‹–”‘†— –‹‘ƒ†‘—–Ž‹‡‘ˆ–Š‡–Š‡•‹• 9   Chapter 2 Š‡ ’”‘‹• —‘—• ‡œ›‡ ‡†‹—Ǧ Šƒ‹ ͵Ǧ‡–‘Ǧƒ ›ŽǦ‘ 39 –Š‹‘Žƒ•‡–”‹‰‰‡”•ƒ˜‹ ‹‘—• › Ž‡‹ˆƒ––›Ǧƒ ‹†„‡–ƒǦ‘š‹†ƒ–‹‘   Chapter 3 ”ƒ• ”‹’–‘‹  ƒƒŽ›•‹• •—‰‰‡•–• ƒ ‘’‡•ƒ–‘”› ”‘Ž‡ ‘ˆ 67 ‘ˆƒ –‘”• ‘‡œ›‡ƒ†Ϊ‹‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡‘ ‘—–‹ ‡   Chapter 4 Š‡”‘Ž‡‘ˆŠ‡’ƒ–‹ ˆƒ––›Ǧƒ ‹†‘š‹†ƒ–‹‘†—”‹‰ ‘Ž†•–”‡••ǣ 127 ”‡‘†‡Ž‹‰‘ˆŠ‡’ƒ–‹ ‡–ƒ„‘Ž‹•‹ƒ‘—•‡™‹–Š‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›   Chapter 5 ‹—Žƒ–‹‰ –Š‡ ‹’ƒ – ‘ˆ ‰‡‡–‹  ƒ† ‡˜‹”‘‡–ƒŽ 157 ‘†‹ˆ‹‡”• ‘  †‡ˆ‹ ‹‡ › ‹ ƒ ‡š–‡†‡† ‘†‡Ž ‘ˆ Š—ƒŽ‹˜‡”ˆƒ––›Ǧƒ ‹† ƒ–ƒ„‘Ž‹•   Chapter 6 ‘™ƒ”†• ‹†‡–‹ˆ›‹‰ in vitro ‡–ƒ„‘Ž‹  ƒ†ƒ’–ƒ–‹‘• –Šƒ– 205 ‡š’Žƒ‹•›’–‘ƒ–‘Ž‘‰›‹‹†‹˜‹†—ƒŽ•Ǥ •Š‘”–”‡’‘”–   Chapter 7 ‡‡”ƒŽ†‹• —••‹‘ 225   Appendix  241  —ƒ”› 243   ƒ‡˜ƒ––‹‰ 249    ‘™Ž‡†‰‡‡–• 255   ‹‘‰”ƒ’Š› 265   ‘”–ˆ‘Ž‹‘ 269   ‹•–‘ˆ’—„Ž‹ ƒ–‹‘• 273 

Chapter 1

General introduction and outline of the thesis

      



General introduction and outline of the thesis

Introduction 1 Š‡ƒƒŽ‹ƒ„‘†›†‡’‡†•‘ƒ™‹†‡”ƒ‰‡‘ˆ ‡ŽŽ—Žƒ”ƒ†•›•–‡‹ ’”‘ ‡••‡•Ǥ•—„•‡– ‘ˆ –Š‡•‡ ’”‘ ‡••‡• ƒ”‡ ’Š›•‹ ‘ Š‡‹ ƒŽ ’”‘ ‡••‡• ƒ† „‹‘ Š‡‹ ƒŽ ”‡ƒ –‹‘• –Šƒ– ƒ”‡ ƒ–ƒŽ›œ‡† „› ‡œ›‡• ƒ† –”ƒ•’‘”–‡”•Ǥ Š‡•‡ ’”‘ ‡••‡• ƒ† ”‡ƒ –‹‘• ˆƒŽŽ ™‹–Š‹ –Š‡ ƒ–‡‰‘”›‘ˆ‡–ƒ„‘Ž‹•Ǥ‰‘‹‰”‡•‡ƒ” Š‹ƒƒŽ‹ƒ‡–ƒ„‘Ž‹•ƒ†‡–ƒ„‘Ž‹ †‹•‡ƒ•‡ Šƒ• ‰‡‡”ƒ–‡† ƒŒ‘” ƒ†˜ƒ ‡• ‹ –Š‡ Žƒ•– †‡ ƒ†‡•Ǥ Š‹• ‹• “—‹–‡ ‹’‘”–ƒ–ǡ ƒ• ƒ “—‹”‡† ‡–ƒ„‘Ž‹ †‹•‡ƒ•‡Šƒ•„‡ ‘‡ƒ™‘”Ž†™‹†‡‡’‹†‡‹ †—‡–‘ƒ—Š‡ƒŽ–Š›Ž‹ˆ‡•–›Ž‡ƒ†ƒ ƒ‰‹‰’‘’—Žƒ–‹‘ȏͳȂͶȐǤ‘”‡‘˜‡”ǡƒ›‘”‡‹„‘”‡–ƒ„‘Ž‹ ‡””‘”•ƒ”‡„‡‹‰†‹• ‘˜‡”‡† †—‡–‘‡ƒ”Ž›• ”‡‡‹‰‡ˆˆ‘”–•ȏͷȂͳ͵ȐǤ†‡”•–ƒ†‹‰‡–ƒ„‘Ž‹•ƒ†‡–ƒ„‘Ž‹ †‹•‡ƒ•‡™‹ŽŽ ‹’”‘˜‡–Š‡ Šƒ ‡•ˆ‘”‡ˆˆ‡ –‹˜‡–”‡ƒ–‡–ƒ†–Š‡Ž‘™‡”‹‰‘ˆ‡˜‡””‹•‹‰Š‡ƒŽ–Š ƒ”‡ ‘•–•Ǥ Š‡ƒŒ‘”“—‡•–‹‘‹–Š‡ˆ‹‡Ž†‹•™Šƒ–ƒ”‡–Š‡‘Ž‡ —Žƒ”‡ Šƒ‹••—†‡”Ž›‹‰‡–ƒ„‘Ž‹  †‹•‡ƒ•‡ȏ͵ǡͳͶȂͳ͹ȐǤ

Common acquired metabolic diseases  “—‹”‡† ‡–ƒ„‘Ž‹  †‹•‡ƒ•‡• ƒ”‡ ‘Ǧ ‘—‹ ƒ„Ž‡ǡ —Ž–‹ˆƒ –‘”‹ƒŽ †‹•‡ƒ•‡• –Šƒ– ƒ› ’”‘‰”‡••‘˜‡”–‹‡ȏͳͺȐǤŠ‡›ƒ”‡‘‡‘ˆ–Š‡‘•– ‘‘‘Ǧ ‘—‹ ƒ„Ž‡†‹•‡ƒ•‡‰”‘—’• ƒˆˆ‡ –‹‰ ‰Ž‘„ƒŽ Š‡ƒŽ–ŠǤ  ‘”†‹‰ –‘ –Š‡ ‘”Ž† ‡ƒŽ–Š ”‰ƒ‹œƒ–‹‘ǡ –Š‡ ’”‡˜ƒŽ‡ ‡ ‘ˆ ‘˜‡”™‡‹‰Š–ƒ†‘„‡•‹–›Šƒ•–”‹’Ž‡†•‹ ‡ͳͻ͹ͷȏͳͻȐǤƒ”–‹ —Žƒ”Ž›‘„‡•‹–›ǡƒˆˆŽ‹ –‹‰‘”‡–Šƒ ͳͻΨ‘ˆ–Š‡‡•–‡”’‘’—Žƒ–‹‘ȋͳʹǤͷΨ‹–Š‡‡–Š‡”Žƒ†•ƒ†͵ͺΨ‹–Š‡ȌȏʹͲȐǡ‹ ”‡ƒ•‡• –Š‡”‹•‘ˆ‡–ƒ„‘Ž‹ †‹•‡ƒ•‡•ǡ‹ Ž—†‹‰–›’‡ʹ†‹ƒ„‡–‡•‡ŽŽ‹–—•ȋʹȌǡƒ†‘ƒŽ ‘Š‘Ž‹  ˆƒ––›Ž‹˜‡”†‹•‡ƒ•‡ȋ ȌȏͳȂͶȐǤʹˆ‘”‡šƒ’Ž‡ǡ‹• Šƒ”ƒ –‡”‹œ‡†„›‹•—Ž‹”‡•‹•–ƒ ‡‘ˆ ’‡”‹’Š‡”ƒŽ –‹••—‡• ƒ† ‹’ƒ‹”‡† ‹•—Ž‹ •‡ ”‡–‹‘ „› –Š‡ ’ƒ ”‡ƒ• —’‘ ‹ ”‡ƒ•‡† „Ž‘‘† ‰Ž— ‘•‡ Ž‡˜‡Ž• ȏʹͳǡʹʹȐǤ Š‡ ”‡•—Ž–‹‰ Š‹‰Š „Ž‘‘† ‰Ž— ‘•‡ Ž‡˜‡Ž• ȋŠ›’‡”‰Ž› ‡‹ƒȌ †ƒƒ‰‡ ’ƒ ”‡ƒ–‹  ‡ŽŽ•ǡ Ǧ ‡ŽŽ•ǡ ‡”˜‡•ǡ ƒ† „Ž‘‘† ˜‡••‡Ž•ǡ ƒ† ‘•‡“—‡–Ž› ƒ—•‡ ƒŒ‘” Š‡ƒŽ–Š ’”‘„Ž‡• ‹ Ž—†‹‰ ƒ”†‹‘˜ƒ• —Žƒ” †‹•‡ƒ•‡•ǡ •–”‘‡ǡ „Ž‹†‡••ǡ ‡—”‘†‡‰‡‡”ƒ–‹˜‡ †‹•‡ƒ•‡ǡ ƒ† ƒ ‡”ȏʹͳǡʹ͵ȂʹͷȐǤ‘ƒŽ ‘Š‘Ž‹ ˆƒ––›Ž‹˜‡” †‹•‡ƒ•‡ȋ ȌŠƒ•ƒ ’”‡˜ƒŽ‡ ‡‘ˆ‘˜‡” ʹͲΨǡƒ‹‰‹––Š‡‘•– ‘‘‹‡•–‡” ‘—–”‹‡•ȏʹ͸ȂʹͻȐǤŠ‡†‹•‡ƒ•‡ ƒ’”‘‰”‡•• ˆ”‘•‹’Ž‡Ž‹’‹†ƒ ——Žƒ–‹‘ȋ•–‡ƒ–‘•‹•Ȍ–‘‘ƒŽ ‘Š‘Ž‹ •–‡ƒ–‘Š‡’ƒ–‹–‹•ȋi.e.‹ˆŽƒƒ–‹‘ ‘ˆ–Š‡Ž‹˜‡”™‹–Š ‘ —””‡–ˆƒ–ƒ ——Žƒ–‹‘‹Ž‹˜‡”Ȍ–‘ ‹””Š‘•‹•–‘Š‡’ƒ–‘ ‡ŽŽ—Žƒ” ƒ” ‹‘ƒ ȏ͵ͲȂ͵͵ȐǤ‘‘ Šƒ”ƒ –‡”‹•–‹ •‹–Š‡•‡†‹•‡ƒ•‡•‹ Ž—†‡‡š ‡••‹˜‡‰Ž— ‘•‡ƒ†ˆƒ––›Ǧƒ ‹† •—’’Ž›–‘–Š‡Ž‹˜‡”ǡ”‡•—Ž–‹‰‹‹ˆŽƒƒ–‹‘ƒ†‘š‹†ƒ–‹˜‡•–”‡••ȏ͵ͶȂ͵͸ȐǤȾǦ ‡ŽŽ•–Šƒ–ƒ”‡ Š”‘‹ ƒŽŽ›‡š’‘•‡†–‘Š‹‰Š‰Ž— ‘•‡ƒ†ˆ”‡‡ˆƒ––›ƒ ‹†Ž‡˜‡Ž•ǡ•Š‘™Š‹‰Š‡””‡ƒ –‹˜‡‘š›‰‡ •’‡ ‹‡•ȋȌ’”‘†— –‹‘Ǥ‹–—””‡†— ‡•‹•—Ž‹‰‡‡‡š’”‡••‹‘ƒ†‹•—Ž‹ ‘–‡–ǡ ƒ†ƒ –‹˜ƒ–‡•— ‘—’Ž‹‰’”‘–‡‹ʹȋʹȌǤŠ‡Žƒ––‡”Ž‘™‡”•’”‘†— –‹‘ƒ†‹Š‹„‹–• ‰Ž— ‘•‡Ǧ•–‹—Žƒ–‡†‹•—Ž‹•‡ ”‡–‹‘ȏ͵͹ȐǤ˜‡”‡š’”‡••‹‘‘ˆƒ–‹‘š‹†ƒ–‡œ›‡•Šƒ•„‡‡ ”‡’‘”–‡†–‘’”‘–‡ –ȾǦ ‡ŽŽ•ˆ”‘†›•ˆ— –‹‘ȏ͵͹ȐǤƒŽ•‘‹†— ‡•‹•—Ž‹”‡•‹•–ƒ ‡‘ˆ–ƒ”‰‡– –‹••—‡•„›‹Š‹„‹–‹‰‹•—Ž‹•‹‰ƒŽ–”ƒ•†— –‹‘ȏ͵ͺǡ͵ͻȐǤ ‘”†‹‰Ž›ǡŠ‹‰ŠǦˆƒ–Ǧ‹†— ‡†‹•—Ž‹ ”‡•‹•–ƒ ‡ Šƒ• „‡‡ ’”‡˜‡–‡† „› ”‡†— ‹‰  –Š”‘—‰Š ƒ–‹‘š‹†ƒ– –”‡ƒ–‡– ‘” ‘˜‡”‡š’”‡••‹‘‘ˆƒ–‹‘š‹†ƒ–‡œ›‡•ȏ͵͹ȐǤš‹†ƒ–‹˜‡•–”‡••ƒŽ•‘’Žƒ›•ƒ‹’‘”–ƒ–”‘Ž‡‹ Ž‹˜‡”†‹•‡ƒ•‡’”‘‰”‡••‹‘ȏͶͲȐǤŠ‹•‹•–‘„‡‡š’‡ –‡†ǡƒ•–Š‡Ž‹˜‡”Šƒ”„‘”•ƒŽ‘–‘ˆ’”‘ ‡••‡•‹ ™Š‹ Š  ‹• ‰‡‡”ƒ–‡†ǡ ‹ Ž—†‹‰ ’”‘–‡‹ •›–Š‡•‹• ȏͶͲȐǡ ‹–‘ Š‘†”‹ƒŽ ”‡•’‹”ƒ–‹‘ ƒ† †‡–‘š‹ˆ‹ ƒ–‹‘ȏͶͲȐǤš ‡••‹˜‡•—’’Ž›‘ˆˆƒ––›ƒ ‹†•‹‘„‡•‹–›™‘—Ž†‘˜‡”™Š‡Ž–Š‡‡Ž‡ –”‘ –”ƒ•’‘”– Šƒ‹ǡ Ž‡ƒ†‹‰ –‘ ‡š ‡••‹˜‡  ’”‘†— –‹‘ ƒ† ‹–‘ Š‘†”‹ƒŽ †›•ˆ— –‹‘Ǥ ‹–‘ Š‘†”‹ƒŽ†›•ˆ— –‹‘™‘—Ž†‹–—”ƒ’Ž‹ˆ›‡š ‡••‹˜‡’”‘†— –‹‘”‡•—Ž–‹‰‹Ž‹’‹† ƒ† ’”‘–‡‹ ’‡”‘š‹†ƒ–‹‘ ƒ† †‡”‡‰—Žƒ–‹‘ ‘ˆ Š‡’ƒ–‹  ‡–ƒ„‘Ž‹• ‹ ‰‡‡”ƒŽǤ  ƒ††‹–‹‘ǡ

ͳͳ Chapter 1

‡š ‡••‹˜‡‹†— ‡•‹ˆŽƒƒ–‘”› ›–‘‹‡•ǡ™Š‹ Š‹–—”‡šƒ ‡”„ƒ–‡’”‘†— –‹‘ƒ† ‹ˆŽƒƒ–‹‘ȏͳͷǡͶͲǡͶͳȐǤ—’’Ž‡‡–ƒ–‹‘‘ˆ–Š‡ƒ–‹‘š‹†ƒ–˜‹–ƒ‹–‘ ’ƒ–‹‡–•ǡ Šƒ•„‡‡•Š‘™–‘ƒ‡Ž‹‘”ƒ–‡ ƒ–•‡˜‡”ƒŽ•–ƒ‰‡•‘ˆ•‡˜‡”‹–›ȏͳͷȐǤ‰‹‰ƒŽ•‘ƒ‡•ƒ ‹†‹˜‹†—ƒŽ ‘”‡ ’”‘‡ –‘ ‡–ƒ„‘Ž‹  †‹•‡ƒ•‡•ǡ ‹ Ž—†‹‰ Ž‘•• ‘ˆ —• Ž‡ ƒ•• ȋ•ƒ” ‘’‡‹ƒȌǡ ʹǡ ƒ† ƒ”†‹‘˜ƒ• —Žƒ”†‹•‡ƒ•‡ȏͶʹȐǤ ƒ††‹–‹‘ǡ‘–Š‡”‡–ƒ„‘Ž‹–‡•ƒ”‡ƒŽ•‘‡‡”‰‹‰ƒ•”‡Ž‡˜ƒ– ‘–”‹„—–‘”•‹‡–ƒ„‘Ž‹ †‹•‡ƒ•‡ǡ ‹ Ž—†‹‰•’‡ ‹ˆ‹ –›’‡•‘ˆŽ‹’‹†•ǡΪǡ ‘‡œ›‡ƒ†ƒ‹‘ƒ ‹†•Ǥ‡Ž‘™ǡ ™‹ŽŽ†‹• —•• ‡ƒ Š‘ˆ–Š‡•‡ȋ Žƒ••‡•‘ˆȌ‡–ƒ„‘Ž‹–‡•‹‘”‡†‡–ƒ‹ŽǤ  Metabolites and common acquired metabolic disease regulators ‹’‹†• ƒ”‡ ’”‡†‘‹ƒ–Ž› ‘™ ƒ• ‡†‹ƒ–‘”• ‘ˆ –‘š‹ ‹–› ƒ† ‹ˆŽƒƒ–‹‘ ‹ ‡–ƒ„‘Ž‹  †‹•‡ƒ•‡Ǥ ‡ ‡–Ž› Š‘™‡˜‡”ǡ •–—†‹‡•Šƒ˜‡ ‹†‡–‹ˆ‹‡† Ž‹’‹† Žƒ••‡• –Šƒ– ƒ’’‡ƒ” –‘ ˆ— –‹‘ ‹ •‹‰ƒŽ‹‰ƒ† ƒ‹†— ‡ƒ–‹Ǧ‹ˆŽƒƒ–‘”›‡ Šƒ‹••Ǥ‹’‘‹‡•ǡˆ‘”‹•–ƒ ‡ǡ ƒˆ— –‹‘ ƒ••‘Ž—„Ž‡•‹‰ƒŽ•‘—–•‹†‡–Š‡ ‡ŽŽ•Ǥ‡šƒ’Ž‡‹•ͳ͸ǣͳ͹Ǧ’ƒŽ‹–‘Ž‡ƒ–‡ȏͶ͵ǡͶͶȐǤƒŽ‹–‘Ž‡ƒ–‡ •›–Š‡•‹•‹•‹ ”‡ƒ•‡†ƒ†‹–•„Ž‘‘†Ž‡˜‡Ž•”‹•‡ƒ•ƒ”‡•—Ž–‘ˆ†‡‘˜‘Ž‹’‘‰‡‡•‹•‹ƒ†‹’‘•‡ –‹••—‡ǤŠ‹•Ž‹’‹†‹•ƒ„Ž‡–‘•—’’”‡••‹ˆŽƒƒ–‹‘ƒ†Ž‹˜‡”Ž‹’‘‰‡‡•‹•ƒ†•–‹—Žƒ–‡•‰Ž— ‘•‡ —’–ƒ‡„›•‡Ž‡–ƒŽ—• Ž‡ȏͶ͵ȂͶ͸ȐǤ ƒ ”‘’Šƒ‰‡•‹–ƒŽŽ‡˜‹ƒ–‡•Ž‹’‘–‘š‹ ‹–›Ǧ‹†— ‡†•–”‡•• ƒ† ‡ŽŽ †‡ƒ–ŠǤ ‘–Š‡” ‡†‘‰‡‘—• Ž‹’‹† Žƒ•• ‘•‹•–• ‘ˆ ˆƒ––›Ǧƒ ‹† Š›†”‘š›Ǧˆƒ––› ƒ ‹†• ȋ  •ȌǤŠ‡•‡  •™‡”‡‹†‡–‹ˆ‹‡†„›Ž‹’‹†‘‹ ƒƒŽ›•‹•‘ˆƒ†‹’‘•‡–‹••—‡ƒ†‹–Š‡ ‹” —Žƒ–‹‘ ‘ˆ ‹ ‡ ™‹–Š ‹ ”‡ƒ•‡† de novo Ž‹’‘‰‡‡•‹•Ǥ  •—„ ƒ–‡‰‘”› ‘ˆ –Š‡•‡  •ǡ ’ƒŽ‹–‹ Ǧƒ ‹†ǦŠ›†”‘š›Ǧ•–‡ƒ”‹ Ǧƒ ‹†• ȋ •Ȍǡ Ž‘™‡” „Ž‘‘† ‰Ž— ‘•‡ǡ ‹’”‘˜‡ ‰Ž— ‘•‡ –‘Ž‡”ƒ ‡ǡ •–‹—Žƒ–‡ ‹•—Ž‹ •‡ ”‡–‹‘ǡ ”‡†— ‡ ƒ†‹’‘•‡ –‹••—‡ ‹ˆŽƒƒ–‹‘ ƒ† ‹’”‘˜‡ ‹•—Ž‹ –‘Ž‡”ƒ ‡ ȏͶ͹ȐǤ ‹ƒŽŽ›ǡ ”‡•‘Ž˜‹• ƒ† ’”‘–‡ –‹•ǡ „‘–Š ‘ˆ ™Š‹ Š ƒ”‡ ’”‘†— ‡† „› ƒ–ƒ„‘Ž‹•‘ˆ‘‡‰ƒǦ͵ˆƒ––›Ǧƒ ‹†•ǡŠƒ˜‡„‡‡”‡’‘”–‡†–‘ƒŽŽ‡˜‹ƒ–‡‘„‡•‹–›Ǧ‹†— ‡†‹•—Ž‹ ”‡•‹•–ƒ ‡ƒ†Š‡’ƒ–‹ •–‡ƒ–‘•‹•ȏͶͺȂͷͲȐǤ  NAD+  Ϊ†‘‡•‘–‘Ž›’Žƒ›ƒ ‡–”ƒŽ”‘Ž‡ƒ•ƒ”‡†‘š‹–‡”‡†‹ƒ–‡‹‡‡”‰› ƒ†ƒ‹‘ƒ ‹† ‡–ƒ„‘Ž‹•ǡ „—– ‹– ‹• ƒŽ•‘ ‹˜‘Ž˜‡† ‹ †‡–‘š‹ˆ‹ ƒ–‹‘ǡ ƒŽ ‹— –”ƒ•’‘”–ǡ  ”‡’ƒ‹”ǡ ƒ† ’”‘–‡‹ †‡ƒ ›Žƒ–‹‘ ȏͷͳȂͷ͵ȐǤ –• ’Š‘•’Š‘”›Žƒ–‡† ˆ‘”ǡ Ϊǡ ‹• ‡••‡–‹ƒŽ ˆ‘” Ž‹’‹† „‹‘•›–Š‡•‹•ȏͷʹȂͷͶȐǤ ‰‡‡”ƒŽǡƒ‹ ”‡ƒ•‡‹ΪŽ‡˜‡Ž•‹’”‘˜‡•‡–ƒ„‘Ž‹ Š‘‡‘•–ƒ•‹• ƒ†’”‡˜‡–•‘”ƒŽŽ‡˜‹ƒ–‡•‡–ƒ„‘Ž‹ †‹•‡ƒ•‡ǤΪ’Žƒ›•ƒ’”‘–‡ –‹˜‡”‘Ž‡‹ʹǡƒ•ΪǦ ‡†‹ƒ–‡† •‹”–—‹ͳ ȋ ͳȌ •‹‰ƒŽ‹‰ ‹’”‘˜‡• Ž‹˜‡” ƒ† —• Ž‡ ‹•—Ž‹ •‡•‹–‹˜‹–› ƒ† ‰Ž— ‘•‡Ǧ•–‹—Žƒ–‡†‹•—Ž‹•‡ ”‡–‹‘„›ȾǦ ‡ŽŽ•Ǥ ͳ•‹‰ƒŽ‹‰ƒŽ•‘’”‘‘–‡•‹–‘ Š‘†”‹ƒŽ „‹‘‰‡‡•‹•Ǥ†‹‹•–”ƒ–‹‘‘ˆΪ’”‡ —”•‘”•Šƒ˜‡•‹‹Žƒ”‡ˆˆ‡ –•ƒ† ƒ‡˜‡ƒ––‡—ƒ–‡ Š‹‰ŠǦˆƒ–Ǧ†‹‡–Ǧ‹†— ‡†‘„‡•‹–›‹‹ ‡ȏͶʹȐǤΪƒ† ͳ’Žƒ›ƒ’”‘–‡ –‹˜‡‘”ƒ‡Ž‹‘”ƒ–‹‰ ”‘Ž‡ ‹  Ǥ š ‡•• Ž‹’‹† •—’’Ž› ‹ –Š‡ Ž‹˜‡” †‡ ”‡ƒ•‡• Š‡’ƒ–‹  Ϊ Ž‡˜‡Ž•ǡ ™Š‹Ž‡ ‘˜‡”‡š’”‡••‹‘ ‘ˆ  ͳ ”‡˜‡”•‡• Š‡’ƒ–‹  •–‡ƒ–‘•‹• ƒ† Ϊ ’”‡ —”•‘”• ƒ––‡—ƒ–‡ ‹–‘ Š‘†”‹ƒŽ†›•ˆ— –‹‘‹ˆƒ––›Ž‹˜‡”‘ˆŠ‹‰ŠǦˆƒ–Ǧ†‹‡–Ǧˆ‡†‹ ‡ȏͶʹȐǤΪŠƒ•ƒŽ•‘‰ƒ‹‡† ‘•‹†‡”ƒ„Ž‡ƒ––‡–‹‘†—‡–‘‹–•”‘Ž‡‹ƒ––‡—ƒ–‹‰ƒ‰‹‰Ǧ”‡Žƒ–‡†‡–ƒ„‘Ž‹ †‹•‡ƒ•‡•ǤΪ †‡ ”‡ƒ•‡•†—”‹‰ƒ‰‹‰ƒ•ƒ”‡•—Ž–‘ˆ†ƒƒ‰‡ǡŠ‹‰Š‡”‡š’”‡••‹‘‘ˆ–Š‡ΪǦ†‡‰”ƒ†‹‰ ‡œ›‡͵ͺǡƒ† Š”‘‹ ‹ˆŽƒƒ–‹‘ǤŠ‡Žƒ––‡””‡†— ‡•–Š‡ΪǦ”‡‰‡‡”ƒ–‹‰ ƒ’ƒ ‹–›Ǥ

ͳʹ General introduction and outline of the thesis

SIRT1‘˜‡”‡š’”‡••‹‘‘”ƒ†‹‹•–”ƒ–‹‘‘ˆΪ’”‡ —”•‘”•Šƒ˜‡„‡‡”‡’‘”–‡†–‘’”‘–‡ – ƒ‰ƒ‹•–ƒ‰‡Ǧ”‡Žƒ–‡††‹ƒ„‡–‡•ǡ ƒ ‡”ǡƒ†‹–‘ Š‘†”‹ƒŽ†›•ˆ— –‹‘ȏͶʹȐǤ 1  Coenzyme A ‘‡œ›‡Šƒ•ƒŽ•‘‰ƒ‹‡†”‡‡™‡†‹–‡”‡•–‹–Š‡‡–ƒ„‘Ž‹ ˆ‹‡Ž†‹”‡ ‡–›‡ƒ”•ȏͷͷȂ͸ͲȐǤ ‘ƒ –‹˜ƒ–‡•ˆƒ––›ƒ ‹†•ˆ‘”‘š‹†ƒ–‹‘ƒ† Šƒ‹‡Ž‘‰ƒ–‹‘Ǥ –‹•ƒŽ•‘ƒ‡••‡–‹ƒŽ ‘‡œ›‡ ˆ‘”de novoŽ‹’‹†•›–Š‡•‹•ǡ†‡–‘š‹ˆ‹ ƒ–‹‘‘ˆš‡‘„‹‘–‹ •ǡ–Š‡ › Ž‡ǡƒ†–Š‡ ƒ–ƒ„‘Ž‹•‘ˆ ƒ‹‘ƒ ‹†•Ǥ –Š‡•‡’ƒ–Š™ƒ›•ǡ‘ ƒ„‡‹˜‘Ž˜‡†ƒ•ˆ”‡‡‘ȋˆ”‘Š‡”‡‘”‡ˆ‡””‡†–‘ƒ• ‘ ǡ„‡ ƒ—•‡‘ˆ‹–• Šƒ”ƒ –‡”‹•–‹ –Š‹‘ŽȋȂ Ȍ‰”‘—’Ȍ‘”ƒ•ƒ–Š‹‘‡•–‡”‹–‡”‡†‹ƒ–‡Ǥ –™ƒ• ”‡ ‡–Ž› •Š‘™ –Šƒ– ‘ Ž‡˜‡Ž• ƒˆˆ‡ – –Š‡ ’Š‡‘–›’‡ ‘ˆ ‘„‡•‹–› ƒ† ʹǤ ‘” ‡šƒ’Ž‡ǡ †‡Ž‡–‹‘‘ˆ–Š‡‘„‹‘•›–Š‡•‹•‡œ›‡PANK1 ƒ—•‡•Ž‘™‡”•‡”—‹•—Ž‹Ž‡˜‡Ž•ǡŠ‹‰Š‡” ‰Ž— ‘•‡–‘Ž‡”ƒ ‡ƒ†‹•—Ž‹•‡•‹–‹˜‹–›ǡƒ†Ž‘™‡”ˆƒ––›Ǧƒ ‹†‘š‹†ƒ–‹‘”ƒ–‡•‹‹ ‡ȏͷͷȐǤ Š‡ob/obʹ‘—•‡‘†‡ŽŠƒ•†‡”‡‰—Žƒ–‡†‘‡–ƒ„‘Ž‹•ƒ†ƒ„‘”ƒŽŽ›Š‹‰ŠŽ‹˜‡” ‘ Ž‡˜‡Ž•ǡ ™Š‹ Š ‘‹ ‹†‡• ™‹–Š Š‹‰Š „Ž‘‘† ‰Ž— ‘•‡ ƒ† ‹•—Ž‹ Ž‡˜‡Ž• ȋŠ›’‡”‰Ž› ‡‹ƒȌǡ ‹•—Ž‹”‡•‹•–ƒ ‡ƒ†‹ ”‡ƒ•‡†‰Ž— ‘‡‘‰‡‡•‹•ȏ͸ͳǡ͸ʹȐǤ‡Ž‡–‹‘‘ˆPANK1‹–Š‹•‘—•‡ ‘†‡Žƒ––‡—ƒ–‡†–Š‡†‹ƒ„‡–‹ ’Š‡‘–›’‡™‹–ŠŽ‘™‡”–‘–ƒŽ‘Ž‡˜‡Ž•ǡ”‡†— ‡†‹–‘ Š‘†”‹ƒŽ ȾǦ‘š‹†ƒ–‹‘ ƒ† ‰Ž— ‘‡‘‰‡‡•‹•ǡ ‘”ƒŽ‹œ‡† ‰Ž— ‘•‡ Ž‡˜‡Ž•ǡ ƒ† Ž‘™‡” ‹•—Ž‹ Ž‡˜‡Ž•Ǥ Š‡ ‹ ‡ǡŠ‘™‡˜‡”ǡ”‡ƒ‹‡†‘„‡•‡ǡ‹•—Ž‹”‡•‹•–ƒ–ƒ†Š‡’ƒ–‘•–‡ƒ–‘–‹ ȏͷͷȐǤŠ‹••—‰‰‡•–•–Šƒ– Ž‘™‡”‹‰ –Š‡ ‘ Ž‡˜‡Ž• ƒ† ˆƒ––›Ǧƒ ‹† ‘š‹†ƒ–‹‘ ”ƒ–‡ ƒ› Šƒ˜‡ •‹’Ž› ”‡†— ‡† –Š‡ ‰Ž— ‘‡‘‰‡‡•‹•”ƒ–‡ƒ†–Š‡”‡„›–Š‡‹•—Ž‹’”‘†— –‹‘ǡ™Š‹ Š†‘‡•‘– ‘’Ž‡–‡Ž›”‡•‘Ž˜‡ –Š‡ˆƒ––›Ǧƒ ‹†‡š ‡••‹••—‡•ƒ†‹‰Š–‡˜‡‡šƒ ‡”„ƒ–‡Ž‹’‹†–‘š‹ ‹–›Ǧ”‡Žƒ–‡†’”‘„Ž‡•Ǥ ‘–Š‡” ’Žƒ›‡” ‹ ‘‡œ›‡  ‡–ƒ„‘Ž‹• ‹˜‘Ž˜‡† ‹ ‡–ƒ„‘Ž‹  †‹•‡ƒ•‡ ‹• ƒ‹ ͳǤ – ƒ–ƒŽ›œ‡• –Š‡ †‡‰”ƒ†ƒ–‹‘ ‘ˆ ’ƒ–‡–Š‡‹‡ǡ ƒ ‹–‡”‡†‹ƒ–‡ ‹ ‘ „‹‘•›–Š‡•‹• ƒ†

†‡‰”ƒ†ƒ–‹‘ǡ –‘ ’ƒ–‘–Š‡ƒ–‡ ȋ‹–ƒ‹ ͷȌǡ ƒ ‘‡œ›‡  •›–Š‡•‹• ’”‡ —”•‘” ƒ†  •—„•–”ƒ–‡Ǥ„›’”‘†— –‘ˆ–Š‹•”‡ƒ –‹‘‹• ›•–‡ƒ‹‡ǡƒƒ–‹‘š‹†ƒ–Ǥƒ‹ͳƒ’’‡ƒ”•–‘Šƒ˜‡ „‘–Š‹ˆŽƒƒ–‘”›ƒ† ›–‘’”‘–‡ –‹˜‡‡ˆˆ‡ –•ǡ†‡’‡†‹‰‘–Š‡ ‘†‹–‹‘•ȏ͸͵ȐǤ ”‡Žƒ–‹‘ –‘ʹǡƒ‹ͳ™ƒ•ƒ„Ž‡–‘’”‘–‡ –’ƒ ”‡ƒ–‹ ‹•Ž‡–„‡–ƒ ‡ŽŽ•ƒ‰ƒ‹•–ˆ”‘•–”‡’–‘œ‘–‘ ‹Ǧ ‹†— ‡††‡ƒ–Šin vitroǡƒ†‹–•†‡Ž‡–‹‘Šƒ•„‡‡ƒ••‘ ‹ƒ–‡†™‹–ŠŠ‹‰Š‡”‹ ‹†‡ ‡‘ˆ†‹ƒ„‡–‡• ƒ†‘”‡•‡˜‡”‡‹ˆŽƒƒ–‘”›‹ˆŽƒƒ–‹‘‘ˆ–Š‡‹•Ž‡–•‘ˆƒ‰‡”Šƒ•ȋ‹•—Ž‹–‹•Ȍ‹–Š‡‘Ǧ ‘„‡•‡†‹ƒ„‡–‹ ȋȌ‘—•‡‘†‡Žȏ͸ͶȐǤ  Amino acids Ž”‡ƒ†›•‡˜‡”ƒŽ†‡ ƒ†‡•ƒ‰‘ǡ‡Ž‡˜ƒ–‡†•‡”—ƒ‹‘ƒ ‹†Ž‡˜‡Ž•Šƒ˜‡„‡‡ƒ••‘ ‹ƒ–‡†™‹–Š ‘„‡•‹–›ƒ†‹•—Ž‹”‡•‹•–ƒ ‡ȏ͸ͷȐǤ ‘™‡˜‡”ǡ”‡ ‡–ƒ†˜ƒ ‡•‹‡–ƒ„‘Ž‘‹ •–‡ Š‹“—‡• Šƒ˜‡•’ƒ”‡†”‡‡™‡†‹–‡”‡•–‹–Š‡”‡Žƒ–‹‘„‡–™‡‡ƒ‹‘ƒ ‹†•ƒ†‡–ƒ„‘Ž‹ †‹•‡ƒ•‡Ǥ ƒ”–‹ —Žƒ”Ž›ǡ ‹” —Žƒ–‹‰„”ƒ Š‡†Ǧ Šƒ‹ƒ‹‘ƒ ‹†•ȋ•Ȍǡ‡–Š‹‘‹‡ǡƒ†–Š‡ƒ”‘ƒ–‹  ƒ‹‘ƒ ‹†•’Š‡›ŽƒŽƒ‹‡ƒ†–”›’–‘’ŠƒŠƒ˜‡„‡‡Ž‹‡†–‘‘„‡•‹–›ƒ†‹•—Ž‹”‡•‹•–ƒ ‡Ǥ ‹ƒŽ‘†‡Ž•ƒ†Š—ƒ•™‹–Š‘„‡•‹–›ƒ†‹•—Ž‹”‡•‹•–ƒ ‡•Š‘™Ž‘™‡”Ž‡˜‡Ž•‘ˆ‡œ›‡• ‹˜‘Ž˜‡†‹ ƒ–ƒ„‘Ž‹•‹™Š‹–‡ƒ†‹’‘•‡–‹••—‡ȋȌƒ†Ž‘™‡”Š‡’ƒ–‹ ƒ –‹˜‹–›‘ˆ–Š‡ •‡ ‘†‡œ›‡‹–Š‡ ƒ–ƒ„‘Ž‹•’ƒ–Š™ƒ›ǡ†‡Š›†”‘‰‡ƒ•‡ ‘’Ž‡šȋ ȌǤ  ƒ††‹–‹‘ǡ‹– ™ƒ• ”‡’‘”–‡† –Šƒ–  ‘˜‡”Ž‘ƒ† ‹ ”‡ƒ•‡• ƒ ›ŽǦ ƒ”‹–‹‡ƒ ——Žƒ–‹‘ ‹ ”ƒ– —• Ž‡ ȏͶͶǡ͸͸ȐǤ ‡–Š‹‘‹‡ ”‡•–”‹ –‹‘ ™ƒ• ”‡’‘”–‡† –‘ ”‡†— ‡ ƒ†‹’‘•‹–›ǡ ‹ ”‡ƒ•‡ ‹•—Ž‹ •‡•‹–‹˜‹–›ƒ†ƒ––‡—ƒ–‡Š‡’ƒ–‘•–‡ƒ–‘•‹•‹‹ ‡ȏͶͶȐǤŠ‡›ŽƒŽƒ‹‡ƒ†–”›’–‘’Šƒƒ’’‡ƒ” –‘„‡’”‡†‹ –‹˜‡ƒ”‡”•ˆ‘”ʹȏͶͶȐǤ 

ͳ͵ Chapter 1

Advances in approaches and methods to study acquired metabolic diseases Bioinformatic approaches Š‡‘–›’‡Ǧ ‘” –”ƒ‹–Ǧ”‡Žƒ–‡† „‹‘‹ˆ‘”ƒ–‹ • ƒ’’”‘ƒ Š‡•ǡ •— Š ƒ• ‰‡‘‡Ǧ™‹†‡ ƒ••‘ ‹ƒ–‡† •–—†‹‡• ȋ Ȍǡ ƒ”‡ ‘ˆ–‡ —•‡† –‘ †‹• ‘˜‡” ‡ Šƒ‹•• —†‡”Ž›‹‰ ƒ “—‹”‡† ‡–ƒ„‘Ž‹  †‹•‡ƒ•‡ȏ͸͹ȐǤ‹–Š‡˜‡”Ǧ‰”‘™‹‰•ƒ’Ž‡•‹œ‡•‹’‘’—Žƒ–‹‘•–—†‹‡•ƒ†™‹–Š–Š‡†‡˜‡Ž‘’‡– ‘ˆˆƒ•–‡”ƒ†‘”‡†‡–ƒ‹Ž‡†•‡“—‡ ‹‰–‡ Š‹“—‡•ǡ‘”‡‰‡‡Ǧ–”ƒ‹–ƒ••‘ ‹ƒ–‹‘•Šƒ˜‡„‡‡ ˆ‘—†ǤŠ‡•‡ƒ••‘ ‹ƒ–‹‘•ǡŠ‘™‡˜‡”ǡ†‘‘–‡ ‡••ƒ”‹Ž›‹’Ž›„‹‘Ž‘‰‹ ƒŽˆ— –‹‘‘” ƒ—•ƒŽ‹–›Ǥ ††‹–‹‘ƒŽ‡š’‡”‹‡–•ƒ†‡–Š‘†•™‘—Ž†„‡”‡“—‹”‡†–‘—”ƒ˜‡Ž‡ Šƒ‹••—†‡”Ž›‹‰ –Š‡‹˜‡•–‹‰ƒ–‡†–”ƒ‹–•ȏ͸͹ȐǤ‡ ‡–ƒ†˜ƒ ‡•Šƒ˜‡„‡‡ƒ†‡‹–Š‡—•‡‘ˆƒ††‹–‹‘ƒŽDzǦ‘‹ •dz †ƒ–ƒ ‹ Ǥ —”–Š‡”‘”‡ǡ •‘Ǧ ƒŽŽ‡† Dz’ƒ–Š™ƒ› ƒƒŽ›•‹•dz ƒŽŽ‘™• „‹‘Ž‘‰‹ ƒŽ ‹–‡”’”‡–ƒ–‹‘ „ƒ•‡† ‘ „‹‘Ž‘‰‹ ƒŽ ’ƒ–Š™ƒ›Ǧ ǡ ‡–™‘”•Ǧǡ ƒ† ‰‡‡ ‘–‘Ž‘‰‹‡• ȋ •Ȍ ƒ†Ȁ‘” †‹•‡ƒ•‡ „‹‘ƒ”‡”‰‡‡•‡–•ȏ͸ͺȐǤ  Experimental models ™‹†‡”ƒ‰‡‘ˆ‡š’‡”‹‡–ƒŽ‘†‡Ž•Šƒ˜‡„‡‡†‡˜‡Ž‘’‡†‘˜‡”–Š‡›‡ƒ”•–‘•–—†›ƒ “—‹”‡† ‡–ƒ„‘Ž‹ †‹•‡ƒ•‡•Ǥ‘•–™‹†‡Ž›—•‡†ƒ”‡‘—•‡‘†‡Ž•ƒ†in vitro ‡ŽŽ —Ž–—”‡‘†‡Ž•Ǥ ‘” ‡šƒ’Ž‡ǡ†‹‡–ƒ”›ǡ Š‡‹ ƒŽƒ†‰‡‡–‹ ‘—•‡‘†‡Ž•Šƒ˜‡„‡‡†‡˜‡Ž‘’‡†ˆ‘”„‘–Šʹ ƒ†   ȏͳͶǡ͸ͻǡ͹ͲȐǤ ‡–ƒ„‘Ž‹  †‹•‡ƒ•‡ ‘”‹‰‹ƒ–‡• ˆ”‘ ƒ ‘’Ž‡š ‹–‡”’Žƒ› „‡–™‡‡ †‹ˆˆ‡”‡–‘”‰ƒ‡ŽŽ‡•ǡ ‡ŽŽ•ƒ†–‹••—‡•‘ˆƒ‘”‰ƒ‹•ǡ™Š‹ Š‘ˆ–‡†‡˜‡Ž‘’•‘˜‡”–‹‡–‘ƒ ’ƒ–Š‘Ž‘‰‹ ƒŽ’”‡•‡–ƒ–‹‘ǤŠ‡”‡ˆ‘”‡ǡ•–—†›‹‰–Š‡™Š‘Ž‡‘—•‡ƒ•‘’’‘•‡†–‘‹†‹˜‹†—ƒŽ ‡ŽŽ• ‘” „‘†› ˆŽ—‹†•ǡ ƒ› ‘ˆˆ‡” ‹•‹‰Š– ‹–‘ ‡ Šƒ‹•• —†‡”Ž›‹‰ ‡–ƒ„‘Ž‹  †‹•‡ƒ•‡ –Šƒ– ƒ”‡ —””‡–Ž›—ƒ––ƒ‹ƒ„Ž‡‹Š—ƒ•Ǥ ‘™‡˜‡”ǡ‘—•‡‘†‡Ž•‹‰Š–‘–„‡ˆ—ŽŽ›–”ƒ•Žƒ–ƒ„Ž‡–‘ Š—ƒ• ȏͳͶǡ͸ͻȐǤ ‘” ‡šƒ’Ž‡ǡ •‡˜‡”ƒŽ   ‘—•‡ ‘†‡Ž• •Š‘™‡† ‰‡‡ ‡š’”‡••‹‘ ’ƒ––‡”•–Šƒ–™‡”‡•‘†‹ˆˆ‡”‡–ˆ”‘–Š‘•‡‘ˆŠ—ƒ•’ƒ–‹‡–•–Šƒ–‘ ‘ Ž—•‹‘•”‡‰ƒ”†‹‰ †‹•‡ƒ•‡•–ƒ‰‡ ‘—Ž†„‡–”ƒ•Žƒ–‡†ˆ”‘‹ ‡–‘Š—ƒ•ȏ͹ͳȐǤ‘”‡”‡ ‡–Ž›ǡŠ—ƒin vitro ƒ† ex vivo ‡ŽŽ ‘” –‹••—‡ ‘†‡Ž•ǡ ™Š‡”‡ ƒ”‡ ‹• –ƒ‡ –‘ —’Š‘Ž† –Š‡ †‹ˆˆ‡”‡–‹ƒ–‡† ‘”‰ƒ ˆ— –‹‘ǡ Šƒ˜‡ ƒŽ•‘ „‡‡ —•‡† –‘ •–—†› ƒ “—‹”‡† ‡–ƒ„‘Ž‹  †‹•‡ƒ•‡ ȏ͸ͻǡ͹ʹȐǤ Š‡•‡ ‹ Ž—†‡ –‹••—‡ •Ž‹ ‡•ǡ —Ž–—”‡† ’”‹ƒ”› Š—ƒ ‡ŽŽ•ǡ ‘Ǧ —Ž–—”‡ ‘ˆ †‹ˆˆ‡”‡– ‡ŽŽ –›’‡• ‘ˆ ƒ •’‡ ‹ˆ‹  –‹••—‡ǡ’ƒ–‹‡–Ǧ†‡”‹˜‡†Š—ƒ’Ž—”‹’‘–‡–•–‡ ‡ŽŽ•ȋŠ•Ȍƒ†‰‡‘‡Ǧ‡†‹–‡†Š—ƒ ‡ŽŽ• ȏ͸ͻǡ͹ʹȂ͹͸ȐǤ  Analytical methods and data analysis –Š‡’ƒ•–†‡ ƒ†‡•ǡƒŽ•‘ƒŒ‘”ƒ†˜ƒ ‡•™‡”‡ƒ†‡‹–Š‡ƒƒŽ›–‹ ƒŽˆ‹‡Ž†ǤŠ‹•Šƒ•‡ƒ„Ž‡† Dz—Ž–‹Ǧ‘‹ •dz ”‡•‡ƒ” Šǡ ™Š‹ Š ‡ ‘’ƒ••‡• Žƒ”‰‡Ǧ• ƒŽ‡ ƒƒŽ›•‹• ‘ˆ †‹ˆˆ‡”‡– Ž‡˜‡Ž• ‘ˆ ”‡‰—Žƒ–‹‘ ȏͳͶǡʹͳǡ͸ͻǡ͹͹ȂͺͲȐǤ ‘–ƒ„Ž›ǡ  •‡“—‡ ‹‰ ƒ† ƒ•• •’‡ –”‘‡–”› ȋȌ –‡ Š‹“—‡•—•‡†ˆ‘”’”‘–‡‘‹ •ǡŽ‹’‹†‘‹ •ƒ†‡–ƒ„‘Ž‘‹ •Šƒ˜‡‡ƒ„Ž‡†Š‹‰ŠǦ–Š”‘—‰Š’—–ǡ “—ƒ–‹–ƒ–‹˜‡ †‡–‡”‹ƒ–‹‘ ‘ˆ ‰‡‡ ‡š’”‡••‹‘ ƒ† ’”‘–‡‹ǡ •ƒŽŽ ‡–ƒ„‘Ž‹–‡ ƒ† Ž‹’‹† ‘ ‡–”ƒ–‹‘•ȏͷǡ͸ǡ͹͹Ȃͺ͵ȐǤ‘•‹†‡”‹‰–Š‡Žƒ”‰‡†ƒ–ƒ•‡–•‰‡‡”ƒ–‡†„›–Š‡•‡ –‡ Š‹“—‡•ǡ ‡–Š‘†•™‡”‡ƒŽ•‘†‡˜‡Ž‘’‡†–‘‹–‡”’”‡––Š‡†ƒ–ƒƒ†–”ƒ•Žƒ–‡–Š‡–‘„‹‘Ž‘‰‹ ƒŽˆ— –‹‘Ǥ ‡šƒ’Ž‡ˆ‘”–Š‡‹–‡”’”‡–ƒ–‹‘‘ˆ•‡“—‡ ‹‰”‡•—Ž–•‹•‰‡‡Ǧ•‡–Ǧ‡”‹ Š‡–ƒƒŽ›•‹• ȋ ȌȏͺͶȐǤ ‡”‡‹ǡ–Š‡‡š’”‡••‹‘‘ˆ‰‡‡•‹•‡˜ƒŽ—ƒ–‡†ƒ––Š‡Ž‡˜‡Ž‘ˆˆ— –‹‘ƒŽŽ›”‡Ž‡˜ƒ– sets‘ˆ‰‡‡•ǤŠ‡•‡•‡–• ‘–ƒ‹‰‡‡•–Šƒ–Šƒ˜‡ƒ„‹‘Ž‘‰‹ ƒŽˆ— –‹‘ǡ Š”‘‘•‘ƒŽŽ‘ ƒ–‹‘ ‘”ƒ••‘ ‹ƒ–‡†’Š‡‘–›’‡•‹ ‘‘Ǥ›‡˜ƒŽ—ƒ–‹‰‰‡‡•‡–•ǡ„‹‘Ž‘‰‹ ƒŽˆ— –‹‘• ƒ„‡ ‘”‡‡ƒ•‹Ž›Ž‹‡†–‘‘Ž‡ —Žƒ”‡ Šƒ‹••Ǥ–Š‡”‘ˆ–‡—•‡†’ƒ––‡””‡ ‘‰‹–‹‘–‡ Š‹“—‡•

ͳͶ General introduction and outline of the thesis

ƒ”‡ ”‹ ‹’Ž‡ ‘’‘‡– ƒŽ›•‹• ȋȌǡ Š‹‡”ƒ” Š‹ ƒŽ Ž—•–‡”‹‰ ƒ† Žƒ••‹ˆ‹ ƒ–‹‘Ǥ ‘” ƒŽŽ –Š‡•‡ –›’‡• ‘ˆ †ƒ–ƒ ƒƒŽ›•‹•ǡ ƒ ”ƒ‰‡ ‘ˆ ‰”ƒ’Š‹ ƒŽǦ—•‡”Ǧ‹–‡”’Šƒ•‡ ȋ  Ȍ ‘Ž‹‡ –‘‘Ž• ƒ† 1 ˆ”‡‡Ž›Ǧ†‘™Ž‘ƒ†ƒ„Ž‡ •‘ˆ–™ƒ”‡ Šƒ• „‡‡ †‡˜‡Ž‘’‡†ǡ „ƒ•‡† ‘ ‘‘ ’”‘‰”ƒ‹‰ Žƒ‰—ƒ‰‡•ǡ•— Šƒ•ǤŠ‹•ƒ‡•’”‘ ‡••‹‰ƒ†‹–‡”’”‡–ƒ–‹‘‘ˆ‘‹ •†ƒ–ƒ‘”‡‡ƒ•‹Ž› ƒ ‡••‹„Ž‡ƒ†—•‡”Ǧˆ”‹‡†Ž›Ǥ‘’—Žƒ”•‘ˆ–™ƒ”‡ƒ†‘Ž‹‡–‘‘Ž•‹ Ž—†‡–Š‡Œƒ˜ƒǦ„ƒ•‡†  †‡•–‘’ •‘ˆ–™ƒ”‡ Ǧǡ ƒ† –Š‡ Ǧ„ƒ•‡† Ž—•–˜‹• ƒ† ‡–ƒ„‘ƒƒŽ›•– ȏͺͶȂͺ͹ȐǤ ‹ƒŽŽ›ǡ ‘’”‡Š‡•‹˜‡‘‹ •†ƒ–ƒ„ƒ•‡•ƒ”‡ƒ ”— ‹ƒŽƒ‹†‹‹–‡”’”‡–‹‰˜ƒ•–ƒ‘—–•‘ˆ‰‡‡”ƒ–‡† †ƒ–ƒǤ‡š––‘–Š‡™‹†‡Ž›‘™†ƒ–ƒ„ƒ•‡••— Šƒ• ȏͺͺȐǡ ‡ƒȏͺͻȐǡ—„ Š‡ȏͻͲȐ ƒ† ‹’”‘– ȏͻͳȐǡ –™‘ Š—ƒǦ•’‡ ‹ˆ‹  †ƒ–ƒ„ƒ•‡•ǡ –Š‡ Dz —ƒ ”‘–‡‹ –Žƒ•dz ȏͻʹȐ ƒ† –Š‡ Dz —ƒ‡–ƒ„‘Ž‘‡ƒ–ƒ„ƒ•‡dzȋ Ȍȏͻ͵ǡͻͶȐƒ”‡”‡Ž‡˜ƒ–ˆ‘”–Š‡‡–ƒ„‘Ž‹ †‹•‡ƒ•‡ˆ‹‡Ž†Ǥ Š‡Dz —ƒ”‘–‡‹–Žƒ•dz‹•ƒ‡ƒ•›–‘—•‡‡„Ǧ„ƒ•‡††ƒ–ƒ„ƒ•‡–Šƒ–’”‘˜‹†‡•ƒ ‘„‹ƒ–‹‘ ‘ˆ “—ƒ–‹–ƒ–‹˜‡  ‡š’”‡••‹‘Ǧ ƒ† ‹—‘Š‹•–‘ Š‡‹•–”›Ǧ„ƒ•‡† ”‡Žƒ–‹˜‡ ’”‘–‡‹ ‘ ‡–”ƒ–‹‘†ƒ–ƒˆ‘”͵ʹ†‹ˆˆ‡”‡–Š—ƒ–‹••—‡•ƒ†‘”‰ƒ•ǡ ‘˜‡”‹‰εͻͲΨ‘ˆ–Š‡’—–ƒ–‹˜‡ ’”‘–‡‹Ǧ ‘†‹‰‰‡‡•ȏͻʹȐǤ ‹•–Š‡Žƒ”‰‡•–ƒ†‘•– ‘’”‡Š‡•‹˜‡™‡„Ǧ„ƒ•‡†Š—ƒ ‡–ƒ„‘Ž‘‹  †ƒ–ƒ„ƒ•‡Ǥ – ‘–ƒ‹• ‹ˆ‘”ƒ–‹‘ ƒ„‘—– Š—ƒ ‡–ƒ„‘Ž‹–‡•ǡ ”‡ƒ –‹‘•ǡ ƒ† ‡œ›‡•ǡ™‹–ŠŽ‹•–‘‡–ƒ„‘Ž‹ ’ƒ–Š™ƒ›•ǡ”‡ˆ‡”‡ ‡ƒ†’ƒ–Š‘Ž‘‰‹  ‘ ‡–”ƒ–‹‘•ǡ†‹•‡ƒ•‡ ƒ••‘ ‹ƒ–‹‘•ǡ”‡ˆ‡”‡ ‡•’‡ –”ƒƒ†‘–Š‡””‡Žƒ–‡†‘‹ •†ƒ–ƒ„ƒ•‡•ǤŠ‡•‡–™‘†ƒ–ƒ„ƒ•‡•™‡”‡ •—ˆˆ‹ ‹‡–Ž› ‘’Ž‡–‡ƒ†•–”— –—”‡†–‘‡ƒ„Ž‡–Š‡ ‘•–”— –‹‘‘ˆ ‘’—–ƒ–‹‘ƒŽ‘†‡Ž•‘ˆ ‡–ƒ„‘Ž‹•„ƒ•‡†‘–Š‡•‡†ƒ–ƒ„ƒ•‡•ȏͻͷȂͳͲͲȐǤ  Systems biology and computational modeling ›•–‡• „‹‘Ž‘‰›ǡ ‹ –Š‡ ™‹†‡•– •‡•‡ ‘ˆ –Š‡ ™‘”†ǡ ˆ”‘ ‘””‡Žƒ–‹‘ ƒƒŽ›•‹• –‘ †‡–ƒ‹Ž‡† ‘’—–ƒ–‹‘ƒŽ ‘†‡Ž‹‰ ‘ˆ ‡–ƒ„‘Ž‹  †›ƒ‹ • ‹• ƒ ’‘™‡”ˆ—Ž –‘‘Ž –‘ ‰ƒ‹ ‹•‹‰Š– ‹–‘ ‡–ƒ„‘Ž‹• ƒ† ƒ “—‹”‡† ‡–ƒ„‘Ž‹  †‹•‡ƒ•‡• ȏͳͲͳȂͳͲͶȐǤ ‡‘‡Ǧ• ƒŽ‡ ‡–ƒ„‘Ž‹  ƒ† †›ƒ‹ ‘†‡Ž•‘ˆƒƒŽ‹ƒ‡–ƒ„‘Ž‹•ƒ”‡‰‡––‹‰‹ ”‡ƒ•‹‰ƒ––‡–‹‘ȏͻͷȂͳͲͲǡͳͲͷȂ ͳͳͲȐǤ ‡‘‡Ǧ• ƒŽ‡ ‡–ƒ„‘Ž‹  ‘†‡Ž• ƒ”‡ •–‘‹ Š‹‘‡–”‹  ‘†‡Ž• –Šƒ– ‘–ƒ‹ ƒŽŽ ‘™ „‹‘ Š‡‹ ƒŽƒ†–”ƒ•’‘”–”‡ƒ –‹‘•‹–Š‡‘†‡Ž‡†‡–™‘”Ǥ‘•–”ƒ‹–Ǧ„ƒ•‡†ƒƒŽ›•‹•‘ˆ –Š‡•‡‘†‡Ž•‡ƒ„Ž‡•–Š‡‡•–‹ƒ–‹‘‘ˆ”‡Žƒ–‹˜‡ˆŽ—š†‹•–”‹„—–‹‘•–Š”‘—‰Š–Š‡‡–™‘”ǡ„ƒ•‡† ‘ ƒ •–‡ƒ†›Ǧ•–ƒ–‡ ƒ••—’–‹‘ ƒ† ‘ˆ–‡ ƒŽ•‘ ‘–Š‡” ‘•–”ƒ‹–•ǡ •— Š ƒ• ”‡ƒ –‹‘ –Š‡”‘†›ƒ‹ •ǡ—–”‹‡–•—’’Ž›ǡ‰‡‡‡š’”‡••‹‘ǡ’”‘–‡‹ ‘ ‡–”ƒ–‹‘‘”‘–Š‡”‘‹ •†ƒ–ƒ ȏͳͳͳȐǤ ‘•‡“—‡–Ž›ǡ –Š‡•‡ ‘†‡Ž• ƒ ”‡˜‡ƒŽ ™Š‹ Š ‡ŽŽ—Žƒ” ‡–ƒ„‘Ž‹  ’ƒ–Š™ƒ›• ƒ› ‘–”‹„—–‡ –‘ ƒ ‘„•‡”˜‡† ‡–ƒ„‘Ž‹  •–ƒ–‡ ‘” †‹•‡ƒ•‡Ǥ Š‡› ƒ”‡ ƒŽ•‘ —•‡† –‘ ‹†‡–‹ˆ› „‹‘ƒ”‡”•‘ˆ†‹•‡ƒ•‡Ǥ‡šƒ’Ž‡‹•iHepatocytes2322ǡƒ‰‡‘‡Ǧ• ƒŽ‡‡–ƒ„‘Ž‹ ‘†‡Ž‘ˆ –Š‡Š—ƒŠ‡’ƒ–‘ ›–‡ǡ™Š‹ Š™ƒ•—•‡†–‘‹†‡–‹ˆ›•‡”‹‡†‡ˆ‹ ‹‡ ›‹ ȏͻͻȐǤ—”‰”‘—’ Šƒ•  —”ƒ–‡† –Š‡ Š—ƒ ‡–ƒ„‘Ž‹  ‘†‡Ž ‡ ‘ʹ ȏͻ͸Ȑ –‘ ƒ —”ƒ–‡Ž› ”‡’”‡•‡– –Š‡ ‡–‹”‡ ˆŽƒ˜‘’”‘–‡‘‡ȏͳͳʹȐǤ‡ ‡–Ž›ǡ–Š‡‡–ƒ„‘Ž‹•‘ˆʹͲ‘”‰ƒ•ǡ•‹š•‡š‘”‰ƒ•ǡ•‹š„Ž‘‘† ‡ŽŽ•ǡ –Š‡‰ƒ•–”‘‹–‡•–‹ƒŽŽ—‡ǡ•›•–‡‹ „Ž‘‘† ‹” —Žƒ–‹‘ǡƒ†–Š‡„Ž‘‘†Ǧ„”ƒ‹„ƒ””‹‡”Šƒ˜‡„‡‡ ‘—’Ž‡†‹–‘ƒ•‹‰Ž‡™Š‘Ž‡Ǧ„‘†›‘†‡Žȋ ƒ”˜‡›ƒ† ƒ”˜‡––ƒȌȏͳͳ͵ȐǤ Ž‹‹–ƒ–‹‘‘ˆ•–‘‹ Š‹‘‡–”‹ ‘†‡Ž•‹•–Šƒ––Š‡›Žƒ ‹‡–‹ ‹ˆ‘”ƒ–‹‘Ǥ›ƒ‹ ‡–ƒ„‘Ž‹  ‘†‡Ž•ƒ”‡‘ˆ–‡”‡’”‡•‡–‡†ƒ••›•–‡•‘ˆ‘”†‹ƒ”›†‹ˆˆ‡”‡–‹ƒŽ‡“—ƒ–‹‘•ˆ‘”–Š‡˜ƒ”‹ƒ„Ž‡ ‡–ƒ„‘Ž‹–‡• ‘ˆ –Š‡ ‘†‡Ž‡† ’ƒ–Š™ƒ›•Ǥ Š‡ ‘”†‹ƒ”› †‹ˆˆ‡”‡–‹ƒŽ ‡“—ƒ–‹‘• ‹ Ž—†‡ ƒŽŽ –Š‡ ”‡ƒ –‹‘•ƒ†–”ƒ•’‘”–’”‘ ‡••‡•‹–Š‡‘†‡Ž–Šƒ– ‘•—‡‘”’”‘†— ‡–Š‡‡–ƒ„‘Ž‹–‡‘ˆ ‹–‡”‡•– ƒ– –Š‡ ‘†‡Ž‡† ‹–”ƒ ‡ŽŽ—Žƒ” Ž‘ ƒ–‹‘ ȏͳͳͳǡͳͳͶȐǤ ‡ƒ –‹‘ ƒ† –”ƒ•’‘”– ”ƒ–‡• ƒ”‡ ’”‡ˆ‡”ƒ„Ž›„ƒ•‡†‘†‡–ƒ‹Ž‡†‹‡–‹ ‘™Ž‡†‰‡‘ˆ‡œ›‡Ǧ ƒ–ƒŽ›œ‡†Ǧǡ–”ƒ•’‘”–‡”Ǧ‡†‹ƒ–‡† ƒ†’Š›•‹ ‘ Š‡‹ ƒŽ’”‘ ‡••‡•‹•‹†‡–Š‡ ‡ŽŽȏͳͳͳȐǤ›ƒ‹ ‘†‡Ž•’”‡†‹ –‡–ƒ„‘Ž‹–‡–‹‡

ͳͷ Chapter 1

‘—”•‡•ǡ „—– ƒŽ•‘ •–‡ƒ†› •–ƒ–‡ ‘ ‡–”ƒ–‹‘• ƒ† ˆŽ—š‡•Ǥ Š‡› ‡ƒ„Ž‡ ‡–ƒ„‘Ž‹  ‘–”‘Ž ƒƒŽ›•‹•ǡƒ†•‡•‹–‹˜‹–›ƒƒŽ›•‹•‹‰‡‡”ƒŽǤ‡–ƒ„‘Ž‹  ‘–”‘ŽƒƒŽ›•‹•ȋȌ‹•–Š‡ƒƒŽ›•‹• ‘ˆ –Š‡ •‡•‹–‹˜‹–› ‘ˆ ‘†‡Ž ˜ƒ”‹ƒ„Ž‡•ǡ •— Š ƒ• ˆŽ—š‡• ƒ† ‡–ƒ„‘Ž‹–‡ ‘ ‡–”ƒ–‹‘•ǡ –‘ ‘†—Žƒ–‹‘‘ˆ–Š‡”ƒ–‡‘ˆƒ”‡ƒ –‹‘‘”–”ƒ•’‘”–’”‘ ‡••ǤƒŽŽ‘™•ƒ‰‡‡”ƒŽ‹œ‡†–Š‡‘”‡–‹ ƒŽ ƒƒŽ›•‹•‘ˆ–Š‡”‡Žƒ–‹‘„‡–™‡‡•‡•‹–‹˜‹–› ‘‡ˆˆ‹ ‹‡–•Ǥ‹’‘”–ƒ–‘—– ‘‡‘ˆ‹•–Šƒ– ˆŽ—š ‘–”‘Ž ƒ„‡†‹•–”‹„—–‡†‘˜‡”ƒŽŽ–Š‡•–‡’•‹–Š‡•›•–‡ƒ††‡’‡†•‘–Š‡‡–ƒ„‘Ž‹  •–ƒ–‡‘ˆ–Š‡•›•–‡ȏͳͳͷȂͳͳͺȐǤŠ‹•‹•‹ ‘–”ƒ•––‘–Š‡ Žƒ••‹ ƒŽƒ†‘ˆ–‡‡””‘‡‘—• ‘ ‡’– ‘ˆ‘‡Dz”ƒ–‡ǦŽ‹‹–‹‰dz•–‡’™‹–Šˆ—ŽŽˆŽ—š ‘–”‘ŽǤ ǡƒŽ•‘–Š‡•‡•‹–‹˜‹–›‘ˆ‘†‡Ž˜ƒ”‹ƒ„Ž‡• –‘‡š–‡”ƒŽ‡ˆˆ‡ –‘”• ƒ„‡ ƒŽ —Žƒ–‡†Ǥš–‡”ƒŽ‡ˆˆ‡ –‘”•ƒ”‡’”‘’‡”–‹‡•‹–Š‡•›•–‡–Šƒ–ƒ”‡ ‘– ‘–”‘ŽŽ‡†„›–Š‡‘†‡Ž‡†•›•–‡ǤŠ‡•‡‹ Ž—†‡ ‘ˆƒ –‘” ‘ ‡–”ƒ–‹‘•–Šƒ–Šƒ˜‡„‡‡ •‡––‘ƒ ‘•–ƒ–˜ƒŽ—‡‘”‡œ›‡ƒˆˆ‹‹–‹‡•ƒ† ƒ–ƒŽ›–‹  ƒ’ƒ ‹–‹‡•–Šƒ–Šƒ˜‡„‡‡•‡––‘ƒ ‘•–ƒ–˜ƒŽ—‡ȏͳͳͻȐǤŠ‡”‡ Žƒ••‹ ƒŽ‹•ˆ‘ —•‡†‘•–Ž›‘–Š‡ƒƒŽ›•‹•‘ˆ•–‡ƒ†›•–ƒ–‡ǡ –Š‡”‡ƒ”‡ƒ›‡š–‡•‹‘•–Šƒ–†‡ƒŽ™‹–Š†›ƒ‹ •ǡ™Š‹ Šƒ›„‡”‡Ž‡˜ƒ––‘‰ƒ‹‹•‹‰Š–‹–‘ ‡ Šƒ‹•• —†‡”Ž›‹‰ †‹•‡ƒ•‡ ’”‘‰”‡••‹‘ ‘˜‡” –‹‡Ǥ  ‹’‘”–ƒ– ‡š–‡•‹‘ ƒŽŽ‘™• –‘ “—ƒ–‹ˆ›–Š‡ ‘–”‹„—–‹‘‘ˆ‹†‹˜‹†—ƒŽ˜ƒ”‹ƒ„Ž‡”‡‰—Žƒ–‘”••— Šƒ•Ǧ‡†‹ƒ–‡†”‡‰—Žƒ–‹‘ ƒ†‡–ƒ„‘Ž‹–‡•–‘–Š‡ Šƒ‰‡‘ˆ”‡ƒ –‹‘”ƒ–‡•‘˜‡”–‹‡ȏͳʹͲȐǤŠ‹•‹•‹’‘”–ƒ–ǡ•‹ ‡‹– ‰‹˜‡• †‡–ƒ‹Ž‡† ‹•‹‰Š– ‹–‘ –Š‡ ‡–‹‘Ž‘‰›‘ˆ ƒ ’ƒ–Š‘Ž‘‰‹ ƒŽ ‘†‹–‹‘Ǥ – ‹• ‹’‘”–ƒ– –‘ ‘–‡ Š‘™‡˜‡”ǡ–Šƒ––Š‡‘—– ‘‡•†‡’‡†‘–Š‡Ž‡˜‡Ž‘ˆ†‡–ƒ‹Žƒ†ƒ —”ƒ ›‘ˆ–Š‡†›ƒ‹ ‘†‡ŽǤ Š‡”‡ˆ‘”‡ǡƒˆ— –‹‘ƒŽ’Š‡‘‡‘†‹• ‘˜‡”‡†in silicoǡ•Š‘—Ž†„‡˜ƒŽ‹†ƒ–‡†in vitro or in vivo ȏͳͳͳȐǤ šƒ’Ž‡•‘ˆ†›ƒ‹ ‘†‡Ž•‘ˆ‡–ƒ„‘Ž‹•ƒ†ƒ “—‹”‡†‡–ƒ„‘Ž‹ †‹•‡ƒ•‡‹ Ž—†‡‘†‡Ž• ‘ˆ Š‡’ƒ–‹  ‰Ž— ‘•‡ ‡–ƒ„‘Ž‹• ™‹–Š Š‘”‘ƒŽ ”‡‰—Žƒ–‹‘ ȏͳʹͳȐǡ ‹–‘ Š‘†”‹ƒŽ ‡‡”‰› ‡–ƒ„‘Ž‹• ȏͳͲ͹ǡͳʹʹȐǡ œ‘ƒ–‹‘ ‘ˆ Š‡’ƒ–‹  ‡‡”‰› ‡–ƒ„‘Ž‹•ǡ ‹•—Ž‹ ”‡•‹•–ƒ ‡ ƒ† •–‡ƒ–‘•‹•ȏͳʹ͵Ȑǡ‰Ž— ‘•‡ƒ†ƒ‹‘ƒ ‹†Ǧ‹†— ‡†‹•—Ž‹•‡ ”‡–‹‘‹ƒ’ƒ ”‡ƒ–‹ ȾǦ ‡ŽŽȏͳʹͶȐǡ ‡–ƒ„‘Ž‹ •›†”‘‡ƒ††›•Ž‹’‹†‡‹ƒȏͳʹͷȐǡƒ†‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧƒ ‹†‘š‹†ƒ–‹‘ȏͳͲͺȐǤ Š‡ •‹—Žƒ–‹‘• ‰‡‡”ƒ–‡† ™‹–Š –Š‡•‡ ‘†‡Ž• •—‰‰‡•– –Šƒ– ™‡ ƒ”‡ —””‡–Ž› ƒ„Ž‡ –‘ ‰ƒ‹ ‘•‹†‡”ƒ„Ž‡‹•‹‰Š–‹–‘–Š‡™‘”‹‰•‘ˆ‡–ƒ„‘Ž‹•ƒ†‡–ƒ„‘Ž‹ †‹•‡ƒ•‡ǡ‡•’‡ ‹ƒŽŽ›™Š‡ —•‡†‹ ‘„‹ƒ–‹‘™‹–Š—Ž–‹Ǧ‘‹ •†ƒ–ƒǤ  Inborn errors of /Inherited metabolic diseases „‘”‡””‘”•‘ˆ‡–ƒ„‘Ž‹•ȋ Ȍƒ”‡‹Š‡”‹–‡††‹•‡ƒ•‡•’”‹ƒ”‹Ž› ƒ—•‡†„›’ƒ–Š‘‰‡‹  —–ƒ–‹‘•‹‰‡‡•–Šƒ– ‘†‡ˆ‘”‡–ƒ„‘Ž‹ ‡œ›‡•‘”–”ƒ•’‘”–’”‘–‡‹•Ǥ •Šƒ˜‡„‡‡ Žƒ••‹ˆ‹‡†‹–‘ͳȌ‹–‘š‹ ƒ–‹‘†‹•‡ƒ•‡•ǡ‹ Ž—†‹‰ƒ‹‘Ǧƒ ‹†‘’ƒ–Š‹‡•ƒ†‘”‰ƒ‹ ƒ ‹†—”‹ƒ•ǡ ʹȌ‹„‘”‡””‘”•‘ˆ‡‡”‰›‡–ƒ„‘Ž‹•ǡ‹ Ž—†‹‰‹–‘ Š‘†”‹ƒŽ”‡•’‹”ƒ–‘”›Ǧ Šƒ‹ƒ†ˆƒ––›Ǧ ƒ ‹†‘š‹†ƒ–‹‘†‡ˆ‹ ‹‡ ‹‡•ǡƒ†͵Ȍ†‹•‡ƒ•‡•”‡Žƒ–‡†–‘‹’ƒ‹”‡††‡‰”ƒ†ƒ–‹‘‘”•›–Š‡•‹•‘ˆ ‘’Ž‡š ‘Ž‡ —Ž‡•ǡ •— Š ƒ• ‰Ž› ‘‰‡ •–‘”ƒ‰‡ †‹•‡ƒ•‡ ƒ† ‘‰‡‹–ƒŽ †‹•‘”†‡”• ‘ˆ ‰Ž› ‘•›Žƒ–‹‘ ȏͳʹ͸ȐǤ ‘‡ ‘ˆ –Š‡•‡ †‡ˆ‡ –• ˆƒŽŽ ‹–‘ —Ž–‹’Ž‡ Žƒ••‡•Ǥ ‘ŽŽ‡ –‹˜‡Ž›ǡ –Š‡  ’”‡˜ƒŽ‡ ‡™ƒ•ͳȀʹͶͲͲ‹ ‡”ƒ›ʹͲͲ͵ƒ†ͳȀͳͺͻͶ‹ –ƒŽ›‹ʹͲͳͷȏ͸ǡͻǡͳͲǡͳͺǡͳͲʹǡͳʹ͹ȐǤ ‡‘ˆ–Š‡‘•– ‘‘ •‹•‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡ȋȌ†‡ˆ‹ ‹‡ › ȋȌȏͺȂͳͲǡͳͺǡͳͲʹǡͳʹ͹ȐǤŠ‡’”‡˜ƒŽ‡ ‡ƒ–„‹”–Š™ƒ•ͳȀͺ͵ͲͲ‹–Š‡‡–Š‡”Žƒ†• „‡–™‡‡ʹͲͲ͹ƒ†ʹͲͳͷȏͳʹͺȐƒ†ͳȀͻͲͻ͵‹ –ƒŽ›‹ʹͲͳͷȏ͸ȐǤ‹•ƒ‡œ›‡‘ˆ–Š‡ ‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧƒ ‹†‘š‹†ƒ–‹‘’ƒ–Š™ƒ›Ǥ•’‡ ‹ƒŽŽ›‡™„‘”•ƒ† Š‹Ž†”‡ ƒ’”‡•‡– ™‹–Š ƒ •‡”‹‘—• ‹ŽŽ‡•• ƒ† †‹•‡ƒ•‡ ”‡Žƒ–‡† –‘ •Ǥ ‘•‡“—‡–Ž›ǡ ‡ƒ”Ž› †‹ƒ‰‘•‹• ƒ† –”‡ƒ–‡–„‡ˆ‘”‡–Š‡‘•‡–‘ˆ•›’–‘•‹•‹’‘”–ƒ––‘’”‡˜‡–•‡˜‡”‡’‡”ƒ‡–†ƒƒ‰‡ ‘”†‡ƒ–ŠȏͳͲǡͳͲʹǡͳʹ͹ȐǤ

ͳ͸ General introduction and outline of the thesis

 Common principles underlying IEMs and acquired metabolic diseases 1 •ƒ”‡–›’‹ ƒŽŽ›‘‘‰‡‹ ǡ•‹‰Ž‡Ǧ‡œ›‡†‡ˆ‹ ‹‡ ‹‡•Ǥ‡˜‡”–Š‡Ž‡••ǡ •‘ˆ–‡†‹•’Žƒ›ƒ •’‡ –”—‘ˆ†‹•‡ƒ•‡’Š‡‘–›’‡•ǡ‡˜‡™‹–Š‹ƒ•—„•‡–‘ˆ‹†‹˜‹†—ƒŽ•™‹–Š–Š‡•ƒ‡—–ƒ–‹‘Ǥ Š‡”‡ˆ‘”‡ǡ •Šƒ˜‡„‡‡˜‹‡™‡†–‘†‹•’Žƒ› ‘’Ž‡š–”ƒ‹–•ǡ‘–—Ž‹‡ ‘‘ƒ “—‹”‡†ǡ —Ž–‹ˆƒ –‘”‹ƒŽ†‹•‡ƒ•‡•ȏͳͺǡͳͲʹǡͳʹͻȐǤ –Šƒ•„‡‡’”‘’‘•‡†–Šƒ–‘†‹ˆ›‹‰ˆƒ –‘”•ǡ‹ Ž—†‹‰ ‡˜‹”‘‡–ƒŽˆƒ –‘”•ȋ•— Šƒ•†‹‡–ƒ†ƒ„‹‡––‡’‡”ƒ–—”‡ȏͳ͵ͲȐȌǡ‡’‹‰‡‡–‹ ˆƒ –‘”•ƒ† ‘–Š‡”‰‡‡•ǡƒ› ‘–”‹„—–‡–‘–Š‡†‹•‡ƒ•‡’”‡•‡–ƒ–‹‘ȏͳͺȐǤ ƒ††‹–‹‘ǡ’Š‡‘–›’‡••‹‹Žƒ” –‘ –Š‘•‡ ‘ˆ ƒ “—‹”‡† ‡–ƒ„‘Ž‹  †‹•‡ƒ•‡•ǡ ˆ‘” ‹•–ƒ ‡ Š‡’ƒ–‘•–‡ƒ–‘•‹•ǡ ‘˜‡”™‡‹‰Š– ƒ† †‹ƒ„‡–‡•ǡŠƒ˜‡„‡‡”‡’‘”–‡†‹ •ȏͳͺǡͳ͵ͳǡͳ͵ʹȐǤ •ƒ›‡˜‡„‡ ‘•‹†‡”‡†ƒ•’ƒ”–‘ˆ –Š‡•ƒ‡‡–ƒ„‘Ž‹ †‹•‡ƒ•‡•’‡ –”—Ǥ–Š‡‘‡‡†‘ˆ–Š‡•’‡ –”—–Š‡†‡ˆ‡ –‹˜‡‰‡‡‘ˆ –Š‡ –‘‰‡–Š‡”™‹–Š‘†‹ˆ›‹‰‰‡‡•ƒ†‘–Š‡”ˆƒ –‘”•†‡–‡”‹‡–Š‡†‹•‡ƒ•‡’”‡•‡–ƒ–‹‘ǡ ‘ˆ–‡ƒ–›‘—‰ƒ‰‡Ǥ–Š‡‘–Š‡”‡†‘ˆ–Š‡•’‡ –”——Ž–‹’Ž‡‰‡‡•–‘‰‡–Š‡”™‹–ŠŽ‹ˆ‡•–›Ž‡ ƒ†Š‹•–‘”›†‡–‡”‹‡–Š‡†‡˜‡Ž‘’‡–‘˜‡”–‹‡‘ˆƒ “—‹”‡†‡–ƒ„‘Ž‹ †‹•‡ƒ•‡ȏͳͺȐǤ –‹• –Š—•‹„‘–Š ƒ•‡•–Š‡ ——Žƒ–‹˜‡‡ˆˆ‡ –‘ˆ†‹ˆˆ‡”‡–‰‡‡˜ƒ”‹ƒ–•’Ž—•‡š–‡”ƒŽˆƒ –‘”•–Šƒ– ”‡•—Ž–‹–Š‡‘„•‡”˜‡†’Š‡‘–›’‡ǤŠ‹•‹•‘–—Ž‹‡–Š‡†‹•–”‹„—–‹‘‘ˆˆŽ—š ‘–”‘Ž‘˜‡”ƒ› ‡œ›‡•‹ƒ‡–ƒ„‘Ž‹ ‡–™‘”Ǥ‘•‡“—‡–Ž›ǡ–Š‡ ‘–”‹„—–‹‘‘ˆƒ‰‡‡–‹ ˜ƒ”‹ƒ–‹‘–‘–Š‡ ’Š‡‘–›’‡†‡–‡”‹‡•™Š‡–Š‡”‹–‹•‹†‡–‹ˆ‹‡†ƒ•ƒ‹„‘”‡””‘”‘”‘–ǤŠ‹•ƒŽ•‘‡ƒ•–Šƒ– Dz™‡ƒ‡” —–ƒ–‹‘•̶ ‹ ƒ ‘–Š‡”™‹•‡ Š‡ƒŽ–Š› ‹†‹˜‹†—ƒŽ ȋ”‡’”‡•‡–‡† „› ƒ ‘„‹ƒ–‹‘ ‘ˆ ‰‡‡–‹ ˜ƒ”‹ƒ–•–Šƒ–”‡•—Ž–‹–Š‡’Š‡‘–›’‡„‡‹‰†‡‘–‡†ƒ•̶Š‡ƒŽ–Š›̶Ȍ™‘—Ž†‘–„‡‡ƒ•‹Ž› †‡–‡ –‡†Ǥ™‡ƒ‡”—–ƒ–‹‘™‘—Ž†„‡ƒ—–ƒ–‹‘–Šƒ–‘‹–•‘™™‘—Ž†‘–”‡•—Ž–‹ƒ Ž‹‹ ƒŽ ’Š‡‘–›’‡Ǥ‡šƒ’Ž‡‹•ƒ •Š‘™‹‰ƒ••‘ ‹ƒ–‹‘‘ˆ•‹‰Ž‡Ǧ— Ž‡‘–‹†‡’‘Ž›‘”’Š‹•• ȋ•Ȍƒ––Š‡Ž‘ —•™‹–Š„Ž‘‘†ͺǦ ƒ”‹–‹‡Ž‡˜‡Ž•ǡƒ„‹‘ƒ”‡”ˆ‘”ȏͳͺǡͳ͵͵ȐǤ ‹ƒŽŽ›ǡ • ”‡•—Ž– ‹ ƒ ‘”‡ ’ƒ–Š‘Ž‘‰‹ ƒŽ ’Š‡‘–›’‡ ‘’ƒ”‡† –‘ –Š‡ ™‡ƒ‡” —–ƒ–‹‘ ’Š‡‘–›’‡ ȋ•‘‡ •‹‹Žƒ” –‘ ƒ “—‹”‡† ‡–ƒ„‘Ž‹  †‹•‡ƒ•‡Ȍ ƒ† ‹‰Š– –Š‡”‡ˆ‘”‡ „‡ ‘”‡ ˜—Ž‡”ƒ„Ž‡ –‘ ƒ††‹–‹‘ƒŽ ™‡ƒ‡••Ǧ‹ ”‡ƒ•‹‰ ˜ƒ”‹ƒ ‡• ‹ ‘–Š‡” ‰‡‡• ƒ† ‘–Š‡” ˆƒ –‘”•Ǥ Š‡”‡ˆ‘”‡ǡ ‘†‹ˆ›‹‰ ˆƒ –‘”• ȋ‹ Ž—†‹‰ ‰‡‡•Ȍ –Šƒ– ‘–”‹„—–‡ –‘ –Š‡ †‡˜‡Ž‘’‡– ƒ† ’”‘‰”‡••‹‘‘ˆƒ “—‹”‡†‡–ƒ„‘Ž‹ †‹•‡ƒ•‡• ƒ„‡‘”‡‡ƒ•‹Ž›‹†‡–‹ˆ‹‡†™Š‡•–—†›‹‰ •Ǥ ‹‹Žƒ”Ž›ǡ †‹ˆˆ‡”‡ ‡• ‹ ‰‡‡ ‡š’”‡••‹‘ „‡–™‡‡ ‡˜‹”‘‡–ƒŽŽ›Ǧ ŠƒŽŽ‡‰‡†  ƒ† ‘–”‘Ž‰”‘—’• ‘—Ž†’‘‹––‘ȋƒŽǦȌƒ†ƒ’–ƒ–‹‘•‹‘†‹ˆ‹‡”‰‡‡•Ǥ‹ ‡’ƒ”–‹ —Žƒ”Ž›‡™„‘” ƒ† Š‹Ž†”‡ ™‹–Š • ƒ ’”‡•‡– ™‹–Š ˜‡”› •‡˜‡”‡ •›’–‘•ǡ ‹– ‹• ˜‡”› ‹’‘”–ƒ– –‘ —†‡”•–ƒ†–Š‡‡–‹‘Ž‘‰›‘ˆ†‹•‡ƒ•‡‹ •Ǥ††‹–‹‘ƒŽŽ›ǡ–Š‡•–—†›‘ˆ •ƒ›‰‹˜‡‹•‹‰Š–• ‹ ‡ Šƒ‹•• –Šƒ– ƒ”‡ ƒŽ•‘ ”‡Ž‡˜ƒ– ˆ‘” ƒ “—‹”‡† ‡–ƒ„‘Ž‹  †‹•‡ƒ•‡Ǥ  –Š‡ ‘–Š‡” Šƒ†ǡ ”‡•‡ƒ” Š‹ƒ “—‹”‡†‡–ƒ„‘Ž‹ †‹•‡ƒ•‡ ƒƒ‹†‹—†‡”•–ƒ†‹‰ •ǤŠ—•ǡ‡–Š‘†•–‘•–—†› „‘–Š–›’‡•‘ˆ†‹•‡ƒ•‡™‘—Ž†‹ Ž—†‡–Š‡—•‡‘ˆ‘‹ •ƒ’’”‘ƒ Š‡•ƒ† ‘’—–ƒ–‹‘ƒŽ‘†‡Ž‹‰Ǥ  ˆƒ –ǡ ‡™„‘” • ”‡‡‹‰ ‡ˆˆ‘”–• —•‡ Ǧ„ƒ•‡† –‡ Š‹“—‡• ˆ‘” –ƒ”‰‡–‡† ‡–ƒ„‘Ž‘‹ •ǡ ‘–ƒ„Ž›ˆ‘”–Š‡‡ƒ•—”‡‡–‘ˆ’Žƒ•ƒƒ‹‘ƒ ‹†ǡ’Žƒ•ƒƒ ›ŽǦ ƒ”‹–‹‡ƒ†—”‹‡‘”‰ƒ‹  ƒ ‹†’”‘ˆ‹Ž‡•ǤŠ‹•Šƒ•‰”‡ƒ–Ž›‹ ”‡ƒ•‡†–Š‡—„‡”‘ˆ •†‹• ‘˜‡”‡†‹–Š‡Žƒ•–ʹͲ›‡ƒ”• ȏ͸ǡͳͲǡͳͺǡͳ͵ͶȐǤŠ‹•ƒŽ•‘Ž‡†–‘–Š‡‹†‡–‹ˆ‹ ƒ–‹‘‘ˆ™‡ƒ‡”—–ƒ–‹‘•ˆ‘”™Š‹ Š‹–™ƒ•— Ž‡ƒ” ‹ˆ–Š‡›‰‹˜‡”‹•‡–‘†‹•‡ƒ•‡Ǥ ‘”‡šƒ’Ž‡ǡƒˆ–‡”–Š‡‹–”‘†— –‹‘‘ˆ‡™„‘”• ”‡‡‹‰ˆ‘”  †‡ˆ‹ ‹‡ ‹‡•ǡ •‡˜‡”ƒŽ ˜ƒ”‹ƒ–• ‘ˆ –Š‡ ACADM ‰‡‡ ™‡”‡ †‹• ‘˜‡”‡† ™‹–Š ‘”‡ –Šƒ ͳͲΨ ”‡•‹†—ƒŽƒ –‹˜‹–›ǡˆ‘”™Š‹ Š–Š‡‡‡†ˆ‘”–”‡ƒ–‡–‹•†‹•’—–‡†ȏͳ͵ͶȐǤŠ‹•™‘—Ž†„‡‹Ž‹‡™‹–Š ACADM•–Šƒ–ƒ••‘ ‹ƒ–‡™‹–ŠͺǦ ƒ”‹–‹‡Ž‡˜‡Ž•‹Š‡ƒŽ–Š›‹†‹˜‹†—ƒŽ•ȏͳͺǡͳ͵͵ȐǤ ƒ›‡–ƒ„‘Ž‹–‡•–Šƒ–’Žƒ›ƒ”‘Ž‡‹ƒ “—‹”‡†‡–ƒ„‘Ž‹ †‹•‡ƒ•‡•Šƒ˜‡ƒŽ•‘„‡‡‹†‡–‹ˆ‹‡†ƒ• ‡›‘†‹ˆ‹‡”•‹ •ǤŠ‡•‡‹ Ž—†‡Ϊǡƒ‹‘ƒ ‹†•ǡƒ† ‘‡œ›‡ǤΪŠƒ•„‡‡

ͳ͹ Chapter 1

‹’Ž‹ ƒ–‡†‹ •„‡ ƒ—•‡‘ˆ‹–•ƒ„‹Ž‹–›–‘ƒ –‹˜ƒ–‡•‹”–—‹•ȋ •Ȍ ȏͳ͵ͷȂͳ͵͹ȐǤ • ƒ †‡ƒ ‡–›Žƒ–‡ƒ††‡ƒ ›Žƒ–‡’”‘–‡‹•ǡ‹ Ž—†‹‰‡œ›‡•ǡ™Š‹ Šƒ›”‡•—Ž–‹ Šƒ‰‡•‹–Š‡ ƒ–ƒŽ›–‹  ’”‘’‡”–‹‡• ‘ˆ ‡œ›‡• ȏͳ͵ͷȂͳͶʹȐǤ ‘” ‡šƒ’Ž‡  ͵Ǧ ƒ–ƒŽ›œ‡† †‡ƒ ‡–›Žƒ–‹‘ ‘ˆ Ž‘‰Ǧ Šƒ‹Ǧƒ†˜‡”›ǦŽ‘‰Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡•ȋƒ†ǡ”‡•’‡ –‹˜‡Ž›Ȍ ƒ†͵Ǧ ›†”‘š›Ǧ͵Ǧ‡–Š›Ž‰Ž—–ƒ”›Ž‘›–Šƒ•‡ʹȋ  ʹȌ‘š‹†ƒ–‹‘‹ ”‡ƒ•‡•–Š‡ƒ –‹˜‹–› ‘ˆ –Š‡•‡ ‡œ›‡• ȏͳ͵ͻǡͳͶͲȐǤ  ʹ ‹• ‹˜‘Ž˜‡† ‹ ‡–‘‰‡‡•‹•Ǥ  ͵ ƒŽ•‘ †‡ƒ ‡–›Žƒ–‡• ƒ––Š‡•ƒ‡ ‘•‡”˜‡†Ž›•‹‡”‡•‹†—‡‡ƒ”–Š‡ƒ –‹˜‡•‹–‡–Šƒ–‹•ƒ ‡–›Žƒ–‡†‹ ƒ†ȏͳͶͲȐǤ‘‘—”‘™Ž‡†‰‡‹–Šƒ•ǡŠ‘™‡˜‡”ǡ‘–„‡‡‹˜‡•–‹‰ƒ–‡†™Š‡–Š‡”–Š‹•ƒˆˆ‡ –• –Š‡ ƒ–ƒŽ›–‹  ƒ’ƒ ‹–›‘ˆǤŠ‡”‘Ž‡‘ˆ •ƒ†Ϊ‹ •ƒ‡•–Š‡’‘–‡–‹ƒŽ –Š‡”ƒ’‡—–‹ –ƒ”‰‡–•ȏͳ͵͹ǡͳͶ͵ǡͳͶͶȐǤŽ–‡”‡†ƒ‹‘ƒ ‹†Ž‡˜‡Ž•Šƒ˜‡ƒŽ•‘„‡‡”‡’‘”–‡†‹ • ‘–Š‡”–Šƒ–Š‡ƒ‹‘ƒ ‹†‘’ƒ–Š‹‡•Ǥ‘‡œ›‡ǡ™Š‹ Š‹•ƒŽ•‘ƒ ‘ˆƒ –‘”‹•‡˜‡”ƒŽ •Šƒ• ”‡ ‡–Ž›‰ƒ‹‡† ‘•‹†‡”ƒ„Ž‡ƒ––‡–‹‘ƒ•ƒ‘†‹ˆ‹‡”‹ •ǡ‡‹–Š‡”˜‹ƒ‹–•ˆ”‡‡ˆ‘”ȋ‘ Ȍ ‘”‹–•‡•–‡”‹ˆ‹‡†ˆ‘”ȋ‘‡•–‡”Ȍȏͷͻǡ͸ͲȐǤ  Computational modeling and omics in IEMs ‘’—–ƒ–‹‘ƒŽ‘†‡Ž‹‰ǡ‹ ‘„‹ƒ–‹‘™‹–Š–Š‡—•‡‘ˆ‘‹ •–‡ Š‹“—‡•™‘—Ž†ƒŽ•‘„‡ƒ ‹’‘”–ƒ––‘‘Ž‹‘”†‡”–‘‰ƒ‹‹•‹‰Š–‹–‘–Š‡‡ Šƒ‹••—†‡”Ž›‹‰†‹•‡ƒ•‡’”‡•‡–ƒ–‹‘ ‹ •Ǥ †‡‡†ǡƒ‡ ‘ͳǦ†‡”‹˜‡†ȏͳͶͷȐ‰‡‘‡Ǧ• ƒŽ‡•–‘‹ Š‹‘‡–”‹ ‘†‡Ž‘ˆ–Š‡Š—ƒ •ƒŽŽ‹–‡•–‹ƒŽ‡’‹–Š‡Ž‹ƒŽ ‡ŽŽ• ‘””‡ –Ž›’”‡†‹ –‡†–Š‡‘™„‹‘ƒ”‡”•‹͹ʹΨ‘ˆ–Š‡͵͸ •‹—Žƒ–‡† •ȏͳͶ͸Ȑǡ™Š‹Ž‡ƒ‘†‡Ž‘ˆ–Š‡Š—ƒŠ‡’ƒ–‘ ›–‡ ‘””‡ –Ž›’”‡†‹ –‡†ƒ„‘—–ŠƒŽˆ ‘ˆ–Š‡͵ͺ•‹—Žƒ–‡† •ȏͻͻȐǤˆ–‡”‘—”‰”‘—’ —”ƒ–‡†–Š‡ˆŽƒ˜‘’”‘–‡‘‡‹‡ ‘ʹǤʹǡ–Š‡ ‘†‡Ž ”‡ƒ Š‡† ͹ͶΨ ƒ —”ƒ › ‹ „‹‘ƒ”‡” ’”‡†‹ –‹‘ ˆ‘” Ͷͷ ˆŽƒ˜‘’”‘–‡‘‡Ǧ”‡Žƒ–‡† • ȏͳͳʹȐǤ‘’—–ƒ–‹‘ƒŽ†›ƒ‹ ‘†‡Ž‹‰‘ˆ •‹•”ƒ”‡Ǥ—”‰”‘—’Šƒ•ǡŠ‘™‡˜‡”ǡ‰‡‡”ƒ–‡† ƒ†›ƒ‹ ‘†‡Ž‘ˆ‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧƒ ‹†‘š‹†ƒ–‹‘™Š‹ Š ‘—Ž†’”‡†‹ ––Š‡„‹‘ Š‡‹ ƒŽ ’Š‡‘–›’‡ ‘ˆ  ƒ† —Ž–‹’Ž‡ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ †‡ˆ‹ ‹‡ › ȏͳͲͶǡͳͲ͹ǡͳͶ͹ȐǤ ‘ ‘†‡Ž ‡–ƒ„‘Ž‹  ƒ†ƒ’–ƒ–‹‘• ‹ • ƒ† ‡˜‡ –Š‡ ’Š‡‘–›’‡ ‘ˆ ‹†‹˜‹†—ƒŽ ’ƒ–‹‡–•ǡ –Š‡ ‹ ‘”’‘”ƒ–‹‘‘ˆ‘‹ •†ƒ–ƒ™‹ŽŽ„‡‹’‘”–ƒ–Ǥ –Šƒ•„‡‡’”‘’‘•‡†–Šƒ–ƒ ‘„‹ƒ–‹‘‘ˆ Š‹‰ŠǦ–Š”‘—‰Š’—–‘‹ •ƒƒŽ›•‹•‹ ‘„‹ƒ–‹‘™‹–Š ‘’—–ƒ–‹‘ƒŽ‘†‡Ž‹‰ ‘—Ž†’”‘˜‹†‡ ˆ‘”ˆƒ•–‡”ƒ†‘”‡ƒ —”ƒ–‡†‹ƒ‰‘•‡•ƒ†•—„•‡“—‡––”‡ƒ–‡–ȏͳͶͺȐǤ  Mitochondrial fatty-ĂĐŝĚɴ-oxidation (mFAO) and mFAO deficiency ”‘ –Š‡ ’”‡˜‹‘—• •‡ –‹‘• ‹– ‹• ‡˜‹†‡– –Šƒ– ‹–‘ Š‘†”‹ƒŽ ˆƒ––›Ǧƒ ‹† ‘š‹†ƒ–‹‘ ‹• ƒ ‹’‘”–ƒ– ’ƒ–Š™ƒ› ‹ „‘–Š ƒ “—‹”‡† ƒ† ‹„‘” ‡–ƒ„‘Ž‹  †‹•‡ƒ•‡Ǥ –—†›‹‰   ‘—Ž† –Š‡”‡ˆ‘”‡”‡˜‡ƒŽ˜ƒŽ—ƒ„Ž‡‹•‹‰Š–•‹–‘–Š‡‡ Šƒ‹••—†‡”Ž›‹‰„‘–Š–›’‡•‘ˆ†‹•‡ƒ•‡•Ǥ ‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧƒ ‹†‘š‹†ƒ–‹‘ǡ‘”‡•’‡ ‹ˆ‹ ƒŽŽ›‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧƒ ‹†ȾǦ‘š‹†ƒ–‹‘‹• –Š‡ › Ž‹  ‘š‹†ƒ–‹‘ ‘ˆ ƒ –‹˜ƒ–‡† ˆƒ––› ƒ ‹†• ȋƒ ›ŽǦ‘ ‡•–‡”•Ȍ –‘ ƒ ‡–›ŽǦ‘Ǥ Š‡ DzȾdz ‹ DzȾǦ ‘š‹†ƒ–‹‘dz”‡Žƒ–‡•–‘–Š‡ˆƒ ––Šƒ––Š‡„‘†„‡–™‡‡–Š‡Ƚȋ–Š‡•‡ ‘† ƒ”„‘Ȍƒ†Ⱦ ƒ”„‘ ȋ–Š‹”† ƒ”„‘Ȍ‘ˆ–Š‡ƒ –‹˜ƒ–‡†ˆƒ––›Ǧƒ ‹†‹•„”‘‡†—”‹‰‡ƒ Š”‘—†‘ˆ–Š‡‘š‹†ƒ–‹‘ › Ž‡•ǡ –Š‡”‡„› ‰‡‡”ƒ–‹‰ ƒ ‡–›ŽǦ‘ ƒ† ƒ •Š‘”–‡‡† ƒ ›ŽǦ‘ ‡•–‡”Ǥ   ‹• ’ƒ”–‹ —Žƒ”Ž› ‹’‘”–ƒ–†—”‹‰’”‘Ž‘‰‡†’‡”‹‘†•‘ˆˆƒ•–‹‰‘”Š‹‰Š‡‡”‰›†‡ƒ†ǡ•— Šƒ•‡š‡” ‹•‡ƒ† ‘Ž†‡š’‘•—”‡Ǥ –Š‡•‡ ‘†‹–‹‘•ǡ ‹–Š‡Ž‹˜‡”’Žƒ›•ƒ’”‘‹‡–”‘Ž‡„›’”‘˜‹†‹‰ ˆ‘” ‰Ž— ‘‡‘‰‡‡•‹• ƒ† ’”‡ —”•‘”• ˆ‘” ‡–‘‰‡‡•‹•Ǥ Š‹•‡ƒ„Ž‡• –Š‡ Ž‹˜‡” –‘ ’”‘˜‹†‡ –Š‡ ’‡”‹’Š‡”ƒŽ–‹••—‡•™‹–Š‰Ž— ‘•‡ƒ†‡–‘‡„‘†‹‡•ƒ•ˆ—‡ŽǤ‡™‹ŽŽ–Š‡”‡ˆ‘”‡Ž‘‘‹–‘Š‡’ƒ–‹   „‹‘ Š‡‹•–”›‹‘”‡†‡–ƒ‹Žȋ ‹‰—”‡ͳȌǤ

ͳͺ General introduction and outline of the thesis

Malonyl-CoA Palmitoyl-CoA Acyl-carnitine 1 carnitine CoASH carnitine CPT1 C16 CACT C4-C16 CPT2 Mitochondrion C4-C16 carnitine CoASH carnitine Acyl-carnitine Acyl-CoA

MCKAT Acetyl-CoA SCAD MCAD LCAD VLCAD FAD C4-C6 C4-C12 C8-C16 C12-C16 C4-C16 CoASH FADH2

Ketoacyl-CoA Enoyl-CoA Acetyl-CoA + NADH + H MTP H2O C4-ketoacyl-CoA NADH + H+ MSCHAD C4-ketoacyl-CoA CROT NAD+ C8-C16 C4-C16 NAD+ C4-C16 CoASH Hydroxyacyl-CoA  Figure 1. Schematic representation of the rodent mFAO pathway. ”‡‡ǣ‡œ›‡•’ƒ”–‹ ‹’ƒ–‹‰‹–Š‡ ƒ”‹–‹‡ › Ž‡Ǣ’—”’Ž‡ǣ‡œ›‡•’ƒ”–‹ ‹’ƒ–‹‰‹–Š‡•Š‘”–Ǧ Šƒ‹„”ƒ Šȋi.e.–Š‡ǦȀ Ǧ „”ƒ ŠȌǢ ‰”‡›ǣ ‡œ›‡• ’ƒ”–‹ ‹’ƒ–‹‰ ‹ –Š‡ ‡†‹—Ǧƒ† Ž‘‰Ǧ Šƒ‹ „”ƒ Š ȋi.e. –Š‡  „”ƒ ŠȌǤ ŽŽ ’”‘ ‡••‡•ƒ”‡”‡˜‡”•‹„Ž‡ƒ†–Š‡•‹œ‡‘ˆ–Š‡ƒ””‘™Š‡ƒ†•‹†‹ ƒ–‡•–Š‡‡–†‹”‡ –‹‘‘ˆ–Š‡”‡ƒ –‹‘•Ǥ  ƒ––›ƒ ‹†•‘”‹‰‹ƒ–‹‰ˆ”‘–Š‡™Š‹–‡ƒ†‹’‘•‡–‹••—‡ȋȌ–›’‹ ƒŽŽ›Šƒ˜‡ƒ Šƒ‹Ž‡‰–Š‘ˆ ͳ͸‘”ͳͺ ƒ”„‘ƒ–‘•ǤŠ‡›ƒ”‡–ƒ‡—’‹–‘–Š‡Ž‹˜‡”ƒ†ƒ –‹˜ƒ–‡†„› ‘†‡•ƒ–‹‘™‹–Š ‘ ǤŠ‹•ƒ –‹˜ƒ–‹‘‹• ƒ–ƒŽ›œ‡†„›ƒ ›ŽǦ‘•›–Š‡–ƒ•‡•‘”ˆƒ––›ƒ ‹†–”ƒ•ˆ‡”’”‘–‡‹• ȋ •Ȍƒ†‰‡‡”ƒ–‡•ƒ ›ŽǦ‘‡•–‡”•ȏͳͶͻǡͳͷͲȐǤŠ‡ƒ ›ŽǦ‘‡•–‡”•ƒ”‡•—„•‡“—‡–Ž›–ƒ‡ —’‹–‘–Š‡‹–‘ Š‘†”‹ƒˆ‘”‘š‹†ƒ–‹‘–‘ƒ ‡–›ŽǦ‘ǤŠ‡ƒ ‡–›ŽǦ‘ ƒ•—„•‡“—‡–Ž›„‡ ‘š‹†‹œ‡†ˆ‘”’”‘†— –‹‘‘”—•‡†ƒ•’”‡ —”•‘”ˆ‘”‡–‘‰‡‡•‹•Ǥ’–ƒ‡‘ˆƒ ›ŽǦ‘‡•–‡”• ‹–‘–Š‡‹–‘ Š‘†”‹ƒŽƒ–”‹š‘ —”•˜‹ƒ–Š‡ ƒ”‹–‹‡•Š—––Ž‡ǤŠ‹••Š—––Ž‡ ‘•‹•–•‘ˆ–Š‡ ‡œ›‡• ƒ”‹–‹‡ ’ƒŽ‹–‘›Ž–”ƒ•ˆ‡”ƒ•‡  ȋͳȌǡ ƒ”‹–‹‡Ǧƒ ›ŽǦ ƒ”‹–‹‡ –”ƒ•Ž‘ ƒ•‡ ȋȌǡ ƒ† ƒ”‹–‹‡ ’ƒŽ‹–‘›Ž–”ƒ•ˆ‡”ƒ•‡  ȋʹȌǤ ‡”‡‹ǡ –Š‡ ›–‘•‘Ž‹  ƒ ›ŽǦ‘ ‹• ‘˜‡”–‡† –‘ ƒ ›ŽǦ ƒ”‹–‹‡ǡ „› ‘•—‹‰ Ǧ ƒ”‹–‹‡ ƒ† ”‡Ž‡ƒ•‹‰ ‘ Ǥ Š‡ ›–‘•‘Ž‹  ƒ ›ŽǦ ƒ”‹–‹‡‹••—„•‡“—‡–Ž›–”ƒ•Ž‘ ƒ–‡†‹–‘–Š‡‹–‘ Š‘†”‹ƒ–Š”‘—‰Š‹‡š Šƒ‰‡ ˆ‘”‹–‘ Š‘†”‹ƒŽǦ ƒ”‹–‹‡Ǥ ‡‹–Š‡‹–‘ Š‘†”‹ƒǡ–Š‡ƒ ›ŽǦ ƒ”‹–‹‡‹• ‘˜‡”–‡†„ƒ  –‘ ƒ ›ŽǦ‘ „› ʹǤ Š‡ ƒ ›ŽǦ‘ ‘Ž‡ —Ž‡ •—„•‡“—‡–Ž› —†‡”‰‘‡• ˆƒ––›Ǧƒ ‹† „‡–ƒǦ ‘š‹†ƒ–‹‘Ǥ‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧƒ ‹†„‡–ƒǦ‘š‹†ƒ–‹‘ȋ Ȍ‘ —”•‹”‡’‡–‹–‹˜‡ˆ‘—”Ǧ”‡ƒ –‹‘ › Ž‡•ȋ ‹‰—”‡ͳȌǤ ‡ƒ Š”‘—†ǡ–Š‡ƒ ›ŽǦ‘‡•–‡”‹••Š‘”–‡‡†„›–™‘ ƒ”„‘ƒ–‘•ƒ†‘‡ ƒ ‡–›ŽǦ‘‹•’”‘†— ‡†ȏͳͷͳȂͳͷ͵ȐǤ‹ˆˆ‡”‡– › Ž‡•ƒ”‡ ƒ–ƒŽ›œ‡†„›–Š‡•ƒ‡•‡–‘ˆ‡œ›‡•Ǥ Š‹•‡ƒ•–Šƒ–‡ƒ Š ‡œ›‡ ƒ–ƒŽ›œ‡•–Š‡ ‘˜‡”•‹‘‘ˆˆƒ––›Ǧƒ ‹††‡”‹˜ƒ–‹˜‡•‘ˆƒ ”ƒ‰‡‘ˆ Šƒ‹Ž‡‰–Š•ȏͳͲͺǡͳͷ͵ȐǤœ›‡•–Šƒ– ƒ–ƒŽ›œ‡–Š‡ ‘˜‡”•‹‘‘ˆ—Ž–‹’Ž‡•—„•–”ƒ–‡• ƒ”‡ ƒŽŽ‡†’”‘‹• —‘—•Ǥ‹ ‡‡ƒ Š ‡œ›‡‹•’”‘‹• —‘—•ǡ–Š‡†‹ˆˆ‡”‡–•—„•–”ƒ–‡• ƒ†”‡•—Ž–‹‰’”‘†— –• ‘’‡–‡ˆ‘”„‹†‹‰–‘–Š‡ƒ –‹˜‡•‹–‡ǤŠ‡”‡ƒ”‡‡˜‡”–Š‡Ž‡••†‹ˆˆ‡”‡– ‹•‘‡œ›‡•ˆ‘”‡ƒ Š”‡ƒ –‹‘ǤŠ‡ˆ‹”•–”‡ƒ –‹‘‘ˆ‡ƒ Š › Ž‡‹• ƒ–ƒŽ›œ‡†„›†‹ˆˆ‡”‡–ƒ ›ŽǦ ‘†‡Š›†”‘‰‡ƒ•‡‹•‘‡œ›‡•ǣ–Š‡‡”›Ǧ‘‰Ǧǡ‘‰Ǧǡ‡†‹—Ǧǡƒ†Š‘”–ǦŠƒ‹ ›ŽǦ‘ ‡Š›†”‘‰‡ƒ•‡• ǡ ǡ ǡ ƒ† Ȍ ȋ•Ȍ ȋǤǤ ͳǤ͵ǤͻͻǤ͵ ƒ† ǤǤͳǤ͵ǤͺǤͳȌǤ ƒ Š‘ˆ –Š‡•‡‹•‘‡œ›‡• ƒ ‡’–•ƒ ”ƒ‰‡ ‘ˆƒ ›ŽǦ‘ •—„•–”ƒ–‡•‘ˆ †‹ˆˆ‡”‡–ǡ ‘˜‡”Žƒ’’‹‰ Šƒ‹Ž‡‰–Š•Ǥ‘†‡–•‡š’”‡••ƒŽŽˆ‘—”ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡‹•‘‡œ›‡•‹–Š‡Ž‹˜‡”ǡ™Š‹Ž‡ ‹Š—ƒŽ‹˜‡”‹•Ž‘™Ž›‡š’”‡••‡†ȏͺͲȐƒ†‘ˆŽ‹‹–‡†‹’‘”–ƒ ‡ˆ‘”Š‡’ƒ–‹   ȏͳͷ͵ȐǤ ”‘†‡–•ǡ Šƒ‹ǦŽ‡‰–Š•’‡ ‹ˆ‹ ‹–‹‡•‘ˆ–Š‡•ƒ”‡ͳʹǦͳ͸ǡͺǦͳ͸ǡͶǦͳʹƒ†ͶǦ͸ ƒ”„‘

ͳͻ Chapter 1

ƒ–‘• ˆ‘” ǡǡ  ƒ†ǡ”‡•’‡ –‹˜‡Ž›ǡ ™Š‹Ž‡ ‹ Š—ƒ• –Š‡ Šƒ‹ǦŽ‡‰–Š •’‡ ‹ˆ‹ ‹–‹‡• ƒ”‡ ͸Ǧͳ͸ǡ ͶǦͳ͸ ƒ† ͶǦ͸ ˆ‘” ǡ  ƒ† ǡ ”‡•’‡ –‹˜‡Ž›Ǥ ‹ ‡ –Š‡ ‹•‘‡œ›‡•Šƒ˜‡‘˜‡”Žƒ’’‹‰ Šƒ‹ǦŽ‡‰–Š•’‡ ‹ˆ‹ ‹–‹‡•ǡ–Š‡› ‘’‡–‡ˆ‘”–Š‡‹”•—„•–”ƒ–‡•ƒ† ’”‘†— –•Ǥ‘‰‡–Š‡”™‹–Š–Š‡ › Ž‹ ƒ–—”‡‘ˆ–Š‡’ƒ–Š™ƒ›ǡ–Š‡˜ƒ•–„‹‘ Š‡‹ ƒŽ ‘’‡–‹–‹‘ ƒ‡• –Š‡   „‹‘ Š‡‹ ƒŽŽ› ’ƒ”–‹ —Žƒ”Ž› ‘’Ž‡šǤ Š‡ ’”‘†— – ‘ˆ –Š‡ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡•‹•‡‘›ŽǦ‘ȋ ‹‰—”‡ͳȌǤ‘›ŽǦ‘‡•–‡”•‘ˆͺ‘”‘”‡ ƒ”„‘ƒ–‘• ƒ„‡ ˆ—”–Š‡”‡–ƒ„‘Ž‹œ‡†„›ƒ•‹‰Ž‡‡œ›‡™‹–Š͵ƒ –‹˜‹–‹‡•‹–‘ Š‘†”‹ƒŽ”‹ˆ— –‹‘ƒŽ”‘–‡‹ ȋȌǤ ”‡ˆ‡”–‘–Š‹•ƒ•–Š‡‡†‹—Ǧƒ†Ž‘‰Ǧ Šƒ‹‘”„”ƒ Š‘ˆ–Š‡ ’ƒ–Š™ƒ›Ǥ Ž–‡”ƒ–‹˜‡Ž›ǡ –Š‡ ‡‘›ŽǦ‘ ‡•–‡” ƒ —†‡”‰‘ ˆ—”–Š‡” Šƒ‹Ǧ•Š‘”–‡‹‰ ƒ–ƒŽ›œ‡† „› ͵ •‡’ƒ”ƒ–‡‡œ›‡•ǣ‡‘›ŽǦ‘Š›†”ƒ–ƒ•‡‘” ”‘–‘ƒ•‡ȋǡǤǤͶǤʹǤͳǤ͹Ͷƒ†ǤǤͶǤʹǤͳǤͳͷͲȌǡ Š›†”‘š›ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡ȋȀ ǡǤǤͳǤͳǤͳǤʹͳͳƒ†ǤǤͳǤͳǤͳǤ͵ͷȌƒ†‡†‹—Ǧ Šƒ‹͵Ǧ‡–‘Ǧƒ ›ŽǦ‘–Š‹‘Žƒ•‡ȋǡǤǤʹǤ͵ǤͳǤͳ͸ȌȌǡ”‡•’‡ –‹˜‡Ž›Ǥ ›ŽǦ‘‡•–‡”•‘ˆƒŽŽ Šƒ‹Ž‡‰–Š• ƒ„‡‘š‹†‹œ‡†˜‹ƒ–Š‹•„”ƒ ŠǤ ”‡ˆ‡”–‘–Š‹•ƒ•–Š‡•Š‘”–Ǧ Šƒ‹„”ƒ Š‘”–Š‡ ǦȀ Ǧ„”ƒ ŠǤŠ‡”‡ƒ –‹‘• ƒ–ƒŽ›œ‡†„›Ȁ ƒ†”‡“—‹”‡ –Š‡ ‘‡œ›‡• Ϊ ƒ† ‘ ǡ ”‡•’‡ –‹˜‡Ž›Ǥ Ȁ  ‘š‹†‹œ‡• Š›†”‘š›ƒ ›ŽǦ‘ –‘ ‡–‘ƒ ›ŽǦ‘™‹–ŠΪƒ•–Š‡‡Ž‡ –”‘ƒ ‡’–‘”ǤŠ‹•”‡ƒ –‹‘‹•”‡˜‡”•‹„Ž‡ƒ†‘’‡”ƒ–‡• Ž‘•‡–‘‡“—‹Ž‹„”‹—ȏͳͲͺȐ†—‡–‘‹–•Š‹‰Š ƒ–ƒŽ›–‹  ƒ’ƒ ‹–›Ǥ –Šƒ•ƒ˜‡”›•ƒŽŽ‡“—‹Ž‹„”‹— ‘•–ƒ–ȋʹǤͳ͹ȉͳͲǦͶȌƒ†–Š‡”‡ˆ‘”‡”‡“—‹”‡•ƒ˜‡”›Ž‘™’”‘†— –‘˜‡”•—„•–”ƒ–‡”ƒ–‹‘–‘”—‹ –Š‡ˆ‘”™ƒ”††‹”‡ –‹‘Ǥ –Š‡Žƒ•–”‡ƒ –‹‘‘ˆ–Š‡ › Ž‡ǡ ƒ–ƒŽ›œ‡†„›ǡ‘‹• ‘•—‡† ƒ†‡–‘ƒ ›ŽǦ‘‹••’Ž‹–‹–‘ƒƒ ›ŽǦ‘‡•–‡”–Šƒ–‹•–™‘ ƒ”„‘•Š‘”–‡”ƒ†ƒ ‡–›ŽǦ‘Ǥ Šƒ•ƒŠ‹‰Š‡“—‹Ž‹„”‹— ‘•–ƒ–‘ˆͳͲ͵ȏͳͷ͵ȐǤŠ‹•ƒŽŽ‘™•–‘ƒ‹–ƒ‹Ž‘™‡–‘ƒ ›ŽǦ ‘ ‘ ‡–”ƒ–‹‘•Ǥ ‘–ƒ„Ž›ǡ ǡ Ȁ  ƒ†  ƒŽ•‘ ƒ–ƒŽ›œ‡ –Š‡ ˆ‹ƒŽ –Š”‡‡ ”‡ƒ –‹‘• ‘ˆ –Š‡ ƒ–ƒ„‘Ž‹• ‘ˆ ‹•‘Ž‡— ‹‡ǣ –‘‰‡–Š‡”ǡ –Š‡•‡ ‡œ›‡• ‘˜‡”– –Š‡ ‹•‘Ž‡— ‹‡ ƒ–ƒ„‘Ž‹•‹–‡”‡†‹ƒ–‡‡–ƒ„‘Ž‹–‡–‹‰Ž›ŽǦ‘ȋ‹Ǥ‡ǤʹǦ‡–Š›Ž„—–ǦʹǦ‡‘›ŽǦ‘Ȍ–‘ʹǦ‡–Š›ŽǦ͵Ǧ Š›†”‘š›„—–›”›ŽǦ‘ǡ •—„•‡“—‡–Ž› –‘ ʹǦ‡–Š›Žƒ ‡–‘ƒ ‡–›ŽǦ‘ ƒ† –Š‡ –‘ ’”‘’‹‘›ŽǦ‘ ƒ†ƒ ‡–›ŽǦ‘ȏͳͷͶȂͳͷ͹ȐǤŠ‹•”‡•—Ž–•‹‡š–”ƒ•—„•–”ƒ–‡ ‘’‡–‹–‹‘ˆ‘”–Š‡•‡‡œ›‡•Ǥ ‘–Š‡” ‘’Ž‡š‹–›‹–Š‡ ’ƒ–Š™ƒ›‹••—„ ‡ŽŽ—Žƒ”Ž‘ ƒŽ‹œƒ–‹‘‘ˆ–Š‡‡œ›‡•ǤŠ‹Ž‡ ͳǡǡʹǡƒ†ƒ”‡Ž‘ ƒ–‡†ƒ––Š‡‹‡”‹–‘ Š‘†”‹ƒŽ‡„”ƒ‡ǡǡ ǡǡǡȀ ǡƒ†ƒ”‡—•—ƒŽŽ› ‘•‹†‡”‡†•‘Ž—„Ž‡ƒ†Ž‘ ƒŽ‹œ‡†‹ –Š‡‹–‘ Š‘†”‹ƒŽƒ–”‹šȏͳͷ͵ȐǤ ‘™‡˜‡”ǡ‡š’‡”‹‡–ƒŽ‡˜‹†‡ ‡‹”ƒ–Ž‹˜‡”‹–‘ Š‘†”‹ƒ Šƒ••Š‘™–Šƒ–ǡƒ† ƒƒŽ•‘„‡’Š›•‹ ƒŽŽ›ƒ••‘ ‹ƒ–‡†–‘–Š‡‡„”ƒ‡Ǧ „‘—† ‘š‹†ƒ–‹˜‡ ’Š‘•’Š‘”›Žƒ–‹‘ ȋ Ȍ •—’‡” ‘’Ž‡š‡• ȏͳͷͺȐǤ – ™ƒ• •—‰‰‡•–‡† –Šƒ– –Š‡‹” ‹–”ƒ ‡ŽŽ—Žƒ” Ž‘ ƒŽ‹œƒ–‹‘ ƒ› „‡ ‘†‹–‹‘ †‡’‡†‡– ȏͳͷͺȐǤ ǡ Ȁ  ƒ† Šƒ˜‡ƒŽ•‘„‡‡•Š‘™–‘„‹††‹”‡ –Ž›‘”‹†‹”‡ –Ž›–‘–Š‡‹–‘ Š‘†”‹ƒŽ‡„”ƒ‡ ȋ™‹–Š •—„•–ƒ–‹ƒŽ Ͷ ƒ –‹˜‹–› ƒ– –Š‡ ‡„”ƒ‡ ˆ‘” ƒŽŽ –Š‡•‡ ‡œ›‡•Ȍ ȏͳͷͻȐǤ –‡”‡•–‹‰Ž›ǡ •—„•–ƒ–‹ƒŽŽ› Š‹‰Š‡” ”‡•’‹”ƒ–‹‘ǦŽ‹‡† ‘š‹†ƒ–‹‘ ‘ˆ „—–›”›ŽǦ‘ǡ „—–‡‘›ŽǦ‘ ƒ† Š›†”‘š›„—–›”›ŽǦ‘ Šƒ• „‡‡ ”‡’‘”–‡† ‹ ‰‡–Ž›Ǧ†‹•”—’–‡† ‘’ƒ”‡† –‘ ‘’Ž‡–‡Ž›Ǧ †‹•”—’–‡†‹–‘ Š‘†”‹ƒȏͳ͸ͲȐǤ —”–Š‡”‘”‡ǡ’Š›•‹ ƒŽƒ••‘ ‹ƒ–‹‘•„‡–™‡‡Ȁ ƒ†  †‡Š›†”‘‰‡ƒ•‡ȋ‘’Ž‡š ȌǡȀ ƒ†–Š‡‡–‘‰‡‡•‹•‡œ›‡  ʹȋ‘’Ž‡š Ȍƒ†„‡–™‡‡ƒ†–Š‡ƒ ‡–›ŽǦ‘Ǧ ‘•—‹‰ › Ž‡‡œ›‡ ‹–”ƒ–‡•›–Šƒ•‡ Šƒ˜‡„‡‡†‡‘•–”ƒ–‡†ȏͳ͸ͳȂͳ͸͵ȐǤŠ‹••—‰‰‡•–•ƒ‡ˆˆ‹ ‹‡–‡ Šƒ‹•ˆ‘”–Š‡”‡‘š‹†ƒ–‹‘ ‘ˆ ǡ–Š‡”‡‰‡‡”ƒ–‹‘‘ˆ‘ ˆ”‘ƒ ‡–›ŽǦ‘„› ‹–”ƒ–‡•›–Šƒ•‡‘”–Š‡‡–‘‰‡‹  ’ƒ–Š™ƒ› ˆ‘” ‹•–ƒ ‡ ƒ† ”‡‘˜ƒŽ ‘ˆ ͶǦ‡–‘ƒ ›ŽǦ‘ ‹–‘ ‡–‘‰‡‡•‹•Ǥ Š—•ǡ ’‘–‡–‹ƒŽŽ›   •‘Ž—„Ž‡ ‡œ›‡• ƒ› •’ƒ–‹ƒŽŽ› ‘”‰ƒ‹œ‡ ‹ •— Š ƒ ƒ‡” –Šƒ– Š‹‰Š‡” ˆƒ––›Ǧƒ ‹† ‘š‹†ƒ–‹‘”ƒ–‡• ƒ„‡ƒ Š‹‡˜‡†ǡ’ƒ”–‹ —Žƒ”Ž›‹ ‘†‹–‹‘•‘ˆŠ‹‰ŠǦ‡‡”‰›†‡ƒ†Ǥ

ʹͲ General introduction and outline of the thesis

 mFAO deficiency 1 ƒ–Š‘Ž‘‰‹ ƒŽ ‹„‘” ‡””‘”• Šƒ˜‡ „‡‡ ‹†‡–‹ˆ‹‡† ˆ‘” ƒŽŽ ‡œ›‡• ‘ˆ –Š‡   ’ƒ–Š™ƒ› ȏͳͷ͵ǡͳ͸ͶȐǤ ˆ —–”‡ƒ–‡†ǡ  Ǧ†‡ˆ‹ ‹‡– ‹†‹˜‹†—ƒŽ• ”— ƒ ”‹• ‘ˆ •‡˜‡”‡ǡ Ž‹ˆ‡Ǧ–Š”‡ƒ–‡‹‰ Š›’‘‡–‘–‹  Š›’‘‰Ž› ‡‹ƒ †—”‹‰ ˆƒ•–‹‰ ‘” ‡‡”‰›Ǧ†‡ƒ†‹‰ ‘†‹–‹‘•Ǥ ǡ ǡ ƒ†  ƒ ‹ ’”‹ ‹’Ž‡ –ƒ‡ ‘˜‡” ‡ƒ Š ‘–Š‡”ǯ• ”‘Ž‡ǡ †—‡ –‘ –Š‡‹” ‘˜‡”Žƒ’’‹‰ •—„•–”ƒ–‡ •’‡ ‹ˆ‹ ‹–‹‡•ȏͳͲͻǡͳͷ͵ȐǡƒŽ„‡‹–™‹–ŠŽ‡••‡”ƒˆˆ‹‹–›ƒ†ƒ––Š‡‡š’‡•‡‘ˆ ƒ’ƒ ‹–›ˆ‘”–Š‡‹”‘™ ˆ— –‹‘Ǥ —”–Š‡”‘”‡ǡƒ†‡ˆ‹ ‹‡ ›‘ˆ‹Š‹„‹–• ƒŽ”‡ƒ†›‹–Š‡„‡‰‹‹‰ǡ™Š‹Ž‡ ‹  †‡ˆ‹ ‹‡ › ƒ†  †‡ˆ‹ ‹‡ › –Š‡ ˆ‹”•– › Ž‡• ‘ˆ –Š‡ EǦ‘š‹†ƒ–‹‘ ƒ ’”‘ ‡‡† ‘”ƒŽŽ›ǤŠ—•ǡ™Š‡‘”ƒ”‡†‡ˆ‹ ‹‡–ǡ•‘‡ ‹„„•‡‡”‰› ƒ•–‹ŽŽ„‡Šƒ”˜‡•–‡† ˆ”‘ ƒ ’ƒ”–‹ƒŽ EǦ‘š‹†ƒ–‹‘Ǥ Š‹• ƒ› ‡š’Žƒ‹ ™Š› Ǧ†‡ˆ‹ ‹‡– ȋȌ Š‹Ž†”‡ǡ ™Š‘ ™‘—Ž†‘Ž›„‡†‡ˆ‹ ‹‡–‹–Š‡Žƒ•–  › Ž‡ǡ‘ˆ–‡Šƒ˜‡‘Ž›‹‘”•›’–‘•ȏͳͷ͵Ȑƒ† ™Š› –Š‡ •‡˜‡”‹–› ‘ˆ †‹•‡ƒ•‡ ’”‡•‡–ƒ–‹‘ ‰‡‡”ƒŽŽ› ‹ ”‡ƒ•‡• ˆ”‘  –‘  –‘  ȏͳͷ͵ȐǤ ƒ•‡† ‘ ”‡’‘”–‡† Ž‹‹ ƒŽ ’”‡•‡–ƒ–‹‘ǡ  ƒ› „‡ ”‡‰ƒ”†‡† ƒ• Ž‡•• •‡˜‡”‡–Šƒƒ†‘”‡•‡˜‡”‡–Šƒȏͳͷ͵ǡͳ͸ͷȐǤŠƒ•„‡‡ƒ••‘ ‹ƒ–‡† ™‹–ŠŠ›’‘‡–‘–‹ Š›’‘‰Ž› ‡‹ƒǡ ƒ”†‹‘›‘’ƒ–Š›ǡ ƒ”†‹ƒ ƒ””›–Š‹ƒ•ƒ† ‘†— –‹‘†‡ˆ‡ –•ǡ •‡Ž‡–ƒŽ ›‘’ƒ–Š› ƒ† ”Šƒ„†‘›‘Ž›•‹•Ǥ  Šƒ• „‡‡ ’”‡†‘‹ƒ–Ž› ƒ••‘ ‹ƒ–‡† ™‹–Š Š›’‘‡–‘–‹  Š›’‘‰Ž› ‡‹ƒǤ  Šƒ• ‘ Ž‡ƒ” ’ƒ–Š‘Ž‘‰‹ ƒŽ ’Š‡‘–›’‡ ƒ† ‹• ‰‡‡”ƒŽŽ› ”‡‰ƒ”†‡†ƒ•ƒ†‡ˆ‹ ‹‡ ›™‹–Š“—‡•–‹‘ƒ„Ž‡ Ž‹‹ ƒŽ•‹‰‹ˆ‹ ƒ ‡ȏͳͷ͵ǡͳ͸ͷǡͳ͸͸ȐǤ ‘™‡˜‡”ǡƒ• ’”‡˜‹‘—•Ž›‹†‹ ƒ–‡†ˆ‘” •‹‰‡‡”ƒŽǡ–Š‡”‡‹•ƒŽƒ”‰‡Š‡–‡”‘‰‡‡‹–›‹†‹•‡ƒ•‡’”‡•‡–ƒ–‹‘ „‡–™‡‡  Ǧ†‡ˆ‹ ‹‡– ‹†‹˜‹†—ƒŽ• ™‹–Š –Š‡ •ƒ‡ ‡œ›‡ †‡ˆ‹ ‹‡ ›ǡ ƒ† ‡˜‡ ™‹–Š –Š‡ •ƒ‡‰‡‡–‹ —–ƒ–‹‘ȏͳ͵Ͷǡͳͷ͵ǡͳ͸ͷǡͳ͸͹Ȃͳ͹ͳȐǤ  Medium-chain acyl-CoA (MCAD) deficiency  †‡ˆ‹ ‹‡ › ȋ  ʹͲͳͶͷͲȌ ‹• –Š‡ ‘•– ‘‘   †‡ˆ‡ –ǡ ™‹–Š ƒ ‘„•‡”˜‡† ’”‡˜ƒŽ‡ ‡ ‘ˆ —’ –‘ ͳ ‹ ͸͸ͲͲ ‡™„‘”• ȏͳͳǡͳ͵ͲȐǤ  —–”‡ƒ–‡† ‹†‹˜‹†—ƒŽ•  Šƒ• ƒ †‹•‡ƒ•‡ •‡˜‡”‹–› –Šƒ– ”ƒ‰‡• ˆ”‘ ‘’Ž‡–‡Ž› ƒ•›’–‘ƒ–‹  –‘ ˆƒ–ƒŽǤ ’ƒ”– ˆ”‘ Šƒ˜‹‰ ƒ Ž‹‹ ƒŽ”‡Ž‡˜ƒ ‡‹‹–•‘™”‹‰Š–ǡƒ›–Š‡”‡ˆ‘”‡ƒŽ•‘‘ˆˆ‡”‹•‹‰Š–•‹–Š‡‘†‹ˆ›‹‰ ˆƒ –‘”• ‘ˆ   ˆ— –‹‘Ǥ Š‹• ™‘—Ž† „‡ ‹’‘”–ƒ– ˆ‘” —†‡”•–ƒ†‹‰ ‘–Š‡”   †‡ˆ‹ ‹‡ ‹‡•ƒ†‡–ƒ„‘Ž‹ †‹•‡ƒ•‡‹‰‡‡”ƒŽǤ †‹˜‹†—ƒŽ• Š‘‘œ›‰‘—• ˆ‘” –Š‡ Ž‘••Ǧ‘ˆǦˆ— –‹‘ Ǥͻͺͷε  —–ƒ–‹‘ ‹ –Š‡ ACADM ‰‡‡ǡ ‡ ‘†‹‰ǡƒ ‘—–ˆ‘”͹͹Ψ‘ˆ–Š‡ ƒ•‡•ȏͳ͵ͶȐǤŠ‹•—–ƒ–‹‘ ƒ—•‡•ƒ Šƒ‰‡ˆ”‘ Ž›•‹‡–‘‰Ž—–ƒƒ–‡ƒ–’‘•‹–‹‘͵ͲͶȋ’Ǥ͵ͲͶȌ‹–Š‡ƒ–—”‡ǤŠ‹•”‡•—Ž–•‹‹’ƒ‹”‡† ’”‘–‡‹ ˆ‘Ž†‹‰ǡ †‡ ”‡ƒ•‡† ’”‘–‡‹ •–ƒ„‹Ž‹–› ƒ† •‡˜‡”‡Ž› ‹’ƒ‹”‡†  ‡œ›‡ ƒ –‹˜‹–› ȏͳ͹ʹȂͳ͹ͷȐǤŠ‹Ž†”‡‹–Š‹•‰”‘—’”—ƒ•‡˜‡”‡”‹•‘ˆŽ‹ˆ‡Ǧ–Š”‡ƒ–‡‹‰Ž‘™„Ž‘‘†‰Ž— ‘•‡ƒ† ‡–‘‡ „‘†‹‡• ‘ ‡–”ƒ–‹‘• ȋŠ›’‘‡–‘–‹  Š›’‘‰Ž› ‡‹ƒȌ ȏͳͷ͵ǡͳ͸ͶȐǤ ††‹–‹‘ƒŽ Ž‹‹ ƒŽ •›’–‘• ‹ Ž—†‡ ‘˜‡”™‡‹‰Š–ǡ —• Ž‡ ’ƒ‹ǡ —• Ž‡ ˆƒ–‹‰—‡ǡ ƒ Ž‘™‡”‡† ‡š‡” ‹•‡ –‘Ž‡”ƒ ‡ǡ Š‡’ƒ–‘‡‰ƒŽ›ǡ –‡’‡”ƒ–—”‡ ‹•–ƒ„‹Ž‹–›ǡ Š›’‘–Š‡”‹ƒǡ †”‘™•‹‡•• ‘” Ž‡–Šƒ”‰› –Šƒ– ƒ› †‡˜‡Ž‘’ ‹–‘ ‘ƒ ‘” ‡˜‡ •—††‡ †‡ƒ–Š ȏͳ͵Ͳǡͳ͵ͳǡͳ͸ͷȐǤ ‹‘ Š‡‹ ƒŽŽ›ǡ  ‹• Šƒ”ƒ –‡”‹œ‡† „› ƒ ——Žƒ–‹‘ ‘ˆ †‹ ƒ”„‘š›Ž‹  ƒ ‹†•ǡ ƒ ›ŽǦ‰Ž› ‹‡• ƒ† ƒ ›ŽǦ ƒ”‹–‹‡• ‘ˆ ƒ”„‘Ǧ Šƒ‹Ž‡‰–Š͸ǦͳͲƒ†ƒŠ‹‰Š‡””ƒ–‹‘‘ˆͺ‘˜‡”ͳͲ Šƒ‹•‹’Žƒ•ƒƒ†—”‹‡ ȏͳ͸ͷǡͳ͹Ͳǡͳ͹͸Ȃͳͺ͵ȐǤ‹†‹˜‹†—ƒŽ•™‹–Šƒ”‡•‹†—ƒŽƒ –‹˜‹–›Š‹‰Š‡”–ŠƒͳͲΨ‘ˆ ‘–”‘Ž• •Š‘™‡† ƒ ‹Ž†‡” „‹‘ Š‡‹ ƒŽ ƒ† Ž‹‹ ƒŽ ’Š‡‘–›’‡ ȏͳ͵ͶȐǤ ‘™‡˜‡”ǡ ‘– ƒŽŽ ˜ƒ”‹ƒ„‹Ž‹–›„‡–™‡‡‹†‹˜‹†—ƒŽ• ƒ„‡‡š’Žƒ‹‡†„›”‡•‹†—ƒŽ‡œ›‡ƒ –‹˜‹–›Ǥ˜‡ƒ•—„•‡–

ʹͳ Chapter 1

‘ˆ Š‹Ž†”‡Š‘‘œ›‰‘—•ˆ‘”–Š‡ Ǥͻͺͷε —–ƒ–‹‘”‡ƒ‹•ƒ•›’–‘ƒ–‹ –Š”‘—‰Š‘—–Ž‹ˆ‡ ȏͳͳǡͳ͵Ͳǡͳ͵ͳǡͳ͵Ͷǡͳ͹ͳǡͳͺͶȂͳͺ͹ȐǤ ‘–ƒ„Ž›ǡ Ǧ†‡ˆ‹ ‹‡– Š‹Ž†”‡ ‘š‹†‹œ‡ ‡†‹—Ǧ Šƒ‹ ˆƒ––› ƒ ‹†• ‘”ƒŽŽ› —†‡” ˆ‡† ‘†‹–‹‘• ȏͳͺͺȐ ƒ† –Š‡› ƒ”‡ ƒ„Ž‡ –‘ ˆƒ•– ˆ‘” ͳͺǦʹͶ Š‘—”• ȏͳ͵ͳǡͳͷ͵ǡͳͺͻǡͳͻͲȐǤŠ‡‡š–‡––‘™Š‹ Š–Š‡›‘—‰‡•–‹ˆƒ–•ȋͲǦʹ›‡ƒ”•‘Ž†Ȍƒ”‡ƒ„Ž‡ –‘ˆƒ•–‹•ǡŠ‘™‡˜‡”ǡ —””‡–Ž›— Ž‡ƒ”Ǥ –‹• —””‡–Ž›–Š‘—‰Š––Šƒ–•’‡ ‹ˆ‹ ƒŽŽ›ƒ ‘„‹ƒ–‹‘‘ˆ ’”‘Ž‘‰‡† ˆƒ•–‹‰ ƒ† ƒ ƒ††‹–‹‘ƒŽ –”‹‰‰‡”ǡ •— Š ƒ• ƒ‹”™ƒ› ‘” ‰ƒ•–”‘‹–‡•–‹ƒŽ ‹ˆ‡ –‹‘•ǡ ƒŽ ‘Š‘Ž—•‡ƒ† ‘Ž†Ǧ‡š’‘•—”‡ǡ‹ ”‡ƒ•‡–Š‡”‹•‘ˆ‡–ƒ„‘Ž‹  ”‹•‹•ƒ†Ž‘™„Ž‘‘†‡–‘‡ƒ† ‰Ž— ‘•‡Ž‡˜‡Ž•ȏͳ͵Ͳǡͳ͸Ͷǡͳ͸ͻǡͳͺ͵ǡͳͻͳȐǤŠ‹••Š‘™•–Šƒ––Š‡‡–‹‘Ž‘‰›‘ˆ–Š‡†‹•‡ƒ•‡’”‡•‡–ƒ–‹‘ ‹• Ž‡ƒ”Ž›‘– ‘’Ž‡–‡Ž›—†‡”•–‘‘†Ǥ  Mouse models of mFAO deficiency ‘—†‡”•–ƒ†–Š‡‡–‹‘Ž‘‰›‘ˆ †‡ˆ‹ ‹‡ ‹‡•ǡ•‡˜‡”ƒŽ‘—•‡‘†‡Ž•Šƒ˜‡„‡‡‰‡‡”ƒ–‡†ǡ ‹ Ž—†‹‰ƒǦǡǦǡǦƒ†Ǧ‹ ‡ȏͳͷ͵ȐǤ –Š‡•‡‘—•‡‘†‡Ž•ǡ –Š‡ Ǧ ‘—•‡ ‘†‡Ž Šƒ• –Š‡ ‘•– •‡˜‡”‡ ’Š‡‘–›’‡ǡ ™‹–Š Š‡’ƒ–‘•–‡ƒ–‘•‹•ǡ ƒ”†‹ƒ  Š›’‡”–”‘’Š›ƒ††›•ˆ— –‹‘ǡ–”‹‰Ž› ‡”‹†‡ƒ† ‡”ƒ‹†‡ƒ ——Žƒ–‹‘ǡƒ†ˆƒ•–‹‰Ǧ‹†— ‡† Š›’‘‡–‘–‹ Š›’‘‰Ž› ‡‹ƒȏͳͷ͵ȐǤŠ‡Ǧ‘—•‡‹• ‘•‹†‡”‡†ƒ Ž‘•‡””‡’”‡•‡–ƒ–‹‘ ‘ˆ –Š‡ Š—ƒ  †‡ˆ‹ ‹‡ › –Šƒ –Š‡ Ǧ ‘—•‡ ‘†‡ŽǤ • ‹†‹ ƒ–‡† ƒ„‘˜‡ǡ ‹ ”‘†‡–• ƒ–ƒŽ›œ‡•–Š‡ͳ͸Ǧͳʹ†‡Š›†”‘‰‡ƒ–‹‘™Š‹Ž‡ ƒ–ƒŽ›œ‡•ƒ„”‘ƒ†‡””ƒ‰‡ ‘ˆͳ͸ǦͺǤ  ‘–”ƒ•–ǡ‹Š—ƒ•–Š‡ ‘ ‡–”ƒ–‹‘‘ˆ‹•˜‡”›Ž‘™ƒ†Š—ƒ ƒ–ƒŽ›œ‡•ͳ͸Ǧ͸†‡Š›†”‘‰‡ƒ–‹‘Ǥ‹•‡ƒ•‡•‡˜‡”‹–›‹Ǧ‹ ‡‹•Š‹‰Š‡•–‹Ǧ ‹ ‡ǡ ˆ‘ŽŽ‘™‡† „› Ǧ ƒ† Ǧ ‹ ‡ ƒ† •—„•‡“—‡–Ž› Ǧ ‹ ‡ ȏͳͷ͵ȐǤ – •‡‡• –Šƒ–  ‹• ‘”‡ ‘” Ž‡•• †‹•’‡•ƒ„Ž‡ ‹ ‹ ‡ǡ †—‡ –‘ –Š‡ ’”‡•‡ ‡ ƒ† „”‘ƒ† •’‡ ‹ˆ‹ ‹–›‘ˆǤŽŽ–Š‡Ǧ‹ ‡Šƒ˜‡„‡‡•Š‘™–‘„‡ ‘Ž†Ǧ‹–‘Ž‡”ƒ–ȏͳͷ͵ǡͳͻʹȂ ͳͻͶȐǤ˜‡‹–Š‡”‡Žƒ–‹˜‡Ž›‹Ž†Ǧ‘—•‡ȏͳͻͷȐˆƒ•–‹‰‹ ‘„‹ƒ–‹‘™‹–Š ‘Ž† ‡š’‘•—”‡”‡•—Ž–‡†‹Ž‘™‡”„Ž‘‘†‰Ž— ‘•‡ ‘ ‡–”ƒ–‹‘•–Šƒ‹‹ ‡ȏͳͻ͸ȂͳͻͺȐǤ ‘ †ƒ–‡ǡ Ǧ ‘—•‡ ‘†‡Ž• ‘ˆ –™‘ †‹ˆˆ‡”‡– „ƒ ‰”‘—†• Šƒ˜‡ „‡‡ ‰‡‡”ƒ–‡† ȏͳͲͻǡͳͻʹǡͳͻͻȐǤ ‹–‹ƒŽŽ›ǡ ƒ  Ǧ ‘—•‡ ‘†‡Ž ™ƒ• ‰‡‡”ƒ–‡† ‹ ƒ ͷ͹Ȁ͸ƒ  Ƭ ͳʹͻʹȀŽƒ •†‹š‡†„ƒ ‰”‘—†ȏͳͻʹȐǤŠ‡‘—•‡•Š‘™‡†•‡˜‡”ƒŽ‘ˆ–Š‡Š—ƒǦ ƒ••‘ ‹ƒ–‡††‹•‡ƒ•‡ Šƒ”ƒ –‡”‹•–‹ •ǡ‹ Ž—†‹‰Š‹‰Š‡”‡‘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”‡†–‘™‹Ž†–›’‡ȋȌ‹ ‡ǤʹͶŠ‘—”•‘ˆˆƒ•–‹‰ ”‡•—Ž–‡† ‹ Š‡’ƒ–‘•–‡ƒ–‘•‹• ‹ –Š‡ Ǧ „—– ‘– ‹ –Š‡  ‹ ‡Ǥ Š‹• ‘””‡•’‘†• –‘ Š‡’ƒ–‹  •–‡ƒ–‘•‹• ƒ† Š‹‰Š‡” „Ž‘‘†   Ž‡˜‡Ž•  •‡‡ ‹ •›’–‘ƒ–‹   ’ƒ–‹‡–•ǡ ȏͳ͵Ͳǡͳ͸Ͷǡͳ͸ͻǡͳͺͻǡͳͻͳȐǤ ‘™‡˜‡”ǡˆƒ•–‹‰†‹†‘–‹†— ‡„Ž‘‘†‰Ž— ‘•‡ƒ†‡–‘‡„‘†›Ž‡˜‡Ž †‹ˆˆ‡”‡ ‡•„‡–™‡‡–Š‡Ǧƒ†‹ ‡Ǥ ƒ•–‹‰‹ ‘„‹ƒ–‹‘™‹–Š ‘Ž†‡š’‘•—”‡ ™ƒ•Ž‡–ŠƒŽ‘”Ž‡†–‘—”‡ ‘˜‡”ƒ„Ž›Ž‘™„‘†›–‡’‡”ƒ–—”‡•‹–Š‡ǡ„—–‘–‹–Š‡‹ ‡ ȏͳͻʹȐǤ ›’‘‰Ž› ‡‹ƒ Šƒ•ǡ Š‘™‡˜‡”ǡ ‡˜‡” „‡‡ ”‡’‘”–‡† ‹ Ǧ ‹ ‡ ȏͺͲǡͳͻʹǡͳͻͻȐǤ ‡’ƒ–‹ ‹ ”‘ƒ””ƒ›ƒƒŽ›•‹•™ƒ•†‘‡‹Ǧ‹ ‡–Šƒ–™‡”‡‡‹–Š‡”ˆƒ•–‡†ˆ‘”ͳʹŠ‘—”• ‘”‰‹˜‡Ž‹’‘’‘Ž›•ƒ Šƒ”‹†‡ȋȌ–‘‹†— ‡ƒƒ —–‡Ǧ’Šƒ•‡‹—‡”‡•’‘•‡Ǥƒ†Ǧ •Š‘™‡†ƒ”‡††‹ˆˆ‡”‡ ‡•‹‡š’”‡••‹‘‘ˆ‰‡‡•‹˜‘Ž˜‡†‹‰Ž— ‘•‡‡–ƒ„‘Ž‹•ȏͳͻͻȐǤ ‡˜‡”ƒŽ ›‡ƒ”• Žƒ–‡”ǡ –Š‡ Ǧ ‘—•‡ ‘†‡Ž ™ƒ• „ƒ  ”‘••‡† –‘ ƒ ͷ͹Ȁ͸  ’—”‡Ǧ „ƒ ‰”‘—†ǤŽ•‘ǡ–Š‹•’—”‡Ǧ„ƒ ‰”‘—†Ǧ‘—•‡•Š‘™‡†Š‹‰Š‡”‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ ƒ”‹–‹‡Ž‡˜‡Ž•‹•‡”—ƒ†ƒŽ‘™‡”ͺǦƒ ›ŽǦ‘‘š‹†ƒ–‹‘ ƒ’ƒ ‹–›–Šƒ–Š‡ƒˆ–‡”ͳʹ Š‘—”•‘ˆˆƒ•–‹‰Ǥ ƒ††‹–‹‘ǡ–Š‡’Š‡›Ž’”‘’”‹‘›ŽǦ‘‘š‹†ƒ–‹‘ ƒ’ƒ ‹–›™ƒ•ƒŽ•‘Ž‘™‡”‹ –Š‡•‡  ‹ ‡ ‘’ƒ”‡† –‘ –Š‡‹”  Ž‹––‡”ƒ–‡•Ǥ Š‡ ƒ’ƒ ‹–› ‘ˆ ’Š‡›Ž’”‘’”‹‘›ŽǦ‘

ʹʹ General introduction and outline of the thesis

‘š‹†ƒ–‹‘‹•ƒ†‹ƒ‰‘•–‹ ƒ”‡”ˆ‘”‹–Š‡ Ž‹‹ ǡ„‡ ƒ—•‡’Š‡›Ž’”‘’”‹‘›ŽǦ‘‹• ‡š Ž—•‹˜‡Ž› ‘š‹†‹œ‡† „› ǡ ™Š‹Ž‡ –Š‡ ‡†‘‰‡‘—• ƒ ›ŽǦ‘ •—„•–”ƒ–‡• ƒ ƒŽ•‘ „‡ 1 ‘š‹†‹œ‡† „› –Š‡ ‘–Š‡” •Ǥ —”–Š‡”‘”‡ǡ ƒŽ•‘ ‹ –Š‡ ͷ͹Ȁ͸   „ƒ ‰”‘—† –Š‡ „Ž‘‘† ‰Ž— ‘•‡ƒ†‡–‘‡„‘†›Ž‡˜‡Ž•™‡”‡•‹‹Žƒ”‹–Š‡Ǧƒ†‹ ‡ƒˆ–‡”ͳʹŠ‘—”•‘ˆ ˆƒ•–‹‰ȏͳͲͻȐǤŠ‡•‡”‡•—Ž–•ƒ›•—‰‰‡•––Šƒ––Š‡Ǧ‹‰Š–‘–„‡ƒ—•‡ˆ—Ž‘†‡Žˆ‘” –Š‡ ƒ —–‡ Š›’‘‰Ž› ‡‹ƒ ‘„•‡”˜‡† ‹ •›’–‘ƒ–‹   ’ƒ–‹‡–•Ǥ ‘™‡˜‡”ǡ –Š‹• ‘—•‡ ‹‰Š–Šƒ˜‡‘–„‡‡•—ˆˆ‹ ‹‡–Ž› ŠƒŽŽ‡‰‡†–‘‹‹ –Š‡–”‹‰‰‡”•–Šƒ– ƒ—•‡Š›’‘‰Ž› ‡‹ƒ‹ Š—ƒ•Ǥ—”‹‰ ‘Ž†‡š’‘•—”‡‰Ž— ‘•‡Šƒ•‘–„‡‡”‡’‘”–‡†ȏͳͻʹȐǤ

MCAD deficiency ŝŶƐŝůŝĐŽ Š‡’Š‡‘–›’‹ Š‡–‡”‘‰‡‡‹–›ǡ ‘’Ž‡š‹–›ǡƒ†‹–‡” ‘‡ –‹˜‹–›‘ˆ ™ƒ””ƒ–ƒ•›•–‡• „‹‘Ž‘‰›ƒ’’”‘ƒ ŠǤŠ‹••Š‘—Ž†Š‡Ž’–‘—”ƒ˜‡ŽŠ‘™–Š‡‹–‡”’Žƒ›„‡–™‡‡†‹•‡ƒ•‡—–ƒ–‹‘ǡ ‘†‹ˆ›‹‰ˆƒ –‘”•ǡƒ†ƒ††‹–‹‘ƒŽ–”‹‰‰‡”•†‡–‡”‹‡™Š‡–Š‡”‘”‘–’ƒ–‹‡–•ƒ”‡ƒ–”‹•‘ˆ Š›’‘‡–‘–‹  Š›’‘‰Ž› ‡‹ƒ ȏͳͺȐǤ ‡‘‡Ǧ• ƒŽ‡ •–‘‹ Š‹‘‡–”‹  ‘†‡Ž• ‘ˆ  †‡ˆ‹ ‹‡ › •Š‘™’”‘‹•‡ˆ‘”–Š‡‹†‡–‹ˆ‹ ƒ–‹‘‘ˆ„‹‘ƒ”‡”•ȏͳͳʹǡʹͲͲȐǤ ‘™‡˜‡”ǡ–Š‡‹‡–‹  ‘’Ž‡š‹–› ‘ˆ –Š‡   ’ƒ–Š™ƒ› ‘—–Ž‹‡† ƒ„‘˜‡ǡ ƒ‘– „‡ ’”‘’‡”Ž› ”‡’”‡•‡–‡† ‹ •–‘‹ Š‹‘‡–”‹  ‘†‡Ž•Ǥ Š‡‹‡–‹ ‘†‡Ž†‡˜‡Ž‘’‡†„›‘—”‰”‘—’†‘‡•‘–‘Ž› ‘–ƒ‹–Š‡•–‘‹ Š‹‘‡–”›‘ˆ–Š‡ ’ƒ–Š™ƒ›ǡ ‹ Ž—†‹‰ –Š‡ ”‡ƒ –‹‘• ‹ ƒŽŽ –Š‡   › Ž‡•ǡ „—– ƒŽ•‘ –Š‡ ’”‘‹• —‹–› ‘ˆ –Š‡ ‡œ›‡•ȏͳͲͺȐǤŠ‹•’”‘‹• —‹–›”‡•—Ž–•‹ ‘’‡–‹–‹‘„‡–™‡‡ƒŽŽ•—„•–”ƒ–‡•ƒ†’”‘†— –• ‘ˆ ƒ ‡œ›‡ǡ •— Š –Šƒ– –Š‡› ‹Š‹„‹– ‡ƒ Š ‘–Š‡”ǯ• ‘˜‡”•‹‘ ‘’‡–‹–‹˜‡Ž›Ǥ ŽŽ‘•–‡”‹  ‹Š‹„‹–‹‘‘ˆͳ„›ƒŽ‘›ŽǦ‘™ƒ•ƒŽ•‘‹ ‘”’‘”ƒ–‡†ƒ•™‡ŽŽƒ• ‘•‡”˜ƒ–‹‘‘ˆ ‘‡œ›‡  ‹ –Š‡ ‹–‘ Š‘†”‹ƒŽ ƒ–”‹š ȏͳͲͺȐǤ Š‡ Žƒ––‡” •–‡• ˆ”‘ –Š‡ ƒ••—’–‹‘ –Šƒ– ‘ „‹‘•›–Š‡•‹• –ƒ‡• ’Žƒ ‡ ƒ– ƒ •Ž‘™‡” –‹‡• ƒŽ‡ –Šƒ –Š‡  Ǥ  ‘•‡“—‡ ‡‹•–Šƒ– ƒ› •‡“—‡•–”ƒ–‹‘‘ˆ‘‹‘‡•–‡”•Ž‡ƒ†•–‘ƒ ‘””‡•’‘†‹‰†”‘’‹ˆ”‡‡‘ ǤŠ‡‹‡–‹  ’ƒ”ƒ‡–‡”•™‡”‡–ƒ‡ˆ”‘in vitro‡œ›‡‹‡–‹ •ƒ˜ƒ‹Žƒ„Ž‡‹–Š‡Ž‹–‡”ƒ–—”‡ǤŠ‡‘†‡Ž •‹—Žƒ–‡•–Š‡ ‘’Ž‡–‡‘š‹†ƒ–‹‘‘ˆ ›–‘•‘Ž‹ ’ƒŽ‹–‘›ŽǦ‘ȋͳ͸Ǧƒ ›ŽǦ‘Ȍ–‘ƒ ‡–›ŽǦ‘Ǥ – Šƒ• „‡‡ ‡š’‡”‹‡–ƒŽŽ› ˜ƒŽ‹†ƒ–‡† ex vivo „› ‡ƒ•—”‹‰ ͵ͲǦ‹—–‡ ƒ ›ŽǦ ƒ”‹–‹‡ –‹‡ ‘—”•‡•‹‹•‘Žƒ–‡†”ƒ–Ž‹˜‡”‹–‘ Š‘†”‹ƒȏͳͲͺȐǤ —ƒƒ†‘—•‡˜‡”•‹‘•‘ˆ–Š‡‘†‡Ž ™‡”‡ ‘•–”— –‡†Žƒ–‡”ǡ„›ˆ‹––‹‰’ƒ”ƒ‡–‡”•–‘”‡Ž‡˜ƒ–‘—•‡ƒ†Š—ƒ†ƒ–ƒ•‡–•ȏͳͲͻȐǤ ‘†‡Ž•‹—Žƒ–‹‘•”‡•—Ž–••Š‘™‡†–Šƒ–‹ ”‡ƒ•‹‰’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘•†‹†‘–Ž‡ƒ† –‘•ƒ–—”ƒ–‹‘‘ˆ–Š‡ ˆŽ—šǡƒ•™‘—Ž†„‡‡š’‡ –‡†ǡ„—–Ž‡†–‘ƒ•–‡‡’†‡ Ž‹‡‘ˆ–Š‡  ˆŽ—šȏͳͲͺȐǤ –™ƒ••Š‘™–Šƒ––Š‹•™ƒ• ƒ—•‡† „›ƒ ›ŽǦ‘‡•–‡”ƒ ——Žƒ–‹‘ƒ†‘  †‡’Ž‡–‹‘ƒ††‹†‘–‘ —”‹ˆ–Š‡‡œ›‡’”‘‹• —‹–›™ƒ• ‘’—–ƒ–‹‘ƒŽŽ›”‡‘˜‡†ȏͳͲͺȐǤ –Š‡—”‹‡˜‡”•‹‘‘ˆ–Š‡‘†‡Ž†‡Ž‡–‹‘‘ˆƒ†‡–Š‡‘†‡Ž‘”‡•—• ‡’–‹„Ž‡–‘–Š‡ ˆŽ—š†‡ Ž‹‡ƒ–Š‹‰Š•—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘•ȏͳͲͻȐǤŠ‡ˆŽ—š†‡ Ž‹‡Šƒ•‘–›‡–„‡‡˜ƒŽ‹†ƒ–‡† ‡š’‡”‹‡–ƒŽŽ›ǤŠ‡Š—ƒ˜‡”•‹‘‘ˆ–Š‡‘†‡ŽŠƒ•„‡‡”‡Žƒ–‡†–‘’ƒ–‹‡–†ƒ–ƒǡŠ‘™‡˜‡” ȏͳͲͻȐǤ  †‡ˆ‹ ‹‡ › •Š‘™‡†ǡ ‘– •—”’”‹•‹‰Ž›ǡ ƒ ——Žƒ–‹‘ ‘ˆ ‡†‹—Ǧ Šƒ‹ ƒ ›Ž ƒ”‹–‹‡•ǡŒ—•–ƒ•‹’ƒ–‹‡–•ȏͳͲͻǡͳ͹Ͳǡͳ͹͸Ȃͳ͹ͺǡͳͺʹǡͳͺ͵ȐǤŠƒ–™ƒ•‘”‡•—”’”‹•‹‰™ƒ•–Šƒ– –Š‡‡Ž‡˜ƒ–‡†Ž‡˜‡Ž•‘ˆƒ„”‘ƒ†”ƒ‰‡‘ˆƒ ›ŽǦ ƒ”‹–‹‡•‹—Ž–‹’Ž‡ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡ǡ ”‡“—‹”‡† –Š‡ ‡œ›‡ ’”‘‹• —‹–› ȏͳͲͻȐǤ Š‡ ‘†‡Ž ”‡’”‘†— ‡† –Š‡ ’Žƒ•ƒ ƒ ›ŽǦ ƒ”‹–‹‡ ’”‘ˆ‹Ž‡•‘ˆ–™‘‹†‹˜‹†—ƒŽ’ƒ–‹‡–•ƒ† ‘””‡ –Ž›’”‡†‹ –‡†–Š‡‹”†‹•‡ƒ•‡•‡˜‡”‹–›„ƒ•‡†‘  ˆŽ—šǡ„—–‘Ž›‹ˆ‡œ›‡’”‘‹• —‹–›™ƒ•‹ Ž—†‡†ȏͳͲͻȐǤ Ž–‘‰‡–Š‡”ǡ –Š‡ ”‡•—Ž–• •—‰‰‡•– –Šƒ– ‡œ›‡ ’”‘‹• —‹–› ƒ‡• –Š‡   ’ƒ–Š™ƒ› ‹–”‹•‹ ƒŽŽ›•‡•‹–‹˜‡–‘•—„•–”ƒ–‡‘˜‡”Ž‘ƒ†ƒ†–Šƒ–•‘‡ †‡ˆ‹ ‹‡ ‹‡•ƒ›ƒ‰‰”ƒ˜ƒ–‡

ʹ͵ Chapter 1

–Š‹• ”‹•Ǥ Š‡ ‡ Šƒ‹• —†‡”Ž›‹‰ –Š‡ ”‡Žƒ–‹‘ „‡–™‡‡ ‡œ›‡ ’”‘‹• —‹–› ƒ† ˆŽ—š †‡ Ž‹‡ƒ–Š‹‰Š•—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘•Šƒ†‘–„‡‡ƒ††”‡••‡†„‡ˆ‘”‡ƒ†™‹ŽŽ„‡ƒƒŽ›œ‡† ‹Chapter 2‘ˆ–Š‹•–Š‡•‹•Ǥ  Chapter 4 ™‹ŽŽƒ††”‡••Š‘™•—””‘—†‹‰’ƒ–Š™ƒ›•ƒˆˆ‡ –  ˆ— –‹‘Ǥ‘–Š•–—†‹‡•™‹ŽŽŠ‡Ž’–‘—†‡”•–ƒ†Š‘™‘†‹ˆ›‹‰ˆƒ –‘”•ƒ›ƒˆˆ‡ –†‹•‡ƒ•‡ ’”‡•‡–ƒ–‹‘Ǥ  Outline of this thesis: a systems medicine approach for MCAD deficiency ›–Š‡•‹• Šƒ˜‡–ƒ‡ƒ•›•–‡•‡†‹ ‹‡ƒ’’”‘ƒ Š–‘‰‡–ƒ„‡––‡”‹•‹‰Š–‹–Š‡ ‘’Ž‡š ‡–‹‘Ž‘‰›‘ˆǤ›‘˜‡”ƒ” Š‹‰“—‡•–‹‘Šƒ•„‡‡™Šƒ–‡Ž‹ ‹–•Š›’‘‡–‘–‹ Š›’‘‰Ž› ‡‹ƒ ‹•‘‡ƒ†‘–Š‡”’ƒ–‹‡–•‘–Š‡‘‡Šƒ†ǡ„—–ƒŽ•‘™Šƒ–’”‘–‡ –•–Š‡Ǥ  ™‘—Ž†Ž‹‡–‘—†‡”•–ƒ†‘–Š‡‘‡Šƒ†™Š‹ Š‘†‹ˆ›‹‰ˆƒ –‘”•™‹–Š‹ƒ†‘—–•‹†‡–Š‡ ’ƒ–Š™ƒ›ƒ›’—•Š‡–ƒ„‘Ž‹•ˆ”‘ƒƒ’’ƒ”‡–Ž›Š‡ƒŽ–Š›•–ƒ–‡‹–‘ƒ†‡–”‹‡–ƒŽ–”ƒŒ‡ –‘”› –‘™ƒ”†•Š›’‘‡–‘–‹ Š›’‘‰Ž› ‡‹ƒǤ–Š‡‘–Š‡”Šƒ†ǡ ƒ‹–‘‹†‡–‹ˆ›–Š‡ˆƒ –‘”•–Šƒ–—†‡” •‘‡ ‘†‹–‹‘•ƒ†Ȁ‘”‹•‘‡’ƒ–‹‡–•‡‡’–Š‡•›•–‡•ƒˆ‡Ž›ƒ™ƒ›ˆ”‘–Š‹•†ƒ‰‡”‘—• •–ƒ–‡Ǥ ƒ‹–‡‰”ƒ–‹˜‡ƒ’’”‘ƒ Šǡ –ƒ‡‹–‘ƒ ‘—–ƒ†ƒ’–ƒ–‹‘•‹‰‡‡‡š’”‡••‹‘ƒ•™‡ŽŽ ƒ•–Š‡”‘Ž‡‘ˆ•—””‘—†‹‰’ƒ–Š™ƒ›•ǡ—–”‹‡–•ǡ ‘ˆƒ –‘”•ƒ†‹–‡”ƒŽ‡–ƒ„‘Ž‹–‡•Ǥ‘–Š‹•‡†  –ƒ‡ ƒ ‘’—–ƒ–‹‘ƒŽ ƒ’’”‘ƒ Š ‹ Chapters 2 and 5 ƒ† ƒ ‡š’‡”‹‡–ƒŽ ƒ’’”‘ƒ Š ‹ Chapters 3, 4, and 6Ǥ  –Š‡ ˆ‹”•– Šƒ’–‡”•  ˆ‘ —• ‘ ”‘†‡–•ǡ „—– ‹ Chapter 5  •Š‹ˆ– ‘’Ž‡–‡Ž›–‘Š—ƒƒ†‘–Š‡”†‡ˆ‹ ‹‡ ‹‡•Ǥ –Š‹••‡ –‹‘ ™‹ŽŽ„”‹‡ˆŽ›‡š’Žƒ‹ –Š‡ƒ’’”‘ƒ Š–Šƒ– ™‹ŽŽ–ƒ‡‹‡ƒ ŠŠƒ’–‡”Ǥ  Chapter 2  ™‹ŽŽ ˆ‘ —• ‘ –Š‡ ’”‘„Ž‡ ‘ˆ –Š‡ •–‡‡’ †‡ Ž‹‡ ‘ˆ ˆŽ—š ƒ• ƒ ˆ— –‹‘ ‘ˆ ƒ ‹ ”‡ƒ•‹‰ ‘ ‡–”ƒ–‹‘‘ˆ–Š‡ •—„•–”ƒ–‡’ƒŽ‹–‘›ŽǦ‘ǤŽ–Š‘—‰Š‹–Šƒ•‡˜‡”„‡‡ ’”‘˜‡–Šƒ––Š‹•†‡ Ž‹‡—†‡”Ž‹‡•–Š‡Š›’‘‡–‘–‹ Š›’‘‰Ž› ‡‹ƒ‹’ƒ–‹‡–•ǡ‹–‹•ƒ•‡”‹‘—• ’‘••‹„‹Ž‹–›Ǥ – Šƒ† „‡‡ •Š‘™ „‡ˆ‘”‡ –Šƒ– –Š‡ ‡œ›‡ ’”‘‹• —‹–› ™ƒ• ‡••‡–‹ƒŽ ˆ‘” –Š‹• „‡Šƒ˜‹‘”ǡ„—––Š‡‡ Šƒ‹•™ƒ•‘–—†‡”•–‘‘†ǤŽŽ™‘”‹–Š‹• Šƒ’–‡”™‹ŽŽ„‡†‘‡‹–Š‡ ’”‡˜‹‘—•Ž›†‡• ”‹„‡† ‘’—–ƒ–‹‘ƒŽ‘†‡Ž‘ˆ ‹”ƒ–‹–‘ Š‘†”‹ƒǤ —•‡†‡••‡–‹ƒŽŽ› –Š”‡‡‡–Š‘†•‘ˆƒƒŽ›•‹•Ǥ ‹”•–ǡ ™‹ŽŽ—•‡‡–ƒ„‘Ž‹ ‘–”‘ŽƒŽ›•‹•ȏͳͳͷȂͳͳͺȐ–‘“—ƒ–‹ˆ› ™Š‹ Š‡œ›‡• ‘–”‘Ž–Š‡ ˆŽ—šƒ†Š‘™–Š‹• Šƒ‰‡•ƒ•ƒˆ— –‹‘‘ˆ–Š‡’ƒŽ‹–‘›ŽǦ ‘ ‘ ‡–”ƒ–‹‘Ǥ‡ ‘†ǡ ™‹ŽŽŽ‘‘‹–‘–Š‡”‘Ž‡‘ˆ’”‘‹• —‹–›‘ˆ‡ƒ Š‹†‹˜‹†—ƒŽ‡œ›‡ •‡’ƒ”ƒ–‡Ž›ǡ„›‡‹–Š‡””‡‘˜‹‰‹–‹‘Ž›–Š‡‡œ›‡‘ˆ‹–‡”‡•–ǡ‘””‡‘˜‹‰‹–‹ƒŽŽ‡œ›‡• „—––Š‡‡œ›‡‘ˆ‹–‡”‡•–Ǥ‘‰‡–Š‡”–Š‡•‡–™‘ƒ’’”‘ƒ Š‡•™‹ŽŽ’‹’‘‹–™Š‹ Š‡œ›‡•ƒ”‡ –Š‡‡›’Žƒ›‡”•Ǥ ‹ƒŽŽ›ǡ ™‹ŽŽ‹•’‡ ––Š‡–‹‡ ‘—”•‡•‘ˆ–Š‡‡–ƒ„‘Ž‹–‡•ƒ†‡œ›‡”ƒ–‡•‹ ‘”‡†‡–ƒ‹ŽǤŠ‡–‹‡ ‘—”•‡•‹ ‘„‹ƒ–‹‘™‹–Š–Š‡”‡‰—Žƒ–‘”›•–”‡‰–Š‘ˆ‡ƒ Š‡–ƒ„‘Ž‹–‡• ȋƒƒŽ›œ‡†ƒ ‘”†‹‰–‘ȏͳʹͲȐ™‹ŽŽ‡Ž— ‹†ƒ–‡–Š‡ ƒ—•ƒŽ Šƒ‹‘ˆ‡˜‡–•–Šƒ– ƒ—•‡†–Š‡ˆŽ—š †‡ Ž‹‡Ǥ Chapter 3 ™‹ŽŽ–ƒ‡–Š‡‘’’‘•‹–‡ƒ’’”‘ƒ Šƒ†ƒ•–Š‡“—‡•–‹‘™Š‹ Š‰‡‡•‘”’ƒ–Š™ƒ›• ‘—Ž† ’”‘–‡ – –Š‡ ‡–ƒ„‘Ž‹  ‡–™‘” ƒ‰ƒ‹•– Š›’‘‡–‘–‹  Š›’‘‰Ž› ‡‹ƒǤ ‘ –Š‹•‡†  ™‹ŽŽ •—„Œ‡ ––Š‡Ǧ‹ ‡–‘‹Ž†Ž› ŠƒŽŽ‡‰‹‰ ‘†‹–‹‘•ȋͳʹǦʹͶŠ”•‘ˆˆƒ•–‹‰ƒ††‹ˆˆ‡”‡– †‹‡–•ȌǤ †‡” –Š‡•‡ ‘†‹–‹‘• –Š‡ ’ƒ”–‹ƒŽ Ž‘•• ‘ˆ   ƒ’ƒ ‹–› †‘‡• ‘– –”‹‰‰‡” Š›’‘‰Ž› ‡‹ƒ‘”‘–Š‡”‘˜‡”–‡–ƒ„‘Ž‹ ’Š‡‘–›’‡•Ǥ ™‹ŽŽ’”‡•‡–ƒ ‘„‹ƒ–‹‘‘ˆ‡“ ƒ† „‹‘‹ˆ‘”ƒ–‹ • –‘ ‹†‡–‹ˆ› ’‘–‡–‹ƒŽ ‘’‡•ƒ–‘”› ’ƒ–Š™ƒ›•Ǥ  ˜‹‡™ ‘ˆ –Š‡ ‹Ž† ƒ†ƒ’–ƒ–‹‘•ǡ ™‹ŽŽ‘–‘Ž›—•‡ Žƒ••‹ ƒŽ‰‡‡Ǧ•‡–‡”‹ Š‡–ƒƒŽ›•‹•ǡ„—–ƒŽ•‘’”‡•‡–ƒ‡™ ƒ’’”‘ƒ Š–‘‹†‡–‹ˆ›‘”‡•—„–Ž‡ƒ†ƒ’–ƒ–‹‘•Ǥ‘•‹†‡”‹‰–Š‡‹’‘”–ƒ ‡‘ˆΪƒ†‘  ™‹ŽŽ‹ Ž—†‡–ƒ‹Ž‘”Ǧƒ†‡‰‡‡•‡–•–‘•–—†›–Š‡‹””‘Ž‡Ǥ

ʹͶ General introduction and outline of the thesis

Chapter 4 ™‹ŽŽ–ƒ‡ƒ‘”‡†”ƒ•–‹ ƒ’’”‘ƒ Š„›•—„Œ‡ –‹‰–Š‡‹ ‡–‘ƒ ‘„‹ƒ–‹‘‘ˆ ˆƒ•–‹‰ƒ† ‘Ž†•–”‡••ǡ™Š‹ Š‹•ƒ•‡˜‡”‡Ž›‡‡”‰›Ǧ†‡ƒ†‹‰ ‘†‹–‹‘Ǥ–‹Ž‘™ǡ–Š‹•Šƒ• 1 „‡‡ –Š‡ ‘Ž› ‘†‹–‹‘ ”‡’‘”–‡† –‘ ƒ—•‡ ƒ •‡˜‡”‡ ’Š‡‘–›’‡ ‹Ǧ‹ ‡ȏͳͻʹȐǤ ‘™‡˜‡”ǡƒ„‹‘ Š‡‹ ƒŽ Šƒ”ƒ –‡”‹œƒ–‹‘‘ˆǦ‹ ‡—†‡” ‘Ž†•–”‡••ǡ‹ Ž—†‹‰„Ž‘‘† ‰Ž— ‘•‡Ž‡˜‡Ž•ǡŠƒ•‡˜‡”„‡‡”‡’‘”–‡†Ǥ –Š‹• Šƒ’–‡” ™‹ŽŽˆ‘ —•‘–Š‡”‘Ž‡‘ˆ–Š‡Ž‹˜‡”Ǥ –‡”‡•–‹‰Ž›ǡ ƒƒ‰‡†–‘•–—†›–Š‡‹ ‡‹ƒ†›ƒ‹  ‘†‹–‹‘™Š‡”‡„‘†›–‡’‡”ƒ–—”‡ ™ƒ•†‡ Ž‹‹‰”ƒ’‹†Ž›ƒ† ˆ‘—†ˆ‘”–Š‡ˆ‹”•––‹‡–Šƒ–’Žƒ•ƒ‰Ž— ‘•‡™ƒ•Ž‘™‡”‹Ǧ  ‹ ‡ –Šƒ Ǥ  ™‹ŽŽ •Š‘™ Š‘™ ‘’”‡Š‡•‹˜‡ ‡–ƒ„‘Ž‘‹  ƒƒŽ›•‹•ǡ ‹ Ž—†‹‰ Ž‹’‹†‘‹ •ǡ”‡˜‡ƒŽ••–”‘‰‡–ƒ„‘Ž‹ ƒ†ƒ’–ƒ–‹‘•ƒ•™‡ŽŽƒ•ƒ‡›”‘Ž‡‘ˆŽ‹˜‡” †—”‹‰ ‘Ž†•–”‡••Ǥ Chapter 5ǡ ™‹ŽŽ•™‹– Š–‘Š—ƒ ǤŽ‹‹–ƒ–‹‘‘ˆ–Š‡‡š‹•–‹‰ ‘’—–ƒ–‹‘ƒŽ‘†‡Ž ‹•–Šƒ–‹–•‹—Žƒ–‡• ‹‹•‘Žƒ–‹‘Ǥ ”‡ƒŽ‹–› †‡’‡†••–”‘‰Ž›‘–Š‡ƒ –‹˜‹–‹‡•‘ˆ •—””‘—†‹‰’ƒ–Š™ƒ›•Ǥ –Š‹• Šƒ’–‡” ™‹ŽŽ”‡˜‹•‹––Š‡ ‘˜‡”•‹‘‘ˆ–Š‡”ƒ–‘†‡Ž–‘ƒŠ—ƒ ˜‡”•‹‘ǡƒ‹‰—•‡‘ˆ‡š–‡•‹˜‡Ž‹–‡”ƒ–—”‡†ƒ–ƒǤ‘”‡‹’‘”–ƒ–Ž›ǡ ™‹ŽŽ‡š–‡†–Š‡‘†‡Ž ™‹–Š’ƒ–Š™ƒ›•–Šƒ–ƒˆˆ‡ ––Š‡ƒ˜ƒ‹Žƒ„‹Ž‹–›‘ˆ‘ ǤŠ‡”‡•—Ž–‹‰‘†‡Ž‹•ƒŽ‘•––™‹ ‡ƒ• Žƒ”‰‡ƒ•–Š‡‘”‹‰‹ƒŽ‘†‡ŽǤ ™‹ŽŽ–‡•–˜ƒ”‹‘—•‡š‹•–‹‰ƒ†Š›’‘–Š‡–‹ ƒŽ‹–‡”˜‡–‹‘•–‘‰‡– ‹•‹‰Š–‹–Š‡•–”‡‰–Š‘ˆ’‘–‡–‹ƒŽ‘†‹ˆ›‹‰ˆƒ –‘”•ƒ†–”‡ƒ–‡–‘’–‹‘•Ǥ  Chapter 6ǡ  ™‹ŽŽ •–—†› •‹ ˆ‹„”‘„Žƒ•–• †‡”‹˜‡† ˆ”‘ •›’–‘ƒ–‹  ƒ† ƒ•›’–‘ƒ–‹  ’ƒ–‹‡–• in vitroǤ•’”‡˜‹‘—•Ž›‹†‹ ƒ–‡†ǡ™‹–Š‹–Š‡•—„’‘’—Žƒ–‹‘‘ˆ‹†‹˜‹†—ƒŽ•™‹–Š ƒ Š‘‘œ›‰‘—• Ž‘••Ǧ‘ˆǦˆ— –‹‘ Ǥͻͺͷε  —–ƒ–‹‘ ‹ –Š‡ Ǧ‡ ‘†‹‰  ‰‡‡ǡ •›’–‘• ƒ”ƒ‰‡ˆ”‘ˆƒ–ƒŽŠ›’‘‡–‘–‹ Š›’‘‰Ž› ‡‹ƒ‡ƒ”Ž›‹Ž‹ˆ‡–‘ƒ ‘’Ž‡–‡Žƒ ‘ˆ •›’–‘•–Š”‘—‰Š‘—–Ž‹ˆ‡ȏ͹Ȃͳ͸ȐǤŠ‡”‡ƒ•‘„‡Š‹†–Š‡Š‡–‡”‘‰‡‡‹–›‹•›’–‘•‹–Š‹• •—„’‘’—Žƒ–‹‘‹••–‹ŽŽ— Ž‡ƒ”ǤŠ‡”‡ˆ‘”‡ǡ ™‹ŽŽ‹˜‡•–‹‰ƒ–‡™Š‡–Š‡”–Š‡”‡ƒ”‡‡–ƒ„‘Ž‹ ‘” ’”‘–‡‘‹  ƒ†ƒ’–ƒ–‹‘• ‹ —Ž–—”‡† •‹ ˆ‹„”‘„Žƒ•–• ‘ˆ  ‹†‹˜‹†—ƒŽ• –Šƒ– †‹•–‹‰—‹•Š •›’–‘ƒ–‹ ˆ”‘ƒ•›’–‘ƒ–‹ ‹†‹˜‹†—ƒŽ•Ǥ ‹ƒŽŽ›ǡ ‹ Chapter 7ǡ –Š‡ ‡‡”ƒŽ ‹• —••‹‘ǡ  ™‹ŽŽ ‡˜ƒŽ—ƒ–‡ › ’”‘‰”‡•• ƒ† †‹• —•• Ž‹‹–ƒ–‹‘•ƒ†‡š–•–‡’•Ǥ’‡ ‹ˆ‹ ƒŽŽ›ǡ ™‹ŽŽ†‹• —••’‘••‹„‹Ž‹–‹‡•–‘Ž‹›•›•–‡•‡†‹ ‹‡ ƒ’’”‘ƒ Š–‘’ƒ–‹‡–†ƒ–ƒ‘”in vitro•–—†‹‡•„ƒ•‡†‘’ƒ–‹‡–Ǧ†‡”‹˜‡† ‡ŽŽ•Ǥ  —ƒ”‹œ‹‰ǡ ™‹ŽŽƒ††”‡••–Š‡ˆ‘ŽŽ‘™‹‰“—‡•–‹‘•‹–Š‹•Š–Š‡•‹•Ǥ ͳǤ Šƒ–‹•–Š‡—†‡”Ž›‹‰‡ Šƒ‹•–Šƒ–Ž‹•‡œ›‡’”‘‹• —‹–›–‘–Š‡ˆŽ—š†‡ Ž‹‡ –Šƒ–‹•‘„•‡”˜‡†ƒ–Š‹‰Š•—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘•‹–Š‡”ƒ– ‘’—–ƒ–‹‘ƒŽ‘†‡Ž‘ˆ  ǫȋChapter 2Ȍ ʹǤ Š‹ Š‡–ƒ„‘Ž‹ ’ƒ–Š™ƒ›• ‘–”‹„—–‡–‘–Š‡”‘„—•–‡••‘ˆǦ‹ ‡—†‡”ˆ‡† ƒ†ˆƒ•–‹‰ ‘†‹–‹‘•‘ƒŠ‹‰ŠǦ‘”Ž‘™Ǧˆƒ–†‹‡–ǫȋChapter 3Ȍ ͵Ǥ ‘™ †‘‡• Š‡’ƒ–‹  ‡–ƒ„‘Ž‹• ”‡•’‘† –‘ •‡˜‡”‡ ‡‡”‰‡–‹  •–”‡•• ‹ ‘Ž†Ǧ‡š’‘•‡†ǡ ˆƒ•–‡†Ǧ‹ ‡ǫȋChapter 4Ȍ ͶǤ ‘™ †‘ –Š‡ ’ƒ–Š™ƒ›• •—””‘—†‹‰ –Š‡  ǡ ‘–ƒ„Ž› –Š‘•‡ –Šƒ– ƒˆˆ‡ – ‘ ǡ ‘†—Žƒ–‡–Š‡’Š‡‘–›’‡‹ƒ ‘’—–ƒ–‹‘ƒŽ‘†‡Ž‘ˆǦ†‡ˆ‹ ‹‡–Š—ƒ ‡ŽŽ•ƒ† ƒ „‡ –Š‡ ‹’ƒ – ‘ˆ ‹–‡”‹†‹˜‹†—ƒŽ ˜ƒ”‹ƒ–‹‘ ƒ† ˜ƒ”‹‘—• –”‡ƒ–‡– ‘’–‹‘•ǫ ȋChapter 5Ȍ ͷǤ ‘ —Ž–—”‡† ˆ‹„”‘„Žƒ•–•‘ˆ •›’–‘ƒ–‹  ‘” ƒ•›’–‘ƒ–‹  ‹†‹˜‹†—ƒŽ• •Š‘™ ƒ†ƒ’–ƒ–‹‘•‹–Š‡‹”ƒ ›ŽǦ ƒ”‹–‹‡’”‘ˆ‹Ž‡•‘”‹–‘ Š‘†”‹ƒŽ’”‘–‡‘‡ǫȋChapter 6Ȍ  References

ʹͷ Chapter 1

ͳǤ ƒˆƒ”ǡŠƒŽ‹“ǡŠƒ† ǡƒœ‘‘”ǡ‘‡Ǥ‡–ƒ„‘Ž‹ •›†”‘‡ǣƒ—’†ƒ–‡‘†‹ƒ‰‘•–‹  ”‹–‡”‹ƒǡ’ƒ–Š‘‰‡‡•‹•ǡƒ†‰‡‡–‹ Ž‹•Ǥ ‘”‘‡•ȋ–Š‡•ȌǤʹͲͳͺ‡’Ǣͳ͹ȋ͵ȌǣʹͻͻȂ͵ͳ͵Ǥ ʹǤ ‹ ǡ ƒ•‹‡ ǡ ‘”± Ǥ ”‘••–ƒŽ „‡–™‡‡ ‘ƒŽ ‘Š‘Ž‹  ˆƒ––› Ž‹˜‡” †‹•‡ƒ•‡ ƒ† ƒ”†‹‘‡–ƒ„‘Ž‹ •›†”‘‡Ǥ„‡•‡˜ǤʹͲͳͺǢȋ—‰—•–ʹͲͳͺȌǣͷͻͻȂ͸ͳͳǤ ͵Ǥ ‘”‹‡”Ǧǡƒ„‡Ž‡ƒǡ ‡”ƒ†‡œǡ‹†•–”‘ǡ–‡‹‰ ǡ–‘„ǡƒ‡Ž–ǡƒ‰ ǡ  ‡Ž ǤŠ‡‡–ƒ„‘Ž‹ •›†”‘‡Ǥ†‘ ”‡˜ǤʹͲͲͺ‡ Ǣʹͻȋ͹Ȍǣ͹͹͹ȂͺʹʹǤ ͶǤ ƒ””‘•‘ ǡ ƒ”–Š› ǤŠ‡ ‡‡–‹ ƒ•‹•‘ˆ‡–ƒ„‘Ž‹ ‹•‡ƒ•‡Ǥ‡ŽŽǤʹͲͳͻƒ”ʹͳǢͳ͹͹ȋͳȌǣͳͶ͸Ȃ ͸ͳǤ ͷǤ —‹‰‡ ǡ‡˜‡”•ǡŽ—‹Œ–ƒ• ǡ˜ƒ†‡” ‡‡ˆ–ǡ˜ƒƒ”‡„‡‡ǡ ‘‡‰‡ǡ ‰‡Ž‡ ǡ™ƒ•– ǡ‘”–ƒǡ†‡‘‡”ǡ‰‡Ž ǡ†‡”‹‡•ǡ Š”‡—†‡” ǡ‘‡‡ ǡ ‡—Žƒ”–• ǡ ˜ƒ †‡ ‘”•– ǡ ‹Ž‹••‡ ǡ ƒ••‡  Ǥ ‡š–Ǧ‰‡‡”ƒ–‹‘ ‡–ƒ„‘Ž‹  • ”‡‡‹‰ǣ –ƒ”‰‡–‡† ƒ† —–ƒ”‰‡–‡† ‡–ƒ„‘Ž‘‹ • ˆ‘” –Š‡ †‹ƒ‰‘•‹• ‘ˆ ‹„‘” ‡””‘”• ‘ˆ ‡–ƒ„‘Ž‹•‹‹†‹˜‹†—ƒŽ’ƒ–‹‡–•Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͺǢͶͳȋ͵Ȍǣ͵͵͹Ȃͷ͵Ǥ ͸Ǥ  ‘Žƒ‹‡”‘ ǡ ‘œœ‘Ž‹‘ ǡ Ž„ƒ‘ ǡ •ƒŽ‘‡ ǡ ƒ–‡”‹‘ ǡ ‘”„‘ ǡ †‹ ‹”‘Žƒ‘  ǡ ‹ –‡ˆƒ‘ ǡ —”ƒ–‡ ǡ ”ƒœ‡•‡ ǡ ”ƒœ‡•‡ ǡ ƒŽŽ‘ ǡ ‹Ž‹„‡”–‹ ǡ ‰‡‹–‘ ǡ ’’‘Ž‹–‘ ǡ ƒŽƒ‹•—”ƒ ǡ ƒœœ‡‘ ǡ ‘”ƒǡ „”‘‡ ǡ ƒ”‡–‹ ǡ ‡ŽŽ‡ Š‹ƒ ǡ ‡ ‡ ǡ ‹‡”— ‹ ǡ ‘ƒ‡ŽŽ‹ǡ‘••‹ǡ‹ƒ‘ǡ–‘†—–‘ǡ‹ŽŽƒ‹ ǡ†”‹ƒ ǡƒŽ˜ƒ–‘”‡ ǡ ”‹••‘ ǡ—‘’’‘Ž‘ Ǥ ƒ”‰‡–‡† ‡–ƒ„‘Ž‘‹ • ‹ –Š‡ ‡š’ƒ†‡† ‡™„‘” • ”‡‡‹‰ ˆ‘” ‹„‘” ‡””‘”• ‘ˆ ‡–ƒ„‘Ž‹•Ǥ‘Ž‹‘•›•–ǤʹͲͳͷǢͳͳȋ͸ȌǣͳͷʹͷȂ͵ͷǤ ͹Ǥ ‹Ž ‡ ǡ ‹Ž‡› ǡ ƒ‘† ǡ ƒ”’‡–‡” Ǥ  ”‡‡‹‰ ‡™„‘”• ˆ‘” „‘” ””‘”• ‘ˆ ‡–ƒ„‘Ž‹•„›ƒ†‡ƒ••’‡ –”‘‡–”›Ǥ‰Ž ‡†ǤʹͲͲ͵Ǣ͵Ͷͺȋʹ͵Ȍǣʹ͵ͲͶȂͳʹǤ ͺǤ ‹Ž ‡ǡ ƒƒ•ǡ ‘›ǡ‹Ž‡›ǡŠƒ’Ž‹ǡŽƒ ǡ Ž‡– Š‡” ǡ ‹ŽŽ ǡ‘‡ŠǤ—– ‘‡‘ˆ ‡‘ƒ–ƒŽ• ”‡‡‹‰ˆ‘”‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›‹—•–”ƒŽ‹ƒǣƒ ‘Š‘”– •–—†›Ǥƒ ‡–ǤʹͲͲ͹Ǣ͵͸ͻȋͻͷͷͷȌǣ͵͹ȂͶʹǤ ͻǤ ‘ŠŽ—ŽŽ‡” ǡ ƒ›ƒ–‡’‡ ǡ  Š—Žœ‡ ǡ ‘ˆˆƒ ǡ ‹†‡” ǡ Ž‰‡‘ŽŽ‡” Ǥ š’ƒ†‡† ‡™„‘” ”‡‡‹‰ˆ‘” „‘”””‘”•‘ˆ‡–ƒ„‘Ž‹•„›Ž‡ –”‘•’”ƒ› ‘‹œƒ–‹‘Ǧƒ†‡ƒ•• ’‡ –”‘‡–”›ǣ‡•—Ž–•ǡ—– ‘‡ǡƒ† ’Ž‹ ƒ–‹‘•Ǥ‡†‹ƒ–”‹ •ǤʹͲͲͶǢͳͳͳȋ͸Ȍǣͳ͵ͻͻȂͶͲ͸Ǥ ͳͲǤ Žƒƒ” ƒ Ǥƒ•••’‡ –”‘‡–”›‹ Ž‹‹ ƒŽ Š‡‹•–”›ǣŠ‡ ƒ•‡‘ˆ‡™„‘”• ”‡‡‹‰Ǥ Šƒ” ‹‘‡†ƒŽǤʹͲͳͶǢͳͲͳǣͳ͹ͶȂͺʹǤ ͳͳǤ ‡”• 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  Š‡”‹–‡–ƒ„‹•ǤʹͲͲͺǢ͵ͳȋͳȌǣͺͺȂͻ͸Ǥ ͳʹǤ ƒ‹Œ– ǡ ƒ‰‡”ǡƒ’”‘•‡ ǡ‡‘‹‰ǡ ‡‹‡”Ǧ ‘‡ƒǡ‡”• Ǥ‡‘ƒ–‡• ƒ–”‹•‘ˆ‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›ǣ’‡”‹ƒ–ƒŽ’”‘–‘ ‘Žˆ‘”—•‡„‡ˆ‘”‡ ’‘’—Žƒ–‹‘‡‘ƒ–ƒŽ• ”‡‡‹‰–‡•–”‡•—Ž–•„‡ ‘‡ƒ˜ƒ‹Žƒ„Ž‡Ǥ ‡‡–‡†ǤʹͲͳ͸ǢͳͺȋͳʹȌǣͳ͵ʹʹȂ͵Ǥ ͳ͵Ǥ Žƒƒ‹ ǡ ƒ”‘ ǡ —––‘ Ǥ ‡™„‘” • ”‡‡‹‰ǣ ƒ ”‡˜‹‡™ ‘ˆ Š‹•–‘”›ǡ ”‡ ‡– ƒ†˜ƒ ‡‡–•ǡƒ†ˆ—–—”‡’‡”•’‡ –‹˜‡•‹–Š‡‡”ƒ‘ˆ‡š–‰‡‡”ƒ–‹‘•‡“—‡ ‹‰Ǥ—””’‹ ‡†‹ƒ–”ǤʹͲͳ͸Ǣʹͺȋ͸Ȍǣ͸ͻͶȂͻǤ ͳͶǤ †ƒ‹Ž˜ƒƒ˜‹‡” ǡ ‘†•‘ Ǥ‘—•‡‘†‡Ž•‘ˆ’‡”‹’Š‡”ƒŽ‡–ƒ„‘Ž‹ †‹•‡ƒ•‡Ǥ‡•–”ƒ –‡•Ž‹ †‘ ”‹‘Ž‡–ƒ„ǤʹͲͳͺǢ͵ʹȋ͵ȌǣʹͻͻȂ͵ͳͷǤ ͳͷǤ ƒ‰ƒ•Š‹ƒ†ƒǡ–ƒǤ‘Ž‡‘ˆ˜‹–ƒ‹‹‘ƒŽ ‘Š‘Ž‹ ˆƒ––›Ž‹˜‡”†‹•‡ƒ•‡Ǥ ‹ˆ‡ǤʹͲͳͻ ’”Ǣ͹ͳȋͶȌǣͷͳ͸ȂʹʹǤ ͳ͸Ǥ ‘•›Ǧ‡•–’ŠƒŽǡ”ƒ—ǡŽ„”‡ Š–ǡòŽŽ‡” Ǥ‡–‡”‹ƒ–•‘ˆ‡ –‘’‹ Ž‹˜‡”ˆƒ–‹‡–ƒ„‘Ž‹  †‹•‡ƒ•‡Ǥ—” Ž‹—–”ǤʹͲͳͺǢ ͳ͹Ǥ ƒ‰‡„‡”‰ ǡ ‘––ƒ Ǥ ‡˜‹‡™ ‡‘‹  ‹•‹‰Š–• ‹–‘ –Š‡ ƒ—•‡• ‘ˆ –›’‡ ʹ †‹ƒ„‡–‡•Ǥ ʹͲͳͺǢʹͶ͸͵Ȃ͹ͶǤ ͳͺǤ ”‰ƒǡ ‘—–‡ǡŠ— ǡ Šƒ†–Ǥ‡š– ‡‡”ƒ–‹‘—Ž–‹• ƒŽ‡‹‡™‘ˆ „‘”””‘”• ‘ˆ‡–ƒ„‘Ž‹•Ǥ‡ŽŽ‡–ƒ„ǤʹͲͳ͸Ǣʹ͵ȋͳȌǣͳ͵Ȃʹ͸Ǥ

ʹ͸ General introduction and outline of the thesis

ͳͻǤ ‘”Ž† ‡ƒŽ–Š ”‰ƒ‹œƒ–‹‘Ǥ „‡•‹–› ƒ† ˜‡”™‡‹‰Š– ȏ –‡”‡–ȐǤ ‘”Ž† ‡ƒŽ–Š ”‰ƒ‹œƒ–‹‘Ǥ ʹͲͳͺ ȏ ‹–‡† ʹͲͳͻ ƒ” ʹͺȐǤ ˜ƒ‹Žƒ„Ž‡ ˆ”‘ǣ Š––’•ǣȀȀ™™™Ǥ™Š‘Ǥ‹–Ȁ‡™•Ǧ”‘‘Ȁˆƒ –Ǧ 1 •Š‡‡–•Ȁ†‡–ƒ‹ŽȀ‘„‡•‹–›Ǧƒ†Ǧ‘˜‡”™‡‹‰Š– ʹͲǤ  „‡•‹–› ’†ƒ–‡ ʹͲͳ͹ ȏ –‡”‡–ȐǤ Ǥ ʹͲͳ͹ ȏ ‹–‡† ʹͲͳͺ ƒ” ʹͺȐǤ ˜ƒ‹Žƒ„Ž‡ ˆ”‘ǣ Š––’ǣȀȀ™™™Ǥ‘‡ †Ǥ‘”‰ȀŠ‡ƒŽ–ŠȀ‘„‡•‹–›Ǧ—’†ƒ–‡ǤŠ– ʹͳǤ ƒŽ‹Ž‹‘‰Ž— ǡ ƒ”ƒ‰‘œ ǡ ‡˜‹‘‰Ž— ǡ ‹Ž‹  ǡ ‘˜ ǡ ”‰ƒ Ǥ ‹••—‡Ǧ’‡ ‹ˆ‹  ‘Ž‡ —Žƒ” ‹‘ƒ”‡” ‹‰ƒ–—”‡• ‘ˆ ›’‡ ʹ ‹ƒ„‡–‡•ǣ  –‡‰”ƒ–‹˜‡ ƒŽ›•‹• ‘ˆ ”ƒ• ”‹’–‘‹ • ƒ† ”‘–‡‹Ǧ”‘–‡‹ –‡”ƒ –‹‘ƒ–ƒǤ ǤʹͲͳͷ‡’ǢͳͻȋͻȌǣͷ͸͵Ȃ͹͵Ǥ ʹʹǤ ƒ‹•‡”ǡ‡–Œ‡Ǥ‘‡–Š‹‰‘Ž†ǡ•‘‡–Š‹‰‡™ƒ†•‘‡–Š‹‰˜‡”›‘Ž†ǣ†”—‰•ˆ‘”–”‡ƒ–‹‰ –›’‡ʹ†‹ƒ„‡–‡•Ǥ” Šƒ”ƒ ‘ŽǤʹͲͳͶ —Ǣͳ͹ͳȋͳʹȌǣʹͻͶͲȂͷͲǤ ʹ͵Ǥ ”‡–‹ǡ‘Žƒ ǡ”‡–‹ǡ‘Žƒ Ǥ •Ž‡–„ ‡ŽŽˆƒ‹Ž—”‡‹–›’‡ʹ†‹ƒ„‡–‡• ‹†–Š‡Žƒ–‡•– ˜‡”•‹‘᩿ǣ‡˜‹‡™•‡”‹‡• •Ž‡–„ ‡ŽŽˆƒ‹Ž—”‡‹–›’‡ʹ†‹ƒ„‡–‡•ǤʹͲͲ͸Ǣͳͳ͸ȋ͹ȌǣͳͺͲʹȂͳʹǤ ʹͶǤ —••‡Žƒǡ‡–ƒǡƒ”•‡ ǡŠ‹ŽŽ‹’•Ǥ‡Žƒ–‹‘•Š‹’‘ˆ†‡’”‡••‹‘–‘†‹ƒ„‡–‡•–›’‡•ͳ ƒ†ʹǣ’‹†‡‹‘Ž‘‰›ǡ„‹‘Ž‘‰›ǡƒ†–”‡ƒ–‡–Ǥ‹‘Ž•› Š‹ƒ–”›ǤʹͲͲ͵ǢͷͶȋ͵Ȍǣ͵ͳ͹ȂʹͻǤ ʹͷǤ ‡–ƒƒ”ƒǡ—ŽŽǡŠ‘”‡ ǡ†Ž‡” ǡ ‘ŽƒǤ‹•ˆƒ –‘”•ˆ‘””‡ƒŽ†›•ˆ— –‹‘‹ –›’‡ʹ†‹ƒ„‡–‡•ǣǤǤ”‘•’‡ –‹˜‡‹ƒ„‡–‡•–—†›͹ͶǤ‹ƒ„‡–‡•ǤʹͲͲ͸Ǣͷͷȋ͸Ȍǣͳͺ͵ʹȂͻǤ ʹ͸Ǥ ƒ”†‹‘‰Ž—ǡŠŽ‡ǡ‘”± Ǥ”‘ƒ†‹‡™•‘ˆ‘ǦƒŽ ‘Š‘Ž‹  ƒ––›‹˜‡”‹•‡ƒ•‡Ǥ‡ŽŽ›•–Ǥ ʹͲͳͺ ƒʹͶǢ͸ȋͳȌǣ͹ȂͻǤ ʹ͹Ǥ ƒ–œ‹— ǡ ‡ŽŽ‡–ƒ‹ ǡ ‘”–‡œǦ‹–‘ ǡ ƒ› ǡ ƒ” Š‡•‹‹ Ǥ  ’‘•‹–‹‘ •–ƒ–‡‡– ‘  Ȁ „ƒ•‡†‘–Š‡ʹͲͲͻ•’‡ ‹ƒŽ ‘ˆ‡”‡ ‡Ǥ  ‡’ƒ–‘ŽǤʹͲͳͲ—‰Ǣͷ͵ȋʹȌǣ͵͹ʹȂͺͶǤ ʹͺǤ ‡”‘ ǡƒ”ƒ‘˜ƒǡ‘—‘••‹Ǥ›•–‡ƒ–‹ ”‡˜‹‡™ǣŠ‡‡’‹†‡‹‘Ž‘‰›ƒ†ƒ–—”ƒŽŠ‹•–‘”› ‘ˆ ‘ǦƒŽ ‘Š‘Ž‹  ˆƒ––› Ž‹˜‡” †‹•‡ƒ•‡ ƒ† ‘ǦƒŽ ‘Š‘Ž‹  •–‡ƒ–‘Š‡’ƒ–‹–‹• ‹ ƒ†—Ž–•Ǥ Ž‹‡– Šƒ”ƒ ‘ŽŠ‡”ǤʹͲͳͳǢ͵Ͷȋ͵Ȍǣʹ͹ͶȂͺͷǤ ʹͻǤ ‘‘„ƒ ǡ ƒ›ƒŽ  Ǥ Š‡ ‰Ž‘„ƒŽ   ‡’‹†‡‹ Ǥ ƒ– ‡˜ ƒ•–”‘‡–‡”‘Ž ‡’ƒ–‘ŽǤ ʹͲͳ͵ǢͳͲȋͳͳȌǣ͸ͺ͸ȂͻͲǤ ͵ͲǤ †ƒ•ǡ›’ ǡ–ƒ—˜‡” ǡƒ†‡”•‘ǡ‹†‘”ǡ ‡Ž†•–‡‹ǡ‰—Ž‘ǤŠ‡ƒ–—”ƒŽ Š‹•–‘”›‘ˆ‘ƒŽ ‘Š‘Ž‹ ˆƒ––›Ž‹˜‡”†‹•‡ƒ•‡ǣƒ’‘’—Žƒ–‹‘Ǧ„ƒ•‡† ‘Š‘”–•–—†›Ǥ ƒ•–”‘‡–‡”‘Ž‘‰›Ǥ ʹͲͲͷ —ŽǢͳʹͻȋͳȌǣͳͳ͵ȂʹͳǤ ͵ͳǤ ”‘™‹‰ ǡœ œ‡’ƒ‹ƒǡ‘„„‹•ǡ—”‡„‡”‰ǡ ‘”–‘ ǡ‘Š‡ ǡ ”—†›ǡ ‘„„• Ǥ ”‡˜ƒŽ‡ ‡ ‘ˆ Š‡’ƒ–‹  •–‡ƒ–‘•‹• ‹ ƒ —”„ƒ ’‘’—Žƒ–‹‘ ‹ –Š‡ ‹–‡† –ƒ–‡•ǣ ‹’ƒ – ‘ˆ ‡–Š‹ ‹–›Ǥ ‡’ƒ–‘Ž‘‰›ǤʹͲͲͶ‡ ǢͶͲȋ͸Ȍǣͳ͵ͺ͹ȂͻͷǤ ͵ʹǤ ‘’‡  ǡ —”• Ǥ ‘ƒŽ ‘Š‘Ž‹  ˆƒ––› Ž‹˜‡” †‹•‡ƒ•‡ǣ ƒ ”‡˜‹‡™ ‘ˆ –Š‡ •’‡ –”— ‘ˆ †‹•‡ƒ•‡ǡ †‹ƒ‰‘•‹•ǡƒ†–Š‡”ƒ’›Ǥ—–”Ž‹”ƒ –ǤʹͲͳͳ –Ǣʹ͸ȋͷȌǣͷ͸ͷȂ͹͸Ǥ ͵͵Ǥ ‘—‘••‹ǡ–‡’ƒ‘˜ƒǡˆ‡†›ǡ ƒ‰ǡ‘—‘••‹ǡ‹” ǡ”‹•Š‘”†ǤŠƒ‰‡•‹–Š‡ ”‡˜ƒŽ‡ ‡‘ˆ–Š‡‘•–‘‘ƒ—•‡•‘ˆŠ”‘‹ ‹˜‡”‹•‡ƒ•‡•‹–Š‡‹–‡†–ƒ–‡• ”‘ ͳͻͺͺ–‘ʹͲͲͺǤŽ‹ ƒ•–”‘‡–‡”‘Ž ‡’ƒ–‘ŽǤʹͲͳͳǢͻȋ͸ȌǣͷʹͶȂͷ͵ͲǤ‡ͳǤ ͵ͶǤ ƒ‘ǡƒ—ˆƒ Ǥƒ”‰‡–‹‰‡†‘’Žƒ•‹ ”‡–‹ —Ž—•–”‡••‹‡–ƒ„‘Ž‹ †‹•‡ƒ•‡Ǥš’‡”–’‹ Š‡”ƒ”‰‡–•ǤʹͲͳ͵’”Ǣͳ͹ȋͶȌǣͶ͵͹ȂͶͺǤ ͵ͷǤ ‡””› ǡ‡‰ǡŽ‹‡ ǡƒ‰ǡƒ„‹Ǧ‘—”–ǡ‘‰ ǡŠƒ‰ǡŠƒ‰ǡ‘œƒ‹ǡ—ˆ‘—” ǡ ‡–‡”•‡  ǡ Š—Žƒ Ǥ ‡ Šƒ‹•• „› ™Š‹ Š ƒ ‡”›Ǧ‘™ǦƒŽ‘”‹‡ ‹‡– ‡˜‡”•‡• ›’‡”‰Ž› ‡‹ƒ‹ƒƒ–‘†‡Ž‘ˆ›’‡ʹ‹ƒ„‡–‡•Ǥ‡ŽŽ‡–ƒ„ǤʹͲͳͺǢʹ͹ȋͳȌǣʹͳͲȂʹͳ͹Ǥ‡͵Ǥ ͵͸Ǥ ‡””› ǡƒ’‘”‡œ Ǧ ǡ—”•ƒ™‡ǡ‹– Š‡‡ŽŽǡŠƒ‰ǡ‡””› ǡ —” œƒ ǡ„—†—ƒ†‹‡” ǡ ƒǡŠƒ‰Ǧǡ—ƒ Ǧǡƒ‰ǡƒ’”‹‘ǡƒ‡ Šǡ—Ž ǡ‹”„ƒ— ǡƒ˜‹• ǡ Ž‹‡ ǡ‡–‡”•‡ ǡŠ—Žƒ Ǥ ‡’ƒ–‹ ƒ ‡–›Ž‘Ž‹•ƒ†‹’‘•‡–‹••—‡‹ˆŽƒƒ–‹‘–‘ Š‡’ƒ–‹ ‹•—Ž‹”‡•‹•–ƒ ‡ƒ†–›’‡ʹ†‹ƒ„‡–‡•Ǥ‡ŽŽǤʹͲͳͷ ‡„ͳʹǢͳ͸ͲȋͶȌǣ͹ͶͷȂͷͺǤ ͵͹Ǥ ƒƒǡ ƒ‹Ǥ„‡•‹–›ǡš‹†ƒ–‹˜‡–”‡••ǡ†‹’‘•‡‹••—‡›•ˆ— –‹‘ǡǤʹͲͳͷǢͳ͵ȋͳͲȌǣͶʹ͵Ȃ ͶͶǤ ͵ͺǤ ƒ”‹„‡›‰‹ ǡ ƒ””‘Š‹ ǡ—–Ž‡”ǡƒŠ‡„ƒ”Ǥ •—Ž‹”‡•‹•–ƒ ‡ǣ‡˜‹‡™‘ˆ–Š‡—†‡”Ž›‹‰ ‘Ž‡ —Žƒ”‡ Šƒ‹••Ǥ ‡ŽŽŠ›•‹‘ŽǤʹͲͳͻ —Ǣʹ͵Ͷȋ͸ȌǣͺͳͷʹȂ͸ͳǤ

ʹ͹ Chapter 1

͵ͻǤ ‘—•–‹•ǡ‘•‡ǡƒ†‡”Ǥ‡ƒ –‹˜‡‘š›‰‡•’‡ ‹‡•Šƒ˜‡ƒ ƒ—•ƒŽ”‘Ž‡‹—Ž–‹’Ž‡ˆ‘”•‘ˆ ‹•—Ž‹”‡•‹•–ƒ ‡Ǥƒ–—”‡ǤʹͲͲ͸ǢͶͶͲȋ͹Ͳͺ͸ȌǣͻͶͶȂͺǤ ͶͲǤ •Š”ƒˆǡŠ‡‹ŠǤ†‘’Žƒ•‹ ”‡–‹ —Ž—•–”‡••ƒ†š‹†ƒ–‹˜‡•–”‡••‹–Š‡’ƒ–Š‘‰‡‡•‹• ‘ˆ‘ǦƒŽ ‘Š‘Ž‹ ˆƒ––›Ž‹˜‡”†‹•‡ƒ•‡Ǥ ”‡‡ƒ†‹ ‡•ǤʹͲͳͷ‡ ʹǢͶͻȋͳʹȌǣͳͶͲͷȂͳͺǤ ͶͳǤ ƒ ǡƒ’‘‹ǡ‘„‹Ž‹ǤŽ‹‹ ƒŽ‹’Ž‹ ƒ–‹‘•‘ˆ—†‡”•–ƒ†‹‰–Š‡ƒ••‘ ‹ƒ–‹‘„‡–™‡‡ ‘š‹†ƒ–‹˜‡•–”‡••ƒ†’‡†‹ƒ–”‹  Ǥš’‡”–‡˜ ƒ•–”‘‡–‡”‘Ž ‡’ƒ–‘ŽǤʹͲͳ͹ǢͳͳȋͶȌǣ͵͹ͳȂͺʹǤ ͶʹǤ ŽŠƒ••ƒǡŠ‹Ž’ǡƒ˜‡”› Ǥƒ”‰‡–‹‰Ϊ‹‡–ƒ„‘Ž‹ ‹•‡ƒ•‡ǣ‡™ •‹‰Š–• –‘ƒ Ž†‘Ž‡ —Ž‡Ǥ †‘ ”‘ ǤʹͲͳ͹Ǣͳȋ͹Ȍǣͺͳ͸Ȃ͵ͷǤ Ͷ͵Ǥ ƒ‘ ǡ ‡”Š‘Ž† ǡ ƒ›‡”• ǡ ‹‡•– ǡ ƒ–‹• Ǥ †‡–‹ˆ‹ ƒ–‹‘ ‘ˆ ƒ ‹’‘‹‡ ǡ ƒ ‹’‹† ‘”‘‡‹‹‰†‹’‘•‡‹••—‡–‘›•–‡‹ ‡–ƒ„‘Ž‹•ǤʹͲͲͺǢͻ͵͵ȂͶͶǤ ͶͶǤ ƒ‰ ǡ ‹Œƒ›ƒ—ƒ” ǡ ƒŠ Ǥ ‡–ƒ„‘Ž‹–‡• ƒ• ”‡‰—Žƒ–‘”• ‘ˆ ‹•—Ž‹ •‡•‹–‹˜‹–› ƒ† ‡–ƒ„‘Ž‹•Ǥƒ–‡˜‘Ž‡ŽŽ‹‘ŽǤʹͲͳͺǢͳͻȋ –‘„‡”ȌǤ ͶͷǤ Šƒ ǡ ‹ŽŽ‘  ǡ ‹˜ƒŽ‘‰ƒƒ–Šƒ ǡ ‘•–ˆ‘”† ǡ ‹— ǡ Š±”‡– ǡ Šƒœƒ—† ǡ Ž‹’ Ǥ ƒŽ‹–‘Ž‡ƒ–‡‡˜‡”•‡• ‹‰Š ƒ–Ǧ‹†— ‡†”‘‹ˆŽƒƒ–‘”›ƒ ”‘’Šƒ‰‡‘Žƒ”‹œƒ–‹‘˜‹ƒǦ ƒ –‹˜ƒ–‡†”‘–‡‹‹ƒ•‡ȋȌǤ ‹‘ŽŠ‡ǤʹͲͳͷ —Ž͵ǢʹͻͲȋʹ͹Ȍǣͳ͸ͻ͹ͻȂͺͺǤ Ͷ͸Ǥ ƒŽ„‘–ǡŠ‡‡Ž‡”ǦŒ‘‡•ǡŽ‡ƒ•„›Ǥ‘Ž‡ —Žƒ”ƒ†‡ŽŽ—Žƒ”†‘ ”‹‘Ž‘‰›ƒŽ‹–‘Ž‡‹  ƒ ‹† ’”‡˜‡–• ’ƒŽ‹–‹  ƒ ‹†Ǧ‹†— ‡† ƒ ”‘’Šƒ‰‡ ƒ –‹˜ƒ–‹‘ ƒ† ‘•‡“—‡– ’͵ͺ Ǧ ‡†‹ƒ–‡†•‡Ž‡–ƒŽ—• Ž‡‹•—Ž‹”‡•‹•–ƒ ‡Ǥ‘Ž‡ŽŽ†‘ ”‹‘ŽǤʹͲͳͶǢ͵ͻ͵ȋͳȂʹȌǣͳʹͻȂͶʹǤ Ͷ͹Ǥ ‘”‡ǡ›‡† ǡ‘”ƒ‡•Ǧ˜‹‡‹”ƒǡŠƒ‰ǡ ‡”ƒǡ ‘ƒǡƒ–‡Žǡ‡‡ ǡŠ‡ǡ ‡”‘‹ ǡ Šƒ‡•Š™ƒ” ǡ ƒƒ”•–‡†– ǡ ‹–Š ǡ  ‰”ƒ™ ǡ ƒ‰Šƒ–‡Ž‹ƒ ǡ ƒŠ Ǥ ‹• ‘˜‡”› ‘ˆ ƒ Žƒ•• ‘ˆ †‘‰‡‘—• ƒƒŽ‹ƒ ‹’‹†• ™‹–Š –‹Ǧ‹ƒ„‡–‹  ƒ† –‹Ǧ ‹ˆŽƒƒ–‘”›ˆˆ‡ –•Ǥ‡ŽŽǤʹͲͳͶǢͳͷͻȋʹȌǣ͵ͳͺȂ͵ʹǤ ͶͺǤ ’‹–‡ǡŽ”‹ƒ ǡ‡”ŠƒǤ ‡•‘Ž˜‹•ǡ ’‡ ‹ƒŽ‹œ‡†”‘”‡•‘Ž˜‹‰ ‹’‹† ‡†‹ƒ–‘”•ǡ ƒ† Š‡‹” ‘–‡–‹ƒŽ‘Ž‡•‹‡–ƒ„‘Ž‹ ‹•‡ƒ•‡•Ǥ‡ŽŽ‡–ƒ„ǤʹͲͳͶ ƒǢͳͻȋͳȌǣʹͳȂ͵͸Ǥ ͶͻǤ ‘œžŽ‡œǦ±”‹œǡ ‘””‹ŽŽ‘ǡ ‡””±ǡ ”‘‡”–ǡ‘‰ǡ‘”žǦƒŽ˜ƒ†‘”ǡ‹–‘•ǡƒ”–À‡œǦ Ž‡‡–‡ǡג‡œǦƒ””ƒǡ””‘›‘ǡŽ”‹ƒ Ǥ„‡•‹–›Ǧ‹†— ‡†‹•—Ž‹”‡•‹•–ƒ ‡ƒ†Š‡’ƒ–‹  •–‡ƒ–‘•‹•ƒ”‡ƒŽŽ‡˜‹ƒ–‡†„›ɘǦ͵ˆƒ––›ƒ ‹†•ǣƒ”‘Ž‡ˆ‘””‡•‘Ž˜‹•ƒ†’”‘–‡ –‹•Ǥ  ǤʹͲͲͻ —Ǣʹ͵ȋ͸ȌǣͳͻͶ͸Ȃͷ͹Ǥ ͷͲǤ —••‘ ǡ ƒ„‹‘ǡƒ••ƒ†‡”ǡƒ• Š‡––ƒǡ‹” ƒƒǤ’‡ ‹ƒŽ‹œ‡†”‘”‡•‘Ž˜‹‰‡†‹ƒ–‘”•᩿ǣ Šƒ ‹‰‘ƒŽ ‘Š‘Ž‹ –‡ƒ–‘Š‡’ƒ–‹–‹•ƒ† ‹„”‘•‹•‡•‘Ž—–‹‘ǤʹͲͳͺǢ͵ͻȋͶȌǣ͵ͺ͹ȂͶͲͳǤ ͷͳǤ ƒ–•›—„ƒ ǡ —™‡”š Ǥ ‘†—Žƒ–‹‰  Ϊ ‡–ƒ„‘Ž‹•ǡ ˆ”‘ „‡ Š –‘„‡†•‹†‡Ǥ  Ǥ ʹͲͳ͹Ǣ͵͸ȋͳͺȌǣ‡ʹͲͳ͹ͻ͹ͳ͵ͷǤ ͷʹǤ ƒ‰ǡƒ—˜‡Ǥڏ‡–ƒ„‘Ž‹•ǣ‹‘‡‡”‰‡–‹ •ǡ•‹‰ƒŽ‹‰ƒ†ƒ‹’—Žƒ–‹‘ˆ‘”–Š‡”ƒ’›Ǥ ‹‘ Š‹‹‘’Š›• –ƒǦ”‘–‡‹•”‘–‡‘‹ •ǤʹͲͳ͸Ǣͳͺ͸ͶȋͳʹȌǣͳ͹ͺ͹ȂͺͲͲǤ ͷ͵Ǥ ‹‹ˆ‘”‘˜ǡ—Ž‹‘˜ƒǡ‹‡‰Ž‡”ǤŠ‡Š—ƒ‡–ƒ„‘Ž‘‡ǣ — –‹‘•ǡ‡–ƒ„‘Ž‹•ƒ† ‘’ƒ”–‡–ƒŽ‹œƒ–‹‘Ǥ”‹–‡˜‹‘ Š‡‘Ž‹‘ŽǤʹͲͳͷǢͷͲȋͶȌǣʹͺͶȂͻ͹Ǥ ͷͶǤ Šƒ‰ ǡ ƒ†ƒ›ƒ––‹ ǡ ‡—‰ Ǥ ”‘–‘Ǧ–”ƒ•Ž‘ ƒ–‹‰ ‹ ‘–‹ƒ‹†‡ — Ž‡‘–‹†‡ –”ƒ•Š›†”‘‰‡ƒ•‡ǣ•–”— –—”ƒŽ’‡”•’‡ –‹˜‡Ǥ ”‘–Š›•‹‘ŽǤʹͲͳ͹ǢͺȋȌǣͳȂ͸Ǥ ͷͷǤ ƒ ‘™•‹ǡ‡‘ƒ”†‹Ǥ‡”‡‰—Žƒ–‡† ‘‡œ›‡ǡŽ‘••‘ˆ‡–ƒ„‘Ž‹ ˆŽ‡š‹„‹Ž‹–›ƒ††‹ƒ„‡–‡•Ǥ ‹‘ Š‡‘ ”ƒ•ǤʹͲͳͶǢͶʹȋͶȌǣͳͳͳͺȂʹʹǤ ͷ͸Ǥ ‡‘ƒ”†‹ ǡ ‘  Ǥ ƒͳ †‡Ž‡–‹‘ ‹ Ž‡’–‹Ǧ†‡ˆ‹ ‹‡– ‹ ‡ ”‡†— ‡• Š›’‡”‰Ž› ƒ‡‹ƒ ƒ† Š›’‡”‹•—Ž‹ƒ‡‹ƒ ƒ† ‘†‹ˆ‹‡• ‰Ž‘„ƒŽ ‡–ƒ„‘Ž‹• ™‹–Š‘—– ƒˆˆ‡ –‹‰ ‹•—Ž‹ ”‡•‹•–ƒ ‡Ǥ ʹͲͳͶǢͳͶ͸͸Ȃ͹ͷǤ ͷ͹Ǥ ”‹‹˜ƒ•ƒ ǡ ‹„‘ Ǥ ‘‡œ›‡ǡ ‘”‡ –Šƒ DzŒ—•–dz ƒ ‡–ƒ„‘Ž‹  ‘ˆƒ –‘”Ǥ ‹‘ Š‡‘  ”ƒ•ǤʹͲͳͶǢͶʹȋͶȌǣͳͲ͹ͷȂͻǤ ͷͺǤ ‡‘ƒ”†‹ ǡ Šƒ‰ ǡ ‘  ǡ ƒ ‘™•‹ Ǥ ‘‡œ›‡ ǣ ƒ  ‹ ƒ –‹‘Ǥ ”‘‰ ‹’‹† ‡•Ǥ ʹͲͲͷǢͶͶȋʹȂ͵ȌǣͳʹͷȂͷ͵Ǥ ͷͻǤ ‹– Š‡ŽŽ ǡ ƒ—–Š‹‡”ǡ‡•‹’Ž‡ǡƒ‰ǡƒ‡”ǡ—”‡•Š‹ Ǥ ‡”‡†‹–ƒ”›ƒ†ƒ “—‹”‡† †‹•‡ƒ•‡•‘ˆƒ ›ŽǦ ‘‡œ›‡‡–ƒ„‘Ž‹•Ǥ‘Ž ‡‡–‡–ƒ„ǤʹͲͲͺǢͻͶȋͳȌǣͶȂͳͷǤ

ʹͺ General introduction and outline of the thesis

ͲǤ ƒ‰ ǡŠƒ‘ǡƒ‰ǡƒ‰ǡ‹– Š‡ŽŽ Ǥ ‡”‡†‹–ƒ”›†‹•‡ƒ•‡•‘ˆ ‘‡œ›‡–Š‹‘‡•–‡” ‡–ƒ„‘Ž‹•Ǥ‹‘ Š‡‘ ”ƒ•ǤʹͲͳͻ ‡„ʹͺǢͶ͹ȋͳȌǣͳͶͻȂͷͷǤ 1 ͳǤ —‹Ž†‹‰ Ǥ Š‡ ‘—•‡ —†–͹ ‰‡‡ ‡ ‘†‡• ƒ ’‡”‘š‹•‘ƒŽ —†‹š Š›†”‘Žƒ•‡ •’‡ ‹ˆ‹  ˆ‘” ‘‡œ›‡ƒ†‹–•†‡”‹˜ƒ–‹˜‡•ǤʹͲͲͳǢ͵ͺǣ͵͵ȂͺǤ ʹǤ ‹†•–”ڏ Ǥ Š‡ Š›•‹‘Ž‘‰› ‘ˆ „‡•‡Ǧ ›’‡”‰Ž› ‡‹  ‹ ‡ ȏ ‘„Ȁ‘„ ‹ ‡ȐǤ  ‹ ‘”Ž† Ǥ ʹͲͲ͹Ǣ͹ǣ͸͸͸ȂͺͷǤ ͵Ǥ ƒ“—‡–ǡ‹–ƒ”‹ ǡ—’”°ǡ ƒŽŽƒ† Ǥ‘Ž‡‘ˆ–Š‡ͳ’ƒ–‡–Š‡‹ƒ•‡‹–‹••—‡–‘Ž‡”ƒ ‡–‘ •–”‡••Ǥ‹‘ Š‡‘ ”ƒ•ǤʹͲͳͶǢͶʹȋͶȌǣͳͲͻͶȂͳͲͲǤ ͶǤ ‘‹•‹Ǧ‘—ˆˆƒ› ǡ ƒ•–‡ŽŽƒ‘ ǡ ƒŽ±”‘ ǡ Šƒ••‘ ǡ ƒŽŽƒ† ǡ ƒ“—‡– Ǥ ‘—•‡ ˜ƒ‹Ǧͳ ‹• ›–‘’”‘–‡ –‹˜‡ ˆ‘” ‹•Ž‡– „‡–ƒ ‡ŽŽ• ƒ† ”‡‰—Žƒ–‡• –Š‡ †‡˜‡Ž‘’‡– ‘ˆ –›’‡ ͳ †‹ƒ„‡–‡•Ǥ ‹ƒ„‡–‘Ž‘‰‹ƒǤʹͲͲͺ —ŽͺǢͷͳȋ͹ȌǣͳͳͻʹȂʹͲͳǤ ͷǤ ‡Ž‹‰ǡƒ”Ž‹••ǡƒŠ‹ŽŽ ǤŽƒ•ƒƒ‹‘ƒ ‹†Ž‡˜‡Ž•ƒ†‹•—Ž‹•‡ ”‡–‹‘‹‘„‡•‹–›Ǥ‰Ž  ‡†Ǥͳͻ͸ͻ –ǢʹͺͳȋͳͷȌǣͺͳͳȂ͸Ǥ ͸Ǥ ”ƒ›ǡ‡‹ƒ•–Ǥ”ƒ Š‡†Šƒ‹‹‘ ‹†•‹‡–ƒ„‘Ž‹ ‹•‡ƒ•‡‘Ž‡˜ƒ–‹‘•‹• ƒ—•‡ •—Ž‹ǤʹͲͳͺǢͳȂͺǤ ͹Ǥ ‹•• Š‡”ǡ”ƒ›ǡŠƒ‰ǡŽƒ”ǡ ƒ”–Š› ǡ”‘™ǡƒ‰ ǤͳͲ‡ƒ”•‘ˆ  ‹• ‘˜‡”›ǣ‹‘Ž‘‰›ǡ — –‹‘ǡƒ†”ƒ•Žƒ–‹‘Ǥ  — ‡‡–ǤʹͲͳ͹ —ŽǢͳͲͳȋͳȌǣͷȂʹʹǤ ͺǤ ƒ‘ǡ —‰ǡŠƒǡ‹‰ǡƒ’ Ǥ‹‘ Š‹‹ ƒ‡–‹‘’Š›•‹ ƒ –ƒƒ–Š™ƒ›ƒƒŽ›•‹• ‘ˆ ‘’Ž‡š †‹•‡ƒ•‡• ˆ‘”  ǡ ‡š–‡†‹‰ –‘ ‘•‹†‡” ”ƒ”‡ ˜ƒ”‹ƒ–• ǡ —Ž–‹Ǧ‘‹ • ƒ† ‹–‡”ƒ –‹‘•ǤʹͲͳ͹Ǣͳͺ͸ͳǣ͵͵ͷȂͷ͵Ǥ ͻǤ ‘Ž‡ǡ ‡ƒ˜‡”ǡƒŠ‘ˆˆǡƒ•Šǡ‘Ž‡ǡ ‡ƒ˜‡”ǡƒŠ‘ˆˆǡƒ•ŠǤš’‡”– ’‹‹‘ ‘ ”—‰ ‹• ‘˜‡”› ‘ǦƒŽ ‘Š‘Ž‹  ˆƒ––› Ž‹˜‡” †‹•‡ƒ•‡ ȋ   Ȍ ‘†‡Ž• ‹ †”—‰ †‹• ‘˜‡”›Ǥš’‡”–’‹”—‰‹• ‘˜ǤʹͲͳͺǢͳ͵ȋʹȌǣͳͻ͵ȂʹͲͷǤ ͲǤ —•ƒ‹ ǡ ƒ‹ ǡ ‡†ƒ Ǥ ‡š–Ǧ‰‡‡”ƒ–‹‘ ƒƒŽ‹ƒ ‰‡‡–‹ • –‘™ƒ”† ‘”‰ƒ‹•ǦŽ‡˜‡Ž •›•–‡•„‹‘Ž‘‰›Ǥ’Œ›•–‹‘Ž’’ŽǤʹͲͳ͹Ǣȋƒ›ȌǣͳȂͳͲǤ ͳǤ ‡—ˆ‡Žǡ –œ‡Žǡ”Šƒ”–ǡ”‘• Šǡƒ‰ǡ‹ǡ ŠÚˆ‡Ž•‘ǡ ‡””ƒǡ”ò ‡” ǡ –‹ ‡Ž ǡ —ˆ‘—” ǡ Šƒ˜ƒ‹• ǡ ‡ŽŽ‡”„”ƒ† ǡ ’ƒ‰ ǡ ƒƒ•• ǡ ‡ ‡” ǡ  Š”‡‹„‡” ǡ  Šƒˆƒ›‡”ǡ Š—’’ƒǡ ƒ’‡ Ǥ‘’ƒ”‹•‘‘ˆ ‡‡š’”‡••‹‘ƒ––‡”•‡–™‡‡‘—•‡ ‘†‡Ž•Ƭ„•’Ǣ‘ˆ ‘ƒŽ ‘Š‘Ž‹  ƒ––› ‹˜‡” ‹•‡ƒ•‡ ƒ† ‹˜‡” ‹••—‡•Ƭ„•’Ǣ ”‘ ƒ–‹‡–•Ǥ ʹͲͳ͸Ǣͷͳ͵ȂʹͷǤ ʹǤ ‡‘ǡ —’–ƒǡ‘”‹ƒǡ—Žƒ”‹Ǥ‹••‡ –‹‰†‹ƒ„‡–‡•Ȁ‡–ƒ„‘Ž‹ †‹•‡ƒ•‡‡ Šƒ‹•• —•‹‰’Ž—”‹’‘–‡–•–‡ ‡ŽŽ•ƒ†‰‡‘‡‡†‹–‹‰–‘‘Ž•Ǥ‘Ž‡–ƒ„ǤʹͲͳͷ‡’ǢͶȋͻȌǣͷͻ͵Ȃ͸ͲͶǤ ͵Ǥ ”ƒˆƒŽǡƒ”••‘ǡ ¡”†ˆ‡Ž†– ǡ”‹••‘ǡƒ”‹‹Ǥ—Ž–—”‹‰‘ˆ ‡’ ʹ ‡ŽŽ•™‹–ŠŠ—ƒ •‡”— ‹’”‘˜‡ –Š‡‹” ˆ— –‹‘ƒŽ‹–› ƒ† •—‹–ƒ„‹Ž‹–› ‹ •–—†‹‡• ‘ˆ Ž‹’‹† ‡–ƒ„‘Ž‹•Ǥ ‹‘ Š‹ ‹‘’Š›• –ƒǦ‘Ž‡ŽŽ‹‘Ž‹’‹†•ǤʹͲͳ͸Ǣͳͺ͸ͳȋͳȌǣͷͳȂͻǤ ͶǤ –‡‡„‡”‰‡ǡ–‹ǡ–‡” ‘”•–ǡƒ–ǡ‡—ˆ‡Ž†–ǡ‡Ž‘˜‘†•‹›ǡŠ—ƒǡŠ‘ ǡ ‘› ‡ǡ —–‹ŽŠ ǡ ›””‡ŽŽ Ǥ •–ƒ„Ž‹•Š‹‰ ‘”ƒŽ ‡–ƒ„‘Ž‹•ƒ† †‹ˆˆ‡”‡–‹ƒ–‹‘ ‹ Š‡’ƒ–‘ ‡ŽŽ—Žƒ” ƒ” ‹‘ƒ ‡ŽŽ•„› —Ž–—”‹‰‹ƒ†—Ž–Š—ƒ•‡”—Ǥ ‹‡’ǤʹͲͳͺ—‰͵ǢͺȋͳȌǣͳͳ͸ͺͷǤ ͷǤ –‡‡„‡”‰‡ ǡ ‘› ‡ǡŠ‘ƒ•ǡ ‘‡•ǡƒ™ ǡ—••‡ŽŽǡ ‘—‰Š–‘ǡ›””‡ŽŽǤ —ƒ•‡”—Ž‡ƒ†•–‘†‹ˆˆ‡”‡–‹ƒ–‹‘‘ˆŠ—ƒŠ‡’ƒ–‘ƒ ‡ŽŽ•ǡ”‡•–‘”ƒ–‹‘‘ˆ˜‡”›ǦŽ‘™Ǧ†‡•‹–› Ž‹’‘’”‘–‡‹ •‡ ”‡–‹‘ǡ ƒ† ƒ ͳͲͲͲǦˆ‘Ž† ‹ ”‡ƒ•‡ ‹  ƒ’ƒ‡•‡ ˆ—Ž‹ƒ– Š‡’ƒ–‹–‹• –›’‡ ͳ –‹–‡”•Ǥ ‡’ƒ–‘Ž‘‰›ǤʹͲͳ͵Ǣͷͺȋ͸ȌǣͳͻͲ͹Ȃͳ͹Ǥ ͸Ǥ — ǡ ”‡‡ ǡ”ƒˆƒŽǡ ‘†•‘Ǥ ˜‹–”‘ ‡ŽŽ—Žƒ”‘†‡Ž•‘ˆŠ—ƒŠ‡’ƒ–‹ ˆƒ––›ƒ ‹† ‡–ƒ„‘Ž‹•ǣ ‹ˆˆ‡”‡ ‡• „‡–™‡‡ —Š͹ ƒ† ‡’ ʹ ‡ŽŽ Ž‹‡• ‹ Š—ƒ ƒ† ˆ‡–ƒŽ „‘˜‹‡ —Ž–—”‹‰•‡”—ǤŠ›•‹‘Ž‡’ǤʹͲͳ͹ǢͷȋʹͶȌǣͳȂͳʹǤ ͹Ǥ ‹Ž ǡ Šƒ‰ ǡ ‘Ž–‡”• ǡ ‡”‡–‹‡” ǡ ‘‡” ǡ ‘”˜ƒ–‘˜‹ Š ǡ ‡‹‡”Ǧ ‘‡ƒ ǡ ‡‹Œ‰‘—† ǡ‹• Š‘ˆˆǤ‡Ǧ˜•–™‘Ǧ’Šƒ•‡‡š–”ƒ –‹‘ǣ”‡Ǧ‡˜ƒŽ—ƒ–‹‘‘ˆ•ƒ’Ž‡’”‡’ƒ”ƒ–‹‘ ’”‘ ‡†—”‡• ˆ‘” —–ƒ”‰‡–‡† Ž‹’‹†‘‹ • ‹ ’Žƒ•ƒ •ƒ’Ž‡•Ǥ ƒŽ ‹‘ƒƒŽ Š‡Ǥ ʹͲͳͺǢͶͳͲȋʹ͵ȌǣͷͺͷͻȂ͹ͲǤ

ʹͻ Chapter 1

͹ͺǤ —ǡ”„ƒǡ‡„‡” ǡ”‡‡ ǡ”‘™ǡ”‡‹–Ž‹‰ǡ ƒ‡‡‹‡”ǡ ‘‘†ƒ ”‡ǡ ‡—ƒǡ‘’ƒ ǡ‹ƒ–Ǥƒ••ƒ’’‡ƒŽǣ‡–ƒ„‘Ž‹–‡‹†‡–‹ˆ‹ ƒ–‹‘‹ƒ•••’‡ –”‘‡–”›Ǧ ˆ‘ —•‡†—–ƒ”‰‡–‡†‡–ƒ„‘Ž‘‹ •Ǥ‡–ƒ„‘Ž‘‹ •ǤʹͲͳ͵ǢͻȋǤͳȌǣͶͶȂ͸͸Ǥ ͹ͻǤ ˜ƒ Ž‹‡• ǡ ‹ƒ ǡ ˜‡”ƒ”• ǡ ‘‘–•ƒ  ǡ —Ž‹ ǡ ƒ†‡”•   ƒǡ ‘‘†  ƒǡ ƒœ Ǥ Šƒ”ƒ –‡”‹œƒ–‹‘ ‘ˆ ƒ”‹–‹‡ ƒ† ˆƒ––› ƒ ‹† ‡–ƒ„‘Ž‹• ‹ –Š‡ Ž‘‰Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡Ǧ†‡ˆ‹ ‹‡–‘—•‡Ǥ‹‘ Š‡ ǤʹͲͲͷǢ͵ͺ͹ȋ–ͳȌǣͳͺͷȂͻ͵Ǥ ͺͲǤ ‘Ž–‡”• ǡ ‹ƒ’ƒ‹–‡ ǡ ƒ —‡ ǡ ‹‡œ‡Ǧ‘‹‰ ǡ ƒ––‘ ǡ ‘”–‡  ǡ ‘”˜ƒ–‘˜‹ Š ǡ ƒ‡” ǡ ‹• Š‘ˆˆ ǡ ‡”‡–‹‡” Ǥ ”ƒ•Žƒ–‹‘ƒŽ ƒ”‰‡–‡† ”‘–‡‘‹ • ”‘ˆ‹Ž‹‰ ‘ˆ ‹–‘ Š‘†”‹ƒŽ ‡”‰› ‡–ƒ„‘Ž‹  ƒ–Š™ƒ›• ‹ ‘—•‡ ƒ† —ƒ ƒ’Ž‡•Ǥ  ”‘–‡‘‡ ‡•Ǥ ʹͲͳ͸ǢͳͷȋͻȌǣ͵ʹͲͶȂͳ͵Ǥ ͺͳǤ ‹‡ǡ ‹Žǡ”‹„‡ ǡ”ƒ‰™›‡ǡ‘‡–– Š‡” Ǥ”‘–‡‹Šƒ•‡‡’ƒ”ƒ–‹‘”‘˜‹†‡•‘‰Ǧ ‡”‡‘”›‘ˆ”ƒ•‹‡–’ƒ–‹ƒŽ–‹—Ž‹Ǥ‡ŽŽ›•–ǤʹͲͳͺǢ͸ͷͷȂ͸͵Ǥ ͺʹǤ ‘ŽŽǡ–‡ǡ‡‹–œǡ‡†ƒǤ—ƒ–‡“͵ǯ•‡“—‡ ‹‰ˆ‘”“—ƒ–‹ˆ‹ ƒ–‹‘Ǥƒ– ‡–Š‘†•ǤʹͲͳͶǢͳͳȋ‘˜‡„‡”ȌǣʹͷǤ ͺ͵Ǥ ‡‹••‡” ǡ ƒ Ǥ —ƒ–‹–ƒ–‹˜‡ •Š‘–‰— ’”‘–‡‘‹ •ǣ ‘•‹†‡”ƒ–‹‘• ˆ‘” ƒ Š‹‰ŠǦ“—ƒŽ‹–› ™‘”ˆŽ‘™‹‹—‘Ž‘‰›Ǥƒ– —‘ŽǤʹͲͳͶǢͳͷȋʹȌǣͳͳʹȂ͹Ǥ ͺͶǤ —„”ƒƒ‹ƒǡƒƒ›‘ǡ‘‘–ŠƒǤ ᩿ǣ ‡‡•‡–‡”‹ Š‡–ƒƒŽ›•‹• ‡‡•‡–‡”‹ Š‡– ƒƒŽ›•‹•᩿ǣ  ‘™Ž‡†‰‡Ǧ„ƒ•‡† ƒ’’”‘ƒ Š ˆ‘” ‹–‡”’”‡–‹‰ ‰‡‘‡Ǧ™‹†‡ ‡š’”‡••‹‘ ’”‘ˆ‹Ž‡•Ǥ ʹͲͲͷǢ ͺͷǤ Š‘‰ ǡ‘—ˆƒǡ‹ǡƒ”ƒ—• ǡ‹ǡ‘—”“—‡ ǡ‹•Šƒ”–ǡ‹ƒ Ǥ‡–ƒ„‘ƒŽ›•–ͶǤͲ᩿ǣ–‘™ƒ”†• ‘”‡–”ƒ•’ƒ”‡–ƒ†‹–‡‰”ƒ–‹˜‡‡–ƒ„‘Ž‘‹ •ƒƒŽ›•‹•Ǥ— Ž‡‹  ‹†•‡•ǤʹͲͳͺǢͶ͸ȋƒ›ȌǣͳȂ ͻǤ ͺ͸Ǥ ‹ƒ ǡ•› Š‘‰‹‘•ǡ‘—‰ǡ‹•Šƒ”–Ǥ‡–ƒ„‘ƒŽ›•–᩿ǣƒ™‡„•‡”˜‡”ˆ‘”‡–ƒ„‘Ž‘‹ †ƒ–ƒ ƒƒŽ›•‹•ƒ†‹–‡”’”‡–ƒ–‹‘ǤʹͲͲͻǢ͵͹ȋƒ›Ȍǣ͸ͷʹȂ͸ͲǤ ͺ͹Ǥ ‡–•ƒŽ— ǡ ‹Ž‘ Ǥ Ž—•–‹•ǣ ƒ ™‡„ –‘‘Ž ˆ‘” ˜‹•—ƒŽ‹œ‹‰ Ž—•–‡”‹‰ ‘ˆ —Ž–‹˜ƒ”‹ƒ–‡ †ƒ–ƒ —•‹‰ ”‹ ‹’ƒŽ‘’‘‡–ƒŽ›•‹•ƒ†Š‡ƒ–ƒ’Ǥ— Ž‡‹  ‹†•‡•ǤʹͲͳͷ —ŽͳǢͶ͵ȋͳȌǣͷ͸͸Ǧ͹ͲǤ ͺͺǤ ƒ‡Š‹•ƒ ǡ ‘–‘ ǡ ƒ–‘ ǡ ƒ™ƒ•Š‹ƒ ǡ —”—‹ Š‹ ǡ ƒƒ„‡ Ǥ ƒ–ƒǡ ‹ˆ‘”ƒ–‹‘ǡ ‘™Ž‡†‰‡ ƒ† ’”‹ ‹’Ž‡ǣ „ƒ  –‘ ‡–ƒ„‘Ž‹• ‹  Ǥ — Ž‡‹   ‹†• ‡•Ǥ ʹͲͳͶ ƒǢͶʹȋͳȌǣͳͻͻȂʹͲͷǤ ͺͻǤ   ‡•‘—” ‡ ‘‘”†‹ƒ–‘”•Ǥ ƒ–ƒ„ƒ•‡ ”‡•‘—” ‡• ‘ˆ –Š‡ ƒ–‹‘ƒŽ ‡–‡” ˆ‘” ‹‘–‡ Š‘Ž‘‰› ˆ‘”ƒ–‹‘Ǥ— Ž‡‹  ‹†•‡•ǤʹͲͳͷ ƒʹͺǢͶ͵ȋƒ–ƒ„ƒ•‡‹••—‡Ȍǣ͸Ǧͳ͹Ǥ ͻͲǤ Š‡‡Ž‡”ǡƒ””‡––ǡ‡•‘ǡ”›ƒ– ǡƒ‡•‡ǡŠ‡–˜‡”‹ǡŠ—” Šǡ‹— ‹‘ǡ †‰ƒ”ǡ ‡†‡”Š‡ǡ ‡‘Ž‘ǡ ‡‡”ǡ ‡Ž„‡”‰ǡƒ’—•–‹ǡŠ‘˜ƒ›‘ǡƒ†•ƒǡ ‹’ƒ  ǡ ƒ††‡ ǡ ƒ‰Ž‘–– ǡ ‹ŽŽ‡” ǡ •–‡ŽŽ ǡ ”—‹–– ǡ  Š—Ž‡” ǡ Š—™ƒ› ǡ ‡“—‡‹”ƒǡŠ‡””›ǡ‹”‘–‹ǡ‘—˜‘”‘˜ǡ–ƒ” Š‡‘ ǡƒ–—•‘˜ǡƒ–—•‘˜ƒǡƒ‰‡” ǡ ƒ• Š‡‘ Ǥ ƒ–ƒ„ƒ•‡ ”‡•‘—” ‡• ‘ˆ –Š‡ ƒ–‹‘ƒŽ ‡–‡” ˆ‘” ‹‘–‡ Š‘Ž‘‰› ˆ‘”ƒ–‹‘Ǥ — Ž‡‹  ‹†•‡•ǤʹͲͲ͹‡ ʹ͵Ǣ͵͸ȋƒ–ƒ„ƒ•‡Ȍǣͳ͵ȂʹͳǤ ͻͳǤ ‹”‘– ‘•‘”–‹—Ǥ ‹”‘–ǣ ƒ Š—„ ˆ‘” ’”‘–‡‹ ‹ˆ‘”ƒ–‹‘Ǥ — Ž‡‹   ‹†• ‡•Ǥ ʹͲͳͷ ƒ ʹͺǢͶ͵ȋƒ–ƒ„ƒ•‡‹••—‡ȌǣʹͲͶǦͳʹǤ ͻʹǤ ŠŽ±ǡ ƒ‰‡”„‡”‰ǡ ƒŽŽ•–”ڏǡ‹†•‘‰ǡ•˜‘Ž†ǡƒ”†‹‘‰Ž—ǡ‹˜‡”–••‘%ǡƒ’ˆ ǡŒÚ•–‡†–ǡ•’Ž—†ǡŽ••‘ ǡ†Ž—†ǡ—†„‡”‰ǡƒ˜ƒ‹ǡœ‹‰›ƒ”–‘ǡ†‡„‡”‰ ǡ Œ—”‡‹‘˜‹ ǡƒƒ‡ ǡ ‘„‡”ǡŽǡ†“˜‹•–ǡ‡”Ž‹‰ ǡ‡‰‡Ž ǡ—Ž†‡” ǡ‘ „‡”‰ ǡ ‹Ž••‘ ǡ  Š™‡ ǡ ƒ•–‡ ǡ ˜‘ ‡‹Ž‹–œ‡ ǡ ‘”•„‡”‰ ǡ ‡”••‘ ǡ ‘Šƒ••‘ ǡ ™ƒŠŽ‡ ǡ ˜‘ ‡‹Œ‡ ǡ ‹‡Ž•‡ ǡ ‘–± Ǥ ”‘–‡‘‹ •Ǥ ‹••—‡Ǧ„ƒ•‡† ƒ’‘ˆ –Š‡ Š—ƒ ’”‘–‡‘‡Ǥ ‹‡ ‡ǤʹͲͳͷ ƒʹ͵Ǣ͵Ͷ͹ȋ͸ʹʹͲȌǣͳʹ͸ͲͶͳͻǤ ͻ͵Ǥ ‹•Šƒ”–ǡœ—”ǡ‘šǡ‹•‡”ǡ —‘ǡ‘—‰ǡŠ‡‰ǡ ‡™‡ŽŽǡ”†–ǡƒ™Š‡›ǡ —‰ǡ‹‘Žƒ‹ǡ‡™‹•ǡ‘—–‘—Ž›ǡ ‘”•›–Š‡ ǡƒ‰ǡŠ”‹˜ƒ•–ƒ˜ƒǡ ‡”‘ ‹ ǡ–‘–Šƒ”† ǡ‡‰„‡› ǡŽ‘ ǡ ƒ—ǡƒ‰‡” ǡ‹‹ƒ ‹ ǡŽ‡‡–•ǡ ‡„”‡‡†Š‹ǡ —‘ǡŠƒ‰ ǡ—‰‰ƒ ǡƒ ‹• ǡ‡ŽŒ‹‡ǡ‘™Žƒ–ƒ„ƒ†‹ǡƒˆ‘”–Š ǡŽ‹˜‡ǡ ”‡‹‡”ǡ‹ǡ

͵Ͳ General introduction and outline of the thesis

ƒ””‹‡ ǡ ›‡• ǡ ‘‰‡Ž ǡ —‡”‡‰‡••‡” Ǥ ǣ Š‡ Š—ƒ ‡–ƒ„‘Ž‘‡ †ƒ–ƒ„ƒ•‡Ǥ — Ž‡‹  ‹†•‡•ǤʹͲͲ͹Ǣ͵ͷȋǤͳȌǣͷʹͳȂ͸Ǥ 1 ͻͶǤ ‹•Šƒ”–ǡ ‡—ƒ‰ǡƒ” —ǡ —‘ǡ‹ƒ‰ǡžœ“—‡œǦ ”‡•‘ǡƒŒ‡†ǡ ‘Š•‘ǡ‹ǡ ƒ”—ǡƒ›‡‡†ƒǡ‘ǡ••‡’‘—”ǡ‡”Œƒ•‹‹ǡ‹‰ŠƒŽǡ”†–ǡ‹ƒ‰ǡƒ†”ƒ ǡ ”ƒ– ǡ‡””ƒǦƒ›—‡Žƒǡ‹—ǡƒ†ƒŽǡ‡˜‡—ǡ‘ǡ‘šǡ‹Ž•‘ǡƒƒ Šǡ ƒŽ„‡”–Ǥ  ͶǤͲǣ –Š‡ Š—ƒ ‡–ƒ„‘Ž‘‡ †ƒ–ƒ„ƒ•‡ ˆ‘” ʹͲͳͺǤ — Ž‡‹   ‹†• ‡•Ǥ ʹͲͳͺ ƒ ͶǢͶ͸ȋͳȌǣ͸ͲͺȂͳ͹Ǥ ͻͷǤ ƒ”†‹‘‰Ž— ǡ ‹‡Ž•‡ Ǥ ‡™ ’ƒ”ƒ†‹‰• ˆ‘” ‡–ƒ„‘Ž‹  ‘†‡Ž‹‰ ‘ˆ Š—ƒ ‡ŽŽ•Ǥ —”” ’‹ ‹‘–‡ Š‘ŽǤʹͲͳͷǢ͵ͶǣͻͳȂ͹Ǥ ͻ͸Ǥ Š‹‡Ž‡ ǡ™ƒ‹•–‘ǡ Ž‡‹‰ǡ ‘’’‡ǡƒŠ‘‘ǡ—”‹ Šǡ ƒ”ƒŽ†•†‘––‹” ǡ‘ǡ ‘Žˆ••‘ǡ–‘„„‡ǡŠ‘”Ž‡‹ˆ••‘ ǡ‰”‡ǡڎŽ‹‰ǡ‘”†‡ŽǡŠƒ˜ƒŽ‹ǡ‘„•‘ǡ —ǡ†Ž‡”ǡ ƒŽƒǡ — ƒǡ —ŽŽǡ ƒ‡•‘ǡ ƒ•Š‹†‹ǡ ‘••‘ ǡ —–›ǡ‡ƒ–‹‰ ǡ‘‘ƒ‡™ ǡ‡‘˜°”‡ǡƒŽ›•ǡƒœ‡‹ǡƒ’‹ ǡ”‹ ‡ǡ‡Ž‘˜ǡ‹‰—”†••‘ ǡ ‹‡‘‹†‹•ǡ‘‡• Š‡‹ǡƒŽŽ„‘‡ǡ‘”‘‹ǡ˜ƒ‡‡ ǡ‡‹ Šƒ”–ǡ ‘”›ƒ‹ ǡ ‹‡Ž•‡ ǡ ‡•–‡”Š‘ˆˆ  ǡ ‡ŽŽ ǡ ‡†‡• ǡ ƒŽ••‘ ]Ǥ  ‘—‹–›Ǧ†”‹˜‡ ‰Ž‘„ƒŽ ”‡ ‘•–”— –‹‘‘ˆŠ—ƒ‡–ƒ„‘Ž‹•Ǥƒ–‹‘–‡ Š‘ŽǤʹͲͳ͵Ǣ͵ͳȋͷȌǣͶͳͻȂʹͷǤ ͻ͹Ǥ ƒ”†‹‘‰Ž— ǡ ‰”‡ ǡ ƒ’ˆ ǡ •’Ž—† ǡ ‘‘ƒ‡™ ǡ ƒ ‘„•‘ ǡ ƒŽŽ‡›  ǡ ”‘‰—‡Ž ǡ ƒ”Ž••‘ ǡ ŠŽ‡ ǡ ‹‡Ž•‡ Ǥ –‡‰”ƒ–‹‘ ‘ˆ Ž‹‹ ƒŽ †ƒ–ƒ ™‹–Š ƒ ‰‡‘‡Ǧ• ƒŽ‡ ‡–ƒ„‘Ž‹  ‘†‡Ž‘ˆ–Š‡Š—ƒƒ†‹’‘ ›–‡Ǥ‘Ž›•–‹‘ŽǤʹͲͳ͵ǢͻȋͳȌǣ͸ͶͻǤ ͻͺǤ ƒŠ‘‘Ǥ›•–‡•„‹‘Ž‘‰›‘ˆ‹„‘”‡””‘”•‘ˆ‡–ƒ„‘Ž‹•ǤʹͲͳ͵ǤʹͶͷ’Ǥ ͻͻǤ ƒ”†‹‘‰Ž— ǡ ‰”‡ ǡ ƒ’ˆ ǡ •’Ž—† ǡ ŠŽ‡ ǡ ‹‡Ž•‡ Ǥ ‡‘‡Ǧ• ƒŽ‡ ‡–ƒ„‘Ž‹  ‘†‡ŽŽ‹‰‘ˆŠ‡’ƒ–‘ ›–‡•”‡˜‡ƒŽ••‡”‹‡†‡ˆ‹ ‹‡ ›‹’ƒ–‹‡–•™‹–Š‘ǦƒŽ ‘Š‘Ž‹ ˆƒ––›Ž‹˜‡” †‹•‡ƒ•‡Ǥƒ–‘—ǤʹͲͳͶǢͷȋƒ›ʹͲͳ͵Ȍǣ͵Ͳͺ͵Ǥ ͳͲͲǤ ƒŠ‘‘ǡ ƒ”ƒŽ†•†×––‹” ǡ Ž‡‹‰ǡŠ‹‡Ž‡ Ǥ‘†‡Ž‹‰–Š‡‡ˆˆ‡ –•‘ˆ ‘‘Ž›—•‡††”—‰• ‘Š—ƒ‡–ƒ„‘Ž‹•Ǥ  ǤʹͲͳͷ ƒǢʹͺʹȋʹȌǣʹͻ͹Ȃ͵ͳ͹Ǥ ͳͲͳǤ ‡‘˜‡”‡Ǥ—ƒ–‹–ƒ–‹˜‡ƒ†Ž‘‰‹ ‘†‡ŽŽ‹‰‘ˆ‘Ž‡ —Žƒ”ƒ†‰‡‡‡–™‘”•Ǥƒ–‡˜ ‡‡–Ǥ ʹͲͳͷǢͳ͸ȋ͵ȌǣͳͶ͸ȂͷͺǤ ͳͲʹǤ ƒŽ†‘ƒ†‘ ǡ ƒŠƒ ǡ ƒŠƒ ǡ ƒŠƒ Ǥ ›•–‡• ‹‘Ž‘‰› ’’”‘ƒ Š‡• ‘™ƒ”† †‡”•–ƒ†‹‰”‹ƒ”›‹–‘ Š‘†”‹ƒŽ‹•‡ƒ•‡•Ǥ ”‘– ‡‡–ǤʹͲͳͻǢͳͲȋ ‡„”—ƒ”›ȌǣͳͻǤ ͳͲ͵Ǥ —‰‹ ǡ ‘‹–ƒ Ǥ  ‰‡‡”ƒŽ ‘’—–ƒ–‹‘ƒŽ ‘†‡Ž ‘ˆ ‹–‘ Š‘†”‹ƒŽ ‡–ƒ„‘Ž‹• ‹ ƒ ™Š‘Ž‡ ‘”‰ƒ‡ŽŽ‡• ƒŽ‡Ǥ‹‘‹ˆ‘”ƒ–‹ •ǤʹͲͲͶǢʹͲȋͳͳȌǣͳ͹ͻͷȂ͸Ǥ ͳͲͶǤ ‘†”‡Ǧ•’”‹ƒǡ•’”‹ƒ ǡ‹Ž‰ǡ Š”‡‹‡” ǡ‡‹„‡”‰‡”ǡ ”ƒ„‡”Ǥ›ƒ‹ •‹—Žƒ–‹‘• ‘–Š‡‹–‘ Š‘†”‹ƒŽˆƒ––›ƒ ‹†„‡–ƒǦ‘š‹†ƒ–‹‘‡–™‘”Ǥ›•–‹‘ŽǤʹͲͲͻǢ͵ǣʹǤ ͳͲͷǤ Šƒ‰ ǡ ƒ•Š ǡ ƒ ‘„• ǡ ƒƒ”ƒ ǡ Ž‘—‰Š  Ǥǡ —†‹  Ǥ –‡‰”ƒ–‡† ‘’—–ƒ–‹‘ƒŽ ‘†‡Ž‘ˆ–Š‡„‹‘‡‡”‰‡–‹ •‘ˆ‹•‘Žƒ–‡†Ž—‰‹–‘ Š‘†”‹ƒǤ‘‡ǤʹͲͳͺǢͳ͵ȋ͸ȌǣͳȂʹͺǤ ͳͲ͸Ǥ —ƒ”ǡ ƒ””‡Ž•‘ǡ‡™‹•ǡ ƒŽŽƒ‰Š‡” ǡ‡‘‹–ŠǡŠ‹Ž‘ƒ Š ǡ‡–‡„ƒ—‰Š Ǥ—Ž–‹Ǧ–‹••—‡ ‘’—–ƒ–‹‘ƒŽ‘†‡Ž‹‰ƒƒŽ›œ‡•’ƒ–Š‘’Š›•‹‘Ž‘‰›‘ˆ–›’‡ʹ†‹ƒ„‡–‡•‹‹ ‡Ǥ‘‡Ǥ ʹͲͳͶǢͻȋ͹ȌǤ ͳͲ͹Ǥ —‰‹ Ǥ ›ƒ‹  ‹‡–‹  ‘†‡Ž‹‰ ‘ˆ ‹–‘ Š‘†”‹ƒŽ ‡”‰› ‡–ƒ„‘Ž‹•Ǥ Ǧ‡ŽŽ ›•– ƒ•‹  ‘ ‡’–•’’ŽǤʹͲͳ͵ǢͳͲͷȂͶʹǤ ͳͲͺǤ ˜ƒ—‡ǡ‹‘• ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ†‡‡•–‡ ǡ‘—™ǡ ‘—–‡ǡ ”‘‡ ǡ”ƒ„ǡ‡‹Œ‰‘—† ǡƒ‡”Ǥ‹‘ Š‡‹ ƒŽ‘’‡–‹–‹‘ƒ‡• ƒ––›Ǧ ‹†ȾǦš‹†ƒ–‹‘ —Ž‡”ƒ„Ž‡–‘—„•–”ƒ–‡˜‡”Ž‘ƒ†Ǥ‘‘’—–‹‘ŽǤʹͲͳ͵ǢͻȋͺȌǣʹȂͻǤ ͳͲͻǤ ˜ƒ—‡ǡ‘Ž‡”Ǧ‘—™ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ‘Ž–‡”• ǡ˜ƒ‹Œ– ǡƒ”–‹‡•Ǧ ǡ ‹‡œ‡Ǧ‘‹‰ǡ ‡‹‡”ǡ‡”‡–‹‡” ǡ ”‘‡ǡ‡‹Œ‰‘—†Ǧ ǡ‡”• ǡƒ‡”Ǥ ‹˜‹‰‘–Š‡‡†‰‡ǣ•—„•–”ƒ–‡ ‘’‡–‹–‹‘‡š’Žƒ‹•Ž‘••‘ˆ”‘„—•–‡••‹‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧ ƒ ‹†‘š‹†ƒ–‹‘†‹•‘”†‡”•Ǥ‹‘ŽǤʹͲͳ͸ǢͳͶȋͳȌǣͳͲ͹Ǥ ͳͳͲǤ ‘”‘Šƒǡ‘†ƒ‹‘ ǡ ƒ”‘•œǡ —‡”ƒ”†ǡ‘’ƒ‹”ƒ ǡ”‡ ‹ƒ– ǡƒÀ‡Ž•†×––‹”ǡ”‡ ‡ǡ ‡”–‡ ǡ ƒ”ƒŽ†•†×––‹” ǡ ‡‹‡ ǡ ‡‹”‡†– ǡ ƒ‰ï•†×––‹”ǡƒ˜ Š‡‡˜ǡƒŠ‘‘ǡ

͵ͳ Chapter 1

ƒ™”‘ǡ ”‹• ‹‘‹ǡ ƒ” ‹ƒǡ”‡†‡”‰ƒ•–ǡ—‡–‡ǡ‘†”‹‰—‡•ǡ‘›ǡ‘—“—ƒ›ƒǡ ‹Ž–‰‡ǡ~ƒ‰ƒ”‡ǡ ‘Šǡ”—‡‰‡”ǡ—’‡”•–‡‹ ǡ‹‘˜›‡˜ǡ Š‡‹†‡”ǡ Ž‡‹‰ǡ Š‹‡Ž‡ Ǥ Š‡ ‹”–—ƒŽ ‡–ƒ„‘Ž‹  —ƒ †ƒ–ƒ„ƒ•‡ǣ ‹–‡‰”ƒ–‹‰ Š—ƒ ƒ† ‰—– ‹ ”‘„‹‘‡ ‡–ƒ„‘Ž‹•™‹–Š—–”‹–‹‘ƒ††‹•‡ƒ•‡Ǥ— Ž‡‹  ‹†•‡•ǤʹͲͳͻ ƒͺǢͶ͹ȋͳȌǣ͸ͳͶȂʹͶǤ ͳͳͳǤ ”—‰‰‡ƒ ǡ ‡•–‡”Š‘ˆˆ  Ǥ Š‡ ƒ–—”‡ ‘ˆ •›•–‡• „‹‘Ž‘‰›Ǥ ”‡†• ‹ ”‘„‹‘ŽǤ ʹͲͲ͹ǢͳͷȋͳȌǣͶͷȂͷͲǤ ͳͳʹǤ ‡‰”œ› ǡ –‘ŽŽ‡ ǡ ‹‡•ƒ ǡ ƒ”–‹• †‘• ƒ–‘• ǡ ƒ‡” ǡ —ƒ”‡œǦ‹‡œ Ǥ ‘ˆƒ –‘”•”‡˜‹•‹–‡†Ȃ”‡†‹ –‹‰–Š‡‹’ƒ –‘ˆˆŽƒ˜‘’”‘–‡‹Ǧ”‡Žƒ–‡††‹•‡ƒ•‡•‘ƒ‰‡‘‡• ƒŽ‡Ǥ ‹‘ Š‹‹‘’Š›• –ƒǦ‘Žƒ•‹•‹•ǤʹͲͳͻǢͳͺ͸ͷȋʹȌǣ͵͸ͲȂ͹ͲǤ ͳͳ͵Ǥ Š‹‡Ž‡ ǡ ƒŠ‘‘ ǡ ‡‹‡ ǡ ‡‹”‡†– ǡ —”‹ Š ǡ ‘”‘Šƒ ǡ Ž‡‹‰ Ǥ Š‡ ‡–ƒ„‘Ž‹•‡‡–•’Š›•‹‘Ž‘‰›ǣ ƒ”˜‡›ƒ† ƒ”˜‡––ƒǤ„‹‘š‹˜ǤʹͲͳͺ ƒͳǢʹͷͷͺͺͷǤ ͳͳͶǤ ‡•–‡”Š‘ˆˆ Ǥǡ‘Ž‘†‹ǡ‘”ƒ†‹‡ǡ‹Ž‹•‘ ǡ”—‰‰‡ƒ ǡ”ƒ„ǡ˜ƒ Š—’’‡ ǡ ƒ”†‹ ǡƒ‡”ǡ‘ǫǫ ǡ›„ƒ‘˜ƒǡ‹Œ‡ǡ˜ƒ‡‡—™‡ ǡ‘‡’ Ǥ›•–‡• „‹‘Ž‘‰›–‘™ƒ”†•Ž‹ˆ‡‹•‹Ž‹ ‘ǣƒ–Š‡ƒ–‹ •‘ˆ–Š‡ ‘–”‘Ž‘ˆŽ‹˜‹‰ ‡ŽŽ•Ǥ ƒ–Š‹‘ŽǤʹͲͲͻǢͷͺȋͳȂ ʹȌǣ͹Ȃ͵ͶǤ ͳͳͷǤ ‘ˆ‡›” Ǥ‡–ƒ„‘Ž‹  ‘–”‘ŽƒƒŽ›•‹•‹ƒ—–•Š‡ŽŽǤ ǣ”‘ ‡‡†‹‰•‘ˆ–Š‡ʹ† –‡”ƒ–‹‘ƒŽ ‘ˆ‡”‡ ‡‘•›•–‡•„‹‘Ž‘‰›ǤʹͲͲͳǤ’ǤʹͻͳȂ͵ͲͲǤ ͳͳ͸Ǥ ‘ˆ‡›” ǡƒ •‡” ǡ˜ƒ†‡”‡”™‡ Ǥ‡–ƒ„‘Ž‹  ‘–”‘ŽƒƒŽ›•‹•‘ˆ‘‹‡–›Ǧ ‘•‡”˜‡† › Ž‡•Ǥ —” ‹‘ Š‡Ǥͳͻͺ͸Ǣͳͷͷȋ͵Ȍǣ͸͵ͳȂͶͳǤ ͳͳ͹Ǥ —”• Ǥ‘–”‘ŽƒƒŽ›•‹•‘ˆ‡–ƒ„‘Ž‹ •›•–‡•Ǥ”‡†•‹‘ Š‡ ‹ǤͳͻͺͷǢͳͲȋͳȌǣͳ͸Ǥ ͳͳͺǤ ‡ŽŽ ǡ ƒ—”‘ Ǥ ‡–ƒ„‘Ž‹  ‘–”‘Ž ƒ† ‹–• ƒƒŽ›•‹•ǣ ††‹–‹‘ƒŽ ”‡Žƒ–‹‘•Š‹’• „‡–™‡‡ ‡Žƒ•–‹ ‹–‹‡•ƒ† ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–•Ǥ—” ‹‘ Š‡ǤͳͻͺͷǢͳͶͺǣͷͷͷȂ͸ͳǤ ͳͳͻǤ Š‘Ž‘†‡‘ǡƒ—”‘ ǡ‡•–‡”Š‘ˆˆ Ǥ‘–”‘Ž„›‡œ›‡•ǡ ‘‡œ›‡•ƒ† ‘•‡”˜‡† ‘‹‡–‹‡•Ǥ  ‰‡‡”ƒŽ‹•ƒ–‹‘ ‘ˆ –Š‡ ‘‡ –‹˜‹–› –Š‡‘”‡ ‘ˆ ‡–ƒ„‘Ž‹  ‘–”‘Ž ƒƒŽ›•‹•Ǥ —”  ‹‘ Š‡ǤͳͻͻͶ –ͳǢʹʹͷȋͳȌǣͳ͹ͻȂͺ͸Ǥ ͳʹͲǤ ”—‰‰‡ƒ ǡ ‘‘‰‡”† ǡ ‡•–‡”Š‘ˆˆ  Ǥ Š‡ —Ž–‹ˆƒ”‹‘—• •Š‘”–Ǧ–‡” ”‡‰—Žƒ–‹‘ ‘ˆ ƒ‘‹— ƒ••‹‹Žƒ–‹‘ ‘ˆ • Š‡”‹ Š‹ƒ ‘Ž‹ǣ ‹••‡ –‹‘ —•‹‰ ƒ ‹ •‹Ž‹ ‘ ”‡’Ž‹ ƒǤ  Ǥ ʹͲͲͷǢʹ͹ʹȋͺȌǣͳͻ͸ͷȂͺͷǤ ͳʹͳǤ ڐ‹‰ ǡ —Ž‹ ǡ ‘ŽœŠò––‡” Ǧ Ǥ —ƒ–‹ˆ›‹‰ –Š‡ ‘–”‹„—–‹‘ ‘ˆ –Š‡ ‹˜‡” –‘ Ž— ‘•‡ ‘‡‘•–ƒ•‹•ǣ‡–ƒ‹Ž‡†‹‡–‹ ‘†‡Ž‘ˆ —ƒ ‡’ƒ–‹  Ž— ‘•‡‡–ƒ„‘Ž‹•Ǥ‘‘’—– ‹‘ŽǤʹͲͳʹǢͺȋ͸Ȍǣ‡ͳͲͲʹͷ͹͹Ǥ ͳʹʹǤ ‘”–ƒ••ƒǡ‘ŽŽ‘–– ǡ‘Ǥ‹–‘ Š‘†”‹ƒŽ”‡•’‹”ƒ–‹‘ƒ†‡‹••‹‘†—”‹‰ȾǦ‘š‹†ƒ–‹‘ ‹–Š‡Š‡ƒ”–ǣ‡š’‡”‹‡–ƒŽǦ ‘’—–ƒ–‹‘ƒŽ•–—†›Ǥ‘‘’—–‹‘ŽǤʹͲͳ͹Ǣͳ͵ȋ͸ȌǣͳȂʹ͵Ǥ ͳʹ͵Ǥ •Š™‘”–Šǡƒ˜‹‡•ǡ‘‰Ž‡ Ǥ‘’—–ƒ–‹‘ƒŽ‘†‡Ž‘ˆ ‡’ƒ–‹ ‡”‰›‡–ƒ„‘Ž‹•ǣ †‡”•–ƒ†‹‰‘ƒ–‡†ƒƒ‰‡ƒ†–‡ƒ–‘•‹•‹ Ǥ‘‘’—–‹‘ŽǤʹͲͳ͸ǢͳʹȋͻȌǤ ͳʹͶǤ ƒŽ˜— ‹ǡ‡—ˆ‡Ž†ǡ‡™•Š‘Ž‡Ǥƒ–Š‡ƒ–‹ ƒŽ‘†‡Ž‘ˆ‡–ƒ„‘Ž‹•ƒ†Ž‡ –”‘’Š›•‹‘Ž‘‰› ‘ˆ‹‘ ‹†ƒ† Ž— ‘•‡–‹—Žƒ–‡† •—Ž‹‡ ”‡–‹‘ǣ ‹–”‘ƒŽ‹†ƒ–‹‘•‹‰ƒȾǦ‡ŽŽ‹‡Ǥ ‘‡ǤʹͲͳ͵Ǣͺȋ͵ȌǤ ͳʹͷǤ ‘œ‡†ƒƒŽ  ǡ ƒ‰ ǡƒƒŽ˜ƒ•– ǡƒ„›”ƒŒƒŠ ǡ‹ ǡ ‹ŽŽ‡• ˜ƒ ‹Œ ǡ‡•‡ ǡ —‹˜‡Š‘˜‡ ǡ ”‘‡ǡ ‹Ž„‡”• ǡ˜ƒ‹‡ŽǤ ˜‹˜‘ƒ†‹•‹Ž‹ ‘†›ƒ‹ •‘ˆ–Š‡ †‡˜‡Ž‘’‡–‘ˆ‡–ƒ„‘Ž‹ ›†”‘‡Ǥ‘‘’—–‹‘ŽǤʹͲͳͺǢͳͶȋ͸ȌǣͳȂͳͻǤ ͳʹ͸Ǥ ‘—‹ŽŽƒ”† ǡ ‡–‹‘ ǡ ‘„„‡Žƒ‡”‡ ǡ ‡‡ƒ— Ǧǡ ƒ—†—„”ƒ› Ǧǡ ƒ–›‰Š‡ ǦǤ ›’‘‰Ž› ƒ‡‹ƒ ”‡Žƒ–‡† –‘ ‹Š‡”‹–‡† ‡–ƒ„‘Ž‹  †‹•‡ƒ•‡• ‹ ƒ†—Ž–•Ǥ ”’Šƒ‡–  ƒ”‡ ‹•Ǥ ʹͲͳʹǢ͹ȋͳȌǣʹ͸Ǥ ͳʹ͹Ǥ ƒ’Š‡”ǡ”—‡––‹Ǧ‹‡””‹ǡ‡‡Ǥ „‘”‡””‘”•‘ˆ‡–ƒ„‘Ž‹•ǣ–Š‡ˆŽ—šˆ”‘‡†‡Ž‹ƒ–‘ ‘’Ž‡š†‹•‡ƒ•‡•Ǥƒ–‡˜ ‡‡–ǤʹͲͲ͸ —Ǣ͹ȋ͸ȌǣͶͶͻȂ͸ͲǤ ͳʹͺǤ ƒ‰‡”ǡ—‹Œ’‡”•ǡ‘• Šǡ—Ž†‡” ǡ—„‹‘Ǧ ‘œƒŽ„‘ǡ‹••‡” ǡ†‡”‹‡•ǡ‹ŽŽ‹ƒ• ǡ ƒ–‡”Šƒ ǡ ˜ƒ ’”‘•‡ ǡ  Š‹‡Ž‡  ǡ ‡”•  Ǥ  ƒ–‹‘™‹†‡ ‡–”‘•’‡ –‹˜‡ „•‡”˜ƒ–‹‘ƒŽ –—†› ˆ ‘’—Žƒ–‹‘ ‡™„‘”  ”‡‡‹‰ ‘” ‡†‹—ǦŠƒ‹  ›ŽǦ‘

͵ʹ General introduction and outline of the thesis

‡Š›†”‘‰‡ƒ•‡ȋȌ‡ˆ‹ ‹‡ › Š‡‡–Š‡”Žƒ†•Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͻ’”ʹ͵Ǣ͵ͳȋͲȌǤ ͳʹͻǤ  ”‹˜‡”Ǥˆ–‡”–Š‡‰‡‘‡ǦǦ–Š‡’Š‡‘‡ǫ  Š‡”‹–‡–ƒ„‹•ǤʹͲͲͶǢʹ͹ȋ͵Ȍǣ͵ͲͷȂͳ͹Ǥ 1 ͳ͵ͲǤ  Šƒ–œƒǤǡ•‡ƒ—‡”ǤŠ‡ Ž‹‹ ƒŽƒ‹ˆ‡•–ƒ–‹‘‘ˆ†‡ˆ‹ ‹‡ ›ǣŠƒŽŽ‡‰‡•–‘™ƒ”†• ƒ†—Ž–Š‘‘†‹–Š‡• ”‡‡‡†’‘’—Žƒ–‹‘Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͲǢ͵͵ȋͷȌǣͷͳ͵ȂʹͲǤ ͳ͵ͳǤ ‡”• 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 ͳ͵ʹǤ ‘—™ ǡ ‡”•  ǡ ƒ‡” ǡ ”‘‡ ǡ ‹– ǡ ‡‹Œ‰‘—†  Ǥ ”‘ ‰‡‘‡ –‘ ’Š‡‘‡Ǧ‹’Ž‡‹„‘”‡””‘”•‘ˆ‡–ƒ„‘Ž‹•ƒ• ‘’Ž‡š–”ƒ‹–•Ǥ‹‘ Š‹‹‘’Š›• –ƒǦ‘Ž ƒ•‹•‹•ǤʹͲͳͶǢͳͺͶʹȋͳͲȌǣʹͲʹͳȂͻǤ ͳ͵͵Ǥ ‡‹‰‡”ǡ‡Ž‹ ǡƒ ‡ǡ—†„‡”‰ǡƒ’ˆǡƒ‰ƒ”ƒŒǡŠŽ‡ǡ‘š ǡƒǤ ‹–‹ƒŽ “—ƒ–‹–ƒ–‹˜‡’”‘–‡‘‹ ƒ’‘ˆʹͺ‘—•‡–‹••—‡•—•‹‰–Š‡ ‘—•‡Ǥ‘Ž‡ŽŽ”‘–‡‘‹ •Ǥ ʹͲͳ͵Ǣͳʹȋ͸Ȍǣͳ͹ͲͻȂʹʹǤ ͳ͵ͶǤ ‘—™ǡ‹– ǡ†‡”‹‡•ǡ†‡Ž‡” ǡ‘• Šǡ‹••‡” ǡ—Ž†‡” ǡ—„‹‘Ǧ ‘œƒŽ„‘ ǡŽ˜‡”•ǡ‹‡œ‡Ǧ‘‹‰ǡƒ†‡”• ǡƒ–‡”Šƒ ǡ‡‹Œ‰‘—†Ǧ ǡ‡”• Ǥ‹• •–”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 ͳ͵ͷǤ —Ž‡”ǡ†‡”••‘ǡ ƒ‘Žƒ ǤŠ‘™ƒ– Š‡•–Š‡™ƒ– Š‡ǫ‡‰—Žƒ–‹‘‘ˆ–Š‡‡š’”‡••‹‘ƒ† ƒ –‹˜‹–›‘ˆ•‹”–—‹•Ǥ  ǤʹͲͳ͸Ǣ͵ͲȋͳʹȌǣˆŒǤʹͲͳ͸ͲͲͶͳͲǤ ͳ͵͸Ǥ ›’Šƒ—•‡ǡ‡‘‘”ǡ—ŠŽƒǡ ‹•Ž‘™•‹ǡš–”ƒǡƒŽ†—•ǡ Šƒ Š‡”Žǡƒ—ƒ ǡ ‡—†‘”ˆ ǡ ƒ‡”• Ǥ •‹‰Š–• ‹–‘ Ž›•‹‡ †‡ƒ ‡–›Žƒ–‹‘ ‘ˆ ƒ–‹˜‡Ž› ˆ‘Ž†‡† •—„•–”ƒ–‡ ’”‘–‡‹•„›•‹”–—‹•Ǥ ‹‘ŽŠ‡ǤʹͲͳ͸ǢʹͻͳȋʹͺȌǣͳͶ͸͹͹ȂͻͶǤ ͳ͵͹Ǥ Ž‡‡‡” ǡ ‘—–‘‘’‡” Ǥ‹”–—‹ƒ –‹˜ƒ–‹‘ƒ•ƒ–Š‡”ƒ’‡—–‹ ƒ’’”‘ƒ Šƒ‰ƒ‹•–‹„‘”‡””‘”• ‘ˆ‡–ƒ„‘Ž‹•Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳ͸Ǣ͵ͻȋͶȌǣͷ͸ͷȂ͹ʹǤ ͳ͵ͺǤ Š‹ƒœ—ǡ ‹”• Š‡›ǡ —ƒǡ‹––‡Šƒˆ‡”Ǧ‡‡†ǡ Š™‡”ǡ‘„ƒ”†ǡ‹ǡ—‡„‘”‰ ǡ Ž– ǡ ‡— ǡ ƒ ‘„•‘ ǡ ‡”†‹ Ǥ  ͵ †‡ƒ ‡–›Žƒ–‡• ‹–‘ Š‘†”‹ƒŽ ͵ǦŠ›†”‘š›Ǧ͵Ǧ ‡–Š›Ž‰Ž—–ƒ”›Ž ‘ •›–Šƒ•‡ ʹ ƒ† ”‡‰—Žƒ–‡• ‡–‘‡ „‘†› ’”‘†— –‹‘Ǥ ‡ŽŽ ‡–ƒ„Ǥ ʹͲͳͲǢͳʹȋ͸Ȍǣ͸ͷͶȂ͸ͳǤ ͳ͵ͻǤ ‹”• Š‡›ǡŠ‹ƒœ—ǡ ‘‡–œƒǡ ‹‰ǡ Š™‡”ǡ‘„ƒ”†ǡ ”—‡–‡”ǡ ƒ””‹•ǡ ‹††‹‰‡”ǡ Žƒ›‡˜ƒǡ–‡˜‡•ǡ‹ǡƒŠƒǡ—†‡”ƒǡƒ‹ ǡ‡™‰ƒ”†ǡ ƒ”‡•‡  Ǥǡ Ž– ǡ ƒŠ ǡ ‡”†‹ Ǥ  ͵ ”‡‰—Žƒ–‡• ‹–‘ Š‘†”‹ƒŽ ˆƒ––›Ǧƒ ‹† ‘š‹†ƒ–‹‘ „› ”‡˜‡”•‹„Ž‡‡œ›‡†‡ƒ ‡–›Žƒ–‹‘Ǥƒ–—”‡ǤʹͲͳͲǢͶ͸Ͷȋ͹ʹͺͷȌǣͳʹͳȂͷǤ ͳͶͲǤ Šƒ”ƒ–Š‹ǡŠƒ‰ǡ‘Š•‡ǡ’’ƒŽƒǡƒŽƒ•—„”ƒƒ‹ǡ Š”‡‹„‡”ǡ‡ Š‹ ǡ‡ ǡ ‘ Ž‡› ǡƒ”†‹ ǡ‡”†‹ǡ ‹„•‘ǡ ‹”• Š‡›ǡ ‘‡–œƒǤ‹”–—‹͵ȋ ͵Ȍ’”‘–‡‹ ”‡‰—Žƒ–‡• Ž‘‰Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ „› †‡ƒ ‡–›Žƒ–‹‰ ‘•‡”˜‡† Ž›•‹‡• ‡ƒ” –Š‡ ƒ –‹˜‡•‹–‡Ǥ ‹‘ŽŠ‡ǤʹͲͳ͵ǢʹͺͺȋͶ͹Ȍǣ͵͵ͺ͵͹ȂͶ͹Ǥ ͳͶͳǤ —ǡŠ‡ǡ’‘–‡ǡƒ––ƒ‰Ž‹ƒǡ — ‡ǡƒ Ǥ”‘Ž‘‰‡†ˆƒ•–‹‰‹†‡–‹ˆ‹‡•Š‡ƒ–•Š‘  ’”‘–‡‹ͳͲƒ•ƒ•‹”–—‹͵•—„•–”ƒ–‡ǣŽ— ‹†ƒ–‹‰ƒ‡™‡ Šƒ‹•Ž‹‹‰‹–‘ Š‘†”‹ƒŽ’”‘–‡‹ ƒ ‡–›Žƒ–‹‘–‘ˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘‡œ›‡ˆ‘Ž†‹‰ƒ†ˆ— –‹‘Ǥ ‹‘ŽŠ‡ǤʹͲͳͷǢʹͻͲȋͶȌǣʹͶ͸͸Ȃ ͹͸Ǥ ͳͶʹǤ Šƒ‰ǡŠƒ”ƒ–Š‹ǡƒ”†‹ ǡ’’ƒŽƒǡ‡”†‹ǡ ‹„•‘ǡ ‘‡–œƒǤ ͵ƒ† ͷ ”‡‰—Žƒ–‡ –Š‡ ‡œ›‡ ƒ –‹˜‹–› ƒ† ƒ”†‹‘Ž‹’‹ „‹†‹‰ ‘ˆ ˜‡”› Ž‘‰Ǧ Šƒ‹  ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡Ǥ‘‡ǤʹͲͳͷǢͳͲȋ͵ȌǣͳȂͳ͵Ǥ ͳͶ͵Ǥ —ƒ”ǡ‘„ƒ”†Ǥ‹–‘ Š‘†”‹ƒŽ‹”–—‹•ƒ†Š‡‹”‡Žƒ–‹‘•Š‹’•™‹–Š‡–ƒ„‘Ž‹ ‹•‡ƒ•‡ ƒ†ƒ ‡”ǤʹͲͳͷǢʹʹȋͳʹȌǤ ͳͶͶǤ ‡”–œǡ–‡‡‰„‘”Ǥ•‹‰‹–‘ Š‘†”‹ƒŽ•‹”–—‹•ƒ•†”—‰–ƒ”‰‡–•ǣ†‹•‡ƒ•‡‹’Ž‹ ƒ–‹‘•ƒ† ƒ˜ƒ‹Žƒ„Ž‡ ‘’‘—†•Ǥ‡ŽŽ‘Ž‹ˆ‡ ‹ǤʹͲͳ͸Ǣ͹͵ȋͳͷȌǣʹͺ͹ͳȂͻ͸Ǥ ͳͶͷǤ —ƒ”–‡ ǡ ‡ ‡” ǡ ƒ•Š‹†‹ ǡ Š‹‡Ž‡ ǡ ‘ ǡ ‘ ǡ ”‹˜ƒ• ǡ ƒŽ••‘ Ǥ Ž‘„ƒŽ ”‡ ‘•–”— –‹‘‘ˆ–Š‡Š—ƒ‡–ƒ„‘Ž‹ ‡–™‘”„ƒ•‡†‘‰‡‘‹ ƒ†„‹„Ž‹‘‹ †ƒ–ƒǤ”‘ ƒ–Ž

͵͵ Chapter 1

 ƒ† ‹ǤʹͲͲ͹ǢͳͲͶȋ͸Ȍǣͳ͹͹͹ȂͺʹǤ ͳͶ͸Ǥ ƒŠ‘‘ǡŠ‹‡Ž‡ Ǥ”‡†‹ –‹‰–Š‡‹’ƒ –‘ˆ†‹‡–ƒ†‡œ›‘’ƒ–Š‹‡•‘Š—ƒ•ƒŽŽ‹–‡•–‹ƒŽ ‡’‹–Š‡Ž‹ƒŽ ‡ŽŽ•Ǥ —‘Ž ‡‡–ǤʹͲͳ͵Ǣʹʹȋͳ͵Ȍǣʹ͹ͲͷȂʹʹǤ ͳͶ͹Ǥ ‘Šǡ ƒ”ˆ‹‡ŽǤ‘’—–‡”•‹—Žƒ–‹‘‘ˆ‡–ƒ„‘Ž‹•‹’ƒŽ‹–ƒ–‡Ǧ’‡”ˆ—•‡†”ƒ–Š‡ƒ”–Ǥ Ǥ ƒŽ‹–ƒ–‡‘š‹†ƒ–‹‘Ǥ‹‘‡†‰Ǥͳͻͺ͵ǢͳͳȋͷȌǣ͵͸ͳȂͺͶǤ ͳͶͺǤ ‡„ƒ‹ǡˆ‘•‘ǡƒ””‡–ǡ‡”‹Ǥ‹ •Ǧ„ƒ•‡†•–”ƒ–‡‰‹‡•‹’”‡ ‹•‹‘‡†‹ ‹‡ǣ‘™ƒ”†ƒ ’ƒ”ƒ†‹‰•Š‹ˆ–‹‹„‘”‡””‘”•‘ˆ‡–ƒ„‘Ž‹•‹˜‡•–‹‰ƒ–‹‘•Ǥ – ‘Ž ‹ǤʹͲͳ͸Ǣͳ͹ȋͻȌǤ ͳͶͻǤ —‹Ǥ‡”‰›‡–ƒ„‘Ž‹•‹–Š‡Ž‹˜‡”Ǥ‘’”Š›•‹‘ŽǤʹͲͳͶ ƒǢͶȋͳȌǣͳ͹͹Ȃͻ͹Ǥ ͳͷͲǤ ƒ–‘Ǥ‘–”‘Ž‘ˆ‹–‘ Š‘†”‹ƒŽ„Ǧ‘š‹†ƒ–‹‘ˆŽ—šǤʹͲͲʹǢͶͳǣͳͻ͹Ȃʹ͵ͻǤ ͳͷͳǤ ‘—–‡ǡƒ†‡”• Ǥ‰‡‡”ƒŽ‹–”‘†— –‹‘–‘–Š‡„‹‘ Š‡‹•–”›‘ˆ‹–‘ Š‘†”‹ƒŽˆƒ––› ƒ ‹†ȾǦ‘š‹†ƒ–‹‘Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͲǢ͵͵ȋͷȌǣͶ͸ͻȂ͹͹Ǥ ͳͷʹǤ ƒ–‘ ǡ ƒ”–Ž‡–– ǡ ‘—”ˆƒ”œƒ Ǥ ƒƒŽ‹ƒ ‹–‘ Š‘†”‹ƒŽ „‡–ƒǦ‘š‹†ƒ–‹‘Ǥ ‹‘ Š‡ Ǥ ͳͻͻ͸Ǣ͵ʹͲǣ͵ͶͷȂͷ͹Ǥ ͳͷ͵Ǥ ‘—–‡ ǡ ‹‘Žƒ–‡ ǡ ‡–—”ƒ  Ǥǡ ƒ†‡”•  Ǥ Š‡ ‹‘ Š‡‹•–”› ƒ† Š›•‹‘Ž‘‰› ‘ˆ ‹–‘ Š‘†”‹ƒŽ ƒ––›  ‹† ȾǦš‹†ƒ–‹‘ ƒ† –• ‡‡–‹  ‹•‘”†‡”•Ǥ — ‡˜ Š›•‹‘ŽǤ ʹͲͳ͸Ǣ͹ͺȋͳȌǣʹ͵ȂͶͶǤ ͳͷͶǤ ‹‘Žƒ–‡ǡ ŒŽ•–ǡ—‹–‡” ǡ‘•–‡” ǡ˜ƒ‡–Š‡ ǡ—”ƒǡ†‡Ž‡‹†ƒ ǡƒ†‡”• ƒǡ ‘—–‡ ǡ‡–—”ƒ Ǥ—„•–”ƒ–‡•’‡ ‹ˆ‹ ‹–›‘ˆŠ—ƒ ƒ”‹–‹‡ƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ǣ ’Ž‹ ƒ–‹‘•ˆ‘” ˆƒ––› ƒ ‹† ƒ† „”ƒ Š‡†Ǧ Šƒ‹ ƒ‹‘ ƒ ‹† ‡–ƒ„‘Ž‹•Ǥ ‹‘ Š‹ ‹‘’Š›•  –ƒǤ ʹͲͳ͵Ǣͳͺ͵ʹȋ͸Ȍǣ͹͹͵ȂͻǤ ͳͷͷǤ ‘‡–œƒ Ǥ †˜ƒ ‡• ‹ –Š‡ †‡”•–ƒ†‹‰ ƒ† ”‡ƒ–‡– ‘ˆ ‹–‘ Š‘†”‹ƒŽ ƒ––›  ‹† š‹†ƒ–‹‘‹•‘”†‡”•ǤʹͲͳ͹Ǣͳ͵ʹȂͶʹǤ ͳͷ͸Ǥ ‹„•‘ǡ‡‡ ǡƒƒŽ‹ǡޘ‹Ǥ ‘—’Ž‡†ƒ••ƒ›†‡–‡ –‹‰†‡ˆ‡ –•‹ˆ‹„”‘„Žƒ•–‹•‘Ž‡— ‹‡ †‡‰”ƒ†ƒ–‹‘†‹•–ƒŽ–‘‡‘›ŽǦ‘Š›†”ƒ–ƒ•‡ǣƒ’’Ž‹ ƒ–‹‘–‘͵Ǧ‘š‘–Š‹‘Žƒ•‡†‡ˆ‹ ‹‡ ›ǤŽ‹Š‹  –ƒǤͳͻͻʹ ƒ͵ͳǢʹͲͷȋͳȂʹȌǣͳʹ͹Ȃ͵ͷǤ ͳͷ͹Ǥ ƒ‹‰— Š‹ǡ‘ƒ‹ǡƒƒ‘ǡ ƒ”ƒ†ƒǡ—”‘ƒ™ƒǡ—‰‹›ƒƒǡ —Œ‹–•—ƒǡŠ‹‘—”ƒ ǡ —–•‘ ǡ ƒ””‹• ǡ ƒƒ‰‹ Ǥ Š‡ ˜ƒŽ‹‡ ƒ–ƒ„‘Ž‹  ’ƒ–Š™ƒ› ‹ Š—ƒ Ž‹˜‡”ǣ ‡ˆˆ‡ – ‘ˆ ‹””Š‘•‹•‘‡œ›‡ƒ –‹˜‹–‹‡•Ǥ ‡’ƒ–‘Ž‘‰›Ǥͳͻͻ͸‡ ǢʹͶȋ͸Ȍǣͳ͵ͻͷȂͺǤ ͳͷͺǤ ƒ‰ǡ‘Š•‡ǡ‹ŠƒŽ‹ ǡ ‘‡–œƒǡ‘ Ž‡› Ǥ˜‹†‡ ‡ˆ‘”’Š›•‹ ƒŽƒ••‘ ‹ƒ–‹‘‘ˆ ‹–‘ Š‘†”‹ƒŽ ˆƒ––› ƒ ‹† ‘š‹†ƒ–‹‘ ƒ† ‘š‹†ƒ–‹˜‡ ’Š‘•’Š‘”›Žƒ–‹‘ ‘’Ž‡š‡•Ǥ  ‹‘Ž Š‡Ǥ ʹͲͳͲǢʹͺͷȋ͵ͻȌǣʹͻͺ͵ͶȂͶͳǤ ͳͷͻǤ —‡‰‹ ǡ ”‡”‡ Ǥ ‹†‹‰ ‘ˆ –Š‡ ‡œ›‡• ‘ˆ ˆƒ––› ƒ ‹† „‡–ƒǦ‘š‹†ƒ–‹‘ ƒ† •‘‡ ”‡Žƒ–‡† ‡œ›‡•–‘’‹‰Š‡ƒ”–‹‡”‹–‘ Š‘†”‹ƒŽ‡„”ƒ‡Ǥ ‹‘ŽŠ‡ǤͳͻͺͶ —ŽʹͷǢʹͷͻȋͳͶȌǣͺ͹ͶͺȂ ͷʹǤ ͳ͸ͲǤ —‡‰‹ǡ‘”’ƒ œ›ǡŽ‘›‹ Ǥ‹‡–‹ ƒ†˜ƒ–ƒ‰‡‘ˆ–Š‡‹–‡”ƒ –‹‘„‡–™‡‡–Š‡ˆƒ––›ƒ ‹†᩿ǫǫǦ ‘š‹†ƒ–‹‘ ‡œ›‡• ƒ† –Š‡ ‘’Ž‡š‡• ‘ˆ –Š‡ ”‡•’‹”ƒ–‘”› Šƒ‹Ǥ ‹‘ Š‹ ‹‘’Š›•  –ƒ ȋȌȀ‹’‹†•‹’‹†‡–ƒ„ǤͳͻͻͳǢͳͲͺͳȋʹȌǣͳʹͳȂͺǤ ͳ͸ͳǤ —‡‰‹ǡ ‹Ž„‡”– ǡ”‡”‡Ǥ –‡”ƒ –‹‘„‡–™‡‡ ‹–”ƒ–‡•›–Šƒ•‡ƒ†–Š‹‘Žƒ•‡Ǥ ‹‘ŽŠ‡Ǥ ͳͻͺͷ ƒǢʹ͸ͲȋͳȌǣͳͺͺȂͻͲǤ ͳ͸ʹǤ —‡‰‹ǡ”‡”‡Ǥ‘’Ž‡š „‹†••‡˜‡”ƒŽ‹–‘ Š‘†”‹ƒŽǦ ‘—’Ž‡††‡Š›†”‘‰‡ƒ•‡•Ǥ ‹‘Ž Š‡ǤͳͻͺͶǢʹͷͻȋʹͶȌǣͳͷͲͶͲȂͷǤ ͳ͸͵Ǥ ƒ”ƒ›ƒǡƒ•–‡”ǡ‹”‡ ‹ǡ‹‡”Žǡ–ƒŽ‡›ǡ‹ǡ–ƒŽ‡›ǡ‡‡–– ǤŠ‘”–Ǧ Šƒ‹ ͵ǦŠ›†”‘š›ƒ ›ŽǦ ‘‡œ›‡ ƒ †‡Š›†”‘‰‡ƒ•‡ ƒ••‘ ‹ƒ–‡• ™‹–Š ƒ ’”‘–‡‹ •—’‡”Ǧ ‘’Ž‡š ‹–‡‰”ƒ–‹‰—Ž–‹’Ž‡‡–ƒ„‘Ž‹ ’ƒ–Š™ƒ›•Ǥ‘‡ǤʹͲͳʹǢ͹ȋͶȌǣͳȂͺǤ ͳ͸ͶǤ ‹ƒŽ†‘ ǡ ƒ–‡” ǡ ‡‡––  Ǥ ƒ––›  ‹† ‘š‹†ƒ–‹‘ ‹•‘”†‡”•Ǥ — ‡˜ Š›•‹‘ŽǤ ʹͲͲʹǢ͸ͶȋͳȌǣͶ͹͹ȂͷͲʹǤ ͳ͸ͷǤ ‡–Ž‡”ǡŠƒ‹ǡŽ•„‡ ‡”ǡ”‘Ž† ǡ—”–‘ǡ‘ Ž‡› ǡƒ‡”‘ǡ ‹‡” ǡ†‹   ǡ‡””›ǡ „‘”””‘”•‘ˆ‡–ƒ„‘Ž‹•‘ŽŽƒ„‘”ƒ–‹˜‡Ǥʹʹͳ‡™„‘”Ǧ• ”‡‡‡†‡‘ƒ–‡•™‹–Š ‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ ‘‡œ›‡†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›ǣ ‹†‹‰•ˆ”‘–Š‡ „‘”””‘”•‘ˆ

͵Ͷ General introduction and outline of the thesis

‡–ƒ„‘Ž‹•‘ŽŽƒ„‘”ƒ–‹˜‡Ǥ‘Ž ‡‡–‡–ƒ„ǤʹͲͳ͸ǢͳͳͻȋͳȂʹȌǣ͹ͷȂͺʹǤ ͳ͸͸Ǥ ‘ Š‹ǡŽ•‡ ǡ ”‡‰‡”•‡ǤŠ‘”–Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›ǣˆ”‘‰‡‡–‘ 1 ‡ŽŽ’ƒ–Š‘Ž‘‰›ƒ†’‘••‹„Ž‡†‹•‡ƒ•‡‡ Šƒ‹••Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳ͹ǢͶͲȋͷȌǣ͸ͶͳȂͷͷǤ ͳ͸͹Ǥ Ž‡‡‡” ǡ‘ ǡ ‡”†‹ƒ†—••‡ǡ˜ƒ†‡”‘Žǡ—’’‡ ǡ‘• Šǡƒ‰‡˜‡Ž†ǡ‡”•  ǡ‹ŽŽ‹ƒ•ǡ†‡”‹‡•ǡ—Ž†‡” ǡ—„‹‘ ‘œƒŽ„‘ǡ˜ƒ†‡”‡Ž†‡ †‡Ǧǡ‡‹‰•  ǡ Š‹‡Ž‡  ǡ‡‡”•ǡ ‘—–‘‘’‡” ǡƒ–‡”Šƒ ǡ”ƒ•Ǧƒ˜‡•ǡƒ†‡”• ǡ˜ƒ ƒ••‡Ž–ǡ Š‘‡ƒ‡”•ǡ‹Œ„—”‰ ǡ‹••‡” Ǥ ’ƒ –‘ˆˆ‘”†‡ˆ‹ ‹‡ ›‘ ‰‡‡–‹ ǡ‡œ›ƒ–‹ ƒ† Ž‹‹ ƒŽ‘—– ‘‡•Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͻ ‡„ͳ͵Ǣ ͳ͸ͺǤ ‡ƒǡ˜ƒƒŽ ƒ”ǡ ƒ•‡ ǡ†‹  ǡƒŽ•Š‘ Ž‡›ǡ‡•Ž‹‡ǡƒ‡”‘ǡ‘Š•‡ ǡ ‡””› ǡ ”‘Ž† ǡ ‘ Ž‡› Ǥ —– ‘‡• ƒ† ‰‡‘–›’‡Ǧ’Š‡‘–›’‡ ‘””‡Žƒ–‹‘• ‹ ͷʹ ‹†‹˜‹†—ƒŽ•™‹–Š†‡ˆ‹ ‹‡ ›†‹ƒ‰‘•‡†„›ƒ†‡”‘ŽŽ‡†‹–Š‡ Ǧ †ƒ–ƒ„ƒ•‡Ǥ‘Ž ‡‡–‡–ƒ„ǤʹͲͳ͸ǢͳͳͺȋͶȌǣʹ͹ʹȂͺͳǤ ͳ͸ͻǤ ‘Ž‡”Ǧ–‘—™Ǥ†‡ˆ‹ ‹‡ ›‘„‡ǡ‘”‘––‘„‡ƒ–”‹•ǤʹͲͳͶǤ ͳ͹ͲǤ ‡”•  Ǥ  †‡ˆ‹ ‹‡ ›᩿ǣ Ž‹‹ ƒŽ ƒ† Žƒ„‘”ƒ–‘”› •–—†‹‡•  Ǧ Š––’•ǣȀȀ”—‰Ǥ‘Ǥ™‘”Ž† ƒ–Ǥ‘”‰Ȁ‘ Ž ȀͳͷͲͶʹͳͷͲͶǤȏȏǤŽǤȐǦǦȐǣȏ•ǤǤȐǢʹͲͲ͹Ǥ ͳ͹ͳǤ ‡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 ͳ͹ʹǤ ”‡‰‡”•‡ǡ†”‡•‡ǡ”‘••ǡ‹–‡”ǡò†‹‰‡”ǡ‰•–ǡ Š‹•ŽƒǡŠ”‹•–‡•‡ǡ‡ŽŽ› ǡ –”ƒ—•• ǡ ގ˜”ƒƒ ǡ ‘Ž—† ǡ Žƒ‡‘”‡ ǡ —”–‹• ǡ ‰‡Ž Ǥ Šƒ”ƒ –‡”‹œƒ–‹‘ ‘ˆ ƒ †‹•‡ƒ•‡Ǧ ƒ—•‹‰ ›•͵ʹͻ –‘ Ž— —–ƒ–‹‘ ‹ ͳ͸ ’ƒ–‹‡–• ™‹–Š ‡†‹—Ǧ Šƒ‹  ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›Ǥ  Š‡”‹–‡–ƒ„‹•ǤͳͻͻͳǢͳͶȋ͵Ȍǣ͵ͳͶȂ͸Ǥ ͳ͹͵Ǥ ƒ‹Œ‘•ǡ‹ŽŽ‹ƒ ǡƒƒƒŠǤ –”ƒ‹–‘ Š‘†”‹ƒŽ ‘Ž†‹‰ƒ†••‡„Ž›‘ˆ‡†‹—Ǧ Šƒ‹ ›ŽǦ ‘‡Š›†”‘‰‡ƒ•‡ȋȌǤͳͻͻͶǢʹ͸ͻȋ͸ȌǣͶͶͲͳȂͺǤ ͳ͹ͶǤ ƒ ǡƒ‹‡”ǡ‡‹„ǡ ƒ•Ž„‡ ǡ‡–‡” ǡ”—‰‡”ǡ‘‡”Š‘ˆˆǡ ‡”†‹ƒ†—••‡ ǡ ƒ†‡”•  ǡ ‡”•–‹‰ ǡ —–ƒ— Ǥ Š‡ †‘ƒ‹Ǧ•’‡ ‹ˆ‹  ƒ† –‡’‡”ƒ–—”‡Ǧ†‡’‡†‡– ’”‘–‡‹‹•ˆ‘Ž†‹‰ ’Š‡‘–›’‡ ‘ˆ ˜ƒ”‹ƒ– ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡Ǥ ‘ ‡Ǥ ʹͲͳͶǢͻȋͶȌǤ ͳ͹ͷǤ ”‘••ǡ ‡•’‡”•‡ǡ ‡•‡ ǡ†”‡•‡ǡ”‹•–‡•‡ ǡ‹–‡”ǡƒ†›ǡ”ƒ—–Ž‡ ǡ Š‹•Žƒǡ‘Ž—†ǡ‹ ǡ ”‡‰‡”•‡Ǥˆˆ‡ –•‘ˆ–™‘—–ƒ–‹‘•†‡–‡ –‡†‹‡†‹— Šƒ‹  ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋȌǦ†‡ˆ‹ ‹‡– ’ƒ–‹‡–• ‘ ˆ‘Ž†‹‰ǡ ‘Ž‹‰‘‡” ƒ••‡„Ž›ǡ ƒ† •–ƒ„‹Ž‹–›‘ˆ‡œ›‡Ǥ‘ŽǤʹ͹Ͳǡ ‘—”ƒŽ‘ˆ‹‘Ž‘‰‹ ƒŽŠ‡‹•–”›ǤͳͻͻͷǤ’ǤͳͲʹͺͶȂͻͲǤ ͳ͹͸Ǥ ƒŽ–‡” ǤǦƒ”‹–‹‡‹‹„‘”‡””‘”•‘ˆ‡–ƒ„‘Ž‹•ǣŠƒ–‹•–Š‡‡˜‹†‡ ‡ǫ  Š‡”‹–‡–ƒ„ ‹•ǤʹͲͲ͵Ǣʹ͸ȋʹȂ͵ȌǣͳͺͳȂͺǤ ͳ͹͹Ǥ ‘‡Ǥ Š‡”‹–‡††‹•‘”†‡”•‘ˆ‹–‘ Š‘†”‹ƒŽˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘ǣ‡™”‡•’‘•‹„‹Ž‹–›ˆ‘”–Š‡ ‡‘ƒ–‘Ž‘‰‹•–Ǥ‡‹‡‘ƒ–‘ŽǤʹͲͲʹǢ͹ȋͳȌǣ͵͹ȂͶ͹Ǥ ͳ͹ͺǤ ‹ƒ‡›Ǧ‹ƒ—†ǡ‹˜”›ǡ ”‡‰‡”•‡ǡƒ–Š‹‡—ǤŠ‡‹„‘”‡””‘”•‘ˆ‹–‘ Š‘†”‹ƒŽˆƒ––›ƒ ‹† ‘š‹†ƒ–‹‘Ǥ  Š‡”‡–ƒ„‹•Ǥͳͻͺ͹ǢͳͲǣͳͷͻȂʹͲͲǤ ͳ͹ͻǤ ”‡‰‡”•‡ǡ‘”–‡•‡ǡގ˜”ƒƒǤ–Š‡„‹‘Ž‘‰‹ ‘”‹‰‹‘ˆ͸ǦͳͲǦ†‹ ƒ”„‘š›Ž‹ ƒ†͸Ǧ ͳͲǦ‘‡‰ƒǦͳǦŠ›†”‘š›‘‘ ƒ”„‘š›Ž‹ ƒ ‹†•‹Š—ƒƒ†”ƒ–™‹–Šƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ–‹‘ †‡ˆ‹ ‹‡ ‹‡•ǣ‹˜‹–”‘•–—†‹‡•‘–Š‡‘‡‰ƒǦƒ†‘‡‰ƒǦͳǦ‘š‹†ƒ–‹‘‘ˆ‡†‹—Ǧ Šƒ‹ȋ͸ǦͳʹȌ ˆƒ––›ƒ ‹†•‹Š—ƒǤ‡†‹ƒ–”‡•Ǥͳͻͺ͵Ǣͳ͹ȋͳͲȌǣͺʹͺȂ͵ͶǤ ͳͺͲǤ ”‡‰‡”•‡ ǡ ‡Ž˜”ƒƒǡƒ•—••‡ ǡ‘”–‡•‡ ǡ ‹˜”› ǡ ƒ˜‹† ǡ ‘„‘Ž–Š Ǥ ‡‡”ƒŽ ȋ‡†‹—Ǧ Šƒ‹Ȍ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ †‡ˆ‹ ‹‡ › ȋ‘Ǧ‡–‘–‹  †‹ ƒ”„‘š›Ž‹  ƒ ‹†—”‹ƒȌǣ “—ƒ–‹–ƒ–‹˜‡—”‹ƒ”›‡š ”‡–‹‘’ƒ––‡”‘ˆʹ͵„‹‘Ž‘‰‹ ƒŽŽ›•‹‰‹ˆ‹ ƒ–‘”‰ƒ‹ ƒ ‹†•‹–Š”‡‡ ƒ•‡•Ǥ Ž‹Š‹ –ƒǤͳͻͺ͵Ǣͳ͵ʹȋʹȌǣͳͺͳȂͻͳǤ ͳͺͳǤ Š‡ƒ† ǡ‡†–ǡ ”‹– Šƒǡ ‡Ž–• Ǥ‹ ƒ”„‘š›Ž‹ ƒ ‹†—”‹ƒǣ†‡ˆ‹ ‹‡–ȏͳǦͳͶȐ‘ –ƒ‘ƒ–‡ ‘š‹†ƒ–‹‘ ƒ† ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ‹ ˆ‹„”‘„Žƒ•–•Ǥ  ‹‡ ‡Ǥ ͳͻͺ͵ —Ž ͳǢʹʹͳȋͶ͸ͲͷȌǣ͹͵ȂͷǤ

͵ͷ Chapter 1

ͳͺʹǤ ‘—ƒǡ Šƒ”’‡–‹‡” Ǥ ‡†‹— Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ †‡ˆ‹ ‹‡ ›Ǥ” Š ‹• Š‹Ž†Ǥ ͳͻͻʹǢ͸͹ǣͳͶʹȂͷǤ ͳͺ͵Ǥ ‡‡––  Ǥ ƒ–Š‘’Š›•‹‘Ž‘‰› ‘ˆ ˆƒ––› ƒ ‹† ‘š‹†ƒ–‹‘ †‹•‘”†‡”•Ǥ  Š‡”‹– ‡–ƒ„ ‹•Ǥ ʹͲͳͲǢ͵͵ȋͷȌǣͷ͵͵Ȃ͹Ǥ ͳͺͶǤ ‘—™ǡ‹– ǡ‹‡œ‡Ǧ‘‹‰ǡ‘•‰”ƒƒˆǦ†‡‘‡”ǡ ‡”†‹‰ǡ‡‹Œ‰‘—†Ǧ ǡ‡”•  Ǥ  ˜‹–”‘ ƒ† ‹ ˜‹˜‘ ‘•‡“—‡ ‡• ‘ˆ ˜ƒ”‹ƒ– ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ‰‡‘–›’‡•Ǥ”’Šƒ‡– ƒ”‡‹•ǤʹͲͳ͵ǢͺȋͳȌǣͶ͵Ǥ ͳͺͷǤ ’‹‡‡”‘‡––‡” ǡ ƒ•–‹ ǡ ‹ŽŽ‹‰Šƒ ǡ ‘””‹• ǡ ‹Œ„—”‰ ǡ ‹Ž ‡ Ǥ —””‡– ‹••—‡• ”‡‰ƒ”†‹‰ –”‡ƒ–‡– ‘ˆ ‹–‘ Š‘†”‹ƒŽ ˆƒ––› ƒ ‹† ‘š‹†ƒ–‹‘ †‹•‘”†‡”•Ǥ  Š‡”‹– ‡–ƒ„ ‹•Ǥ ʹͲͳͲǢ͵͵ȋͷȌǣͷͷͷȂ͸ͳǤ ͳͺ͸Ǥ ”‘‡–›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 ͳͺ͹Ǥ —”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 ͳͺͺǤ ‡ƒŽ‡• ǡŠ‘’•‘ ǡƒ••‘—† ǡƒŠ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  Š‡”‹–‡–ƒ„‹•ǤͳͻͻͶǢͳ͹ȋͳȌǣ͹ͶȂͺͲǤ ͳͺͻǤ ‡”• ǡƒ’”‘•‡ ǡƒ‡ ǡƒ‡” ‹Ž•–ǡ’ƒǡ‹– Ǥ ƒˆ‡ƒ†—•ƒˆ‡ †—”ƒ–‹‘‘ˆˆƒ•–‹‰ˆ‘” Š‹Ž†”‡™‹–Š†‡ˆ‹ ‹‡ ›Ǥ—” ‡†‹ƒ–”ǤʹͲͲ͹Ǣͳ͸͸ȋͳȌǣͷȂͳͳǤ ͳͻͲǤ ƒŽ–‡” Ǥ ‘Ž‡”ƒ ‡ –‘ ˆƒ•–ǣ ƒ–‹‘ƒŽ ƒ† ’”ƒ –‹ ƒŽ ‡˜ƒŽ—ƒ–‹‘ ‹ Š‹Ž†”‡ ™‹–Š Š›’‘‡–‘ƒ‡‹ƒǤ  Š‡”‹–‡–ƒ„‹•ǤʹͲͲͻǢ͵ʹȋʹȌǣʹͳͶȂ͹Ǥ ͳͻͳǤ ‹•Š™ƒƒ–Š Ǥ ƒ––› ƒ ‹† „‡–ƒǦ‘š‹†ƒ–‹‘ †‹•‘”†‡”•ǣ  „”‹‡ˆ ”‡˜‹‡™Ǥ  ‡—”‘• ‹Ǥ ʹͲͳ͸Ǣʹ͵ȋͳȌǣͷͳȂͷǤ ͳͻʹǤ ‘Ž™ƒ‹ ǡ ƒǡ‹ƒǡŠƒ”‡” ǡ‘ Ž‡› ǡ‹ƒŽ†‘ǡƒ–‡”ǡ Š‘‡„ǡ‘‘†Ǥ ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ †‡ˆ‹ ‹‡ › ‹ ‰‡‡Ǧ–ƒ”‰‡–‡† ‹ ‡Ǥ ‘ ‡‡–Ǥ ʹͲͲͷǢͳȋʹȌǣͲʹͲͷȂͳʹǤ ͳͻ͵Ǥ —‡””ƒ ǡ ‘œƒ ǡ ƒŽ•Š ǡ —”–œ ǡ ‘‘† ǡ ‘œƒ Ǥ „‘”ƒŽ ‘•Š‹˜‡”‹‰ –Š‡”‘‰‡‡•‹• ‹ ‹ ‡ ™‹–Š ‹Š‡”‹–‡† †‡ˆ‡ –• ‘ˆ ˆƒ––› ƒ ‹† ‘š‹†ƒ–‹‘Ǥ  Ž‹ ˜‡•–Ǥ ͳͻͻͺǢͳͲʹȋͻȌǣͳ͹ʹͶȂ͵ͳǤ ͳͻͶǤ  Š—‹‡”ǡ‘‘†Ǥ‘—•‡‘†‡Ž•ˆ‘”‹•‘”†‡”•‘ˆ‹–‘ Š‘†”‹ƒŽ ƒ––› ‹†Ǧš‹†ƒ–‹‘Ǥ  ǤʹͲͲʹǢͶ͵ȋʹȌǣͷ͹Ȃ͸ͷǤ ͳͻͷǤ ’‹‡‡”‘‡––‡”ǡ‘‘†Ǥ‹–‘ Š‘†”‹ƒŽˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘†‹•‘”†‡”•ǣƒ–Š‘’Š›•‹‘Ž‘‰‹ ƒŽ •–—†‹‡•‹‘—•‡‘†‡Ž•Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͲǢ͵͵ȋͷȌǣͷ͵ͻȂͶ͸Ǥ ͳͻ͸Ǥ ‹‘‰ǡ ‡ ǡ ƒ‡• ǡ‘—ƒ‰ƒǡ‘™‡”•ǡ —ƒ‰ǡ•‹•ƒ ǡ‘™„‹ ǡ—”‡˜Œƒ˜ǡ ƒŽ• Š‹ ǡ ƒ˜ƒ†‘˜ǡ ‘™ƒ ǡ–”ƒ—••ǡŠ— Š—ƒǤƒ”†‹ƒ Ǧ•’‡ ‹ˆ‹ †‡ˆ‹ ‹‡ › ‹†— ‡• †‹Žƒ–‡† ƒ”†‹‘›‘’ƒ–Š› ƒ† ‘Ž† ‹–‘Ž‡”ƒ ‡Ǥ   ‡ƒ” ‹”  Š›•‹‘ŽǤ ʹͲͳͶǢ͵Ͳ͸ȋ͵Ȍǣ ͵ʹ͸Ȃ͵ͺǤ ͳͻ͹Ǥ ’‹‡‡”‘‡––‡”ǡ‘—ƒ‰ƒǡ‡†‡Žǡƒ›ƒ–‡’‡ǡš‹Žǡ—”ƒǡ‹Œ„—”‰ ǡƒ†‡”•  ǡ–”ƒ—••ǤŠƒ‰‡•‹„Ž‘‘† ƒ”‹–‹‡ƒ†ƒ ›Ž ƒ”‹–‹‡’”‘ˆ‹Ž‡•‘ˆ˜‡”›Ž‘‰Ǧ Šƒ‹ƒ ›ŽǦ ‘†‡Š›†”‘‰‡ƒ•‡Ǧ†‡ˆ‹ ‹‡–‹ ‡•—„Œ‡ –‡†–‘•–”‡••Ǥ—” Ž‹ ˜‡•–ǤʹͲͲͶǢ͵Ͷȋ͵ȌǣͳͻͳȂ͸Ǥ ͳͻͺǤ š‹Ž Ǥ„‘”ƒŽ‹–‘ Š‘†”‹ƒŽ„‹‘‡‡”‰‡–‹ •ƒ†Š‡ƒ”–”ƒ–‡†›•ˆ— –‹‘‹‹ ‡Žƒ ‹‰˜‡”›Ǧ Ž‘‰Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡Ǥ  ‡ƒ”‹” Š›•‹‘ŽǤʹͲͲͷǢʹͻͲȋ͵Ȍǣ ͳʹͺͻȂͻ͹Ǥ ͳͻͻǤ ‡””‡ƒ ǡ‡”• ǡƒ‹Œ ǡŽ‘•ǡ ‡”†‹‰ǡ ƒ˜‹‰ƒǡ‹‡–‰‡ ǡòŽŽ‡”ǡ‹– ǡ —‹’‡”• ǡ ‡‹Œ‰‘—†  Ǥ ‹•–—”„‡† Š‡’ƒ–‹  ƒ”„‘Š›†”ƒ–‡ ƒƒ‰‡‡– †—”‹‰ Š‹‰Š ‡–ƒ„‘Ž‹  †‡ƒ† ‹ ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋȌǦ†‡ˆ‹ ‹‡– ‹ ‡Ǥ ‡’ƒ–‘Ž‘‰›ǤʹͲͲͺǢͶ͹ȋ͸ȌǣͳͺͻͶȂͻͲͶǤ ʹͲͲǤ ƒŠ‘‘ǡ ”ƒœ•‘ǡ ‘••‘ ǡŠ‹‡Ž‡ Ǥ ‘’‡†‹—‘ˆ‹„‘”‡””‘”•‘ˆ‡–ƒ„‘Ž‹•ƒ’’‡†

͵͸ ‘–‘–Š‡Š—ƒ‡–ƒ„‘Ž‹ ‡–™‘”Ǥ‘Ž‹‘•›•–ǤʹͲͳʹǢͺȋͳͲȌǣʹͷͶͷȂͷͺǤ  



Chapter 2

The promiscuous medium-chain 3-keto-acyl-CoA triggers a vicious cycle in fatty-acid beta-oxidation

Anne-Claire M.F. Martines1,2, Karen van Eunen1,2, Dirk-Jan Reijngoud1,2, and Barbara M. Bakker1,2,*

1 Laboratory of Pediatrics, University of Groningen, University Medical Center Groningen, The Netherlands 2 Systems Biology Centre for Energy Metabolism and Ageing, University of Groningen, University Medical Center Groningen, The Netherlands

PLoS Comput Biol. 2017;13(4):1–22

The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation

Abstract ‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧƒ ‹†„‡–ƒǦ‘š‹†ƒ–‹‘ȋ Ȍ’Žƒ›•ƒ ‡–”ƒŽ”‘Ž‡‹ƒƒŽ‹ƒ‡‡”‰› ‡–ƒ„‘Ž‹•Ǥ—Ž–‹’Ž‡•‡˜‡”‡†‹•‡ƒ•‡•ƒ”‡ƒ••‘ ‹ƒ–‡†™‹–Š†‡ˆ‡ –•‹–Š‹•’ƒ–Š™ƒ›Ǥ –•‹‡–‹  •–”— –—”‡ ‹• Šƒ”ƒ –‡”‹œ‡† „› ƒ ‘’Ž‡š ™‹”‹‰ ‘ˆ ™Š‹ Š –Š‡ ˆ— –‹‘ƒŽ ‹’Ž‹ ƒ–‹‘• Šƒ˜‡ 2 Šƒ”†Ž› „‡‡ ‡š’Ž‘”‡†Ǥ ‡’‡–‹–‹˜‡ › Ž‡• ‘ˆ ”‡˜‡”•‹„Ž‡ ”‡ƒ –‹‘•ǡ ‡ƒ Š › Ž‡ •Š‘”–‡‹‰ –Š‡ ˆƒ––›ƒ ‹†„›–™‘ ƒ”„‘ƒ–‘•ǡ‡˜‘‡ ‘’‡–‹–‹‘„‡–™‡‡‹–‡”‡†‹ƒ–‡•‘ˆ†‹ˆˆ‡”‡– Šƒ‹ Ž‡‰–Š•ˆ‘”ƒ ‘‘•‡–‘ˆǮ’”‘‹• —‘—•ǯ‡œ›‡•ȋ‡œ›‡•™‹–Šƒ –‹˜‹–›–‘™ƒ”†•—Ž–‹’Ž‡ •—„•–”ƒ–‡•ȌǤ ‘—”˜ƒŽ‹†ƒ–‡†‹‡–‹ ‘†‡Ž‘ˆ–Š‡’ƒ–Š™ƒ›ǡ•—„•–”ƒ–‡‘˜‡”Ž‘ƒ† ƒ—•‡•ƒ•–‡‡’ ƒ††‡–”‹‡–ƒŽ ˆŽ—š †‡ Ž‹‡Ǥ ‡”‡ǡ ™‡ —”ƒ˜‡Ž–Š‡ —†‡”Ž›‹‰ ‡ Šƒ‹• ƒ†–Š‡ ”‘Ž‡ ‘ˆ ‡œ›‡’”‘‹• —‹–›‹‹–Ǥ ‘’ƒ”‹•‘‘ˆƒŽ–‡”ƒ–‹˜‡‘†‡Ž˜‡”•‹‘•‡Ž— ‹†ƒ–‡†–Š‡”‘Ž‡‘ˆ’”‘‹• —‹–›‘ˆ‹†‹˜‹†—ƒŽ ‡œ›‡•Ǥ ”‘‹• —‹–› ‘ˆ –Š‡ Žƒ•– ‡œ›‡ ‘ˆ –Š‡ ’ƒ–Š™ƒ›ǡ ‡†‹—Ǧ Šƒ‹ ‡–‘ƒ ›ŽǦ‘ –Š‹‘Žƒ•‡ȋȌǡ™ƒ•„‘–Š‡ ‡••ƒ”›ƒ†•—ˆˆ‹ ‹‡––‘‡Ž‹ ‹––Š‡ˆŽ—š†‡ Ž‹‡Ǥ—„•‡“—‡–Ž›ǡ ‡–ƒ„‘Ž‹ ‘–”‘ŽƒŽ›•‹•”‡˜‡ƒŽ‡†–Šƒ–Šƒ†‹•—ˆˆ‹ ‹‡– ƒ’ƒ ‹–›–‘ ‘’‡™‹–ŠŠ‹‰Š •—„•–”ƒ–‡‹ˆŽ—šǤ‡š–ǡ ™‡“—ƒ–‹ˆ‹‡†–Š‡‹–‡”ƒŽ‡–ƒ„‘Ž‹ ”‡‰—Žƒ–‹‘ǡ”‡˜‡ƒŽ‹‰ƒ˜‹ ‹‘—• › Ž‡ƒ”‘—†Ǥ’‘•—„•–”ƒ–‡‘˜‡”Ž‘ƒ†ǡǯ•‡–‘ƒ ›ŽǦ‘•—„•–”ƒ–‡••–ƒ”–‡†–‘ ƒ ——Žƒ–‡Ǥ Š‡ —ˆƒ˜‘—”ƒ„Ž‡ ‡“—‹Ž‹„”‹— ‘•–ƒ– ‘ˆ –Š‡ ’”‡ ‡†‹‰ ‡œ›‡ǡ ‡†‹—Ȁ•Š‘”–Ǧ Šƒ‹ Š›†”‘š›ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ǡ ™‘”‡† ƒ• ƒ ƒ’Ž‹ˆ‹‡”ǡ Ž‡ƒ†‹‰ –‘ ƒ ——Žƒ–‹‘‘ˆ—’•–”‡ƒ‘‡•–‡”•ǡ‹ Ž—†‹‰ƒ ›ŽǦ‘‡•–‡”•ǤŠ‡•‡ƒ ›ŽǦ‘‡•–‡”•ƒ”‡ ƒ––Š‡•ƒ‡–‹‡’”‘†— –•‘ˆƒ†‹Š‹„‹–‡†‹–•ƒŽ”‡ƒ†›Ž‘™ƒ –‹˜‹–›ˆ—”–Š‡”Ǥ ‹ƒŽŽ›ǡ –Š‡ƒ ——Žƒ–‹‘‘ˆ‘‡•–‡”•Ž‡†–‘ƒ•‡“—‡•–”ƒ–‹‘‘ˆˆ”‡‡‘Ǥ‘„‡‹‰ƒ ‘ˆƒ –‘”ˆ‘” ǡ ‹–• •‡“—‡•–”ƒ–‹‘ Ž‹‹–‡† –Š‡  ƒ –‹˜‹–› ‡˜‡ ˆ—”–Š‡”ǡ –Š—• ‘’Ž‡–‹‰ –Š‡ ˜‹ ‹‘—• › Ž‡Ǥ‹ ‡‘‹•ƒŽ•‘ƒ•—„•–”ƒ–‡ˆ‘”†‹•–ƒ–‡œ›‡•ǡ‹–‡ˆˆ‹ ‹‡–Ž› ‘—‹ ƒ–‡† –Š‡Ǯ–”ƒˆˆ‹ Œƒǯƒ––‘–Š‡‡–‹”‡’ƒ–Š™ƒ›Ǥ Š‹•‘˜‡Ž‡ Šƒ‹•’”‘˜‹†‡•ƒ„ƒ•‹•–‘‡š’Ž‘”‡–Š‡”‘Ž‡‘ˆ ‹†‹•‡ƒ•‡ƒ†‡Ž— ‹†ƒ–‡ •‹‹Žƒ”’”‹ ‹’Ž‡•‹‘–Š‡”’ƒ–Š™ƒ›•‘ˆŽ‹’‹†‡–ƒ„‘Ž‹•Ǥ 

 

Ͷͳ Chapter 2

Introduction Š‡ ‹–‘ Š‘†”‹ƒŽ ˆƒ––›Ǧƒ ‹† ‘š‹†ƒ–‹‘ ȋ Ȍ ‹ –Š‡ Ž‹˜‡” ‹• ‘ˆ ‡› ‹’‘”–ƒ ‡ ˆ‘” –Š‡ ’”‘†— –‹‘ ‘ˆ  ƒ† ‡–‘‡ „‘†‹‡• †—”‹‰ ˆƒ•–‹‰Ǥ – ƒŽŽ‘™• ‰Ž— ‘•‡ –‘ „‡ •’ƒ”‡† ˆ‘” —–‹Ž‹œƒ–‹‘ ‹ –Š‡ ’‡”‹’Š‡”›ǡ ƒ •—’’Ž› –Š‡ ‡‡”‰› ˆ‘” †‡ ‘˜‘ ‰Ž— ‘•‡ ’”‘†— –‹‘ǡ ƒ† ’”‘˜‹†‡• ’”‡ —”•‘”• ˆ‘” ‡–‘‡Ǧ„‘†› •›–Š‡•‹•Ǥ Š—•ǡ   ‹• ‹–”‹ ƒ–‡Ž› ‘‡ –‡† ™‹–Š ‘–Š‡” ’ƒ–Š™ƒ›• ‘ˆ ‡‡”‰› ‡–ƒ„‘Ž‹•Ǥ ‘” ƒŽ‘•– ƒŽŽ ‡œ›‡• ‘ˆ –Š‡ ’ƒ–Š™ƒ›ǡ ‹„‘” †‡ˆ‹ ‹‡ ‹‡• ƒ••‘ ‹ƒ–‡†™‹–Š Š—ƒ †‹•‡ƒ•‡ Šƒ˜‡ „‡‡ ‹†‡–‹ˆ‹‡† ȏͳǡʹȐǤ ŽŽ –Š‡•‡ †‹•‘”†‡”• •Šƒ”‡ –Š‡ ”‹• ‘ˆ ǮŠ›’‘‡–‘–‹  Š›’‘‰Ž› ‡‹ƒǯǡ ƒ Ž‹ˆ‡Ǧ–Š”‡ƒ–‡‹‰ ‘†‹–‹‘ ™‹–Š Ž‘™ „Ž‘‘† Ž‡˜‡Ž•‘ˆ‰Ž— ‘•‡ƒ†‡–‘‡„‘†‹‡•ȏͳǡʹȐǤŠ‡•‡˜‡”‹–›‘ˆ–Š‡†‹•‡ƒ•‡•›’–‘•†‡’‡†•‘ ™Š‹ Š‡œ›‡‹•‹••‹‰ǡ™Š‡–Š‡”–Š‡”‡‹•”‡•‹†—ƒŽƒ –‹˜‹–›ǡ„—––Š‡”‡‹•ƒŽ•‘ƒ—‡š’Žƒ‹‡† ˜ƒ”‹ƒ„‹Ž‹–›„‡–™‡‡‹†‹˜‹†—ƒŽ•Ǥ ‘”‡šƒ’Ž‡ǡ•‘‡ Š‹Ž†”‡™‹–ŠƒŽ‘••Ǧ‘ˆǦˆ— –‹‘—–ƒ–‹‘ ‹ –Š‡ ‡œ›‡ ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋȌ ’”‡•‡– ™‹–Š ƒ Ž‹ˆ‡Ǧ –Š”‡ƒ–‡‹‰ †”‘’ ‹ „Ž‘‘† ‰Ž— ‘•‡ǡ ™Š‹Ž‡ ‘–Š‡”• ™‹–Š –Š‡ •ƒ‡ —–ƒ–‹‘ ‡˜‡” •Š‘™ ƒ› ’Š‡‘–›’‡ ȏ͵Ȃ͸ȐǤ ‘”‡‘˜‡”ǡ Ǧ†‡ˆ‹ ‹‡– Š‹Ž†”‡ †‘ ‘š‹†‹œ‡ ‡†‹—Ǧ Šƒ‹ ˆƒ––› ƒ ‹†• ‘”ƒŽŽ› —†‡” ‘Ǧ ŠƒŽŽ‡‰‹‰ ‘†‹–‹‘• ȏ͹ȐǤ – •‡‡• –Š‡”‡ˆ‘”‡ –Šƒ– ‘– –Š‡ ‘š‹†ƒ–‹‘ ƒ’ƒ ‹–›ǡ „—– –Š‡ ƒ’ƒ„‹Ž‹–› –‘ —•‡ –Š‹• ƒ’ƒ ‹–› —†‡” ƒŽŽ ‹” —•–ƒ ‡•ǡ Š‡ ‡ –Š‡ ”‘„—•–‡••‘ˆ–Š‡ ’ƒ–Š™ƒ›ǡ‹• ‘’”‘‹•‡†‹–Š‡•‡’ƒ–‹‡–•Ǥ ‡ ‡–Ž›ǡ™‡ ‘•–”— –‡†ƒ†‡š’‡”‹‡–ƒŽŽ›˜ƒŽ‹†ƒ–‡†ƒ†›ƒ‹  ‘’—–ƒ–‹‘ƒŽ‘†‡Ž‘ˆ  ’ƒ–Š™ƒ›‹”ƒ–Ž‹˜‡”ȏͺȐǤ‘†‡Ž•‹—Žƒ–‹‘••Š‘™‡†–Šƒ––Š‡’ƒ–Š™ƒ›‹•‹–”‹•‹ ƒŽŽ› •‡•‹–‹˜‡–‘„‡ ‘‹‰‘˜‡”Ž‘ƒ†‡†™‹–Š‹–••—„•–”ƒ–‡’ƒŽ‹–‘›ŽǦ‘ǡƒ•‹š–‡‡Ǧ ƒ”„‘ȋͳ͸Ȍǡ •ƒ–—”ƒ–‡† ƒ ›ŽǦ‘ †‡”‹˜ƒ–‹˜‡ ‘ˆ –Š‡ †‹‡–ƒ”› ˆƒ––› ƒ ‹† ’ƒŽ‹–‹  ƒ ‹†Ǥ – Š‹‰Š •—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘•–Š‡ ˆŽ—š†‡ Ž‹‡†ǡ‹–‡”‡†‹ƒ–‡ƒ ›ŽǦ‘‡•–‡”•ƒ ——Žƒ–‡†ƒ†–Š‡ ƒ ›Ž ƒ””‹‡” ‘‡œ›‡  ȋ‘Ȍ ™ƒ• †‡’Ž‡–‡†Ǥ —„•‡“—‡– •‹—Žƒ–‹‘• ‘ˆ   ‹ Ǧ †‡ˆ‹ ‹‡– ‘—•‡ Ž‹˜‡” ȏͻȐǡ „ƒ•‡† ‘ †‡–ƒ‹Ž‡† ‡š’‡”‹‡–ƒŽ Šƒ”ƒ –‡”‹œƒ–‹‘ ‘ˆ –Š‡•‡ ‹ ‡ǡ •Š‘™‡† –Šƒ– –Š‡ ‡œ›‡ †‡ˆ‹ ‹‡ › ”‡†— ‡† –Š‡ –‘Ž‡”ƒ–‡† •—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘ ƒ† ƒ‰‰”ƒ˜ƒ–‡†–Š‡”‹•‘ˆ‘˜‡”Ž‘ƒ†ȏͻȐǤ‡Š›’‘–Š‡•‹œ‡–Šƒ–‹’ƒ–‹‡–•™‹–Šƒ †‹•‘”†‡”ƒ •‹‹Žƒ” ’Š‡‘‡‘ ƒ› „‡ ‹†— ‡† †—”‹‰ ˆƒ•–‹‰ǡ †—‡ –‘ –Š‡ ”‡ ”—‹–‡– ‘ˆ ˆƒ––› ƒ ‹†• ˆ”‘ ƒ†‹’‘•‡ •–‘”‡•ǡ ’‘••‹„Ž› ‹ ‘„‹ƒ–‹‘ ™‹–Š ƒ ‹†‡–ƒŽ ’‡”–—”„ƒ–‹‘• ‘ˆ ‘–Š‡” ‘Ǧ †‡’‡†‡–’”‘ ‡••‡•Ǥ‡Žƒ–‡†–‘–Š‹•ǡ‹–Šƒ•„‡‡’”‘’‘•‡†–Šƒ–ƒ‹„ƒŽƒ ‡„‡–™‡‡ˆƒ––› ƒ ‹† —’–ƒ‡ ‹–‘ –Š‡ ‹–‘ Š‘†”‹ƒ ƒ†  ǡ ƒ ——Žƒ–‹‘ ‘ˆ   ‹–‡”‡†‹ƒ–‡•ǡ ƒ† ‹ ‘’Ž‡–‡ Ž‹‡ƒ––Š‡„ƒ•‹•‘ˆ‹•—Ž‹”‡•‹•–ƒ ‡‹–Š‡—• Ž‡ȏͳͲȐǤ‘‰‡–‘”‡‹•‹‰Š– ‹–‘–Š‡‡ Šƒ‹••—†‡”Ž›‹‰–Š‡•‡†‹ˆˆ‡”‡–†‹•‡ƒ•‡•ǡ‹–‹•‹’‘”–ƒ––‘—†‡”•–ƒ†Š‘™ –Š‡ ‹„ƒŽƒ ‡ ƒ”‹•‡•ǡ ƒ† –Š‡ ‘’—–ƒ–‹‘ƒŽ ‘†‡Ž ‰‹˜‡• —• •‘‡ ˆ‹”•– Š‹–•ǡ ƒ• ‘—–Ž‹‡† „‡Ž‘™Ǥ •’‡ ‹ƒŽˆ‡ƒ–—”‡‘ˆ–Š‡ „‹‘ Š‡‹•–”›‹•–Š‡’”‘‹• —‹–›‘ˆ‹–•‡œ›‡•ǡ‡ƒ‹‰–Šƒ– ‡ƒ Š‡œ›‡ ƒ–ƒŽ›œ‡•–Š‡ ‘˜‡”•‹‘‘ˆˆƒ––›Ǧƒ ‹††‡”‹˜ƒ–‹˜‡•‘ˆƒ”ƒ‰‡‘ˆ Šƒ‹Ž‡‰–Š• ȏʹǡͺȐǤš‹†ƒ–‹‘‘ˆƒŽ‘‰Ǧ Šƒ‹ˆƒ––›ƒ ‹†”‡“—‹”‡•—’–ƒ‡‹–‘–Š‡‹–‘ Š‘†”‹ƒŽƒ–”‹š„› –Š‡ƒ ›ŽǦ ƒ”‹–‹‡•Š—––Ž‡ƒ†—Ž–‹’Ž‡”‘—†•‘ˆ ȋ ‹‰ͳȌǤ ‡ƒ Š”‘—†ǡ–Š‡ƒ ›ŽǦ‘ ‡•–‡”‹••Š‘”–‡‡†„›–™‘ ƒ”„‘ƒ–‘•ƒ†‘‡ƒ ‡–›ŽǦ‘‘Ž‡ —Ž‡‹•’”‘†— ‡†Ǥƒ Š › Ž‡ ‘•‹•–• ‘ˆ ˆ‘—” ‘•‡ —–‹˜‡ ”‡ƒ –‹‘• ƒ–ƒŽ›œ‡† „› ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋǤǤ ͳǤ͵ǤͻͻǤ͵ ƒ†ǤǤͳǤ͵ǤͺǤͳȌǡ‡‘›ŽǦ‘Š›†”ƒ–ƒ•‡‘” ”‘–‘ƒ•‡ȋǡǤǤͶǤʹǤͳǤ͹Ͷƒ†ǤǤͶǤʹǤͳǤͳͷͲȌǡ Š›†”‘š›ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡ȋǤǤͳǤͳǤͳǤʹͳͳƒ†ǤǤͳǤͳǤͳǤ͵ͷȌƒ†‡–‘ƒ ›ŽǦ‘–Š‹‘Žƒ•‡ ȋǤǤ ʹǤ͵ǤͳǤͳ͸ȌȌǤ ‘—” ‹•‘‡œ›‡• ȋ–Š‡ ‡”›Ǧ‘‰Ǧǡ ‘‰Ǧǡ ‡†‹—Ǧǡ ƒ† Š‘”–ǦŠƒ‹  ›ŽǦ ‘‡Š›†”‘‰‡ƒ•‡•ǡǡǡƒ†Ȍ ƒ–ƒŽ›œ‡–Š‡ˆ‹”•–”‡ƒ –‹‘‘ˆ–Š‡ › Ž‡ǡ ƒ ‡’–‹‰ƒ”ƒ‰‡‘ˆƒ ›ŽǦ‘•—„•–”ƒ–‡•‘ˆ†‹ˆˆ‡”‡–ǡ‘˜‡”Žƒ’’‹‰ Šƒ‹Ž‡‰–Š•Ǥ

Ͷʹ The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation

Malonyl-CoA Palmitoyl-CoA Palmitoyl-carnitine carnitine CoASH carnitine 2 CPT1 C16 CACT C4-C16 CPT2 Mitochondrion C4-C16 carnitine CoASHcarnitine Palmitoyl-carnitine Acyl-CoA

MCKAT Acetyl-CoA SCAD MCAD LCAD VLCAD FAD C4-C6 C4-C12 C8-C16 C12-C16 C4-C16 CoASH FADH2

Ketoacyl-CoA Enoyl-CoA Acetyl-CoA + NADH + H MTP C4-ketoacyl-CoA NADH + H+ C8-C16 MSCHAD C4-ketoacyl-CoA CROT NAD+ C4-C16 NAD+ C4-C16 CoASH Hydroxyacyl-CoA  Fig 1. Schematic representation of the modelled mFAO pathway in rat. ‡–ƒ„‘Ž‹–‡•‹„Ž—‡ƒ”‡ˆ‹š‡† ’ƒ”ƒ‡–‡”•ǡ™Š‹Ž‡‘–Š‡”‡–ƒ„‘Ž‹–‡•ƒ”‡ˆ”‡‡˜ƒ”‹ƒ„Ž‡•ǤŠ‡•—‘ˆ˜ƒ”‹ƒ„Ž‡‘‡•–‡”•ƒ†ˆ”‡‡‘‹•ƒ ‘•‡”˜‡†‘‹‡–›Ǥ ”‡‡ǣ‡œ›‡•’ƒ”–‹ ‹’ƒ–‹‰‹–Š‡ ƒ”‹–‹‡ › Ž‡Ǣ’—”’Ž‡ǣ‡œ›‡•’ƒ”–‹ ‹’ƒ–‹‰‹ –Š‡•Š‘”–Ǧ Šƒ‹„”ƒ ŠǢ‰”‡›ǣ‡œ›‡•’ƒ”–‹ ‹’ƒ–‹‰‹–Š‡‡†‹—Ǧƒ†Ž‘‰Ǧ Šƒ‹„”ƒ ŠǤŽŽ’”‘ ‡••‡• ƒ”‡”‡˜‡”•‹„Ž‡ƒ†–Š‡•‹œ‡‘ˆ–Š‡ƒ””‘™Š‡ƒ†•‹†‹ ƒ–‡•–Š‡‡–†‹”‡ –‹‘‘ˆ–Š‡ˆŽ—šǤŠ‹•‘†‡Ž‹•‡šƒ –Ž› –Š‡•ƒ‡ƒ•’—„Ž‹•Š‡†„‡ˆ‘”‡ȏͺȐǡ™‹–Š‘—–‘†‹ˆ‹ ƒ–‹‘•Ǥ  ‹–‘ Š‘†”‹ƒŽ ”‹ˆ— –‹‘ƒŽ ”‘–‡‹ ȋȌ –Š‡ ƒ–ƒŽ›œ‡• –Š‡ ‡š– –Š”‡‡ ”‡ƒ –‹‘• ˆ‘” •—„•–”ƒ–‡•‘ˆͺ ƒ”„‘ƒ–‘•‘”Ž‘‰‡”ǡ™Š‹Ž‡•Š‘”–‡”•—„•–”ƒ–‡•ȋ͸ƒ†ͶȌƒ”‡‡š Ž—•‹˜‡Ž› ‘˜‡”–‡† „› ƒ †‹•–‹ – ǡ ‡†‹—Ȁ•Š‘”–Ǧ Šƒ‹ Š›†”‘š›ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋȀ Ȍǡ ƒ† ‡†‹—Ǧ Šƒ‹ ‡–‘ƒ ›ŽǦ‘ –Š‹‘Žƒ•‡ ȋȌǤ ‹ ‡ ‡ƒ Š ‡œ›‡ ‹• ’”‘‹• —‘—• ƒ† ƒ–ƒŽ›œ‡• ”‡ƒ –‹‘• ‹ —Ž–‹’Ž‡ ”‘—†•ǡ –Š‡ †‹ˆˆ‡”‡– •—„•–”ƒ–‡• ƒ† ”‡•—Ž–‹‰’”‘†— –• ‘’‡–‡ˆ‘”„‹†‹‰–‘–Š‡ƒ –‹˜‡•‹–‡Ǥ –Š‡ ‘•–”— –‹‘‘ˆ–Š‡‘†‡ŽȏͺȐǡ–Š‡–‘–ƒŽ ‘ ‡–”ƒ–‹‘‘ˆ‘Šƒ•„‡‡ ‘•‹†‡”‡†ƒ•ƒ ‘•‡”˜‡†‘‹‡–›ǡ‹Ǥ‡Ǥˆ”‡‡‘ƒ†‘‡•–‡”•ƒ”‡‘Ž›‹–‡” ‘˜‡”–‡†ƒ†‘•›–Š‡•‹•‹• ‘—–•‹†‡ –Š‡ • ‘’‡ ‘ˆ –Š‡ ‘†‡ŽǤ ŽŽ ”‡ƒ –‹‘• ™‡”‡ –”‡ƒ–‡† ƒ• ”‡˜‡”•‹„Ž‡ ƒ† ’”‘†— – •‡•‹–‹˜‡Ǥƒ‹‰‹–‘ƒ ‘—––Š‡’”‘‹• —‹–›‘ˆ–Š‡‡œ›‡•ǡ”‡˜‡”•‹„Ž‡‹ Šƒ‡Ž‹•Ǧ‡–‡ ‡“—ƒ–‹‘•™‡”‡‘†‹ˆ‹‡†–‘‹ Ž—†‡ƒŽŽ‘‡•–‡”•–Šƒ–ƒ‡œ›‡ ƒ’”‘†— ‡‘” ‘•—‡ ƒ• ‘’‡–‹–‹˜‡‹Š‹„‹–‘”•ǡ™‹–Š–Š‡‹”ƒˆˆ‹‹–› ‘•–ƒ– Km•‡”˜‹‰ƒ• ‘’‡–‹–‹˜‡‹Š‹„‹–‹‘ ‘•–ƒ–Ǥ  ‡šƒ’Ž‡ ‘ˆ •— Š ƒ ”ƒ–‡ ‡“—ƒ–‹‘ǡ ˆ‘” –Š‡ ‘˜‡”•‹‘ ‘ˆ ͳ͸Ǧƒ ›ŽǦ‘ „› –Š‡ ‡œ›‡ǡ‹•ǣ

ሾ಴భలషೌ೎೤೗ష಴೚ಲሿ ሾಷಲವሿ ሾ಴భలష೐೙೚೤೗ష಴೚ಲሿ ሾಷಲವಹమሿ ௦௙ೇಽ಴ಲವǡ಴భలή௏೘ೌೣǡೇಽ಴ಲವήቌ ή ି ή ቍ ಼೘ೇಽ಴ಲವǡ಴భలషೌ೎೤೗ష಴೚ಲ ಼೘ೇಽ಴ಲವǡಷಲವ ಼೘ೇಽ಴ಲವǡ಴భలషೌ೎೤೗ష಴೚ಲ ಼೘ ೇಽ಴ಲವǡಷಲವήܭ݁ݍǡܸܮܥܣܦ ݒ௏௅஼஺஽ǡ஼ଵ଺ ൌ ሾ಴భలషೌ೎೤೗ష಴೚ಲሿ ሾ಴భలష೐೙೚೤೗ష಴೚ಲሿ ሾ಴೙షೌ೎೤೗ష಴೚ಲሿ ሾ಴೙ష೐೙೚೤೗ష಴೚ಲሿ ሾಷಲವሿ ሾಷಲವಹ ሿ ήቆଵା ା మ ቇۊ ଵା ା ା σ೙సభర ାۇ ಼೘ ಼೘ ೙సభమ಼೘ ಼೘ ಼೘ ಼೘ ೇಽ಴ಲವǡ಴భలషೌ೎೤೗ష಴೚ಲ ᇣᇧᇧೇಽ಴ಲವǡ಴భలష೐೙೚೤೗ష಴೚ಲᇧᇧᇧᇧᇧᇧᇤᇧᇧᇧᇧᇧᇧᇧᇧᇥ ᇣᇧᇧᇧᇧᇧᇧೇಽ಴ಲವǡ಴೙షೌ೎೤೗ష಴೚ಲᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇤᇧᇧᇧᇧᇧೇಽ಴ಲವǡ಴೙ష೐೙೚೤೗ష಴೚ಲᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇧᇥ ೇಽ಴ಲವǡಷಲವ ೇಽ಴ಲವǡಷಲವಹమ ی೔೙೓೔್೔೟೔೚೙್೤ೝ೐ೌ೎೟೔೚೙೛ೝ೚೏ೠ೎೟ ೔೙೓೔್೔೟೔೚೙್೤ೞೠ್ೞ೟ೝೌ೟೐ೞೌ೙೏೛ೝ೚೏ೠ೎೟ೞ೚೑೚೟೓೐ೝ೎೓ೌ೔೙೗೐೙೒೟೓ೞ ۉ ȋͳȌ  –Š‹• ‡“—ƒ–‹‘ǡ –Š‡ ‡–ƒ„‘Ž‹–‡• ‹ –Š‡ ‰”‡› „‘š ƒ”‡ –Š‡ •—„•–”ƒ–‡• ƒ† ’”‘†— –• ‘ˆ ‘’‡–‹‰”‡ƒ –‹‘•ƒ†ƒ –ƒ•‹Š‹„‹–‘”•Ǥ ˆ–Š‹•’”‘‹• —‹–›™ƒ•”‡‘˜‡†in silico„›–ƒ‹‰ ‘—–ƒŽŽ ‘’‡–‹–‹˜‡‹Š‹„‹–‹‘–‡”•‡š ‡’–†‹”‡ –’”‘†— –‹Š‹„‹–‹‘ȋ‹Ǥ‡ǤƒŽŽ–‡”•‹–Š‡‰”‡› „‘šȌǡ –Š‡ ˆŽ—š ‡šŠ‹„‹–‡† ‘”ƒŽ •ƒ–—”ƒ–‹‘ „‡Šƒ˜‹‘—”Ǥ Š‹• •—‰‰‡•–• –Šƒ– –Š‡ ‡œ›‡

Ͷ͵ Chapter 2

’”‘‹• —‹–›ƒ†–Š‡”‡•—Ž–‹‰‡–ƒ„‘Ž‹  ‘’‡–‹–‹‘ ƒ—•‡–Š‡ˆŽ—š†‡ Ž‹‡ƒ–Š‹‰Š•—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘•ȏͺǡͻȐǤHow ‘’‡–‹–‹‘‡†‹ƒ–‡•–Š‹•‡‡”‰‡–ƒ†’‘–‡–‹ƒŽŽ›†‡Ž‡–‡”‹‘—• ˆŽ—š†‡ Ž‹‡’Š‡‘‡‘Šƒ•ǡŠ‘™‡˜‡”ǡ‘–„‡‡•–—†‹‡†›‡–Ǥ‘„‡ƒ„Ž‡–‘‡š’Ž‘”‡‹–•”‘Ž‡‹ †‹•‡ƒ•‡ ƒ† ‰‡– ƒ „‡––‡” ‹•‹‰Š– ‹–‘ –Š‡ ‘”ƒŽ ˆ— –‹‘‹‰ ‘ˆ –Š‡   ’ƒ–Š™ƒ›ǡ ‹– ‹• ‹’‘”–ƒ– –‘ —”ƒ˜‡Ž –Š‡ ‘’Ž‡–‡ •‡“—‡ ‡ ‘ˆ ‡˜‡–•Ǥ Š‹•™‹ŽŽ „‡ ”— ‹ƒŽ –‘ —†‡”•–ƒ† Š‘™ƒŠ‡ƒŽ–Š› ‹•’”‘–‡ –‡†ƒ‰ƒ‹•––Š‹•’Š‡‘‡‘‘–Š‡‘‡Šƒ†ƒ†Š‘™–Š‡ ”‹•ˆ‘”’ƒ–‹‡–• ƒ„‡‹‹‹œ‡†‘–Š‡‘–Š‡”Šƒ†Ǥ Š‡ ƒ‹ ‘ˆ –Š‹• •–—†› ‹• –‘ †‹••‡ – –Š‡ ‡ Šƒ‹• ˜‹ƒ ™Š‹ Š ‡œ›‡ ’”‘‹• —‹–› ƒ† –Š‡ ”‡•—Ž–‹‰ ‡–ƒ„‘Ž‹  ‘’‡–‹–‹‘ ‹†— ‡• ˆŽ—š †‡ Ž‹‡ ƒ– Š‹‰Š •—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘•Ǥ ’‡ ‹ˆ‹ ƒŽŽ›ǡ™‡™‹ŽŽ†‹••‡ –ȋ‹Ȍ–Š‡”‘Ž‡‘ˆ’”‘‹• —‹–›‘ˆ‡ƒ Š‹†‹˜‹†—ƒŽ‡œ›‡ƒ†ȋ‹‹Ȍ–Š‡ ”‘Ž‡‘ˆ‡ƒ Š‘ˆ–Š‡‡–ƒ„‘Ž‹–‡•‹–Š‡‹–‡”ƒŽ”‡‰—Žƒ–‹‘‘ˆ–Š‡’ƒ–Š™ƒ›Ǥ‘–Š‹•‡†™‡—•‡ ‘’—–ƒ–‹‘ƒŽ ‘†‡ŽŽ‹‰ ƒ† ‡–ƒ„‘Ž‹  ‘–”‘Ž ƒƒŽ›•‹• ȏͳͳȐǡƒ† “—ƒ–‹ˆ› –Š‡ ”‡‰—Žƒ–‘”› •–”‡‰–Š‘ˆ–Š‡‡–ƒ„‘Ž‹–‡•„›ƒ’”‡˜‹‘—•Ž›’—„Ž‹•Š‡†‡–Š‘†ȏͳʹȐǤ‡™‹ŽŽ”‡˜‡ƒŽƒ˜‹ ‹‘—• › Ž‡ –Šƒ– •–ƒ”–• ƒ– –Š‡ ˜‡”› ‡† ‘ˆ –Š‡ ’ƒ–Š™ƒ› ƒ”‘—†  ƒ† ‹• ƒ’Ž‹ˆ‹‡† ƒ† –”ƒ•‹––‡† –Š”‘—‰Š‘—– –Š‡ ‡–‹”‡ ’ƒ–Š™ƒ›Ǥ ‡ ™‹ŽŽ †‹• —•• Š‘™ ƒ–—”ƒŽ ”‡‰—Žƒ–‹‘ ƒ† ’”‘’‡” ‡„‡††‹‰ ‹ •—””‘—†‹‰ ’ƒ–Š™ƒ›• ƒ› ’”‡˜‡– –Š‹• ’Š‡‘‡‘ ‹ Š‡ƒŽ–Š› ‹†‹˜‹†—ƒŽ•ƒ†Š‘™‹–ƒ›’‘–‡–‹ƒŽŽ›„‡ƒ––‡—ƒ–‡†‘”ƒ‰‰”ƒ˜ƒ–‡†‹†‹•‡ƒ•‡Ǥ  Results Description of the phenotype  ‘—” ’”‡˜‹‘—• •–—†‹‡• ™‡ •Š‘™‡† –Šƒ– –Š‡ ˆŽ—š †‡ Ž‹‡ ƒ– Š‹‰Š ›–‘•‘Ž‹  ’ƒŽ‹–‘›ŽǦ‘

‘ ‡–”ƒ–‹‘•ȋȏ’ƒŽ‹–‘›ŽǦ‘ȐȌȋ ‹‰ʹȌ™ƒ•†—‡–‘–Š‡†‡’Ž‡–‹‘‘ˆ–Š‡ˆ”‡‡‘’‘‘Žǡ ™Š‹ Š”‡•—Ž–‡†ˆ”‘–Š‡ƒ ——Žƒ–‹‘‘ˆ‘‡•–‡”•ȋ ‹‰ʹȌǤ AB 2.0 6000 ) -1 free CoA 1.6 5000

M) Intermediate ȝ 4000

.gProtein CoA esters

-1 1.2 3000 C6/C4 CoA esters 0.8 2000 C4-CoA (μmol.min [Metabolite] ( esters 0.4 1000 C6-CoA uptake

J esters 0.0 0 0 50 100 150 200 250 0 50 100 150 200 250

[Palmitoyl-CoA]CYT (ȝM) [Palmitoyl-CoA]CYT (ȝM) CD 8 8 vmtpC8 vmckatC4 ) 7 vmtpC10 ) 7

-1 vmckatC6 -1 6 vmtpC12 6 vmckatC8 vmtpC14 5 5 vmckatC10 vmtpC16 vmckatC12 .gProtein .gProtein -1 4 -1 4 vmckatC14 3 3 vmckatC16 2 2

J (μmol.min 1 1 J (μmol.min 0 0 0 50 100 150 200 250 0 50 100 150 200 250 [Palmitoyl-CoA] ȝ0 [Palmitoyl-CoA]CYT ȝ0 CYT  Fig 2. Simulated steady-state fluxes and concentrations in the mFAO model. (A) Š‡ ‡ˆˆ‡ – ‘ˆ ›–‘•‘Ž‹  ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘ ȋȏ’ƒŽ‹–‘›ŽǦ‘ȐȌ ‘ –Š‡ •–‡ƒ†›Ǧ•–ƒ–‡ ˆŽ—šǤ Š‡ •–‡ƒ†›Ǧ•–ƒ–‡ —’–ƒ‡ˆŽ—š‘ˆ’ƒŽ‹–‘›ŽǦ‘ȋ —’–ƒ‡Ȍ‹•’Ž‘––‡†ȋ ƒŽ —Žƒ–‡†ƒ•–Š‡•–‡ƒ†›Ǧ•–ƒ–‡ˆŽ—š‘ˆ’ƒŽ‹–‘›ŽǦ ƒ”‹–‹‡

ͶͶ The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation

–Š”‘—‰Šǡ‹Ǥ‡Ǥ–Š‡—’–ƒ‡‘ˆ’ƒŽ‹–‘›ŽǦ ƒ”‹–‹‡‹–‘–Š‡‹–‘ Š‘†”‹ƒȌǡ‹ ‘–”ƒ•––‘˜ƒ—‡et al. ʹͲͳ͵ȏͺȐ‹™Š‹ Š–Š‡ ˆŽ—š™ƒ•’Ž‘––‡†ǤŠ‡–™‘ƒ”‡—‹“—‡Ž›Ž‹‡†™‹–Š–Š‡Žƒ––‡”„‡‹‰͹Ǧˆ‘Ž† Š‹‰Š‡”Ǥ (B) Š‡ •–‡ƒ†›Ǧ•–ƒ–‡ ‘ ‡–”ƒ–‹‘• ‘ˆ ˆ”‡‡ ‘ ȋ‹†‡–‹ ƒŽ –‘ †ƒ–ƒ ˆ”‘ ȏͺȐȌǡ –Š‡ •— ‘ˆ ƒŽŽ ‹–‡”‡†‹ƒ–‡‘‡•–‡”•ǡ–Š‡•—‘ˆƒŽŽͶǦƒ†͸‘‡•–‡”•ƒ†–Š‡•—„•‡–‘ˆ ͶǦƒ†͸‘‡•–‡”• 2 ȋ‡™ ”‡•—Ž–•ȌǤ (C-D) ‹•–”‹„—–‹‘ ‘ˆ •–‡ƒ†›Ǧ•–ƒ–‡ ˆŽ—š‡• ȋ Ȍ ‘ˆ †‹ˆˆ‡”‡– Šƒ‹ǦŽ‡‰–Š •—„•–”ƒ–‡• –Š”‘—‰Š ƒ†ǡ”‡•’‡ –‹˜‡Ž›ǤŽ›–Š‡ Šƒ‹Ž‡‰–Š•™Š‹ Šƒ”‡ ‘˜‡”–‡†„›–Š‡•‡‡œ›‡•ȋ ˆǤ ‹‰ͳȌƒ”‡ ‹ Ž—†‡†Ǥ

‡ˆ‹”•– ƒŽ —Žƒ–‡†™Š‹ Š‘‡•–‡”•ƒ ——Žƒ–‡†ǡƒ†–Š‡•‡–—”‡†‘—––‘„‡’”‡†‘‹ƒ–Ž› –Š‡ ‘ ‡•–‡”• ‘ˆ Šƒ‹ Ž‡‰–Š• Ͷ ƒ† ͸ ȋ ‹‰ ʹȌǤ ”‘ ‡‘›ŽǦ‘ ‘™ƒ”† –Š‡   ’ƒ–Š™ƒ› ‘•‹•–• ‘ˆ –™‘ „”ƒ Š‡•ǣ –Š‡  „”ƒ Š ȋ‰”‡› ‹ ‹‰ ͳȌǡ ™Š‹ Š ‡š Ž—•‹˜‡Ž› ‘˜‡”–• –Š‡ Ž‘‰‡” Šƒ‹ Ž‡‰–Š• ‘ˆ ͺ ƒ† Š‹‰Š‡”ǡ ƒ† –Š‡ „”‘ƒ† •’‡ ‹ˆ‹ ‹–› Ǧ Ȁ Ǧ „”ƒ Š ȋ’—”’Ž‡ ‹ ‹‰ ͳȌǡ ™Š‹ Š ‘˜‡”• –Š‡ ‘’Ž‡–‡”ƒ‰‡‘ˆ Šƒ‹ Ž‡‰–Š•ˆ”‘Ͷ–Š”‘—‰Šͳ͸ǤŠ—•ǡ–Š‡ͶǦƒ†͸‘‡•–‡”• ƒ‘Ž›„‡‘š‹†‹œ‡†˜‹ƒ–Š‡ ǦȀ Ǧ „”ƒ Šǡ ™Š‹Ž‡ Ž‘‰‡” Šƒ‹ Ž‡‰–Š• ƒ „‡ ‘š‹†‹œ‡† ˜‹ƒ „‘–Š „”ƒ Š‡•Ǥ ‘™‡˜‡”ǡ –Š‡ ƒ –—ƒŽ ˆŽ—š ‘ˆ ͺ ƒ† Ž‘‰‡” ‡‘›ŽǦ‘ ‡•–‡”• ™‡– ƒŽ‘•– ‡š Ž—•‹˜‡Ž› ˜‹ƒ –Š‡  ”‘—–‡ ȋ ‹‰ ʹȌǤ Ž› Ͷ ƒ† ͸ ‘ ‡•–‡”•ǡ ™Š‹ Š Šƒ† ‘ ‘–Š‡” ‘’–‹‘ǡ–‘‘–Š‡ǦȀ Ǧ„”ƒ Šȋ ‹‰ʹȌǤŠ‡”‡ˆ‘”‡ǡ™‡”‡ˆ‡”–‘–Š‡Žƒ––‡” ƒ• –Š‡ •Š‘”–Ǧ Šƒ‹ „”ƒ ŠǤ Š‡ ˆƒ – –Šƒ– ’”‡ ‹•‡Ž› –Š‡ ‡•–‡”• ‹ –Š‡ •Š‘”–Ǧ Šƒ‹ „”ƒ Š ƒ ——Žƒ–‡†™ƒ•ƒˆ‹”•–‹†‹ ƒ–‹‘ˆ‘”ƒ„‘––Ž‡‡ ‹–Š‹•„”ƒ ŠǤ

The flux decline originates at the promiscuous MCKAT enzyme ‘ ‰ƒ‹ ‘”‡ ‹•‹‰Š– ‹–‘ –Š‡ ‡ Šƒ‹• ˜‹ƒ ™Š‹ Š ‡œ›‡ ’”‘‹• —‹–› ‹†— ‡• –Š‡ ˆŽ—š †‡ Ž‹‡ǡ™‡ˆ‹”•–†‹••‡ –‡†–Š‡”‘Ž‡‘ˆ’”‘‹• —‹–›‘ˆ ‡ƒ Š‹†‹˜‹†—ƒŽ‡œ›‡Ǥ‘–Š‹•‡†ǡ ‡œ›‡ ’”‘‹• —‹–› ™ƒ• ”‡‘˜‡† ‘’—–ƒ–‹‘ƒŽŽ› Ȃ ‡‹–Š‡” ˆ”‘ ƒŽŽ ‡œ›‡• •‹—Ž–ƒ‡‘—•Ž› ‘” ˆ”‘ ‡ƒ Š ‡œ›‡ ‹†‹˜‹†—ƒŽŽ› Ȃ „› ’ƒ”–‹–‹‘‹‰ ƒ ‡œ›‡ ’‘‘Ž –‘ †‹•–‹ –ˆ”ƒ –‹‘•†‡†‹ ƒ–‡†–‘ƒ•‹‰Ž‡ Šƒ‹Ž‡‰–Šȋ•‡‡‡–Š‘†•ȌǤ –Š‡‘Ǧ’”‘‹• —‘—• ”ƒ–‡‡“—ƒ–‹‘ǡ–Š‡‡œ›‡™ƒ••–‹ŽŽ‹Š‹„‹–‡†„›‹–•‘™’”‘†— –ǡ„—–‘–„›’”‘†— –•ƒ† •—„•–”ƒ–‡• ‘ˆ ‘–Š‡” Šƒ‹ Ž‡‰–Š•Ǥ – –Š‡ •ƒ‡ –‹‡ –Š‡ –‘–ƒŽ ƒ–ƒŽ›–‹  ƒ’ƒ ‹–› ȋVmaxȌ ™ƒ• ’ƒ”–‹–‹‘‡† ‘˜‡” –Š‡ †‹ˆˆ‡”‡– ”‡ƒ –‹‘• ƒ–ƒŽ›œ‡† „› ‡ƒ Š ‡œ›‡ ȋ•‡‡ ‡–Š‘†• ˆ‘” ƒ ƒ–Š‡ƒ–‹ ƒŽ †‡• ”‹’–‹‘ȌǤ Š‡ ˆŽ—š †‡ Ž‹‡ ‹ –Š‡ ’—„Ž‹•Š‡† ‘†‡Ž ™‹–Š ˆ—ŽŽ ’”‘‹• —‹–› ™ƒ• ’Ž‘––‡† ƒ• ƒ ”‡ˆ‡”‡ ‡ ȋ ‹‰ ͵ȌǤ ˆ ’”‘‹• —‹–› ™ƒ• ”‡‘˜‡† ˆ”‘ ƒŽŽ ‡œ›‡• •‹—Ž–ƒ‡‘—•Ž›ǡ–Š‡ˆŽ—š†‹†‘–†‡ Ž‹‡ƒ›‘”‡ǡ„—–ƒ’’”‘ƒ Š‡†ƒƒš‹—ƒ–•ƒ–—”ƒ–‹‰ ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘ ȋ ‹‰ ͵ȌǤ  ‘–”ƒ•–ǡ ”‡‘˜‹‰ –Š‡ ’”‘‹• —‹–› ˆ”‘ ƒ› ‡œ›‡ ‹†‹˜‹†—ƒŽŽ› †‹† ‘– ’”‡˜‡– ˆŽ—š †‡ Ž‹‡ ȋˆ‹‰—”‡  ‹ ͳ ‹‰Ȍǡ ‡š ‡’– ™Š‡ ’”‘‹• —‹–›™ƒ•”‡‘˜‡†ˆ”‘–Š‡”‡ƒ –‹‘ȋ ‹‰͵ȌǤ‘˜‡”•‡Ž›ǡ™Š‡’”‘‹• —‹–› ™ƒ•”‡‘˜‡†ˆ”‘ƒŽŽ‡œ›‡•exceptǡ–Š‡ˆŽ—š†”‘’’‡†‡˜‡‘”‡•–‡‡’Ž›–Šƒ‹ –Š‡‘”‹‰‹ƒŽ‘†‡Ž™‹–Šˆ—ŽŽ’”‘‹• —‹–›ȋ ‹‰͵ȌǤŠ‡•‡”‡•—Ž–••Š‘™–Šƒ–Ȃ—†‡”–Š‡‰‹˜‡ ‘†‹–‹‘•Ǧ’”‘‹• —‹–›‘ˆ‹•„‘–Š‡ ‡••ƒ”›ƒ†•—ˆˆ‹ ‹‡–ˆ‘”–Š‡ˆŽ—š†‡ Ž‹‡ƒ–ƒ ”‹–‹ ƒŽ ‘ ‡–”ƒ–‹‘ ‘ˆ ƒ„‘—– ͷͲ ρ ‘ˆ ’ƒŽ‹–‘›ŽǦ‘Ǥ ˆ ‘–‡ǡ ™Š‡ –Š‡ ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘™ƒ•‹ ”‡ƒ•‡†–‘•—’”ƒ’Š›•‹‘Ž‘‰‹ ƒŽ ‘ ‡–”ƒ–‹‘•ƒ„‘˜‡ʹͷͲρǡ‡˜‡–Š‡ ‘†‡ŽŽƒ ‹‰’”‘‹• —‹–›‡šŠ‹„‹–‡†ƒˆŽ—š†‡ Ž‹‡ȋˆ‹‰—”‡‹ͳ ‹‰ȌǤ –Š‡‘†‡Ž ™‹–Š’”‘‹• —‹–›‹‘Ž›ǡ–Š‡ˆŽ—š†”‘’’‡†ƒ–ƒŠ‹‰Š‡”’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘ –Šƒ ‹ –Š‡ ‘†‡Ž ™‹–Š ˆ—ŽŽ ’”‘‹• —‹–› ȋ ‹‰ ͵ȌǤ ‘‰‡–Š‡”ǡ –Š‹• †‡‘•–”ƒ–‡• –Šƒ– –Š‡ ’”‘‹• —‹–› ‘ˆ –Š‡ ‘–Š‡” ‡œ›‡• ‘—Ž† ‹ ’”‹ ‹’Ž‡ ‘–”‹„—–‡ –‘ ˆŽ—š †‡ Ž‹‡ǡ „—– —†‡” ’Š›•‹‘Ž‘‰‹ ƒŽ ‘†‹–‹‘•–Š‡’”‘‹• —‹–›‘ˆ‹•„›ˆƒ”–Š‡‘•–‹’‘”–ƒ–Ǥ

Ͷͷ Chapter 2

AB 2.0 4 ) -1 CPT1 1.6 Published model 2

.gProtein 1.2 No promiscuity at MCAD -1 MCKAT 0 0.8 No promiscuity SCAD except at MCKAT -2 (μmol.min 0.4 No promiscuity MCKAT Flux control coefficient (-) uptake

J 0.0 -4 0 50 100 150 200 250 0 50 100 150 200 250

[Palmitoyl-CoA]CYT ȝ0 [Palmitoyl-CoA]CYT (ȝM)

CDJuptake Juptake E Rp Rp -2.5 0.0 2.5 -2.5 0.0 2.5 3 sfcpt1C16 Keqmschad vmschadC4 Vcpt1 Vmckat

]/[NADH] vmschadC6 CarCYT [NAD+]/[NADH] + 2 vmtpC8 Kmcpt1CarCYT CoACYT uptake [NAD J CoACYT Keqcrot vmtpC10 sfmckatC4 Kmcpt1CoACYT vmtpC12 C16AcylCoACYT sfcpt1C16 1 Kmcpt1C16AcylCoACYT Vcpt1 vmtpC14 Keqcpt1 KmmckatC4ketoacylCoA vmtpC16 Keqcact Kmcpt1CarCYT 0 Vfcact CarCYT sfcactC16 Kmcpt1CoACYT [NAD+]/[NADH] C16AcylCoACYT -1

Vmckat KmmckatC4acylCoA Reaction-partitioned R 5 15 25 53.37 60 250 Keqmschad KmmckatCoAMAT [Palmitoyl-CoA] (μM) CYT  Fig 3. The role of the short-chain branch. (A) –‡ƒ†›Ǧ•–ƒ–‡ ˆŽ—š ˜‡”•—• ȏ’ƒŽ‹–‘›ŽǦ‘Ȑ‹–Š‡ ’—„Ž‹•Š‡†‘†‡Žȋ„Žƒ Ž‹‡Ȍǡƒ‘†‡Ž™‹–Š‘—–ƒ›‡œ›ƒ–‹ ’”‘‹• —‹–›ȋ„Žƒ †ƒ•Š‡†Ž‹‡ǡ”‡’”‘†— ‹‰ ”‡•—Ž–•ˆ”‘ȏͺȐƒ•ƒ”‡ˆ‡”‡ ‡Ȍǡƒ‘†‡Ž™‹–Š‘—–’”‘‹• —‹–›‘ˆȋ‰”‡‡Ž‹‡Ȍǡƒ†ˆ‹ƒŽŽ›ƒ‘†‡Ž ™‹–Š‘Ž›’”‘‹• —‹–›‘ˆǡ„—–‘–‘ˆƒ›‘ˆ–Š‡‘–Š‡”‡œ›‡•ȋ’—”’Ž‡Ž‹‡ȌǤ (B)Š‡ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–• ‹ –Š‡ ‘”‹‰‹ƒŽ ‘†‡Ž ƒ• ƒ ˆ— –‹‘ ‘ˆ ȏ’ƒŽ‹–‘›ŽǦ‘ȐǤ Ž› ‡œ›‡• ™‹–Š ƒ •—„•–ƒ–‹ƒŽ ‘–”‹„—–‹‘ƒ”‡‹ Ž—†‡†Ǣˆ‘”ƒ ‘’Ž‡–‡•‡–‘ˆˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–•ǡ•‡‡ˆ‹‰—”‡‹ʹ ‹‰Ǥ(C-D)‘’ ͳͷˆŽ—š”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–•™‹–Š–Š‡Š‹‰Š‡•–ƒ„•‘Ž—–‡˜ƒŽ—‡•ƒ–ʹͷɊȋȌƒ†͸ͲɊȋȌ‘ˆȏ’ƒŽ‹–‘›ŽǦ ‘ȐǤŠ‡’ƒ”ƒ‡–‡”•p ˆ‘”™Š‹ Š–Š‡ˆŽ—š”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–™ƒ• ƒŽ —Žƒ–‡†‹•‹†‹ ƒ–‡†‘–Š‡Ǧƒš‹•Ǥ ƒ” ‰”‡› „ƒ”• ”‡’”‡•‡– ͳǦ”‡Žƒ–‡† ’ƒ”ƒ‡–‡”•ǡ ’‹ „ƒ”• ”‡’”‡•‡– Ȁ Ǧ”‡Žƒ–‡† ’ƒ”ƒ‡–‡”•ǡ ’—”’Ž‡„ƒ”•”‡’”‡•‡–Ǧ”‡Žƒ–‡†’ƒ”ƒ‡–‡”•ƒ†–Š‡Ž‹‰Š–„Ž—‡„ƒ””‡’”‡•‡–•ƒ ”‘–‘ƒ•‡Ǧ”‡Žƒ–‡† (E) Ϊ ୎୳୮୲ୟ୩ୣ ’ƒ”ƒ‡–‡”Ǥ  Ž—š”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–‘ˆȏ ȐȀȏ Ȑȋሾ୒୅ୈశሿȀሾ୒୅ୈୌሿȌ’ƒ”–‹–‹‘‡†‹ ‘–”‹„—–‹‘• Ϊ ௃ ‘ˆ‹†‹˜‹†—ƒŽ Ǧ†‡’‡†‡–”‡ƒ –‹‘•ƒ–ͷ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘•ƒ ‘”†‹‰–‘ ܴሾே஺஽శሿȀሾே஺஽ுሿ ൌ σ ௃ ௩೔ . (cf. Eq. 4). ୎୳୮୲ୟ୩ୣ  ௜ ܥ௜ ήߝሾே஺஽శሿȀሾே஺஽ுሿ Š‡ ‘–”‹„—–‹‘•‘ˆȀ ”‡ƒ –‹‘•ͺǦͳ͸–‘ሾ୒୅ୈశሿȀሾ୒୅ୈୌሿ™‡”‡ ‡‰Ž‹‰‹„Ž‡ƒ†–Š‡”‡ˆ‘”‡‘–•Š‘™‹–Š‡Ž‡‰‡†Ǥ  MCKAT becomes the most important flux-controlling enzyme when the flux declines ƒŽ‹–‘›ŽǦ‘‹•–Š‡•—„•–”ƒ–‡‘ˆͳǡ–Š‡ˆ‹”•–‡œ›‡‘ˆ–Š‡–”ƒ•’‘”–•Š—––Ž‡ǡ ‘•‹•–‹‰ ‘ˆͳǡǡƒ†ʹȋ ‹‰ͳȌǤŠ‡†‡ Ž‹‡‘ˆˆŽ—šƒ•ƒˆ— –‹‘‘ˆ’ƒŽ‹–‘›ŽǦ‘•—‰‰‡•–• –Šƒ–ͳƒ –‹˜‹–›Šƒ•ƒ‡‰ƒ–‹˜‡ ‘–”‘Ž‘˜‡”–Š‡’ƒ–Š™ƒ›ˆŽ—šǤ‘‹˜‡•–‹‰ƒ–‡Š‘™–Š‡ˆŽ—š ‹• ‘–”‘ŽŽ‡†—†‡”–Š‡•‡ ‘†‹–‹‘•ǡ–Š‡ ‘–”‘Ž‡š‡”–‡†„›‡ƒ Š‡œ›‡‘˜‡”–Š‡ˆŽ—š™ƒ• ௃ “—ƒ–‹ˆ‹‡†Ǥ  ‡–ƒ„‘Ž‹  ‘–”‘Ž ƒŽ›•‹• ȏͳͳǡͳ͵ȂͳͷȐǡ –Š‡ ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡– ܥ௜  ‘ˆ ƒ ‡œ›‡i‘˜‡”–Š‡•–‡ƒ†›Ǧ•–ƒ–‡ˆŽ—šJ ‹•†‡ˆ‹‡†ƒ•ǣ ௃ ௗ௟௡௃Ȁௗ௣ ܥ௜ ൌ ȋʹȌ డ௟௡௩೔Τడ௣ ‹™Š‹ Šp‹•ƒ’ƒ”ƒ‡–‡”–Šƒ–‘Ž›ƒˆˆ‡ –•–Š‡”ƒ–‡v‘ˆ‡œ›‡iǤˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡– ‡“—ƒŽ–‘ͳ‡ƒ•–Šƒ––Š‡†‹”‡ –‡ˆˆ‡ –‘ˆp ȋ‡Ǥ‰Ǥ–Š‡‡œ›‡ ‘ ‡–”ƒ–‹‘‘”VmaxȌ‘–Š‡ ‡œ›‡‹•ˆ—ŽŽ›–”ƒ•Žƒ–‡†–‘–Š‡•–‡ƒ†›Ǧ•–ƒ–‡ˆŽ—šǤ ˆ–Š‡ƒ„•‘Ž—–‡ ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–•‘ˆ–Š‡

Ͷ͸ The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation

‘–Š‡” ‡œ›‡• ƒ”‡ Ž‘•‡ –‘ œ‡”‘ǡ –Š‹• ‹†‹ ƒ–‡• –Šƒ– –Š‡ ‡œ›‡ ‹• ‘’Ž‡–‡Ž› ”ƒ–‡Ǧ ‘–”‘ŽŽ‹‰Ǥ  Ž‘™ ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡– ‹†‹ ƒ–‡• –Šƒ– –Š‡ ’”‹ƒ”› ‡ˆˆ‡ – ‘ˆ p‹• ‘—–‡”ƒ –‡†„›ƒ†ƒ’–ƒ–‹‘•‘ˆ‡–ƒ„‘Ž‹–‡ ‘ ‡–”ƒ–‹‘•ǡ•— Š–Šƒ––Š‡ˆ‹ƒŽ‡ˆˆ‡ –‘–Š‡ •–‡ƒ†›Ǧ•–ƒ–‡ ˆŽ—š ‹• •ƒŽŽǤ Š‡ ”‡•—Ž–• ƒ”‡ •Š‘™ ‹ ‹‰ ͵Ǥ – Ž‘™ ’ƒŽ‹–‘›ŽǦ‘ 2 ‘ ‡–”ƒ–‹‘• ͳ ˆ—ŽŽ› ‘–”‘ŽŽ‡† –Š‡ ˆŽ—šǡ ƒ• ™ƒ• ƒ’’ƒ”‡– ˆ”‘ ‹–• ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡– ‡“—ƒŽ –‘ ͳǡ ™Š‹Ž‡ ƒŽŽ ‘–Š‡” ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–• ™‡”‡ ‡‰Ž‹‰‹„Ž‡ȋ ‹‰ ͵ ƒ† ʹȌǤ–Š‹‰Š‡”’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘•ǡŠ‘™‡˜‡”ǡ–Š‡ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–‘ˆͳ „‡ ƒ‡ •–”‘‰Ž› ‡‰ƒ–‹˜‡ǡ ‹†‡‡† ‹†‹ ƒ–‹‰ –Šƒ– ƒ •–‹—Žƒ–‹‘ ‘ˆ ‹–• ƒ–ƒŽ›–‹  ƒ’ƒ ‹–› ”‡†— ‡†–Š‡ˆŽ—š•–‡‡’Ž›Ǥ ‘”†‹‰–‘–Š‡•—ƒ–‹‘–Š‡‘”‡ȏͳͳǡͳͶȐ–Š‡•—‘ˆƒŽŽˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–•‹•‡“—ƒŽ–‘ͳǡ‹’Ž›‹‰–Šƒ––Š‡‡‰ƒ–‹˜‡ ‘–”‘Ž‡š‡”–‡†„›ͳ•Š‘—Ž† „‡ ‘’‡•ƒ–‡†„›Š‹‰Š’‘•‹–‹˜‡ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–•‘ˆ‘‡‘”‘”‡‘–Š‡”‡œ›‡•ǤŠ‡ ƒŒ‘”•Šƒ”‡‘ˆ–Š‹•’‘•‹–‹˜‡ˆŽ—š ‘–”‘Ž™ƒ•ƒ†‘’–‡†„›ǡƒ†ƒ‹‘”’ƒ”–„› ƒ†  ȋ ‹‰ ͵ ƒ† ʹȌǤ ‹••‡ –‹‰ –Š‡ ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡– ‘ˆ  ‹ –Šƒ– ‘ˆ ‹†‹˜‹†—ƒŽǡ Šƒ‹ǦŽ‡‰–ŠǦ•’‡ ‹ˆ‹  ”‡ƒ –‹‘• •Š‘™‡† –Šƒ– ‘Ž› ‹–• Ͷ ƒ† ͸ ”‡ƒ –‹‘• ‘–”‹„—–‡† •—„•–ƒ–‹ƒŽŽ› ȋˆ‹‰—”‡  ‹ ʹ ‹‰Ȍǡ ƒ‰ƒ‹ †‡‘•–”ƒ–‹‰ ƒ ’‹˜‘–ƒŽ ”‘Ž‡ ‘ˆ –Š‡ •Š‘”–Ǧ Šƒ‹ „”ƒ Š ‘ˆ –Š‡ ’ƒ–Š™ƒ›Ǥ –Š‡” ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–• ™‡”‡ •ƒŽŽ —†‡” ƒŽŽ ‘†‹–‹‘•ȋˆ‹‰—”‡‹ʹ ‹‰ȌǤ  The accumulation of intermediates is amplified by the short-chain branch ‡ –Š‡ ™‘†‡”‡† ™Š‡–Š‡” ‘–Š‡” ”‡ƒ –‹‘•ǡ „‡•‹†‡• ǡ ͳǡ ǡ ƒ†  ‘†—Žƒ–‡† –Š‡ ’Š‡‘–›’‡Ǥ Š‡”‡ˆ‘”‡ǡ ™‡ “—ƒ–‹ˆ‹‡† –Š‡ ˆŽ—š ”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–• ˆ‘” ƒŽŽ ’ƒ”ƒ‡–‡”•p‹–Š‡‘†‡Žƒ––™‘ ›–‘•‘Ž‹ ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘•ǣ„‡Ž‘™–Š‡ ”‹–‹ ƒŽ ‘ ‡–”ƒ–‹‘ˆ‘”ˆŽ—š†‡ Ž‹‡ȋʹͷɊȌƒ†„‡›‘†–Š‹• ”‹–‹ ƒŽ ‘ ‡–”ƒ–‹‘™Š‡–Š‡ˆŽ—š ௃ ‹•„‡Ž‘™‹–•ƒš‹— ƒ’ƒ ‹–›ȋ͸ͲɊȌǤŠ‡ˆŽ—š”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–ܴ௣ȏͳͷȐ“—ƒ–‹ˆ‹‡•–Š‡ ”‡Žƒ–‹˜‡‡ˆˆ‡ –‘ˆƒ•ƒŽŽ Šƒ‰‡‘ˆƒ’ƒ”ƒ‡–‡”p‘–Š‡•–‡ƒ†›Ǧ•–ƒ–‡ˆŽ—šƒ ‘”†‹‰–‘ǣ ௃ ܴ௣ ൌ  ݈݀݊ܬΤ ݈݀݊݌ ȋ͵Ȍ ‹‰ ͵ ƒ† ͵ •Š‘™ –Š‡ –‘’ ͳͷ ’ƒ”ƒ‡–‡”• ™‹–Š –Š‡ Š‹‰Š‡•– ƒ„•‘Ž—–‡ ˆŽ—š ”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–•ƒ–ʹͷɊƒ†͸ͲɊ‘ˆ’ƒŽ‹–‘›ŽǦ‘ǡ”‡•’‡ –‹˜‡Ž›Ǥ ƒ‰”‡‡‡–™‹–Š–Š‡Š‹‰Š ˆŽ—š ‘–”‘Ž‘ˆͳǡ–Š‡‡‹‰Š–Š‹‰Š‡•–”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–•ƒ–ʹͷρ ‘’”‹•‡†‡š Ž—•‹˜‡Ž› ’ƒ”ƒ‡–‡”• –Šƒ– ƒˆˆ‡ – –Š‡ ”ƒ–‡ ‘ˆ ͳǡ ‹ Ž—†‹‰ ‹–• ȋˆ‹š‡†Ȍ •—„•–”ƒ–‡Ǧ ƒ† ’”‘†— –

‘ ‡–”ƒ–‹‘• ȋƒ”ǡ ‘ǡ ͳ͸ ›Ž‘Ȍǡ ƒ–ƒŽ›–‹  ƒ’ƒ ‹–› ȋ•ˆͳǡͳ͸ƒ† ’–ͳȌǡ ƒ†

ƒˆˆ‹‹–› ‘•–ƒ–• ȋǡͳǡƒ”ǡ ǡͳǡ‘ǡ ǡͳǡͳ͸ ›Ž‘Ȍ ȋ ‹‰ ͵Ȍǡ ƒ† –Š‡ ‡š– ”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–• ™‡”‡ ƒŽ”‡ƒ†› –‘‘ Ž‘™ –‘ ˜‹•—ƒŽ‹œ‡Ǥ – ͸Ͳ ρǡ Š‘™‡˜‡”ǡ ƒ–‹ ‹’ƒ–‡†

’ƒ”ƒ‡–‡”•”‡Žƒ–‹‰–‘ƒ’’‡ƒ”‡†ȋ‹Ǥ‡ǤͶǡ•ˆǡͶǡǡ ƒ–ǡͶǦ‡–‘ƒ ›ŽǦ‘ǡǡ ƒ–ǡͶǦ Ϊ ƒ ›ŽǦ‘ƒ†ǡ‘ǡǡƒ••Š‘™„›–Š‡’—”’Ž‡„ƒ”•ȌǤ ƒ††‹–‹‘ǡ–Š‡ȏ ȐȀȏ Ȑ”ƒ–‹‘‹ –Š‡ ‹–‘ Š‘†”‹ƒŽ ƒ–”‹š ƒ† –Š‡ ‡“—‹Ž‹„”‹— ‘•–ƒ– ‘ˆ Ȁ  ȋ•Š‘™ „› –Š‡ ’‹ „ƒ”•Ȍ ƒ†  ȋ•Š‘™ „› –Š‡ Ž‹‰Š– „Ž—‡ „ƒ”Ȍ ™‡”‡ ‹ –Š‡ –‘’ Ͷ ‘ˆ Š‹‰Š‡•– ”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–•ȋ ‹‰͵ȌǤ ‹ ‡ Ϊ ‹• ƒ •—„•–”ƒ–‡ ‘ˆ Ȁ ǡ ‹– •‡‡• Ž‹‡Ž› –Šƒ– –Š‡ Š‹‰Š ˆŽ—š ”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–• ‘ˆ ȏΪȐȀȏ Ȑ ƒ† –Š‡ ‡“—‹Ž‹„”‹— ‘•–ƒ– ƒ”‡ —–—ƒŽŽ› ”‡Žƒ–‡†Ǥ ‡ –Š‡”‡ˆ‘”‡•‡–‘—––‘‡Ž— ‹†ƒ–‡–Š‡‡ Šƒ‹•˜‹ƒ™Š‹ Š–Š‡›ƒˆˆ‡ –‡†–Š‡ˆŽ—šǤŠ‡”‘Ž‡‘ˆ Ϊ‹• ‘’Ž‡šǡ•‹ ‡‹–‹•ƒŽ•‘ƒ•—„•–”ƒ–‡ˆ‘”–Š‡”‡ƒ –‹‘ƒ†„‘–ŠȀ ƒ†  ƒ–ƒŽ›œ‡”‡ƒ –‹‘•‘ˆ•—„•–”ƒ–‡•™‹–Šƒ”ƒ‰‡‘ˆˆƒ––›Ǧƒ ‹† Šƒ‹Ž‡‰–Š•ǤŠ‡”‡ˆ‘”‡ǡ™‡

Ͷ͹ Chapter 2

†‹••‡ –‡†–Š‡”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–‘ˆȏΪȐȀȏ Ȑ‹–‘ ‘–”‹„—–‹‘•‘ˆƒŽŽ–Š‡†‹ˆˆ‡”‡– ”‡ƒ –‹‘•–Šƒ–ƒ”‡‹ˆŽ—‡ ‡†„›–Š‹•”ƒ–‹‘ǡ„ƒ•‡†‘–Š‡ ‘„‹‡†”‡•’‘•‡–Š‡‘”‡ȏͳͷȐǣ

௃ ௃ ௩೔ ܴሾே஺஽శሿȀሾே஺஽ுሿ ൌσ௜ ܥ௜ ήߝሾே஺஽శሿȀሾே஺஽ுሿ ȋͶȌ

௜ ‹™Š‹ Š–Š‡•—ƒ–‹‘‹•‘˜‡”ƒŽŽ”‡ƒ –‹‘•iǡƒ†ߝே஺஽ାȀே஺஽ு†‡‘–‡•–Š‡‡Žƒ•–‹ ‹–›ȋ‹Ǥ‡Ǥ–Š‡ †‹”‡ –•‡•‹–‹˜‹–›Ȍ‘ˆ–Š‡”ƒ–‡‘ˆ”‡ƒ –‹‘i–‘™ƒ”†•–Š‡ΪǦ ”ƒ–‹‘ǡƒ ‘”†‹‰–‘ǣ ௩೔ ା ሿሻ ȋͷȌܪܦܣሿΤ ሾܰ ܦܣߝሾே஺஽శሿΤ ሾே஺஽ுሿ ൌ  ߲݈݊ݒ௜Τ߲Žሺሾܰ ௃ ௩೔ Š‡†‹••‡ –‹‘‘ˆ“ǤͶ‹‹–•”‡ƒ –‹‘Ǧ•’‡ ‹ˆ‹ –‡”•ܥ௜ ήߝሾே஺஽శሿȀሾே஺஽ுሿȋ ‹‰͵Ȍ”‡˜‡ƒŽ‡†–™‘ Žƒ”‰‡ƒ†’‘•‹–‹˜‡–‡”•ǡ”‡’”‡•‡–‹‰–Š‡Ͷƒ†͸”‡ƒ –‹‘• ƒ–ƒŽ›œ‡†„›Ȁ ǤŠ‡ –‡”•”‡’”‡•‡–‹‰™‡”‡•ƒŽŽƒ†‡‰ƒ–‹˜‡Ǥ‡ ‘ Ž—†‡ˆ”‘–Š‹•–Šƒ––Š‡ ’‘•‹–‹˜‡ ‡ˆˆ‡ – ‘ˆ ȏΪȐȀȏ Ȑ ‘ –Š‡ ˆŽ—š ƒ „‡ ƒ––”‹„—–‡† ‡š Ž—•‹˜‡Ž› –‘ –Š‡ •Š‘”–Ǧ Šƒ‹ ”‡ƒ –‹‘•ȋͶƒ†͸Ȍ‘ˆȀ ǡ™Š‹Ž‡‡˜‡‡š‡”–‡†ƒ•ƒŽŽ‡‰ƒ–‹˜‡ ‘–”‹„—–‹‘Ǥ Š‡ Ȁ  ”‡ƒ –‹‘ ‹• Ž‘•‡ –‘ –Š‡”‘†›ƒ‹  ‡“—‹Ž‹„”‹— ™Š‡ –Š‡ ’ƒ–Š™ƒ› ‹• ƒ– •–‡ƒ†›•–ƒ–‡ȋ ˆǤ–Š‡•‹‹Žƒ”•Šƒ’‡•‘ˆȀ •—„•–”ƒ–‡•ƒ†’”‘†— –•‹ˆ‹‰—”‡ƒ†‹ ͵ ‹‰ǡƒ•‘’’‘•‡†–‘–Š‘•‡‘ˆ•—„•–”ƒ–‡•ƒ†’”‘†— –•‹ˆ‹‰—”‡ƒ†‹͵ ‹‰ȌǤ ‹ ”‡ƒ•‡‘ˆ–Š‡ȏΪȐȀȏ Ȑ”ƒ–‹‘•Š‹ˆ–•–Š‡‡“—‹Ž‹„”‹—‘ˆ–Š‡”‡ƒ –‹‘‹–Š‡ˆ‘”™ƒ”† †‹”‡ –‹‘ǡ‡š’Žƒ‹‹‰™Š›‹–Šƒ•–Š‡•ƒ‡‡ˆˆ‡ –ƒ•ƒ Šƒ‰‡‘ˆ–Š‡‡“—‹Ž‹„”‹— ‘•–ƒ–ǤŠ‡ ”‡ƒ•‘–Šƒ––Š‡‡“—‹Ž‹„”‹— ‘•–ƒ–•ƒ”‡•‘‹’‘”–ƒ–ˆ‘”–Š‡ˆŽ—š‹•–Šƒ––Š‡‡“—‹Ž‹„”‹— ‘•–ƒ–‘ˆȀ ‹•‡š–”‡‡Ž›—ˆƒ˜‘—”ƒ„Ž‡ƒ–ʹǤͳ͹ȉͳͲǦͶȋ†‹‡•‹‘Ž‡••ǡ ˆǤ ‹‰ͳȌ‹–Š‡ ˆ‘”™ƒ”††‹”‡ –‹‘Ǥ•ƒŽŽƒ ——Žƒ–‹‘‘ˆͶǦƒ†͸‡–‘ƒ ›ŽǦ‘ǡ–Š‡†‹”‡ –•—„•–”ƒ–‡•‘ˆ ǡ™‹ŽŽ–Š‡”‡ˆ‘”‡‡–”ƒ‹–Š‡Š›†”‘š›ƒ ›ŽǦ‘•—„•–”ƒ–‡•‘ˆȀ –‘ƒ ——Žƒ–‡ •‡˜‡”ƒŽ‘”†‡”•‘ˆƒ‰‹–—†‡‘”‡ǡ–Š‡‡šƒ –”ƒ–‹‘†‡’‡†‹‰‘–Š‡ȏΪȐȀȏ Ȑ”ƒ–‹‘ ȋ ‹‰—”‡‹͵ ‹‰ȌǤŠ‡‡“—‹Ž‹„”‹— ‘•–ƒ–‘ˆ‹•͵Ǥͳ͵ǡ–Š—•‹†— ‹‰ƒ‡‘›ŽǦ‘ ƒ ——Žƒ–‹‘‘ˆƒŽ‘•––Š‡•ƒ‡‘”†‡”‘ˆƒ‰‹–—†‡ȋ ‹‰—”‡‹͵ ‹‰ȌǤŠ—•ǡȀ  ƒ†  ƒ’Ž‹ˆ› –Š‡ ƒ ——Žƒ–‹‘ ‘ˆ ‘ ‡•–‡”• ‡‘”‘—•Ž›ǡ –Š‡”‡„› †”ƒ‹‹‰ –Š‡ ˆ”‡‡ ‘’‘‘ŽǤ

‹ƒŽŽ›ǡ‹ ‹‰͵‘Ž›’ƒ”ƒ‡–‡”•”‡Žƒ–‡†–‘–Š‡Ͷ”‡ƒ –‹‘ƒ’’‡ƒ”ȋ‹Ǥ‡Ǥ•ˆǡͶǡ

ǡ ƒ–ǡͶǦ‡–‘ƒ ›ŽǦ‘Ȍǡ „—– Šƒ”†Ž› ƒ› ’ƒ”ƒ‡–‡”• ”‡Žƒ–‡† –‘ –Š‡ ͸ ”‡ƒ –‹‘ǡ ‡š ‡’– ˆ‘” –Š‡

ƒˆˆ‹‹–›–‘™ƒ”†‹–•’”‘†— –ͶǦƒ ›ŽǦ‘ ȋ‹Ǥ‡Ǥǡ ƒ–ǡͶǦƒ ›ŽǦ‘Ȍǡ•‹ ‡–Š‡Žƒ––‡” Šƒ˜‡Ž‘™‡” ”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–•Ǥ Š‹• ƒ› „‡ ƒ––”‹„—–‡† –‘ †‹ˆˆ‡”‡ ‡• ‹ –Š‡ ƒ–ƒŽ›–‹  ‘•–ƒ–• „‡–™‡‡ –Š‡ Ͷ ƒ† ͸ ”‡ƒ –‹‘•ǡ ‘–ƒ„Ž› ȋ‹Ȍ –Š‡ ͶǦ•’‡ ‹ˆ‹   ƒ–ƒŽ›–‹  ƒ’ƒ ‹–›

ȋ•ˆͶǡƒ Šƒ‹ǦŽ‡‰–ŠǦ•’‡ ‹ˆ‹ ‘†‹ˆ›‹‰ˆƒ –‘”ˆ‘”ƒšȌ„‡‹‰ʹǦˆ‘Ž†Ž‘™‡”–Šƒ‹–•͸Ǧ

•’‡ ‹ˆ‹  ‘—–‡”’ƒ”– ȋ•ˆ͸Ȍ ȋͲǤͶͻ ˜‡”•—•ͳȌ ƒ† ȋ‹‹Ȍ –Š‡ ƒŽ‘•– ʹǦˆ‘Ž† Ž‘™‡” ƒˆˆ‹‹–› ‘ˆ ˆ‘”‹–•ͶǦ‡–‘ƒ ›ŽǦ‘•—„•–”ƒ–‡–Šƒˆ‘”‹–•͸Ǧ‡–‘ƒ ›ŽǦ‘•—„•–”ƒ–‡ǡ“—ƒ–‹ˆ‹‡†

„›ƒʹǦˆ‘Ž†Š‹‰Š‡” ‘””‡•’‘†‹‰‹ Šƒ‡Ž‹•Ǧ‡–‡ ‘•–ƒ–ȋȌȋͳʹǤͶ˜‡”•—•͸Ǥ͹ɊȌǤ  Internal regulation reveals a vicious cycle around MCKAT ‘ —†‡”•–ƒ† –Š‡ •‡“—‡ ‡ ‘ˆ ‡˜‡–• Ž‡ƒ†‹‰ –‘ ˆŽ—š †‡ Ž‹‡ ƒ† –Š‡ ’‘–‡–‹ƒŽ ”‘Ž‡ ‘ˆ ‘’‡–‹–‹˜‡‹Š‹„‹–‹‘–Š‡”‡‹ǡ™‡‹•’‡ –‡†–‹‡ ‘—”•‡•‘ˆ‡–ƒ„‘Ž‹–‡ ‘ ‡–”ƒ–‹‘•ƒ† ‡œ›‡ ”ƒ–‡•Ǥ –ƒ”–‹‰ ˆ”‘ ƒ •–‡ƒ†› •–ƒ–‡ ƒ– ͲǤͳ ρǡ –Š‡ ‘ ‡–”ƒ–‹‘ ‘ˆ –Š‡ •—„•–”ƒ–‡ ’ƒŽ‹–‘›ŽǦ‘ ™ƒ• ‹ ”‡ƒ•‡† ƒ– –‹‡ ’‘‹– œ‡”‘ –‘ ʹͷ ‘” ͸Ͳ ρ ƒ† –Š‡ ”‡•’‘•‡ ‘ˆ –Š‡ ’ƒ–Š™ƒ›™ƒ•ˆ‘ŽŽ‘™‡†—–‹Ž–Š‡•›•–‡”‡Žƒš‡†‹–‘ƒ‡™•–‡ƒ†›•–ƒ–‡Ǥ–„‘–Š•—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘•‡–ƒ„‘Ž‹–‡•ƒ ——Žƒ–‡†‘”†‡ Ž‹‡†‰”ƒ†—ƒŽŽ›ǡ ™‹–ŠŠƒ”†Ž›ƒ›†‡–‡ –ƒ„Ž‡

Ͷͺ The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation

‘˜‡”Ǧ‘”—†‡”•Š‘‘–•ȋ ‹‰—”‡‹Ͷ ‹‰ȌǤ–ƒŽ‘™ ‘ ‡–”ƒ–‹‘‘ˆ’ƒŽ‹–‘›ŽǦ‘ȋʹͷρȌ –Š‡ ”ƒ–‡• ‘ˆ ƒŽŽ ‡œ›‡• ƒŽ•‘ ‹ ”‡ƒ•‡† ‰”ƒ†—ƒŽŽ› ‹ –‹‡ ȋ ‹‰—”‡ ‹ ͷ ‹‰ȌǤ – ͸Ͳ ρǡ Š‘™‡˜‡”ǡ–Š‡’ƒ––‡”™ƒ• ‘•’‹ —‘—•Ž›†‹ˆˆ‡”‡–ǡƒ•‡š‡’Ž‹ˆ‹‡†„›‡œ›‡”ƒ–‡•ƒ”‘—† –Š‡Ͷ‡–ƒ„‘Ž‹–‡•ȋ ‹‰ͶȌǤ 2 AB 1.5 0.01 C16-Ketoacyl-CoA ) ) vMCKATC6 C14-Ketoacyl-CoA í -1 vMCADC4 + C12-Ketoacyl-CoA 1.4 vSCADC4 C10-Ketoacyl-CoA C8-Ketoacyl-CoA vMCKATC4 C6-Ketoacyl-CoA 1.3

.gProtein C4-Ketoacyl-CoA .mgProtein -1

2 0 C16-Acyl-CoA í j C14-Acyl-CoA 1.2 vmckatC4 X

Ĭ C12-Acyl-CoA C10-Acyl-CoA 1.1 C8-Acyl-CoA C6-Acyl-CoA (μmol.min v (μmol.min C4-Acyl-CoA 0.0 -0.01 Free CoA 0 100 200 300 0 100 200 300 time (min) time (min)  Fig 4. Time course of key short-chain reaction rates and regulation of the MCKAT-C4 reaction. (A) ‹‡ ‘—”•‡ ‘ˆ –Š‡ ”‡ƒ –‹‘ ”ƒ–‡• ‘ˆ  ˆ‘” ‹–• ͸ ƒ† Ͷ •—„•–”ƒ–‡• ȋ˜͸ ƒ† ˜Ͷǡ ”‡•’‡ –‹˜‡Ž›Ȍ ƒ† ‘ˆ –Š‡ •—‡† ƒ –‹˜‹–‹‡• ‘ˆ  ƒ†  ‘ –Š‡‹” Ͷ •—„•–”ƒ–‡ ȋ˜Ͷ Ϊ ˜ͶȌƒˆ–‡”ƒ•—††‡—’•Š‹ˆ–‘ˆȏ’ƒŽ‹–‘›ŽǦ‘Ȑˆ”‘ͲǤͳ–‘͸ͲɊǤ(B) ‹‡ ‘—”•‡‘ˆ”‡‰—Žƒ–‘”› ‡–ƒ„‘Ž‹–‡ ‘–”‹„—–‹‘•–‘˜Ͷƒˆ–‡”ƒ•—††‡ȏ’ƒŽ‹–‘›ŽǦ‘Ȑ‹ ”‡ƒ•‡ˆ”‘ͲǤͳ–‘͸ͲɊǤ   Š‡”ƒ–‡‘ˆ‘‹–•ͶǦ‡–‘ƒ ›ŽǦ‘•—„•–”ƒ–‡Žƒ‰‰‡†•—„•–ƒ–‹ƒŽŽ›„‡Š‹†–Š‡”ƒ–‡ƒ– ™Š‹ Š –Š‡ ͶǦƒ ›ŽǦ‘ ‡–‡”‡† –Š‡ •Š‘”–Ǧ Šƒ‹ „”ƒ Š ˜‹ƒ  ƒ† Ǥ ‘–Š ”ƒ–‡• ‹ ”‡ƒ•‡† ‹ –‹‡ ƒ• –Š‡‹” •—„•–”ƒ–‡ ’‘‘Ž• ˆ‹ŽŽ‡†ǡ —–‹Ž ƒ– –‹‡ ’‘‹– ʹͺ ‹ǡ ™Š‡ –Š‡› •–ƒ”–‡†–‘†‡ Ž‹‡ǤŠ‡•ƒ‡Šƒ’’‡‡†ƒ––Š‡Ž‡˜‡Ž‘ˆ–Š‡͸”‡ƒ –‹‘•‘ˆȀƒ† ǡƒŽ„‡‹––‘ƒŽ‡••‡”‡š–‡–ȋ ‹‰—”‡‹͸ ‹‰ȌǤŠ‹••—‰‰‡•–‡†–Šƒ–‘‡‘”‘”‡‘ˆ–Š‡ ‡–ƒ„‘Ž‹–‡•‹Š‹„‹–‡†–Š‡•‡‡œ›‡•‘”„‡ ƒ‡Ž‹‹–‹‰Ǥ ‡ ™‘†‡”‡† ™Š‹ Š ‡–ƒ„‘Ž‹–‡• ƒ—•‡† –Š‡ ‡œ›‡ ”ƒ–‡• –‘ †‡ Ž‹‡Ǥ ‹ ‡ ‡ƒ Š ”‡ƒ –‹‘ ”ƒ–‡†‡’‡†•‘–Š‡ ‘ ‡–”ƒ–‹‘•‘ˆ—Ž–‹’Ž‡’”‹ƒ”›•—„•–”ƒ–‡•ƒ†’”‘†— –•ƒ•™‡ŽŽƒ• ‘’‡–‹‰‡–ƒ„‘Ž‹–‡•‘ˆ‘–Š‡” Šƒ‹Ž‡‰–Š•ǡ–Š‡ ‘–”‹„—–‹‘‘ˆ‡ƒ Š‹†‹˜‹†—ƒŽ‡–ƒ„‘Ž‹–‡ –‘–Š‡”‡‰—Žƒ–‹‘‘ˆ–Š‡”‡ƒ –‹‘”ƒ–‡•™ƒ•“—ƒ–‹ˆ‹‡†ǡƒ ‘”†‹‰–‘–Š‡‡–Š‘†‘ˆ”—‰‰‡ƒ ‡–ƒŽǤʹͲͲͷȏͳʹȐǤŠ‡ ‘ ‡’–„‡Š‹†–Š‹•ƒƒŽ›•‹•‹•–Šƒ–ƒ Šƒ‰‡‹‡œ›‡”ƒ–‡v‹•–Š‡ ƒ–Š‡ƒ–‹ ƒŽ ’”‘†— – ‘ˆ ƒ Šƒ‰‡ ‹ ƒ ‡–ƒ„‘Ž‹–‡ ‘ ‡–”ƒ–‹‘ Xj —Ž–‹’Ž‹‡† „› –Š‡ ‡Žƒ•–‹ ‹–› ‘ˆ –Š‡ ‡œ›‡ ”ƒ–‡ –‘ –Š‹• ‡–ƒ„‘Ž‹–‡ ‘ ‡–”ƒ–‹‘ǡ ƒ† •—‡† ‘˜‡” ƒŽŽ ‡–ƒ„‘Ž‹–‡•j–Šƒ–ƒˆˆ‡ ––Š‡‡œ›‡ǣ ௗ௟௡௩ డ௟௡௩ ௗ௟௡௑ೕ ௩ ሺݐሻ ൌ σ௝ ሺݐሻ ή ሺݐሻ ؠ σ௝ ȣ௑ ሺݐሻ ȋ͸ƒȌ ௗ௧ డ௟௡௑ೕ ௗ௧ ೕ

௩ డ௟௡௩ ௗ௟௡௑ೕ ǣ ȣ௑ ሺݐሻ ؠ ሺݐሻ ή ሺݐሻ ȋ͸„ȌŠ–‹™ ೕ డ௟௡௑ೕ ௗ௧ –‡‰”ƒ–‹‘ ‘˜‡” –Š‡ ‡–‹”‡ ”‡Žƒšƒ–‹‘ –‹‡ ˆ”‘ ‘‡ •–‡ƒ†› •–ƒ–‡ –‘ ƒ‘–Š‡” ƒŽŽ‘™• –Š‡

†‹••‡ –‹‘ ‘ˆ –Š‡ ‘˜‡”ƒŽŽ ”‡‰—Žƒ–‹‘ ‘ˆ –Š‡ ”ƒ–‡ ‹–‘ ƒ˜‡”ƒ‰‡ ‘–”‹„—–‹‘• 4തതതఫ ‘ˆ ‡ƒ Š ଵ ௧ ௗ௟௡௩ ଵ ௧ డ௟௡௩ ௗ௟௡௑ೕ Xjǣ ׬ ሺ߬ሻ݀߬ ൌ σ௝ ׬ ሺ߬ሻ ή ሺ߬ሻ݀߬ ൌ‡–‹Ž‘„ƒ–‡Žƒ—†‹˜‹†‹ ௧ ଴ ௗ௧ ௧ ଴ డ௟௡௑ೕ ௗ௧ ଵ ௧         σ ׬ ȣ௩ ሺ߬ሻ݀߬ ൌ σ ȣഥ௩ ௝ ௧ ଴ ௑ೕ ௝ ௑ೕ ȋ͹Ȍ Š‡ƒ˜‡”ƒ‰‡ƒ„•‘Ž—–‡ ‘–”‹„—–‹‘‘ˆ‡ƒ Š‡–ƒ„‘Ž‹–‡‹•‰‹˜‡„›ǣ

Ͷͻ Chapter 2

ଵ ௧ ȁȣഥȁ௩ ൌ ׬ ቚȣ௩ ሺ߬ሻቚ ݀߬ ȋͺȌ ௑ೕ ௧ ଴ ௑ೕ ௩ ‘˜‡”•‹‘ ‘ˆ ͶǦ ‡Š– – ‡ˆˆƒ –ƒŠ– •‡–‹Ž‘„ƒ–‡ ŽŽƒ ˆ‘ ‘–”‹„—–‹‘ȣ௑ೕሺݐሻ ‡Š– •™‘Š• Ͷ ‰‹ ‡–‘ƒ ›ŽǦ‘„›’Ž‘––‡†ƒ•ƒˆ— –‹‘‘ˆ–‹‡ˆ‘”ƒ–”ƒ•‹–‹‘ˆ”‘ͲǤͳ–‘͸Ͳρ‘ˆ ’ƒŽ‹–‘›ŽǦ‘ǡ ƒŽ —Žƒ–‡† ƒ ‘”†‹‰ –‘ “Ǥ ͸„Ǥ Š‡ ƒ„•‘Ž—–‡ ƒ”‡ƒ —†‡” ‡ƒ Š —”˜‡ ‹• ഥ ௩ெ஼௄஺்ି஼ସ j ȁȣȁ௑ೕ ƒ††‡‘–‡•–Š‡ƒ˜‡”ƒ‰‡ƒ„•‘Ž—–‡ ‘–”‹„—–‹‘‘ˆƒ•’‡ ‹ˆ‹ ‡–ƒ„‘Ž‹–‡ –‘–Š‡ Šƒ‰‡‘ˆ–Š‡”ƒ–‡‘ˆ–Š‡Ͷ”‡ƒ –‹‘‘ˆ†—”‹‰–Š‡”‡Žƒšƒ–‹‘–‘–Š‡‡™•–‡ƒ†›•–ƒ–‡Ǥ  Žƒ”‰‡ ’‘•‹–‹˜‡ ‘–”‹„—–‹‘ ‹• ƒ†‡ „› –Š‡ •—„•–”ƒ–‡ ͶǦ‡–‘ƒ ›ŽǦ‘Ǥ ‡‰ƒ–‹˜‡ ‘–”‹„—–‹‘•™‡”‡ƒ†‡„›ƒ ›ŽǦ‘‡•–‡”•‘ˆ˜ƒ”‹‘—• Šƒ‹Ž‡‰–Š•ǡ™Š‹ Šƒ”‡–Š‡’”‘†— –• ‘ˆƒŽŽ–Š‡‘–Š‡””‡ƒ –‹‘• ƒ–ƒŽ›œ‡†„›Ǥ‘–ƒ„Ž›ƒŽ•‘–Š‡ˆ”‡‡‘ ‘ ‡–”ƒ–‹‘ƒ†‡ ƒ •—„•–ƒ–‹ƒŽ ‡‰ƒ–‹˜‡ ‘–”‹„—–‹‘Ǥ ”‡‡ ‘ ‹• ƒ •—„•–”ƒ–‡ ‘ˆ  ƒ† –Š‡”‡ˆ‘”‡ ‹–• డ௟௡௩ ‡Žƒ•–‹ ‹–› ‘‡ˆˆ‹ ‹‡– ಾ಴಼ಲ೅ష಴ర‹•’‘•‹–‹˜‡ǡ„—–•‹ ‡–Š‡ ‘ ‡–”ƒ–‹‘†‡ ”‡ƒ•‡†™‹–Š–‹‡ǡ డ୪୬ሾ஼௢஺ሿ –Š‡‘˜‡”ƒŽŽ ‘–”‹„—–‹‘„‡ ƒ‡‡‰ƒ–‹˜‡Ǥ  ‹‰ ͷǦ  –Š‡•‡ ƒ˜‡”ƒ‰‡ ‘–”‹„—–‹‘• ™‡”‡ ’Ž‘––‡† ƒ• ƒ ˆ— –‹‘ ‘ˆ –Š‡ •—„•–”ƒ–‡ ’ƒŽ‹–‘›ŽǦ‘ˆ‘”ƒ–”ƒ•‹–‹‘ˆ”‘ͲǤͳρ–‘–Š‡‹†‹ ƒ–‡† ‘ ‡–”ƒ–‹‘‘ˆ’ƒŽ‹–‘›ŽǦ‘Ǥ ‡•–ƒ”–‡†ˆ”‘–Š‡”‡ƒ –‹‘•ˆ‘”‹–••—„•–”ƒ–‡•͸ƒ†ͶǦ‡–‘ƒ ›ŽǦ‘ǡƒ–™Š‹ Š –Š‡ˆŽ—š†‡ Ž‹‡ƒ’’‡ƒ”‡†–‘‘”‹‰‹ƒ–‡ȋ ‹‰ͷƒ†ȌǤ‹‡ˆ‘”–Š‡–”ƒ•‹–‹‘ˆ”‘ͲǤͳ–‘͸Ͳ ρǡƒŽ•‘ƒ–‘–Š‡”’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘•–Š‡”‡ƒ –‹‘™ƒ•‘•–Ž›”‡‰—Žƒ–‡† „›–Š‡‡–‘ƒ ›ŽǦ‘•—„•–”ƒ–‡‘ˆ–Š‡”‡ƒ –‹‘–Šƒ–‹•‹•’‡ –‡†ȋ‹Ǥ‡Ǥ–Š‡ͶǦ‡–‘ƒ ›ŽǦ‘ˆ‘” –Š‡ ͶǦ ”‡ƒ –‹‘ ƒ† –Š‡ ͸Ǧ‡–‘ƒ ›ŽǦ‘ ˆ‘” –Š‡ ͸Ǧ ”‡ƒ –‹‘Ȍǡ ‹–• ‘Ǧ •—„•–”ƒ–‡ ˆ”‡‡ ‘ǡ ƒ† ƒ ›ŽǦ‘ ‡•–‡”• ‘ˆ ˜ƒ”‹‘—• Šƒ‹ Ž‡‰–Š•ǡ ‹ Ž—†‹‰ –Š‡ ƒ ›ŽǦ‘ ’”‘†— –‘ˆ–Š‡”‡ƒ –‹‘‹–•‡ŽˆǤŠ‡‘Ž›‘˜‡”ƒŽŽ’‘•‹–‹˜‡ ‘–”‹„—–‹‘™ƒ•„›–Š‡‡–‘ƒ ›ŽǦ‘ •—„•–”ƒ–‡ǡ†—‡–‘–Š‡‹ ”‡ƒ•‡‘ˆ‹–• ‘ ‡–”ƒ–‹‘‹–‹‡ȋ ˆ ‹‰—”‡‹͹ȌǤŠ‡†‡ ”‡ƒ•‡‘ˆ –Š‡ ‘Ǧ•—„•–”ƒ–‡ ‘ ›‹‡Ž†‡† ƒ ‡‰ƒ–‹˜‡ ‘–”‹„—–‹‘Ǥ Š‡ ”‘Ž‡ ‘ˆ –Š‡ ƒ ›ŽǦ‘ ‡•–‡”• ‹• ’ƒ”ƒ†‘š‹ ƒŽǤŠ‡›ƒ”‡’”‘†— –•‘ˆƒ†–Š‡”‡ˆ‘”‡‹Š‹„‹––Š‡‡œ›‡ǡƒ• ƒ„‡•‡‡ ˆ”‘–Š‡‡‰ƒ–‹˜‡”‡‰—Žƒ–‘”› ‘–”‹„—–‹‘‘ˆ–Š‡ƒ ›ŽǦ‘‡•–‡”•‹ ‹‰ͶǤ––Š‡•ƒ‡–‹‡ –Š‡͸Ǧƒ†ͶǦƒ ›ŽǦ‘‡•–‡”•ƒ”‡•—„•–”ƒ–‡•ˆ‘”–Š‡•Š‘”–Ǧ Šƒ‹„”ƒ Šƒ†ƒ”‡ ‘˜‡”–‡† –‘ ͸Ǧ ƒ† ͶǦ‡–‘ƒ ›Ž •—„•–”ƒ–‡• ˆ‘” Ǥ › ‘„‹‹‰ ƒŽŽ †ƒ–ƒǡ ™‡ †‡†— ‡† –Š‡ ˆ‘ŽŽ‘™‹‰‡ Šƒ‹•ȋ•—ƒ”‹œ‡†‹ ‹‰͸ȌǤ—’•Š‹ˆ–‘ˆ’ƒŽ‹–‘›ŽǦ‘ˆ‹ŽŽ•–Š‡ƒ ›ŽǦ‘ ’‘‘Ž•ǡ™Š‹ Šƒ”‡ ‘˜‡”–‡†‹–‘‡‘›ŽǦ‘‡•–‡”•ƒ†Žƒ”‰‡Ž›ˆ‘ŽŽ‘™–Š‡”‘—–‡†‘™–‘ ͸Ǧƒ ›ŽǦ‘Ǥ͸Ǧƒ†ͶǦƒ ›ŽǦ‘–Š‡ˆ‘ŽŽ‘™–Š‡•Š‘”–Ǧ Šƒ‹„”ƒ Š’”‘˜‹†‹‰•—„•–”ƒ–‡–‘ ǡ™Š‹ ŠŠƒ•‹•—ˆˆ‹ ‹‡– ƒ’ƒ ‹–›–‘‡–ƒ„‘Ž‹•‡–Š‡ƒ––Š‡•ƒ‡”ƒ–‡ǤŠ‹•Ž‡ƒ†•–‘ƒ ƒ ——Žƒ–‹‘ ‘ˆ ǯ• •—„•–”ƒ–‡• ͶǦ ƒ† ͸Ǧ‡–‘ƒ ›ŽǦ‘Ǥ • ™‡ Šƒ˜‡ •‡‡ǡ –Š‡ ƒ„•‘Ž—–‡ ‘ ‡–”ƒ–‹‘•‘ˆ–Š‡‡–‘ƒ ›ŽǦ‘‡•–‡”•ƒ”‡•ƒŽŽǡ„—––Š‡‹”‡ˆˆ‡ –•ƒ”‡ƒ’Ž‹ˆ‹‡† „›‡“—‹Ž‹„”ƒ–‹‰™‹–ŠŠ›†”‘š›ƒ ›ŽǦ‘‡•–‡”•ƒ†‡‘›ŽǦ‘‡•–‡”•Ǥ Š‡•‡‹Š‹„‹––Š‡ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡•ƒ†–Š—•–Š‡—’•–”‡ƒƒ ›ŽǦ‘‡•–‡”••–ƒ”––‘ ƒ ——Žƒ–‡ǤŠ‡•‡ƒ ›ŽǦ‘‡•–‡”•ƒ”‡ƒ––Š‡•ƒ‡–‹‡’”‘†— –•‘ˆƒ†–Š‡”‡ˆ‘”‡ ‹Š‹„‹––Š‹•‡œ›‡ǤŠ‹•ƒ‰‰”ƒ˜ƒ–‡•–Š‡„‘––Ž‡‡ ƒ–Ǥ‘”‡‘˜‡”ǡ–Š‡ƒ ——Žƒ–‹‰ ‘‡•–‡”••‡“—‡•–‡”–Š‡ˆ”‡‡‘ƒ†–Š‡‘˜‡”ƒŽŽ”‡•—Ž–‹•ƒ†”ƒ•–‹ ”‡†— –‹‘‘ˆ–Š‡ˆ”‡‡‘ ’‘‘ŽǤ‹ ‡‘‹•ƒ ‘Ǧ•—„•–”ƒ–‡‘ˆǡ–Š‹•Ž‹‹–•–Š‡”‡ƒ –‹‘‡˜‡ˆ—”–Š‡”ƒ† –”ƒ’•–Š‡’ƒ–Š™ƒ›‹ƒ˜‹ ‹‘—• › Ž‡Ǥ –Š‡–‹‡ ‘—”•‡ȋ ‹‰ͶȌ–Š‡†‡ ”‡ƒ•‡‘ˆ–Š‡”ƒ–‡•‡–•‹™Š‡–Š‡‡‰ƒ–‹˜‡ ‘–”‹„—–‹‘• ‘ˆ‘†‡ Ž‹‡ƒ†ƒ ›ŽǦ‘ƒ ——Žƒ–‹‘•—”’ƒ••–Š‡’‘•‹–‹˜‡ ‘–”‹„—–‹‘‘ˆ–Š‡‹ ”‡ƒ•‡† ‡–‘ƒ ›ŽǦ‘•—„•–”ƒ–‡ȋ ‹‰ͶȌǤ‘•—’’‘”––Š‡‘–‹‘‘ˆ‹Š‹„‹–‹‘„›–Š‡ƒ ›ŽǦ‘

ͷͲ The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation

‡•–‡”•ǡ–Š‡‘ —’ƒ ›‘ˆ™‹–Š’”‘†— –•ƒ†•—„•–”ƒ–‡•‘ˆ†‹ˆˆ‡”‡– Šƒ‹ǦŽ‡‰–Š•™ƒ• ƒŽ —Žƒ–‡†ȋ ‹‰—”‡‹ͺ ‹‰ȌǤ †‡‡†ǡ–Š‡†‘‹ƒ–‘ —’ƒ–•‘ˆ–Š‡ƒ –‹˜‡•‹–‡™‡”‡ͶǦ ƒ† ͸Ǧƒ ›ŽǦ‘ǡ –‘‰‡–Š‡” ‘ —’›‹‰ ͸͸ǤͶ Ψ ‘ˆ –Š‡ ƒ˜ƒ‹Žƒ„Ž‡ ƒ –‹˜‡ •‹–‡• ƒ– ͸Ͳ ρ ‘ˆ ’ƒŽ‹–‘›ŽǦ‘ȋ ‹‰—”‡‹ͺ ‹‰ȌǤ 2  A B vMCKATC6 vMCKATC4 100% Free CoA Free CoA Free CoA C4-Acyl-CoA Free CoA C4-Acyl-CoA C6-Acyl-CoA C6-Acyl-CoA 75% C8-Acyl-CoA C8-Acyl-CoA C10-Acyl-CoA C10-Acyl-CoA C12-Acyl-CoA C12-Acyl-CoA Â

j C14-Acyl-CoA C14-Acyl-CoA j v X

| 50% C16-Acyl-CoA C16-Acyl-CoA v X | ഥ Ĭ

| C4-Ketoacyl-CoA C4-Ketoacyl-CoA ഥ Ĭ j | C6-Ketoacyl-CoA C6-Ketoacyl-CoA σ C6-ketoacyl-CoA C4-ketoacyl-CoA 25% C8-Ketoacyl-CoA C8-Ketoacyl-CoA () C10-Ketoacyl-CoA (substrate) C10-Ketoacyl-CoA C12-Ketoacyl-CoA C12-Ketoacyl-CoA C14-Ketoacyl-CoA C14-Ketoacyl-CoA 0% C16-Ketoacyl-CoA C16-Ketoacyl-CoA 0 50 100 150 200 250 0 50 100 150 200 250

[Palmitoyl-CoA]CYT ȝ0 [Palmitoyl-CoA]CYT ȝ0 C vMCADC6 D vSCADC4 100% C4-Enoyl-CoA C6-Enoyl-CoA C4-enoyl-CoA 75% C8-Enoyl-CoA C10-Enoyl-CoA Â j C12-Enoyl-CoA C4-Enoyl-CoA j v X

| 50% v X | ഥ Ĭ C4-Acyl-CoA C6-Enoyl-CoA | ഥ Ĭ j |

σ C4-Acyl-CoA C6-acyl-CoA C8-Acyl-CoA C4-acyl-CoA 25% (substrate) C10-Acyl-CoA (substrate) C6-Acyl-CoA C12-Acyl-CoA C6-Acyl-CoA 0% 0 50 100 150 200 250 0 50 100 150 200 250

[Palmitoyl-CoA]CYT ȝ0 [Palmitoyl-CoA]CYT ȝ0

E vMTPC8 F vCPT2C16 100% Free CoA Free CoA C4-Acyl-CoA C4-Ketoacyl-CoA Free CoA C6-Acyl-CoA Free CoA C6-Acyl-CoA 75% C8-Acyl-CoA C8-Acyl-CoA C10-Acyl-CoA

 C10-Acyl-CoA

j C12-Acyl-CoA j v X C12-Acyl-CoA | C14-Acyl-CoA v X | ഥ Ĭ 50% C14-Acyl-CoA | C16-Acyl-CoA ഥ Ĭ j | C16-Acyl-CoA C4-Acyl-carnitine σ C8-Enoyl-CoA C6-Acyl-carnitine 25% C8-enoyl-CoA C10-Enoyl-CoA C16-acyl-carnitine C8-Acyl-carnitine C10-Acyl-carnitine (substrate) C12-Enoyl-CoA (substrate) C14-Enoyl-CoA C12-Acyl-carnitine C16-Enoyl-CoA C14-Acyl-carnitine 0% C16-Acyl-carnitine 0 50 100 150 200 250 0 50 100 150 200 250

[Palmitoyl-CoA]CYT ȝ0 [Palmitoyl-CoA]CYT ȝ0  Fig 5.Regulation of key reactions by their substrates and products. (A-F) ‡Žƒ–‹˜‡ƒ˜‡”ƒ‰‡ƒ„•‘Ž—–‡ ࢜ ȁࢨഥȁࢄ ‘–”‹„—–‹‘ǡ‹Ǥ‡Ǥ ࢐ ή ૚૙૙Ψ,‘ˆ‡–ƒ„‘Ž‹–‡•–‘–Š‡–”ƒ•‹–‹‘ˆ”‘–Š‡•–‡ƒ†›•–ƒ–‡ƒ–ͲǤͳɊ–‘–Š‡ σ ȁࢨഥȁ࢜ ࢐ ࢄ࢐ ‹†‹ ƒ–‡† ‘ ‡–”ƒ–‹‘‘ˆ’ƒŽ‹–‘›ŽǦ‘ǡ ƒŽ —Žƒ–‡†ˆ‘”˜͸ȋ’ƒ‡ŽȌǡ˜Ͷȋ’ƒ‡ŽȌǡ ˜͸ȋ’ƒ‡ŽȌǡ˜Ͷȋ’ƒ‡ŽȌǡ˜ͺȋ’ƒ‡ŽȌǡ˜ʹͳ͸ȋ’ƒ‡Ž Ȍ.  Ž„‡‹– ‹†‹˜‹†—ƒŽŽ› Ž‡•• ‹’‘”–ƒ–ǡ ‘ŽŽ‡ –‹˜‡Ž› –Š‡ ‘–Š‡” ƒ ›ŽǦ‘ǯ• ‘ —’‹‡† ͵ͲǤͶΨ‘ˆ –Š‡ ƒ –‹˜‡•‹–‡•—†‡”–Š‡•ƒ‡ ‘†‹–‹‘Ǥ––Šƒ–’‘‹–ǡ–Š‡‡–‘ƒ ›ŽǦ‘•—„•–”ƒ–‡•‘ —’‹‡† ‘Ž›ͳǤ͸ΨƒŽ–‘‰‡–Š‡”Ǥ ‹ƒŽŽ›ǡ ™‡ ™‘†‡”‡† Š‘™ –Š‡ Ž‘‰‰‹‰ ‘ˆ ‡–ƒ„‘Ž‹–‡• ‹ –Š‡ •Š‘”–Ǧ Šƒ‹ „”ƒ Š ™ƒ• –”ƒ•‹––‡†–Š”‘—‰Š–Š‡‡–™‘”–‘‡˜‡–—ƒŽŽ›•Ž‘™†‘™–Š‡‡–‹”‡’ƒ–Š™ƒ›Ǥ‘–Š‹•‡† –Š‡‹–‡”ƒŽ‡–ƒ„‘Ž‹ ”‡‰—Žƒ–‹‘‘ˆǡǡƒ†ʹ™‡”‡ ‘’—–‡†Ǥƒ† ǡ –Š‡ ‡œ›‡• ƒ– –Š‡ ‰ƒ–‡™ƒ› ‘ˆ –Š‡ •Š‘”–Ǧ Šƒ‹ „”ƒ Šǡ ”‡•’‘†‡† „› ƒ •–”‘‰

ͷͳ Chapter 2

”‡‰—Žƒ–‹‘ „› –Š‡‹” ’”‘†— –• ͶǦƒ† ͸Ǧ‡‘›ŽǦ‘ ȋ ‹‰ ͷ ƒ† ȌǤ Š‡•‡ ”‡‰—Žƒ–‹‘• ƒ”‡ ‘˜‡”ƒŽŽ‡‰ƒ–‹˜‡ƒ”‘—†–Š‡’‘‹–‘ˆˆŽ—š†‡ Ž‹‡ǡ•‹ ‡–Š‡‹” ‘ ‡–”ƒ–‹‘•‹ ”‡ƒ•‡ȋ ‹‰—”‡ ‹͵ ‹‰Ȍƒ†–Š‡›‹Š‹„‹––Š‡‡œ›‡ǤŠ‹•”‡‰—Žƒ–‹‘™ƒ••—’’‘”–‡†„›–Š‡ˆƒ ––Šƒ––Š‡ ƒ –‹˜‡•‹–‡•‘ˆƒ†™‡”‡’”‡†‘‹ƒ–Ž›‘ —’‹‡†„›–Š‡‹”’”‘†— –•ͶǦƒ†͸Ǧ ‡‘›ŽǦ‘ȋ ‹‰—”‡ƒ†‹ͺ ‹‰ȌǤŠ‡ƒ ›ŽǦ‘•—„•–”ƒ–‡•‘ˆƒ†‘ —’‹‡† ‘Ž›ƒ‹‘”ˆ”ƒ –‹‘‘ˆ–Š‡ƒ –‹˜‡•‹–‡•ƒ–•–‡ƒ†›•–ƒ–‡Ǥ‘”‡†‹•–ƒ–‡œ›‡•ƒ†ʹ ”‡•’‘†‡† –‘ –Š‡‹” Ž‘ ƒŽ •—„•–”ƒ–‡• ƒ† ’”‘†— –•ǡ „—– ƒŽ•‘ •–”‘‰Ž› ˜‹ƒ –Š‡ ‘‡ –‹‰ ‡–ƒ„‘Ž‹–‡ˆ”‡‡‘ȋ ‹‰ͷƒ† ȌǤ  Discussion Š‡‘ǦŽ‹‡ƒ”†›ƒ‹ ‘†‡Ž—•‡†‹–Š‹•’ƒ’‡”‹•–Š‡‘•–•–”— –—”‡†ƒ†“—ƒ–‹–ƒ–‹˜‡ ”‡’”‡•‡–ƒ–‹‘‘ˆ‘—” ‘ŽŽ‡ –‹˜‡‘™Ž‡†‰‡‘ Ǥ ‘’—–ƒ–‹‘ƒŽ‘†‡Ž‹• ‘’Ž‡–‡Ž› –”ƒ•’ƒ”‡–ǡƒ’”‘’‡”–›–Šƒ–™‡‡š’Ž‘‹–‡†–‘†‹••‡ –ƒ˜‹ ‹‘—• › Ž‡Ž‡ƒ†‹‰–‘ ˆŽ—š†‡ Ž‹‡ —’‘•—„•–”ƒ–‡‘˜‡”Ž‘ƒ†ȋ ‹‰͸ȌǤ Palmitoyl-CoA

Mitochondrion 5. Acyl-CoA 2. 3. MCKAT Acetyl-CoA SCAD MCAD LCAD FAD 4. C4-C6 C4-C12 VLCAD C4-C16 CoASH FADH2

Ketoacyl-CoA 1. Enoyl-CoA 2.

MSCHAD NADH + H+ CROT Acetyl-CoA C4-ketoacyl-CoA MTP C4-C16 NAD+ C4-C16 CoASH K = 5. eq Hydroxyacyl-CoA 0.0002 2.  Fig 6.Elucidating the mechanism of flux decline. 1. ’‘•—„•–”ƒ–‡‘˜‡”Ž‘ƒ†™‹–Š ›–‘•‘Ž‹ ’ƒŽ‹–‘›ŽǦ ‘ǡǯ•‡–‘ƒ ›ŽǦ‘•—„•–”ƒ–‡••–ƒ”––‘ƒ ——Žƒ–‡Ǥ2. Š‡—ˆƒ˜‘—”ƒ„Ž‡‡“—‹Ž‹„”‹— ‘•–ƒ–‘ˆ –Š‡ ’”‡ ‡†‹‰ ‡œ›‡ǡ ‡†‹—Ȁ•Š‘”–Ǧ Šƒ‹ Š›†”‘š›ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ǡ ™‘” ƒ• ƒ ƒ’Ž‹ˆ‹‡”ǡ Ž‡ƒ†‹‰–‘ƒ ——Žƒ–‹‘‘ˆ—’•–”‡ƒ‘‡•–‡”•ǡ‹ Ž—†‹‰ƒ ›ŽǦ‘‡•–‡”•Ǥ3.Š‡•‡ƒ ›ŽǦ‘‡•–‡”•ƒ”‡ƒ– –Š‡•ƒ‡–‹‡’”‘†— –•‘ˆƒ†‹Š‹„‹–‹–•ƒŽ”‡ƒ†›Ž‘™ƒ –‹˜‹–›ˆ—”–Š‡”Ǥ4. ‹ƒŽŽ›ǡ–Š‡ƒ ——Žƒ–‹‘ ‘ˆ ‘ ‡•–‡”• Ž‡ƒ†• –‘ ƒ •‡“—‡•–”ƒ–‹‘ ‘ˆ ˆ”‡‡ ‘Ǥ ‘ „‡‹‰ ƒ ‘ˆƒ –‘” ˆ‘” ǡ ‹–• •‡“—‡•–”ƒ–‹‘ Ž‹‹–‡†–Š‡ƒ –‹˜‹–›‡˜‡ˆ—”–Š‡”ǡ–Š—• ‘’Ž‡–‹‰–Š‡˜‹ ‹‘—• › Ž‡Ǥ5Ǥ‹ ‡‘‹•ƒŽ•‘ƒ•—„•–”ƒ–‡ ˆ‘”†‹•–ƒ– ‡œ›‡•ȋ•— Š ƒ•Ȍǡ‹–‡ˆˆ‹ ‹‡–Ž› ‘—‹ ƒ–‡•–Š‡ Ǯ–”ƒˆˆ‹ Œƒǯƒ– –‘–Š‡ ‡–‹”‡ ’ƒ–Š™ƒ›Ǥ  •‹‰‡–ƒ„‘Ž‹ ‘–”‘ŽƒŽ›•‹•ƒ†”‡Žƒ–‡†ƒ’’”‘ƒ Š‡•ǡ™‡‹†‡–‹ˆ‹‡†ƒ’‹˜‘–ƒŽ”‘Ž‡ˆ‘”–Š‡ Žƒ•–‡œ›‡‘ˆ–Š‡’ƒ–Š™ƒ›ǡǡˆ‘”•‡“—‡•–”ƒ–‹‘‘ˆ‘ƒ† ‘’‡–‹–‹˜‡‹Š‹„‹–‹‘‘ˆ „›‹–•ƒ ›ŽǦ‘’”‘†— –•ǤŠ‹•ˆ‹†‹‰‹•‹•Šƒ”’ ‘–”ƒ•––‘‘—”‡ƒ”Ž‹‡”•’‡ —Žƒ–‹‘ –Šƒ––Š‡—–—ƒŽ ‘’‡–‹–‹‘„‡–™‡‡‡œ›‡substrates‹‰Š– ƒ—•‡–Š‡ˆŽ—š†‡ Ž‹‡ȏͺȐǤ Š‡ ‡Ž— ‹†ƒ–‡† ‡ Šƒ‹• ƒ› Šƒ˜‡ ‡†‹ ƒŽ ‹’Ž‹ ƒ–‹‘•ǡ „‘–Š ˆ‘” ’ƒ–‹‡–• ™‹–Š ‹„‘” †‡ˆ‡ –•‹–Š‡ ƒ†ˆ‘”—Ž–‹ˆƒ –‘”‹ƒŽ†‹•‡ƒ•‡•‹™Š‹ Š ‹•‹’Ž‹ ƒ–‡†Ǥ‡Šƒ˜‡ ’”‡˜‹‘—•Ž› •Š‘™ –Šƒ– †‡ˆ‹ ‹‡ › ƒ‡• –Š‡   ’ƒ–Š™ƒ› •—• ‡’–‹„Ž‡ –‘ „‡ ‘‡ ‘˜‡”Ž‘ƒ†‡†„›ƒŠ‹‰Š•—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘ȏͻȐǤŠ‹• ƒ„‡—†‡”•–‘‘†‹–Š‡Ž‹‰Š–‘ˆ–Š‡ ˜‹ ‹‘—• › Ž‡Ǥ ‹–•‡Žˆǡ–Š‡‡œ›‡‹•‘–‡••‡–‹ƒŽǡ•‹ ‡”‡ƒ –‹‘• ƒ–ƒŽ›œ‡†„› ƒ”‡–ƒ‡‘˜‡”„›ǡǡƒ†ȋ”‘†‡–•ǡ ‹‰ͳȌ‘”„›ƒ†ȋŠ—ƒ•Ȍ ȏͳ͸ȐǤ ‘™‡˜‡”ǡ–Š‡Žƒ––‡”ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡•Šƒ˜‡ƒ™‡ƒ‡”ƒˆˆ‹‹–›ˆ‘”–Š‡‡†‹—Ǧ

ͷʹ The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation

Šƒ‹ ƒ ›ŽǦ‘ ‡•–‡”• –Šƒ  ȏͳ͹ǡͳͺȐǡ –Š‡”‡„› ‡Šƒ ‹‰ ƒ ›ŽǦ‘ ƒ ——Žƒ–‹‘ ƒ† ’”‘†— – ‹Š‹„‹–‹‘ ‘ˆ Ǥ ƒŽ‘‰‘—•Ž›ǡ –Š‡ ˜‹ ‹‘—• › Ž‡ ƒ› ’Žƒ› ƒ ”‘Ž‡ ‹ ‘„‡•‹–›Ǧ ”‡Žƒ–‡†‡–ƒ„‘Ž‹ †‹•‡ƒ•‡•ǡ™Š‡”‡ƒ‹–‘ Š‘†”‹ƒŽ•—„•–”ƒ–‡‘˜‡”Ž‘ƒ†Šƒ•„‡‡’”‘’‘•‡†–‘ Ž‡ƒ†–‘ƒ‡–ƒ„‘Ž‹ ‰”‹†Ž‘ ȏͳͻȐǤŠ‡˜‹ ‹‘—• › Ž‡™‘—Ž††‡ˆ‹‹–‡Ž›‡Šƒ ‡–Š‡”‹•‘ˆ•— Š 2 ‡–ƒ„‘Ž‹  ‘’Ž‹ ƒ–‹‘•‘ˆŠ‹‰Š ‹” —Žƒ–‹‰ˆƒ––›Ǧƒ ‹† ‘ ‡–”ƒ–‹‘•‹‘„‡•‹–›Ǥ –Š‹••‡ –‹‘™‡™‹ŽŽ—•‡‘—”‡™‹•‹‰Š–•–‘„‡––‡”—†‡”•–ƒ†Š‘™–Š‡’ƒ–Š™ƒ›•–”— –—”‡ ƒ› ’Žƒ› ƒ ”‘Ž‡ ‹ †‹•‡ƒ•‡ǡ ’ƒ”–‹ —Žƒ”Ž› ”‡‰ƒ”†‹‰ –Š‡ Žƒ  ‘ˆ ”‘„—•–‡•• ‘ˆ ‰Ž— ‘•‡ ƒ† ‡–‘‡Ǧ„‘†› Š‘‡‘•–ƒ•‹•‹ ’ƒ–‹‡–• ™‹–Š ‹Š‡”‹–‡†   †‡ˆ‡ –•Ǥ ”‘†— –• ‘ˆ –Š‡   ƒ”‡ ”‡“—‹”‡† ƒ• ‡‡”‰› •‘—” ‡ ˆ‘” de novo ‰Ž— ‘•‡ ’”‘†— –‹‘ ƒ† ƒ• •—„•–”ƒ–‡ ˆ‘” ‡–‘‰‡‡•‹•Ǥ‡Š›’‘–Š‡•‹œ‡–Š‡”‡ˆ‘”‡–Šƒ––Š‡•–‡‡’ˆŽ—š†‡ Ž‹‡—’‘•—„•–”ƒ–‡‘˜‡”Ž‘ƒ† ’Žƒ›• ƒ ‹’‘”–ƒ– ”‘Ž‡ ‹ –Š‹• ‡–ƒ„‘Ž‹  ˆ”ƒ‰‹Ž‹–›Ǥ ‡› ‡Ž‡‡–• ‘ˆ –Š‡ ‡Ž— ‹†ƒ–‡† ˜‹ ‹‘—• › Ž‡ ƒ”‡ ȋ‹Ȍ –Š‡ Š‹‰Š ˆŽ—š ‘–”‘Ž ‘ˆ  ƒ† ’”‘†— – ‹Š‹„‹–‹‘ ‘ˆ –Š‹• ‡œ›‡ „› ƒ ‘’‡–‹‰ •‡– ‘ˆ ƒ ›ŽǦ‘ ‡•–‡”•ǡ Ž‡ƒ†‹‰ –‘ ‡–‘ƒ ›ŽǦ‘ ƒ ——Žƒ–‹‘Ǣ ȋ‹‹Ȍ –Š‡ ƒ’Ž‹ˆ‹ ƒ–‹‘‘ˆ‘Ǧ‡•–‡”ƒ ——Žƒ–‹‘„›Ȁ ƒ†–Š‡”‘Ž‡‘ˆ–Š‡ȏΪȐȀȏ Ȑ ”ƒ–‹‘–Š‡”‡‹Ǣƒ†ȋ‹‹‹Ȍ–Š‡”‡•—Ž–‹‰†‡’Ž‡–‹‘‘ˆ–Š‡ˆ”‡‡‘ ‘ ‡–”ƒ–‹‘™Š‹ Šˆ—”–Š‡” Ž‹‹–•–Š‡ƒ –‹˜‹–›Ǥ‡™‹ŽŽ•–”— –—”‡‘—”†‹• —••‹‘ƒ”‘—†–Š‡•‡–Š”‡‡‡Ž‡‡–•ƒ† –Š‡‹” ‘‡ –‹‘ –‘ –Š‡ •—””‘—†‹‰ ‡–ƒ„‘Ž‹  ‡–™‘”Ǥ Š‹• ™‹ŽŽ ‰‹˜‡ ‹•‹‰Š– ‹–‘ Š‘™ ’‡”–—”„ƒ–‹‘• ‹ –Š‡ •—””‘—†‹‰ ‡–ƒ„‘Ž‹  ‡–™‘” ƒ› •–ƒ„‹Ž‹œ‡ ‘” †‡•–ƒ„‹Ž‹œ‡ –Š‡ ’ƒ–Š™ƒ›ƒ†–Š‡”‡„›ƒ––‡—ƒ–‡‘”ƒ‰‰”ƒ˜ƒ–‡–Š‡”‹•‘ˆ‡–ƒ„‘Ž‹ ˆƒ‹Ž—”‡Ǥ MCKAT – Š‡ ‡Ž— ‹†ƒ–‡† ˜‹ ‹‘—• › Ž‡ •–ƒ”–‡† ƒ– Ǥ – Ž‘™ ’ƒŽ‹–‘›ŽǦ‘ •—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘• –Š‡ ˆ‹”•– ‡œ›‡ ‘ˆ –Š‡ ’ƒ–Š™ƒ›ǡ ͳ ‡š‡”–‡† ƒ Š‹‰Š ˆŽ—š ‘–”‘Žǡ ‹ ƒ‰”‡‡‡– ™‹–Š ‡ƒ•—”‡‡–• ‹ ”ƒ– Ž‹˜‡” ȏʹͲǡʹͳȐǤ ‘™‡˜‡”ǡ ‘ ‡ –Š‡ ’ƒ–Š™ƒ› „‡ ƒ‡ ‘˜‡”Ž‘ƒ†‡†™‹–Š’ƒŽ‹–‘›Ž‘ǡ–‘‘‘˜‡” ‘–”‘Ž‘˜‡”–Š‡•–‡ƒ†›Ǧ•–ƒ–‡ˆŽ—š‹–Š‡ ‘†‡ŽǤ  ƒ ‘’”‡Š‡•‹˜‡ Ž‹–‡”ƒ–—”‡ ”‡˜‹‡™ ƒ„‘—– ‘–”‘Ž ‘ˆ –Š‡ ȾǦ‘š‹†ƒ–‹‘ ’ƒ–Š™ƒ›ǡ ƒ–‘ ȏʹͳȐ •Š‘™‡† –Šƒ–  ‹‰Š– ‹†‡‡† Ž‹‹– –Š‡   ˆŽ—š ‹ Ž‹˜‡” ‹–‘ Š‘†”‹ƒ —†‡” ‡”–ƒ‹ ‘†‹–‹‘•Ǥ Š‹• ‘ Ž—•‹‘ ™ƒ• „ƒ•‡† ‘ •–—†‹‡• ™‹–Š  ‹Š‹„‹–‘”• ™Š‹ Š ™‡”‡ ˆ‘—† –‘ ƒˆˆ‡ – –Š‡   ˆŽ—š ȏʹʹǡʹ͵ȐǤ  ‡š’‡”‹‡–ƒŽ “—ƒ–‹ˆ‹ ƒ–‹‘ ‘ˆ ˆŽ—š ‘–”‘Ž•Š‘™‡†–Šƒ–•–ƒ”–‡†–‘ ‘–”‘Ž–Š‡ˆŽ—š‘Ž›ƒ–Š‹‰Š•—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘• ȏʹͶȐǤŠ‹• ‘ˆ‹”•‘—””‡•—Ž––Šƒ– ‘–”‘Ž•Š‹ˆ–•†‡’‡†‹‰‘–Š‡•—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘ ƒ†–Š‡”‡„›‰‹˜‡•ˆ—”–Š‡” ”‡†‹„‹Ž‹–›–‘–Š‡ ‘’—–ƒ–‹‘ƒŽ‘†‡ŽǤ ‡ ‡–Ž›ǡ –Š‡ ‹–‘ Š‘†”‹ƒŽ Š  ‹•‘ˆ‘” ’Ͷ͸Š  ™ƒ• ‹†‡–‹ˆ‹‡† ƒ• ƒ •’‡ ‹ˆ‹ ǡ ‡†‘‰‡‘—• ‹Š‹„‹–‘” ‘ˆ  ȏʹͷȐǤ Š  ‘ †‘™ ‹ ”‡ƒ•‡† ‹–‘ Š‘†”‹ƒŽ ’ƒŽ‹–ƒ–‡ ‘š‹†ƒ–‹‘ǡ ˆ—”–Š‡” ‘””‘„‘”ƒ–‹‰ –Š‡ ‘–‹‘ –Šƒ–  ƒ› Ž‹‹– –Š‡ ˆŽ—š in vivoǤ –‡”‡•–‹‰Ž›ǡ Š  ‘ †‘™ƒŽ•‘Ž‡†–‘ƒ–‡†‡ ›‘ˆ‹ ”‡ƒ•‡†Ž‹ˆ‡•’ƒ‹‹ ‡ȏʹͷȐƒ†–Š‡ ‘””‡•’‘†‹‰ SHC1Ž‘ —•Šƒ•„‡‡ƒ••‘ ‹ƒ–‡†™‹–ŠŽ‘‰‡˜‹–›‹ƒǤƒ•‡†‘‘—””‡•—Ž–•ǡ–Š‹•ƒ›„‡†—‡ –‘ ‹ ”‡ƒ•‡†   ’‡” •‡ ‘” –‘ ƒ ”‡Ž‹‡ˆ ‘ˆ ȋ–‘š‹ Ȍ ‘Ǧ‡•–‡” ƒ ——Žƒ–‹‘Ǥ – ™‹ŽŽ „‡ ‹–‡”‡•–‹‰ –‘ ‹˜‡•–‹‰ƒ–‡ ‹ˆ –Š‡ ‡š’”‡••‹‘ ‘ˆ –Š‡ ‰‡‡• ‡ ‘†‹‰  ȋʹȌ ‘” ‹–• ‹Š‹„‹–‘” SHC1 ‹‰Š– †‹ˆˆ‡”‡–‹ƒ–‡ „‡–™‡‡ Ǧ†‡ˆ‹ ‹‡– ’ƒ–‹‡–• –Šƒ– ’”‡•‡–‡† ™‹–Š Š›’‘‡–‘–‹ Š›’‘‰Ž› ‡‹ƒƒ†–Š‘•‡–Šƒ–‡˜‡”†‹†Ǥ M/SCHAD and the mitochondrial redox state – Š‡ ‹–‘ Š‘†”‹ƒŽ ȏΪȐȀȏ Ȑ ”ƒ–‹‘ ‘‡ –• –Š‡   ’ƒ–Š™ƒ› –‘ –Š‡ ”‡•’‹”ƒ–‘”› Šƒ‹ ƒ† –‘ ‘–Š‡”   ’”‘†— ‹‰ ’ƒ–Š™ƒ›•Ǥ‡•Š‘™‡†–Šƒ––Š‡ȏΪȐȀȏ Ȑ”ƒ–‹‘ƒˆˆ‡ –•–Š‡ƒ ——Žƒ–‹‘‘ˆ‘‡•–‡”• ˜‹ƒ –Š‡ Ž‘•‡Ǧ–‘Ǧ‡“—‹Ž‹„”‹— ”‡ƒ –‹‘ Ȁ Ǥ – ‹• ’Žƒ—•‹„Ž‡ǡ –Š‡”‡ˆ‘”‡ǡ –Šƒ– ‹†‹˜‹†—ƒŽ †‹ˆˆ‡”‡ ‡•‹–Š‡ƒ –‹˜‹–›‘ˆ–Š‡”‡•’‹”ƒ–‘”› Šƒ‹‘†—Žƒ–‡–Š‡”‹•‘ˆ•—„•–”ƒ–‡‘˜‡”Ž‘ƒ†Ǥ

ͷ͵ Chapter 2

A link to CoA metabolism – Š‡†‡’Ž‡–‹‘‘ˆˆ”‡‡‘ Ž‘•‡†–Š‡˜‹ ‹‘—• › Ž‡ǤŠ‡”‡Žƒ–‹˜‡Ž› Ž‘™ ƒ’ƒ ‹–› ‘ˆ ǡ ƒ ——Žƒ–‹‘ ‘ˆ ‹–• ‡–‘ƒ ›ŽǦ‘ •—„•–”ƒ–‡•ǡ ƒ† ƒ’Ž‹ˆ‹ ƒ–‹‘ ‘ˆ ‘Ǧ‡•–‡”ƒ ——Žƒ–‹‘„›–Š‡Ž‘™‡“—‹Ž‹„”‹— ‘•–ƒ–‘ˆȀ Ž‡†–‘ƒ•‡“—‡•–”ƒ–‹‘ ‘ˆ‘‹–Š‡•‡‡•–‡”•ǤŠ‹•Ž‹‹–‡†ˆ—”–Š‡”ǡŽ‡ƒ†‹‰–‘‡˜‡‘”‡•‡“—‡•–”ƒ–‹‘‘ˆ ‘Ǥ ‡–ǡ ™‡ ‡˜‡” ‘„•‡”˜‡† ƒ ‘’Ž‡–‡ †‡’Ž‡–‹‘ ‘ˆ ‘ ‹ –Š‡ ‘†‡ŽǤ  ˆƒ –ǡ ‘’Ž‡–‡ †‡’Ž‡–‹‘™‘—Ž†„‡‹ ‘’ƒ–‹„Ž‡™‹–Š ˆŽ—šǡ•‹ ‡„‘–Šƒ†ʹ”‡“—‹”‡‘ ƒ•ƒ•—„•–”ƒ–‡ǤŠ‹• ”— ‹ƒŽ”‘Ž‡‘ˆ‘•—‰‰‡•–•–Šƒ–‘Ǧ”‡Ž‡ƒ•‹‰”‡ƒ –‹‘•ƒ›’”‘–‡ ––Š‡   ’ƒ–Š™ƒ›Ǥ šƒ’Ž‡• ‘ˆ ‘ ”‡Ž‡ƒ•‹‰ ’”‘ ‡••‡• ƒ”‡ –Š‡ ƒ ›ŽǦ‘ Š›†”‘Ž›•‹• „› –Š‹‘‡•–‡”ƒ•‡• ȏʹ͸ȂʹͺȐ ‘” –Š‡ ‡–‘‰‡‡•‹• ’ƒ–Š™ƒ› „”ƒ Š‹‰ ˆ”‘ ͶǦ‡–‘ƒ ›Ž‘ ȋƒ ‡–‘ƒ ‡–›ŽǦ‘Ȍ˜‹ƒ–Š‡‡œ›‡  Ǧ‘•›–Šƒ•‡–‘™ƒ”†•‡–‘‡„‘†‹‡•Ǥ‹–Š”‡•’‡ – –‘–Š‹‘‡•–‡”ƒ•‡•ǡƒ–‘ƒŽ”‡ƒ†›†‡• ”‹„‡†–Š‡‹”’‘–‡–‹ƒŽ Ǧ’”‘–‡ –‹‰”‘Ž‡ǡ„—–™‹–Š‘—– ƒ ˜‹ ‹‘—• › Ž‡ Š‡ Šƒ† ‘ ”‡ƒ•‘ –‘ „‡Ž‹‡˜‡ –Šƒ– –Š‡ ‘ ‘ ‡–”ƒ–‹‘ ‘—Ž† †‡ ”‡ƒ•‡ •‘ †”ƒƒ–‹ ƒŽŽ› ȏʹͳȐǤ š’”‡••‹‘ ‘ˆ ‹–‘ Š‘†”‹ƒŽ ƒ ›ŽǦ‘ –Š‹‘‡•–‡”ƒ•‡• ‹ –Š‡ Ž‹˜‡” ‹• —’”‡‰—Žƒ–‡† ‘ ƒ Š‹‰ŠǦˆƒ– †‹‡–ǡ ˆƒ•–‹‰ ƒ† ”‡‰—Žƒ–‡† „› –Š‡ ’‡”‘š‹•‘‡Ǧ’”‘Ž‹ˆ‡”ƒ–‘” ƒ –‹˜ƒ–‡†”‡ ‡’–‘”•ȋ•Ȍȏʹ͹ǡʹͻȐǡ•—‰‰‡•–‹‰–Šƒ––Š‡›‹†‡‡†’Žƒ›ƒ”‘Ž‡—†‡”ˆƒ––›Ǧƒ ‹† ‘˜‡”Ž‘ƒ† ‘†‹–‹‘•Ǥ‘‰–Š‡‹–‘ Š‘†”‹ƒŽƒ ›ŽǦ‘–Š‹‘‡•–‡”ƒ•‡•ȋ•Ȍ–Š‡ͻ ‹•‘‡œ›‡Šƒ•–Š‡„”‘ƒ†‡•– Šƒ‹ǦŽ‡‰–Š•’‡ ‹ˆ‹ ‹–›Ǥ –‹•–Š‡‘Ž›–Šƒ– ƒ–ƒŽ›œ‡•–Š‡ Š›†”‘Ž›•‹•‘ˆ•Š‘”–Ǧƒ†‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘‡•–‡”•ȏʹͺȐǡƒ‹‰‹–‘’–‹ƒŽŽ›•—‹–‡†–‘ ’”‘–‡ – ƒ‰ƒ‹•– –Š‡ ˜‹ ‹‘—• › Ž‡Ǥ ‘”‡‘˜‡”ǡ ͻ ‹• •–”‘‰Ž› ‹Š‹„‹–‡† „› ‘ ȏʹͺȐǡ ‡•—”‹‰ –Šƒ– –Š‡ Ǧ†‡’‡†‡– ˆ—–‹Ž‡ › Ž‡ ‘ˆ ƒ ›ŽǦ‘ •›–Š‡•‹• ƒ†Š›†”‘Ž›•‹• ‹• ‘Ž› ƒ –‹˜ƒ–‡†™Š‡‘‹•Ž‘™Ǥ—”’”‹•‹‰Ž›ǡͻ‡š’”‡••‹‘‹Ž‹˜‡”‹•”‡Žƒ–‹˜‡Ž›Ž‘™ǡƒ–Ž‡ƒ•– ‹‹ ‡‘ƒ Š‘™†‹‡–ȏʹͺȐǤ –™‹ŽŽ„‡‹–‡”‡•–‹‰–‘•‡‡‹ˆ‹–‹•‹†— ‡†‘ƒŠ‹‰ŠǦˆƒ–†‹‡–ǡ †—”‹‰ˆƒ•–‹‰ǡ‘”‹ †‹•‘”†‡”•Ǥ‹–Š”‡•’‡ ––‘‡–‘‰‡‡•‹•ǡ–Š‡’”‘†— –‹‘‘ˆ‡–‘‡ „‘†‹‡•ˆ”‘ƒ ‡–‘ƒ ‡–›ŽǦ‘‘”ƒ ‡–›ŽǦ‘ƒ†–Š‡‹”•—„•‡“—‡–‡š’‘”–ˆ”‘–Š‡Ž‹˜‡”ƒŽ•‘ Ž‹„‡”ƒ–‡‘Ǥ ’‘”–ƒ–Ž›ǡ‡œ›‡• ƒ–ƒŽ›•‹‰”‡ƒ –‹‘•‘ˆ–Š‡‡–‘‰‡‹ ’ƒ–Š™ƒ›ǡƒ”‡„‘–Š —’”‡‰—Žƒ–‡†ƒ†’‘•–Ǧ–”ƒ• ”‹’–‹‘ƒŽŽ›ƒ –‹˜ƒ–‡††—”‹‰ˆƒ•–‹‰ȏ͵ͲȐ™Š‡–Š‡Ž‹˜‡”†‡’‡†• ‘  Ǥ ‹ƒŽŽ›ǡ –Š‡ ˆ‹”•– ‡œ›‡ ‘ˆ –Š‡ ‡–‘‰‡‡•‹• ’ƒ–Š™ƒ› •–ƒ”–‹‰ ˆ”‘ ƒ ‡–›ŽǦ‘ ȋƒ ‡–‘ƒ ‡–›ŽǦ‘–Š‹‘Žƒ•‡ǡ‡ ‘†‡†„›ͳǡ™Š‹ Š‹•Š‹‰ŠŽ›‡š’”‡••‡†‹–Š‡Ž‹˜‡”ȏ͵ͳȐȌǡ‹• ”‡˜‡”•‹„Ž‡ ƒ† ƒ› ƒŽ•‘ ™‘” ‹ –Š‡ †‹”‡ –‹‘ ‘ˆ „‡–ƒǦ‘š‹†ƒ–‹‘ȏ͵ʹǡ͵͵Ȑǡ –Š—• ’”‘˜‹†‹‰ ƒ „›’ƒ••ˆ‘”Ǥ  ‘–”ƒ•–ǡƒ –‹˜ƒ–‹‘‘ˆ‘Ǧ—–‹Ž‹œ‹‰’ƒ–Š™ƒ›•‹–Š‡‹–‘ Š‘†”‹ƒƒ›†‡ ”‡ƒ•‡–Š‡ˆ”‡‡ ‘ ‘ ‡–”ƒ–‹‘ ƒ† –Š‡”‡„› –”‹‰‰‡” ƒ —–‡ ˆƒ‹Ž—”‡ ‘ˆ  Ǥ ‹– Š‡ŽŽ ȏ͵ͶȐ Š›’‘–Š‡•‹œ‡† –Šƒ––Š‹•ƒ›„‡ƒ ‘‘†‡‘‹ƒ–‘”‹ƒŽŽ‡œ›‡†‡ˆ‡ –•–Šƒ–Ž‡ƒ†–‘ƒ ——Žƒ–‹‘‘ˆ ‘‡•–‡”•Ǥ ‡–‡”‡†–Š‹•‰”‘—’‘ˆ†‹•‡ƒ•‡•C‘AS‡“—‡•–”ƒ–‹‘ǡT‘š‹ ‹–›ƒ†R‡†‹•–”‹„—–‹‘ ȋȌ†‹•‡ƒ•‡•ƒ†ƒ”‰—‡†–Šƒ––Š‡›ƒŽŽ•Šƒ”‡–Š‡”‹•‘ˆŠ›’‘‰Ž› ‡‹ƒǤ•—”’Ž—•‘ˆƒ ‘Ǧ†‡’‡†‡–•—„•–”ƒ–‡ƒ›–Š‡„‡•—ˆˆ‹ ‹‡––‘–”‹‰‰‡”–Š‡˜‹ ‹‘—• › Ž‡‹’ƒ–‹‡–•™‹–Š ƒ †‹•‡ƒ•‡Ǥ – ‹• –‡’–‹‰ –‘ •’‡ —Žƒ–‡ –Šƒ– –Š‡ ƒ –‹˜‹–› ‘ˆ –Š‡ ‘ •›–Š‡•‹• ’ƒ–Š™ƒ› ȏ͵ͷȂ͵͹Ȑ ƒ› ‘†—Žƒ–‡ –Š‡ ”‹• ‘ˆ ‘ †‡’Ž‡–‹‘Ǥ †‡‡†ǡ ‹ ”‡ƒ•‹‰ –Š‡ –‘–ƒŽ ‘ ’‘‘Ž ȋ‡•–‡”‹ˆ‹‡† ’Ž—• ˆ”‡‡Ȍ†‘‡•‹ ”‡ƒ•‡–Š‡–Š”‡•Š‘Ž† ‘ ‡–”ƒ–‹‘‘ˆ’ƒŽ‹–‘›ŽǦ‘ƒ–™Š‹ Š–Š‡ˆŽ—š†‡ Ž‹‡ •‡–• ‹ ȋ ‹‰—”‡ ‹ ͻ ‹‰ȌǤ ‘™‡˜‡”ǡ –Š‹• ‘‡• ƒ– –Š‡ ‘•– ‘ˆ ‹ ”‡ƒ•‡† ‘Ǧ‡•–‡” ‘ ‡–”ƒ–‹‘•Ǥ•–Š‡•‡ƒ›„‡ ‘‡–‘š‹ ǡ™‡†‘—„––Šƒ–‘„‹‘•›–Š‡•‹•ƒ›’”‘˜‹†‡ƒ •–”‘‰ ’”‘–‡ –‹˜‡ ‡ Šƒ‹•Ǥ ‹ƒŽŽ›ǡ –Š‡ –”ƒ•‡•–‡”‹ˆ‹ ƒ–‹‘ ‘ˆ ‘ ‡•–‡”• ‹–‘ ƒ”‹–‹‡ ‡•–‡”• ƒ† –Š‡‹” •—„•‡“—‡– ‡š ”‡–‹‘ ƒ› ’”‘–‡ – ‡ŽŽ• ƒ‰ƒ‹•– ‘ †‡’Ž‡–‹‘Ǥ †‡‡†ǡ ƒ”‹–‹‡ ‡•–‡”• †‡”‹˜‡† ˆ”‘ ‘•–   ‹–‡”‡†‹ƒ–‡•ǡ ‹ Ž—†‹‰ Š›†”‘š›ƒ ›ŽǦ ƒ”‹–‹‡

ͷͶ The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation

‡•–‡”•ǡ™‡”‡ˆ‘—†‹•‡”—ƒ†—”‹‡‘ˆ ’ƒ–‹‡–•ȏ͵ǡͶȐǤ –Šƒ•„‡‡•Š‘™–Šƒ––Š‹• ƒŽ‡ƒ†–‘Ž‘™ ‹” —Žƒ–‹‰ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘•‹‹ ‡ƒ†’ƒ–‹‡–•™‹–Š †‡ˆ‡ –• ȏ͵ͺȂͶͳȐǤŠ‡”‡ˆ‘”‡ǡ’ƒ–‹‡–••‘‡–‹‡•”‡ ‡‹˜‡ ƒ”‹–‹‡•—’’Ž‡–‹‘Ǥš–”ƒ ƒ”‹–‹‡•Š‘—Ž† •–‹—Žƒ–‡ –Š‡ ‡š’‘”– ‘ˆ   ‹–‡”‡†‹ƒ–‡• ‹ –Š‡ ˆ‘” ‘ˆ ƒ”‹–‹‡ ‡•–‡”• ˜‹ƒ –Š‡  2 –”ƒ•’‘”–‡” ƒ† –Š‡”‡„› •–‹—Žƒ–‡ ”‡‰‡‡”ƒ–‹‘ ‘ˆ ‘Ǥ Š‡ ‡ˆˆ‡ –‹˜‡‡•• ‘ˆ ƒ”‹–‹‡ •—’’Ž‡‡–ƒ–‹‘–‘’ƒ–‹‡–•‹•†‡„ƒ–‡†ǡ–Š‘—‰Š ȏͶʹȐǤŠ‡ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘‹„Ž‘‘† †‘‡• ‘– ”‡ˆŽ‡ – –Šƒ– ‹ –‹••—‡• ȏͶ͵Ȑ ƒ† –Š‡”‡ ƒ”‡ ƒŽ•‘ ”‡’‘”–• ‘ˆ ƒ ›ŽǦ ƒ”‹–‹‡Ǧ‡†‹ƒ–‡† –‘š‹ ‹–›ȏͶͶȐǤ‘”‡‘˜‡”ǡ ƒ”‹–‹‡•—’’Ž‡‡–ƒ–‹‘‹‰Š–ƒŽ•‘•–‹—Žƒ–‡‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧ ƒ ‹†—’–ƒ‡˜‹ƒ–Š‡ͳǦ†‡’‡†‡–•Š—––Ž‡ǡ–Š‡”‡„›ƒ‰‰”ƒ˜ƒ–‹‰–Š‡•‹–—ƒ–‹‘Ǥ Formation of enzyme supercomplexes – ‹–Š–Š‡‡š ‡’–‹‘‘ˆ–Š‡”‡ƒ –‹‘• ƒ–ƒŽ›œ‡†„› ȏͶͷȂͷͳȐǡ‘—” ‘’—–ƒ–‹‘ƒŽ‘†‡Ž†‹†‘–‹ Ž—†‡–Š‡ˆ‘”ƒ–‹‘‘ˆ‡œ›‡ ‘’Ž‡š‡•Ǥ  •— Š ‘’Ž‡š‡• ‹–‡”‡†‹ƒ–‡ ‡–ƒ„‘Ž‹–‡• ƒ› „‡ Šƒ‡ŽŽ‡† ˆ”‘ ‘‡ ƒ –‹˜‡ •‹–‡ –‘ ƒ‘–Š‡”Ǥ ”‡ƒŽ‹–›ǡŠ‘™‡˜‡”ǡƒŽ•‘‘–Š‡”‡œ›‡•ƒ›ˆ‘” ‘’Ž‡š‡•ƒ†–Š‡•‡ƒ›ƒˆˆ‡ – –Š‡’ƒ–Š™ƒ›‹‡–‹ •Ǥ ‘”‹•–ƒ ‡Ȁ Šƒ•„‡‡ˆ‘—†‹ƒ”‡•’‹”ƒ–‘”›•—’‡” ‘’Ž‡š ™‹–Š –Š‡   †‡Š›†”‘‰‡ƒ•‡ ‘’Ž‡š  ȏͷʹǡͷ͵ȐǤ  –Š‡ Ž‹‰Š– ‘ˆ ‘—” ‘†‡ŽŽ‹‰ ‹– •‡‡• ’Žƒ—•‹„Ž‡ –Šƒ– –Š‹• ˆƒ ‹Ž‹–ƒ–‡• ƒ †‹”‡ – Šƒ‡ŽŽ‹‰ ‘ˆ   –‘ –Š‡ ”‡•’‹”ƒ–‘”› Šƒ‹Ǥ Š‹• ™‘—Ž†’—•Š–Š‡Ȁ ‡“—‹Ž‹„”‹—‹–Š‡ˆ‘”™ƒ”††‹”‡ –‹‘ƒ†”‡Ž‹‡˜‡–Š‡ƒ ——Žƒ–‹‘ ‘ˆ‘‡•–‡”•Ǥ —”–Š‡”‘”‡ǡ™‡ ƒ‘–‡š Ž—†‡–Šƒ–ƒ ›ŽǦ‘‡•–‡”•ƒ”‡’ƒ”–Ž› Šƒ‡ŽŽ‡† ˆ”‘  –‘ –Š‡ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡•ǡ •‹ ‡ –Š‡•‡ ‡œ›‡• Šƒ˜‡ ƒŽŽ „‡‡ ˆ‘—† ‹ •—’‡” ‘’Ž‡š‡• ȏͷͶȐǤ Š‹• ™‘—Ž† ”‡†— ‡ –Š‡ •‘Ž—„Ž‡ ƒ ›ŽǦ‘ ‘ ‡–”ƒ–‹‘• ‹ –Š‡ ‹–‘ Š‘†”‹ƒŽƒ–”‹šƒ†–Š‡”‡„›”‡Ž‹‡˜‡–Š‡’”‘†— –‹Š‹„‹–‹‘‘ˆǤŠ‡”‡‡š‹•–•–‘ ‘—”‘™Ž‡†‰‡ǡŠ‘™‡˜‡”ǡ‘†‹”‡ –‡˜‹†‡ ‡ˆ‘”•— Š Šƒ‡Ž‹‰ƒ†–Š‡‘„•‡”˜ƒ–‹‘‘ˆŠ‹‰Š ƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘•‹’ƒ–‹‡–•ǯ•‡”—‹•”ƒ–Š‡”‹–‡”’”‡–‡†ƒ•ƒ”‡ˆŽ‡ –‹‘‘ˆŠ‹‰Š ƒ ›ŽǦ‘ ‘ ‡–”ƒ–‹‘•‹–Š‡–‹••—‡•Ǥ ‹ƒŽŽ›ǡȀ Šƒ•„‡‡ˆ‘—†‹ ‘’Ž‡š™‹–Š ‹–‘ Š‘†”‹ƒŽ  Ǧ‘ •›–Šƒ•‡ǡ –Š‡ ˆ‹”•– ‡œ›‡ ‘ˆ ‡–‘‰‡‡•‹• ȏͷͷȐǤ Š‹• ƒ••‘ ‹ƒ–‹‘ ‹‰Š– Šƒ‡Ž ‡–‘ƒ ›ŽǦ‘ ‡•–‡”• †‹”‡ –Ž› ‹–‘ ‡–‘‡ „‘†‹‡•Ǥ — Š ƒ ‡ Šƒ‹• ™‘—Ž† •ƒˆ‡‰—ƒ”†‡–‘‡Ǧ„‘†›•›–Š‡•‹•ƒ†ƒ––Š‡•ƒ‡–‹‡”‡†— ‡–Š‡’”‡••—”‡‘Ǥ Concluding remarks – – ‹• Ž‡ƒ” –Šƒ– –Š‡   ’ƒ–Š™ƒ› ‹• ‘‡ –‡† –‘ ‘–Š‡” ‡–ƒ„‘Ž‹  ’ƒ–Š™ƒ›• ƒ– ”— ‹ƒŽ „”ƒ Š ’‘‹–•Ǥ ‘ ‰‡– ƒ ˆ—ŽŽ ‘˜‡”˜‹‡™ ‘ˆ –Š‡ ’‘–‡–‹ƒŽ „‹‘‡†‹ ƒŽ ‘•‡“—‡ ‡•‘ˆ–Š‡ ‘‡ –‹‘•ƒ†–‘ƒ‡–Š‡‘†‡Ž•—‹–ƒ„Ž‡ˆ‘” Ž‹‹ ƒŽƒ’’Ž‹ ƒ–‹‘ǡ‹– ‡‡†•–‘„‡‡š–‡†‡†ƒ†ˆ—”–Š‡”˜ƒŽ‹†ƒ–‡†—†‡”ƒ”ƒ‰‡‘ˆ Ž‹‹ ƒŽŽ›”‡Ž‡˜ƒ– ‘†‹–‹‘•Ǥ ‡˜‡”–Š‡Ž‡••ǡ‡˜‡–Š‡‘†‡Ž‘ˆ–Š‡‹•‘Žƒ–‡† ’ƒ–Š™ƒ›‰‹˜‡•—•—’”‡ ‡†‡–‡†‹•‹‰Š– ‹–‘’‘–‡–‹ƒŽ”‹•ˆƒ –‘”•ƒ†’”‘–‡ –‹‘‡ Šƒ‹••Ǥ —–—”‡”‡•‡ƒ” Š•Š‘—Ž†‡Ž— ‹†ƒ–‡–‘ ™Šƒ–‡š–‡––Š‡•‡ƒ›‡š’Žƒ‹–Š‡’ƒ–‹‡–Ǧ–‘Ǧ’ƒ–‹‡–˜ƒ”‹ƒ–‹‘ƒ†–Š‡ƒ —–‡–”‹‰‰‡”•Ž‡ƒ†‹‰ –‘‡–ƒ„‘Ž‹ ˆƒ‹Ž—”‡Ǥ  Methods Computational models ŽŽƒƒŽ›•‡•™‡”‡†‘‡™‹–Š–Š‡ ‘’—–ƒ–‹‘ƒŽ‘†‡Ž‘ˆ ƒ•’—„Ž‹•Š‡†‹ȏͺȐǤƒŽ —Žƒ–‹‘•™‡”‡ †‘‡‹‘Žˆ”ƒƒ–Š‡ƒ–‹ ƒȋ‘Žˆ”ƒ‡•‡ƒ” Šǡ  ǤǡŠƒ’ƒ‹‰ǡ ǡȌǡ˜‡”•‹‘ͳͲǤʹǤͲǤͲǤˆ—ŽŽ †‡• ”‹’–‹‘ ‘ˆ –Š‡ ‘†‡Ž ‡“—ƒ–‹‘• ‹• ‰‹˜‡ ‹ ȏͺȐǤ Š‡ ‘†‡Ž ‹–•‡Žˆ ƒ „‡ ˆ‘—† ƒ– Š––’•ǣȀȀŒŒŒǤ„‹‘Ǥ˜—ǤŽȀ‘†‡Ž•Ȁ˜ƒ‡—‡͸Ǥ ‹–‹ƒŽ ‘†‹–‹‘• ˆ‘” •–‡ƒ†›Ǧ•–ƒ–‡ ƒŽ —Žƒ–‹‘• ™‡”‡ –Š‡ •–‡ƒ†›Ǧ•–ƒ–‡ ‡–ƒ„‘Ž‹–‡ ‘ ‡–”ƒ–‹‘• ƒ– ƒ ›–‘•‘Ž‹  ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘ ȋȏ’ƒŽ‹–‘›ŽǦ

‘ȐȌ ‘ˆ ͲǤͳ ɊǤ Š‡ ƒ–Š‡ƒ–‹ ƒ ˆ— –‹‘• ‘Ž˜‡ ƒ† ‹†‘‘– ™‡”‡ —•‡† ˆ‘” –Š‡ –‹‡ •‹—Žƒ–‹‘•ƒ†–‘ˆ‹†•–‡ƒ†›•–ƒ–‡•ǡ”‡•’‡ –‹˜‡Ž›ǡƒ•†‘‡‹ȏͺȐǤ• ˆŽ—š™‡–‘‘–Š‡—’–ƒ‡

ͷͷ Chapter 2

ˆŽ—š‘ˆ’ƒŽ‹–‘›ŽǦ ƒ”‹–‹‡‹–‘–Š‡‹–‘ Š‘†”‹ƒȋ —’–ƒ‡Ȍǡ™Š‹ Š‹•–Š‡•–‡ƒ†›Ǧ•–ƒ–‡”ƒ–‡‘ˆ–Š‡ͳ͸Ǧ ’ƒŽ‹–‘›ŽǦ ƒ”‹–‹‡—’–ƒ‡–Š”‘—‰ŠǤŠ‡• ”‹’–•ˆ‘””‡‘˜‹‰’”‘‹• —‹–›ƒ†ˆ‘” ‘’—–‹‰ ഥ ௩ –Š‡”‡‰—Žƒ–‘”› ‘–”‹„—–‹‘•ȁȣȁ௑ೕ ƒ”‡‰‹˜‡‹ͳ’’‡†‹šƒ†ʹ’’‡†‹šǤ  Removal of promiscuity ”‘‹• —‹–›™ƒ•”‡‘˜‡†‡‹–Š‡”ˆ”‘ƒŽŽ‡œ›‡•‹–Š‡‘†‡Ž•‹—Ž–ƒ‡‘—•Ž›‘”’‡”‡œ›‡ǡƒ• ‹†‹ ƒ–‡† ‹ –Š‡ –‡š–Ǥ  •‹‹Žƒ” ’”‘ ‡†—”‡ ™ƒ• ˆ‘ŽŽ‘™‡† ‹ ȏͺȐ ˆ‘” ƒŽŽ ‡œ›‡• •‹—Ž–ƒ‡‘—•Ž›Ǥ ‘ ”‡‘˜‡’”‘‹• —‹–›ˆ”‘ƒ‡œ›‡ǡ™‡ˆ‹”•–”‡‘˜‡†–Š‡•—„•–”ƒ–‡•ƒ†’”‘†— –•‘ˆ‘–Š‡” Šƒ‹ Ž‡‰–Š•ǡ™Š‹ Šƒ”‡‹–Š‡‡“—ƒ–‹‘ƒ• ‘’‡–‹–‹˜‡‹Š‹„‹–‘”•ȋ–Š‡‰”‡›ǦŠ‹‰ŠŽ‹‰Š–‡†‡–ƒ„‘Ž‹–‡•‹“Ǥ ͻȌǤ ‡ ‘†Ž›ǡ –Š‡ ‡œ›‡ ‘ ‡–”ƒ–‹‘ ™ƒ• ’ƒ”–‹–‹‘‡† ‡˜‡Ž› ‘˜‡” –Š‡ Šƒ‹ǦŽ‡‰–ŠǦ•’‡ ‹ˆ‹  ”‡ƒ –‹‘• –Šƒ– ‹– ƒ–ƒŽ›œ‡• „› ƒ††‹‰ ƒ ’ƒ”–‹–‹‘ ˆƒ –‘”D –‘ –Š‡ ”ƒ–‡ ‡“—ƒ–‹‘Ǥ Š‡ ˜ƒŽ—‡ ‘ˆ D ™ƒ• ‡šƒ –Ž›–Š‡•ƒ‡ˆ‘”ƒŽŽ–Š‡”‡ƒ –‹‘• ƒ–ƒŽ›œ‡†„›ƒ‡œ›‡Ǣ‹–•˜ƒŽ—‡‹•ͳ†‹˜‹†‡†„›–Š‡—„‡”‘ˆ ”‡ƒ –‹‘• ƒ–ƒŽ›œ‡†„›–Š‡‡œ›‡Ǥ ‘—”’”‡˜‹‘—••–—†›ȏͺȐ™‡Šƒ†„ƒ•‡†–Š‡’ƒ”–‹–‹‘ˆƒ –‘”•D‘ –Š‡ˆŽ—š†‹•–”‹„—–‹‘‹–Š‡‘”‹‰‹ƒŽ‘†‡Žǡƒ–ƒ”‡ˆ‡”‡ ‡ ‘ ‡–”ƒ–‹‘‘ˆʹͷρ‘ˆ’ƒŽ‹–‘›ŽǦ‘Ǥ Š‡–™‘™ƒ›•‘ˆ’ƒ”–‹‘‹‰›‹‡Ž†‡†˜‡”›•‹‹Žƒ””‡•—Ž–•Ǥ‡šƒ’Ž‡™‹ŽŽ„‡‰‹˜‡ˆ‘”–Š‡ͳ͸Ǧ•’‡ ‹ˆ‹  ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ–‹‘ ”‡ƒ –‹‘ ƒ–ƒŽ›œ‡† Ǥ Š‡ ”ƒ–‡ ‡“—ƒ–‹‘ ˆ‘” –Š‹• ”‡ƒ –‹‘ ‹ –Š‡ ‘”‹‰‹ƒŽ‘†‡Ž™‹–Š’”‘‹• —‹–›‹•ǣ

ݒ௏௅஼஺஽ǡ஼ଵ଺ ൌ

ሾ಴భలషೌ೎೤೗ష಴೚ಲሿ ሾಷಲವሿ ሾ಴భలష೐೙೚೤೗ష಴೚ಲሿ ሾಷಲವಹమሿ ௦௙ೇಽ಴ಲವǡ಴భలή௏೘ೌೣǡೇಽ಴ಲವήቆ ή ି ή ቇ ಼೘ೇಽ಴ಲವǡ಴భలషೌ೎೤೗ష಴೚ಲ ಼೘ೇಽ಴ಲವǡಷಲವ ಼೘ೇಽ಴ಲವǡ಴భలషೌ೎೤೗ష಴೚ಲ ಼೘ೇಽ಴ಲವǡಷಲವή಼೐೜ೇಽ಴ಲವ ሾ಴భలషೌ೎೤೗ష಴೚ಲሿ ሾ಴భలష೐೙೚೤೗ష಴೚ಲሿ ሾ಴೙షೌ೎೤೗ష಴೚ಲሿ ሾ಴೙ష೐೙೚೤೗ష಴೚ಲሿ ሾಷಲವሿ ሾಷಲವಹ ሿ ȋͻȌ ቆଵା ା ାσ೙సభర ା ቇήቆଵା ା మ ቇ ಼೘ ಼೘ ೙సభమ಼೘ ಼೘ ಼೘ ಼೘ ೇಽ಴ಲವǡ಴భలషೌ೎೤೗ష಴೚ಲ ೇಽ಴ಲವǡ಴భలష೐೙೚೤೗ష಴೚ಲ ೇಽ಴ಲವǡ಴೙షೌ೎೤೗ష಴೚ಲ ೇಽ಴ಲವǡ಴೙ష೐೙೚೤೗ష಴೚ಲ ೇಽ಴ಲವǡಷಲವ ೇಽ಴ಲವǡಷಲವಹమ Š‡”ƒ–‡‡“—ƒ–‹‘ˆ‘”–Š‡•ƒ‡”‡ƒ –‹‘‹–Š‡‘†‡Ž™‹–Š‘—–’”‘‹• —‹–›„‡ ƒ‡ǣ

ሾ಴భలషೌ೎೤೗ష಴೚ಲሿ ሾಷಲವሿ ሾ಴భలష೐೙೚೤೗ష಴೚ಲሿ ሾಷಲವಹ ሿ ఈή௦௙ ή௏ ήቆ ή ି ή మ ቇ ೇಽ಴ಲವǡ಴భల ೘ೌೣǡೇಽ಴ಲವ ಼೘ ಼೘ ಼೘ ಼೐೜ ݒ ൌ ೇಽ಴ಲವǡ಴భలషೌ೎೤೗ష಴೚ಲ ೇಽ಴ಲವǡಷಲವ ೇಽ಴ಲವǡ಴భలషೌ೎೤೗ష಴೚ಲ ೇಽ಴ಲವ ௏௅஼஺஽ǡ஼ଵ଺ ሾ಴భలషೌ೎೤೗ష಴೚ಲሿ ሾ಴భలష೐೙೚೤೗ష಴೚ಲሿ ሾಷಲವሿ ሾಷಲವಹ ሿ ȋͳͲȌ ቆଵା ା ቇήቆଵା ା మ ቇ ಼೘ ಼೘ ಼೘ ಼೘ ೇಽ಴ಲವǡ಴భలషೌ೎೤೗ష಴೚ಲ ೇಽ಴ಲವǡ಴భలష೐೙೚೤೗ష಴೚ಲ ೇಽ಴ಲವǡಷಲವ ೇಽ಴ಲವǡಷಲವಹమ  Metabolic control analysis ŽŽ‡œ›‡”ƒ–‡‡“—ƒ–‹‘•‹–Š‡‘†‡Žƒ”‡‘ˆ–Š‡•Šƒ’‡ǣ

஼௡ ήܸ௠௔௫ ή݂ሺࢄሻ ȋͳͳȌ݂ݏݒൌ

‹™Š‹ Šv‹•–Š‡”ƒ–‡‘ˆ–Š‡‡œ›‡ǡsfCn ƒ•’‡ ‹ˆ‹ ‹–›ˆƒ –‘”™Š‹ Š ‘””‡ –•–Š‡ƒš‹ƒŽ˜‡Ž‘ ‹–›Vmax ˆ‘”–Š‡ ƒ”„‘ Šƒ‹Ž‡‰–Š‘ˆ–Š‡•—„•–”ƒ–‡ǡƒ†f()‹•ƒˆ— –‹‘‘ˆ–Š‡‡–ƒ„‘Ž‹–‡ ‘ ‡–”ƒ–‹‘•‹ ˜‡ –‘”Ǥ Ž—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–•™‡”‡ƒ’’”‘š‹ƒ–‡†ƒ•ˆ‘ŽŽ‘™•ǣ

௃ ௗ௟௡௃Ȁௗ௣ ௗ௃ ௏೘ೌೣǡ೐೙೥೤೘೐ ο௃ ௏೘ೌೣǡ೐೙೥೤೘೐ ܥ௘௡௭௬௠௘ ൌ ൌ ή ൎ ή ȋͳʹȌ డ௟௡௩Ȁడ௣ ௗ௏೘ೌೣǡ೐೙೥೤೘೐ ௃ ο௏೘ೌೣǡ೐೙೥೤೘೐ ௃ ‡”‡ p †‡‘–‡• ƒ ’ƒ”ƒ‡–‡” –Šƒ– ‘Ž› ƒˆˆ‡ –• –Š‡ ”ƒ–‡ ‘ˆ –Š‡ ‡œ›‡ –Šƒ– ‹• ƒƒŽ›œ‡†Ǥ ‘” –Š‹• ’—”’‘•‡ǡ™‡—•‡†–Š‡Vmaxǡ•‹ ‡–Š‡”ƒ–‡•‘ˆƒŽŽ”‡ƒ –‹‘•™‡”‡’”‘’‘”–‹‘ƒŽ–‘–Š‹•’ƒ”ƒ‡–‡”ȋ“Ǥ ͳͳȌǤ ‘”–Š‡ˆ‹‹–‡ Šƒ‰‡†‡‘–‡†„›'„‘–ŠΪͲǤͲͲͲͳΨƒ†ǦͲǤͲͲͲͳΨ™‡”‡–ƒ‡ǡƒ†–Š‡”‡•—Ž–™ƒ• ƒ ‡’–‡† ‹ˆ –Š‡ –™‘ ‰ƒ˜‡ ‹†‡–‹ ƒŽ ”‡•—Ž–• ™‹–Š‹ ͳΨǤ Š‡ Šƒ‹ǦŽ‡‰–ŠǦ•’‡ ‹ˆ‹  ˆƒ –‘”• ™‡”‡ ƒŽ —Žƒ–‡†ƒ•ǣ

௃ ο௃ ௦௙಴೙ ܥ௘௡௭௬௠௘ǡ஼௡ ൎ ή   ȋͳ͵Ȍ ο௦௙಴೙ ௃

‹™Š‹ Šοݏ݂஼௡™ƒ•ƒ‰ƒ‹ Šƒ‰‡†„›ΪͲǤͲͲͲͳΨƒ†ǦͲǤͲͲͲͳΨǤ –™ƒ• Š‡ ‡†–Šƒ––Š‡•—‘ˆ–Š‡ Šƒ‹ǦŽ‡‰–ŠǦ•’‡ ‹ˆ‹ ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–•™ƒ•‡“—ƒŽ–‘–Š‡‘˜‡”ƒŽŽˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–‘ˆ–Š‡ ‡œ›‡ǡƒ†–Šƒ––Š‡•—‘ˆƒŽŽˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–•‹–Š‡‘†‡Ž™ƒ•‡“—ƒŽ–‘ͳȏͳͳǡͳͶȐǤ Ž—š”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–•™‡”‡ƒ’’”‘š‹ƒ–‡†ƒ•ǣ

ௗ௟௡௃ ο௃ ௣ ܴ௃ ൌ ൎ ή ȋͳͶȌ ௣ ௗ௟௡௣ ο௣ ௃

ͷ͸ The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation

‹™Š‹ Š’ƒ‰ƒ‹†‡‘–‡•ƒ‘†‡Ž’ƒ”ƒ‡–‡”Ǥ –Š‹• ƒ•‡–Š‡ˆ‹‹–‡ Šƒ‰‡‘ˆ–Š‡’ƒ”ƒ‡–‡”˜ƒŽ—‡ ™ƒ• –ƒ‡ –‘ „‡ ΪͲǤͲͲͲͳΨ ƒ† ǦͲǤͲͲͲͳΨǤ Š‡ ˜ƒŽ‹†‹–› ‘ˆ –Š‡ ƒŽ —Žƒ–‡† ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–• ™‡”‡ Š‡ ‡† ™‹–Š –Š‡ •—ƒ–‹‘ –Š‡‘”‡ ȏͳͳǡͳͶȐ ƒ† –Š‡ ”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡– ‘ˆ –Š‡ ȏΪȐȀȏ Ȑ ”ƒ–‹‘™ƒ• Š‡ ‡†™‹–Š–Š‡ ‘‡ –‹˜‹–›–Š‡‘”‡ȏͳͳǡͳͶȐǤŠ‡”‡•’‘•‡ ‘‡ˆˆ‹ ‹‡–•™‡”‡ ƒŽ —Žƒ–‡† 2 ˆ‘”‡ƒ Š’ƒ”ƒ‡–‡”‹–Š‡‘†‡Žǡ„—–‘Ž›–Š‡Š‹‰Š‡•–˜ƒŽ—‡•™‡”‡•Š‘™Ǥ

Quantification of regulation of enzyme rates by individual variable metabolites Š‡ ‘–”‹„—–‹‘ ‘ˆ ‡ƒ Š ‡–ƒ„‘Ž‹–‡ –‘ –Š‡ ”‡‰—Žƒ–‹‘ ‘ˆ ƒ •’‡ ‹ˆ‹  ‡œ›‡ ”ƒ–‡ ™ƒ• •–‡’™‹•‡ ƒŽ —Žƒ–‡†ƒ ‘”†‹‰–‘ȏͳʹȐƒ•ˆ‘ŽŽ‘™•Ǥ ‹”•–ǡ™‡ ƒŽ —Žƒ–‡†ǣ ௗ௟௡௩ ௗ௟௡௑ ఋ௩ ௑ ሺ௧ሻ ௗ௑ ሺ௧ሻ ଵ ೔ቚ ሺݐሻ ൌߝ௩೔ ሺݐሻ ή ೕ ൌ൬ ೔ ሺݐሻ ή ೕ ൰ή ೕ ή   ȋͳͷȌ ௗ௧ ௑ೕ ௗ௧ ఋ௑ ௩ሺ௧ሻ ௗ௧ ௑ ሺ௧ሻ ௑ೕ ೕ ೕ

™Š‡”‡˜‹†‡‘–‡•–Š‡”ƒ–‡ƒ–™Š‹ Šƒ•’‡ ‹ˆ‹ ‡œ›‡ ƒ–ƒŽ›œ‡•”‡ƒ –‹‘‹ǤŠ‡‘—– ‘‡™ƒ•’Ž‘––‡†‹ ‹‰Ͷƒ† ‹‰—”‡‹͹ ‹‰Ǥ ‘” ‹‰ͷǦ ǡ–Š‡ ‘””‡•’‘†‹‰ƒ„•‘Ž—–‡‘ˆ–Š‡•‡˜ƒŽ—‡•ȋ ƒŽ —Žƒ–‡†™‹–Š–Š‡ƒ–Š‡ƒ–‹ ƒˆ— –‹‘ Dz„•dzȌ™‡”‡‹–‡‰”ƒ–‡†‘˜‡”–‹‡ȋˆ”‘–αͲ–Š”‘—‰ŠͶͲͲ‹—–‡•ǡ™Š‹ Š™ƒ•‡‘—‰Š–‘”‡ƒ Šƒ‡™ •–‡ƒ†›•–ƒ–‡—†‡”ƒŽŽ‹•’‡ –‡† ‘†‹–‹‘•Ȍ™‹–Š–Š‡ƒ–Š‡ƒ–‹ ƒˆ— –‹‘Dz –‡‰”ƒ–‡dzƒ††‹˜‹†‡† ഥ ௩ „›ͶͲͲ‹—–‡•ǤŠ‹•‰‹˜‡•–Š‡ƒ˜‡”ƒ‰‡ƒ„•‘Ž—–‡ȋ–‘–ƒŽȌ ‘–”‹„—–‹‘‘ˆ‡ƒ Š‡–ƒ„‘Ž‹–‡ȋȁȣȁ௑ೕȌǤ –Š‡ ‡– ‘–”‹„—–‹‘‘ˆƒ‡–ƒ„‘Ž‹–‡–Š‡‡‰ƒ–‹˜‡ƒ†’‘•‹–‹˜‡ ‘–”‹„—–‹‘•’ƒ”–‹ƒŽŽ› ƒ ‡Ž‡ƒ Š‘–Š‡” ‘—–ǡ•‹ ‡–Š‡’‘•‹–‹˜‡ƒ”‡ƒ•—†‡” —”˜‡ƒ†–Š‡‡‰ƒ–‹˜‡ƒ”‡ƒ•—†‡” —”˜‡ƒ”‡•—‡†Ǥ ‘™‡˜‡”ǡ ‹ –Š‡ ƒ˜‡”ƒ‰‡ ƒ„•‘Ž—–‡ ‘–”‹„—–‹‘ ȋƒ• ƒŽ•‘ ƒŽ —Žƒ–‡† „› –Š‡ ƒ—–Š‘”• ‘ˆ –Š‹• ‡–Š‘†Ȍǡ ƒ ”‡ƒŽ ‹’”‡••‹‘‹•‰‹˜‡‘Š‘™— Šƒ‡–ƒ„‘Ž‹–‡Šƒ•ƒ –—ƒŽŽ› ‘–”‹„—–‡†–‘–Š‡–”ƒ•‹–‹‘ˆ”‘–Š‡ •–ƒ–‡ƒ––αͲ–‘–Š‡‡™•–‡ƒ†›•–ƒ–‡•‹ ‡„‘–Š’‘•‹–‹˜‡ƒ†‡‰ƒ–‹˜‡ƒ”‡ƒ•ƒ”‡ƒ†‡’‘•‹–‹˜‡ƒ†–Š‡ •—‡†Ǥ Š‹• –‘–ƒŽ ‘–”‹„—–‹‘ Šƒ• „‡‡ —•‡† ˆ‘” ‹‰ ͷǦ Ǥ  ‹‰ ͷǦ  –Š‡› ƒ”‡ †‡’‹ –‡† ƒ• ’‡” ‡–ƒ‰‡•‘ˆ–Š‡•—‘ˆƒŽŽ–‘–ƒŽ ‘–”‹„—–‹‘•‘ˆ–Š‡‡–ƒ„‘Ž‹–‡•–Šƒ– ‘–”‹„—–‡†–‘–Š‡”ƒ–‡‹ “—‡•–‹‘ǡƒ ‘”†‹‰–‘ǣ

ഥ ೡ ȁ஀ȁ೉ ೕ ȋͳ͸Ȍ σ ȁ஀ഥȁೡ ೕ ೉ೕ Š‡”‡ˆ‘”‡ǡ–Š‡”‡Žƒ–‹˜‡ƒ˜‡”ƒ‰‡ƒ„•‘Ž—–‡ ‘–”‹„—–‹‘•‘ˆƒŽŽ–Š‡ ‘–”‹„—–‹‰‡–ƒ„‘Ž‹–‡•’‡””‡ƒ –‹‘ •——’–‘‘‡ǣ ഥ ೡ ȁ஀ȁ೉ σ ೕ = 1 ȋͳ͹Ȍ ௝ σ ȁ஀ഥȁೡ ೕ ೉ೕ

Acknowledgements Š‡ƒ—–Š‘”•Ž‹‡–‘–Šƒ‰‹‡•œƒ‡‰”œ›ˆ‘” ƒ”‡ˆ—Žƒ†‹†‡’‡†‡– Š‡ ‹‰‘ˆ–Š‡ ‘’—–ƒ–‹‘ƒŽƒƒŽ›•‹•Ǥ  References ͳǤ ‹ƒŽ†‘ ǡ ƒ–‡” ǡ ‡‡––  Ǥ ƒ––›  ‹† ‘š‹†ƒ–‹‘ ‹•‘”†‡”•Ǥ — ‡˜ Š›•‹‘ŽǤ ʹͲͲʹǢ͸ͶȋͳȌǣͶ͹͹ȂͷͲʹǤ ʹǤ ‘—–‡ ǡ ‹‘Žƒ–‡ ǡ ‡–—”ƒ  Ǥǡ ƒ†‡”•  Ǥ Š‡ ‹‘ Š‡‹•–”› ƒ† Š›•‹‘Ž‘‰› ‘ˆ ‹–‘ Š‘†”‹ƒŽ ƒ––›  ‹† ȾǦš‹†ƒ–‹‘ ƒ† –• ‡‡–‹  ‹•‘”†‡”•Ǥ — ‡˜ Š›•‹‘ŽǤ ʹͲͳ͸Ǣ͹ͺȋͳȌǣʹ͵ȂͶͶǤ ͵Ǥ ‡”• ǡ‡‹Œ‰‘—† ǡƒ–‡”Šƒ ǡ ‡”˜‡” ǡ˜ƒ†‡‡”‰ǡƒ—‡” ǡ‹– Ǥ Š‡ƒ–—”ƒŽŠ‹•–‘”›‘ˆ‡†‹—Ǧ Šƒ‹ƒ ›Ž‘†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›‹–Š‡‡–Š‡”Žƒ†•ǣ Ž‹‹ ƒŽ’”‡•‡–ƒ–‹‘ƒ†‘—– ‘‡Ǥ ‡†‹ƒ–”ǤʹͲͲ͸ǢͳͶͺȋͷȌǤ ͶǤ ‡”• ǡ‘‡”ǡ••‡ǡ‘•ǡ—‹–‡” ǡƒ–‡”Šƒ ǡ‹‡œ‡Ǧ‘‹‰ǡƒ†‡”• ǡ ‘†‡‡Ž ǡ‘‡„‡” ǡ‡ƒ–‡ǡ‹– ǡ‡‹Œ‰‘—† Ǥ‡‘ƒ–ƒŽ• ”‡‡‹‰ˆ‘”‡†‹—Ǧ

ͷ͹ Chapter 2

Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋȌ †‡ˆ‹ ‹‡ › ‹ Š‡ ‡–Š‡”Žƒ†•ǣ Š‡ ‹’‘”–ƒ ‡ ‘ˆ ‡œ›‡ƒƒŽ›•‹•–‘ƒ• ‡”–ƒ‹–”—‡†‡ˆ‹ ‹‡ ›Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͲͺǢ͵ͳȋͳȌǣͺͺȂͻ͸Ǥ ͷǤ ‘—™ǡ‹– ǡ‹‡œ‡Ǧ‘‹‰ǡ‘•‰”ƒƒˆǦ†‡‘‡”ǡ ‡”†‹‰ǡ‡‹Œ‰‘—†Ǧ ǡ‡”•  Ǥ  ˜‹–”‘ ƒ† ‹ ˜‹˜‘ ‘•‡“—‡ ‡• ‘ˆ ˜ƒ”‹ƒ– ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ‰‡‘–›’‡•Ǥ”’Šƒ‡– ƒ”‡‹•ǤʹͲͳ͵ǢͺȋͳȌǣͶ͵Ǥ ͸Ǥ ‘—™ǡ‹– ǡ†‡”‹‡•ǡ†‡Ž‡” ǡ‘• Šǡ‹••‡” ǡ—Ž†‡” ǡ—„‹‘Ǧ ‘œƒŽ„‘ ǡŽ˜‡”•ǡ‹‡œ‡Ǧ‘‹‰ǡƒ†‡”• ǡƒ–‡”Šƒ ǡ‡‹Œ‰‘—†Ǧ ǡ‡”• Ǥ‹• •–”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 ͹Ǥ ‡ƒŽ‡• ǡŠ‘’•‘ ǡƒ••‘—† ǡƒŠ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  Š‡”‹–‡–ƒ„‹•ǤͳͻͻͶǢͳ͹ȋͳȌǣ͹ͶȂͺͲǤ ͺǤ ˜ƒ—‡ǡ‹‘• ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ†‡‡•–‡ ǡ‘—™ǡ ‘—–‡ǡ ”‘‡ ǡ ”ƒ„ ǡ ‡‹Œ‰‘—†  ǡ ƒ‡” Ǥ ‹‘ Š‡‹ ƒŽ ‘’‡–‹–‹‘ ƒ‡• ƒ––›Ǧ ‹† ‡–ƒǦ š‹†ƒ–‹‘—Ž‡”ƒ„Ž‡–‘—„•–”ƒ–‡˜‡”Ž‘ƒ†Ǥ‘‘’—–‹‘ŽǤʹͲͳ͵ǢͻȋͺȌǣʹȂͻǤ ͻǤ ˜ƒ—‡ǡ‘Ž‡”Ǧ‘—™ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ‘Ž–‡”• ǡ˜ƒ‹Œ– ǡƒ”–‹‡•Ǧ  ǡ ‹‡œ‡Ǧ‘‹‰ ǡ ‡‹‡” ǡ ‡”‡–‹‡” ǡ ”‘‡ ǡ ‡‹Œ‰‘—† Ǧ ǡ ‡”•  ǡ ƒ‡” Ǥ ‹˜‹‰ ‘ –Š‡ ‡†‰‡ǣ •—„•–”ƒ–‡ ‘’‡–‹–‹‘ ‡š’Žƒ‹• Ž‘•• ‘ˆ ”‘„—•–‡•• ‹ ‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧƒ ‹†‘š‹†ƒ–‹‘†‹•‘”†‡”•Ǥ‹‘ŽǤʹͲͳ͸ǢͳͶȋͳȌǣͳͲ͹Ǥ ͳͲǤ ‘˜‡•ǡ••Š‡” ǡ‘Žƒ†ǡŽ‡–œǡ‘•‡†ƒŽ‡ǡ Žƒ›‡˜ƒǡƒ‹ ǡ–‡˜‡•ǡ›  ǡ ‡™‰ƒ”†ǡ ‘’ƒ• Š— ǡ —‘‹‘Ǥ ‹–‘ Š‘†”‹ƒŽ ˜‡”Ž‘ƒ† ƒ†  ‘’Ž‡–‡ ƒ––›  ‹† š‹†ƒ–‹‘‘–”‹„—–‡–‘‡Ž‡–ƒŽ—• Ž‡ •—Ž‹‡•‹•–ƒ ‡Ǥ‡ŽŽ‡–ƒ„ǤʹͲͲͺǢ͹ȋͳȌǣͶͷȂͷ͸Ǥ ͳͳǤ ‘ˆ‡›” Ǥ‡–ƒ„‘Ž‹  ‘–”‘ŽƒƒŽ›•‹•‹ƒ—–•Š‡ŽŽǤ ǣ”‘ ‡‡†‹‰•‘ˆ–Š‡ʹ† –‡”ƒ–‹‘ƒŽ ‘ˆ‡”‡ ‡‘•›•–‡•„‹‘Ž‘‰›ǤʹͲͲͳǤ’ǤʹͻͳȂ͵ͲͲǤ ͳʹǤ ”—‰‰‡ƒ ǡ ‘‘‰‡”† ǡ ‡•–‡”Š‘ˆˆ  Ǥ Š‡ —Ž–‹ˆƒ”‹‘—• •Š‘”–Ǧ–‡” ”‡‰—Žƒ–‹‘ ‘ˆ ƒ‘‹— ƒ••‹‹Žƒ–‹‘ ‘ˆ • Š‡”‹ Š‹ƒ ‘Ž‹ǣ ‹••‡ –‹‘ —•‹‰ ƒ ‹ •‹Ž‹ ‘ ”‡’Ž‹ ƒǤ  Ǥ ʹͲͲͷǢʹ͹ʹȋͺȌǣͳͻ͸ͷȂͺͷǤ ͳ͵Ǥ —”• Ǥ‘–”‘ŽƒƒŽ›•‹•‘ˆ‡–ƒ„‘Ž‹ •›•–‡•Ǥ”‡†•‹‘ Š‡ ‹ǤͳͻͺͷǢͳͲȋͳȌǣͳ͸Ǥ ͳͶǤ ‡ŽŽǤ‡–ƒ„‘Ž‹  ‘–”‘ŽƒƒŽ›•‹•ǣƒ•—”˜‡›‘ˆ‹–•–Š‡‘”‡–‹ ƒŽƒ†‡š’‡”‹‡–ƒŽ†‡˜‡Ž‘’‡–Ǥ ‹‘ Š‡ ǤͳͻͻʹǢʹͺ͸ȋ–ʹǣ͵ͳ͵Ȃ͵ͲǤ ͳͷǤ Š‘Ž‘†‡‘ǡƒ—”‘ ǡ‡•–‡”Š‘ˆˆ Ǥ‘–”‘Ž„›‡œ›‡•ǡ ‘‡œ›‡•ƒ† ‘•‡”˜‡† ‘‹‡–‹‡•Ǥ  ‰‡‡”ƒŽ‹•ƒ–‹‘ ‘ˆ –Š‡ ‘‡ –‹˜‹–› –Š‡‘”‡ ‘ˆ ‡–ƒ„‘Ž‹  ‘–”‘Ž ƒƒŽ›•‹•Ǥ —”  ‹‘ Š‡ǤͳͻͻͶ –ͳǢʹʹͷȋͳȌǣͳ͹ͻȂͺ͸Ǥ ͳ͸Ǥ ‘—–‡ǡƒ†‡”• Ǥ‰‡‡”ƒŽ‹–”‘†— –‹‘–‘–Š‡„‹‘ Š‡‹•–”›‘ˆ‹–‘ Š‘†”‹ƒŽˆƒ––› ƒ ‹†ȾǦ‘š‹†ƒ–‹‘Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͲǢ͵͵ȋͷȌǣͶ͸ͻȂ͹͹Ǥ ͳ͹Ǥ œƒ‹ǡ Š‹†ƒǡ”‹‹ǡƒƒ‘–‘ǡ ƒ•Š‹‘–‘Ǥ‘˜‡Žˆƒ––›ƒ ‹†„‡–ƒǦ‘š‹†ƒ–‹‘‡œ›‡•‹ ”ƒ– Ž‹˜‡” ‹–‘ Š‘†”‹ƒǤ Ǥ —”‹ˆ‹ ƒ–‹‘ ƒ† ’”‘’‡”–‹‡• ‘ˆ ˜‡”›ǦŽ‘‰Ǧ Šƒ‹ ƒ ›ŽǦ ‘‡œ›‡  †‡Š›†”‘‰‡ƒ•‡Ǥ ‹‘ŽŠ‡Ǥͳͻͻʹ ƒͳͷǢʹ͸͹ȋʹȌǣͳͲʹ͹Ȃ͵͵Ǥ ͳͺǤ ‡†ƒǡƒ—”ƒǦ‹‡†ƒ•ǡƒƒƒǤ—”‹ˆ‹ ƒ–‹‘ƒ†Šƒ”ƒ –‡”‹œƒ–‹‘‘‰Ǧ Šƒ‹ ›ŽǦ‘ ‡Š›†”‘‰‡ƒ•‡•ˆ”‘ƒ–‹˜‡”‹–‘ Š‘†”‹ƒǤŽ‘‘†ǤͳͻͺͷǢ ͳͻǤ —‘‹‘Ǥ‡–ƒ„‘Ž‹ ‹ˆŽ‡š‹„‹Ž‹–›ǣŠ‡‹–‘ Š‘†”‹ƒŽ‹†‡ ‹•‹‘Ž‡ƒ†•–‘‡–ƒ„‘Ž‹ ‰”‹†Ž‘ Ǥ ‡ŽŽǤʹͲͳͶǢͳͷͻȋ͸Ȍǣͳʹͷ͵Ȃ͸ʹǤ ʹͲǤ ’—”™ƒ› ǡ Š‡””ƒ––  ƒǡ ‘‰•‘  ǡ ‰‹—• Ǥ ȝǦš‹†ƒ–‹‘ ‹ ƒ– ‡’ƒ–‘ ›–‡ —Ž–—”‡•Ǥ ‹‘ Š‡ Ǥͳͻͻ͹Ǣ͵ʹ͵ǣͳͳͻȂʹʹǤ ʹͳǤ ƒ–‘Ǥ‘–”‘Ž‘ˆ‹–‘ Š‘†”‹ƒŽ„Ǧ‘š‹†ƒ–‹‘ˆŽ—šǤʹͲͲʹǢͶͳǣͳͻ͹Ȃʹ͵ͻǤ ʹʹǤ ‹ ǡ  Š—Žœ Ǥ ͶǦ”‘‘ǦʹǦ‘ –‡‘‹  ƒ ‹† •’‡ ‹ˆ‹ ƒŽŽ› ‹ƒ –‹˜ƒ–‡• ͵Ǧ‡–‘ƒ ›ŽǦ‘ –Š‹‘Žƒ•‡ ƒ† –Š‡”‡„›ˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘‹”ƒ–Ž‹˜‡”‹–‘ Š‘†”‹ƒǤ‹‘ Š‡‹•–”›ǤͳͻͺͺǢʹ͹ȋͳ͸ȌǣͷͻͻͷȂ͸ͲͲͲǤ ʹ͵Ǥ Ž‘™‡ǡ Š—Žœ ǤͶǦ”‘‘ ”‘–‘‹  ‹†ǡƒˆˆ‡ –‹˜‡ Š‹„‹–‘”‘ˆ ƒ––› ‹†š‹†ƒ–‹‘ƒ† ‡–‘‡‘†›‡‰”ƒ†ƒ–‹‘‹ƒ– ‡ƒ”–‹–‘ Š‘†”‹ƒǤ ‹‘ŽŠ‡ǤͳͻͺʹǢȋͳͲȌǣͷͶͲͺȂͳ͵Ǥ

ͷͺ The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation

ʹͶǤ —œ Ǥ ˜ƒŽ—ƒ–‹‘ ‘ˆ ‡Ž‡ –”‘Ǧ–”ƒ•ˆ‡” ˆŽƒ˜‘’”‘–‡‹ ƒ† ƒŽ’ŠƒǦŽ‹’‘ƒ‹†‡ †‡Š›†”‘‰‡ƒ•‡ ”‡†‘š •–ƒ–‡• „› –™‘Ǧ Šƒ‡Ž ˆŽ—‘”‹‡–”› ƒ† ‹–• ƒ’’Ž‹ ƒ–‹‘ –‘ –Š‡ ‹˜‡•–‹‰ƒ–‹‘ ‘ˆ „‡–ƒǦ ‘š‹†ƒ–‹‘ǤǦ‹‘‡‡”‰ǤͳͻͺͺǢͻ͵ʹȋȌǣͺȂͳ͸Ǥ ʹͷǤ ‘‹Ž‘˜ǡ‘‹Ž‘˜ƒǡŠƒǡ ƒ‰‘’‹ƒǡ‡––ƒ‹‡„ǡ‹ǡ‡Ž‹ ‹ ǡ ƒŒ ǡƒ•‡› ǡ 2 ‘”–‘’ƒ••‹ Ǥ ’Ͷ͸Š  Š‹„‹–• Š‹‘Žƒ•‡ ƒ† ‹’‹† š‹†ƒ–‹‘ ‹ ‹–‘ Š‘†”‹ƒǤ  ‹‘Ž Š‡Ǥ ʹͲͳ͸ǢʹͻͳȋʹͶȌǣͳʹͷ͹ͷȂͺͷǤ ʹ͸Ǥ ‘ˆˆƒ–ǡŠƒ–‹ƒǡ‰—›‡ǡ› Šǡƒ‰ǡƒ‰ǡ Žƒ›‡˜ƒǡ ƒǡ ‹”• Š‡›ǡ Žƒ›’‘‘Žǡ‡‹ˆ‡”–Ǥ ›ŽǦ‘–Š‹‘‡•–‡”ƒ•‡Ǧʹˆƒ ‹Ž‹–ƒ–‡•‹–‘ Š‘†”‹ƒŽˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘ ‹–Š‡Ž‹˜‡”Ǥ ‹’‹†‡•ǤʹͲͳͶǢͷͷȋͳʹȌǣʹͶͷͺȂ͹ͲǤ ʹ͹Ǥ —– ǡ Ž‡š•‘ Ǥ Š‡ ”‘Ž‡  ›ŽǦ‘ –Š‹‘‡•–‡”ƒ•‡• ’Žƒ› ‹ ‡†‹ƒ–‹‰ ‹–”ƒ ‡ŽŽ—Žƒ” Ž‹’‹† ‡–ƒ„‘Ž‹•Ǥ”‘‰‹’‹†‡•ǤʹͲͲʹǢͶͳȋʹȌǣͻͻȂͳ͵ͲǤ ʹͺǤ ‹ŽŽƒ†‡” ǡ ‘”†•–”ڏ ǡ ‡‹ŽŽ› ǡ –”‘œ› ǡ ƒ ‡Ž†Š‘˜‡ ǡ —– ǡ Ž‡š•‘  Ǥ  ›ŽǦ‘–Š‹‘‡•–‡”ƒ•‡ͻȋͻȌ‹‘—•‡ƒ›’”‘˜‹†‡ƒ‘˜‡ŽŽ‹„‡–™‡‡ˆƒ––›ƒ ‹†ƒ† ƒ‹‘ƒ ‹†‡–ƒ„‘Ž‹•‹‹–‘ Š‘†”‹ƒǤ‡ŽŽ‘Ž‹ˆ‡ ‹ǤʹͲͳͶǢ͹ͳȋͷȌǣͻ͵͵ȂͶͺǤ ʹͻǤ ŽŽ‹• ǡ ‘™ƒ ǡ ‘Žˆ‰ƒ‰  Ǥ ‡–ƒ„‘Ž‹  ƒ† ‹••—‡Ǧ’‡ ‹ˆ‹  ‡‰—Žƒ–‹‘ ‘ˆ  ›ŽǦ‘ ‡–ƒ„‘Ž‹•Ǥ‘‡ǤʹͲͳͷǢͳͲȋ͵Ȍǣ‡Ͳͳͳ͸ͷͺ͹Ǥ ͵ͲǤ ”ƒ„ƒ ƒǡ‹‡”œ ŠƒŽ•ƒǡ‡ƒǡ‡‹••Ǥ‡‰—Žƒ–‹‘‘ˆ‡–‘‡‘†›‡–ƒ„‘Ž‹•ƒ†–Š‡ ‘Ž‡‘ˆȽǤ – ‘Ž ‹ǤʹͲͳ͸Ǣͳ͹ȋͳʹȌǣʹͲͻ͵Ǥ ͵ͳǤ ‘ ‹–Ž‡ ȏ –‡”‡–ȐǤ ȏ ‹–‡† ʹͲͳ͹ ‡„ ʹ͸ȐǤ ˜ƒ‹Žƒ„Ž‡ ˆ”‘ǣ Š––’ǣȀȀ™™™Ǥ’”‘–‡‹ƒ–Žƒ•Ǥ‘”‰Ȁ ͲͲͲͲͲͲ͹ͷʹ͵ͻǦͳȀ–‹••—‡ȀŽ‹˜‡” ͵ʹǤ ‹††Ž‡–‘ Ǥ Š‡ ‘š‘ƒ ›ŽǦ ‘‡œ›‡  –Š‹‘Žƒ•‡• ‘ˆ ƒ‹ƒŽ –‹••—‡•Ǥ ‹‘ Š‡ Ǥ ͳͻ͹͵Ǣͳ͵ʹȋͶȌǣ͹ͳ͹Ȃ͵ͲǤ ͵͵Ǥ ‹††Ž‡–‘ǤŠ‡‹‡–‹ ‡ Šƒ‹•ƒ†’”‘’‡”–‹‡•‘ˆ–Š‡ ›–‘’Žƒ•‹ ƒ ‡–‘ƒ ‡–›ŽǦ ‘‡œ›‡ –Š‹‘Žƒ•‡ˆ”‘”ƒ–Ž‹˜‡”Ǥ‹‘ Š‡ Ǥͳͻ͹ͶǢͳ͵ͻǣͳͲͻȂʹͳǤ ͵ͶǤ ‹– Š‡ŽŽ ǡ ƒ—–Š‹‡”ǡ‡•‹’Ž‡ǡƒ‰ǡƒ‡”ǡ—”‡•Š‹ Ǥ ‡”‡†‹–ƒ”›ƒ†ƒ “—‹”‡† †‹•‡ƒ•‡•‘ˆƒ ›ŽǦ ‘‡œ›‡‡–ƒ„‘Ž‹•Ǥ‘Ž ‡‡–‡–ƒ„ǤʹͲͲͺǢͻͶȋͳȌǣͶȂͳͷǤ ͵ͷǤ ‹‡”‘–‡ ǡ ƒ”ƒ†‹‡• ǡ ‘†‹• ‘ ǡ ƒ”‘„„‹‘ ǡ ƒŽ‹‡”‹ Ǥ  ‘˜‡Ž ‡„‡” ‘ˆ ‘Ž—–‡ ƒ””‹‡” ƒ‹Ž› ʹͷ ȋʹͷͶʹȌ • ƒ ”ƒ•’‘”–‡” ‘ˆ ‘‡œ›‡  ƒ† †‡‘•‹‡ ͵ ǯǡͷ ǯǦ ‹’Š‘•’Šƒ–‡‹ —ƒ‹–‘ Š‘†”‹ƒǤ ‹‘ŽŠ‡ǤʹͲͲͻǢʹͺͶȋʹ͹ȌǣͳͺͳͷʹȂͻǤ ͵͸Ǥ ”‹‹˜ƒ•ƒǡƒ”ƒ–ƒ•Š˜‹Ž‹ǡ™ƒƒ‰ƒ‡”ǡƒ‘ǡ‘Ž‘„‡ŽŽ‹ǡ Šƒƒ’ǡ‘ŽŽ‡ǡ ‘†‰‘”懍ǡ‘•‡  ǡ‡–‘˜‹ ǡ ƒ›ˆŽ‹ Ǥš–”ƒ ‡ŽŽ—Žƒ”ͶǯǦŠ‘•’Š‘’ƒ–‡–Š‡‹‡‹•ƒ•‘—” ‡ ˆ‘”‹–”ƒ ‡ŽŽ—Žƒ”‘‡œ›‡•›–Š‡•‹•Ǥƒ–Š‡‹‘ŽǤʹͲͳͷǢͳͳȋ—‰—•–ȌǣͳȂʹ͸Ǥ ͵͹Ǥ Šƒ•‡Ž†‹ ǡ‹–Šǡ‡–ƒ„ǡŽŠƒŽ‹†‹ ǡ—‡”•ǡŽ•‡†ƒ‹”› ǡ‹‘‰ǡ —’–ƒǡ Ž—”ƒ›ƒ Ǥ —–ƒ–‹‘ ‘ˆ –Š‡ ‹–‘ Š‘†”‹ƒŽ ƒ””‹‡” ʹͷͶʹ ƒ—•‡• ƒ ‘˜‡Ž ˆ‘” ‘ˆ ‹–‘ Š‘†”‹ƒŽ›‘’ƒ–Š›‹Š—ƒ•Ǥ — ‡‡–ǤʹͲͳ͸Ǣͳ͵ͷȋͳȌǣʹͳȂ͵ͲǤ ͵ͺǤ –ƒŽ‡› ǡ ƒŽ‡ ǡ ‘ƒ–‡• ǡ ƒŽŽ ǡ ‘”‡› ǡ ƒ‰ ǡ ‡ŽŽ‡›  ǡ ‘œƒŽ‡• ǡ ‹ŽŽ‹ƒ•‘ ǡ ƒ‡” Ǥ ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ †‡ˆ‹ ‹‡ › ‹ Š‹Ž†”‡ ™‹–Š ‘Ǧ‡–‘–‹ Š›’‘‰Ž› ‡‹ƒƒ†Ž‘™ ƒ”‹–‹‡Ž‡˜‡Ž•Ǥ‡†‹ƒ–”‡•Ǥͳͻͺ͵Ǣͳ͹ȋͳͳȌǣͺ͹͹ȂͺͶǤ ͵ͻǤ ƒŽ‡ǡƒ–•Šƒ™ǡ‘ƒ–‡•ǡ ”‡”ƒ ǡ ‘‘†ƒ ǡ‹‰Š ǡ–ƒŽ‡›ƒǤ‘‰Ǧ Šƒ‹ ƒ ›Ž ‘‡œ›‡†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›ǣƒ‹Š‡”‹–‡† ƒ—•‡‘ˆ‘‡–‘–‹ Š›’‘‰Ž› ‡‹ƒǤ ‡†‹ƒ–”‡•ǤͳͻͺͷǢͳͻǣ͸͸͸Ȃ͹ͳǤ ͶͲǤ ”‡‡ ǡ –ƒŽ‡›  ƒǡ ƒŽ‡ ǡ ‡‘’‘Ž† ǡ ›ƒ• Ǥ ›’‘‰Ž› ‡‹ƒǡ Š›’‘–‘‹ƒǡ ƒ† ƒ”†‹‘›‘’ƒ–Š›ǣ –Š‡ ‡˜‘Ž˜‹‰ Ž‹‹ ƒŽ ’‹ –—”‡ ‘ˆ Ž‘‰Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ †‡ˆ‹ ‹‡ ›Ǥ‡†‹ƒ–”‹ •ǤͳͻͻͳǢͺ͹ȋ͵Ȍǣ͵ʹͺȂ͵͵Ǥ ͶͳǤ ƒ‡”ƒ•  ǡ ƒ ‡‡‰Š‡Ž ǡ ‡‹• ǡ ‹ ‘Žƒ› ǡ ”‘’‡”• ǡ ‘—–‡ Ǥ ƒ”‹–‹‡ •—’’Ž‡‡–ƒ–‹‘ ƒ––‡—ƒ–‡• ›‘ ƒ”†‹ƒŽ Ž‹’‹† ƒ ——Žƒ–‹‘ ‹ Ž‘‰Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡‘ ‘—–‹ ‡Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳ͵Ǣ͵͸ȋ͸Ȍǣͻ͹͵ȂͺͳǤ ͶʹǤ ’‹‡‡”‘‡––‡” ǡ ƒ•–‹ ǡ ‹ŽŽ‹‰Šƒ ǡ ‘””‹• ǡ ‹Œ„—”‰ ǡ ‹Ž ‡ Ǥ —””‡– ‹••—‡• ”‡‰ƒ”†‹‰ –”‡ƒ–‡– ‘ˆ ‹–‘ Š‘†”‹ƒŽ ˆƒ––› ƒ ‹† ‘š‹†ƒ–‹‘ †‹•‘”†‡”•Ǥ  Š‡”‹– ‡–ƒ„ ‹•Ǥ

ͷͻ Chapter 2

ʹͲͳͲǢ͵͵ȋͷȌǣͷͷͷȂ͸ͳǤ Ͷ͵Ǥ ’‹‡‡”‘‡––‡”ǡ‘‘†Ǥ‹–‘ Š‘†”‹ƒŽˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘†‹•‘”†‡”•ǣƒ–Š‘’Š›•‹‘Ž‘‰‹ ƒŽ •–—†‹‡•‹‘—•‡‘†‡Ž•Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͲǢ͵͵ȋͷȌǣͷ͵ͻȂͶ͸Ǥ ͶͶǤ  Š‘‘‡ƒ ǡ Š–‡”ƒ’ǡ”‰ƒǡ‘‡–‡”•ǡ ‘—–‡ǤŽƒ•ƒƒ ›Ž ƒ”‹–‹‡• ‹ƒ†‡“—ƒ–‡Ž› ”‡ˆŽ‡ – –‹••—‡ ƒ ›Ž ƒ”‹–‹‡ ‡–ƒ„‘Ž‹•Ǥ ‹‘ Š‹ ‹‘’Š›•  –ƒ Ǧ ‘Ž ‡ŽŽ ‹‘Ž ‹’‹†•ǤʹͲͳͶǢͳͺͶͳȋ͹Ȍǣͻͺ͹ȂͻͶǤ ͶͷǤ ƒ–‘ ǡ —”•„› ǡ ‹††Ž‡–‘ ǡ ‘—”ˆƒ”œƒ ǡ ‹ŽŽ• ǡ ‘Š•‘ ǡ ƒ”–Ž‡  Ǥ Š‡ ‹–‘ Š‘†”‹ƒŽ–”‹ˆ— –‹‘ƒŽ’”‘–‡‹ǣ ‡–”‡‘ˆƒȾǦ‘š‹†ƒ–‹‘‡–ƒ„‘Ž‘ǫ‹‘ Š‡‘ ”ƒ•Ǥ ʹͲͲͲ ‡„ͳǢʹͺȋʹȌǣͳ͹͹ǦͳͺʹǤ Ͷ͸Ǥ  Š‹†ƒ ǡ œƒ‹ ǡ ”‹‹ ǡ ƒ•Š‹‘–‘ Ǥ ‘˜‡Ž ˆƒ––› ƒ ‹† ȾǦ‘š‹†ƒ–‹‘ ‡œ›‡• ‹ ”ƒ– Ž‹˜‡” ‹–‘ Š‘†”‹ƒǣ Ǥ —”‹ˆ‹ ƒ–‹‘ ƒ† ’”‘’‡”–‹‡• ‘ˆ ‡‘›ŽǦ ‘‡œ›‡  ȋ‘Ȍ Š›†”ƒ–ƒ•‡Ȁ͵Ǧ Š›†”‘š›ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡Ȁ͵Ǧ‡–‘ƒ ›ŽǦ‘ –Š‹‘Žƒ•‡ –”‹ˆ— –‹‘ƒŽ ’”‘–‡‹Ǥ  ‹‘Ž Š‡Ǥ ͳͻͻʹǢʹ͸͹ȋʹȌǣͳͲ͵ͶȂͶͳǤ Ͷ͹Ǥ •Š‹ƒ™ƒǡ•— Š‹›ƒǡ›ƒƒǡ•—ƒƒǡ‘”‹ƒ™ƒǤ–”— –—”ƒŽ„ƒ•‹•ˆ‘” Šƒ‡ŽŽ‹‰ ‡ Šƒ‹•‘ˆƒˆƒ––›ƒ ‹†ȾǦ‘š‹†ƒ–‹‘—Ž–‹‡œ›‡ ‘’Ž‡šǤ ǤʹͲͲͶǢʹ͵ȋͳͶȌǣʹ͹ͶͷȂͷͶǤ ͶͺǤ —‡‰‹ ǡ ”‡”‡ Ǥ ‹†‹‰ ‘ˆ –Š‡ ‡œ›‡• ‘ˆ ˆƒ––› ƒ ‹† „‡–ƒǦ‘š‹†ƒ–‹‘ ƒ† •‘‡ ”‡Žƒ–‡† ‡œ›‡•–‘’‹‰Š‡ƒ”–‹‡”‹–‘ Š‘†”‹ƒŽ‡„”ƒ‡Ǥ ‹‘ŽŠ‡ǤͳͻͺͶ —ŽʹͷǢʹͷͻȋͳͶȌǣͺ͹ͶͺȂ ͷʹǤ ͶͻǤ ƒ†ƒǡŠ‡ƒ† ǡ’”‡ Š‡” ǡ Š—Žœ ǡ‘‡Ǥ˜‹†‡ ‡ˆ‘”‹–‡”‡†‹ƒ–‡ Šƒ‡Ž‹‰‹ ‹–‘ Š‘†”‹ƒŽȾǦ‘š‹†ƒ–‹‘Ǥ‘ŽǤʹ͹Ͳǡ ‘—”ƒŽ‘ˆ‹‘Ž‘‰‹ ƒŽŠ‡‹•–”›ǤͳͻͻͷǤ’Ǥͷ͵ͲȂͷǤ ͷͲǤ ‹ƒ‰ǡ‡ǡŠƒ‰ǡ Š—Žœ Ǥ ’ƒ –‘ˆ–Š‡‹–”ƒ‹–‘ Š‘†”‹ƒŽ‡œ›‡‘”‰ƒ‹œƒ–‹‘‘ ˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘Ǥ‹‘ Š‡‘ ”ƒ•ǤʹͲͲͳǢʹͻȋ–ʹȌǣʹ͹ͻȂͺʹǤ ͷͳǤ ƒ–‘ ǡ ƒ”–Ž‡–– ǡ ‘—”ˆƒ”œƒ Ǥ –‡”‡†‹ƒ–‡• ‘ˆ ›‘ ƒ”†‹ƒŽ ‹–‘ Š‘†”‹ƒŽ ȾǦ‘š‹†ƒ–‹‘ǣ ‘••‹„Ž‡ Šƒ‡ŽŽ‹‰‘ˆ ƒ†‘ˆ‘‡•–‡”•Ǥ‹‘ Š‹‹‘’Š›• –ƒǦ‘Ž‡ŽŽ‹‘Ž‹’‹†•Ǥ ͳͻͻͻǢͳͶ͵͹ȋ͵ȌǣͶͲʹȂͺǤ ͷʹǤ ——•Š‹ƒ ǡ ‡ ‡”  Ǥǡ †‡”•‘ ǡ ’‹˜‡› Ǥ —„•–”ƒ–‡ Šƒ‡Ž‹‰ ‘ˆ   ƒ† „‹†‹‰‘ˆ†‡Š›†”‘‰‡ƒ•‡•–‘ ‘’Ž‡š Ǥ ‹‘ŽŠ‡ǤͳͻͺͻǢʹ͸ͶȋʹͺȌǣͳ͸Ͷͺ͵ȂͺǤ ͷ͵Ǥ —‡‰‹ǡ”‡”‡Ǥ‘’Ž‡š „‹†••‡˜‡”ƒŽ‹–‘ Š‘†”‹ƒŽǦ ‘—’Ž‡††‡Š›†”‘‰‡ƒ•‡•Ǥ  ‹‘ŽŠ‡ǤͳͻͺͶǢʹͷͻȋʹͶȌǣͳͷͲͶͲȂͷǤ ͷͶǤ ƒ‰ǡ‘Š•‡ǡ‹ŠƒŽ‹ ǡ ‘‡–œƒǡ‘ Ž‡› Ǥ˜‹†‡ ‡ˆ‘”’Š›•‹ ƒŽƒ••‘ ‹ƒ–‹‘‘ˆ ‹–‘ Š‘†”‹ƒŽ ˆƒ––› ƒ ‹† ‘š‹†ƒ–‹‘ ƒ† ‘š‹†ƒ–‹˜‡ ’Š‘•’Š‘”›Žƒ–‹‘ ‘’Ž‡š‡•Ǥ  ‹‘Ž Š‡Ǥ ʹͲͳͲǢʹͺͷȋ͵ͻȌǣʹͻͺ͵ͶȂͶͳǤ ͷͷǤ ƒ”ƒ›ƒǡƒ•–‡”ǡ‹”‡ ‹ǡ‹‡”Žǡ–ƒŽ‡›ǡ‹ǡ–ƒŽ‡›ǡ‡‡–– ǤŠ‘”–Ǧ Šƒ‹ ͵ǦŠ›†”‘š›ƒ ›ŽǦ ‘‡œ›‡ ƒ †‡Š›†”‘‰‡ƒ•‡ ƒ••‘ ‹ƒ–‡• ™‹–Š ƒ ’”‘–‡‹ •—’‡”Ǧ ‘’Ž‡š ‹–‡‰”ƒ–‹‰—Ž–‹’Ž‡‡–ƒ„‘Ž‹ ’ƒ–Š™ƒ›•Ǥ‘‡ǤʹͲͳʹǢ͹ȋͶȌǣͳȂͺǤ

͸Ͳ The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation

Supplementary information

AB

2.0 Published model ) 2.0 ) -1 -1 No prom CPT2 2 1.6 No prom VLCAD 1.6 No prom LCAD Published model .gProtein .gProtein

1.2 No prom MCAD -1 1.2 -1 No prom SCAD No prom CROT 0.8 0.8 No promiscuity No prom MSCHAD at MCKAT (μmol.min (μmol.min 0.4 No prom MCKAT 0.4 No prom MTP uptake uptake No promiscuity J J 0.0 0.0 0 50 100 150 200 250 0 100 200 300 400 500 [Palmitoyl-CoA] [Palmitoyl-CoA] CYT ȝ0 CYT ȝ0 S1 Fig. Model versions with full or limited promiscuity. (A)–‡ƒ†›Ǧ•–ƒ–‡—’–ƒ‡ˆŽ—šȋ —’–ƒ‡ǡ‹Ǥ‡Ǥ ƒ –ͳ͸Ȍ ˜‡”•—•ȏ’ƒŽ‹–‘›ŽǦ‘Ȑ‹–Š‡’—„Ž‹•Š‡†‘†‡Žǡ–Š‡‘†‡Ž™‹–Š‘—–’”‘‹• —‹–›ƒ†–Š‡ͻ‘–Š‡”‘†‡Ž• ™‹–Š‡ƒ Š‘‡‡œ›‡™‹–Š‘—–’”‘‹• —‹–›Ǥ(B) —’–ƒ‡˜‡”•—•ȏ’ƒŽ‹–‘›ŽǦ‘Ȑ‹–Š‡’—„Ž‹•Š‡†‘†‡Ž ƒ†–Š‡‘†‡Ž™‹–Š‘—–’”‘‹• —‹–›ƒ–ˆ‘”ƒ ‘ ‡–”ƒ–‹‘”ƒ‰‡ˆ”‘Ͳ–‹ŽŽͷͲͲɊǤ

AB 4 CPT1 4.0 CACT 3.5 vmckatC4 2 CPT2 3.0 vmckatC6 VLCAD 2.5 vmckatC8 LCAD 2.0 vmckatC10 0 MCAD 1.5 vmckatC12 SCAD vMCKATC4 vmckatC14 CROT 1.0 -2 MSCHAD 0.5 vmckatC16 vMCKATC6 MCKAT 0.0 Flux control coefficient (-) Flux control coefficient Flux control coefficient (-) Flux control coefficient -4 MTP -0.5 0 50 100 150 200 250 0 50 100 150 200 250 [Palmitoyl-CoA] (ȝM) [Palmitoyl-CoA]CYT ȝ0 CYT

S2 Fig. Flux control coefficients. (A) Ž—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–•‘ˆƒŽŽ‡œ›‡•‹–Š‡‘†‡Ž‘ —’–ƒ‡Ǥ(B) Ž—š ‘–”‘Ž‘ˆ Šƒ‹ǦŽ‡‰–Š•’‡ ‹ˆ‹ Ǧ ƒ–ƒŽ›œ‡†”‡ƒ –‹‘•‘ —’–ƒ‡ǤŠ‡•—‘ˆ–Š‡•‡ Šƒ‹ǦŽ‡‰–Š ௃ೠ೛೟ೌೖ೐ •’‡ ‹ˆ‹  ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–•‡“—ƒŽ•–Š‡‘˜‡”ƒŽŽܥெ௄஺் ǡ™Š‹ Š‹•’Ž‘––‡†‹’ƒ‡ŽǤ  ABC D [Acyl-CoA@ ȝ0 [Enoyl-CoA@ ȝ0 [Hydroxyacyl-CoA@ ȝ0 [Ketoacyl-CoA@ ȝ0 400 800 2500 8 C16 C14 C12 2000 C10 C16 C16 300 C8 600 6 C16 C14 C14 C14 C6 C12 C4 C12 C12 C10 1500 C10 C10 C8 200 400 C8 4 C8 C6 C6 C6 C4 1000 C4 C4

100 200 2 500

0 0 0 0 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 [Palmitoyl-CoA] ȝ0 [Palmitoyl-CoA] ȝ0 [Palmitoyl-CoA] ȝ0 [Palmitoyl-CoA] ȝ0 CYT CYT CYT CYT S3 Fig. Steady-state CoA ester concentrations. –‡ƒ†›Ǧ•–ƒ–‡ ‘ ‡–”ƒ–‹‘•‘ˆ‹–‘ Š‘†”‹ƒŽƒ ›ŽǦ‘ (A)ǡ‡‘›ŽǦ‘(B)ǡŠ›†”‘š›ƒ ›ŽǦ‘(C)ƒ†‡–‘ƒ ›ŽǦ‘‡•–‡”•(D)ƒ•ƒˆ— –‹‘‘ˆȏ’ƒŽ‹–‘›ŽǦ‘Ȑǡ •’‡ ‹ˆ‹‡†’‡” Šƒ‹Ž‡‰–ŠǤ 

͸ͳ Chapter 2

A 25 ȝM [Acyl-CoA@ ȝ0 [Enoyl-CoA@ ȝ0 [Hydroxyacyl-CoA@ ȝ0 [Ketoacyl-CoA@ ȝ0 400 800 2500 8 C16 C16 C16 C16 C14 C14 C14 C14 C12 C12 2000 C12 C12 300 C10 600 C10 C10 6 C10 C8 C8 C8 C8 C6 C6 1500 C6 C6 200 C4 400 C4 C4 4 C4 1000

100 200 2 500

0 0 0 0 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 time (min) time (min) time (min) time (min) B 60 ȝM 400 800 2500 8

C16 C16 C16 C16 C14 C14 2000 C14 C14 300 C12 600 C12 C12 6 C12 C10 C10 C10 C10 C8 C8 1500 C8 C8 C6 C6 C6 C6 200 C4 400 C4 C4 4 C4 1000

100 200 2 500

0 0 0 0 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 time (min) time (min) time (min) time (min)

S4 Fig. Time course of mitochondrial CoA ester concentrations. ‹‡ ‘—”•‡ ‘ˆ ‹–‘ Š‘†”‹ƒŽ ‘ ‡–”ƒ–‹‘•‘ˆƒ ›ŽǦ‘ǡ‡‘›ŽǦ‘ǡŠ›†”‘š›ƒ ›ŽǦ‘ƒ†‡–‘ƒ ›ŽǦ‘ƒˆ–‡”ƒ—’•Š‹ˆ–‘ˆȏ’ƒŽ‹–‘›ŽǦ ‘Ȑˆ”‘ͲǤͳ–‘ʹͷɊ(A) ‘”͸ͲɊ(B)Ǥ

͸ʹ The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation

1.5 vCPT2C16 vMTPC16 vMTPC14 + vMCKATC14 )

-1 vVLCAD16 + vLCADC16 vVLCAD14 + vLCADC14 1.4 vVLCAD12 + vLCADC12 + vMTPC16 vMCKATC14 + vMTPC14 vMCADC12 vMTPC12 + vMCKATC12 1.3 2 .gProtein -1

1.2

1.1 v (μmol.min v

1.0 0 100 200 300 0 100 200 300 0 100 200 300 time (min) time (min) time (min)

1.5 vMTPC12 + MCKATC12 vMCKATC10 + vMTPC10 vMCKATC8 + vMTPC8 )

-1 1.4 vLCADC10 + vMCADC10 vLCADC8 + vMCADC8 vMCADC6 + vSCADC6 vMTPC10 + MCKATC10 vMTPC8 + vMCKATC8 vMCKATC6 1.3 .gProtein -1 1.2

1.1 v (μmol.min v

1.0 0 100 200 300 0 100 200 300 0 100 200 300 time (min) time (min) time (min)

1.5 vMCKATC6 )

-1 vMCADC4 + vSCADC4 1.4 vMCKATC4

1.3 .gProtein -1

1.2

1.1 v (μmol.min v

1.0 0 100 200 300 time (min)

S5 Fig. Time course of the rates of Cn-acyl-CoA production, dehydrogenation and Cn-2-acyl-CoA production per chain length (thus per oxidation cycle) after an upshift of [palmitoyl-CoA]CYT 0.1 to ʹͷɊǤ

1.5 ) -1 1.4 vMCKATC8 + vMTPC8 vMCADC6 + vSCADC6 1.3 vMCKATC6 .gProtein -1 1.2

1.1 v (μmol.min v 1.0 0 100 200 300 time (min) S6 Fig. vMTPC8+vMCKATC6, vMCADC4+vSCADC4 and vMCKATC6 after an upshift of [palmitoyl- CoA]CYT ˆ”‘ͲǤͳ–‘͸ͲɊǤ

͸͵ Chapter 2

) 0.01 C16-Ketoacyl-CoA í C14-Ketoacyl-CoA C12-Ketoacyl-CoA C10-Ketoacyl-CoA C8-Ketoacyl-CoA C6-Ketoacyl-CoA C4-Ketoacyl-CoA

.mgProtein 0 C16-Acyl-CoA 2 í j C14-Acyl-CoA vmckatC6 X C12-Acyl-CoA Ĭ C10-Acyl-CoA C8-Acyl-CoA C6-Acyl-CoA C4-Acyl-CoA (μmol.min -0.01 Free CoA 0 100 200 300 time (min) S7 Fig. Metabolite contributions to vMCKATC6 after a sudden [palmitoyl-CoA]CYT increase from 0.1 –‘͸ͲɊǤ

A 1 B 1 Acetyl-CoA C4-Ketoacyl-CoA C4-Enoyl-CoA C4-Acyl-CoA C4-Acyl-CoA C6-Ketoacyl-CoA C6-Enoyl-CoA C4-acyl-CoA C6-Acyl-CoA C8-Ketoacyl-CoA C4-enoyl-CoA C6-Acyl-CoA C8-Acyl-CoA C8-Enoyl-CoA 0.5 C10-Ketoacyl-CoA 0.5 C10-Acyl-CoA C8-Acyl-CoA C6-acyl-CoA C12-Ketoacyl-CoA C10-Enoyl-CoA C12-Acyl-CoA C10-Acyl-CoA C14-Ketoacyl-CoA C6-enoyl-CoA C14-Acyl-CoA C12-Enoyl-CoA C16-Ketoacyl-CoA C12-Acyl-CoA

C16-Acyl-CoA per chain-length occupancy MCAD

MCKAT occupancy per chain-length occupancy MCKAT 0 0 0 50 100 150 200 250 0 50 100 150 200 250

[Palmitoyl-CoA]CYT ȝ0 [Palmitoyl-CoA]CYT ȝ0 C 1

C4-enoyl-CoA C4-Enoyl-CoA C4-Acyl-CoA 0.5 C6-Enoyl-CoA C6-Acyl-CoA

C6-enoyl-CoA

SCAD occupancy per chain-length per occupancy SCAD 0 0 50 100 150 200 250

[Palmitoyl-CoA]CYT ȝ0 S8 Fig. Fraction of the occupied by each of the indicated metabolites, calculated for MCKAT (A), MCAD (B), and SCAD (C) as a function of [palmitoyl-CoA]CYT.Š‡‘ —’ƒ ›ȋ Ȍ‘ˆƒ ‡œ›‡ȋȌ„›ƒ‡–ƒ„‘Ž‹–‡Œ™ƒ• ƒŽ —Žƒ–‡†„›ǣ ሾ೉ೕሿ  ಼೘ ಶǡ೉ೕ ܱܿܿாǡ௑ ൌ ȋͳͺȌ ೕ ሾ೉ೕሿ ሾ೉ ሿ ൭ଵା ାσ ೖ ൱ ಼೘ ೖ಼೘ ಶǡ೉ೕ ಶǡ೉ೖ ™Š‡”‡†‡‘–‡•ƒ‡–ƒ„‘Ž‹–‡–Šƒ––Š‡‡œ›‡ ƒƒŽ•‘„‹†‹–Š‡•ƒ‡’‘ ‡–ƒ•ŒǤ

͸Ͷ The promiscuous enzyme MCKAT triggers a vicious cycle in fatty-acid beta-oxidation

) 2.0 -1 0.5x CoAt,m 1x CoAt,m 1.6 2x CoAt,m 3x CoAt,m .gProtein -1 1.2 4x CoAt,m 2 5x CoAt,m 0.8 6x CoAt,m 7x CoAt,m (μmol.min 0.4 8x CoAt,m 9x CoAt,m uptake

J 10x CoAt,m 0.0 0 50 100 150 200 250

[Palmitoyl-CoA]CYT ȝ0

S9 Fig. The effect of varying total mitochondrial CoA concentration (CoAt,m) on flux decline.

S1 Appendix. Model without promiscuity. Confer PLoS Comput Biol. 2017;13(4):1–22 for the corresponding script.

S12 Appendix. Calculation of average absolute regulatory contribution.  Confer PLoS Comput Biol. 2017;13(4):1–22 for the corresponding script. 

͸ͷ

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

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 3 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‹•‘—•‡Ǥƒ†‹ ‡™‡”‡‰‹˜‡ƒŠ‹‰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  –Š‡ –”ƒ• ”‹’–‘‡ǡ ™‡ ‹†‡–‹ˆ‹‡† •—„–Ž‡ ƒ†ƒ’–ƒ–‹‘•‹–Š‡‡š’”‡••‹‘‘ˆ‰‡‡•‡ ‘†‹‰‡œ›‡• ƒ–ƒŽ›œ‹‰‘Ǧƒ†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 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‡”‡“—‹”‡†ǤŠ‹Ž†”‡™‹–ŠƒŽ‘••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 ‡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 ”‘†‡–•ǡ–Š‡•‡ƒ”‡˜‡”›ǦŽ‘‰Ǧǡ Ž‘‰Ǧǡ‡†‹—Ǧǡƒ†•Š‘”–Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡ȋǡǡǡƒ†ȌǤ Š—ƒ•ǡ–Š‡’”‘–‡‹ ‘ ‡–”ƒ–‹‘‹•Ž‘™‹–Š‡Ž‹˜‡”ƒ†’Žƒ›•ƒ‹‘””‘Ž‡‹–Š‡   ’ƒ–Š™ƒ›ȏͳǡʹͲȐǤ Š‡ ‘‡œ›‡•‹ ‘–‹ƒ‹†‡ ƒ†‡‹‡ †‹— Ž‡‘–‹†‡ ȋΪȌ 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 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  –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 –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 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  ƒ††‹–‹‘ǡ Š‡’ƒ–‹  ‹–‘ Š‘†”‹ƒŽ ˆ— –‹‘ ™ƒ• 3 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 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 AB 50 4 LF fed ‡‡ LF fasted ‡‡ ‡‡ ‡‡ HF fed HF fasted ‡‡ 3 40 LF fasted HF fasted * # 2 30 #

1

Body weight (g) weight Body 20 20 0 0

WT KO decrease (g) weight Body WT KO

C 15 ‡ LF fed HF fed ## LF fasted 10 ## ## ## HF fasted

5 Blood glucose (mM) glucose Blood 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 Ȍ‘”ʹͷΨ’‡” ‡–‹Ž‡‹—•ͳǤͷȉ 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”†• ƒŠ‹‰Š‡”†‡‰”‡‡‘ˆ

͹ͳ 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

High-fat fasted MCAD KO

CD

High-fat fasted WT

Figure 2. Similar steatosis grade (A, C) and difference in lobular inflammation (B, D) between MCAD- KO 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 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 Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

A B 150 250 LF fed LF fed HF fed 200 HF fed 100 LF fasted LF fasted HF fasted 150 HF fasted

100 50 50 Pyruvate-induced

consumption rate (St3) rate consumption (St3) rate consumption 3 C16-acyl-CoA-induced 2 0 2 0 (μmol/(min*gmitProtein)) (μmol/(min*gmitProtein)) O O WT KO WT KO

C 150 LF fed HF fed i 100 LF fasted * HF fasted

50 consumption rate (St3) rate consumption C8-acyl-CoA-induced

2 0 (μmol/(min*gmitProtein))

O 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 Ȍ‘”ʹͷΨ’‡” ‡–‹Ž‡‹—•ͳǤͷȉ 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 

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‹ Šƒ”‡‘™–‘„‡‡š’”‡••‡†‹‘—•‡Ž‹˜‡”ȏʹͻȐǤ –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 ‹‰—”‡ ͶǡȌƒ†–Š‡› ‘—Ž†–Š—•‡š’Žƒ‹–Š‡’”‡•‡ ‡‘ˆƒ”‡•‹†—ƒŽˆŽ—š‘ˆͺǦƒ ›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 

͹͵ Chapter 3

AB SCAD MCAD 150 150 LF fed LF fed HF fed ‡‡ HF fed 100 LF fasted 100 LF fasted g mito prot) g mito HF fasted prot) g mito P

P HF fasted

50 50

**** 0 0 WT KO WT KO [] (fmol/ [Protein] (fmol/

CD LCAD VLCAD 150 80 LF fed LF fed HF fed HF fed 60 100 LF fasted LF fasted g mito prot) g mito HF fasted prot) g mito P

P HF fasted 40

50 20

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/ Figure 4. Absolute protein levels of the mFAO SCAD (A), MCAD (B), LCAD (C) and VLCAD (D). Š‡‘–Š‡”‹–‘ Š‘†”‹ƒŽ‡‡”‰›‡–ƒ„‘Ž‹•’”‘–‡‹•ƒ”‡•Š‘™‹•—’’Ž‡‡–ƒ”› ‹‰—”‡͸ǤŠ‡”‡•—Ž–• ƒ”‡”‡’”‡•‡–‡†ƒ•—‡›„‘šƒ†™Š‹•‡”’Ž‘–•ǡ™Š‡”‡–Š‡„Žƒ Ǧˆ‹ŽŽ‡† ‹” Ž‡•‹†‹ ƒ–‡‹†‹˜‹†—ƒŽ‹ ‡ ˆƒŽŽ‹‰‘—–•‹†‡–Š‡͹ͷΨ’‡” ‡–‹Ž‡’Ž—•ͳǤͷȉ‹–‡”Ǧ“—ƒ”–‹Ž‡”ƒ‰‡ȋ Ȍ‘”ʹͷΨ’‡” ‡–‹Ž‡‹—•ͳǤͷȉ 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 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 B HF Fa LF Fe HF Fa LF Fe

42 47 28 63 110 000 82 44 84 27 00 00

210 200 0 0 0 0 0 0 LF Fa HF Fe LF Fa HF Fe Figure 5. Venn diagrams showing overlap between conditions of upregulated (A) and downregulated (B) between WT and KO ˆ‘” ƒ ’δͲǤͲͳǤ Š‡ †‡’‹ –‡† ”‡•—Ž–• ƒ”‡ „ƒ•‡† ‘ –Š‡ ‡‡“ʹ‡–Š‘†Ǥ

͹Ͷ 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  -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 3 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 –Š‡Ž‘™Ǧˆƒ–ˆƒ•–‡†ƒ†Š‹‰ŠǦ ˆƒ–ˆ‡† ‘†‹–‹‘‘‡–ƒ„‘Ž‹ ‰‡‡•‡–™ƒ•—’Ǧ‘”†‘™”‡‰—Žƒ–‡† ‘’ƒ”‡†–‘™‹–Šƒ ˆƒŽ•‡†‹• ‘˜‡”›”ƒ–‡ȋ Ȍ‘ˆδͲǤʹͷǤ –Š‡Ž‘™Ǧˆƒ–ˆ‡† ‘†‹–‹‘ʹ‰‡‡•‡–•™‡”‡—’”‡‰—Žƒ–‡† ƒ† ͳͲ ™‡”‡ †‘™”‡‰—Žƒ–‡† ȋƒ„Ž‡ ͳȌǤ  –Š‡ Š‹‰ŠǦˆƒ– ˆƒ•–‡† ‘†‹–‹‘ ͺ ‰‡‡ •‡–• ™‡”‡ †‘™”‡‰—Žƒ–‡†Ǥ  –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 biosynthesis - Sulfur metabolism Glutathione metabolism ‹•–‹†‹‡‡–ƒ„‘Ž‹•  ȋȌȋ Ȍƒ‹  ›–Š‡•‹•ƒ††‡‰”ƒ†ƒ–‹‘‘ˆ‡–‘‡„‘†‹‡• ȗ –‡”‘‹†Š‘”‘‡„‹‘•›–Š‡•‹• ȗ –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 Chapter 3

 –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  –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 •—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•ƒŽ‹‹–ƒ–‹‘Ǥ –‘Ž›‹†‡–‹ˆ‹‡•‰‡‡•‡–•–Šƒ– ‡‹–Š‡” ‘–ƒ‹ ’”‡†‘‹ƒ–Ž›—’”‡‰—Žƒ–‡†‘”’”‡†‘‹ƒ–Ž›†‘™”‡‰—Žƒ–‡†‰‡‡•ǡ™Š‡”‡ƒ•ƒ‹š–—”‡‘ˆ —’Ǧƒ††‘™”‡‰—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  ƒ††‹–‹‘ǡ ™‡ •—„•‡“—‡–Ž› ƒŽ•‘ ‹†‡–‹ˆ‹‡†‰‡‡•‡–•–Šƒ–ƒ”‡ȋ‘˜‡”Ȍ”‡’”‡•‡–‡†—†‡”‘”‡–Šƒ‘‡ ‘†‹–‹‘Ǥ •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 

͹͸ 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 3 ‡–‹‘Ž‡–ƒ„‘Ž‹• ȗ —”‹‡‡–ƒ„‘Ž‹• ȗǡș –‡”‘‹†Š‘”‘‡„‹‘•›–Š‡•‹• ȗ Pyruvate metabolism ȗǡș Ž› ‡”‘Ž‹’‹†‡–ƒ„‘Ž‹• ȗ ‘•‹–‘Ž’Š‘•’Šƒ–‡‡–ƒ„‘Ž‹• ȗ ”— –‘•‡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 ›”‹‹†‹‡‡–ƒ„‘Ž‹• ȗ ȗǡș ‡’‹ –‡†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͵ʹȐǤŠ‹•‹• ‘’ƒ–‹„Ž‡™‹–Šƒ”‘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 AB mRNA expression of Acot2 mRNA expression of Acot2 6 p=0.02 2.5 p=0.05

2.0 4 1.5

1.0 2 0.5 Normalized readcount Normalized expression 0 0.0 WT KO WT KO  Figure 6. Acot2 . Acot2‰‡‡‡š’”‡••‹‘‹–Š‡ ˆƒ•–‡† ‘†‹–‹‘„›‡“ȋAȌƒ†‹  ˆƒ•–‡† ‘†‹–‹‘„›”‡ƒŽǦ–‹‡ȋBȌǤŠ‡‡“”‡•—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

͹͹ Chapter 3

Š‡•‡”‡•—Ž–••—‰‰‡•––Šƒ–‡š–”ƒ•–‹—Žƒ–‹‘‘ˆ–Š‡ „›–Š‡Acot2†—”‹‰ˆƒ•–‹‰ ƒ› ‘’‡•ƒ–‡ˆ‘”–Š‡Ž‹‹–‡†  ƒ’ƒ ‹–›‹–Š‡Ǧ‘—•‡Ǥ

Discussion  –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 ’”‹ ‹’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 –‡”‡•–‹‰Ž›ǡ –Š‡ –”ƒ• ”‹’–‘‹  †ƒ–ƒ •—‰‰‡•– ‹’ƒ‹”‡† †‡–‘š‹ˆ‹ ƒ–‹‘ ‡ Šƒ‹•• ‹ –Š‡ Ǧ‘—•‡Ǥ ˆ–Š‹•ƒŽ•‘ƒ’’Ž‹‡•–‘Š—ƒ•ǡ‹–ƒ›„‡†‡–”‹‡–ƒŽ–‘’ƒ–‹‡–•ǡ •‹ ‡Š—ƒ•‹‰‡‡”ƒŽƒŽ”‡ƒ†›Šƒ˜‡Ž‡••Ͷƒ†Ͷ ‰‡‡•–Šƒ‹ ‡Ǥ 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‹‰‘ˆŠ‡’ƒ–‹   ”‡˜‡ƒŽ‡†ƒ’‘–‡–‹ƒŽ˜‹ ‹‘—• › Ž‡‘ˆ‘‡•–‡”ƒ ——Žƒ–‹‘ǡ‘ †‡’Ž‡–‹‘ǡƒ†  ˆŽ—š†‡ Ž‹‡ǡ™Š‹ Š™ƒ•ƒ‰‰”ƒ˜ƒ–‡†„›Ž‘••‘ˆȏͶͷȐǤ –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„•‡ ‡‘ˆǤ ˆ—–—”‡•–—†‹‡•™‡‡š’‡ ––‘•–—†›–Š‡ ”‘Ž‡•‘ˆ‘ ƒ†ȋȌȋ Ȍ‡–ƒ„‘Ž‹•‹–Š‡”‘„—•–‡••‘ˆ–Š‡Ǧ‘—•‡Ǥ   Materials and Methods

͹ͺ 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‹‡– 3 ‡”˜‹ ‡•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 ʹŠ‘—”•„‡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 (TG) concentration ”‡‡œ‡Ǧ Žƒ’‡†Ž‹˜‡”™ƒ• ”—•Š‡†‹Ž‹“—‹†‹–”‘‰‡ƒ†ͳͷΨȋ™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͹ʹͲͲƒ†͹ʹͲͲ‰‹•–‡ƒ†‘ˆ͹ͷͲǡ͹ͲͲͲƒ†͹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 •„”— 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 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 ‘Ȍȋ‘Ž›—ƒ– „ ǡƒ†„„ƒ Šǡ ‡”ƒ›Ȍƒ ‘”†‹‰–‘‘Ž–‡”•‡–ǤƒŽǤȏʹͻȐǤ  RNA isolation, RNASeq analysis and quantitative reverse transcriptase 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† •–‹–—–‡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 –‹‘•ǤŠ‡ †‡•–‘’ ƒ˜ƒƒ’’Ž‹ ƒ–‹‘•‘ˆ–™ƒ”‡™ƒ••‡––‘’‡”ˆ‘”ͳͲͲͲ’‡”—–ƒ–‹‘•ǡ—•‡ ™‡‹‰Š‡† ‡”‹ Š‡– •–ƒ–‹•–‹ Ǥ —”–Š‡”‘”‡ǡ ˆ‘” ‡ƒ Š ‰‡‡ •‡– ™‹–ŠƒˆƒŽ•‡†‹• ‘˜‡”›”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 ‘”†‹‰–‘ ͓ܥܩ஼௢஺̴ே஺஽௉ுȀ͓ܥܩ ܴܴ஼௢஺̴ே஺஽௉ு ൌ  ሺ͓݃݁݊݁ݏ஼௢஺̴ே஺஽௉ு െ͓ܥܩ஼௢஺̴ே஺஽௉ுሻȀሺ͓݃݁݊݁ݏ െ ͓ܥܩሻ  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 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†‰‡‡•‡–•™‹–Šƒ’‡” ‡–ƒ‰‡‘ˆœ‡”‘ ”‡ ‡‹˜‡† –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• 3 †‡–‡”‹‡† ™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 ͓ܦܧܩ௠௘௧௔௕௢௟௜௖ǡ௚௘௡௘௦௘௧Ȁ͓ܦܧܩ௠௘௧௔௕௢௟௜௖ǡ௔௟௟ ܴܴ௚௘௡௘௦௘௧ ൌ  ௠௘௧௔௕௢௟௜௖ǡ௔௟௟ሻܩܧܦ͓ െ ݏ௠௘௧௔௕௢௟௜௖ǡ௚௘௡௘௦௘௧ሻȀሺ͓݉݁ݐܾܽ݋݈݅ܿ݃݁݊݁ܩܧܦ௚௘௡௘௦௘௧ െ͓ݏሺ͓݃݁݊݁ 

™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–‡•–ǤŠ‡•‡ƒƒŽ›•‡• ™‡”‡ ’‡”ˆ‘”‡† —•‹‰   –ƒ–‹•–‹ • ˜‡”•‹‘ ʹʹǤͲ ȋ  Ǥǡ Š‹ ƒ‰‘ǡ 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 ‘—–‡ ǡ ‹‘Žƒ–‡ ǡ ‡–—”ƒ  Ǥǡ ƒ†‡”•  Ǥ Š‡ ‹‘ Š‡‹•–”› ƒ† Š›•‹‘Ž‘‰› ‘ˆ ‹–‘ Š‘†”‹ƒŽ ƒ––›  ‹† ȾǦš‹†ƒ–‹‘ ƒ† –• ‡‡–‹  ‹•‘”†‡”•Ǥ — ‡˜ Š›•‹‘ŽǤ ʹͲͳ͸Ǣ͹ͺȋͳȌǣʹ͵ȂͶͶǤ ʹǤ ‹ƒŽ†‘ ǡ ƒ–‡” ǡ ‡‡––  Ǥ ƒ––›  ‹† ‘š‹†ƒ–‹‘ ‹•‘”†‡”•Ǥ — ‡˜ Š›•‹‘ŽǤ ʹͲͲʹǢ͸ͶȋͳȌǣͶ͹͹ȂͷͲʹǤ ͵Ǥ ‡”• 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 ͶǤ ‡”• 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  Š‡”‹–‡–ƒ„‹•ǤʹͲͲͺǢ͵ͳȋͳȌǣͺͺȂͻ͸Ǥ ͷǤ ‘—™ǡ‹– ǡ‹‡œ‡Ǧ‘‹‰ǡ‘•‰”ƒƒˆǦ†‡‘‡”ǡ ‡”†‹‰ǡ‡‹Œ‰‘—†Ǧ ǡ‡”•  Ǥ  ˜‹–”‘ ƒ† ‹ ˜‹˜‘ ‘•‡“—‡ ‡• ‘ˆ ˜ƒ”‹ƒ– ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ‰‡‘–›’‡•Ǥ”’Šƒ‡– ƒ”‡‹•ǤʹͲͳ͵ǢͺȋͳȌǣͶ͵Ǥ ͸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 Chapter 3

•–”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 ͹Ǥ ’‹‡‡”‘‡––‡” ǡ ƒ•–‹ ǡ ‹ŽŽ‹‰Šƒ ǡ ‘””‹• ǡ ‹Œ„—”‰ ǡ ‹Ž ‡ Ǥ —””‡– ‹••—‡• ”‡‰ƒ”†‹‰ –”‡ƒ–‡– ‘ˆ ‹–‘ Š‘†”‹ƒŽ ˆƒ––› ƒ ‹† ‘š‹†ƒ–‹‘ †‹•‘”†‡”•Ǥ  Š‡”‹– ‡–ƒ„ ‹•Ǥ ʹͲͳͲǢ͵͵ȋͷȌǣͷͷͷȂ͸ͳǤ ͺǤ ”‘‡–›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 ͻǤ ‡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 ͳͲǤ —”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 ͳͳǤ  Šƒ–œƒǤǡ•‡ƒ—‡”ǤŠ‡ Ž‹‹ ƒŽƒ‹ˆ‡•–ƒ–‹‘‘ˆ†‡ˆ‹ ‹‡ ›ǣŠƒŽŽ‡‰‡•–‘™ƒ”†• ƒ†—Ž–Š‘‘†‹–Š‡• ”‡‡‡†’‘’—Žƒ–‹‘Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͲǢ͵͵ȋͷȌǣͷͳ͵ȂʹͲǤ ͳʹǤ ‡ƒŽ‡• ǡŠ‘’•‘ ǡƒ••‘—† ǡƒŠ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  Š‡”‹–‡–ƒ„‹•ǤͳͻͻͶǢͳ͹ȋͳȌǣ͹ͶȂͺͲǤ ͳ͵Ǥ ‡”• ǡƒ’”‘•‡ ǡƒ‡ ǡƒ‡” ‹Ž•–ǡ’ƒǡ‹– Ǥ ƒˆ‡ƒ†—•ƒˆ‡ †—”ƒ–‹‘‘ˆˆƒ•–‹‰ˆ‘” Š‹Ž†”‡™‹–Š†‡ˆ‹ ‹‡ ›Ǥ—” ‡†‹ƒ–”ǤʹͲͲ͹Ǣͳ͸͸ȋͳȌǣͷȂͳͳǤ ͳͶǤ ƒŽ–‡” Ǥ ‘Ž‡”ƒ ‡ –‘ ˆƒ•–ǣ ƒ–‹‘ƒŽ ƒ† ’”ƒ –‹ ƒŽ ‡˜ƒŽ—ƒ–‹‘ ‹ Š‹Ž†”‡ ™‹–Š Š›’‘‡–‘ƒ‡‹ƒǤ  Š‡”‹–‡–ƒ„‹•ǤʹͲͲͻǢ͵ʹȋʹȌǣʹͳͶȂ͹Ǥ ͳͷǤ ‡‡––  Ǥ ƒ–Š‘’Š›•‹‘Ž‘‰› ‘ˆ ˆƒ––› ƒ ‹† ‘š‹†ƒ–‹‘ †‹•‘”†‡”•Ǥ  Š‡”‹– ‡–ƒ„ ‹•Ǥ ʹͲͳͲǢ͵͵ȋͷȌǣͷ͵͵Ȃ͹Ǥ ͳ͸Ǥ ‹•Š™ƒƒ–Š Ǥ ƒ––› ƒ ‹† „‡–ƒǦ‘š‹†ƒ–‹‘ †‹•‘”†‡”•ǣ  „”‹‡ˆ ”‡˜‹‡™Ǥ  ‡—”‘• ‹Ǥ ʹͲͳ͸Ǣʹ͵ȋͳȌǣͷͳȂͷǤ ͳ͹Ǥ ‘Ž‡”Ǧ–‘—™Ǥ†‡ˆ‹ ‹‡ ›‘„‡ǡ‘”‘––‘„‡ƒ–”‹•ǤʹͲͳͶǤ ͳͺǤ ‘—–‡ǡƒ†‡”• Ǥ‰‡‡”ƒŽ‹–”‘†— –‹‘–‘–Š‡„‹‘ Š‡‹•–”›‘ˆ‹–‘ Š‘†”‹ƒŽˆƒ––› ƒ ‹†ȾǦ‘š‹†ƒ–‹‘Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͲǢ͵͵ȋͷȌǣͶ͸ͻȂ͹͹Ǥ ͳͻǤ ƒ–‘ ǡ ƒ”–Ž‡–– ǡ ‘—”ˆƒ”œƒ Ǥ ƒƒŽ‹ƒ ‹–‘ Š‘†”‹ƒŽ „‡–ƒǦ‘š‹†ƒ–‹‘Ǥ ‹‘ Š‡ Ǥ ͳͻͻ͸Ǣ͵ʹͲǣ͵ͶͷȂͷ͹Ǥ ʹͲǤ ˜ƒ—‡ǡ‘Ž‡”Ǧ‘—™ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ‘Ž–‡”• ǡ˜ƒ‹Œ– ǡƒ”–‹‡• ǡ ‹‡œ‡Ǧ‘‹‰ǡ ‡‹‡”ǡ‡”‡–‹‡” ǡ ”‘‡ǡ‡‹Œ‰‘—† ǡ‡”• ǡƒ‡”Ǥ ‹˜‹‰‘–Š‡‡†‰‡ǣ—„•–”ƒ–‡ ‘’‡–‹–‹‘‡š’Žƒ‹•Ž‘••‘ˆ”‘„—•–‡••‹‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧ ƒ ‹†‘š‹†ƒ–‹‘†‹•‘”†‡”•Ǥ‹‘ŽǤʹͲͳ͸ǢͳͶȋͳȌǣͳȂͳͷǤ ʹͳǤ ƒ–•›—„ƒ ǡ —™‡”š Ǥ ‘†—Žƒ–‹‰  Ϊ ‡–ƒ„‘Ž‹•ǡ ˆ”‘ „‡ Š –‘„‡†•‹†‡Ǥ  Ǥ ʹͲͳ͹Ǣ͵͸ȋͳͺȌǣ‡ʹͲͳ͹ͻ͹ͳ͵ͷǤ ʹʹǤ ƒ‰ǡƒ—˜‡Ǥڏ‡–ƒ„‘Ž‹•ǣ‹‘‡‡”‰‡–‹ •ǡ•‹‰ƒŽ‹‰ƒ†ƒ‹’—Žƒ–‹‘ˆ‘”–Š‡”ƒ’›Ǥ ‹‘ Š‹‹‘’Š›• –ƒǦ”‘–‡‹•”‘–‡‘‹ •ǤʹͲͳ͸Ǣͳͺ͸ͶȋͳʹȌǣͳ͹ͺ͹ȂͺͲͲǤ ʹ͵Ǥ ‹‹ˆ‘”‘˜ǡ—Ž‹‘˜ƒǡ‹‡‰Ž‡”ǤŠ‡Š—ƒ‡–ƒ„‘Ž‘‡ǣ — –‹‘•ǡ‡–ƒ„‘Ž‹•ƒ† ‘’ƒ”–‡–ƒŽ‹œƒ–‹‘Ǥ”‹–‡˜‹‘ Š‡‘Ž‹‘ŽǤʹͲͳͷǢͷͲȋͶȌǣʹͺͶȂͻ͹Ǥ ʹͶǤ Šƒ‰ ǡ ƒ†ƒ›ƒ––‹ ǡ ‡—‰ Ǥ ”‘–‘Ǧ–”ƒ•Ž‘ ƒ–‹‰ ‹ ‘–‹ƒ‹†‡ — Ž‡‘–‹†‡ –”ƒ•Š›†”‘‰‡ƒ•‡ǣ•–”— –—”ƒŽ’‡”•’‡ –‹˜‡Ǥ ”‘–Š›•‹‘ŽǤʹͲͳ͹ǢͺȋȌǣͳȂ͸Ǥ ʹͷǤ ‹– Š‡ŽŽ ǡ ƒ—–Š‹‡”ǡ‡•‹’Ž‡ǡƒ‰ǡƒ‡”ǡ—”‡•Š‹ Ǥ ‡”‡†‹–ƒ”›ƒ†ƒ “—‹”‡† †‹•‡ƒ•‡•‘ˆƒ ›ŽǦ ‘‡œ›‡‡–ƒ„‘Ž‹•Ǥ‘Ž ‡‡–‡–ƒ„ǤʹͲͲͺǢͻͶȋͳȌǣͶȂͳͷǤ ʹ͸Ǥ ‘Ž™ƒ‹ ǡ ƒǡ‹ƒǡŠƒ”‡” ǡ‘ Ž‡› ǡ‹ƒŽ†‘ǡƒ–‡”ǡ Š‘‡„ǡ‘‘†Ǥ ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ †‡ˆ‹ ‹‡ › ‹ ‰‡‡Ǧ–ƒ”‰‡–‡† ‹ ‡Ǥ ‘ ‡‡–Ǥ ʹͲͲͷǢͳȋʹȌǣͲʹͲͷȂͳʹǤ

ͺʹ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

ʹ͹Ǥ ‡””‡ƒ ǡ‡”• ǡƒ‹Œ ǡŽ‘•ǡ ‡”†‹‰ǡ ƒ˜‹‰ƒǡ‹‡–‰‡ ǡǫǫŽŽ‡”ǡ‹– ǡ —‹’‡”• ǡ ‡‹Œ‰‘—†  Ǥ ‹•–—”„‡† Š‡’ƒ–‹  ƒ”„‘Š›†”ƒ–‡ ƒƒ‰‡‡– †—”‹‰ Š‹‰Š ‡–ƒ„‘Ž‹  †‡ƒ† ‹ ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋȌǦ†‡ˆ‹ ‹‡– ‹ ‡Ǥ ‡’ƒ–‘Ž‘‰›ǤʹͲͲͺǢͶ͹ȋ͸ȌǣͳͺͻͶȂͻͲͶǤ ʹͺǤ ˜ƒ—‡ǡ‘Ž‡”Ǧ‘—™ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ‘Ž–‡”• ǡ˜ƒ‹Œ– ǡƒ”–‹‡•Ǧ ǡ ‹‡œ‡Ǧ‘‹‰ǡ ‡‹‡”ǡ‡”‡–‹‡” ǡ ”‘‡ǡ‡‹Œ‰‘—†Ǧ ǡ‡”• ǡƒ‡”Ǥ ‹˜‹‰‘–Š‡‡†‰‡ǣ•—„•–”ƒ–‡ ‘’‡–‹–‹‘‡š’Žƒ‹•Ž‘••‘ˆ”‘„—•–‡••‹‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧ 3 ƒ ‹†‘š‹†ƒ–‹‘†‹•‘”†‡”•Ǥ‹‘ŽǤʹͲͳ͸ǢͳͶȋͳȌǣͳͲ͹Ǥ ʹͻǤ ‘Ž–‡”• ǡ ‹ƒ’ƒ‹–‡ ǡ ˜ƒ —‡ ǡ ‹‡œ‡Ǧ‘‹‰ ǡ ƒ––‘ ǡ ‘”–‡  ǡ ‘”˜ƒ–‘˜‹ Š ǡ ƒ‡” ǡ ‹• Š‘ˆˆ ǡ ‡”‡–‹‡” Ǥ ”ƒ•Žƒ–‹‘ƒŽ –ƒ”‰‡–‡† ’”‘–‡‘‹ • ’”‘ˆ‹Ž‹‰ ‘ˆ ‹–‘ Š‘†”‹ƒŽ ‡‡”‰› ‡–ƒ„‘Ž‹  ’ƒ–Š™ƒ›• ‹ ‘—•‡ ƒ† Š—ƒ •ƒ’Ž‡•Ǥ —„‹–– –‘ —„ŽǤ ʹͲͳ͸Ǣ ͵ͲǤ ‘—™ ǡ ‡”•  ǡ ƒ‡” ǡ ”‘‡ ǡ ‹– ǡ ‡‹Œ‰‘—†  Ǥ ”‘ ‰‡‘‡ –‘ ’Š‡‘‡Ǧ‹’Ž‡‹„‘”‡””‘”•‘ˆ‡–ƒ„‘Ž‹•ƒ• ‘’Ž‡š–”ƒ‹–•Ǥ‹‘ Š‹‹‘’Š›• –ƒǦ‘Ž ƒ•‹•‹•ǤʹͲͳͶǢͳͺͶʹȋͳͲȌǣʹͲʹͳȂͻǤ ͵ͳǤ ‘ˆˆƒ–ǡŠƒ–‹ƒǡ‰—›‡ǡ› Šǡƒ‰ǡƒ‰ǡ Žƒ›‡˜ƒǡ ƒǡ ‹”• Š‡›ǡ Žƒ›’‘‘Žǡ‡‹ˆ‡”–Ǥ ›ŽǦ‘–Š‹‘‡•–‡”ƒ•‡Ǧʹˆƒ ‹Ž‹–ƒ–‡•‹–‘ Š‘†”‹ƒŽˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘ ‹–Š‡Ž‹˜‡”Ǥ ‹’‹†‡•ǤʹͲͳͶǢͷͷȋͳʹȌǣʹͶͷͺȂ͹ͲǤ ͵ʹǤ ŽŽ‹• ǡ ‘™ƒ ǡ ‘Žˆ‰ƒ‰  Ǥ ‡–ƒ„‘Ž‹  ƒ† ‹••—‡Ǧ’‡ ‹ˆ‹  ‡‰—Žƒ–‹‘ ‘ˆ  ›ŽǦ‘ ‡–ƒ„‘Ž‹•Ǥ‘‡ǤʹͲͳͷǢͳͲȋ͵Ȍǣ‡Ͳͳͳ͸ͷͺ͹Ǥ ͵͵Ǥ ‹ŽŽƒ†‡”ǡŽ‡š•‘ ǡ‘Š‡Ǥ‡ƒ –‹˜ƒ–‹‰ ƒ––› ‹†•ǣ ›ŽǦ‘Š‹‘‡•–‡”ƒ•‡Ǧ‡†‹ƒ–‡† ‘–”‘Ž‘ˆ‹’‹†‡–ƒ„‘Ž‹•Ǥ”‡†•†‘ ”‹‘Ž‡–ƒ„ǤʹͲͳ͹Ǣʹͺȋ͹ȌǣͶ͹͵ȂͺͶǤ ͵ͶǤ —– ǡ Ž‡š•‘ Ǥ Š‡ ”‘Ž‡  ›ŽǦ‘ –Š‹‘‡•–‡”ƒ•‡• ’Žƒ› ‹ ‡†‹ƒ–‹‰ ‹–”ƒ ‡ŽŽ—Žƒ” Ž‹’‹† ‡–ƒ„‘Ž‹•Ǥ”‘‰‹’‹†‡•ǤʹͲͲʹǢͶͳȋʹȌǣͻͻȂͳ͵ͲǤ ͵ͷǤ  ƒ‹‹ ǡ‹‘ǡ‘‹ǡ—•ƒ‡ŽŽ‘ǡ‘—”ƒǡ ‡””‡‹”ƒ ǡƒŒ‡”ǡ–”‡ ǡ  Š—   Ǥ ‘š‹ ‹–› ‘ˆ ‘ –ƒ‘ƒ–‡ ƒ† †‡ ƒ‘ƒ–‡ ‹ ”ƒ– ’‡”‹’Š‡”ƒŽ –‹••—‡•ǣ ‡˜‹†‡ ‡ ‘ˆ „‹‘‡‡”‰‡–‹ †›•ˆ— –‹‘ƒ†‘š‹†ƒ–‹˜‡†ƒƒ‰‡‹†— –‹‘‹Ž‹˜‡”ƒ†•‡Ž‡–ƒŽ—• Ž‡Ǥ‘Ž‡ŽŽ ‹‘ Š‡ǤʹͲͳʹǢ͵͸ͳȋͳȂʹȌǣ͵ʹͻȂ͵ͷǤ ͵͸Ǥ ƒ—‡”ǡ— ǡ ‘ˆˆƒ ǡ‘‡Ž‡”ǡ‘”ƒ–ŠǤ ’ƒ –‘ˆ•Š‘”–Ǧƒ†‡†‹—Ǧ Šƒ‹ ‘”‰ƒ‹  ƒ ‹†•ǡ ƒ ›Ž ƒ”‹–‹‡•ǡ ƒ† ƒ ›ŽǦ‘• ‘‹–‘ Š‘†”‹ƒŽ ‡‡”‰› ‡–ƒ„‘Ž‹•Ǥ ‹‘ Š‹ ‹‘’Š›• –ƒǦ‹‘‡‡”‰ǤʹͲͲͺǢͳ͹͹͹ȋͳͲȌǣͳʹ͹͸ȂͺʹǤ ͵͹Ǥ  Š—  ǡ†ƒ‘•–ƒ ‡””‡‹”ƒ ǡ‘‹ǡ‹‡‰ƒ•ǡ—•ƒ‡ŽŽ‘ǡ‘—”ƒǡƒƒ––ƒ ƹ ǡ Žƒ– ǡƒŒ‡”Ǥ˜‹†‡ ‡–Šƒ––Š‡ƒŒ‘”‡–ƒ„‘Ž‹–‡•ƒ ——Žƒ–‹‰‹‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ ‘†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›†‹•–—”„‹–‘ Š‘†”‹ƒŽ‡‡”‰›Š‘‡‘•–ƒ•‹•‹”ƒ–„”ƒ‹Ǥ”ƒ‹ ‡•ǤʹͲͲͻǢͳʹͻ͸ǣͳͳ͹Ȃʹ͸Ǥ ͵ͺǤ ƒ”ƒŽ ǡ ‡ ƒ––‘ ǡ †ƒ ‹Ž˜ƒ ǡ ƒŒ‡” ǡ ƒŒ‡” Ǥ ‡ Šƒ‹•–‹  ƒ•‡• ‘ˆ ‡—”‘–‘š‹ ‹–› ”‘˜‘‡†„› ƒ––› ‹†• ——Žƒ–‹‰‹ƒ† ‡ˆ‹ ‹‡ ‹‡•Ǥ  „‘”””‘”•‡–ƒ„  ”‡‡ǤʹͲͳ͹Ǣͷǣʹ͵ʹ͸ͶͲͻͺͳ͹͹ͲͳͶ͹Ǥ ͵ͻǤ ‘—‰‘˜‹ƒǡ‡”‹‡ ǡƒ†‡”• ǡ ‘—–‡ǡ†‡‘‡” Ǥ„‡””ƒ–’”‘–‡‹ƒ ›Žƒ–‹‘ ‹• ƒ ‘‘ ‘„•‡”˜ƒ–‹‘ ‹ ‹„‘” ‡””‘”• ‘ˆ ƒ ›ŽǦ‘ ‡–ƒ„‘Ž‹•Ǥ  Š‡”‹– ‡–ƒ„ ‹•Ǥ ʹͲͳͶǢ͵͹ȋͷȌǣ͹ͲͻȂͳͶǤ ͶͲǤ ‡•ŠǤ ƒ––›ƒ ›Žƒ–‹‘‘ˆ’”‘–‡‹•ǣŠ‡Ž‘‰ƒ†–Š‡•Š‘”–‘ˆ‹–Ǥ”‘‰‹’‹†‡•ǤʹͲͳ͸Ǣ͸͵ǣͳʹͲȂ ͵ͳǤ ͶͳǤ — ‹ ǡ ”‹ƒ••‹ ǡ ‡” ‡Ž† ǡ ’‹‡‡”‘‡––‡” Ǥ ‡†‹—Ǧ Šƒ‹ –”‹‰Ž› ‡”‹†‡• ‹’ƒ‹” Ž‹’‹† ‡–ƒ„‘Ž‹•ƒ†‹†— ‡Š‡’ƒ–‹ •–‡ƒ–‘•‹•‹˜‡”›Ž‘‰Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡ȋȌǦ †‡ˆ‹ ‹‡–‹ ‡Ǥ‘Ž ‡‡–‡–ƒ„ǤʹͲͳͲǢͳͲͳȋͳȌǣͶͲȂ͹Ǥ ͶʹǤ ƒ†‡”• ǡ‘‡ ǡ‡’Ǥ ƒ––›ƒ ‹†‘‡‰ƒǦ‘š‹†ƒ–‹‘ƒ•ƒ”‡• —‡’ƒ–Š™ƒ›ˆ‘”ˆƒ––›ƒ ‹† ‘š‹†ƒ–‹‘†‹•‘”†‡”•‹Š—ƒ•Ǥ  ǤʹͲͳͳǢʹ͹ͺȋʹȌǣͳͺʹȂͻͶǤ Ͷ͵Ǥ ‡Ž• ǡ ‡•–Š—‹œ‡  ƒ ‡”ǡ ”‡–‘”‹—•  ǡ ”ƒ•—• Ǥ „ƒŽƒ ‡† „‹‘–”ƒ•ˆ‘”ƒ–‹‘

ͺ͵ Chapter 3

‡–ƒ„‘Ž‹•ƒ†‘š‹†ƒ–‹˜‡•–”‡•••–ƒ–—•᩿ǣ‹’Ž‹ ƒ–‹‘•ˆ‘”†‡ˆ‹ ‹‡–ˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘Ǥ”ƒ•—•Ǥ ʹͲͳͳǢ͵ȋͳȌǣͶ͵ȂͺǤ ͶͶǤ ‡”• ǡ‘—™ǡ‹„ƒ• ǡ‹ƒ ‹‹ ǡƒœ‹ǡ‡‰”‡––‘ ǡ‡• ƒǡ‡‹Œ‰‘—† ƒǤǡ ‹– ǡ ƒŒ‡” ǡ ƒ”‰ƒ• Ǥ š’‡”‹‡–ƒŽ ‡˜‹†‡ ‡ ˆ‘” ’”‘–‡‹ ‘š‹†ƒ–‹˜‡ †ƒƒ‰‡ ƒ† ƒŽ–‡”‡†ƒ–‹‘š‹†ƒ–†‡ˆ‡•‡‹’ƒ–‹‡–•™‹–Š‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͶǢ͵͹ȋͷȌǣ͹ͺ͵ȂͻǤ ͶͷǤ ˜ƒ—‡ǡ‹‘• ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ†‡‡•–‡ ǡ‘—™ǡ ‘—–‡ǡ ”‘‡ ǡ ”ƒ„ ǡ ‡‹Œ‰‘—†  ǡ ƒ‡” Ǥ ‹‘ Š‡‹ ƒŽ ‘’‡–‹–‹‘ ƒ‡• ƒ––›Ǧ ‹† ‡–ƒǦ š‹†ƒ–‹‘—Ž‡”ƒ„Ž‡–‘—„•–”ƒ–‡˜‡”Ž‘ƒ†Ǥ‘‘’—–‹‘ŽǤʹͲͳ͵ǢͻȋͺȌǣʹȂͻǤ Ͷ͸Ǥ ƒ”–‹‡•Ǧ ǡ˜ƒ—‡ǡ‡‹Œ‰‘—†Ǧ ǡƒ‡”ǤŠ‡’”‘‹• —‘—•‡œ›‡‡†‹—Ǧ Šƒ‹ ͵Ǧ‡–‘Ǧƒ ›ŽǦ‘ –Š‹‘Žƒ•‡ –”‹‰‰‡”• ƒ ˜‹ ‹‘—• › Ž‡ ‹ ˆƒ––›Ǧƒ ‹† „‡–ƒǦ‘š‹†ƒ–‹‘Ǥ  ‘’—–‹‘ŽǤʹͲͳ͹Ǣͳ͵ȋͶȌǣͳȂʹʹǤ Ͷ͹Ǥ ‘‹Ž‘˜ǡ‘‹Ž‘˜ƒǡŠƒǡ ƒ‰‘’‹ƒǡ‡––ƒ‹‡„ǡ‹ǡ‡Ž‹ ‹ ǡ ƒŒ ǡƒ•‡› ǡ ‘”–‘’ƒ••‹ Ǥ ’Ͷ͸Š  Š‹„‹–• Š‹‘Žƒ•‡ ƒ† ‹’‹† š‹†ƒ–‹‘ ‹ ‹–‘ Š‘†”‹ƒǤ  ‹‘Ž Š‡Ǥ ʹͲͳ͸ǢʹͻͳȋʹͶȌǣͳʹͷ͹ͷȂͺͷǤ ͶͺǤ ƒ”„‘•ƒǡ‘ƒ”‡•ǡ‘˜ƒǡ‹ Žƒ‹”ǡ‡˜‹‡ ǡ•‘›ǡŠ‹‹ǤŠ‡‡œ›‡͵ͺ ȋƒ‰Ž› ‘Š›†”‘Žƒ•‡ǡ͵ǤʹǤʹǤͷȌ‹•‡ ‡••ƒ”›ˆ‘”–Š‡†‡˜‡Ž‘’‡–‘ˆ†‹‡–Ǧ‹†— ‡†‘„‡•‹–›Ǥ  ǤʹͲͲ͹Ǣʹͳȋͳ͵Ȍǣ͵͸ʹͻȂ͵ͻǤ ͶͻǤ †‡‹– ǡ‘• ŠǦ‡”‡—Ž‡ ǡ†‡ ”‘‘– ǡ ‘‘‹˜‡Ž† ǡ”‘Šƒƒ” ǡ ƒ•‡ ǡòŽŽ‡”ǡ ˜ƒ†‡”‡‡”ǤŠ‡”‘Ž‡‘ˆ–Š‡•ƒŽŽ‹–‡•–‹‡‹–Š‡†‡˜‡Ž‘’‡–‘ˆ†‹‡–ƒ”›ˆƒ–Ǧ‹†— ‡†‘„‡•‹–› ƒ†‹•—Ž‹”‡•‹•–ƒ ‡‹ͷ͹Ȁ͸ ‹ ‡Ǥ‡† ‡‘‹ •ǤʹͲͲͺǢͳȋͳȌǣͳͶǤ ͷͲǤ Ž‡‹‡”ǡ”—–ǡƒƒ––ƒǡ‡ŠŽ‹‰ǡ‘–‘• ǡ—‹‰•ǡ ‡””‡ŽŽǡ‹—ǡ ‘”„‡•‘ǡ ƒŽ’Ǧ”‹†ƒ ǡ ‡Š ǡ  —ŽŽ‘—‰Š  ǡ ƒ›ƒŽ  Ǥ ‡•‹‰ƒ† ˜ƒŽ‹†ƒ–‹‘‘ˆƒ Š‹•–‘Ž‘‰‹ ƒŽ• ‘”‹‰•›•–‡ˆ‘”‘ƒŽ ‘Š‘Ž‹ ˆƒ––›Ž‹˜‡”†‹•‡ƒ•‡Ǥ ‡’ƒ–‘Ž‘‰›ǤʹͲͲͷǢͶͳȋ͸Ȍǣͳ͵ͳ͵Ȃ ʹͳǤ ͷͳǤ Ž‹‰Š ǡ›‡” Ǥƒƒ†‹ƒ ‘—”ƒŽ‘ˆǤƒ–Ž‡•‘— ƒƒ†ƒǤͳͻͷͻǢ͵͹ȋͺȌǤ ͷʹǤ ‹Ž†ƒœ‹‡‡ǡƒ— ‹‡‡ǡƒ‹‡‡ǡ ”‹‰‹‡‡ Ǥ—Ž–‹’Ž‡‡ˆˆ‡ –•‘ˆʹǡʹǯǡͷǡͷǯǦ–‡–”ƒ ŠŽ‘”‘„‹’Š‡›Ž ‘‘š‹†ƒ–‹˜‡’Š‘•’Š‘”›Žƒ–‹‘‹”ƒ–Ž‹˜‡”‹–‘ Š‘†”‹ƒǤ‘š‹ ‘Ž ‹ǤʹͲͲʹǢ͸ͷȋʹȌǣʹʹͲȂ͹Ǥ ͷ͵Ǥ ƒ‹‰‡”ǡ—œ‡–•‘˜ƒǡ Š‡‡„‡”‰‡”ǡ‡‹Ž‡”ǡ”ƒ†ƒ Š‡” ǡ–‡—”‡”ǡƒ”‰”‡‹–‡”Ǥ ‹–‘ Š‘†”‹ƒ‹–Š‡ ‘Ž†Ǥ‹ˆ‡‘Ž†ǤʹͲͲͲǢͶ͵ͳȂͶʹǤ ͷͶǤ —„”ƒƒ‹ƒǡƒƒ›‘ǡ‘‘–ŠƒǤ ᩿ǣ ‡‡•‡–‡”‹ Š‡–ƒƒŽ›•‹• ‡‡•‡–‡”‹ Š‡– ƒƒŽ›•‹•᩿ǣ  ‘™Ž‡†‰‡Ǧ„ƒ•‡† ƒ’’”‘ƒ Š ˆ‘” ‹–‡”’”‡–‹‰ ‰‡‘‡Ǧ™‹†‡ ‡š’”‡••‹‘ ’”‘ˆ‹Ž‡•Ǥ ʹͲͲͷǢ ͷͷǤ ƒ™ǡŠ‡ǡŠ‹ǡ›–Š Ǥ‘‘ǣ”‡ ‹•‹‘™‡‹‰Š–•—Ž‘ Ž‹‡ƒ”‘†‡ŽƒƒŽ›•‹•–‘‘Ž• ˆ‘”Ǧ•‡“”‡ƒ† ‘—–•Ǥ ‡‘‡‹‘ŽǤʹͲͳͶǢͳͷȋʹȌǣͳȂͳ͹Ǥ ͷ͸Ǥ ‘—–‡ǡ ‡””‡ƒ ǡ”‹‡ ‡ǡ‡‹•ǡ—‹–‡” ǡ˜ƒ‹Œ ǡ”‰ƒǡ––‡Š‘ˆˆ ǡǫǫŽŽ‡”ǡ ”‘‡ǡ—‹’‡”• ǡ‡‹Œ‰‘—† ǡƒ†‡”• Ǥ ’ƒ‹”‡†ƒ‹‘ƒ ‹†‡–ƒ„‘Ž‹• ‘–”‹„—–‡•–‘ˆƒ•–‹‰Ǧ‹†— ‡†Š›’‘‰Ž› ‡‹ƒ‹ˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘†‡ˆ‡ –•Ǥ —‘Ž ‡‡–Ǥ ʹͲͳ͵ǢʹʹȋʹͷȌǣͷʹͶͻȂ͸ͳǤ  Acknowledgements Š‹• ™‘” ™ƒ• •—’’‘”–‡† „›ǣ ‹˜‡”•‹–ƒ‹” ‡†‹• Š ‡–”— ”‘‹‰‡ ȋŠ––’ǣȀȀ™™™Ǥ— ‰ǤŽȀ–‘ ȌǢ‘•ƒŽ‹† ”ƒŽ‹ ‡ŽŽ‘™•Š‹’ǡ‹˜‡”•‹–›‘ˆ ”‘‹‰‡ȋȌ ȋŠ––’•ǣȀȀ™™™Ǥ— ‰ǤŽȀȀ‡•‡ƒ” ŠȀ‡•‡ƒ” Š‡”•Ȁƒ”‡‡” —†‹‰Ȁ‘•ƒŽ‹† ”ƒŽ‹ ‡ŽŽ‘™ •Š‹’Ȁƒ‰‹ƒ•Ȁ†‡ˆƒ—Ž–Ǥƒ•’š–‘ǤŠ‡ˆ—†‡”•Šƒ†‘”‘Ž‡‹•–—†›†‡•‹‰ǡ†ƒ–ƒ ‘ŽŽ‡ –‹‘ ƒ†ƒƒŽ›•‹•ǡ†‡ ‹•‹‘–‘’—„Ž‹•Šǡ‘”’”‡’ƒ”ƒ–‹‘‘ˆ–Š‡ƒ—• ”‹’–Ǥ  Author Contributions

ͺͶ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

 ǡ Ǧ  ƒ†  †‡•‹‰‡† –Š‡ ‡š’‡”‹‡–•Ǥ  ǡ   ƒ†   ’‡”ˆ‘”‡† –Š‡ ‡š’‡”‹‡–•Ǥ ǡ ǡǡ ǡ ǡǡǡǡ ™‡”‡‹˜‘Ž˜‡†‹•ƒ’Ž‡’”‡’ƒ††ƒ–ƒ ‰‡‡”ƒ–‹‘Ǥ ǡƒ†™‡”‡‹˜‘Ž˜‡†‹†ƒ–ƒƒƒŽ›•‡•Ǥ ǡǡ ǡǡǡǡ ǡǦ ǡ™‡”‡‹˜‘Ž˜‡†‹‹–‡”’”‡–ƒ–‹‘‘ˆ”‡•—Ž–•Ǥ ™”‘–‡–Š‡ƒ—• ”‹’–Ǥ ǡ ǡǡǡǡǡǦ ƒ†‡†‹–‡†–Š‡ƒ—• ”‹’–Ǥ  3 Competing interests Š‡ƒ—–Š‘”•†‡ Žƒ”‡‘ ‘’‡–‹‰‹–‡”‡•–•Ǥ 

ͺͷ Chapter 3

^ƵƉƉůĞŵĞŶƚĂƌLJĨŝŐƵƌĞƐ͕ƚĂďůĞƐĂŶĚŵĞƚŚŽĚƐ Acyl-CoA

Mitochondrion Acyl-CoA

MCKAT Acetyl-CoA SCAD MCAD LCAD VLCAD FAD C4-C6 C4-C12 C8-C16 C12-C16 C4-C16 CoASH FADH2

Ketoacyl-CoA Enoyl-CoA

MSCHAD NADH + H+ Acetyl-CoA C4-C16 NAD+ CoASH Hydroxyacyl-CoA  Supplementary Figure 1. Schematic representation of the murine mFAO pathway. ‘ ǣ ˆ”‡‡ ‘‡œ›‡Ǣ‘š‡•”‡’”‡•‡–‹†‹˜‹†—ƒŽ‡œ›‡•ǤŽ›‡œ›‡•–Šƒ–ƒ”‡‡–‹‘‡†‹–Š‡ƒ‹–‡š–ƒ”‡ •’‡ ‹ˆ‹ ƒŽŽ›’‘”–”ƒ›‡†™‹–Šƒƒ‡ƒ† Šƒ‹Ž‡‰–Š•’‡ ‹ˆ‹ ‹–›Ǣǡǡƒ†ǣ˜‡”›ǦŽ‘‰Ǧ ǡŽ‘‰Ǧǡ‡†‹—Ǧǡƒ†•Š‘”–Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡ǡ”‡•’‡ –‹˜‡Ž›Ǣǣ‡†‹—Ǧ Šƒ‹‡–‘ƒ ›ŽǦ ‘–Š‹‘Žƒ•‡Ǣ ǣ‡†‹—Ȁ•Š‘”–Ǧ Šƒ‹Š›†”‘š›ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡

AB 3 8 LF fed LF fed HF fed HF fed ΐ ΐ 6 ΐΐ LF fasted ηη LF fasted 2 ηη HF fasted ηη͕ΐΐ HF fasted ηη͕ΐ 4 ηη ηη 1 2 Liver weight (g) weight Liver

0 0 WT KO Relative liver weight (%) WT KO

C 250 LF fed 200 HF fed LF fasted 150 HF fasted

100 mol/g liver) P ( 50 [Liver triglyceride] [Liver 0 WT KO Supplementary Figure S2. Liver weight (A), relative liver weight (B) and liver triglyceride content (C)Ǥ Š‡ ”‡•—Ž–• ƒ”‡ ”‡’”‡•‡–‡† ƒ• —‡› „‘š ƒ† ™Š‹•‡” ’Ž‘–•ǡ ™Š‡”‡ –Š‡ „Žƒ Ǧˆ‹ŽŽ‡† Œƒ‰‰‡† ‹” Ž‡• ‹†‹ ƒ–‡ ‹†‹˜‹†—ƒŽ ‹ ‡ ˆƒŽŽ‹‰ ‘—–•‹†‡ –Š‡ ͹ͷΨ ’‡” ‡–‹Ž‡ ’Ž—• ͳǤͷȉ‹–‡”Ǧ“—ƒ”–‹Ž‡ ”ƒ‰‡ ȋ Ȍ ‘” ʹͷΨ ’‡” ‡–‹Ž‡‹—•ͳǤͷȉ Ǥα͸Ǧͺˆ‘”„‘–Šƒ†Ǥȗǣ’δͲǤͲͷ ‘’ƒ”‡†–‘ǡ͓ƒ†͓͓ǣ’δͲǤͲͷƒ† ’δͲǤͲͳ ‘’ƒ”‡† –‘ ˆ‡†ǡ ”‡•’‡ –‹˜‡Ž›ǡ ș ƒ† șșǣ ’δͲǤͲͷ ƒ† ’δͲǤͲͳ ‘’ƒ”‡† –‘ Ž‘™Ǧˆƒ– †‹‡– ȋ Ȍǡ ”‡•’‡ –‹˜‡Ž›Ǥ 

ͺ͸ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

AWTBKO

3

C WT D

E WT F

G WT H

Supplementary Figure S3. Liver tissue integrity determined by H&E stainingǤǦǣ‘™Ǧˆƒ–ˆ‡†ǡǦǡ ‹‰ŠǦˆƒ– ˆ‡†ǡ Ǧ ǣ ‘™Ǧˆƒ– ˆƒ•–‡†ǡ Ǧ ǣ ‹‰ŠǦˆƒ– ˆƒ•–‡†Ǥ ǡ ǡ ǡ ǣ Ǥ ǡǡ ǡ ǣ Ǥ Š‘™ Š‡”‡ ƒ”‡ ”‡’”‡•‡–ƒ–‹˜‡’‹ –—”‡•ƒ–ͳšƒ‰‹ˆ‹ ƒ–‹‘

ͺ͹ Chapter 3

AWTBKO

C WT D

E WT F

G WT H

Supplementary Figure S4. Extent of steatosis determined by Oil Red O staining. Ǧǣ‘™Ǧˆƒ–ˆ‡†ǡǦǡ ‹‰ŠǦˆƒ– ˆ‡†ǡ Ǧ ǣ ‘™Ǧˆƒ– ˆƒ•–‡†ǡ Ǧ ǣ ‹‰ŠǦˆƒ– ˆƒ•–‡†Ǥ ǡ ǡ ǡ ǣ Ǥ ǡǡ ǡ ǣ Ǥ Š‘™ Š‡”‡ ƒ”‡ ”‡’”‡•‡–ƒ–‹˜‡’‹ –—”‡•ƒ–ͳšƒ‰‹ˆ‹ ƒ–‹‘

ͺͺ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

Supplementary table ST1: Histological semi-quantitative scoring of liver health NAFLD Oil Red O Vacuolar Steatosis Lobular Portal Ballooning activity staining changes grade inflammation inflammation score Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM    ‡† ͲǤ͵ ͲǤ͵ ͳǤͲ ͲǤͲ ͲǤ͵ ͲǤʹ ͲǤͲ ͲǤͲ ͲǤͷ ͲǤ͵ ͲǤ͹ ͲǤͷ ͲǤͺ ͲǤͶ    ƒ•–‡† ͵ǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ͳǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͶ ͲǤʹ ͲǤ͸ ͲǤ͵ ͳǤͶ ͲǤʹ   ‡† ʹǤͷ ͲǤ͵ ͳǤͲ ͲǤͲ ʹǤ͵ ͲǤʹ ͲǤͲ ͲǤͲ ͲǤͷ ͲǤʹ ͳǤͲ ͲǤͶ ʹǤͺ ͲǤ͵ 3   ƒ•–‡† ʹǤ͵ ͲǤ͵ ͲǤͲ ͲǤͲ ʹǤ͸ ͲǤʹ ͲǤͳ ͲǤͳ ͲǤͻ ͲǤͳ ͳǤͻ ͲǤͷ ͵Ǥ͸ ͲǤ͵    ‡† ͲǤͺ ͲǤ͵ ͳǤͲ ͲǤͲ ͲǤͷ ͲǤʹ ͲǤͲ ͲǤͲ ͲǤͷ ͲǤʹ ͳǤͲ ͲǤͶ ͳǤͲ ͲǤ͵    ƒ•–‡† ʹǤͺ ͲǤ͵ ͲǤͲ ͲǤͲ ͳǤͲ ͲǤʹ ͲǤͲ ͲǤͲ ͲǤͶ ͲǤʹ ͲǤ͸ ͲǤͶ ͳǤͶ ͲǤ͵   ‡† ʹǤ͵ ͲǤ͵ ͳǤͲ ͲǤͲ ʹǤʹ ͲǤ͵ ͲǤʹ ͲǤʹ ͲǤͷ ͲǤʹ ͲǤͺ ͲǤͷ ʹǤͺ ͲǤ͸   ƒ•–‡† ʹǤͷ ͲǤ͵ ͲǤͲ ͲǤͲ ʹǤ͸ ͲǤʹ ͲǤ͸ ͲǤͶ ͳǤ͵͓ ͲǤʹ ͵ǤͲ ͲǤͺ ͶǤͶ ͲǤͷ ˆ–‡” –‡”‹ƒ–‹‘ǡ Ž‹˜‡”• ™‡”‡ ”‡‘˜‡† “—‹ Ž› ƒ† ƒ ’‹‡ ‡ ™ƒ• ‹‡”•‡† ‹ ͳͲΨ „—ˆˆ‡”‡† ˆ‘”ƒŽ‹Ǥ —„•‡“—‡–Ž›ǡ–Š‡’‹‡ ‡•™‡”‡’”‘ ‡••‡†ˆ‘”’ƒ”ƒˆˆ‹•‡ –‹‘‹‰ƒ†•–ƒ‹‡†™‹–ŠŠ‡ƒ–‘š›Ž‹ƒ†‡‘•‹Ǥ Š‡•Ž‹†‡•™‡”‡• ‘”‡†ƒ ‘”†‹‰–‘Ž‡‹‡”‡–ƒŽȏͶʹȐǤ͓ǣ’δͲǤͳ„‡–™‡‡ƒ†Ǥ 

AB10 40 LF fed LF fed HF fed HF fed 8 30 LF fasted LF fasted 6 HF fasted HF fasted 20

RCI (-) 4

10 2 Pyruvate-induced Pyruvate-induced consumption rate (St4) rate consumption

2 0 0 (μmol/(min*gmitProtein))

O WT KO WT KO

CD 10 40 LF fed LF fed HF fed HF fed 8 30 LF fasted LF fasted 6 HF fasted HF fasted 20

RCI (-) 4

10 2 consumption rate (St4) rate consumption C16-acyl-CoA-induced C16-acyl-CoA-induced 2 0 0 (μmol/(min*gmitProtein))

O WT KO WT KO

EF 10 40 LF fed LF fed HF fed HF fed 8 30 LF fasted LF fasted 6 HF fasted HF fasted 20

RCI (-) 4

10 2 consumption rate (St4) rate consumption C8-acyl-CoA-induced C8-acyl-CoA-induced

2 0 0 (μmol/(min*gmitProtein)) O WT KO WT KO  Supplementary Figure S5. Pyruvate- (A-B), C16-acyl-CoA – (C-D) and C8-acyl-CoA-induced (E-F) state 4 oxygen consumption flux (A,C,E) and RCI (B, D,F) in liver mitochondria. State 4 is the basal oxygen consumption. RCI is the respiratory control index, calculated as State 3 O2 consumption rate divided by State 4 O2 consumption rate. Š‡”‡•—Ž–•ƒ”‡”‡’”‡•‡–‡†ƒ•—‡›„‘šƒ†™Š‹•‡”’Ž‘–•ǡ™Š‡”‡–Š‡ „Žƒ Ǧˆ‹ŽŽ‡†Œƒ‰‰‡† ‹” Ž‡•‹†‹ ƒ–‡‹†‹˜‹†—ƒŽ‹ ‡ˆƒŽŽ‹‰‘—–•‹†‡–Š‡͹ͷΨ’‡” ‡–‹Ž‡’Ž—•ͳǤͷȉ‹–‡”Ǧ“—ƒ”–‹Ž‡ ”ƒ‰‡ ȋ Ȍ ‘” ʹͷΨ ’‡” ‡–‹Ž‡ ‹—• ͳǤͷȉ Ǥα͸ ˆ‘” „‘–Š  ƒ† Ǥ  ǣ ‘™Ǧˆƒ–ǡ ǣ ‹‰ŠǦˆƒ–ǡ  ǣ ‡•’‹”ƒ–‘”› ‘–”‘Ž ‹†‡šǢ –Š‹• ‹• ƒŽ —Žƒ–‡† „› †‹˜‹†‹‰ –Š‡ ʹ ‘•—’–‹‘ ”ƒ–‡ ‹ –ƒ–‡ ͵ „› –Š‡ ʹ ‘•—’–‹‘”ƒ–‡‹–ƒ–‡ͶǤ

ͺͻ Chapter 3

AB Atp5b Cox5a 800 150 LF fed LF fed HF fed HF fed 600 LF fasted 100 LF fasted g mito prot) g mito HF fasted prot) g mito HF fasted P P 400

50 200

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

CDCycs Gpx4 80 10 LF fed LF fed HF fed 8 HF fed 60 LF fasted LF fasted g mito prot) g mito HF fasted prot) g mito 6

P HF fasted P 40 4 20 2

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

EF Gsr Mtnd5 6 10 LF fed LF fed HF fed 8 HF fed 4 LF fasted LF fasted g mito prot) g mito HF fasted prot) g mito 6 P HF fasted P

4 2 2

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

GH Ndufs1 Prdx6/Prdx6b 60 10 LF fed LF fed HF fed 8 HF fed 40 LF fasted LF fasted g mito prot) g mito HF fasted prot) g mito 6 HF fasted P P

4 20 2

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/ 

ͻͲ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

IJ Sdha Sdhb 150 100 LF fed LF fed HF fed 80 HF fed 100 LF fasted LF fasted g mito prot) g mito HF fasted prot) g mito 60 HF fasted P P

40 50 3 20

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

KL Slc25a3 Slc25a4 200 50 LF fed LF fed HF fed HF fed 150 40 LF fasted LF fasted g mito prot) g mito HF fasted prot) g mito 30 P HF fasted 100 P 20 50 10

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

MNSlc25a5 Sod2 600 100 LF fed LF fed HF fed 80 HF fed 400 LF fasted LF fasted g mito prot) g mito HF fasted prot) g mito 60 HF fasted P P

40 200 20

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

OP Ucp2 Ucp3 2.5 2.0 LF fed LF fed HF fed HF fed 2.0 1.5 LF fasted LF fasted g mito prot) g mito g mito prot) g mito HF fasted 1.5 HF fasted P P 1.0 1.0 0.5 0.5

0.0 0.0 WT KO

WT KO [Protein] (fmol/ [Protein] (fmol/ 

ͻͳ Chapter 3

QR Uqcrc2 Aco2 150 150 LF fed LF fed HF fed HF fed LF fasted 100 LF fasted 100 g mito prot) g mito g mito prot) g mito HF fasted HF fasted P P

50 50

0 0 WT KO

WT KO [Protein] (fmol/ [Protein] (fmol/

STCs Dlat 150 60 LF fed LF fed HF fed HF fed 100 LF fasted 40 LF fasted g mito prot) g mito HF fasted prot) g mito HF fasted P P

50 20

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

UVDld Dlst 100 100 LF fed LF fed 80 HF fed 80 HF fed LF fasted LF fasted g mito prot) g mito 60 HF fasted prot) g mito 60 HF fasted P P

40 40

20 20

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

WX Fh (Fh1) ldh2 80 80 LF fed LF fed HF fed HF fed 60 60 LF fasted LF fasted g mito prot) g mito HF fasted prot) g mito P HF fasted P 40 40

20 20

0 0 WT KO

[Protein] (fmol/ WT KO [Protein] (fmol/ 

ͻʹ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

YZ ldh3a Mdh2 40 250 LF fed LF fed HF fed 200 HF fed 30 LF fasted LF fasted

g mito prot) g mito prot) g mito 150

P HF fasted P HF fasted 20 100 3 10 50

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

AAOgdh/Ogdhl AB Pdha1 40 80 LF fed LF fed HF fed HF fed 30 60 LF fasted LF fasted g mito prot) g mito HF fasted prot) g mito P

P HF fasted 20 40

10 20

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

AC AD Pdk1 Slc25a1 6 100 LF fed LF fed HF fed 80 HF fed 4 LF fasted LF fasted g mito prot) g mito g mito prot) g mito 60 P HF fasted HF fasted P

40 2 20

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

AESlc25a10 AF Slc25a11 80 60 LF fed LF fed HF fed HF fed 60 LF fasted 40 LF fasted g mito prot) g mito HF fasted prot) g mito P

P HF fasted 40 ΐΐ

20 20

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/ 

ͻ͵ Chapter 3

AG AH Slc25a22 Sucla2 40 80 LF fed LF fed HF fed HF fed 30 60 LF fasted LF fasted g mito prot) g mito HF fasted prot) g mito HF fasted P P 20 40

10 20

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

AI AJ Suclg1 Suclg2 200 150 LF fed LF fed HF fed HF fed 150 LF fasted 100 LF fasted g mito prot) g mito g mito prot) g mito HF fasted HF fasted P P 100 η͕ΐΐ η͕ΐ 50 50

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

AKAcaa2 AL Acadl 1000 150 LF fed LF fed 800 HF fed HF fed LF fasted 100 LF fasted g mito prot) g mito 600 HF fasted prot) g mito HF fasted P P

400 50 200

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

AMAcadm AN Acads 150 150 LF fed LF fed HF fed HF fed ΐΐ 100 LF fasted 100 LF fasted g mito prot) g mito HF fasted prot) g mito HF fasted P P

50 50

ΎΎΎΎ 0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/ 

ͻͶ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

AO AP Acadvl Cpt1a 80 60 LF fed LF fed HF fed HF fed 60 LF fasted 40 LF fasted g mito prot) g mito HF fasted prot) g mito P

P HF fasted 40 20 3 20

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

AQ AR Cpt1b Cpt2 4 80 LF fed LF fed HF fed HF fed 3 60 LF fasted LF fasted g mito prot) g mito g mito prot) g mito

P HF fasted HF fasted P 2 40

1 20

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

ASDecr1 AT Echs1 150 200 LF fed LF fed HF fed HF fed 150 LF fasted 100 LF fasted g mito prot) g mito g mito prot) g mito HF fasted P

P HF fasted 100

50 50

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

AU AV Eci1 Etfa 150 400 LF fed LF fed HF fed HF fed 300 100 LF fasted LF fasted g mito prot) g mito HF fasted prot) g mito HF fasted P P ΐΐ 200

50 η 100

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

ͻͷ Chapter 3

AW AX Etfb Etfdh 300 100 LF fed LF fed HF fed 80 HF fed LF fasted LF fasted

200 prot) g mito g mito prot) g mito 60 HF fasted HF fasted P P

40 100 20

0 0 WT KO

WT KO [Protein] (fmol/ [Protein] (fmol/

AYHadh AZ Hadha 300 200 LF fed LF fed HF fed HF fed 150 200 LF fasted LF fasted g mito prot) g mito HF fasted prot) g mito HF fasted P P 100 ΐΐ 100 50

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

BAHadhb BB Slc25a29 250 4 LF fed LF fed 200 HF fed HF fed 3 LF fasted LF fasted g mito prot) g mito 150 HF fasted prot) g mito HF fasted P P 2 100

1 50

0 0 WT KO WT KO [Protein] (fmol/ [Protein] (fmol/

BC Slc25a20 15 LF fed HF fed 10 LF fasted g mito prot) g mito HF fasted P

5

0 WT KO [Protein] (fmol/  Supplementary Figure S6. Absolute levels of mitochondrial liver proteins. In the figure, the gene names corresponding to the proteins are given. Š‡”‡•—Ž–•ƒ”‡”‡’”‡•‡–‡†ƒ•—‡›„‘šƒ†™Š‹•‡” ’Ž‘–•ǡ™Š‡”‡–Š‡„Žƒ Ǧˆ‹ŽŽ‡†Œƒ‰‰‡† ‹” Ž‡•‹†‹ ƒ–‡‹†‹˜‹†—ƒŽ‹ ‡ˆƒŽŽ‹‰‘—–•‹†‡–Š‡͹ͷΨ’‡” ‡–‹Ž‡’Ž—• ͳǤͷȉ‹–‡”Ǧ“—ƒ”–‹Ž‡”ƒ‰‡ȋ Ȍ‘”ʹͷΨ’‡” ‡–‹Ž‡‹—•ͳǤͷȉ Ǥ ǣ‘™Ǧˆƒ– ǣ ‹‰ŠǦˆƒ–Ǥα͸Ǧͺˆ‘”„‘–Š ƒ†Ǥȗǣ’δͲǤͲͷ ‘’ƒ”‡†–‘ǡ͓ƒ†͓͓ǣ’δͲǤͲͷƒ†’δͲǤͲͳ ‘’ƒ”‡†–‘ˆ‡†ǡ”‡•’‡ –‹˜‡Ž›ǡșƒ†șșǣ ’δͲǤͲͷƒ†’δͲǤͲͳ ‘’ƒ”‡†–‘ ǡ”‡•’‡ –‹˜‡Ž›Ǥ

ͻ͸ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

10

0

-10 3

PC2 (23%) -20

-30

-40 -20 -10 0 10 20 30 PC1 (39%) Supplementary figure S7. Principal Component Analysis of mRNA expression data. ”‡›Ǧˆ‹ŽŽ‡†ǣ ‡†Ǣ Š‹–‡Ǧˆ‹ŽŽ‡†ǣˆƒ•–‡†Ǣ ”‹ƒ‰Ž‡ǣǡ ‹” Ž‡ǣǢ Ž—‡ǣ‘™Ǧˆƒ–†‹‡–Ǣ ‡†ǣ ‹‰ŠǦˆƒ–†‹‡–Ǥ α͵ǦͶˆ‘”ƒ†Ǥ

Low-fat fasted High-fat fasted A B 100 100 50 50

5 5 4 4 3 3

Fold Change 2 Change Fold 2 1 1 0 0 Low-fat fed High-fat fed C D 100 100 50 50

5 5 4 4 3 3

Fold Change Fold 2 Change Fold 2 1 1 0 0

Voom_up Voom_up DeSeq2_up DeSeq2_up Voom_down Voom_down DeSeq2_down DeSeq2_down Method Method

EFUp Down ,&&Ă >&&Ğ ,&&Ă >&&Ğ

ϮϮ Ϯϵ ϭϴ ϯϬ ϭϬϬ ϭϬϬ ϰϵ Ϯϱ ϳϱ ϭϯ ϬϬ ϬϬ

ϭϬϬ ϭϭϬ Ϭ Ϭ Ϭ Ϭ Ϭ Ϭ >&&Ă ,&&Ğ >&&Ă ,&&Ğ

Supplementary figure S8. KO vs WT fold changes for the differential gene expression analysis methods DeSeq2 and Voom (A-D) and Venn diagrams for the Voom method (E-F) (p<0.01)ǣ α‘™Ǧ ˆƒ–†‹‡–ǡ α ‹‰ŠǦˆƒ–†‹‡–ǡ ƒα ƒ•–‡†ǡ ‡α ‡†Ǥ α͵ǦͶ’‡”‰”‘—’Ǥ

ͻ͹ Chapter 3

Supplementary table ST2. Differentially-expressed genes (p<0.01)a Low-fat fasted KO/WT (DeSeq2) High-fat fasted KO/WT (DeSeq2) Upregulated Downregulated Upregulated Downregulated Gene FC padj Gene FC padj Gene FC padj Gene FC padj Star 2.3 0.05 Eif4ebp2 0.5 0.01 ƒƒͳ ʹǤ͹ ͲǤ͵ͳ „ ƒ͵ ͲǤͷ ͲǤ͵Ͷ Akr1b7 2.0 0.05 ƒ–†ͳ ͲǤͶ ͲǤʹͲ ‰„ ͳǤ͸ ͲǤ͵ͳ Ž ͻƒ͵”ʹ ͲǤͷ ͲǤ͹ʹ ͳ͹ ͸ͷ͵ ʹǤʹ ͲǤʹͲ —’ ͲǤ͸ ͲǤ͵͸ Ž Ͷͳƒʹ ʹǤͳ ͲǤ͵ͳ ‘’͵ƒ ͲǤͷ ͲǤ͹ʹ ‘—ʹˆͳ ʹǤͶ ͲǤͶͷ ‡ͳͳͲ ͲǤͷ ͲǤͶͷ  ʹ ʹǤͷ ͲǤͶͲ ‡š͸ ͲǤ͸ ͲǤ͹ʹ  ͵ ʹǤͲ ͲǤͶͷ •’ƒͳͶ ͲǤ͸ ͲǤͶͷ ‡͵ͻƒ ʹǤͲ ͲǤ͸Ͳ ‘—͸ˆͳ ͲǤͷ ͲǤ͹͵  –ƒʹ ʹǤʹ ͲǤͶͷ ʹͷͳͲͲ͵ͻͳͺ‹ ͲǤ͸ ͲǤͶͺ ‰ƒ ͳǤͷ ͲǤ͹ʹ •–ͳ ͲǤ͸ ͲǤͻͶ ”‡„„’ ͳǤͶ ͲǤͶͺ ˆ‘†ͳ ͲǤͶ ͲǤͶͺ ’ • ʹǤͲ ͲǤ͹ʹ Ž ʹͷƒͳ ͲǤ͹ ͲǤͻͶ „ ͳ†ͳͶ ͳǤͻ ͲǤͶͺ ƒ„ʹͺ ͲǤ͸ ͲǤͶͺ •ͳ„ ͳǤͺ ͲǤ͹͵ Šƒ’͵ ͲǤͷ ͳǤͲͲ †—ˆƒ„ͳ ͳǤ͸ ͲǤͶͺ ”‡šͳ ͲǤͷ ͲǤͶͺ –‹Š͵ ͳǤ͹ ͲǤ͹ͷ Ž ‘ͳƒͶ ͲǤͷ ͳǤͲͲ • ͳǤͷ ͲǤͶͺ ‰„ ͲǤͶ ͲǤͶͺ ‰Žͳ ʹǤʹ ͲǤ͹ͷ ’”ͳ ͲǤͷ ͳǤͲͲ –ͳͻ ͳǤͺ ͲǤͶͺ ͻ•ˆʹ ͲǤ͹ ͲǤͶͺ –‰ʹ ʹǤͳ ͲǤͻͶ „–„͹„ ͲǤ͸ ͳǤͲͲ †ƒ’ͳ ͳǤ͸ ͲǤͶͺ ’ͳ ͲǤ͹ ͲǤͶͺ Ž„Šͺ ͳǤͺ ͲǤͻͶ †’ʹ ͲǤ͸ ͳǤͲͲ ‹͵ ʹǤͲ ͲǤͶͺ –‡” ͲǤ͸ ͲǤͶͺ ’‹‰ ͳǤͶ ͳǤͲͲ Žƒ– ͲǤͷ ͳǤͲͲ ™Šƒ„ ͳǤ͵ ͲǤͶͺ ‘ •͸ ͲǤ͸ ͲǤͶͺ ˆƒͳ ͳǤ͹ ͳǤͲͲ ͳ͵ͳͻͻ ͲǤͷ ͳǤͲͲ ”š‡•ʹ ͳǤͻ ͲǤͶͺ ƒ’œ„ ͲǤ͹ ͲǤͶͺ ‘—ʹˆʹ ͳǤͻ ͳǤͲͲ ‹ʹͳ ͲǤͷ ͳǤͲͲ ’•Ͷ ͳǤͻ ͲǤͶͺ ”” ͸ͳ ͲǤͷ ͲǤͶͺ ”ͳ ͳǤͷ ͳǤͲͲ ”’„ ͲǤ͹ ͳǤͲͲ •†ͳ͹„ͳͳ ͳǤ͸ ͲǤͶͺ ƒ•‹ʹ ͲǤͷ ͲǤͶͺ ‹’Ͷʹƒ ͳǤͻ ͳǤͲͲ ‘ʹʹ ͲǤ͹ ͳǤͲͲ ʹͲͲͲ ͳǤ͸ ͲǤͶͺ •ͳ‰ʹ ͲǤ͹ ͲǤͶͺ ’’ͳ”ͻƒ ͳǤ͸ ͳǤͲͲ Š’ ͲǤ͸ ͳǤͲͲ ’’ͳ”͵‰ ʹǤͳ ͲǤͶͺ ‹’͹ ͲǤ͸ ͲǤͶͺ ›’͵ƒͳ͵ ͳǤͶ ͳǤͲͲ ›š ͲǤ͸ ͳǤͲͲ ‡ͳͳͷ ͳǤ͹ ͲǤͶͺ † ʹ͵ ͲǤ͸ ͲǤͶͺ ‘ Ͷ ʹǤͲ ͳǤͲͲ ‘„ʹ ͲǤ͸ ͳǤͲͲ ˆ’– ͳǤͻ ͲǤͶͺ ‰ ͲǤ͸ ͲǤͶͺ ƒͳͳ͹ƒ ʹǤͲ ͳǤͲͲ ›’ʹ ͵ͺ ͲǤ͸ ͳǤͲͲ ”ƒͳ ͳǤͶ ͲǤͶͺ ƒ Šʹ ͲǤͷ ͲǤͶͺ ˆ’ͻ͵͹ ʹǤͲ ͳǤͲͲ ‹ƒ•͵ ͲǤ͸ ͳǤͲͲ ˆ–ͳͶͲ ʹǤͳ ͲǤͶͺ  † ͻ͵ ͲǤ͸ ͲǤͶͺ ˆ† ʹͳ ͳǤ͵ ͳǤͲͲ –„’Ͷ ͲǤ͸ ͳǤͲͲ ˆ’ͳʹͲ ͳǤͺ ͲǤͶͺ ”•ˆͳͲ ͲǤ͸ ͲǤͶͺ †‹ƒͷ ͳǤͷ ͳǤͲͲ †‹ͳ ͲǤ͹ ͳǤͲͲ ””ͳͶŽ ͳǤ͸ ͲǤͶͺ ‹’ʹ ͲǤͷ ͲǤͶͺ —’ʹͳ ͳǤ͹ ͳǤͲͲ ’ͷ ͲǤͷ ͳǤͲͲ ˆͳʹͳ ͳǤ͸ ͲǤͶͺ ””‘• ͲǤͷ ͲǤͶͺ ƒƒʹ ʹǤͲ ͳǤͲͲ ͳͳͳͲͲͷͳʹͲ‹ ͲǤͷ ͳǤͲͲ ”‹–ͳ ͳǤͻ ͲǤͶͺ ƒŽ„ ͲǤ͸ ͲǤͶͺ ˆŽƒ” ͳǤͶ ͳǤͲͲ ‹Ž‹ͳ ͲǤ͸ ͳǤͲͲ Ž”šʹ ͳǤͷ ͲǤͶͻ ƒ„ͷ„ ͲǤ͸ ͲǤͶͺ Ž ʹʹƒʹͺ ͳǤͻ ͳǤͲͲ ‡ƒͳ ͲǤ͸ ͳǤͲͲ „ˆͳ ʹǤͳ ͲǤͶͻ ”‘•  ͲǤ͹ ͲǤͶͺ „ʹͲ ʹǤͲ ͳǤͲͲ    ‹’„Ž ͳǤͶ ͲǤͶͻ  † ͲǤ͸ ͲǤͶͺ †ƒ’ʹ ͳǤͷ ͳǤͲͲ    •ƒ† ͳǤ͹ ͲǤͶͻ „’ʹ ͲǤͷ ͲǤͶͺ ‡Žˆƒ ͳǤ͸ ͳǤͲͲ    ‡‡’ͷ ͳǤ͹ ͲǤͶͻ ƒƒͶͲ ͲǤͷ ͲǤͶͺ ͷͻͲͲ ͳǤͺ ͳǤͲͲ    ›’ʹͳƒͳ ͳǤ͸ ͲǤͶͻ •‰ͳͷ ͲǤ͸ ͲǤͶͺ –ʹ ʹǤͲ ͳǤͲͲ    Š•Žͳ ʹǤͲ ͲǤͶͻ šš ͷ ͲǤ͹ ͲǤͶͻ ƒͺͳƒ ͳǤͻ ͳǤͲͲ    •†ͳͲ ͳǤͷ ͲǤͶͻ ƒͳ† ͲǤ͹ ͲǤͶͻ –‡ƒ’Ͷ ͳǤ͹ ͳǤͲͲ    † ͳ͸ ͳǤ͹ ͲǤͶͻ šͳ ͲǤ͸ ͲǤͶͻ –„†ͳͻ ͳǤͻ ͳǤͲͲ    †—ˆ˜͵ ͳǤͶ ͲǤͷʹ ‰Žʹ ͲǤͷ ͲǤͶͻ ‹ˆ͵ƒ ͳǤͶ ͳǤͲͲ    ””ͷŽ ʹǤͲ ͲǤͷʹ ƒ’ͷ ͲǤ͸ ͲǤͶͻ  ‡”ʹ ͳǤ͹ ͳǤͲͲ    ‡” ʹǤͲ ͲǤͷʹ —–ˆʹ ͲǤͷ ͲǤͷʹ ͺ„ ͳǤͶ ͳǤͲͲ    †šͶͻ ͳǤ͹ ͲǤͷʹ  ”Ž ͲǤ͸ ͲǤͷʹ ‡ ™ʹ ͳǤͺ ͳǤͲͲ    ’” ͳǤͶ ͲǤͷʹ ’–ͳ ͲǤ͸ ͲǤͷ͵ ‡”’‹ƒͳͲ ͳǤͷ ͳǤͲͲ    ‡ͳͶ͹ ͳǤ͵ ͲǤͷͶ –‘Žͳ ͲǤͷ ͲǤͷͶ ‰‰ ͳǤͶ ͳǤͲͲ     ‘Ž ‡ ʹǤͳ ͲǤͷͶ ”Š‰†‹„ ͲǤͷ ͲǤͷͶ ’–›ʹ†ͳ ͳǤͷ ͳǤͲͲ    ƒ”• ͳǤͷ ͲǤͷͶ –͵ ͲǤ͸ ͲǤͷͶ ‡š†  ͳǤͻ ͳǤͲͲ    †ͳ ͳǤͺ ͲǤͷͶ ˆ†ʹ ͲǤ͹ ͲǤͷͶ       ”’ˆ͵ͺ„ ͳǤͶ ͲǤͷͶ ƒ•Žͳ ͲǤͷ ͲǤͷͶ        ͳƒ ͳǤͶ ͲǤͷͶ ƒʹ ͲǤͺ ͲǤͷͶ       ’”ͳ ͳǤͻ ͲǤͷͷ ‡ʹͷ ͲǤ͹ ͲǤͷͶ       ›”‘š†ͳ ͳǤ͸ ͲǤͷͷ Š„ ͲǤ͹ ͲǤͷͶ       ͳ͹ͲͲͲʹ͹ ͳͻ‹ ʹǤͲ ͲǤͷ͸ †›Ž ͲǤ͸ ͲǤͷͷ       ’–›ʹ†ͳ ͳǤͷ ͲǤͷ͸ †‡Ͷ„ ͲǤ͸ ͲǤͷͷ       ’ʹ ͳǤͷ ͲǤͷ͸ ͳͲ͹͸͹ ͲǤͷ ͲǤͷ͸       ’Š„͵ ʹǤͲ ͲǤͷ͸ –›Šʹ ͲǤ͹ ͲǤͷ͸       Ž‘ ͳ•ͷ ͳǤͷ ͲǤͷ͸ ‡”–ƒ†ʹ ͲǤ͹ ͲǤͷ͸       ’•Ͷͷ ͳǤͻ ͲǤͷ͹  ƒ’ ͲǤ͹ ͲǤͷ͸       †—ˆƒͷ ͳǤͷ ͲǤͷͺ ”’ͶͶ ͲǤ͹ ͲǤͷ͸       ‘•Ž ͳǤͶ ͲǤͷͺ ’‘’–ͳ ͲǤ͸ ͲǤͷ͸        ƒƒͳ„ ͳǤͺ ͲǤͷͺ ƒ’ʹ͸ ͲǤͷ ͲǤͷ͸       † ͳͶ„ ͳǤͶ ͲǤͷͺ ˆ’͹ʹͻƒ ͲǤ͸ ͲǤͷ͸       ‹ˆʹ„ͷ ͳǤͷ ͲǤͷͺ ‡š‹ͳ ͲǤ͹ ͲǤͷ͸       ’Ž͵͹ ͳǤͶ ͲǤͷͻ •†͵„͹ ͲǤͺ ͲǤͷ͹       ʹ͵Ͳʹͳͻʹʹ‹ ͳǤ͵ ͲǤ͸Ͳ Š’‘ ͲǤ͸ ͲǤͷ͹       ‘”–ͳ ͳǤͺ ͲǤ͸ͳ ˆ’ʹͺͲ  ͲǤͷ ͲǤͷ͹       ‹•” ͳǤͶ ͲǤ͸ʹ ƒ•’ͳ ͲǤͺ ͲǤͷͺ       Š•›ͳ ͳǤ͹ ͲǤ͸͵ Ž• ”ʹ ͲǤ͹ ͲǤ͸Ͳ       Šͳ ͳǤ͵ ͲǤ͸͵ œˆͷ ͲǤ͹ ͲǤ͸ʹ       ͳͲ͸Ͷ͵ ͳǤͺ ͲǤ͸͵ ƒ” ͲǤ͸ ͲǤ͸ʹ       • ͳ ͳǤ͵ ͲǤ͸͵ †Žʹ ͲǤ͹ ͲǤ͸͵       Ž‡ ͳʹƒ ͳǤͺ ͲǤ͸͵ „‡ʹ‰ʹ ͲǤ͸ ͲǤ͸͵        –ͺ ͳǤ͵ ͲǤ͸͵  Ͷ ͲǤ͸ ͲǤ͸͵      

ͻͺ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

Low-fat fasted KO/WT (DeSeq2)- continued  Upregulated Downregulated   Gene FC padj Gene FC padj —ŽͶ„ ͳǤ͸ ͲǤ͸͵ –’ʹ„ʹ ͲǤͷ ͲǤ͸͵        –ͳ ͳǤ͸ ͲǤ͸͵ –”ʹ ͲǤͷ ͲǤ͸͵       ‡ˆͳ„ʹ ͳǤͶ ͲǤ͸͵ †ŠŠ ͳ͹ ͲǤ͸ ͲǤ͸͵       ŽŽ–ͳͲ ͳǤͶ ͲǤ͸͵ ›ʹ ͲǤ͹ ͲǤ͸͵       ‹ˆ͵ ͳǤͷ ͲǤ͸͵ ƒͳͳ͹„ ͲǤ͸ ͲǤ͸͵       –’†ʹ ͳǤͻ ͲǤ͸͵ ‹ƒ’ͳ ͲǤ͹ ͲǤ͸͵       „š͵ ͳǤ͹ ͲǤ͸͵ ‡„’ͳ ͲǤ͹ ͲǤ͸͵       3 —–ˆʹǦ’•ͳ ͳǤ͹ ͲǤ͸͵ ”ͳ ͲǤ͹ ͲǤ͸͵       †’ʹ ͳǤͷ ͲǤ͸͵ •‰ʹ ͲǤ͹ ͲǤ͸͵       ‹•–ʹŠ͵ ʹ ͳǤͻ ͲǤ͸͵ Ž†Šͳ„ͳ ͲǤ͹ ͲǤ͸͵       „ƒ• ͳǤͺ ͲǤ͸͵ ’•ͳͷŽͳ ͲǤ͸ ͲǤ͸͵       ˆ’ͷͻͷ ͳǤͻ ͲǤ͸͵ ƒ‰‡†ʹ ͲǤͷ ͲǤ͸͵        ʹ†ͳ„ ͳǤ͹ ͲǤ͸͵ –š͹Ž͵„ ͲǤͺ ͲǤ͸͵       ‡ƒͷ„ ͳǤͻ ͲǤ͸͵ „Š†ͺ ͲǤͷ ͲǤ͸͵       ͵ ͳǤͻ ͲǤ͸͵ Ž„Šͳ ͲǤ͸ ͲǤ͸͵          Š•‰ ͲǤͺ ͲǤ͸͵        Low-fat fed KO/WT (DeSeq2) High-fat fed KO/WT (DeSeq2) Upregulated Downregulated Upregulated Downregulated Gene FC padj Gene FC padj Gene FC padj Gene FC padj ”’͸ͺ ͳǤͷ ͲǤͷʹ ͵‰ƒŽ–ͳ ͲǤͶ ͲǤʹͲ Šƒ‹ ʹǤ͸ ͲǤ͸ͳ Acnat2 0.4 0.02 ”‘ͳŽ„ ͳǤͻ ͲǤͺͶ ƒ Šʹ ͲǤͶ ͲǤ͵Ͳ  ͳƒ ͳǤͺ ͲǤͻ͸ ‡ƒͷ„ ͲǤͶ ͲǤ͸ͳ ›‘͹ƒ ʹǤͲ ͳǤͲͲ  ͵Šͳ͵ ͲǤͷ ͲǤ͵͵ ƒ‰Žͳ ʹǤͳ ͳǤͲͲ Ž ʹʹƒʹͻ ͲǤͷ  ͲʹͻʹͳͶ ʹǤͳ ͳǤͲͲ ‘‰ͷ ͲǤͶ ͲǤ͵Ͷ ›’Ͷƒͳʹ„ ͳǤͺ ͳǤͲͲ • ƒͳ ͲǤ͸ ͳǤͲͲ ‘š“ͳ ʹǤͳ ͳǤͲͲ › Ž ͲǤͷ ͲǤ͵Ͷ • ƒͶ ʹǤʹ ͳǤͲͲ ”‘ ƒͳ ͲǤͷ ͳǤͲͲ †ŠŠ ͹ ͳǤͺ ͳǤͲͲ ˆ’͵ͺ͸ ͲǤͷ ͲǤͷʹ Žœˆͳ ͳǤͺ ͳǤͲͲ ”’•͵ͷ ͲǤ͹ ͳǤͲͲ ‡”Ž͵ ʹǤͲ ͳǤͲͲ š”‰ ͲǤͷ ͲǤͺͶ ‘ͳ ʹǤͳ ͳǤͲͲ – ͲǤ͹ ͳǤͲͲ ƒ„‰‡ˆͳ ͳǤͻ ͳǤͲͲ  Š ͳͳ ͲǤ͸ ͲǤͻͻ ‡†ͳ„ ͳǤ͸ ͳǤͲͲ ”†ʹͶ ͲǤͷ ͳǤͲͲ ‡ʹͳͺ ͳǤͻ ͳǤͲͲ Ž ͳ͸ƒͳ ͲǤ͸ ͲǤͻͻ † ͷ ͳǤͻ ͳǤͲͲ ˆšʹ ͲǤ͹ ͳǤͲͲ ‡ʹͶͺ ͳǤͶ ͳǤͲͲ ‡š ͲǤͶ ͳǤͲͲ  •– ͳǤ͸ ͳǤͲͲ ›’ʹ„ͻ ͲǤ͸ ͳǤͲͲ „ˆͳ ͳǤ͹ ͳǤͲͲ ™Šƒœ ͲǤ͹ ͳǤͲͲ ”ƒ† ͳ ʹǤͳ ͳǤͲͲ ’ͳ‰ͳ ͲǤ͹ ͳǤͲͲ ”’ˆ͵ͳ ͳǤͻ ͳǤͲͲ †šͷͷ ͲǤͷ ͳǤͲͲ † ͳͶƒ ʹǤͳ ͳǤͲͲ –ƒ–•ˆͳ ͲǤ͹ ͳǤͲͲ ’ʹ ͳǤ͵ ͳǤͲͲ †”Ͷʹ‡ͳ ͲǤ͹ ͳǤͲͲ ‹ˆ” ͳǤ͸ ͳǤͲͲ †šͷʹ ͲǤ͸ ͳǤͲͲ Ž ͳͳƒʹ ͳǤ͸ ͳǤͲͲ „ʹ͸ ͲǤ͹ ͳǤͲͲ ”‹•’Ž†ʹ ʹǤͳ ͳǤͲͲ ƒš ͲǤ͸  „ „ͳ„ ͳǤͻ ͳǤͲͲ  Š Ͷ ͲǤ͸ ͳǤͲͲ Ž’‹ ʹǤͳ ͳǤͲͲ ’’‡ͳ ͲǤͷ ͳǤͲͲ ƒʹ͸ˆ ʹǤͲ ͳǤͲͲ ”’ʹ ͲǤ͸ ͳǤͲͲ „Žͳ ͳǤͻ ͳǤͲͲ ‘ͳʹͳ ͲǤ͸ ͳǤͲͲ  „’Ͷ ͳǤͺ ͳǤͲͲ †šͶʹ ͲǤ͹ ͳǤͲͲ ͺ„ ͳǤͷ ͳǤͲͲ Ž ͳʹƒ͸ ͲǤ͸ ͳǤͲͲ „‘ͳ ͳǤͶ ͳǤͲͲ –ˆʹ‹”†ʹ ͲǤͷ ͳǤͲͲ ˆ’͸ͷͷ ͳǤ͹ ͳǤͲͲ ‡’ƒ ͲǤ͸ ͳǤͲͲ ‡šͳͳ„ ͳǤͷ ͳǤͲͲ ‹͵ † ͲǤͷ ͳǤͲͲ ˆ’͸ͺͻ ͳǤͻ ͳǤͲͲ ‹’ ͲǤ͹ ͳǤͲͲ •ͶƒͶ† ͳǤͻ ͳǤͲͲ ŠŠ ͲǤ͸ ͳǤͲͲ Šˆͺ ͳǤ͸ ͳǤͲͲ ƒŽŽʹ ͲǤͷ ͳǤͲͲ „ƒ• ͳǤͻ ͳǤͲͲ ‘Ž‰ƒŽ–ʹ ͲǤͷ ͳǤͲͲ ʹ͸ͳͲͲʹͳͲͳ‹ ʹǤͲ ͳǤͲͲ Ž•ͳ ͲǤͷ ͳǤͲͲ ”„ͳ ʹǤͲ ͳǤͲͲ ’‘„” ͲǤͷ ͳǤͲͲ ƒƒʹͷ ͳǤͷ ͳǤͲͲ †ͳͲ ͲǤ͸ ͳǤͲͲ ”’•ͳͺ  ʹǤͲ ͳǤͲͲ „Ͷ ͲǤͷ ͳǤͲͲ ˆ’ͳ͸ͻ ʹǤͲ ͳǤͲͲ ˆͳͶͻ ͲǤ͹ ͳǤͲͲ • ͳǤͶ ͳǤͲͲ ‘Ž͹ ͲǤ͹ ͳǤͲͲ ƒ– ͳǤ͸  ’͵•ͳ ͲǤͷ ͳǤͲͲ †‰ˆ† ͳǤͻ ͳǤͲͲ ‹ˆʹ„͵ ͲǤͷ ͳǤͲͲ ŽˆͶ ʹǤͲ ͳǤͲͲ Š†ͳ ͲǤ͹ ͳǤͲͲ ƒ‰Žͳ ͳǤͻ ͳǤͲͲ „Š†ͳ͵ ͲǤ͹ ͳǤͲͲ ͺʹ͸ ͳǤͻ ͳǤͲͲ ’Žͻ ͲǤ͸ ͳǤͲͲ ’—•†͵ ͳǤ͸ ͳǤͲͲ ʹͲ͸ͻ͸ ͲǤͷ ͳǤͲͲ ‡˜͵Ž ͳǤͷ ͳǤͲͲ ‹•–ʹŠʹ„‡ ͲǤ͸ ͳǤͲͲ ‡ ’”ͳ ͳǤ͸ ͳǤͲͲ ˆ’ͻͲ ͲǤͷ ͳǤͲͲ ŠŽ͵ ͳǤͻ ͳǤͲͲ    †›Š˜ͳ ͳǤ͹ ͳǤͲͲ  ƒ’ʹͻ ͲǤ͸ ͳǤͲͲ ƒ’‰‡ˆ͸ ͳǤͷ ͳǤͲͲ     š” ͳǤͷ ͳǤͲͲ ‹ʹ ͲǤͷ ͳǤͲͲ ˆ’͵͸ͻ ͳǤ͸ ͳǤͲͲ     ƒ‹’ ͳǤͻ ͳǤͲͲ –Ǧ†ͷ ͲǤͺ ͳǤͲͲ  –’’ͳ ͳǤͷ ͳǤͲͲ    ”‹–ʹ ʹǤͲ ͳǤͲͲ ‡͵ͻƒ ͲǤ͸ ͳǤͲͲ ’’Ͷ ʹǤͲ ͳǤͲͲ    ‹•ʹ ͳǤͷ ͳǤͲͲ  ͵ ͲǤͷ ͳǤͲͲ ƒŒ ͳ͹ ʹǤͲ ͳǤͲͲ    Š‘ ʹǤͲ ͳǤͲͲ ‘‰œ ͲǤ͹ ͳǤͲͲ ƒ’ͳͲ ͳǤ͹ ͳǤͲͲ    ƒ”˜‰ ͳǤͺ ͳǤͲͲ ƒ’͹ ͲǤ͸ ͳǤͲͲ Œƒͳ ͳǤͶ ͳǤͲͲ    ‰Š„’ʹ ͳǤͻ ͳǤͲͲ ˆƒͷ ͲǤ͸ ͳǤͲͲ ͵„–Ž͵ ͳǤͺ ͳǤͲͲ    ‘ƒ–ʹ ͳǤ͹ ͳǤͲͲ ‡ͳͺ ͲǤ͸ ͳǤͲͲ  ”•ͳ ͳǤͺ     ŠˆͳͲ ͳǤͷ ͳǤͲͲ ƒ„„”ʹ ͲǤ͸ ͳǤͲͲ ’•„Ͷ ͳǤͻ ͳǤͲͲ    –ˆ͵ƒ ͳǤͺ ͳǤͲͲ —•’͸ ͲǤ͸ ͳǤͲͲ ƒ•’͸ ͳǤͶ ͳǤͲͲ    ‘” ͳ ͳǤ͵ ͳǤͲͲ Ž • ͲǤ͸ ͳǤͲͲ ˆ’ͺͳͲ ͳǤͺ ͳǤͲͲ    ‡”’—†ͳ ͳǤͷ ͳǤͲͲ ‡’ͺͷŽ ͲǤͷ ͳǤͲͲ ™ƒͻ ͳǤ͹ ͳǤͲͲ    ’†ʹ ͳǤͷ ͳǤͲͲ •ƒ͵ ͲǤ͹ ͳǤͲͲ  ƒͳ ͳǤ͸ ͳǤͲͲ    ˆ’͹Ͳͻ ͳǤͻ ͳǤͲͲ ˆ’ ͲǤ͸ ͳǤͲͲ ‹ˆͳͺƒ ͳǤͺ     ƒ†ͳ ͳǤ͹ ͳǤͲͲ ƒͳʹ͸„ ͲǤ͹ ͳǤͲͲ ‡††ʹ ͳǤͻ ͳǤͲͲ    ›„ƒ ͳǤ͸ ͳǤͲͲ ƒ“”Ͷ ͲǤͷ ͳǤͲͲ † ʹͷƒ ͳǤ͹ ͳǤͲͲ    ‰’Žͳ ͳǤͶ ͳǤͲͲ –—†͵ ͲǤͷ ͳǤͲͲ ‰ˆ ͳǤͻ ͳǤͲͲ    ‡͸ ͳǤ͹ ͳǤͲͲ ”‹ͳʹ  ͲǤ͸ ͳǤͲͲ       —•†Ͷ ͳǤ͸ ͳǤͲͲ ˆ’ͶͶͷ ͲǤͷ ͳǤͲͲ       —‘ ͳǤͷ ͳǤͲͲ ŽͶ ͲǤͷ ͳǤͲͲ       Ž ʹ͸ƒͳͲ ͳǤͻ ͳǤͲͲ ˆƒ’ͳʹ͸ ͲǤͷ ͳǤͲͲ      

ͻͻ Chapter 3

Low-fat fed KO/WT (DeSeq2)- continued  Downregulated   Gene FC padj    ’•ͺ ͲǤͷ ͳǤͲͲ          ƒŽ–ͳ ͲǤͷ ͳǤͲͲ          ƒ•–†ʹ ͲǤ͸ ͳǤͲͲ          •—ͷ ͲǤ͸ ͳǤͲͲ          ”ƒ’’ ͺ ͲǤ͸ ͳǤͲͲ          ‘ •͸ ͲǤ͸ ͳǤͲͲ           Œʹ ͲǤͷ ͳǤͲͲ          ‘ŽͶŽ ͲǤͷ ͳǤͲͲ          ƒ” Šͻ ͲǤͷ ͳǤͲͲ          š–Žͳ ͲǤ͸ ͳǤͲͲ          ‹”ƒ ͲǤͷ ͳǤͲͲ          ʹǦͶ ͲǤ͸ ͳǤͲͲ          Ž•–ͺ ͲǤ͸ ͳǤͲͲ          ”‘•‡”ͳ ͲǤ͸ ͳǤͲͲ        Low-fat fasted KO/WT (Voom) High-fat fasted KO/WT (Voom) Upregulated Downregulated Upregulated Downregulated Gene FC padj Gene FC padj Gene FC padj Gene FC padj –Žͳȁ ʹͲ͹Ͷ͸ ͳǤͻ ͲǤͶ͸ ‹ˆͶ‡„’ʹ ͳǤͻ ͲǤͳ͵ •ͳ„ ͳǤͻ ͳǤͲͲ Ž ͻƒ͵”ʹ ͳǤͻ ͳǤͲͲ Ž”šʹ ͳǤͶ ͲǤͷͻ —’ ͳǤͺ ͲǤͷͻ ‡͵ͻƒ ʹǤʹ ͳǤͲͲ œœ͵ ͳǤͷ ͳǤͲͲ ’Žͳʹ ͳǤͺ ͲǤͷͻ ’ͳ ͳǤͶ ͲǤͷͻ ”ͳ ͳǤͷ ͳǤͲͲ Ž ʹͷƒͳ ͳǤͶ ͳǤͲͲ • ͳǤͷ ͲǤͷͻ ƒ„ͷ„ ͳǤ͸ ͲǤͷͻ ’‹‰ ͳǤͷ ͳǤͲͲ ‰”ʹ„ ͳǤͷ ͳǤͲͲ  ͳǤ͸ ͲǤͷͻ ͵͵ͲͲ͸ͻͳ͸‹ ͷǤ͸ ͲǤͷͻ  ͳǤͺ ͳǤͲͲ ͻͲ͵Ͳ͸ͳͻͲͺ‹ ʹǤ͵ ͳǤͲͲ ˆ’ͳʹͲ ʹǤͲ ͲǤͷͻ ‡ͳͳͲ ʹǤͳ ͲǤͷͻ ˆƒͳ ͳǤͻ ͳǤͲͲ Šƒ’ͳ ͳǤͻ ͳǤͲͲ ͳ͹ ͸ͷ͵ ʹǤͲ ͲǤͷͻ –‡” ͳǤͷ ͲǤͷͻ ƒ ͳ ͳǤͺ ͳǤͲͲ •–ͳ ͳǤ͸ ͳǤͲͲ ”‘† ͳǤͶ ͲǤͷͻ ͻ•ˆʹ ͳǤͷ ͲǤͷͻ ‰„ ͳǤͷ ͳǤͲͲ ‡ƒͳ ͳǤ͸ ͳǤͲͲ  ͳǤ͸ ͲǤͷͻ Š’‘ ͳǤ͹ ͲǤͷͻ ͺ„ ͳǤ͹ ͳǤͲͲ †‹ͳ ͳǤͶ ͳǤͲͲ †”͹Ͳ ͳǤͻ ͲǤͷͻ •’ƒͳͶ ͳǤ͸ ͲǤͷͻ ͳ͹•—ͳͲͶ‡ ͳǤͶ ͳǤͲͲ ‡š͸ ͳǤ͵ ͳǤͲͲ •†ͳͲ ͳǤͶ ͲǤͷͻ ”•ˆͳͲ ͳǤͷ ͲǤͷͻ ͳͲͲƒͻ ͵Ǥͺ ͳǤͲͲ ”͹ƒͷ ͳǤ͵ ͳǤͲͲ —Ž–ͳ ʹ ͶǤͲ ͲǤͷͻ ‰ ͳǤ͸ ͲǤͷͻ ˆŽƒ” ͳǤͶ ͳǤͲͲ ͸Ͷ͵Ͳͷ͹͵ ͳͳ‹ ͳǤͺ ͳǤͲͲ „ ͳ†ͳͶ ͳǤͺ ͲǤͷͻ ƒ’œ„ ͳǤͶ ͲǤͷͻ ‰Žͳ ʹǤ͹ ͳǤͲͲ ”’„ ͳǤͶ ͳǤͲͲ ”‹–ͳ ʹǤʹ ͲǤͷͻ ƒŽ„ ͳǤ͸ ͲǤͷͻ ˆ† ʹͳ ͳǤ͵ ͳǤͲͲ ’‡’Žͳ ͳǤͶ ͳǤͲͲ ͵”–†ʹͷͶ‡ ʹǤ͸ ͲǤͷͻ ‹͵ ͳǤͻ ͲǤͷͻ ‡––Žͳ ͳǤͻ ͳǤͲͲ Ž ‘ͳƒͶ ʹǤͳ ͳǤͲͲ ’” ͳǤͶ ͲǤͷͻ •ͳ‰ʹ ͳǤͷ ͲǤͷͻ Š › ͳǤ͹ ͳǤͲͲ „ ƒ͵ ͳǤ͹ ͳǤͲͲ ”’’ͳͻ ͳǤ͵ ͲǤͷͻ ƒƒͶͲ ʹǤ͸ ͲǤͷͻ ‡††ʹ ͳǤͺ ͳǤͲͲ ‹Ž‹ͳ ͳǤͻ ͳǤͲͲ ™Šƒ„ ͳǤ͵ ͲǤͷͻ ‘ •͸ ͳǤ͸ ͲǤͷͻ  ʹǤͳ ͳǤͲͲ ‡ͳͺͶ„ ͳǤ͸ ͳǤͲͲ ”‡„„’ ͳǤ͵ ͲǤͷͻ ‹’͹ ͳǤ͹ ͲǤͷͻ œ’ ͳǤͶ ͳǤͲͲ  ‡ͳ ʹǤʹ ͳǤͲͲ •ƒͶ ͳǤ͵ ͲǤͷͻ ƒͳ͵͸ƒ ͳǤͶ ͲǤͷͻ ’–›ʹ†ͳ ͳǤͶ ͳǤͲͲ ›’ʹ ͵ͺ ͳǤͺ ͳǤͲͲ ‹ͺƒͳ ͳǤ͸ ͲǤͷͻ Ž ͳ͸ƒ͸ ͳǤͺ ͲǤͷͻ –‰ʹ ʹǤ͹ ͳǤͲͲ    ’•ʹͲ ͳǤͶ ͲǤͷͻ „’͵ ʹǤͶ ͲǤͷͻ –‹Š͵ ͳǤ͸ ͳǤͲͲ    ʹʹͲͲͲͲʹͲͳ‹ ʹǤͲ ͲǤͷͻ †ŠŠ ͳ͹ ʹǤ͸ ͲǤͷͻ Ž Ͷͳƒʹ ʹǤͲ ͳǤͲͲ    ˆ’– ʹǤͳ ͲǤͷͻ  ƒ”ˆͳ ʹǤ͵ ͲǤͷͻ  ͵Šͳ͵ ͳǤͷ ͳǤͲͲ     ʹ†ͳ„ ͳǤ͸ ͲǤͷͻ ƒͳ† ͳǤͷ ͲǤͷͻ ’‹ŽͶ ͳǤ͸ ͳǤͲͲ    •†ͳ͹„ͳͳ ͳǤ͸ ͲǤͷͻ † ʹ͵ ͳǤ͹ ͲǤͷͻ       „ ˆʹ ͳǤ͹ ͲǤͷͻ ƒŽ†ͳ ͳǤ͹ ͲǤͷͻ       ʹƒˆͳŽͶ ͳǤ͸ ͲǤͷͻ Ž†Šͳ„ͳ ͳǤ͸ ͲǤͷͻ       ™ ͳͷ ͳǤ͵ ͲǤͷͻ š‹ʹ ʹǤͷ ͲǤͷͻ       ’•Ͷ ͳǤͻ ͲǤͷͻ ”‡šͳ ʹǤͳ ͲǤͷͻ       ƒʹͲͺƒ ͳǤͶ ͲǤͷͻ ”‘•  ͳǤͶ ͲǤͷͻ       ”Žͺ„ ͳǤͶ ͲǤͷͻ ”’ͶͶ ͳǤͶ ͲǤͷͻ       „–„ͳͺ ͳǤ͸ ͲǤͷͻ  ›’ͳ ͳǤͺ ͲǤͷͻ       † ͳͶ„ ͳǤͶ ͲǤͷͻ ‡ʹͷ ͳǤͷ ͲǤͷͻ       ‘Ž‰ƒͳ ͳǤͷ ͲǤͷͻ ƒ„ʹͺ ͳǤ͸ ͲǤͷͻ       ƒͳ͸ ͳǤͷ ͲǤͷͻ ‡ͳͺͶ„ ͳǤ͸ ͲǤͷͻ       †ͳ ͳǤͶ ͲǤͷͻ –͵ƒ ͳǤͷ ͲǤͷͻ       „ƒ’ʹ ͳǤͷ ͲǤͷͻ †‡Ͷ„ ͳǤͻ ͲǤͷͻ       ƒ”•ʹ ͳǤ͸ ͲǤͷͻ Ž”’͸ ͳǤͶ ͲǤͷͻ       ‘Žͳƒͳ ͶǤͶ ͲǤͷͻ ƒ Šʹ ʹǤʹ ͲǤͷͻ       ’›ͳͻŽ͵ ʹǤͲ ͲǤͷͻ ƒ††Ͷͷƒ ͷǤͳ ͲǤͷͻ       ’–›ʹ†ͳ ͳǤͶ ͲǤͷͻ ƒ’ͻ ͳǤͶ ͲǤͷͻ       ƒ ‡ͳ ͳǤ͹ ͲǤͷͻ ’–ͳ ͳǤͺ ͲǤͷͻ        ͳǤͷ ͲǤͷͻ †ŠŠ ͷ ͳǤ͸ ͲǤͷͻ       ˆ†͸ ͳǤͶ ͲǤͷͻ ƒ” • ͳǤ͵ ͲǤͷͻ       ‘šͶ ͵Ǥ͸ ͲǤͷͻ ‡š‹ͳ ͳǤ͸ ͲǤͷͻ       ‰ʹͲƒ ͳǤͶ ͲǤͷͻ  † ͷ͵ ͳǤ͹ ͲǤͷͻ       Ž–’†ʹ ͳǤͶ ͲǤͷͻ „‡ʹ‰ʹ ͳǤͺ ͲǤͷͻ       †ƒ ͳǤͶ ͲǤͷͻ ”’ ͳǤͷ ͲǤͷͻ       ‡––Ž͹„ ͳǤͶ ͲǤͷͻ ƒ†’•ʹ ͳǤͷ ͲǤͷͻ       ‡ͳͲ͸  ͳǤ͹ ͲǤͷͻ ‡’ͳ ͳǤ͹ ͲǤͷͻ          †›Ž ʹǤͲ ͲǤͷͻ      

ͳͲͲ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

Low-fat fasted KO/WT (Voom)- continued  Upregulated Downregulated   Gene FC padj    ‡’ͶͶ ͳǤͷ ͲǤͷͻ          ˆƒ ͳǤͶ ͲǤͷͻ          ˆͶ͵ ʹǤͲ ͲǤͷͻ          † ƒ‰͵ ͳǤͻ ͲǤͷͻ          ‰Žͳ ͳǤͷ ͲǤͷͻ          ƒ’ʹ ʹǤͷ ͲǤͷͻ          Ͳͳ͹͸Ͷ͵ ͳǤ͸ ͲǤͷͻ       3    –͸ ͳǤ͹ ͲǤͷͻ          •Ͷƒ͸  ʹǤͳ ͲǤͷͻ          –ͷ ͳǤͺ ͲǤͷͻ          ‡”–ƒ†ʹ ͳǤ͵ ͲǤͷͻ          ͸͵ͲͲ͵ͻͲ͵‹ ͵Ǥʹ ͲǤͷͻ          „šŽͶ ʹǤ͵ ͲǤͷͻ          ‘‘ͳ ͳǤͶ ͲǤͷͻ          ”ͳ ͳ͵ ͳǤ͵ ͲǤͷͻ          ˆ’ʹͳ͹ ͳǤͷ ͲǤͷͻ          ˆ’ͷͳ͸ ʹǤͷ ͲǤͷͻ          Ͷ„ ͳǤ͵ ͲǤͷͻ          ” ‹ ͳǤ͹ ͲǤͷͻ          Ž„Šͳ ͳǤͺ ͲǤͷͻ          šͳ ͳǤͶ ͲǤͷͻ          Ž• ”ʹ ͳǤ͸ ͲǤͷͻ          ͹͹ͲͺͲ ͳǤͶ ͲǤͷͻ          ƒ•Žͳ ʹǤͲ ͲǤͷͻ        Low-fat fed KO/WT (Voom) High-fat fed KO/WT (Voom) Upregulated Downregulated Upregulated Downregulated Gene FC padj Gene FC padj Gene FC padj Gene FC padj ”’͸ͺ ͳǤͷ ͳǤͲͲ ͵‰ƒŽ–ͳ ͶǤͲ ͲǤ͹ͻ —’ʹͳͶ ͳǤ͹ ͳǤͲͲ  ƒ–ʹ ͵Ǥͳ ͲǤʹ͸ †”͹Ͷ ͳǤͻ ͳǤͲͲ  Š Ͷ ͳǤͺ ͲǤͻʹ ‡†ͳ„ ͳǤ͹ ͳǤͲͲ ”’•͵ͷ ͳǤͶ ͳǤͲͲ †ŠŠ ͹ ʹǤͲ ͳǤͲͲ › Ž ʹǤʹ ͳǤͲͲ ʹˆͷ ʹǤͲ ͳǤͲͲ ͷͻ͵ͲͶ͵ͲͲͳ‹ ͳǤ͸ ͳǤͲͲ ȁƒ–ƒŽͳ „ˆͳ ͳǤ͸ ͳǤͲͲ ƒ Šʹ ͵ǤͲ ͳǤͲͲ ‘–ͳͳ ͳǤ͸ ͳǤͲͲ ‘ͳʹͳ ͳǤͷ ͳǤͲͲ ƒ—•Ͷ ʹǤͳ ͳǤͲͲ †”Ͷʹ‡ͳ ͳǤͷ ͳǤͲͲ ‡ƒ–”͸ ͳǤͷ ͳǤͲͲ ’ͳ‰ͳ ͳǤ͵ ͳǤͲͲ ‡ʹͶͺ ͳǤͷ ͳǤͲͲ ͳͺͳͲͲʹͳʹʹ‹ ʹǤͳ ͳǤͲͲ Ž‰ƒ’Ͷ ͳǤͻ ͳǤͲͲ ‡’ƒ ʹǤͲ ͳǤͲͲ ‹•ʹ ͳǤͷ ͳǤͲͲ ”‹ͳʹ  ͳǤ͸ ͳǤͲͲ ‡”‹ ͷ ͳǤ͸ ͳǤͲͲ ’Žͻȁ ͷͶͷͳ ͳǤͺ ͳǤͲͲ ’•ͳͷ ͳǤ͵ ͳǤͲͲ Ž•–͵ ͵Ǥͳ ͳǤͲͲ ƒ†ͳ ͳǤ͹ ͳǤͲͲ ˆͳͶͻ ͳǤͷ ͳǤͲͲ ‡šͳͳ„ ͳǤͷ ͳǤͲͲ ”’ʹ ͳǤ͸ ͳǤͲͲ  ͳƒ ͳǤ͹ ͳǤͲͲ ’͵•ͳ ʹǤ͵ ͳǤͲͲ • ͳǤͶ ͳǤͲͲ  ƒ’ʹͻ ͳǤͻ ͳǤͲͲ Ͳ͸ͳͲͲͶͲ Ͳͳ‹ ͳǤͶ ͳǤͲͲ ›’ʹ ͵ͺ ͳǤͺ ͳǤͲͲ ƒ†͵ ͳǤͺ ͳǤͲͲ  ͳǤͻ ͳǤͲͲ ƒ„‰‡ˆͳ ͳǤͻ ͳǤͲͲ Ž ͳʹƒ͸ ʹǤͳ ͳǤͲͲ ”‘ͳŽ„ ʹǤͲ ͳǤͲͲ Ž ͳ͸ƒͳ ͳǤ͹ ͳǤͲͲ •‹ʹ ͳǤͶ ͳǤͲͲ ’’ʹ”ͷ† ͳǤͷ ͳǤͲͲ ‡ʹͳͻ ͳǤͶ ͳǤͲͲ š”‰ ͳǤ͹ ͳǤͲͲ —•–ͳ ʹǤ͵ ͳǤͲͲ  ‘š͵ ͳǤͷ ͳǤͲͲ ‘•ͳƒ’ ʹǤͶ ͳǤͲͲ ‡͵ͻƒ ʹǤͲ ͳǤͲͲ ””ˆ ͳǤͷ ͳǤͲͲ ‹”’ƒ ͳǤͷ ͳǤͲͲ Ž†͵ ͳǤ͹ ͳǤͲͲ ”Š‰‡ˆʹ ͳǤͻ ͳǤͲͲ ›’Ͷƒͳʹ„ ͳǤͻ ͳǤͲͲ    Ž ͷ ͳǤͻ ͳǤͲͲ ‡š ͵Ǥ͵ ͳǤͲͲ ’”ͳͶ͸ ͳǤͷ ͳǤͲͲ    Š‹–ʹ ͳǤ͸ ͳǤͲͲ „Š†ͳ͵ ͳǤͷ ͳǤͲͲ ͸͵͵ͲͶͳ͸ ͳ͵‹ ͳǤ͹ ͳǤͲͲ    ‹ˆ͵‰ ͳǤͶ ͳǤͲͲ Ž ʹƒͷ ʹǤʹ ͳǤͲͲ ŽˆͶ ʹǤ͸ ͳǤͲͲ    ͳ ͳǤͺ ͳǤͲͲ ’‹ŽͶ ͳǤ͸ ͳǤͲͲ ƒ•’͸ ͳǤͶ ͳǤͲͲ    ”’”„ ͳǤͺ ͳǤͲͲ ͷ ͳǤ͵ ͳǤͲͲ † ʹͷƒ ʹǤͲ ͳǤͲͲ    †”Ͷͷ ͳǤ͸ ͳǤͲͲ ‹͵ ʹ‰ ʹǤͶ ͳǤͲͲ ‘Žͳͳ ͳǤ͵ ͳǤͲͲ    ’ʹ ͳǤ͵ ͳǤͲͲ –š͸ ͳǤͺ ͳǤͲͲ Šˆͺ ͳǤͷ ͳǤͲͲ    ‡ ͳ͸ƒ ͳǤ͵ ͳǤͲͲ †‡Ͷ† ͶǤ͵ ͳǤͲͲ  ʹǤ͸ ͳǤͲͲ    Ž‘ ͳ•͸ ͳǤͺ ͳǤͲͲ ʹͶͳͲͲͳ͸Ͳ͸‹ ͳǤ͹ ͳǤͲͲ „ƒ ͳǤͶ ͳǤͲͲ    –ƒ”†ͳ͵ ͳǤͷ ͳǤͲͲ —•’͸ ͳǤͺ ͳǤͲͲ ‡†ͳͷ ͳǤͷ ͳǤͲͲ –ƒ ͳǤ͵ ͳǤͲͲ ”†ͳ͸ ͳǤ͹ ͳǤͲͲ „–ͳ ͻͺǤͻ ͳǤͲͲ    —’–ͷ ͳǤ͵ ͳǤͲͲ  ͵Šͳ͵ ͳǤͷ ͳǤͲͲ       Ž„†ʹ ͳǤ͵ ͳǤͲͲ ͲʹͷͶͶ͸ ͳǤ͹ ͳǤͲͲ       š›Ž’ͳ ͳǤͺ ͳǤͲͲ • ƒͳ ͳǤͶ ͳǤͲͲ       – ͳ͹ ͳǤ͸ ͳǤͲͲ †šͷͷ ͳǤͺ ͳǤͲͲ          •ƒ͵ ͳǤͶ ͳǤͲͲ       •”„ʹ ͳǤ͸ ͳǤͲͲ ƒǣ”ƒ™’Ǧ˜ƒŽ—‡•Šƒ˜‡„‡‡‘‹––‡†ˆ”‘–Š‹•–ƒ„Ž‡†—‡–‘•’ƒ ‡ ‘•–”ƒ‹–•ǤŽ‡ƒ•‡ ‘ˆ‡”’”‘ˆ‡••‘”ƒ”„ƒ”ƒǤƒ‡” ƒ–„ǤǤ„ƒ‡”Ͳͳ̷— ‰ǤŽˆ‘”–Š‡ ‘’Ž‡–‡–ƒ„Ž‡Ǥ  

ͳͲͳ Chapter 3

Supplementary table ST3. CoA and NAD(P)(H) gene sets CoA gene set NAD(P)(H) main gene set NAD(P)(H) redox gene set ƒ • ”‘– –ͳ ƒ•†Š ›’ʹƒͷ †š” ƒ•†Š ›’ʹ ͵ͺ ƒ’†Š ƒƒ– • –ʹ ƒ•• ›’ʹ„ͳͲ ‘ͳ ƒ•• ›’ʹ ͵ͻ ƒ’†Š• ƒ•†Š „– —†–ͳͻ †Šͳ ›’ʹ„ͳͻ ‘ʹ †Šͳ ›’ʹ ͶͲ •Š ƒ•†Š’’– ‰ƒ–ͳ —†–͹ †ŠͶ ›’ʹ„ͻ ‘͵ †ŠͶ ›’ʹ ͷͲ Ž—†ͳ „Š†ͷ ‰ƒ–ʹ —†–ͺ †Šͷ ›’ʹ ʹͻ ‘Ͷ †Šͷ ›’ʹ ͷͶ ͳͲ͸ͺͳ  ƒƒͳƒ ‰ƒ–ʹŽ͸ ‰†Š †Š͸ƒ ›’ʹ ͵͹ ‘ͷ †Š͸ƒ ›’ʹ ͷͷ ͶͶͷͲ  ƒƒͳ„ Žƒ– ‰†ŠŽ †Š͸„ ›’ʹ ͵ͺ ‘͸ †Š͸„ ›’ʹ ͸ͷ ’”  ƒƒʹ Ž† š• †Š͹ ›’ʹ ͵ͻ ͸’†š †Š͹ ›’ʹ ͸͸ ’”ʹ  ƒ–ͳ Ž•– †Šƒͳ ”ͳƒͳ ›’ʹ ͶͲ ƒ’†Š ”ͳƒͳ ›’ʹ ͹Ͳ ’†ͳ  ƒ–ʹ Ž‘˜Žͳ †Šƒʹ ”ͳ„ͳͲ ›’ʹ ͷͲ ƒ’†Š• ”ͳ„ͳͲ ›’ʹ†ͳͲ ’†ͳŽ  Ž› Ž‘˜Žʹ †Š„ ”ͳ„͵ ›’ʹ ͷͶ •Š ”ͳ„͵ ›’ʹ†ͳͳ ”Š’”  ƒ–ͳ Ž‘˜Ž͵ ‹‰™ ”ͳ„͹ ›’ʹ ͷͷ Ž—†ͳ ”ͳ„͹ ›’ʹ†ʹʹ •”  ƒ–ʹ Ž‘˜ŽͶ ’–ͳ ”ͳ„ͺ ›’ʹ ͸ͷ ͳͲ͸ͺͳ ”ͳ„ͺ ›’ʹ†ʹ͸ ʹǦ‡͸  ‘–ͳ Ž‘˜Žͷ ’–ʹ ”ͳ†ͳ ›’ʹ ͸͸ ͶͶͷͲ ”ͳ†ͳ ›’ʹ†ͻ ͸’†  ‘–ͳͲ Ž‘˜Ž͸ ƒ–ͳ ”ͳ‡ͳ ›’ʹ ͹Ͳ ’” ”ͳ‡ͳ ›’ʹ‡ͳ ƒ†Š  ‘–ͳͳ Ž‘˜Ž͹ ƒ–ʹ ”͹ƒͷ ›’ʹ†ͳͲ ’”ʹ ”͹ƒͷ ›’ʹŒͷ ƒ†Šƒ  ‘–ͳʹ Ž’͵  ’ʹ Ž†Šͳͺƒͳ ›’ʹ†ͳͳ ’†ͳ Ž†Šͳͺƒͳ ›’ʹŒ͸ ƒ†Š„  ‘–ͳ͵ ’͵ͲͲ Ž ʹͷƒͳ͸ Ž†Šͳƒͳ ›’ʹ†ʹʹ ’†ͳŽ Ž†Šͳƒͳ ›’ʹ”ͳ ‹„ƒ†Š  ‘–ʹ ƒ”ͳ Ž ʹ͹ƒʹ Ž†Šͳƒʹ ›’ʹ†ʹ͸ ”Š’” Ž†Šͳƒʹ ›’ʹ•ͳ ‰ ”  ‘–͵ ƒ”ʹ Ž ʹ͹ƒͷ Ž†Šͳƒ͵ ›’ʹ†ͻ •” Ž†Šͳƒ͵ ›’ʹ—ͳ ’‰†  ‘–Ͷ ƒ• Ž ͵͵ƒͳ Ž†Šͳƒ͹ ›’ʹ‡ͳ ʹǦ‡͸ Ž†Šͳƒ͹ ›’͵ͻƒͳ •†ͳͳ„ͳ  ‘–͸ ƒ– ‘ƒ–ͳ Ž†Šͳ„ͳ ›’ʹŒͷ ͸’† Ž†Šͳ„ͳ ›’͵ƒͳͳ •†ͳͳ„ʹ  ‘–͹ Ž›ƒ– ‘ƒ–ʹ Ž†ŠͳŽͳ ›’ʹŒ͸ ƒ†Š Ž†ŠͳŽͳ ›’͵ƒͳ͵ •†ͳ͹„ͳ  ‘–ͺ ’ƒ– ’–Ž ͳ Ž†ŠͳŽʹ ›’ʹ”ͳ ƒ†Šƒ Ž†ŠͳŽʹ ›’͵ƒͳ͸ •†ͳ͹„ͳͲ  ‘–ͻ ’ƒ–ͳ ’–Ž ʹ Ž†Šʹ ›’ʹ•ͳ ƒ†Š„ Ž†Šʹ ›’͵ƒʹͷ •†ͳ͹„ͳͳ  •„‰ͳ ’ƒ ’–Ž ͵ Ž†Š͵ƒͳ ›’ʹ—ͳ ‹„ƒ†Š Ž†Š͵ƒͳ ›’͵ƒͶͳƒ •†ͳ͹„ͳʹ  •„‰ʹ ’ƒ–ʹ — Žƒʹ Ž†Š͵ƒʹ ›’͵ͻƒͳ ‰ ” Ž†Š͵ƒʹ ›’͵ƒͶͳ„ •†ͳ͹„ͳͶ  •ˆʹ ’ƒ–Ͷ — Ž‰ͳ Ž†Š͵„ͳ ›’͵ƒͳͳ ’‰† Ž†Š͵„ͳ ›’Ͷ͸ƒͳ •†ͳ͹„ʹ  •ˆ͵ –ˆ͵ Ͷ — Ž‰ʹ Ž†Š͵„ʹ ›’͵ƒͳ͵ •†ͳͳ„ͳ Ž†Š͵„ʹ ›’ͶƒͳͲ •†ͳ͹„͵  •Žͳ ƒ†Š„ Š‡Ͷ Ž†Š͵„͵ ›’͵ƒͳ͸ •†ͳͳ„ʹ Ž†Š͵„͵ ›’Ͷƒͳʹƒ •†ͳ͹„Ͷ  •Ž͵ ƒ–ͳ Š‡ͷ Ž†ŠͶƒͳ ›’͵ƒʹͷ •†ͳ͹„ͳ Ž†ŠͶƒͳ ›’ͶƒͳͶ •†ͳ͹„͸  •ŽͶ ‰•ƒ– –͸ Ž†Šͷƒͳ ›’͵ƒͶͳƒ •†ͳ͹„ͳͲ Ž†Šͷƒͳ ›’Ͷƒ͵ʹ •†ͳ͹„͹  •Žͷ Šƒ– †ŠŠ ͳ Ž†Š͸ƒͳ ›’͵ƒͶͳ„ •†ͳ͹„ͳͳ Ž†Š͸ƒͳ ›’Ͷ„ͳ •†͵„ͳ  •Ž͸ ‹„ Š †ŠŠ ͳͳ Ž†Š͹ƒͳ ›’Ͷ͸ƒͳ •†ͳ͹„ͳʹ Ž†Š͹ƒͳ ›’ͶˆͳͶ •†͵„ʹ  •ͳ ‰ ” †ŠŠ ͳʹ Ž†Šͺƒͳ ›’ͶƒͳͲ •†ͳ͹„ͳͶ Ž†Šͺƒͳ ›’Ͷˆͳͺ •†͵„͵  •ʹ ‰ •ͳ †ŠŠ ͳ͵ Ž†Šͻƒͳ ›’Ͷƒͳʹƒ •†ͳ͹„ʹ Ž†Šͻƒͳ ›’Ͷ˜͵ •†͵„Ͷ  •͵ ‰ •ʹ †ŠŠ ͳͶ ”–ͳ ›’ͶƒͳͶ •†ͳ͹„͵ •’†Š ›’Ͷšͳ •†͵„ͷ  •Ͷ ƒ–ʹƒ †ŠŠ ͳͷ ”–͵ ›’Ͷƒ͵ʹ •†ͳ͹„Ͷ  †Šƒ ›’ͷͳ •†͵„͸  •ͷ ƒ–ʹ„ †ŠŠ ͳ͸ ”–Ͷ ›’Ͷ„ͳ •†ͳ͹„͸  †Š„ ›’͹ƒͳ •†͵„͹  ••ͳ ƒ–ͷ †ŠŠ ͳ͹ ”–ͷ ›’ͶˆͳͶ •†ͳ͹„͹ †Šͳ ›’͹„ͳ †Šͳ  ••ʹ ƒ–͸ƒ †ŠŠ ͳͺ •’†Š ›’Ͷˆͳͺ •†͵„ͳ †Šʹ ›’ͺ„ͳ †Šʹ  ••͵ ƒ–͸„ †ŠŠ ͳͻ  †Šƒ ›’Ͷ˜͵ •†͵„ʹ Ž˜”ƒ  š” †Š͵ƒ ‰’ƒ–ͳ ƒ–͹ †ŠŠ ʹ  †Š„ ›’Ͷšͳ •†͵„͵ Ž˜”„ ‡ ”ͳ †Š͵„ ‰’ƒ–ʹ ƒ–ͺ †ŠŠ ʹͲ †Šͳ ›’ͷͳ •†͵„Ͷ „”ͳ ‡ ”ʹ †Š͵‰ ‰’ƒ–͵  Žƒ–ͳ †ŠŠ ʹͳ †Šʹ ›’͹ƒͳ •†͵„ͷ „”͵ Š ”ʹͶ ’†Šͳ ‰’ƒ–Ͷ ’ ƒ–ͳ †ŠŠ ʹʹ Ž˜”ƒ ›’͹„ͳ •†͵„͸ ‘“͹ Š ”͹ ’†Šʹ ‰’ƒ–ͷ ’ ƒ–ʹ †ŠŠ ʹ͵ Ž˜”„ ›’ͺ„ͳ •†͵„͹ ”›Žͳ Š†Š ›†  ͸ ’ ƒ–͵ †ŠŠ ʹͶ •–ͳ  š” †Šͳ ”›œ Šˆ”  ƒ„ʹ  ͻ ’ ƒ–Ͷ †ŠŠ ͵ „”ͳ ‡ ”ͳ †Šʹ ›„ͷ”ͳ Š’• †•” Žƒ•ͳ ’‰ƒ–ͳ †ŠŠ Ͷ „”͵ ‡ ”ʹ †Š͵ƒ ›„ͷ”ʹ Š”•͵ ‘ Žƒ•ʹ „‘ƒ–ͳ †ŠŠ ͷ †͵ͺ Š ”ʹͶ †Š͵„ ›„ͷ”͵ Š”•Ͷ †Šƒ Ž†Š͸ƒͳ „‘ƒ–ʹ †ŠŠ ͸ ‘“͹ Š ”͹ †Š͵‰ ›„ͷ”Ͷ Š”•ͻ †ŠƒŽ͸„ –ƒ–ͳ „‘ƒ–͹ †ŠŠ ͹ ”›Žͳ Š†Š ’†Šͳ ›„ͷ”Ž Žƒ– †Š„ ™ƒ–ͳ  ƒ– †ŠŠ ͺ ”›œ Šˆ” ’†Šʹ ›’ͳ͹ƒͳ Ž† †Š  ™ƒ–ʹ ‰‡ƒͷ †ŠŠ ͻ ›„ͷ”ͳ Š’• ›† ›’ͳͻƒͳ ’›† †Šͳ ƒƒ– ‘‰ƒ–ͳ  ›„ͷ”ʹ Š”•͵  ƒ„ʹ ›’ͳƒͳ —‘šͳ †Šʹ †›Ž ‘‰ƒ–ʹ  ›„ͷ”͵ Š”•Ͷ †•” ›’ͳƒʹ —‘šʹ ‡ͳ ‡”•ͳ ƒƒͳͲ  ›„ͷ”Ͷ Š”•ͻ ‘ ›’ͳ„ͳ ŠŠƒ†Š ‡ʹ ‡”•ʹ ƒƒͳͳ  ›„ͷ”Ž Žƒ– †Šƒ ›’ʹͳƒͳ ‘šͳ ‡͵ ‡”•͵ ƒƒͳͷ  ›’ͳ͹ƒͳ Ž† †ŠƒŽ͸„ ›’ʹͶƒͳ ‘šʹ ‡ ” ‡”•Ͷ ƒƒʹͲ  ›’ͳͻƒͳ ’›† †Š„ ›’ʹ͸ƒͳ ƒ”ͳ ‹ ƒŽͳ ‡”•ͷ ƒƒ͵Ͳ  ›’ͳƒͳ —‘šͳ †Š  ›’ʹ͸ ͳ ƒ”ʹ ‹ ƒŽʹ ‡”•͸ ƒƒͶͲ  ›’ͳƒʹ —‘šʹ †Šͳ ›’ʹ͹ƒͳ ƒ• ‹ ƒŽ͵ Šƒ– ƒƒ͸Ͳ  ›’ͳ„ͳ ŠŠƒ†Š †Šʹ ›’ʹ͹„ͳ †ˆ–ͳ •‘ͳ Ž‘  ƒ‰•  ›’ʹͳƒͳ ‘šͳ ‡ͳ ›’ʹƒͳʹ †š” –Šˆ†ͳ ‘ƒ•› ƒ–ͳ  ›’ʹͶƒͳ ‘šʹ ‡ʹ ›’ʹƒͶ ‘ͳ –Šˆ†ʹ ’–ͳƒ ƒ–ʹ  ›’ʹ͸ƒͳ ’’ͳ ‡͵ ›’ʹƒͷ ‘ʹ –Šˆ†ʹŽ ’–ͳ„ ƒ–͸  ›’ʹ͸ ͳ ’’͵ ‡ ” ›’ʹ„ͳͲ ‘͵ –Šˆ” ’–ͳ  ƒ–ͺ  ›’ʹ͹ƒͳ ƒ”ͳ ‹ ƒŽͳ ›’ʹ„ͳͻ ‘Ͷ –Ǧ†ͳ ’–ʹ ƒ–ͺŽ  ›’ʹ͹„ͳ ƒ”ʹ ‹ ƒŽʹ ›’ʹ„ͻ ‘ͷ –Ǧ†ʹ ”ƒ–  ‘ƒͳ  ›’ʹƒͳʹ ƒ• ‹ ƒŽ͵ ›’ʹ ʹͻ ‘͸ –Ǧ†͵ ”‡„„’  ‘ƒ͵  ›’ʹƒͶ †ˆ–ͳ •‘ͳ ›’ʹ ͵͹ ͸’†š –Ǧ†Ͷ

ͳͲʹ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

CoA gene set NAD(P)(H) main gene set -continued NAD(P)(H) redox gene set - continued    –Šˆ†ͳ †—ˆ•͵ › ”Ž –Ǧ†ͶŽ “‘ͳ š”†ͳ    –Šˆ†ʹ †—ˆ•Ͷ †’” –Ǧ†ͷ •†ŠŽ š”†ʹ    –Šˆ†ʹŽ †—ˆ•ͷ †Šͳ –Ǧ†͸ š š”†͵    –Šˆ” †—ˆ•͸ †ŠͳͲ –”” ‰†Š ›”    –”ͳ †—ˆ•͹ †Šͳͳ ƒ†•›ͳ ‰†ŠŽ ‰†Š    –”ʹ †—ˆ•ͺ †Šͳʹ †‘”ͳ †Šƒͳ †Š    –”͸ †—ˆ˜ͳ †Šͳ͵ †—ˆƒͳ †Šƒʹ ƒ†Šʹ    –”͹ †—ˆ˜ʹ †ŠͳͶ †—ˆƒͳͲ †Š„     –Ǧ†ͳ †—ˆ˜͵ †Šͳ͸ †—ˆƒͳͳ †Šš  3    –Ǧ†ʹ ƒ–ͳ †Šͳ͸ˆʹ †—ˆƒͳʹ ‡ ”     –Ǧ†͵ ƒ–ʹ †Šͳͻ †—ˆƒͳ͵ ‰†     –Ǧ†Ͷ ƒ–͵ †Šͷ †—ˆƒʹ Š‰†Š     –Ǧ†ͶŽ – †Š͹ †—ˆƒ͵ ‘”     –Ǧ†ͷ ‘•ͳ †Šͺ †—ˆƒͶ ”‘†Š     –Ǧ†͸ ‘•ʹ †Šͻ †—ˆƒͶŽʹ –‰”ͳ     –”” ‘•͵  ͷ† †—ˆƒͷ –‰”ʹ     ƒ† ‘šͳ  ’†Š †—ˆƒ͸ › ”ͳ     ƒ†ʹ “‘ͳ †”ͳ͸ ͷ †—ˆƒ͹ › ”ʹ     ƒ†•›ͳ •†ŠŽ ‹”–ͳ †—ˆƒͺ › ”Ž     †‘”ͳ —†–ͳʹ ‹”–ʹ †—ˆƒͻ †’”     †—ˆƒͳ š ‹”–͵ †—ˆƒ„ͳ †Šͳ     †—ˆƒͳͲ ‰†Š ‹”–Ͷ †—ˆ„ͳͲ †ŠͳͲ     †—ˆƒͳͳ ‰†ŠŽ ‹”–ͷ †—ˆ„ͳͳ †Šͳͳ     †—ˆƒͳʹ ƒ”’ͳ ‹”–͸ †—ˆ„ʹ †Šͳʹ     †—ˆƒͳ͵ ƒ”’ͳͲ ‹”–͹ †—ˆ„͵ †Šͳ͵     †—ˆƒʹ ƒ”’ͳͳ ‘”† †—ˆ„Ͷ †ŠͳͶ     †—ˆƒ͵ ƒ”’ͳʹ ’” †—ˆ„ͷ †Šͳ͸     †—ˆƒͶ ƒ”’ͳͶ “Ž‡ †—ˆ„͸ †Šͳ͸ˆʹ     †—ˆƒͶŽʹ ƒ”’ͳ͸ ”†ͷƒͳ †—ˆ„͹ †Šͳͻ     †—ˆƒͷ ƒ”’ʹ ”†ͷƒʹ †—ˆ„ͺ †Šͷ     †—ˆƒ͸ ƒ”’͵ ”†ͷƒ͵ †—ˆ„ͻ †Š͹     †—ˆƒ͹ ƒ”’Ͷ –‡ƒ’ͳ †—ˆ ͳ †Šͺ     †—ˆƒͺ ƒ”’͸ –‡ƒ’ʹ †—ˆ ʹ †Šͻ     †—ˆƒͻ ƒ”’ͺ –‡ƒ’͵ †—ˆ•ͳ  ͷ†     †—ˆƒ„ͳ ƒ”’ͻ –‡ƒ’Ͷ †—ˆ•ʹ  ’†Š     †—ˆ„ͳͲ †Šƒͳ ‡ ” †—ˆ•͵ †”ͳ͸ ͷ     †—ˆ„ͳͳ †Šƒʹ ‹’ƒ”’ †—ˆ•Ͷ ‘”†     †—ˆ„ʹ †Š„ ͹•ˆʹ †—ˆ•ͷ ’”     †—ˆ„͵ †Šš • †—ˆ•͸ “Ž‡     †—ˆ„Ͷ ‡ ” •ʹ †—ˆ•͹ ”†ͷƒͳ     †—ˆ„ͷ ‰† ”’–ͳ †—ˆ•ͺ ”†ͷƒʹ     †—ˆ„͸ Š‰†Š •–ƒ͵ †—ˆ˜ͳ ”†ͷƒ͵     †—ˆ„͹ Š’–ͳ š”†ͳ †—ˆ˜ʹ –‡ƒ’ͳ     †—ˆ„ͺ ‘” š”†ʹ †—ˆ˜͵ –‡ƒ’ʹ     †—ˆ„ͻ ”‘†Š š”†͵ – –‡ƒ’͵     †—ˆ ͳ –‰”ͳ ›” ‘•ͳ –‡ƒ’Ͷ     †—ˆ ʹ –‰”ʹ ‰†Š ‘•ʹ ‡ ”     †—ˆ•ͳ › ”ͳ †Š ‘•͵ ͹•ˆʹ     †—ˆ•ʹ › ”ʹ ƒ†Šʹ ‘šͳ •–ƒ͵ 

Supplementary table ST4: Contributing genes in upregulated metabolic gene sets in the low-fat fed condition TAURINE AND HYPOTAURINE METABOLISM FDR = 0.13 αλ  ʹǦƒ‹‘‡–Šƒ‡–Š‹‘Ž†‹‘š›‰‡ƒ•‡    ›•–‡‹‡•—Žˆ‹‹ ƒ ‹††‡ ƒ”„‘š›Žƒ•‡    ‹Ž‡ƒ ‹†Ǧ‘ǣƒ‹‘ƒ ‹†Ǧƒ ›Ž–”ƒ•ˆ‡”ƒ•‡  

METABOLISM OF XENOBIOTICS BY CYTOCHROME P450 FDR = 0.25 RR = 1.03  Ͷ ƒŽ ‘Š‘Ž†‡Š›†”‘‰‡ƒ•‡Ͷȋ Žƒ•• Ȍǡ’‹’‘Ž›’‡’–‹†‡ ͹ͷ ƒŽ†‘Ǧ‡–‘”‡†— –ƒ•‡ˆƒ‹Ž›͹ǡ‡„‡”ͷȋƒˆŽƒ–‘š‹ƒŽ†‡Š›†‡”‡†— –ƒ•‡Ȍ ͳ ƒ”„‘›Ž”‡†— –ƒ•‡ͳ ʹ ʹ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›ˆǡ’‘Ž›’‡’–‹†‡ʹ  ͳ ‡’‘š‹†‡Š›†”‘Žƒ•‡ͳǡ‹ ”‘•‘ƒŽ Ͷ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡƒŽ’ŠƒͶ ͳ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ—ͳ ͷ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ—ͷ ͳ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡‘‡‰ƒͳ  ʹͷ ‰Ž— —”‘‘•›Ž–”ƒ•ˆ‡”ƒ•‡ʹˆƒ‹Ž›ǡ’‘Ž›’‡’–‹†‡ͷ  εͳǣ‰‡‡•‡–•ǡ™Š‡”‡‘ƒ†ȋȌȋ Ȍ‰‡‡•ƒ”‡‘˜‡””‡’”‡•‡–‡†‹–Š‡‹”Ž‹•–‘ˆ ‘–”‹„—–‹‰‰‡‡• ‘’ƒ”‡†–‘ –Š‡ ‘’Ž‡–‡‰‡‡•‡–Ǥ  

ͳͲ͵ Chapter 3

Supplementary table ST5: Contributing genes in downregulated metabolic gene sets in the low-fat fed condition  PYRUVATE METABOLISM FDR = 0.003 RR = 0.90  †‹Š›†”‘Ž‹’‘ƒ‹†‡†‡Š›†”‘‰‡ƒ•‡    ’›”—˜ƒ–‡‹ƒ•‡ǡ—• Ž‡   ͳ ƒ ›Ž’Š‘•’Šƒ–ƒ•‡ͳǡ‡”›–Š”‘ ›–‡ȋ ‘‘Ȍ–›’‡   ʹ ƒ ‡–›ŽǦ‘‡œ›‡ƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ʹ    ʹ ƒŽƒ–‡†‡Š›†”‘‰‡ƒ•‡ʹǡȋ‹–‘ Š‘†”‹ƒŽȌ    ’›”—˜ƒ–‡‹ƒ•‡Ž‹˜‡”ƒ†”‡†„Ž‘‘† ‡ŽŽ    ƒ ‡–›ŽǦ‘‡œ›‡ ƒ”„‘š›Žƒ•‡„‡–ƒ    ͳͳ ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡ͳˆƒ‹Ž›ǡ‡„‡”ͳ    ʹ ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡ʹǡ‹–‘ Š‘†”‹ƒŽ    †‹Š›†”‘Ž‹’‘ƒ‹†‡Ǧƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ȋʹ ‘’‘‡–‘ˆ’›”—˜ƒ–‡†‡Š›†”‘‰‡ƒ•‡ ‘’Ž‡šȌ ͳ ƒŽ‹ ‡œ›‡ͳǡȋΪȌǦ†‡’‡†‡–ǡ ›–‘•‘Ž‹     ƒ ‡–›ŽǦ‘‡œ›‡ ƒ”„‘š›Žƒ•‡ƒŽ’Šƒ    ͳ ’›”—˜ƒ–‡†‡Š›†”‘‰‡ƒ•‡ͳƒŽ’Šƒͳ    FATTY ACID BIOSYNTHESIS FDR = 0.04 RR = 1.00  ˆƒ––›ƒ ‹†•›–Šƒ•‡   ͳ ƒ ›ŽǦ‘•›–Š‡–ƒ•‡Ž‘‰Ǧ Šƒ‹ˆƒ‹Ž›‡„‡”ͳ   ͷ ƒ ›ŽǦ‘•›–Š‡–ƒ•‡Ž‘‰Ǧ Šƒ‹ˆƒ‹Ž›‡„‡”ͷ    ƒ ‡–›ŽǦ‘‡œ›‡ ƒ”„‘š›Žƒ•‡„‡–ƒ   Ͷ ƒ ›ŽǦ‘•›–Š‡–ƒ•‡Ž‘‰Ǧ Šƒ‹ˆƒ‹Ž›‡„‡”Ͷ    ƒ ‡–›ŽǦ‘‡œ›‡ ƒ”„‘š›Žƒ•‡ƒŽ’Šƒ    SULFUR METABOLISM FDR = 0.04 RR = n.a. ʹ ‹ˆ— –‹‘ƒŽ͵̵Ǧ’Š‘•’Š‘ƒ†‡‘•‹‡ͷ̵Ǧ’Š‘•’Š‘•—Žˆƒ–‡•›–Šƒ•‡ʹ   ͳ ‹‘•‹–‘Ž‘‘’Š‘•’Šƒ–ƒ•‡†‘ƒ‹ ‘–ƒ‹‹‰ͳ    ͳ ‡–Š›ŽƒŽ‘‹ ‡ ‡’ŠƒŽ‘’ƒ–Š›ͳ    ‡” ƒ’–‘’›”—˜ƒ–‡•—Žˆ—”–”ƒ•ˆ‡”ƒ•‡    –Š‹‘•—Žˆƒ–‡•—Žˆ—”–”ƒ•ˆ‡”ƒ•‡ǡ‹–‘ Š‘†”‹ƒŽ    ›–‘ Š”‘‡ ǡ•‘ƒ–‹     HISTIDINE METABOLISM FDR = 0.09 RR = 0.79  ƒ•’ƒ”–‘ƒ ›Žƒ•‡    ˆ‘”‹‹‘–”ƒ•ˆ‡”ƒ•‡ › Ž‘†‡ƒ‹ƒ•‡    Š‹•–ƒ‹‡Ǧ‡–Š›Ž–”ƒ•ˆ‡”ƒ•‡    ‘‘ƒ‹‡‘š‹†ƒ•‡    ͳͳ ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡ͳˆƒ‹Ž›ǡ‡„‡”ͳ    ʹ ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡ʹǡ‹–‘ Š‘†”‹ƒŽ    OTHER TYPES OF O-GLYCAN BIOSYNTHESIS FDR = 0.18 RR = n.a. ͵ ’”‘ ‘ŽŽƒ‰‡ǦŽ›•‹‡ǡʹǦ‘š‘‰Ž—–ƒ”ƒ–‡ͷǦ†‹‘š›‰‡ƒ•‡͵      †‘ƒ‹Ǧ•’‡ ‹ˆ‹ ǦŽ‹‡†Ǧƒ ‡–›Ž‰Ž— ‘•ƒ‹‡ȋ Ž  Ȍ–”ƒ•ˆ‡”ƒ•‡       Ǧˆ— ‘•›Ž’‡’–‹†‡͵Ǧ„‡–ƒǦǦƒ ‡–›Ž‰Ž— ‘•ƒ‹›Ž–”ƒ•ˆ‡”ƒ•‡   Ͷ ͳ Ǧ ƒŽǣ„‡–ƒ Ž  „‡–ƒͳǡͶǦ‰ƒŽƒ –‘•›Ž–”ƒ•ˆ‡”ƒ•‡ǡ’‘Ž›’‡’–‹†‡ͳ  ǦŽ‹‡†Ǧƒ ‡–›Ž‰Ž— ‘•ƒ‹‡ȋ Ž  Ȍ–”ƒ•ˆ‡”ƒ•‡ȋǦǦƒ ‡–›Ž‰Ž— ‘•ƒ‹‡ǣ’‘Ž›’‡’–‹†‡ǦǦƒ ‡–›Ž‰Ž— ‘•ƒ‹›Ž   –”ƒ•ˆ‡”ƒ•‡Ȍ  ʹ ’”‘–‡‹Ǧˆ— ‘•›Ž–”ƒ•ˆ‡”ƒ•‡ʹ   ͳ ’”‘–‡‹ǦǦƒ‘•›Ž–”ƒ•ˆ‡”ƒ•‡ͳ       SYNTHESIS AND DEGRADATION OF KETONE BODIES FDR = 0.18 RR = 2.40  ʹ ͵ǦŠ›†”‘š›Ǧ͵Ǧ‡–Š›Ž‰Ž—–ƒ”›ŽǦ‘‡œ›‡•›–Šƒ•‡ʹ    ͳ ͵ǦŠ›†”‘š›„—–›”ƒ–‡†‡Š›†”‘‰‡ƒ•‡ǡ–›’‡ͳ    ͳ ͵ǦŠ›†”‘š›Ǧ͵Ǧ‡–Š›Ž‰Ž—–ƒ”›ŽǦ‘‡œ›‡•›–Šƒ•‡ͳ   ʹ ƒ ‡–›ŽǦ‘‡œ›‡ƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ʹ   ͳ ͵Ǧ‘š‘ƒ ‹†‘–”ƒ•ˆ‡”ƒ•‡ͳ    BIOSYNTHESIS OF UNSATURATED FATTY ACIDS FDR = 0.18 RR = 1.88 ͳ ƒ ‡–›ŽǦ‘‡œ›‡ƒ ›Ž–”ƒ•ˆ‡”ƒ•‡ͳ   ͳ ƒ ‡–›ŽǦ‘‡œ›‡ƒ ›Ž–”ƒ•ˆ‡”ƒ•‡ͳ   ͳ ƒ ›ŽǦ‘–Š‹‘‡•–‡”ƒ•‡ͳ   ͵ ƒ ›ŽǦ‘–Š‹‘‡•–‡”ƒ•‡͵   Ͷ ƒ ›ŽǦ‘–Š‹‘‡•–‡”ƒ•‡Ͷ   ͹ ƒ ›ŽǦ‘–Š‹‘‡•–‡”ƒ•‡͹   ͳ ƒ ›ŽǦ‘‡œ›‡‘š‹†ƒ•‡ͳǡ’ƒŽ‹–‘›Ž   ͵ ƒ ›ŽǦ‘‡œ›‡‘š‹†ƒ•‡͵ǡ’”‹•–ƒ‘›Ž   ʹ ‡Ž‘‰ƒ–‹‘‘ˆ˜‡”›Ž‘‰ Šƒ‹ˆƒ––›ƒ ‹†•ȋ ͳȀŽ‘ʹǡͶȀŽ‘͵ǡ›‡ƒ•–ȌǦŽ‹‡ʹ  ͷ ˆƒ‹Ž›‡„‡”ͷǡ‡Ž‘‰ƒ–‹‘‘ˆŽ‘‰ Šƒ‹ˆƒ––›ƒ ‹†•ȋ›‡ƒ•–Ȍ  ͸ ˆƒ‹Ž›‡„‡”͸ǡ‡Ž‘‰ƒ–‹‘‘ˆŽ‘‰ Šƒ‹ˆƒ––›ƒ ‹†•ȋ›‡ƒ•–Ȍ  ͳ ˆƒ––›ƒ ‹††‡•ƒ–—”ƒ•‡ͳ   ʹ ˆƒ––›ƒ ‹††‡•ƒ–—”ƒ•‡ʹ   ͳ͹ͳʹ   

ͳͲͶ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

BIOSYNTHESIS OF UNSATURATED FATTY ACIDS - continued   ͳ •–‡ƒ”‘›ŽǦ‘‡œ›‡†‡•ƒ–—”ƒ•‡ͳ   ʹ •–‡ƒ”‘›ŽǦ‘‡œ›‡†‡•ƒ–—”ƒ•‡ʹ  

FATTY ACID METABOLISM FDR = 0.20 RR = 1.08 ͳ ƒ ‡–›ŽǦ‘‡œ›‡ƒ ›Ž–”ƒ•ˆ‡”ƒ•‡ͳ   ͳ ˆƒ––›ƒ ‹††‡•ƒ–—”ƒ•‡ͳ    ˆƒ––›ƒ ‹†•›–Šƒ•‡   ͳ ƒ ›ŽǦ‘•›–Š‡–ƒ•‡Ž‘‰Ǧ Šƒ‹ˆƒ‹Ž›‡„‡”ͳ   3 ʹ ƒ ‡–›ŽǦ‘‡œ›‡ƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ʹ   ͳ͹ͳʹ Š›†”‘š›•–‡”‘‹†ȋͳ͹Ǧ„‡–ƒȌ†‡Š›†”‘‰‡ƒ•‡ͳʹ   ͷ ƒ ›ŽǦ‘•›–Š‡–ƒ•‡Ž‘‰Ǧ Šƒ‹ˆƒ‹Ž›‡„‡”ͷ    ƒ ›ŽǦ‘‡œ›‡†‡Š›†”‘‰‡ƒ•‡ǡŽ‘‰Ǧ Šƒ‹   ͳ •–‡ƒ”‘›ŽǦ‘‡œ›‡†‡•ƒ–—”ƒ•‡ͳ   ʹ ‡Ž‘‰ƒ–‹‘‘ˆ˜‡”›Ž‘‰ Šƒ‹ˆƒ––›ƒ ‹†•ȋ ͳȀŽ‘ʹǡͶȀŽ‘͵ǡ›‡ƒ•–ȌǦŽ‹‡ʹ  ʹ •–‡ƒ”‘›ŽǦ‘‡œ›‡†‡•ƒ–—”ƒ•‡ʹ   Ͷ ƒ ›ŽǦ‘•›–Š‡–ƒ•‡Ž‘‰Ǧ Šƒ‹ˆƒ‹Ž›‡„‡”Ͷ   ͸ ˆƒ‹Ž›‡„‡”͸ǡ‡Ž‘‰ƒ–‹‘‘ˆŽ‘‰ Šƒ‹ˆƒ––›ƒ ‹†•ȋ›‡ƒ•–Ȍ 

GLYCOSPHINGOLIPID BIOSYNTHESIS - LACTO AND NEOLACTO SERIES FDR = 0.21 RR = n.a. ͵ ͵ ǦǦƒ ‡–›Ž‡—”ƒ‹ƒ–‡Ǧ„‡–ƒǦͳǡͶǦ‰ƒŽƒ –‘•‹†‡ƒŽ’ŠƒǦʹǡ͵Ǧ•‹ƒŽ›Ž–”ƒ•ˆ‡”ƒ•‡  ʹ Ǧƒ ‡–›ŽŽƒ –‘•ƒ‹‹†‡„‡–ƒǦͳǡ͸ǦǦƒ ‡–›Ž‰Ž— ‘•ƒ‹›ŽǦ–”ƒ•ˆ‡”ƒ•‡  Ͷ ͳ ‡–ƒǦͳǡͶǦ‰ƒŽƒ –‘•›Ž–”ƒ•ˆ‡”ƒ•‡ͳ   ͵ ͸ ›’‡ʹŽƒ –‘•ƒ‹‡ƒŽ’ŠƒǦʹǡ͵Ǧ•‹ƒŽ›Ž–”ƒ•ˆ‡”ƒ•‡   ͵ ͳ ‡–ƒǦͳǡ͵Ǧ‰ƒŽƒ –‘•›Ž–”ƒ•ˆ‡”ƒ•‡ͳ    TCA FDR = 0.24 RR = 1.08  †‹Š›†”‘Ž‹’‘ƒ‹†‡†‡Š›†”‘‰‡ƒ•‡    ʹ ƒŽƒ–‡†‡Š›†”‘‰‡ƒ•‡ʹǡȋ‹–‘ Š‘†”‹ƒŽȌ    ƒ‡• ‹–”ƒ–‡ˆ”‘ƒ ‡–›ŽǦ‘ǡ–Š‡”‡„›”‡Ž‡ƒ•‹‰‘    Ǧ ‹–”ƒ–‡•›–Šƒ•‡    †‹Š›†”‘Ž‹’‘ƒ‹†‡Ǧƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ȋʹ ‘’‘‡–‘ˆ’›”—˜ƒ–‡†‡Š›†”‘‰‡ƒ•‡ ‘’Ž‡šȌ  ͳ ’›”—˜ƒ–‡†‡Š›†”‘‰‡ƒ•‡ͳƒŽ’Šƒͳ   εͳǣ‰‡‡•‡–•ǡ™Š‡”‡‘ƒ†ȋȌȋ Ȍ‰‡‡•ƒ”‡‘˜‡””‡’”‡•‡–‡†‹–Š‡‹”Ž‹•–‘ˆ ‘–”‹„—–‹‰‰‡‡• ‘’ƒ”‡†–‘ –Š‡ ‘’Ž‡–‡‰‡‡•‡–ǤǤƒǤǣ‘–ƒ’’Ž‹ ƒ„Ž‡ǡ„‡ ƒ—•‡–Š‡ ‘’Ž‡–‡‰‡‡•‡–†‘‡•‘– ‘–ƒ‹‘‘”ȋȌȋ Ȍ‰‡‡•Ǥ

Supplementary table ST6: Contributing genes in metabolic gene sets in the high-fat fasted condition METABOLISM OF XENOBIOTICS BY CYTOCHROME P450 FDR = 0.000 RR = 0.97 ͳʹ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ͳǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ʹ   ʹ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ—ʹ   ʹ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡƒŽ’Šƒʹȋ ʹȌ   ͳ ƒ”„‘›Ž”‡†— –ƒ•‡ͳ    ʹͷ ‰Ž— —”‘‘•›Ž–”ƒ•ˆ‡”ƒ•‡ʹˆƒ‹Ž›ǡ’‘Ž›’‡’–‹†‡ͷ   ͵ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ—͵    ʹ͵͸ ‰Ž— —”‘‘•›Ž–”ƒ•ˆ‡”ƒ•‡ʹˆƒ‹Ž›ǡ’‘Ž›’‡’–‹†‡͵͸    Ͷ ƒŽ ‘Š‘Ž†‡Š›†”‘‰‡ƒ•‡Ͷȋ Žƒ•• Ȍǡ’‹’‘Ž›’‡’–‹†‡   ʹͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›‡ǡ’‘Ž›’‡’–‹†‡ͳ   ͵ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ–Š‡–ƒ͵   ͳ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ–Š‡–ƒͳ   Ͷ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡƒŽ’ŠƒͶ    ͳ ‡’‘š‹†‡Š›†”‘Žƒ•‡ͳǡ‹ ”‘•‘ƒŽ    ͵ ‹ ”‘•‘ƒŽ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡͵   ͸ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ—͸   ͵ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡƒŽ’Šƒ͵   ͳͳͳ Š›†”‘š›•–‡”‘‹†ͳͳǦ„‡–ƒ†‡Š›†”‘‰‡ƒ•‡ͳ    ʹ͵ͷ ‰Ž— —”‘‘•›Ž–”ƒ•ˆ‡”ƒ•‡ʹˆƒ‹Ž›ǡ’‘Ž›’‡’–‹†‡͵ͷ   ͳ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ƒ’’ƒͳ   ͹ͷ ƒŽ†‘Ǧ‡–‘”‡†— –ƒ•‡ˆƒ‹Ž›͹ǡ‡„‡”ͷȋƒˆŽƒ–‘š‹ƒŽ†‡Š›†‡”‡†— –ƒ•‡Ȍ  ͳ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ—ͳ    DRUG METABOLISM - CYTOCHROME P450 FDR = 0.001 RR = 0.91 ͳʹ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ͳǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ʹ   ʹ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ—ʹ   ʹ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡƒŽ’Šƒʹȋ ʹȌ    ʹͷ ‰Ž— —”‘‘•›Ž–”ƒ•ˆ‡”ƒ•‡ʹˆƒ‹Ž›ǡ’‘Ž›’‡’–‹†‡ͷ   ͵ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ—͵    ʹ͵͸ ‰Ž— —”‘‘•›Ž–”ƒ•ˆ‡”ƒ•‡ʹˆƒ‹Ž›ǡ’‘Ž›’‡’–‹†‡͵͸   ͳ ƒŽ†‡Š›†‡‘š‹†ƒ•‡ͳ    Ͷ ƒŽ ‘Š‘Ž†‡Š›†”‘‰‡ƒ•‡Ͷȋ Žƒ•• Ȍǡ’‹’‘Ž›’‡’–‹†‡   Ͷ ˆŽƒ˜‹ ‘–ƒ‹‹‰‘‘‘š›‰‡ƒ•‡Ͷ   ʹͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›‡ǡ’‘Ž›’‡’–‹†‡ͳ   ͵ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ–Š‡–ƒ͵  

ͳͲͷ Chapter 3

DRUG METABOLISM - CYTOCHROME P450 - continued   ͳ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ–Š‡–ƒͳ   Ͷ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡƒŽ’ŠƒͶ   ͳ ˆŽƒ˜‹ ‘–ƒ‹‹‰‘‘‘š›‰‡ƒ•‡ͳ    ͵ ‹ ”‘•‘ƒŽ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡͵    ‘‘ƒ‹‡‘š‹†ƒ•‡   ͸ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ—͸   ͵ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡƒŽ’Šƒ͵    ʹ͵ͷ ‰Ž— —”‘‘•›Ž–”ƒ•ˆ‡”ƒ•‡ʹˆƒ‹Ž›ǡ’‘Ž›’‡’–‹†‡͵ͷ   ͳ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ƒ’’ƒͳ   ͳ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ—ͳ    GLUTATHIONE METABOLISM FDR = 0.028 RR = 0.50 ͳ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡‘‡‰ƒͳ   ͳ ‘”‹–Š‹‡†‡ ƒ”„‘š›Žƒ•‡ǡ•–”— –—”ƒŽͳ    ‰Ž—–ƒƒ–‡Ǧ ›•–‡‹‡Ž‹‰ƒ•‡ǡ ƒ–ƒŽ›–‹ •—„—‹–   ͳ ‰Ž—–ƒ–Š‹‘‡’‡”‘š‹†ƒ•‡ͳ   Ͷ ‰Ž—–ƒ–Š‹‘‡’‡”‘š‹†ƒ•‡Ͷ   ʹ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ—ʹ   ʹ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡƒŽ’Šƒʹȋ ʹȌ   ͳʹ –Š‹‘”‡†‘š‹†‘ƒ‹ ‘–ƒ‹‹‰ͳʹȋ‡†‘’Žƒ•‹ ”‡–‹ —Ž—Ȍ   ͵ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ—͵    ʹ ‹•‘ ‹–”ƒ–‡†‡Š›†”‘‰‡ƒ•‡ʹȋΪȌǡ‹–‘ Š‘†”‹ƒŽ   ͸ ‰ƒƒǦ‰Ž—–ƒ›Ž–”ƒ•ˆ‡”ƒ•‡͸   ͵ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ–Š‡–ƒ͵   ͳ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ–Š‡–ƒͳ   Ͷ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡƒŽ’ŠƒͶ    ͵ ‹ ”‘•‘ƒŽ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡͵   ͸ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ—͸   ͵ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡƒŽ’Šƒ͵   ͳ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ƒ’’ƒͳ   ͳ ‰Ž—–ƒ–Š‹‘‡Ǧ–”ƒ•ˆ‡”ƒ•‡ǡ—ͳ    OXIDATIVE PHOSPHORYLATION FDR = 0.08 RR = 1.44 ͷͳ •›–Šƒ•‡ǡ Ϊ–”ƒ•’‘”–‹‰ǡ‹–‘ Š‘†”‹ƒŽ ͳ ‘’Ž‡šǡƒŽ’Šƒ•—„—‹–ͳ  ͷ •›–Šƒ•‡ǡ Ϊ–”ƒ•’‘”–‹‰‹–‘ Š‘†”‹ƒŽ ͳ ‘’Ž‡šǡ„‡–ƒ•—„—‹–  ͷ •›–Šƒ•‡ǡ Ϊ–”ƒ•’‘”–‹‰ǡ‹–‘ Š‘†”‹ƒŽ ͳ ‘’Ž‡šǡ†‡Ž–ƒ•—„—‹–  ͷ •›–Šƒ•‡ǡ Ϊ–”ƒ•’‘”–‹‰ǡ‹–‘ Š‘†”‹ƒŽ ͳ ‘’Ž‡šǡ‡’•‹Ž‘•—„—‹–  ͷ ͳ •›–Šƒ•‡ǡ Ϊ–”ƒ•’‘”–‹‰ǡ‹–‘ Š‘†”‹ƒŽ Ͳ ‘’Ž‡šǡ•—„—‹–ͳ  ͷ ͳ •›–Šƒ•‡ǡ Ϊ–”ƒ•’‘”–‹‰ǡ‹–‘ Š‘†”‹ƒŽ Ͳ ‘’Ž‡šǡ•—„—‹–ͳȋ•—„—‹–ͻȌ  ͷ  •›–Šƒ•‡ǡ Ϊ–”ƒ•’‘”–‹‰ǡ‹–‘ Š‘†”‹ƒŽ Ͳ ‘’Ž‡šǡ•—„—‹–  ͷ  •›–Šƒ•‡ǡ Ϊ–”ƒ•’‘”–‹‰ǡ‹–‘ Š‘†”‹ƒŽ Ͳ ‘’Ž‡šǡ•—„—‹–   ͷ ʹ •›–Šƒ•‡ǡ Ϊ–”ƒ•’‘”–‹‰ǡ‹–‘ Š‘†”‹ƒŽ Ͳ ‘’Ž‡šǡ•—„—‹– ʹ  ͷ •›–Šƒ•‡ǡ Ϊ–”ƒ•’‘”–‹‰ǡ‹–‘ Š‘†”‹ƒŽ Ͳ ‘’Ž‡šǡ•—„—‹–   ͸Ͳͳ ƒ•‡ǡ Ϊ–”ƒ•’‘”–‹‰ǡŽ›•‘•‘ƒŽͲ•—„—‹–ͳ   ͸Ͳ ƒ•‡ǡ Ϊ–”ƒ•’‘”–‹‰ǡŽ›•‘•‘ƒŽͲ•—„—‹–   ͸ͳ ƒ•‡ǡ Ϊ–”ƒ•’‘”–‹‰ǡŽ›•‘•‘ƒŽͳ•—„—‹–   ͸ͳͳ ƒ•‡ǡ Ϊ–”ƒ•’‘”–‹‰ǡŽ›•‘•‘ƒŽͳ•—„—‹–ͳ   ͸ͳ ƒ•‡ǡ Ϊ–”ƒ•’‘”–‹‰ǡŽ›•‘•‘ƒŽͳ•—„—‹–   ͸ͳͳ ƒ•‡ǡ Ϊ–”ƒ•’‘”–‹‰ǡŽ›•‘•‘ƒŽͳ•—„—‹–ͳ   ͸ͳ ͳ ƒ•‡ǡ Ϊ–”ƒ•’‘”–‹‰ǡŽ›•‘•‘ƒŽͳ•—„—‹– ͳ   ʹ ›–‘ Š”‘‡ ‘š‹†ƒ•‡•—„—‹–ʹ   ͵ ›–‘ Š”‘‡ ‘š‹†ƒ•‡•—„—‹–͵   Ͷ ͳ ›–‘ Š”‘‡ ‘š‹†ƒ•‡•—„—‹– ‹•‘ˆ‘”ͳ   ͷ ›–‘ Š”‘‡ ‘š‹†ƒ•‡•—„—‹–ƒ   ͸ͳ ›–‘ Š”‘‡ ‘š‹†ƒ•‡•—„—‹– ƒ’‘Ž›’‡’–‹†‡ͳ   ͸ͳ ›–‘ Š”‘‡ ‘š‹†ƒ•‡ǡ•—„—‹– „’‘Ž›’‡’–‹†‡ͳ   ͹ʹ ›–‘ Š”‘‡ ‘š‹†ƒ•‡•—„—‹– ƒʹ   ͹ ›–‘ Š”‘‡ ‘š‹†ƒ•‡•—„—‹– „   ͺ ›–‘ Š”‘‡ ‘š‹†ƒ•‡•—„—‹– ƒ   ͳ ›–‘ Š”‘‡ Ǧͳ     ’Š‘•’Š‘Ž›•‹‡’Š‘•’Š‘Š‹•–‹†‹‡‹‘”‰ƒ‹ ’›”‘’Š‘•’Šƒ–‡’Š‘•’Šƒ–ƒ•‡  ͳ  Ǧ—„‹“—‹‘‡‘š‹†‘”‡†— –ƒ•‡ Šƒ‹ͳ    ͳ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌͳƒŽ’Šƒ•—„ ‘’Ž‡šǡͳ    ͳͳ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌͳƒŽ’Šƒ•—„ ‘’Ž‡šͳͳ    ͳʹ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌͳƒŽ’Šƒ•—„ ‘’Ž‡šǡͳʹ    ͵  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌͳƒŽ’Šƒ•—„ ‘’Ž‡šǡ͵    ͸  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌͳƒŽ’Šƒ•—„ ‘’Ž‡šǡ͸ȋͳͶȌ    ͹  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌͳƒŽ’Šƒ•—„ ‘’Ž‡šǡ͹ȋͳͶǤͷƒȌ   ͳͲ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍͳ„‡–ƒ•—„ ‘’Ž‡šǡͳͲ    ͳͳ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍͳ„‡–ƒ•—„ ‘’Ž‡šǡͳͳ    ͸  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍͳ„‡–ƒ•—„ ‘’Ž‡šǡ͸    ͻ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍͳ„‡–ƒ•—„ ‘’Ž‡šǡͻ  

ͳͲ͸ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

OXIDATIVE PHOSPHORYLATION - continued    ʹ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍͳǡ•—„ ‘’Ž‡š—‘™ǡʹ    ͳ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍ ‡Ǧ’”‘–‡‹ͳ    ʹ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍ ‡Ǧ’”‘–‡‹ʹ    ͸  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍ ‡Ǧ’”‘–‡‹͸    ͹  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍ ‡Ǧ’”‘–‡‹͹    ͺ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍ ‡Ǧ’”‘–‡‹ͺ    ͳ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌˆŽƒ˜‘’”‘–‡‹ͳ    ʹ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌˆŽƒ˜‘’”‘–‡‹ʹ    ͵  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌˆŽƒ˜‘’”‘–‡‹͵   3 ʹ ’›”‘’Š‘•’Šƒ–ƒ•‡ȋ‹‘”‰ƒ‹ Ȍʹ     •— ‹ƒ–‡†‡Š›†”‘‰‡ƒ•‡ ‘’Ž‡šǡ•—„—‹–ǡˆŽƒ˜‘’”‘–‡‹ȋ ’Ȍ     ͳ  ‡ŽŽǡ‹—‡”‡‰—Žƒ–‘”ͳǡƒ•‡ǡ Ϊ–”ƒ•’‘”–‹‰ǡŽ›•‘•‘ƒŽͲ’”‘–‡‹͵  ͳͲ —„‹“—‹‘ŽǦ ›–‘ Š”‘‡ ”‡†— –ƒ•‡ǡ ‘’Ž‡š •—„—‹–   ͳͳ —„‹“—‹‘ŽǦ ›–‘ Š”‘‡ ”‡†— –ƒ•‡ǡ ‘’Ž‡š •—„—‹–     —„‹“—‹‘ŽǦ ›–‘ Š”‘‡ ”‡†— –ƒ•‡„‹†‹‰’”‘–‡‹    —„‹“—‹‘ŽǦ ›–‘ Š”‘‡ ”‡†— –ƒ•‡ǡ ‘’Ž‡š •—„—‹–     NADPH_MAIN FDR = 0.08 RR = 1  Ͷ ƒŽ ‘Š‘Ž†‡Š›†”‘‰‡ƒ•‡Ͷȋ Žƒ•• Ȍǡ’‹’‘Ž›’‡’–‹†‡   ͳͳ ƒŽ†‘Ǧ‡–‘”‡†— –ƒ•‡ˆƒ‹Ž›ͳǡ‡„‡”ͳȋƒŽ†‡Š›†‡”‡†— –ƒ•‡Ȍ   ͳͳ ƒŽ†‘Ǧ‡–‘”‡†— –ƒ•‡ˆƒ‹Ž›ͳǡ‡„‡”ͳ   ͹ͷ ƒŽ†‘Ǧ‡–‘”‡†— –ƒ•‡ˆƒ‹Ž›͹ǡ‡„‡”ͷȋƒˆŽƒ–‘š‹ƒŽ†‡Š›†‡”‡†— –ƒ•‡Ȍ   ͳͳ ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡ˆƒ‹Ž›ͳǡ•—„ˆƒ‹Ž›ͳ    ͳͳ ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡ͳˆƒ‹Ž›ǡ‡„‡”ͳ    ͵ʹ ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡ˆƒ‹Ž›͵ǡ•—„ˆƒ‹Ž›ʹ    Ͷͳ ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡Ͷˆƒ‹Ž›ǡ‡„‡”ͳ    ͸ͳ ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡ˆƒ‹Ž›͸ǡ•—„ˆƒ‹Ž›ͳ    ͹ͳ ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡ˆƒ‹Ž›͹ǡ‡„‡”ͳ     ƒ•’ƒ”–ƒ–‡†‡Š›†”‘‰‡ƒ•‡†‘ƒ‹ ‘–ƒ‹‹‰     „”ƒ Š‡† Šƒ‹‡–‘ƒ ‹††‡Š›†”‘‰‡ƒ•‡ͳǡƒŽ’Šƒ’‘Ž›’‡’–‹†‡    „‹Ž‹˜‡”†‹”‡†— –ƒ•‡ȋˆŽƒ˜‹”‡†— –ƒ•‡ȋ ȌȌ   ͳ ƒ”„‘›Ž”‡†— –ƒ•‡ͳ   ͳ ”›•–ƒŽŽ‹ǡŽƒ„†ƒͳ    ”›•–ƒŽŽ‹ǡœ‡–ƒ   ͷ͵ ›–‘ Š”‘‡„ͷ”‡†— –ƒ•‡͵   ͳ͹ͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ͳ͹ǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ͳ   ͳʹ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ͳǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ʹ   ʹʹͻ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡ʹͻ   ʹ͵͹ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǤ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡͵͹   ʹ͵ͺ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡͵ͺ   ʹͷͲ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡ͷͲ   ʹͷͶ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡ͷͶ   ʹͳͲ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›†ǡ’‘Ž›’‡’–‹†‡ͳͲ   ʹʹʹ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›†ǡ’‘Ž›’‡’–‹†‡ʹʹ   ʹʹ͸ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›†ǡ’‘Ž›’‡’–‹†‡ʹ͸   ʹͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›‡ǡ’‘Ž›’‡’–‹†‡ͳ   ʹͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›”ǡ’‘Ž›’‡’–‹†‡ͳ   ʹͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›—ǡ’‘Ž›’‡’–‹†‡ͳ   ͵ͻͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›͵ͻǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ͳ   ͵ͳͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›͵ǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ͳͳ   ͵ʹͷ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›͵ǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ʹͷ   Ͷͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›Ͷǡ•—„ˆƒ‹Ž›„ǡ’‘Ž›’‡’–‹†‡ͳ   Ͷ ͳͶ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›Ͷǡ•—„ˆƒ‹Ž›ˆǡ’‘Ž›’‡’–‹†‡ͳͶ    †‹ ƒ”„‘›ŽǦš›Ž—Ž‘•‡”‡†— –ƒ•‡    ͵ †‡Š›†”‘‰‡ƒ•‡Ȁ”‡†— –ƒ•‡ȋˆƒ‹Ž›Ȍ‡„‡”͵    †‹Š›†”‘Ž‹’‘ƒ‹†‡Ǧƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ȋʹ ‘’‘‡–‘ˆ’›”—˜ƒ–‡†‡Š›†”‘‰‡ƒ•‡ ‘’Ž‡šȌ ʹ ‡ –‘Ǧ†‹•—Žˆ‹†‡Ǧ–Š‹‘Ž‡š Šƒ‰‡”ʹ   ͳ ‡ –‘— Ž‡‘–‹†‡’›”‘’Š‘•’Šƒ–ƒ•‡Ȁ’Š‘•’Š‘†‹‡•–‡”ƒ•‡ͳ   ͵ ‡ –‘— Ž‡‘–‹†‡’›”‘’Š‘•’Šƒ–ƒ•‡Ȁ’Š‘•’Š‘†‹‡•–‡”ƒ•‡͵    ˆƒ––›ƒ ‹†•›–Šƒ•‡   ͳ ˆŽƒ˜‹ ‘–ƒ‹‹‰‘‘‘š›‰‡ƒ•‡ͳ   Ͷ ˆŽƒ˜‹ ‘–ƒ‹‹‰‘‘‘š›‰‡ƒ•‡Ͷ     Š›†”‘š›’”‘•–ƒ‰Žƒ†‹†‡Š›†”‘‰‡ƒ•‡ͳͷȋȌ   ͳͳͳ Š›†”‘š›•–‡”‘‹†ͳͳǦ„‡–ƒ†‡Š›†”‘‰‡ƒ•‡ͳ   ͳ͹Ͷ Š›†”‘š›•–‡”‘‹†ȋͳ͹Ǧ„‡–ƒȌ†‡Š›†”‘‰‡ƒ•‡Ͷ   ͵ͷ Š›†”‘š›Ǧ†‡Ž–ƒǦͷǦ•–‡”‘‹††‡Š›†”‘‰‡ƒ•‡ǡ͵„‡–ƒǦƒ†•–‡”‘‹††‡Ž–ƒǦ‹•‘‡”ƒ•‡ͷ  ͵͹ Š›†”‘š›Ǧ†‡Ž–ƒǦͷǦ•–‡”‘‹††‡Š›†”‘‰‡ƒ•‡ǡ͵„‡–ƒǦƒ†•–‡”‘‹††‡Ž–ƒǦ‹•‘‡”ƒ•‡͹   ͳ ‹•‘ ‹–”ƒ–‡†‡Š›†”‘‰‡ƒ•‡ͳȋΪȌǡ•‘Ž—„Ž‡    ʹ ‹•‘ ‹–”ƒ–‡†‡Š›†”‘‰‡ƒ•‡ʹȋΪȌǡ‹–‘ Š‘†”‹ƒŽ    ͵ ‹•‘ ‹–”ƒ–‡†‡Š›†”‘‰‡ƒ•‡͵ȋΪȌ„‡–ƒ    ͵  ‹•‘ ‹–”ƒ–‡†‡Š›†”‘‰‡ƒ•‡͵ȋΪȌǡ‰ƒƒ   ͳ ƒŽ‹ ‡œ›‡ͳǡȋΪȌǦ†‡’‡†‡–ǡ ›–‘•‘Ž‹       

ͳͲ͹ Chapter 3

NADPH_MAIN - continued    ͵ ‹ ”‘–—„—Ž‡ƒ••‘ ‹ƒ–‡†‘‘‘š›‰‡ƒ•‡ǡ ƒŽ’‘‹ƒ† †‘ƒ‹ ‘–ƒ‹‹‰͵  ‡–Š›Ž‡‡–‡–”ƒŠ›†”‘ˆ‘Žƒ–‡†‡Š›†”‘‰‡ƒ•‡ȋΪ†‡’‡†‡–Ȍǡ  ͳ ‡–Š‡›Ž–‡–”ƒŠ›†”‘ˆ‘Žƒ–‡ › Ž‘Š›†”‘Žƒ•‡ǡˆ‘”›Ž–‡–”ƒŠ›†”‘ˆ‘Žƒ–‡•›–Šƒ•‡    ‹ƒ•‡   ʹ ‹ƒ•‡ʹǡ‹–‘ Š‘†”‹ƒŽ   ͳ •›–Š‡–ƒ•‡ͳ    ͳͳ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌͳƒŽ’Šƒ•—„ ‘’Ž‡šͳͳ    ͳʹ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌͳƒŽ’Šƒ•—„ ‘’Ž‡šǡͳʹ    ͵  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌͳƒŽ’Šƒ•—„ ‘’Ž‡šǡ͵    ͸  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌͳƒŽ’Šƒ•—„ ‘’Ž‡šǡ͸ȋͳͶȌ    ͹  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌͳƒŽ’Šƒ•—„ ‘’Ž‡šǡ͹ȋͳͶǤͷƒȌ   ͳͲ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍͳ„‡–ƒ•—„ ‘’Ž‡šǡͳͲ    ͳͳ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍͳ„‡–ƒ•—„ ‘’Ž‡šǡͳͳ    ͸  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍͳ„‡–ƒ•—„ ‘’Ž‡šǡ͸    ͻ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍͳ„‡–ƒ•—„ ‘’Ž‡šǡͻ    ʹ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍͳǡ•—„ ‘’Ž‡š—‘™ǡʹ    ͳ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍ ‡Ǧ’”‘–‡‹ͳ    ʹ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍ ‡Ǧ’”‘–‡‹ʹ   ͸  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍ ‡Ǧ’”‘–‡‹͸   ͹  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍ ‡Ǧ’”‘–‡‹͹   ͳ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌˆŽƒ˜‘’”‘–‡‹ͳ   ʹ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌˆŽƒ˜‘’”‘–‡‹ʹ   ͵  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌˆŽƒ˜‘’”‘–‡‹͵  ͳ ‹ ‘–‹ƒ‹†‡— Ž‡‘–‹†‡ƒ†‡›Ž›Ž–”ƒ•ˆ‡”ƒ•‡ͳ  ͳ ȋȌ †‡Š›†”‘‰‡ƒ•‡ǡ“—‹‘‡ͳ  ͳ ’‘Ž›ȋǦ”‹„‘•‡Ȍ’‘Ž›‡”ƒ•‡ˆƒ‹Ž›ǡ‡„‡”ͳ ͳͲ ’‘Ž›ȋǦ”‹„‘•‡Ȍ’‘Ž›‡”ƒ•‡ˆƒ‹Ž›ǡ‡„‡”ͳͲ ͳʹ ’‘Ž›ȋǦ”‹„‘•‡Ȍ’‘Ž›‡”ƒ•‡ˆƒ‹Ž›ǡ‡„‡”ͳʹ  Ͷ ’‘Ž›ȋǦ”‹„‘•‡Ȍ’‘Ž›‡”ƒ•‡ˆƒ‹Ž›ǡ‡„‡”Ͷ   ͳ ’›”—˜ƒ–‡†‡Š›†”‘‰‡ƒ•‡ͳƒŽ’Šƒͳ    ’›”—˜ƒ–‡†‡Š›†”‘‰‡ƒ•‡ȋŽ‹’‘ƒ‹†‡Ȍ„‡–ƒ   ’‡”‘š‹•‘ƒŽ–”ƒ•ǦʹǦ‡‘›ŽǦ‘”‡†— –ƒ•‡   ͳ ’Š‘•’Š‘Š‹•–‹†‹‡’Š‘•’Šƒ–ƒ•‡ͳ    ’”‘Ž‹‡†‡Š›†”‘‰‡ƒ•‡   ͹ ”‡–‹‘Ž†‡Š›†”‘‰‡ƒ•‡͹  ͳ •‹”–—‹ͳ   ʹ •‹”–—‹ʹ   ͵ •‹”–—‹͵   ͹ •‹”–—‹͹  ͷͳ •–‡”‘‹†ͷƒŽ’ŠƒǦ”‡†— –ƒ•‡ͳ  ͷ͵ •–‡”‘‹†ͷƒŽ’ŠƒǦ”‡†— –ƒ•‡͵  ʹ •‹š–”ƒ•‡„”ƒ‡‡’‹–Š‡Ž‹ƒŽƒ–‹‰‡‘ˆ’”‘•–ƒ–‡ʹ   –”ƒ•Ǧʹǡ͵Ǧ‡‘›ŽǦ‘”‡†— –ƒ•‡ ͹ ʹ –”ƒ•‡„”ƒ‡͹•—’‡”ˆƒ‹Ž›‡„‡”ʹ  ͵ –‹••—‡•’‡ ‹ˆ‹ –”ƒ•’Žƒ–ƒ–‹‘ƒ–‹‰‡͵ͷ  ͵ –Š‹‘”‡†‘š‹”‡†— –ƒ•‡͵    STEROID HORMONE BIOSYNTHESIS FDR = 0.09 RR = 1.5 ͳͳ ƒŽ†‘Ǧ‡–‘”‡†— –ƒ•‡ˆƒ‹Ž›ͳǡ‡„‡”ͳ   ͳ͹ͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ͳ͹ǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ͳ   ͳʹ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ͳǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ʹ   ʹʹͻ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡ʹͻ   ʹ͵͹ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǤ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡͵͹   ʹ͵ͺ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡͵ͺ   ʹͷͲ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡ͷͲ   ʹͷͶ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡ͷͶ   ʹ͸ͺ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡͸ͺ   ʹ͹Ͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡͹Ͳ   ʹͳͲ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›†ǡ’‘Ž›’‡’–‹†‡ͳͲ   ʹʹʹ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›†ǡ’‘Ž›’‡’–‹†‡ʹʹ   ʹʹ͸ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›†ǡ’‘Ž›’‡’–‹†‡ʹ͸   ʹͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›‡ǡ’‘Ž›’‡’–‹†‡ͳ   ͵ͳͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›͵ǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ͳͳ   ͵ʹͷ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›͵ǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ʹͷ   ͹ͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›͹ǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ͳ   ͳͳͳ Š›†”‘š›•–‡”‘‹†ͳͳǦ„‡–ƒ†‡Š›†”‘‰‡ƒ•‡ͳ   ͵ͷ Š›†”‘š›Ǧ†‡Ž–ƒǦͷǦ•–‡”‘‹††‡Š›†”‘‰‡ƒ•‡ǡ͵„‡–ƒǦƒ†•–‡”‘‹††‡Ž–ƒǦ‹•‘‡”ƒ•‡ͷ  ͷͳ •–‡”‘‹†ͷƒŽ’ŠƒǦ”‡†— –ƒ•‡ͳ   ͷ͵ •–‡”‘‹†ͷƒŽ’ŠƒǦ”‡†— –ƒ•‡͵    ʹ͵Ͷ ‰Ž— —”‘‘•›Ž–”ƒ•ˆ‡”ƒ•‡ʹˆƒ‹Ž›ǡ’‘Ž›’‡’–‹†‡͵Ͷ    ʹ͵ͷ ‰Ž— —”‘‘•›Ž–”ƒ•ˆ‡”ƒ•‡ʹˆƒ‹Ž›ǡ’‘Ž›’‡’–‹†‡͵ͷ    ʹ͵͸ ‰Ž— —”‘‘•›Ž–”ƒ•ˆ‡”ƒ•‡ʹˆƒ‹Ž›ǡ’‘Ž›’‡’–‹†‡͵͸    ʹͷ ‰Ž— —”‘‘•›Ž–”ƒ•ˆ‡”ƒ•‡ʹˆƒ‹Ž›ǡ’‘Ž›’‡’–‹†‡ͷ  

ͳͲͺ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

 NADPH_REDOX FDR = 0.09 RR = 1  Ͷ ƒŽ ‘Š‘Ž†‡Š›†”‘‰‡ƒ•‡Ͷȋ Žƒ•• Ȍǡ’‹’‘Ž›’‡’–‹†‡   ͳͳ ƒŽ†‘Ǧ‡–‘”‡†— –ƒ•‡ˆƒ‹Ž›ͳǡ‡„‡”ͳȋƒŽ†‡Š›†‡”‡†— –ƒ•‡Ȍ   ͳͳ ƒŽ†‘Ǧ‡–‘”‡†— –ƒ•‡ˆƒ‹Ž›ͳǡ‡„‡”ͳ   ͹ͷ ƒŽ†‘Ǧ‡–‘”‡†— –ƒ•‡ˆƒ‹Ž›͹ǡ‡„‡”ͷȋƒˆŽƒ–‘š‹ƒŽ†‡Š›†‡”‡†— –ƒ•‡Ȍ   ͳͳ ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡ˆƒ‹Ž›ͳǡ•—„ˆƒ‹Ž›ͳ    ͳͳ ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡ͳˆƒ‹Ž›ǡ‡„‡”ͳ    ͵ʹ ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡ˆƒ‹Ž›͵ǡ•—„ˆƒ‹Ž›ʹ   3  Ͷͳ ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡Ͷˆƒ‹Ž›ǡ‡„‡”ͳ    ͸ͳ ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡ˆƒ‹Ž›͸ǡ•—„ˆƒ‹Ž›ͳ    ͹ͳ ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡ˆƒ‹Ž›͹ǡ‡„‡”ͳ     ƒ•’ƒ”–ƒ–‡†‡Š›†”‘‰‡ƒ•‡†‘ƒ‹ ‘–ƒ‹‹‰     „”ƒ Š‡† Šƒ‹‡–‘ƒ ‹††‡Š›†”‘‰‡ƒ•‡ͳǡƒŽ’Šƒ’‘Ž›’‡’–‹†‡    „‹Ž‹˜‡”†‹”‡†— –ƒ•‡ȋˆŽƒ˜‹”‡†— –ƒ•‡ȋ ȌȌ   ͳ ƒ”„‘›Ž”‡†— –ƒ•‡ͳ   ͳ ”›•–ƒŽŽ‹ǡŽƒ„†ƒͳ    ”›•–ƒŽŽ‹ǡœ‡–ƒ   ͷ͵ ›–‘ Š”‘‡„ͷ”‡†— –ƒ•‡͵   ͳ͹ͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ͳ͹ǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ͳ   ͳʹ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ͳǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ʹ   ʹͷ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ͷ   ʹʹͻ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡ʹͻ   ʹ͵͹ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǤ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡͵͹   ʹ͵ͺ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡͵ͺ   ʹͷͲ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡ͷͲ   ʹͷͶ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡ͷͶ   ʹ͹Ͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡͹Ͳ   ʹͳͲ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›†ǡ’‘Ž›’‡’–‹†‡ͳͲ   ʹʹʹ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›†ǡ’‘Ž›’‡’–‹†‡ʹʹ   ʹʹ͸ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›†ǡ’‘Ž›’‡’–‹†‡ʹ͸   ʹͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›‡ǡ’‘Ž›’‡’–‹†‡ͳ   ʹͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›”ǡ’‘Ž›’‡’–‹†‡ͳ   ʹͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›—ǡ’‘Ž›’‡’–‹†‡ͳ   ͵ͻͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›͵ͻǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ͳ   ͵ͳͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›͵ǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ͳͳ   ͵ʹͷ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›͵ǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ʹͷ   Ͷͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›Ͷǡ•—„ˆƒ‹Ž›„ǡ’‘Ž›’‡’–‹†‡ͳ   Ͷ ͳͶ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›Ͷǡ•—„ˆƒ‹Ž›ˆǡ’‘Ž›’‡’–‹†‡ͳͶ   ͹ͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›͹ǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ͳ    †‹ ƒ”„‘›ŽǦš›Ž—Ž‘•‡”‡†— –ƒ•‡    ͹ ͹Ǧ†‡Š›†”‘ Š‘Ž‡•–‡”‘Ž”‡†— –ƒ•‡    ͵ †‡Š›†”‘‰‡ƒ•‡Ȁ”‡†— –ƒ•‡ȋˆƒ‹Ž›Ȍ‡„‡”͵    †‹Š›†”‘Ž‹’‘ƒ‹†‡Ǧƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ȋʹ ‘’‘‡–‘ˆ’›”—˜ƒ–‡†‡Š›†”‘‰‡ƒ•‡ ‘’Ž‡šȌ ʹ ‡ –‘Ǧ†‹•—Žˆ‹†‡Ǧ–Š‹‘Ž‡š Šƒ‰‡”ʹ    ˆƒ––›ƒ ‹†•›–Šƒ•‡   ͳ ˆŽƒ˜‹ ‘–ƒ‹‹‰‘‘‘š›‰‡ƒ•‡ͳ   Ͷ ˆŽƒ˜‹ ‘–ƒ‹‹‰‘‘‘š›‰‡ƒ•‡Ͷ     Š›†”‘š›’”‘•–ƒ‰Žƒ†‹†‡Š›†”‘‰‡ƒ•‡ͳͷȋȌ   ͳͳͳ Š›†”‘š›•–‡”‘‹†ͳͳǦ„‡–ƒ†‡Š›†”‘‰‡ƒ•‡ͳ   ͳ͹Ͷ Š›†”‘š›•–‡”‘‹†ȋͳ͹Ǧ„‡–ƒȌ†‡Š›†”‘‰‡ƒ•‡Ͷ   ͵ͷ Š›†”‘š›Ǧ†‡Ž–ƒǦͷǦ•–‡”‘‹††‡Š›†”‘‰‡ƒ•‡ǡ͵„‡–ƒǦƒ†•–‡”‘‹††‡Ž–ƒǦ‹•‘‡”ƒ•‡ͷ  ͵͹ Š›†”‘š›Ǧ†‡Ž–ƒǦͷǦ•–‡”‘‹††‡Š›†”‘‰‡ƒ•‡ǡ͵„‡–ƒǦƒ†•–‡”‘‹††‡Ž–ƒǦ‹•‘‡”ƒ•‡͹   ͳ ‹•‘ ‹–”ƒ–‡†‡Š›†”‘‰‡ƒ•‡ͳȋΪȌǡ•‘Ž—„Ž‡    ʹ ‹•‘ ‹–”ƒ–‡†‡Š›†”‘‰‡ƒ•‡ʹȋΪȌǡ‹–‘ Š‘†”‹ƒŽ    ͵ ‹•‘ ‹–”ƒ–‡†‡Š›†”‘‰‡ƒ•‡͵ȋΪȌ„‡–ƒ    ͵  ‹•‘ ‹–”ƒ–‡†‡Š›†”‘‰‡ƒ•‡͵ȋΪȌǡ‰ƒƒ   ͳ ƒŽ‹ ‡œ›‡ͳǡȋΪȌǦ†‡’‡†‡–ǡ ›–‘•‘Ž‹     ͵ ‹ ”‘–—„—Ž‡ƒ••‘ ‹ƒ–‡†‘‘‘š›‰‡ƒ•‡ǡ ƒŽ’‘‹ƒ† †‘ƒ‹ ‘–ƒ‹‹‰͵  ‡–Š›Ž‡‡–‡–”ƒŠ›†”‘ˆ‘Žƒ–‡†‡Š›†”‘‰‡ƒ•‡ȋΪ†‡’‡†‡–Ȍǡ‡–Š‡›Ž–‡–”ƒŠ›†”‘ˆ‘Žƒ–‡ › Ž‘Š›†”‘Žƒ•‡ǡ  ͳ ˆ‘”›Ž–‡–”ƒŠ›†”‘ˆ‘Žƒ–‡•›–Šƒ•‡ ͳ •›–Š‡–ƒ•‡ͳ    ͳ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌͳƒŽ’Šƒ•—„ ‘’Ž‡šǡͳ    ͳͳ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌͳƒŽ’Šƒ•—„ ‘’Ž‡šͳͳ    ͳʹ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌͳƒŽ’Šƒ•—„ ‘’Ž‡šǡͳʹ    ͵  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌͳƒŽ’Šƒ•—„ ‘’Ž‡šǡ͵    ͸  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌͳƒŽ’Šƒ•—„ ‘’Ž‡šǡ͸ȋͳͶȌ    ͹  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌͳƒŽ’Šƒ•—„ ‘’Ž‡šǡ͹ȋͳͶǤͷƒȌ   ͳͲ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍͳ„‡–ƒ•—„ ‘’Ž‡šǡͳͲ    ͳͳ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍͳ„‡–ƒ•—„ ‘’Ž‡šǡͳͳ    ͸  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍͳ„‡–ƒ•—„ ‘’Ž‡šǡ͸    ͻ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍͳ„‡–ƒ•—„ ‘’Ž‡šǡͻ    ʹ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍͳǡ•—„ ‘’Ž‡š—‘™ǡʹ    ͳ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍ ‡Ǧ’”‘–‡‹ͳ  

ͳͲͻ Chapter 3

 ʹ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍ ‡Ǧ’”‘–‡‹ʹ    ͸  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍ ‡Ǧ’”‘–‡‹͸    ͹  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡Ȍ ‡Ǧ’”‘–‡‹͹    ͳ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌˆŽƒ˜‘’”‘–‡‹ͳ    ʹ  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌˆŽƒ˜‘’”‘–‡‹ʹ    ͵  †‡Š›†”‘‰‡ƒ•‡ȋ—„‹“—‹‘‡ȌˆŽƒ˜‘’”‘–‡‹͵   ͳ ȋȌ †‡Š›†”‘‰‡ƒ•‡ǡ“—‹‘‡ͳ    ͳ ’›”—˜ƒ–‡†‡Š›†”‘‰‡ƒ•‡ͳƒŽ’Šƒͳ     ’›”—˜ƒ–‡†‡Š›†”‘‰‡ƒ•‡ȋŽ‹’‘ƒ‹†‡Ȍ„‡–ƒ    ’‡”‘š‹•‘ƒŽ–”ƒ•ǦʹǦ‡‘›ŽǦ‘”‡†— –ƒ•‡     ’”‘Ž‹‡†‡Š›†”‘‰‡ƒ•‡    ͳ͸ ”‡–‹‘Ž†‡Š›†”‘‰‡ƒ•‡ͳ͸    ͹ ”‡–‹‘Ž†‡Š›†”‘‰‡ƒ•‡͹   ͷͳ •–‡”‘‹†ͷƒŽ’ŠƒǦ”‡†— –ƒ•‡ͳ   ͷ͵ •–‡”‘‹†ͷƒŽ’ŠƒǦ”‡†— –ƒ•‡͵   ʹ •‹š–”ƒ•‡„”ƒ‡‡’‹–Š‡Ž‹ƒŽƒ–‹‰‡‘ˆ’”‘•–ƒ–‡ʹ    –”ƒ•Ǧʹǡ͵Ǧ‡‘›ŽǦ‘”‡†— –ƒ•‡   ͹ ʹ –”ƒ•‡„”ƒ‡͹•—’‡”ˆƒ‹Ž›‡„‡”ʹ   ͵ –‹••—‡•’‡ ‹ˆ‹ –”ƒ•’Žƒ–ƒ–‹‘ƒ–‹‰‡͵ͷ   ͵ –Š‹‘”‡†‘š‹”‡†— –ƒ•‡͵      Ǧ‰Ž— ‘•‡†‡Š›†”‘‰‡ƒ•‡    TAURINE AND HYPOTAURINE METABOLISM FDR = 0.23 RR = 0  ʹǦƒ‹‘‡–Šƒ‡–Š‹‘Žȋ ›•–‡ƒ‹‡Ȍ†‹‘š›‰‡ƒ•‡ȏ‘—” ‡ǣ ›„‘ŽǢ ǣ ǣʹ͸ͺͷͲͺ͵Ȑ   ͸ ‰ƒƒǦ‰Ž—–ƒ›Ž–”ƒ•ˆ‡”ƒ•‡͸ȏ‘—” ‡ǣ ›„‘ŽǢ ǣ ǣͳͻͳͺ͹͹ʹȐ    ›•–‡‹‡•—Žˆ‹‹ ƒ ‹††‡ ƒ”„‘š›Žƒ•‡ȏ‘—” ‡ǣ ›„‘ŽǢ ǣ ǣʹͳͺͲͲͻͺȐ   εͳǣ‰‡‡•‡–•ǡ™Š‡”‡‘ƒ†ȋȌȋ Ȍ‰‡‡•ƒ”‡‘˜‡””‡’”‡•‡–‡†‹–Š‡‹”Ž‹•–‘ˆ ‘–”‹„—–‹‰‰‡‡• ‘’ƒ”‡†–‘ –Š‡ ‘’Ž‡–‡‰‡‡•‡–Ǥ

Supplementary Table ST7. Gene-set enrichment analysis (GSEA) results for metabolic gene sets for mixed- background MCAD-KO vs WT mice Upregulated gene sets Downregulated gene sets Š‡›ŽƒŽƒ‹‡ǡ–›”‘•‹‡ƒ†–”›’–‘’Šƒ„‹‘•›–Š‡•‹• Pyruvate metabolism Ž› ‘•ƒ‹‘‰Ž› ƒ†‡‰”ƒ†ƒ–‹‘ ƒ––›ƒ ‹†„‹‘•›–Š‡•‹• ƒŽ‹‡ǡŽ‡— ‹‡ƒ†‹•‘Ž‡— ‹‡„‹‘•›–Š‡•‹• Glycophingolipid biosynthesis – lacto and neolacto series  ‘‰‡‡•‡–  ƒ––›ƒ ‹†‡–ƒ„‘Ž‹•  ”‘’ƒ‘ƒ–‡‡–ƒ„‘Ž‹•  ‹–”ƒ–‡ › Ž‡ȋ › Ž‡Ȍ  ‡–‹‘Ž‡–ƒ„‘Ž‹• ‡‡•‡–•†‡’‹ –‡†Š‡”‡Šƒ˜‡ƒ ‘ˆδͲǤʹͷȋ‡š’Ž‘”ƒ–‹˜‡ȌǤ ‡‡•‡–•™‹–Šƒ ‘ˆδͲǤͲͷƒ”‡†‡’‹ –‡†‹„‘Ž†Ǥƒ–ƒ ˆ”‘ ‡””‡ƒet al.ȏͳȐǤ  Supplementary table ST8: Contributing genes in the upregulated metabolic gene sets in the Chow fasted condition GLYCOSAMINOGLYCAN DEGRADATION FDR = 0.19 RR = λ ͳ ‰ƒŽƒ –‘•‹†ƒ•‡ǡ„‡–ƒͳ    Š‡’ƒ”ƒƒ•‡    ‰Ž— —”‘‹†ƒ•‡ǡ„‡–ƒ    ‰Ž— ‘•ƒ‹‡ȋǦƒ ‡–›ŽȌǦ͸Ǧ•—Žˆƒ–ƒ•‡    Š‡š‘•ƒ‹‹†ƒ•‡    Š‡’ƒ”ƒǦƒŽ’ŠƒǦ‰Ž— ‘•ƒ‹‹†‡Ǧƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡     ƒŽ’ŠƒǦǦƒ ‡–›Ž‰Ž— ‘•ƒ‹‹†ƒ•‡ȋƒˆ‹Ž‹’’‘†‹•‡ƒ•‡ Ȍ    ‹†—”‘ƒ–‡ʹǦ•—Žˆƒ–ƒ•‡  

PHENYLALANINE, TYROSINE AND TRYPTOPHAN BIOSYNTHESIS FDR = 0.22 RR = n.a. ͳ ‰Ž—–ƒ‹ Ǧ‘šƒŽ‘ƒ ‡–‹ –”ƒ•ƒ‹ƒ•‡ͳǡ•‘Ž—„Ž‡     ’Š‡›ŽƒŽƒ‹‡Š›†”‘š›Žƒ•‡    –›”‘•‹‡ƒ‹‘–”ƒ•ˆ‡”ƒ•‡    VALINE, LEUCINE AND ISOLEUCINE BIOSYNTHESIS FDR = 0.24 RR = n.a.  •‡”‹‡†‡Š›†”ƒ–ƒ•‡    •‡”‹‡†‡Š›†”ƒ–ƒ•‡ǦŽ‹‡   ʹ „”ƒ Š‡† Šƒ‹ƒ‹‘–”ƒ•ˆ‡”ƒ•‡ʹǡ‹–‘ Š‘†”‹ƒŽ   εͳǣ‰‡‡•‡–•ǡ™Š‡”‡‘ƒ†ȋȌȋ Ȍ‰‡‡•ƒ”‡‘˜‡””‡’”‡•‡–‡†‹–Š‡‹”Ž‹•–‘ˆ ‘–”‹„—–‹‰‰‡‡• ‘’ƒ”‡†–‘ –Š‡ ‘’Ž‡–‡‰‡‡•‡–ǤǤƒǤǣ‘–ƒ’’Ž‹ ƒ„Ž‡ǡ„‡ ƒ—•‡–Š‡ ‘’Ž‡–‡‰‡‡•‡–†‘‡•‘– ‘–ƒ‹‘‘”ȋȌȋ Ȍ‰‡‡•Ǥ

ͳͳͲ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

Supplementary table ST9: Contributing genes in the downregulated metabolic gene sets in the Chow fasted condition PYRUVATE METABOLISM FDR = 0.04 RR = 0.67 ʹ ƒ ›ŽǦ‘•›–Š‡–ƒ•‡•Š‘”–Ǧ Šƒ‹ˆƒ‹Ž›‡„‡”ʹ   ͳ ƒ ‡–›ŽǦ‘‡œ›‡ƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ͳ    †‹Š›†”‘Ž‹’‘ƒ‹†‡†‡Š›†”‘‰‡ƒ•‡    ’›”—˜ƒ–‡ ƒ”„‘š›Žƒ•‡    ͸ Žƒ –ƒ–‡†‡Š›†”‘‰‡ƒ•‡ǦŽ‹‡͸     Žƒ –ƒ–‡†‡Š›†”‘‰‡ƒ•‡   3 ͳ ƒ ›Ž’Š‘•’Šƒ–ƒ•‡ͳǡ‡”›–Š”‘ ›–‡ȋ ‘‘Ȍ–›’‡    ͳ ’›”—˜ƒ–‡†‡Š›†”‘‰‡ƒ•‡ͳƒŽ’Šƒͳ    ƒ ‡–›ŽǦ‘‡œ›‡ ƒ”„‘š›Žƒ•‡„‡–ƒ    ƒ ‡–›ŽǦ‘‡œ›‡ ƒ”„‘š›Žƒ•‡ƒŽ’Šƒ    ’›”—˜ƒ–‡‹ƒ•‡Ž‹˜‡”ƒ†”‡†„Ž‘‘† ‡ŽŽ   ͳ ƒŽ‹ ‡œ›‡ͳǡȋΪȌǦ†‡’‡†‡–ǡ ›–‘•‘Ž‹    ͳ ‰Ž›‘šƒŽƒ•‡ͳ     ’›”—˜ƒ–‡†‡Š›†”‘‰‡ƒ•‡ȋŽ‹’‘ƒ‹†‡Ȍ„‡–ƒ    GLYCOSPHINGOLIPID BIOSYNTHESIS - LACTO AND NEOLACTO SERIES FDR = 0.04 RR = n.a. ʹ ‰Ž— ‘•ƒ‹›ŽȋǦƒ ‡–›ŽȌ–”ƒ•ˆ‡”ƒ•‡ʹǡ Ǧ„”ƒ Š‹‰‡œ›‡   ͵ Ͷ Ǧ Ž  ǣ„‡–ƒ ƒŽ„‡–ƒǦͳǡ͵ǦǦƒ ‡–›Ž‰Ž— ‘•ƒ‹›Ž–”ƒ•ˆ‡”ƒ•‡Ͷ   Ͷ ͳ Ǧ ƒŽǣ„‡–ƒ Ž  „‡–ƒͳǡͶǦ‰ƒŽƒ –‘•›Ž–”ƒ•ˆ‡”ƒ•‡ǡ’‘Ž›’‡’–‹†‡ͳ  ͵ Ͷ ͵„‡–ƒǦ‰ƒŽƒ –‘•‹†‡ƒŽ’ŠƒǦʹǡ͵Ǧ•‹ƒŽ›Ž–”ƒ•ˆ‡”ƒ•‡Ͷ   ͵ ͸ ͵„‡–ƒǦ‰ƒŽƒ –‘•‹†‡ƒŽ’ŠƒǦʹǡ͵Ǧ•‹ƒŽ›Ž–”ƒ•ˆ‡”ƒ•‡͸   ͵ ͳ Ǧ ƒŽǣ„‡–ƒ Ž  „‡–ƒͳǡ͵Ǧ‰ƒŽƒ –‘•›Ž–”ƒ•ˆ‡”ƒ•‡ǡ’‘Ž›’‡’–‹†‡ͳ   COA_GENE_SET FDR = 0.06 RR =  †‹Š›†”‘Ž‹’‘ƒ‹†‡Ǧ•— ‹›Ž–”ƒ•ˆ‡”ƒ•‡ȋʹ ‘’‘‡–‘ˆʹǦ‘š‘Ǧ‰Ž—–ƒ”ƒ–‡ ‘’Ž‡šȌ  ͳͳ œ‹ ˆ‹‰‡”ǡ †‘ƒ‹ ‘–ƒ‹‹‰ͳͳ   ͵ ‡”ƒ‹†‡•›–Šƒ•‡͵    ͵Ͷ ‰‡‡”ƒŽ–”ƒ• ”‹’–‹‘ˆƒ –‘” ǡ’‘Ž›’‡’–‹†‡Ͷ    ͷ œ‹ ˆ‹‰‡”ǡ †‘ƒ‹ ‘–ƒ‹‹‰ͷ   ͳͷ ȋƒŽ’ŠƒȌǦƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ͳͷǡƒ–ƒ—š‹Ž‹ƒ”›•—„—‹–   ʹ ȋŽ›•‹‡Ȍƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ʹ     ƒ‹‘ƒ†‹’ƒ–‡Ǧ•‡‹ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡Ǧ’Š‘•’Š‘’ƒ–‡–Š‡‹›Ž–”ƒ•ˆ‡”ƒ•‡   ͵ ƒ ›ŽǦ‘•›–Š‡–ƒ•‡ˆƒ‹Ž›‡„‡”͵    ͳͻ œ‹ ˆ‹‰‡”ǡ †‘ƒ‹ ‘–ƒ‹‹‰ͳͻ     ͵ǦŠ›†”‘š›‹•‘„—–›”›ŽǦ‘‡œ›‡Š›†”‘Žƒ•‡   ʹ ‡”ƒ‹†‡•›–Šƒ•‡ʹ     ͵ǦŠ›†”‘š›Ǧ͵Ǧ‡–Š›Ž‰Ž—–ƒ”›ŽǦ‘‡œ›‡”‡†— –ƒ•‡   ͳ ƒŽ’Šƒ–—„—Ž‹ƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ͳ    ͳͷ œ‹ ˆ‹‰‡”ǡ †‘ƒ‹ ‘–ƒ‹‹‰ͳͷ   Ͷ ƒ ›ŽǦ‘–Š‹‘‡•–‡”ƒ•‡Ͷ   ͸ ƒ ›ŽǦ‘•›–Š‡–ƒ•‡Ž‘‰Ǧ Šƒ‹ˆƒ‹Ž›‡„‡”͸     ‹–”ƒ–‡Ž›ƒ•‡    ͳ ͳǦƒ ›Ž‰Ž› ‡”‘ŽǦ͵Ǧ’Š‘•’Šƒ–‡Ǧƒ ›Ž–”ƒ•ˆ‡”ƒ•‡ͳȋŽ›•‘’Š‘•’Šƒ–‹†‹ ƒ ‹†ƒ ›Ž–”ƒ•ˆ‡”ƒ•‡ǡƒŽ’ŠƒȌ ͺ ƒ ›ŽǦ‘–Š‹‘‡•–‡”ƒ•‡ͺ    ʹ œ‹ ˆ‹‰‡”ǡ †‘ƒ‹ ‘–ƒ‹‹‰ʹ   ʹ ƒ ›ŽǦ‘•›–Š‡–ƒ•‡•Š‘”–Ǧ Šƒ‹ˆƒ‹Ž›‡„‡”ʹ   ͳ ƒ ‡–›ŽǦ‘‡œ›‡ƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ͳ    „‹Ž‡ƒ ‹†Ǧ‘‡œ›‡ǣƒ‹‘ƒ ‹†Ǧƒ ›Ž–”ƒ•ˆ‡”ƒ•‡    ƒŽ‘›Ž‘ǣƒ ›Ž–”ƒ•ˆ‡”ƒ•‡ȋ‹–‘ Š‘†”‹ƒŽȌ   ͵ ƒ ›ŽǦ‘–Š‹‘‡•–‡”ƒ•‡͵    †‹Š›†”‘Ž‹’‘ƒ‹†‡†‡Š›†”‘‰‡ƒ•‡    ʹ •— ‹ƒ–‡Ǧ‘‡œ›‡Ž‹‰ƒ•‡ǡ Ǧˆ‘”‹‰ǡ„‡–ƒ•—„—‹–   ʹ Ǧƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ʹȋƒ”›Žƒ‹‡Ǧƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡Ȍ   ʹ •–‡”‘ŽǦƒ ›Ž–”ƒ•ˆ‡”ƒ•‡ʹ   ͳ ‡Ž‘‰ƒ–‹‘‘ˆ˜‡”›Ž‘‰ Šƒ‹ˆƒ––›ƒ ‹†•ȋ ͳȀŽ‘ʹǡͶȀŽ‘͵ǡ›‡ƒ•–ȌǦŽ‹‡ͳ   ʹͳ œ‹ ˆ‹‰‡”ǡ †‘ƒ‹ ‘–ƒ‹‹‰ʹͳ   ͹ —†‹šȋ— Ž‡‘•‹†‡†‹’Š‘•’Šƒ–‡Ž‹‡†‘‹‡–›ȌǦ–›’‡‘–‹ˆ͹    ͳ ’›”—˜ƒ–‡†‡Š›†”‘‰‡ƒ•‡ͳƒŽ’Šƒͳ    ͳ͵ œ‹ ˆ‹‰‡”ǡ †‘ƒ‹ ‘–ƒ‹‹‰ͳ͵    ͳ Ž›•‘’Š‘•’Šƒ–‹†›Ž‰Ž› ‡”‘Žƒ ›Ž–”ƒ•ˆ‡”ƒ•‡ͳ   ʹ͹ͷ •‘Ž—–‡ ƒ””‹‡”ˆƒ‹Ž›ʹ͹ȋˆƒ––›ƒ ‹†–”ƒ•’‘”–‡”Ȍǡ‡„‡”ͷ   ͷ ˆƒ‹Ž›‡„‡”ͷǡ‡Ž‘‰ƒ–‹‘‘ˆŽ‘‰ Šƒ‹ˆƒ––›ƒ ‹†•ȋ›‡ƒ•–Ȍ  ͳ •‡”‹‡’ƒŽ‹–‘›Ž–”ƒ•ˆ‡”ƒ•‡ǡŽ‘‰ Šƒ‹„ƒ•‡•—„—‹–ͳ    ˆƒ––›ƒ ‹†•›–Šƒ•‡    ͵Ǧ‘š‘ƒ ›ŽǦ•›–Šƒ•‡ǡ‹–‘ Š‘†”‹ƒŽ   ʹ •‡”‹‡’ƒŽ‹–‘›Ž–”ƒ•ˆ‡”ƒ•‡ǡŽ‘‰ Šƒ‹„ƒ•‡•—„—‹–ʹ   ͸ ˆƒ‹Ž›‡„‡”͸ǡ‡Ž‘‰ƒ–‹‘‘ˆŽ‘‰ Šƒ‹ˆƒ––›ƒ ‹†•ȋ›‡ƒ•–Ȍ  ͳ Ǧƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ͳ   ͵ ƒ ›ŽǦ‘•›–Š‡–ƒ•‡Ž‘‰Ǧ Šƒ‹ˆƒ‹Ž›‡„‡”͵    ƒ ‡–‘ƒ ‡–›ŽǦ‘•›–Š‡–ƒ•‡  

ͳͳͳ Chapter 3

COA_GENE_SET - continued    ͻ ͳǦƒ ›Ž‰Ž› ‡”‘ŽǦ͵Ǧ’Š‘•’Šƒ–‡Ǧƒ ›Ž–”ƒ•ˆ‡”ƒ•‡ͻ   ͳͳ ƒ ›ŽǦ‘–Š‹‘‡•–‡”ƒ•‡ͳͳ   ʹ ƒ ›ŽǦ ‘‡œ›‡ƒ‹‘ƒ ‹†Ǧƒ ›Ž–”ƒ•ˆ‡”ƒ•‡ʹ     ’›”—˜ƒ–‡†‡Š›†”‘‰‡ƒ•‡ȋŽ‹’‘ƒ‹†‡Ȍ„‡–ƒ        FATTY ACID BIOSYNTHESIS FDR = 0.06 RR = 0.75 ͸ ƒ ›ŽǦ‘•›–Š‡–ƒ•‡Ž‘‰Ǧ Šƒ‹ˆƒ‹Ž›‡„‡”͸    ƒŽ‘›Ž‘ǣƒ ›Ž–”ƒ•ˆ‡”ƒ•‡ȋ‹–‘ Š‘†”‹ƒŽȌ    ƒ ‡–›ŽǦ‘‡œ›‡ ƒ”„‘š›Žƒ•‡„‡–ƒ    ƒ ‡–›ŽǦ‘‡œ›‡ ƒ”„‘š›Žƒ•‡ƒŽ’Šƒ    ˆƒ––›ƒ ‹†•›–Šƒ•‡    ͵Ǧ‘š‘ƒ ›ŽǦ•›–Šƒ•‡ǡ‹–‘ Š‘†”‹ƒŽ   ͳ͹ͲͲͲ͸ͳ ͳͻ    ͳ͹ͲͲͲ͸ͳ ͳͻ‰‡‡   ͵ ƒ ›ŽǦ‘•›–Š‡–ƒ•‡Ž‘‰Ǧ Šƒ‹ˆƒ‹Ž›‡„‡”͵    FATTY ACID METABOLISM FDR = 0.09 RR = 1.61   Š›†”‘š›ƒ ›ŽǦ‘‡œ›‡†‡Š›†”‘‰‡ƒ•‡   ͸ ƒ ›ŽǦ‘•›–Š‡–ƒ•‡Ž‘‰Ǧ Šƒ‹ˆƒ‹Ž›‡„‡”͸   ͳ ƒ ‡–›ŽǦ‘‡œ›‡ƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ͳ    ƒŽ‘›Ž‘ǣƒ ›Ž–”ƒ•ˆ‡”ƒ•‡ȋ‹–‘ Š‘†”‹ƒŽȌ   ͳ͹ͳʹ Š›†”‘š›•–‡”‘‹†ȋͳ͹Ǧ„‡–ƒȌ†‡Š›†”‘‰‡ƒ•‡ͳʹ    ’‡”‘š‹•‘ƒŽ–”ƒ•ǦʹǦ‡‘›ŽǦ‘”‡†— –ƒ•‡    ƒ ‡–›ŽǦ‘‡œ›‡ ƒ”„‘š›Žƒ•‡ƒŽ’Šƒ   ͷ ˆƒ‹Ž›‡„‡”ͷǡ‡Ž‘‰ƒ–‹‘‘ˆŽ‘‰ Šƒ‹ˆƒ––›ƒ ‹†•ȋ›‡ƒ•–Ȍ   ˆƒ––›ƒ ‹†•›–Šƒ•‡    ͵Ǧ‘š‘ƒ ›ŽǦ•›–Šƒ•‡ǡ‹–‘ Š‘†”‹ƒŽ   ͳ͹ͲͲͲ͸ͳ ͳͻ    ͳ͹ͲͲͲ͸ͳ ͳͻ‰‡‡   ͸ ˆƒ‹Ž›‡„‡”͸ǡ‡Ž‘‰ƒ–‹‘‘ˆŽ‘‰ Šƒ‹ˆƒ––›ƒ ‹†•ȋ›‡ƒ•–Ȍ  ͵ ƒ ›ŽǦ‘•›–Š‡–ƒ•‡Ž‘‰Ǧ Šƒ‹ˆƒ‹Ž›‡„‡”͵    PROPANOATE METABOLISM FDR = 0.16 RR = 0.67  ’”‘’‹‘›Ž‘‡œ›‡ ƒ”„‘š›Žƒ•‡ǡ„‡–ƒ’‘Ž›’‡’–‹†‡   ʹ ƒ ‡–›ŽǦ‘‡œ›‡ƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ʹ    ‡–Š›ŽƒŽ‘›ŽǦ‘‡œ›‡—–ƒ•‡    ƒŽ‘›ŽǦ‘†‡ ƒ”„‘š›Žƒ•‡    ‡–Š›ŽƒŽ‘›Ž‘‡’‹‡”ƒ•‡    ͳ ‡‘›Ž‘‡œ›‡Š›†”ƒ–ƒ•‡†‘ƒ‹ ‘–ƒ‹‹‰ͳ     ͵ǦŠ›†”‘š›‹•‘„—–›”›ŽǦ‘‡œ›‡Š›†”‘Žƒ•‡   ʹ ƒ ›ŽǦ‘•›–Š‡–ƒ•‡•Š‘”–Ǧ Šƒ‹ˆƒ‹Ž›‡„‡”ʹ   ͳ ƒ ‡–›ŽǦ‘‡œ›‡ƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ͳ    †‹Š›†”‘Ž‹’‘ƒ‹†‡†‡Š›†”‘‰‡ƒ•‡    ʹ •— ‹ƒ–‡Ǧ‘‡œ›‡Ž‹‰ƒ•‡ǡ Ǧˆ‘”‹‰ǡ„‡–ƒ•—„—‹–    ͸ Žƒ –ƒ–‡†‡Š›†”‘‰‡ƒ•‡ǦŽ‹‡͸    ƒ ‡–›ŽǦ‘‡œ›‡ ƒ”„‘š›Žƒ•‡„‡–ƒ    ƒ ‡–›ŽǦ‘‡œ›‡ ƒ”„‘š›Žƒ•‡ƒŽ’Šƒ    CITRATE CYCLE (TCA CYCLE) FDR = 0.23 RR = 0.95   ‹–”ƒ–‡Ž›ƒ•‡     •— ‹ƒ–‡†‡Š›†”‘‰‡ƒ•‡ ‘’Ž‡šǡ•—„—‹–ǡˆŽƒ˜‘’”‘–‡‹ȋ ’Ȍ   ͳ ƒ ‘‹–ƒ•‡ͳ     •— ‹ƒ–‡†‡Š›†”‘‰‡ƒ•‡ ‘’Ž‡šǡ•—„—‹–ǡ‹”‘•—Žˆ—”ȋ ’Ȍ    ͳ ‹•‘ ‹–”ƒ–‡†‡Š›†”‘‰‡ƒ•‡ͳȋΪȌǡ•‘Ž—„Ž‡    †‹Š›†”‘Ž‹’‘ƒ‹†‡†‡Š›†”‘‰‡ƒ•‡    ʹ •— ‹ƒ–‡Ǧ‘‡œ›‡Ž‹‰ƒ•‡ǡ Ǧˆ‘”‹‰ǡ„‡–ƒ•—„—‹–    ’›”—˜ƒ–‡ ƒ”„‘š›Žƒ•‡   Ͷͻ͵͵ͶͲͷʹͲ    Ͷͻ͵͵ͶͲͷʹͲ‰‡‡    ͳ ’›”—˜ƒ–‡†‡Š›†”‘‰‡ƒ•‡ͳƒŽ’Šƒͳ     ’›”—˜ƒ–‡†‡Š›†”‘‰‡ƒ•‡ȋŽ‹’‘ƒ‹†‡Ȍ„‡–ƒ    RETINOL METABOLISM FDR = 0.24 RR = 1.39 ʹ͹Ͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡͹Ͳ   Ͷͳʹ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›Ͷǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ͳʹƒ   ͳ ƒŽ†‡Š›†‡‘š‹†ƒ•‡ͳ   ʹͷͷ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡ͷͷ   ͸ͷ ”‡–‹ƒŽ’‹‰‡–‡’‹–Š‡Ž‹—͸ͷ   ͳ͸ͷ •Š‘”– Šƒ‹†‡Š›†”‘‰‡ƒ•‡Ȁ”‡†— –ƒ•‡ˆƒ‹Ž›ͳ͸ǡ‡„‡”ͷ   ʹͷͲ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡ͷͲ   ͵ͳͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›͵ǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ͳͳ    Ͷ ƒŽ ‘Š‘Ž†‡Š›†”‘‰‡ƒ•‡Ͷȋ Žƒ•• Ȍǡ’‹’‘Ž›’‡’–‹†‡    ͳͳ ”‡–‹‘Ž†‡Š›†”‘‰‡ƒ•‡ͳͳ  

ͳͳʹ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

RETINOL METABOLISM - continued   ʹ͵͹ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǤ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡͵͹    ”‡–‹‘Ž•ƒ–—”ƒ•‡ȋƒŽŽ–”ƒ•”‡–‹‘Žͳ͵ǡͳͶ”‡†— –ƒ•‡Ȍ    ͳ͹ ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡ˆƒ‹Ž›ͳǡ•—„ˆƒ‹Ž›͹    ͵ †‡Š›†”‘‰‡ƒ•‡Ȁ”‡†— –ƒ•‡ȋˆƒ‹Ž›Ȍ‡„‡”͵   ʹ͵ͻ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡͵ͻ    ͳͳ ƒŽ†‡Š›†‡†‡Š›†”‘‰‡ƒ•‡ˆƒ‹Ž›ͳǡ•—„ˆƒ‹Ž›ͳ    ʹ͵͹ ‰Ž— —”‘‘•›Ž–”ƒ•ˆ‡”ƒ•‡ʹˆƒ‹Ž›ǡ’‘Ž›’‡’–‹†‡͵͹   ʹͻ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›„ǡ’‘Ž›’‡’–‹†‡ͻ   ͵ͶͶ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›͵ǡ•—„ˆƒ‹Ž›ƒǡ’‘Ž›’‡’–‹†‡ͶͶ   3 ʹ͵ͺ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž› ǡ’‘Ž›’‡’–‹†‡͵ͺ    ʹ͵ͷ ‰Ž— —”‘‘•›Ž–”ƒ•ˆ‡”ƒ•‡ʹˆƒ‹Ž›ǡ’‘Ž›’‡’–‹†‡͵ͷ    ͳͲ ”‡–‹‘Ž†‡Š›†”‘‰‡ƒ•‡ͳͲȋƒŽŽǦ–”ƒ•Ȍ   ʹͳͲ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ʹǡ•—„ˆƒ‹Ž›„ǡ’‘Ž›’‡’–‹†‡ͳͲ    STEROID BIOSYNTHESIS FDR = 0.24 RR = 1.20   Ž›•‘•‘ƒŽƒ ‹†Ž‹’ƒ•‡    ʹͶ ʹͶǦ†‡Š›†”‘ Š‘Ž‡•–‡”‘Ž”‡†— –ƒ•‡   ͹ ʹ –”ƒ•‡„”ƒ‡͹•—’‡”ˆƒ‹Ž›‡„‡”ʹ    ͹ ͹Ǧ†‡Š›†”‘ Š‘Ž‡•–‡”‘Ž”‡†— –ƒ•‡    Žƒ‘•–‡”‘Ž•›–Šƒ•‡   ͳ͹͹ Š›†”‘š›•–‡”‘‹†ȋͳ͹Ǧ„‡–ƒȌ†‡Š›†”‘‰‡ƒ•‡͹    •“—ƒŽ‡‡‡’‘š‹†ƒ•‡     ȋȌ†‡’‡†‡–•–‡”‘‹††‡Š›†”‘‰‡ƒ•‡ǦŽ‹‡   ʹ •–‡”‘ŽǦƒ ›Ž–”ƒ•ˆ‡”ƒ•‡ʹ   ͷͳ ›–‘ Š”‘‡ͶͷͲǡˆƒ‹Ž›ͷͳ   „„”‡˜‹ƒ–‹‘•ƒ† ‘’ƒ”‹•‘ƒ”‡–Š‡•ƒ‡ƒ••—’’Ž‡‡–ƒ”›–ƒ„Ž‡ͺǤ 

ͳͳ͵ Chapter 3

Supplementary table ST10: Differentially-expressed metabolic genes LFD Fed KO/WT (metabolic genes) Upregulated Downregulated DeSeq FC Voom FC DeSeq FC Voom FC † ›͹ ͳǤͺ ‰ͳ ʹǤͳ B3galt1 ʹǤ͹ Pde4d ͶǤ͵ ‹‰„ ͳǤͺ Zdhhc7 ʹǤͲ Colgalt2 ʹǤͲ  ‘–͵ ͶǤʹ Zdhhc7 ͳǤͺ ˆƒ• ͳǤͺ Pik3cd ͳǤͺ B3galt1 ͶǤͲ • ͳǤ͹ ’”ͳ ͳǤͺ ‹͵ ʹ‰ ͳǤͺ Pik3c2g ʹǤͶ Soat2 ͳǤ͹ ’’„ ͳǤ͹ Hlcs ͳǤͺ š–Žͳ ʹǤʹ Nme6 ͳǤ͹ Pld3 ͳǤ͹ Extl1 ͳǤͺ ’’ ͳǤͻ ˆƒ• ͳǤ͹ •–ƒͶ ͳǤ͸ ͻͳ͵ͲͶͲͻ ʹ͵‹ ͳǤͺ Ž‘˜Ž͸ ͳǤͻ †ŠŠ ͳͶ ͳǤ͸ — ͳǤ͸ ›’Ͷ͸ƒͳ ͳǤ͹ ‘Ͷ ͳǤͺ ƒ† ͳǤ͸ ‡͸ ͳǤ͸ ‰–ƒͳ ͳǤ͹ Ž „Ͷ ͳǤͺ •–ƒͶ ͳǤ͸ †‡͹ƒ ͳǤ͸ ’’ ͳǤ͹ Ž„ͳ ͳǤͺ ‰ͳ ͳǤ͸ •‰ƒŽƒ –ʹ ͳǤͷ š –ͳ ͳǤ͹ Ž • ͳǤͺ –Ͷ ͳǤͷ ”›Žͳ ͳǤͷ  ƒ ƒ ͳǤ͸  ƒ ƒ ͳǤ͹ ƒ• ͳǤͷ ƒ–ʹ ͳǤͷ ƒ–͵ ͳǤ͸  †ʹ ͳǤ͹ ’’„ ͳǤͷ •ƒ† ͳǤͷ †‡ͷƒ ͳǤ͸ –͵‰ƒŽ͸ ͳǤ͹ Ž†ͳ ͳǤͷ †•–ʹ ͳǤͷ  ••͵ ͳǤ͸ ‡’Š•ʹ ͳǤ͸ Ampd2 ͳǤͷ  ‹”‰ͳ ͳǤͷ –”͹ ͳǤ͸ ‡ͳ ͳǤ͸ Dcxr ͳǤͷ ›’ʹ ͵͹ ͳǤͷ ‘‰ƒ–ͳ ͳǤ͸  Ž› ͳǤ͸ ͸ ͳǤͷ ƒ†•›ͳ ͳǤͶ Ž „Ͷ ͳǤ͸ Ž „͵ ͳǤ͸ — ͳǤͷ •• ͳǤͶ ’š͸ ͳǤ͸ †Šͳͳ ͳǤ͸ •• ͳǤͶ ’†ʹ ͳǤͶ –’͸˜Ͳƒʹ ͳǤ͸ Ž˜”ƒ ͳǤͷ ‹ ƒŽʹ ͳǤͶ ˆ’–ͳ ͳǤͶ ƒ’ ͳǤ͸ – ͳǤͷ ”›Žͳ ͳǤͶ ’”– ͳǤͶ ‰–ʹ„͵͹ ͳǤ͸ Š– ͳǤͷ ˆ’–ͳ ͳǤͶ ‡ ͳǤ͵ Žƒʹ‰Ͷƒ ͳǤͷ †•Ž ͳǤͷ •‰ƒŽƒ –ʹ ͳǤͶ •’†Š ͳǤ͵ Ž‘˜Ž͸ ͳǤͷ –‡ƒ’͵ ͳǤͷ ›’ʹ ͵͹ ͳǤͶ ‰’Žͳ ͳǤ͵ ’Žƒ͵ ͳǤͷ Š ”ʹͶ ͳǤͶ †‡͹ƒ ͳǤͶ Fnta ͳǤ͵ ‰’•ͳ ͳǤͷ •ƒŠʹ ͳǤͶ ’Ššͳ ͳǤͶ •–‘ͳ ͳǤ͵ Ž„ͳ ͳǤͷ  •ŽͶ ͳǤͶ ‡ ͳǤͶ Rpn2 ͳǤ͵ ͵ ͳǤͷ  ͳ ͳǤͶ †‘ ͳǤͶ ‘‰• ͳǤ͵ ƒʹ„ʹ ͳǤͷ Š’ˆʹ ͳǤͶ Sgpl1 ͳǤͶ ›’ʹ ͶͶ ͳǤ͵ ‡ͳ ͳǤͷ ND5 ͳǤ͵ Rpn2 ͳǤ͵ ƒ†ͳ ͳǤʹ  †ʹ ͳǤͷ Š• ͳŽͳ ͳǤ͵ ‹ˆ›˜‡ ͳǤ͵ —†–ͳͻ ͳǤʹ †•Ž ͳǤͷ ʹ ͳǤ͵ Vkorc1 ͳǤ͵ †—ˆ„͹ ͳǤʹ •’ƒ ͳǤͷ •ŠͳŽ ͳǤʹ ‘Ž”ʹ‡ ͳǤ͵ ‡ Š ͳǤʹ Žƒʹ‰͸ ͳǤͷ †Šƒͳ ͳǤʹ ›’ʹ†ʹ͸ ͳǤ͵ ‡‰•ͳ ͳǤʹ ƒ‘ƒ ͳǤͷ   ›’ʹ ͶͶ ͳǤ͵    •ŽͶ ͳǤͷ   „• ͳǤ͵   –͵‰ƒŽͷ ͳǤͶ   †—ˆ„ͳͳ ͳǤ͵   ‘šʹ ͳǤͶ   ‘Ž‰ƒŽ–ͳ ͳǤ͵   Ž†Šͳ„ͳ ͳǤͶ   —†–ͳͻ ͳǤ͵   –Ǧ†ʹ ͳǤ͵   ‡‰•ͳ ͳǤʹ   ’†ʹ ͳǤ͵       Š• ͳŽͳ ͳǤ͵       mt-Nd5 ͳǤ͵  

HFD Fed KO/WT (metabolic genes) Upregulated Downregulated DeSeq FC Voom FC DeSeq FC Voom FC ‰–ʹ„͵ͺ ͳǤͻ ‰–ʹ„͵ͺ ͶǤͲ Acnat2 ʹǤͶ ›’ͶƒͳͶ ͵Ǥ͸ Cyp4a12b ͳǤͺ Cyp4a12b ͳǤͻ –Šˆ†ͳŽ ͳǤͺ  ƒ–ʹ ͵Ǥͳ ƒ †Ͷ ͳǤͺ ˆˆ„͵ ͳǤͻ †‡ͺ„ ͳǤ͹ Cyp2c38 ͳǤͺ ‰ƒ•‡ ͳǤ͹ Ž†ͳ ͳǤͺ Cyp2b9 ͳǤ͸ –͵‰ƒŽͷ ͳǤ͹ ”ƒ† ͳǤ͹ •†͵„ͷ ͳǤͺ ›’͵ͻƒͳ ͳǤ͸ ‘šͳͷ ͳǤ͸ ”ͳ„ͳͲ ͳǤ͹ ƒŽ–͹ ͳǤ͹  ͳ ͳǤ͸  •Ž͵ ͳǤ͸ †‡͵ƒ ͳǤ͸ ƒ–ͺ ͳǤ͸ –’ƒ ͳǤͷ  ƒ–ͳ ͳǤͷ ƒ”’ʹ ͳǤ͸ –͵‰ƒŽ͸ ͳǤ͸ ‘šͳͷ ͳǤͷ †Šͳ͸ ͳǤͷ ‘š͹ƒͳ ͳǤ͸ †’ʹ ͳǤ͸ ƒ–ͳ ͳǤͷ Acox3 ͳǤͷ ‘͵ ͳǤ͸ ƒ”’Ͷ ͳǤͷ  •Ž͵ ͳǤͷ › ”ʹ ͳǤͶ „‘ƒ–ͳ ͳǤ͸ ‡˜͵Ž ͳǤͷ Nnt ͳǤͷ ’ƒ– ͳǤͶ ƒ”’Ͷ ͳǤͷ ‡͸ ͳǤͷ ‹‰  ͳǤͷ  ‹”‰ͳ ͳǤͶ Rev3l ͳǤͷ š ͳǤͷ ›’ʹ ͵ͺ ͳǤͷ – ͳǤ͵ –͵‰ƒŽʹ ͳǤͷ ‰† ͳǤͶ ’ƒ– ͳǤͶ Ž†Šͷƒͳ ͳǤʹ •†͵„ͷ ͳǤͷ ‹”–ͳ ͳǤͶ ‡ͳ ͳǤͶ ‘ •ʹ ͳǤʹ Ž‘†ʹ ͳǤͷ Gba ͳǤͶ –͵‰ƒŽͶ ͳǤͶ   ƒŽ–͹ ͳǤͷ ‰—‘ ͳǤ͵ –ƒ’ ͳǤͶ   ‡͸ ͳǤͷ Š• ͳŽͳ ͳǤʹ  ‘š͵ ͳǤͶ   Dctpp1 ͳǤͷ   ’†ͳ ͳǤ͵   ”ͳ ͳǤͷ   Ž†Šͷƒͳ ͳǤʹ  

ͳͳͶ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

HFD Fed KO/WT (metabolic genes) - continued Upregulated DeSeq FC ͳ ͳǤͷ       †’ʹ ͳǤͷ       Ž†ͳ ͳǤͷ       ‡‰ͳ ͳǤͶ       •– ͳǤͶ       ƒ”’ͻ ͳǤͶ       †ŠŠ ͸ ͳǤ͵       3  •ͷ ͳǤ͵       •’†Š ͳǤ͵        Ž› ͳǤʹ      

Low-fat fasted KO/WT Upregulated Downregulated DeSeq FC Voom FC DeSeq FC Voom FC Akr1b7 ʹǤͲ •†͵„ʹ ʹǤʹ Naa40 ʹǤͲ Naa40 ʹǤ͸ Cerk ʹǤͲ ‡” ʹǤʹ Ocrl ͳǤͺ Zdhhc17 ʹǤ͸ Entpd2 ͳǤͻ ‡”•ͷ ʹǤͲ Zdhhc17 ͳǤͺ †Š† ʹǤͳ Npr1 ͳǤͻ  ‘–ʹ ʹǤͲ †ƒ ͳǤͺ †• ʹǤͳ ‹‰„ ͳǤͺ ͵‰ƒŽ–ͳ ͳǤͺ †ŠŠ ͺ ͳǤͺ †ŠŠ ͳ͵ ʹǤͳ Acaa1b ͳǤͺ ’”ͳ ͳǤ͹ Cdyl ͳǤͺ ‹‰‰ ʹǤͲ  ‘–ʹ ͳǤͺ ’‘š ͳǤ͹ Pde4b ͳǤ͹ Cdyl ʹǤͲ Chsy1 ͳǤ͹ ‘Ͷ ͳǤ͹ †ŠŠ ͳ͵ ͳǤ͹ ‹‰ƒ ʹǤͲ Csad ͳǤ͹ •–ͷ ͳǤ͸ Pnpt1 ͳǤ͹ Pde4b ͳǤͻ  ͳǤ͸ Š•›ͳ ͳǤ͸ † ›͹ ͳǤ͸ •͵•–͵„ͳ ͳǤͻ ƒ–ͳ ͳǤ͸ ’‹ʹ ͳǤ͸ ˆˆ„ʹ ͳǤ͸ ›• ͳǤͻ ‘Ž”͵‰Ž ͳǤ͸  ƒƒͳ„ ͳǤ͸ –ͷ ͳǤ͸ ‘Ž”ʹŠ ͳǤͺ ‘ ͵ ͳǤ͸ Hsd17b11 ͳǤ͸ ‡–†ͳƒ ͳǤ͸ ’’ ͳǤͺ ‡”•ͷ ͳǤ͸ ƒ–ͳ ͳǤ͸ †ŠŠ ʹ ͳǤ͸ Acyp1 ͳǤͺ Cyp21a1 ͳǤ͸ •ƒ† ͳǤ͸ ‹Ͷ„ ͳǤ͸ † ›͸ ͳǤͺ ”ͳ„ͺ ͳǤ͸  ʹ ͳǤͷ † ›͸ ͳǤ͸ Pnpt1 ͳǤͺ Hsd17b11 ͳǤ͸ •– ͳǤͷ ƒͳ ͳ ͳǤͷ Nt5m ͳǤͺ †‡͵ƒ ͳǤ͸  ͳǤͷ  ›’ͳ ͳǤͷ –’ͷŒ ͳǤͺ Ndufab1 ͳǤ͸ †•Ž ͳǤͷ ’‹ͳ ͳǤͷ  ”Ž ͳǤ͹ ”ͳ„ͳͲ ͳǤ͸ Gda ͳǤͶ ’”ʹ ͳǤͷ ’ʹ ͳǤ͹ ƒ’ͳŽͳ ͳǤͷ Urod ͳǤͶ ͵ ͳǤͷ ‹’ƒ”’ ͳǤ͹ †ŠŠ ͳ ͳǤͷ ‘Ž‡͵ ͳǤͶ •–ƒʹ ͳǤͷ ƒ †ʹ ͳǤ͹ ƒ„ ͳǤͷ ’Ššͳ ͳǤͶ †••Žͳ ͳǤͷ   ͳǤ͹ ͳ‰ƒŽ–ͳ ͳǤͷ –‰•ͳ ͳǤͶ ‘Ž”ʹ† ͳǤͷ †ŠŠ ͺ ͳǤ͹ Ndufa5 ͳǤͷ Crebbp ͳǤ͵ ‹‰ƒ” ͳǤͷ –͸‰ƒŽͳ ͳǤ͹ ’‘š ͳǤͷ ’”ʹ ͳǤ͵ –ƒͶŠ ͳǤͷ ‘‰–ͳ ͳǤ͸ •– ͳǤͷ †—ˆƒ„ͳ ͳǤ͵ –͸‰ƒŽͳ ͳǤͷ Aldh1b1 ͳǤ͸ † ›͵ ͳǤͶ Ž›ƒ– ͳǤ͵ Aldh1b1 ͳǤͷ •” ͳǤ͸ Ž ‡ͳ ͳǤͶ †—ˆ˜͵ ͳǤ͵ †ŠŠ ͷ ͳǤͶ ”‘• ͳǤ͸ ’ƒʹ ͳǤͶ —†–ͳʹ ͳǤʹ ‹’ƒ”’ ͳǤͶ  Žͳ ͳǤ͸ ‹ˆ ͳǤͶ ’” ͳǤʹ ‘‰–ͳ ͳǤͶ Zdhhc5 ͳǤ͸ Žƒ† ͳǤͶ ’”• ͳǤʹ Ž†͵ ͳǤͶ –ƒͶŠ ͳǤ͸ › • ͳǤͶ ”ƒŠ ͳǤʹ ‡–†„ͳ ͳǤͶ –ʹ„ ͳǤ͸ Crebbp ͳǤͶ †—ˆƒʹ ͳǤʹ ”‘• ͳǤͶ ‘šͳ͹ ͳǤ͸ ’Ššͳ ͳǤͶ   †‘ ͳǤͶ ›’Ͷ„ͳ ͳǤͷ †ƒ ͳǤͶ    Žͳ ͳǤͶ Ž ͵͵ƒͳ ͳǤͷ Ndufv3 ͳǤͶ   Ͷ ͳǤͶ ‡”•͸ ͳǤͷ ‘Ž‡͵ ͳǤͶ   ›’ͶˆͳͶ ͳǤͶ ‹’’ͳ ͳǤͷ ’ŽƒŠ ͳǤͶ   Ž ‰ͳ ͳǤͶ ‡–†ͳƒ ͳǤͷ †—ˆ ͳ ͳǤ͵   ‹Ͷʹ„ ͳǤ͵ ƒŠ ͳǤͷ ›Ž„ ͳǤ͵   ’ƒ–ͳ ͳǤ͵ Ͷ ͳǤͷ ’” ͳǤ͵   –͸ ͳǤ͵ Dnmt3a ͳǤͷ ’–ʹ ͳǤ͵   Hsd3b7 ͳǤ͵ †‘ ͳǤͷ ”‘† ͳǤ͵   ƒ‰Š ͳǤʹ ͹•ˆʹ ͳǤͷ ’ ƒ–͵ ͳǤ͵   ’ƒͳ ͳǤʹ ’”ʹ ͳǤͷ ƒƒ͸Ͳ ͳǤ͵     —†–ͷ ͳǤͶ ‘ͷ ͳǤ͵     Ž†Š͸ƒͳ ͳǤͶ †—ˆƒͳͳ ͳǤ͵     — Ž‰ʹ ͳǤͶ  ƒƒʹ ͳǤ͵     ‹„ Š ͳǤͶ Ž›ƒ– ͳǤ͵     †—ˆƒͳ ͳǤ͵ †—ˆ˜ͳ ͳǤ͵     ‰ƒ– ͳǤ͵ ”ͳƒͳ ͳǤʹ     –͵‰ƒŽͳ ͳǤ͵ †—ˆƒͳ͵ ͳǤʹ     ’ƒ–ͳ ͳǤ͵ –Ǧ†Ͷ ͳǤʹ     Ž ‰ͳ ͳǤ͵

ͳͳͷ Chapter 3

Low-fat fasted KO/WT - continued Upregulated Downregulated DeSeq FC Voom FC DeSeq FC Voom FC ”ƒŠ ͳǤʹ     •†͵„͹ ͳǤ͵ †—ˆƒʹ ͳǤʹ     ’ƒͳ ͳǤʹ       ‘ •ʹ ͳǤʹ

High-fat fasted KO/WT Upregulated Downregulated DeSeq FC Voom FC DeSeq FC Voom FC Pip4k2a ͳǤͻ ‹’Ͷʹƒ ͳǤͻ Npr1 ʹǤͲ Rce1 ʹǤʹ Hexdc ͳǤͻ  ‡”ʹ ͳǤͺ  ‡ͳ ͳǤͺ Cyp2c38 ͳǤͺ Ͷ‰ƒŽ–͸ ͳǤ͹ ›’Ͷƒͳʹƒ ͳǤͺ Cyp2c38 ͳǤ͹ ’”ͳ ͳǤ͹ Steap4 ͳǤ͹ ͵‰ƒŽ–ͳ ͳǤͺ Cndp2 ͳǤ͹ Gstm1 ͳǤ͸ Acer2 ͳǤ͹ ›’Ͷƒͳʹ„ ͳǤͺ ›’ʹ ͵ͻ ͳǤ͹ ’†ƒͳ ͳǤͷ ›’Ͷƒ͵ͳ ͳǤ͹ Ž „Ͷ ͳǤ͹ ‰ʹŽͳ ͳǤ͸ †••Žͳ ͳǤͷ ›’Ͷƒͳʹ„ ͳǤ͸ Ahcy ͳǤ͹ Š•–ͳͶ ͳǤ͸ ‰Š ͳǤͷ ‘•͵ ͳǤ͸ Ž‰ʹ ͳǤ͸ Žƒʹ‰Ͷƒ ͳǤ͸ š–Žʹ ͳǤͷ Ž „Ͷ ͳǤ͸ •ƒŠʹ ͳǤͷ Gstm1 ͳǤ͸ ‘Ž”ʹŒ ͳǤͷ ͵‰ƒŽ–ͳ ͳǤ͸ ‘šͳ ͳǤͷ ‰Š ͳǤ͸ •–ƒ͵ ͳǤͷ  •† ͳǤͷ  ‘ƒ͵ ͳǤͶ –‰‡•ʹ ͳǤͷ †’ʹ ͳǤͶ †  ͳǤͷ ›’͵ƒͳ͵ ͳǤͶ ‰ͳ ͳǤͷ •ƒ† ͳǤͶ ‹‰– ͳǤͷ š• ͳǤ͵ •–ƒͶ ͳǤͷ Adi1 ͳǤͶ Ž‰ʹ ͳǤͷ Ž›ƒ– ͳǤ͵ š”†͵ ͳǤͷ ’”ʹ ͳǤͶ ›’Ͷƒͳʹƒ ͳǤͷ ›’ʹƒͳʹ ͳǤ͵ ‘ƒ–ʹ ͳǤͷ ’Šʹ ͳǤͶ –͵‰ƒŽͷ ͳǤͷ ‹͵ ʹƒ ͳǤ͵ „‘ƒ–͹ ͳǤͷ ‰•ʹ ͳǤͶ ˆŽ ͳǤͶ ’”• ͳǤʹ ›’ʹ ͸ͺ ͳǤͷ Š”•͵ ͳǤͶ ‰’ƒ–ͷ ͳǤͶ   Ͷ‰ƒŽ–Ͷ ͳǤͷ Akr7a5 ͳǤ͵ ƒ‰Ž— ͳǤͶ    ͳǤͶ •’†Š ͳǤ͵ Cyp3a13 ͳǤͶ   Žƒʹ‰ͳͷ ͳǤͶ Ž—Ž ͳǤ͵ –͵‰ƒŽͶ ͳǤͶ    ͳ ͳǤͶ ‰–ʹ„͵ͷ ͳǤ͵  ‘ƒ͵ ͳǤͶ   ‘Ž”ʹŒ ͳǤͶ •–ͳ ͳǤ͵ Ž ʹ͹ƒʹ ͳǤ͵   –ʹ ͳǤͶ  ›–ʹ ͳǤ͵ –’ͷ ͳ ͳǤ͵   Adi1 ͳǤͶ Š–ʹ ͳǤ͵ ›’ʹƒͳʹ ͳǤ͵    †Šƒ ͳǤͶ •†͵„͹ ͳǤʹ     ’–ʹ ͳǤ͵ ›’ʹ†ͳͲ ͳǤʹ     ”͹ƒͷ ͳǤ͵ ‘š͸ƒͳ ͳǤʹ     •–ͳ ͳǤ͵       ›’ʹ†ͳͲ ͳǤ͵       ‰•ʹ ͳǤ͵       Š–ʹ ͳǤ͵       ‘š͸„ͳ ͳǤʹ       •†͵„͹ ͳǤʹ   Ž—‡Š‹‰ŠŽ‹‰Š–ǣ—”‹‡‰‡‡Ǣ‡†„‘”†‡”ǣȋȌȋ Ȍ‰‡‡ȋ‹ Ž”‡†‘šȌǢ ”‡‡ǣ‘˜‡”Žƒ’™‹–Š‘‡‘”‘”‡‘–Š‡” ‘†‹–‹‘Ǣ†‡”Ž‹‡†ǣ‘Ǧ‹˜‘Ž˜‹‰‰‡‡Ǣ‘Ž†ǣ’δͲǤͲͳǤŠ‡”‡•—Ž–•’”‡•‡–‡†Š‡”‡ƒ”‡ˆ”‘ ‘’ƒ”‹•‘•„‡–™‡‡ ƒ†Ǥ 

ͳͳ͸ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

Supplementary table ST11: Metabolic gene sets ranked according to the percentage of genes they contain that fit the “(abs(log2(fold change))>0.5 AND p<0.1) OR p<0.05”criteria Gene set with upregulated genes LF Fa HF Fa LF Fe HF Fe ȋȌȋ Ȍƒ‹ ͳ ͳ ʹ ͳ ȋȌȋ Ȍ”‡†‘š ʹ ͳ ʹ ʹ —”‹‡‡–ƒ„‘Ž‹• Ͷ ͻͲ ͳ ʹ ‘‰‡‡•‡– ͵ Ͷ ͵ ʹ ‡–‹‘Ž‡–ƒ„‘Ž‹• ͻͲ ͳ ͳͲ ͷ –‡”‘‹†Š‘”‘‡„‹‘•›–Š‡•‹• ʹʹ ͳͲ ͸ ͷ 3 Ž› ‡”‘Ž‹’‹†‡–ƒ„‘Ž‹• ͹ ͳͲ ͻͲ ͷ ”— –‘•‡ƒ†ƒ‘•‡‡–ƒ„‘Ž‹• ͻ ͳͲ ͳͲ ͷ ƒŽƒ –‘•‡‡–ƒ„‘Ž‹• ͻ ͳͲ ͻͲ ͷ ›”‹‹†‹‡‡–ƒ„‘Ž‹• ͳͶ ͻͲ ͸ ͷ Ž› ‘Ž›•‹•Ȁ Ž— ‘‡‘‰‡‡•‹• ͳͶ ͳͲ ͳͻ ͷ ’Š‹‰‘Ž‹’‹†‡–ƒ„‘Ž‹• ͳͶ ͹ ͳͲ ͻͲ ƒ––›ƒ ‹††‡‰”ƒ†ƒ–‹‘ ͻ Ͷ ͻͲ ͳ͸ ‡–‘•‡ƒ†‰Ž— —”‘ƒ–‡‹–‡” ‘˜‡”•‹‘• ͸ ͻͲ ͳͲ ͳ͸ š‹†ƒ–‹˜‡’Š‘•’Š‘”›Žƒ–‹‘ ͷ ͳͲ ͳͻ ͳ͸ ”ƒ Š‹†‘‹ ƒ ‹†‡–ƒ„‘Ž‹• ͻͲ Ͷ ͳͲ ͳ͸ ‹‘•›–Š‡•‹•‘ˆƒ‹‘ƒ ‹†• ʹʹ ͳͲ ͳͲ ͳ͸ Ž› ‡”‘’Š‘•’Š‘Ž‹’‹†‡–ƒ„‘Ž‹• ʹʹ ͳͲ ͳͻ ͷ ƒ”„‘‡–ƒ„‘Ž‹• ʹʹ ͳͲ ͳͻ ͷ ‘•‹–‘Ž’Š‘•’Šƒ–‡‡–ƒ„‘Ž‹• ͳͶ ͹ ͳͻ ͻͲ ”‰‹‹‡ƒ†’”‘Ž‹‡‡–ƒ„‘Ž‹• ͻͲ ͳͲ ͳͻ ͳ͸ ‹‘•—‰ƒ”ƒ†— Ž‡‘–‹†‡•—‰ƒ”‡–ƒ„‘Ž‹• ʹʹ ͻͲ ͵ ͳ͸ „‡–ƒǦŽƒ‹‡‡–ƒ„‘Ž‹• ʹʹ ͻͲ ͳͻ ͳ͸ ‹‘Ž‡‹ ƒ ‹†‡–ƒ„‘Ž‹• ͻͲ ͳͲ ͳͲ ͻͲ ƒ––›ƒ ‹†‡Ž‘‰ƒ–‹‘ ͳͶ ͻͲ ͻͲ ͳ͸ ›•–‡‹‡ƒ†‡–Š‹‘‹‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͸ ͳ͸ ‹•–‹†‹‡‡–ƒ„‘Ž‹• ͻͲ ͳͲ ͻͲ ͳ͸ Š‡›ŽƒŽƒ‹‡‡–ƒ„‘Ž‹• ͻ ͻͲ ͻͲ ͳ͸ –Š‡”‰Ž› ƒ†‡‰”ƒ†ƒ–‹‘ ͻͲ ͳͲ ͻͲ ͳ͸ Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•Ǧ‰ƒ‰Ž‹‘•‡”‹‡• ͻͲ ͳͲ ͻͲ ͳ͸ ‹‘•›–Š‡•‹•‘ˆ—•ƒ–—”ƒ–‡†ˆƒ––›ƒ ‹†• ͳͶ ͻͲ ͻͲ ͳ͸ ƒ––›ƒ ‹†‡–ƒ„‘Ž‹• ͻ ͻͲ ͻͲ ͳ͸ ›•‹‡†‡‰”ƒ†ƒ–‹‘ ͻͲ ͻͲ ͳͻ ͳ͸ Ǧ Ž› ƒ„‹‘•›–Š‡•‹• ͻͲ ͳͲ ͳͻ ͻͲ — ‹–›’‡Ǧ Ž› ƒ„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͳͻ ͷ –Š‡”Ž‹’‹†‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͳͻ ͳ͸ Ž› ‹‡ǡ•‡”‹‡ƒ†–Š”‡‘‹‡‡–ƒ„‘Ž‹• ʹʹ ͻͲ ͳͻ ͻͲ ƒ—”‹‡ƒ†Š›’‘–ƒ—”‹‡‡–ƒ„‘Ž‹• ʹʹ ͻͲ ͳͻ ͻͲ ‡Ž‡‘ ‘’‘—†‡–ƒ„‘Ž‹• ʹʹ ͻͲ ͻͲ ͳ͸ Ž—–ƒ–Š‹‘‡‡–ƒ„‘Ž‹• ʹʹ ͻͲ ͸ ͻͲ Ž› ‘•ƒ‹‘‰Ž› ƒ„‹‘•›–Š‡•‹•Ǧ Š‘†”‘‹–‹•—Žˆƒ–‡Ȁ†‡”ƒ–ƒ•—Žˆƒ–‡ ʹʹ ͻͲ ͳͻ ͻͲ Ž› ‘•›Ž’Š‘•’Šƒ–‹†›Ž‹‘•‹–‘Žȋ  ȌǦƒ Š‘”„‹‘•›–Š‡•‹• ʹʹ ͻͲ ͳͻ ͻͲ ‹ ‘–‹ƒ–‡ƒ†‹ ‘–‹ƒ‹†‡‡–ƒ„‘Ž‹• ʹʹ ͻͲ ͻͲ ͳ͸ ‡–ƒ„‘Ž‹•‘ˆš‡‘„‹‘–‹ •„› ›–‘ Š”‘‡ͶͷͲ ʹʹ ͻͲ ͳͲ ͻͲ ”—‰‡–ƒ„‘Ž‹•Ǧ ›–‘ Š”‘‡ͶͷͲ ʹʹ ͻͲ ͳͻ ͻͲ ‹–”ƒ–‡ › Ž‡ȋ › Ž‡Ȍ ͻͲ ͻͲ ͻͲ ͻͲ ‡–‘•‡’Š‘•’Šƒ–‡’ƒ–Š™ƒ› ͻͲ ͳͲ ͻͲ ͻͲ • ‘”„ƒ–‡ƒ†ƒŽ†ƒ”ƒ–‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ ƒ––›ƒ ‹†„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͻͲ ͻͲ ›–Š‡•‹•ƒ††‡‰”ƒ†ƒ–‹‘‘ˆ‡–‘‡„‘†‹‡• ͻͲ ͻͲ ͻͲ ͻͲ –‡”‘‹†„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͳͻ ͻͲ ”‹ƒ”›„‹Ž‡ƒ ‹†„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͻͲ ͻͲ „‹“—‹‘‡ƒ†‘–Š‡”–‡”’‡‘‹†Ǧ“—‹‘‡„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͳͻ ͻͲ ƒˆˆ‡‹‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ Žƒ‹‡ǡƒ•’ƒ”–ƒ–‡ƒ†‰Ž—–ƒƒ–‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͳͲ ͻͲ ƒŽ‹‡ǡŽ‡— ‹‡ƒ†‹•‘Ž‡— ‹‡†‡‰”ƒ†ƒ–‹‘ ͳͶ ͻͲ ͻͲ ͻͲ ƒŽ‹‡ǡŽ‡— ‹‡ƒ†‹•‘Ž‡— ‹‡„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͻͲ ͻͲ ›•‹‡„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͻͲ ͻͲ ›”‘•‹‡‡–ƒ„‘Ž‹• ͳͶ ͻͲ ͻͲ ͻͲ ”›’–‘’Šƒ‡–ƒ„‘Ž‹• ͻͲ ͳͲ ͻͲ ͻͲ Š‡›ŽƒŽƒ‹‡ǡ–›”‘•‹‡ƒ†–”›’–‘’Šƒ„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͻͲ ͻͲ Ǧ Ž—–ƒ‹‡ƒ†Ǧ‰Ž—–ƒƒ–‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ Ǧ”‰‹‹‡ƒ†Ǧ‘”‹–Š‹‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ –ƒ” Šƒ†•— ”‘•‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͷ –Š‡”–›’‡•‘ˆǦ‰Ž› ƒ„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͳͻ ͻͲ —–‹”‘•‹ƒ†‡‘› ‹„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͻͲ ͳ͸ Ž› ‘•ƒ‹‘‰Ž› ƒ†‡‰”ƒ†ƒ–‹‘ ͻͲ ͳͲ ͻͲ ͻͲ Ž› ‘•ƒ‹‘‰Ž› ƒ„‹‘•›–Š‡•‹•Ǧ‡”ƒ–ƒ•—Žˆƒ–‡ ͻͲ ͻͲ ͻͲ ͳ͸ Ž› ‘•ƒ‹‘‰Ž› ƒ„‹‘•›–Š‡•‹•ǦŠ‡’ƒ”ƒ•—Žˆƒ–‡ȀŠ‡’ƒ”‹ ͻͲ ͻͲ ͻͲ ͻͲ ƒŽ’ŠƒǦ‹‘Ž‡‹ ƒ ‹†‡–ƒ„‘Ž‹• ʹʹ ͻͲ ͻͲ ͻͲ Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•ǦŽƒ –‘ƒ†‡‘Žƒ –‘•‡”‹‡• ͻͲ ͹ ͻͲ ͻͲ

ͳͳ͹ Chapter 3

Gene set with upregulated genes - continued LF Fa HF Fa LF Fe HF Fe Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•Ǧ‰Ž‘„‘•‡”‹‡• ͻͲ ͻͲ ͻͲ ͳ͸ ›”—˜ƒ–‡‡–ƒ„‘Ž‹• ʹʹ ͻͲ ͻͲ ͻͲ Ž›‘š›Žƒ–‡ƒ††‹ ƒ”„‘š›Žƒ–‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ ”‘’ƒ‘ƒ–‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ —–ƒ‘ƒ–‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͳ͸ ‡ ƒ”„‘’‘‘Ž„›ˆ‘Žƒ–‡ ͻͲ ͻͲ ͻͲ ͻͲ Š‹ƒ‹‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ ‹„‘ˆŽƒ˜‹‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ ‹–ƒ‹͸‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ ƒ–‘–Š‡ƒ–‡ƒ†‘„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͻͲ ͻͲ ‹‘–‹‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ ‹’‘‹ ƒ ‹†‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ ‘Žƒ–‡„‹‘•›–Š‡•‹• ʹʹ ͻͲ ͻͲ ͻͲ ‘”’Š›”‹ƒ† ŠŽ‘”‘’Š›ŽŽ‡–ƒ„‘Ž‹• ͹ ͻͲ ͻͲ ͻͲ ‡”’‡‘‹†„ƒ „‘‡„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͻͲ ͻͲ ‹–”‘‰‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ —Žˆ—”‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͳ͸ ”—‰‡–ƒ„‘Ž‹•Ǧ‘–Š‡”‡œ›‡• ͻͲ ͻͲ ͻͲ ͻͲ ʹǦš‘ ƒ”„‘š›Ž‹ ƒ ‹†‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ

Gene set with downregulated genes LFD Fa HFD Fa LFD Fe HFD Fe ȋȌȋ Ȍƒ‹ ͵ ͳ ͳ ͳ ȋȌȋ Ȍ”‡†‘š ͷ ͳ ʹ ͳ ‘‰‡‡•‡– ʹ ͹ Ͷ ͵ ”ƒ Š‹†‘‹ ƒ ‹†‡–ƒ„‘Ž‹• ͳͲ ͵ ͸ Ͷ —”‹‡‡–ƒ„‘Ž‹• ͳ ͹ ͳͳ Ͷ ›”—˜ƒ–‡‡–ƒ„‘Ž‹• ͸ ͳͺ ͸ Ͷ ‘•‹–‘Ž’Š‘•’Šƒ–‡‡–ƒ„‘Ž‹• ͵ ͻͲ ͵ ͳʹ Ž› ‡”‘’Š‘•’Š‘Ž‹’‹†‡–ƒ„‘Ž‹• ͳͲ ͹ ͸ ͻͲ Žƒ‹‡ǡƒ•’ƒ”–ƒ–‡ƒ†‰Ž—–ƒƒ–‡‡–ƒ„‘Ž‹• ͳͲ ͻͲ ͳͳ Ͷ ƒ––›ƒ ‹†‡–ƒ„‘Ž‹• ͻͲ ͳͺ Ͷ Ͷ Ž› ‘Ž›•‹•Ȁ Ž— ‘‡‘‰‡‡•‹• ͳͲ ͳͷ ͳͳ ͳʹ ‹‘Ž‡‹ ƒ ‹†‡–ƒ„‘Ž‹• ͻͲ ͵ ͳͳ ͳʹ ƒ”„‘‡–ƒ„‘Ž‹• ͳ͹ ͹ ͳͳ ͳʹ Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•ǦŽƒ –‘ƒ†‡‘Žƒ –‘•‡”‹‡• ͻͲ ͳͷ ͳͳ ͳʹ ƒ––›ƒ ‹††‡‰”ƒ†ƒ–‹‘ ͳ͹ ͻͲ ͳͳ Ͷ ”—‰‡–ƒ„‘Ž‹•Ǧ ›–‘ Š”‘‡ͶͷͲ ͳ͹ ͹ ͳͳ ͻͲ ’Š‹‰‘Ž‹’‹†‡–ƒ„‘Ž‹• ͻͲ ͳͺ ͳͳ ͳʹ ƒŽ‹‡ǡŽ‡— ‹‡ƒ†‹•‘Ž‡— ‹‡†‡‰”ƒ†ƒ–‹‘ ͳ͹ ͳͺ ͳͳ ͻͲ ”‰‹‹‡ƒ†’”‘Ž‹‡‡–ƒ„‘Ž‹• ͳ͹ ͳͺ ͳͳ ͻͲ ‹•–‹†‹‡‡–ƒ„‘Ž‹• ͳ͹ ͳͺ ͸ ͻͲ –ƒ” Šƒ†•— ”‘•‡‡–ƒ„‘Ž‹• ͳ͹ ͳͺ ͳͳ ͻͲ –Š‡”Ž‹’‹†‡–ƒ„‘Ž‹• ͳ͹ ͳͺ ͳͳ ͻͲ ”—‰‡–ƒ„‘Ž‹•Ǧ‘–Š‡”‡œ›‡• ͳ͹ ͳͺ ͵͸ ͳʹ –‡”‘‹†Š‘”‘‡„‹‘•›–Š‡•‹• ͻͲ ͵ ͵͸ Ͷ ‡–‹‘Ž‡–ƒ„‘Ž‹• ͻͲ ͹ ͵͸ Ͷ Ž—–ƒ–Š‹‘‡‡–ƒ„‘Ž‹• ͳ͹ ͳͷ ͵͸ ͻͲ ‘”’Š›”‹ƒ† ŠŽ‘”‘’Š›ŽŽ‡–ƒ„‘Ž‹• ͳ͹ ͻͲ ͵͸ ͳʹ ‡–ƒ„‘Ž‹•‘ˆš‡‘„‹‘–‹ •„› ›–‘ Š”‘‡ͶͷͲ ͳ͹ ͵ ͵͸ ͻͲ š‹†ƒ–‹˜‡’Š‘•’Š‘”›Žƒ–‹‘ ͻͲ ͳͺ ͵͸ ͳʹ „‡–ƒǦŽƒ‹‡‡–ƒ„‘Ž‹• ͳ͹ ͳͺ ͵͸ ͻͲ ›•‹‡†‡‰”ƒ†ƒ–‹‘ ͸ ͻͲ ͸ ͻͲ ›”‹‹†‹‡‡–ƒ„‘Ž‹• ͷ ͹ ͻͲ ͻͲ ‹‘•—‰ƒ”ƒ†— Ž‡‘–‹†‡•—‰ƒ”‡–ƒ„‘Ž‹• ͳͲ ͻͲ ͳͳ ͻͲ ƒ––›ƒ ‹†„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͳͳ ͳʹ ‹‘•›–Š‡•‹•‘ˆ—•ƒ–—”ƒ–‡†ˆƒ––›ƒ ‹†• ͻͲ ͻͲ ͳͳ ͳʹ ‡–‘•‡ƒ†‰Ž— —”‘ƒ–‡‹–‡” ‘˜‡”•‹‘• ͳ͹ ͻͲ ͳͳ ͻͲ ƒŽƒ –‘•‡‡–ƒ„‘Ž‹• ͳ͹ ͻͲ ͳͳ ͻͲ • ‘”„ƒ–‡ƒ†ƒŽ†ƒ”ƒ–‡‡–ƒ„‘Ž‹• ͳ͹ ͻͲ ͳͳ ͻͲ ”›’–‘’Šƒ‡–ƒ„‘Ž‹• ͳ͹ ͻͲ ͳͳ ͻͲ Ž› ‡”‘Ž‹’‹†‡–ƒ„‘Ž‹• ͳ͹ ͻͲ ͳͳ ͻͲ ›•–‡‹‡ƒ†‡–Š‹‘‹‡‡–ƒ„‘Ž‹• ͻͲ ͳͺ ͻͲ ͳʹ ƒŽ’ŠƒǦ‹‘Ž‡‹ ƒ ‹†‡–ƒ„‘Ž‹• ͻͲ ͳͺ ͳͳ ͻͲ ”‘’ƒ‘ƒ–‡‡–ƒ„‘Ž‹• ͻͲ ͳͺ ͳͳ ͻͲ ‡ ƒ”„‘’‘‘Ž„›ˆ‘Žƒ–‡ ͻͲ ͳͺ ͻͲ ͳʹ ”— –‘•‡ƒ†ƒ‘•‡‡–ƒ„‘Ž‹• ͸ ͻͲ ͵͸ ͻͲ ƒ––›ƒ ‹†‡Ž‘‰ƒ–‹‘ ͻͲ ͳͺ ͵͸ ͻͲ ”‹ƒ”›„‹Ž‡ƒ ‹†„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͵͸ ͳʹ Ž› ‹‡ǡ•‡”‹‡ƒ†–Š”‡‘‹‡‡–ƒ„‘Ž‹• ͻͲ ͳͺ ͵͸ ͻͲ –Š‡”–›’‡•‘ˆǦ‰Ž› ƒ„‹‘•›–Š‡•‹• ͳͲ ͻͲ ͵͸ ͻͲ —–‹”‘•‹ƒ†‡‘› ‹„‹‘•›–Š‡•‹• ͳ͹ ͻͲ ͵͸ ͻͲ —–ƒ‘ƒ–‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͵͸ ͳʹ ‹ ‘–‹ƒ–‡ƒ†‹ ‘–‹ƒ‹†‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͵͸ ͳʹ

ͳͳͺ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

Gene set with downregulated genes - continued LFD Fa HFD Fa LFD Fe HFD Fe ‡”’‡‘‹†„ƒ „‘‡„‹‘•›–Š‡•‹• ͻͲ ͳͺ ͵͸ ͻͲ ‹–”ƒ–‡ › Ž‡ȋ › Ž‡Ȍ ͻͲ ͻͲ ͻͲ ͳʹ ‡–‘•‡’Š‘•’Šƒ–‡’ƒ–Š™ƒ› ͻͲ ͻͲ ͻͲ ͻͲ ›–Š‡•‹•ƒ††‡‰”ƒ†ƒ–‹‘‘ˆ‡–‘‡„‘†‹‡• ͻͲ ͻͲ ͵͸ ͻͲ –‡”‘‹†„‹‘•›–Š‡•‹• ͻͲ ͳͺ ͻͲ ͻͲ „‹“—‹‘‡ƒ†‘–Š‡”–‡”’‡‘‹†Ǧ“—‹‘‡„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͻͲ ͻͲ ƒˆˆ‡‹‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͳʹ ƒŽ‹‡ǡŽ‡— ‹‡ƒ†‹•‘Ž‡— ‹‡„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͻͲ ͻͲ ›•‹‡„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͻͲ ͻͲ 3 ›”‘•‹‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͵͸ ͻͲ Š‡›ŽƒŽƒ‹‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͵͸ ͻͲ Š‡›ŽƒŽƒ‹‡ǡ–›”‘•‹‡ƒ†–”›’–‘’Šƒ„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͻͲ ͻͲ ƒ—”‹‡ƒ†Š›’‘–ƒ—”‹‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ ‡Ž‡‘ ‘’‘—†‡–ƒ„‘Ž‹• ͻͲ ͳͺ ͻͲ ͻͲ Ǧ Ž—–ƒ‹‡ƒ†Ǧ‰Ž—–ƒƒ–‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ Ǧ”‰‹‹‡ƒ†Ǧ‘”‹–Š‹‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ Ǧ Ž› ƒ„‹‘•›–Š‡•‹• ͳͲ ͻͲ ͻͲ ͻͲ –Š‡”‰Ž› ƒ†‡‰”ƒ†ƒ–‹‘ ͻͲ ͻͲ ͳͳ ͻͲ — ‹–›’‡Ǧ Ž› ƒ„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͻͲ ͻͲ Ž› ‘•ƒ‹‘‰Ž› ƒ†‡‰”ƒ†ƒ–‹‘ ͻͲ ͻͲ ͵͸ ͻͲ Ž› ‘•ƒ‹‘‰Ž› ƒ„‹‘•›–Š‡•‹•Ǧ Š‘†”‘‹–‹•—Žˆƒ–‡Ȁ†‡”ƒ–ƒ•—Žˆƒ–‡ ͻͲ ͳͺ ͻͲ ͻͲ Ž› ‘•ƒ‹‘‰Ž› ƒ„‹‘•›–Š‡•‹•Ǧ‡”ƒ–ƒ•—Žˆƒ–‡ ͻͲ ͳͺ ͻͲ ͻͲ Ž› ‘•ƒ‹‘‰Ž› ƒ„‹‘•›–Š‡•‹•ǦŠ‡’ƒ”ƒ•—Žˆƒ–‡ȀŠ‡’ƒ”‹ ͻͲ ͻͲ ͵͸ ͻͲ Ž› ‘•›Ž’Š‘•’Šƒ–‹†›Ž‹‘•‹–‘Žȋ  ȌǦƒ Š‘”„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͻͲ ͳʹ Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•Ǧ‰Ž‘„‘•‡”‹‡• ͻͲ ͻͲ ͻͲ ͻͲ Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•Ǧ‰ƒ‰Ž‹‘•‡”‹‡• ͻͲ ͻͲ ͳͳ ͻͲ Ž›‘š›Žƒ–‡ƒ††‹ ƒ”„‘š›Žƒ–‡‡–ƒ„‘Ž‹• ͻͲ ͳͺ ͻͲ ͻͲ Š‹ƒ‹‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ ‹„‘ˆŽƒ˜‹‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ ‹–ƒ‹͸‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ ƒ–‘–Š‡ƒ–‡ƒ†‘„‹‘•›–Š‡•‹• ͻͲ ͻͲ ͻͲ ͻͲ ‹‘–‹‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ ‹’‘‹ ƒ ‹†‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ ‘Žƒ–‡„‹‘•›–Š‡•‹• ͻͲ ͳͺ ͻͲ ͻͲ ‹–”‘‰‡‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ —Žˆ—”‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ ʹǦš‘ ƒ”„‘š›Ž‹ ƒ ‹†‡–ƒ„‘Ž‹• ͻͲ ͻͲ ͻͲ ͻͲ ‹‘•›–Š‡•‹•‘ˆƒ‹‘ƒ ‹†• ͻͲ ͹ ͻͲ ͻͲ Š‡”‡•—Ž–•Š‡”‡ƒ”‡ˆ”‘ ‘’ƒ”‹•‘•„‡–™‡‡ƒ†Ǥ ‹‰ŠŽ‹‰Š–‡†‹‰”‡›ǣƒ‘—–‘ˆ†‹ˆˆ‡”‡–‹ƒŽŽ›‡š’”‡••‡† ‰‡‡•‘ˆ–Š‡‰‹˜‡‰‡‡•‡–‹•‘˜‡””‡’”‡•‡–‡†‹–Š‡‰‹˜‡ ‘†‹–‹‘Ǥ α‘™Ǧˆƒ–†‹‡–ǡ α ‹‰ŠǦˆƒ–†‹‡–ǡ ƒα ƒ•–‡†ǡ ‡α ‡†

Supplementary table ST12: Risk ratios of the ranked metabolic gene sets in ST11 Gene set with upregulated genes LF Fa HF Fa LF Fe HF Fe ȋȌȋ Ȍƒ‹ ͳǤ͹ ͳǤͲ ͲǤ͹ ͳǤͲ ȋȌȋ Ȍ”‡†‘š ͳǤͺ ͳǤʹ ͲǤͺ ͲǤ͸ —”‹‡‡–ƒ„‘Ž‹• ͳǤ͹ ͲǤͲ ͳǤͺ ͳǤͲ ‘‰‡‡•‡– ͳǤ͹ ͳǤͳ ͲǤͻ ͲǤͻ ‡–‹‘Ž‡–ƒ„‘Ž‹• ͲǤͲ ͶǤͶ ͳǤͲ ͳǤͶ –‡”‘‹†Š‘”‘‡„‹‘•›–Š‡•‹• ͲǤͶ ͲǤͻ ͳǤ͸ ͳǤͶ Ž› ‡”‘Ž‹’‹†‡–ƒ„‘Ž‹• ʹǤͶ ͳǤ͵ ͲǤͲ ʹǤʹ ”— –‘•‡ƒ†ƒ‘•‡‡–ƒ„‘Ž‹• ͵ǤͲ ʹǤʹ ʹǤ͹ ͵Ǥ͹ ƒŽƒ –‘•‡‡–ƒ„‘Ž‹• ͵Ǥ͵ ʹǤͶ ͲǤͲ ͶǤͳ ›”‹‹†‹‡‡–ƒ„‘Ž‹• ͲǤ͸ ͲǤͲ ͳǤ͵ ͳǤʹ Ž› ‘Ž›•‹•Ȁ Ž— ‘‡‘‰‡‡•‹• ͳǤͲ ͳǤͳ ͲǤ͹ ͳǤͻ ’Š‹‰‘Ž‹’‹†‡–ƒ„‘Ž‹• ͳǤͶ ͵Ǥʹ ͳǤͻ ͲǤͲ ƒ––›ƒ ‹††‡‰”ƒ†ƒ–‹‘ ʹǤʹ ͶǤͻ ͲǤͲ ͳǤ͵ ‡–‘•‡ƒ†‰Ž— —”‘ƒ–‡‹–‡” ‘˜‡”•‹‘• ͷǤʹ ͲǤͲ ʹǤ͸ ͳǤͺ š‹†ƒ–‹˜‡’Š‘•’Š‘”›Žƒ–‹‘ ʹǤͳ ͲǤ͸ ͲǤ͵ ͲǤͷ ”ƒ Š‹†‘‹ ƒ ‹†‡–ƒ„‘Ž‹• ͲǤͲ ʹǤ͸ ͳǤͲ ͲǤ͹ ‹‘•›–Š‡•‹•‘ˆƒ‹‘ƒ ‹†• ͲǤͶ ͳǤͲ ͳǤʹ ͲǤͺ Ž› ‡”‘’Š‘•’Š‘Ž‹’‹†‡–ƒ„‘Ž‹• ͲǤ͵ ͲǤͺ ͲǤͷ ͳǤ͵ ƒ”„‘‡–ƒ„‘Ž‹• ͲǤ͵ ͲǤ͸ ͲǤͶ ͳǤͳ ‘•‹–‘Ž’Š‘•’Šƒ–‡‡–ƒ„‘Ž‹• ͳǤͲ ʹǤʹ ͲǤ͸ ͲǤͲ ”‰‹‹‡ƒ†’”‘Ž‹‡‡–ƒ„‘Ž‹• ͲǤͲ ͳǤͷ ͲǤͻ ͳǤ͵ ‹‘•—‰ƒ”ƒ†— Ž‡‘–‹†‡•—‰ƒ”‡–ƒ„‘Ž‹• ͲǤ͹ ͲǤͲ ͶǤͲ ͳǤ͵ „‡–ƒǦŽƒ‹‡‡–ƒ„‘Ž‹• ͳǤͲ ͲǤͲ ͳǤͶ ʹǤͲ ‹‘Ž‡‹ ƒ ‹†‡–ƒ„‘Ž‹• ͲǤͲ ͳǤͷ ͳǤͻ ͲǤͲ ƒ––›ƒ ‹†‡Ž‘‰ƒ–‹‘ ʹǤ͸ ͲǤͲ ͲǤͲ ʹǤͶ ›•–‡‹‡ƒ†‡–Š‹‘‹‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͵ǤͲ ͳǤ͵ ‹•–‹†‹‡‡–ƒ„‘Ž‹• ͲǤͲ ͵Ǥͳ ͲǤͲ ʹǤ͸

ͳͳͻ Chapter 3

Gene set with upregulated genes - continued LF Fa HF Fa LF Fe HF Fe Š‡›ŽƒŽƒ‹‡‡–ƒ„‘Ž‹• ͶǤͻ ͲǤͲ ͲǤͲ ʹǤͺ –Š‡”‰Ž› ƒ†‡‰”ƒ†ƒ–‹‘ ͲǤͲ ͶǤ͵ ͲǤͲ ͵Ǥ͸ Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•Ǧ‰ƒ‰Ž‹‘•‡”‹‡• ͲǤͲ ͷǤ͵ ͲǤͲ ͶǤͶ ‹‘•›–Š‡•‹•‘ˆ—•ƒ–—”ƒ–‡†ˆƒ––›ƒ ‹†• ʹǤͷ ͲǤͲ ͲǤͲ ʹǤ͵ ƒ––›ƒ ‹†‡–ƒ„‘Ž‹• ʹǤͲ ͲǤͲ ͲǤͲ ͳǤʹ ›•‹‡†‡‰”ƒ†ƒ–‹‘ ͲǤͲ ͲǤͲ ͲǤͻ ͳǤʹ Ǧ Ž› ƒ„‹‘•›–Š‡•‹• ͲǤͲ ͳǤͷ ͲǤͻ ͲǤͲ — ‹–›’‡Ǧ Ž› ƒ„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͳǤ͹ ͶǤ͹ –Š‡”Ž‹’‹†‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͳǤͲ ͳǤͶ Ž› ‹‡ǡ•‡”‹‡ƒ†–Š”‡‘‹‡‡–ƒ„‘Ž‹• ͲǤͺ ͲǤͲ ͳǤͳ ͲǤͲ ƒ—”‹‡ƒ†Š›’‘–ƒ—”‹‡‡–ƒ„‘Ž‹• ͵Ǥʹ ͲǤͲ ͶǤͷ ͲǤͲ ‡Ž‡‘ ‘’‘—†‡–ƒ„‘Ž‹• ʹǤͲ ͲǤͲ ͲǤͲ ͵Ǥͺ Ž—–ƒ–Š‹‘‡‡–ƒ„‘Ž‹• ͲǤ͸ ͲǤͲ ʹǤͷ ͲǤͲ Ž› ‘•ƒ‹‘‰Ž› ƒ„‹‘•›–Š‡•‹•Ǧ Š‘†”‘‹–‹•—Žˆƒ–‡Ȁ†‡”ƒ–ƒ•—Žˆƒ–‡ ͳǤ͹ ͲǤͲ ʹǤ͵ ͲǤͲ Ž› ‘•›Ž’Š‘•’Šƒ–‹†›Ž‹‘•‹–‘Žȋ  ȌǦƒ Š‘”„‹‘•›–Š‡•‹• ͳǤ͵ ͲǤͲ ͳǤͻ ͲǤͲ ‹ ‘–‹ƒ–‡ƒ†‹ ‘–‹ƒ‹†‡‡–ƒ„‘Ž‹• ͳǤͲ ͲǤͲ ͲǤͲ ͳǤͺ ‡–ƒ„‘Ž‹•‘ˆš‡‘„‹‘–‹ •„› ›–‘ Š”‘‡ͶͷͲ ͲǤͷ ͲǤͲ ͳǤͶ ͲǤͲ ”—‰‡–ƒ„‘Ž‹•Ǧ ›–‘ Š”‘‡ͶͷͲ ͲǤͷ ͲǤͲ ͲǤ͹ ͲǤͲ ‹–”ƒ–‡ › Ž‡ȋ › Ž‡Ȍ ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ‡–‘•‡’Š‘•’Šƒ–‡’ƒ–Š™ƒ› ͲǤͲ ʹǤͶ ͲǤͲ ͲǤͲ • ‘”„ƒ–‡ƒ†ƒŽ†ƒ”ƒ–‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ƒ––›ƒ ‹†„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ›–Š‡•‹•ƒ††‡‰”ƒ†ƒ–‹‘‘ˆ‡–‘‡„‘†‹‡• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ –‡”‘‹†„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ʹǤͷ ͲǤͲ ”‹ƒ”›„‹Ž‡ƒ ‹†„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ „‹“—‹‘‡ƒ†‘–Š‡”–‡”’‡‘‹†Ǧ“—‹‘‡„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͶǤͷ ͲǤͲ ƒˆˆ‡‹‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ Žƒ‹‡ǡƒ•’ƒ”–ƒ–‡ƒ†‰Ž—–ƒƒ–‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ʹǤͷ ͲǤͲ ƒŽ‹‡ǡŽ‡— ‹‡ƒ†‹•‘Ž‡— ‹‡†‡‰”ƒ†ƒ–‹‘ ͳǤʹ ͲǤͲ ͲǤͲ ͲǤͲ ƒŽ‹‡ǡŽ‡— ‹‡ƒ†‹•‘Ž‡— ‹‡„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ›•‹‡„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ›”‘•‹‡‡–ƒ„‘Ž‹• ͳǤͺ ͲǤͲ ͲǤͲ ͲǤͲ ”›’–‘’Šƒ‡–ƒ„‘Ž‹• ͲǤͲ ͳǤ͸ ͲǤͲ ͲǤͲ Š‡›ŽƒŽƒ‹‡ǡ–›”‘•‹‡ƒ†–”›’–‘’Šƒ„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ Ǧ Ž—–ƒ‹‡ƒ†Ǧ‰Ž—–ƒƒ–‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ Ǧ”‰‹‹‡ƒ†Ǧ‘”‹–Š‹‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ –ƒ” Šƒ†•— ”‘•‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ʹǤͶ –Š‡”–›’‡•‘ˆǦ‰Ž› ƒ„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͳǤͷ ͲǤͲ —–‹”‘•‹ƒ†‡‘› ‹„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͲǤͲ ͳͷǤ͵ Ž› ‘•ƒ‹‘‰Ž› ƒ†‡‰”ƒ†ƒ–‹‘ ͲǤͲ ͵Ǥ͹ ͲǤͲ ͲǤͲ Ž› ‘•ƒ‹‘‰Ž› ƒ„‹‘•›–Š‡•‹•Ǧ‡”ƒ–ƒ•—Žˆƒ–‡ ͲǤͲ ͲǤͲ ͲǤͲ ͶǤͶ Ž› ‘•ƒ‹‘‰Ž› ƒ„‹‘•›–Š‡•‹•ǦŠ‡’ƒ”ƒ•—Žˆƒ–‡ȀŠ‡’ƒ”‹ ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ƒŽ’ŠƒǦ‹‘Ž‡‹ ƒ ‹†‡–ƒ„‘Ž‹• ͳǤ͵ ͲǤͲ ͲǤͲ ͲǤͲ Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•ǦŽƒ –‘ƒ†‡‘Žƒ –‘•‡”‹‡• ͲǤͲ ͸Ǥͳ ͲǤͲ ͲǤͲ Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•Ǧ‰Ž‘„‘•‡”‹‡• ͲǤͲ ͲǤͲ ͲǤͲ ͶǤͶ ›”—˜ƒ–‡‡–ƒ„‘Ž‹• ͲǤͻ ͲǤͲ ͲǤͲ ͲǤͲ Ž›‘š›Žƒ–‡ƒ††‹ ƒ”„‘š›Žƒ–‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ”‘’ƒ‘ƒ–‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ —–ƒ‘ƒ–‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ʹǤͶ ‡ ƒ”„‘’‘‘Ž„›ˆ‘Žƒ–‡ ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ Š‹ƒ‹‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ‹„‘ˆŽƒ˜‹‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ‹–ƒ‹͸‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ƒ–‘–Š‡ƒ–‡ƒ†‘„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ‹‘–‹‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ‹’‘‹ ƒ ‹†‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ‘Žƒ–‡„‹‘•›–Š‡•‹• ʹǤ͹ ͲǤͲ ͲǤͲ ͲǤͲ ‘”’Š›”‹ƒ† ŠŽ‘”‘’Š›ŽŽ‡–ƒ„‘Ž‹• ͵Ǥͷ ͲǤͲ ͲǤͲ ͲǤͲ ‡”’‡‘‹†„ƒ „‘‡„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ‹–”‘‰‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ —Žˆ—”‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͸Ǥͳ ”—‰‡–ƒ„‘Ž‹•Ǧ‘–Š‡”‡œ›‡• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ʹǦš‘ ƒ”„‘š›Ž‹ ƒ ‹†‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ

Gene set with downregulated genes LF Fa HF Fa LF Fe HF Fe ȋȌȋ Ȍƒ‹ ͲǤ͸ ͳǤͳ ͳǤͲ ͳǤ͸ ȋȌȋ Ȍ”‡†‘š ͲǤͷ ͳǤʹ ͲǤͺ ͳǤͺ ‘‰‡‡•‡– ͳǤͻ ͲǤͻ ͲǤͻ ͳǤͶ ”ƒ Š‹†‘‹ ƒ ‹†‡–ƒ„‘Ž‹• ͲǤͻ ʹǤ͸ ͳǤͷ ʹǤͳ

ͳʹͲ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

Gene set with downregulated genes LF Fa HF Fa LF Fe HF Fe —”‹‡‡–ƒ„‘Ž‹• ʹǤͶ ͲǤͻ ͲǤͷ ͳǤͲ ›”—˜ƒ–‡‡–ƒ„‘Ž‹• ͵Ǥͷ ͳǤͷ ͵Ǥ͸ ͷǤͳ ‘•‹–‘Ž’Š‘•’Šƒ–‡‡–ƒ„‘Ž‹• ͵Ǥͳ ͲǤͲ ͵Ǥ͵ ͳǤ͵ Ž› ‡”‘’Š‘•’Š‘Ž‹’‹†‡–ƒ„‘Ž‹• ͲǤͻ ͳǤͺ ͳǤͶ ͲǤͲ Žƒ‹‡ǡƒ•’ƒ”–ƒ–‡ƒ†‰Ž—–ƒƒ–‡‡–ƒ„‘Ž‹• ʹǤ͵ ͲǤͲ ʹǤͶ ͷǤ͵ ƒ––›ƒ ‹†‡–ƒ„‘Ž‹• ͲǤͲ ͳǤͳ ͵Ǥ͸ ͵Ǥͺ Ž› ‘Ž›•‹•Ȁ Ž— ‘‡‘‰‡‡•‹• ͳǤ͵ ͳǤ͹ ͳǤ͵ ͳǤͶ ‹‘Ž‡‹ ƒ ‹†‡–ƒ„‘Ž‹• ͲǤͲ ͶǤͺ ͳǤͺ ͳǤͻ 3 ƒ”„‘‡–ƒ„‘Ž‹• ͲǤ͵ ͳǤͷ ͲǤ͹ ͲǤͺ Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•ǦŽƒ –‘ƒ†‡‘Žƒ –‘•‡”‹‡• ͲǤͲ ͶǤ͸ ͵Ǥͷ ͵Ǥ͹ ƒ––›ƒ ‹††‡‰”ƒ†ƒ–‹‘ ͲǤͻ ͲǤͲ ͳǤͺ ͶǤͲ ”—‰‡–ƒ„‘Ž‹•Ǧ ›–‘ Š”‘‡ͶͷͲ ͲǤ͸ ʹǤ͸ ͳǤ͵ ͲǤͲ ’Š‹‰‘Ž‹’‹†‡–ƒ„‘Ž‹• ͲǤͲ ͳǤʹ ͳǤͺ ʹǤͲ ƒŽ‹‡ǡŽ‡— ‹‡ƒ†‹•‘Ž‡— ‹‡†‡‰”ƒ†ƒ–‹‘ ͲǤͺ ͳǤͲ ͳǤ͸ ͲǤͲ ”‰‹‹‡ƒ†’”‘Ž‹‡‡–ƒ„‘Ž‹• ͲǤͺ ͳǤͳ ͳǤͺ ͲǤͲ ‹•–‹†‹‡‡–ƒ„‘Ž‹• ͳǤ͹ ʹǤ͵ ͷǤͺ ͲǤͲ –ƒ” Šƒ†•— ”‘•‡‡–ƒ„‘Ž‹• ͲǤͺ ͳǤͲ ͳǤ͸ ͲǤͲ –Š‡”Ž‹’‹†‡–ƒ„‘Ž‹• ͲǤͻ ͳǤ͵ ʹǤͲ ͲǤͲ ”—‰‡–ƒ„‘Ž‹•Ǧ‘–Š‡”‡œ›‡• ͲǤͺ ͳǤͳ ͲǤͺ ͳǤͺ –‡”‘‹†Š‘”‘‡„‹‘•›–Š‡•‹• ͲǤͲ ʹǤ͹ ͲǤͷ ʹǤʹ ‡–‹‘Ž‡–ƒ„‘Ž‹• ͲǤͲ ͳǤͻ ͲǤͷ ʹǤͳ Ž—–ƒ–Š‹‘‡‡–ƒ„‘Ž‹• ͲǤ͹ ʹǤͲ ͲǤͺ ͲǤͲ ‘”’Š›”‹ƒ† ŠŽ‘”‘’Š›ŽŽ‡–ƒ„‘Ž‹• ͳǤͲ ͲǤͲ ͳǤͳ ʹǤ͵ ‡–ƒ„‘Ž‹•‘ˆš‡‘„‹‘–‹ •„› ›–‘ Š”‘‡ͶͷͲ ͲǤ͸ ͵Ǥ͸ ͲǤ͹ ͲǤͲ š‹†ƒ–‹˜‡’Š‘•’Š‘”›Žƒ–‹‘ ͲǤͲ ͲǤͶ ͲǤ͵ ͲǤ͹ „‡–ƒǦŽƒ‹‡‡–ƒ„‘Ž‹• ͳǤ͵ ͳǤͺ ͳǤͶ ͲǤͲ ›•‹‡†‡‰”ƒ†ƒ–‹‘ ʹǤͶ ͲǤͲ ʹǤͷ ͲǤͲ ›”‹‹†‹‡‡–ƒ„‘Ž‹• ͳǤ͸ ͳǤ͹ ͲǤͲ ͲǤͲ ‹‘•—‰ƒ”ƒ†— Ž‡‘–‹†‡•—‰ƒ”‡–ƒ„‘Ž‹• ͳǤ͹ ͲǤͲ ͳǤͺ ͲǤͲ ƒ––›ƒ ‹†„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͹Ǥͳ ͹Ǥͳ ‹‘•›–Š‡•‹•‘ˆ—•ƒ–—”ƒ–‡†ˆƒ––›ƒ ‹†• ͲǤͲ ͲǤͲ ͵Ǥ͵ ͵ǤͶ ‡–‘•‡ƒ†‰Ž— —”‘ƒ–‡‹–‡” ‘˜‡”•‹‘• ͳǤʹ ͲǤͲ ʹǤͷ ͲǤͲ ƒŽƒ –‘•‡‡–ƒ„‘Ž‹• ͳǤ͵ ͲǤͲ ʹǤͺ ͲǤͲ • ‘”„ƒ–‡ƒ†ƒŽ†ƒ”ƒ–‡‡–ƒ„‘Ž‹• ͳǤ͸ ͲǤͲ ͵ǤͶ ͲǤͲ ”›’–‘’Šƒ‡–ƒ„‘Ž‹• ͲǤͻ ͲǤͲ ͳǤͻ ͲǤͲ Ž› ‡”‘Ž‹’‹†‡–ƒ„‘Ž‹• ͲǤ͹ ͲǤͲ ͳǤͷ ͲǤͲ ›•–‡‹‡ƒ†‡–Š‹‘‹‡‡–ƒ„‘Ž‹• ͲǤͲ ͳǤʹ ͲǤͲ ʹǤͲ ƒŽ’ŠƒǦ‹‘Ž‡‹ ƒ ‹†‡–ƒ„‘Ž‹• ͲǤͲ ʹǤ͵ ͵Ǥ͹ ͲǤͲ ”‘’ƒ‘ƒ–‡‡–ƒ„‘Ž‹• ͲǤͲ ͳǤͻ ͵ǤͲ ͲǤͲ ‡ ƒ”„‘’‘‘Ž„›ˆ‘Žƒ–‡ ͲǤͲ ͵Ǥͳ ͲǤͲ ͷǤͳ ”— –‘•‡ƒ†ƒ‘•‡‡–ƒ„‘Ž‹• ͵Ǥͺ ͲǤͲ ͳǤʹ ͲǤͲ ƒ––›ƒ ‹†‡Ž‘‰ƒ–‹‘ ͲǤͲ ʹǤͳ ͳǤ͸ ͲǤͲ ”‹ƒ”›„‹Ž‡ƒ ‹†„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ʹǤͺ ͸Ǥʹ Ž› ‹‡ǡ•‡”‹‡ƒ†–Š”‡‘‹‡‡–ƒ„‘Ž‹• ͲǤͲ ͳǤͶ ͳǤͳ ͲǤͲ –Š‡”–›’‡•‘ˆǦ‰Ž› ƒ„‹‘•›–Š‡•‹• ʹǤͺ ͲǤͲ ͳǤͶ ͲǤͲ —–‹”‘•‹ƒ†‡‘› ‹„‹‘•›–Š‡•‹• ͳͲǤͳ ͲǤͲ ͳͲǤ͸ ͲǤͲ —–ƒ‘ƒ–‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͳǤ͸ ͵Ǥ͸ ‹ ‘–‹ƒ–‡ƒ†‹ ‘–‹ƒ‹†‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͳǤʹ ʹǤ͹ ‡”’‡‘‹†„ƒ „‘‡„‹‘•›–Š‡•‹• ͲǤͲ ʹǤͷ ͳǤͻ ͲǤͲ ‹–”ƒ–‡ › Ž‡ȋ › Ž‡Ȍ ͲǤͲ ͲǤͲ ͲǤͲ ͵ǤͲ ‡–‘•‡’Š‘•’Šƒ–‡’ƒ–Š™ƒ› ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ›–Š‡•‹•ƒ††‡‰”ƒ†ƒ–‹‘‘ˆ‡–‘‡„‘†‹‡• ͲǤͲ ͲǤͲ ͶǤʹ ͲǤͲ –‡”‘‹†„‹‘•›–Š‡•‹• ͲǤͲ ͵Ǥͳ ͲǤͲ ͲǤͲ „‹“—‹‘‡ƒ†‘–Š‡”–‡”’‡‘‹†Ǧ“—‹‘‡„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ƒˆˆ‡‹‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͳͺǤͷ ƒŽ‹‡ǡŽ‡— ‹‡ƒ†‹•‘Ž‡— ‹‡„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ›•‹‡„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ›”‘•‹‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͳǤͳ ͲǤͲ Š‡›ŽƒŽƒ‹‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͳǤͻ ͲǤͲ Š‡›ŽƒŽƒ‹‡ǡ–›”‘•‹‡ƒ†–”›’–‘’Šƒ„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ƒ—”‹‡ƒ†Š›’‘–ƒ—”‹‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ‡Ž‡‘ ‘’‘—†‡–ƒ„‘Ž‹• ͲǤͲ ͵Ǥͷ ͲǤͲ ͲǤͲ Ǧ Ž—–ƒ‹‡ƒ†Ǧ‰Ž—–ƒƒ–‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ Ǧ”‰‹‹‡ƒ†Ǧ‘”‹–Š‹‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ Ǧ Ž› ƒ„‹‘•›–Š‡•‹• ͳǤ͹ ͲǤͲ ͲǤͲ ͲǤͲ –Š‡”‰Ž› ƒ†‡‰”ƒ†ƒ–‹‘ ͲǤͲ ͲǤͲ ͷǤ͵ ͲǤͲ — ‹–›’‡Ǧ Ž› ƒ„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ Ž› ‘•ƒ‹‘‰Ž› ƒ†‡‰”ƒ†ƒ–‹‘ ͲǤͲ ͲǤͲ ʹǤͳ ͲǤͲ Ž› ‘•ƒ‹‘‰Ž› ƒ„‹‘•›–Š‡•‹•Ǧ Š‘†”‘‹–‹•—Žˆƒ–‡Ȁ†‡”ƒ–ƒ•—Žˆƒ–‡ ͲǤͲ ʹǤͻ ͲǤͲ ͲǤͲ

ͳʹͳ Chapter 3

Gene set with downregulated genes - continued LF Fa HF Fa LF Fe HF Fe Ž› ‘•ƒ‹‘‰Ž› ƒ„‹‘•›–Š‡•‹•Ǧ‡”ƒ–ƒ•—Žˆƒ–‡ ͲǤͲ ͶǤͲ ͲǤͲ ͲǤͲ Ž› ‘•ƒ‹‘‰Ž› ƒ„‹‘•›–Š‡•‹•ǦŠ‡’ƒ”ƒ•—Žˆƒ–‡ȀŠ‡’ƒ”‹ ͲǤͲ ͲǤͲ ͳǤͺ ͲǤͲ Ž› ‘•›Ž’Š‘•’Šƒ–‹†›Ž‹‘•‹–‘Žȋ  ȌǦƒ Š‘”„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͲǤͲ ͵Ǥͻ Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•Ǧ‰Ž‘„‘•‡”‹‡• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•Ǧ‰ƒ‰Ž‹‘•‡”‹‡• ͲǤͲ ͲǤͲ ͸Ǥͷ ͲǤͲ Ž›‘š›Žƒ–‡ƒ††‹ ƒ”„‘š›Žƒ–‡‡–ƒ„‘Ž‹• ͲǤͲ ʹǤͲ ͲǤͲ ͲǤͲ Š‹ƒ‹‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ‹„‘ˆŽƒ˜‹‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ‹–ƒ‹͸‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ƒ–‘–Š‡ƒ–‡ƒ†‘„‹‘•›–Š‡•‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ‹‘–‹‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ‹’‘‹ ƒ ‹†‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ‘Žƒ–‡„‹‘•›–Š‡•‹• ͲǤͲ ͶǤ͸ ͲǤͲ ͲǤͲ ‹–”‘‰‡‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ —Žˆ—”‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ʹǦš‘ ƒ”„‘š›Ž‹ ƒ ‹†‡–ƒ„‘Ž‹• ͲǤͲ ͲǤͲ ͲǤͲ ͲǤͲ ‹‘•›–Š‡•‹•‘ˆƒ‹‘ƒ ‹†• ͲǤͲ ʹǤʹ ͲǤͲ ͲǤͲ Š‡”‡•—Ž–•’”‡•‡–‡†Š‡”‡ƒ”‡ˆ”‘ ‘’ƒ”‹•‘•„‡–™‡‡ƒ†Ǥ α‘™Ǧˆƒ–†‹‡–ǡ α ‹‰ŠǦˆƒ–†‹‡–ǡ ƒα ƒ•–‡†ǡ ‡α ‡†  Supplementary table ST13: (Differentially-expressed) metabolic gene sets ranked by their CoA/NAD(P)(H) gene content CoA/NAD(P)(H) gene HF Fa LF Fa Top 10 gene Gene set content (%) FDR<0.25 FDR<0.25 sets ̴ ̴ ͳͲͲ   ‡•  ̴  ͳͲͲ ‡•  ‡•  ̴ ͳͲͲ ‡•  ‡• ”‹ƒ”›„‹Ž‡ƒ ‹†„‹‘•›–Š‡•‹• ͺͳ    –‡”‘‹†„‹‘•›–Š‡•‹• ͹ͻ ‡•  ‡• ƒ––›ƒ ‹†‡Ž‘‰ƒ–‹‘ ͹ͺ    ƒ––›ƒ ‹†„‹‘•›–Š‡•‹• ͹ͳ  ‡•  ƒ––›ƒ ‹††‡‰”ƒ†ƒ–‹‘ ͸ͻ    ƒˆˆ‡‹‡‡–ƒ„‘Ž‹• ͸͹    ”‘’ƒ‘ƒ–‡‡–ƒ„‘Ž‹• ͸͹    ›”—˜ƒ–‡‡–ƒ„‘Ž‹• ͸͸  ‡• ‡• ƒ––›ƒ ‹†‡–ƒ„‘Ž‹• ͸͵  ‡• ‡• ‹–”ƒ–‡ › Ž‡ȋ › Ž‡Ȍ ͸͵  ‡•  –‡”‘‹†Š‘”‘‡„‹‘•›–Š‡•‹• ͸ͳ    ‡–‹‘Ž‡–ƒ„‘Ž‹• ͸Ͳ   ‡• —–ƒ‘ƒ–‡‡–ƒ„‘Ž‹• ͷͻ    ›–Š‡•‹•ƒ††‡‰”ƒ†ƒ–‹‘‘ˆ‡–‘‡„‘†‹‡• ͷͷ  ‡•  ‹‘•›–Š‡•‹•‘ˆ—•ƒ–—”ƒ–‡†ˆƒ––›ƒ ‹†• ͷͶ  ‡•  ›•‹‡„‹‘•›–Š‡•‹• ͷͲ    ‹„‘ˆŽƒ˜‹‡–ƒ„‘Ž‹• ͷͲ    ƒŽ‹‡ǡŽ‡— ‹‡ƒ†‹•‘Ž‡— ‹‡†‡‰”ƒ†ƒ–‹‘ Ͷ͹    „‡–ƒǦŽƒ‹‡‡–ƒ„‘Ž‹• Ͷ͹    Ž› ‡”‘Ž‹’‹†‡–ƒ„‘Ž‹• ͶͶ   ‡• Ž› ‘Ž›•‹•Ȁ Ž— ‘‡‘‰‡‡•‹• Ͷʹ    ‹‘Ž‡‹ ƒ ‹†‡–ƒ„‘Ž‹• Ͷʹ    ‹•–‹†‹‡‡–ƒ„‘Ž‹• ͶͲ  ‡•  ”›’–‘’Šƒ‡–ƒ„‘Ž‹• ͵͹    ʹǦš‘ ƒ”„‘š›Ž‹ ƒ ‹†‡–ƒ„‘Ž‹• ͵͹    ‡ ƒ”„‘’‘‘Ž„›ˆ‘Žƒ–‡ ͵͹    ‡–‘•‡ƒ†‰Ž— —”‘ƒ–‡‹–‡” ‘˜‡”•‹‘• ͵͸    ‹ ‘–‹ƒ–‡ƒ†‹ ‘–‹ƒ‹†‡‡–ƒ„‘Ž‹• ͵Ͷ    ‹‘–‹‡–ƒ„‘Ž‹• ͵͵    Ǧ Ž—–ƒ‹‡ƒ†Ǧ‰Ž—–ƒƒ–‡‡–ƒ„‘Ž‹• ͵͵    ƒ”„‘‡–ƒ„‘Ž‹• ͵ʹ    ”‰‹‹‡ƒ†’”‘Ž‹‡‡–ƒ„‘Ž‹• ͵ʹ    ‡–ƒ„‘Ž‹•‘ˆš‡‘„‹‘–‹ •„› ›–‘ Š”‘‡ͶͷͲ ʹͻ ‡• ‡•  ”ƒ Š‹†‘‹ ƒ ‹†‡–ƒ„‘Ž‹• ʹͻ   ‡• š‹†ƒ–‹˜‡’Š‘•’Š‘”›Žƒ–‹‘ ʹͺ ‡•   ƒ–‘–Š‡ƒ–‡ƒ†‘„‹‘•›–Š‡•‹• ʹͺ    Ž›‘š›Žƒ–‡ƒ††‹ ƒ”„‘š›Žƒ–‡‡–ƒ„‘Ž‹• ʹͺ    ›•‹‡†‡‰”ƒ†ƒ–‹‘ ʹ͸    Š‡›ŽƒŽƒ‹‡‡–ƒ„‘Ž‹• ʹ͸    ›”‘•‹‡‡–ƒ„‘Ž‹• ʹ͸    ”—‰‡–ƒ„‘Ž‹•Ǧ ›–‘ Š”‘‡ͶͷͲ ʹͷ ‡• ‡•  Ž› ‡”‘’Š‘•’Š‘Ž‹’‹†‡–ƒ„‘Ž‹• ʹ͵   ‡• ‘Žƒ–‡„‹‘•›–Š‡•‹• ʹ͵    • ‘”„ƒ–‡ƒ†ƒŽ†ƒ”ƒ–‡‡–ƒ„‘Ž‹• ʹʹ   

ͳʹʹ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

CoA/NAD(P)(H) gene HF Fa LF Fa Top 10 gene Gene set content (%) FDR<0.25 FDR<0.25 sets ‡”’‡‘‹†„ƒ „‘‡„‹‘•›–Š‡•‹• ʹʹ    ’Š‹‰‘Ž‹’‹†‡–ƒ„‘Ž‹• ʹͳ    Ž› ‹‡ǡ•‡”‹‡ƒ†–Š”‡‘‹‡‡–ƒ„‘Ž‹• ʹͲ    „‹“—‹‘‡ƒ†‘–Š‡”–‡”’‡‘‹†Ǧ“—‹‘‡„‹‘•›–Š‡•‹• ͳͺ    ‹‘•›–Š‡•‹•‘ˆƒ‹‘ƒ ‹†• ͳͺ    ‡Ž‡‘ ‘’‘—†‡–ƒ„‘Ž‹• ͳͺ    ”— –‘•‡ƒ†ƒ‘•‡‡–ƒ„‘Ž‹• ͳ͹   ‡• ‹‘•—‰ƒ”ƒ†— Ž‡‘–‹†‡•—‰ƒ”‡–ƒ„‘Ž‹• ͳ͸    3 ›•–‡‹‡ƒ†‡–Š‹‘‹‡‡–ƒ„‘Ž‹• ͳ͵    ƒŽƒ –‘•‡‡–ƒ„‘Ž‹• ͳ͵   ‡• ”—‰‡–ƒ„‘Ž‹•Ǧ‘–Š‡”‡œ›‡• ͳʹ    Žƒ‹‡ǡƒ•’ƒ”–ƒ–‡ƒ†‰Ž—–ƒƒ–‡‡–ƒ„‘Ž‹• ͳͳ   ‡• ‘”’Š›”‹ƒ† ŠŽ‘”‘’Š›ŽŽ‡–ƒ„‘Ž‹• ͳͲ    ‡–‘•‡’Š‘•’Šƒ–‡’ƒ–Š™ƒ› ͻ    ƒ—”‹‡ƒ†Š›’‘–ƒ—”‹‡‡–ƒ„‘Ž‹• ͻ ‡• ‡•  Ž—–ƒ–Š‹‘‡‡–ƒ„‘Ž‹• ͻ ‡•   ƒŽ’ŠƒǦ‹‘Ž‡‹ ƒ ‹†‡–ƒ„‘Ž‹• ͺ    ‘•‹–‘Ž’Š‘•’Šƒ–‡‡–ƒ„‘Ž‹• ͹   ‡• –Š‡”Ž‹’‹†‡–ƒ„‘Ž‹• ͹    Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•Ǧ‰ƒ‰Ž‹‘•‡”‹‡• ͹    ‹–”‘‰‡‡–ƒ„‘Ž‹• ͸    –ƒ” Šƒ†•— ”‘•‡‡–ƒ„‘Ž‹• ͸    Ž› ‘•ƒ‹‘‰Ž› ƒ†‡‰”ƒ†ƒ–‹‘ ͷ    Ž› ‘•›Ž’Š‘•’Šƒ–‹†›Ž‹‘•‹–‘Žȋ  ȌǦƒ Š‘”„‹‘•›–Š‡•‹• Ͷ    —”‹‡‡–ƒ„‘Ž‹• Ͷ   ‡• ›”‹‹†‹‡‡–ƒ„‘Ž‹• Ͷ   ‡• —–‹”‘•‹ƒ†‡‘› ‹„‹‘•›–Š‡•‹• Ͳ    Ǧ”‰‹‹‡ƒ†Ǧ‘”‹–Š‹‡‡–ƒ„‘Ž‹• Ͳ    Ž› ‘•ƒ‹‘‰Ž› ƒ„‹‘•›–Š‡•‹•Ǧ Š‘†”‘‹–‹•—Žˆƒ–‡Ȁ †‡”ƒ–ƒ•—Žˆƒ–‡ Ͳ    Ž› ‘•ƒ‹‘‰Ž› ƒ„‹‘•›–Š‡•‹•ǦŠ‡’ƒ”ƒ•—Žˆƒ–‡Ȁ Š‡’ƒ”‹ Ͳ    Ž› ‘•ƒ‹‘‰Ž› ƒ„‹‘•›–Š‡•‹•Ǧ‡”ƒ–ƒ•—Žˆƒ–‡ Ͳ    Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•Ǧ‰Ž‘„‘•‡”‹‡• Ͳ    Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•ǦŽƒ –‘ƒ†‡‘Žƒ –‘ •‡”‹‡• Ͳ  ‡•  ‹’‘‹ ƒ ‹†‡–ƒ„‘Ž‹• Ͳ    — ‹–›’‡Ǧ Ž› ƒ„‹‘•›–Š‡•‹• Ͳ    Ǧ Ž› ƒ„‹‘•›–Š‡•‹• Ͳ    –Š‡”‰Ž› ƒ†‡‰”ƒ†ƒ–‹‘ Ͳ    –Š‡”–›’‡•‘ˆǦ‰Ž› ƒ„‹‘•›–Š‡•‹• Ͳ  ‡•  Š‡›ŽƒŽƒ‹‡ǡ–›”‘•‹‡ƒ†–”›’–‘’Šƒ„‹‘•›–Š‡•‹• Ͳ    —Žˆ—”‡–ƒ„‘Ž‹• Ͳ  ‡•  Š‹ƒ‹‡‡–ƒ„‘Ž‹• Ͳ    ƒŽ‹‡ǡŽ‡— ‹‡ƒ†‹•‘Ž‡— ‹‡„‹‘•›–Š‡•‹• Ͳ    ‹–ƒ‹͸‡–ƒ„‘Ž‹• Ͳ    ƒ—”‹‡ƒ†Š›’‘–ƒ—”‹‡‡–ƒ„‘Ž‹• ͻ ‡• ‡•  Ž—–ƒ–Š‹‘‡‡–ƒ„‘Ž‹• ͻ ‡•   ƒŽ’ŠƒǦ‹‘Ž‡‹ ƒ ‹†‡–ƒ„‘Ž‹• ͺ    ‘•‹–‘Ž’Š‘•’Šƒ–‡‡–ƒ„‘Ž‹• ͹   ‡• –Š‡”Ž‹’‹†‡–ƒ„‘Ž‹• ͹    Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•Ǧ‰ƒ‰Ž‹‘•‡”‹‡• ͹    ‹–”‘‰‡‡–ƒ„‘Ž‹• ͸    –ƒ” Šƒ†•— ”‘•‡‡–ƒ„‘Ž‹• ͸    Ž› ‘•ƒ‹‘‰Ž› ƒ†‡‰”ƒ†ƒ–‹‘ ͷ    Ž› ‘•›Ž’Š‘•’Šƒ–‹†›Ž‹‘•‹–‘Žȋ  ȌǦƒ Š‘”„‹‘•›–Š‡•‹• Ͷ    —”‹‡‡–ƒ„‘Ž‹• Ͷ   ‡• ›”‹‹†‹‡‡–ƒ„‘Ž‹• Ͷ   ‡• —–‹”‘•‹ƒ†‡‘› ‹„‹‘•›–Š‡•‹• Ͳ    Ǧ”‰‹‹‡ƒ†Ǧ‘”‹–Š‹‡‡–ƒ„‘Ž‹• Ͳ    Ž› ‘•ƒ‹‘‰Ž› ƒ„‹‘•›–Š‡•‹•Ǧ Š‘†”‘‹–‹•—Žˆƒ–‡Ȁ †‡”ƒ–ƒ•—Žˆƒ–‡ Ͳ    Ž› ‘•ƒ‹‘‰Ž› ƒ„‹‘•›–Š‡•‹•ǦŠ‡’ƒ”ƒ•—Žˆƒ–‡Ȁ Š‡’ƒ”‹ Ͳ    Ž› ‘•ƒ‹‘‰Ž› ƒ„‹‘•›–Š‡•‹•Ǧ‡”ƒ–ƒ•—Žˆƒ–‡ Ͳ    Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•Ǧ‰Ž‘„‘•‡”‹‡• Ͳ    Ž› ‘•’Š‹‰‘Ž‹’‹†„‹‘•›–Š‡•‹•ǦŽƒ –‘ƒ†‡‘Žƒ –‘ •‡”‹‡• Ͳ  ‡•  ‹’‘‹ ƒ ‹†‡–ƒ„‘Ž‹• Ͳ    — ‹–›’‡Ǧ Ž› ƒ„‹‘•›–Š‡•‹• Ͳ    Ǧ Ž› ƒ„‹‘•›–Š‡•‹• Ͳ    –Š‡”‰Ž› ƒ†‡‰”ƒ†ƒ–‹‘ Ͳ   

ͳʹ͵ Chapter 3

CoA/NAD(P)(H) gene HF Fa LF Fa Top 10 gene Gene set content (%) FDR<0.25 FDR<0.25 sets –Š‡”–›’‡•‘ˆǦ‰Ž› ƒ„‹‘•›–Š‡•‹• Ͳ ‡• Š‡›ŽƒŽƒ‹‡ǡ–›”‘•‹‡ƒ†–”›’–‘’Šƒ„‹‘•›–Š‡•‹• Ͳ —Žˆ—” ‡–ƒ„‘Ž‹• Ͳ ‡• Š‹ƒ‹‡‡–ƒ„‘Ž‹• Ͳ ƒŽ‹‡ǡŽ‡— ‹‡ƒ†‹•‘Ž‡— ‹‡„‹‘•›–Š‡•‹• Ͳ ‹–ƒ‹͸‡–ƒ„‘Ž‹• Ͳ ‘’ƒ”‹•‘ƒ†ƒ„„”‡˜‹ƒ–‹‘•ƒ”‡ƒ•‹—’’Ž‡‡–ƒ”›–ƒ„Ž‡ͳ͵Ǥ

A mRNA expression of 1600 WT LF fed WT LF fasted 400 KO LF fed 400 Ύ KO LF fasted 300

200

100 Normalized readcount 0

Acot1 Acot2 Acot3 Acot4 Acot5 Acot6 Acot7 Acot8 Acot9 Acot10 Acot11 Acot12 Acot13 Them4 Them5

B mRNA expression of thioesterases 700 WT HF fed 600 WT HF fasted KO HF fed 500 KO HF fasted 400 300 200 100 Normalized readcount 0

Acot1 Acot2 Acot3 Acot4 Acot5 Acot6 Acot7 Acot8 Acot9 Acot10 Acot11 Acot12 Acot13 Them4 Them5 Supplementary figure S9. Acyl-CoA thioesterases gene expression in LFD fed and LFD fasted (A) and in HFD fed and HFD fasted (B) conditions. α͵ ƒ†Ͷ ˆ‘”ƒ† ˆ‘”Š‹‰ŠǦˆƒ–†‹‡– ȋ ȌǦ ƒ†Ž‘™Ǧˆƒ– †‹‡– ȋ Ȍ ‘†‹–‹‘•ǡ”‡•’‡ –‹˜‡Ž›ǤŠ‡”‡•—Ž–•ƒ”‡”‡’”‡•‡–‡†ƒ•—‡›„‘šƒ†™Š‹•‡”’Ž‘–•ǡ™Š‡”‡–Š‡ „Žƒ Ǧˆ‹ŽŽ‡†Œƒ‰‰‡† ‹” Ž‡•‹†‹ ƒ–‡‹†‹˜‹†—ƒŽ‹ ‡ˆƒŽŽ‹‰‘—–•‹†‡–Š‡͹ͷΨ’‡” ‡–‹Ž‡’Ž—•ͳǤͷȉ‹–‡”Ǧ“—ƒ”–‹Ž‡ ”ƒ‰‡ȋ Ȍ‘”ʹͷΨ’‡” ‡–‹Ž‡‹—•ͳǤͷȉ Ǥα͸Ǧͺˆ‘”„‘–Šƒ†Ǥȗǣ’δͲǤͲͷ ‘’ƒ”‡†–‘ǡ͓ƒ† ͓͓ǣ’δͲǤͲͷƒ†’δͲǤͲͳ ‘’ƒ”‡†–‘ˆ‡†ǡ”‡•’‡ –‹˜‡Ž›ǡșƒ†șșǣ’δͲǤͲͷƒ†’δͲǤͲͳ ‘’ƒ”‡†–‘Ž‘™Ǧˆƒ–†‹‡– ȋ Ȍǡ”‡•’‡ –‹˜‡Ž›Ǥ

Supplementary Text 1. RNA isolation, RNASeq analysis and quantitative reverse transcriptase polymerase chain (qRT-PCR) ™ƒ•‹•‘Žƒ–‡†ˆ”‘ ”—•Š‡†ˆ”‡‡œ‡Ǧ Žƒ’‡†Ž‹˜‡”™‹–Š”‹œ‘Ž”‡ƒ‰‡–ȋ ˜‹–”‘‰‡ǡƒ”Ž•„ƒ†ǡǡ ȌǤŠ‡’—”‹–›ƒ† ‘ ‡–”ƒ–‹‘‘ˆ™ƒ•†‡–‡”‹‡†™‹–Šƒ‘”‘’ǦͳͲͲͲȋ  ‹ˆ‡  ‹‡ ‡ǡ‡‡‡”ǡŠ‡‡–Š‡”Žƒ†•ȌǤ—„•‡“—‡–Ž›ǡ‹–‡‰”‹–›ƒ† ‘ ‡–”ƒ–‹‘™‡”‡ †‡–‡”‹‡† „› ƒ’‹ŽŽƒ”›‡Ž‡ –”‘’Š‘”‡•‹•—•‹‰‡”‹Ž‡”ƒ„Š‹’ ȋ‡”‹Ž‡”ǡƒŽ–ŠƒǡǡȌǤ •ƒ’Ž‡•™‹–Šƒ‹–‡‰”‹–›• ‘”‡ƒ„‘˜‡͹ǡƒ•ƒŽŽͷ’‡ƒǡƒ† Ž‡ƒ”Ž›†‹•–‹‰—‹•Šƒ„Ž‡ͳͺƒ†ʹͺ’‡ƒ• ™‡”‡ —•‡† ˆ‘” •—„•‡“—‡– •‡“—‡ ‹‰ ƒƒŽ›•‹•Ǥ ‡“—‡ ‡ Ž‹„”ƒ”‹‡• ™‡”‡ ‰‡‡”ƒ–‡† —•‹‰ –Š‡ ͵ǯ—ƒ–‡“•ƒ’Ž‡’”‡’ƒ”ƒ–‹‘‹–•ȋ‡š‘‰‡ȌǤŠ‡‘„–ƒ‹‡† ˆ”ƒ‰‡–Ž‹„”ƒ”‹‡•™‡”‡•‡“—‡ ‡† ‘ƒ ŽŽ—‹ƒ ‹‡“ʹͷͲͲ•‡“—‡ ‡”—•‹‰†‡ˆƒ—Ž–’ƒ”ƒ‡–‡”•ȋ•‹‰Ž‡”‡ƒ†ͳšͷͲ„’Ȍ‹’‘‘Ž•‘ˆ‡‹‰Š– •ƒ’Ž‡•’‡”Žƒ‡ǤŠ‡‘„–ƒ‹‡†•‡“—‡ ‹‰”‡ƒ†•ȋˆƒ•–ˆ‹Ž‡•Ȍ™‡”‡ƒ’’‡†–‘„—‹Ž†—•̴—• —Ž—• ͵ͺ‡•‡„Ž‡‡Ž‡ƒ•‡ͺʹ”‡ˆ‡”‡ ‡‰‡‘‡—•‹‰Š‹•ƒ–ȀͲǤͳǤͷǦ„‡–ƒǦ‰‘‘ŽˆǦͳǤ͹ǤʹͲȏʹȐ ™‹–Š†‡ˆƒ—Ž– •‡––‹‰•Ǥ —„•‡“—‡–Ž›ǡ –‘‘Ž•ȀͳǤʹǦ ‰‘‘ŽˆǦͳǤ͹ǤʹͲ ™ƒ• —•‡† –‘ •‘”– –Š‡ ƒŽ‹‰‡† ”‡ƒ†•ȏ͵ȐǤ —ƒ–‹ˆ‹ ƒ–‹‘ ‘ˆ ‰‡‡ ”‡ƒ† ‘—–• ™ƒ• ’‡”ˆ‘”‡† „› ‡“Ǧ ‘—– ‡“ȀͲǤ͸Ǥͳ’ͳ —•‹‰ Ȃ ‘†‡α—‹‘ȏͶȐǤ ƒŽ›•‹• ‘ˆ †‹ˆˆ‡”‡–‹ƒŽ ‰‡‡ ‡š’”‡••‹‘ ȋ Ȍ ™ƒ• ’‡”ˆ‘”‡† —•‹‰ ‘‘ –”ƒ•ˆ‘”ƒ–‹‘’”‘˜‹†‡†„›–Š‡‹ƒ’ƒ ƒ‰‡ȏͷȐ ƒ†ˆ‘” ‘’ƒ”‹•‘ƒŽ•‘™‹–Š–Š‡‡“ʹ’ƒ ƒ‰‡ ȋ˜‡”•‹‘ͳǤͳͲǤͳȌ‹ȋ˜‡”•‹‘͵ǤʹǤͷȌǡƒ•‹†‹ ƒ–‡†‹–Š‡–‡š–Ǥ ”‹ ‹’Ž‡ ‘’‘‡–ƒƒŽ›•‹•ȋȌ™ƒ• ’‡”ˆ‘”‡†‘”‡‰—Žƒ”‹œ‡†Ž‘‰–”ƒ•ˆ‘”‡†”ƒ™ ‘—–•ȏ͸Ȑ —•‹‰–Š‡ˆ— –‹‘ˆ”‘–Š‡‡“ʹ

ͳʹͶ Transcriptomic analysis suggests a compensatory role of CoA and NAD+ in MCAD-KO mice

’ƒ ƒ‰‡ȋ˜‡”•‹‘ͳǤͳͲǤͳȌ‹ȋ˜‡”•‹‘͵ǤʹǤͷȌǤ ‘”Acot2‰‡‡‡š’”‡••‹‘ƒƒŽ›•‹•„›“Ǧǡ  ™ƒ••›–Š‡•‹œ‡†ˆ”‘‹•‘Žƒ–‡†Ž‹˜‡”—•‹‰”ƒ• ”‹’–‹‘‹˜‡”•ƒŽ ƒ•–‡”ȋ‘ Š‡ȌǤ ‡š’”‡••‹‘™ƒ•ƒ••ƒ›‡†„›“—ƒ–‹–ƒ–‹˜‡’‘Ž›‡”ƒ•‡ Šƒ‹”‡ƒ –‹‘—•‹‰‰”‡‡ȋ‘ Š‡Ȍƒ†–Š‡ ”‡ƒŽǦ–‹‡ ’‘Ž›‡”ƒ•‡ Šƒ‹ ”‡ƒ –‹‘ › Ž‡” ȋ—ƒ––—†‹‘ ͹ Ž‡š ‡ƒŽǦ‹‡  ›•–‡ǡ ’’Ž‹‡† ‹‘•›•–‡•Ȍ —•‹‰ –Š‡ ’”‹‡”•       ȋˆ‘”™ƒ”†Ȍ ƒ†    ȋ”‡˜‡”•‡Ȍ ȋŠ‡”‘• ‹‡–‹ˆ‹  ˜‹–”‘‰‡Ȍȏ͹ȐǤ Acot2 ‰‡‡ ‡š’”‡••‹‘ ™ƒ• ‘”ƒŽ‹œ‡† ƒ‰ƒ‹•– ’‡’–‹†›ŽǦ’”‘Ž›ŽǦ ‹•Ǧ–”ƒ• ‹•‘‡”ƒ•‡  ȋPpiaȌ —•‹‰ –Š‡ ’”‹‡”•   ȋˆ‘”™ƒ”†Ȍ ƒ†      ȋ”‡˜‡”•‡Ȍ 3 ȋŠ‡”‘• ‹‡–‹ˆ‹  ˜‹–”‘‰‡ȌǤ š’”‡••‹‘ †ƒ–ƒ ™‡”‡ ƒƒŽ›œ‡† —•‹‰ —ƒ––—†‹‘ ‡ƒŽ ‹‡  •‘ˆ–™ƒ”‡˜‡”•‹‘ͳǤʹȋ’’Ž‹‡†‹‘•›•–‡•Ȍƒ†–Š‡•–ƒ†ƒ”† —”˜‡‡–Š‘†‘ˆ ƒŽ —Žƒ–‹‘Ǥ‡•—Ž–•™‡”‡ ’”‡•‡–‡†ƒ•‡š’”‡••‹‘‘”ƒŽ‹œ‡†–‘–Š‡‡š’”‡••‹‘‘ˆ Ppiaȋ ‘†‹‰ˆ‘”‡’–‹†›ŽǦ’”‘Ž›Ž ‹•Ǧ–”ƒ• ‹•‘‡”ƒ•‡ǡƒŽ•‘‘™ƒ•› Ž‘’Š‹Ž‹Ȍ  ZĞĨĞƌĞŶĐĞƐ ͳǤ ‡””‡ƒ ǡ‡”• ǡƒ‹Œ ǡŽ‘•ǡ ‡”†‹‰ǡ ƒ˜‹‰ƒǡ‹‡–‰‡ ǡǫǫŽŽ‡”ǡ‹– ǡ —‹’‡”• ǡ ‡‹Œ‰‘—†  Ǥ ‹•–—”„‡† Š‡’ƒ–‹  ƒ”„‘Š›†”ƒ–‡ ƒƒ‰‡‡– †—”‹‰ Š‹‰Š ‡–ƒ„‘Ž‹  †‡ƒ† ‹ ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋȌǦ†‡ˆ‹ ‹‡– ‹ ‡Ǥ ‡’ƒ–‘Ž‘‰›ǤʹͲͲͺǢͶ͹ȋ͸ȌǣͳͺͻͶȂͻͲͶǤ ʹǤ ‘„‹ǡƒ˜‹•ǡ ŠŽ‡•‹‰‡” ǡ”‡‘™ ǡƒŽ‡•‹ǡ Šƒǡƒ–—–ǡŠƒ‹••‘ǡ ‹‰‡”ƒ•Ǥ ǣŽ–”ƒˆƒ•–—‹˜‡”•ƒŽǦ•‡“ƒŽ‹‰‡”Ǥ‹‘‹ˆ‘”ƒ–‹ •ǤʹͲͳ͵ǢʹͻȋͳȌǣͳͷȂʹͳǤ ͵Ǥ ‹ ǡ ƒ†•ƒ‡”ǡ›•‘‡”ǡ ‡‡ŽŽǡ—ƒ ǡ ‘‡”ǡƒ”–Š ǡ„‡ ƒ•‹• ǡ—”„‹ǤŠ‡ ‡“—‡ ‡Ž‹‰‡–Ȁƒ’ˆ‘”ƒ–ƒ†–‘‘Ž•Ǥ‹‘‹ˆ‘”ƒ–‹ •ǤʹͲͲͻǢʹͷȋͳ͸ȌǣʹͲ͹ͺȂͻǤ ͶǤ †‡”• ǡ ›Ž ǡ —„‡” Ǥ ‡“Ǧ ›–Š‘ ˆ”ƒ‡™‘” –‘ ™‘” ™‹–Š Š‹‰ŠǦ–Š”‘—‰Š’—– •‡“—‡ ‹‰†ƒ–ƒǤ‹‘‹ˆ‘”ƒ–‹ •ǤʹͲͳͷǢ͵ͳȋʹȌǣͳ͸͸ȂͻǤ ͷǤ ƒ™ǡŠ‡ǡŠ‹ǡ›–Š Ǥ‘‘ǣ”‡ ‹•‹‘™‡‹‰Š–•—Ž‘ Ž‹‡ƒ”‘†‡ŽƒƒŽ›•‹•–‘‘Ž• ˆ‘”Ǧ•‡“”‡ƒ† ‘—–•Ǥ ‡‘‡‹‘ŽǤʹͲͳͶǢͳͷȋʹȌǣͳȂͳ͹Ǥ ͸Ǥ ‘˜‡ ǡ —„‡”ǡ†‡”•Ǥ‘†‡”ƒ–‡†‡•–‹ƒ–‹‘‘ˆˆ‘Ž† Šƒ‰‡ƒ††‹•’‡”•‹‘ˆ‘”Ǧ•‡“ †ƒ–ƒ™‹–Š‡“ʹǤ ‡‘‡‹‘ŽǤʹͲͳͶǢͳͷȋͳʹȌǣͳȂʹͳǤ ͹Ǥ ŽŽ‹• ǡ ‘™ƒ ǡ ‘Žˆ‰ƒ‰  Ǥ ‡–ƒ„‘Ž‹  ƒ† ‹••—‡Ǧ’‡ ‹ˆ‹  ‡‰—Žƒ–‹‘ ‘ˆ  ›ŽǦ‘ ‡–ƒ„‘Ž‹•Ǥ‘‡ǤʹͲͳͷǢͳͲȋ͵Ȍǣ‡Ͳͳͳ͸ͷͺ͹Ǥ

ͳʹͷ

Chapter 4

The role of hepatic fatty-acid oxidation during cold stress: remodeling of hepatic metabolism in a mouse with medium-chain acyl-CoA dehydrogenase deficiency

A.M.F. Martines1,#, Wenxuan Zhang1,2,#, Albert Gerding1,3, Maaike Goris4, Marcel de Vries1, Mirjam H. Koster1,4, Laura Bongiovanni5, Alain de Bruin1,5, Rob H. Henning4, Terry G. J. Derks1,6, Rainer Bischoff2, Dirk-Jan Reijngoud1,2, Barbara M. Bakker1,*

#Authors contributed equally to this work.

1 Laboratory of Pediatrics, Center of Liver, Digestive and Metabolic Diseases, University of Groningen, Groningen, University Medical Center Groningen, Groningen, The Netherlands, 2 Department of Analytical Biochemistry, University of Groningen, Groningen, The Netherlands 3 Department of Laboratory Medicine, University of Groningen, University Medical Center Groningen, The Netherlands 4 Department of Anesthesiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 5 Department of Pathobiology, Faculty of Veterinary Medicine, Dutch Molecular Pathology Center, Utrecht University, The Netherlands 6 Section of Metabolic Diseases, Beatrix Children's Hospital, University Medical Center of Groningen, University of Groningen.

Journal publication in preparation

Effect of fasting and cold stress on hepatic metabolism in MCAD-KO mice

Abstract Š‹Ž†”‡ ™‹–Š ƒ †‡ˆ‹ ‹‡ › ‹ –Š‡ ‹–‘ Š‘†”‹ƒŽ ˆƒ––›Ǧƒ ‹† ȾǦ‘š‹†ƒ–‹‘ ȋ Ȍ ‡œ›‡ ‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘‡œ›‡†‡Š›†”‘‰‡ƒ•‡ȋȌƒ”‡ƒ–”‹•‘ˆŽ‹ˆ‡Ǧ–Š”‡ƒ–‡‹‰Ž‘™ „Ž‘‘†‰Ž— ‘•‡Ž‡˜‡Ž•Ǥ‘‡Ž— ‹†ƒ–‡–Š‡”‘Ž‡‘ˆŽ‹˜‡”‹–Š‡”‡•’‘•‡–‘‡‡”‰‡–‹ •–”‡••ǡ ˆƒ•–‡†Ǧ†‡ˆ‹ ‹‡–ȋǦȌƒ†™‹Ž†Ǧ–›’‡ȋȌ‹ ‡™‡”‡‡š’‘•‡†–‘ ‘Ž†ǤǦ ‹ ‡ •Š‘™‡† Ž‘™‡” „Ž‘‘† ‰Ž— ‘•‡ Ž‡˜‡Ž• ƒ† Š‡’ƒ–‹  ƒ‹‘ ƒ ‹† Ž‡˜‡Ž•ǡ ƒ† Š‹‰Š‡” Ž‹˜‡” ™‡‹‰Š–•ǡŽ‹˜‡”–”‹‰Ž› ‡”‹†‡•ƒ†„Ž‘‘†‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ ƒ”‹–‹‡Ž‡˜‡Ž•Ǥ‡–ƒ‹Ž‡†Ž‹’‹†‘‹ • ƒƒŽ›•‹•”‡˜‡ƒŽ‡†ƒƒ ——Žƒ–‹‘‘ˆ–”‹‰Ž› ‡”‹†‡•’‡ ‹‡• ‘–ƒ‹‹‰‡†‹—Ǧ Šƒ‹ˆƒ––›Ǧƒ ›Ž 4 „”ƒ Š‡•‹–Š‡Ž‹˜‡”Ǥ —””‡•—Ž–•ƒ”‡ ‘•‹•–‡–™‹–Šƒ‡›”‘Ž‡‘ˆŽ‹˜‡” ‹‰Ž— ‘•‡Š‘‡‘•–ƒ•‹•†—”‹‰ ‘Ž† •–”‡••ǤǦ‹ ‡‡šŠ‹„‹–ƒ ‘’‡•ƒ–‘”›”‡•’‘•‡‹™Š‹ Šƒ‹‘ƒ ‹†•’ƒ”–Ž›–ƒ‡ ‘˜‡”–Š‡”‘Ž‡‘ˆˆƒ––›ƒ ‹†•‹–Š‡‰‡‡”ƒ–‹‘‘ˆǡ‰Ž— ‘•‡ǡƒ†‡–‘‡„‘†‹‡•ǡ™Š‹Ž‡‡š ‡•• ‡†‹—Ǧ Šƒ‹ˆƒ––›Ǧƒ ‹†‹–‡”‡†‹ƒ–‡•ƒ”‡‡š’‘”–‡†ƒ†”‡”‘—–‡†‹–‘–”‹‰Ž› ‡”‹†‡•Ǥ  

ͳʹͻ Chapter 4

Introduction ‡’ƒ–‹ ‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧƒ ‹†„‡–ƒǦ‘š‹†ƒ–‹‘ȋ Ȍ’Žƒ›•ƒ‹’‘”–ƒ–”‘Ž‡‹ ‡‡”‰› Š‘‡‘•–ƒ•‹• †—”‹‰ ˆƒ•–‹‰ ƒ† ‘†‹–‹‘• ‘ˆ Š‹‰Š ‡‡”‰› †‡ƒ†Ǥ Š‡ ’ƒ–Š™ƒ› ’”‘˜‹†‡•‡‡”‰›ˆ‘”de novo‰Ž— ‘•‡’”‘†— –‹‘ƒ†•—„•–”ƒ–‡ˆ‘”‡–‘‡„‘†›’”‘†— –‹‘ȏͳȂ ͵ȐǤ‘•‡“—‡–Ž›ǡ‹†‹˜‹†—ƒŽ•™‹–Šƒ†‡ˆ‡ –‹ ‘ˆ–‡’”‡•‡–™‹–ŠŽ‘™„Ž‘‘†‰Ž— ‘•‡ƒ† ‡–‘‡ „‘†› ‘ ‡–”ƒ–‹‘• ȋŠ›’‘‡–‘–‹  Š›’‘‰Ž› ‡‹ƒȌ ȏͳȂ͵ȐǤ Š‡‘•– ’”‡˜ƒŽ‡–   †‹•‘”†‡” ‹• ƒ ‘’Ž‡–‡ †‡ˆ‹ ‹‡ › ‘ˆ –Š‡ ‡œ›‡ ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋȌǤ Š‹Ž‡ Ǧ†‡ˆ‹ ‹‡– ȋȌ Š‹Ž†”‡ ƒ› –‘Ž‡”ƒ–‡ ‘˜‡”‹‰Š– ˆƒ•–‹‰ ȏͶǡͷȐǡ ƒ ‘„‹ƒ–‹‘‘ˆ’”‘Ž‘‰‡†ˆƒ•–‹‰ƒ†ƒƒ††‹–‹‘ƒŽ–”‹‰‰‡”ǡ•— Šƒ•ȋ ‘„‹ƒ–‹‘•‘ˆȌˆ‡˜‡”ǡ †‡ ”‡ƒ•‡† ‰ƒ•–”‘‹–‡•–‹ƒŽ ‹–ƒ‡ ‘” ‹ ”‡ƒ•‡† Ž‘••‡•ǡ ‹ ”‡ƒ•‡• –Š‡ ”‹• ‘ˆ Š›’‘‡–‘–‹  Š›’‘‰Ž› ‡‹ƒƒ†•—††‡Ǧ‹ˆƒ–†‡ƒ–Šȏͳǡ͸ȂͻȐǤ ‘‹˜‡•–‹‰ƒ–‡–Š‡‡–‹‘Ž‘‰›‘ˆǡƒǦ†‡ˆ‹ ‹‡–ȋǦȌ‘—•‡‘†‡Ž Šƒ•„‡‡‰‡‡”ƒ–‡†ȏͳͲǡͳͳȐǤŠ‡Ǧ‹ ‡‡šŠ‹„‹–‡†–Š‡‡Ž‡˜ƒ–‡†„Ž‘‘†‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ ƒ”‹–‹‡• –Šƒ– ƒ”‡ ƒŽ•‘ Šƒ”ƒ –‡”‹•–‹  ‘ˆ Š—ƒ ǡ ƒ† ƒ ‡Šƒ ‡† ”ƒ–‡ ‘ˆ ‡‘ƒ–ƒŽ†‡ƒ–ŠȏͳͲȐǤ —•–ƒ• Š‹Ž†”‡ǡǦ‹ ‡™‡”‡“—‹–‡”‡•‹Ž‹‡––‘ˆƒ•–‹‰ ƒŽ‘‡ƒ†ƒ‹–ƒ‹‘”ƒŽ„Ž‘‘†‰Ž— ‘•‡Ž‡˜‡Ž•†—”‹‰ʹͶŠ‘—”•‘ˆˆƒ•–‹‰ȏͳͲǡͳʹȐǤ ‘™‡˜‡”ǡ ƒ ‘„‹ƒ–‹‘ ‘ˆ ˆƒ•–‹‰ ƒ† ‘Ž† ‡š’‘•—”‡ ȋͶι Ȍ ™ƒ• Ž‡–ŠƒŽ ‘” Ž‡† –‘ ˜‡”› Ž‘™ „‘†› –‡’‡”ƒ–—”‡•‹–Š‡ǡ„—–‘–‹–Š‡™‹Ž†–›’‡ȋȌ‹ ‡ȏͳͲȐǤ‘Ž†‹–‘Ž‡”ƒ ‡‹•ƒ ‘‘ ˆ‡ƒ–—”‡‘ˆ †‡ˆ‹ ‹‡–‘—•‡‘†‡Ž•ƒ†™ƒ•ƒŽ•‘‘„•‡”˜‡†™Š‡‡‹–Š‡”‘ˆ–Š‡‰‡‡• ‡ ‘†‹‰•Š‘”–ǦǡŽ‘‰Ǧǡ‘”˜‡”›ǦŽ‘‰Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡ȋǡƒ†Ȍ ™‡”‡†‡Ž‡–‡†ȋ”‡˜‹‡™‡†‹ȏͳ͵ȐȌǤ Š‡ ‹ ‡ ƒ”‡ ‡š’‘•‡† –‘ ‘Ž† •–”‡••ǡ „”‘™ ƒ†‹’‘•‡ –‹••—‡ ȋȌ —•‡• ‰Ž— ‘•‡ǡ ƒ‹‘Ǧƒ ‹†ǡƒ†ˆƒ––›Ǧƒ ‹† ƒ–ƒ„‘Ž‹•ˆ‘”‘Ǧ•Š‹˜‡”‹‰–Š‡”‘‰‡‡•‹•ȏͳͶȂͳ͸ȐǤ‘Ž†•–”‡•• ‹ ”‡ƒ•‡•–Š‡‡š’”‡••‹‘‘ˆ‰‡‡•‹˜‘Ž˜‡†‹ˆƒ––›Ǧƒ ‹†—’–ƒ‡ƒ† ‹ȏͳ͹ȐǤ –Š‹• ‘†‹–‹‘ǡ–Š‡Ž‹˜‡”’Žƒ›•ƒ‡›”‘Ž‡‹–Š‡•—’’Ž›‘ˆ•—„•–”ƒ–‡„›‹ ”‡ƒ•‡†‰Ž— ‘‡‘‰‡‡•‹• ȏͳͺȐƒ†ƒ ›ŽǦ ƒ”‹–‹‡’”‘†— –‹‘ƒ†•‡ ”‡–‹‘ȏͳͻǡʹͲȐǤ Ž‹‡™‹–Š–Š‹•ǡ–Š‡Ž‹˜‡”‡šŠ‹„‹–•ƒ Š‹‰Š‡”‡š’”‡••‹‘‘ˆ‰‡‡•‹˜‘Ž˜‡†‹ ƒ†ƒŽ‘™‡”‡š’”‡••‹‘‘ˆ‰‡‡•‹ˆƒ––›Ǧƒ ‹† •›–Š‡•‹•ȏʹͳȂʹ͵ȐǤŠ‡ ‘”ˆƒ––›Ǧƒ ‹†—’–ƒ‡‹•†‡ˆ‡ –‹˜‡ǡ–Š‡”‡Ž‹‡•‘”‡Š‡ƒ˜‹Ž› ‘‰Ž— ‘•‡ƒ†ƒ‹‘ƒ ‹†•ǤŠ‹•Ž‡ƒ†•–‘ƒŠ‹‰Š‡””‡“—‹”‡‡–ˆ‘”de novo‰Ž— ‘•‡’”‘†— –‹‘ „›–Š‡Ž‹˜‡”Ǥ ‘”†‹‰Ž›ǡˆƒ•–‹‰‹ ‘„‹ƒ–‹‘™‹–Š ‘Ž†‡š’‘•—”‡”‡•—Ž–‡†‹Ž‘™‡”„Ž‘‘† ‰Ž— ‘•‡ ‘ ‡–”ƒ–‹‘•‹Ǧ ‘’ƒ”‡†–‘‹ ‡ȏʹͶȂʹ͸ȐǤ‘†ƒ–‡Š‘™‡˜‡”ǡ–Š‡”‡ ƒ”‡‘”‡’‘”–•‘–Š‡Ž‹˜‡”ˆ— –‹‘‘ˆǦ‘—•‡†—”‹‰ ‘Ž†•–”‡••ǡ‘”‘ˆƒ›‘–Š‡” ’Š›•‹‘Ž‘‰‹ ƒŽ’ƒ”ƒ‡–‡”•„—–”‡ –ƒŽ–‡’‡”ƒ–—”‡ȏͳͲǡʹ͹ȐǤŠ‡”‡ˆ‘”‡ǡ–Š‡ƒ‹‘ˆ–Š‹••–—†›™ƒ• –‘‹˜‡•–‹‰ƒ–‡Ž‹˜‡”ˆ— –‹‘‹Ǧ‹ ‡†—”‹‰ ‘„‹‡† ‘Ž†‡š’‘•—”‡ƒ†ˆƒ•–‹‰Ǥ  –Š‹• •–—†›ǡ Ǧ ‹ ‡ ™‡”‡ ˆƒ•–‡† ˆ‘” ͳͶ Š ƒ– ʹͳ ι ƒ† •—„•‡“—‡–Ž› ˆ‘” ƒ‘–Š‡”ͶǦ͸Šƒ–ͶιǤŠ‡’Š‡‘–›’‡‘ˆ›‘—‰‹ ‡ȋͺ™‡‡•Ȍ™ƒ•‘”‡ƒˆˆ‡ –‡†–Šƒ–Šƒ–‘ˆ ƒ†—Ž–‹ ‡ȋͳ͸ǦʹͲ™‡‡•ȌǤ––Š‡‡†‘ˆ–Š‡ ŠƒŽŽ‡‰‡ǡ–Š‡„Ž‘‘†‰Ž— ‘•‡ ‘ ‡–”ƒ–‹‘‘ˆ ›‘—‰Ǧ‹ ‡™ƒ•Ž‘™‡”–Šƒ–Šƒ–‘ˆ–Š‡‹” ‘—–‡”’ƒ”–•ǡ™Š‹Ž‡‡–‘‡Ǧ„‘†›Ž‡˜‡Ž• ™‡”‡ •‹‹Žƒ”Ǥ ‹˜‡” ƒ‹‘Ǧƒ ‹† Ž‡˜‡Ž• ™‡”‡ Ž‘™‡” ‹ Ǧ ‹ ‡ ‘’ƒ”‡† –‘ ǡ ‘•‹•–‡– ™‹–Š ƒ Š‹‰Š‡” •—„•–”ƒ–‡ —•‡ ˆ‘” ‰Ž— ‘‡‘‰‡‡•‹•Ǥ  ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ ƒ”‹–‹‡ Ž‡˜‡Ž•ȋ‘–ƒ„Ž›ͳͲǣͳȌ™‡”‡‡Ž‡˜ƒ–‡†‹–Š‡„Ž‘‘†‘ˆ–Š‡‹ ‡ǡ„—–—ƒˆˆ‡ –‡†‹–Š‡Ž‹˜‡”Ǥ Ǧ†‡’–Š Š‡’ƒ–‹  Ž‹’‹†‘‹  ƒƒŽ›•‹• ”‡˜‡ƒŽ‡† ƒ ——Žƒ–‹‘ ‘ˆ –”‹‰Ž› ‡”‹†‡ ȋ Ȍ •’‡ ‹‡• ‘–ƒ‹‹‰ ͸ǦͳͶ ƒ ›Ž Šƒ‹•ǡ •—‰‰‡•–‹‰ –Šƒ– ‡†‹—Ǧ Šƒ‹   ‹–‡”‡†‹ƒ–‡• ™‡”‡ ”‡”‘—–‡†‹–‘ȋ’ƒ”–‹ƒŽȌ Šƒ‹‡Ž‘‰ƒ–‹‘ƒ†Ȁ‘”•–‘”ƒ‰‡ǤŠ‹•‹•–Š‡ˆ‹”•––‹‡–Šƒ––Š‡Ž‹˜‡” ‡–ƒ„‘Ž‹•‘ˆ–Š‡Ǧ‘—•‡™ƒ••–—†‹‡†—†‡”•‡˜‡”‡‡‡”‰‡–‹ •–”‡••Ǥ‡ ‘ Ž—†‡

ͳ͵Ͳ Effect of fasting and cold stress on hepatic metabolism in MCAD-KO mice

–Šƒ–‹–‹•ƒ˜ƒŽ—ƒ„Ž‡‘†‡Ž–‘‰ƒ‹‹•‹‰Š–‹–‘‡–ƒ„‘Ž‹ ƒ†ƒ’–ƒ–‹‘•‹ƒ†’”‘˜‹†‡ ˆ—”–Š‡”‡˜‹†‡ ‡ˆ‘”–Š‡ ”— ‹ƒŽ”‘Ž‡‘ˆŽ‹˜‡” ‹’”‘–‡ –‹‘ƒ‰ƒ‹•– ‘Ž†•–”‡••Ǥ  Results Young MCAD-KO mice are more vulnerable to cold exposure than adult MCAD-KO mice. ‘‡š’‘•‡ƒ†—Ž–ȋͳ͸ǦʹͲ™‡‡•‘Ž†ȌǦ‹ ‡–‘•‡˜‡”‡‡‡”‰‡–‹ •–”‡••ǡ–Š‡›™‡”‡ˆƒ•–‡† ˆ‘”ͳͶŠ‘—”•ƒ†•—„•‡“—‡–Ž›‡š’‘•‡†–‘ƒͶ釐˜‹”‘‡–ǡ•–‹ŽŽ‹–Š‡ƒ„•‡ ‡‘ˆˆ‘‘†Ǥ‘Ž† ‡š’‘•—”‡ˆ‘”ͶǦ͸Š‘—”•‹†— ‡†ƒ•—„•–ƒ–‹ƒŽ†”‘’‹–Š‡„‘†›–‡’‡”ƒ–—”‡‘ˆͶ‘—–‘ˆͺˆƒ•–‡† 4 ‹ ‡ǡ™Š‹Ž‡ƒŽŽ͹†‡–‡ –‡†‹ ‡ƒ‹–ƒ‹‡†ƒ•–ƒ„Ž‡–‡’‡”ƒ–—”‡ȋ ‹‰—”‡ͳȌǤ

ABC 40 i 40 15

C) i WT q KO 35 30 10 16-20 w.o. 30

5 20 WT 25 20 KO (g) Body weight Blood glucose (mM) glucose Blood

Body temperature ( Body temperature 0 20 0 BF AF AFC BF AF AFC BF AF AFC

DEF 6 1.6 6 * *

1.2 5 4 16-20 w.o. 0.8 4 2 0.4

Liver weight (g) weight Liver 3 3 Ketone bodies (mM) bodies Ketone 0 0.0 0 BF AF AFC WT KO Relative liver weight (%) WT KO

GHI J 40 40 30 40 C) C) C)

q WT q q WT KO 35 KO 25 30 30 30 r=0.84 p<0.0001 8 w.o. 25 20 20 WT 20 20 20 20 20 KO (g) Body weight Body temperature ( Body temperature

0 ( temperature Rectal 0 15 ( temperature Rectal 0 BF AF AFC WT KO BF AF AFC 0 10 20 Body weight AF (g) KL MN 15 4 1.6 6 ** * 3 1.2 5 10 * i 2 0.8 8 w.o. 4 5 1 0.4

Liver weight (g) weight Liver 3 3 Blood glucose (mM) glucose Blood (mM) bodies Ketone 0 0 0.0 0 BF AF AFC BF AF AFC WT KO Relative liver weight (%) WT KO

Figure 1. Biometric results of 8- and 16-20-weeks-old mice. ‘†› –‡’‡”ƒ–—”‡ ‡ƒ•—”‡† „› ‹–”ƒ’‡”‹–‘‡ƒŽŽ›Ǧ‹’Žƒ–‡†Ž‘‰‰‡”ȋA,GȌǡ„‘†›™‡‹‰Š–ȋB,IȌǡ„Ž‘‘†‰Ž— ‘•‡ȋC,KȌƒ†‡–‘‡„‘†‹‡•ȋD,LȌ „‡ˆ‘”‡ˆƒ•–‹‰ȋ Ȍǡƒˆ–‡”ͳͶŠ”•‘ˆˆƒ•–‹‰ǡ„‡ˆ‘”‡ ‘Ž†‡š’‘•—”‡ȋ Ȍƒ†ƒˆ–‡”ˆƒ•–‹‰’Ž—• ‘Ž†‡š’‘•—”‡ ȋ Ȍǡƒ„•‘Ž—–‡Ž‹˜‡”™‡‹‰Š–ȋE,MȌƒ†”‡Žƒ–‹˜‡Ž‹˜‡”™‡‹‰Š–ȋΨ‘ˆ„‘†›™‡‹‰Š–ȌȋF,NȌ‘ˆ–Š‡ͳ͸ǦʹͲ™‡‡•Ǧ ‘Ž† ȋA-FȌ ƒ† ͺ ™‡‡•Ǧ‘Ž† ‹ ‡ ȋG,I,K-NȌ ƒ† ”‡ –ƒŽ –‡’‡”ƒ–—”‡ ȋHȌǡ ”‡ –ƒŽ –‡’‡”ƒ–—”‡ ˜‡”•—• „‘†› ™‡‹‰Š– ƒˆ–‡” ͳͶŠ”•‘ˆ ˆƒ•–‹‰ȋJȌ‘ˆ–Š‡ͺǦ ™‡‡•Ǧ‘Ž† ‹ ‡Ǥ α͸Ǧͺˆ‘” „‘–Šƒ† Ǥ Š‡”‡•—Ž–•ƒ”‡ ”‡’”‡•‡–‡†ƒ•—‡›„‘šƒ†™Š‹•‡”’Ž‘–•ǡ™Š‡”‡–Š‡„Žƒ Ǧˆ‹ŽŽ‡†Œƒ‰‰‡† ‹” Ž‡•‹†‹ ƒ–‡‹†‹˜‹†—ƒŽ‹ ‡ ˆƒŽŽ‹‰‘—–•‹†‡ –Š‡͹ͷΨ’‡” ‡–‹Ž‡’Ž—•ͳǤͷȉ‹–‡”Ǧ“—ƒ”–‹Ž‡”ƒ‰‡ȋ Ȍ‘”ʹͷΨ’‡” ‡–‹Ž‡‹—•ͳǤͷȉ Ǥ †‹ ƒ–‹˜‡ǡiǣ’δͲǤͳǡȗǣ’δͲǤͲͷǡȗȗǣ’δͲǤͲͳǡȗȗȗǣ’δͲǤͲͲͳƒ†ȗȗȗȗǣ’δͲǤͲͲͲͳ ‘’ƒ”‡†–‘ǤŽŽ„‘š’Ž‘–•ƒ† ‹†‹ ƒ–‹‘•‘ˆ•‹‰‹ˆ‹ ƒ ‡‹–Š‡”‡ƒ‹†‡”‘ˆ–Š‹•”‡’‘”–ƒ”‡”‡’”‡•‡–‡†‹–Š‡•ƒ‡™ƒ›ƒ•–Š‹•ˆ‹‰—”‡Ǥ 

ͳ͵ͳ Chapter 4

Š‡ ˜ƒ”‹ƒ–‹‘ ‹ –Š‡ Ǧ ‰”‘—’ ™ƒ• •—„•–ƒ–‹ƒŽǡ ’‘••‹„Ž› „‡ ƒ—•‡ –Š‡ •–ƒ”– ‘ˆ –Š‡ –‡’‡”ƒ–—”‡†‡ Ž‹‡™ƒ••Š‹ˆ–‡†‹–‹‡„‡–™‡‡‹†‹˜‹†—ƒŽ•ȋ—’’Ž‡‡–ƒ”›ˆ‹‰—”‡ͳȌǤ Š‡„‘†›™‡‹‰Š–†‡ ”‡ƒ•‡†–‘ƒ•‹‹Žƒ”‡š–‡–‹„‘–Š‰”‘—’•ǡƒ†™ƒ••‹‹Žƒ”‹Ǧ ƒ†‹ ‡ȋ ‹‰—”‡ͳȌǤ ƒ††‹–‹‘ǡ„Ž‘‘†‰Ž— ‘•‡ƒ†‡–‘‡Ǧ„‘†› ‘ ‡–”ƒ–‹‘•™‡”‡ •‹‹Žƒ”„‡–™‡‡ƒ†‹ ‡ȋ ‹‰—”‡ͳǦȌǤŠ‡ƒ„•‘Ž—–‡ƒ†”‡Žƒ–‹˜‡Ž‹˜‡”™‡‹‰Š–•™‡”‡ǡ Š‘™‡˜‡”ǡ•‹‰‹ˆ‹ ƒ–Ž›Š‹‰Š‡”‹Ǧ–Šƒ‹ ‡ƒ––Š‡‡†‘ˆ–Š‡‡š’‡”‹‡–ȋ ‹‰—”‡ ͳǡ ȌǤ ”‡˜‹‘—•Ž›ǡƒ•‹‰‹ˆ‹ ƒ–Ž›Ž‘™‡”„‘†›–‡’‡”ƒ–—”‡Šƒ•„‡‡”‡’‘”–‡†‹ͺǦ™‡‡•‘Ž†ǡˆƒ•–‡†ǡ ‘Ž†Ǧ‡š’‘•‡†Ǧ‹ ‡”‡Žƒ–‹˜‡–‘–Š‡‹” ‘—–‡”’ƒ”–•ȏͳͲȐǤŠ‡™‡”‡’‡ƒ–‡†–Š‡ ‡š’‡”‹‡–‹ͺ™‡‡•‘Ž†‹ ‡ǡ‘”‡ƒ‹ƒŽ••Š‘™‡†ƒ†”‘’‹„‘†›–‡’‡”ƒ–—”‡ǡ‹–Š‹• ƒ•‡ƒŽ•‘‹–Š‡‰”‘—’ȋ—’’Ž‡‡–ƒ”›ˆ‹‰—”‡ͳȌǤŠ‹•‡–ƒ‹Ž‡†ǡ͵͵Ǥ͵ΨȋʹȀ͸Ȍƒ†͹ͳΨ ȋͷȀ͹Ȍ‘ˆ–Š‡ƒ†‹ ‡ǡ”‡•’‡ –‹˜‡Ž›ǡ‘ˆ™Š‹ Š–Š‡„‘†›–‡’‡”ƒ–—”‡ ‘—Ž†„‡‡ƒ•—”‡† ȋ ‹‰—”‡ͳ ǡ Ǣ—’’Ž‡‡–ƒ”›ˆ‹‰—”‡ͳȌǤ‘•‹‰‹ˆ‹ ƒ–†‹ˆˆ‡”‡ ‡„‡–™‡‡–Š‡‰‡‘–›’‡• ™ƒ•‘„•‡”˜‡†ȋ ‹‰—”‡ͳ ǡ ȌǤ —”–Š‡”‘”‡ǡ–Š‡„‘†›™‡‹‰Š–†‡ ”‡ƒ•‡†‹„‘–Š‰‡‘–›’‡•–‘ƒ •‹‹Žƒ”‡š–‡–ǡƒ†ƒŽ•‘Š‡”‡™ƒ••‹‹Žƒ”Ǧƒ†‹ ‡ȋ ‹‰—”‡ͳ ȌǤ ‘””‡Žƒ–‹‘ ƒƒŽ›•‹• ”‡˜‡ƒŽ‡† –Šƒ– –Š‡ ƒ‹ƒŽ• ™‹–Š ƒ Ž‘™‡” „‘†› ™‡‹‰Š– ƒŽ•‘ Šƒ† ƒ Ž‘™‡” „‘†› –‡’‡”ƒ–—”‡ƒˆ–‡” ‘Ž†‡š’‘•—”‡ȋ ‹‰—”‡ͳ ƒ†—’’Ž‡‡–ƒ”› ‹‰—”‡ͳȌǤ –‡”‡•–‹‰Ž›ǡ ƒˆ–‡” ‘Ž†‡š’‘•—”‡–Š‡„Ž‘‘†Ǧ‰Ž— ‘•‡ ‘ ‡–”ƒ–‹‘™ƒ••‹‰‹ˆ‹ ƒ–Ž›Ž‘™‡”‹›‘—‰Ǧ –Šƒ‹‹ ‡ȋ ‹‰—”‡ͳȌǡƒŽ„‡‹–™‹–Š‘—–ƒ›‡ˆˆ‡ –‘–Š‡Ž‡˜‡Ž‘ˆ‡–‘‡„‘†‹‡•ȋ ‹‰—”‡ ͳȌǤ’’ƒ”‡–Ž›ǡ†‡ˆ‹ ‹‡ ›ƒˆˆ‡ –•–Š‡„Ž‘‘†‰Ž— ‘•‡ ‘ ‡–”ƒ–‹‘‘Ž›—†‡”•‡˜‡”‡ ‡‡”‰‡–‹ •–”‡••ǡ‹Ǥ‡Ǥ—’‘ƒ ‘„‹ƒ–‹‘‘ˆˆƒ•–‹‰ƒ† ‘Ž†•–”‡••‹›‘—‰ƒ‹ƒŽ•ǤŠ‹•‹• •‹‹Žƒ”–‘–Š‡•‹–—ƒ–‹‘‹’ƒ–‹‡–•ǡ™Š‘ƒŽ•‘’”‡•‡–™‹–ŠŠ›’‘‰Ž› ‡‹ƒ‘Ž›ƒ–ƒ›‘—‰ƒ‰‡ ȋ–›’‹ ƒŽŽ› ͲǦʹ ›‡ƒ”•Ȍǡ †—”‹‰ ‘˜‡”‹‰Š– ˆƒ•–‹‰ ‹ ‘„‹ƒ–‹‘ ™‹–Š ƒ‘–Š‡” •–”‡•• ˆƒ –‘”Ǥ ‡Žƒ–‹˜‡Ž‹˜‡”™‡‹‰Š–•™‡”‡•‹‰‹ˆ‹ ƒ–Ž›Š‹‰Š‡”‹›‘—‰ ‘’ƒ”‡†–‘–Š‡‹ ‡ȋ ‹‰—”‡ ͳǡȌǡƒ•‘„•‡”˜‡†ˆ‘”ƒ†—Ž–‹ ‡„—––Š‡†‹ˆˆ‡”‡ ‡™ƒ•‡˜‡‘”‡’”‘‘— ‡†‹›‘—‰ ‹ ‡ǤŽ–‘‰‡–Š‡”ǡ„ƒ•‡†‘–Š‡‡–ƒ„‘Ž‹ ”‡•—Ž–•ǡ–Š‡•‡”‡•—Ž–••Š‘™–Šƒ–›‘—‰ Ǧ ‹ ‡ƒ”‡‘”‡˜—Ž‡”ƒ„Ž‡–‘ ‘Ž†‡š’‘•—”‡–Šƒƒ†—Ž–‹ ‡Ǥ  Higher plasma acyl-carnitines and NEFAs in fasted, cold-exposed MCAD-KO mice ”‡˜‹‘—•Ž›ǡ•‹‰‹ˆ‹ ƒ–Ž›Ž‘™‡”’Žƒ•ƒƒŽƒ‹‡Ž‡˜‡Ž•Šƒ˜‡„‡‡”‡’‘”–‡†‹ˆƒ•–‡†Ǧ ‘’ƒ”‡†–‘  ‹ ‡ǡ™Š‹Ž‡ ˜ƒŽ‹‡ǡ Ž‡— ‹‡ ƒ†‹•‘Ž‡— ‹‡ ™‡”‡ •‹‹Žƒ” ȏʹͺȐǤ ‡ †‹† ‘– ‘„•‡”˜‡•‹‰‹ˆ‹ ƒ–†‹ˆˆ‡”‡ ‡•‹ƒ‹‘ƒ ‹† ‘ ‡–”ƒ–‹‘•„‡–™‡‡ͺ™‡‡•‘Ž† Ǧ ƒ†‹ ‡ǡ‡š ‡’–ˆ‘”ȽǦƒ‹‘„—–›”‹ ƒ ‹†ǡ™Š‹ Š™ƒ•Ž‘™‡”‹–Š‡Ǧ‰”‘—’ȋ ‹‰—”‡ ʹƒ†—’’Ž‡‡–ƒ”›ˆ‹‰—”‡ʹȌǤŽ‡˜ƒ–‡†„Ž‘‘†‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ ƒ”‹–‹‡Ž‡˜‡Ž•ƒ”‡ƒ ‹’‘”–ƒ– „‹‘ƒ”‡” ˆ‘”  †‡ˆ‹ ‹‡ › ȏʹͻȂ͵͵ȐǤ †‡‡†ǡ –Š‡ ͸Ǧ –‘ ͳͲǦƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘•™‡”‡Š‹‰Š‡”‹Ǧ ‘’ƒ”‡†–‘‹ ‡ǡ—†‡”ƒŽŽ ‘†‹–‹‘•ƒ†‹ „‘–Šƒ‰‡‰”‘—’•ȋ ‹‰—”‡ʹǦƒ†—’’Ž‡‡–ƒ”›ˆ‹‰—”‡ʹǦȌǤ ƒ‰”‡‡‡–™‹–Š’”‡˜‹‘—• ˆ‹†‹‰•‹Ǧ‹ ‡ȏͳͲǡʹͺǡ͵ͶȐǡ’ƒ”–‹ —Žƒ”Ž›ͳͲǣͳ™ƒ•‡Ž‡˜ƒ–‡†ǤŠ‹•‹•‹ ‘–”ƒ•––‘ Š—ƒ’ƒ–‹‡–•ǡ‹™Š‹ ŠͺǦƒ ›ŽǦ ƒ”‹–‹‡ƒ ——Žƒ–‡•‘•–ȏʹͻȂ͵͵ȐǤŠ‡†‹ˆˆ‡”‡ ‡ „‡–™‡‡Ǧƒ†™ƒ•‘•–’”‘‘— ‡†ƒˆ–‡”ˆƒ•–‹‰ǡ‹””‡•’‡ –‹˜‡‘ˆ ‘Ž†‡š’‘•—”‡ ȋ ‹‰—”‡ ʹǦǡ —’’Ž‡‡–ƒ”› ˆ‹‰—”‡ ʹǦ ƒ† —’’Ž‡‡–ƒ”› –ƒ„Ž‡ ͳȌǤ ‹ƒŽŽ›ǡ ’Žƒ•ƒ  Ž‡˜‡Ž•ƒˆ–‡” ‘Ž†‡š’‘•—”‡™‡”‡•‹‰‹ˆ‹ ƒ–Ž›Š‹‰Š‡”‹–Š‡›‘—‰Ǧ‹ ‡ȋ ‹‰—”‡ ʹȌǤ—”‹‰ ‘Ž†•–”‡••‡Šƒ ‡†–”‹‰Ž› ‡”‹†‡Ž‹’‘Ž›•‹•„›ƒ†‹’‘•‡–‹••—‡”‡•—Ž–•‹‹ ”‡ƒ•‡† ”‡Ž‡ƒ•‡‘ˆ •ȏ͵ͷȐǤ

ͳ͵ʹ Effect of fasting and cold stress on hepatic metabolism in MCAD-KO mice

A 1500 M) P

1000

500

[Blood amino acid] ( 0

Serine Valine Alanine Cystine Glycine Lysine Proline Arginine Histidine Leucine Tyrosine GlutamateGlutamine Isoleucine Threonine Asparagine Methionine Tryptophan Phenylalanine 4

BDC before fasting 14-hr fasted after cold exposure 0.4

M) 0.4 0.4

P *** *** 0.3 0.3 *** 0.3 * **** *** 0.2 0.2 i 0.2 ** ** ** ** * -acyl-carnitine ( -acyl-carnitine n 0.1 0.1 * 0.1

0.0 0.0 0.0 Blood C C6 C8 C10:1 C10 C12:1 C12 C6 C8 C10:1 C10 C12:1 C12 C6 C8 C10:1 C10 C12:1 C12 E 1500 ** M) P 1000

500 Serum NEFA ( NEFA Serum 0 16-20 w.o. 8 w.o. Figure 2. Blood metabolite concentrations.Žƒ•ƒƒ‹‘ƒ ‹† ‘ ‡–”ƒ–‹‘•ƒˆ–‡” ‘Ž†‡š’‘•—”‡ȋAȌǡ „Ž‘‘† ƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘• ȋB-DȌ ‘ˆ –Š‡ ͺǦ™‡‡•Ǧ‘Ž† ‹ ‡ǡ ƒ† •‡”—   Ž‡˜‡Ž• ƒˆ–‡” ‘Ž† ‡š’‘•—”‡ȋEȌǤ  Š‡†‹ˆˆ‡”‡ ‡‹’Žƒ•ƒ Ž‡˜‡Ž•ƒ›–Š‡”‡ˆ‘”‡‡‹–Š‡”„‡†—‡–‘–Š‡Š‹‰Š‡””‡Ž‡ƒ•‡‘ˆ  •„›ƒ†‹’‘•‡–‹••—‡ǡ‘”–‘–Š‡†‡ˆ‡ –‹˜‡ ’ƒ–Š™ƒ›‹–Š‡Ž‹˜‡”ǡ‘”ƒ ‘„‹ƒ–‹‘‘ˆ „‘–ŠǤ  Altered amino-acid and lipid handling in the liver of cold-exposed MCAD-KO mice —„•‡“—‡–Ž›ǡ ™‡ ˆ‘ —•‡† ‘ ‡–ƒ„‘Ž‹  ƒ†ƒ’–ƒ–‹‘• ‹ –Š‡ Ž‹˜‡” ‘ˆ ˆƒ•–‡†ǡ ‘Ž†Ǧ‡š’‘•‡† ƒ‹ƒŽ•Ǥ ‡’ƒ–‹ ƒ‹‘ƒ ‹†•ƒ›•‡”˜‡ƒ•ƒŽ–‡”ƒ–‹˜‡•—„•–”ƒ–‡•ƒ†ˆ—‡Ž•ˆ‘”‡–‘‰‡‡•‹• ƒ†‰Ž— ‘‡‘‰‡‡•‹•‹–Š‡Ǧ‹ ‡Ǥ Ž‹‡™‹–Š–Š‹•ǡ™‡‘„•‡”˜‡†•‹‰‹ˆ‹ ƒ–Ž›Ž‘™‡” ƒ‹‘Ǧƒ ‹† ‘ ‡–”ƒ–‹‘• ‹ Ž‹˜‡”• ‘ˆ ‘Ž†Ǧ‡š’‘•‡† ˆƒ•–‡† Ǧ ‹ ‡ ȋ ‹‰—”‡ ͵ǡ —’’Ž‡‡–ƒ”› ˆ‹‰—”‡ ͵ ƒ† —’’Ž‡‡–ƒ”› –ƒ„Ž‡ ͳȌǤ Š‡ †‹ˆˆ‡”‡ ‡ ™ƒ• ƒ‰ƒ‹ ‘”‡ ’”‘‘— ‡† ‹ –Š‡ ›‘—‰ ‹ ‡Ǥ  –Š‡•‡ ‹ ‡ǡ –Š‡ Ž‡˜‡Ž• ™‡”‡ •‹‰‹ˆ‹ ƒ–Ž› Ž‘™‡” ˆ‘” ‰Ž—–ƒ‹‡ǡ ‰Ž› ‹‡ǡ Ž‡— ‹‡ǡ Ž›•‹‡ǡ •‡”‹‡ǡ –Š”‡‘‹‡ǡ –›”‘•‹‡ ƒ† Ǧ ›•–ƒ–Š‹‘‹‡ ƒ† ‹†‹ ƒ–‹˜‡ȋiǡ’δͲǤͳȌˆ‘”ƒŽƒ‹‡ǡ‰Ž—–ƒ‹ ƒ ‹†ǡȽǦƒ‹‘„—–›”‹ ƒ ‹†ƒ†‘”‹–Š‹‡Ǥ –Š‡ƒ†—Ž– ‹ ‡ǡ‘Ž›‰Ž› ‹‡Ž‡˜‡Ž•™‡”‡•‹‰‹ˆ‹ ƒ–Ž›Ž‘™‡”ǡƒŽ–Š‘—‰Š–Š‡•ƒ‡–”‡†™ƒ•‘„•‡”˜‡† ˆ‘”‘–Š‡”ƒ‹‘ƒ ‹†•ȋ—’’Ž‡‡–ƒ”›ˆ‹‰—”‡͵ǡȌǤ‹˜‡”ƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘•™‡”‡ •‹‹Žƒ” „‡–™‡‡  ƒ† ‹ ‡ ȋ ‹‰—”‡ ͵ǡ ƒ† —’’Ž‡‡–ƒ”› ƒ„Ž‡ ͳȌǤ ”‡˜‹‘—•Ž›ǡ •‘‡™Šƒ–Š‹‰Š‡” ‘ ‡–”ƒ–‹‘•‘ˆ‡†‹—Ǧ Šƒ‹Ž‹˜‡”ƒ ›ŽǦ ƒ”‹–‹‡•Šƒ˜‡„‡‡”‡’‘”–‡†‹ ˆƒ•–‡†Ǧ‹ ‡ǡƒŽ„‡‹––Š‡†‹ˆˆ‡”‡ ‡™ƒ•Ž‡••’”‘‘— ‡†–Šƒ‹„Ž‘‘†ȏ͵ͶȐǤ

ͳ͵͵ Chapter 4

A 8 i ** 6 ** * *** ** i 4 **

2 i *

[Liver amino acid] (nmol/g)0

Serine Valine Alanine Glycine Lysine Proline Arginine Histidine Leucine Tyrosine Glutamine Isoleucine Threonine Asparagine Methionine Aspartic acid Glutamic acid Phenylalanine BC 1.0 100 * **

0.8 80

0.6 60

0.4 mol/g liver) 40 P -acyl-carnitine (nmol/g)

n 0.2 20 TG ( TG

0.0 0

Liver C C6 C8 C10:1 C10 C12:1 C12 16-20 w.o. 8 w.o.  Figure 3. Hepatic metabolite profiles.‹˜‡”ƒ‹‘Ǧƒ ‹†ȋAȌƒ†͸Ǧͳʹƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘•ˆ‘” ͺǦ™‡‡•‘Ž†‹ ‡ȋBȌǡƒ†Ž‹˜‡”–”‹‰Ž› ‡”‹†‡ȋ Ȍ ‘ ‡–”ƒ–‹‘‘ˆͳ͸ǦʹͲǦƒ†ͺǦ™‡‡•Ǧ‘Ž†‹ ‡ȋCȌǤ  ’’ƒ”‡–Ž›ǡ ‘Ž†Ǧ‡š’‘•—”‡–”‹‰‰‡”•ƒƒ†ƒ’–ƒ–‹‘‘ˆŽ‹’‹†‡–ƒ„‘Ž‹•‹–Š‡Ǧ‹ ‡ǡ Ž‡ƒ†‹‰ –‘ ƒ ›ŽǦ ƒ”‹–‹‡ Š‘‡‘•–ƒ•‹•Ǥ ‹ƒŽŽ›ǡ Ž‹˜‡” –”‹‰Ž› ‡”‹†‡ ȋ Ȍ ‘ ‡–”ƒ–‹‘• ™‡”‡ Š‹‰Š‡”‹–Š‡Ǧ–Šƒ‹–Š‡‹ ‡ǡ‹„‘–Šƒ‰‡‰”‘—’•ȋ ‹‰—”‡͵ȌǡƒŽ•‘•—‰‰‡•–‹‰ ƒŽ–‡”‡†Ž‹’‹†Šƒ†Ž‹‰‹–Š‡Ž‹˜‡”Ǥ  Rerouting of medium-chain acyl-CoAs into chain-elongation and triglyceride synthesis Š‡Š‹‰Š‡”Ž‹˜‡”  ‘ ‡–”ƒ–‹‘•‹–Š‡Ž‹˜‡”ƒ† ‘’ƒ”ƒ„Ž‡Ž‹˜‡”ƒ ›ŽǦ ƒ”‹–‹‡’”‘ˆ‹Ž‡• •—‰‰‡•–ƒƒŽ–‡”‡†Ž‹’‹†‡–ƒ„‘Ž‹•‹–Š‡Ǧ‹ ‡ǤŠ‡”‡ˆ‘”‡ǡ™‡ƒƒŽ›œ‡†–Š‡Ž‹’‹†• ‡š–”ƒ –‡† ˆ”‘ –Š‡ Ž‹˜‡” „› —–ƒ”‰‡–‡† Ž‹’‹†‘‹ •Ǥ ˜‡” ͸ͲͲ •‹‰Ž‡ Ž‹’‹† •’‡ ‹‡• ‘—Ž† „‡ ƒ‘–ƒ–‡†ǡ ‘˜‡”‹‰ ƒŽŽ ƒŒ‘” Ž‹’‹† Žƒ••‡• ȋ ‹‰—”‡ Ͷ ƒ† —’’Ž‡‡–ƒ”› ƒ„Ž‡ ʹȌǤ ”‹ ‹’ƒŽ‘’‘‡–ƒŽ›•‹•ȋȌ•Š‘™‡†ƒ Ž‡ƒ”•‡’ƒ”ƒ–‹‘„‡–™‡‡ƒ†Ǧ ‹ ‡ „ƒ•‡† ‘ –Š‡‹” Š‡’ƒ–‹  Ž‹’‹† ’”‘ˆ‹Ž‡• ȋ ‹‰—”‡ ͶȌǤ Š‡ Ž‹’‹†• –Šƒ– „”‘—‰Š– ƒ„‘—– –Š‡ •‡’ƒ”ƒ–‹‘ ‘ˆ –Š‡ ‰”‘—’• ™‡”‡ ƒŽ‘•– ‡š Ž—•‹˜‡Ž›  •Ǥ Š‡ ‰”‘—’ ‘ˆ Ž‹’‹†• ™‹–Š ‘•– ’”‘‘— ‡††‹ˆˆ‡”‡ ‡•„‡–™‡‡ƒ†ȋ’δͲǤͲͷƒ†ˆ‘Ž† Šƒ‰‡εʹǢ ‹‰—”‡Ͷ—’’‡” ”‹‰Š–“—ƒ†”ƒ–Ȍ ‘•‹•–‡†‘ˆ͵ͻ •ǡͳ†‹‰Ž› ‡”‹†‡ǡƒ†ͳ•’Š‹‰‘Ž‹’‹†ȋ—’’Ž‡‡–ƒ”›–ƒ„Ž‡ ʹȌǤŠ‡ •™‹–Š–Š‡Š‹‰Š‡•–ˆ‘Ž†Ǧ Šƒ‰‡•ǡ‡˜‡—’–‘ʹͶǦˆ‘Ž†ǡŠƒ†ƒ•—‘ˆ ƒ”„‘ƒ–‘• ‹–Š‡ƒ ›Ž Šƒ‹•‹–Š‡”ƒ‰‡ͶͲǦͶͺȋ ‹‰—”‡ͶǦ ȌǤŠ‹••—‰‰‡•–•–Šƒ–‘•–‘ˆ–Š‡ ‘–ƒ‹‡† ƒ–Ž‡ƒ•–‘‡ƒ ›Ž Šƒ‹•Š‘”–‡”–Šƒͳ͸Ǥ—„•‡“—‡–Ž›ǡ•–”— –—”ƒŽƒƒŽ›•‹•”‡˜‡ƒŽ‡†–Š‡ƒ ›ŽǦ Šƒ‹ ‘’‘•‹–‹‘‘ˆ͵ͳ‘—–‘ˆ–Š‡ͶʹŽ‹’‹†•™Š‹ Š™‡”‡•‹‰‹ˆ‹ ƒ–Ž›‡Ž‡˜ƒ–‡†‹Ǧ ‹ ‡ ȋ—’’‡” ”‹‰Š– “—ƒ†”ƒ–ǡ ‹‰—”‡ ͶǤ Š‡ sn-’‘•‹–‹‘ ‘ˆ –Š‡ ƒ ›Ž Šƒ‹• ‘—Ž† ‘– „‡ †‡–‡”‹‡†Ǥ˜‡”ͳ͵Ͳ ‹•‘‡”•ƒ†ͳ ™‡”‡ƒ‘–ƒ–‡†ȋ—’’Ž‡‡–ƒ”›–ƒ„Ž‡͵ȌǤͻͷΨ ‘ˆ–Š‡‹†‡–‹ˆ‹‡† ‹•‘‡”• ‘–ƒ‹‡†ƒ–Ž‡ƒ•–‘‡ˆƒ––›Ǧƒ ›Ž Šƒ‹™‹–Šƒ ƒ”„‘—„‡”‹ –Š‡”ƒ‰‡͸–‘ͳͶǤʹ͸Ψ‘ˆ–Š‡•‡ ‘–ƒ‹‡†ƒ•‡ ‘†ͳͶˆƒ––›ƒ ›ŽǦ Šƒ‹ȋ ‹‰—”‡Ͷ ȌǤŠ‡ ƒŒ‘”‹–›‘ˆ • ‘–ƒ‹‡†ƒ–Ž‡ƒ•–‘‡ Šƒ‹ˆƒ––›ƒ ›ŽǦ”‡•‹†—‡tͳ͸ǤŠ‡†‡ˆ‹ ‹‡ ›‘ˆ–Š‡ ‡œ›‡‘•–Ž›ƒˆˆ‡ –•–Š‡‘š‹†ƒ–‹‘‘ˆˆƒ––›ƒ ‹†•‘ˆ Šƒ‹Ž‡‰–Š͸ǦͳͲǤ

ͳ͵Ͷ Effect of fasting and cold stress on hepatic metabolism in MCAD-KO mice

A B C 10 6 TG (240) WT n.s. PC (124) KO TG DG (80) 5 PE (49) 4 Other SP (48) PS (18) 0 CE (18) MG (13) 2 PC2 (25.5%) PG (12) -5 (p-value) -log PI (11) CL (11) -10 0 Total=624 -10 -5 0 5 10 -5 0 5 PC1 (35%) log2(fold change) 4 D E F GH TG(48:8) TG(44:2) TG(42:4) TG(42:2) DG(28:2)

1.5 100 1.8 12 1.5 )

6 **** **** *** **** ****

10 80 ˜ 1.0 1.2 1.0 60 6 40 0.5 0.6 0.5 20 Liver TG (n.s.i. TG Liver 0.0 0 0.0 0 0.0 WT KO WT KO WT KO WT KO WT KO I TG composition

C6 C8 100 C9 C10 C12 50 C14 C15

Number ofNumber isomers C16 0 >C16 a b c  Figure 4. Hepatic lipidomics profile of 8-weeks old mice. ‹‡ Šƒ”–‘ˆ‹†‡–‹ˆ‹‡†Ž‹’‹† Žƒ••‡•ȋAȌǡ ’Ž‘–‘ˆ‹†‡–‹ˆ‹‡†Ž‹’‹†•’‡ ‹‡•ˆ‘”Ž‹˜‡”ǡ™‹–Šƒ†‹ ‡†‡’‹ –‡†‹”‡†ƒ†„Žƒ ǡ”‡•’‡ –‹˜‡Ž›ȋBȌǡ ‘Ž ƒ‘’Ž‘–‘ˆ–Š‡‹†‡–‹ˆ‹‡†Ž‹’‹†•’‡ ‹‡•ˆ‘”˜•ȋCȌǤ‹’‹†•ˆ‘”™Š‹ Š–Š‡–Š”‡•Š‘Ž†‘ˆ’δͲǤͲͷ™‡”‡ ‘–”‡ƒ Š‡†ƒ”‡†‡’‹ –‡†‹„Žƒ Ǥ ‘”–Š‡‘–Š‡”Ž‹’‹†•ǡ •ƒ”‡†‡’‹ –‡†‹”‡†ƒ†–Š‡”‡ƒ‹‹‰Ž‹’‹†•‹ „Ž—‡—‡›’Ž‘–•‘ˆ–Š‡–‘’ͷŽ‹’‹†•ȋ™‹–Š–Š‡Žƒ”‰‡•–ƒ„—†ƒ ‡†‹ˆˆ‡”‡ ‡„‡–™‡‡ƒ†ȋD-HȌǡƒ† –Š‡ƒ ›ŽǦ Šƒ‹ ‘’‘•‹–‹‘‘ˆ†‹ˆˆ‡”‡–‹ƒŽŽ›‡š’”‡••‡† ‹•‘‡”•ȋIȌǤ‹’‹†•‹’ƒ‡ŽI™‡”‡ƒ‘–ƒ–‡†ƒ• ȋǣȌǡ™Š‡”‡‹†‹ ƒ–‡•Ž‹’‹† Žƒ••ǡ–Š‡–‘–ƒŽ ƒ”„‘—„‡”••—‡†‘˜‡”ƒŽŽƒ ›Ž Šƒ‹•ǡƒ†–Š‡ —„‡”‘ˆ†‘—„Ž‡„‘†•ȏ͵͸ȐǤŠ‡ƒ ›Ž Šƒ‹•‹‡ƒ Š‹•‘‡”™‡”‡‘”†‡”‡†ˆ”‘ƒ–‘ ǡˆ”‘•Š‘”–‡•––‘ Ž‘‰‡•– Šƒ‹Ž‡‰–ŠǤ•ǣ‘–•‹‰‹ˆ‹ ƒ–ǡȗȗȗ’δͲǤͲͲͳǡȗȗȗȗ’δͲǤͲͲͲͳǤαͺ’‡”‰”‘—’ǤǤ•Ǥ‹Ǥ‘”ƒŽ‹œ‡†•‹‰ƒŽ ‹–‡•‹–›Ǥ ‡‡ ‹‰—”‡ ͳ ˆ‘” ˆ—”–Š‡” ‡š’Žƒƒ–‹‘ ‘ˆ ’Ž‘–•Ǥ ‡‡ —’’Ž‡‡–ƒ”› ƒ„Ž‡ ʹ ˆ‘” ƒ††‹–‹‘ƒŽ Ž‹’‹†‘‹ ”‡•—Ž–•Ǥ  Š‡Ž‹’‹†‘‹ •”‡•—Ž–••—‰‰‡•––Šƒ–•‘‡‘ˆ–Š‡ƒ„‡””ƒ–‡†‹—Ǧ Šƒ‹ˆƒ––›ƒ ›ŽǦ”‡•‹†—‡• ™‡”‡‹ ‘”’‘”ƒ–‡†‹–‘ •ǡ‡‹–Š‡”†‹”‡ –Ž›‘”‹†‹”‡ –Ž›˜‹ƒ’ƒ”–‹ƒŽ Šƒ‹‡Ž‘‰ƒ–‹‘ǡ–Š‡”‡„› ‘–”‹„—–‹‰–‘–Š‡’”‡˜‡–‹‘‘ˆ–Š‡‹”ƒ ——Žƒ–‹‘‹–Š‡ ‡ŽŽ•Ǥ  Zooming into an MCAD-KO mouse with a severe liver phenotype ‹ƒŽŽ›ǡ ™‡ •–—†‹‡† –Š‡  ‘—•‡ ™‹–Š –Š‡ ‘•– ‡š–”‡‡ ’Š‡‘–›’‡ ‹ ‘”‡ †‡’–Šǡ ‘•‹†‡”‹‰–Šƒ–‹–ƒ›„‡ƒ‘†‡Žˆ‘”ƒŠ—ƒ’ƒ–‹‡– Ž‘•‡–‘ƒ‡–ƒ„‘Ž‹  ”‹•‹•Ǥ‡ ˆ‘ —•‡† ‘ •‡˜‡”‡ Š‡’ƒ–‘•–‡ƒ–‘•‹• ȋ‘„•‡”˜‡† ‹  ’ƒ–‹‡–•™Š‘ †‹‡† ‘ˆ ƒ ‡–ƒ„‘Ž‹  ”‹•‹• ȏ͵͹ȐȌ ƒ† ‘ –Š‡ ‡ˆˆ‡ – ‘ˆ ‘Ž† ‡š’‘•—”‡ ‘ „Ž‘‘† ‰Ž— ‘•‡Ǥ ”‘ “—ƒŽ‹–ƒ–‹˜‡ ƒ† “—ƒ–‹–ƒ–‹˜‡Š‡’ƒ–‘Š‹•–‘Ž‘‰‹ ƒŽƒƒŽ›•‡•ƒ†„Ž‘‘†‰Ž— ‘•‡ Šƒ‰‡†—”‹‰ ‘Ž†‡š’‘•—”‡ǡ™‡ ‹†‡–‹ˆ‹‡†ƒ‘—•‡ȋͳͲȌ™‹–Š„‘–Š•‡˜‡”‡•–‡ƒ–‘Š‡’ƒ–‹–‹•ȋi.e.‹ˆŽƒƒ–‹‘‘ˆ–Š‡Ž‹˜‡”

ͳ͵ͷ Chapter 4

†—‡–‘ˆƒ–ƒ ——Žƒ–‹‘Ȍȋ ‹‰—”‡ͷƒ†—’’Ž‡‡–ƒ”›–ƒ„Ž‡ͶȌƒ† ‘Ž†Ǧ‹†— ‡††‡ ”‡ƒ•‡ ‘ˆ–Š‡„Ž‘‘†‰Ž— ‘•‡ ‘ ‡–”ƒ–‹‘ǤŠ‹•’Š‡‘–›’‡™ƒ••’‡ ‹ˆ‹ ˆ‘”ͳͲǡ•‹ ‡‡‹–Š‡”ƒ Š‡’ƒ–‘’ƒ–Š‘Ž‘‰‹ ƒŽ ’Š‡‘–›’‡ ȋ—’’Ž‡‡–ƒ”› ˆ‹‰—”‡ ͶȌ ‘” ƒ ”‡†— –‹‘ ‘ˆ –Š‡ „Ž‘‘† ‰Ž— ‘•‡ ‘ ‡–”ƒ–‹‘†—”‹‰ ‘Ž†‡š’‘•—”‡ȋ—’’Ž‡‡–ƒ”›ˆ‹‰—”‡ͶȌ™ƒ•–›’‹ ƒŽ‘ˆ–Š‡ Ǧ‰”‘—’ǤͳͲ™ƒ•–Š‡•ƒŽŽ‡•–‘—•‡„‡ˆ‘”‡ ‘Ž†‡š’‘•—”‡ȋͳͺǤͶ‰Ȍƒ†Šƒ†–Š‡ Ž‘™‡•–„‘†›–‡’‡”ƒ–—”‡ƒ––‡”‹ƒ–‹‘ȋʹͳǤ͵ιȌǤ™‘Ǧ†‹‡•‹‘ƒŽ Ž—•–‡”‹‰‘ˆ•‡Ž‡ –‡† •‡–•‘ˆ‘ǦŽ‹’‹†‘‹ ‡–ƒ„‘Ž‹–‡•ȋ ˆǤƒ–‡”‹ƒŽ•ƒ†‡–Š‘†•Ȍ•Š‘™‡†–Šƒ–’ƒ”–‹ —Žƒ”Ž›–Š‡ ‘ ‡–”ƒ–‹‘•‘ˆŠ‡’ƒ–‹ ƒ‹‘ƒ ‹†•ƒ†Š‡’ƒ–‹ ˆ”‡‡ƒ†–‘–ƒŽ ƒ”‹–‹‡™‡”‡–Š‡Ž‘™‡•–‹ ͳͲ ‘’ƒ”‡† –‘ –Š‡ ‘–Š‡” Ǧ ‹ ‡ ȋ ‹‰—”‡ ͷȌǤ  ŽŽ –Š‡ ƒ‹‘ ƒ ‹†• ‹ –Š‡ ‘””‡•’‘†‹‰ Ž—•–‡”ǡ ‡š ‡’– ˆ‘” Ž‡— ‹‡ǡ ƒ”‡ ‰Ž— ‘‡‘‰‡‹ Ǥ Š‹• •—‰‰‡•–• ƒ •Š‘”–ƒ‰‡ ‘ˆ •—„•–”ƒ–‡ˆ‘”‰Ž— ‘‡‘‰‡‡•‹•ƒ†‹•‹ƒ‰”‡‡‡–™‹–Š–Š‡Ž‘™‡”„Ž‘‘†‰Ž— ‘•‡Ž‡˜‡Ž‹–Š‹• ‘—•‡Ǥ ƒ††‹–‹‘ǡ–Š‡‘—•‡Šƒ•–Š‡Ž‘™‡•– ‘ ‡–”ƒ–‹‘•‘ˆ‡–Š‹‘‹‡ǡŠ‹•–‹†‹‡ƒ† Ǧ ›•–ƒ–Š‹‘‡ǡƒŽŽ‘ˆ™Š‹ Š ‘–”‹„—–‡–‘–Š‡ƒ–‹‘š‹†ƒ– ƒ’ƒ ‹–›Ǥ ‹ƒŽŽ›ǡ–Š‹•‘—•‡Šƒ†–Š‡ Ž‘™‡•–ƒ•’ƒ”–ƒ–‡ƒ†•‡”‹‡Ž‡˜‡Ž•‘ˆƒŽŽǦ‹ ‡ǤŠ‡•‡ƒ‹‘ƒ ‹†•’”‘˜‹†‡„—‹Ž†‹‰ „Ž‘ •ˆ‘”’—”‹‡•ǡ™Š‹ Šƒ”‡‹–—”„—‹Ž†‹‰„Ž‘ •ˆ‘”„‘–Š‘ ƒ†ΪǤ‘–Š‘  ƒ† Ϊ ƒ”‡ ‹’‘”–ƒ– ‘ˆƒ –‘”• ˆ‘”   ƒ† ƒ”‡ ƒŽ•‘ ‹˜‘Ž˜‡† ‹ ƒ–‹‘š‹†ƒ– ƒ† †‡–‘š‹ˆ‹ ƒ–‹‘‡ Šƒ‹••Ǥ  Discussion  –Š‹• •–—†›ǡ ™‡ ‹˜‡•–‹‰ƒ–‡† Ž‹˜‡” ‡–ƒ„‘Ž‹• ‹   †‡ˆ‹ ‹‡– Ǧ ‹ ‡ †—”‹‰ˆƒ•–‹‰ƒ† ‘Ž† ŠƒŽŽ‡‰‡ǡƒ•ƒ‘†‡Žˆ‘”•‡˜‡”‡‡‡”‰‡–‹ •–”‡••Ǥ–‹Ž‘™ǡǦ ‹ ‡™‡”‡ˆ‘—†–‘Šƒ˜‡ƒ”‡Žƒ–‹˜‡Ž›‹Ž†’Š‡‘–›’‡ƒ†–‘ƒ‹–ƒ‹‘”ƒŽ„Ž‘‘†‰Ž— ‘•‡ Ž‡˜‡Ž•†—”‹‰ˆƒ•–‹‰Ǥ”‡˜‹‘—•‡“ƒ†’”‘–‡‘‹ ƒƒŽ›•‹•‘ˆŽ‹˜‡”–‹••—‡‘ˆˆƒ•–‡†Ǧ Ž‹˜‡”–‹••—‡‹ ‡•Š‘™‡†‘Ž›‹‘”ƒ†ƒ’–ƒ–‹‘•ȏƒ”–‹‡•‡–ƒŽǤScientific Reportsǡ—†‡” ”‡˜‹•‹‘Ȑǡ•—‰‰‡•–‹‰–Šƒ––Š‡‘”ƒŽ ƒ’ƒ ‹–›‘ˆ•Š‘”–Ǧƒ†Ž‘‰Ǧ Šƒ‹‹•‘‡œ›‡•ȋǡ ǡȌ™ƒ••—ˆˆ‹ ‹‡––‘‡‡––Š‡Š‡’ƒ–‹ ‡‡”‰›†‡ƒ††—”‹‰ˆƒ•–‹‰ǤŠ‡‘”‡ •‡˜‡”‡‡‡”‰‡–‹ •–”‡••‹’‘•‡†‹–Š‹••–—†› Ȃƒ ‘„‹ƒ–‹‘‘ˆˆƒ•–‹‰ƒ† ‘Ž†•–”‡•• Ȃ ”‡˜‡ƒŽ‡†ˆ‘”–Š‡ˆ‹”•––‹‡•‹‰‹ˆ‹ ƒ–Ž›Ž‘™‡”„Ž‘‘†‰Ž— ‘•‡Ž‡˜‡Ž•‹Ǧ ‘’ƒ”‡†–‘ ‹ ‡Ǥ  ‘–”ƒ•––‘ƒ‡ƒ”Ž‹‡”•–—†›ȏͳͲȐǡ™‡†‹†‘–Ž‡ƒ˜‡–Š‡‹ ‡‹–Š‡ ‘Ž†—–‹Ž–Š‡› †‹‡†ǡ•‹ ‡™‡™‡”‡‹–‡”‡•–‡†‹–Š‡‹”‡–ƒ„‘Ž‹ ”‡•’‘•‡„‡ˆ‘”‡–Š‡‡‡”‰‡–‹ •–”‡••™‘—Ž† „‡Ž‡–ŠƒŽǤ’ƒ”–ˆ”‘–Š‡Ž‘™‡”„Ž‘‘†‰Ž— ‘•‡Ž‡˜‡Ž•ǡ ‘’ƒ”‡†–‘–Š‡‹ ‡ǡ–Š‡Ǧ ‹ ‡•Š‘™‡†Ž‘™‡”ƒ‹‘Ǧƒ ‹†Ž‡˜‡Ž•‹–Š‡Ž‹˜‡”„—–‘–‹„Ž‘‘†ǡŠ‹‰Š‡”„Ž‘‘† Ž‡˜‡Ž•ǡ ƒ†Š‹‰Š‡”•–‘”ƒ‰‡‘ˆ • ‘–ƒ‹‹‰͸ǦͳͶƒ ›Ž Šƒ‹•‹–Š‡Ž‹˜‡”Ǥ –Š‹••‡ –‹‘™‡™‹ŽŽ ‹–‡”’”‡–‘—”ˆ‹†‹‰•‹–Š‡Ž‹‰Š–‘ˆ™Šƒ–‹•‘™ƒ„‘—––Š‡”‘Ž‡‘ˆ–Š‡Ž‹˜‡”†—”‹‰ ‘Ž† •–”‡••ǤŠ‡™‡™‹ŽŽ†‹• —••Š‘™–Š‡‘„•‡”˜‡††‹ˆˆ‡”‡ ‡•‹ƒ‹‘ƒ ‹†ƒ†Ž‹’‹†‡–ƒ„‘Ž‹• „‡–™‡‡ –Š‡  ƒ†  ‹ ‡ ‘„˜‹ƒ–‡ –Š‡ ‡–ƒ„‘Ž‹  ‘•‡“—‡ ‡• ‘ˆ  †‡ˆ‹ ‹‡ ›Ǥ ‹ƒŽŽ›ǡ™‡™‹ŽŽ†‹• —••–Š‡‹’ƒ –‘ˆ‘—”ˆ‹†‹‰•ˆ‘”’ƒ–‹‡–’ƒ–Š‘’Š›•‹‘Ž‘‰›Ǥ ‘•–•–—†‹‡•‘–Š‡”‘Ž‡‘ˆ †—”‹‰ ‘Ž†•–”‡••ˆ‘ —•‘ǡ–Š‡’”‹ƒ”›–‹••—‡ ”‡•’‘•‹„Ž‡ˆ‘”‘Ǧ•Š‹˜‡”‹‰–Š‡”‘‰‡‡•‹•ȏͳͶȂͳ͸ǡʹͲȐǤŠ‡”‡‹•‡˜‹†‡ ‡ǡŠ‘™‡˜‡”ǡˆ‘”ƒ †‹”‡ –”‘Ž‡‘ˆ—• Ž‡ȏ͵ͺȐƒ†Ž‹˜‡”ȏ͵ͻȐ‹‘Ǧ•Š‹˜‡”‹‰–Š‡”‘‰‡‡•‹•ƒ•™‡ŽŽǤŠ‡”‘Ž‡‘ˆ –Š‡Ž‹˜‡”–‘•—’’Ž›—–”‹‡–•ˆ‘”ˆ— –‹‘Šƒ•Šƒ”†Ž›„‡‡•–—†‹‡†ǡ„—–”‡ ‡–Ž›‰ƒ‹‡† ”‡‡™‡†‹–‡”‡•–ȏͳͻǡʹͲȐǤ‹ ‘š‡–ƒŽǤȏͳͻǡʹͲȐ”‡˜‡ƒŽ‡†Š‘™ ‘Ž†‡š’‘•—”‡‡Šƒ ‡•Ž‹’‘Ž›•‹• ‹™Š‹–‡ƒ†‹’‘•‡–‹••—‡ƒ†•—’’Ž‹‡•–Š‡Ž‹˜‡”™‹–Š •Ǥ

ͳ͵͸ Effect of fasting and cold stress on hepatic metabolism in MCAD-KO mice

A H&E ORO

KO typical

KO severe (KO10) 4 B

Ń Severe (KO10)  Figure 5. KO individual with extreme phenotype. ‹•–‘Ž‘‰‹ ƒŽ”‡’”‡•‡–ƒ–‹‘ ‘ˆƒ”‡’”‡•‡–ƒ–‹˜‡ ‘—•‡Ž‹˜‡”ƒ†‘ˆ–Š‡Ž‹˜‡”‘ˆ–Š‡‘—•‡™‹–Š–Š‡‘•–‡š–”‡‡’Š‡‘–›’‡ȋͳͲȌȋAȌƒ†Š‡ƒ–ƒ’‘ˆ ƒ•‡Ž‡ –‡†•‡–‘ˆ‡–ƒ„‘Ž‹–‡•‹ͺǦ™‡‡•‘Ž†Ǧ‹ ‡ȋBȌǤ‹˜‡”•™‡”‡•–ƒ‹‡†„› ƒ‡‘–‘š›Ž‹ƒ† ‘•‹ȋŽ‡ˆ–Ȍƒ†‹Ž‡†ȋ”‹‰Š–ȌǤ 

ͳ͵͹ Chapter 4

 –—”ǡ –Š‡  • ƒ –‹˜ƒ–‡ Ž‹˜‡”   ƒ† ‡Šƒ ‡ •‡ ”‡–‹‘ ‘ˆ ƒ ›ŽǦ ƒ”‹–‹‡• ‹–‘ –Š‡ „Ž‘‘†•–”‡ƒǤŠ‡•‡ƒ ›ŽǦ ƒ”‹–‹‡•’”‘˜‡†–‘„‡ ”‹–‹ ƒŽ•—„•–”ƒ–‡•ˆ‘”–Š‡”‘‰‡‡•‹•„› ȏͳͻǡʹͲȐǤ ‘—”‹ ‡ǡMcad†‡ˆ‹ ‹‡ ›Ž‡†–‘Š‹‰Š‡”Ž‡˜‡Ž•‘ˆƒ ›ŽǦ ƒ”‹–‹‡•‹–Š‡„Ž‘‘†Ǥ –‹• Ž‹‡Ž›–Šƒ––Š‡›•‡”˜‡ƒ••—„•–”ƒ–‡•ˆ‘”ǡ‡˜‡–Š‘—‰Š–Š‡›ƒ›„‡‘Ž›’ƒ”–Ž›‘š‹†‹œ‡††—‡ –‘–Š‡Mcad†‡ˆ‹ ‹‡ ›ǤŠ‹Ž‡™‡Šƒ˜‡‘‹ˆ‘”ƒ–‹‘ƒ„‘—–‡†‹—Ǧ Šƒ‹  ƒ’ƒ ‹–›‹ ‘ˆ–Š‡Mcad-†‡ˆ‹ ‹‡–‹ ‡ǡ‹–Šƒ•„‡‡”‡’‘”–‡†–Šƒ– ‰‡‡•ǡ‹ Ž—†‹‰Acadvl, Acadl and Acadm‰‡‡•ǡƒ”‡—’”‡‰—Žƒ–‡†‹ƒ†Ž‹˜‡”•‘ˆǦƒ†Ǧ‹ ‡–Šƒ–Šƒ˜‡ „‡‡ˆƒ•–‡†ƒ† ‘Ž†Ǧ‡š’‘•‡†ȏʹʹȐǤ Š‡–‹‡ ‘—”•‡•‘ˆ–Š‡„‘†›–‡’‡”ƒ–—”‡•Ȃ™‹–Š•‘‡‹ ‡•Š‘™‹‰‹‹ƒŽƒ† •‘‡•—„•–ƒ–‹ƒŽ–‡’‡”ƒ–—”‡†”‘’•‹„‘–Š‰”‘—’•ȋ—’’Ž‡‡–ƒ”›ˆ‹‰—”‡•ͳǦȌȂ•—‰‰‡•– –Šƒ–™‡ƒ”‡ƒ„Ž‡–‘„”‹‰‘Ž›ƒ•—„•‡–‘ˆǦ‹ ‡–‘–Š‡„”‹‘ˆƒ‡–ƒ„‘Ž‹  ‘ŽŽƒ’•‡Ǥ Š‹•™‘—Ž†‡š’Žƒ‹™Š›™‡‘„•‡”˜‡ƒ˜ƒ•–Š‡–‡”‘‰‡‡‹–›‘ˆ„‘†›–‡’‡”ƒ–—”‡•ƒ––Š‡‡†‘ˆ –Š‡ ‘Ž† ’‡”‹‘† ȋ ‹‰—”‡• ͳ ƒ† ȌǤ  –Š‹• ‘†‹–‹‘ǡ ™‡ ‘„•‡”˜‡† ƒ”‡† †‹ˆˆ‡”‡ ‡• ‹ ƒ‹‘Ǧƒ ‹†ƒ†Ž‹’‹†‡–ƒ„‘Ž‹•„‡–™‡‡–Š‡ƒ†‹ ‡ǤŽ‹‹–ƒ–‹‘‘ˆ–Š‹••–—†›‹• –Šƒ–™‡†‹†‘–‡ƒ•—”‡‡–ƒ„‘Ž‹ ˆŽ—š‡•„›‹•‘–‘’‡Žƒ„‡ŽŽ‹‰Ǥ ‘™‡˜‡”ǡ–Š‡ ‘’”‡Š‡•‹˜‡ ƒƒŽ›•‹• ‘ˆ „Ž‘‘† ƒ† Ž‹˜‡” ‡–ƒ„‘Ž‹–‡• ƒŽ”‡ƒ†› ›‹‡Ž†• ‹–‡”‡•–‹‰ ‹•‹‰Š–• ‹ ‡–ƒ„‘Ž‹  ƒ†ƒ’–ƒ–‹‘•ǡƒ•†‹• —••‡†„‡Ž‘™Ǥ Š‡–Š‡‘š‹†ƒ–‹‘‘ˆˆƒ––›ƒ ‹†•‹•†‡ˆ‹ ‹‡–ǡƒ‹‘ƒ ‹†•ƒ›•‡”˜‡ƒ•ƒƒŽ–‡”ƒ–‹˜‡ •—„•–”ƒ–‡ˆ‘”‘š‹†ƒ–‹‘ƒ†’”‘†— –‹‘Ǥ’‡ ‹ˆ‹ ‡–‘‰‡‹ ƒ‹‘ƒ ‹†•ȋ‘–ƒ„Ž›Ž‡— ‹‡ ƒ†Ž›•‹‡Ȍ ƒƒŽ•‘•‡”˜‡–‘”‡’Žƒ ‡ˆƒ––›ƒ ‹†•ƒ•ƒ•—„•–”ƒ–‡ˆ‘”‡–‘‡Ǧ„‘†›’”‘†— –‹‘ †—”‹‰ˆƒ•–‹‰Ǥ‘•‹•–‡–™‹–Š•— Š‹ ”‡ƒ•‡†—–‹Ž‹œƒ–‹‘ǡ‘•–ƒ‹‘ƒ ‹† ‘ ‡–”ƒ–‹‘•ǡ ‹ Ž—†‹‰Ž‡— ‹‡ƒ†Ž›•‹‡ǡ™‡”‡•—„•–ƒ–‹ƒŽŽ›Ž‘™‡”‹ǦŽ‹˜‡”• ‘’ƒ”‡†–‘ ‹ ‡ǡ’ƒ”–‹ —Žƒ”Ž›‹›‘—‰‹ ‡ȋ ‹‰—”‡͵ƒ†—’’Ž‡‡–ƒ”›ˆ‹‰—”‡͵ȌǤŽ‘‘†‡–‘‡Ǧ „‘†› Ž‡˜‡Ž• ™‡”‡ ‘– ƒŽ–‡”‡† ‹ Ǧ ‹ ‡ǡ •—‰‰‡•–‹‰ –Šƒ– ƒ‹‘ ƒ ‹†• ‡ˆˆ‹ ‹‡–Ž› ”‡’Žƒ ‡† ˆƒ––› ƒ ‹†• ƒ• •—„•–”ƒ–‡• ˆ‘” ‡–‘‰‡‡•‹•Ǥ Š‡ ˆƒ – –Šƒ– ’Žƒ•ƒ ƒ‹‘ ƒ ‹†• ™‡”‡ ‘”ƒŽ‹–Š‡Ǧ‹ ‡ǡ•—‰‰‡•–•–Šƒ––Š‡Š‡’ƒ–‹ ƒ‹‘Ǧƒ ‹††‡ˆ‹ ‹–‘”‹‰‹ƒ–‡•‹–Š‡ Ž‹˜‡”ƒ†ƒ›„‡ ƒ—•‡†„›ƒ‹ ”‡ƒ•‡††‡ƒ†Ǥ Š‡ Š‹‰Š‡” ’Žƒ•ƒ   ‘ ‡–”ƒ–‹‘• ‹ Ǧ ‘’ƒ”‡† –‘  ‹ ‡ ‹• ‘’ƒ–‹„Ž‡ ™‹–Š ‘Ž†Ǧ‹†— ‡† Ž‹’‘Ž›•‹• ȏͳͻǡʹͲȐ ‹ ‘„‹ƒ–‹‘ ™‹–Š ƒ ”‡†— ‡† ˆƒ––›Ǧƒ ‹† —–‹Ž‹œƒ–‹‘†—‡–‘–Š‡†‡ˆ‹ ‹‡ ›Ǥ†‡ˆ‹ ‹‡ › ‘ˆ‡”•ƒ”‹•‘ˆƒ ——Žƒ–‹‘‘ˆ ‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘‡•–‡”•ƒ††‡’Ž‡–‹‘‘ˆ‘Ǥ –Šƒ•„‡‡Š›’‘–Š‡•‹œ‡†–Šƒ––Š‹•ƒ› ƒ—•‡ –Š‡ ‡–ƒ„‘Ž‹  ‘ŽŽƒ’•‡ ‘ˆ  ’ƒ–‹‡–• ȏͶͲȂͶʹȐǤ –‡”‡•–‹‰Ž›ǡ ƒŽŽ Ž‹’‹†‘‹  ƒŽ–‡”ƒ–‹‘•–Šƒ–™‡‘„•‡”˜‡‹Ǧ‹ ‡ǡƒ›„‡‹–‡”’”‡–‡†ƒ•ƒ’”‘–‡ –‹‘ƒ‰ƒ‹•–•— Š ‘†‡’Ž‡–‹‘Ǥ ‹”•–ǡ™‡‘„•‡”˜‡†–Šƒ–Ž‹˜‡”ƒ ›ŽǦ ƒ”‹–‹‡•™‡”‡‘”ƒŽ‹Ǧ‹ ‡Ǥ  ›ŽǦ ƒ”‹–‹‡•ƒ”‡‘ˆ–‡˜‹‡™‡†ƒ•ƒ”‡ˆŽ‡ –‹‘‘ˆ–Š‡‹” ‘‰ƒ–‡ƒ ›ŽǦ‘‡•–‡”•ǤŠ‹•™‘—Ž† •—‰‰‡•––Šƒ–Ǧ‹ ‡ƒ‹–ƒ‹ƒ ›ŽǦ‘Š‘‡‘•–ƒ•‹•—†‡” ‘Ž†•–”‡••Ǥ‡ ‘†ǡ™‡ ‘„•‡”˜‡†‹ ”‡ƒ•‡†ƒ ›Ž ƒ”‹–‹‡Ž‡˜‡Ž•‹–Š‡„Ž‘‘†‘ˆǦ‹ ‡ȋ ‹‰—”‡ʹǦȌǤŠ‹•‹• ‘’ƒ–‹„Ž‡™‹–Š‡Šƒ ‡† ‘Ž†Ǧ‹†— ‡†ƒ ›Ž ƒ”‹–‹‡•‡ ”‡–‹‘ˆ”‘–Š‡Ž‹˜‡”ȏͳͻǡʹͲȐƒ† ™‘—Ž† ‘–”‹„—–‡–‘ƒŽŽ‡˜‹ƒ–‹‰Š‡’ƒ–‹ ƒ ›ŽǦ ƒ”‹–‹‡ƒ†ƒ ›ŽǦ‘ƒ ——Žƒ–‹‘Ǥ  ‘–”ƒ•–ǡ Ǧ‹ ‡–Šƒ–™‡”‡‘Ž›ˆƒ•–‡†ǡ„—–‘–‡š’‘•‡†–‘ ‘Ž†ǡ†‹†‘–ƒ‹–ƒ‹Š‡’ƒ–‹ ƒ ›ŽǦ ƒ”‹–‹‡ Š‘‡‘•–ƒ•‹• ȏͶ͵Ȑǡ •—‰‰‡•–‹‰ –Šƒ– ƒ ›ŽǦ ƒ”‹–‹‡ •‡ ”‡–‹‘ †—”‹‰ ‘Ž†Ǧ‡š’‘•—”‡ ƒ††•–‘–Š‡Š‘‡‘•–ƒ•‹•Ǥ‡š–ǡ™‡‘„•‡”˜‡†Š‹‰Š‡” Ž‡˜‡Ž•ǡ™Š‹ Š™ƒ•‘•–’”‘‘— ‡† ˆ‘” • ‘–ƒ‹‹‰͸ǦͳͶƒ ›Ž Šƒ‹•ȋ ‹‰—”‡ͶǦ ȌǤ  ‘”’‘”ƒ–‹‘‘ˆ‡š ‡••ƒ ›Ž‘‹‡–‹‡•‹  • ™‘—Ž† ƒŽ•‘ ƒ†† –‘ –Š‡ ’”‡˜‡–‹‘ ‘ˆ ƒ ›ŽǦ‘ ƒ ——Žƒ–‹‘ ƒ† •—„•‡“—‡– ‘ †‡’Ž‡–‹‘Ǥ͸ǦͳͲƒ ›ŽǦ‘‡•–‡”•ƒ”‡–Š‡’”‡ˆ‡””‡†•—„•–”ƒ–‡•‘ˆ–Š‡‡œ›‡ƒ†ƒ”‡

ͳ͵ͺ Effect of fasting and cold stress on hepatic metabolism in MCAD-KO mice

ƒ’’ƒ”‡–Ž›ƒŽ•‘”‡”‘—–‡†‹–‘ •›–Š‡•‹•ǤŠ‡‹ ”‡ƒ•‡†ͳʹǦͳͶƒ ›Ž ‘–‡–‘ˆ–Š‡ ’‘‘Ž ƒ„‡‡š’Žƒ‹‡†‡‹–Š‡”„›†‹”‡ –”‡”‘—–‹‰‘ˆ–Š‡•‡‹–‡”‡†‹ƒ–‡•ˆ”‘ ‘š‹†ƒ–‹‘‘”„› ’ƒ”–‹ƒŽ Šƒ‹‡Ž‘‰ƒ–‹‘‘ˆ͸ǦͳͲ‹–‡”‡†‹ƒ–‡•’”‹‘”–‘–Š‡‹”‹ ‘”’‘”ƒ–‹‘‹–‘ •Ǥ ‹ƒŽŽ›ǡ ™‡ ™‘†‡”‡† ™Šƒ– ‹• –Š‡ ”‡Ž‡˜ƒ ‡ ‘ˆ –Š‹• •–—†› ˆ‘” –Š‡ —†‡”•–ƒ†‹‰ ‘ˆ Š—ƒǤŠ‡ˆƒ ––Šƒ–‘Ž››‘—‰Ǧ‹ ‡‡šŠ‹„‹–‡†Ž‘™‡”„Ž‘‘†‰Ž— ‘•‡Ž‡˜‡Ž• –Šƒ–Š‡‹”™‹Ž†Ǧ–›’‡ ‘—–‡”’ƒ”–•†—”‹‰•‡˜‡”‡‡‡”‰‡–‹ •–”‡••ǡ•—‰‰‡•–•–Šƒ––Š‡›ƒ”‡ƒ‰‘‘† ‘†‡Žˆ‘”Š—ƒ†‹•‡ƒ•‡ǤŽ•‘‹Š—ƒǡ—–”‡ƒ–‡† Š‹Ž†”‡ƒ”‡–›’‹ ƒŽŽ›ƒ–”‹•‘ˆ Š›’‘‰Ž› ‡‹ƒ†—”‹‰–Š‡ˆ‹”•–›‡ƒ”•‘ˆŽ‹ˆ‡™Š‡ˆƒ•–‡†‹ ‘„‹ƒ–‹‘™‹–Šƒƒ††‹–‹‘ƒŽ 4 –”‹‰‰‡”ǡ „—– Ž‡•• ˜—Ž‡”ƒ„Ž‡ ™Š‡ –Š‡› ‰”‘™ ‘Ž†‡” ȏͶͶȐǤ  Š—ƒ  Š›’‘‡–‘–‹  Š›’‘‰Ž› ‡‹ƒ‹•‘•–‘ˆ–‡”‡’‘”–‡†–‘‘ —”—’‘ƒ ‘„‹ƒ–‹‘‘ˆˆƒ•–‹‰ƒ†‹–‡” —””‡– ‹ŽŽ‡••ȏͳǡ͸ȂͻȐǤŠ‹•‹•ǡŠ‘™‡˜‡”ǡ†‹ˆˆ‹ —Ž––‘‹‹ ‹–Š‡Žƒ„ǤŠ‡”‡ƒ”‡ƒŽ•‘”‡’‘”–•‘ˆ ‘Ž† •‡•‹–‹˜‹–›‘ˆ’ƒ–‹‡–•ȏͺǡ͵ͲǡͶͷȐǡƒ‹‰ ‘Ž†•–”‡••ƒ”‡Ž‡˜ƒ–ƒ†‡š’‡”‹‡–ƒŽŽ› ƒ ‡••‹„Ž‡ Žƒ„‘”ƒ–‘”› ‹–‡”˜‡–‹‘Ǥ ›’‘–Š‡•‹œ‹‰ –Šƒ– –Š‡ ‘—•‡ ™‹–Š –Š‡ ‘•– •‡˜‡”‡ ’Š‡‘–›’‡ ‘—Ž†›‹‡Ž†‹•‹‰Š–•‹–‘–Š‡’”‘ ‡••‡•†—”‹‰‘” Ž‘•‡–‘ƒ‡–ƒ„‘Ž‹  ”‹•‹•ǡ™‡ œ‘‘‡†‹‘Ǧ‘—•‡ͳͲȋͳͲȌǤŠ‹•‘—•‡•Š‘™‡†„‘–ŠŠ‡’ƒ–‘•–‡ƒ–‘•‹•ƒ†ƒ †‡ ”‡ƒ•‡ ‘ˆ „Ž‘‘† ‰Ž— ‘•‡ ‘ ‡–”ƒ–‹‘ †—”‹‰ ‘Ž† ‡š’‘•—”‡Ǥ Š‹• ‹• ‹ Ž‹‡ ™‹–Š Š‡’ƒ–‘•–‡ƒ–‘•‹•‘„•‡”˜‡†‹’ƒ–‹‡–•™Š‘†‹‡†ˆ”‘‡–ƒ„‘Ž‹  ”‹•‹•ȋ„‹‘ Š‡‹ ƒŽŽ› Šƒ”ƒ –‡”‹œ‡†„›Š›’‘‡–‘–‹ Š›’‘‰Ž› ‡‹ƒȌǤͳͲ•Š‘™‡†Ž‘™‡”Ž‡˜‡Ž•‘ˆ–Š‡ƒ‹‘ƒ ‹†• –Šƒ– ‘–”‹„—–‡–‘–Š‡ƒ–‹‘š‹†ƒ– ƒ’ƒ ‹–›Ǥ ‘”†‹‰Ž›ǡŽ‘™‡”ƒ–‹‘š‹†ƒ– ƒ’ƒ ‹–›Šƒ•„‡‡ ”‡’‘”–‡†‹’ƒ–‹‡–•ȏͶ͸ȐǤŠ‡”‡ˆ‘”‡ǡ™Š‡–Š‡„Ž‘‘†‰Ž— ‘•‡ ‘ ‡–”ƒ–‹‘‹ ‘Ž†Ǧ ‡š’‘•‡† Ǧ ‹ ‡ †”‘’•ǡ ƒ• ‘„•‡”˜‡† ‹ ͳͲǡ •— Š ‹ ‡ ƒ› „‡ ƒ†‡“—ƒ–‡ ƒ‹ƒŽ ‘†‡Ž•ˆ‘”–Š‡‘•‡–‘ˆ–Š‡‡–ƒ„‘Ž‹  ”‹•‹•‹Š—ƒ†‹•‡ƒ•‡Ǥ ‹ƒŽŽ›ǡͳͲŠƒ†–Š‡Ž‘™‡•– „‘†› –‡’‡”ƒ–—”‡ ƒ– –‡”‹ƒ–‹‘Ǥ‹ ‡ –‡’‡”ƒ–—”‡ ƒ „‡ ˆ‘ŽŽ‘™‡† ‘–‹—‘—•Ž› „› –Š‡ ‹’Žƒ–‡†Ž‘‰‰‡”•ǡ‹–•‡‡•ˆ‡ƒ•‹„Ž‡–‘—•‡‹–ƒ•ƒ”‡ƒ†‘—––‘•–—†›‹ ‡‹ƒ ‘–”‘ŽŽ‡†™ƒ› †—”‹‰‘”‡•‡˜‡”‡‡–ƒ„‘Ž‹  ”‹•‹•Ǥ ‡ ‘ Ž—†‡–Šƒ–›‘—‰ǡˆƒ•–‡†ǡƒ† ‘Ž†‡š’‘•‡†Ǧ‹ ‡ƒ”‡ƒ”‡Ž‡˜ƒ–‘†‡Žˆ‘”–Š‡ ‘•‡–‘ˆ‡–ƒ„‘Ž‹  ”‹•‹•‹’ƒ–‹‡–•Ǥ‡‘„•‡”˜‡†ƒ”‡†‡–ƒ„‘Ž‹ ƒŽ–‡”ƒ–‹‘•‹ Ž‹˜‡” ‡–ƒ„‘Ž‹• ‘ˆ ‘Ž†Ǧ‡š’‘•‡† ˆƒ•–‡† Ǧ ‹ ‡Ǥ Š‡ ”‡•—Ž–• ‘””‘„‘”ƒ–‡ ”‡ ‡– ˆ‹†‹‰• ‘ˆ –Š‡ ‹’‘”–ƒ ‡ ‘ˆ Š‡’ƒ–‹  ˆƒ––›Ǧƒ ‹† ‡–ƒ„‘Ž‹• †—”‹‰ ‘Ž† •–”‡•• ȏͳͻǡʹͲȐǤ Ž–‡”ƒ–‹‘•‹ƒ‹‘ƒ ‹†ƒ†Ž‹’‹†‡–ƒ„‘Ž‹•ƒ”‡‹Ž‹‡™‹–Šƒ ‘’‡•ƒ–‘”›”‡•’‘•‡‹ ™Š‹ Šƒ‹‘ƒ ‹†•’ƒ”–Ž›–ƒ‡‘˜‡”–Š‡”‘Ž‡‘ˆˆƒ––›ƒ ‹†•ƒ••—„•–”ƒ–‡•ˆ‘”ƒ†Ȁ‘”‡–‘‡ „‘†›•›–Š‡•‹•ǡƒ† •›–Š‡•‹••‡”˜‡•ƒ•ƒ•‹ˆ‘”‡š ‡••ƒ ›Ž‘‹‡–‹‡•ǡ–Š‡”‡„›’”‡˜‡–‹‰ †ƒ‰‡”‘—•ƒ ›ŽǦ‘ƒ ——Žƒ–‹‘ȏͶͲȂͶʹȐǤ

Materials and Methods , Experimental design and Tissue sampling ƒŽ‡Ǧƒ†™‹Ž†Ǧ–›’‡ȋȌ‹ ‡‘ƒͷ͹Ȁ͸ „ƒ ‰”‘—†ȋ„ƒ  ”‘••‡†ˆ‘”ͳͲ‰‡‡”ƒ–‹‘• ȏͶ͹ȐȌ™‡”‡Š‘—•‡†‹ƒ–‡’‡”ƒ–—”‡ǦȋʹͳιȌƒ†Ž‹‰Š–Ǧ ‘–”‘ŽŽ‡†ˆƒ ‹Ž‹–›Ǥ‹ ‡™‡”‡ͳ͸ǦʹͲ™‡‡•‘ˆ ƒ‰‡ȋ›‘—‰ƒ†—Ž–‹ ‡Ȍ‘”ͺ™‡‡•‘ˆƒ‰‡ȋ›‘—‰‹ ‡Ȍƒ––Š‡•–ƒ”–‘ˆ–Š‡‡š’‡”‹‡–ǤŽŽ‡š’‡”‹‡–• ™‡”‡ƒ’’”‘˜‡†„›–Š‡–Š‹ •‘‹––‡‡ˆ‘”‹ƒŽš’‡”‹‡–•‘ˆ–Š‡‹˜‡”•‹–›‘ˆ ”‘‹‰‡Ǥ”‹‘” –‘–Š‡•–ƒ”–‘ˆ–Š‡‡š’‡”‹‡–ǡ‹ ‡™‡”‡ˆ‡†ƒ Š‘™ǦŽ‹‡•‡‹Ǧ•›–Š‡–‹ †‹‡–ȋͳʹͶͷͲǡ‡•‡ƒ” Š‹‡– ‡”˜‹ ‡•ǡ‹Œ‹Œ——”•–‡†‡ǡŠ‡‡–Š‡”Žƒ†•Ȍˆ‘”ͶǦ͸™‡‡•ǡƒ†Šƒ†ˆ”‡‡ƒ ‡••–‘†”‹‹‰™ƒ–‡”Ǥ —„•‡“—‡–Ž›ǡ–Š‡‹ ‡™‡”‡ˆƒ•–‡†ˆ‘”ͳͶŠ”•‘˜‡”‹‰Š–ƒ†–Š‡”‡ƒˆ–‡”’Žƒ ‡†‹ƒͷ釐˜‹”‘‡– ˆ‘”ͶǦ͸Š”•ǤŠ‡‹ ‡™‡”‡–‡”‹ƒ–‡†„› ƒ”†‹ƒ ’— –—”‡—†‡”‹•‘ˆŽ—”ƒ‡ƒ‡•–Š‡•‹ƒǤ™‘™‡‡• „‡ˆ‘”‡ ˆƒ•–‹‰ǡ ‹‹ƒ–—”‹œ‡† ‹’‹ŽŽ ‡’‡”ƒ–—”‡ ’Žƒ– Ž‘‰‰‡”• ȋƒ–ƒ  ‹‡ ‡• –‡”ƒ–‹‘ƒŽǡ

ͳ͵ͻ Chapter 4

‹‡•‘–ƒǡ Ȍ ™‡”‡ ‹’Žƒ–‡† ‹–”ƒ’‡”‹–‘‡ƒŽŽ› —†‡” ‹•‘ˆŽ—”ƒ‡ ƒ† ƒƒŽ‰‡•‹ƒ ™‹–Š ˆŽ—‹š‹Ȁ‡‰Ž—‹‡ ȋʹ ‰Ȁ‰ȌǤ – –Š‡ ‡† ‘ˆ –Š‡ ‘Ž† ‡š’‘•—”‡ ƒ† „‡ˆ‘”‡ –‡”‹ƒ–‹‘ǡ –Š‡ „‘†› –‡’‡”ƒ–—”‡™ƒ•ƒŽ•‘‡ƒ•—”‡†™‹–Šƒ”‡ –ƒŽ’”‘„‡ȋŠ›•‹–‡’ •–”—‡–•ǡŽ‹ˆ–‘ǡ ǡȌǤ—”‹‰ –Š‡ ‡š’‡”‹‡–ǡ „Ž‘‘† ‰Ž— ‘•‡ ƒ† ‡–‘‡ „‘†› ‘ ‡–”ƒ–‹‘• ™‡”‡ ‡ƒ•—”‡† „› ƒ ‡‘— Š Ž–”ƒƒ•›̺‘‹–‘”ȋ‹ˆ‡ ƒ‡‡Ž—šǡ‡‡”•‡ǡ‡Ž‰‹—Ȍƒ†’–‹— ‡‡†‘‹–‘”ȋ„‘––‹ƒ„‡–‡• ƒ”‡–†Ȍǡ”‡•’‡ –‹˜‡Ž›Ǥˆ–‡”–‡”‹ƒ–‹‘–Š‡Ž‹˜‡”™ƒ•”‡‘˜‡†“—‹ Ž›ǡ™‡‹‰Š‡†ǡƒ†ƒ’‹‡ ‡‘ˆ –Š‡Ž‹˜‡”ˆ”‡‡œ‡Ǧ Žƒ’‡†Ǣƒ•‡ ‘†’‹‡ ‡™ƒ•‹‡”•‡†‹ͳͲΨ„—ˆˆ‡”‡†ˆ‘”ƒŽ‹ƒ†ƒ–Š‹”†’‹‡ ‡™ƒ• ˆ‹šƒ–‡†‹‹••—‡Ǧ‡̺ƒ†ˆ”‘œ‡ǤŽ‘‘†•’‘–•™‡”‡–ƒ‡‹‡†‹ƒ–‡Ž›„‡ˆ‘”‡ˆƒ•–‹‰ǡƒ–ͳͶŠ‘—”• ƒˆ–‡”–Š‡•–ƒ”–‘ˆˆƒ•–‹‰ȋ‹Ǥ‡Ǥ„‡ˆ‘”‡‡š’‘•‹‰–Š‡‹ ‡–‘ ‘Ž†Ȍƒ†ƒ––‡”‹ƒ–‹‘ȋƒˆ–‡” ‘Ž†‡š’‘•—”‡ȌǤ  Metabolite analysis ‘ †‡–‡”‹‡ Š‡’ƒ–‹  –”‹‰Ž› ‡”‹†‡ Ž‡˜‡Ž•ǡ ˆ”‡‡œ‡Ǧ Žƒ’‡† Ž‹˜‡” ™ƒ• ”—•Š‡† ‹ Ž‹“—‹† ‹–”‘‰‡Ǥ —„•‡“—‡–Ž›ʹͲΨȋ™Ȁ˜ȌŽ‹˜‡”Š‘‘‰‡ƒ–‡•™‡”‡’”‡’ƒ”‡†‹ȋ’ ͹ǤͶȌǤ‘–ƒŽŽ‹˜‡”Ž‹’‹†•™‡”‡ ‡š–”ƒ –‡†ƒ ‘”†‹‰–‘ȏͶͺȐǡ”‡†‹••‘Ž˜‡†‹ʹΨ”‹–‘ǦͳͲͲƒ†ƒƒŽ›•‡†™‹–Šƒ ‘‡” ‹ƒŽŽ›ƒ˜ƒ‹Žƒ„Ž‡ ‹– ˆ‘” –”‹‰Ž› ‡”‹†‡ ȋ‘ Š‡ ‹ƒ‰‘•–‹ •ǡ ƒŠ‡‹ǡ ‡”ƒ›Ȍ ƒ ‘”†‹‰ –‘ –Š‡ ƒ—ˆƒ –—”‡”ǯ• ‹•–”— –‹‘•Ǥ‹‘Ǧƒ ‹†Ž‡˜‡Ž•™‡”‡‡ƒ•—”‡†ƒ ‘”†‹‰–‘‘‘”‡et al.ȏͶͻȐǤ ›ŽǦ ƒ”‹–‹‡Ž‡˜‡Ž• ™‡”‡ ‡ƒ•—”‡† ƒ ‘”†‹‰ –‘ ‡”• et al. ȏͷͲȐǤ   ‘ ‡–”ƒ–‹‘• ™‡”‡ †‡–‡”‹‡† ™‹–Š ƒ ‘‡” ‹ƒŽŽ›ƒ˜ƒ‹Žƒ„Ž‡‹ƒ•›• ‹–ȋ‹ƒ•›•‹ƒ‰‘•–‹ ›•–‡• „ ǡ ‡”ƒ›ȌǤ  Liver lipid extraction ‹˜‡”–‹••—‡™ƒ•Š‘‘‰‡‹œ‡†‹ͲǤͻΨƒŽ–‘’”‡’ƒ”‡ƒʹͲΨȋ™Ȁ˜ȌŠ‘‘‰‡ƒ–‡Ǥ‡—•‡†ƒ‰”‘—’‘ˆ Ž‹’‹†• ȋ’—” Šƒ•‡† ˆ”‘ ˜ƒ–‹ ‘Žƒ” Ž‹’‹†•ǡ Žƒ„ƒ•–‡”ǡ Ȍ ƒ• ‹–‡”ƒŽ •–ƒ†ƒ”†• ˆ‘” ‘””‡ –‹‰ –Š‡ ˜ƒ”‹ƒ–‹‘‹Ž‹’‹†‡š–”ƒ –‹‘ƒ†ƒƒŽ›–‹ ƒŽƒƒŽ›•‹•Ǥ –‡”ƒŽ•–ƒ†ƒ”†• ‘–ƒ‹Ͷ‘Ž‘ˆ‡ƒ Š‘ˆ–Š‡ ˆ‘ŽŽ‘™‹‰ Ž‹’‹†•  ȋͳͶǣͲȌǡ  ȋͳͶǣͲȌǡȋͳ͹ǣͳȌǡ  ȋͳ͹ǣͲȀͳ͹ǣͲȌǡ ȋʹ͵ǣͲȀʹ͵ǣͲȌǡ  ȋͳͶǣͲȀͳͶǣͲȌǡ

ȋͳ͹ǣͲȀͳ͹ǣͲȌǡ  ȋͳ͹ǣͲȀͳ͹ǣͲȌǡ   ȋͳ͹ǣͲȀͳ͹ǣͲȌǡ  ȋͳͶǣͲȌͶǡ   ȋͳͻǣʹȀͳͻǣʹȀͳͻǣʹȌǡ  ȋͳ͹ǣͲȌǡ 

ȋͳͶǣͲȌƒ†ͺ‘Ž‘ˆ ȋͳͶǣͲȌǤ –‡”ƒŽ•–ƒ†ƒ”†•™‡”‡†‹••‘Ž˜‡†‹ʹͲͲɊ Ž͵ǣ‡ ȋͳǣͳǡ˜Ȁ˜Ȍ ƒ† ™‡”‡ ƒ††‡† –‘ ‡ƒ Š ’’‡†‘”ˆ –—„‡ ƒ† ’”‡Ǧ†”‹‡† ‹ ˜ƒ —— ‡–”‹ˆ—‰‡ ƒ– Ͷͷι „‡ˆ‘”‡ Ž‹’‹† ‡š–”ƒ –‹‘Ǥ‹˜‡”Š‘‘‰‡ƒ–‡ȋͷͲɊ ‘””‡•’‘†‹‰–‘γͳͲ‰Ȍ™ƒ•ƒ††‡†–‘–Š‡–—„‡• ‘–ƒ‹‹‰–Š‡ ’”‡Ǧ†”‹‡†Ž‹’‹†•–ƒ†ƒ”†•Ǥ‹’‹†‡š–”ƒ –‹‘™ƒ•’‡”ˆ‘”‡†ƒ ‘”†‹‰–‘ȏͷͳȐ™‹–Š•Ž‹‰Š–‘†‹ˆ‹ ƒ–‹‘Ǥ  „”‹‡ˆǡ ͵ͲͲ Ɋ ‡  ™ƒ• ‹š‡† ™‹–Š ͷͲ Ɋ Ž‹˜‡” Š‘‘‰‡ƒ–‡ ƒ† „ƒ–ŠǦ•‘‹ ƒ–‡† ˆ‘” ͷ ‹Ǥ —„•‡“—‡–Ž›ǡͳͲͲͲɊ™ƒ•ƒ††‡†–‘–Š‡‹š–—”‡ƒ†‹ —„ƒ–‡†‘ƒ•Šƒ‡”ƒ–ͻͲͲ”’ˆ‘” ʹͲ‹ƒ–”‘‘–‡’‡”ƒ–—”‡Ǥƒ’Ž‡•™‡”‡ ‡–”‹ˆ—‰‡†ƒ–͵ͲͲͲ‰ˆ‘”ͳͲ‹–‘‹†— ‡ ‘’Ž‡–‡’Šƒ•‡ •‡’ƒ”ƒ–‹‘ǤŠ‡—’’‡”’Šƒ•‡ȋͻͲͲɊȌ™ƒ•–”ƒ•ˆ‡””‡†–‘ƒ‡™–—„‡ǤŠ‡Ž‘™‡”’Šƒ•‡™ƒ•”‡Ǧ‡š–”ƒ –‡†

™‹–Š͸ͲͲɊȀ‡ Ȁ ʹȋͳͲǣ͵ǣʹǤͷǡ˜Ȁ˜Ȁ˜ȌǤŠ‡ ‘„‹‡†ͳͶͲͲɊ—’’‡”’Šƒ•‡™ƒ•†”‹‡†‹ƒ

˜ƒ —— ‡–”‹ˆ—‰‡ƒ–ͶͷιǤŠ‡Ž‹’‹†’‡ŽŽ‡–™ƒ•†‹••‘Ž˜‡†‹ Ž͵Ȁ‡ ȋͳǣͳǡ˜Ȁ˜Ȍ‹‡†‹ƒ–‡Ž›’”‹‘” –‘Ǧ‡ƒ•—”‡‡–Ǥ  LC-MS measurement ‹’‹†‘‹ ƒƒŽ›•‹•™ƒ•’‡”ˆ‘”‡†‘Ž–‹ƒ–‡͵ͲͲͲŠ‹‰Š’‡”ˆ‘”ƒ ‡Ž‹“—‹† Š”‘ƒ–‘‰”ƒ’Š›ȋ Ȍ ‘—’Ž‡† ™‹–Š Ǧšƒ –‹˜‡ ”„‹–”ƒ’ ‹‰Š ‡•‘Ž—–‹‘ ƒ•• •’‡ –”‘‡–”› ȋȌ ȋŠ‡”‘  ‹‡–‹ˆ‹ ǡ ƒŽ–ŠƒǡȌǤ•‡’ƒ”ƒ–‹‘™ƒ•’‡”ˆ‘”‡†ƒ•†‡• ”‹„‡†’”‡˜‹‘—•Ž›ȏͷʹȐǤ†‡–‡ –‹‘™ƒ•†‘‡‹ ’‘•‹–‹˜‡‡Ž‡ –”‘•’”ƒ›‹‘‹œƒ–‹‘‘†‡™‹–Š†ƒ–ƒ†‡’‡†‡–Ȁƒ “—‹•‹–‹‘ƒ ”‘••–Š‡’”‡ —”•‘” ‹‘”ƒ‰‡ȀœͷͲΫͳ͹ͷͲǤ‡• ƒ™ƒ•’‡”ˆ‘”‡†ˆ‘ŽŽ‘™‡††‹”‡ –Ž›„›ͳͲȀ• ƒ•”‡•—Ž–‹‰ ‹ƒ†—–› › Ž‡‘ˆ̱ͳǤʹ•Ǥ‡•‘Ž—–‹‘ˆ‘”ˆ—ŽŽ• ƒƒ†ˆ”ƒ‰‡–•’‡ –”ƒƒ “—‹•‹–‹‘™‡”‡•‡–ƒ–͹ͲǡͲͲͲ ƒ†ͳ͹ǡͷͲͲ  ǡ”‡•’‡ –‹˜‡Ž›ǤŠ‡•‘—” ‡’ƒ”ƒ‡–‡”•™‡”‡•‡–ƒ•ˆ‘ŽŽ‘™•ǣ•‘—” ‡–‡’‡”ƒ–—”‡ͳͷͲ ιǡ•’”ƒ›˜‘Ž–ƒ‰‡ǣ͵Ǥͷǡ ƒ’‹ŽŽƒ”›–‡’‡”ƒ–—”‡ǣ͵ͲͲιǤ 

ͳͶͲ Effect of fasting and cold stress on hepatic metabolism in MCAD-KO mice

Lipidomic data analysis Š‡”‘  ƒŽ‹„—”̺ •‘ˆ–™ƒ”‡ ȋ˜‡”•‹‘ ͵ǤʹǤ͸͵ ȋŠ‡”‘  ‹‡–‹ˆ‹ ǡ ƒŽ–Šƒǡ Ȍ ™ƒ• —•‡† ˆ‘” †ƒ–ƒ ƒ “—‹•‹–‹‘Ǥ ”‘‰‡‡•‹•  ̺ •‘ˆ–™ƒ”‡ ȋƒ–‡”• ‘”’‘”ƒ–‹‘ǡ ‹Žˆ‘”†ǡ Ȍ ™ƒ• —•‡† ˆ‘” †ƒ–ƒ ’”‡’”‘ ‡••‹‰‹ Ž—†‹‰”‡–‡–‹‘–‹‡ƒŽ‹‰‡–ǡ’‡ƒ’‹ ‹‰ƒ†ƒ‘–ƒ–‹‘‘ˆ–Š‡Ǧ†ƒ–ƒǤ ‡ƒ’‹ ‹‰™ƒ•’‡”ˆ‘”‡†™‹–Šƒƒ„•‘Ž—–‡‹‘‹–‡•‹–›ˆ‹Ž–‡”‘ˆʹͲͲǡͲͲͲ ‘—–•Ǥ‹’‹†ƒ‘–ƒ–‹‘ ™ƒ• ’‡”ˆ‘”‡† „› •‡ƒ” Š‹‰ ’—„Ž‹•Š‡† Ž‹’‹† †ƒ–ƒ„ƒ•‡• ȋ —ƒ ‡–ƒ„‘Ž‘‡ ƒ–ƒ„ƒ•‡ǡ ‹’‹†ƒ’•ǡ ‹’‹†„Žƒ•–Ȍ„ƒ•‡†‘ƒ••ƒ —”ƒ ›ȋδͷ’’Ȍǡ‹•‘–‘’‹ •‹‹Žƒ”‹–‹‡•ǡƒ††— ––›’‡ǡȀ•’‡ –”ƒƒ† ‡Ž—–‹‘„‡Šƒ˜‹‘”‹ ‘’ƒ”‹•‘™‹–Š‘™Ž‹’‹†•‘ˆƒŠ‘‡Ǧƒ†‡†ƒ–ƒ„ƒ•‡ǤƒŽ›•‹•‘ˆ–Š‡ˆƒ––›Ǧƒ ›Ž ‘’‘•‹–‹‘‘ˆ ™ƒ•’‡”ˆ‘”‡†—•‹‰‹’‹†Š—–‡”ʹ•‘ˆ–™ƒ”‡ȏͷ͵Ȑ™‹–Šƒ’”‡†‡ˆ‹‡†–ƒ„Ž‡‘ˆ’‘••‹„Ž‡ 4 ˆ”ƒ‰‡–• ȋ—’’Ž‡‡–ƒ”› –ƒ„Ž‡ ͷȌǤ ‹’‹† ‹–‡•‹–› ™ƒ• ‘”ƒŽ‹œ‡† –‘ –Š‡ ‹–‡•‹–› ‘ˆ –Š‡ ‘””‡•’‘†‹‰Ž‹’‹†•–ƒ†ƒ”†•‹–Š‡•ƒ‡ Žƒ••Ǥ  ƒ•‡‘ˆ–Š‡ƒ„•‡ ‡‘ˆŽ‹’‹†•–ƒ†ƒ”†•ǡ–Š‡‹–‡•‹–› ™ƒ•‘”ƒŽ‹œ‡†–‘–Š‡ƒ˜‡”ƒ‰‡‘ˆƒŽŽ•–ƒ†ƒ”†•Ǥ”‹ ‹’Ž‡ ‘’‘‡–ƒƒŽ›•‹•ȋȌ™ƒ•’‡”ˆ‘”‡† —•‹‰‡–ƒ„‘ƒŽ›•–ȏͷͶȐǤ  Histology ‘”ƒŽ‹Ǧˆ‹š‡†Ž‹˜‡”•™‡”‡’”‘ ‡••‡†ˆ‘”’ƒ”ƒˆˆ‹•‡ –‹‘‹‰ǡƒ†•–ƒ‹‡†™‹–ŠŠ‡ƒ–‘š›Ž‹ƒ†‡‘•‹Ǥ ”‘œ‡ǡ‹••—‡Ǧ‡̺ˆ‹š‡†Ž‹˜‡”•™‡”‡’”‘ ‡••‡†—•‹‰•–ƒ†ƒ”†‡–Š‘†•ƒ†•‡ –‹‘•™‡”‡•–ƒ‹‡† ™‹–Š ‹ŽǦ‡†ǦǤ Š‡ •Ž‹†‡• ™Š‡”‡  • ‘”‡† ˆ‘” ‘ƒŽ ‘Š‘Ž‹  ˆƒ––› Ž‹˜‡” †‹•‡ƒ•‡ ȋ Ȍ ƒ ‘”†‹‰ –‘ Ž‡‹‡”‡–ƒŽǤȏͷͷȐǤ  Statistical analysis Š‡•‹‰‹ˆ‹ ƒ ‡‘ˆ†‹ˆˆ‡”‡ ‡•ǡ‡š ‡’–ˆ‘”Ž‹’‹†‘‹ †ƒ–ƒǡ™‡”‡ƒ••‡••‡†—•‹‰’ƒ”ƒ‡–”‹ ȋ–—†‡–ǯ•–Ǧ –‡•–Ȍ‘”‘’ƒ”ƒ‡–”‹ –‡•–•ȋƒŠ‹–‡›–‡•–Ȍ†‡’‡†‹‰‘–Š‡‹”†‹•–”‹„—–‹‘Ǥ ‘” Šƒ‰‡•‹ –‹‡ǡ”‡’‡ƒ–‡†Ǧ‡ƒ•—”‡•™ƒ•—•‡†Ǥ–ƒ”‰‡–‡†Ž‹’‹†‘‹ †ƒ–ƒ™‡”‡ˆ‹”•–Ž‘‰Ǧ–”ƒ•ˆ‘”‡†ƒ† •—„•‡“—‡–Ž›†‹ˆˆ‡”‡ ‡•™‡”‡ƒ••‡••‡†™‹–Š–—†‡–ǯ•–Ǧ–‡•–ƒ†ƒ†Œ—•–‡†ˆ‘”—Ž–‹’Ž‡ ‘’ƒ”‹•‘Ǥ ’˜ƒŽ—‡‘ˆδͲǤͲͷ™ƒ• ‘•‹†‡”‡†•–ƒ–‹•–‹ ƒŽŽ›•‹‰‹ˆ‹ ƒ–Ǥ–ƒ–‹•–‹ ƒŽƒƒŽ›•‡•™‡”‡’‡”ˆ‘”‡†—•‹‰ ȋ˜ʹʹȌƒ†–Š‡†ƒ–ƒ™ƒ•˜‹•—ƒŽ‹œ‡†—•‹‰ ”ƒ’Šƒ†”‹••‘ˆ–™ƒ”‡ȋ ”ƒ’Šƒ†‘ˆ–™ƒ”‡  Ǥǡ˜‡”•‹‘ ͷǤͲͲǡʹͲͲ͹Ȍƒ†–Š‡‡–ƒ„‘ƒŽ›•–™‡„•‡”˜‡”ȏͷͶȐǤ  Author contributions  ǡǡ ǡ ǡ ƒ††‡•‹‰‡†–Š‡‡š’‡”‹‡–•Ǥ ǡ ƒ† ’‡”ˆ‘”‡†–Š‡ƒ‹ƒŽ ‡š’‡”‹‡–•Ǥ ǡǡ ǡ ǡǡǡǡǡ ’”‡’ƒ”‡†ƒ†ƒƒŽ›œ‡†–Š‡•ƒ’Ž‡•Ǥ ǡ ƒ†  ƒƒŽ›œ‡† –Š‡ †ƒ–ƒǤ  ǡ ǡ ǡ ǡ ǡ  ǡ  ǡ ǡ  ǡ  ™‡”‡ ‹˜‘Ž˜‡† ‹ ‹–‡”’”‡–ƒ–‹‘ ‘ˆ ”‡•—Ž–•Ǥ  ǡ ǡ  ǡ ƒ†  ™”‘–‡ –Š‡ ƒ—• ”‹’–Ǥ ŽŽ ƒ—–Š‘”• ‡†‹–‡† –Š‡ ƒ—• ”‹’–Ǥ  Acknowledgements Š‹•™‘”™ƒ••—’’‘”–‡†„›–Š‡‹˜‡”•‹–›‡†‹ ƒŽ‡–‡” ”‘‹‰‡ǤŠ‡ˆ—†‡”•Šƒ†‘”‘Ž‡‹•–—†› †‡•‹‰ǡ†ƒ–ƒ ‘ŽŽ‡ –‹‘ƒ†ƒƒŽ›•‹•ǡ†‡ ‹•‹‘–‘’—„Ž‹•Šǡ‘”’”‡’ƒ”ƒ–‹‘‘ˆ–Š‡ƒ—• ”‹’–ǤŠ‡ƒ—–Š‘”• Ž‹‡–‘–Šƒ‹ ‡–ǤŽ‘•ˆ‘” ƒ”‡ˆ—Žƒ†‹†‡’‡†‡– Š‡ ‹‰‘ˆ–Š‡•–ƒ–‹•–‹ ƒŽƒƒŽ›•‹•ƒ† ƒ”‹‡‡‹–ˆ‘”ƒ••‹•–‹‰™‹–Š–‡”‹ƒ–‹‘•Ǥ  Declaration of Interests Š‡ƒ—–Š‘”•†‡ Žƒ”‡‘ ‘’‡–‹‰‹–‡”‡•–•Ǥ  References ͳǤ ‹ƒŽ†‘ ǡ ƒ–‡” ǡ ‡‡––  Ǥ ƒ––›  ‹† ‘š‹†ƒ–‹‘ ‹•‘”†‡”•Ǥ — ‡˜ Š›•‹‘ŽǤ ʹͲͲʹǢ͸ͶȋͳȌǣͶ͹͹ȂͷͲʹǤ

ͳͶͳ Chapter 4

ʹǤ ‘—–‡ ǡ ‹‘Žƒ–‡ ǡ ‡–—”ƒ  Ǥǡ ƒ†‡”•  Ǥ Š‡ ‹‘ Š‡‹•–”› ƒ† Š›•‹‘Ž‘‰› ‘ˆ ‹–‘ Š‘†”‹ƒŽ ƒ––›  ‹† ȾǦš‹†ƒ–‹‘ ƒ† –• ‡‡–‹  ‹•‘”†‡”•Ǥ — ‡˜ Š›•‹‘ŽǤ ʹͲͳ͸Ǣ͹ͺȋͳȌǣʹ͵ȂͶͶǤ ͵Ǥ ‘––‡”—• ǡŽ‡‡‡” ǡò•– ǡ ‡”†‹ƒ†—••‡ǡ ŒŽ•–ǡ‹Œ„—”‰ ǡƒ†‡”• ǡ‹••‡” ǡ ‘—–‘‘’‡” Ǥ‹•‘”†‡”•‘ˆ‹–‘ Š‘†”‹ƒŽŽ‘‰Ǧ Šƒ‹ˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘ƒ†–Š‡ ƒ”‹–‹‡ •Š—––Ž‡ǤʹͲͳͺǢ ͶǤ ‡”• ǡƒ’”‘•‡ ǡƒ‡ ǡƒ‡” ‹Ž•–ǡ’ƒǡ‹– Ǥ ƒˆ‡ƒ†—•ƒˆ‡ †—”ƒ–‹‘‘ˆˆƒ•–‹‰ˆ‘” Š‹Ž†”‡™‹–Š†‡ˆ‹ ‹‡ ›Ǥ—” ‡†‹ƒ–”ǤʹͲͲ͹Ǣͳ͸͸ȋͳȌǣͷȂͳͳǤ ͷǤ ƒŽ–‡” Ǥ ‘Ž‡”ƒ ‡ –‘ ˆƒ•–ǣ ƒ–‹‘ƒŽ ƒ† ’”ƒ –‹ ƒŽ ‡˜ƒŽ—ƒ–‹‘ ‹ Š‹Ž†”‡ ™‹–Š Š›’‘‡–‘ƒ‡‹ƒǤ  Š‡”‹–‡–ƒ„‹•ǤʹͲͲͻǢ͵ʹȋʹȌǣʹͳͶȂ͹Ǥ ͸Ǥ ‡‡––  Ǥ ƒ–Š‘’Š›•‹‘Ž‘‰› ‘ˆ ˆƒ––› ƒ ‹† ‘š‹†ƒ–‹‘ †‹•‘”†‡”•Ǥ  Š‡”‹– ‡–ƒ„ ‹•Ǥ ʹͲͳͲǢ͵͵ȋͷȌǣͷ͵͵Ȃ͹Ǥ ͹Ǥ ‹•Š™ƒƒ–Š Ǥ ƒ––› ƒ ‹† „‡–ƒǦ‘š‹†ƒ–‹‘ †‹•‘”†‡”•ǣ  „”‹‡ˆ ”‡˜‹‡™Ǥ  ‡—”‘• ‹Ǥ ʹͲͳ͸Ǣʹ͵ȋͳȌǣͷͳȂͷǤ ͺǤ  Šƒ–œƒǤǡ•‡ƒ—‡”ǤŠ‡ Ž‹‹ ƒŽƒ‹ˆ‡•–ƒ–‹‘‘ˆ†‡ˆ‹ ‹‡ ›ǣŠƒŽŽ‡‰‡•–‘™ƒ”†• ƒ†—Ž–Š‘‘†‹–Š‡• ”‡‡‡†’‘’—Žƒ–‹‘Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͲǢ͵͵ȋͷȌǣͷͳ͵ȂʹͲǤ ͻǤ ‘Ž‡”Ǧ–‘—™Ǥ†‡ˆ‹ ‹‡ ›‘„‡ǡ‘”‘––‘„‡ƒ–”‹•ǤʹͲͳͶǤ ͳͲǤ ‘Ž™ƒ‹ ǡ ƒǡ‹ƒǡŠƒ”‡” ǡ‘ Ž‡› ǡ‹ƒŽ†‘ǡƒ–‡”ǡ Š‘‡„ǡ‘‘†Ǥ ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ †‡ˆ‹ ‹‡ › ‹ ‰‡‡Ǧ–ƒ”‰‡–‡† ‹ ‡Ǥ ‘ ‡‡–Ǥ ʹͲͲͷǢͳȋʹȌǣͲʹͲͷȂͳʹǤ ͳͳǤ ˜ƒ—‡ǡ‘Ž‡”Ǧ‘—™ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ‘Ž–‡”• ǡ˜ƒ‹Œ– ǡƒ”–‹‡•Ǧ ǡ ‹‡œ‡Ǧ‘‹‰ǡ ‡‹‡”ǡ‡”‡–‹‡” ǡ ”‘‡ǡ‡‹Œ‰‘—†Ǧ ǡ‡”• ǡƒ‡”Ǥ ‹˜‹‰‘–Š‡‡†‰‡ǣ•—„•–”ƒ–‡ ‘’‡–‹–‹‘‡š’Žƒ‹•Ž‘••‘ˆ”‘„—•–‡••‹‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧ ƒ ‹†‘š‹†ƒ–‹‘†‹•‘”†‡”•Ǥ‹‘ŽǤʹͲͳ͸ǢͳͶȋͳȌǣͳͲ͹Ǥ ͳʹǤ ‡””‡ƒ ǡ‡”• ǡƒ‹Œ ǡŽ‘•ǡ ‡”†‹‰ǡ ƒ˜‹‰ƒǡ‹‡–‰‡ ǡǫǫŽŽ‡”ǡ‹– ǡ —‹’‡”• ǡ ‡‹Œ‰‘—†  Ǥ ‹•–—”„‡† Š‡’ƒ–‹  ƒ”„‘Š›†”ƒ–‡ ƒƒ‰‡‡– †—”‹‰ Š‹‰Š ‡–ƒ„‘Ž‹  †‡ƒ† ‹ ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋȌǦ†‡ˆ‹ ‹‡– ‹ ‡Ǥ ‡’ƒ–‘Ž‘‰›ǤʹͲͲͺǢͶ͹ȋ͸ȌǣͳͺͻͶȂͻͲͶǤ ͳ͵Ǥ ’‹‡‡”‘‡––‡”ǡ‘‘†Ǥ‹–‘ Š‘†”‹ƒŽˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘†‹•‘”†‡”•ǣƒ–Š‘’Š›•‹‘Ž‘‰‹ ƒŽ •–—†‹‡•‹‘—•‡‘†‡Ž•Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͲǢ͵͵ȋͷȌǣͷ͵ͻȂͶ͸Ǥ ͳͶǤ Š‹„ƒ–ƒ ǡ±”—••‡ ǡƒŽŽ‡”ƒ†ƒǡ—‘™‹‡ ‹ Ǥ‘Ž†‡š’‘•—”‡”‡˜‡”•‡•‹Š‹„‹–‘”›‡ˆˆ‡ –•‘ˆ ˆƒ•–‹‰‘’‡”‹’Š‡”ƒŽ‰Ž— ‘•‡—’–ƒ‡‹”ƒ–•Ǥ Š›•‹‘ŽǤͳͻͺͻǢʹͷ͹ȋͳ–ʹȌǣͻ͸ȂͳͲͳǤ ͳͷǤ ג‡œǦ‘”‹ƒ‘ ǡ ‡”ž†‡œǦג‡œ ǡƒ’‡Žǡ‹ŽŽƒ””‘›ƒ ǡ ‰Ž‡•‹ƒ•ǡŽ‡ƒ›Ǥ‹‘ ƒ ‹†ƒ†‰Ž— ‘•‡—’–ƒ‡„›”ƒ–„”‘™ƒ†‹’‘•‡–‹••—‡Ǥˆˆ‡ –‘ˆ ‘Ž†Ǧ‡š’‘•—”‡ƒ†ƒ Ž‹ƒ–‹‘Ǥ ‹‘ Š‡ ǤͳͻͺͺǢʹͷʹȋ͵ȌǣͺͶ͵ȂͻǤ ͳ͸Ǥ ‡‹” ǡƒƒ‰‡ǡ›‘ŽǡŠ‘†‡• ǡ›Ž‡ ǡ Ž‡– Š‡”ǡ”ƒ˜‡ ǡƒ‡Ž‹ ǡ”ƒ‡ ǡ ”‡‰‘”‹ƒ†‡•ǡ•Š–‘ǡ‡‹”ǡ˜ƒ‡‡ ǡƒ”’‡ ǡƒŽ‡”ǡ–‹•‘ Ǥ—„•–ƒ–‹ƒŽ ‡–ƒ„‘Ž‹  –‹˜‹–›‘ˆ —ƒ”‘™†‹’‘•‡‹••—‡†—”‹‰ƒ”‘†‹–‹‘•ƒ†‘Ž†Ǧ †— ‡† ‹’‘Ž›•‹•‘ˆ‘ ƒŽ”‹‰Ž› ‡”‹†‡•Ǥ‡ŽŽ‡–ƒ„ǤʹͲͳͺǢʹ͹ȋ͸Ȍǣͳ͵ͶͺǦͳ͵ͷͷǤ‡ͶǤ ͳ͹Ǥ ƒ”–‡Ž–ǡ”—•ǡ‡‹‡”ǡ ‘Š‡„‡”‰ ǡ ––”‹ Š ǡ‡Ž†• Š—•ǡƒ—Ž ǡ”‘•†‘”ˆ ǡ ‡ŽŽ‡” ǡ ƒ—”‹• Š ǡ › ŠòŽŽ‡” ǡ ‘”†–• ǡ ‹‹‰‡” ǡ ”—‡‰‡Žƒ ǡ ”‡—† ǡ ‹‡Ž•‡ǡ‡”‡Žǡ ‡‡”‡ Ǥ”‘™ƒ†‹’‘•‡–‹••—‡ƒ –‹˜‹–› ‘–”‘Ž•–”‹‰Ž› ‡”‹†‡ Ž‡ƒ”ƒ ‡Ǥƒ– ‡†ǤʹͲͳͳǢͳ͹ȋʹȌǣʹͲͲȂ͸Ǥ ͳͺǤ Š‹‘–ƒǡƒƒƒǡ—‰ƒ‘Ǥˆˆ‡ –‘ˆ‘”‡’‹‡’Š”‹‡‘‰Ž— ‘‡‘‰‡‡•‹•‹’‡”ˆ—•‡†Ž‹˜‡”• ‘ˆ ‘Ž†Ǧ‡š’‘•‡†”ƒ–•Ǥ Š›•‹‘Ž‡–ƒ„ǤʹͲͳ͹ǢʹͶͻȋ͵ȌǣʹͺͳȂ͸Ǥ ͳͻǤ ‹ ‘š ǡ ‡‘‰Š‡‰ƒ ǡƒ• Š‡ ǡ‡•ƒ”†ǡƒ•“—ƒŽ‹ǡ‹ƒ‘ǡ‡‡ǡ ‹ƒ‰ǡ —  ǡ ‡”•Šƒ™ǡ‘ƒ–‘ ǡ’–‡ǡ‘‰‘ǡ—––‡” ǡ Š”‡‹„‡”ǡ‡ Š‡”ǡ‘š ǡ‹ŽŽƒ—‡˜ƒ Ǥ Ž‘„ƒŽƒŽ›•‹•‘ˆŽƒ•ƒ‹’‹†• †‡–‹ˆ‹‡•‹˜‡”Ǧ‡”‹˜‡† ›Ž ƒ”‹–‹‡•ƒ•ƒ —‡Ž‘—” ‡ˆ‘” ”‘™ ƒ–Š‡”‘‰‡‡•‹•Ǥ‡ŽŽ‡–ƒ„ǤʹͲͳ͹Ǣʹ͸ȋ͵ȌǣͷͲͻǦͷʹʹǤ‡͸Ǥ

ͳͶʹ Effect of fasting and cold stress on hepatic metabolism in MCAD-KO mice

ʹͲǤ „—”ƒ†ǤŠ‡‹˜‡”ƒ•ƒ —„‹Š‡”‘‰‡‡•‹•Ǥ‡ŽŽ‡–ƒ„ǤʹͲͳ͹Ǣʹ͸ȋ͵ȌǣͶͷͶȂͷǤ ʹͳǤ ”‡ˆŠ‘”•–ǡ˜ƒ†‡‡—‡Ž ǡ‹Œǡ–‡‡„‡”‰‡ ǡ‘‘”–ƒ ǡ‡‡—™‡„—”‰Šǡ‹••‡” ǡ ‡”•–‡ǡ ”‹‡•‡ƒ ǡŠ‡‡ǡ‹••‡” Ǥ—Ž–‹’Ž‡‡ˆˆ‡ –•‘ˆ ‘Ž†‡š’‘•—”‡‘Ž‹˜‡”• ‘ˆƒŽ‡‹ ‡Ǥ †‘ ”‹‘ŽǤʹͲͳͺ—‰Ǣʹ͵ͺȋʹȌǣͻͳȂͳͲ͸Ǥ ʹʹǤ ‘‡–œƒǡ‹ƒǡ‘‘†Ǥ‹ˆˆ‡”‡–‹ƒŽ‹†— –‹‘‘ˆ‰‡‡•‹Ž‹˜‡”ƒ†„”‘™ƒ†‹’‘•‡–‹••—‡ ”‡‰—Žƒ–‡†„›’‡”‘š‹•‘‡’”‘Ž‹ˆ‡”ƒ–‘”Ǧƒ –‹˜ƒ–‡†”‡ ‡’–‘”ǦȽ†—”‹‰ˆƒ•–‹‰ƒ† ‘Ž†‡š’‘•—”‡‹ ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡Ǧ†‡ˆ‹ ‹‡–‹ ‡Ǥ‘Ž ‡‡–‡–ƒ„ǤʹͲͲͷǢͺͶȋͳȌǣ͵ͻȂͶ͹Ǥ ʹ͵Ǥ Š‘”‡ǡƒ”ƒ‹–”‹ǡ‡’ǡ’‡ƒƒ ǡ ”ƒŠƒǡ‘ƒšǤ‘Ž†Ǧ †— ‡†Šƒ‰‡•‹ ‡‡š’”‡••‹‘‹”‘™†‹’‘•‡‹••—‡ǡŠ‹–‡†‹’‘•‡‹••—‡ƒ†‹˜‡”Ǥ‡„—”‰ǡ‡†‹–‘”Ǥ 4 ‘‡ǤʹͲͳ͵ —ŽʹʹǢͺȋ͹Ȍǣ‡͸ͺͻ͵͵Ǥ ʹͶǤ ‹‘‰ǡ ‡ ǡ ƒ‡• ǡ‘—ƒ‰ƒǡ‘™‡”•ǡ —ƒ‰ǡ•‹•ƒ ǡ‘™„‹ ǡ—”‡˜Œƒ˜ǡ ƒŽ• Š‹ ǡ ƒ˜ƒ†‘˜ǡ ‘™ƒ ǡ–”ƒ—••ǡŠ— Š—ƒǤƒ”†‹ƒ Ǧ•’‡ ‹ˆ‹ †‡ˆ‹ ‹‡ › ‹†— ‡• †‹Žƒ–‡† ƒ”†‹‘›‘’ƒ–Š› ƒ† ‘Ž† ‹–‘Ž‡”ƒ ‡Ǥ   ‡ƒ” ‹”  Š›•‹‘ŽǤ ʹͲͳͶǢ͵Ͳ͸ȋ͵Ȍǣ ͵ʹ͸Ȃ͵ͺǤ ʹͷǤ ’‹‡‡”‘‡––‡”ǡ‘—ƒ‰ƒǡ‡†‡Žǡƒ›ƒ–‡’‡ǡš‹Žǡ—”ƒǡ‹Œ„—”‰ ǡƒ†‡”•  ǡ–”ƒ—••ǤŠƒ‰‡•‹„Ž‘‘† ƒ”‹–‹‡ƒ†ƒ ›Ž ƒ”‹–‹‡’”‘ˆ‹Ž‡•‘ˆ˜‡”›Ž‘‰Ǧ Šƒ‹ƒ ›ŽǦ ‘†‡Š›†”‘‰‡ƒ•‡Ǧ†‡ˆ‹ ‹‡–‹ ‡•—„Œ‡ –‡†–‘•–”‡••Ǥ—” Ž‹ ˜‡•–ǤʹͲͲͶǢ͵Ͷȋ͵ȌǣͳͻͳȂ͸Ǥ ʹ͸Ǥ š‹Ž Ǥ„‘”ƒŽ‹–‘ Š‘†”‹ƒŽ„‹‘‡‡”‰‡–‹ •ƒ†Š‡ƒ”–”ƒ–‡†›•ˆ— –‹‘‹‹ ‡Žƒ ‹‰˜‡”›Ǧ Ž‘‰Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡Ǥ  ‡ƒ”‹” Š›•‹‘ŽǤʹͲͲͷǢʹͻͲȋ͵Ȍǣ ͳʹͺͻȂͻ͹Ǥ ʹ͹Ǥ ‘Ž–‡”• ǡ ‹ƒ’ƒ‹–‡ ǡ ƒ —‡ ǡ ‹‡œ‡Ǧ‘‹‰ ǡ ƒ––‘ ǡ ‘”–‡  ǡ ‘”˜ƒ–‘˜‹ Š ǡ ƒ‡” ǡ ‹• Š‘ˆˆ ǡ ‡”‡–‹‡” Ǥ ”ƒ•Žƒ–‹‘ƒŽ ƒ”‰‡–‡† ”‘–‡‘‹ • ”‘ˆ‹Ž‹‰ ‘ˆ ‹–‘ Š‘†”‹ƒŽ ‡”‰› ‡–ƒ„‘Ž‹  ƒ–Š™ƒ›• ‹ ‘—•‡ ƒ† —ƒ ƒ’Ž‡•Ǥ  ”‘–‡‘‡ ‡•Ǥ ʹͲͳ͸ǢͳͷȋͻȌǣ͵ʹͲͶȂͳ͵Ǥ ʹͺǤ ‡””‡ƒ ǡ‡”• ǡƒ‹Œ ǡŽ‘•ǡ ‡”†‹‰ǡ ƒ˜‹‰ƒǡ‹‡–‰‡ ǡòŽŽ‡”ǡ‹– ǡ —‹’‡”• ǡ ‡‹Œ‰‘—†  Ǥ ‹•–—”„‡† Š‡’ƒ–‹  ƒ”„‘Š›†”ƒ–‡ ƒƒ‰‡‡– †—”‹‰ Š‹‰Š ‡–ƒ„‘Ž‹  †‡ƒ† ‹ ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋȌǦ†‡ˆ‹ ‹‡– ‹ ‡Ǥ ‡’ƒ–‘Ž‘‰›ǤʹͲͲͺǢͶ͹ȋ͸ȌǣͳͺͻͶȂͻͲͶǤ ʹͻǤ ‹ƒ‡›Ǧ‹ƒ—†ǡ‹˜”›ǡ ”‡‰‡”•‡ǡƒ–Š‹‡—ǤŠ‡‹„‘”‡””‘”•‘ˆ‹–‘ Š‘†”‹ƒŽˆƒ––›ƒ ‹† ‘š‹†ƒ–‹‘Ǥ  Š‡”‡–ƒ„‹•Ǥͳͻͺ͹ǢͳͲǣͳͷͻȂʹͲͲǤ ͵ͲǤ ‡–Ž‡”ǡŠƒ‹ǡŽ•„‡ ‡”ǡ”‘Ž† ǡ—”–‘ǡ‘ Ž‡› ǡƒ‡”‘ǡ ‹‡” ǡ†‹   ǡ ‡””› Ǥ ʹʹͳ ‡™„‘”Ǧ• ”‡‡‡† ‡‘ƒ–‡• ™‹–Š ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ ‘‡œ›‡  †‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›ǣ ‹†‹‰•ˆ”‘–Š‡ „‘”””‘”•‘ˆ‡–ƒ„‘Ž‹•‘ŽŽƒ„‘”ƒ–‹˜‡Ǥ‘Ž ‡‡–‡–ƒ„ǤʹͲͳ͸ǢͳͳͻȋͳȂʹȌǣ͹ͷȂͺʹǤ ͵ͳǤ ƒŽ–‡” ǤǦƒ”‹–‹‡‹‹„‘”‡””‘”•‘ˆ‡–ƒ„‘Ž‹•ǣŠƒ–‹•–Š‡‡˜‹†‡ ‡ǫ  Š‡”‹–‡–ƒ„ ‹•ǤʹͲͲ͵Ǣʹ͸ȋʹȂ͵ȌǣͳͺͳȂͺǤ ͵ʹǤ ‘‡Ǥ Š‡”‹–‡††‹•‘”†‡”•‘ˆ‹–‘ Š‘†”‹ƒŽˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘ǣ‡™”‡•’‘•‹„‹Ž‹–›ˆ‘”–Š‡ ‡‘ƒ–‘Ž‘‰‹•–Ǥ‡‹‡‘ƒ–‘ŽǤʹͲͲʹǢ͹ȋͳȌǣ͵͹ȂͶ͹Ǥ ͵͵Ǥ ‡”•  Ǥ  †‡ˆ‹ ‹‡ ›᩿ǣ Ž‹‹ ƒŽ ƒ† Žƒ„‘”ƒ–‘”› •–—†‹‡•  Ǧ Š––’•ǣȀȀ”—‰Ǥ‘Ǥ™‘”Ž† ƒ–Ǥ‘”‰Ȁ‘ Ž ȀͳͷͲͶʹͳͷͲͶǤȏȏǤŽǤȐǦǦȐǣȏ•ǤǤȐǢʹͲͲ͹Ǥ ͵ͶǤ ˜ƒ—‡ǡ‘Ž‡”Ǧ‘—™ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ‘Ž–‡”• ǡ˜ƒ‹Œ– ǡƒ”–‹‡•Ǧ ǡ ‹‡œ‡Ǧ‘‹‰ǡ ‡‹‡”ǡ‡”‡–‹‡” ǡ ”‘‡ǡ‡‹Œ‰‘—†Ǧ ǡ‡”• ǡƒ‡”Ǥ ‹˜‹‰‘–Š‡‡†‰‡ǣ•—„•–”ƒ–‡ ‘’‡–‹–‹‘‡š’Žƒ‹•Ž‘••‘ˆ”‘„—•–‡••‹‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧ ƒ ‹†‘š‹†ƒ–‹‘†‹•‘”†‡”•Ǥ‹‘ŽǤʹͲͳ͸ǢͳͶȋͳȌǣͳͲ͹Ǥ ͵ͷǤ ƒ”’‡–‹‡”ǡŽ‘†‹ǡ‹”–ƒ‡ǡ‹ Šƒ”†ǡ ƒƒ ǡ—” ‘––‡2Ǥ”‘™ƒ†‹’‘•‡–‹••—‡ ‡‡”‰›‡–ƒ„‘Ž‹•‹Š—ƒ•Ǥ ”‘–†‘ ”‹‘Žȋƒ—•ƒ‡ȌǤʹͲͳͺǢͻȋ ȌǣͳȂʹͳǤ ͵͸Ǥ ƒŠ›ǡ—„”ƒƒ‹ƒǡ—”’Š›ǡ‹•Š‹Œ‹ƒǡƒ‡–œ ǡŠ‹‹œ—ǡ’‡‡” ǡ˜ƒ‡‡” ǡ ƒ‡Žƒ ǡ‡‹•Ǥ’†ƒ–‡‘ˆ–Š‡   ‘’”‡Š‡•‹˜‡ Žƒ••‹ˆ‹ ƒ–‹‘•›•–‡ˆ‘” Ž‹’‹†•Ǥ ‹’‹†‡•ǤʹͲͲͻǢͷͲȋ—’’Ž‡‡–ȌǣͻȂͳͶǤ ͵͹Ǥ  Šƒ–œǡ•‡ƒ—‡”ǤŠ‡ Ž‹‹ ƒŽƒ‹ˆ‡•–ƒ–‹‘‘ˆ†‡ˆ‹ ‹‡ ›ǣŠƒŽŽ‡‰‡•–‘™ƒ”†•

ͳͶ͵ Chapter 4

ƒ†—Ž–Š‘‘†‹–Š‡• ”‡‡‡†’‘’—Žƒ–‹‘Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͲǢ͵͵ȋͷȌǣͷͳ͵ȂʹͲǤ ͵ͺǤ ˜ƒ ƒ”‡ ‹ Š–‡„‡Ž– ǡ  Š”ƒ—™‡ Ǥ ’Ž‹ ƒ–‹‘• ‘ˆ ‘•Š‹˜‡”‹‰ –Š‡”‘‰‡‡•‹• ˆ‘” ‡‡”‰›„ƒŽƒ ‡”‡‰—Žƒ–‹‘‹Š—ƒ•Ǥ Š›•‹‘Ž –‡‰”‘’Š›•‹‘ŽǤʹͲͳͳǢ͵ͲͳȋʹȌǣʹͺͷȂ ͻ͸Ǥ ͵ͻǤ –‘‡” Ǥ Š‡ ”‘Ž‡ ‘ˆ –Š‡ Ž‹˜‡” ‹ ‘Ǧ•Š‹˜‡”‹‰ –Š‡”‘‰‡‡•‹• ‹ –Š‡ ”ƒ–Ǥ  Š›•‹‘ŽǤ ͳͻ͹͵ —ŽǢʹ͵ʹȋʹȌǣʹͺͷȂͻ͸Ǥ ͶͲǤ ƒ”–‹‡• ǡ˜ƒ—‡ǡ‡‹Œ‰‘—† ǡƒ‡”ǤŠ‡’”‘‹• —‘—•‡œ›‡‡†‹—Ǧ Šƒ‹ ͵Ǧ‡–‘Ǧƒ ›ŽǦ‘ –Š‹‘Žƒ•‡ –”‹‰‰‡”• ƒ ˜‹ ‹‘—• › Ž‡ ‹ ˆƒ––›Ǧƒ ‹† „‡–ƒǦ‘š‹†ƒ–‹‘Ǥ ‘ ‘’—–‹‘ŽǤʹͲͳ͹Ǣͳ͵ȋͶȌǣͳȂʹʹǤ ͶͳǤ ‹– Š‡ŽŽ ǡ ƒ—–Š‹‡”ǡ‡•‹’Ž‡ǡƒ‰ǡƒ‡”ǡ—”‡•Š‹ Ǥ ‡”‡†‹–ƒ”›ƒ†ƒ “—‹”‡† †‹•‡ƒ•‡•‘ˆƒ ›ŽǦ ‘‡œ›‡‡–ƒ„‘Ž‹•Ǥ‘Ž ‡‡–‡–ƒ„ǤʹͲͲͺǢͻͶȋͳȌǣͶȂͳͷǤ ͶʹǤ ƒ‰ ǡŠƒ‘ǡƒ‰ǡƒ‰ǡ‹– Š‡ŽŽ Ǥ ‡”‡†‹–ƒ”›†‹•‡ƒ•‡•‘ˆ ‘‡œ›‡–Š‹‘‡•–‡” ‡–ƒ„‘Ž‹•Ǥ‹‘ Š‡‘ ”ƒ•ǤʹͲͳͻ ‡„ʹͺǢͶ͹ȋͳȌǣͳͶͻȂͷͷǤ Ͷ͵Ǥ ˜ƒ—‡ǡ‘Ž‡”Ǧ‘—™ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ‘Ž–‡”• ǡ˜ƒ‹Œ– ǡƒ”–‹‡•Ǧ ǡ ‹‡œ‡Ǧ‘‹‰ǡ ‡‹‡”ǡ‡”‡–‹‡” ǡ ”‘‡ǡ‡‹Œ‰‘—†Ǧ ǡ‡”• ǡƒ‡”Ǥ ‹˜‹‰‘–Š‡‡†‰‡ǣ•—„•–”ƒ–‡ ‘’‡–‹–‹‘‡š’Žƒ‹•Ž‘••‘ˆ”‘„—•–‡••‹‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧ ƒ ‹†‘š‹†ƒ–‹‘†‹•‘”†‡”•Ǥ‹‘ŽǤʹͲͳ͸ǢͳͶȋͳȌǣͳͲ͹Ǥ ͶͶǤ ‡”• Ǥ†‡ˆ‹ ‹‡ ›ǣ Ž‹‹ ƒŽƒ†Žƒ„‘”ƒ–‘”›•–—†‹‡•Ǥ•ǤǤǢʹͲͲ͹Ǥͳ͸͵’Ǥ ͶͷǤ —‹–‡„‡‡ǡ‘‡Ž• ǡ—„—ŽŽǡ ƒ”ƒ˜ƒ‰Ž‹ƒǡŠƒŽ‡”•ǡƒ›Ž‘”Ǥ‘•‡–‡†‹— Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›ǤͳͻͻͷǢʹͲͻȂͳͶǤ Ͷ͸Ǥ ‡”• ǡ‘—™ǡ‹„ƒ• ǡ‹ƒ ‹‹ ǡƒœ‹ǡ‡‰”‡––‘ ǡ‡• ƒǡ‡‹Œ‰‘—† ǡ ‹– ǡ ƒŒ‡” ǡ ƒ”‰ƒ• Ǥ š’‡”‹‡–ƒŽ ‡˜‹†‡ ‡ ˆ‘” ’”‘–‡‹ ‘š‹†ƒ–‹˜‡ †ƒƒ‰‡ ƒ† ƒŽ–‡”‡†ƒ–‹‘š‹†ƒ–†‡ˆ‡•‡‹’ƒ–‹‡–•™‹–Š‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͶǢ͵͹ȋͷȌǣ͹ͺ͵ȂͻǤ Ͷ͹Ǥ ˜ƒ—‡ǡ‘Ž‡”Ǧ‘—™ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ‘Ž–‡”• ǡ˜ƒ‹Œ– ǡƒ”–‹‡• ǡ ‹‡œ‡Ǧ‘‹‰ǡ ‡‹‡”ǡ‡”‡–‹‡” ǡ ”‘‡ǡ‡‹Œ‰‘—† ǡ‡”• ǡƒ‡”Ǥ ‹˜‹‰‘–Š‡‡†‰‡ǣ—„•–”ƒ–‡ ‘’‡–‹–‹‘‡š’Žƒ‹•Ž‘••‘ˆ”‘„—•–‡••‹‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧ ƒ ‹†‘š‹†ƒ–‹‘†‹•‘”†‡”•Ǥ‹‘ŽǤʹͲͳ͸ǢͳͶȋͳȌǣͳȂͳͷǤ ͶͺǤ Ž‹‰Š ǡ›‡” Ǥƒƒ†‹ƒ ‘—”ƒŽ‘ˆǤƒ–Ž‡•‘— ƒƒ†ƒǤͳͻͷͻǢ͵͹ȋͺȌǤ ͶͻǤ ‘‘”‡ǡ’ƒ ƒ ǡ–‡‹ ǤŠ”‘ƒ–‘‰”ƒ’Š›‘ˆ‹‘ ‹†•‘—Žˆ‘ƒ–‡†‘Ž›•–›”‡‡ ‡•‹•Ǥ ’”‘˜‡†›•–‡ǤƒŽŠ‡Ǥͳͻͷͺ —ŽǢ͵Ͳȋ͹ȌǣͳͳͺͷȂͻͲǤ ͷͲǤ ‡”• 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  Š‡”‹–‡–ƒ„‹•ǤʹͲͲͺǢ͵ͳȋͳȌǣͺͺȂͻ͸Ǥ ͷͳǤ ƒ–›ƒ•Šǡ‹‡„‹• Š ǡ—”œ ŠƒŽ‹ƒǤǡŠ‡˜ Š‡‘ǡ Š™—†‡Ǥ‹’‹†‡š–”ƒ –‹‘„›‡–Š›ŽǦ tertǦ„—–›Ž‡–Š‡”ˆ‘”Š‹‰ŠǦ–Š”‘—‰Š’—–Ž‹’‹†‘‹ •Ǥ ‹’‹†‡•ǤʹͲͲͺǢͶͻȋͷȌǣͳͳ͵͹ȂͶ͸Ǥ ͷʹǤ ‹Ž ǡ Šƒ‰ ǡ ‘Ž–‡”• ǡ ‡”‡–‹‡” ǡ ‘‡” ǡ ‘”˜ƒ–‘˜‹ Š ǡ ‡‹‡”Ǧ ‘‡ƒ ǡ ‡‹Œ‰‘—† ǡ‹• Š‘ˆˆǤ‡Ǧ˜•–™‘Ǧ’Šƒ•‡‡š–”ƒ –‹‘ǣ”‡Ǧ‡˜ƒŽ—ƒ–‹‘‘ˆ•ƒ’Ž‡’”‡’ƒ”ƒ–‹‘ ’”‘ ‡†—”‡• ˆ‘” —–ƒ”‰‡–‡† Ž‹’‹†‘‹ • ‹ ’Žƒ•ƒ •ƒ’Ž‡•Ǥ ƒŽ ‹‘ƒƒŽ Š‡Ǥ ʹͲͳͺǢͶͳͲȋʹ͵ȌǣͷͺͷͻȂ͹ͲǤ ͷ͵Ǥ ‹ ǡ ‰‡Ž‹†‘— ǡ ƒ‰‡ ǡ ‡†‘”‘˜ƒ Ǥ ‹’‹† —–‡” †‡–‹ ˆ‹ ‡• Š‘•’Š‘Ž‹’‹†• „› ‹‰ŠǦ Š”‘—‰Š’—–”‘ ‡••‹‰‘ˆǦƒ†Š‘–‰—‹’‹†‘‹ •ƒ–ƒ•‡–•ǤʹͲͳ͹Ǣ ͷͶǤ Š‘‰ ǡ‘—ˆƒǡ‹ǡƒ”ƒ—• ǡ‹ǡ‘—”“—‡ ǡ‹•Šƒ”–ǡ‹ƒ Ǥ‡–ƒ„‘ƒŽ›•–ͶǤͲ᩿ǣ–‘™ƒ”†• ‘”‡–”ƒ•’ƒ”‡–ƒ†‹–‡‰”ƒ–‹˜‡‡–ƒ„‘Ž‘‹ •ƒƒŽ›•‹•Ǥ— Ž‡‹  ‹†•‡•ǤʹͲͳͺǢͶ͸ȋƒ›ȌǣͳȂ ͻǤ ͷͷǤ Ž‡‹‡”ǡ”—–ǡƒƒ––ƒǡ‡ŠŽ‹‰ǡ‘–‘• ǡ—‹‰•ǡ ‡””‡ŽŽǡ‹—ǡ ‘”„‡•‘ǡ ƒŽ’Ǧ”‹†ƒ ǡ ‡Š ǡ  —ŽŽ‘—‰Š  ǡ ƒ›ƒŽ  Ǥ ‡•‹‰ ƒ† ˜ƒŽ‹†ƒ–‹‘ ‘ˆ ƒ Š‹•–‘Ž‘‰‹ ƒŽ• ‘”‹‰•›•–‡ˆ‘”‘ƒŽ ‘Š‘Ž‹ ˆƒ––›Ž‹˜‡”†‹•‡ƒ•‡Ǥ ‡’ƒ–‘Ž‘‰›ǤʹͲͲͷǢͶͳȋ͸Ȍǣͳ͵ͳ͵Ȃ

ͳͶͶ Effect of fasting and cold stress on hepatic metabolism in MCAD-KO mice

ʹͳǤ  

4

ͳͶͷ Chapter 4

Supplementary figures and tables

A B C 40 16-20 w.o. 40 8 w.o. 40 WT WT WT young

C) KO C) C) MCAD KO young

q q KO q WT old 30 30 30 r=0.64 MCAD KO old p<0.001

20 20 20 Temperature ( Temperature 20 ( Temperature 20 ( Temperature 20 0 0 0 0 2 4 6 0 2 4 6 0 8 16 24 32 Time (h) Time (h) Body weight before cold exposure (g)

Suppl. Fig. S1. Rectal and body temperatures. ‘†› –‡’‡”ƒ–—”‡ ‡ƒ•—”‡† „› ‹–”ƒ’‡”‹–‘‡ƒŽŽ›Ǧ ‹’Žƒ–‡†Ž‘‰‰‡”‹–Š‡ͳ͸ǦʹͲȋAȌƒ†ͺ™‡‡•‘Ž†Ǥ‹ ‡ȋBȌǡ‘†›–‡’‡”ƒ–—”‡˜‡”•—•„‘†›™‡‹‰Š–„‡ˆ‘”‡ ‘Ž†‡š’‘•—”‡ȋCȌǤ 

ͳͶ͸ Effect of fasting and cold stress on hepatic metabolism in MCAD-KO mice

A 8 w.o. 10000 WT M) P KO 1000

* 100 ** [Blood amino acid] ( 10

Total Taurine Citrulline Ornithine

Aminoadipic acid -Aminobutyric acid D BCDbefore fasting 14-hr fasted after cold exposure 50

M) 50 50

P WT 4 40 40 40 KO 30 30 i 30 20 20 20 ** * 10 10 10 16-20 w.o. -acyl-carnitine ( -acyl-carnitine n 1 1 * 1 **

0 0 0 Blood C Blood Ctotal C0 C2 C3 C4 C5:1 C5 Ctotal C0 C2 C3 C4 C5:1 C5 Ctotal C0 C2 C3 C4 C5:1 C5 EF G

M) 50 50 50 P 40 40 40 30 30 30 20 20 20 10 10 10 8 w.o. -acyl-carnitine ( -acyl-carnitine n 1 1 1 i 0 0 0 Blood C Blood Ctotal C0 C2 C3 C4 C5:1 C5 Ctotal C0 C2 C3 C4 C5:1 C5 Ctotal C0 C2 C3 C4 C5:1 C5 HI J

M) 2.5 2.5 2.5 P

2.0 2.0 2.0

1.5 1.5 1.5 16-20 w.o. 1.0 1.0 1.0 -acyl-carnitine ( -acyl-carnitine n 0.5 0.5 i 0.5

0.0 0.0 0.0 Blood C Blood C14:1 C14 C16:1 C16 C18:2 C18:1 C18 C14:1 C14 C16:1 C16 C18:2 C18:1 C18 C14:1 C14 C16:1 C16 C18:2 C18:1 C18 K L M 2.5 2.5

M) 2.5 P 2.0 2.0 2.0 * 1.5 1.5 1.5 8 w.o. 1.0 1.0 1.0 -acyl-carnitine ( i n 0.5 0.5 0.5

0.0 0.0 0.0 Blood C C14:1 C14 C16:1 C16 C18:2 C18:1 C18 C14:1 C14 C16:1 C16 C18:2 C18:1 C18 C14:1 C14 C16:1 C16 C18:2 C18:1 C18 NPO

M) 0.5 0.5 0.5 P

0.4 0.4 0.4

0.3 0.3 0.3 16-20 w.o. 0.2 0.2 0.2

-acyl-carnitine ( i n 0.1 0.1 0.1 * 0.0 0.0 0.0 Blood C

C5DC C5DC C5DC C12-OHC14-OHC16-OH C18-OH C12-OHC14-OHC16-OH C18-OH C12-OHC14-OHC16-OH C18-OH C18:1-OH C18:1-OH C18:1-OH C4OH+C3DCC5OH+C4DC C4OH+C3DCC5OH+C4DC C4OH+C3DCC5OH+C4DC QR S 0.5 0.5

M) 0.5 P

0.4 0.4 0.4 8 w.o. 0.3 0.3 0.3 i 0.2 0.2 0.2 -acyl-carnitine ( n 0.1 i 0.1 0.1 *

0.0 0.0 0.0 Blood C

C5DC C5DC C5DC C12-OHC14-OHC16-OH C18-OH C12-OHC14-OHC16-OH C18-OH C12-OHC14-OHC16-OH C18-OH C18:1-OH C18:1-OH C18:1-OH C4OH+C3DCC5OH+C4DC C4OH+C3DCC5OH+C4DC C4OH+C3DCC5OH+C4DC  Suppl. Fig. S2. Blood non-proteomic amino acids and acyl-carnitine concentration.

ͳͶ͹ Chapter 4

‹‘ƒ ‹† ‘ ‡–”ƒ–‹‘ͺ™Ǥ‘ǡ‹ ‡ȋAȌǡŽ‘‘†ƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘•„‡ˆ‘”‡ˆƒ•–‹‰ȋB,E,H,K,N,QȌǡ ƒˆ–‡”ͳͶŠ”•‘ˆˆƒ•–‹‰ǡ„‡ˆ‘”‡ ‘Ž†‡š’‘•—”‡ȋC,F,I,L,O,RȌǡƒ†ƒˆ–‡”ˆƒ•–‹‰’Ž—• ‘Ž†‡š’‘•—”‡ȋǡ ǡ ǡǡǡȌ ‹ͳ͸ǦʹͲǦ™‡‡•Ǧ‘Ž†ȋB-D, H-J, N-PȌƒ†ͺǦ™‡‡•Ǧ‘Ž†‹ ‡ȋB-D, H-J, N-PȌǤ A 8 w.o. 100 **

10 i

i 1

0.1 *

[Liver amino acid] (nmol/g) 0.01

Total Taurine Citrulline Ornithine Sarcosine saccharopine l-cystathionine Aminoadipic acid -Aminobutyric acid D B C 16-20 w.o. 16-20 w.o. 8 100 *

mol/g) 10 mol/g) P

P 6 1 4 0.1 i i i i 2 i i 0.01

[Liver amino acid] ( 0.001 [Liver amino acid] ( 0

Total Serine Valine Alanine Glycine Lysine Proline Taurine Arginine Cysteine Histidine Leucine Tyrosine Citrulline Ornithine GlutamateGlutamine Threonine Sarcosine AsparagineAsparagine Iso-leucine Methionine Saccharopine Phenylalanine l-cystathionine Aminoadipic acid -Aminobutyric acid D  Suppl. Fig. S3. Non-proteomic liver amino acid levels of 8-weeks-old mice (A) and proteomic (B) and non-proteomic liver amino acid levels 16-20-weeks-old mice (C). A B H&E ORO 3 C) q WT 2 1

0

KO BG change by cold ( -1 n Severe (KO10) Suppl. Fig S4. Histological representation of representative WT and KO livers of 8-weeks-old mice (A) and blood glucose change during cold exposure in 8-weeks-old mice (B).‹˜‡”•™‡”‡•–ƒ‹‡†„› ƒ‡‘–‘š›Ž‹ƒ†‘•‹ȋŽ‡ˆ–Ȍƒ†‹Ž‡†ȋ”‹‰Š–ȌǤ  Supplementary Table ST1. Biometric results and non-lipidomic metabolite concentrations of the WT and KO mice  16-20 weeks-old   8 weeks-old    WT KO WT KO  Tissue ‹‘‡–”‹ •    ʹͻǤͳͲ ά ͲǤͷͻ  ʹͻǤͶͺ ά ͲǤͻͷ   ʹʹǤͷͳ ά ͲǤ͹͹  ʹʹǤͺͷ ά ͲǤͷͳ     ʹ͸Ǥͳͻ ά ͲǤ͸͵  ʹ͸Ǥ͵Ͳ ά ͲǤͻͶ   ͳͻǤͷͻ ά ͲǤ͹ͻ  ʹͲǤ͵Ͳ ά ͲǤͷͶ     ʹͷǤʹͺ ά ͲǤ͸ͳ  ʹͷǤ͹ͻ ά ͲǤͻͻ   ͳͺǤͺ͵ ά ͲǤ͸ͺ  ͳͻǤͷͲ ά ͲǤͷͲ      ͺǤ͸͸ ά ͲǤʹ͹  ͹Ǥͺͺ ά ͲǤͶͻ   ͻǤͺ͹ ά ͲǤ͵ͻ  ͻǤͲ͸ ά ͲǤ͵͹     ͷǤͷͷ ά ͲǤ͵Ͳ  ͶǤͺͶ ά ͲǤͶʹ   ͷǤ͹ͻ ά ͲǤͷͺ  ͶǤͷͺ ά ͲǤʹͳ i Ž‘‘†    ͸Ǥ͹Ͷ ά ͲǤ͸Ͳ ͸Ǥͷ͵ ά ͲǤͶͲ   ͹Ǥ͵ͻ ά ͲǤͷʹ ͷǤ͸͵ ά ͲǤ͵ͷ ȗ Ž‘‘†   ͲǤ͵ͳ ά ͲǤͲͶ  ͲǤ͵ͺ ά ͲǤͲ͵   ͲǤͶͷ ά ͲǤͲ͵  ͲǤͷ͵ ά ͲǤͲ͹ Ž‘‘†   ͳǤͻ͸ ά ͲǤʹ͹  ͳǤͶͺ ά ͲǤͳ͵   ʹǤʹͶ ά ͲǤͳͻ  ͳǤͺͺ ά ͲǤͳͻ  Ž‘‘†   ͵ǤͳͶ ά ͲǤ͵͸  ʹǤ͹Ͳ ά ͲǤ͵ͻ   ʹǤ͸Ͷ ά ͲǤͳͶ  ʹǤ͵ͷ ά ͲǤʹͳ  Ž‘‘† ‘‰‰‡”̴‡’  ͵͹Ǥ͸ͺ ά ͲǤ͵ͳ  ͵͸Ǥͺ͸ ά ͲǤʹͻ   ͵͹Ǥ͸Ͷ ά ͲǤʹͷ  ͵͸Ǥͻ͵ ά ͲǤʹͻ   ‘‰‰‡”̴‡’  ͵͸Ǥʹ͹ ά ͲǤʹͺ  ͵͸ǤͳͲ ά ͲǤʹͷ i  ͵ͷǤͶ͵ ά ͲǤͷͻ  ͵ͷǤ͹Ͳ ά ͲǤʹͻ  ‘‰‰‡”̴‡’  ͵͵Ǥ͵Ͳ ά ͲǤ͹ͺ  ʹͺǤ͹Ͳ ά ʹǤʹͳ   ʹͷǤͲͻ ά ʹǤͺͶ  ʹͺǤͳͺ ά ʹǤʹͶ  

ͳͶͺ Effect of fasting and cold stress on hepatic metabolism in MCAD-KO mice

 16-20 weeks-old   8 weeks-old   WT  KO   WT  WT Tissue ‡ –ƒŽ–‡’‡”ƒ–—”‡ ͵ͳǤʹͲ ά ǤƒǤ  ʹͺǤʹͲ ά ǤƒǤ   ʹͳǤͶͻ ά ʹǤ͵͸  ʹͷǤ͵ͻ ά ͳǤͷͻ   ‹˜‡”™‡‹‰Š– ͳǤͲ͸ ά ͲǤͲͶ  ͳǤʹ͵ ά ͲǤͲͷ ȗ  ͲǤͺͻ ά ͲǤͲͶ  ͳǤͲͶ ά ͲǤͲ͵ ȗ  ‡Žƒ–‹˜‡Ž‹˜‡”™‡‹‰Š– ͶǤʹͲ ά ͲǤͳͶ  ͶǤ͹ͻ ά ͲǤͳͷ ȗ  ͶǤ͸ͳ ά ͲǤͳʹ  ͷǤ͵Ͳ ά ͲǤͳͶ ȗȗ  ‹˜‡””‹‰Ž› ‡”‹†‡• ͷ͸Ǥ͹ͷ ά ͶǤ͹͸  ͹ͳǤʹͷ ά ͶǤ͸ͷ ȗ  ͸ͲǤ͸͵ ά ͵Ǥͷͺ  ͹͸Ǥ͵ͺ ά ͵ǤͲͷ ȗȗ ‹˜‡” Žƒ•ƒ  ͺͷͷ ά ͻͳ  ͻͷͲ ά ͷʹ   ͺͳͺ ά ʹͻ  ͳͲ͵͸ ά ͸ʹ ȗȗ Žƒ•ƒ                    Ž‘‘†ƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘•„‡ˆ‘”‡ˆƒ•–‹‰              Ž‘‘†–‘–ƒŽ  ʹͷǤʹͲ ά ͳǤ͸ʹ  ʹͳǤͲͲ ά ʹǤͷͳ   ͵ͲǤͺͺ ά ʹǤ͸ͷ  ʹͻǤͳͶ ά ͳǤʹͺ  Ž‘‘† Ž‘‘†Ͳ  ͳͷǤͲͲ ά ͳǤͳͶ  ͳʹǤͲͲ ά ͳǤʹ͸   ͳͷǤͺͺ ά ͳǤ͸ʹ  ͳͷǤͲͲ ά ͲǤ͸ʹ Ž‘‘† Ž‘‘†ʹ  ͹ǤͶ͹ ά ͲǤͶ͸  ͸Ǥʹͺ ά ͳǤͳʹ   ͳͳǤ͵͵ ά ͲǤͻ͹  ͳͲǤ͸Ͷ ά ͲǤͷ͸  Ž‘‘† 4 Ž‘‘†͵  ͲǤ͵ͷ ά ͲǤͲʹ  ͲǤ͵ͺ ά ͲǤͲ͹   ͲǤͷ͵ ά ͲǤͲ͹  ͲǤͷ͹ ά ͲǤͲ͹ Ž‘‘† Ž‘‘†Ͷ  ͲǤʹͷ ά ͲǤͲ͸  ͲǤʹ͸ ά ͲǤͲͷ   ͲǤʹͷ ά ͲǤͲ͸  ͲǤʹͷ ά ͲǤͲͶ  Ž‘‘† Ž‘‘†ͷǣͳ  ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ   ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͷ  ͲǤͲͻ ά ͲǤͲʹ  ͲǤͲͻ ά ͲǤͲͳ   ͲǤͳͶ ά ͲǤͲʹ ͲǤͳͳ ά ͲǤͲͳ  Ž‘‘† Ž‘‘†͸  ͲǤͲ͵ ά ͲǤͲͲ  ͲǤͲ͹ ά ͲǤͲʹ ȗ  ͲǤͲ͵ ά ͲǤͲͳ  ͲǤͳͲ ά ͲǤͲʹ ȗ Ž‘‘† Ž‘‘†ͺ  ͲǤͲͶ ά ͲǤͲͲ  ͲǤͲ͸ ά ͲǤͲͳ ȗ  ͲǤͲͷ ά ͲǤͲͳ  ͲǤͲͻ ά ͲǤͲͳ ȗȗ Ž‘‘† Ž‘‘†ͳͲǣͳ  ͲǤͲʹ ά ͲǤͲͲ  ͲǤͲͷ ά ͲǤͲʹ ȗȗ  ͲǤͲʹ ά ͲǤͲͲ  ͲǤͲͺ ά ͲǤͲͳ ȗȗ Ž‘‘† Ž‘‘†ͳͲ  ͲǤͲ͵ ά ͲǤͲͳ  ͲǤͲͶ ά ͲǤͲͳ   ͲǤͲ͵ ά ͲǤͲͳ  ͲǤͲͳ ά ͲǤͲͳ  Ž‘‘† Ž‘‘†ͳʹǣͳ  ͲǤͲʹ ά ͲǤͲͲ  ͲǤͲ͵ ά ͲǤͲͲ   ͲǤͲʹ ά ͲǤͲͲ  ͲǤͲʹ ά ͲǤͲͲ Ž‘‘† Ž‘‘†ͳʹ  ͲǤͲ͹ ά ͲǤͲͲ  ͲǤͲ͸ ά ͲǤͲͳ   ͲǤͲͺ ά ͲǤͲͳ  ͲǤͲ͹ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͳͶǣͳ  ͲǤͲͻ ά ͲǤͲͳ  ͲǤͲͺ ά ͲǤͲʹ   ͲǤͳͳ ά ͲǤͲͳ  ͲǤͳͳ ά ͲǤͲͳ  Ž‘‘† Ž‘‘†ͳͶ  ͲǤʹ͵ ά ͲǤͲ͵  ͲǤʹͲ ά ͲǤͲʹ   ͲǤʹ͵ ά ͲǤͲʹ  ͲǤʹͶ ά ͲǤͲʹ  Ž‘‘† Ž‘‘†ͳ͸ǣͳ  ͲǤͳͲ ά ͲǤͲʹ  ͲǤͲͺ ά ͲǤͲʹ   ͲǤͳʹ ά ͲǤͲʹ  ͲǤͳʹ ά ͲǤͲͳ  Ž‘‘† Ž‘‘†ͳ͸  ͲǤͺͲ ά ͲǤͲͺ  ͲǤ͸ͻ ά ͲǤͲͺ   ͲǤͻͷ ά ͲǤͲͷ  ͲǤͻͲ ά ͲǤͲͶ Ž‘‘† Ž‘‘†ͳͺǣʹ  ͲǤͲͷ ά ͲǤͲͳ  ͲǤͲͶ ά ͲǤͲͳ   ͲǤͲ͸ ά ͲǤͲͲ  ͲǤͲ͸ ά ͲǤͲͲ Ž‘‘† Ž‘‘†ͳͺǣͳ  ͲǤ͵Ͷ ά ͲǤͲͶ  ͲǤ͵ͳ ά ͲǤͲͺ   ͲǤͶͷ ά ͲǤͲͶ ͲǤͶʹ ά ͲǤͲͶ  Ž‘‘† Ž‘‘†ͳͺ  ͲǤͳͻ ά ͲǤͲʹ  ͲǤͳͺ ά ͲǤͲʹ   ͲǤʹͶ ά ͲǤͲͳ ͲǤʹʹ ά ͲǤͲͳ  Ž‘‘† Ž‘‘†Ͷ Ϊ͵  ͲǤͲ͵ ά ͲǤͲͳ  ͲǤͲʹ ά ͲǤͲͳ   ͲǤͲͷ ά ͲǤͲͳ  ͲǤͲͶ ά ͲǤͲͲ i Ž‘‘† Ž‘‘†ͷ ΪͶ  ͲǤͳͳ ά ͲǤͲͳ  ͲǤͲ͹ ά ͲǤͲʹ   ͲǤͳͶ ά ͲǤͲͳ  ͲǤͳ͵ ά ͲǤͲͳ  Ž‘‘† Ž‘‘†ͷ  ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ   ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͳʹ   ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ   ͲǤͲͳ ά ͲǤͲͲ  ͲǤͲͳ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͳͶ   ͲǤͲͳ ά ͲǤͲͲ  ͲǤͲͳ ά ͲǤͲͲ   ͲǤͲʹ ά ͲǤͲͲ  ͲǤͲʹ ά ͲǤͲͲ Ž‘‘† Ž‘‘†ͳ͸   ͲǤͲ͵ ά ͲǤͲͳ  ͲǤͲʹ ά ͲǤͲͲ   ͲǤͲ͵ ά ͲǤͲͲ  ͲǤͲʹ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͳͺǣͳ   ͲǤͲʹ ά ͲǤͲͲ  ͲǤͲͳ ά ͲǤͲͲ   ͲǤͲ͵ ά ͲǤͲͲ  ͲǤͲʹ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͳͺ   ͲǤͲͳ ά ͲǤͲͲ  ͲǤͲͳ ά ͲǤͲͲ   ͲǤͲͳ ά ͲǤͲͲ  ͲǤͲͳ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͲȀ–‘–ƒŽ  ͲǤͷͻ ά ͲǤͲʹ  ͲǤͷͺ ά ͲǤͲʹ   ͲǤͷʹ ά ͲǤͲʹ  ͲǤͷͲ ά ͲǤͲͳ  Ž‘‘†                    Ž‘‘†ƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘•ͷͺŠ”•‘ˆˆƒ•–‹‰             Ž‘‘†–‘–ƒŽ  ʹͳǤͺ͸ ά ͳǤ͹ͻ  ͳͺǤͳ͵ ά ͲǤ͸ͻ i  ʹ͵Ǥ͹ͷ ά ʹǤͲʹ  ʹͶǤʹͷ ά ͳǤ͸ʹ Ž‘‘† Ž‘‘†Ͳ  ͺǤͲͻ ά ͲǤͷͶ  ͷǤ͹ͷ ά ͲǤʹͷ ȗȗ  ͹ǤͲͲ ά ͲǤ͵͵  ͹ǤͲͲ ά ͲǤͷ͵  Ž‘‘† Ž‘‘†ʹ  ͳͲǤ͵ͳ ά ͳǤͳͶ ͺǤ͵ͳ ά ͲǤ͸ͳ   ͳʹǤʹ͵ ά ͳǤͷͲ  ͳʹǤͲ͵ ά ͲǤͻͲ  Ž‘‘† Ž‘‘†͵  ͲǤͶ͸ ά ͲǤͲͷ  ͲǤʹ͸ ά ͲǤͲͶ ȗȗ  ͲǤ͸ͺ ά ͲǤͳ͸  ͲǤͷͳ ά ͲǤͲͶ  Ž‘‘† Ž‘‘†Ͷ  ͲǤͲͷ ά ͲǤͲͲ  ͲǤͲͷ ά ͲǤͲͳ   ͲǤͲͷ ά ͲǤͲͳ  ͲǤͲͶ ά ͲǤͲͳ  Ž‘‘† Ž‘‘†ͷǣͳ  ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ   ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͷ  ͲǤͲͷ ά ͲǤͲͲ  ͲǤͲͷ ά ͲǤͲͳ   ͲǤͲͻ ά ͲǤͲͳ ͲǤͲ͹ ά ͲǤͲͳ i Ž‘‘† Ž‘‘†͸  ͲǤͲʹ ά ͲǤͲͲ  ͲǤͳ͵ ά ͲǤͲͳ ȗȗȗȗ ͲǤͲ͵ ά ͲǤͲͲ  ͲǤͳʹ ά ͲǤͲ͵ ȗȗȗ Ž‘‘† Ž‘‘†ͺ  ͲǤͲͶ ά ͲǤͲͳ  ͲǤͳͳ ά ͲǤͲͳ ȗȗȗ  ͲǤͲ͸ ά ͲǤͲͲ ͲǤͳͷ ά ͲǤͲͳ ȗȗȗȗ Ž‘‘† Ž‘‘†ͳͲǣͳ  ͲǤͲ͵ ά ͲǤͲͲ ͲǤͳͻ ά ͲǤͲʹ ȗȗȗȗ ͲǤͲʹ ά ͲǤͲͲ  ͲǤʹʹ ά ͲǤͲʹ ȗȗȗ Ž‘‘† Ž‘‘†ͳͲ  ͲǤͲͶ ά ͲǤͲͲ  ͲǤͲ͹ ά ͲǤͲͲ ȗȗȗ  ͲǤͲ͵ ά ͲǤͲͳ  ͲǤͲͺ ά ͲǤͲͳ ȗȗ Ž‘‘† Ž‘‘†ͳʹǣͳ  ͲǤͲ͵ ά ͲǤͲͲ ͲǤͲͷ ά ͲǤͲͳ ȗ  ͲǤͲ͵ ά ͲǤͲͲ  ͲǤͲͷ ά ͲǤͲͳ ȗ Ž‘‘† Ž‘‘†ͳʹ  ͲǤͲ͸ ά ͲǤͲͳ  ͲǤͲͻ ά ͲǤͲͳ i  ͲǤͲͺ ά ͲǤͲͳ  ͲǤͳͳ ά ͲǤͲͳ i Ž‘‘† Ž‘‘†ͳͶǣͳ  ͲǤͳ͹ ά ͲǤͲʹ  ͲǤʹ͵ ά ͲǤͲʹ i  ͲǤʹͳ ά ͲǤͲ͵  ͲǤʹͷ ά ͲǤͲʹ  Ž‘‘† Ž‘‘†ͳͶ  ͲǤʹ͹ ά ͲǤͲͶ  ͲǤ͵͵ ά ͲǤͲ͵   ͲǤ͵ͳ ά ͲǤͲͶ ͲǤͶͳ ά ͲǤͲʹ i Ž‘‘† Ž‘‘†ͳ͸ǣͳ  ͲǤͳͻ ά ͲǤͲʹ  ͲǤʹʹ ά ͲǤͲ͵   ͲǤʹͶ ά ͲǤͲ͵  ͲǤ͵Ͳ ά ͲǤͲʹ  Ž‘‘† Ž‘‘†ͳ͸  ͲǤͺ͹ ά ͲǤͲͻ  ͲǤͻͷ ά ͲǤͲͷ   ͳǤͲͻ ά ͲǤͳ͵  ͳǤͶͲ ά ͲǤͲ͸ ȗ Ž‘‘† Ž‘‘†ͳͺǣʹ  ͲǤͲͻ ά ͲǤͲͳ ͲǤͲͻ ά ͲǤͲͳ   ͲǤͳͳ ά ͲǤͲʹ  ͲǤͳʹ ά ͲǤͲͳ  Ž‘‘† Ž‘‘†ͳͺǣͳ  ͲǤ͸Ͳ ά ͲǤͲͻ ͲǤ͸ͷ ά ͲǤͲ͸   ͲǤ͹͸ ά ͲǤͳͲ  ͲǤͺͻ ά ͲǤͲͷ  Ž‘‘† Ž‘‘†ͳͺ  ͲǤͳ͹ ά ͲǤͲʹ  ͲǤͳͺ ά ͲǤͲͳ   ͲǤʹ͵ ά ͲǤͲ͵ ͲǤʹ͹ ά ͲǤͲͳ Ž‘‘† Ž‘‘†Ͷ Ϊ͵  ͲǤͳ͵ ά ͲǤͲ͵  ͲǤͲ͹ ά ͲǤͲͳ i  ͲǤͳ͸ ά ͲǤͲʹ  ͲǤͳͲ ά ͲǤͲʹ i Ž‘‘† Ž‘‘†ͷ ΪͶ  ͲǤͲ͹ ά ͲǤͲͳ ͲǤͲ͸ ά ͲǤͲͳ   ͲǤͲͻ ά ͲǤͲͳ  ͲǤͳͲ ά ͲǤͲͳ  Ž‘‘† Ž‘‘†ͷ  ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ   ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͳʹ   ͲǤͲͳ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ   ͲǤͲͳ ά ͲǤͲͲ  ͲǤͲͳ ά ͲǤͲͲ  Ž‘‘†

ͳͶͻ Chapter 4

 16-20 weeks-old   8 weeks-old   WT  KO   WT  WT Tissue Ž‘‘†ͳͶ   ͲǤͲʹ ά ͲǤͲͲ  ͲǤͲʹ ά ͲǤͲͲ   ͲǤͲʹ ά ͲǤͲͲ  ͲǤͲʹ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͳ͸   ͲǤͲ͵ ά ͲǤͲͲ  ͲǤͲ͵ ά ͲǤͲͲ   ͲǤͲͶ ά ͲǤͲͳ ͲǤͲͶ ά ͲǤͲͲ Ž‘‘† Ž‘‘†ͳͺǣͳ   ͲǤͲ͵ ά ͲǤͲͳ  ͲǤͲ͵ ά ͲǤͲͲ   ͲǤͲͷ ά ͲǤͲͳ  ͲǤͲͶ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͳͺ   ͲǤͲͳ ά ͲǤͲͲ  ͲǤͲͳ ά ͲǤͲͲ   ͲǤͲͳ ά ͲǤͲͲ  ͲǤͲʹ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͲȀ–‘–ƒŽ  ͲǤ͵ͺ ά ͲǤͲʹ  ͲǤ͵͵ ά ͲǤͲʹ   ͲǤ͵ʹ ά ͲǤͲͷ  ͲǤʹͻ ά ͲǤͲͳ  Ž‘‘†                    Ž‘‘†ƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘•ƒˆ–‡”ˆƒ•–‹‰ƒ† ‘Ž†‡š’‘•—”‡           Ž‘‘†–‘–ƒŽ  ʹʹǤ͹Ͳ ά ʹǤʹͲ  ͳͺǤ͸͵ ά ͳǤͶͲ   ʹ͸Ǥ͸͵ ά ʹǤͳ͹  ʹͶǤͷͲ ά ʹǤͳ͸  Ž‘‘† Ž‘‘†Ͳ  ͸Ǥ͸͹ ά ͲǤͷ͵  ͷǤͲͲ ά ͲǤͷ͵ ȗ  ͷǤͺͺ ά ͲǤͶͶ  ͷǤ͹ͷ ά ͲǤͷ͸  Ž‘‘† Ž‘‘†ʹ  ͳͳǤ͸ͷ ά ͳǤͶͺ  ͺǤ͹͸ ά ͲǤ͸͹   ͳ͵ǤͻͲ ά ͳǤͲ͵  ͳʹǤͳ͵ ά ͳǤ͵ͳ  Ž‘‘† Ž‘‘†͵  ͲǤͶͺ ά ͲǤͲͶ  ͲǤ͵ͳ ά ͲǤͲͷ ȗ  ͲǤ͸Ͷ ά ͲǤͳͲ  ͲǤͶ͹ ά ͲǤͲ͹ Ž‘‘† Ž‘‘†Ͷ  ͲǤͲͷ ά ͲǤͲͳ  ͲǤͲͷ ά ͲǤͲͳ   ͲǤͲͺ ά ͲǤͲʹ  ͲǤͲͷ ά ͲǤͲͳ  Ž‘‘† Ž‘‘†ͷǣͳ  ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ   ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͷ  ͲǤͲ͵ ά ͲǤͲͳ  ͲǤͲʹ ά ͲǤͲͲ   ͲǤͲ͹ ά ͲǤͲͳ  ͲǤͲͷ ά ͲǤͲͳ  Ž‘‘† Ž‘‘†͸  ͲǤͲʹ ά ͲǤͲͲ  ͲǤͲͺ ά ͲǤͲͳ  ͲǤͲʹ ά ͲǤͲͳ  ͲǤͲͺ ά ͲǤͲʹ ȗȗ Ž‘‘† Ž‘‘†ͺ  ͲǤͲͶ ά ͲǤͲͳ  ͲǤͲͻ ά ͲǤͲͳ   ͲǤͲͷ ά ͲǤͲͳ  ͲǤͳͶ ά ͲǤͲͳ ȗȗȗ Ž‘‘† Ž‘‘†ͳͲǣͳ  ͲǤͲʹ ά ͲǤͲͲ  ͲǤͳͺ ά ͲǤͲʹ   ͲǤͲʹ ά ͲǤͲͲ  ͲǤʹ͵ ά ͲǤͲʹ ȗȗȗ Ž‘‘† Ž‘‘†ͳͲ  ͲǤͲͶ ά ͲǤͲͲ  ͲǤͲ͹ ά ͲǤͲͳ   ͲǤͲͶ ά ͲǤͲͳ  ͲǤͲͺ ά ͲǤͲͳ ȗ Ž‘‘† Ž‘‘†ͳʹǣͳ  ͲǤͲ͵ ά ͲǤͲͲ  ͲǤͲͶ ά ͲǤͲͲ   ͲǤͲͶ ά ͲǤͲͲ  ͲǤͲͷ ά ͲǤͲͳ Ž‘‘† Ž‘‘†ͳʹ  ͲǤͲ͹ ά ͲǤͲͳ  ͲǤͲͻ ά ͲǤͲͳ   ͲǤͳͳ ά ͲǤͲͳ ͲǤͳʹ ά ͲǤͲͳ  Ž‘‘† Ž‘‘†ͳͶǣͳ  ͲǤʹʹ ά ͲǤͲʹ  ͲǤʹ͸ ά ͲǤͲ͵   ͲǤ͵ͺ ά ͲǤͲͷ  ͲǤ͵͹ ά ͲǤͲ͵  Ž‘‘† Ž‘‘†ͳͶ  ͲǤ͵ͻ ά ͲǤͲͷ ͲǤͶ͸ ά ͲǤͲ͸  ͲǤ͸ͳ ά ͲǤͲ͹  ͲǤ͸͸ ά ͲǤͲͶ  Ž‘‘† Ž‘‘†ͳ͸ǣͳ  ͲǤʹ͹ ά ͲǤͲ͵ ͲǤ͵ͳ ά ͲǤͲͷ  ͲǤͶ͸ ά ͲǤͲͷ  ͲǤͶ͸ ά ͲǤͲͶ  Ž‘‘† Ž‘‘†ͳ͸  ͳǤͳʹ ά ͲǤͳͳ ͳǤʹ͸ ά ͲǤͳ͵   ͳǤ͸Ͳ ά ͲǤͳ͸  ͳǤ͹ͺ ά ͲǤͲ͸  Ž‘‘† Ž‘‘†ͳͺǣʹ  ͲǤͳͶ ά ͲǤͲͳ ͲǤͳͶ ά ͲǤͲʹ   ͲǤʹ͵ ά ͲǤͲ͵  ͲǤͳͺ ά ͲǤͲͳ  Ž‘‘† Ž‘‘†ͳͺǣͳ  ͲǤͺ͹ ά ͲǤͲͺ ͳǤͲʹ ά ͲǤͳ͵   ͳǤͶ͹ ά ͲǤͳͻ  ͳǤ͵ͺ ά ͲǤͲͷ  Ž‘‘† Ž‘‘†ͳͺ  ͲǤʹʹ ά ͲǤͲʹ  ͲǤʹͶ ά ͲǤͲ͵ ȗ  ͲǤ͵͹ ά ͲǤͲͶ  ͲǤ͵͸ ά ͲǤͲʹ  Ž‘‘† Ž‘‘†Ͷ Ϊ͵  ͲǤͳͷ ά ͲǤͲͶ  ͲǤͲͷ ά ͲǤͲʹ   ͲǤʹʹ ά ͲǤͲͶ  ͲǤͳͷ ά ͲǤͲͳ  Ž‘‘† Ž‘‘†ͷ ΪͶ  ͲǤͲ͹ ά ͲǤͲͳ  ͲǤͲ͹ ά ͲǤͲͳ   ͲǤͲͻ ά ͲǤͲͳ  ͲǤͲͺ ά ͲǤͲͳ Ž‘‘† Ž‘‘†ͷ  ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ   ͲǤͲͶ ά ͲǤͲͶ  ͲǤͲͲ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͳʹ   ͲǤͲͳ ά ͲǤͲͲ  ͲǤͲͳ ά ͲǤͲͲ   ͲǤͲʹ ά ͲǤͲͲ  ͲǤͲʹ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͳͶ   ͲǤͲʹ ά ͲǤͲͲ  ͲǤͲʹ ά ͲǤͲͲ   ͲǤͲͶ ά ͲǤͲͳ  ͲǤͲ͵ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͳ͸   ͲǤͲͶ ά ͲǤͲͲ  ͲǤͲͶ ά ͲǤͲͳ   ͲǤͲ͹ ά ͲǤͲͳ  ͲǤͲͷ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͳͺǣͳ   ͲǤͲͷ ά ͲǤͲͲ ͲǤͲͷ ά ͲǤͲͳ   ͲǤͳͲ ά ͲǤͲʹ  ͲǤͲ͹ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͳͺ   ͲǤͲͳ ά ͲǤͲͲ ͲǤͲͳ ά ͲǤͲͲ   ͲǤͲʹ ά ͲǤͲͲ  ͲǤͲʹ ά ͲǤͲͲ  Ž‘‘† Ž‘‘†ͲȀ–‘–ƒŽ  ͲǤ͵Ͳ ά ͲǤͲͳ  ͲǤʹ͹ ά ͲǤͲʹ   ͲǤʹ͵ ά ͲǤͲͳ ͲǤʹ͵ ά ͲǤͲͲ  Ž‘‘†                    ‹˜‡”ƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘•ȋƒˆ–‡”ˆƒ•–‹‰ƒ† ‘Ž†‡š’‘•—”‡Ȍ          ‹˜‡”–‘–ƒŽ ͵ͲͲ ά ͳͳ  ʹͺͶ ά ʹͳ   ͵͵͸ ά ͳ͹ ͵ʹͶ ά ͳʹ ‹˜‡” ‹˜‡”Ͳ ʹͺͶ ά ͻ  ʹ͹ͳ ά ʹͳ   ͵ͳͳ ά ͳͺ  ͵Ͳ͵ ά ͳ͵  ‹˜‡” ‹˜‡”ʹ ͶǤͺͶ ά ͳǤ͹ʹ  ʹǤ͹͵ ά ͳǤ͵ͷ   ͻǤʹ͸ ά ʹǤʹͳ  ͷǤͷͳ ά ͳǤ͸ʹ  ‹˜‡” ‹˜‡”͵ ͳǤͻ͵ ά ͲǤ͵ͺ  ͳǤͺͻ ά ͲǤ͵͸   ͵ǤͶͺ ά ͲǤͷͺ  ͵Ǥʹ͸ ά ͲǤ͵ͷ  ‹˜‡” ‹˜‡”Ͷ ͲǤʹͶ ά ͲǤͲͷ  ͲǤͳͲ ά ͲǤͲʹ   ͲǤ͵Ͳ ά ͲǤͲͷ  ͲǤʹͻ ά ͲǤͲ͸  ‹˜‡” ‹˜‡”ͷǣͳ ͲǤͲͺ ά ͲǤͲͳ  ͲǤͲͶ ά ͲǤͲͳ   ͲǤͲͻ ά ͲǤͲͳ  ͲǤͲ͸ ά ͲǤͲͳ  ‹˜‡” ‹˜‡”ͷ ͲǤʹͳ ά ͲǤͳͳ  ͲǤͲͺ ά ͲǤͲͳ   ͲǤͳ͸ ά ͲǤͲ͵  ͲǤͳʹ ά ͲǤͲͳ ‹˜‡” ‹˜‡”͸ ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ   ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ  ‹˜‡” ‹˜‡”ͺ ͲǤͲͺ ά ͲǤͲͳ  ͲǤͲͷ ά ͲǤͲͳ i  ͲǤͲͺ ά ͲǤͲͳ  ͲǤͲͻ ά ͲǤͲͳ  ‹˜‡” ‹˜‡”ͳͲǣͳ ͲǤʹͶ ά ͲǤͲͷ  ͲǤʹͲ ά ͲǤͲʹ   ͲǤ͵Ͷ ά ͲǤͳͲ  ͲǤʹʹ ά ͲǤͲʹ  ‹˜‡” ‹˜‡”ͳͲ ͲǤͳͳ ά ͲǤͲͳ  ͲǤͳͳ ά ͲǤͲʹ   ͲǤͳ͵ ά ͲǤͲͳ  ͲǤͳͶ ά ͲǤͲͳ  ‹˜‡” ‹˜‡”ͳʹǣͳ ͲǤͲͷ ά ͲǤͲͳ  ͲǤͲ͵ ά ͲǤͲͳ   ͲǤͲͷ ά ͲǤͲͳ  ͲǤͲͶ ά ͲǤͲͳ  ‹˜‡” ‹˜‡”ͳʹ ͲǤͲ͸ ά ͲǤͲͳ  ͲǤͲ͸ ά ͲǤͲͳ   ͲǤͲͺ ά ͲǤͲͳ  ͲǤͲͺ ά ͲǤͲͳ  ‹˜‡” ‹˜‡”ͳͶǣͳ ͲǤͲͻ ά ͲǤͲͳ  ͲǤͲͺ ά ͲǤͲͳ   ͲǤͲͻ ά ͲǤͲͳ  ͲǤͲͺ ά ͲǤͲͳ  ‹˜‡” ‹˜‡”ͳͶ ͲǤͲͶ ά ͲǤͲͳ  ͲǤͲ͵ ά ͲǤͲͳ   ͲǤͲͺ ά ͲǤͲͳ  ͲǤͲͷ ά ͲǤͲͲ  ‹˜‡” ‹˜‡”ͳ͸ǣͳ ͲǤͲ͸ ά ͲǤͲʹ  ͲǤͲͷ ά ͲǤͲʹ   ͲǤͲͺ ά ͲǤͲͳ  ͲǤͲ͸ ά ͲǤͲͳ  ‹˜‡” ‹˜‡”ͳ͸ ͲǤͳ͸ ά ͲǤͲʹ  ͲǤͳ͵ ά ͲǤͲ͵   ͲǤʹ͸ ά ͲǤͲͶ  ͲǤͳͻ ά ͲǤͲ͵  ‹˜‡” ‹˜‡”ͳͺǣʹ ͲǤͲͻ ά ͲǤͲͳ  ͲǤͲͺ ά ͲǤͲͳ   ͲǤͲͻ ά ͲǤͲͳ  ͲǤͳͲ ά ͲǤͲͳ  ‹˜‡” ‹˜‡”ͳͺǣͳ ͲǤʹͳ ά ͲǤͲ͵  ͲǤͳ͸ ά ͲǤͲͶ   ͲǤʹͶ ά ͲǤͲ͵  ͲǤʹʹ ά ͲǤͲ͵ ‹˜‡” ‹˜‡”ͳͺ ͲǤͲ͹ ά ͲǤͲͳ  ͲǤͲ͹ ά ͲǤͲͳ   ͲǤͳʹ ά ͲǤͲͳ  ͲǤͲͺ ά ͲǤͲͳ ȗ ‹˜‡” ‹˜‡”Ͷ Ϊ͵ ͵Ǥͳ͹ ά ͲǤ͵ʹ  ͳǤͺ͸ ά ͲǤͳ͵   ͵Ǥͻͺ ά ͲǤʹͷ ʹǤʹͷ ά ͲǤͲͺ ȗȗȗ ‹˜‡” ‹˜‡”ͷ ΪͶ ͲǤ͹͹ ά ͲǤͲ͸  ͲǤͷͺ ά ͲǤͲͶ   ͲǤͺͻ ά ͲǤͲͷ  ͲǤ͹Ͳ ά ͲǤͲͶ ȗ ‹˜‡” ‹˜‡”ͷ‹˜‡” ʹǤͻʹ ά ͲǤ͵͵  ͶǤ͹͸ ά ͲǤͻ͸   ͶǤͻ͸ ά ͲǤ͵Ͷ  ͹Ǥ͹Ͳ ά ͲǤ͹͸ ȗȗ ‹˜‡” ‹˜‡”ͳʹ  ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ   ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ  ‹˜‡”

ͳͷͲ Effect of fasting and cold stress on hepatic metabolism in MCAD-KO mice

 16-20 weeks-old   8 weeks-old    WT  KO   WT  WT  Tissue ‹˜‡”ͳͶ  ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ   ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ  ‹˜‡” ‹˜‡”ͳ͸  ͲǤͲͶ ά ͲǤͲͳ  ͲǤͲͲ ά ͲǤͲͲ   ͲǤͲ͸ ά ͲǤͲͳ  ͲǤͲͶ ά ͲǤͲͳ  ‹˜‡” ‹˜‡”ͳͺǣͳ  ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ   ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ  ‹˜‡” ‹˜‡”ͳͺ  ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ   ͲǤͲͲ ά ͲǤͲͲ  ͲǤͲͲ ά ͲǤͲͲ  ‹˜‡” ‹˜‡”ͲȀ–‘–ƒŽ ͲǤͻͷ ά ͲǤͲͳ  ͲǤͻͷ ά ͲǤͲͳ   ͲǤͻʹ ά ͲǤͲͳ ͲǤͻ͵ ά ͲǤͲͳ  ‹˜‡”                    ‹˜‡”ƒ‹‘ƒ ‹† ‘ ‡–”ƒ–‹‘•ȋƒˆ–‡”ˆƒ•–‹‰ƒ† ‘Ž†‡š’‘•—”‡Ȍ           Žƒ‹‡ ͷͻͳͶ ά ͷͲͺ  ͶͶ͸͸ ά ͸ʹͲ   ͷ͸Ͷ͵ ά ͵ͻͻ  Ͷ͹Ͳ͸ ά ʹͷʹ i ‹˜‡” ”‰‹‹‡ Ǥ†Ǥ ά Ǥ†Ǥ  Ǥ†Ǥ ά Ǥ†Ǥ   ͵͹ ά ʹͳ  ͳͺ ά ͵  ‹˜‡” •’ƒ”ƒ‰‹‡ ͳ͵͸͹ ά ͳʹʹ  ͳͳ͵ʹ ά ͸͸   ͳʹͲͲ ά ͳ͵ͳ  ͻͶ͹ ά Ͷʹ  ‹˜‡” 4 •’ƒ”–‹ ƒ ‹† ͳͲ͸Ͷ ά ͹͵  ͻͶͳ ά Ͷ͹   ͳʹͺͻ ά ͸ͳ  ͳͳ͸ͳ ά Ͷ͹  ‹˜‡” ›•–‡‹‡ ʹ͵Ǥ͸ ά ͳͲǤͶ  ͶͳǤͲ ά ʹͲǤʹ   ͵ͳǤͷ ά ͻǤͻ  ͳʹǤ͵ ά ͷǤʹ  ‹˜‡” Ž—–ƒ‹‡ ʹ͸ͷʹ ά Ͷ͹Ͷ  ͳͻͳͺ ά ʹͷʹ   ͷͲͳʹ ά ʹͶͺ  ͵͹͸Ͳ ά ʹͳͶ ȗȗ ‹˜‡” Ž—–ƒ‹ ƒ ‹† ͵͸ʹͷ ά ͵͸͸  ͵͵Ͷ͵ ά ʹ͹͵   ʹ͸͹ͷ ά ʹ͸͹ ʹͲͻͷ ά ͳ͹͵ i ‹˜‡” Ž› ‹‡ ͷͺͲͷ ά ʹ͸͸  Ͷ͹ͻͻ ά ͳ͹͹ ȗȗ  ͷͷ͸Ͳ ά ͳͻͳ  Ͷ͸Ͳͷ ά ͳʹͶ ȗȗ ‹˜‡” ‹•–‹†‹‡ ͳʹͲͲ ά ͸͸  ͻͲͲ ά ͳ͵͵ ȗ  ͳͳͻͻ ά ͻ͵  ͳͲ͸͸ ά ͶͶ  ‹˜‡” •‘Ž‡— ‹‡ ͳʹ͹ͷ ά ͺͺ  ͳ͵ͳͷ ά ͳͺ͹   ͳ͵͸ͺ ά ͳͳʹ  ͳͳͶͻ ά ͹Ͳ  ‹˜‡” ‡— ‹‡ ͵Ͳͷͻ ά ʹͳʹ  ʹ͸ͻͳ ά ͳ͸ͻ   ͵͵ͻͷ ά ʹ͵͵  ʹ͹ͺͲ ά ͳͳ͵ ȗȗ ‹˜‡” ›•‹‡ ͵ͻͶͶ ά ʹͷͷ  ͵ʹͻ͸ ά ͶͶͳ   ͷͶͻͲ ά ʹͻ͹  Ͷ͵͵ͺ ά ʹ͸ͷ ȗ ‹˜‡” ‡–Š‹‘‹‡ ͺ͸͸ ά ͸Ͷ  ͸Ͷͷ ά ͻͷ i  ͻͳͶ ά ͺͶ  ͹ͷͳ ά Ͷͳ i ‹˜‡” Š‡›ŽƒŽƒ‹‡ ͳ͵ͺͷ ά ͻ͹  ͳͶ͹Ͳ ά ʹͺ͸   ͳͶʹ͸ ά ͳ͸͵  ͳͳͺͳ ά ͷ͹  ‹˜‡” ”‘Ž‹‡ ͳ͵ʹͻ ά ͳͷʹ  ͻͺͲ ά ͷ͸ ȗ  ͳͳͺ͵ ά ͳ͸ͻ  ͳͲͲ͸ ά Ͷʹ  ‹˜‡” ‡”‹‡ ͵͹ʹͲ ά ͳͻͺ  ͵͵͸Ͳ ά ͳͻͻ   Ͷ͵͸Ͷ ά ͳͻͶ  ͵Ͷ͸͸ ά ͳͳ͵ ȗȗȗ ‹˜‡” Š”‡‘‹‡ ʹͶͳͷ ά ͳͶͺ  ʹͳͻͶ ά ͳͶ͸   ʹͺͶͲ ά ͳͶͳ ʹʹ͹ʹ ά ͺͳ ȗȗ ‹˜‡” ›”‘•‹‡ ͳʹͲ͵ ά ͹Ͳ  ͳͲ͵Ͳ ά Ͷ͹ i  ͳʹ͸ʹ ά ͳͲʹ  ͳͲ͵Ͷ ά ͵ʹ ȗ ‹˜‡” ƒŽ‹‡ ʹ͹ͳͺ ά ͳͺʹ  ʹͳͲͲ ά ͵Ͷͷ   ͵Ͳ͸ͻ ά ͳ͸ͻ  ʹ͹Ͷ͸ ά ͳͷͷ  ‹˜‡” ‹‘ƒ†‹’‹ ƒ ‹† ͷ͹ͷͳ ά ͳͶͶͺ  ͷ͹Ͷ͵ ά ͳͷ͹͵   ͳͲͶ͹͵ ά ͳ͵ͻͲ  ͻͲ͹ͷ ά ͻ͸ͻ  ‹˜‡” ȽǦ‹‘„—–›”‹ ƒ ‹† ͵͵Ͷ ά ͸Ͷ  ͷͳʹ ά ͵Ͳͷ   ͹͸͹ ά ͳʹͶ  Ͷ͵ͺ ά ͹͸ i ‹˜‡” ‹–”—ŽŽ‹‡ ʹͶ͹ ά ʹͷ  ʹ͹ͻ ά ͹Ͷ   Ͷʹ͸ ά ͷͶ  ͵͵ͻ ά ͸ͺ  ‹˜‡” ŽǦ ›•–ƒ–Š‹‘‹‡ ͳͻ ά ͻ  ͳʹ͹ ά ͳͲ͸   ͹͸ ά ͳͷ  ͵Ͷ ά ͸ ȗ ‹˜‡” ”‹–Š‹‡ ʹͻ͵ͻ ά ͳ͸ͻ  ʹͻʹ͹ ά ʹͻʹ   ͵͹ͷͻ ά ͳ͹͹  ͵ʹ͹ͷ ά ͳͻͲ i ‹˜‡” ƒ Šƒ”‘’‹‡ Ͷ͹ ά ͳ͵  ͳʹͻ ά ͹ͷ   ͳͷʹ ά ʹ͹  ͳͲͺ ά ͳͺ  ‹˜‡” ƒ” ‘•‹‡ ͵Ͳ͸ͷ ά ͷ͵ͷ  ʹ͹ͺ͹ ά ͵ͷʹ   ʹͳʹͲ ά ͵ͳ͸  ʹ͹ͷͲ ά ʹͷͻ  ‹˜‡” ƒ—”‹‡ ͹ͳͳͲ ά ͳͷ͵  ͸ͺ͸͵ ά ͳ͸Ͷ   ͸ͷͻͳ ά ʹͲͻ ͸͸ͷ͸ ά ͳͷͺ i ‹˜‡” ‘–ƒŽƒ‹‘ƒ ‹†• ͸͵Ͳ͹ͷ ά ʹͻͲ͸  ͷͷͻͺ͹ ά ͵ʹ͹ͷ   ͹ʹʹͻ͹ ά ʹ͹Ͷͻ  ͸ͳ͹ͷ͹ ά ͳͺͷ͹ ȗȗ ‹˜‡”                    Žƒ•ƒƒ‹‘ƒ ‹† ‘ ‡–”ƒ–‹‘•ȋƒˆ–‡”ˆƒ•–‹‰ƒ† ‘Ž†‡š’‘•—”‡Ȍ           Žƒ‹‡          ͵͵͵ ά ͹ʹ  ʹͺ͵ ά ͷͲ  Žƒ•ƒ ”‰‹‹‡          ͺ͸ ά ͳͷ  ͹ͷ ά ͺ  Žƒ•ƒ •’ƒ”ƒ‰‹‡          ͷͺ ά ͳͲ  ͷͳ ά ͹  Žƒ•ƒ ›•–‹‡          ͺ ά ͷ  ͹ ά ʹ  Žƒ•ƒ Ž—–ƒƒ–‡          ʹͷǤͷ ά ͸Ǥ͵  ʹ͵ǤͶ ά ʹǤ͵  Žƒ•ƒ Ž—–ƒ‹‡          ͸͵ͻ ά ͹͵  ͸ͳͳ ά Ͷͻ  Žƒ•ƒ Ž› ‹‡          ʹʹʹ ά Ͷͳ  ͳͻͳ ά ͵͹  Žƒ•ƒ ‹•–‹†‹‡          ͻͶ ά ʹͷ  ͳͲ͵ ά ʹͲ  Žƒ•ƒ •‘Ž‡— ‹‡          ͳͺ͵ ά ͳ͹  ͳ͹ͷ ά ͸  Žƒ•ƒ ‡— ‹‡          Ͷͻͺ ά ͺͻ  ͶͲʹ ά Ͷͺ  Žƒ•ƒ ›•‹‡          ͷͻͻ ά ͳʹͳ  Ͷ͵͸ ά ͸ͻ  Žƒ•ƒ ‡–Š‹‘‹‡          ͷʹ ά ͻ  ͵ͺ ά Ͷ  Žƒ•ƒ Š‡›ŽƒŽƒ‹‡         ͳ͵ͷ ά ͵͵  ͳ͵ͺ ά ʹͶ  Žƒ•ƒ ”‘Ž‹‡          ͺͺ ά ͳͻ  ͹Ͷ ά ͳͲ  Žƒ•ƒ ‡”‹‡          ͳͶ͹ ά ͵Ͳ  ͳʹ͹ ά ʹ͵  Žƒ•ƒ Š”‡‘‹‡          ʹ͸͹ ά ͶͲ  ʹͲͲ ά ͳͺ  Žƒ•ƒ ”›’–‘’Šƒ          Ͷͳ ά ͺ  Ͷʹ ά Ͷ  Žƒ•ƒ ›”‘•‹‡         ͳͺͺ ά ͸ͷ  ͳ͵Ͳ ά ͵Ͷ  Žƒ•ƒ ƒŽ‹‡          ͷͻͳ ά ͻʹ  ͷʹͶ ά ͷ͹  Žƒ•ƒ ‹‘ƒ†‹’‹ ƒ ‹†          ͻͶ ά ͳͺ  ͺͳ ά ͳͲ  Žƒ•ƒ ȽǦ‹‘„—–›”‹ ƒ ‹†          ͶͲ ά ͸  ʹͲ ά ʹ ȗ Žƒ•ƒ ‹–”—ŽŽ‹‡          ͷͻ ά ͹  ͸͸ ά ͺ  Žƒ•ƒ ”‹–Š‹‡          ͳ͵ͺ ά ͵Ͷ  ͳ͵Ͳ ά ʹͷ  Žƒ•ƒ ƒ—”‹‡          ͺͳ͵ ά ͷʹ  ͹ͻͻ ά ʹ͸ Žƒ•ƒ ‘–ƒŽƒ‹‘ƒ ‹†•          ͷ͵ͻ͹ ά ͺ͵ͷ  Ͷ͹ʹ͸ ά Ͷͻͷ  Žƒ•ƒ

ͳͷͳ Chapter 4

 ǣ‡ˆ‘”‡ˆƒ•–‹‰ǡ ǣˆ–‡”ͳͶŠ‘—”•‘ˆˆƒ•–‹‰ǡ ǣˆ–‡”ˆƒ•–‹‰ƒ† ‘Ž†‡š’‘•—”‡ǡǣ‘†›™‡‹‰Š–•ǡ ǣŽ‘‘† ‰Ž— ‘•‡ǡǣ‡–‘‡„‘†‹‡•ǡ‹ǣ’δͲǤͳ˜•ǡȗǣ’δͲǤͲͷ˜•ǡȗȗǣ’δͲǤͲͳ˜•ǡȗȗȗǣ’δͲǤͲͲͳ˜•ǡȗȗȗȗǣ’δͲǤͲͲͲͳ˜• ǤŠ‡˜ƒŽ—‡•ƒ”‡‡š’”‡••‡†ƒ•ƒ˜‡”ƒ‰‡άǤαͷǦͺǤ  Supplementary Table ST2. Lipidomic profile results in livers of 8-week-old mice.  ‹’‹† ‡ƒ ‡ƒ †Œ—•–‡† ‘Ž†  ƒ‡ ’Ǧ˜ƒŽ—‡ Š”‡•Š‘Ž† Žƒ•• Ǧ  ’Ǧ˜ƒŽ—‡ Šƒ‰‡    ʹͲ  ȋͶ͸ǣ͸Ȍ ʹǤʹΪͲ͸ ͶǤͳΪͲͷ ͵ǤʹǦͲͺ ͸Ǥ͹ǦͲ͸ ͷǤʹͺ ’  ͳ͹ ‡š‡”ȋ†Ͷ͸ǣ͵Ȍ ͳǤͳΪͲ͸ ͳǤͳΪͲͷ ʹǤͷǦͲͺ ͸Ǥ͹ǦͲ͸ ͳͲǤ͹͹ ’    Ͷ͵  ȋͶͶǣʹȌ ͸ǤʹΪͲ͹ ͶǤ͵ΪͲ͸ ʹǤʹǦͲͺ ͸Ǥ͹ǦͲ͸ ͳͶǤͶ͸ ’    ʹͷ  ȋͶͺǣ͹Ȍ ͻǤʹΪͲ͸ ͻǤʹΪͲͷ ͷǤͳǦͲͺ ͺǤͲǦͲ͸ ͳͲǤͲͳ ’    ͳ͵  ȋͶͲǣʹȌ ͵ǤʹΪͲ͸ ͵Ǥ͹ΪͲͷ ͳǤ͹ǦͲ͹ ͳǤͺǦͲͷ ͺǤ͸ͷ ’    ͳ͸  ȋͶ͸ǣ͸Ȍ ʹǤ͹ΪͲ͸ ͵ǤͲΪͲͷ ͳǤͻǦͲ͹ ͳǤͺǦͲͷ ͻǤͳ͸ ’    ͳͷ  ȋͶʹǣ͵Ȍ ͺǤ͸ΪͲ͸ ͺǤͺΪͲͷ ʹǤͲǦͲ͹ ͳǤͺǦͲͷ ͻǤͺͺ ’    ʹ͹  ȋͶͲǣͲȌ ͶǤͲΪͲͷ ͳǤͲΪͲͷ ʹǤ͹ǦͲ͹ ʹǤͲǦͲͷ ͵Ǥͻ͹ ’    ʹͺ  ȋͶͶǣ͵Ȍ ͹Ǥ͸ΪͲ͹ ͹ǤʹΪͲ͸ ͵ǤʹǦͲ͹ ʹǤͲǦͲͷ ͳͲǤͶͻ ’    ͳͻ  ȋͶͶǣͶȌ ͳǤͻΪͲ͹ ͳǤ͸ΪͲ͸ ͵ǤͲǦͲ͹ ʹǤͲǦͲͷ ͳͳǤ͸ͻ ’    ʹͶ  ȋͶʹǣʹȌ ͹Ǥ͹ΪͲ͸ ͶǤͳΪͲͷ ͶǤͶǦͲ͹ ʹǤͷǦͲͷ ͳͺǤͻͷ ’    ͳͶ  ȋͶͶǣͷȌ ͵ǤͳΪͲ͸ ʹǤ͵ΪͲͷ ͷǤͳǦͲ͹ ʹǤ͸ǦͲͷ ͳ͵ǤͲ͵ ’    ʹ͵  ȋͶ͸ǣͷȌ ͳǤ͵ΪͲ͹ ʹǤͲΪͲ͸ ͸ǤͶǦͲ͹ ͵ǤͳǦͲͷ ͸Ǥ͵͹ ’    Ͷͷ  ȋͶ͵ǣͳȌ ͵Ǥ͹ΪͲͷ ͺǤ͸ΪͲͶ ͺǤʹǦͲ͹ ͵Ǥ͸ǦͲͷ ͶǤ͵ͷ ’    ͳͺ  ȋͶͲǣͳȌ ͶǤʹΪͲ͸ ͷǤͲΪͲͷ ͳǤͲǦͲ͸ ͶǤͳǦͲͷ ͺǤͷͲ ’    ʹʹ  ȋͶʹǣʹȌ ͳǤ͸ΪͲ͹ ʹǤ͸ΪͲ͸ ͳǤʹǦͲ͸ ͶǤͷǦͲͷ ͸ǤͲͷ ’    ͳ͹  ȋͶͺǣͺȌ ͸ǤͻΪͲͷ ʹǤͻΪͲͶ ͳǤ͵ǦͲ͸ ͶǤͻǦͲͷ ʹͶǤͲ͸ ’    ͵  ȋʹͺǣʹȌ ͹ǤͺΪͲͷ ͶǤ͹ΪͲͶ ʹǤͻǦͲ͸ ͳǤͲǦͲͶ ͳ͸Ǥͷ͵ ’    ͵Ͳ  ȋͶ͸ǣͶȌ ͷǤͳΪͲ͹ ͳǤʹΪͲ͹ ͵ǤʹǦͲ͸ ͳǤͲǦͲͶ ͶǤ͵ͻ ’    ͵ʹ  ȋͶʹǣͳȌ ʹǤͲΪͲ͹ ͶǤͲΪͲ͸ ͶǤʹǦͲ͸ ͳǤ͵ǦͲͶ ͷǤͳͳ ’    ͵ͳ  ȋͶͺǣ͸Ȍ ͸ǤͻΪͲ͸ ʹǤͳΪͲ͸ ͷǤͺǦͲ͸ ͳǤ͹ǦͲͶ ͵Ǥ͵ͳ ’    ͷͷ  ȋͶʹǣͲȌ ͳǤ͵ΪͲ͸ ͷǤ͸ΪͲͷ ͸ǤʹǦͲ͸ ͳǤ͹ǦͲͶ ʹǤ͵͹ ’    ͵ͷ  ȋͶͶǣʹȌ ͶǤͷΪͲ͹ ͳǤͶΪͲ͹ ͹ǤͶǦͲ͸ ʹǤͲǦͲͶ ͵Ǥͳͺ ’    ʹ͸  ȋͷͲǣͺȌ ʹǤͻΪͲ͸ ͻǤͻΪͲͷ ͳǤͷǦͲͷ ͵ǤͻǦͲͶ ʹǤͻ͸ ’    ͳʹ  ȋͶʹǣͶȌ ͳǤͳΪͲ͸ ͸Ǥ͸ΪͲͶ ͳǤ͸ǦͲͷ ͵ǤͻǦͲͶ ͳͷǤͻͷ ’    ͶͶ  ȋͶ͸ǣ͵Ȍ ͳǤͻΪͲ͹ ͸ǤͺΪͲ͸ ʹǤͳǦͲͷ ͷǤͲǦͲͶ ʹǤͺͲ ’    ͵͵  ȋͶͺǣ͸Ȍ ͵ǤͶΪͲ͸ ͳǤ͵ΪͲ͸ ʹǤͻǦͲͷ ͸Ǥ͹ǦͲͶ ʹǤͷʹ ’    ͷ͵  ȋͶ͸ǣ͵Ȍ ͵ǤͺΪͲ͹ ͳǤ͵ΪͲ͹ ͵ǤͳǦͲͷ ͸ǤͺǦͲͶ ʹǤͻͲ ’    ͸ͳ  ȋͶͶǣͳȌ ͶǤ͸ΪͲ͹ ͳǤ͹ΪͲ͹ ͶǤ͹ǦͲͷ ͻǤͺǦͲͶ ʹǤ͹ͷ ’    ʹͳ  ȋͶͺǣ͹Ȍ ͵ǤͲΪͲ͸ ͺǤ͵ΪͲͷ ͶǤ͹ǦͲͷ ͻǤͺǦͲͶ ͵Ǥ͸ͷ ’    ͷʹ  ȋͶ͹ǣ͸Ȍ ʹǤ͸ΪͲͷ ͺǤͺΪͲͶ ͷǤʹǦͲͷ ͳǤͲǦͲ͵ ʹǤͻͶ ’    ͷͶ  ȋͶͷǣʹȌ ʹǤͶΪͲ͸ ͳǤͳΪͲ͸ ͹Ǥ͹ǦͲͷ ͳǤͷǦͲ͵ ʹǤʹͲ ’    ͵Ͷ  ȋͶͷǣ͵Ȍ ͷǤͲΪͲͷ ͻǤ͸ΪͲͶ ͺǤͻǦͲͷ ͳǤ͹ǦͲ͵ ͷǤͳ͸ ’  ͸͹ ȋͶͶǣͲȌ ͵ǤʹΪͲͷ ʹǤͷΪͲͷ ͵ǤʹǦͲͶ ͷǤͺǦͲ͵ ͳǤʹ͸ ‘–    ͵͸  ȋͶ͹ǣͷȌ ʹǤͻΪͲ͸ ͸ǤͷΪͲͷ ͶǤͻǦͲͶ ͺǤͺǦͲ͵ ͶǤͶͺ ’    ͸͸  ȋͶ͸ǣʹȌ ʹǤͲΪͲͺ ͳǤͲΪͲͺ ͷǤͶǦͲͶ ͻǤ͵ǦͲ͵ ʹǤͲͳ ’  ͹ͺ ȋǦ͵ͺǣͳȌ ͺǤ͵ΪͲͶ ͳǤ͹ΪͲͷ ͸ǤͳǦͲͶ ͳǤͲǦͲʹ ͲǤͶͺ ‘™  ͹ ȋ͵ʹǣʹȌ ͹ǤͶΪͲ͸ ͶǤͶΪͲ͸ ͸Ǥ͹ǦͲͶ ͳǤͳǦͲʹ ͳǤ͹Ͳ ‘–    ͵͹  ȋͶͺǣͷȌ ͵Ǥ͵ΪͲ͹ ͳǤͺΪͲ͹ ͸ǤͺǦͲͶ ͳǤͳǦͲʹ ͳǤͺ͸ ‘–    ͶͲ  ȋͷͲǣ͹Ȍ ͶǤͶΪͲ͸ ʹǤͳΪͲ͸ ͸Ǥ͹ǦͲͶ ͳǤͳǦͲʹ ʹǤͲ͹ ’  ʹ ȋ͵ͲǣͳȌ ͸ǤͻΪͲͷ ͵ǤͺΪͲͷ ͹ǤͻǦͲͶ ͳǤʹǦͲʹ ͳǤͺͳ ‘–    ͷͳ  ȋͷͲǣ͸Ȍ ͳǤ͸ΪͲ͹ ͹ǤͺΪͲ͸ ͺǤ͸ǦͲͶ ͳǤ͵ǦͲʹ ʹǤͲͻ ’    ͹͵  ȋͶͷǣͳȌ ʹǤͳΪͲ͸ ͳǤͲΪͲ͸ ͻǤͲǦͲͶ ͳǤ͵ǦͲʹ ʹǤͲ͸ ’    ͷͻ  ȋͶͺǣͶȌ ͹ǤͲΪͲ͹ ͵ǤͷΪͲ͹ ͳǤ͵ǦͲ͵ ͳǤͺǦͲʹ ʹǤͲͳ ’  ͺ ȋ͵ʹǣ͵Ȍ ʹǤʹΪͲͷ ͳǤͶΪͲͷ ͳǤͶǦͲ͵ ͳǤͻǦͲʹ ͳǤ͸Ͳ ‘–    ͺͺ  ȋͶ͸ǣͳȌ ͳǤͻΪͲͺ ͻǤ͹ΪͲ͹ ͳǤͷǦͲ͵ ʹǤͲǦͲʹ ͳǤͻͳ ‘–  ͻ ȋ͹ͲǣͷȌ ʹǤͲΪͲ͸ ͳǤ͵ΪͲ͸ ͳǤ͹ǦͲ͵ ʹǤ͵ǦͲʹ ͳǤͷͺ ‘–  ͳͺ ȋ͵ͶǣͶȌ ʹǤ͹ΪͲ͸ ͳǤͺΪͲ͸ ʹǤʹǦͲ͵ ʹǤͺǦͲʹ ͳǤͷͷ ‘–    ͺͶ  ȋͶͶǣͲȌ ͷǤ͹ΪͲ͸ ͵Ǥ͵ΪͲ͸ ʹǤͶǦͲ͵ ͵ǤͲǦͲʹ ͳǤ͹Ͷ ‘–    ͵ͻ  ȋͷͲǣ͹Ȍ ͺǤͶΪͲ͸ ͷǤͲΪͲ͸ ʹǤͷǦͲ͵ ͵ǤͳǦͲʹ ͳǤ͸͹ ‘–    ͹Ͷ  ȋͶͺǣ͵Ȍ ͳǤ͸ΪͲͺ ͳǤͲΪͲͺ ʹǤͻǦͲ͵ ͵ǤͷǦͲʹ ͳǤͷͷ ‘–    ͺʹ  ȋͶ͹ǣʹȌ ͳǤͳΪͲ͹ ͸Ǥ͸ΪͲ͸ ͵ǤͶǦͲ͵ ͶǤͲǦͲʹ ͳǤ͹͵ ‘–    ͻ͹  ȋͶͺǣʹȌ ͳǤͳΪͲͻ ͸ǤʹΪͲͺ ͵Ǥ͵ǦͲ͵ ͶǤͲǦͲʹ ͳǤ͹Ͷ ‘–    ͸ͷ  ȋͶ͹ǣ͵Ȍ ͵ǤʹΪͲ͸ ͳǤ͹ΪͲ͸ ͵ǤͶǦͲ͵ ͶǤͲǦͲʹ ͳǤͻʹ ‘–    ͸ʹ  ȋͷͲǣ͸Ȍ ͺǤͳΪͲ͸ ͶǤ͸ΪͲ͸ ͵ǤͺǦͲ͵ ͶǤ͵ǦͲʹ ͳǤ͹ͷ ‘–    ͹͹  ȋͷʹǣ͸Ȍ ͵Ǥ͵ΪͲͺ ʹǤͲΪͲͺ ͶǤͲǦͲ͵ ͶǤͶǦͲʹ ͳǤ͸͵ ‘–    ͸Ͳ  ȋͶͺǣͶȌ ͸ǤͻΪͲ͹ ͶǤͷΪͲ͹ ͶǤʹǦͲ͵ ͶǤͷǦͲʹ ͳǤͷ͵ ‘–    ͸Ͷ  ȋͷʹǣ͹Ȍ ͷǤͲΪͲ͹ ʹǤͻΪͲ͹ ͶǤ͵ǦͲ͵ ͶǤͷǦͲʹ ͳǤ͹ͳ ‘–    Ͷʹ  ȋͶ͹ǣͶȌ ͸ǤʹΪͲͷ ʹǤͷΪͲͷ ͶǤ͵ǦͲ͵ ͶǤͷǦͲʹ ʹǤͶ͸ ’  ͳͻ ȋ͵ͶǣͶȌ ͳǤͻΪͲ͸ ͳǤ͵ΪͲ͸ ͶǤͶǦͲ͵ ͶǤ͸ǦͲʹ ͳǤͶ͵ ‘–    ͳͳͷ  ȋͶ͸ǣͲȌ ʹǤͳΪͲ͹ ͳǤ͵ΪͲ͹ ͶǤ͸ǦͲ͵ ͶǤ͸ǦͲʹ ͳǤ͸ͳ ‘–    ͷ͹  ȋͶͻǣͷȌ ͶǤͳΪͲ͸ ʹǤͳΪͲ͸ ͶǤͷǦͲ͵ ͶǤ͸ǦͲʹ ͳǤͻʹ ‘–  ͹ͷ ȋǦ͵͸ǣͳȌ ͳǤͺΪͲͷ ͵ǤͳΪͲͷ ͶǤͻǦͲ͵ ͶǤͺǦͲʹ ͲǤͷͺ ‘–  ’ǡ’ƒ†ŒδͲǤͲͷ  ‘™ǡ’ƒ†ŒδͲǤͲͷ     ’ǡ’ƒ†ŒδͲǤͲͷǡ εʹ  ‘™ǡ’ƒ†ŒδͲǤͲͷǡ δͲǤͷ   

ͳͷʹ ͳͷ͵    in MCAD-KO mice mice in MCAD-KO  ‡”‰”‘—’ǤŽ‡ƒ•‡ ‘–ƒ –’”‘ˆ 4 ̴ͺǣͲȌ ̴ͳͺǣ͵Ȍ  ȋͳ͸ǣʹ̴ͳ͸ǣʹ̴ͳͺǣʹȌ ̴ͳͺǣͳȌ  ȋͳͶǣͳ̴ͳͶǣͳ̴ͳͺǣʹȌ ̴ͳͺǣͳȌ  ȋͳ͸ǣͲ̴ͳ͸ǣʹ̴ͳ͸ǣʹȌ  ȋͳ͸ǣͳ̴ͳ͸ǣͳ̴ͳ͸ǣʹȌ ̴ͳͺǣͳȌ  ȋͳͶǣͳ̴ͳ͸ǣͲ̴ͳ͸ǣʹȌ  ȋͳͶǣͳ̴ͳ͸ǣͳ̴ͳ͸ǣͳȌ ̴ͳ͸ǣͳȌ  ȋͳͶǣͳ̴ͳͶǣͳ̴ͳͺǣͲȌ  ȋͳͶǣͳ̴ͳ͸ǣͲ̴ͳ͸ǣͳȌ ̴ͳ͸ǣͳȌ  ȋͳͶǣͲ̴ͳͶǣͳ̴ͳ͸ǣͲȌ ̴ͳ͸ǣͳȌ  ȋͳͶǣͳ̴ͳͶǣͳ̴ͳ͸ǣͲȌ  ȋͳͺǣͳ̴ͳͺǣͳ̴ͺǣͲȌ hepatic metabolism hepatic metabolism    †‘‘–ˆ‹–‘‘‡’ƒ‰‡Ǥαͺ’    •—Ž–•ǡƒ•–Š‡›     ‘Ǧ•‹‰‹ˆ‹ ƒ–”‡ Effect of fasting and cold stress on stress cold and Effect of fasting   ͳͺǣͳ̴͸ǣͲȌ ͳ͸ǣͳ̴ͺǣͲȌ  ȋͳ͸ǣͲ̴ͳͺǣʹ̴͸ǣͲȌ  ȋͳ͸ǣͳ̴ͳͺǣͳ̴͸ǣͲȌ ̴ͳͺǣͳ̴ͺǣͲȌ  ȋͳͺǣͳ̴ͳͺǣʹ̴͸ǣͲȌ Ͳ̴ͳ͸ǣͲ̴ͺǣͲȌ Ͳ̴ͳͺǣͳ̴ͺǣͲȌ Ͳ̴ͳͺǣʹ̴ͺǣͲȌ  ȋͳ͸ǣͳ̴ͳͺǣͳ̴ͺǣͲȌ Ͳ̴ͳͺǣ͵̴ͳͺǣ͵Ȍ  ȋͳͶǣͳ̴ͳ͸ǣͳ̴ʹͲǣͶȌ  ȋͳͶǣͳ̴ͳͺǣʹ̴ͳͺǣ͵Ȍ  ȋͳ͸ǣͳ̴ͳ͸ǣʹ ͳ̴ͳ͸ǣͳ̴ͳ͸ǣʹȌ  ȋͳͺǣͳ̴ͳͺǣ͵̴ͺǣͲȌ  ȋͳͺǣʹ̴ͳͺǣʹ̴ͺǣͲȌ ͳ̴ͳͺǣ͵̴ͳͺǣ͵Ȍ  ȋͳ͸ǣʹ̴ͳ͸ǣʹ̴ͳͺǣ͵Ȍ ʹ̴ͳ͸ǣʹ̴ͳ͸ǣʹȌ Ͳ̴ͳ͸ǣʹ̴ͳͺǣʹȌ  ȋͳʹǣͳ̴ͳ͸ǣͲ̴ͳͺǣ͵Ȍ  ȋͳʹǣͳ̴ͳ͸ǣͳ̴ͳͺǣʹȌͳ̴ͳ͸ǣʹ̴ͳͺǣʹȌ  ȋͳʹǣͳ̴ͳ͸ǣʹ  ȋͳ͸ǣͲ̴ʹʹǣͷ̴ͺǣͲȌ  ȋͳͺǣͳ̴ʹͲǣͶ̴ͺǣͲȌ Ͳ̴ͳ͸ǣʹ̴ͳͺǣʹȌ  ȋͳͶǣͳ̴ͳ͸ǣͲ̴ͳͺǣ͵Ȍ  ȋͳͶǣͳ̴ͳ͸ǣͳ̴ͳͺǣʹȌ  ȋͳͶǣͳ̴ͳ͸ǣʹ ͳ̴ͳ͸ǣͲ̴ͳͺǣʹȌ  ȋͳʹǣͳ̴ͳ͸ǣͳ̴ͳͺǣͳȌ  ȋͳͶǣͲ̴ͳͶǣͳ̴ͳͺǣʹȌ  ȋͳͶǣͳ̴ͳͶǣͳ ͳ̴ͳ͸ǣͲ̴ͳͺǣͳȌ  ȋͳʹǣͳ̴ͳ͸ǣͳ̴ͳͺǣͲȌ  ȋͳͶǣͲ̴ͳͶǣͳ̴ͳͺǣͳȌ  ȋͳͶǣͲ̴ͳ͸ǣͳ ͳ̴ͳͶǣͲ̴ͳͺǣʹȌ  ȋͳʹǣͳ̴ͳͶǣͳ̴ͳͺǣͳȌ  ȋͳʹǣͳ̴ͳ͸ǣͳ̴ͳ͸ǣͳȌ  ȋͳͺǣͳ̴ͳͺǣʹ Ͳ̴ͳ͸ǣͲ̴ͳ͸ǣͳȌ  ȋͳʹǣͳ̴ͳͶǣͲ̴ͳͺǣͲȌ  ȋͳʹǣͳ̴ͳ͸ǣͲ̴ͳ͸ǣͲȌͲ̴ͳͶǣͳ̴ͳͺǣͳȌ  ȋͳͶǣͲ̴ͳͶǣͲ  ȋͳʹǣͲ̴ͳ͸ǣͳ̴ͳ͸ǣͳȌ  ȋͳʹǣͳ̴ͳͶǣͲ̴ͳͺǣͳȌ  ȋͳʹǣͳ̴ͳ͸ǣͲ

‡ –Š‡‹†‹˜‹†—ƒŽ˜ƒŽ—‡•ƒ†–Š‡                     ͳ‹•‘‡”•™‡”‡ ‘„‹‡†Ǥ –ƒ”›ƒ„Ž‡ʹ†‘‡•‘– ‘–ƒ‹ ‘Ȁ ‘Ȁ ‘Ȁ ‘Ȁ  ȋͷͶǣͷ̸ͷ;ǣͷȌ Ž‘™ ‘ˆ‹†‡ ‡ Ž‘™ ‘ˆ‹†‡ ‡  ȋͳ͸ǣͲ̴ͳ͸ǣͳ̴ͺǣͲȌ  ȋͳͶǣͲ̴ͳͺǣͳ̴ͺǣͲȌ  ȋͳͲǣͲ̴ͳͶǣͲ̴ͳ͸ǣͳȌ  ȋͳ͸ǣͲ̴  ȋͳ͸ǣͲ̴ʹʹǣ͸̴ͺǣͲȌ  ȋͳͺǣʹ̴ͳͺǣ͵̴ͺǣͲȌ  ȋͳ͸ǣͳ̴ʹͲǣͶ̴ͺǣͲȌ  ȋͿǣͶ̸ͷͼǣͶ̸ͷ;ǣͷȌ  ȋͷͶǣͶ̸ͷͻǣͶ̸ͷ;ǣͷȌ  ȋͳͲǣͲ̴ͳͺǣͳ̴ʹʹǣ͸Ȍ  ȋͳʹǣͲ̴ͳ͸ǣͳ̴ʹʹǣ͸Ȍ  ȋͳʹǣͳ̴ͳ͸ǣͲ̴ʹʹǣ͸Ȍ  ȋͳͶǣ  ȋͳͲǣͲ̴ͳͶǣͳ̴ͳ͸ǣͳȌ  ȋͳͶǣͲ̴ͳͺǣʹ̴ͺǣͲȌ  ȋͳͶǣͳ̴ͳͺǣͳ̴ͺǣͲȌ  ȋͳ͸ǣͳ̴  ȋͳͲǣͲ̴ͳͶǣͲ̴ͳ͸ǣͲȌ  ȋͳʹǣͲ̴ͳʹǣͲ̴ͳ͸ǣͲȌ  ȋͳʹǣͲ̴ͳͶǣͲ̴ͳͶǣͲȌ  ȋͳ͸ǣ  ȋͳͲǣͲ̴ͳͶǣͲ̴ͳͺǣͳȌ  ȋͳͲǣͲ̴ͳ͸ǣͲ̴ͳ͸ǣͳȌ  ȋͳʹǣͳ̴ͳͶǣͲ̴ͳ͸ǣͲȌ  ȋͳ͸ǣ  ȋͳͲǣͲ̴ͳ͸ǣͲ̴ͳ͸ǣͲȌ  ȋͳʹǣͲ̴ͳͶǣͲ̴ͳ͸ǣͲȌ  ȋͳͶǣͲ̴ͳͶǣͲ̴ͳͶǣͲȌ  ȋͳͲǣͲ̴ͳͶǣͲ̴ͳͺǣʹȌ  ȋͳͲǣͲ̴ͳ͸ǣͳ̴ͳ͸ǣͳȌ  ȋͳʹǣͳ̴ͳͶǣͳ̴ͳ͸ǣͲȌ  ȋͳ͸ǣ  ȋͳʹǣͲ̴ͳ͸ǣͲ̴ʹʹǣ͸Ȍ  ȋͳʹǣͳ̴ͳͺǣͳ̴ʹͲǣͶȌ  ȋͳͶǣͲ̴ͳͶǣͲ̴ʹʹǣ͸Ȍ  ȋͳͶǣ  ȋͳͲǣͲ̴ͳ͸ǣͳ̴ʹʹǣ͸Ȍ  ȋͳͲǣͲ̴ͳ͸ǣͲ̴ʹʹǣ͸Ȍ  ȋͳͲǣͲ̴ͳͺǣʹ̴ʹͲǣͶȌ  ȋͳͶǣͳ̴ͳͶǣͳ̴ʹͲǣͶȌ  ȋͳ͸ǣ  ȋͳͲǣͲ̴ͳͺǣʹ̴ͳͺǣ͵Ȍ  ȋͳʹǣͲ̴ͳ͸ǣʹ̴ͳͺǣ͵Ȍ  ȋͳʹǣͳ̴ͳ͸ǣͳ̴ͳͺǣ͵Ȍ  ȋͳʹǣ  ȋͳʹǣͲ̴ͳͺǣʹ̴ͳͺǣʹȌ  ȋͳʹǣͳ̴ͳͺǣͳ̴ͳͺǣʹȌ  ȋͳͶǣͲ̴ͳ͸ǣͳ̴ͳͺǣ͵Ȍ  ȋͳͶǣ  ȋͳͲǣͲ̴ͳ͸ǣͲ̴ʹͲǣͶȌ  ȋͳͲǣͲ̴ͳͺǣͳ̴ͳͺǣ͵Ȍ  ȋͳʹǣͲ̴ͳ͸ǣͳ̴ͳͺǣ͵Ȍ  ȋͳʹǣ  ȋͳͲǣͲ̴ͳͺǣͳ̴ͳͺǣʹȌ  ȋͳʹǣͲ̴ͳ͸ǣͳ̴ͳͺǣʹȌ  ȋͳʹǣͲ̴ͳ͸ǣʹ̴ͳͺǣͳȌ  ȋͳʹǣ  ȋͳʹǣͲ̴ͳͷǣͲ̴ͳͺǣͳȌ  ȋͳͶǣͲ̴ͳͷǣͲ̴ͳ͸ǣͳȌ  ȋͳͶǣͳ̴ͳͷǣͲ̴ͳ͸ǣͲȌ  ȋͳʹǣͲ̴ͳͷǣͲ̴ͳͺǣʹȌ  ȋͳͲǣͳ̴ͳ͹ǣͲ̴ͳͺǣʹȌ  ȋͳͲǣͲ̴ͳͺǣͳ̴ͳͺǣͳȌ  ȋͳʹǣͲ̴ͳ͸ǣͲ̴ͳͺǣʹȌ  ȋͳʹǣͲ̴ͳ͸ǣͳ̴ͳͺǣͳȌ  ȋͳʹǣ  ȋͳͲǣͲ̴ͳ͸ǣͳ̴ͳͺǣ͵Ȍ  ȋͳͲǣͲ̴ͳ͸ǣʹ̴ͳͺǣʹȌ  ȋͳʹǣͳ̴ͳͶǣͳ̴ͳͺǣʹȌ  ȋͳʹǣ  ȋͳͲǣͲ̴ͳ͸ǣͲ̴ͳͺǣ͵Ȍ  ȋͳͲǣͲ̴ͳ͸ǣͳ̴ͳͺǣʹȌ  ȋͳͲǣͲ̴ͳ͸ǣʹ̴ͳͺǣͳȌ  ȋͳʹǣ  ȋͳͲǣͲ̴ͳ͸ǣͲ̴ͳͺǣʹȌ  ȋͳͲǣͲ̴ͳ͸ǣͳ̴ͳͺǣͳȌ  ȋͳʹǣͲ̴ͳͶǣͲ̴ͳͺǣʹȌ  ȋͳʹǣ  ȋͳͲǣͲ̴ͳ͸ǣͲ̴ͳͺǣͳȌ  ȋͳʹǣͲ̴ͳͶǣͲ̴ͳͺǣͳȌ  ȋͳʹǣͲ̴ͳͶǣͳ̴ͳͺǣͲȌ  ȋͳʹǣ  ȋͳͲǣͲ̴ͳͶǣͲ̴ͳͺǣ͵Ȍ  ȋͳͶǣͳ̴ͳͶǣͳ̴ͳͶǣͳȌ  ȋͳ͸ǣͳ̴ͳͺǣʹ̴ͺǣͲȌ  ȋͳ͸ǣʹ Name Isomer1 Isomer2 Isomer3 Isomer4Isomer5 Isomer6 Isomer7 Isomer8 Isomer9 Name TG(50:7) TG(50:7) TG(60:5) TG(60:5) TG(50:8) TG(50:8) TG(40:2) TG(40:2) TG(40:0) TG(40:0) TG(40:1) TG(42:1) TG(42:1) TG(42:0) TG(42:0) TG(42:2) TG(48:8) TG(48:8) TG(50:6) TG(48:7) TG(48:7) TG(48:6) TG(48:6) TG(46:5) TG(46:5) TG(47:4) TG(47:4) TG(47:5) TG(47:6) TG(48:4) TG(46:4) TG(46:4) TG(46:6) TG(46:3) TG(46:3) TG(45:1) TG(45:1) TG(45:2) TG(45:3) TG(45:3) TG(46:2) TG(44:5) TG(44:5) TG(44:4) TG(44:4) TG(44:3) TG(44:3) TG(44:2) TG(44:2) TG(44:1) TG(44:1) TG(42:3) TG(42:3) TG(43:1) TG(43:1) DG(28:2) DG(28:2) Š‡–Š‡•‹•˜‡”•‹‘‘ˆ—’’Ž‡‡ ‘Ž†ƒ†‹–ƒŽ‹ ǣƒ—ƒŽŽ› Š‡ ‡†Ǥ  mice 8-week-old of livers from isomers DG and TG Annotated ST3. Table Supplementary ƒ”„ƒ”ƒǤƒ‡”ƒ–„ǤǤ„ƒ‡”Ͳͳ̷— ‰ǤŽˆ‘”–Š‡ ‘’Ž‡–‡–ƒ„Ž Chapter 4

Supplementary Table ST4. Histological examination scores for 16-20-week old and 8-week-old mice  16-20 weeks-old   8 weeks-old  WT  KO   WT  KO •–ƒ‹‹‰ ͲǤͻ ά ͲǤͲ  ͲǤͺ ά ͲǤͳ ȗ  ͲǤͻ ά ͲǤͲ  ͲǤͺ ά ͲǤͳ –‡ƒ–‘•‹•‰”ƒ†‡ ͵ǤͲ ά ͲǤͲ  ʹǤͻ ά ͲǤͳ   ͵ǤͲ ά ͲǤͲ  ʹǤͺ ά ͲǤʹ †‹‡•‹‘‘ˆ˜‡•‹ Ž‡• ͳǤͶ ά ͲǤʹ  ͳǤ͹ ά ͲǤͳ   ʹǤͲ ά ͲǤͳ  ͳǤͺ ά ͲǤʹ ‹•‘ƒ”›‘•‹• ͲǤ͵ ά ͲǤʹ  ͲǤ͵ ά ͲǤ͵   ͲǤͳ ά ͲǤͳ  ͲǤ͸ ά ͲǤ͵ –‡ƒ–‘•‹•‰”ƒ†‡ ʹǤͺ ά ͲǤʹ  ͳǤ͸ ά ͲǤ͵ ȗ  ͳǤͻ ά ͲǤ͵  ʹǤͲ ά ͲǤ͵ ‘ ƒ–‹‘•–‡ƒ–‘•‹• ʹǤͺ ά ͲǤʹ  ʹǤͳ ά ͲǤͳ ȗ  ʹǤͳ ά ͲǤͳ  ʹǤͲ ά ͲǤͲ ‹” ‘˜‡•‹ —Žƒ”•–‡ƒ–‘•‹• ͳǤͲ ά ͲǤͲ  ͳǤͲ ά ͲǤͲ   ͳǤͲ ά ͲǤͲ  ͲǤͻ ά ͲǤͳ ƒŽŽ‘‘‹‰ ͲǤͳ ά ͲǤͳ  ͲǤͲ ά ͲǤͲ   ͲǤͲ ά ͲǤͲ  ͲǤͲ ά ͲǤͲ ‘„—Žƒ”‹ˆŽƒƒ–‹‘ ͳǤͳ ά ͲǤ͵  ͳǤͳ ά ͲǤʹ  ͲǤͺ ά ͲǤ͵  ͳǤͲ ά ͲǤ͵ ‘”–ƒŽ‹ˆŽƒƒ–‹‘ ͲǤͳ ά ͲǤͳ  ͲǤ͵ ά ͲǤʹ   ͲǤͳ ά ͲǤͳ  ͲǤͷ ά ͲǤʹ ‹‰‡–‡†ƒ ”‘’Šƒ‰‡• ͲǤͷ ά ͲǤʹ  ͲǤͳ ά ͲǤͳ  ͲǤͲ ά ͲǤͲ  ͲǤͲ ά ͲǤͲ  ƒ –‹˜‹–›• ‘”‡ ͶǤͲ ά ͲǤͷ  ʹǤͺ ά ͲǤͶ i  ʹǤ͸ ά ͲǤͶ  ͵ǤͲ ά ͲǤͷ ••Ǥ‡•‹‘•• ‘”‡ ͳǤͶ ά ͲǤͶ  ͲǤͷ ά ͲǤͷ i  ͲǤͻ ά ͲǤͷ  ͳǤͲ ά ͲǤ͹ ‘’ƒ”‹•‘•ǡ•–ƒ–‹•–‹ •ƒ†—„‡”‘ˆ‹ ‡’‡”‰”‘—’ƒ•‹—’’Ž‡‡–ƒ”›–ƒ„Ž‡͵Ǥ  Supplementary Table ST5. Table of possible lipid fragments  ƒ––›ƒ ›Ž‡ƒ” Š‹‰†ƒ–ƒ„ƒ•‡      Fatty acid fa1 fa2 fa3 PL TG DG FA6:0       FA8:0       FA9:0       FA10:0       FA12:0       FA12:1       FA14:0       FA14:1       FA15:0       FA15:1       FA16:0       FA16:1       FA16:2       FA17:0       FA18:0       FA18:1       FA18:2       FA18:3       FA19:0       FA20:3       FA20:4       FA20:5       FA21:0       FA22:3       FA22:4       FA22:5       FA22:6       O-16:0       O-18:0       O-20:0       P-16:0       P-18:0       P-20:0             ”ƒ‰‡–• ‘”‡™‡‹‰Š–       Type Weight Group     FA1_[FA-H2O+H]+ ͳǤ͸ ͳ     FA2_[FA-H2O+H]+ ͳǤ͸ ͳ     FA3_[FA-H2O+H]+ ͳǤ͸ ͳ     [MG(FA1)-H2O+H]+ ͳǤ͸ ʹ     [MG(FA2)-H2O+H]+ ͳǤ͸ ʹ     [MG(FA3)-H2O+H]+ ͳǤ͸ ʹ     [M-(FA1)+H]+ ͵Ͳ ʹ     [M-(FA2)+H]+ ͵Ͳ ʹ     [M-(FA3)+H]+ ͵Ͳ ʹ      

ͳͷͶ 

Chapter 5

Simulating the impact of genetic and environmental modifiers on MCAD deficiency in an extended computational model of human liver fatty-acid catabolism

ۥAnne-Claire M.F. Martines1,#, Dirk-Jan Reijngoud1,2, Barbara M. Bakkerƌϭ͕

1 Laboratory of Pediatrics, Center of Liver, Digestive and Metabolic Diseases, University of Groningen, Groningen, University Medical Center Groningen, Groningen, The Netherlands, 2 Department of Analytical Biochemistry, University of Groningen, Groningen, The Netherlands.

Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

Abstract ‹–‘ Š‘†”‹ƒŽ ˆƒ––›Ǧƒ ‹† „‡–ƒǦ‘š‹†ƒ–‹‘ ȋ Ȍ ‹ –Š‡ Ž‹˜‡” ‹• ‘ˆ ”‹–‹ ƒŽ ‹’‘”–ƒ ‡ ˆ‘” ‡‡”‰›‡–ƒ„‘Ž‹•†—”‹‰ˆƒ•–‹‰ƒ†—†‡”‘–Š‡”‡‡”‰›Ǧ†‡ƒ†‹‰ ‘†‹–‹‘•ǤŠƒ’–‡”• ͵ ƒ† Ͷ Šƒ˜‡ •Š‘™ –Š‡ ‹’‘”–ƒ ‡ ‘ˆ •—””‘—†‹‰ ’ƒ–Š™ƒ›• ƒ† ’‡”‹’Š‡”ƒŽ –‹••—‡• ‘ Š‡’ƒ–‹  ˆ— –‹‘‹†‡ˆ‹ ‹‡ ›Ǥ –Š‹••–—†›ǡ™‡—•‡† ‘’—–ƒ–‹‘ƒŽ‘†‡Ž‹‰ –‘‰ƒ‹‘”‡‹•‹‰Š–‹–‘–Š‡”‘Ž‡‘ˆ–Š‡•—””‘—†‹‰‡–™‘”‘ ˆ— –‹‘‹ †‡ˆ‹ ‹‡ ›Ǥ ‘ –Š‹• ‡†ǡ ™‡ Š—ƒ‹œ‡† –Š‡ ”ƒ– †›ƒ‹  ‘†‡Ž ‘ˆ –Š‡   ’ƒ–Š™ƒ› ƒ† ‡š–‡†‡†‹–™‹–Š”‡Ž‡˜ƒ–’ƒ–Š™ƒ›•‘ˆ‡‡”‰›ƒ††‡–‘š‹ˆ‹ ƒ–‹‘‡–ƒ„‘Ž‹•ǤŠ‡•‹—Žƒ–‡† ˆŽ—š‡• ƒ† ‡–ƒ„‘Ž‹–‡ ’”‘ˆ‹Ž‡• ”‡ˆŽ‡ – –Š‡ ”‡Žƒ–‹˜‡ †‹•‡ƒ•‡ •‡˜‡”‹–‹‡• ‘ˆ ǡ ǡ ƒ†  †‡ˆ‹ ‹‡ › ‹ Š—ƒ•Ǥ —”–Š‡”‘”‡ǡ ™‡ •‹—Žƒ–‡† Šƒ‰‡• ‹ –Š‡ •—””‘—†‹‰ 5 ‡–™‘”ǡ ‹ Ž—†‹‰ –Šƒ– ‘ˆ –Š‡ ˆ—–‹Ž‡ › Ž‡ ‘ˆ ƒ ›ŽǦ‘ Š›†”‘Ž›•‹• ƒ† ”‡ƒ –‹˜ƒ–‹‘Ǥ ‡’”‡••‹‘‘ˆ–Š‹• › Ž‡„›‘†—Žƒ–‹‰–Š‡•—„•–”ƒ–‡•’‡ ‹ˆ‹ ‹–›‘ˆͳǡ‹’ƒ‹”‡†–Š‡  ˆŽ—š‹–Š‡†‡ˆ‹ ‹‡–‘†‡Žǡ„—–Šƒ”†Ž›‹–Š‡‘”†‡ˆ‹ ‹‡–‘†‡Ž•ǤŠ‡•‡ ”‡•—Ž–• ‰‹˜‡ ƒ ˆ‹”•– ‹†‹ ƒ–‹‘ Š‘™ †‹ˆˆ‡”‡ ‡• ‹ ‰‡‡–‹  „ƒ ‰”‘—† ”‡Žƒ–‡† –‘ –Š‡ •—””‘—†‹‰’ƒ–Š™ƒ›• ‘—Ž†ƒˆˆ‡ ––Š‡˜—Ž‡”ƒ„‹Ž‹–›‘ˆ’ƒ–‹‡–•–‘‡–ƒ„‘Ž‹  ”‹•‹•Ǥ 

ͳͷͻ Chapter 5

Introduction ‹–‘ Š‘†”‹ƒŽˆƒ––›ƒ ‹†„‡–ƒǦ‘š‹†ƒ–‹‘ȋ Ȍ‹–Š‡Ž‹˜‡”‹•‘ˆ‡›‹’‘”–ƒ ‡ˆ‘”‡‡”‰› Š‘‡‘•–ƒ•‹•†—”‹‰ˆƒ•–‹‰‘” ‘†‹–‹‘•‘ˆŠ‹‰Š‡‡”‰›†‡ƒ†Ǥ –Š‡ ‡ŽŽǡˆƒ––›ƒ ‹†•ƒ”‡ ˆ‹”•–ƒ –‹˜ƒ–‡†„›ˆ‘”ƒ–‹‘‘ˆƒˆƒ––›Ǧƒ ‹†Ǧ ‘‡œ›‡ȋ‘Ȍ‡•–‡”ȋƒ ›ŽǦ‘Ȍ‹–Š‡ ›–‘•‘ŽǤ Š‡”‡•—Ž–‹‰ ›–‘•‘Ž‹ ƒ ›ŽǦ‘‡•–‡”•ƒ”‡–ƒ‡—’‹–‘–Š‡‹–‘ Š‘†”‹ƒƒ†‘š‹†‹œ‡†–‘ ƒ ‡–›ŽǦ‘ǡ ™Š‹ Š ‹• ˆ—”–Š‡” ‘š‹†‹œ‡† ‹ –Š‡ ‹–”‹  ƒ ‹† › Ž‡ ˆ‘” –Š‡ ˆ‘”ƒ–‹‘ ‘ˆ Ǥ  ƒ††‹–‹‘ǡ –Š‡ Ž‹˜‡” ƒ ‘˜‡”– ƒ ‡–›ŽǦ‘ –‘ ‡–‘‡ „‘†‹‡•ǡ ™Š‹ Šƒ”‡—•‡† „› ’‡”‹’Š‡”ƒŽ –‹••—‡•†—”‹‰ˆƒ•–‹‰ǡ–Š—••ƒ˜‹‰‰Ž— ‘•‡Ǥ Š‡ˆƒ––›Ǧƒ ‹†„‡–ƒǦ‘š‹†ƒ–‹‘•–ƒ”–•™‹–Š–Š‡–”ƒ•’‘”–‘ˆ–Š‡ƒ ›ŽǦ Šƒ‹‘ˆ ›–‘•‘Ž‹ ƒ ›ŽǦ‘ ‹–‘ ‹–‘ Š‘†”‹ƒ „› –Š‡ ƒ”‹–‹‡Ǧ•Š—––Ž‡ ȋFigure 1ǡ ™‹–Š –Š‡ •Š—––Ž‡ ‡œ›‡• ‰‹˜‡ ‹ ‰”‡‡ȌǤŠ‹••Š—––Ž‡ ‘’”‹•‡•–Š”‡‡ ‘•‡ —–‹˜‡”‡ƒ –‹‘•ǡ–”ƒ•ˆ‡”‘ˆ–Š‡ƒ ›Ž Šƒ‹ˆ”‘ ƒ ›ŽǦ‘ ‘–‘ ƒ”‹–‹‡ ƒ–ƒŽ›œ‡† „› ƒ”‹–‹‡ ’ƒŽ‹–‘›Ž–”ƒ•ˆ‡”ƒ•‡ ȋȌͳǡ –”ƒ•’‘”– ‘ˆ ƒ ›ŽǦ ƒ”‹–‹‡ ƒ ”‘•• –Š‡ ‹–‘ Š‘†”‹ƒŽ ‹‡” ‡„”ƒ‡ ‹ ‡š Šƒ‰‡ ˆ‘” ƒ”‹–‹‡ „› ƒ”‹–‹‡ ƒ ›ŽǦ ƒ”‹–‹‡ –”ƒ•’‘”–‡” ȋȌǡ ƒ† –”ƒ•ˆ‡” ‘ˆ –Š‡ ƒ ›Ž Šƒ‹ ˆ”‘ ƒ ›ŽǦ ƒ”‹–‹‡‘–‘‘ „›ʹǤ‡š––Š‹•ƒ ›ŽǦ‘‹••Š‘”–‡‡†‹”‡’‡–‹–‹˜‡ˆ‘—”Ǧ”‡ƒ –‹‘ › Ž‡•ǡ–Š‡”‡„›’”‘†— ‹‰ƒƒ ‡–›ŽǦ‘‘Ž‡ —Ž‡ȏͳȂ͵Ȑ‹‡ƒ Š › Ž‡ǤŠ‡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 Š—ƒ•ǡ–Š‡•‡ƒ”‡˜‡”›ǦŽ‘‰Ǧǡ‡†‹—Ǧǡƒ† •Š‘”–Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡ȋǡǡƒ†Ȍȏ͵ǡͶȐǤ –Š‹•”‡ƒ –‹‘ǡƒ ›ŽǦ ‘‹•†‡Š›†”‘‰‡ƒ–‡†ǡˆ‘”‹‰‡‘›ŽǦ‘ǡƒ† „‘—†‹–Š‡ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡

‹•”‡†— ‡†–‘  ʹǤŠ‡”‡†— ‹‰‡“—‹˜ƒŽ‡–•ƒ”‡•—„•‡“—‡–Ž›–”ƒ•ˆ‡””‡†–‘–Š‡’”‘–‡‹ ‡Ž‡ –”‘ –”ƒ•ˆ‡” ˆŽƒ˜‘’”‘–‡‹ ȋ Ȍ ƒ† ˆ‹ƒŽŽ› –‘   †‡Š›†”‘‰‡ƒ•‡ ȋi.e.  —„‹“—‹‹‘‡ ‘š‹†‘”‡†— –ƒ•‡  Ȍ –”ƒ•ˆ‡”•–Š‡ ”‡†— ‹‰ ‡“—‹˜ƒŽ‡–• ˆ”‘  Ǧ„‘—†

 ʹ–‘‘‹–Š‡‹–‘ Š‘†”‹ƒŽ‡Ž‡ –”‘–”ƒ•’‘”– Šƒ‹Ǥ –Š”‡‡ ‘•‡ —–‹˜‡”‡ƒ –‹‘•ǡ ‡‘›ŽǦ‘‹•Š›†”ƒ–‡†ǡƒ‰ƒ‹†‡Š›†”‘‰‡ƒ–‡†ǡ–Š‡”‡„›”‡†— ‹‰Ϊ–‘ ǡƒ†ˆ‹ƒŽŽ› –Š‹‘Ž›œ‡†ǤŠ‡–Š‹‘Žƒ•‡”‡ƒ –‹‘’”‘†— ‡•ƒ•Š‘”–‡‡†ƒ ›ŽǦ‘‡•–‡”ƒ†ƒ ‡–›ŽǦ‘ǡ™‹–Š –Š‡ ‘ ‘‹–ƒ– ‘•—’–‹‘‘ˆƒˆ”‡‡‘‘Ž‡ —Ž‡ȋ”‡ˆ‡””‡†–‘ƒ•‘ ǡ„‡ ƒ—•‡‘ˆ‹–• ˆ”‡‡ –Š‹‘Ž ‰”‘—’ȌǤ Š‡ ’”‘†— –• ‘ˆ  ǡƒ ‡–›ŽǦ‘ǡ   ƒ†ƒ ›ŽǦ‘Ǧ†‡Š›†”‘‰‡ƒ•‡Ǧ

„‘—†  ʹǡ ƒ„‡ˆ—”–Š‡”‘š‹†‹œ‡†–‘’”‘†— ‡‹–Š‡–”‹ ƒ”„‘š›Ž‹ ƒ ‹†ȋȌ › Ž‡ ƒ†‘š‹†ƒ–‹˜‡’Š‘•’Š‘”›Žƒ–‹‘ȋ ȌǤ—”‹‰ˆƒ•–‹‰‘”‹–‡•‡‡š‡” ‹•‡ǡ–Š‡Ž‹˜‡”—•‡• –Š‹•ˆ‘”–Š‡’”‘†— –‹‘‘ˆ‰Ž— ‘•‡„›‰Ž— ‘‡‘‰‡‡•‹•Ǥ „‘”†‡ˆ‹ ‹‡ ‹‡•ƒ••‘ ‹ƒ–‡†™‹–ŠŠ—ƒ†‹•‡ƒ•‡Šƒ˜‡„‡‡‹†‡–‹ˆ‹‡†ˆ‘”ƒŽ‘•–ƒŽŽ–Š‡   ‡œ›‡•  ȏ͵ǡͷȐǤ †‡” ‡‡”‰›Ǧ†‡ƒ†‹‰ ‘†‹–‹‘•ǡ ‹†‹˜‹†—ƒŽ• ™‹–Š ƒ   †‡ˆ‹ ‹‡ › ”— ƒ ”‹• ‘ˆ •‡˜‡”‡ǡ Ž‹ˆ‡Ǧ–Š”‡ƒ–‡‹‰ Š›’‘‡–‘–‹  Š›’‘‰Ž› ‡‹ƒǤ ‹ ‡ –Š‡ ‹•‘‡œ›‡•ǡǡƒ†Šƒ˜‡‘˜‡”Žƒ’’‹‰•—„•–”ƒ–‡•’‡ ‹ˆ‹ ‹–‹‡•ȏ͵ǡͶȐǡ–Š‡› ƒ ‹ ’”‹ ‹’Ž‡ –ƒ‡ ‘˜‡” ‡ƒ Š ‘–Š‡”ǯ• ”‘Ž‡Ǥ Š‹• ƒ› ‡š’Žƒ‹ ™Š› Ǧ†‡ˆ‹ ‹‡– ȋȌ Š‹Ž†”‡‘ˆ–‡Šƒ˜‡‘Ž›‹‘”•›’–‘•ȏ͸ȐǤŠ‡†‹•‡ƒ•‡’”‡•‡–ƒ–‹‘‰‡‡”ƒŽŽ›„‡ ‘‡• ‘”‡•‡˜‡”‡ˆ”‘–‘–‘ȏ͵ȐǤ ‘™‡˜‡”ǡ–Š‡”‡‹•ƒŽƒ”‰‡Š‡–‡”‘‰‡‡‹–› ‹ †‹•‡ƒ•‡ ’”‡•‡–ƒ–‹‘ „‡–™‡‡  Ǧ†‡ˆ‹ ‹‡– ȋ Ȍ ‹†‹˜‹†—ƒŽ• ™‹–Š –Š‡ •ƒ‡ ‡œ›‡ †‡ˆ‹ ‹‡ ›ǡ ƒ† ‡˜‡ ™‹–Š –Š‡ •ƒ‡ ‰‡‡–‹  —–ƒ–‹‘Ǥ ‘” ‡šƒ’Ž‡ǡ ƒ •—„•‡– ‘ˆ Š‹Ž†”‡™‹–ŠƒŠ‘‘œ›‰‘—•Ž‘••Ǧ‘ˆǦˆ— –‹‘ Ǥͻͺͷε —–ƒ–‹‘‹–Š‡ACADM‰‡‡ǡ™Š‹ Š ‡ ‘†‡• ǡ ƒ ’”‡•‡– ™‹–Š Ž‹ˆ‡Ǧ–Š”‡ƒ–‡‹‰ Š›’‘‰Ž› ‡‹ƒȏ͵ǡͷȐǡ ™Š‹Ž‡ ƒ †‹ˆˆ‡”‡– •—„•‡– ‘ˆ –Š‡•‡ Š‹Ž†”‡ ‡˜‡” †‡˜‡Ž‘’• ƒ› •›’–‘• ȏ͹ȂͳͷȐǤ – ™ƒ• ”‡’‘”–‡† –Šƒ– ‹†‹˜‹†—ƒŽ• Š‘‘œ›‰‘—• ˆ‘” –Š‡ Ǥͻͺͷε  —–ƒ–‹‘ ƒ”‡ ƒ„Ž‡ –‘ ˆƒ•– ˆ‘” ͳͺǦʹͶ Š‘—”• ȏ͵ǡ͹ǡͳ͸ǡͳ͹ȐǤ ‘„‹ƒ–‹‘‘ˆ’”‘Ž‘‰‡†ˆƒ•–‹‰ƒ†ƒƒ††‹–‹‘ƒŽ–”‹‰‰‡”ǡ•— Šƒ•ƒƒ‹”™ƒ›

ͳ͸Ͳ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

‘”‰ƒ•–”‘‹–‡•–‹ƒŽ‹ˆ‡ –‹‘ǡƒŽ ‘Š‘Ž—•‡ǡ‘” ‘Ž†Ǧ‡š’‘•—”‡ǡ‹ ”‡ƒ•‡•–Š‡”‹•‘ˆŠ›’‘‡–‘–‹  Š›’‘‰Ž› ‡‹ƒȏͷǡͳͷǡͳͺȂʹͲȐǤ ‘ ‹˜‡•–‹‰ƒ–‡ –Š‡ „‹‘ Š‡‹ ƒŽ „ƒ•‹• ˆ‘” –Š‡ †‹ˆˆ‡”‡ ‡ ‹ ‡–ƒ„‘Ž‹  ”‘„—•–‡•• „‡–™‡‡ ‹†‹˜‹†—ƒŽ•ǡ ™‡ Šƒ˜‡ ‘•–”— –‡† ƒ †›ƒ‹  ‘’—–ƒ–‹‘ƒŽ ‘†‡Ž ‘ˆ –Š‡  ʹ͸Ǥ ‘†‡Ž •‹—Žƒ–‹‘•”‡˜‡ƒŽ‡†ƒ’‘–‡–‹ƒŽ˜‹ ‹‘—• › Ž‡‘ˆ‘‡•–‡”ƒ ——Žƒ–‹‘ǡ‘ †‡’Ž‡–‹‘ǡ ƒ†   ˆŽ—š †‡ Ž‹‡ ȏʹͳȐǡ ™Š‹ Š ™ƒ• ƒ—•‡† „› ƒ Š‹‰Š ‘ ‡–”ƒ–‹‘ ‘ˆ –Š‡ •—„•–”ƒ–‡ ’ƒŽ‹–‘›ŽǦ‘ ƒ† ƒ‰‰”ƒ˜ƒ–‡† „› Ž‘•• ‘ˆ  ȏͶȐǤ Š‡‘ Ǧ ‘•—‹‰ ‡œ›‡ ‘ˆ –Š‡ Žƒ•– ”‡ƒ –‹‘ǡ‡†‹—Ǧ Šƒ‹‡–‘ƒ ›ŽǦ‘–Š‹‘Žƒ•‡ȋȌƒ†–Š‡ΪǦ”‡†— ‹‰ ‡œ›‡ ‡†‹—Ǧ Šƒ‹ Š›†”‘š›ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋ Ȍ ’Žƒ›‡† ƒ ‹’‘”–ƒ– ”‘Ž‡‹–Š‡—†‡”Ž›‹‰‡ Šƒ‹•ȏʹͳȐȋChapter 2ȌǤŠ‡‘†‡Ž™ƒ•„ƒ•‡†‘„‹‘ Š‡‹ ƒŽ †ƒ–ƒ–Šƒ–Šƒ†„‡‡ ‘ŽŽ‡ –‡†‘•–Ž›ˆ”‘”ƒ–‹–‘ Š‘†”‹ƒǤŠ—ƒ˜‡”•‹‘‘ˆ–Š‡‘†‡Ž 5 Šƒ• „‡‡ †‡”‹˜‡† „› ˆ‹––‹‰ ‘ˆ ƒ •—„•‡– ‘ˆ ‘†‡Ž ’ƒ”ƒ‡–‡”• –‘ ’Žƒ•ƒ ƒ ›ŽǦ ƒ”‹–‹‡• ‘ˆ Š‡ƒŽ–Š›‹†‹˜‹†—ƒŽ•ȏʹʹȐǤŠ‹•Š—ƒ‘†‡Ž˜‡”•‹‘ ‘—Ž†‡š’Žƒ‹–Š‡†‹ˆˆ‡”‡ ‡‹•‡˜‡”‹–› ‘ˆ †‹•‡ƒ•‡ ’”‡•‡–ƒ–‹‘ „‡–™‡‡ –™‘ ‹†‹˜‹†—ƒŽ• ™‹–Š —Ž–‹’Ž‡Ǧƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ †‡ˆ‹ ‹‡ ›ȋȌǡƒ ‘†‹–‹‘ ƒ—•‡†„›ƒ†‡ˆ‡ –‹ ‘” †‡Š›†”‘‰‡ƒ•‡ȏʹʹȐǤŠ‡ ‘†‡ŽŠƒ•Š‘™‡˜‡”ǡ‘–›‡–„‡‡ƒ’’Ž‹‡†–‘‹˜‡•–‹‰ƒ–‡Š‡–‡”‘‰‡‡‘—•†‹•‡ƒ•‡’”‡•‡–ƒ–‹‘ ‘ˆ‘–Š‡”‡œ›‡†‡ˆ‡ –•Ǥ ‘–‡–‹ƒŽŽ›ǡ•—„–Ž‡†‹ˆˆ‡”‡ ‡•‹‰‡‡–‹ „ƒ ‰”‘—†‡š’Žƒ‹†‹ˆˆ‡”‡ ‡•‹˜—Ž‡”ƒ„‹Ž‹–›‘ˆ ”‘„—•–‡••‘ˆ ’ƒ–‹‡–•ǤŠ‹•ƒ›„‡”‡Žƒ–‡†–‘†‹ˆˆ‡”‡ ‡•™‹–Š‹–Š‡ ’ƒ–Š™ƒ›ǡ ‡Ǥ‰Ǥ†—‡–‘†‹ˆˆ‡”‡ ‡•‹ ‘ ‡–”ƒ–‹‘•‘”ƒ –‹˜‹–›‘ˆ–Š‡”‡ƒ‹‹‰‡œ›‡•Ǥ ‘™‡˜‡”ǡ–Š‡ ‘ˆƒ –‘”• Ϊ ƒ† ‘  ƒ˜ƒ‹Žƒ„‹Ž‹–› ƒŽ•‘ ’Žƒ›• ƒ ”‹–‹ ƒŽ ”‘Ž‡ ‹  Ǥ Š‡”‡ˆ‘”‡ǡ ™‡ Ž‘‘‡†‹–‘–Š‡‹–”‹ ƒ–‡ ‘‡ –‹‘‘ˆ–Š‡ ’ƒ–Š™ƒ›™‹–Š‘–Š‡”‡–ƒ„‘Ž‹ ’ƒ–Š™ƒ›•‘ˆ ‡–ƒ„‘Ž‹•ǡ’ƒ”–‹ —Žƒ”Ž›–Š‘•‡‹™Š‹ Š–Š‡•‡ ‘ˆƒ –‘”•ƒ”‡‹˜‘Ž˜‡†Ǥ‹ ‡ƒŠ‹‰Š ˆŽ—š ”‡“—‹”‡• Š‹‰Š ‘ ‡–”ƒ–‹‘• ‘ˆ Ϊ ƒ† ‘ ǡ ™‡ Š›’‘–Š‡•‹œ‡ –Šƒ– ’ƒ–Š™ƒ›• –Šƒ– ‹ ”‡ƒ•‡–Š‡•‡ ‘ ‡–”ƒ–‹‘•ǡƒ›ƒŽŽ‡˜‹ƒ–‡ Ž‹‹ ƒŽ•›’–‘•Ǥ  ‘–”ƒ•–ǡ’ƒ–Š™ƒ›•–Šƒ– †‡ ”‡ƒ•‡Ϊ‘”‘ ǡƒ›ƒ‰‰”ƒ˜ƒ–‡–Š‡”‹•‘ˆƒ‡–ƒ„‘Ž‹  ”‹•‹•Ǥ Š‡’ƒ–Š™ƒ›•–Šƒ–—–‹Ž‹œ‡–Š‡’”‘†— –•‘ˆ ƒ”‡–›’‹ ƒŽŽ›‹–Š‡ ƒ–‡‰‘”›‘ˆƒŽŽ‡˜‹ƒ–‹‰ ’ƒ–Š™ƒ›•Ǥ –Š‹• ƒ–‡‰‘”›ǡ‡–‘‰‡‡•‹•‹•–Š‡’”‘†— –‹‘‘ˆ–Š‡‡–‘‡„‘†‹‡•EǦŠ›†”‘š›Ǧ „—–›”ƒ–‡ƒ†ƒ ‡–‘ƒ ‡–ƒ–‡‹–Š‡Ž‹˜‡”ȋFigure 1ȌǤŠ‡•‡‡–‘‡„‘†‹‡•ƒ”‡•‡ ”‡–‡†‹–‘–Š‡ ’Žƒ•ƒˆ‘” ‘•—’–‹‘ƒ•ˆ—‡Ž„›’‡”‹’Š‡”ƒŽ–‹••—‡•ƒ†„”ƒ‹ǤŠ‡‡–‘‰‡‡•‹•’ƒ–Š™ƒ› —•‡• ƒ ‡–›ŽǦ‘ ƒ† ƒ ‡–‘ƒ ‡–›ŽǦ‘ ȋͶǦ‡–‘ƒ ›ŽǦ‘Ȍ ƒ• •—„•–”ƒ–‡• ƒ† ”‡‰‡‡”ƒ–‡• ‘ Ǥ  –Š‡ ‘˜‡”•‹‘ ‘ˆ ƒ ‡–‘ƒ ‡–ƒ–‡ –‘ EǦŠ›†”‘š›Ǧ„—–›”ƒ–‡ ȋ”‘—‰ŠŽ› ʹͷǦͶͺΨ ‘ˆ –Š‡ –‘–ƒŽ‡–‘‡Ǧ„‘†›’‘‘Žȏʹ͵ǡʹͶȐȌǡ ‹•”‡‘š‹†‹œ‡†‹–‘ΪǤ›‹ ”‡ƒ•‹‰„‘–Š‘  ƒ†Ϊǡ–Š‡‡–‘‰‡‡•‹•’ƒ–Š™ƒ›•Š‘—Ž† •–‹—Žƒ–‡–Š‡ ˆŽ—šǤ‡ ‘†ǡ–Š‡  •›•–‡ ‹• –Š‡ ƒŒ‘” Ǧ’”‘†— ‹‰ •›•–‡ –Šƒ– ”‡‘š‹†‹œ‡•   –‘ Ϊ ƒ† ’”‘–‡‹Ǧ Ϊ „‘—†  ʹ –‘  Ǥ  ƒ –‹˜‡   ’ƒ–Š™ƒ› ™‘—Ž† –Š‡”‡ˆ‘”‡ •–‹—Žƒ–‡ –Š‡  Ǥ ‡ ‡–Ž›ǡ Š‘™‡˜‡”ǡ ‹– ™ƒ• ”‡’‘”–‡† –Šƒ– –Š‡   •›•–‡ ˆ— –‹‘• •—„‘’–‹ƒŽŽ› ‹ ˆ‹„”‘„Žƒ•–•‘ˆ’ƒ–‹‡–•ȏʹͷȐǡ™Š‹ Š‹‰Š–ƒ‰‰”ƒ˜ƒ–‡–Š‡‹” Ž‹‹ ƒŽ•›’–‘•ȏͶǡʹ͸ȐǤ Š‹”†ǡ –Š‡ ‡š’‘”– ‘ˆ ƒ ›ŽǦ ƒ”‹–‹‡• ƒ ”‡Ž‹‡˜‡ –Š‡ ‹–‘ Š‘†”‹ƒ ˆ”‘ ƒ ——Žƒ–‹‰ ‘ ‡•–‡”• ƒ† –Š‡”‡„› Ž‹„‡”ƒ–‡ ‘ Ǥ ‹ƒŽŽ›ǡ –Š‡ Š›†”‘Ž›•‹• ‘ˆ ƒ ›ŽǦ‘ ‡•–‡”• „› ‹–‘ Š‘†”‹ƒŽ ƒ ›ŽǦ‘ –Š‹‘‡•–‡”ƒ•‡• ȋȌ ”‡Ž‡ƒ•‡• ‘  ƒ† ƒ› –Š‡”‡„› •–‹—Žƒ–‡ –Š‡ ǤŠ‡”‡•—Ž–‹‰ˆ”‡‡ˆƒ––›ƒ ‹†•ȋ •Ȍƒ›„‡•‡ ”‡–‡†ƒ†”‡ƒ –‹˜ƒ–‡†„› ›–‘•‘Ž‹  ƒ ›ŽǦ‘ •›–Š‡–ƒ•‡• ȋ•Ȍ ȋFigure 1ȌǤ Š‹• ˆ—–‹Ž‡ › Ž‡ Šƒ• ‹†‡‡† „‡‡ ”‡’‘”–‡† –‘ ‘–”‹„—–‡–‘–Š‡ ˆŽ—š ȏʹ͹Ȃ͵ͲȐƒ†ƒ› –Š‡”‡ˆ‘”‡ƒŽŽ‡˜‹ƒ–‡†‹•‡ƒ•‡•›’–‘•ǤŠ‡ ˆ‹ƒŽ ƒŽŽ‡˜‹ƒ–‹‰ ’ƒ–Š™ƒ› ™‘—Ž† „‡ –Š‡ ‹ ”‘•‘ƒŽ ɘǦ‘š‹†ƒ–‹‘ ’ƒ–Š™ƒ›ǡ ™Š‹ Š ‘š‹†‹œ‡•

ͳ͸ͳ Chapter 5

‡†‹—Ǧ Šƒ‹ˆƒ––›ƒ ‹†•–‘–Š‡‹” ‘””‡•’‘†‹‰†‹ ƒ”„‘š›Ž‹ ƒ ‹†•ǤŠ‹•’ƒ–Š™ƒ›™‘—Ž†„‡ ƒ•‹ˆ‘”‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘‡•–‡”•–Šƒ––‡†–‘ƒ ——Žƒ–‡™Š‡‹•†‡ˆ‹ ‹‡–Ǥ †‡‡†ǡ‹†‹˜‹†—ƒŽ••Š‘™Š‹‰Š‡”‡†‹—Ǧ Šƒ‹†‹ ƒ”„‘š›Ž‹ ƒ ‹†Ž‡˜‡Ž•‹„Ž‘‘†ƒ† —”‹‡ ‘’ƒ”‡† –‘ —ƒˆˆ‡ –‡† ‹†‹˜‹†—ƒŽ•ǡ ‹†‹ ƒ–‹‰ –Šƒ– –Š‡ ‘‡‰ƒǦ‘š‹†ƒ–‹‘ ƒ „‡ ‘•‹†‡”‡†ƒ’‘–‡–‹ƒŽ”‡• —‡’ƒ–Š™ƒ›‹ ȏ͵ͳȐǤ ‘”†‹‰–‘‘—””ƒ–‹‘ƒŽ‡ǡ’”‘ ‡••‡• –Šƒ– ‘’‡–‡ˆ‘”–Š‡ƒ˜ƒ‹Žƒ„Ž‡‘ ‘”Ϊ™‘—Ž†‹Š‹„‹––Š‡ ƒ†ƒ›ƒ‰‰”ƒ˜ƒ–‡ –Š‡”‹•‘ˆ Ž‹‹ ƒŽ•›’–‘•ȏ͵ʹǡ͵͵ȐǤ‹—Ž–ƒ‡‘—•‘š‹†ƒ–‹‘‘ˆ—Ž–‹’Ž‡•—„•–”ƒ–‡•™‘—Ž† „‡‹–Š‹• ƒ–‡‰‘”›Ǥ—”‹‰ˆƒ•–‹‰ǡ ‘š‹†ƒ–‹‘‘ˆ‰Ž— ‘•‡•Š‘—Ž†„‡‹‹ƒŽǤ”‡ƒ†‘™‘ˆ ’”‘–‡‹•ǡŠ‘™‡˜‡”ǡ™‘—Ž†’”‘†— ‡Š‹‰Š‡”Ž‡˜‡Ž•‘ˆƒ‹‘ƒ ‹†•Ǥƒ”–‹ —Žƒ”Ž›–Š‡ ƒ–ƒ„‘Ž‹• ‘ˆ „”ƒ Š‡†Ǧ Šƒ‹ ƒ‹‘ ƒ ‹†• ”‡“—‹”‡• ‘ Ǥ  ƒ††‹–‹‘ǡ ƒ–ƒ„‘Ž‹• ‘ˆ š‡‘„‹‘–‹ • ”‡“—‹”‡•‘ Ǥ Š‡ ƒ‹ ‘ˆ –Š‹• •–—†› ™ƒ• –‘ ‡š’Ž‘”‡ –Š‡ ‹’ƒ – ‘ˆ ’‘–‡–‹ƒŽ ƒŽŽ‡˜‹ƒ–‹‰ ’ƒ–Š™ƒ›• ‘ –Š‡ Š‡’ƒ–‹    ˆŽ—šǡ ‹ ‘”†‡” –‘ ‰ƒ‹ ‹•‹‰Š– ‹–‘ ‡ Šƒ‹•• —†‡”Ž›‹‰ –Š‡ †‹•‡ƒ•‡ ’”‡•‡–ƒ–‹‘‘ˆǡƒ†Ǥ‘–Š‹•‡†ǡ™‡”‡˜‹•‹–‡†–Š‡ ‘˜‡”•‹‘‘ˆ–Š‡ ”ƒ–   ‘†‡Ž ‹–‘ ƒ Š—ƒ ˜‡”•‹‘ǡ ƒ† ‡š–‡†‡† ‹– ™‹–Š –Š‡ ƒŽŽ‡˜‹ƒ–‹‰ ’ƒ–Š™ƒ›• †‹• —••‡† ƒ„‘˜‡Ǥ ‘Ǧ ƒ–ƒ„‘Ž‹• ‘ˆ ‘’‡–‹‰ •—„•–”ƒ–‡• ‹• ‘– ’ƒ”– ‘ˆ –Š‹• •–—†›Ǥ  –Š‡ ‡š–‡†‡† ‘†‡Ž ™‡ •‹—Žƒ–‡† †‡ˆ‹ ‹‡ ‹‡• ‘ˆ –Š‡ †‹ˆˆ‡”‡– ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡• ȋ•Ȍ ƒ† ƒ†‡ •—„–Ž‡ ˜ƒ”‹ƒ–‹‘• ‹ ‘ˆƒ –‘” ƒ˜ƒ‹Žƒ„‹Ž‹–‹‡• ‘” ƒ –‹˜‹–‹‡• ‘ˆ –Š‡ ƒ††‡† ’ƒ–Š™ƒ›•Ǥ‡•Š‘™–Šƒ––Š‡‘†‡Ž”‡’”‘†— ‡•–Š‡†‹ˆˆ‡”‡ ‡•‹†‹•‡ƒ•‡•‡˜‡”‹–›–Šƒ–ƒ”‡ ‰‡‡”ƒŽŽ› ‘„•‡”˜‡† „‡–™‡‡ ǡ ǡ ƒ†  ’ƒ–‹‡–•Ǥ  ƒ††‹–‹‘ǡ –Š‡ •‹—Žƒ–‡†‹–‡”˜‡–‹‘••Š‘™–Šƒ–†‹ˆˆ‡”‡ ‡•‹ ‘ˆƒ –‘”ƒ˜ƒ‹Žƒ„‹Ž‹–›ƒ†‡œ›‡ƒ –‹˜‹–‹‡• ƒ›‰‹˜‡”‹•‡–‘†‹ˆˆ‡”‡–ˆŽ—š‡•‹’ƒ–‹‡–•™‹–Š–Š‡•ƒ‡—–ƒ–‹‘Ǥ‡™‹ŽŽ†‹• —••Š‘™–Š‡ ‘†‡Žƒ›„‡—•‡†ƒ• ƒ•–ƒ”–‹‰’‘‹––‘‹˜‡•–‹‰ƒ–‡–Š‡†‹ˆˆ‡”‡ ‡•‹ Ž‹‹ ƒŽ•›’–‘• „‡–™‡‡‹†‹˜‹†—ƒŽ’ƒ–‹‡–•Ǥ  Results Model construction and characterization •ƒ•–ƒ”–‹‰’‘‹–ˆ‘”‘†‡Ž‡š–‡•‹‘™‡ˆ‹”•– ‘•‹†‡”‡†–Š‡’”‡˜‹‘—•Ž›’—„Ž‹•Š‡†Š—ƒ ˜‡”•‹‘ ‘ˆ –Š‡   ‘†‡Ž ȏʹʹȐǤ Š‡ ™‡ •‹—Žƒ–‡†  †‡ˆ‹ ‹‡ › ‹ –Š‹• ‘†‡Žǡ ‘ †‹ˆˆ‡”‡ ‡‹ ˆŽ—š™ƒ•‘„•‡”˜‡†„‡–™‡‡–Š‡‘ ‘—–ȋȌƒ†‘†‡Žƒ– ƒ‹ ”‡ƒ•‹‰ ‘ ‡–”ƒ–‹‘‘ˆ–Š‡•—„•–”ƒ–‡’ƒŽ‹–‘›ŽǦ‘ȋͳ͸Ǧƒ ›ŽǦ‘Ȍȋ—’’Ž‡‡–ƒ”› ˆ‹‰—”‡ͳȌǤ –Š‡‘†‡Ž ƒ””‹‡†Ž‹––Ž‡ˆŽ—šȋ—’’Ž‡‡–ƒ”› ‹‰—”‡ͳȌǤ‡†‘—„– –Šƒ––Š‹•‹•”‡ƒŽ‹•–‹ Ǥ –‹‰Š–”ƒ–Š‡”„‡ƒƒ”–‹ˆƒ –‘ˆ’ƒ”ƒ‡–‡”ˆ‹––‹‰™Š‡–Š‡”ƒ–‘†‡Ž ™ƒ• ‘˜‡”–‡†‹–‘ƒŠ—ƒ˜‡”•‹‘ȏʹʹȐǤŠ‡”‡ˆ‘”‡ǡ™‡™‡–„ƒ –‘–Š‡‘”‹‰‹ƒŽ”ƒ–‘†‡Žǡ ™Š‹ Š ™ƒ• „ƒ•‡† ‘ ’”‹ƒ”› ‡œ›‡ ‹‡–‹  †ƒ–ƒǤ Š‡”‡ ’‘••‹„Ž‡ǡ ’ƒ”ƒ‡–‡”• ™‡”‡ ”‡’Žƒ ‡†„›˜ƒŽ—‡•†‹”‡ –Ž›‡ƒ•—”‡†‘Š—ƒ‡œ›‡•ǤŠ‡Ž‘‰Ǧ Šƒ‹ƒ ›ŽǦ‘ȋȌ ‹•‘‡œ›‡™ƒ•”‡‘˜‡†ǡ•‹ ‡‹–‹•‘–‡š’”‡••‡†‹Š—ƒŽ‹˜‡”ȏ͵ͶȐǤ‡š–ǡ–Š‡‘†‡Ž™ƒ• ‡š’ƒ†‡†™‹–Š–Š‡’ƒ–Š™ƒ›•†‡’‹ –‡†‹ ‹‰—”‡ͳǡ‘–ƒ„Ž›‡–‘‰‡‡•‹•ǡ †‡Š›†”‘‰‡ƒ•‡ǡ ‹–‘ Š‘†”‹ƒŽǡ ›–‘•‘Ž‹  ”‡ƒ –‹˜ƒ–‹‘„›ǡƒ†‹ ”‘•‘ƒŽ‘‡‰ƒǦ‘š‹†ƒ–‹‘Ǥ —”–Š‡”‘”‡ǡ –Š‡ ‡š’‘”– ‘ˆ ƒ ›ŽǦ ƒ”‹–‹‡•ǡ • ƒ††‹ ƒ”„‘š›Ž‹  ƒ ‹†• ȋ•Ȍ ‘—– ‘ˆ –Š‡ ‡ŽŽ™‡”‡‹ ‘”’‘”ƒ–‡†Ǥ ‘”–Š‡‡–‘‰‡‡•‹•’ƒ–Š™ƒ›ǡƒŽŽ”‡ƒ –‹‘•™‡”‡‹ Ž—†‡†ǡ•‹ ‡–Š‡› †‹”‡ –Ž› ‹˜‘Ž˜‡ ‘ –Š‹‘‡•–‡”•ǡ ‘  ƒ† ΪǤ ‘” –Š‡   †‡Š›†”‘‰‡ƒ•‡Ǧ ƒ–ƒŽ›œ‡†

”‡ƒ –‹‘ǡ ǡ‘ƒ†‹–•”‡†— ‡†‡“—‹˜ƒŽ‡–ȋ‘ ʹȌ™‡”‡‹ Ž—†‡†Ǥ‘”‡ › Ž‡ ›–‘•‘Ž‹  ƒ ›ŽǦ‘‡•–‡”•‰‡‡”ƒ–‡†„›‹–‘–Š‡ ǡ–Š‡‹‡–‹ •‘ˆͳŠƒ†–‘„‡ƒ†ƒ’–‡†Ǥ  ’”‡˜‹‘—•‘†‡Ž˜‡”•‹‘•‹–•‘Ž›•—„•–”ƒ–‡™ƒ•’ƒŽ‹–‘›ŽǦ‘ǡ„—–‹”‡ƒŽ‹–›‹–ƒŽ•‘ ‘˜‡”–•

ͳ͸ʹ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

‘–Š‡” ƒ ›ŽǦ‘ ‡•–‡”• –‘ ƒ ›ŽǦ ƒ”‹–‹‡• ȏ͵ͷȐǤ Š‡ ‘‡‰ƒǦ‘š‹†ƒ–‹‘ ™ƒ• ”‡’”‡•‡–‡† „› ƒ •‹‰Ž‡ ”‡ƒ –‹‘ǡ ˆ‘” ™Š‹ Š –Š‡ ‹‡–‹ • ‘ˆ Ͷͳͳ ȏ͵͸Ȑ ™‡”‡ —•‡†Ǥ  ƒ–Š‡ƒ–‹ ƒŽ †‡• ”‹’–‹‘‘ˆ–Š‡‡š–‡†‡†‘†‡Ž‹•‰‹˜‡‹—’’Ž‡‡–ƒ”›–‡š–ͳǤ ‹‰—”‡ʹ•Š‘™•–Š‡ ‡–•–‡ƒ†›Ǧ•–ƒ–‡ ’”‘†— –‹‘ˆŽ—šƒ•ƒˆ— –‹‘‘ˆ–Š‡ ‘ ‡–”ƒ–‹‘‘ˆ–Š‡•—„•–”ƒ–‡ ›–‘•‘Ž‹  ’ƒŽ‹–‘›ŽǦ‘Ǥ  ‹ ”‡ƒ•‹‰ ›–‘•‘Ž‹  ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘ ”‡ˆŽ‡ –• –Š‡ ˆƒ•–‹‰ ‘†‹–‹‘™Š‡–”‹‰Ž› ‡”‹†‡•ˆ”‘–Š‡ƒ†‹’‘•‡•–‘”‡•ƒ”‡Š›†”‘Ž›œ‡†ƒ†•‡ ”‡–‡† ‹–‘ –Š‡ ‹” —Žƒ–‹‘ǡ –Š—• ’”‘˜‹†‹‰ ‘”‡ •—„•–”ƒ–‡ ˆ‘” –Š‡ Ž‹˜‡”  Ǥ Š‡   ’”‘†— –‹‘ ˆŽ—š •Š‘™‡† ƒ •ƒ–—”ƒ–‹‘ —”˜‡ǡ ‹ ™Š‹ Š –Š‡ ƒš‹— ˆŽ—š ™ƒ• ”‡ƒ Š‡† ƒ– ƒ’’”‘š‹ƒ–‡Ž› ͵ͲɊ ›–‘•‘Ž‹  ’ƒŽ‹–‘›ŽǦ‘ ȋ ‹‰—”‡ ʹȌǤ – Š‹‰Š‡” •—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘•–Š‡ˆ”‡‡‘ Ž‡˜‡Ž•†‡ Ž‹‡†ǡ‹””‘”‹‰–Š‡‹ ”‡ƒ•‡‘ˆ–Š‡–‘–ƒŽ‘‡•–‡” ‘ ‡–”ƒ–‹‘‹–Š‡ƒ–”‹šȋ ‹‰—”‡ʹȌǤŠ‡–‘–ƒŽ‘ ‘ ‡–”ƒ–‹‘™ƒ•ˆ‹š‡†ƒ–ƒ ‘•–ƒ– 5 ˜ƒŽ—‡ǡƒ•‹’”‡˜‹‘—•‘†‡Ž˜‡”•‹‘•ȏʹʹǡ͵͹Ȑǡ”‡ˆŽ‡ –‹‰–Šƒ–‘„‹‘•›–Š‡•‹•–ƒ‡•’Žƒ ‡ƒ–ƒ •Ž‘™‡”–‹‡• ƒŽ‡Ǥ 

Cytosol DCA Palmitoyl-CoA (C -Acyl-CoA) чϭϰ DCA ʘ-oxidation Malonyl-CoA Acyl-carnitine FFA C4-C16 Acyl-CoA FFA carnitine CoASH CoASH Microsome carnitine CPT1 ACS C4-C16 CACT C4-C16 CoQ CoQH C4-C16 CPT2 2 Mitochondrion C4-C16 ETFox 2 Acetyl-CoA ACOT FFA ETFQO carnitine C4-C16 ETF CoASH carnitine red ACAT1 Acyl-carnitine CoASH CoASH Acyl-CoA C4-ketoacyl-CoA Acetyl-CoA ETF HMGCS2 MCKAT Acetyl-CoA SCAD MCAD VLCAD ox CoASH C4-C16 C4-C6 C4-C16 C6-C16 ETF CoASH red HMG-CoA

Ketoacyl-CoA Enoyl-CoA Acetyl-CoA HMGCL + NADH + H MTP Acetyl-CoA H2O C4-ketoacyl-CoA NADH + H+ Acetoacetate MSCHAD C4-ketoacyl-CoA CROT NAD+ C8-C16 C4-C16 NAD+ C4-C16 CoASH + NADH + H BDH1 Hydroxyacyl-CoA NAD+ ɴ-OH-butyrate  Figure 1. Schematic representation of the extended human model.‡–ƒ„‘Ž‹–‡•‹„Ž—‡ƒ”‡ ‘•‹†‡”‡† ƒ• „‘—†ƒ”› ‡–ƒ„‘Ž‹–‡• ™‹–Š ˆ‹š‡† ‘ ‡–”ƒ–‹‘•Ǥ Š‡ —†‡”Ž›‹‰ ƒ••—’–‹‘ ‹• –Šƒ– –Š‡‹” ‘ ‡–”ƒ–‹‘• ƒ”‡ †‡–‡”‹‡† Žƒ”‰‡Ž› „› –Š‡ •—””‘—†‹‰ ‡–™‘”Ǥ Š‡ ‘–Š‡” ‡–ƒ„‘Ž‹–‡• ƒ”‡ ˆ”‡‡ ˜ƒ”‹ƒ„Ž‡•ˆ‘”™Š‹ Š‘”†‹ƒ”›†‹ˆˆ‡”‡–‹ƒŽ‡“—ƒ–‹‘•ȋ•ȌŠƒ˜‡„‡‡†‡ˆ‹‡†ȋ•‡‡—’’Ž‡‡–ƒŽ–‡š–ͳ ˆ‘”–Š‡ ‘’Ž‡–‡‘†‡Ž†‡• ”‹’–‹‘ȌǤ›–‘•‘Ž‹ ’ƒŽ‹–‘›ŽǦ‘ȋ‰‹˜‡‹„Ž—‡Ȍ‹•–Š‡•—„•–”ƒ–‡ƒ†‡’–ƒ–ƒ ˆ‹š‡† ‘ ‡–”ƒ–‹‘ǡ™Š‹Ž‡ ›–‘•‘Ž‹ ƒ ›ŽǦ‘‡•–‡”•‘ˆ ƒ”„‘ Šƒ‹Ž‡‰–Š•‘ˆͳͶ‘”Ž‘™‡”ȋ‹†‹ ƒ–‡†„›

dͳͶǡ ‹ „Žƒ Ȍ ƒ”‡ ˜ƒ”‹ƒ„Ž‡Ǥ Š‡› ƒ”‡ ‹–‡”‡†‹ƒ–‡ ‡–ƒ„‘Ž‹–‡• †‡”‹˜‡† ˆ”‘ –Š‡ ’ƒ”–‹ƒŽ ‘š‹†ƒ–‹‘ ‘ˆ ’ƒŽ‹–‘›ŽǦ‘ƒ†”‡ › Ž‹‰„›Ǥ   ‘’ƒ”‹•‘ –‘ Chapter 2ǡ ™‡ ‘–‡ –Šƒ– –Š‡‘  ‘ ‡–”ƒ–‹‘ †‘‡• ‘– †”‘’ –‘ –Š‡ ’‘‹–™Š‡”‡–Š‡ˆŽ—š•–ƒ”–‡†–‘†‡ Ž‹‡‹–Š‡‡ƒ”Ž‹‡”‘†‡Ž˜‡”•‹‘Ǥ –‹•’‘••‹„Ž‡–Šƒ––Š‹•‹• ƒ—•‡†„›–Š‡ƒ††‹–‹‘‘ˆ’”‘ ‡••‡•–Šƒ–”‡‰‡‡”ƒ–‡‘ ƒ†”‡†— ‡–Š‡ƒ ——Žƒ–‹‘‘ˆ ‘‡•–‡”•ǤŠ‡†‹ˆˆ‡”‡–’ƒ”ƒ‡–‡”•ˆ‘”–Š‡Š—ƒ‡œ›‡•ƒ›ǡŠ‘™‡˜‡”ǡƒŽ•‘ ‘–”‹„—–‡Ǥ ‡†‹†‘–‹˜‡•–‹‰ƒ–‡–Š‹•ˆ—”–Š‡”Ǥ 

ͳ͸͵ Chapter 5

A B C 14 5000 3.0 )

-1 free CoA Leak * 12 vExAc-carnCn * 4000 Intermediate CoA esters ɇ 10 C4-CoA esters ɇvExFFACn * ;ʅDͿ 2.0 .gProtein vExDCACn * -1 3000 C6-CoA esters ɇ

8 MAT vacsC16 * 6 C8-CoA esters 2000 C10-CoA esters 4 1.0 (μmol.min C12-CoA esters (%) leak Substrate 2 1000

[Metabolite] C14-CoA esters nadhsink J 0 0 C16-CoA esters 0.0 0501000 50 100 0 50 100 [Palmitoyl-CoA] [Palmitoyl-CoA] [Palmitoyl-CoA] CYT ;ʅDͿ CYT ;ʅDͿ CYT ;ʅDͿ  Figure 2. The net NADH production flux in the mitochondrionȋ‹Ǥ‡’”‘†— –‹‘„›Ȁ ƒ† ‹—• ‘•—’–‹‘ „› ‡–‘‰‡‡•‹•Ȍ ȋ†‡‘–‡† ‹ –Š‡ ‰”ƒ’Š ƒ• ƒ†Š•‹ǡ •‹ ‡ ƒ– •–‡ƒ†› •–ƒ–‡ –Š‹• ˆŽ—š ‹’Ž‹ ‹–›ƒŽ•‘”‡’”‡•‡–•–Š‡”ƒ–‡‘ˆ  ‘•—’–‹‘„›‘š‹†ƒ–‹˜‡’Š‘•’Š‘”›Žƒ–‹‘ǡ–Š‡•‹ȋAȌǢƒ† –Š‡‹–‘ Š‘†”‹ƒŽ‘ ‡•–‡” ‘ ‡–”ƒ–‹‘•ǡ•—‡†‘˜‡”ƒ ›ŽǦ‘ǡ‡‘›ŽǦ‘ǡŠ›†”‘š›Ǧ‡‘›ŽǦ‘ƒ† ‡–‘ƒ ›ŽǦ‘ǡ •’‡ ‹ˆ‹‡† ’‡” ƒ”„‘Ǧ Šƒ‹ Ž‡‰–Š ȋBȌ ȋ ˆǤ ‹‰—”‡ ͳȌǤ ƒ‡Ž C †‡’‹ –• ǮŽ‡ƒǯ ˆŽ—š‡• ‘ˆ ƒ ›ŽǦ ‰”‘—’•–Šƒ–Ž‡ƒ˜‡–Š‡•›•–‡˜‹ƒ‡š’‘”–‘ˆƒ ›ŽǦ ƒ”‹–‹‡•ƒ† ‘”˜‹ƒ ZǦ‘š‹†ƒ–‹‘ǡƒ†–Š‡ͳ͸Ǧƒ ›ŽǦ ”‡ › Ž‹‰ˆŽ—š˜‹ƒǤŠ‡ˆŽ—š‡•™‡”‡ˆ‹”•– ƒŽ —Žƒ–‡†‹—‹–•‘ˆ –Šƒ–™‘—Ž†Šƒ˜‡„‡‡’”‘†— ‡†‹ˆ –Š‡›Šƒ†„‡‡ˆ—ŽŽ›‘š‹†‹œ‡†ǡ‹Ǥ‡Ǥƒͳ͸Ǧƒ ›ŽˆŽ—š™‘—Ž†„‡—Ž–‹’Ž‹‡†„›͹ǡƒͳͶǦƒ ›ŽˆŽ—š„›͸ǡ‡– ǤŠ‹• ƒŽŽ‘™• †‹”‡ – ‘’ƒ”‹•‘ ‘ˆ –Š‡ ˆŽ—š‡• –‘ –Š‡ ‡–     ˆŽ—š ‹ ’ƒ‡Ž  ƒ† –‘ ‡ƒ Š ‘–Š‡” ȋSupplementary Figure S2ȌǤ—„•‡“—‡–Ž›ǡ–Š‡•‡ˆŽ—š‡•™‡”‡ ‘˜‡”–‡†–‘ƒ’‡” ‡–ƒ‰‡‘ˆ–Š‡’ƒŽ‹–‘›ŽǦ ‘ ‘•—’–‹‘ˆŽ—š‡š’”‡••‡†ƒ• ’”‘†— –‹‘‡“—‹˜ƒŽ‡–•ȋͳͲͲΨȗˆŽ—šȀȋ͹ȗ˜ ’–ͳͳ͸ȌȌǤ  ‡ƒƒ‰‡ ‘ˆ ƒ ›ŽǦ‘‹‡–‹‡• ‘—– ‘ˆ –Š‡ •›•–‡ ˜‹ƒ ‡š’‘”– ‘” ZǦ‘š‹†ƒ–‹‘ ™ƒ• ˜‡”› •ƒŽŽ ȋ–‘‰‡–Š‡”ƒš‹ƒŽŽ›ʹǤ͹Ψ‘ˆ–Š‡–‘–ƒŽ’ƒŽ‹–‘›ŽǦ‘ ‘•—’–‹‘ˆŽ—šǢȋ ‹‰—”‡ʹǡȌǤŠ‹• ”‡•—Ž–•‹ƒ‡– ’”‘†— –‹‘ˆŽ—š•‹‹Žƒ”–‘–Š‡‡š’‡ –‡† ’”‘†— –‹‘ˆŽ—š‹ˆƒŽŽ ’ƒŽ‹–‘›ŽǦ‘ ™‘—Ž† „‡ ˆ—ŽŽ› ‘š‹†‹œ‡† ‹ –Š‡  ǡ ‹Ǥ‡Ǥ „ƒ•‡† ‘ ͹š –Š‡ ͳ͸Ǧƒ ›ŽǦ‘ ‘•—’–‹‘ˆŽ—šȋ—’’Ž‡‡–ƒ”›ˆ‹‰—”‡ʹȌǤ ‹ƒŽŽ›ǡ‡–ƒ„‘Ž‹ ‘–”‘ŽƒŽ›•‹•ȋ ˆǤŠƒ’–‡” ʹȌ •Š‘™‡† –Šƒ– ͳ ƒ†   †‡Š›†”‘‰‡ƒ•‡ ȋ Ȍ •Šƒ”‡ ˆŽ—š ‘–”‘Ž ƒ– ʹͷ Ɋ

’ƒŽ‹–‘›ŽǦ‘ǡ „—– ƒ– ƒ– ͳͲͲ Ɋ ’ƒŽ‹–‘›ŽǦ‘ ͳ †‘‡• ‘– ‡š‡”– ƒ› ˆŽ—š ‘–”‘Ž ƒ›‘”‡Ǥ•‡š’‡ –‡†ǡͳŠƒ•ƒ‡‰ƒ–‹˜‡ ‘–”‘Ž‘‘ ǡ™Š‹ Š™ƒ•‘”‡’”‘‘— ‡† ƒ– ͳͲͲ Ɋ ’ƒŽ‹–‘›ŽǦ‘Ǥ Š‹• ‹• „ƒŽƒ ‡† „› ƒ ’‘•‹–‹˜‡ ‘–”‘Ž ‡š‡”–‡† „›   ȋ—’’Ž‡‡–ƒ”›–ƒ„Ž‡ͳȌǤ  Simulating the impact of ACAD deficiencies on the mitochondrial mFAO flux ‡š–ǡ–Š‡‹’ƒ –‘ˆƒ ‘’Ž‡–‡‹ƒ –‹˜ƒ–‹‘‘ˆ‡ƒ Š‘ˆ–Š‡•™ƒ••‹—Žƒ–‡†•‡’ƒ”ƒ–‡Ž›ǡ –‘ ‹‹  –Š‡ ‘””‡•’‘†‹‰ ‡œ›‡ †‡ˆ‹ ‹‡ ‹‡• ‹ ’ƒ–‹‡–• ȏͳͲǡ͵ͺǡ͵ͻȐ ȋ ‹‰—”‡ ͵ȌǤ  ‘’Ž‡–‡ ‹ƒ –‹˜ƒ–‹‘ ‘ˆ –Š‡ ‡œ›‡• ‹• ”‡ƒŽ‹•–‹ ǣ „‘–Š ‹  ȏ͵ͺȐ ƒ†  ȏͶͲȐ ”‡•‹†—ƒŽ ƒ –‹˜‹–‹‡• Ž‘™‡” –Šƒ ͲǤͷΨ Šƒ˜‡ „‡‡ ”‡’‘”–‡† ƒ† ‹  ƒ ”‡•‹†—ƒŽ ƒ –‹˜‹–› Ž‘™‡” –Šƒ ͶΨ Šƒ• „‡‡ ”‡’‘”–‡† ȏͶͳȐǤ Š‡   ‡šŠ‹„‹–‡† –Š‡ Ž‘™‡•–   ’”‘†— –‹‘ˆŽ—šǡˆ‘ŽŽ‘™‡†„›–Š‡ǡ™Š‹Ž‡–Š‡ˆŽ—š‹–Š‡‘†‡Ž™ƒ•‘Ž› ƒ”‰‹ƒŽŽ›Ž‘™‡”–Šƒ–Šƒ–‘ˆ–Š‡ȋ ‹‰—”‡͵ȌǤŠ‹•”‡ˆŽ‡ –•–Š‡‰‡‡”ƒŽ‘”†‡”‘ˆ†‹•‡ƒ•‡ •‡˜‡”‹–› ‘ˆ –Š‡ Š—ƒ  †‡ˆ‹ ‹‡ ‹‡•ǣ  ‹• —•—ƒŽŽ› ƒ•›’–‘ƒ–‹ ȏ͸Ȑǡ  ’”‡•‡–•‘Ž›†—”‹‰‹–‡” —””‡–‹ŽŽ‡••‘”—’‘ƒ‘–Š‡”–”‹‰‰‡”ȋ•‡‡ƒ„‘˜‡Ȍǡ™Š‹Ž‡ Šƒ•–Š‡‘•–•‡˜‡”‡•›’–‘•ǤŽ‡ƒ”Ž›ǡ–Š‹•‘†‡Ž•‹—Žƒ–‡•–Š‡Š—ƒ†‹•‡ƒ•‡•„‡––‡”–Šƒ –Š‡’”‡˜‹‘—•Ž›’—„Ž‹•Š‡†Š—ƒ‘†‡Žȋ ‹‰—”‡͵ȌǤ‹‡–Š‡ȋ ‹‰—”‡ʹȌƒŽ•‘–Š‡ †‡ˆ‹ ‹‡– ‘†‡Ž• •Š‘™‡†ƒ †‡ Ž‹‡ ‘ˆ ‘  ™‹–Š ‹ ”‡ƒ•‹‰ ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘ ȋ ‹‰—”‡͵ǦȌǤ –Š‡‘†‡Ž–Š‡†‡ Ž‹‡‘ˆ‘ ™ƒ•Ž‡••–Šƒ‹–Š‡Ǥ 

ͳ͸Ͷ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

A )

-1 14 12 10 WT

.gProtein -1 8 SCAD KO 6 MCAD KO 4

(μmol.min VLCAD KO 2

0 nadhsink J  B C D 5000 VLCAD KO MCAD KO SCAD KO free CoA 4000 Intermediate CoA esters

;ʅDͿ C4-CoA esters

MAT 3000 C6-CoA esters 5 C8-CoA esters 2000 C10-CoA esters C12-CoA esters 1000 [Metabolite] C14-CoA esters C16-CoA esters 0   E 40 F 0 G VLCAD KO MCAD KO SCAD KO Leak * 30 0 ɇvExAc-carnCn *

20 0 ɇvExFFACn *

ɇvExDCACn * 10 0 Substrate leak (%) Substrate vacsC16 *

0 0 050100050100050100

[Palmitoyl-CoA]CYT ;ʅDͿ [Palmitoyl-CoA]CYT ;ʅDͿ [Palmitoyl-CoA]CYT ;ʅDͿ Ǥ  Figure 3. The net NADH production flux in the mFAOȋAȌǡ ‹–‘ Š‘†”‹ƒŽ ‘ ‡•–‡” ‘ ‡–”ƒ–‹‘•ǡ •—‡† ‘˜‡” ƒ ›ŽǦ‘ǡ ‡‘›ŽǦ‘ǡ Š›†”‘š›Ǧ‡‘›ŽǦ‘ ƒ† ‡–‘ƒ ›ŽǦ‘ǡ •’‡ ‹ˆ‹‡† ’‡” ƒ”„‘Ǧ Šƒ‹ Ž‡‰–Š‘ˆȋŠ‡ƒŽ–Š›Ȍƒ†•‹‰Ž‡Ǧ‡œ›‡˜‡”•‹‘•‘ˆ–Š‡‡š–‡†‡†‘†‡ŽȋB-DȌǤƒ‡Ž•E-G†‡’‹ –ǡ ˆ‘”‡ƒ Š•‹‰Ž‡Ǧ‡œ›‡‘†‡Žǡ–Š‡ǮŽ‡ƒǯˆŽ—š‡•‘ˆƒ ›ŽǦ‰”‘—’•–Šƒ–Ž‡ƒ˜‡–Š‡•›•–‡˜‹ƒ‡š’‘”–‘ˆƒ ›ŽǦ ƒ”‹–‹‡• ƒ†  ‘” ˜‹ƒ ZǦ‘š‹†ƒ–‹‘ǡ ƒ† –Š‡ ͳ͸Ǧƒ ›ŽǦ”‡ › Ž‹‰ ˆŽ—š ˜‹ƒ  ƒ• ƒ ’‡” ‡–ƒ‰‡ ‘ˆ –Š‡ ’ƒŽ‹–‘›ŽǦ‘ ‘•—’–‹‘ˆŽ—š‡š’”‡••‡†ƒ• ’”‘†— –‹‘‡“—‹˜ƒŽ‡–•Ǥ  Š‹• •Š‘—Ž† „‡ ‡š’‡ –‡†ǡ •‹ ‡  ‹• ‘‡ ‘ˆ –Š‡ ˆ‹”•– ‡œ›‡• ‹ –Š‡ ’ƒ–Š™ƒ› ƒ† ƒ †‡ˆ‹ ‹‡ ›’”‡˜‡–•ƒ ——Žƒ–‹‘‘ˆ‘‡•–‡”•†‘™•–”‡ƒǤ ‘”†‹‰Ž›ǡ‹–Š‡ ‘†‡Ž‘Ž›ͳ͸Ǧ‘‡•–‡”•ƒ ——Žƒ–‡†ȋ ‹‰—”‡͵ȌǤ  ‘–”ƒ•–ǡ‹–Š‡‘†‡Ž–Š‡ †‡ Ž‹‡‘ˆ‘ ™ƒ•Žƒ”‰‡”–Šƒ‹–Š‡‘†‡Žƒ†‹–™ƒ•‡˜‡Žƒ”‰‡”‹–Š‡ ‘†‡ŽǤ –Š‡•‡ ƒ•‡•ǡ–Š‡†‡ˆ‡ –‹•ˆ—”–Š‡”†‘™•–”‡ƒǡ•— Š–Šƒ–•—„•–”ƒ–‡‡–‡”•‘”ƒŽŽ› ƒ† –Š‡ •–ƒ”–• ƒ ——Žƒ–‹‰Ǥ  –Š‡   ‘†‡Ž ‘•–Ž› ͺ ƒ† ͳͲ ‘ ‡•–‡”• ƒ ——Žƒ–‡† ȋ ‹‰—”‡ ͵Ȍǡ ™Š‹ Š ‹• ‘•‹•–‡– ™‹–Š –Š‡ ƒ ——Žƒ–‹‘ ‘ˆ ͺ ƒ† ͳͲ ƒ ›ŽǦ ƒ”‹–‹‡•‹„Ž‘‘†•ƒ’Ž‡•ˆ”‘’ƒ–‹‡–•ȏͶʹȂͶ͸ȐǤ –Š‡‘†‡Žǡƒ ——Žƒ–‹‘‘ˆͶǦ‘‡•–‡”•™ƒ•‘•–’”‘‹‡–ȋ ‹‰—”‡͵Ȍǡ‹ Ž‹‡ ™‹–Š ƒ †‡ˆ‡ – ‹ –Š‡ ‡–ƒ„‘Ž‹• ‘ˆ •Š‘”–Ǧ Šƒ‹ ƒ ›ŽǦ‘ ‡•–‡”• ƒ† ‘„•‡”˜ƒ–‹‘• ‹ ’ƒ–‹‡–•ǤŠ‡Ž‡ƒˆŽ—š‡•ǡi.e.–Š‡•—‘ˆ–Š‡ˆŽ—š‡•‘ˆ‹ ‘’Ž‡–‡Ž›‘š‹†‹œ‡†’ƒŽ‹–‘›ŽǦ‘ǡ ™ƒ•˜‡”›•ƒŽŽ‹–Š‡ƒ†‘†‡Ž•ǡƒ•‹–™ƒ•‹–Š‡Š‡ƒŽ–Š›‘†‡Žȋ ˆǤ ‹‰—”‡ ʹǦ ƒ† ‹‰—”‡ ͵ Ǧ ȌǤ  ‘–”ƒ•–ǡ ‹ –Š‡   ‘†‡Ž –Š‡ Ž‡ƒ ™ƒ• “—‹–‡ ƒ Žƒ”‰‡ ˆ”ƒ –‹‘ ‘ˆ –Š‡ –‘–ƒŽ ’ƒŽ‹–‘›ŽǦ‘ ‘•—’–‹‘ ˆŽ—šǤ Š‡ Žƒ”‰‡” ˆ”ƒ –‹‘ ‘ˆ –Š‡ –‘–ƒŽ Ž‡ƒ ™ƒ•ǡŠ‘™‡˜‡”ǡ”‡ › Ž‡†–Š”‘—‰Šȋ’‹Ž‹‡‹ ‹‰—”‡͵ȌǤŠ‹•”‡ˆŽ‡ –•–Š‡‰‡‡”ƒŽ‘”†‡” ‘ˆ †‹•‡ƒ•‡ •‡˜‡”‹–› ‘ˆ –Š‡ Š—ƒ  †‡ˆ‹ ‹‡ ‹‡•ǣ  ‹• —•—ƒŽŽ› ƒ•›’–‘ƒ–‹ ȏ͸Ȑǡ ’”‡•‡–•‘Ž›†—”‹‰‹–‡” —””‡–‹ŽŽ‡••‘”—’‘ƒ‘–Š‡”–”‹‰‰‡”ȋ•‡‡ƒ„‘˜‡Ȍǡ™Š‹Ž‡

ͳ͸ͷ Chapter 5

Šƒ•–Š‡‘•–•‡˜‡”‡•›’–‘•ǤŽ‡ƒ”Ž›ǡ–Š‹•‘†‡Ž•‹—Žƒ–‡•–Š‡Š—ƒ†‹•‡ƒ•‡• „‡––‡” –Šƒ –Š‡ ’”‡˜‹‘—•Ž› ’—„Ž‹•Š‡† Š—ƒ ‘†‡Ž ȋ ‹‰—”‡ ͵ȌǤ ‹‡ –Š‡  ȋ ‹‰—”‡ ʹȌ ƒŽ•‘–Š‡†‡ˆ‹ ‹‡–‘†‡Ž••Š‘™‡†ƒ†‡ Ž‹‡‘ˆ‘ ™‹–Š‹ ”‡ƒ•‹‰’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘ȋ ‹‰—”‡͵ǦȌǤ –Š‡‘†‡Ž–Š‡†‡ Ž‹‡‘ˆ‘ ™ƒ•Ž‡••–Šƒ‹ –Š‡ǤŠ‹••Š‘—Ž†„‡‡š’‡ –‡†ǡ•‹ ‡‹•‘‡‘ˆ–Š‡ˆ‹”•–‡œ›‡•‹–Š‡’ƒ–Š™ƒ›ƒ† ƒ†‡ˆ‹ ‹‡ ›’”‡˜‡–•ƒ ——Žƒ–‹‘‘ˆ‘‡•–‡”•†‘™•–”‡ƒǤ ‘”†‹‰Ž›ǡ‹–Š‡ ‘†‡Ž‘Ž›ͳ͸Ǧ‘‡•–‡”•ƒ ——Žƒ–‡†ȋ ‹‰—”‡͵ȌǤ  ‘–”ƒ•–ǡ‹–Š‡‘†‡Ž –Š‡†‡ Ž‹‡‘ˆ‘ ™ƒ•Žƒ”‰‡”–Šƒ‹–Š‡‘†‡Žƒ†‹–™ƒ•‡˜‡Žƒ”‰‡”‹–Š‡ ‘†‡ŽǤ –Š‡•‡ ƒ•‡•ǡ–Š‡†‡ˆ‡ –‹•ˆ—”–Š‡”†‘™•–”‡ƒǡ•— Š–Šƒ–•—„•–”ƒ–‡‡–‡”•‘”ƒŽŽ› ƒ† –Š‡ •–ƒ”–• ƒ ——Žƒ–‹‰Ǥ  –Š‡   ‘†‡Ž ‘•–Ž› ͺ ƒ† ͳͲ ‘ ‡•–‡”• ƒ ——Žƒ–‡† ȋ ‹‰—”‡ ͵Ȍǡ ™Š‹ Š ‹• ‘•‹•–‡– ™‹–Š –Š‡ ƒ ——Žƒ–‹‘ ‘ˆ ͺ ƒ† ͳͲ ƒ ›ŽǦ ƒ”‹–‹‡• ‹ „Ž‘‘† •ƒ’Ž‡• ˆ”‘  ’ƒ–‹‡–• ȏͶʹȂͶ͸ȐǤ  –Š‡   ‘†‡Žǡ ƒ ——Žƒ–‹‘‘ˆͶǦ‘‡•–‡”•™ƒ•‘•–’”‘‹‡–ȋ ‹‰—”‡͵Ȍǡ‹Ž‹‡™‹–Šƒ†‡ˆ‡ –‹–Š‡ ‡–ƒ„‘Ž‹•‘ˆ•Š‘”–Ǧ Šƒ‹ƒ ›ŽǦ‘‡•–‡”•ƒ†‘„•‡”˜ƒ–‹‘•‹’ƒ–‹‡–•ǤŠ‡Ž‡ƒˆŽ—š‡•ǡi.e. –Š‡•—‘ˆ–Š‡ˆŽ—š‡•‘ˆ‹ ‘’Ž‡–‡Ž›‘š‹†‹œ‡†’ƒŽ‹–‘›ŽǦ‘ǡ™ƒ•˜‡”›•ƒŽŽ‹–Š‡ ƒ†‘†‡Ž•ǡƒ•‹–™ƒ•‹–Š‡Š‡ƒŽ–Š›‘†‡Žȋ ˆǤ ‹‰—”‡ʹǦƒ† ‹‰—”‡͵ Ǧ ȌǤ  ‘–”ƒ•–ǡ‹–Š‡‘†‡Ž–Š‡Ž‡ƒ™ƒ•“—‹–‡ƒŽƒ”‰‡ˆ”ƒ –‹‘‘ˆ–Š‡–‘–ƒŽ’ƒŽ‹–‘›ŽǦ ‘ ‘•—’–‹‘ˆŽ—šǤŠ‡Žƒ”‰‡”ˆ”ƒ –‹‘‘ˆ–Š‡–‘–ƒŽŽ‡ƒ™ƒ•ǡŠ‘™‡˜‡”ǡ”‡ › Ž‡†–Š”‘—‰Š ȋ’‹Ž‹‡‹ ‹‰—”‡͵ȌǤ  Impact of individual variations on the mFAO flux †‹˜‹†—ƒŽ ˜ƒ”‹ƒ–‹‘• ‹ –Š‡ ‡–ƒ„‘Ž‹  ’ƒ–Š™ƒ›• –Šƒ– •—””‘—† –Š‡   ƒ› ƒˆˆ‡ – –Š‡  ˆŽ—šƒ†–Š‡”‡„›†‹•‡ƒ•‡’”‡•‡–ƒ–‹‘ǤŠ‡”‡ˆ‘”‡ǡƒ—„‡”‘ˆ˜ƒ”‹ƒ–‹‘•™‡”‡–‡•–‡† ‹–Š‡‘†‡Žǡ™‹–Šƒˆ‘ —•‘”‡†— –‹‘‘ˆ–Š‡ƒ˜ƒ‹Žƒ„‹Ž‹–›‘ˆ‘ ‘”ΪǤ‡•‹—Žƒ–‡† –Š‡ ‹’ƒ – ‘ˆ Ž‘™‡” –‘–ƒŽ ‘ ‘ ‡–”ƒ–‹‘ ‹ –Š‡ ƒ–”‹š ȋ”‡ˆŽ‡ –‹‰ ƒ Ž‡•• ƒ –‹˜‡ ‘ „‹‘•›–Š‡•‹• ‘” Ž‘™‡” ‘ ‡–”ƒ–‹‘ ‘ˆ ‹–• ’”‡ —”•‘” ˜‹–ƒ‹ ͷȌǤ ‡ ƒŽ•‘ •‹—Žƒ–‡† –Š‡ ‹’ƒ – ‘ˆ Ž‘™‡” ΪǦ  ”ƒ–‹‘ ȋ”‡ˆŽ‡ –‹‰ ƒ Ž‡•• ƒ –‹˜‡  Ȍǡ –Š‡ ‡ˆˆ‡ – ‘ˆ ƒ Ž‘™‡” ›–‘•‘Ž‹  ƒ† ‹–‘ Š‘†”‹ƒŽ Ǧ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘ ȋ”‡ˆŽ‡ –‹‰ ’‘–‡–‹ƒŽŽ› Ž‘™‡” Ǧ ƒ”‹–‹‡Ž‡˜‡Ž•‹’ƒ–‹‡–•Ȍƒ†ƒŽƒ ‘ˆƒ ›ŽǦ ƒ”‹–‹‡‡š’‘”–‹–‘–Š‡„Ž‘‘†ǡƒ•™ƒ• –Š‡ ƒ•‡ ‹ –Š‡ ’—„Ž‹•Š‡† ‹•‘Žƒ–‡†   ‘†‡Ž• ȏʹʹǡͶ͹ȐǤ —”–Š‡”‘”‡ǡ ™‡ •‹—Žƒ–‡† –Š‡ ‡ˆˆ‡ –‘ˆ–Š‡ƒ„•‡ ‡‘ˆͳǡ–Š‡ˆ‹”•–‡œ›‡‘ˆ–Š‡‡–‘‰‡‡•‹•’ƒ–Š™ƒ›„›•‡––‹‰‹–ǯ•

ƒš–‘œ‡”‘ȋͳαͲȌƒ†–Š‡‹’ƒ –‘ˆ‘ †‘™‘ˆ–Š‡–Š‹‘‡•–‡”ƒ•‡•„›”‡†— ‹‰

‹–• ƒš –‘ ͳΨ ‘ˆ ‹–• ”‡ˆ‡”‡ ‡ ˜ƒŽ—‡Ǥ ‹ƒŽŽ›ǡ ™‡ •‹—Žƒ–‡† –Š‡ ‡ˆˆ‡ – ‘ˆ ƒ ‘ †‘™ ‘ˆ

„›Ž‘™‡”‹‰‹–•ƒš–‘ͳΨ‘ˆ‹–•”‡ˆ‡”‡ ‡˜ƒŽ—‡ȋ ‹‰—”‡ͶȌǤ‘•–‘ˆ–Š‡˜ƒ”‹ƒ–‹‘• –Šƒ–™‡‹–”‘†— ‡†ǡŠƒ†‘Ž›ƒ‹‘”‹’ƒ –‘–Š‡‡– ’”‘†— –‹‘ˆŽ—šǡƒ• ƒ„‡ •‡‡ ˆ”‘ –Š‡ ”‡Žƒ–‹˜‡Ž› •ƒŽŽ †‹ˆˆ‡”‡ ‡ „‡–™‡‡ –Š‡ •‘Ž‹† Ž‹‡• ƒ† –Š‡ ‘””‡•’‘†‹‰ †ƒ•Š‡†Ž‹‡•‹ ‹‰—”‡ͶǦ ǤŠ‹•ƒ›”‡ˆŽ‡ ––Šƒ––Š‡Žƒ”‰‡—„‡”‘ˆ‘†‡Ž‡š–‡•‹‘•Šƒ• ƒ†‡–Š‡‘†‡Ž”‡Žƒ–‹˜‡Ž›”‘„—•–ƒ‰ƒ‹•––Š‡•‡˜ƒ”‹ƒ–‹‘•Ǥ ‡†‹†‘––‡•–™Š‡–Š‡”ƒŠ—ƒ‘†‡Ž™‹–Š‘—––Š‡•‡‡š–‡•‹‘•™‘—Ž†„‡‘”‡•‡•‹–‹˜‡ –‘ ”‡†— –‹‘ ‘ˆ –Š‡ ‘‡œ›‡•Ǥ ‡ ‘„•‡”˜‡† ’”‡˜‹‘—•Ž›ǡ Š‘™‡˜‡”ǡ –Šƒ– –Š‡ ”ƒ– ‘†‡Ž ™‹–Š‘—––Š‡‡š–‡•‹‘•ǡ™ƒ•˜‡”›•‡•‹–‹˜‡–‘ Šƒ‰‡•‹–Š‡ΪȀ ”ƒ–‹‘ȏʹ͸ǡͶͺȐǤŠƒ– ͳŠƒ†Ž‹––Ž‡‡ˆˆ‡ –ȋ ‹‰—”‡ͶȌƒ›„‡†—‡–‘–Š‡ˆƒ ––Šƒ–‹– ƒ–ƒŽ›œ‡•ƒ”‡ƒ –‹‘–Šƒ–‹• ƒŽ•‘ ƒ–ƒŽ›œ‡†„›Ǥ†‡ˆ‹ ‹‡ ›Šƒ†ƒŽƒ”‰‡”‡ˆˆ‡ ––Šƒͳ†‡ˆ‹ ‹‡ ›ȋ ˆǤ ‹‰—”‡Ͷ ƒ† ǡ”‡•’‡ –‹˜‡Ž›ȌǤ 

ͳ͸͸ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

 A B C D ) 14 -1 12

10

.gProtein -1 8 6 4 (μmol.min 2 0 nadhsink J 050100050100050100050100 [Palmitoyl-CoA] [Palmitoyl-CoA] [Palmitoyl-CoA] [Palmitoyl-CoA] CYT ;ʅDͿ CYT ;ʅDͿ CYT ;ʅDͿ CYT ;ʅDͿ  A B C ) 14 -1 12 WT 5 10 .gProtein

-1 8 SCAD KO

6 MCAD KO 4

(μmol.min VLCAD KO 2 0 nadhsink J 050100050100050100

[Palmitoyl-CoA]CYT ;ʅDͿ [Palmitoyl-CoA]CYT ;ʅDͿ [Palmitoyl-CoA]CYT ;ʅDͿ   Figure 4. Net NADH production fluxȋ–‡”‡†ƒ†Š•‹Ȍ‘ˆ ƒ†•‹‰Ž‡Ǧ‡œ›‡ ˜‡”•‹‘•‘ˆ–Š‡ ‘†‡Ž•—„Œ‡ –‡†–‘ƒ††‹–‹‘ƒŽ‹–‡”˜‡–‹‘•ǤŠ‡‹–‡”˜‡–‹‘•ƒ”‡Ž‘™‡”–‘–ƒŽ‘ ‘ ‡–”ƒ–‹‘‹–Š‡ ƒ–”‹šȋʹͷΨ‘ˆ–Š‡”‡ˆ‡”‡ ‡•–ƒ–‡ȌȋAȌǡŽ‘™‡”ΪȀ ”ƒ–‹‘ȋʹͷΨ‘ˆ–Š‡”‡ˆ‡”‡ ‡•–ƒ–‡ȌȋBȌǡŽ‘™‡” ›–‘•‘Ž‹ ƒ†‹–‘ Š‘†”‹ƒŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘ȋʹͷΨ‘ˆ–Š‡”‡ˆ‡”‡ ‡•–ƒ–‡ȌȋCȌǡ‘ƒ ›ŽǦ ƒ”‹–‹‡ ‡š’‘”–‹–‘–Š‡„Ž‘‘†ȋDȌǡ‘ͳȋEȌǡŽ‘™‡”ȋͳΨ‘ˆ–Š‡”‡ˆ‡”‡ ‡•–ƒ–‡ȌȋFȌǡŽ‘™‡”ȋʹͷΨ‘ˆ –Š‡”‡ˆ‡”‡ ‡•–ƒ–‡ȌȋGȌǤƒ•Š‡†Ž‹‡•”‡’”‡•‡––Š‡”‡ˆ‡”‡ ‡•–ƒ–‡‘ˆ–Š‡‘”–Š‡‹†‹ ƒ–‡† ȋ–Š‡•ƒ‡†ƒ–ƒƒ•‹ ‹‰—”‡͵Ȍǡ™Š‹Ž‡•‘Ž‹†Ž‹‡•”‡’”‡•‡––Š‡‹†‹ ƒ–‡†˜ƒ”‹ƒ–‹‘‹–Š‡•‡‘†‡Ž•Ǥ  ǡŠ‘™‡˜‡”ǡ ‘˜‡”–••—„•–”ƒ–‡•‘ˆƒ”ƒ‰‡‘ˆ ƒ”„‘Ǧ Šƒ‹Ž‡‰–Š•˜ƒ”›‹‰ˆ”‘Ͷ–‹ŽŽ ͳ͸Ǥ  —„•–”ƒ–‡• †‘™ –‘ ͺ ƒ ƒŽ•‘ „‡ ‘˜‡”–‡† „›  ȋ ‹‰—”‡ ͳȌǡ „—–  ‹• ƒ ‡••‡–‹ƒŽ‡œ›‡ˆ‘” ‘˜‡”•‹‘‘ˆ͸Ǧ‡–‘ƒ ›ŽǦ‘ǤˆƒŽŽ˜ƒ”‹ƒ–‹‘•–Šƒ–™‡”‡‹’‘•‡†ǡ–Š‡ ”‡†— –‹‘ ‘ˆ ‡ŽŽ—Žƒ” Ǧ ƒ”‹–‹‡ Ž‡˜‡Ž• Šƒ† –Š‡ •–”‘‰‡•– ‡ˆˆ‡ – ‘   ˆ— –‹‘ ȋ ‹‰—”‡ ͶȌǤ ƒ”‹–‹‡ ‹•”‡“—‹”‡† ˆ‘” –Š‡ —’–ƒ‡ ‘ˆ ƒ ›ŽǦ‘ ‡•–‡”• –Š”‘—‰Š –Š‡ ͳǦǦʹ •Š—––Ž‡ ȋ ‹‰—”‡ ͳȌ ƒ† ˆ‘” –Š‡ ‡š’‘”– ‘ˆ ƒ ›Ž ‘‹‡–‹‡• ‹ –Š‡ ˆ‘” ‘ˆ ƒ ›ŽǦ ƒ”‹–‹‡•Ǥ ’ƒ‹”‡– ‘ˆ –Š‡ ‡š’‘”– ‘ˆ ƒ ›ŽǦ ƒ”‹–‹‡• †‹† ‘– ƒˆˆ‡ – –Š‡   ˆŽ—š ȋ ‹‰—”‡ ͶȌǤ Š‡”‡ˆ‘”‡ǡ ƒ† „‡ ƒ—•‡ ͳ Šƒ† •—„•–ƒ–‹ƒŽ ‘–”‘Ž ‘˜‡” –Š‡ ˆŽ—šǡ ™‡ ‘ Ž—†‡ –Šƒ– –Š‡ ”‡†— –‹‘‘ˆ ‡ŽŽ—Žƒ” ƒ”‹–‹‡Ž‡˜‡Ž•ƒˆˆ‡ –•–Š‡ ˆŽ—š–Š”‘—‰Š‹–•”‘Ž‡‹–Š‡•Š—––Ž‡Ǥ  Impact of acyl-CoA recycling on the mFAO flux Š‡–Š‹‘‡•–‡”ƒ•‡•ȋ•ȌŠƒ˜‡„‡‡”‡’‘”–‡†–‘ ‘–”‹„—–‡–‘–Š‡ ˆŽ—šȏʹ͹Ȃ͵ͲȐǤŠ‡ •–Šƒ–ƒ”‡’”‘†— ‡†‹–Š‹•”‡ƒ –‹‘ ƒ„‡‡š’‘”–‡†‘””‡ƒ –‹˜ƒ–‡†„›ǤŠ‡”‡ˆ‘”‡ǡ‹– ™ƒ•‹˜‡•–‹‰ƒ–‡†™Š‡–Š‡”–Š‡ ”‡ƒ –‹˜ƒ–‹‘ƒ†ƒ ›ŽǦ‘”‡ › Ž‹‰Šƒ•ƒ‹’ƒ –‘–Š‡

 ˆŽ—šǤ ‘”–Š‹•ǡ™‡Ž‘™‡”‡†–Š‡ƒš‘ˆ–‘ͳΨ‘ˆ‹–•”‡ˆ‡”‡ ‡˜ƒŽ—‡Ǥ ˆ™‡‘Ž›Ž‘™‡” –Š‡  ƒ’ƒ ‹–›ǡ  ƒ •–‹ŽŽ „‡ ’”‘†— ‡† „›  ƒ† ‡š’‘”–‡†ǡ „—– ‘– ”‡ƒ –‹˜ƒ–‡†Ǥ ‘™‡”‹‰Š‘™‡˜‡”ǡ†‹†‘–ƒˆˆ‡ ––Š‡ˆŽ—š‡••—„•–ƒ–‹ƒŽŽ›‹–Š‡‘”ƒ›‘ˆ–Š‡ •ȋ ‹‰—”‡ͷȌǤ ƒ††‹–‹‘ǡ‡–ƒ„‘Ž‹ ‘–”‘ŽƒŽ›•‹••Š‘™‡†–Šƒ–‡š‡”–‡†‘ˆŽ—š ‘–”‘Ž ‹ –Š‡ ”‡ˆ‡”‡ ‡ ‘†‡Ž ƒ† –Š‹• ™ƒ• ‘– Šƒ‰‡† ™Š‡ ‹– ™ƒ• ‹’ƒ‹”‡† ȋ ˆǤ —’’Ž‡‡–ƒ”›–ƒ„Ž‡•ͳƒ†ʹȌǤŠ‡‘ †‘™‘ˆ‡Šƒ ‡†–Š‡ƒ ——Žƒ–‹‘‘ˆ ‘‡•–‡”•ƒ†–Š‡ ‘””‡•’‘†‹‰”‡†— –‹‘‘ˆˆ”‡‡‘ ‹–Š‡ƒ† ‘†‡Ž•ȋ ‹‰—”‡ͷǦȌǤ

ͳ͸͹ Chapter 5

A

) 14 -1 12

10 WT

.gProtein

-1 8 SCAD KO

6 MCAD KO 4

(μmol.min VLCAD KO 2

0 nadhsink  J 050100  WT VLCAD KO MCAD KO SCAD KO B C D E 5000 Ϳ 4000 ʅD ; free CoA

MAT 3000 ]

2000 Intermediate

Metabolite 1000 CoA esters [

0 050100050100050100050100 [Palmitoyl-CoA] [Palmitoyl-CoA] [Palmitoyl-CoA] [Palmitoyl-CoA] CYT ;ʅDͿ CYT ;ʅDͿ CYT ;ʅDͿ CYT ;ʅDͿ  ‹‰—”‡ ͷǤ ‹—Žƒ–‹‘ ”‡•—Ž–• ‘ˆ –Š‡ Ǧ‘ †‘™ –‘ ͳΨ ‘ˆ ‹–• ”‡ˆ‡”‡ ‡ ˜ƒŽ—‡Ǥ ‡’‘”–‡† ƒ”‡ –Š‡ ‡–   ’”‘†— –‹‘ ˆŽ—š ‹ –Š‡   ȋȌǡ ‹–‘ Š‘†”‹ƒŽ ˆ”‡‡ ‘ ƒ† ‘ ‡•–‡” ‘ ‡–”ƒ–‹‘• ȋǦȌ  ’ƒ‡Ž–Š‡†ƒ•Š‡†Ž‹‡•”‡’”‡•‡––Š‡”‡ˆ‡”‡ ‡•–ƒ–‡‘ˆ–Š‡‘”–Š‡‹†‹ ƒ–‡†ȋ–Š‡•ƒ‡†ƒ–ƒ ƒ•‹ ‹‰—”‡͵Ȍǡ™Š‹Ž‡•‘Ž‹†Ž‹‡•”‡’”‡•‡––Š‡‘ †‘™Ǥ  ’’ƒ”‡–Ž›–Š‡–Š‹‘‡•–‡”ƒ•‡Ȃ › Ž‡”‡Ž‹‡˜‡†–Š‡•‡“—‡•–”ƒ–‹‘‘ˆ‘ ǤŠ‡ˆŽ—š‡•‘ˆ –Š‡Ž‡ƒƒ‰‡”‡ƒ –‹‘”ƒ–‡•”‡ƒ‹‡†Ž‘™‹–Š‹•‘†‡Žȋ—’’Ž‡‡–ƒ”› ‹‰—”‡͵ȌǤ‘–Š‡” ™ƒ›–‘‹˜‡•–‹‰ƒ–‡™Š‡–Š‡”–Š‡ ”‡ƒ –‹˜ƒ–‹‘ƒ†ƒ ›ŽǦ‘”‡ › Ž‹‰Šƒ•ƒ‹’ƒ –‘–Š‡  ˆŽ—š‹•„›‹’ƒ‹”‹‰–Š‡ ‘˜‡”•‹‘‘ˆ–Š‡”‡ƒ –‹˜ƒ–‡† •ȋ ›–‘•‘Ž‹ ͳͶǦͶƒ ›ŽǦ‘ ‡•–‡”•Ȍ–‘ƒ ›ŽǦ ƒ”‹–‹‡•„›ͳǤŠ‹•™ƒ•†‘‡„›ƒ‹‰ͳ‘”‡ͳ͸Ǧ•’‡ ‹ˆ‹ ǡi.e. „›

Ž‘™‡”‹‰ –Š‡ ƒš ˜ƒŽ—‡• ‘ˆ ͳ ˆ‘” –Š‡ ͳͶǦͶ ”‡ƒ –‹‘• ƒ† „› Ž‘™‡”‹‰ ‹–• ƒˆˆ‹‹–› –‘™ƒ”†•ͳͶǦͶƒ ›ŽǦ‘‡•–‡”•ȋ•‡‡—’’Ž‡‡–ƒ”›–‡š–ͳˆ‘”–Š‡ ‘””‡•’‘†‹‰‹‡–‹ 

’ƒ”ƒ‡–‡”•ȌǤ Š‡ Žƒ––‡” ™ƒ• ƒ Š‹‡˜‡† „› ‹ ”‡ƒ•‹‰ –Š‡ ‘””‡•’‘†‹‰  ˜ƒŽ—‡•Ǥ Š‹• ‘†‹ˆ‹ ƒ–‹‘Šƒ†Ž‹––Ž‡‡ˆˆ‡ –‘–Š‡ ˆŽ—š‹–Š‡ǡƒ†ȋ ‹‰—”‡ ͸ȌǤŠ‡ ˆŽ—š‘ˆ–Š‡™ƒ••—„•–ƒ–‹ƒŽŽ›”‡†— ‡†Š‘™‡˜‡”„›–Š‡‹’ƒ‹”‡– ‘ˆƒ ›ŽǦ‘”‡ › Ž‹‰–Š”‘—‰Šͳȋ ‹‰—”‡͸ȌǤ  ‘–”ƒ•––‘–Š‡”‡ˆ‡”‡ ‡‘†‡Žǡ–Š‡ͳ͸Ǧ •’‡ ‹ˆ‹  ͳ ƒ‹–ƒ‹‡† ͳͲͲΨ ‘–”‘Ž ‘˜‡” –Š‡   ˆŽ—š ‘˜‡” –Š‡ ™Š‘Ž‡ ”ƒ‰‡ ‘ˆ ›–‘•‘Ž‹  ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘• ȋ—’’Ž‡‡–ƒ”› ƒ„Ž‡ ͵ȌǤ —”’”‹•‹‰Ž›ǡ –Š‡ ƒ ——Žƒ–‹‘‘ˆ‹–‘ Š‘†”‹ƒŽ‘‡•–‡”•™ƒ• ‘’Ž‡–‡Ž›ƒ„‘Ž‹•Š‡†‹–Š‡ƒ†  ‘†‡Ž ™‹–Š ƒ ͳ͸Ǧ•’‡ ‹ˆ‹  ͳ ȋ ‹‰—”‡ ͸ǦȌǤ ‘–ƒ„Ž›ǡ –Š‡ ›–‘•‘Ž‹  ‘ ‡•–‡” ‘ ‡–”ƒ–‹‘• ‘ˆ ƒŽŽ –Š‡ ‘†‡Ž• ƒ ——Žƒ–‡† –‘ ˜‡”› Š‹‰Š Ž‡˜‡Ž•ǡ •‹ ‡ ‘ ‘‹‡–› ‘•‡”˜ƒ–‹‘ˆ‘” ›–‘•‘Ž‹ ‘™ƒ•‹’‘•‡†ȋ—’’Ž‡‡–ƒ”›–ƒ„Ž‡ͶȌǤŠ‡ˆŽ—š‡•‘ˆ–Š‡ Ž‡ƒƒ‰‡”‡ƒ –‹‘”ƒ–‡•”‡ƒ‹‡†Ž‘™‹–Š‹•‘†‡Žȋ—’’Ž‡‡–ƒ”› ‹‰—”‡ͶȌǤŠ‡ ‘†‡Ž™‹–Šƒͳ͸Ǧ•’‡ ‹ˆ‹ ͳ™ƒ•— Š‘”‡•‡•‹–‹˜‡–‘ƒ††‹–‹‘ƒŽ–”‹‰‰‡”•ǣ™Š‡ ‡‹–Š‡”–Š‡–‘–ƒŽ‹–‘ Š‘†”‹ƒŽ‘‘”–Š‡–‘–ƒŽǦ ƒ”‹–‹‡Ž‡˜‡Ž™ƒ•”‡†— ‡†„›ͳͲΨǡ–Š‹•Ž‡† –‘ƒ•–”‘‰”‡†— –‹‘‘ˆ–Š‡ ˆŽ—šǡ†‘™–‘–Š‡Ž‡˜‡Ž‘ˆ–Š‡‘†‡Žȋ ‹‰—”‡ ͸ ǡ ȌǤ ‘•‹†‡”‹‰ –Š‡ •‡˜‡”‡Ž› ‘ˆ ǡ ‹• ‹– Ž‹‡Ž› –Šƒ– –Š‹• ‹• ƒ ˆŽ—š ”‡†— –‹‘ –Šƒ– ‘””‡•’‘†•–‘ƒ Ž‹‹ ƒŽ’Š‡‘–›’‡Ǥ 

ͳ͸ͺ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

A

) 14 -1 12

10 WT

.gProtein

-1 8 SCAD KO

6 MCAD KO 4

(μmol.min VLCAD KO 2 0 nadhsink  J 050100  WT VLCAD KO MCAD KO SCAD KO  B C D E 5000 4000

;ʅDͿ free CoA 5 MAT 3000

2000

Intermediate 1000 CoA esters [Metabolite] 0 050100050100050100050100 [Palmitoyl-CoA] [Palmitoyl-CoA] [Palmitoyl-CoA] [Palmitoyl-CoA] CYT ;ʅDͿ CYT ;ʅDͿ CYT ;ʅDͿ CYT ;ʅDͿ   F G

) 14 -1 12 10 WT

.gProtein

-1 8 SCAD KO

6 MCAD KO 4

(μmol.min VLCAD KO 2 0 nadhsink J 050100050100

[Palmitoyl-CoA]CYT ;ʅDͿ [Palmitoyl-CoA]CYT ;ʅDͿ   Figure 6. Simulation results of the extended model with a CPT1 with reduced activity and affinity towards C14-C4 acyl-CoA substrates. Š‡’ƒ”ƒ‡–‡”˜ƒŽ—‡•–Šƒ–™‡”‡ Šƒ‰‡†–‘‘„–ƒ‹–Š‹•ͳǡƒ”‡ ‰‹˜‡‹–Š‡•‡ ‘†–ƒ„Ž‡‹•—’’Ž‡‡–ƒ”›–‡š–ͳǤ‡’‘”–‡†ƒ”‡–Š‡–‘–ƒŽ ’”‘†— –‹‘ˆŽ—š‹–Š‡   ȋAȌ ƒ† –Š‡ ‹–‘ Š‘†”‹ƒŽ ‘  ƒ† ‘ ‡•–‡” ‘ ‡–”ƒ–‹‘• ȋͳ͸ǦͶ ƒ ›ŽǦ‘ ‡•–‡”•Ȍ ȋB-EȌǤ ƒ‡Ž• F-G †‹•’Žƒ› –Š‡ ‡–   ’”‘†— –‹‘ ˆŽ—š ȋ–‡”‡† ƒ†Š•‹Ȍ ‘ˆ  ƒ† •‹‰Ž‡Ǧ‡œ›‡  ˜‡”•‹‘• ‘ˆ –Š‡ ‘†‡Ž •—„Œ‡ –‡† –‘ ƒ††‹–‹‘ƒŽ ‹–‡”˜‡–‹‘•Ǥ Š‡ ‹–‡”˜‡–‹‘• ƒ”‡ Ž‘™‡” –‘–ƒŽ ‘ ‘ ‡–”ƒ–‹‘ ‹ –Š‡ ƒ–”‹š ȋͻͲΨ ‘ˆ –Š‡ ”‡ˆ‡”‡ ‡ •–ƒ–‡Ȍ ȋFȌ ƒ† Ž‘™‡” ›–‘•‘Ž‹  ƒ† ‹–‘ Š‘†”‹ƒŽ Ǧ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘ȋͻͲΨ‘ˆ–Š‡”‡ˆ‡”‡ ‡•–ƒ–‡ȌȋGȌǤƒ•Š‡†Ž‹‡•”‡’”‡•‡––Š‡”‡ˆ‡”‡ ‡•–ƒ–‡‘ˆ–Š‡ ‘”–Š‡‹†‹ ƒ–‡†ȋ–Š‡•ƒ‡†ƒ–ƒƒ•‹ Figure 3Ȍǡ™Š‹Ž‡•‘Ž‹†Ž‹‡•”‡’”‡•‡––Š‡‹†‹ ƒ–‡† ˜ƒ”‹ƒ–‹‘‹–Š‡•‡‘†‡Ž•Ǥ  Discussion Š‡   ’ƒ–Š™ƒ› ‹• ‹–”‹ ƒ–‡Ž› ‘‡ –‡† –‘ •‡˜‡”ƒŽ ’ƒ–Š™ƒ›• ‘ˆ ‡‡”‰› ‡–ƒ„‘Ž‹•Ǥ Š‡•‡ ’ƒ–Š™ƒ›• ƒ› ’Žƒ› ƒ ’ƒ”–‹ —Žƒ”Ž› ”— ‹ƒŽ ”‘Ž‡ ‹ –Š‡ ˆ— –‹‘ ‘ˆ   ‹ –Š‡ Ž‹˜‡” †—”‹‰ˆƒ•–‹‰‘” ‘†‹–‹‘•‘ˆŠ‹‰Š‡‡”‰›†‡ƒ†Ǥ –Š‹••–—†›ǡ™‡‰‡‡”ƒ–‡†ˆ‘”–Š‡ˆ‹”•– –‹‡ǡ ƒ †›ƒ‹  ‘’—–ƒ–‹‘ƒŽ ‘†‡Ž ‘ˆ   ‹ Š—ƒ Ž‹˜‡” ™‹–Š ‡š–‡•‹‘• ‹–‘ –Š‡ ƒŒ‘” ’ƒ–Š™ƒ›• –Šƒ– ƒˆˆ‡ – –Š‡ ƒ ›ŽǦ‘ ƒ† ‘  ‘ ‡–”ƒ–‹‘•Ǥ — Š ƒ ‘†‡Ž ‹• ƒ ‹’‘”–ƒ––‘‘Ž–‘‰‡–ƒ‘”‡”‡ƒŽ‹•–‹ ‹’”‡••‹‘‘ˆ–Š‡‡ Šƒ‹••Ž‡ƒ†‹‰–‘”‘„—•–‡•• ƒ†˜—Ž‡”ƒ„‹Ž‹–›‹ Ǥ ƒ”–‹ —Žƒ”Ž›ǡ‹ǡ†‹•‡ƒ•‡•‡˜‡”‹–› ƒ‘–„‡’”‡†‹ –‡† ‡˜‡ ™‹–Š‹ –Š‡ •ƒ‡ —–ƒ–‹‘ ȏ͹ȂͳͷȐǤ —” ƒ‹ ™ƒ• –‘ ‹˜‡•–‹‰ƒ–‡ ™Š‡–Š‡” –Š‡ †‹ˆˆ‡”‡– †‹•‡ƒ•‡’”‡•‡–ƒ–‹‘•‘ˆ’ƒ–‹‡–• ‘—Ž† –Š‡‘”‡–‹ ƒŽŽ›„‡ ƒ—•‡†„›‹†‹˜‹†—ƒŽ˜ƒ”‹ƒ–‹‘•‹ –Š‡ •—””‘—†‹‰ ‡–™‘”Ǥ  ‘’—–ƒ–‹‘ƒŽ ‘†‡Ž †‘‡• ‘– ’”‘˜‡ –Šƒ– •— Š ˜ƒ”‹ƒ–‹‘•

ͳ͸ͻ Chapter 5

ƒ –—ƒŽŽ›’Žƒ›ƒ”‘Ž‡‹’ƒ–‹‡–•ǡ„—––Š‡›‰‹˜‡‹•‹‰Š–‹™Š‹ Š˜ƒ”‹ƒ–‹‘•ǡ‹ˆ–Š‡›‘ —””‡†ǡ ‘—Ž†Šƒ˜‡–Š‡•–”‘‰‡•–‹’ƒ –in vivoǤ ‘”–Š‹•™‡ Šƒ”ƒ –‡”‹œ‡†–Š‡‘†‡Žƒ†‹–”‘†— ‡† ’‡”–—”„ƒ–‹‘•–Šƒ–™‡”‡‡š’‡ –‡†–‘„‡”‡Ž‡˜ƒ–‹Ǥ ‡–ƒ„‘Ž‹  ‘–”‘ŽƒƒŽ›•‹••Š‘™‡†–Šƒ–ͳ•Šƒ”‡† ‘–”‘Ž™‹–Š †‡Š›†”‘‰‡ƒ•‡‹–Š‡ ‘†‡ŽǤ ͳ Ž‘•– ˆŽ—š ‘–”‘Ž ƒ– Š‹‰Š ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘•Ǥ Š‹•‹•†‹ˆˆ‡”‡– ˆ”‘ –Š‡’”‡˜‹‘—•Ž›’—„Ž‹•Š‡†”ƒ–‘†‡Žȋ™‹–Š‘—–‡š–‡•‹‘•Ȍ‹™Š‹ Šͳ‰‘–ƒ‡‰ƒ–‹˜‡ˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–ƒ–Š‹‰Š•—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘•ȏͶͺȐǤŠ‡”‡Žƒ–‹˜‡ˆŽ—š‡•‘ˆ–Š‡ ‘†‡Ž•™‡”‡ ‹ ƒ‰”‡‡‡– ™‹–Š ‰‡‡”ƒŽ †‹•‡ƒ•‡ •‡˜‡”‹–› ‘ˆ  ‹ Š—ƒ• ȋ”‡ˆ‡”‡ ‡  ƒ†  ‘†‡Ž•ȌǤ  ƒ††‹–‹‘ǡ –Š‡   ‘†‡Ž• •Š‘™‡† ƒ ›ŽǦ‘ ’”‘ˆ‹Ž‡• ‹ –Š‡ ‹–‘ Š‘†”‹ƒŽ ƒ–”‹š –Šƒ– ƒ”‡ •‹‹Žƒ” –‘ –Š‡ ‘””‡•’‘†‹‰ ƒ ›ŽǦ ƒ”‹–‹‡ ’”‘ˆ‹Ž‡• ‹ ’ƒ–‹‡–•Ǥƒ•‡†‘‡š’‡ –‡†‹’‘”–ƒ ‡‘ˆ•‡˜‡”ƒŽ‡œ›‡•ƒ†‡–ƒ„‘Ž‹–‡•‹ǡ™‡ †‡–‡”‹‡†–Š‡‡ˆˆ‡ –‘ˆ Šƒ‰‹‰•‡Ž‡ –”‡Žƒ–‡†’ƒ”ƒ‡–‡”•‘–Š‡ˆŽ—š‘ˆ–Š‡ƒ†  ‘†‡Ž•Ǥ ‘•– ‘ˆ –Š‡•‡ ’ƒ”ƒ‡–‡”• Šƒ† ƒ •ƒŽŽ ‡ˆˆ‡ – ‘ –Š‡ ˆŽ—š‡• ‹ –Š‡   ‘†‡Ž•Ǥ ‘™‡”‹‰ Ǧ ƒ”‹–‹‡ǡ Š‘™‡˜‡”ǡ Šƒ† ƒ ‹†‹• ”‹‹ƒ–‡Ž› •–”‘‰ ‡ˆˆ‡ – ‘ ƒŽŽ –Š‡ ‘†‡Ž•Ǥ ‡‡š–‹˜‡•–‹‰ƒ–‡†–Š‡‹’‘”–ƒ ‡‘ˆ ”‡ › Ž‹‰‹–Š‹•‘†‡Žǡ–Š”‘—‰Š–Š‡ˆ—–‹Ž‡ › Ž‡ ‘ˆ ‹–‘ Š‘†”‹ƒŽ ƒ ›ŽǦ‘ Š›†”‘Ž›•‹•ǡ ‡š’‘”– ‹–‘ –Š‡ ›–‘•‘Žǡ ”‡ƒ –‹˜ƒ–‹‘ „›  ƒ† —’–ƒ‡„ƒ ‹–‘–Š‡‹–‘ Š‘†”‹ƒǤ ‘™‡˜‡”ǡ‹’ƒ‹”‡†ƒ –‹˜‹–›†‹†‘–ƒˆˆ‡ ––Š‡  ˆŽ—š‹ƒ›‘ˆ–Š‡‘†‡Ž•Ǥ‘–‡–‹ƒŽŽ›ǡ–Š‡‹’ƒ –‘ˆ–Š‹• › Ž‡™ƒ•‹‹ƒŽ†—‡–‘ –Š‡”‡Žƒ–‹˜‡Ž›•ƒŽŽˆŽ—š–Š”‘—‰ŠǤ Ž—šƒƒŽ›•‹•‡š’‡”‹‡–•in vitro ‘—Ž†’”‘˜‹†‡‘”‡ ‹ˆ‘”ƒ–‹‘‘–Š‡”ƒ–‹‘‘ˆ˜‡”•—• ˆŽ—š‹ ‡ŽŽ•ǡ‹‘”†‡”–‘„‡ƒ„Ž‡–‘•‹—Žƒ–‡–Š‡ ”‘Ž‡‘ˆ–Š‡Ǧ‡†‹ƒ–‡†”‡ƒ –‹˜ƒ–‹‘•›•–‡‘”‡”‡ƒŽ‹•–‹ ƒŽŽ›Ǥ ‹ƒŽŽ›ǡ™‡‹˜‡•–‹‰ƒ–‡†–Š‡‡ˆˆ‡ –‘ˆ Šƒ‰‹‰ͳ‹‡–‹ •ǡ–‘•–—†›‹–•”‘Ž‡‹–Š‡”‡ › Ž‹‰ ‘ˆ ˜‹ƒǤ –‡”‡•–‹‰Ž›ǡ’ƒ”–‹ —Žƒ”Ž›–Š‡˜‡”•‹‘‘ˆ–Š‡‘†‡Ž™ƒ••‡•‹–‹˜‡ –‘ƒŽ–‡”ƒ–‹‘•‘ˆ–Š‡’ƒ”ƒ‡–‡”•‹˜‘Ž˜‡†‹”‡ › Ž‹‰‘ˆͶǦͳͶƒ ›ŽǦ‘‡•–‡”•Ǥ ƒ††‹–‹‘ǡ in silico ’‡”–—”„ƒ–‹‘•–Šƒ–Šƒ†‘’Š‡‘–›’‡‹–Š‡”‡ˆ‡”‡ ‡‘†‡Žǡ”‡•—Ž–‡†‹ƒ •–”‘‰†‡ ”‡ƒ•‡‘ˆ–Š‡ ˆŽ—š‹–Š‡‘†‡Ž™‹–Š–Š‡ƒŽ–‡”‡†ͳ‹‡–‹ •Ǥ‡ —””‡–Ž›Šƒ˜‡‘‡˜‹†‡ ‡ˆ‘”–Š‡‡š‹•–‡ ‡‘ˆ—–ƒ–‹‘•‹ͳ–Šƒ–Šƒ˜‡•— Š‡ˆˆ‡ –•‘ ͳ ‹‡–‹ •Ǥ ‹˜‡ –Šƒ– ͳ ‹• ‘™ –‘ Šƒ˜‡ ƒ Š‹‰Š ‘–”‘Ž ‘˜‡”   ˆŽ—š —†‡” •’‡ ‹ˆ‹  ‘†‹–‹‘• ȏͶͻǡͷͲȐ ƒ† –Šƒ– ‘—” ‘†‡Ž ’”‡†‹ –• •— Š ƒ •–”‘‰ ‡ˆˆ‡ – ‘ˆ ƒ ͳ ‹‡–‹ • ‹ –Š‡   ‘†‡Žǡ ‹– ƒ› „‡ ‹–‡”‡•–‹‰ –‘ ‹˜‡•–‹‰ƒ–‡ ™Š‡–Š‡” ͳ ‹‰Š– ’Žƒ›ƒ”‘Ž‡‹–Š‡˜—Ž‡”ƒ„‹Ž‹–›‘ˆ•‘‡•›’–‘ƒ–‹ ‹†‹˜‹†—ƒŽ•Ǥ‘‹˜‡•–‹‰ƒ–‡–Š‹•ǡ ‹–™‘—Ž†„‡”‡Ž‡˜ƒ––‘‹˜‡•–‹‰ƒ–‡—–ƒ–‹‘•‹–Š‡ͳ‰‡‡‹–Š‡‰‡‡”ƒŽ’‘’—Žƒ–‹‘ƒ† ‹ •›’–‘ƒ–‹  ˜‡”•—• ƒ•›’–‘ƒ–‹   †‡ˆ‹ ‹‡– Š‹Ž†”‡Ǥ ‘”‡‘˜‡”ǡ ‡š‘‡ •‡“—‡ ‹‰‘ˆ•›’–‘ƒ–‹ ˜‡”•—•ƒ•›’–‘ƒ–‹  Š‹Ž†”‡ƒ››‹‡Ž† ƒ†‹†ƒ–‡‰‡‡• –Šƒ– ƒ„‡–‡•–‡†ˆ— –‹‘ƒŽŽ›‹–Š‡ ‘’—–ƒ–‹‘ƒŽ‘†‡ŽǤŠ‹••Š‘—Ž†ƒŽŽ‘™—•–‘•‡Ž‡ ––Š‡ ‘•–’”‘‹•‹‰ ƒ†‹†ƒ–‡•ˆ‘”ˆ—”–Š‡”‹˜‹–”‘–‡•–‹‰Ǥ –Š‹•‹‹–‹ƒŽ•–—†›™‡Šƒ˜‡‘–›‡– ‘•‹†‡”‡† ‡”–ƒ‹ˆƒ –‘”•ǡ”‡ƒ –‹‘•‘”’ƒ–Š™ƒ›•Ǥ ‘” ‹•–ƒ ‡ǡ™‡†‹†‘–ƒŽŽ‘™ˆ‘” ‘•‡”˜‡†‘‹‡–‹‡•‘ˆ ƒ”‹–‹‡‹–Š‡ƒ–”‹šƒ† ›–‘•‘Žƒ† –‘–ƒŽ‘ȋˆ”‡‡ƒ†ƒ ›Žƒ–‡†‘Ȍ‹–Š‡ ›–‘•‘Žǡƒ•–Š‡•‡ ‘—Ž†Ž‹‹––Š‡ƒ ›ŽǦ ƒ”‹–‹‡ƒ† ‘ ‡•–‡” ‘ ‡–”ƒ–‹‘• ‹ –Š‡ ‘†‡ŽǤ ‡ †‘ ‘– –Š‹ –Šƒ– –Š‹• ™‹ŽŽ ‹˜ƒŽ‹†ƒ–‡ –Š‡ ‘ Ž—•‹‘• ‘ˆ –Š‹• •–—†›Ǥ ‡ Šƒ˜‡ ƒŽ•‘ ‘– ”‡’‘”–‡† ™Š‹ Š ’ƒ”ƒ‡–‡”• ƒ› Šƒ˜‡ ‘–”‹„—–‡† –‘ ƒ ƒ„•‡ ‡ ‹ ˆŽ—š †‡ Ž‹‡ ƒ• ™ƒ• ‘„•‡”˜‡† ‹ –Š‡  ”ƒ– ‘†‡Ž ‘ˆ –Š‡ ‹•‘Žƒ–‡† ǡ„—–‘–‹‹–Š‡˜‡”•‹‘‘ˆ–Š‡ˆ‹––‡†‘—•‡‘†‡Ž‘ˆ–Š‡‹•‘Žƒ–‡† Ǥ ‡‘–‡–Šƒ–‹ȏʹʹȐǡ™Š‡”‡ƒ‘—•‡ƒ†Š—ƒ‘†‡Ž‘ˆ‹•‘Žƒ–‡ ™ƒ•’”‡•‡–‡†–Š‹•

ͳ͹Ͳ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

†‹ˆˆ‡”‡ ‡™ƒ•ƒŽ•‘‘–‹˜‡•–‹‰ƒ–‡†ǡ’‘–‡–‹ƒŽŽ›–‘ˆ‘ —•‘ƒ–Š‡”‘Ž‡‘ˆ‡œ›‡’”‘‹• —‹–› ‘ –Š‡ ˆŽ—š ƒ† ‡–ƒ„‘Ž‹–‡ ’”‘ˆ‹Ž‡•Ǥ ‡”‡ ™‡ ˆ‘ —•‡† ‘ –Š‡ ‡ˆˆ‡ – ‘ˆ ‰‡‡–‹  ƒ† ‡˜‹”‘‡–ƒŽ‘†‹ˆ‹‡”•‘ ˆŽ—š‹ƒ†‘–‡–Šƒ–‹–™ƒ•‘–‡ ‡••ƒ”›ˆ‘”–Š‡ ‘†‡Ž•–‘•Š‘™ˆŽ—š†‡ Ž‹‡‹‘”†‡”–‘ƒ•™‡”‘—””‡•‡ƒ” Š“—‡•–‹‘Ǥ‡Šƒ˜‡–Š‡”‡ˆ‘”‡ †‡‡‡†‹–‘–‡ ‡••ƒ”›–‘”‡’‘”–™Š‹ Š’ƒ”ƒ‡–‡”•‘” ‘„‹ƒ–‹‘‘ˆ’ƒ”ƒ‡–‡”•ƒˆˆ‡ – –Š‡ ’”‡•‡ ‡ ‘” ƒ„•‡ ‡ ‘ˆ ˆŽ—š †‡ Ž‹‡ ‹ –Š‹• •–—†›Ǥ ‡ ƒŽ•‘ ƒ‡ –Š‡ ’”‡Ž‹‹ƒ”› ‘ Ž—•‹‘–Šƒ–ˆŽ—š†‡ Ž‹‡‹•‘–ƒ‡ ‡••ƒ”›‘”’‡”•‹•–‡–’Š‡‘‡‘‡˜‡‹‹•‘Žƒ–‡†   ‘†‡Ž•Ǥ ‡ Šƒ˜‡ ƒŽ•‘ ‘– ‹ Ž—†‡† ‹–‘ Š‘†”‹ƒŽ ‡†‹—Ǧ Šƒ‹  ”‡ƒ –‹˜ƒ–‹‘ǡ ›–‘•‘Ž‹  ƒ ›ŽǦ‘ Š›†”‘Ž›•‹• ƒ† ’‡”‘š‹•‘ƒŽ ˆƒ––›Ǧƒ ‹† „‡–ƒ ‘š‹†ƒ–‹‘ ‘ˆ ‡†‹—Ǧ Šƒ‹ •Ǥ ‘™‡˜‡”ǡ‹–Š‹•‹‹–‹ƒŽ•–—†›™‡Šƒ˜‡†‡ ‹†‡†–‘ˆ‘ —•‘–Š‡ Šƒ”ƒ –‡”‹œƒ–‹‘‘ˆƒ Š—ƒ‘†‡Ž ‘–ƒ‹‹‰–Š‡‘•–‹’‘”–ƒ–”‡ƒ –‹‘•ƒ†’ƒ–Š™ƒ›•ˆ‘”™Š‹ Š•—„•–ƒ–‹ƒŽ 5 ‡˜‹†‡ ‡Šƒ•„‡‡‰‹˜‡ˆ‘”–Š‡‹””‘Ž‡‹ ˆŽ—šǤŠ‹•’”‡Ž‹‹ƒ”›‘†‡Ž‹•–Š‡”‡ˆ‘”‡ƒŽ•‘ •ƒŽŽ‡”ƒ†‡ƒ•‹‡”–‘ Šƒ”ƒ –‡”‹œ‡Ǥ•‹†‹ ƒ–‡†ǡ–Š‡ƒ„•‡ ‡‘ˆ‡Ž‡‡–•‹–Š‡‘†‡Ž†‘‡• ‘–‡ ‡••ƒ”‹Ž›‹˜ƒŽ‹†ƒ–‡–Š‡ ‘ Ž—•‹‘•‹–Š‹••–—†›ǡ„—–‹•–‡ƒ†‘ˆˆ‡”‡™ƒ˜‡—‡•ˆ‘” ‹†‡–‹ˆ›‹‰‘†‹ˆ‹‡”•–Šƒ– ‘—Ž†ƒˆˆ‡ –•›’–‘ƒ–‘Ž‘‰›‹‹•’‡ ‹ˆ‹ ƒ† ‹ ‰‡‡”ƒŽǤ  Materials and Methods Model construction and simulation Š‡ ‘†‡Ž ™ƒ• „—‹Ž– ƒ† –Š‡ •‹—Žƒ–‹‘• ƒ† ƒŽ —Žƒ–‹‘• ™‡”‡ †‘‡ ‹ ‘Žˆ”ƒ ƒ–Š‡ƒ–‹ ƒ ȋ‘Žˆ”ƒ ‡•‡ƒ” Šǡ  Ǥǡ Šƒ’ƒ‹‰ǡ ǡ Ȍǡ ˜‡”•‹‘ ͳͳǤ͵ǤͲǤͲǤ  ˆ—ŽŽ †‡• ”‹’–‹‘ ‘ˆ –Š‡ ‘†‡Ž ‡“—ƒ–‹‘• ‹• ‰‹˜‡ ‹ —’’Ž‡‡–ƒ”› ‡š– ͳǤ ŽŽ –Š‡ •–‡ƒ†›Ǧ•–ƒ–‡ †ƒ–ƒ ™‡”‡ ‰‡‡”ƒ–‡† ˆ”‘ –Š‡

•–‡ƒ†›Ǧ•–ƒ–‡‹‹–‹ƒŽ ‘†‹–‹‘ƒ–ƒ ›–‘•‘Ž‹ ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘ȋȏ’ƒŽ‹–‘›ŽǦ‘ȐȌ‘ˆͲǤͳ Ɋǡ™Š‹ Š‹•‡šƒ –Ž›ƒ•†‘‡‹‘—”’”‡˜‹‘—•’ƒ’‡”ȏʹͳȐǤŠ‡ƒ–Š‡ƒ–‹ ƒˆ— –‹‘•‘Ž˜‡ƒ† ‹†‘‘–™‡”‡—•‡†–‘‰‡‡”ƒ–‡–Š‡•‹—Žƒ–‹‘”‡•—Ž–•ǡƒ•†‘‡‹ȏ͵͹ǡͷͳȐǤŠ‡‘†‡ŽŠƒ•—Ž–‹’Ž‡ ‹†‡’‡†‡–ˆŽ—š‡•Ǥ ‡”‡ǡ™‡ˆ‘ —•‡†‘–Š‡‡–‹–‘ Š‘†”‹ƒŽ ’”‘†— –‹‘ˆŽ—šǡ™Š‹ Š‡“—ƒŽ• –Š‡‹’Ž‹‡†‡š–‡”ƒŽ ”‡‘š‹†ƒ–‹‘”ƒ–‡–Šƒ–™‘—Ž†”‡•—Ž–‹–Š‡ƒ‹–‡ƒ ‡‘ˆƒ ‘•–ƒ–ΪǦ  ”ƒ–‹‘ǤŠ‡”‡ˆ‘”‡ǡ™‡—•‡†–Š‡–‡”ƒ†Š•‹–‘†‡’‹ ––Š‡‡– ’”‘†— –‹‘ˆŽ—š‡•‹›Ǧ

ƒš‡•‘ˆ–Š‡‰”ƒ’Š•‹–Š‹••–—†›ȋ ƒ†Š•‹Ȍǡƒ•™ƒ•†‘‡‹‘—”’”‡˜‹‘—•’ƒ’‡”•ȏʹ͸ǡͷͳȐǤ‡†‹†‘– Š‡ ‹ˆ–Š‡•–‡ƒ†›•–ƒ–‡™ƒ•—‹“—‡Ǥ  Flux control analysis Ž—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–•™‡”‡ƒ’’”‘š‹ƒ–‡†ƒ ‘”†‹‰–‘ǣ

௃ ௗ௟௡௃Ȁௗ௣ ο௃ ௏೘ೌೣǡ೐೙೥೤೘೐   ܥ௘௡௭௬௠௘ ൌ ൎ ή     ȋͳȌ డ௟௡௩Ȁడ௣ ο௏೘ೌೣǡ೐೙೥೤೘೐ ௃

™Š‡”‡p†‡‘–‡•ƒ’ƒ”ƒ‡–‡”–Šƒ–‘Ž›ƒˆˆ‡ –•–Š‡”ƒ–‡‘ˆ–Š‡‡œ›‡–Šƒ–‹•ƒƒŽ›œ‡†ǤVmax™ƒ•—•‡† •‹ ‡–Š‡”ƒ–‡•‘ˆƒŽŽ”‡ƒ –‹‘•™‡”‡’”‘’‘”–‹‘ƒŽ–‘–Š‹•’ƒ”ƒ‡–‡”Ǥ ‘”–Š‡ˆ‹‹–‡ Šƒ‰‡†‡‘–‡†„› 'ƒ’‡” ‡–ƒ‰‡„‡Ž‘™͵Ψ–ƒ‡ǡƒ†–Š‡”‡•—Ž–™ƒ•ƒ ‡’–‡†‹ˆ–Š‡•—‘ˆƒŽŽˆŽ—š ‘–”‘Ž ‘‡ˆˆ‹ ‹‡–• ‹–Š‡‘†‡Ž™ƒ•‡“—ƒŽ–‘ͳȏͷʹǡͷ͵ȐǤ  References ͳǤ ‘—–‡ǡƒ†‡”• Ǥ‰‡‡”ƒŽ‹–”‘†— –‹‘–‘–Š‡„‹‘ Š‡‹•–”›‘ˆ‹–‘ Š‘†”‹ƒŽˆƒ––› ƒ ‹†ȾǦ‘š‹†ƒ–‹‘Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͲǢ͵͵ȋͷȌǣͶ͸ͻȂ͹͹Ǥ ʹǤ ƒ–‘ ǡ ƒ”–Ž‡–– ǡ ‘—”ˆƒ”œƒ Ǥ ƒƒŽ‹ƒ ‹–‘ Š‘†”‹ƒŽ „‡–ƒǦ‘š‹†ƒ–‹‘Ǥ ‹‘ Š‡ Ǥ ͳͻͻ͸Ǣ͵ʹͲǣ͵ͶͷȂͷ͹Ǥ ͵Ǥ ‘—–‡ ǡ ‹‘Žƒ–‡ ǡ ‡–—”ƒ  Ǥǡ ƒ†‡”•  Ǥ Š‡ ‹‘ Š‡‹•–”› ƒ† Š›•‹‘Ž‘‰› ‘ˆ

ͳ͹ͳ Chapter 5

‹–‘ Š‘†”‹ƒŽ ƒ––›  ‹† ȾǦš‹†ƒ–‹‘ ƒ† –• ‡‡–‹  ‹•‘”†‡”•Ǥ — ‡˜ Š›•‹‘ŽǤ ʹͲͳ͸Ǣ͹ͺȋͳȌǣʹ͵ȂͶͶǤ ͶǤ ˜ƒ—‡ǡ‘Ž‡”Ǧ‘—™ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ‘Ž–‡”• ǡ˜ƒ‹Œ– ǡƒ”–‹‡• ǡ ‹‡œ‡Ǧ‘‹‰ǡ ‡‹‡”ǡ‡”‡–‹‡” ǡ ”‘‡ǡ‡‹Œ‰‘—† ǡ‡”• ǡƒ‡”Ǥ ‹˜‹‰‘–Š‡‡†‰‡ǣ—„•–”ƒ–‡ ‘’‡–‹–‹‘‡š’Žƒ‹•Ž‘••‘ˆ”‘„—•–‡••‹‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧ ƒ ‹†‘š‹†ƒ–‹‘†‹•‘”†‡”•Ǥ‹‘ŽǤʹͲͳ͸ǢͳͶȋͳȌǣͳȂͳͷǤ ͷǤ ‹ƒŽ†‘ ǡ ƒ–‡” ǡ ‡‡––  Ǥ ƒ––›  ‹† ‘š‹†ƒ–‹‘ ‹•‘”†‡”•Ǥ — ‡˜ Š›•‹‘ŽǤ ʹͲͲʹǢ͸ͶȋͳȌǣͶ͹͹ȂͷͲʹǤ ͸Ǥ ‘—–‡ ǡ ‹‘Žƒ–‡ ǡ ‡–—”ƒ  ǡ ƒ†‡”•  Ǥ Š‡ ‹‘ Š‡‹•–”› ƒ† Š›•‹‘Ž‘‰› ‘ˆ ‹–‘ Š‘†”‹ƒŽ ƒ––›  ‹† ȾǦš‹†ƒ–‹‘ ƒ† –• ‡‡–‹  ‹•‘”†‡”•Ǥ — ‡˜ Š›•‹‘ŽǤ ʹͲͳͷǢ͹ͺǣʹ͵ȂͶͶǤ ͹Ǥ ‡”• ǡ‡‹Œ‰‘—† ǡƒ–‡”Šƒ ǡ ‡”˜‡” ǡ˜ƒ†‡‡”‰ǡƒ—‡” ǡ‹– Ǥ Š‡ƒ–—”ƒŽŠ‹•–‘”›‘ˆ‡†‹—Ǧ Šƒ‹ƒ ›Ž‘†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›‹–Š‡‡–Š‡”Žƒ†•ǣ Ž‹‹ ƒŽ’”‡•‡–ƒ–‹‘ƒ†‘—– ‘‡Ǥ ‡†‹ƒ–”ǤʹͲͲ͸ǢͳͶͺȋͷȌǤ ͺǤ ‡”• 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  Š‡”‹–‡–ƒ„‹•ǤʹͲͲͺǢ͵ͳȋͳȌǣͺͺȂͻ͸Ǥ ͻǤ ‘—™ǡ‹– ǡ‹‡œ‡Ǧ‘‹‰ǡ‘•‰”ƒƒˆǦ†‡‘‡”ǡ ‡”†‹‰ǡ‡‹Œ‰‘—†Ǧ ǡ‡”•  Ǥ  ˜‹–”‘ ƒ† ‹ ˜‹˜‘ ‘•‡“—‡ ‡• ‘ˆ ˜ƒ”‹ƒ– ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ‰‡‘–›’‡•Ǥ”’Šƒ‡– ƒ”‡‹•ǤʹͲͳ͵ǢͺȋͳȌǣͶ͵Ǥ ͳͲǤ ‘—™ǡ‹– ǡ†‡”‹‡•ǡ†‡Ž‡” ǡ‘• Šǡ‹••‡” ǡ—Ž†‡” ǡ—„‹‘Ǧ ‘œƒŽ„‘ ǡŽ˜‡”•ǡ‹‡œ‡Ǧ‘‹‰ǡƒ†‡”• ǡƒ–‡”Šƒ ǡ‡‹Œ‰‘—†Ǧ ǡ‡”• Ǥ‹• •–”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 ͳͳǤ ’‹‡‡”‘‡––‡” ǡ ƒ•–‹ ǡ ‹ŽŽ‹‰Šƒ ǡ ‘””‹• ǡ ‹Œ„—”‰ ǡ ‹Ž ‡ Ǥ —””‡– ‹••—‡• ”‡‰ƒ”†‹‰ –”‡ƒ–‡– ‘ˆ ‹–‘ Š‘†”‹ƒŽ ˆƒ––› ƒ ‹† ‘š‹†ƒ–‹‘ †‹•‘”†‡”•Ǥ  Š‡”‹– ‡–ƒ„ ‹•Ǥ ʹͲͳͲǢ͵͵ȋͷȌǣͷͷͷȂ͸ͳǤ ͳʹǤ ”‘‡–›ǡ ƒ•‘—”‹  ƒǡ ‘‡ˆ‘– ǡ ‘—”–‘‹• ǡ —‹ Š  ƒǡ ƒ„‹‡” ǡ ‡••ƒ›”‡Ǥ ‘•– ƒ•‡•‘ˆ‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›‡• ƒ’‡†‡–‡ –‹‘‹ ”ƒ ‡Ǥ — ‡‡–Ǥͳͻͻ͸Ǣͻ͹ȋ͵Ȍǣ͵͸͹ȂͺǤ ͳ͵Ǥ ‡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 ͳͶǤ —”ƒ ǡ ‘ˆƒ’ ǡ Š‡ƒ†  ǡ ƒ—†—„”ƒ› Ǧǡ ƒ†ƒ Ǥ —††‡ Š‹Ž† ‡ƒ–Š ƒ† Ʈ ‡ƒŽ–Š›ǯ ˆˆ‡ –‡† ƒ‹Ž› ‡„‡”• ‹–Š ‡†‹—ǦŠƒ‹  ›ŽǦ‘‡œ›‡  ‡Š›†”‘‰‡ƒ•‡ ‡ˆ‹ ‹‡ ›Ǥ‡†‹ƒ–”‹ •Ǥͳͻͺ͸Ǣ͹ͺȋ͸ȌǣͳͲͷʹȂ͹Ǥ ͳͷǤ  Šƒ–œƒǤǡ•‡ƒ—‡”ǤŠ‡ Ž‹‹ ƒŽƒ‹ˆ‡•–ƒ–‹‘‘ˆ†‡ˆ‹ ‹‡ ›ǣŠƒŽŽ‡‰‡•–‘™ƒ”†• ƒ†—Ž–Š‘‘†‹–Š‡• ”‡‡‡†’‘’—Žƒ–‹‘Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͲǢ͵͵ȋͷȌǣͷͳ͵ȂʹͲǤ ͳ͸Ǥ ‡”• ǡƒ’”‘•‡ ǡƒ‡ ǡƒ‡” ‹Ž•–ǡ’ƒǡ‹– Ǥƒˆ‡ƒ†—•ƒˆ‡ †—”ƒ–‹‘‘ˆˆƒ•–‹‰ˆ‘” Š‹Ž†”‡™‹–Š†‡ˆ‹ ‹‡ ›Ǥ—” ‡†‹ƒ–”ǤʹͲͲ͹Ǣͳ͸͸ȋͳȌǣͷȂͳͳǤ ͳ͹Ǥ ƒŽ–‡” Ǥ ‘Ž‡”ƒ ‡ –‘ ˆƒ•–ǣ ƒ–‹‘ƒŽ ƒ† ’”ƒ –‹ ƒŽ ‡˜ƒŽ—ƒ–‹‘ ‹ Š‹Ž†”‡ ™‹–Š Š›’‘‡–‘ƒ‡‹ƒǤ  Š‡”‹–‡–ƒ„‹•ǤʹͲͲͻǢ͵ʹȋʹȌǣʹͳͶȂ͹Ǥ ͳͺǤ ‡‡––  Ǥ ƒ–Š‘’Š›•‹‘Ž‘‰› ‘ˆ ˆƒ––› ƒ ‹† ‘š‹†ƒ–‹‘ †‹•‘”†‡”•Ǥ  Š‡”‹– ‡–ƒ„ ‹•Ǥ ʹͲͳͲǢ͵͵ȋͷȌǣͷ͵͵Ȃ͹Ǥ ͳͻǤ ‹•Š™ƒƒ–Š Ǥ ƒ––› ƒ ‹† „‡–ƒǦ‘š‹†ƒ–‹‘ †‹•‘”†‡”•ǣ  „”‹‡ˆ ”‡˜‹‡™Ǥ  ‡—”‘• ‹Ǥ ʹͲͳ͸Ǣʹ͵ȋͳȌǣͷͳȂͷǤ ʹͲǤ ‘Ž‡”Ǧ–‘—™Ǥ†‡ˆ‹ ‹‡ ›‘„‡ǡ‘”‘––‘„‡ƒ–”‹•ǤʹͲͳͶǤ ʹͳǤ ƒ”–‹‡•Ǧ ǡ˜ƒ—‡ǡ‡‹Œ‰‘—†Ǧ ǡƒ‡”ǤŠ‡’”‘‹• —‘—•‡œ›‡‡†‹—Ǧ

ͳ͹ʹ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

Šƒ‹ ͵Ǧ‡–‘Ǧƒ ›ŽǦ‘ –Š‹‘Žƒ•‡ –”‹‰‰‡”• ƒ ˜‹ ‹‘—• › Ž‡ ‹ ˆƒ––›Ǧƒ ‹† „‡–ƒǦ‘š‹†ƒ–‹‘Ǥ  ‘’—–‹‘ŽǤʹͲͳ͹Ǣͳ͵ȋͶȌǣͳȂʹʹǤ ʹʹǤ ˜ƒ—‡ǡ‘Ž‡”Ǧ‘—™ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ‘Ž–‡”• ǡ˜ƒ‹Œ– ǡƒ”–‹‡• ǡ ‹‡œ‡Ǧ‘‹‰ǡ ‡‹‡”ǡ‡”‡–‹‡” ǡ ”‘‡ǡ‡‹Œ‰‘—† ǡ‡”• ǡƒ‡”Ǥ ‹˜‹‰‘–Š‡‡†‰‡ǣ—„•–”ƒ–‡ ‘’‡–‹–‹‘‡š’Žƒ‹•Ž‘••‘ˆ”‘„—•–‡••‹‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧ ƒ ‹†‘š‹†ƒ–‹‘†‹•‘”†‡”•Ǥ‹‘ŽǤʹͲͳ͸ǢͳͶȋͳȌǣͳȂͳͷǤ ʹ͵Ǥ ƒƒ‘ƒ ǡ ƒƒ›ƒƒ ǡ ƒŒ‹”‹ ǡ ƒƒ‡ ǡ ƒ”—‘ ǡ ƒ–‘ Ǥ Ž‹‹ ƒŽ •‹‰‹ˆ‹ ƒ ‡ ‘ˆ ƒ”–‡”‹ƒŽ‡–‘‡„‘†›”ƒ–‹‘‹ Š”‘‹ Ž‹˜‡”†‹•‡ƒ•‡Ǥ‹‰‡•–‹‘ǤʹͲͲ͵ǢͷͻȋͶȌǣ͵͸ͲȂ͵Ǥ ʹͶǤ ‹—•ƒ ǡ œƒ™ƒ ǡ Š‹ƒŠƒ”ƒ ǡ •ƒ‘ ǡ ƒƒ–ƒ‹ ǡ ‘„‡ Ǥ Šƒ‰‡• ‹ „Ž‘‘† ‡–‘‡ „‘†›”ƒ–‹‘ǣ–Š‡‹”•‹‰‹ˆ‹ ƒ ‡ƒˆ–‡”ƒŒ‘”Š‡’ƒ–‹ ”‡•‡ –‹‘Ǥ” Š—”‰Ǥͳͻͺͳ —Ǣͳͳ͸ȋ͸Ȍǣ͹ͺͳȂͷǤ ʹͷǤ ‹ǡƒŒ‹ƒǡŠ‹—”ƒǡƒ”‡›ǡ–”‘—†ǡ—”ƒ›ƒƒǡŠ–ƒ‡ǡ ‡œ‹‡Ǥ‘•• ‘ˆ –Š‡ ‹–‘ Š‘†”‹ƒŽ ƒ––›  ‹† ȾǦš‹†ƒ–‹‘ ”‘–‡‹ ‡†‹—ǦŠƒ‹  ›ŽǦ‘‡œ›‡  5 ‡Š›†”‘‰‡ƒ•‡‹•”—’–•š‹†ƒ–‹˜‡Š‘•’Š‘”›Žƒ–‹‘”‘–‡‹‘’Ž‡š–ƒ„‹Ž‹–›ƒ† — –‹‘Ǥ  ‹‡’ǤʹͲͳͺǢȋ‡ ‡„‡”ʹͲͳ͹ȌǣͳȂͳ͹Ǥ ʹ͸Ǥ ˜ƒ—‡ǡ‹‘• ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ†‡‡•–‡ ǡ‘—™ǡ ‘—–‡ǡ ”‘‡ ǡ”ƒ„ǡ‡‹Œ‰‘—† ǡƒ‡”Ǥ‹‘ Š‡‹ ƒŽ‘’‡–‹–‹‘ƒ‡• ƒ––›Ǧ ‹†ȾǦš‹†ƒ–‹‘ —Ž‡”ƒ„Ž‡–‘—„•–”ƒ–‡˜‡”Ž‘ƒ†Ǥ‘‘’—–‹‘ŽǤʹͲͳ͵ǢͻȋͺȌǣʹȂͻǤ ʹ͹Ǥ ‘ˆˆƒ–ǡŠƒ–‹ƒǡ‰—›‡ǡ› Šǡƒ‰ǡƒ‰ǡ Žƒ›‡˜ƒǡ ƒǡ ‹”• Š‡›ǡ Žƒ›’‘‘Žǡ‡‹ˆ‡”–Ǥ ›ŽǦ‘–Š‹‘‡•–‡”ƒ•‡Ǧʹˆƒ ‹Ž‹–ƒ–‡•‹–‘ Š‘†”‹ƒŽˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘ ‹–Š‡Ž‹˜‡”Ǥ ‹’‹†‡•ǤʹͲͳͶǢͷͷȋͳʹȌǣʹͶͷͺȂ͹ͲǤ ʹͺǤ ŽŽ‹• ǡ ‘™ƒ ǡ ‘Žˆ‰ƒ‰  Ǥ ‡–ƒ„‘Ž‹  ƒ† ‹••—‡Ǧ’‡ ‹ˆ‹  ‡‰—Žƒ–‹‘ ‘ˆ  ›ŽǦ‘ ‡–ƒ„‘Ž‹•Ǥ‘‡ǤʹͲͳͷǢͳͲȋ͵ȌǤ ʹͻǤ ‹ŽŽƒ†‡” ǡ Ž‡š•‘  ǡ ‘Š‡ Ǥ ‡ƒ –‹˜ƒ–‹‰ ƒ––›  ‹†•ǣ  ›ŽǦ‘ Š‹‘‡•–‡”ƒ•‡Ǧ ‡†‹ƒ–‡†‘–”‘Ž‘ˆ‹’‹†‡–ƒ„‘Ž‹•Ǥ”‡†•†‘ ”‹‘Ž‡–ƒ„ǤʹͲͳ͹Ǣʹͺȋ͹ȌǣͶ͹͵ȂͺͶǤ ͵ͲǤ —– ǡ Ž‡š•‘ Ǥ Š‡ ”‘Ž‡  ›ŽǦ‘ –Š‹‘‡•–‡”ƒ•‡• ’Žƒ› ‹ ‡†‹ƒ–‹‰ ‹–”ƒ ‡ŽŽ—Žƒ” Ž‹’‹† ‡–ƒ„‘Ž‹•Ǥ”‘‰‹’‹†‡•ǤʹͲͲʹǢͶͳȋʹȌǣͻͻȂͳ͵ͲǤ ͵ͳǤ ƒ†‡”• ǡ‘‡ ǡ‡’Ǥ ƒ––›ƒ ‹†‘‡‰ƒǦ‘š‹†ƒ–‹‘ƒ•ƒ”‡• —‡’ƒ–Š™ƒ›ˆ‘”ˆƒ––›ƒ ‹† ‘š‹†ƒ–‹‘†‹•‘”†‡”•‹Š—ƒ•Ǥ  ǤʹͲͳͳǢʹ͹ͺȋʹȌǣͳͺʹȂͻͶǤ ͵ʹǤ ‹– Š‡ŽŽ ǡ ƒ—–Š‹‡”ǡ‡•‹’Ž‡ǡƒ‰ǡƒ‡”ǡ—”‡•Š‹ Ǥ ‡”‡†‹–ƒ”›ƒ†ƒ “—‹”‡† †‹•‡ƒ•‡•‘ˆƒ ›ŽǦ ‘‡œ›‡‡–ƒ„‘Ž‹•Ǥ‘Ž ‡‡–‡–ƒ„ǤʹͲͲͺǢͻͶȋͳȌǣͶȂͳͷǤ ͵͵Ǥ ƒ‰ ǡŠƒ‘ǡƒ‰ǡƒ‰ǡ‹– Š‡ŽŽ Ǥ ‡”‡†‹–ƒ”›†‹•‡ƒ•‡•‘ˆ ‘‡œ›‡–Š‹‘‡•–‡” ‡–ƒ„‘Ž‹•Ǥ‹‘ Š‡‘ ”ƒ•ǤʹͲͳͻ ‡„ʹͺǢͶ͹ȋͳȌǣͳͶͻȂͷͷǤ ͵ͶǤ ‘Ž–‡”• ǡ ‹ƒ’ƒ‹–‡ ǡ ƒ —‡ ǡ ‹‡œ‡Ǧ‘‹‰ ǡ ƒ––‘ ǡ ‘”–‡  ǡ ‘”˜ƒ–‘˜‹ Š ǡ ƒ‡” ǡ ‹• Š‘ˆˆ ǡ ‡”‡–‹‡” Ǥ ”ƒ•Žƒ–‹‘ƒŽ ƒ”‰‡–‡† ”‘–‡‘‹ • ”‘ˆ‹Ž‹‰ ‘ˆ ‹–‘ Š‘†”‹ƒŽ ‡”‰› ‡–ƒ„‘Ž‹  ƒ–Š™ƒ›• ‹ ‘—•‡ ƒ† —ƒ ƒ’Ž‡•Ǥ  ”‘–‡‘‡ ‡•Ǥ ʹͲͳ͸ǢͳͷȋͻȌǣ͵ʹͲͶȂͳ͵Ǥ ͵ͷǤ ‹‘ Š‹ƒ”‘ ǡ ‘Ž‘„‘ ǡ ‹‘ƒ–‘ Ǥ —”‹ˆ‹ ƒ–‹‘ǡ Šƒ”ƒ –‡”‹œƒ–‹‘ ƒ† ’ƒ”–‹ƒŽ ƒ‹‘ ƒ ‹† •‡“—‡ ‡• ‘ˆ ƒ”‹–‹‡ ’ƒŽ‹–‘›ŽǦ–”ƒ•ˆ‡”ƒ•‡ ˆ”‘ Š—ƒ Ž‹˜‡”Ǥ  ‡––Ǥ ͳͻͻͲǢʹ͹ͶȋͳȂ ʹȌǣͳ͸͵Ȃ͸Ǥ ͵͸Ǥ ”‡‰‡”•‡ǡ‘”–‡•‡ǡގ˜”ƒƒǤ–Š‡„‹‘Ž‘‰‹ ‘”‹‰‹‘ˆ͸ǦͳͲǦ†‹ ƒ”„‘š›Ž‹ ƒ†͸Ǧ ͳͲǦ‘‡‰ƒǦͳǦ Š›†”‘š› ‘‘ ƒ”„‘š›Ž‹  ƒ ‹†• ‹ Š—ƒ ƒ† ”ƒ– ™‹–Š ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ–‹‘ †‡ˆ‹ ‹‡ ‹‡•ǣ ‹ ˜‹–”‘ •–—†‹‡• ‘ –Š‡ ‘‡‰ƒǦ ƒ† ‘‡‰ƒǦͳǦ‘š‹†ƒ–‹‘ ‘ˆ ‡†‹—Ǧ Šƒ‹ȋ͸ǦͳʹȌˆƒ––›ƒ ‹†•‹Š—ƒǤ‡†‹ƒ–”‡•Ǥͳͻͺ͵Ǣͳ͹ȋͳͲȌǣͺʹͺȂ͵ͶǤ ͵͹Ǥ ˜ƒ—‡ǡ‹‘• ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ†‡‡•–‡ ǡ‘—™ǡ ‘—–‡ǡ ”‘‡ ǡ ”ƒ„ ǡ ‡‹Œ‰‘—†  ǡ ƒ‡” Ǥ ‹‘ Š‡‹ ƒŽ ‘’‡–‹–‹‘ ƒ‡• ƒ––›Ǧ ‹† ‡–ƒǦ š‹†ƒ–‹‘—Ž‡”ƒ„Ž‡–‘—„•–”ƒ–‡˜‡”Ž‘ƒ†Ǥ‘‘’—–‹‘ŽǤʹͲͳ͵ǢͻȋͺȌǣʹȂͻǤ ͵ͺǤ –‡” ‡Ž† ǡ —‡ŽŽ‡” ǡ ”ƒ‡” ǡ ƒ—••ƒ ǡ ‡”‡„‹ƒ ǡ ƒ›ƒ–‡’‡ ǡ ƒ”›‡ƒ ǡ ”‹ƒ••‹ ǡ ’‹‡‡”‘‡––‡” Ǥ  ‘˜‡Ž –ƒ†‡ ƒ•• •’‡ –”‘‡–”› ‡–Š‘† ˆ‘” ”ƒ’‹† ‘ˆ‹”ƒ–‹‘ ‘ˆ ‡†‹—Ǧ ƒ† ˜‡”› Ž‘‰Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ †‡ˆ‹ ‹‡ › ‹

ͳ͹͵ Chapter 5

‡™„‘”•Ǥ‘‡ǤʹͲͲͻǢͶȋ͹ȌǤ ͵ͻǤ ‘ǡ”‡‡ǡ‹Œ„—”‰ ǡƒ†‡”• ǡ ”‡‰‡”•‡ǡ‘”›†‘ ǡƒ–‡”Šƒ ǡ—”ƒ ǤŠ‘”–ǦŠƒ‹ ›ŽǦ‘‡Š›†”‘‰‡ƒ•‡‡ˆ‹ ‹‡ ›ǣ–—†‹‡•‹ƒƒ”‰‡ ƒ‹Ž›††‹‰–‘–Š‡ ‘’Ž‡š‹–›‘ˆ–Š‡‹•‘”†‡”Ǥ‡†‹ƒ–”‹ •ǤʹͲͲ͵ǢͳͳʹȋͷȌǣͳͳͷʹȂͷǤ ͶͲǤ ‘—™ǡ‹– ǡ†‡”‹‡•ǡ†‡Ž‡” ǡ‘• Šǡ‹••‡” ǡ—Ž†‡” ǡ—„‹‘Ǧ ‘œƒŽ„‘ ǡŽ˜‡”•ǡ‹‡œ‡Ǧ‘‹‰ǡƒ†‡”• ǡƒ–‡”Šƒ ǡ‡‹Œ‰‘—†Ǧ ǡ‡”• Ǥ‹• •–”ƒ–‹ˆ‹ ƒ–‹‘„›”‡•‹†—ƒŽ‡œ›‡ƒ –‹˜‹–›ƒˆ–‡”‡™„‘”• ”‡‡‹‰ˆ‘”‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘ †‡Š›”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›ǣ†ƒ–ƒˆ”‘ƒ ‘Š‘”–•–—†›Ǥ”’Šƒ‡– ƒ”‡‹•ǤʹͲͳʹƒ›ʹͷǢ͹ȋͳȌǣ͵ͲǤ ͶͳǤ ‘ǡ”‡‡ǡ‹Œ„—”‰ ǡƒ†‡”• ǡ ”‡‰‡”•‡ǡ‘”›†‘ ǡƒ–‡”Šƒ ǡ—”ƒ Ǥ Š‘”–Ǧ Šƒ‹  ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ †‡ˆ‹ ‹‡ ›ǣ •–—†‹‡• ‹ ƒ Žƒ”‰‡ ˆƒ‹Ž› ƒ††‹‰ –‘ –Š‡ ‘’Ž‡š‹–›‘ˆ–Š‡†‹•‘”†‡”Ǥ‡†‹ƒ–”‹ •ǤʹͲͲ͵‘˜ǢͳͳʹȋͷȌǣͳͳͷʹȂͷǤ ͶʹǤ ‹ƒ‡›Ǧ‹ƒ—† ǡ ‹˜”› ǡ ”‡‰‡”•‡ ǡ ƒ–Š‹‡— Ǥ Š‡ ‹„‘” ‡””‘”• ‘ˆ ‹–‘ Š‘†”‹ƒŽ ˆƒ––› ƒ ‹†‘š‹†ƒ–‹‘Ǥ  Š‡”‡–ƒ„‹•Ǥͳͻͺ͹ǢͳͲǣͳͷͻȂʹͲͲǤ Ͷ͵Ǥ ‘—ƒǡ Šƒ”’‡–‹‡” Ǥ ‡†‹— Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ †‡ˆ‹ ‹‡ ›Ǥ” Š ‹• Š‹Ž†Ǥ ͳͻͻʹǢ͸͹ǣͳͶʹȂͷǤ ͶͶǤ ‡–Ž‡”ǡŠƒ‹ǡŽ•„‡ ‡”ǡ”‘Ž† ǡ—”–‘ǡ‘ Ž‡› ǡƒ‡”‘ǡ ‹‡” ǡ†‹   ǡ‡””›ǡŠ‘ƒ• ǡ‘†‰‡ǡ‹‰Šǡƒ•Šƒǡ‘ƒŽ‡›ǡ–‡„”‹†‰‡ǡ—••‹ǡ Š‹ŽŽ‹’•ǡ—”–‘ǡ†ƒ‘ǡŠ”‡•–Šƒǡ ‘‰ƒ•‘ ǡ™›‡”ǡ ƒ‹Ž‹‡ǡ‘‹‡ǡ ƒ‹Ž‹‡ ǡ•ƒ‘ƒŠǡ ‘‘†‹ǡƒŒƒƒ”—ƒǡ ƒ •‘ǡ ƒ‘•Šǡ‡”‘ ǡ‹–ŠǡŠƒ†ǡ ‹’‹•‹ǡ ‡Ž†ƒ ǡ‡””›ǡŽ•„‡ ‡”ǡ‡–Ž‡”ǡ ‘–Ǧ‘–‰‘‡”›ǡ‡ ǡ‡ƒǡ ‘‡„‡”Žǡ ‹ƒ‰Š—‹ǡ‹•Šƒ‹ǡ‹œœ‘ǡƒ™•‘ǡ„”‘•‡ǡ‡˜›ǡ”‘ǡ ‘‰ –‘ǡǯ ‘ǡ ƒ”–ǡ”„‡ǡƒ‘•ǡ‡•Ž‹‡ǡ‘™‡”•ǡƒ”–Š‘Ž‘‡™ǡ ‘ˆˆǡ˜ƒƒŽ ƒ”ǡ ƒ•‡ ǡ”‘Ž† ǡ‘ Ž‡› ǡƒŽ•ŠǦ‘ Ž‡›ǡŠ‡ƒ†ǡ‹‘ ǡ‰‡Ž‹‰ǡ‹”†ǡ ™ƒǡ Š™‘‡”‡” ǡ ‡”›ǡƒ”—ƒ Š‹ǡ —‡Žǡ‹Ž‹• ǡƒ˜‹•Ǧ‡’’‡ǡ–‡‹ ǡ‘ƒǡƒ‡”‘ǡ†‹  ǡ ‹‡” ǡ —•–‹ ‡ǡŠƒ‹Ǥʹʹͳ‡™„‘”Ǧ• ”‡‡‡†‡‘ƒ–‡• ™‹–Š ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ ‘‡œ›‡  †‡Š›†”‘‰‡ƒ•‡ †‡ˆ‹ ‹‡ ›ǣ ‹†‹‰• ˆ”‘ –Š‡ „‘” ””‘”•‘ˆ‡–ƒ„‘Ž‹•‘ŽŽƒ„‘”ƒ–‹˜‡Ǥ‘Ž ‡‡–‡–ƒ„ǤʹͲͳ͸ǢͳͳͻȋͳȂʹȌǣ͹ͷȂͺʹǤ ͶͷǤ ƒŽ–‡” ǤǦƒ”‹–‹‡‹‹„‘”‡””‘”•‘ˆ‡–ƒ„‘Ž‹•ǣŠƒ–‹•–Š‡‡˜‹†‡ ‡ǫ  Š‡”‹–‡–ƒ„ ‹•ǤʹͲͲ͵Ǣʹ͸ȋʹȂ͵ȌǣͳͺͳȂͺǤ Ͷ͸Ǥ ‘‡Ǥ Š‡”‹–‡††‹•‘”†‡”•‘ˆ‹–‘ Š‘†”‹ƒŽˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘ǣ‡™”‡•’‘•‹„‹Ž‹–›ˆ‘”–Š‡ ‡‘ƒ–‘Ž‘‰‹•–Ǥ‡‹‡‘ƒ–‘ŽǤʹͲͲʹǢ͹ȋͳȌǣ͵͹ȂͶ͹Ǥ Ͷ͹Ǥ ˜ƒ—‡ǡ‹‘• ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ†‡‡•–‡ ǡ‘—™ǡ ‘—–‡ǡ ”‘‡ ǡ”ƒ„ǡ‡‹Œ‰‘—† ǡƒ‡”Ǥ‹‘ Š‡‹ ƒŽ‘’‡–‹–‹‘ƒ‡• ƒ––›Ǧ ‹†ȾǦš‹†ƒ–‹‘ —Ž‡”ƒ„Ž‡–‘—„•–”ƒ–‡˜‡”Ž‘ƒ†Ǥ‘‘’—–‹‘ŽǤʹͲͳ͵ǢͻȋͺȌǣʹȂͻǤ ͶͺǤ ƒ”–‹‡• ǡ˜ƒ—‡ǡ‡‹Œ‰‘—† ǡƒ‡”ǤŠ‡’”‘‹• —‘—•‡œ›‡‡†‹—Ǧ Šƒ‹ ͵Ǧ‡–‘Ǧƒ ›ŽǦ‘ –Š‹‘Žƒ•‡ –”‹‰‰‡”• ƒ ˜‹ ‹‘—• › Ž‡ ‹ ˆƒ––›Ǧƒ ‹† „‡–ƒǦ‘š‹†ƒ–‹‘Ǥ ‘ ‘’—–‹‘ŽǤʹͲͳ͹Ǣͳ͵ȋͶȌǣͳȂʹʹǤ ͶͻǤ ƒ–‘Ǥ‘–”‘Ž‘ˆ‹–‘ Š‘†”‹ƒŽ„Ǧ‘š‹†ƒ–‹‘ˆŽ—šǤʹͲͲʹǢͶͳǣͳͻ͹Ȃʹ͵ͻǤ ͷͲǤ ’—”™ƒ› ǡ Š‡””ƒ––  ƒǡ ‘‰•‘  ǡ ‰‹—• Ǥ ȝǦš‹†ƒ–‹‘ ‹ ƒ– ‡’ƒ–‘ ›–‡ —Ž–—”‡•Ǥ ‹‘ Š‡ Ǥͳͻͻ͹Ǣ͵ʹ͵ǣͳͳͻȂʹʹǤ ͷͳǤ ˜ƒ—‡ǡ‘Ž‡”Ǧ‘—™ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ‘Ž–‡”• ǡ˜ƒ‹Œ– ǡƒ”–‹‡•Ǧ  ǡ ‹‡œ‡Ǧ‘‹‰ ǡ ‡‹‡” ǡ ‡”‡–‹‡” ǡ ”‘‡ ǡ ‡‹Œ‰‘—† Ǧ ǡ ‡”•  ǡ ƒ‡” Ǥ ‹˜‹‰ ‘ –Š‡ ‡†‰‡ǣ •—„•–”ƒ–‡ ‘’‡–‹–‹‘ ‡š’Žƒ‹• Ž‘••‘ˆ”‘„—•–‡••‹ ‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧƒ ‹†‘š‹†ƒ–‹‘†‹•‘”†‡”•Ǥ‹‘ŽǤʹͲͳ͸ǢͳͶȋͳȌǣͳͲ͹Ǥ ͷʹǤ ‘ˆ‡›” Ǥ‡–ƒ„‘Ž‹  ‘–”‘ŽƒƒŽ›•‹•‹ƒ—–•Š‡ŽŽǤ ǣ”‘ ‡‡†‹‰•‘ˆ–Š‡ʹ† –‡”ƒ–‹‘ƒŽ ‘ˆ‡”‡ ‡‘•›•–‡•„‹‘Ž‘‰›ǤʹͲͲͳǤ’ǤʹͻͳȂ͵ͲͲǤ ͷ͵Ǥ ‡ŽŽǤ‡–ƒ„‘Ž‹  ‘–”‘ŽƒƒŽ›•‹•ǣƒ•—”˜‡›‘ˆ‹–•–Š‡‘”‡–‹ ƒŽƒ†‡š’‡”‹‡–ƒŽ†‡˜‡Ž‘’‡–Ǥ ‹‘ Š‡ ǤͳͻͻʹǢʹͺ͸ȋ–ʹǣ͵ͳ͵Ȃ͵ͲǤ 

ͳ͹Ͷ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

^ƵƉƉůĞŵĞŶƚĂƌLJƚĞdžƚ^dϭ͗DŽĚĞůĚĞƐĐƌŝƉƚŝŽŶ Š‡ •–ƒ”–‹‰ ’‘‹– ˆ‘” –Š‡ ‘†‡Ž †‡• ”‹„‡† Š‡”‡ ™ƒ• –Šƒ– ‘ˆ „‡–ƒǦ‘š‹†ƒ–‹‘ ‹ ”ƒ– Ž‹˜‡” ‹–‘ Š‘†”‹ƒȏͳȐǤŠ‹•‘†‡ŽŠƒ•„‡‡ƒ†ƒ’–‡†–‘”‡’”‡•‡–Š—ƒŽ‹˜‡”„›”‡‘˜‹‰–Š‡ ”‡ƒ –‹‘ǡ•‹ ‡–Š‹•‡œ›‡‹•Šƒ”†Ž›‡š’”‡••‡†‹Š—ƒŽ‹˜‡”ȏʹȐǤ‘”‡‘˜‡”ǡ™Š‡”‡ ƒ˜ƒ‹Žƒ„Ž‡ǡ’ƒ”ƒ‡–‡”•ˆ‘”Š—ƒ‡œ›‡•Šƒ˜‡„‡‡—•‡†Ǥ ƒ††‹–‹‘ǡ–Š‡‘”‹‰‹ƒŽ‘†‡Ž Šƒ•—†‡”‰‘‡ƒ ‘’”‡Š‡•‹˜‡‡š–‡•‹‘ǡ™‹–Šƒˆ‘ —•‘–Š‡”‡ƒ –‹‘•ƒ”‘—† ‘‡œ›‡  ƒ† –Š‡ ƒ ›ŽǦ‘ ‡•–‡”•Ǥ  ˜‹•—ƒŽ ”‡’”‡•‡–ƒ–‹‘ ‘ˆ –Š‡ ”‡ƒ –‹‘• ‹ Ž—†‡† ‹ –Š‡ ‡™ ‘†‡Ž‹•‰‹˜‡‹ ‹‰—”‡ͳǤŠ‡‡˜‡”’ƒ”ƒ‡–‡”•™‡”‡—•‡†ˆ”‘–Š‡‘”‹‰‹ƒŽ‘†‡Žǡ–Š‡ ”‡ˆ‡”‡ ‡–‘–Š‡ ‘””‡•’‘†‹‰’ƒ’‡”ȏͳȐ‹•‰‹˜‡”ƒ–Š‡”–Šƒ–‘–Š‡—†‡”Ž›‹‰„‹‘ Š‡‹ ƒŽ Ž‹–‡”ƒ–—”‡ǤŠ‡‘–Š‡””‡ˆ‡”‡ ‡•ƒ”‡‰‹˜‡ǡ–Š‹•‹†‹ ƒ–‡•–Šƒ–‹– ‘ ‡”•ƒ‡š–‡•‹‘‘” 5 ƒ ƒ†ƒ’–ƒ–‹‘ –‘ –Š‡ Š—ƒ •‹–—ƒ–‹‘Ǥ ‹š‡† „‘—†ƒ”› ‡–ƒ„‘Ž‹–‡• ‹ –Š‡ ‘”‹‰‹ƒŽ ‘†‡Ž ™‡”‡ ‡’– ˆ‹š‡† ‹ –Š‹• ‡š–‡†‡† ‘†‡ŽǤ ƒ› ‘ˆ –Š‡ ˜ƒ”‹ƒ„Ž‡ ‡–ƒ„‘Ž‹–‡• †‡’‡† ‘ –Š‡ ”‡ƒ –‹‘• –Šƒ– ™‡”‡ ƒ††‡† ‹ –Š‡ ‡š–‡•‹‘ǡ Š‡ ‡ –Š‡ ”†‹ƒ”› ‹ˆˆ‡”‡–‹ƒŽ “—ƒ–‹‘• ‰‘˜‡”‹‰–Š‡Šƒ˜‡ Šƒ‰‡†ƒ•†‡• ”‹„‡†„‡Ž‘™Ǥ ƒ††‹–‹‘ǡƒ ‘—’Ž‡‘ˆ‡™†‹ˆˆ‡”‡–‹ƒŽ ‡“—ƒ–‹‘•Šƒ˜‡„‡‡‹–”‘†— ‡†ˆ‘”˜ƒ”‹ƒ„Ž‡‡–ƒ„‘Ž‹–‡•–Šƒ–™‡”‡‡™Ž›‹ ‘”’‘”ƒ–‡†Ǥ–Š‡ ‹ ”‘•‘‡™ƒ•‹ ‘”’‘”ƒ–‡†–‘ ‘–ƒ‹–Š‡‘‡‰ƒ‘š‹†ƒ–‹‘ǤŠ‡‹ ”‘•‘‡™ƒ•‹ Ž—†‡ƒ• ‹–Šƒ•‹–•‘™Ž‘ ƒŽ ‘ ‡–”ƒ–‹‘‘ˆ Ǥ

KƌĚŝŶĂƌLJĚŝĨĨĞƌĞŶƚŝĂůĞƋƵĂƚŝŽŶƐ;KƐͿ Š‡ • ‹ –Š‡ ‘†‡Ž ƒ”‡ ‰‹˜‡ „‡Ž‘™Ǥ ›–‘•‘Ž‹ ǡ ‹–‘ Š‘†”‹ƒŽ ƒ–”‹š ƒ† ‹ ”‘•‘‡ Ž‘ ƒŽ‹œƒ–‹‘•ƒ”‡†‡‘–‡†ƒ•ǡƒ† ǡ”‡•’‡ –‹˜‡Ž›Ǥ‡ƒ –‹‘”ƒ–‡•ƒ”‡†‡‘–‡† ™‹–Šƒ•ƒŽŽ˜ƒ† ›–‘•‘Ž‹ ǡ‹–‘ Š‘†”‹ƒŽƒ–”‹šƒ†‹ ”‘•‘‡˜‘Ž—‡•ƒ”‡†‡‘–‡†™‹–Š ǡƒ† ǡ”‡•’‡ –‹˜‡Ž›ǤŠ‡ˆ‘”™ƒ”††‹”‡ –‹‘‹•ǡˆ‘” ǡ–Š‡†‹”‡ –‹‘‘ˆ ‘š‹†ƒ–‹‘ ƒ† ˆ‘” ‡–‘‰‡‡•‹•ǡ –Š‡ †‹”‡ –‹‘ ‘ˆ ‡–‘‡ „‘†› ’”‘†— –‹‘ ȋ™Š‹ Š ‹• ™Š› –Š‡”‡ˆ‘”‡ ˆ‘” ͳǡ –Š‡ ˆ‘”™ƒ”† †‹”‡ –‹‘ ‹•ǡ ‹ ‘–”ƒ•– –‘ ǡ ‘– –Š‹‘Ž›•‹• „—– ‘†‡•ƒ–‹‘ȌǤŠ‡ˆ‘”™ƒ”††‹”‡ –‹‘ˆ‘”‡ƒ Š”‡ƒ –‹‘‹•ƒŽ•‘‰‹˜‡„›–Š‡†‹”‡ –‹‘‘ˆ–Š‡ Žƒ”‰‡ƒ””‘™‹ ‹‰—”‡ͳǤ•‘•–‡œ›‡• ƒ–ƒŽ›œ‡”‡ƒ –‹‘•ˆ”‘•—„•–”ƒ–‡•‘ˆ‘”‡–Šƒ ‘‡ Šƒ‹Ž‡‰–Šǡ–Š‡ƒ„„”‡˜‹ƒ–‹‘ˆ‘”–Š‡”ƒ–‡‘ˆƒ”‡ƒ –‹‘ ƒ–ƒŽ›œ‡†„›ƒ‡œ›‡ǡˆ‹‹•Š‡• ™‹–Š  ƒ† –Š‡ —„‡” ‘ˆ ƒ”„‘• ‹ –Š‡ ƒ ›ŽǦ Šƒ‹ ‘ˆ –Š‡ •—„•–”ƒ–‡ ‘ˆ –Š‡ ”‡ƒ –‹‘ ‹ “—‡•–‹‘Ǥ ‘”‡šƒ’Ž‡ǡ˜ ’–ͳͳ͸”‡’”‡•‡–•–Š‡”ƒ–‡‘ˆ–Š‡ ‘˜‡”•‹‘‘ˆͳ͸Ǧƒ ›ŽǦ‘ȋ‹Ǥ‡Ǥ ’ƒŽ‹–‘›ŽǦ‘Ȍ–‘ͳ͸Ǧƒ ›ŽǦ ƒ”‹–‹‡„›ͳǤ  ODE’s from metabolites that were already in the rat model. ƒ–‡•–Šƒ–ƒ”‡†‹ˆˆ‡”‡– ‘’ƒ”‡†–‘–Š‡”ƒ–‘†‡ŽŠƒ˜‡„‡‡‹†‹ ƒ–‡†‹„‘Ž†Ǥ ᇱ ି୴ୡୟୡ୲େଵ଺ା୴ୡ୮୲ଵେଵ଺ି୴୉୶୅ୡୡେଵ଺  ሾݐሿ ൌൌ      ȋͳȌ”ƒŽ› ͳ͸ ୚େଢ଼୘

ᇱ ୴ୡୟୡ୲େଵ଺ି୴ୡ୮୲ଶେଵ଺ ›Žƒ” ሾݐሿ ൌൌ  ȋʹȌ ͳ͸ ୚୑୅୘

۱૚૟ି୴୴୪ୡୟୢେଵ଺܌܉܋ܕܞ۱૚૟ା୴ୡ୮୲ଶେଵ଺ିܜܗ܋܉ܞି ᇱ ›Ž‘ ሾݐሿ ൌൌ   ȋ͵Ȍ ͳ͸ ୚୑୅୘

۱૚૟ି୴୫୲୮େଵ଺ା୴୴୪ୡୟୢେଵ଺܌܉܋ܕᇱ ି୴ୡ୰୭୲େଵ଺ା୴ ͳ͸‘›Ž‘ ሾݐሿ ൌൌ    ȋͶȌ ୚୑୅୘

ᇱ ୴ୡ୰୭୲େଵ଺ି୴୫ୱୡ୦ୟୢେଵ଺ ›Ž‘ ሾݐሿ ൌൌ     ȋͷȌ ƒ›š‘”†› ͳ͸ ୚୑୅୘

ͳ͹ͷ Chapter 5

ᇱ ି୴୫ୡ୩ୟ୲େଵ଺ା୴୫ୱୡ୦ୟୢେଵ଺ ›Ž‘ ሾݐሿ ൌൌ      ȋ͸Ȍ ƒ‘–‡ͳ͸ ୚୑୅୘

େଵସ ૚۱૚૝ି୴୉୶ܜܘ܋ܞᇱ ି୴ୡୟୡ୲େଵସା  ሾݐሿ ൌൌ      ȋ͹Ȍ”ƒŽ› ͳͶ ୚େଢ଼୘

ᇱ ୴ୡୟୡ୲େଵସି୴ୡ୮୲ଶେଵସ ›Žƒ” ሾݐሿ ൌൌ       ȋͺȌ ͳͶ ୚୑୅୘

۱૚૝ା୴୫ୡ୩ୟ୲େଵ଺ା୴୫୲୮େଵ଺ି୴୴୪ୡୟୢେଵସ܌܉܋ܕܞ۱૚૝ା୴ୡ୮୲ଶେଵସିܜܗ܋܉ܞି ᇱ ›Ž‘ ሾݐሿ ൌൌ  ȋͻȌ ͳͶ ୚୑୅୘

۱૚૝ି୴୫୲୮େଵସା୴୴୪ୡୟୢେଵସ܌܉܋ܕܞᇱ ି୴ୡ୰୭୲େଵସା ͳͶ‘›Ž‘ ሾݐሿ ൌൌ    ȋͳͲȌ ୚୑୅୘

ᇱ ୴ୡ୰୭୲େଵସି୴୫ୱୡ୦ୟୢେଵସ ›Ž‘ ሾݐሿ ൌൌ      ȋͳͳȌ ƒ›š‘”†› ͳͶ ୚୑୅୘

ᇱ ି୴୫ୡ୩ୟ୲େଵସା୴୫ୱୡ୦ୟୢେଵସ ›Ž‘ ሾݐሿ ൌൌ      ȋͳʹȌ ƒ‘–‡ͳͶ ୚୑୅୘

େଵଶ ૚۱૚૛ି୴୉୶ܜܘ܋ܞᇱ ି୴ୡୟୡ୲େଵଶା  ሾݐሿ ൌൌ      ȋͳ͵Ȍ”ƒŽ› ͳʹ ୚େଢ଼୘

૛۱૚૛ܜܘ܋ܞᇱ ୴ୡୟୡ୲େଵଶି ›Žƒ” ሾݐሿ ൌൌ       ȋͳͶȌ ͳʹ ୚୑୅୘

۱૚૛ା୴ୡ୮୲ଶେଵଶି୴୫ୡୟୢେଵଶା୴୫ୡ୩ୟ୲େଵସା୴୫୲୮େଵସି୴୴୪ୡୟୢେଵଶܜܗ܋܉ܞି ᇱ ›Ž‘ ሾݐሿ ൌൌ  ȋͳͷȌ ͳʹ ୚୑୅୘

ᇱ ି୴ୡ୰୭୲େଵଶା୴୫ୡୟୢେଵଶି୴୫୲୮େଵଶା୴୴୪ୡୟୢେଵଶ ͳʹ‘›Ž‘ ሾݐሿ ൌൌ    ȋͳ͸Ȍ ୚୑୅୘

ᇱ ୴ୡ୰୭୲େଵଶି୴୫ୱୡ୦ୟୢେଵଶ ›Ž‘ ሾݐሿ ൌൌ      ȋͳ͹Ȍ ƒ›š‘”†› ͳʹ ୚୑୅୘

ᇱ ି୴୫ୡ୩ୟ୲େଵଶା୴୫ୱୡ୦ୟୢେଵଶ ›Ž‘ ሾݐሿ ൌൌ      ȋͳͺȌ ƒ‘–‡ͳʹ ୚୑୅୘

ᇱ ି୴ୡୟୡ୲େଵ଴ା୴ୡ୮୲ଵେଵ଴ି୴୉୶ େଵ଴  ሾݐሿ ൌൌ      ȋͳͻȌ”ƒŽ› ͳͲ ୚େଢ଼୘

૛۱૚૙ܜܘ܋ܞᇱ ୴ୡୟୡ୲େଵ଴ି ›Žƒ” ሾݐሿ ൌൌ       ȋʹͲȌ ͳͲ ୚୑୅୘

۱૚૙܌܉܋ܔܞܞ۱૚૙ା୴ୡ୮୲ଶେଵ଴ି୴୫ୡୟୢେଵ଴ା୴୫ୡ୩ୟ୲େଵଶା୴୫୲୮େଵଶିܜܗ܋܉ܞି ᇱ ›Ž‘ ሾݐሿ ൌൌ  ȋʹͳȌ ͳͲ ୚୑୅୘

۱૚૙܌܉܋ܔܞܞᇱ ି୴ୡ୰୭୲େଵ଴ା୴୫ୡୟୢେଵ଴ି୴୫୲୮େଵ଴ା ͳͲ‘›Ž‘ ሾݐሿ ൌൌ    ȋʹʹȌ ୚୑୅୘

ᇱ ୴ୡ୰୭୲େଵ଴ି୴୫ୱୡ୦ୟୢେଵ଴ ›Ž‘ ሾݐሿ ൌൌ     ȋʹ͵Ȍ ƒ›š‘”†› ͳͲ ୚୑୅୘

ᇱ ି୴୫ୡ୩ୟ୲େଵ଴ା୴୫ୱୡ୦ୟୢେଵ଴ ›Ž‘ ሾݐሿ ൌൌ      ȋʹͶ ƒ‘–‡ͳͲ ୚୑୅୘

େ଼ ૚۱ૡି୴୉୶ܜܘ܋ܞᇱ ି୴ୡୟୡ୲େ଼ା  ሾݐሿ ൌൌ      ȋʹͷȌ”ƒŽ› ͺ ୚େଢ଼୘

૛۱ૡܜܘ܋ܞᇱ ୴ୡୟୡ୲େ଼ି ›Žƒ” ሾݐሿ ൌൌ ȋʹ͸Ȍ ͺ ୚୑୅୘

۱ૡ܌܉܋ܔܞܞ۱ૡା୴ୡ୮୲ଶେ଼ି୴୫ୡୟୢେ଼ା୴୫ୡ୩ୟ୲େଵ଴ା୴୫୲୮େଵ଴ିܜܗ܋܉ܞି ᇱ ›Ž‘ ሾݐሿ ൌൌ   ȋʹ͹Ȍ ͺ ୚୑୅୘

۱ૡ܌܉܋ܔܞܞᇱ ି୴ୡ୰୭୲େ଼ା୴୫ୡୟୢେ଼ି୴୫୲୮େ଼ା ͺ‘›Ž‘ ሾݐሿ ൌൌ ȋʹͺȌ ୚୑୅୘

ͳ͹͸ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

ᇱ ୴ୡ୰୭୲େ଼ି୴୫ୱୡ୦ୟୢେ଼ ›Ž‘ ሾݐሿ ൌൌ       ȋʹͻȌ ƒ›š‘”†› ͺ ୚୑୅୘

ᇱ ି୴୫ୡ୩ୟ୲େ଼ା୴୫ୱୡ୦ୟୢେ଼ ›Ž‘ ሾݐሿ ൌൌ      ȋ͵ͲȌ ƒ‘–‡ͺ ୚୑୅୘

େ଺ ૚۱૟ି୴୉୶ܜܘ܋ܞᇱ ି୴ୡୟୡ୲େ଺ା  ሾݐሿ ൌൌ      ȋ͵ͳȌ”ƒŽ› ͸ ୚େଢ଼୘

ᇱ ୴ୡୟୡ୲େ଺ି୴ୡ୮୲ଶେ଺ ›Žƒ” ሾݐሿ ൌൌ       ȋ͵ʹȌ ͸ ୚୑୅୘

۱૟܌܉܋ܔܞܞ۱૟ା୴ୡ୮୲ଶେ଺ି୴୫ୡୟୢେ଺ା୴୫ୡ୩ୟ୲େ଼ା୴୫୲୮େ଼ି୴ୱୡୟୢେ଺ିܜܗ܋܉ܞି ᇱ ›Ž‘ ሾݐሿ ൌൌ  ȋ͵͵Ȍ ͸ ୚୑୅୘

۱଺ 5܌܉܋ܔܞܞᇱ ି୴ୡ୰୭୲େ଺ା୴୫ୡୟୢେ଺ା୴ୱୡୟୢେ଺ା ͸‘›Ž‘ ሾݐሿ ൌൌ     ȋ͵ͶȌȌ ୚୑୅୘

ᇱ ୴ୡ୰୭୲େ଺ି୴୫ୱୡ୦ୟୢେ଺ ›Ž‘ ሾݐሿ ൌൌ      ȋ͵ͷȌ ƒ›š‘”†› ͸ ୚୑୅୘

ᇱ ି୴୫ୡ୩ୟ୲େ଺ା୴୫ୱୡ୦ୟୢେ଺ ›Ž‘ ሾݐሿ ൌൌ      ȋ͵͸Ȍ ƒ‘–‡͸ ୚୑୅୘

ᇱ ି୴ୡୟୡ୲େସା୴ୡ୮୲ଵେସି୴୉୶ େସ  ሾݐሿ ൌൌ      ȋ͵͹Ȍ”ƒŽ› Ͷ ୚େଢ଼୘

ᇱ ୴ୡୟୡ୲େସି୴ୡ୮୲ଶେସ ›Žƒ” ሾݐሿ ൌൌ ȋ͵ͺȌ Ͷ ୚୑୅୘

۱૝ା୴ୡ୮୲ଶେସି୴୫ୡୟୢେସା୴୫ୡ୩ୟ୲େ଺ି୴ୱୡୟୢେସܜܗ܋܉ܞି ᇱ ›Ž‘ ሾݐሿ ൌൌ    ȋ͵ͻȌ Ͷ ୚୑୅୘

ᇱ ି୴ୡ୰୭୲େସା୴୫ୡୟୢେସା୴ୱୡୟୢେସ Ͷ‘›Ž‘ ሾݐሿ ൌൌ      ȋͶͲȌ ୚୑୅୘

ᇱ ୴ୡ୰୭୲େସି୴୫ୱୡ୦ୟୢେସ ›Ž‘ ሾݐሿ ൌൌ      ȋͶͳ ƒ›š‘”†› Ͷ ୚୑୅୘

ᇱ ୴ୟୡୟ୲ଵି୴୦୫୥ୡୱଶି୴୫ୡ୩ୟ୲େସା୴୫ୱୡ୦ୟୢେସ ›Ž‘ ሾݐሿ ൌൌ    ȋͶʹȌ ƒ‘–‡ Ͷ ୚୑୅୘

 ODEs from newly introduced metabolites.

˜˜Ž ƒ†ͳ͸ ൅ ˜˜Ž ƒ†ͳͶ ൅ ˜˜Ž ƒ†ͳʹ ൅ ˜˜Ž ƒ†ͳͲ ൅ ˜˜Ž ƒ†ͺ ൅ ˜˜Ž ƒ†͸ ൅ ˜ ƒ†ͳ͸ ൅˜ ƒ†ͳͶ ൅ ˜ ƒ†ͳʹ ൅ ˜ ƒ†ͳͲ ൅ ˜ ƒ†ͺ ൅ ˜ ƒ†͸ ൅ ˜ ƒ†Ͷ ൅ ˜• ƒ†͸ ൅ ˜• ƒ†Ͷ െ ˜‡–ˆ“‘  ᇱሾݐሿ ൌൌ“Š    ȋͶ͵

ᇱ ୴ୟୡ୭୲େଵ଺ି୴୫ୡ୊୊୅େଵ଺  ሾݐሿ ൌൌ ȋͶͶȌ ͳ͸ ୚୑୅୘

ᇱ ୴ୟୡ୭୲େଵସି୴୫ୡ୊୊୅େଵସ  ሾݐሿ ൌൌ ȋͶͷȌ ͳͶ ୚୑୅୘

ᇱ ୴ୟୡ୭୲େଵଶି୴୫ୡ୊୊୅େଵଶ  ሾݐሿ ൌൌ ȋͶ͸Ȍ ͳʹ ୚୑୅୘

ᇱ ୴ୟୡ୭୲େଵ଴ି୴୫ୡ୊୊୅େଵ଴  ሾݐሿ ൌൌ ȋͶ͹Ȍ ͳͲ ୚୑୅୘

ᇱ ୴ୟୡ୭୲େ଼ି୴୫ୡ୊୊୅େ଼  ሾݐሿ ൌൌ ȋͶͺȌ ͺ ୚୑୅୘

ᇱ ୴ୟୡ୭୲େ଺ି୴୫ୡ୊୊୅େ଺  ሾݐሿ ൌൌ ȋͶͻȌ ͸ ୚୑୅୘

ͳ͹͹ Chapter 5

ᇱ ୴ୟୡ୭୲େସି୴୫ୡ୊୊୅େସ  ሾݐሿ ൌൌ ȋͷͲȌ Ͷ ୚୑୅୘

ᇱ ି୴୦୫୥ୡ୪ା୴୦୫୥ୡୱଶ ሾݐሿ ൌൌ ȋͷͳȌ ‘  ୚୑୅୘

ᇱ ି୴ୠୢ୦ଵି୴୉୶୅ୡୣ୲୭ୟୡୣ୲ୟ୲ୣ୑୅୘ା୴୦୫୥ୡ୪ ሾݐሿ ൌൌ     ȋͷʹȌ ‡–ƒ–‡ ƒ‘–‡  ୚୑୅୘

ᇱ ୴ୠୢ୦ଵି୴୉୶୆୓ୌ୆ ሾݐሿ ൌൌ      ȋͷ͵Ȍ ‡–ƒ”›–—„›š‘”†›Šƒ–‡ ୚୑୅୘

ᇱ ି୴ୟୡୱେଵ଺ି୴ୡ୫୧୊୊୅େଵ଺ି୴୉୶୊୊୅େଵ଺ା୴୫ୡ୊୊୅େଵ଺  ሾݐሿ ൌൌ    ȋͷͶȌ ͳ͸ ୚େଢ଼୘

ᇱ ି୴ୟୡୱେଵସି୴ୡ୫୧୊୊୅େଵସି୴୉୶୊୊୅େଵସା୴୫ୡ୊୊୅େଵସ  ሾݐሿ ൌൌ   ȋͷͷȌ ͳͶ ୚େଢ଼୘

ᇱ ି୴ୟୡୱେଵଶି୴ୡ୫୧୊୊୅େଵଶି୴୉୶୊୊୅େଵଶା୴୫ୡ୊୊୅େଵଶ  ሾݐሿ ൌൌ     ȋͷ͸Ȍ ͳʹ ୚େଢ଼୘

ᇱ ି୴ୟୡୱେଵ଴ି୴ୡ୫୧୊୊୅େଵ଴ି୴୉୶୊୊୅େଵ଴ା୴୫ୡ୊୊୅େଵ଴  ሾݐሿ ൌൌ     ȋͷ͹Ȍ ͳͲ ୚େଢ଼୘

ᇱ ି୴ୟୡୱେ଼ି୴ୡ୫୧୊୊୅େ଼ି୴୉୶୊୊୅େ଼ା୴୫ୡ୊୊୅େ଼  ሾݐሿ ൌൌ     ȋͷͺȌ ͺ ୚େଢ଼୘

ᇱ ି୴ୟୡୱେ଺ି୴ୡ୫୧୊୊୅େ଺ି୴୉୶୊୊୅େ଺ା୴୫ୡ୊୊୅େ଺  ሾݐሿ ൌൌ     ȋͷͻȌ ͸ ୚େଢ଼୘

ᇱ ି୴ୟୡୱେସି୴ୡ୫୧୊୊୅େସି୴୉୶୊୊୅େସା୴୫ୡ୊୊୅େସ  ሾݐሿ ൌൌ     ȋ͸ͲȌ Ͷ ୚େଢ଼୘

ᇱ ୴ୟୡୱେଵସି୴ୡ୮୲ଵେଵସ  ሾݐሿ ൌൌ ȋ͸ͳȌ‘Ž› ͳͶ ୚େଢ଼୘

ᇱ ୴ୟୡୱେଵଶି୴ୡ୮୲ଵେଵଶ  ሾݐሿ ൌൌ ȋ͸ʹȌ‘Ž› ͳʹ ୚େଢ଼୘

ᇱ ୴ୟୡୱେଵ଴ି୴ୡ୮୲ଵେଵ଴  ሾݐሿ ൌൌ ȋ͸͵Ȍ‘Ž› ͳͲ ୚େଢ଼୘

ᇱ ୴ୟୡୱେ଼ି୴ୡ୮୲ଵେ଼  ሾݐሿ ൌൌ ȋ͸ͶȌ‘Ž› ͺ ୚େଢ଼୘

ᇱ ୴ୟୡୱେ଺ି୴ୡ୮୲ଵେ଺  ሾݐሿ ൌൌ ȋ͸ͷȌ‘Ž› ͸ ୚େଢ଼୘

ᇱ ୴ୟୡୱେସି୴ୡ୮୲ଵେସ  ሾݐሿ ൌൌ ȋ͸͸Ȍ‘Ž› Ͷ ୚େଢ଼୘

ᇱ ୴ୡ୫୧୊୊୅େଵ଺ି୴ன୭୶େଵ଺   ሾݐሿ ൌൌ ȋ͸͹Ȍ ͳ͸ ୚୑୍ୈ

ᇱ ୴ୡ୫୧୊୊୅େଵସି୴ன୭୶େଵସ   ሾݐሿ ൌൌ ȋ͸ͺȌ ͳͶ ୚୑୍ୈ

ᇱ ୴ୡ୫୧୊୊୅େଵଶି୴ன୭୶େଵଶ   ሾݐሿ ൌൌ ȋ͸ͻȌ ͳʹ ୚୑୍ୈ

ᇱ ୴ୡ୫୧୊୊୅େଵ଴ି୴ன୭୶େଵ଴   ሾݐሿ ൌൌ ȋ͹ͲȌ ͳͲ ୚୑୍ୈ

ᇱ ୴ୡ୫୧୊୊୅େ଼ି୴ன୭୶େ଼   ሾݐሿ ൌൌ        ȋ͹ͳȌ ͺ ୚୑୍ୈ

ᇱ ୴ୡ୫୧୊୊୅େ଺ି୴ன୭୶େ଺   ሾݐሿ ൌൌ        ȋ͹ʹȌ ͸ ୚୑୍ୈ

ͳ͹ͺ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

ᇱ ୴ୡ୫୧୊୊୅େସି୴ன୭୶େସ   ሾݐሿ ൌൌ        ȋ͹͵Ȍ Ͷ ୚୑୍ୈ

ᇱ ି୴୫୧ୡୈେ୅େଵ଺ା୴ன୭୶େଵ଺ ͳ͸  ሾݐሿ ൌൌ       ȋ͹ͶȌ ୚୑୍ୈ

ᇱ ି୴୫୧ୡୈେ୅େଵସା୴ன୭୶େଵସ ͳͶ  ሾݐሿ ൌൌ       ȋ͹ͷȌ ୚୑୍ୈ

ᇱ ି୴୫୧ୡୈେ୅େଵଶା୴ன୭୶େଵଶ ͳʹ  ሾݐሿ ൌൌ       ȋ͹͸Ȍ ୚୑୍ୈ

ᇱ ି୴୫୧ୡୈେ୅େଵ଴ା୴ன୭୶େଵ଴ ͳͲ  ሾݐሿ ൌൌ       ȋ͹͹Ȍ ୚୑୍ୈ

ᇱ ି୴୫୧ୡୈେ୅େ଼ା୴ன୭୶େ଼ 5 ͺ  ሾݐሿ ൌൌ       ȋ͹ͺȌ ୚୑୍ୈ

ᇱ ି୴୫୧ୡୈେ୅େ଺ା୴ன୭୶େ଺ ͸  ሾݐሿ ൌൌ       ȋ͹ͻȌ ୚୑୍ୈ

ᇱ ି୴୫୧ୡୈେ୅େସା୴ன୭୶େସ Ͷ  ሾݐሿ ൌൌ       ȋͺͲȌ ୚୑୍ୈ

ᇱ ି୴୉୶ୈେ୅େଵ଺ା୴୫୧ୡୈେ୅େଵ଺  ሾݐሿ ൌൌ       ȋͺͳȌͳ͸ ୚େଢ଼୘

ᇱ ି୴୉୶ୈେ୅େଵସା୴୫୧ୡୈେ୅େଵସ  ሾݐሿ ൌൌ       ȋͺʹȌͳͶ ୚େଢ଼୘

ᇱ ି୴୉୶ୈେ୅େଵଶା୴୫୧ୡୈେ୅େଵଶ  ሾݐሿ ൌൌ       ȋͺ͵Ȍͳʹ ୚େଢ଼୘

ᇱ ି୴୉୶ୈେ୅େଵ଴ା୴୫୧ୡୈେ୅େଵ଴  ሾݐሿ ൌൌ       ȋͺͶȌͳͲ ୚େଢ଼୘

ᇱ ି୴୉୶ୈେ୅େ଼ା୴୫୧ୡୈେ୅େ଼  ሾݐሿ ൌൌ        ȋͺͷȌͺ ୚େଢ଼୘

ᇱ ି୴୉୶ୈେ୅େ଺ା୴୫୧ୡୈେ୅େ଺  ሾݐሿ ൌൌ       ȋͺ͸Ȍ͸ ୚େଢ଼୘

ᇱ ି୴୉୶ୈେ୅େସା୴୫୧ୡୈେ୅େସ  ሾݐሿ ൌൌ       ȋͺ͹ȌͶ ୚େଢ଼୘ 

ͳ͹ͻ Chapter 5

ZĂƚĞĞƋƵĂƚŝŽŶƐ ‘•–‘ˆ–Š‡‡œ›‡•‹–Š‹•‘†‡Ž ƒ–ƒŽ›œ‡”‡ƒ –‹‘•‘ˆ•—„•–”ƒ–‡•‘ˆ‘”‡–Šƒ‘‡ Šƒ‹Ž‡‰–ŠǤ ‘”•— Š”‡ƒ –‹‘•–Š‡”ƒ–‡‡“—ƒ–‹‘•”‡ƒ‹ –Š‡•ƒ‡Ǥ •— Š ƒ•‡•ǡ–Š‡‰‡‡”ƒŽ”ƒ–‡‡“—ƒ–‹‘™ƒ•‰‹˜‡ǡ™Š‡”‡‹†‹ ƒ–‡•–Š‡ ƒ”„‘Ǧ Šƒ‹Ž‡‰–Š‘ˆƒ•—„•–”ƒ–‡ǤŠ‡’ƒ”ƒ‡–‡”•‹–Š‡

‡“—ƒ–‹‘• ƒ”‡ ‘•– ‘ˆ–‡ Šƒ‹ǦŽ‡‰–ŠǦ•’‡ ‹ˆ‹ ǡ ƒ• ‹†‹ ƒ–‡† ‹ –Š‡ •—„• ”‹’– –‘ –Š‡ ’ƒ”ƒ‡–‡” ȋ‡Ǥ‰Ǥ Km ’–ͳͳ͸ ›Ž‘ †‡‘–‹‰ –Š‡  ‘ˆ –Š‡ ‡œ›‡ͳ–‘™ƒ”†•ƒƒ ›ŽǦ‘•—„•–”ƒ–‡™‹–Šƒƒ ›Ž Šƒ‹ ‘–ƒ‹‹‰ͳ͸ ƒ”„‘ƒ–‘•ǡŽ‘ ƒŽ‹œ‡†‹–Š‡ ›–‘•‘ŽȌǤ‘•–‡“—ƒ–‹‘•ƒ”‡‘ˆ–Š‡ ʹǦ•—„•–”ƒ–‡ ”‡˜‡”•‹„Ž‡ ‹ Šƒ‡Ž‹•Ǧ‡–‡ –›’‡ ‘” ‹ ‡š ‡’–‹‘ƒŽ ƒ•‡• „ƒ•‡† ‘ ƒ••Ǧƒ –‹‘ ‹‡–‹ •Ǥ Š‡ ”‡ƒ –‹‘ ”ƒ–‡• ‹ –Š‡ ‘†‡Ž ƒ”‡ ‡š’”‡••‡†‹Ɋ‘Žȉ‹Ǧͳȉ‰‹–‘ Š‘†”‹ƒŽ”‘–‡‹Ǧͳǡƒ†–Š‡”ƒ–‡‘ˆ‡–ƒ„‘Ž‹–‡ ‘ ‡–”ƒ–‹‘ Šƒ‰‡‹–Š‡•ƒ”‡‡š’”‡••‡†ƒ•Ɋȉ‹ǦͳǤ  Ϊ –Š‡‘†‡Žǡ–Š‡ ‘ˆƒ –‘”•ǡ‘ǡ‘ ʹǡ ǡ ƒ†ƒ ‡–›ŽǦ‘ƒ”‡ ‘•‹†‡”‡†‡š–‡”ƒŽȀ„‘—†ƒ”›’ƒ”ƒ‡–‡”•ƒ†Šƒ˜‡„‡‡ˆ‹š‡††‹”‡ –Ž›Ǥ Š‹• ‹• ‹ ‘–”ƒ•– –‘ –Š‡ —‡ et alǤ ʹͲͳ͵ ‘†‡Žǡ ™Š‡”‡ ƒ ‡–›ŽǦ‘ ƒ† –Š‡ ‘ˆƒ –‘”• ‹ –Šƒ– ‘†‡Ž Šƒ† „‡‡ ˆ‹š‡† ‹†‹”‡ –Ž› ˜‹ƒ ƒ •‹ ”‡ƒ –‹‘–Šƒ–Šƒ†‘ˆŽ—š ‘–”‘Žǡ„—– ‘–ƒ‹‡†ƒ’ƒ”ƒ‡–‡”–Šƒ–‡ˆˆ‡ –‹˜‡Ž›ˆ‹š‡†–Š‡•‡‡–‹–‹‡•Ǥ‹”‡ –Ž›ˆ‹š‹‰–Š‡•‡‡–ƒ„‘Ž‹–‡•‹’”‘˜‡•–Š‡

•–ƒ„‹Ž‹–›‘ˆ–Š‡‘†‡Žƒ††‡ ”‡ƒ•‡•–Š‡•–‹ˆˆ‡••‘ˆ–Š‡•Ǥ –Š‡”ƒ–‡‡“—ƒ–‹‘•ǡ–Š‡ƒš˜ƒŽ—‡•ƒ”‡†‡‘–‡†ƒ•ǡƒ†–Š‡ ƒ”„‘Ǧ Šƒ‹

Ž‡‰–Š‘ˆ•—„•–”ƒ–‡•‘”’”‘†— –•ƒ”‡‰‹˜‡„›Ǥ˜ƒŽ—‡•†‡‘–‡–Š‡ƒˆˆ‹‹–›‘ˆ–Š‡‡œ›‡ˆ‘”–Š‡‹†‹ ƒ–‡†”‡ƒ –ƒ–ǡ‹˜ƒŽ—‡•ƒ”‡‹Š‹„‹–‹‘

‘•–ƒ–‘ˆ–Š‡‡œ›‡ˆ‘”–Š‡‹†‹ ƒ–‡†‡–ƒ„‘Ž‹–‡ǡƒ†‡“˜ƒŽ—‡•†‡‘–‡–Š‡‡“—‹Ž‹„”‹— ‘•–ƒ–‘ˆ–Š‡”‡ƒ –‹‘Ǥ††‹–‹‘ƒŽƒ„„”‡˜‹ƒ–‹‘• ƒ”‡ǣ  •ˆ ’‡ ‹ˆ‹ ‹–›ˆƒ –‘”Ǥ—Ž–‹’Ž›‹‰–Š‡•ˆ™‹–ŠVmax of the enzyme ™‹ŽŽ‰‹˜‡–Š‡ Šƒ‹ǦŽ‡‰–ŠǦ•’‡ ‹ˆ‹ ƒš‘ˆ–Šƒ–‡œ›‡  ’–ͳ ‹ŽŽ ‘‡ˆˆ‹ ‹‡–‘ˆˆ‘”–Š‡ ‘‘’‡”ƒ–‹˜‡‹Š‹„‹–‹‘‘ˆͳ„›ƒŽ‘›ŽǦ‘ – ‘–ƒŽ ‘ ‡–”ƒ–‹‘‘ˆ‘š‹†‹œ‡†ƒ†”‡†— ‡†‹–Š‡‹†‹ ƒ–‡†‘”‰ƒ‡ŽŽ‡ ‘– ‘–ƒŽ ‘ ‡–”ƒ–‹‘ˆ”‡‡‘ƒ†‘‡•–‡”•‹–Š‡‹†‹ ƒ–‡†‘”‰ƒ‡ŽŽ‡ ƒ” ƒ”‹–‹‡ ͳ ƒ”‹–‹‡Ǧ’ƒŽ‹–‘›Ž–”ƒ•ˆ‡”ƒ•‡ͳ  ƒ”‹–‹‡Ǧƒ ›ŽǦ ƒ”‹–‹‡–”ƒ•Ž‘ ƒ•‡ ʹ ƒ”‹–‹‡Ǧ’ƒŽ‹–‘›Ž–”ƒ•ˆ‡”ƒ•‡ʹ  Š‘”–Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡  ‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡  ‡”›ǦŽ‘‰Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡  ”‘–‘ƒ•‡‘”‘›Ž‘‡œ›‡ ›†”ƒ–ƒ•‡ Ȁ  ‡†‹—Ȁ•Š‘”–Ǧ Šƒ‹Š›†”‘š›ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡  ‡†‹—Ǧ Šƒ‹‡–‘ƒ ›ŽǦ‘–Š‹‘Žƒ•‡  ‹–‘ Š‘†”‹ƒŽ–”‹ˆ— –‹‘ƒŽ’”‘–‡‹    †‡Š›†”‘‰‡ƒ•‡‘” “—‹‘‡‘š‹†‘”‡†— –ƒ•‡

ͳͺͲ ͳͺͳ Ž‡ǡ Šƒ‹Ǧ †ƒ’–‡†Ǥ -acid catabolism -acid catabolism Ͷͳͳ 

5   ȋͺͺȌ ሻ ቁ ency in human liver fatty –‹‘”‡ƒ –‹‘ ƒ–ƒŽ›œ‡†„› ‡ ‹ˆ‹ ’ƒ”ƒ‡–‡”˜ƒŽ—‡•Ǥ ‘”‡šƒ’ ి౤ఽౙ౯ౢి౥ఽిౕ౐ሾ೟ሿ ሻ ేౣౙ౦౪భి౤ఽౙ౯ౢి౥ఽిౕ౐ ା ˆ‘”‘ ˆ‘”Ǥ       ȋͺͻȌ ሿ ȋͻͳȌ ௧ ሻ ሾ ሻ ቁ ‹ˆ‹  ȋͻͲȌ ሻ modifiers on MCAD defici modifiers ి౤ఽౙ౯ౢి౗౨ిౕ౐ሾ೟ሿ ሻ ቁ ేౣౙ౦౪భి౤ఽౙ౯ౢి౗౨ిౕ౐ ቀ ŽȌ ‡–›ŽǦ‘–Š‹‘Žƒ•‡ ˆ’—–ƒ–‹˜‡”ƒ–‡ǦŽ‹‹–‹‰Š›†”‘š›Žƒ ೙సభర ೙సర ሻ σ ା ి౤ఽౙ౯ౢి౗౨ిౕ౐ሾ೟ሿήి౥ఽిౕ౐ ి౤ు౤౥౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ”‘•‘‡–‘–Š‡ ›–‘•‘Ž ƒŽŽ”ƒ–‡‡“—ƒ–‹‘•ƒ”‡‡‹–Š‡”‡™ǡ†—‡–‘ƒ††‡†”‡ƒ –‹‘•ǡ‘”ƒ ‘ƒ ‘‘†ƒ–‡ Šƒ‹ǦŽ‡‰–ŠǦ•’ ˆ”‘–Š‡‹–‘ Š‘†”‹‘–‘–Š‡ ›–‘•‘Ž –Š‡ ›–‘•‘Ž‹–‘–Š‡„Ž‘‘† ‘Ž–‘–Š‡‹ ”‘•‘‡ ేౣ౬ౢౙ౗ౚి౤ు౤౥౯ౢి౥ఽ౉ఽ౐ ౤ౙ౦౪భ ሻ ା ి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ା୏୫ୡୟୡ୲େୟ୰୑୅୘ήେ୬୅ୡ୷୪େୟ୰େଢ଼୘ ሿ ే౛౧ౙ౦౪భήేౣౙ౦౪భి౤ఽౙ౯ౢి౥ఽిౕ౐ήేౣౙ౦౪భి౗౨ిౕ౐ ేౣౙ౦౪మి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ ௧ ే౛౧ౙ౗ౙ౪ ି ሾ ା ి౤ు౤౥౯ౢి౥ఽ౉ఽ౐ሾ೟ሿήు౐ూ౞౧౉ఽ౐ሾ೟ሿ ౉౗ౢి౥ఽిౕ౐ ి౗౨౉ఽ౐ήిభలఽౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ి౗౨ిౕ౐ήి౤ఽౙ౯ౢి౗౨౉ఽ౐ሾ೟ሿ Simulating the impact of ే౟ౙ౦౪భή౉౗ౢి౥ఽిౕ౐ ే౛౧౬ౢౙ౗ౚήేౣ౬ౢౙ౗ౚి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ήేౣ౬ౢౙ౗ౚు౐ూ౥౮ ାሺ ି ి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ేౣ౬ౢౙ౗ౚి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ ቀ ‘”ͳƒ•™ƒ• Šƒ‹ǦŽ‡‰–ŠǦ•’‡ ే౛౧ౙ౦౪మήేౣౙ౦౪మిభలఽౙ౯ౢి౗౨౉ఽ౐ήేౣౙ౦౪మి౥ఽ౉ఽ౐ ‹•”‡’”‡•‡–‡†„›–Š‡”ƒ–‡‘ ି ి౤ఽౙ౯ౢి౗౨౉ఽ౐ሾ೟ሿ ‡ͳȋ‹–‘ Š‘†”‹ƒŽȌ‘” ‡–‘ƒ ŽǦ‘Ž›ƒ•‡‘”  Ǧ‘Ž›ƒ•‡ ేౣౙ౦౪మి౤ఽౙ౯ౢి౗౨౉ఽ౐ ೙సభల ೙సల ቀ σ ౜౒ౙ౗ౙ౪ήే౛౧ౙ౗ౙ౪ ‡ˆƒ––›ƒ ‹†•‘ˆ Šƒ‹Ž‡‰–Š ೙సభల ೙సర —–ƒ”›ŽǦ‘•›–Šƒ•‡ʹȋ‹–‘ Š‘†”‹ƒ ሻήሺଵା ి౤ఽౙ౯ౢి౥ఽిౕ౐ήి౗౨ిౕ౐ σ ˆ”‡‡ˆƒ––›ƒ ‹†•ˆ”‘–Š‡ ›–‘• †‹ ƒ”„‘š›Ž‹ ƒ ‹†•ˆ”‘–Š‡‹ ిభలఽౙ౯ౢి౥ఽిౕ౐ ƒ”‹–‹‡‘ˆ Šƒ‹Ž‡‰–Šˆ”‘ ሻήి౤ఽౙ౯ౢి౗౨౉ఽ౐ሾ೟ሿశి౗౨ిౕ౐ήሺేౣౙ౗ౙ౪ి౤ఽౙ౯ౢి౗౨౉ఽ౐శి౤ఽౙ౯ౢి౗౨౉ఽ౐ሾ೟ሿሻ ሻሺଵା ేౣౙ౦౪భిభలఽౙ౯ౢి౥ఽిౕ౐ ‘ˆ’ƒ”ƒ‡–‡”•ƒ†‡š’ƒ•‹‘– ǡǡƒ†Ȁ ǡ ేౣౙ౦౪భి౤ఽౙ౯ౢి౥ఽిౕ౐ήేౣౙ౦౪భి౗౨ిౕ౐ ሻሺଵା ‘ˆˆ”‡‡ˆƒ––›ƒ ‹†• ు౐ూ౞౧౉ఽ౐ሾ೟ሿ ేౣ౬ౢౙ౗ౚు౐ూ౞౧ ା ి౥ఽ౉ఽ౐ ቁή୏୫ୡୟୡ୲େ୬୅ୡ୷୪େୟ୰େଢ଼୘ାେୟ୰୑୅୘ήେ୬୅ୡ୷୪େୟ୰େଢ଼୘ ేౣౙ౦౪మి౥ఽ౉ఽ౐ ి౤ఽౙ౯ౢి౗౨ిౕ౐ሾ೟ሿ ి౥ఽిౕ౐ ା ి౥ఽ౉ఽ౐ήిభలఽౙ౯ౢి౗౨౉ఽ౐ሾ೟ሿ ୱ୤ୡ୮୲ଵେ୬ή୚ୡ୮୲ଵሺ ే౟ౙ౗ౙ౪ήి౤ఽౙ౯ౢి౗౨ిౕ౐ ేౣ౬ౢౙ౗ౚి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ήేౣ౬ౢౙ౗ౚు౐ూ౥౮ ୱ୤ୡୟୡ୲େ୬ή୚୤ୡୟୡ୲ήሺେୟ୰୑୅୘ήେ୬୅ୡ୷୪େୟ୰େଢ଼୘ሾ௧ሿି ేౣౙ౦౪భి౥ఽిౕ౐ ి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿήሺు౐ూ౪౉ఽ౐షు౐ూ౞౧౉ఽ౐ሾ೟ሿሻ ା ేౣౙ౦౪మిభలఽౙ౯ౢి౗౨౉ఽ౐ήేౣౙ౦౪మి౥ఽ౉ఽ౐ ి౗౨ిౕ౐ ే౟ౙ౗ౙ౪ి౗౨ిౕ౐ ˆ‘”Ǧ ƒ”‹–‹‡™ƒ•‹ Ž—†‡†ˆ ి౗౨౉ఽ౐ ేౣ౬ౢౙ౗ౚు౐ూ౥౮  ేౣౙ౦౪మి౗౨౉ఽ౐ ి౗౨ిౕ౐ ు౐ూ౪౉ఽ౐షు౐ూ౞౧౉ఽ౐ሾ೟ሿ ሺଵା ేౣౙ౗ౙ౪ి౗౨ిౕ౐ήሺభశ ేౣౙ౦౪భి౗౨ిౕ౐ ሺଵା ା ୱ୤୴୪ୡୟୢେ୬ή୚୴୪ୡୟୢሺ ୱ୤ୡ୮୲ଶେ୬ή୚ୡ୮୲ଶሺ ሺଵା େୟ୰୑୅୘ήቀଵା †ƒ’–ƒ–‹‘•‹ Ž—†‡†Š—ƒ‹œƒ–‹‘ Ž‡‰–ŠǦ•’‡ ‹ˆ‹  ˜ ƒ – ൌ ͳ  ‡–›ŽǦ‘ƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•  š ‡’–ˆ‘”–Š‡”ƒ–‡‡“—ƒ–‹‘•ˆ‘” ˜ ’–ͳ ൌ ˜˜Ž ƒ† ൌ   ʹ ͵ǦŠ›†”‘š›Ǧ͵Ǧ‡–Š›Ž‰Ž ˜ ˜š ‡–‘ƒ ‡–ƒ–‡˜š ˜š  š’‘”–”ƒ–‡‘ˆƒ ‡–‘ƒ ‡–ƒ–‡ ˜ ‹  ”ƒ•Ž‘ ƒ–‹‘”ƒ–‡‘ˆˆ”‡ ˜‹ ˜š š’‘”–”ƒ–‡‘ˆȾǦŠ›†”‘š›„—–›”ƒ–‡ š’‘”–”ƒ–‡ ”ƒ•Ž‘ ƒ–‹‘”ƒ–‡‘ˆ ”ƒ•Ž‘ ƒ–‹‘”ƒ–‡‘ˆ š’‘”–”ƒ–‡‘ˆ†‹ ƒ”„‘š›Ž‹ ƒ ‹†•   ͳɘ‘š˜š  ͵ǦŠ›†”‘š›Ǧ͵Ǧ‡–Š›Ž‰Ž—–ƒ”› Ǧ„‡–ƒǦŠ›†”‘š›„—–›”ƒ–‡†‡Š›†”‘‰‡ƒ•‡ȋ‹–‘ Š‘†”‹ƒŽȌ  ›ŽǦ‘–Š‹‘‡•–‡”ƒ•‡  ›ŽǦ‘•›–Š‡–ƒ•‡ š’‘”–”ƒ–‡‘ˆƒ ›ŽǦ ɘǦ‘š‹†ƒ–‹‘‘ˆ™Š‹ Š–Š‡”ƒ–‡ ˜ ’–ʹ ൌ Chapter 5

ి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿήሺు౐ూ౪౉ఽ౐షు౐ూ౞౧౉ఽ౐ሾ೟ሿሻ ి౤ు౤౥౯ౢి౥ఽ౉ఽ౐ሾ೟ሿήు౐ూ౞౧౉ఽ౐ሾ೟ሿ ୱ୤୫ୡୟୢେ୬ή୚୫ୡୟୢሺ ି ሻ ేౣౣౙ౗ౚి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ήేౣౣౙ౗ౚు౐ూ౥౮ ే౛౧ౣౙ౗ౚήేౣౣౙ౗ౚి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ήేౣౣౙ౗ౚు౐ూ౥౮ ˜ ƒ† ൌ ు౐ూ౪౉ఽ౐షు౐ూ౞౧౉ఽ౐ሾ೟ሿ ు౐ూ౞౧౉ఽ౐ሾ೟ሿ ి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ి౤ు౤౥౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ȋͻʹȌ ሺଵା ା ሻήሺଵାσ೙సభలቀ ା ቁሻ ేౣౣౙ౗ౚు౐ూ౥౮ ేౣౣౙ౗ౚు౐ూ౞౧ ೙సర ేౣౣౙ౗ౚి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ ేౣౣౙ౗ౚి౤ు౤౥౯ౢి౥ఽ౉ఽ౐

ి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿήሺు౐ూ౪౉ఽ౐షు౐ూ౞౧౉ఽ౐ሾ೟ሿሻ ి౤ు౤౥౯ౢి౥ఽ౉ఽ౐ሾ೟ሿήు౐ూ౞౧౉ఽ౐ሾ೟ሿ ୱ୤୫ୱୡୟୢେ୬ή୚ୱୡୟୢሺ ି ሻ ేౣ౩ౙ౗ౚి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ήేౣ౩ౙ౗ౚు౐ూ౥౮ ే౛౧౩ౙ౗ౚήేౣ౩ౙ౗ౚి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ήేౣ౩ౙ౗ౚు౐ూ౥౮ ˜• ƒ† ൌ ు౐ూ౪౉ఽ౐షు౐ూ౞౧౉ఽ౐ሾ೟ሿ ు౐ూ౞౧౉ఽ౐ሾ೟ሿ ి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ి౤ు౤౥౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ      ȋͻ͵Ȍ ሺଵା ା ሻήሺଵାσ೙సలቀ ା ቁሻ ేౣ౩ౙ౗ౚు౐ూ౥౮ ేౣ౩ౙ౗ౚు౐ూ౞౧ ೙సర ేౣ౩ౙ౗ౚి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ ేౣ౩ౙ౗ౚి౤ు౤౥౯ౢి౥ఽ౉ఽ౐

ి౤ు౤౥౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ి౤ౄ౯ౚ౨౥౮౯౗ౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ୱ୤ୡ୰୭୲େ୬ή୚ୡ୰୭୲ήሺ ି ሻ ేౣౙ౨౥౪ి౤ు౤౥౯ౢి౥ఽ౉ఽ౐ ే౛౧ౙ౨౥౪ేౣౙ౨౥౪ήి౤ు౤౥౯ౢి౥ఽ౉ఽ౐ ˜ ”‘– ൌ ఽౙ౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ి౤ు౤౥౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ి౤ౄ౯ౚ౨౥౮౯౗ౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ       ȋͻͶȌ ଵା ାσ೙సభలቀ ା ቁ ే౟ౙ౨౥౪ఽౙ౛౪౥౗ౙ౯ౢి౥ఽ ೙సర ేౣౙ౨౥౪ి౤ు౤౥౯ౢి౥ఽ౉ఽ౐ ేౣౙ౨౥౪ి౤ౄ౯ౚ౨౥౮౯౗ౙ౯ౢి౥ఽ౉ఽ౐

ሺషొఽీౄ౉ఽ౐శొఽీ౪౉ఽ౐ሻήి౤ౄ౯ౚ౨౥౮౯౗ౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ొఽీౄ౉ఽ౐ήి౤ే౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ୱ୤୫ୱୡ୦ୟୢେ୬ή୚୫ୱୡ୦ୟୢሺ ି ሻ ేౣౣ౩ౙ౞౗ౚి౤ౄ౯ౚ౨౥౮౯౗ౙ౯ౢి౥ఽ౉ఽ౐ήేౣౣ౩ౙ౞౗ౚొఽీ౉ఽ౐ ే౛౧ౣ౩ౙ౞౗ౚήేౣౣ౩ౙ౞౗ౚి౤ౄ౯ౚ౨౥౮౯౗ౙ౯ౢి౥ఽ౉ఽ౐ήేౣౣ౩ౙ౞౗ౚొఽీ౉ఽ౐ ˜• Šƒ† ൌ ొఽీౄ౉ఽ౐ షొఽీౄ౉ఽ౐శొఽీ౪౉ఽ౐ ి౤ౄ౯ౚ౨౥౮౯౗ౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ి౤ే౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ  ȋͻͷȌ ሺଵା ା ሻήሺଵାσ೙సభలቀ ା ቁሻ ేౣౣ౩ౙ౞౗ౚొఽీౄ౉ఽ౐ ేౣౣ౩ౙ౞౗ౚొఽీ౉ఽ౐ ೙సర ేౣౣ౩ౙ౞౗ౚి౤ౄ౯ౚ౨౥౮౯౗ౙ౯ౢి౥ఽ౉ఽ౐ ేౣౣ౩ౙ౞౗ౚి౤ే౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐

ి౥ఽ౉ఽ౐ήి౤ే౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ి౤Ǧʹήఽౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ୱ୤୫ୡ୩ୟ୲େ୬ή୚୫ୡ୩ୟ୲ήሺ ି ሻ ేౣౣౙౡ౗౪ి౤ే౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐ήేౣౣౙౡ౗౪ి౥ఽ౉ఽ౐ి౤ ే౛౧ౣౙౡ౗౪ήేౣౣౙౡ౗౪ి౤ే౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐ήేౣౣౙౡ౗౪ి౥ఽ౉ఽ౐ి౤ ˜ ƒ– ൌ ఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ ి౥ఽ౉ఽ౐ ఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ ి౤Ǧʹ ›Ž‘ሾ೟ሿ ి౤ే౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ  ȋͻ͸Ȍ ሺଵା ା ሻήሺଵା ାσ೙సభలቀ ା ቁሻ ేౣౣౙౡ౗౪ఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ ేౣౣౙౡ౗౪ి౥ఽ౉ఽ౐ి౤ ేౣౣౙౡ౗౪ఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ ೙సర ేౣౣౙౡ౗౪ి౤Ǧʹ ›Ž‘ ేౣౣౙౡ౗౪ి౤ే౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐

ఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ήొఽీౄ౉ఽ౐ήిభరఽౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ి౥ఽ౉ఽ౐ήሺషొఽీౄ౉ఽ౐శొఽీ౪౉ఽ౐ሻήిభలు౤౥౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ୱ୤୫୲୮େଵ଺ή୚୫୲୮ሺି ା ሻ ే౛౧ౣ౪౦ήేౣౣ౪౦ిభలు౤౥౯ౢి౥ఽ౉ఽ౐ήేౣౣ౪౦ి౥ఽ౉ఽ౐ήేౣౣ౪౦ొఽీ౉ఽ౐ ేౣౣ౪౦ిభలు౤౥౯ౢి౥ఽ౉ఽ౐ήేౣౣ౪౦ి౥ఽ౉ఽ౐ήేౣౣ౪౦ొఽీ౉ఽ౐ ˜–’ ൌή ఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ ి౥ఽ౉ఽ౐ ొఽీౄ౉ఽ౐ షొఽీౄ౉ఽ౐శొఽీ౪౉ఽ౐ ి౤Ǧʹ ›Ž‘ሾ೟ሿ ి౤ు౤౥౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ȋͻ͹Ȍ ሺଵା ା ሻήሺଵା ା ሻήሺଵାσ೙సభలቀ ା ቁሻ ేౣౣ౪౦ఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ ేౣౣ౪౦ి౥ఽ౉ఽ౐ ేౣౣ౪౦ొఽీౄ౉ఽ౐ ేౣౣ౪౦ొఽీ౉ఽ౐ ೙సఴ ేౣౣ౪౦ి౤Ǧʹ ›Ž‘ ేౣౣ౪౦ి౤ు౤౥౯ౢి౥ఽ౉ఽ౐

ి౥్ౄమሺు౐ూ౪౉ఽ౐షు౐ూ౞౧౉ఽ౐ሾ೟ሿሻ ୚୤ୣ୲୤୯୭ήሺି ାሺେ୭୕୲ିେ୭୕ୌଶሻή୉୘୊୦୯୑୅୘ሾ௧ሿሻ ే౛౧ు౪౜౧౥ ˜‡–ˆ“‘ ൌ ి౥్ౄమ ȋͻͺȌ ሺିେ୭୕ୌଶାେ୭୕୲ሻήቀଵା ቁή୏୫୉୲୤୯୭୉୲୤୦୯ାሺିେ୭୕ୌଶାେ୭୕୲ሻή୉୘୊୦୯୑୅୘ሾ௧ሿା୏୫୉୲୤୯୭େ୭୕ή୉୘୊୦୯୑୅୘ሾ௧ሿ ే౟ు౪౜౧౥ి౥్ౄమ ు౐ూ౞౧౉ఽ౐ሾ೟ሿ ి౥్ౄమήሺు౐ూ౪౉ఽ౐శేౣు౪౜౧౥ు౪౜౥౮షు౐ూ౞౧౉ఽ౐ሾ೟ሿሻశేౣు౪౜౧౥ి౥్ౄమήሺు౐ూ౪౉ఽ౐షు౐ూ౞౧౉ఽ౐ሾ೟ሿሻήሺభశ ሻ ା ే౟ు౪౜౧౥ు౪౜౞౧ ే౛౧ు౪౜౧౥

ి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ి౥ఽ౉ఽ౐ήి౤ూూఽ౉ఽ౐ሾ೟ሿ ୱ୤ୟୡ୭୲େ୬ή୚ୟୡ୭୲ήሺ ି ሻ ేౣ౗ౙ౥౪ి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ ే౛౧౗ౙ౥౪ి౤ఽౙ౯ౢి౥ఽήేౣ౗ౙ౥౪ి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ ˜ƒ ‘– ൌ ి౥ఽ౉ఽ౐ ి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ి౤ూూఽ౉ఽ౐ሾ೟ሿ ȋͻͻȌ ሺଵା ሻሺଵାσ೙సభలቀ ା ቁሻ ేౣ౗ౙ౥౪ి౥ఽ౉ఽ౐ ೙సర ేౣ౗ౙ౥౪ి౤ఽౙ౯ౢి౥ఽ౉ఽ౐ ేౣ౗ౙ౥౪ి౤ూూఽ౉ఽ౐

ሾݐሿȋͳͲͲȌ”ƒŽ›  ൌ ‡ ή š˜

ሾݐሿሻȋͳͲͳȌ  ሾݐሿ െ  ή ሺ  ൌ  ˜

ి౥ఽ౉ఽ౐ήిరఽౙ౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ ఽౙ౛౪౯ౢి౥ఽ౉ఽ౐మ ୚ୟୡୟ୲ଵήሺ ି ሻ ేౣ౗ౙ౗౪భిరఽౙ౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐ήేౣ౗ౙ౗౪భి౥ఽ౉ఽ౐ ే౛౧౗ౙ౗౪భήేౣ౗ౙ౗౪భిరఽౙ౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐ήేౣ౗ౙ౗౪భి౥ఽ౉ఽ౐ ˜ƒ ƒ–ͳ ൌ െ ఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ ి౥ఽ౉ఽ౐ ఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ ిరఽౙ౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ      ȋͳͲʹȌ ሺଵା ା ሻήሺଵା ା ሻ ేౣ౗ౙ౗౪భఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ ేౣ౗ౙ౗౪భి౥ఽ౉ఽ౐ ేౣ౗ౙ౗౪భఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ ేౣ౗ౙ౗౪భిరఽౙ౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐

ͳͺʹ  ͳͺ͵ ሿ ݐ ሾ  -acid catabolism -acid catabolism  ȋͳͲͶȌ ȋͳͲͻȌ ሻሻ ቁ ሻ ሿ ݐ ሾ 5 ి౤ూూఽిౕ౐ሾ೟ሿ ేౣ౗ౙ౩ి౤ూూఽిౕ౐ ቀ ency in human liver fatty ೙సభల ೙సర ఽౙ౛౪౥౗ౙ౛౪౗౪౛౉ఽ౐ሾ೟ሿήౄ౉ృి౥ఽ౉ఽ౐ሾ೟ሿ ȋͳͲ͵Ȍ σ ሻ ሻήሺ ”‡ƒ –‹‘™‹–Š͵•—„•–”ƒ–‡•Ǥ ేౣ౞ౣౝౙౢఽౙ౛౪౥౗ౙ౛౪౗౪౛౉ఽ౐ήేౣ౞ౣౝౙౢౄ౉ృి౥ఽ౉ఽ౐ ା „†Šͳ ή  ‡–ƒŠ›†”‘š›„—–›”ƒ–‡ ‹„†Šͳ ‡–‘ƒ ‡–ƒ–‡ ή ‹„†Šͳ ή „†Šͳ‡–ƒŠ›†”‘š›„—–›”ƒ–‡ ‡–ƒ–‡ሾݐሿ ή ‡–ƒŠ›†”‘š›„—–›”ƒ–‡ሾݐሿ ƒ‘–‡   ൅ –ሻ ή ሺെ   ή  ‡–‘ƒ ‡–ƒ–‡ ሻ ൅ ሻ  ‹„†Šͳ  ή ‹„†Šͳ ή „†Šͳ‡–ƒŠ›†”‘š›„—–›”ƒ–‡ ൅ ‹„†Šͳ  ή „†Šͳ ‡–‘ƒ ‡–ƒ–‡ ൅ ሿ ఽ౐ౌిౕ౐ήి౥ఽిౕ౐ ݐ ሿ ሾ ݐ ሾ ేౣ౗ౙ౩ఽ౐ౌిౕ౐ήేౣ౗ౙ౩ి౥ఽిౕ౐ ౄ౉ృి౥ఽ౉ఽ౐ሾ೟ሿ ା ేౣ౞ౣౝౙౢౄ౉ృి౥ఽ౉ఽ౐ ା ‹„†Šͳ ή „†Šͳ‡–ƒŠ›†”‘š›„—–›”ƒ–‡ ౄ౉ృి౥ఽ౉ఽ౐ሾ೟ሿ modifiers on MCAD defici modifiers  ൅ –ሻ ή ‡–ƒŠ›†”‘š›„—–›”ƒ–‡ሾݐሿ ሺെ ൅ ేౣ౞ౣౝౙ౩మౄ౉ృి౥ఽ౉ఽ౐ ి౥ఽిౕ౐ ା ేౣ౗ౙ౩ి౥ఽిౕ౐ ȋͳͳͳȌ ሻ ା ి౥ఽ౉ఽ౐ౄ౉ృి౥ఽ౉ఽ౐ሾ೟ሿ ే౛౧౞ౣౝౙౢήేౣ౞ౣౝౙౢౄ౉ృి౥ఽ౉ఽ౐ ఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ήఽౙ౛౪౥౗ౙ౛౪౗౪౛౉ఽ౐ሾ೟ሿ ఽ౐ౌిౕ౐ ି „†Šͳ ‡–‘ƒ ‡–ƒ–‡ ేౣ౗ౙ౩ఽ౐ౌిౕ౐ ାሺ ి౤ూూఽ౉౅ి౎ሾ೟ሿ ేౣ౭౥౮ి౤ూూఽ౉౅ి౎ „†Šͳ  ή  ‡–‘ƒ ‡–ƒ–‡ ൅ ȋͳͲͷȌ ೙సభల ೙సర „†Šͳ ή ‡–ƒŠ›†”‘š›„—–›”ƒ–‡ ిరఽౙ౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ σ ȋͳ͹ʹȌ ‹„†Šͳ ή „†Šͳ‡–ƒŠ›†”‘š›„—–›”ƒ–‡ “—ƒ–‹‘ǣ ే౛౧౞ౣౝౙ౩మేౣ౞ౣౝౙ౩మఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ేౣ౞ౣౝౙ౩మిరఽౙ౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐ ౄ౉ృి౥ఽ౉ఽ౐ሾ೟ሿ ൅ ేౣ౞ౣౝౙ౩మిరఽౙ౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐ ି ሿ Simulating the impact of ሻήሺଵା ݐ ሾ ేౣ౞ౣౝౙౢౄ౉ృి౥ఽ౉ఽ౐ ఽ౐ౌిౕ౐ήి౥ఽిౕ౐ ሻሺଵା ఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ήఽౙ౛౪౥౗ౙ౛౪౗౪౛౉ఽ౐ሾ೟ሿ –‡‡“—ƒ–‹‘‹•ƒ‹ Šƒ‡Ž‹•Ǧ‡–‡‡“—ƒ–‹‘ˆ‘”ƒ‹””‡˜‡”•‹„Ž‡ ేౣ౗ౙ౩ఽ౐ౌిౕ౐ήేౣ౗ౙ౩ి౥ఽిౕ౐ ୱ୤ୟୡୱେ୬ή୚ୟୡୱήେ୬୊୊୅େଢ଼୘ሾ௧ሿή୅୘୔େଢ଼୘ήେ୭୅େଢ଼୘ ሾݐሿ ή ‡–ƒŠ›†”‘š›„—–›”ƒ–‡ሾݐሿ‡–ƒ–‡ ƒ‘–‡   ା ሾݐሿ‡–ƒ–‡ ƒ‘–‡   ‹„†Šͳ ୚୦୫୥ୡ୪ήሺ ేౣ౞ౣౝౙౢఽౙ౛౪౥౗ౙ౛౪౗౪౛౉ఽ౐ήేౣ౞ౣౝౙౢఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ ା െ  ൅ – ొఽీౌౄ౉౅ి౎ ి౥ఽ౉ఽ౐ ȋͳͳͲȌ ൅ ‹„†Šͳ‡–ƒŠ›†”‘š›„—–›”ƒ–‡ ή ‹„†Šͳ  ή „†Šͳ ‡–‘ƒ ‡–ƒ–‡ ή  ‡–‘ƒ ‡–ƒ–‡ ి౥ఽిౕ౐ ేౣ౭౥౮ొఽీౌౄ౉౅ి౎ ሻ ൅ ేౣ౗ౙ౩ి౥ఽిౕ౐ ేౣ౞ౣౝౙ౩మి౥ఽ౉ఽ౐ ା ‡“„†Šͳ ή ‹„†Šͳ ή „†Šͳ‡–ƒŠ›†”‘š›„—–›”ƒ–‡   ή ‡–ƒŠ›†”‘š›„—–›”ƒ–‡ሾݐሿ ”†‹‰–‘–Š‡”†‡”‡†‹‹‡ „†Šͳሺെ ‹„†Šͳ  ఽౙ౛౪౥౗ౙ౛౪౗౪౛౉ఽ౐ሾ೟ሿ ͳ൅ ȋͳͲͺȌ ేౣ౞ౣౝౙౢఽౙ౛౪౥౗ౙ౛౪౗౪౛౉ఽ౐ ୚୵୭୶ήେ୬୊୊୅୑୍ୈሾ௧ሿή୒୅ୈ୔ୌ୑୍ୈήୱ୤୵୭୶େ୬ ା ఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ െ  ൅ – ሺ ఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ిరఽౙ౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐ሾ೟ሿ  ሾݐሿሻ  ሾݐሿ െ ేౣ౞ౣౝౙ౩మఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ ሺଵା ేౣ౞ౣౝౙ౩మఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ేౣ౞ౣౝౙ౩మిరఽౙ౛౪౥౗ౙ౯ౢి౥ఽ౉ఽ౐ ఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ ‹„†Šͳ  ή ‹„†Šͳ ή „†Šͳ ‡–‘ƒ ‡–ƒ–‡ „†Šͳ  ή ేౣ౞ౣౝౙౢఽౙ౛౪౯ౢి౥ఽ౉ఽ౐ ୚୦୫୥ୡୱଶሺ ୏୫୵୭୶େ୬୊୊୅୑୍ୈή୏୫୵୭୶୒୅ୈ୔ୌ୑୍ୈήሺଵା ൅ ଵା ୏୫ୟୡୱ୅୘୔େଢ଼୘ή୏୫ୟୡୱେ୬୊୊୅େଢ଼୘ή୏୫ୟୡୱେ୭୅େଢ଼୘ήሺ ‹„†Šͳ ή „†Šͳ‡–ƒŠ›†”‘š›„—–›”ƒ–‡  ൅ –ሻ ή ‡–ƒŠ›†”‘š›„—–›”ƒ–‡ሾݐሿ ሺെ ൅ ˜Š‰ Ž ൌ ‡–ƒ–‡ሾݐሿ ˜„†Šͳ ൌെ ˜™‘š ൌ ƒ‘–‡  ‡–ƒ–‡ ή ƒ‘–‡ ‡ ‡–ƒ–‡ ൌ ƒ‘–‡ š˜ ൌ ʹ• ‰Š˜  ȋͳͲ͸Ȍ ˜ ‹  ൌ  ‹ ή ሺ  ሾݐሿ  ή ‡ ൌ  š˜ ˜š  ൌ ‡‡–ƒŠ›†”‘š›„—–›”ƒ–‡ ˜ƒ • ൌ  Š‡”‡ƒ –‹‘‹•‘†‡Ž‡†ƒ•ƒ‹””‡˜‡”•‹„Ž‡”‡ƒ –‹‘ǤŠ‡”ƒ  Š‡ ͳǦ ƒ–ƒŽ›œ‡†”‡ƒ –‹‘‹•ƒ ‘ Chapter 5

ሾݐሿ ൅  ሾݐሿሻȋͳͳʹȌ ή ሺെ ‹ ൌ  ‹˜

ሾݐሿȋͳͳ͵Ȍ ൌ ‡ ή š˜

ŽŶƐĞƌǀĞĚŵŽŝĞƚLJĞƋƵĂƚŝŽŶƐ ሼ‘ ൌ ‘–Ǧͳ͸ ›Ž‘ሾ–ሿǦͳ͸‘›Ž‘ሾ–ሿǦͳ͸ ›†”‘š›ƒ ›Ž‘ሾ–ሿǦͳ͸‡–‘ƒ ›Ž‘ሾ–ሿǦͳͶ ›Ž‘ሾ–ሿǦͳͶ‘›Ž‘ሾ–ሿǦͳͶ ›†”‘š›ƒ ›Ž‘ሾ–ሿǦͳͶ‡–‘ƒ ›Ž‘ሾ–ሿǦͳʹ ›Ž‘ሾ–ሿǦ ͳʹ‘›Ž‘ሾ–ሿǦͳʹ ›†”‘š›ƒ ›Ž‘ሾ–ሿǦͳʹ‡–‘ƒ ›Ž‘ሾ–ሿǦͳͲ ›Ž‘ሾ–ሿǦͳͲ‘›Ž‘ሾ–ሿǦͳͲ ›†”‘š›ƒ ›Ž‘ሾ–ሿǦͳͲ‡–‘ƒ ›Ž‘ሾ–ሿǦͺ ›Ž‘ሾ–ሿǦͺ‘›Ž‘ሾ–ሿǦ ͺ ›†”‘š›ƒ ›Ž‘ሾ–ሿǦͺ‡–‘ƒ ›Ž‘ሾ–ሿǦ͸ ›Ž‘ሾ–ሿǦ͸‘›Ž‘ሾ–ሿǦ͸ ›†”‘š›ƒ ›Ž‘ሾ–ሿǦ͸‡–‘ƒ ›Ž‘ሾ–ሿǦͶ ›Ž‘ሾ–ሿǦͶ‘›Ž‘ሾ–ሿǦͶ ›†”‘š›ƒ ›Ž‘ሾ–ሿǦ Ͷ ‡–‘ƒ ›Ž‘ሾ–ሿǦ ‡–›Ž‘Ǧ  ‘ሾ–ሿሽ ȋͳͳͶȌ

ͳͺͶ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

DŽĚĞůƉĂƌĂŵĞƚĞƌƐ Š‡˜ƒŽ—‡•‘ˆ–Š‡’ƒ”ƒ‡–‡”•—•‡†ˆ‘”–Š‡‘†‡Ž•‹—Žƒ–‹‘•ƒ”‡‰‹˜‡‹ƒ„Ž‡ͳǤ‡˜‹ƒ–‹‘• ˆ”‘–Š‡•‡˜ƒŽ—‡•ƒ”‡‰‹˜‡‹–Š‡ˆ‹‰—”‡Ž‡‰‡†•™Š‡”‡ƒ’’Ž‹ ƒ„Ž‡ǤŠ‡’ƒ”ƒ‡–”‹œƒ–‹‘‘ˆ –Š‡ ‘†‡Ž ™ƒ• „ƒ•‡† ‘ in vitro ‹‡–‹  †ƒ–ƒ ‘ˆ ‹•‘Žƒ–‡† ‡œ›‡• ‘” ‹•‘Žƒ–‡† ‘”‰ƒ‡ŽŽ‡ ‘” ‘’—–ƒ–‹‘ƒŽ‘†‡Ž‹‰Ž‹–‡”ƒ–—”‡ǤŠ‡”‡ƒ•‹‹Žƒ”˜ƒŽ—‡™ƒ•—•‡†ƒ•‹—‡et al.ʹͲͳ͵ ȏͳȐǡ–Š‹•”‡ˆ‡”‡ ‡’ƒ’‡”™ƒ•‰‹˜‡ƒ•”‡ˆ‡”‡ ‡Ǥ   5

ͳͺͷ Chapter 5

Parameter Value unit Used references CPT1  sf ’–ͳͳ͸ ͳ ȏ͵Ȑ  sf ’–ͳͳͶ ͳǤ͵Ͷ ȏ͵Ȑ  sf ’–ͳͳʹ ͳǤͻ͵ ȏ͵Ȑ  sf ’–ͳͳͲ ͳǤͷͶ ȏ͵Ȑ  sf ’–ͳͺ ͲǤͺ͵ ȏ͵Ȑ  sf ’–ͳ͸ ͲǤͷͳ ȏ͵Ȑ  sf ’–ͳͶ ͲǤʹͷ •–‹ƒ–‡†„ƒ•‡†‘ȏ͵ǡͶȐ ρ‘ŽǤ‹Ǧ V ’–ͳ ͲǤͲʹͺ •–‹ƒ–‡†„ƒ•‡†‘ȏ͵ǡͷǡ͸Ȑ ͳǤ‰”‘–‡‹Ǧͳ Km ’–ͳͳ͸ ›Ž‘ ͳʹǤʹ ρ ȏ͵Ȑ

Km ’–ͳͳͶ ›Ž‘ ͵ͲǤͺ ρ ȏ͵Ȑ

Km ’–ͳͳʹ ›Ž‘ ͳͳ ρ ȏ͵Ȑ

Km ’–ͳͳͲ ›Ž‘ ͳ͸Ǥ͹ ρ ȏ͵Ȑ

Km ’–ͳͺ ›Ž‘ ʹʹǤͻ ρ ȏ͵Ȑ

Km ’–ͳ͸ ›Ž‘ ͳͷͳǤͷ ρ ȏ͵Ȑ

Km ’–ͳͶ ›Ž‘ ͸ͷ͵Ǥͷ ρ š–”ƒ’‘Žƒ–‡†„ƒ•‡†ȏ͵ǡͶȐ

Km ’–ͳƒ”ͳ͸ ͵ͺ͸ ρ ȏ͵Ȑ

Km ’–ͳƒ”ͳͶ ͷͻ͵ ρ ȏ͵Ȑ

Km ’–ͳƒ”ͳʹ ͶͲͲ ρ ȏ͵Ȑ

Km ’–ͳƒ”ͳͲ ͳͷͷͶ ρ –”ƒ’‘Žƒ–‡†

Km ’–ͳƒ”ͺ ͷͲͳͶ ρ ȏ͵Ȑ

Km ’–ͳƒ”͸ ͳ͸ͳͺ͵ ρ ȏ͵Ȑ

Km ’–ͳƒ”Ͷ ͷʹʹ͵ͳ ρ ȏ͵Ȑ

Km ’–ͳͳ͸ ›Žƒ” ͳʹ͵ ρ ȏ͵Ȑ

Km ’–ͳͳͶ ›Žƒ” ͵͹͹ ρ ȏ͵Ȑ

Km ’–ͳͳʹ ›Žƒ” ͸͵ͳ ρ ȏ͵Ȑ

Km ’–ͳͳͲ ›Žƒ” ͺͺͷ ρ ȏ͵Ȑ

Km ’–ͳͺ ›Žƒ” ͳͳ͵ͻ ρ ȏ͵Ȑ

Km ’–ͳ͸ ›Žƒ” ͳ͵ͻ͵ ρ ȏ͵Ȑ

Km ’–ͳͶ ›Žƒ” ͳ͸Ͷ͹ ρ ȏ͵Ȑ

Km ’–ͳ‘ ͶͲǤ͹ ρ ȏͳȐ

Ki ’–ͳƒŽ‘ ͻǤͳ ρ ȏͳȐ  Keq ’–ͳ ͲǤͶͷ ȏͳȐ  n ’–ͳ ʹǤͶ͹ͻͻ ȏͳȐ  

CACT    sf ƒ –ͳ ͳ ȏͳȐ ρ‘ŽǤ‹Ǧ Vf ƒ – ͲǤͶʹ ȏͳȐ ͳǤ‰”‘–‡‹Ǧͳ Km ›Žƒ” ͳͷ ρ ȏͳȐ

Kmƒ” ͳ͵Ͳ ρ ȏͳȐ

Km ›Žƒ” ͳͷ ρ ȏͳȐ

Kmƒ” ͳ͵Ͳ ρ ȏͳȐ

Ki ›Žƒ” ͷ͸ ρ ȏͳȐ

Kiƒ” ʹͲͲ ρ ȏͳȐ  Keq ƒ – ͳ ȏͳȐ

ͳͺ͸ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

Parameter Value unit References CPT2  sf ’–ʹͳ͸ ͲǤͺͷ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ  sf ’–ʹͳͶ ͳǤͲͲ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ  sf ’–ʹͳʹ ͲǤͻ͸ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ  sf ’–ʹͳͲ ͳǤͲͲ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ  sf ’–ʹͺ ͲǤͶʹ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ  sf ’–ʹ͸ ͲǤʹͷ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ  sf ’–ʹͶ ͲǤͳͻ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ ρ‘ŽǤ‹Ǧ V ’–ʹ ͲǤ͵ͻʹ ͳǤ‰”‘–‡‹Ǧͳ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ 5 Km ’–ʹͳ͸ ›Žƒ” ͸͹ǤͲͲ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ Km ’–ʹͳͶ ›Žƒ” ͶǤ͸Ͳ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ Km ’–ʹͳʹ ›Žƒ” ͳͷ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ Km ’–ʹͳͲ ›Žƒ” Ͷ͵ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ Km ’–ʹͺ ›Žƒ” ͳͺ͹ǤͶ͸ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ Km ’–ʹ͸ ›Žƒ” ͳʹͷǤͷͶ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ Km ’–ʹͶ ›Žƒ” Ͷ͵Ǥͷ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ Km ’–ʹ‘ ͵Ͳ ρ ȏͳȐ Km ’–ʹͳ͸ ›Ž‘ ͸Ǥ͵ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ Km ’–ʹͳͶ ›Ž‘ ͹Ǥͺ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ Km ’–ʹͳʹ ›Ž‘ ʹʹǤͶ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ Km ’ʹͳͲ ›Ž‘ ͵͹ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ Km ’–ʹͺ ›Ž‘ ͷͲǤ͵ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ Km ’–ʹ͸ ›Ž‘ ͷͶǤͷ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏ͹ǡͺȐ Km ’–ʹͶ ›Ž‘ Ͷ͵Ǥͷ ρ ȏͳȐ

Km ’–ʹƒ” ͵ͷͲ ρ ȏͳȐ  Keq ’–ʹ ʹǤʹʹ  

VLCAD    sf˜Ž ƒ†ͳ͸ ͳǤͲͲ •–‹ƒ–‡†„ƒ•‡†‘ȏ͸ǡͻǡͳͲȐ  sf˜Ž ƒ†ͳͶ ͲǤͶ͵ •–‹ƒ–‡†„ƒ•‡†‘ȏ͸ǡͻǡͳͲȐ  sf˜Ž ƒ†ͳʹ ͲǤʹ͸ •–‹ƒ–‡†„ƒ•‡†‘ȏ͸ǡͻǡͳͲȐ  sf˜Ž ƒ†ͳͲ ͲǤͲ͸ •–‹ƒ–‡†„ƒ•‡†‘ȏ͸ǡͻȂͳͳȐ  sf˜Ž ƒ†ͺ ͲǤͲ͵ •–‹ƒ–‡†„ƒ•‡†‘ȏ͸ǡͻȂͳͳȐ  sf˜Ž ƒ†͸ ͲǤͲͳ •–‹ƒ–‡†„ƒ•‡†‘ȏ͸ǡͻȂͳͳȐ  V˜Ž ƒ† ͲǤͳͲ͵ •–‹ƒ–‡†„ƒ•‡†‘ȏ͸ǡͻȂͳͳȐ ρ‘ŽǤ‹Ǧ Kmͳ͸ ›Ž‘ ͳǤ͸ͷ •–‹ƒ–‡†„ƒ•‡†‘ȏͳͳǡͳʹȐ ͳǤ‰”‘–‡‹Ǧͳ KmͳͶ ›Ž‘ ͳǤͳͷ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏͳͳǡͳʹȐ

Kmͳʹ ›Ž‘ ͲǤͺͲ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏͳͳǡͳʹȐ

KmͳͲ ›Ž‘ ͳͲ ρ ȏͳͲȐ

Kmͺ ›Ž‘ ͺ ρ ȏͳͲȐ

Km͸ ›Ž‘ ʹͻ ρ ȏͳͲȐ

Km ‘ ͲǤͳʹ ρ ȏͳȐ

Km‘›Ž‘ ͳǤͲͺ ρ ȏͳȐ

Km ”‡† ʹͶǤʹ ρ ȏͳȐ  Keq˜Ž ƒ† ͸ ȏͳȐ

ͳͺ͹ Chapter 5

Parameter Value unit References MCAD  sf ƒ†ͳ͸ ͲǤͲͳ͸ •–‹ƒ–‡†„ƒ•‡†‘ȏͳͳǡͳ͵Ȑ  sf ƒ†ͳͶ ͲǤʹͳ ȏͳ͵Ȑ  sf ƒ†ͳʹ ͲǤͶͷ ȏͳ͵Ȑ  sf ƒ†ͳͲ ͲǤͷʹ ȏͳ͵Ȑ  sf ƒ†ͺ ͲǤ͹ͷ ȏͳ͵Ȑ  sf ƒ†͸ ͳǤͲͲ ȏͳ͵Ȑ  sf ƒ†Ͷ ͲǤͳͶ ȏͳ͵Ȑ  V ƒ† ͲǤͲ͹Ͷ •–‹ƒ–‡†„ƒ•‡†‘ȏͻǡͳ͵Ȑ ρ‘ŽǤ‹Ǧ Kmͳ͸ ›Ž‘ ʹ͵Ǥͺ ͳǤ‰”‘–‡‹Ǧͳ ȏͳ͵Ȑ KmͳͶ ›Ž‘ ͳͲǤͲ ρ ȏͳ͵Ȑ Kmͳʹ ›Ž‘ ͻǤ͵ ρ ȏͳ͵Ȑ KmͳͲ ›Ž‘ ͻǤͳ ρ ȏͳ͵Ȑ Kmͺ ›Ž‘ ͺ ρ ȏͳ͵Ȑ Km͸ ›Ž‘ ʹͳǤ͸ ρ ȏͳ͵Ȑ KmͶ ›Ž‘ ͹ͳǤͶ ρ ȏͳ͵Ȑ Km ‘š ͲǤͳʹ ρ ȏͳȐ

Km‘›Ž‘ ͳǤͲͺ ρ ȏͳȐ

Km ”‡† ʹͶǤʹ ρ ȏͳȐ  Keq˜Ž ƒ† ͸ ȏͳȐ  

SCAD    sf• ƒ†͸ ͲǤͺͺ ȏͳ͵Ȑ  sf• ƒ†Ͷ ͳ ȏͳ͵Ȑ ρ‘ŽǤ‹Ǧ V• ƒ† ͲǤͲ͸Ͳ ͳǤ‰”‘–‡‹Ǧͳ •–‹ƒ–‡†„ƒ•‡†‘ȏͻǡͳ͵Ȑ Km͸ ›Ž‘ ͵͵Ǥͻ ρ ȏͳ͵Ȑ KmͶ ›Ž‘ ͳʹǤͻ ρ ȏͳ͵Ȑ Km ‘š ͲǤͳʹ ρ ȏͳȐ

Km‘›Ž‘ ͳǤͲͺ ρ ȏͳȐ

Km ”‡† ʹͶǤʹ ρ ȏͳȐ  Keq• ƒ† ͸ ȏͳȐ   CROT  ȏͳȐ  sf ”‘–ͳ͸ ͲǤͳ͵ ȏͳȐ  sf ”‘–ͳͶ ͲǤʹ ȏͳȐ  sf ”‘–ͳʹ ͲǤʹͷ ȏͳȐ  sf ”‘–ͳͲ ͲǤ͵͵ ȏͳȐ  sf ”‘–ͺ ͲǤͷͺ ȏͳȐ  sf ”‘–͸ ͲǤͺ ȏͳȐ  sf ”‘–Ͷ ͳ ȏͳȐ ρ‘ŽǤ‹Ǧ V ”‘– ͵Ǥ͸ ȏͳȐ ͳǤ‰”‘–‡‹Ǧͳ Kmͳ͸‘›Ž‘ ͳͷͲ ρ ȏͳȐ

KmͳͶ‘›Ž‘ ͳͲͲ ρ ȏͳȐ

Kmͳʹ‘›Ž‘ ʹͷ ρ ȏͳȐ

ͳͺͺ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

Parameter Value unit References

KmͳͲ‘›Ž‘ ʹͷ ρ ȏͳȐ

Kmͺ‘›Ž‘ ʹͷ ρ ȏͳȐ

Km͸‘›Ž‘ ʹͷ ρ ȏͳȐ

KmͶ‘›Ž‘ ͶͲ ρ ȏͳȐ

Km ›†”‘š›ƒ ›Ž‘ Ͷͷ ρ ȏͳȐ

Kiƒ ‡–‘ƒ ‡–›Ž‘ ͳǤ͸ ρ ȏͳȐ  Keq ”‘– ͵Ǥͳ͵ ȏͳȐ  

MSCHAD    sf• Šƒ†ͳ͸ ͲǤ͸ ȏͳȐ 5  sf• Šƒ†ͳͶ ͲǤͷ ȏͳȐ  sf• Šƒ†ͳʹ ͲǤͶ͵ ȏͳȐ  sf• Šƒ†ͳͲ ͲǤ͸Ͷ ȏͳȐ  sf• Šƒ†ͺ ͲǤͺͻ ȏͳȐ  sf• Šƒ†͸ ͳ ȏͳȐ  sf• Šƒ†Ͷ ͲǤ͸͹ ȏͳȐ ρ‘ŽǤ‹Ǧ V• Šƒ† ͳ ȏͳȐ ͳǤ‰”‘–‡‹Ǧͳ Kmͳ͸ ›†”‘š›ƒ ›Ž‘ ͳǤͷ ρ ȏͳȐ

KmͳͶ ›†”‘š›ƒ ›Ž‘ ͳǤͺ ρ ȏͳȐ

Kmͳʹ ›†”‘š›ƒ ›Ž‘ ͵Ǥ͹ ρ ȏͳȐ

KmͳͲ ›†”‘š›ƒ ›Ž‘ ͺǤͺ ρ ȏͳȐ

Kmͺ ›†”‘š›ƒ ›Ž‘ ͳ͸Ǥ͵ ρ ȏͳȐ

Km͸ ›†”‘š›ƒ ›Ž‘ ʹͺǤ͸ ρ ȏͳȐ

KmͶ ›†”‘š›ƒ ›Ž‘ ͸ͻǤͻ ρ ȏͳȐ

Km ͷͺǤͷ ρ ȏͳȐ

Kmͳ͸‡–‘ƒ ›Ž‘ ͳǤͶ ρ ȏͳȐ

KmͳͶ‡–‘ƒ ›Ž‘ ͳǤͶ ρ ȏͳȐ

Kmͳʹ‡–‘ƒ ›Ž‘ ͳǤ͸ ρ ȏͳȐ

KmͳͲ‡–‘ƒ ›Ž‘ ʹǤ͵ ρ ȏͳȐ

Kmͺ‡–‘ƒ ›Ž‘ ͶǤͳ ρ ȏͳȐ

Km͸‡–‘ƒ ›Ž‘ ͷǤͺ ρ ȏͳȐ

KmͶ ‡–‘ƒ ›Ž‘ ͳ͸Ǥͻ ρ ȏͳȐ

Km  ͷǤͶ ρ ȏͳȐ  Keq• Šƒ† ʹǤͳ͹ȉͳͲǦͶ ȏͳȐ   

MCKAT     sf ƒ–ͳ͸ ͲǤͲͳ ”„‹–”ƒ”›‘Ǧœ‡”‘˜ƒŽ—‡  sf ƒ–ͳͶ ͲǤʹ ȏͳȐ  sf ƒ–ͳʹ ͲǤ͵ͺ ȏͳȐ  sf ƒ–ͳͲ ͲǤ͸ͷ ȏͳȐ  sf ƒ–ͺ ͲǤͺͳ ȏͳȐ  sf ƒ–͸ ͳ ȏͳȐ  sf ƒ–Ͷ ͲǤͶͻ ȏͳȐ ρ‘ŽǤ‹Ǧ V ƒ– ͲǤͶ͸͵ •–‹ƒ–‡†„ƒ•‡†‘ȏ͸ǡͳͶȐ ͳǤ‰”‘–‡‹Ǧͳ Km ƒ–ͳ͸‡–‘ƒ ›Ž‘ ͲǤͷ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏͳͶǡͳͷȐ

ͳͺͻ Chapter 5

Parameter Value unit References

Km ƒ–ͳͶ‡–‘ƒ ›Ž‘ ͲǤͷͷ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏͳͶǡͳͷȐ

Km ƒ–ͳʹ‡–‘ƒ ›Ž‘ ͲǤ͸ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏͳͶǡͳͷȐ

Km ƒ–ͳͲ‡–‘ƒ ›Ž‘ ͲǤͻ͹ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏͳͶǡͳͷȐ

Km ƒ–ͺ‡–‘ƒ ›Ž‘ ͳǤʹͻ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏͳͶǡͳͷȐ

Km ƒ–͸‡–‘ƒ ›Ž‘ ͶǤͶͷ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏͳͶǡͳͷȐ Km  ƒ–Ͷ ‡–‘ƒ ‡–›Ž‘ ͻǤʹ ρ ȏͳͶȐ  Km ƒ–‘ͳ͸ ͳͳ͵Ǥͳ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏͳͷǡͳ͸Ȑ

Km ƒ–‘ͳͶ ͳ͵ʹǤͷ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏͳͷǡͳ͸Ȑ

Km ƒ–‘ͳʹ ͳͷͳǤͻ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏͳͷǡͳ͸Ȑ

Km ƒ–‘ͳͲ ͳͶͳǤʹ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏͳͷǡͳ͸Ȑ

Km ƒ–‘ͺ ͳͶͲǤͶ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏͳͷǡͳ͸Ȑ

Km ƒ–‘͸ ͹ͶǤ͹ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏͳͷǡͳ͸Ȑ

Km ƒ–‘Ͷ ͺǤ͹ ρ ȏͳͷȐ

Km ƒ–ͳ͸ ›Ž‘ ͸ͷǤ͸ ρ •–‹Ǥ—•‹‰ ƒŽ†ƒ‡‡ŽǤ™ǤˆȀ”αͳͲ

Km ƒ–ͳͶ ›Ž‘ ͸ͷǤ͸ ρ •–‹Ǥ—•‹‰ ƒŽ†ƒ‡‡ŽǤ™ǤˆȀ”αͳͲ

Km ƒ–ͳʹ ›Ž‘ ͺ͵Ǥͺ ρ •–‹Ǥ—•‹‰ ƒŽ†ƒ‡‡ŽǤ™ǤˆȀ”αͳͲ

Km ƒ–ͳͲ ›Ž‘ ͳͲͶ ρ •–‹Ǥ—•‹‰ ƒŽ†ƒ‡‡ŽǤ™ǤˆȀ”αͳͲ

Km ƒ–ͺ ›Ž‘ ͳͷ͸Ǥʹ ρ •–‹Ǥ—•‹‰ ƒŽ†ƒ‡‡ŽǤ™ǤˆȀ”αͳͲ

Km ƒ–͸ ›Ž‘ ʹͲ͹Ǥͳ ρ •–‹Ǥ—•‹‰ ƒŽ†ƒ‡‡ŽǤ™ǤˆȀ”αͳͲ

Km ƒ–Ͷ ›Ž‘ ͵ͺͳǤ͵ ρ •–‹Ǥ—•‹‰ ƒŽ†ƒ‡‡ŽǤ™ǤˆȀ”αͳͲ

Km ƒ– ‡–›Ž‘ ͻͳǤ͹ ρ •–‹Ǥ—•‹‰ ƒŽ†ƒ‡‡ŽǤ™ǤˆȀ”αͳͲ  Keq ƒ– ͳͲͷͳ ȏͳȐ   

MTP     sf–’ͳ͸ ͳ ȏͳȐ  sf–’ͳͶ ͲǤͻ ȏͳȐ  sf–’ͳʹ ͲǤͺͳ ȏͳȐ  sf–’ͳͲ ͲǤ͹͵ ȏͳȐ  sf–’ͺ ͲǤ͵Ͷ ȏͳȐ ρ‘ŽǤ‹Ǧ V–’ ʹǤͺͶ ȏͳȐ ͳǤ‰”‘–‡‹Ǧͳ Km‘›Ž‘ ʹͷ ρ ȏͳȐ

Km ͸Ͳ ρ ȏͳȐ

Km‘ ͵Ͳ ρ ȏͳȐ

Km ›Ž‘ ͳ͵Ǥͺ͵ ρ ȏͳȐ

Km  ͷͲ ρ ȏͳȐ

Km ‡–›Ž‘ ͵Ͳ ρ ȏͳȐ  Keq–’ ͲǤ͹ͳ ȏͳȐ    ETFQO ρ‘ŽǤ‹Ǧ V‡–ˆ“‘ ͲǤͲ͹͵ͷ ͳǤ‰”‘–‡‹Ǧͳ •–‹ƒ–‡†„ƒ•‡†‘ȏ͸ǡͳ͹ǡͳͺȐ Km–ˆ“‘–ˆŠ“ ͳǤͻ͹ ρ ȏͳ͹Ȑ Km–ˆ“‘‘ ʹͲǤ͵ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏͳ͹ǡͳͻȐ Km–ˆ“‘–ˆ‘š ͳǤͻ͹ ρ •–‹ƒ–‡†„ƒ•‡†‘ȏͳ͹ǡʹͲȐ Km–ˆ“‘‘ ʹ ͵ͺǤͳ ρ •–‹Ǥ—•‹‰ ƒŽ†ƒ‡‡ŽǤ™‹–ŠˆȀ”αͳ

ͳͻͲ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

Parameter Value unit References

Ki–ˆ“‘–ˆŠ“ ͳǤͻ͹ ρ ••—‡†‡“—ƒŽ–‘Km–ˆ“‘–ˆŠ“

Ki–ˆ“‘‘ ʹ ͵ͺǤͳ ρ ••—‡†‡“—ƒŽ–‘Km–ˆ“‘‘ ʹ  Keq–ˆ“‘ ͳͻͻǤͷ •–‹ƒ–‡†„ƒ•‡†‘ȏʹͳǡʹʹȐ     ACAT1    ρ‘ŽǤ‹Ǧ Vͳ •–‹ƒ–‡†„ƒ•‡†‘ȏͷǡ͸Ȑ ͲǤ͵Ͷͻ ͳǤ‰”‘–‡‹Ǧͳ Kmͳǡ ‡–›Ž‘ ͸Ǥʹ ρ ȏʹ͵Ȑ

Kmͳǡ ‡–‘ƒ ‡–›Ž‘ ͹ ρ ȏʹͶȐ

Kmͳǡ‘ ʹͳ ρ ȏʹͶȐ  5 Keqͳ ͳͲͷͳ ȏͳȐȋ‹–Š‡–Š‹‘Žƒ•‡†‹”‡ –‹‘Ȍ   

HMGCS2    ρ‘ŽǤ‹Ǧ V  ʹ ͲǤͲʹͳ ͳǤ‰”‘–‡‹Ǧͳ ȏʹͷȐ Km ‰ •ʹͶ ‡–‘ƒ ›Ž‘ ͲǤ͵ͷ ρ   KmŠ‰ •ʹ ‡–›Ž‘ ͷͳ ρ ȏʹ͸Ȑ

KmŠ‰ •ʹ  ‘ ͵ͷ ρ ȏʹ͸Ȑ

KmŠ‰ •ʹ‘ ͷͲͲ ρ ȏʹ͸Ȑ  Keq  ʹ ͵ͲͲͲ ȏʹ͹Ȑ    HMGCL ρ‘ŽǤ‹Ǧ Vf   ͲǤͳ͵͵ ȏʹͷȐ ͳǤ‰”‘–‡‹Ǧͳ KmŠ‰ Ž  ‘ ͺ ρ ȏʹͺȐ  KmŠ‰ Ž ‡–›Ž‘ ͵Ͳ ρ ••—‡†•ƒ‡ƒ• KmŠ‰ Ž ‡–‘ƒ ‡–ƒ–‡ ʹͳ ρ ȏʹͻȐ Keq   ʹͲͲͲ ρ ȏ͵ͲȐ  BDH1 ρ‘ŽǤ‹Ǧ V ͳ ͳǤͻͻ ȏ͵ͳȐ ͳǤ‰”‘–‡‹Ǧͳ Km„†Šͳ ‡–‘ƒ ‡–ƒ–‡ ͳͶͻ ρ ȏ͵ͳȐ

Km„†Šͳ  ͳͶ ρ ȏ͵ͳȐ Km „†Šͳ‡–ƒŠ›†”‘š›„—–›”ƒ–‡ ͺ͸͸ ρ ȏ͵ͳȐ  Km„†Šͳ ͹ͳ ρ ȏ͵ͳȐ

Ki„†Šͳ ‡–‘ƒ ‡–ƒ–‡ ͷͺͶ ρ ȏ͵ͳȐ

Ki„†Šͳ  ͹ͷ ρ ȏ͵ͳȐ Ki „†Šͳ‡–ƒŠ›†”‘š›„—–›”ƒ–‡ ͳͲͲͲͲ ρ ••—‡†Š‹‰Š„ƒ•‡†‘ȏ͵ͳȐ  Ki„†Šͳ ͵Ͳͳ ρ ȏ͵ͳȐ ƒŽ —Žƒ–‡†™‹–Šǣ Keq  „†Šͳ ͳǤʹͷͷ ȋͳȀʹȌȗȋȋ’ȗ‹“ȌȀȋ‹ƒȗ„ȌȌ

ACOT    •ˆƒ ‘–ͳ͸ ͲǤͺ͵ ȏ͵ʹȐ  •ˆƒ ‘–ͳͶ ͲǤͺͻ ȏ͵ʹȐ

ͳͻͳ Chapter 5

Parameter Value unit References  •ˆƒ ‘–ͳʹͳȏ͵ʹȐ  •ˆƒ ‘–ͳͲ ͲǤͶ͹ ȏ͵ʹȐ  •ˆƒ ‘–ͺ ͲǤͷͳ ȏ͵ʹȐ  •ˆƒ ‘–͸ ͲǤʹ͸ ȏ͵ʹȐ  •ˆƒ ‘–Ͷ ͲǤͲͶ͹ ȏ͵ʹȐ ρ‘ŽǤ‹Ǧ ƒš̴”‡ˆ ͲǤͲͶ͵͹ ȏ͵ʹȂ͵͹Ȑ ͳǤ‰”‘–‡‹Ǧͳ ƒ ‘–ͳ͸‘ ʹǤ͸ ρ ȏ͵ʹȐ

ƒ ‘–ͳͶ‘ ʹǤͶ ρ ȏ͵ʹȐ

ƒ ‘–ͳͲ‘ ͸Ǥͳ ρ ȏ͵ʹȐ

ƒ ‘–ͺ‘ ͺǤ͵ ρ ȏ͵ʹȐ

ƒ ‘–͸‘ ͳ͵ ρ ȏ͵ʹȐ

ƒ ‘–Ͷ‘ ͳ͸ ρ ȏ͵ʹȐ

ƒ ‘–ͳ͸  ͸ʹͶͲͲ ρ ƒ ‘–‘ȉ‡“ƒ ‘–

ƒ ‘–ͳͶ  ͳͺʹͶͲͲ ρ DzDz

ƒ ‘–ͳʹ  ʹͷͲͺͲͲ ρ DzDz

ƒ ‘–ͳͲ  Ͷ͸͵͸ͲͲ ρ DzDz

ƒ ‘–ͺ  ͸͵ͲͺͲͲ ρ DzDz

ƒ ‘–͸  ͻͺͺͲͲͲ ρ DzDz

ƒ ‘–Ͷ  ͶͺͲͲͲͲͲͲ ρ DzDz

ƒ ‘–ʹ  ͳ͵ͳͳͲͲͲͲ ρ DzDz ̴‘ ͻ ρ ȏ͵ͷȐ  ‡“ƒ ‘–ͳ͸ ʹͶͲͲͲ ȏ͵ͺȐ  ‡“ƒ ‘–ͳͶ ͹͸ͲͲͲ ȏ͵ͺȐ  ‡“ƒ ‘–ͳʹ ͹͸ͲͲͲ ȏ͵ͺȐ  ‡“ƒ ‘–ͳͲ ͹͸ͲͲͲ ȏ͵ͺȐ  ‡“ƒ ‘–ͺ ͹͸ͲͲͲ ȏ͵ͺȐ  ‡“ƒ ‘–͸ ͹͸ͲͲͲ ȏ͵ͺȐ  ‡“ƒ ‘–Ͷ ͵ͲͲͲͲͲͲ ȏ͵ͺȐ  ‡“ƒ ‘–ʹ ͷ͹ͲͲͲͲ ȏ͵ͺȐ  ‡“ƒ ‘–  Ͷ͵ͲͲͲͲ ȏ͵ͺȐ  ‡“ƒ ‘–   ͳ͹ͲͲͲ ȏ͵ͺȐ  ACS  sfƒ •ͳ͸ ͲǤͻͳ ȏ͵ͻȐ  sfƒ •ͳͶ ͲǤͻ͹ ȏ͵ͻȐ  sfƒ •ͳʹ ͳ ȏ͵ͻȐ  sfƒ •ͳͲ ͲǤͶ ȏ͵ͻȐ  sfƒ •ͺ ͲǤͲ͸ͺ ȏ͵ͻȐ  sfƒ •͸ ͲǤͲͲ͵ͳ ȏ͵ͻȐ  sfƒ •Ͷ ͲǤͲͲͲͳͶ š–”ƒ’‘Žƒ–‡†

Kmͳʹ  ͶǤͷͷ ρ ȏ͵ͻȐ

KmͳͲ  ͹Ǥ͸ͻ ρ ȏ͵ͻȐ

Kmͺ  ͳͳǤͳ ρ ȏ͵ͻȐ

Km͸  ʹͲǤͺ ρ ȏ͵ͻȐ

KmͶ  ͹ͷǤͳ ρ š–”ƒ’‘Žƒ–‡†

ͳͻʹ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

Parameter Value unit References

Km‘ ͲǤͷ͵ ρ ȏͶͲȐ

Km ͷͲͲ ρ ȏͶͲȐ   Omega-oxidation    sf™‘šͳ͸ ͲǤͲͳ ȏͶͳȐ  sf™‘šͳͶ ͲǤͲͳ ȏͶͳȐ  sf™‘šͳʹ ͲǤͲͷ ȏͶͳȐ  sf™‘šͳͲ ͲǤʹʹ ȏͶͳȐ  sf™‘šͺ ͲǤ͵ͺ ȏͶͳȐ  sf™‘š͸ ͺ ȏͶͳȐ 5  sf™‘šͶ ͲǤͲͳ ȏͶͳȐ ρ‘ŽǤ‹Ǧ Vɘ‘š ͳǤͶ͸ •–‹ƒ–‡†„ƒ•‡†‘ȏͶʹȂͶͷȐ ͳǤ‰”‘–‡‹Ǧͳ Km™‘šͳ͸   ʹͲͲͲͲ ρ ȏͶͳȐ

Km™‘šͳͶ   ʹͲͲͲͲ ρ ȏͶͳȐ

Km™‘šͳʹ   ͵Ǥ͸ ρ ȏͶͳȐ

Km™‘šͳͲ   ͷʹʹ ρ ȏͶͳȐ

Km™‘šͺ   Ͷͺ͸ͳ ρ ȏͶͳȐ

Km™‘š͸   ͸ͺʹͷ ρ ȏͶͳȐ

Km™‘šͶ   ʹͲͲͲͲ ρ ȏͶͳȐ

Km™‘š   ͵͹ ρ ȏͶ͸Ȑ  Transmembranal transport parameters  Ǧͳ Ǧͳ k ͵ Ǥ‹ Ǥ‰”‘–‡‹  •–‹ƒ–‡†ƒ”„‹–”ƒ”‹Ž› Ǧͳ Ǧͳ k ‹ ͵ Ǥ‹ Ǥ‰”‘–‡‹  DzDz Ǧͳ Ǧͳ k‹ ͵ Ǥ‹ Ǥ‰”‘–‡‹  DzDz Ǧͳ Ǧͳ k‡  ͳǦͲ͸ Ǥ‹ Ǥ‰”‘–‡‹  DzDz Ǧͳ Ǧͳ k‡ ͳǦͲ͸ Ǥ‹ Ǥ‰”‘–‡‹  DzDz Ǧͳ Ǧͳ k‡ ‡–‘ƒ ‡–ƒ–‡ ͳǦͲ͸ Ǥ‹ Ǥ‰”‘–‡‹  DzDz Ǧͳ Ǧͳ k‡‡–ƒŠ›†”‘š›„—–›”ƒ–‡ ͳǦͲ͸ Ǥ‹ Ǥ‰”‘–‡‹  DzDz k‡ ͳǦͲ͸ Ǥ‹ǦͳǤ‰”‘–‡‹Ǧͳ DzDz

Boundary metabolites and cofactors  ƒ” ʹͲͲ ρ ȏͳȐ ‘ ͳͶͲ ρ ȏͳȐ ƒ” ͻͷͲ ρ ȏͳȐ  ‡–›Ž‘ ͹Ͳ ρ ȏͳȐ  – ͲǤ͹͹ ρ ȏͳȐ – ʹͷͲ ρ ȏͳȐ   ͳ͸ ρ ȏͳȐ ‘– ʹͺͺ ρ ȏͶ͹Ȑ ‘ ʹ ʹͺ ρ ȏͶ͹Ȑ  ͶͻͲͲ ρ ȏͶͺȐ    ͵ͷ͸ ρ ȏͶͻȐ  

ͳͻ͵ Chapter 5

Parameter Value unit References Conserved moieties ‘– ͷͲͲͲ ρ ȏͳȐ  Compartment volumes    ʹǤʹȉͳͲǦ͸ ˜‰”‘–‡‹Ǧͳ ȏͳȐ  ͳǤͺȉͳͲǦ͸ ˜‰”‘–‡‹Ǧͳ ȏͳȐ   ͳǤͲȉͳͲǦ͸ ˜‰”‘–‡‹Ǧͳ •–‹ƒ–‡†„ƒ•‡†‘ȏͷͲȐ  CPT1 parameters used for Figure 6. Parameter Value Reference value unit CPT1    sf ’–ͳͳͶ ͲǤͳ͵Ͷ ͳǤ͵Ͷ   sf ’–ͳͳʹ ͲǤͳͻ͵ ͳǤͻ͵   sf ’–ͳͳͲ ͲǤͳͷͶ ͳǤͷͶ   sf ’–ͳͺ ͲǤͲͺ͵ ͲǤͺ͵   sf ’–ͳ͸ ͲǤͲͷͳ ͲǤͷͳ   sf ’–ͳͶ ͲǤͲʹͷ ͲǤʹͷ  

Km ’–ͳͳͶ ›Ž‘ ͵ͲͺͲͲ ͵ͲǤͺ ρ 

Km ’–ͳͳʹ ›Ž‘ ͳͳͲͲͲ ͳͳ ρ 

Km ’–ͳͳͲ ›Ž‘ ͳ͸͹ͲͲ ͳ͸Ǥ͹ ρ 

Km ’–ͳͺ ›Ž‘ ʹʹͻͲͲ ʹʹǤͻ ρ 

Km ’–ͳ͸ ›Ž‘ ͳͷͳͷͲͲ ͳͷͳǤͷ ρ 

Km ’–ͳͶ ›Ž‘ ͸ͷ͵ͷͲͲ ͸ͷ͵Ǥͷ ρ  ‡—•‡†–Š‡•‡–‘•‹—Žƒ–‡–Š‡‡ˆˆ‡ –‘ˆ”‡†— ‡†ƒ –‹˜‹–›ƒ†ƒˆˆ‹‹–›–‘™ƒ”†•ͳͶǦͶƒ ›ŽǦ‘•—„•–”ƒ–‡•Ǥ  ZĞĨĞƌĞŶĐĞƐ ͳǤ ˜ƒ—‡ǡ‹‘• ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ†‡‡•–‡ ǡ‘—™ǡ ‘—–‡ǡ ”‘‡ ǡ”ƒ„ǡ‡‹Œ‰‘—† ǡƒ‡”Ǥ‹‘ Š‡‹ ƒŽ‘’‡–‹–‹‘ƒ‡• ƒ––›Ǧ ‹†ȾǦš‹†ƒ–‹‘ —Ž‡”ƒ„Ž‡–‘—„•–”ƒ–‡˜‡”Ž‘ƒ†Ǥ‘‘’—–‹‘ŽǤʹͲͳ͵ǢͻȋͺȌǣʹȂͻǤ ʹǤ ‘Ž–‡”• ǡ ‹ƒ’ƒ‹–‡ ǡ ˜ƒ —‡ ǡ ‹‡œ‡Ǧ‘‹‰ ǡ ƒ––‘ ǡ ‘”–‡  ǡ ‘”˜ƒ–‘˜‹ Š ǡ ƒ‡” ǡ ‹• Š‘ˆˆ ǡ ‡”‡–‹‡” Ǥ ”ƒ•Žƒ–‹‘ƒŽ –ƒ”‰‡–‡† ’”‘–‡‘‹ • ’”‘ˆ‹Ž‹‰ ‘ˆ ‹–‘ Š‘†”‹ƒŽ ‡‡”‰› ‡–ƒ„‘Ž‹  ’ƒ–Š™ƒ›• ‹ ‘—•‡ ƒ† Š—ƒ •ƒ’Ž‡•Ǥ —„‹–– –‘ —„ŽǤ ʹͲͳ͸Ǣ ͵Ǥ ‹‘ Š‹ƒ”‘ ǡ ‘Ž‘„‘ ǡ ‹‘ƒ–‘ Ǥ —”‹ˆ‹ ƒ–‹‘ǡ Šƒ”ƒ –‡”‹œƒ–‹‘ ƒ† ’ƒ”–‹ƒŽ ƒ‹‘ ƒ ‹† •‡“—‡ ‡• ‘ˆ ƒ”‹–‹‡ ’ƒŽ‹–‘›ŽǦ–”ƒ•ˆ‡”ƒ•‡ ˆ”‘ Š—ƒ Ž‹˜‡”Ǥ  ‡––Ǥ ͳͻͻͲǢʹ͹ͶȋͳȂ ʹȌǣͳ͸͵Ȃ͸Ǥ ͶǤ ƒ•ƒ› ǡ ‡””‹  ǡ ”‹‡† ǡ —„„• Ǥ —”‹ˆ‹ ƒ–‹‘ ƒ† ’”‘’‡”–‹‡• ‘ˆ –Š‡ •‘Ž—„Ž‡ ƒ”‹–‹‡ ’ƒŽ‹–‘›Ž–”ƒ•ˆ‡”ƒ•‡ ˆ”‘ „‘˜‹‡ Ž‹˜‡” ‹–‘ Š‘†”‹ƒǤ ‹‘ Š‡ Ǥ ͳͻͺ͹ — ͳǢʹͶͶȋʹȌǣʹ͹ͳȂͺǤ ͷǤ ‹Ž„‡”– ǡ ‡‘š  ǡ ‘••ƒ ǡ ƒ”Ž‡ Ǥ Š‡ ”‡Žƒ–‹‘ ‘ˆ ƒ ›Ž –”ƒ•ˆ‡” –‘ –Š‡ ‘˜‡”ƒŽŽ ”‡ƒ –‹‘‘ˆ–Š‹‘Žƒ•‡ ˆ”‘’‘” ‹‡Š‡ƒ”–Ǥ ‹‘ŽŠ‡ǤͳͻͺͳǢʹͷ͸ȋͳͶȌǣ͹͵͹ͳȂ͹Ǥ ͸Ǥ ‹ä‹‡™•‹ ǡ ‹Ž†Š‡†‡ ǡ ‘”± ǡ ”–—”••‘ Ǥ Ǧ†‡’–Š “—ƒ–‹–ƒ–‹˜‡ ƒƒŽ›•‹• ƒ† ‘’ƒ”‹•‘‘ˆ–Š‡Š—ƒŠ‡’ƒ–‘ ›–‡ƒ†Š‡’ƒ–‘ƒ ‡ŽŽŽ‹‡ ‡’ ʹ’”‘–‡‘‡•Ǥ ”‘–‡‘‹ •Ǥ ʹͲͳ͸Ǣͳ͵͸ǣʹ͵ͶȂͶ͹Ǥ ͹Ǥ    ǡ  ǡ    ǡ    ǡ   ǡ   Ǥ —”‹ˆ‹ ƒ–‹‘ ƒ† ’”‘’‡”–‹‡• ‘ˆ ƒ”‹–‹‡ ƒ ‡–›Ž–”ƒ•ˆ‡”ƒ•‡ ˆ”‘Š—ƒ Ž‹˜‡”Ǥ —”  ‹‘ Š‡Ǥ

ͳͻͶ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

ͳͻͻͲǢͳͺͻȋ͵Ȍǣͷ͵ͻȂͶ͸Ǥ ͺǤ ‹›ƒœƒ™ƒ ǡ œƒ•ƒ ǡ •—‹ ǡ ƒ•Š‹‘–‘ Ǥ —”‹ˆ‹ ƒ–‹‘ ƒ† ’”‘’‡”–‹‡• ‘ˆ ƒ”‹–‹‡ ‘ –ƒ‘›Ž–”ƒ•ˆ‡”ƒ•‡ ƒ† ƒ”‹–‹‡ ’ƒŽ‹–‘›Ž–”ƒ•ˆ‡”ƒ•‡ ˆ”‘ ”ƒ– Ž‹˜‡”Ǥ  ‹‘ Š‡Ǥ ͳͻͺ͵ǢͻͶȋʹȌǣͷʹͻȂͶʹǤ ͻǤ ‘›ƒƒǡ‘—”‹ǡ•Š‹—„‘ǡƒ‹Œ‘ǡƒƒ‰— Š‹ǡ‡ŽŽ‡› ǡŠ‡ƒ† ǡ‡–ƒ‡ǡƒƒƒ ǡ ƒ•Š‹‘–‘Ǥ—”‹ˆ‹ ƒ–‹‘‘ˆŠ—ƒ˜‡”›ǦŽ‘‰Ǧ Šƒ‹ƒ ›ŽǦ ‘‡œ›‡†‡Š›†”‘‰‡ƒ•‡ƒ† Šƒ”ƒ –‡”‹œƒ–‹‘‘ˆ‹–•†‡ˆ‹ ‹‡ ›‹•‡˜‡’ƒ–‹‡–•Ǥ Ž‹ ˜‡•–ǤͳͻͻͷǢͻͷȋ͸ȌǣʹͶ͸ͷȂ͹͵Ǥ ͳͲǤ ƒ†› ǡ ‹‡™‡‰ ǡ ”ƒ—–Ž‡ ǡ ‘  ǡ ò ŠŽ‡” ǡ ”‘•• ǡ ‹ ǡ ƒ• Š‡† ǡ Š‹•Žƒ Ǥ ‡†‹—ǦŽ‘‰Ǧ Šƒ‹ Š‹‡”‹  Š—ƒ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ǣ ‡†‹—Ǧ Šƒ‹ ‡œ›‡ ™‹–Š –Š‡ ƒ –‹˜‡ ‡–‡” „ƒ•‡ ƒ””ƒ‰‡‡– ‘ˆ Ž‘‰Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡Ǥ ‹‘ Š‡‹•–”›Ǥ ͳͻͻ͸Ǣ͵ͷȋ͵ͺȌǣͳʹͶͲʹȂͳͳǤ ͳͳǤ ˜ƒ—‡ǡ‘Ž‡”Ǧ‘—™ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ‘Ž–‡”• ǡ˜ƒ‹Œ– ǡƒ”–‹‡• ǡ 5 ‹‡œ‡Ǧ‘‹‰ǡ ‡‹‡”ǡ‡”‡–‹‡” ǡ ”‘‡ǡ‡‹Œ‰‘—† ǡ‡”• ǡƒ‡”Ǥ ‹˜‹‰‘–Š‡‡†‰‡ǣ—„•–”ƒ–‡ ‘’‡–‹–‹‘‡š’Žƒ‹•Ž‘••‘ˆ”‘„—•–‡••‹‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧ ƒ ‹†‘š‹†ƒ–‹‘†‹•‘”†‡”•Ǥ‹‘ŽǤʹͲͳ͸ǢͳͶȋͳȌǣͳȂͳͷǤ ͳʹǤ œƒ‹ǡ Š‹†ƒǡ”‹‹ǡƒƒ‘–‘ǡ ƒ•Š‹‘–‘Ǥ‘˜‡Žˆƒ––›ƒ ‹†„‡–ƒǦ‘š‹†ƒ–‹‘‡œ›‡•‹ ”ƒ– Ž‹˜‡” ‹–‘ Š‘†”‹ƒǤ Ǥ —”‹ˆ‹ ƒ–‹‘ ƒ† ’”‘’‡”–‹‡• ‘ˆ ˜‡”›ǦŽ‘‰Ǧ Šƒ‹ ƒ ›ŽǦ ‘‡œ›‡  †‡Š›†”‘‰‡ƒ•‡Ǥ ‹‘ŽŠ‡Ǥͳͻͻʹ ƒͳͷǢʹ͸͹ȋʹȌǣͳͲʹ͹Ȃ͵͵Ǥ ͳ͵Ǥ ‹‘ Š‹ƒ”‘ ǡ –‘ǡƒƒƒǤ—”‹ˆ‹ ƒ–‹‘ƒ†’”‘’‡”–‹‡•‘ˆ•Š‘”–Ǧ Šƒ‹ƒ ›ŽǦ‘ǡ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ǡ ƒ† ‹•‘˜ƒŽ‡”›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡• ˆ”‘ Š—ƒ Ž‹˜‡”Ǥ †˜ ‡—”‘ŽǤ ͳͻͺͺǢͶͺȋͳ͹ȌǣʹʹͳȂ͵ͲǤ ͳͶǤ ‹‡ƒǡ ƒ”‹Œƒǡ–”‘œ›ǡ —ƒ‘ǡŽ‡š•‘ ǡ‹‡”‡‰ƒǤŠ‡ ”›•–ƒŽ•–”— –—”‡‘ˆ Š—ƒ‹–‘ Š‘†”‹ƒŽ͵Ǧ‡–‘ƒ ›ŽǦ‘–Š‹‘Žƒ•‡ȋͳȌǣ •‹‰Š–‹–‘–Š‡”‡ƒ –‹‘‡ Šƒ‹•‘ˆ‹–• –Š‹‘Žƒ•‡ ƒ† –Š‹‘‡•–‡”ƒ•‡ ƒ –‹˜‹–‹‡•Ǥ  –ƒ ”›•–ƒŽŽ‘‰” ‡ –  ‹‘Ž ”›•–ƒŽŽ‘‰”Ǥ ʹͲͳͶǢ͹ͲȋͳʹȌǣ͵ʹͳʹȂʹͷǤ ͳͷǤ –ƒƒ  ǡ‹•–‘  ǡ Š—Žœ Ǥ—”‹ˆ‹ ƒ–‹‘ƒ†’”‘’‡”–‹‡•‘ˆƒ’‹‰Š‡ƒ”––Š‹‘Žƒ•‡™‹–Š„”‘ƒ† Šƒ‹Ž‡‰–Š•’‡ ‹ˆ‹ ‹–›ƒ† ‘’ƒ”‹•‘‘ˆ–Š‹‘Žƒ•‡•ˆ”‘’‹‰Š‡ƒ”–ƒ†• Š‡”‹ Š‹ƒ ‘Ž‹Ǥ ‹‘Ž Š‡Ǥͳͻ͹ͺǢʹͷ͵ȋ͸Ȍǣͳͺʹ͹Ȃ͵ͳǤ ͳ͸Ǥ ‹›ƒœƒ™ƒǡ —”—–ƒǡ•—‹ǡ ƒ•Š‹‘–‘ǡ‹Ǥ”‘’‡”–‹‡•‘ˆ’‡”‘š‹•‘ƒŽ͵Ǧ‡–‘ƒ ›ŽǦ ‘ –Š‹‘Žƒ•‡ˆ”‘”ƒ–Ž‹˜‡”Ǥ ‹‘ Š‡Ǥͳͻͺͳ—‰ǢͻͲȋʹȌǣͷͳͳȂͻǤ ͳ͹Ǥ ƒ•ƒ› ǡ –‡‡ƒ’  ǡ —•ƒ‹ Ǥ ‡ƒ –‹‘• ‘ˆ ‡Ž‡ –”‘Ǧ–”ƒ•ˆ‡” ˆŽƒ˜‘’”‘–‡‹ ƒ† ‡Ž‡ –”‘Ǧ–”ƒ•ˆ‡”ˆŽƒ˜‘’”‘–‡‹ǣ—„‹“—‹‘‡‘š‹†‘”‡†— –ƒ•‡Ǥ‹‘ Š‡ Ǥͳͻͺ͹ǢʹͶͳǣͺͺ͵ȂͻʹǤ ͳͺǤ ‡ ƒ ǡ ”‡”ƒ ǤŽ‡ –”‘Ǧ”ƒ•ˆ‡” Žƒ˜‘’”‘–‡‹Ǧ„‹“—‹‘‡š‹†‘”‡†— –ƒ•‡ˆ”‘ ‹‰ ‹˜‡”ǣ —”‹ˆ‹ ƒ–‹‘ ƒ† ‘Ž‡ —Žƒ”ǡ ‡†‘šǡ ƒ† ƒ–ƒŽ›–‹  ”‘’‡”–‹‡•Ǥ ‹‘ Š‡‹•–”›Ǥ ͳͻͺͷǢʹͶȋͳͷȌǣ͵ͻͳ͵ȂʹͳǤ ͳͻǤ ƒ–‘—‰Š ǡ‘‡Š” ǡ”ƒ‡ǡ ”‡”ƒ Ǥ”›’–‘’ŠƒˆŽ—‘”‡• ‡ ‡‹‡Ž‡ –”‘Ǧ–”ƒ•ˆ‡” ˆŽƒ˜‘’”‘–‡‹ǣ—„‹“—‹‘‡ ‘š‹†‘”‡†— –ƒ•‡ǣ ˆŽ—‘”‡• ‡ ‡ “—‡ Š‹‰ „› ƒ „”‘‹ƒ–‡† ’•‡—†‘•—„•–”ƒ–‡Ǥ‹‘ Š‡‹•–”›ǤͳͻͻͳǢ͵ͲȋͷȌǣͳ͵ͳ͹Ȃʹ͵Ǥ ʹͲǤ ”‡”ƒ Ǥ‡ƒ –‹‘‘ˆ ‡Ž‡ –”‘Ǧ–”ƒ•ˆ‡” ˆŽƒ˜‘’”‘–‡‹—„‹“—‹‘‡‘š‹†‘”‡†— –ƒ•‡ ™‹–Š –Š‡ ‹–‘ Š‘†”‹ƒŽ”‡•’‹”ƒ–‘”› Šƒ‹ǤǦ‹‘‡‡”‰Ǥͳͻͺ͹Ǣͺͻ͵ȋʹȌǣͳ͸ͳȂͻǤ ʹͳǤ —•ƒ‹ǡ–ƒ‘˜‹ Šǡ ‘š Ǥ‡ƒ•—”‡‡–‘ˆ–Š‡‘š‹†ƒ–‹‘Ǧ”‡†— –‹‘’‘–‡–‹ƒŽ•ˆ‘”‘‡Ǧ ‡Ž‡ –”‘ƒ†–™‘Ǧ‡Ž‡ –”‘”‡†— –‹‘‘ˆ‡Ž‡ –”‘Ǧ–”ƒ•ˆ‡”ˆŽƒ˜‘’”‘–‡‹ˆ”‘’‹‰Ž‹˜‡”Ǥ‹‘ Š‡ ǤͳͻͺͶǢʹͳͻȋ͵ȌǣͳͲͶ͵Ȃ͹Ǥ ʹʹǤ ƒ”’ Ǥ ‘ ‹–Ž‡ ȏ –‡”‡–ȐǤ ƒ„Ž‡ ͷǤͳ ‹ ƒ”’Ǣ ‡”ƒŽ†Ǣ ‡ŽŽ ƒ† ‘Ž‡ —Žƒ” ‹‘Ž‘‰›Ǣ ͷ–Š †ǤǢ ‹Ž‡›Ǣ ʹͲͲͺǤ ʹͲͲͺǤ ˜ƒ‹Žƒ„Ž‡ ˆ”‘ǣ Š––’ǣȀȀŠ›’‡”’Š›•‹ •Ǥ’Š›Ǧ ƒ•–”Ǥ‰•—Ǥ‡†—ȀŠ„ƒ•‡ȀŠ‡‹ ƒŽȀ”‡†‘š’ǤŠ–Ž ʹ͵Ǥ —–Šǡ‹‡”‹ Šǡ‡›Šƒ—•‡˜Ǥǡ‡—„‡”–Ǥ—Ž–‹’Ž‡‹–‘ Š‘†”‹ƒŽˆ‘”•‘ˆƒ ‡–‘ƒ ‡–›ŽǦ ‘ –Š‹‘Žƒ•‡ ‹ ”ƒ– Ž‹˜‡”ǣ ‘••‹„Ž‡ ”‡‰—Žƒ–‘”› ”‘Ž‡ ‹ ‡–‘‰‡‡•‹•Ǥ ‹‘ Š‡ ‹‘’Š›• ‡• ‘—Ǥͳͻ͹ͶǢͷ͸ȋͶȌǣͳͲ͸ͻȂ͹͹Ǥ

ͳͻͷ Chapter 5

ʹͶǤ ‹††Ž‡–‘ Ǥ Š‡ ‘š‘ƒ ›ŽǦ ‘‡œ›‡  –Š‹‘Žƒ•‡• ‘ˆ ƒ‹ƒŽ –‹••—‡•Ǥ ‹‘ Š‡ Ǥ ͳͻ͹͵Ǣͳ͵ʹȋͶȌǣ͹ͳ͹Ȃ͵ͲǤ ʹͷǤ ‡‡† ǡ Ž‹‡„‡ƒ”† ǡ ƒ‡ Ǥ ‘Ž‡ —Žƒ” ƒ† ƒ–ƒŽ›–‹  ’”‘’‡”–‹‡• ‘ˆ ‹–‘ Š‘†”‹ƒŽ ȋ‡–‘‰‡‹ Ȍ ͵ǦŠ›†”‘š›Ǧ͵Ǧ‡–Š›Ž‰Ž—–ƒ”›Ž ‘‡œ›‡  •›–Šƒ•‡ ‘ˆ Ž‹˜‡”Ǥ  ‹‘Ž Š‡Ǥ ͳͻ͹ͷǢʹͷͲȋͺȌǣ͵ͳͳ͹Ȃʹ͵Ǥ ʹ͸Ǥ ‘™‡ ǡ —„„• Ǥ ͵Ǧ ›†”‘š›Ǧ͵Ǧ‡–Š›Ž‰Ž—–ƒ”›ŽǦ ‘‡œ›‡  •›–Šƒ•‡ ˆ”‘ ‘š Ž‹˜‡”Ǥ —”‹ˆ‹ ƒ–‹‘ǡ‘Ž‡ —Žƒ”ƒ† ƒ–ƒŽ›–‹ ’”‘’‡”–‹‡•Ǥ‹‘ Š‡ ǤͳͻͺͷǢʹʹ͹ǣͷͻͳȂͻǤ ʹ͹Ǥ ‡“  ʹǤ ʹͺǤ –‡‰‹ǡ‘‘ Ǥ–‡”‡‘•’‡ ‹ˆ‹ ‹–›ƒ†‘–Š‡”’”‘’‡”–‹‡•‘ˆŠ‹‰ŠŽ›’—”‹ˆ‹‡†„‡–ƒǦŠ›†”‘š›Ǧ „‡–ƒǦ‡–Š›Ž‰Ž—–ƒ”›Ž ‘‡œ›‡  Ž‡ƒ˜ƒ‰‡ ‡œ›‡ ˆ”‘ „‘˜‹‡ Ž‹˜‡”Ǥ  ‹‘Ž Š‡Ǥ ͳͻ͸ͺǢʹͶ͵ȋʹͲȌǣͷʹ͹ʹȂͻǤ ʹͻǤ —‹•–”ƒǡƒ‰ǡ‹– Š‡ŽŽ ǡ‹œ‹‘”‘ Ǥ˜ƒŽ—ƒ–‹‘‘ˆ͵Ǧ ›†”‘š›Ǧ͵Ǧ‡–Š›Ž‰Ž—–ƒ”›ŽǦ ‘‡œ›‡  ›ƒ•‡ ”‰‹‹‡ǦͶͳ ƒ• ƒ ƒ–ƒŽ›–‹  ‡•‹†—‡ǣ •‡ ‘ˆ  ‡–›Ž†‹–Š‹‘Ǧ‘‡œ›‡  –‘ ‘‹–‘””‘†— –‘Ž‹œƒ–‹‘Ǥ‹‘ Š‡‹•–”›ǤʹͲͲͶǢͶ͵ȋͳͺȌǣͷʹͺ͹ȂͻͷǤ ͵ͲǤ ‡“  Ǥ ͵ͳǤ — ‡”  ǡ ƒ™•‘ Ǥ Š‡ ‹‡–‹ • ‘ˆ ƒ– ‹˜‡” ƒ† ‡ƒ”– ‹–‘ Š‘†”‹ƒŽ Ǧ ›†”‘š›„—–›”ƒ–‡‡Š›†”‘‰‡ƒ•‡Ǥͳͻ͹ͻǢͳ͹ͻǣͷ͹ͻȂͺͳǤ ͵ʹǤ Šƒ‘ ǡƒ”–‹ǡ‹•‘ˆˆ‹ǡ—ƒ™ƒ›Ǧƒ”‹ƒ‘ǤŠ‡–Ǧ–‡”‹ƒŽ‘†—Žƒ–‘”’”‘–‡‹‹• ƒ ›ŽǦ‘–Š‹‘‡•–‡”ƒ•‡‘ˆ–Š‡Š‘–†‘‰Ǧˆ‘Ž†ˆƒ‹Ž›Ǥ‹‘ Š‡‹•–”›ǤʹͲͲͻǢͶͺȋʹͶȌǣͷͷͲ͹ȂͻǤ ͵͵Ǥ ƒ‰Ž‹ƒ”‹‹ ǡƒŽ˜‘ǡŠƒ‰ǡŠ‡–Šǡƒˆƒ‹ǡ‰ǡƒŽˆ‘”† ǡ—‰‹ƒƒǡ‘‡Šǡ Š‡ ǡ ‹ŽŽ ǡ ‹†ƒŽ ǡ ˜ƒ• ǡ Š‘”„—” ǡ ƒ”” ǡ ‘‘–ŠƒǤ‹–‘ Š‘†”‹ƒŽ ”‘–‡‹‘’‡†‹—Ž— ‹†ƒ–‡•‘’Ž‡š ‹•‡ƒ•‡‹‘Ž‘‰›Ǥ‡ŽŽǤʹͲͲͺǢͳ͵ͶȋͳȌǣͳͳʹȂʹ͵Ǥ ͵ͶǤ ŽŽ‹• ǡ ‘™ƒ ǡ ‘Žˆ‰ƒ‰  Ǥ ‡–ƒ„‘Ž‹  ƒ† ‹••—‡Ǧ’‡ ‹ˆ‹  ‡‰—Žƒ–‹‘ ‘ˆ  ›ŽǦ‘ ‡–ƒ„‘Ž‹•Ǥ‘‡ǤʹͲͳͷǢͳͲȋ͵ȌǤ ͵ͷǤ ƒ‘ ǡ — ǡ Šƒ‘ ǡ ‘‰ ǡ —ƒ™ƒ›Ǧƒ”‹ƒ‘ Ǥ Š‡ ‡ Šƒ‹•• ‘ˆ Š—ƒ Š‘–†‘‰Ǧˆ‘Ž† –Š‹‘‡•–‡”ƒ•‡ ʹȋŠͲͲͷͶ ʹȌ •—„•–”ƒ–‡ ”‡ ‘‰‹–‹‘ ƒ† ƒ–ƒŽ›•‹• ‹ŽŽ—‹ƒ–‡†„› ƒ •–”— –—”‡ ƒ†ˆ— –‹‘„ƒ•‡†ƒƒŽ›•‹•Ǥ‹‘ Š‡‹•–”›ǤʹͲͲͻǢͶͺȋ͸Ȍǣͳʹͻ͵Ȃ͵ͲͶǤ ͵͸Ǥ ‘ˆˆƒ–ǡŠƒ–‹ƒǡ‰—›‡ǡ› Šǡƒ‰ǡƒ‰ǡ Žƒ›‡˜ƒǡ ƒǡ ‹”• Š‡›ǡ Žƒ›’‘‘Žǡ‡‹ˆ‡”–Ǥ ›ŽǦ‘–Š‹‘‡•–‡”ƒ•‡Ǧʹˆƒ ‹Ž‹–ƒ–‡•‹–‘ Š‘†”‹ƒŽˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘ ‹–Š‡Ž‹˜‡”Ǥ ‹’‹†‡•ǤʹͲͳͶǢͷͷȋͳʹȌǣʹͶͷͺȂ͹ͲǤ ͵͹Ǥ ‹ŽŽƒ†‡” ǡ ‘”†•–”ڏ ǡ ‡‹ŽŽ› ǡ –”‘œ› ǡ ƒ ‡Ž†Š‘˜‡ ǡ —– ǡ Ž‡š•‘  Ǥ  ›ŽǦ‘–Š‹‘‡•–‡”ƒ•‡ͻȋͻȌ‹‘—•‡ƒ›’”‘˜‹†‡ƒ‘˜‡ŽŽ‹„‡–™‡‡ˆƒ––›ƒ ‹†ƒ† ƒ‹‘ƒ ‹†‡–ƒ„‘Ž‹•‹‹–‘ Š‘†”‹ƒǤ‡ŽŽ‘Ž‹ˆ‡ ‹ǤʹͲͳͶǢ͹ͳȋͷȌǣͻ͵͵ȂͶͺǤ ͵ͺǤ ŽƒŠ‘Žœ ǡ ‘‘” ǡ ƒ”Ǧ˜‡ ǡ ‹Ž‘ Ǥ —‹Ž‹„”ƒ–‘” Ǧ Š‡ „‹‘ Š‡‹ ƒŽ –Š‡”‘†›ƒ‹ • ƒŽ —Žƒ–‘”Ǥ— Ž‡‹  ‹†•‡•ǤʹͲͳʹǢͶͲȋͳȌǣ͹͹ͲȂͷǤ ͵ͻǤ ƒ” ‡Ž ǡ —œ—‡ Ǥ ‹‡–‹  –—†‹‡• ‘ –Š‡ ’‡ ‹ˆ‹ ‹–› ‘ˆ ‘‰ Šƒ‹  ›Ž ‘‡œ›‡  ›–Š‡–ƒ•‡ˆ”‘ƒ–‹˜‡”‹ ”‘•‘‡•‹‡–‹ ‘‡œ›‡–—†‹‡•‘–Š‡’‡ ‹ˆ‹ ‹–›ˆ”‘ƒ– ‘ˆ‘‰‹˜‡”Šƒ‹ ›Ž›–Š‡–ƒ•‡‹ ”‘•‘‡•Ǥ ‹‘ŽŠ‡Ǥͳͻ͹ʹǢʹͶ͹ȋʹͳȌǣͶͶ͵͵Ȃ͸Ǥ ͶͲǤ —Œ‹‘ ǡ ƒǦ ‘‰ ǡ —œ—— ǡ ‹Œ‹ƒ ǡ ƒƒ‘–‘ Ǥ ‘Ž‡ —Žƒ” Šƒ”ƒ –‡”‹œƒ–‹‘ ƒ† š’”‡••‹‘‘ˆƒ– ›ŽǦ‘›–Š‡–ƒ•‡͵Ǥ ‹‘ŽŠ‡Ǥͳͻͻ͸Ǣʹ͹ͳȋʹͺȌǣͳ͸͹ͶͺȂͷʹǤ ͶͳǤ ”‡‰‡”•‡ǡ‘”–‡•‡ǡގ˜”ƒƒǤ–Š‡„‹‘Ž‘‰‹ ‘”‹‰‹‘ˆ͸ǦͳͲǦ†‹ ƒ”„‘š›Ž‹ ƒ†͸Ǧ ͳͲǦ‘‡‰ƒǦͳǦ Š›†”‘š› ‘‘ ƒ”„‘š›Ž‹  ƒ ‹†• ‹ Š—ƒ ƒ† ”ƒ– ™‹–Š ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ–‹‘ †‡ˆ‹ ‹‡ ‹‡•ǣ ‹ ˜‹–”‘ •–—†‹‡• ‘ –Š‡ ‘‡‰ƒǦ ƒ† ‘‡‰ƒǦͳǦ‘š‹†ƒ–‹‘ ‘ˆ ‡†‹—Ǧ Šƒ‹ȋ͸ǦͳʹȌˆƒ––›ƒ ‹†•‹Š—ƒǤ‡†‹ƒ–”‡•Ǥͳͻͺ͵Ǣͳ͹ȋͳͲȌǣͺʹͺȂ͵ͶǤ ͶʹǤ ƒ™ƒ•Š‹ƒ ǡ ƒ‰ƒ—ƒ ǡ —•—‘•‡ ǡ ‘‘ ǡ ƒ•—‘–‘ ǡ —‰‹—”ƒ ǡ ‹•Š‹‘–‘ Ǥ —ƒˆƒ––›ƒ ‹†ɘǦŠ›†”‘š›Žƒ•‡ǡͶͳͳǣ‡–‡”‹ƒ–‹‘‘ˆ ‘’Ž‡–‡‰‡‘‹ •‡“—‡ ‡ƒ† Šƒ”ƒ –‡”‹œƒ–‹‘‘ˆ’—”‹ˆ‹‡†”‡ ‘„‹ƒ–’”‘–‡‹Ǥ” Š‹‘ Š‡‹‘’Š›•ǤʹͲͲͲǢ͵͹ͺȋʹȌǣ͵͵͵ȂͻǤ Ͷ͵Ǥ ‘ ŠǡŠƒ‰ǡ”‘‡–œǡ”–‹œ†‡‘–‡ŽŽƒ‘Ǥ–”— –—”ƒŽ†‡–‡”‹ƒ–‹‘‘ˆ–Š‡•—„•–”ƒ–‡ •’‡ ‹ˆ‹ ‹–‹‡•ƒ†”‡‰‹‘•‡Ž‡ –‹˜‹–‹‡•‘ˆ–Š‡”ƒ–ƒ†Š—ƒˆƒ––›ƒ ‹†‘‡‰ƒǦŠ›†”‘š›Žƒ•‡•Ǥ” Š

ͳͻ͸ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

‹‘ Š‡‹‘’Š›•ǤʹͲͲͲǢ͵͹͵ȋͳȌǣ͸͵Ȃ͹ͳǤ ͶͶǤ ƒ‹‡”  Ǥǡ ‡ŽŽƒ‹‡ ǡ ƒ™•‘ ǡ ‘„Ž‡ ǡ ”ƒ– ǡ ƒ‰ ǡ —’’Ž‡• ǡ —‘ ǡ ‡‹••‹‡ ǡ ǯ‘‡ŽŽ  ǡ ”‘™  ǡ ƒ–‡”ƒ ǡ ƒ’†‡˜‹Žƒ Ǥ — –‹‘ƒŽ ˜ƒ”‹ƒ– ‘ˆ ͶͳͳʹͲǦŠ›†”‘š›‡‹ ‘•ƒ–‡–”ƒ‡‘‹ ƒ ‹†•›–Šƒ•‡‹•ƒ••‘ ‹ƒ–‡†™‹–Š‡••‡–‹ƒŽŠ›’‡”–‡•‹‘Ǥ ‹” —Žƒ–‹‘ǤʹͲͲͷǢͳͳͳȋͳȌǣ͸͵ȂͻǤ ͶͷǤ ‹‡”• ǡ Šƒ‰ ǡ ‘Š•‘  ǡ ‡ ”–‹œ ‘–‡ŽŽƒ‘ Ǥ Š‡ ƒ–ƒŽ›–‹  •‹–‡ ‘ˆ ›–‘ Š”‘‡ ͶͷͲͶͳͳȋͶͳͳȌƒ†‹–•ͳ͵ͳ —–ƒ–Ǥ ‹‘ŽŠ‡ǤͳͻͻͺǢʹ͹͵ȋ͵͸Ȍǣʹ͵ͲͷͷȂ͸ͳǤ Ͷ͸Ǥ ‹—”ƒǡ ‹•ƒ‹ ǡ‡–ƒǤ‘‡‰ƒǦƒ†ȋ‘‡‰ƒǦǦͳȌǦŠ›†”‘š›Žƒ–‹‘‘ˆˆƒ––›ƒ ‹†•„›ˆ”‘‰Ž‹˜‡” ‹ ”‘•‘‡•Ǥ —„•–”ƒ–‡ •’‡ ‹ˆ‹ ‹–› ƒ† ‘–Š‡” ’”‘’‡”–‹‡•Ǥ ‹‘ Š‹ ‹‘’Š›•  –ƒǤ ͳͻ͹ͺ ‘˜ ʹʹǢͷ͵ͳȋʹȌǣͳͶͻȂͷ͹Ǥ Ͷ͹Ǥ —‰‹ Ǥ ›ƒ‹  ‹‡–‹  ‘†‡Ž‹‰ ‘ˆ ‹–‘ Š‘†”‹ƒŽ ‡”‰› ‡–ƒ„‘Ž‹•Ǥ Ǧ‡ŽŽ ›•– ƒ•‹  ‘ ‡’–•’’ŽǤʹͲͳ͵ǢͳͲͷȂͶʹǤ 5 ͶͺǤ ‘„‘ŽŽǡ‡‹–œ ǡ‹‡• ǡ‹‡‰Ž‡”ǡ Š‘ŽœǡŠ‡‹‡ǡ‹—••‡Ž†‘”ˆ ǡ—••‡Ž†‘”ˆǦǤˆˆ‡ –‘ˆ Ž‘‰Ǧ Šƒ‹ˆƒ––›ƒ ›ŽǦ‘‘‹–‘ Š‘†”‹ƒŽƒ† ›–‘•‘Ž‹ Ȁ”ƒ–‹‘•‹–Š‡‹–ƒ –Ž‹˜‡” ‡ŽŽǤʹͲͲͲǢʹʹͲȋͳͻͺͶȌǣ͵͹ͳȂ͸Ǥ ͶͻǤ ‹• ŠŽ‡”ǡ ”‹‡†”‹ Š•ǡ‘ŽŽǡ‹ŽŽ‹ƒ•‘ Ǥ›”‹†‹‡— Ž‡‘–‹†‡†‹•–”‹„—–‹‘•ƒ†‡œ›‡ ƒ•• ƒ –‹‘ ”ƒ–‹‘• ‹ Š‡’ƒ–‘ ›–‡• ˆ”‘ ˆ‡† ƒ† •–ƒ”˜‡† ”ƒ–•Ǥ ” Š ‹‘ Š‡ ‹‘’Š›•Ǥ ͳͻ͹͹ ‘˜ǢͳͺͶȋͳȌǣʹʹʹȂ͵͸Ǥ ͷͲǤ Ž„‡”–•ǡ ‘Š•‘ǡ‡™‹• ǡƒˆˆǡ‘„‡”–•ǡƒŽ–‡”ǤŠ‡‘’ƒ”–‡–ƒŽ‹œƒ–‹‘‘ˆ‡ŽŽ•Ǥ ǣ‘Ž‡ —Žƒ”‹‘Ž‘‰›‘ˆ–Š‡‡ŽŽǤͷ–Š‡†Ǥ‡™‘”ǣ ƒ”Žƒ† ‹‡ ‡ǢʹͲͲʹǤ 

ͳͻ͹ Chapter 5

Supplementary figures and tables

A B C D 14 ) vmcadC4 -1 vvlcadC6 12 vvlcadC8 vmcadC6 vscadC4 vmcadC8 10 vvlcadC10 vmcadC10 .gProtein

-1 vvlcadC12 8 vmcadC12 vvlcadC14 vscadC6 6 vmcadC14 vvlcadC16 vmcadC16 4 (μmol.min 2 nadhsnk J 0 0501000501000501000 50 100 150 200 250

[Palmitoyl-CoA]CYT ;ʅDͿ Palmitoyl-CoA]CYT ;ʅDͿ Palmitoyl-CoA]CYT ;ʅDͿ Palmitoyl-CoA]CYT ;ʅDͿ Supplementary figure S1, mFAO flux of WT and MCAD KO versions of the published fitted human isolated mFAO dynamic model [Eunen ‡–al. 2016] (A) and steady-state fluxes through VLCAD (B), MCAD (C) and SCAD (D). Žƒ ǣǢ”ƒ‰‡ǣǤ

Supplementary Table ST1. Flux and concentration control coefficients of the model as described in Supplementary Text ST1. The same model version (healthy, without any deficiencies) was used in Figure 2 in the main text. The control coeffients were calculated over the net NAD production flux (M/SCHAD plus MTP minus ketogenesis) , the consumption flux of palmitoyl-CoA by CPT1 (vCPT1 C16) and the CoASH concentration in the mitochondrial matrix (MAT).

JNAD production JCPT1C16 [CoASH]MAT [palmitoyl-CoA]CYT ʹͷɊ ͳͲͲɊ ʹͷɊ ͳͲͲɊ ʹͷɊ ͳͲͲɊ Vcpt1 0.59 0.00 0.61 0.06 -0.24 -1.11 Vfcact ͲǤͲͳ ͲǤͲͲ ͲǤͲͳ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vcpt2 ͲǤͲͳ ͲǤͲͲ ͲǤͲͳ ͲǤͲͲ ͲǤͲͲ ǦͲǤͲͺ Vvlcad ͲǤͲͳ ͲǤͲͳ ͲǤͲͲ ͲǤͲͳ ͲǤͲͲ ǦͲǤͲͶ Vmcad ͲǤͲ͵ ͲǤͲʹ ͲǤͲ͵ ͲǤͲʹ ͲǤͲͳ ͲǤͲͲ Vscad ͲǤͲͳ ͲǤͲͲ ͲǤͲͳ ͲǤͲͲ ͲǤͲͲ ͲǤͲͷ Vcrot ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vmschad ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vmckat ͲǤͲ͵ ͲǤͲͳ ͲǤͲʹ ͲǤͲͳ ͲǤͲʹ ͲǤͲ͸ Vmtp ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vacot ǦͲǤͲͶ ͲǤͲͲ ǦͲǤͲͶ ͲǤͲͲ ͲǤͲʹ ͲǤͲͻ Vacat1 ͲǤͲͳ ͲǤͲͳ ͲǤͲͳ ͲǤͲͲ ͲǤͲͳ ͲǤͲ͵ Vhmgcs2 ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vhmgcl ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vbdh1 ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vfetfqo 0.35 0.95 0.33 0.88 0.18 0.98 kmc ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vacs ǦͲǤͲͳ ͲǤͲͲ ǦͲǤͲͳ ͲǤͲͲ ͲǤͲͳ ͲǤͲʹ kcmi ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vwox ͲǤͲͳ ͲǤͲͲ ͲǤͲͳ ͲǤͲͲ ͲǤͲͲ ǦͲǤͲͳ kmic ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ keFFA ͲǤͲͳ ͲǤͲͲ ͲǤͲͳ ͲǤͲͲ ͲǤͲͲ ǦͲǤͲͳ keCn ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͳ ͲǤͲͲ ͲǤͲͳ keDCA ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ keAcetoacetateMAT ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͳ keBetahydroxybutyrateMAT ͲǤͲͲ ǦͲǤͲͳ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ

ͳͻͺ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

14

) 12 -1 10 Leak 8

.gProtein vC16-CoA_cons * -1 6 vnadhsink ɇvExAc-carnCn * 4 ɇvExFFACn * 2 ɇvExDCACn * vacsC16 * J (μmol.min 0 050100 [Palmitoyl-CoA] CYT ;ʅDͿ Supplementary Figure S2. Net NADH production fluxes in the mitochondria (vnadhsink), palmitoyl- CoA consumption flux in nadh production equivalents, leakage of incompletely oxidized intermediate metabolites (“Leak”) expressed in nadh production equivalents and reaction rates which the leakage consists of, namely acyl- ƒ”‹–‹‡‡š’‘”–ȋȭ˜ExAc-carnCn), free fatty acid export 5 ȋȭ˜ Ȍǡ†‹ ƒ”„‘š›Ž‹  ƒ ‹†‡š’‘”–ȋȭ˜Ȍ ƒ†-catalyzed return of palmitoyl-CoA to its constant pool (also in nadh production equivalents). 

ͳͻͻ Chapter 5

Supplementary Table ST2. Flux control coefficients of model with ACS knockdown to 1% of its reference value JNAD production JCPT1C16 [CoASH]MAT [palmitoyl-CoA]CYT ʹͷɊ ͳͲͲɊ ʹͷɊ ͳͲͲɊ ʹͷɊ ͳͲͲɊ Vcpt1 0.31 ͲǤͲͲ 0.34 ͲǤͲ͹ -0.35 -1.04 Vfcact ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vcpt2 ͲǤͲͳ ͲǤͲͲ ͲǤͲͳ ͲǤͲͲ ǦͲǤͲͳ ǦͲǤͲͻ Vvlcad ͲǤͲʹ ͲǤͲͳ ͲǤͲͳ ͲǤͲͳ ͲǤͲͲ ǦͲǤͲͻ Vmcad ͲǤͲ͵ ͲǤͲʹ ͲǤͲ͵ ͲǤͲʹ ͲǤͲͳ ͲǤͲʹ Vscad ͲǤͲͲ ͲǤͲͲ ͲǤͲͳ ͲǤͲͲ ͲǤͲͳ ͲǤͲ͸ Vcrot ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vmschad ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͳ Vmckat ͲǤͲʹ ͲǤͲͳ ͲǤͲʹ ͲǤͲͳ ͲǤͲ͵ ͲǤͲͺ Vmtp ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vacot ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲʹ ͲǤͲͳ ͲǤͲͶ Vacat1 ͲǤͲͳ ͲǤͲͳ ͲǤͲͲ ͲǤͲͲ ͲǤͲͳ ͲǤͲͶ Vhmgcs2 ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vhmgcl ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vbdh1 ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vfetfqo 0.60 0.96 0.56 0.86 0.30 0.95 kmc ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vacs ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ kcmi ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vwox ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ kmic ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Ͳ ͲǤͲͲ keFFA ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ keCn ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͳ ͲǤͲͲ ͲǤͲͳ keDCA ͲǤͲͲ ͲǤͲͲ Ͳ Ͳ Ͳ ͲǤͲͲ keAcetoacetateMAT ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͳ keBetahydroxybutyrateMAT ͲǤͲͲ ǦͲǤͲͳ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ

   WT VLCAD KO MCAD KO SCAD KO  A 40 B C D Leak *

30 ɇvExAc-carnCn *

ɇvExFFACn * 20 ɇvExDCACn * 10 leak (%) Substrate vacsC16 *

0 050100050100050100050100 [Palmitoyl-CoA]CYT ;ʅDͿ [Palmitoyl-CoA]CYT ;ʅDͿ [Palmitoyl-CoA]CYT ;ʅDͿ [Palmitoyl-CoA]CYT ;ʅDͿ   Supplementary Figure S3Ǥ‹—Žƒ–‹‘”‡•—Ž–•‘ˆ–Š‡‘†‡Ž™‹–ŠǦ‘ †‘™–‘ͳΨ‘ˆ‹–•”‡ˆ‡”‡ ‡ ˜ƒŽ—‡Ǥ ƒ‡Ž• A-D †‹•’Žƒ› •—„•–”ƒ–‡ Ž‡ƒƒ‰‡ ”ƒ–‡• ƒ• ’‡” ‡–ƒ‰‡ ‘ˆ ’ƒŽ‹–‘›ŽǦ‘ ‘•—’–‹‘ ˆŽ—š ‡š’”‡••‡†‹ ’”‘†— –‹‘‡“—‹˜ƒŽ‡–•ˆ‘”–Š‡ȋAȌǡȋBȌǡȋCȌƒ†ȋDȌ ˜‡”•‹‘‘ˆ–Š‡‘†‡ŽǤ 

ʹͲͲ Simulating the impact of modifiers on MCAD deficiency in human liver fatty-acid catabolism

Supplementary Table ST3. Flux control coefficients of WT model with C16-specific CPT1 JNAD production JCPT1C16 [CoASH]MAT [palmitoyl-CoA]CYT ʹͷɊ ͳͲͲɊ ʹͷɊ ͳͲͲɊ ʹͷɊ ͳͲͲɊ Vcpt1 1.02 1.01 1.02 1.00 -0.02 -0.11 Vfcact ͲǤͲͳ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vcpt2 ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vvlcad ͲǤͲͳ ͲǤͲͲ ͲǤͲͳ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vmcad ͲǤͳ͸ ͲǤͳ͹ ͲǤͳ͸ ͲǤͳ͹ ͲǤͲͲ ǦͲǤͲͳ Vscad ǦͲǤͲʹ ǦͲǤͲͶ ǦͲǤͲʹ ǦͲǤͲͶ ͲǤͲͲ ͲǤͲͳ Vcrot ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vmschad ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vmckat ͲǤͲͺ ͲǤͲͷ ͲǤͲͺ ͲǤͲͷ ͲǤͲͳ ͲǤͲͳ Vmtp ͲǤͲͲ ͲǤͲͳ ͲǤͲͲ ͲǤͲͳ ͲǤͲͲ ͲǤͲͲ 5 Vacot -0.33 -0.36 -0.33 -0.36 ͲǤͲͳ ͲǤͲͶ Vacat1 ͲǤͲͳ ͲǤͲͳ ͲǤͲͳ ͲǤͲͳ ͲǤͲͲ ͲǤͲͲ Vhmgcs2 ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vhmgcl ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vbdh1 ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vfetfqo ͲǤͲͶ ͲǤͳ͸ ͲǤͲͶ ͲǤͳ͹ ͲǤͲͳ ͲǤͲ͸ kmc ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vacs ǦͲǤͲͷ ǦͲǤͲͶ ǦͲǤͲͷ ǦͲǤͲͶ ͲǤͲͲ ͲǤͲͲ kcmi ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ Vwox ͲǤͲͶ ͲǤͲ͵ ͲǤͲͶ ͲǤͲ͵ ͲǤͲͲ ͲǤͲͲ kmic ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ keFFA ͲǤͲͳ ͲǤͲͲ ͲǤͲͳ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ keCn ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ keDCA ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ keAcetoacetateMAT ͲǤͲͲ ͲǤͲͳ ͲǤͲͲ ͲǤͲͳ ͲǤͲͲ ͲǤͲͲ keBetahydroxybutyrateMAT ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ ͲǤͲͲ –Š‹•‘†‡Ž˜‡”•‹‘–Š‡˜ƒŽ—‡•ˆ‘”ͶǦͳ͸ƒ ›ŽǦ‘•—„•–”ƒ–‡•™‡”‡‹ ”‡ƒ•‡†ǡ™Š‹Ž‡–Š‡ ‘””‡•’‘†‹‰ƒš˜ƒŽ—‡•™‡”‡†‡ ”‡ƒ•‡†ǤŠ‡’ƒ”ƒ‡–‡”•‘ˆ–Š‡ͳ͸Ǧ•’‡ ‹ˆ‹ ͳƒ”‡‰‹˜‡‹ —’’Ž‡‡–ƒ”›‡š–ͳǤ

Supplementary table ST4. Total C16-C4 CoA ester concentrations in ACAD KO en WT models with C16-specific CPT1. Reference model Model with C16-specific CPT1 Model   ȏǦ‘Ȑƒ– ȏǦ‘Ȑ ›–   ȏǦ‘Ȑƒ– ȏǦ‘Ȑ ›–  ͳʹǤͺ ͳͶͻͷ ͳ͹ͻ ͳͳǤͷ ͳͳʹ ͳͺͻͻ͵ʹ  ͶǤͳ ͳ͸ͳͳ ͷ ͶǤͳ ͳ͸ͳͳ ͻ͸ʹͲ  ͺǤͲ ʹ͵͸ͺ ʹͲ͹ ͶǤͻ Ͷͺ ͸ͺͲͳͳ͸  ͳͳǤ͸ ʹͷͺ͸ ͳʹͻͲ ͳͳǤ͸ ʹͷͲͷ ͸͹͸ͻ͵ͷ †‡‘–‡•ͳ͸ǦͶ Šƒ‹Ž‡‰–Š•ǡ•‘ȏǦ‘Ȑ–Š‡–‘–ƒŽ‘‡•–‡” ‘ ‡–”ƒ–‹‘‹–Š‡ƒ–”‹šƒ†ȏǦ ‘Ȑ ›––Š‡–‘–ƒŽ‘‡•–‡” ‘ ‡–”ƒ–‹‘‹–Š‡ ›–‘•‘ŽǤŠ‡’ƒ”ƒ‡–‡”•‘ˆ–Š‡ͳ͸Ǧ•’‡ ‹ˆ‹ ͳƒ”‡‰‹˜‡ ‹—’’Ž‡‡–ƒ”›‡š–ͳǤ 

ʹͲͳ Chapter 5

 WT VLCAD KO MCAD KO SCAD KO   A B C D 40 Leak *

30 ɇvExAc-carnCn *

ɇvExFFACn * 20 ɇvExDCACn * 10 leak (%) Substrate vacsC16 *

0 050100050100050100050100 [Palmitoyl-CoA]CYT ;ʅDͿ [Palmitoyl-CoA]CYT ;ʅDͿ [Palmitoyl-CoA]CYT ;ʅDͿ [Palmitoyl-CoA]CYT ;ʅDͿ  Supplementary Figure S4Ǥ‹—Žƒ–‹‘”‡•—Ž–•‘ˆ–Š‡‡š–‡†‡†‘†‡Ž™‹–Šƒ‘”‡ͳ͸Ǧ•’‡ ‹ˆ‹ ͳǡi.e.ƒ ͳ™Š‹ Š‹•Ž‡•••‡•‹–‹˜‡ͳͶǦͶ Šƒ‹Ž‡‰–Š”‡ƒ –ƒ–•ǡˆ‘”ƒ†•‹‰Ž‡Ǧ‡œ›‡Ǥƒ‡Ž•A- D†‹•’Žƒ›••—„•–”ƒ–‡Ž‡ƒƒ‰‡”ƒ–‡•ƒ•’‡” ‡–ƒ‰‡‘ˆ’ƒŽ‹–‘›ŽǦ‘ ‘•—’–‹‘ˆŽ—š‡š’”‡••‡†‹  ’”‘†— –‹‘‡“—‹˜ƒŽ‡–•ˆ‘”–Š‡ȋAȌǡȋBȌǡȋCȌƒ†ȋDȌ˜‡”•‹‘‘ˆ–Š‡‘†‡ŽǤ 

ʹͲʹ

Chapter 6

Towards identifying in vitro metabolic adaptations that explain symptomatology in MCADD individuals A short report

Anne-Claire M.F. Martines1,#, Emmalie A. Jager1,2,#, Albert Gerding1,3, Justina C. Wolters1,2, ۥBarbara M. Bakkerƌϭ͕ ,ۥDirk-Jan Reijngoud1,2, Terry G.J. Derksϭ͕Ϯ͕ϰ͕

1 Laboratory of Pediatrics, Center of Liver, Digestive and Metabolic Diseases, University of Groningen, Groningen, University Medical Center Groningen, Groningen, The Netherlands, 2 Department of Pediatrics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 3 Department of Laboratory Medicine, University of Groningen, University Medical Center Groningen, The Netherlands 4 Section of Metabolic Diseases, Beatrix Children's Hospital, University Medical Center of Groningen, University of Groningen.

.These authors contributed equallyۥη͕

Journal publication in preparation

In vitro metabolic adaptations that explain symptomatology in MCADD individuals

Abstract  –Š‹• •–—†› ™‡ ‹˜‡•–‹‰ƒ–‡† —Ž–—”‡† •‹ ˆ‹„”‘„Žƒ•–• ‘ˆ ›‘—‰ •›’–‘ƒ–‹  ȋαͶȌ ƒ† ƒ•›’–‘ƒ–‹  Ǧ†‡ˆ‹ ‹‡– ȋȌ ‹†‹˜‹†—ƒŽ• ȋα͸Ȍǡ ‹ Ž—†‹‰ ƒ ‘Ž†‡”  ‹†‹˜‹†—ƒŽȋ͵Ͳ›‡ƒ”•Ȍ™Š‘”‡ƒ‹‡†ƒ•›’–‘ƒ–‹ ǤŠ‡ ‘–”‘Ž•‹ˆ‹„”‘„Žƒ•–ȋα͸Ȍ™‡”‡ ‘„–ƒ‹‡† ˆ”‘ ‹†‹˜‹†—ƒŽ• ƒ’’ƒ”‡–Ž› ˆ”‡‡ ‘ˆ ƒ ‡–ƒ„‘Ž‹  †‹•‡ƒ•‡Ǥ †‡” „ƒ•ƒŽ —Ž–—”‹‰ ‘†‹–‹‘•ǡ ͸ǦͳͲ ƒ ›ŽǦ ƒ”‹–‹‡ •‡ ”‡–‹‘ ƒ† ‹–”ƒ ‡ŽŽ—Žƒ” ͸Ǧ ƒ† ͺǦƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘•™‡”‡Š‹‰Š‡”‹ˆ‹„”‘„Žƒ•–•–Šƒ‹ ‘–”‘Žˆ‹„”‘„Žƒ•–•Ǥ ƒ††‹–‹‘ǡ–Š‡ ˆ‹„”‘„Žƒ•–• ‘ˆ –Š‡ ‡Ž†‡•– ƒ•›’–‘ƒ–‹   ‹†‹˜‹†—ƒŽ •Š‘™‡† Š‹‰Š‡” ƒ ›ŽǦ ƒ”‹–‹‡ •‡ ”‡–‹‘ –Šƒ –Š‘•‡ ‘ˆ –Š‡ ‘–Š‡”  ‹†‹˜‹†—ƒŽ•Ǥ —ƒ–‹–ƒ–‹˜‡ǡ –ƒ”‰‡–‡† ’”‘–‡‘‹ • •Š‘™‡†ƒ—’”‡‰—Žƒ–‹‘‘ˆ‹–Š‡•ƒ‡ƒ•›’–‘ƒ–‹ ‹†‹˜‹†—ƒŽǤ–Š‡”™‹•‡‘ ƒ†ƒ’–ƒ–‹‘•‹–Š‡’”‘–‡‘‡™‡”‡ˆ‘—†‹–Š‡ˆ‹„”‘„Žƒ•–•ǤŠ‡”‡•—Ž–•‹ŽŽ—•–”ƒ–‡–Š‡ ’‘–‡–‹ƒŽƒ†–Š‡ ŠƒŽŽ‡‰‡•‘ˆƒ’‡”•‘ƒŽ‹œ‡†‡†‹ ‹‡•–”ƒ–‡‰›Ǥ‡†‹• —••Š‘™–Š‡ —Ž–—”‡ ‘†‹–‹‘•ƒ›„‡‘’–‹‹œ‡†–‘”‡˜‡ƒŽ‡–ƒ„‘Ž‹ ƒ†ƒ’–ƒ–‹‘•ǡˆ‘”‹•–ƒ ‡„›‹’‘•‹‰Ž‘™ 6 ‰Ž— ‘•‡ ‘ ‡–”ƒ–‹‘•Ǥ 

ʹͲ͹ Chapter 6

Introduction  †‡ˆ‹ ‹‡ › ‹• ‘‡ ‘ˆ –Š‡ ‘•– ’”‡˜ƒŽ‡– ‹Š‡”‹–‡† ‡–ƒ„‘Ž‹  †‹•‡ƒ•‡• ƒ† –Š‡ ‘•– ‘‘‹„‘”‡””‘”‘ˆ–Š‡‹–‘ Š‘†”‹ƒŽˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘ȋ Ȍ’ƒ–Š™ƒ›ȏͳȂ͸ȐǤŠ‡ ’”‡˜ƒŽ‡ ‡ƒ–„‹”–Š™ƒ•ͳȀͺ͵ͲͲ‹–Š‡‡–Š‡”Žƒ†•„‡–™‡‡ʹͲͲ͹ƒ†ʹͲͳͷȏ͹ȐǤ  ’Žƒ›•ƒ‹’‘”–ƒ–”‘Ž‡‹‡‡”‰›Š‘‡‘•–ƒ•‹•†—”‹‰ˆƒ•–‹‰ƒ† ‘†‹–‹‘•‘ˆŠ‹‰ŠǦ ‡‡”‰›†‡ƒ†ǤǦ†‡ˆ‹ ‹‡–ȋȌ‹†‹˜‹†—ƒŽ•”—ƒ”‹•‘ˆ‡–ƒ„‘Ž‹  ”‹•‹•—†‡” •— Šƒ ‘†‹–‹‘ǡ‡•’‡ ‹ƒŽŽ›‹ˆ’”‘Ž‘‰‡†ˆƒ•–‹‰‘ —”•–‘‰‡–Š‡”™‹–Š‹–‡” —””‡–‹ŽŽ‡••ǡ•— Š ƒ• ƒ‹”™ƒ› ‘” ‰ƒ•–”‘‹–‡•–‹ƒŽ ‹ˆ‡ –‹‘• ȏͺȂͳͳȐǤ  —–‡ •›’–‘• ‹ Ž—†‡ Ž‹ˆ‡Ǧ–Š”‡ƒ–‡‹‰ Š›’‘‡–‘–‹ Š›’‘‰Ž› ‡‹ƒǡ ‘ƒƒ†•‡‹œ—”‡•Ǥ –‡”‡•–‹‰Ž›ǡ‡˜‡™‹–Š‹–Š‡•—„’‘’—Žƒ–‹‘ ‘ˆ‹†‹˜‹†—ƒŽ•™‹–ŠƒŠ‘‘œ›‰‘—•Ž‘••Ǧ‘ˆǦˆ— –‹‘ Ǥͻͺͷε —–ƒ–‹‘‹–Š‡Ǧ‡ ‘†‹‰ ACADM ‰‡‡ǡ •›’–‘• ƒ ”ƒ‰‡ ˆ”‘ ˆƒ–ƒŽ Š›’‘‡–‘–‹  Š›’‘‰Ž› ‡‹ƒ ‡ƒ”Ž› ‹ Ž‹ˆ‡ –‘ ƒ ‘’Ž‡–‡Žƒ ‘ˆ•›’–‘•–Š”‘—‰Š‘—–Ž‹ˆ‡ȏͳͲǡͳʹȂʹͲȐǤ ‡‡–‹ ƒ†‡˜‹”‘‡–ƒŽ‘†‹ˆ‹‡”• ‘—Ž† ’Žƒ› ƒ ”‘Ž‡ ‹ –Š‡ Š‡–‡”‘‰‡‡‹–› ‹ ˜—Ž‡”ƒ„‹Ž‹–› –‘ ‘” ”‘„—•–‡•• ƒ‰ƒ‹•– ‡–ƒ„‘Ž‹  •–”‡••Ǥ ‘™–Š‹•˜—Ž‡”ƒ„‹Ž‹–›‘””‘„—•–‡••ƒ”‹•‡•‹•ƒ•›‡–— Ž‡ƒ”Ǥ •ƒŽ”‡ƒ†›‹†‹ ƒ–‡†‹–Š‡ ‡‡”ƒŽ –”‘†— –‹‘‹Šƒ’–‡”ͳ‘ˆ–Š‹•–Š‡•‹•ǡ‹–Š‡’ƒ•–†‡ ƒ†‡• ”‡Ž‹ƒ„Ž‡—–ƒ”‰‡–‡†ƒ†–ƒ”‰‡–‡†—Ž–‹Ǧ‘‹ •‡–Š‘†•Šƒ˜‡„‡‡†‡˜‡Ž‘’‡†ǤŠ‹•‘ˆˆ‡”•–Š‡ ‘’’‘”–—‹–›–‘•–—†›‹„‘”‡””‘”•‘ˆ‡–ƒ„‘Ž‹•‘Ž‡˜‡Ž•‘–’”‡˜‹‘—•Ž›’‘••‹„Ž‡ȏʹͳȂʹ͵ȐǤ Š‡ ƒ’’Ž‹ ƒ–‹‘ ‘ˆ –Š‡•‡ —Ž–‹Ǧ‘‹ •–‡ Š‘Ž‘‰‹‡•‘ —Ž–—”‡† •‹ ˆ‹„”‘„Žƒ•–•‘”‹‰‹ƒ–‹‰ ˆ”‘•›’–‘ƒ–‹ ƒ†ƒ•›’–‘ƒ–‹ ‹†‹˜‹†—ƒŽ• ƒ„‡ƒˆ‹”•–•–‡’–‘‰ƒ‹‹•‹‰Š– ‹–‘”‡‰—Žƒ–‘”›‡ Šƒ‹••—†‡”Ž›‹‰•›’–‘ƒ–‘Ž‘‰›‹†‡ˆ‹ ‹‡ ›Ǥ ƒ††‹–‹‘ǡ‹– ‹‰Š–‡˜‡ƒŽŽ‘™—•–‘‹†‡–‹ˆ›‡ƒ‹‰ˆ—Ž„‹‘ƒ”‡”•–Šƒ–’”‡†‹ –™Š‡–Š‡”ƒ’ƒ–‹‡– ‘—Ž† †‡˜‡Ž‘’ •›’–‘• ƒ† ™Š‹ Š ƒ†ƒ’–ƒ–‹‘• ‹ ‡‡”‰› ‡–ƒ„‘Ž‹• ‘—Ž† —†‡”Ž‹‡ –Š‡‹” ”‘„—•–‡•• ‘” ˜—Ž‡”ƒ„‹Ž‹–›Ǥ ”‡˜‹‘—•Ž›ǡ ™‡ ‘„•‡”˜‡† ƒ†ƒ’–ƒ–‹‘• ‹ –Š‡ ’”‘–‡‘‡ ‘ˆ ‹–‘ Š‘†”‹ƒŽ ‡‡”‰› ‡–ƒ„‘Ž‹• ‹ —Ž–—”‡† •‹ ˆ‹„”‘„Žƒ•–• ‘ˆ  ‹†‹˜‹†—ƒŽ• ‘’ƒ”‡†–‘ ‘–”‘Ž•ǤŠ‡Ž‡˜‡Ž•‘ˆ•‡˜‡”ƒŽ ’”‘–‡‹•ǡ‹ Ž—†‹‰ǡǡ •—„—‹–ƒ† •—„—‹–ǡ™‡”‡Š‹‰Š‡”‹‹–‘ Š‘†”‹ƒ‹•‘Žƒ–‡†ˆ”‘•‹ˆ‹„”‘„Žƒ•–•‘ˆ –Š”‡‡’ƒ–‹‡–•™‹–Š–Š‡Š‘‘œ›‰‘—• Ǥͻͺͷε ACADM—–ƒ–‹‘–Šƒ‹‹–‘ Š‘†”‹ƒ ˆ”‘ ‘–”‘Žˆ‹„”‘„Žƒ•–•ȏʹͶȐǤŠ‡Žƒ––‡”•–—†›ˆ‘ —•‡†‘–Š‡†‡˜‡Ž‘’‡–‘ˆ–Š‡–ƒ”‰‡–‡† ’”‘–‡‘‹ •‡–Š‘†‘Ž‘‰›ǤŠ‡’ƒ–‹‡–ˆ‹„”‘„Žƒ•–•‹ŽŽ—•–”ƒ–‡†–Š‡’‘–‡–‹ƒŽ‘ˆ–Š‡–‡ Š‹“—‡Ǥ —‡–‘–Š‡˜‡”›Ž‘™—„‡”‘ˆ‹†‹˜‹†—ƒŽ•ǡŠ‘™‡˜‡”ǡ‘ ‘ Ž—•‹‘•‘ˆƒ› Ž‹‹ ƒŽ ”‡Ž‡˜ƒ ‡ ‘—Ž†„‡†”ƒ™Ǥ ‘”†‡”–‘•–—†›–Š‡‡ˆˆ‡ –‘ˆ‘†‹ˆ‹‡”•‘–Š‡•›’–‘ƒ–‘Ž‘‰›‹ ǡ ƒ Žƒ”‰‡” —„‡” ‘ˆ „‘–Š •›’–‘ƒ–‹  ƒ† ƒ•›’–‘ƒ–‹   ‹†‹˜‹†—ƒŽ• ‹• ”‡“—‹”‡†Ǥ –Š‡•–—†›’”‡•‡–‡†Š‡”‡ǡ™‡‡š’Ž‘”‡†™Š‡–Š‡”•›’–‘ƒ–‹ ƒ†ƒ•›’–‘ƒ–‹  ˆ‹„”‘„Žƒ•–• ƒ† ‘–”‘Ž ˆ‹„”‘„Žƒ•–• •Š‘™ †‹ˆˆ‡”‡ ‡• ‹ –Š‡‹” ‹–‘ Š‘†”‹ƒŽ ‡‡”‰› ‡–ƒ„‘Ž‹•—†‡”„ƒ•‡Ž‹‡ ‘†‹–‹‘•ȋ•–ƒ†ƒ”† —Ž–—”‡‡†‹—™‹–Š͸Ǥͳ‰Ž— ‘•‡ȌǤ‡ ‡ƒ•—”‡†–Š‡’”‘–‡‘‡‘ˆ‹–‘ Š‘†”‹ƒŽ‡‡”‰›‡–ƒ„‘Ž‹•ȏʹͶȐƒ†–Š‡‹–”ƒ ‡ŽŽ—Žƒ”ƒ† ‡š–”ƒ ‡ŽŽ—Žƒ”ƒ ›ŽǦ ƒ”‹–‹‡’”‘ˆ‹Ž‡•‘ˆˆ‹„”‘„Žƒ•–•‘ˆͶ•›’–‘ƒ–‹ ǡ͸ƒ•›’–‘ƒ–‹ ƒ†͸ ‹†‹˜‹†—ƒŽ•ƒ’’ƒ”‡–Ž›ˆ”‡‡‘ˆƒ‡–ƒ„‘Ž‹ †‹•‡ƒ•‡Ǥ –Š‡ˆ‹”•–’ƒ”–‘ˆ–Š‹••–—†›ǡ‘‡„‹‘Ž‘‰‹ ƒŽ ”‡’Ž‹ ƒ–‡ ˆ‘” ‡ƒ Š •‹ ˆ‹„”‘„Žƒ•– ‡ŽŽ Ž‹‡ ™ƒ• ‰‡‡”ƒ–‡†ǡ ƒ• ™‡ •‡ƒ” Š‡† ˆ‘” †‹ˆˆ‡”‡ ‡• „‡–™‡‡ –Š‡ ‰”‘—’•Ǥ Š‡ ”‡•—Ž–• ‘ˆ –Š‡ ƒƒŽ›•‹• •—‰‰‡•–‡† ƒŒ‘” ƒ†ƒ’–ƒ–‹‘• ‹ ‘Ž› ‘‡ ‹†‹˜‹†—ƒŽǣ „‘–Š –Š‡ ’”‘–‡‘‡ ƒ† –Š‡ ƒ ›ŽǦ ƒ”‹–‹‡ ’”‘ˆ‹Ž‡ ‘ˆ –Š‡ ‡Ž†‡•– ƒ•›’–‘ƒ–‹   ‹†‹˜‹†—ƒŽ †‹ˆˆ‡”‡† •—„•–ƒ–‹ƒŽŽ› ˆ”‘ –Šƒ– ‘ˆ ƒŽŽ ‘–Š‡”  ‹†‹˜‹†—ƒŽ•Ǥ Š‡•‡ ’‘–‡–‹ƒŽŽ› ˆ— –‹‘ƒŽ ƒ†ƒ’–ƒ–‹‘• ‘—Ž† —†‡”Ž‹‡ –Š‡ Žƒ  ‘ˆ •›’–‘• ‹ –Š‹• ‹†‹˜‹†—ƒŽǤ Š‡”‡ˆ‘”‡ǡ ™‡ ‡š–‡†‡† –Š‡ •–—†› ƒ† ”‡’‡ƒ–‡† –Š‡ ƒƒŽ›•‹• ‘ˆ –Š‡ ’”‘–‡‘‡ ƒ† ƒ ›ŽǦ

ʹͲͺ In vitro metabolic adaptations that explain symptomatology in MCADD individuals

ƒ”‹–‹‡’”‘ˆ‹Ž‡‹ƒ–‘–ƒŽ‘ˆ͵ǦͶ„‹‘Ž‘‰‹ ƒŽ”‡’Ž‹ ƒ–‡•ˆ‹„”‘„Žƒ•– —Ž–—”‡•‘ˆ‡ƒ Š‹†‹˜‹†—ƒŽǡ–‘ ˜ƒŽ‹†ƒ–‡–Š‡‹‹–‹ƒŽ‡š’‡”‹‡–ƒŽˆ‹†‹‰•ǤŠ‡ƒ‹”‡•—Ž–•ƒ”‡’”‡•‡–‡†‹–Š‹• Šƒ’–‡”Ǥ

Results Cohort and their characteristics ‡—•‡†’”‹ƒ”›Š—ƒ•‹ˆ‹„”‘„Žƒ•–•ˆ”‘‹†‹˜‹†—ƒŽ•Š‘‘œ›‰‘—•ˆ‘”–Š‡Ž‘••Ǧ ‘ˆǦˆ— –‹‘ Ǥͻͺͷε —–ƒ–‹‘ȋƒ„Ž‡ͳȌǤ  Table 1: Summary of patient characteristics Patient Gender Gene Manifes- Age of Clinical manifestation ID mutation tation onset/ Diagnosis (years) ͳ  Ǥͻͺͷε  ›’– ʹ ‘—† •—„Ǧ ‘ƒ–‘•‡ ‹ „‡†ǡ ™‹–Š 6 Š›’‘‡–‘–‹  Š›’‘‰Ž› ‡‹ƒǡ ƒˆ–‡” ƒ ‡’‹•‘†‡‘ˆ†‹ƒ””Š‡ƒǤ ʹ  Ǥͻͺͷε  •›’– ͳ Ž†‡” •‹„Ž‹‰ ’”‡•‡–‡† ƒ– ƒ‰‡ ͳ ™‹–Š Ǧ”‡Žƒ–‡† ‡–ƒ„‘Ž‹  ”‹•‹•Ǥ Š‡”‡ƒˆ–‡”ǡ–ʹ™ƒ•–”‡ƒ–‡†™‹–Šˆ”‡“—‡– ˆ‡‡†‹‰• ƒ† ƒ ˆƒ–Ǧ”‡•–”‹ –‡† †‹‡–Ǥ †‹––‡† •‡˜‡”ƒŽ –‹‡• ƒ• ƒ ’”‡ ƒ—–‹‘ †—”‹‰ ‹–‡” —””‡– ‹ŽŽ‡••ǡ „—– ™‹–Š‘—– †‘ —‡–‡† Š›’‘‰Ž› ‡‹ƒ ‘” ‘–Š‡” •‡˜‡”‡•›’–‘•Ǥ ͵  Ǥͻͺͷε  •›’– ͳǤͷ ‹ƒ‰‘•‡†ƒˆ–‡”–™‹•‹„Ž‹‰†‹‡†ƒ–ͳǤͷ› †—”‹‰ ƒ ˆŽ— ‡’‹•‘†‡Ǥ ”‡ƒ–‡† ™‹–Š ˆ”‡“—‡–ˆ‡‡†‹‰•ƒ†Ǧ ƒ”Ǥ Ͷ  Ǥͻͺͷε  •›’– ͳͳ Ž†‡” •‹„Ž‹‰ ‘ˆ –͵Ǥ ‘ †‘ —‡–‡† †‡”ƒ‰‡‡–•Ǥ ͷ  Ǥͻͺͷε  ›’– ʹ ”‡•‡–‡† ™‹–Š ’‘•–Ǧ‘’‡”ƒ–‹˜‡ Š›’‘‰Ž› ‡‹  ‘ƒǡ Ž‡ƒ†‹‰ –‘ ”‡ƒ‹ƒ–‹‘ƒ†•‡˜‡”‡†‹•ƒ„‹Ž‹–›Ǥ ͸  Ǥͻͺͷε  •›’– Ͳ ‘—‰‡”•‹„Ž‹‰‘ˆ–ͷǤ”‡ƒ–‡†ˆ”‘‡ƒ” „‹”–Š ™‹–Š ˆ”‡“—‡– ˆ‡‡†‹‰•Ǥ ‘ †‘ —‡–‡††‡”ƒ‰‡‡–•Ǥ ͹  Ǥͻͺͷε  ›’– ʹ ‘—† Ž‡–Šƒ”‰‹  ƒ† Š›’‘–‘‹  ‹ „‡†ǡ ƒ ‘’ƒ‹‡† „› •‡‹œ—”‡• ƒ† Š›’‘‰Ž› ‡‹ƒǤ ͺ  Ǥͻͺͷε  ›’– ͳ ‘ƒ–‘•‡ ƒ† Š›’‘‰Ž› ‡‹  ƒˆ–‡” –™‘ †ƒ›•‘ˆ˜‘‹–‹‰ƒ†Š‹‰Šˆ‡˜‡”Ǥ ͻ  Ǥͻͺͷε  •›’– Ͳ ‘—‰‡”•‹„Ž‹‰‘ˆ–ͺǤ”‡ƒ–‡†ˆ”‘‡ƒ” „‹”–Š ™‹–Š ˆ”‡“—‡– ˆ‡‡†‹‰•ǡ Ǧ ƒ” ƒ† ˜‹–ƒ‹ ʹǤ ‘ †‘ —‡–‡† †‡”ƒ‰‡‡–•Ǥ ͳͲ  Ǥͻͺͷε  •›’– ͵Ͳ ‹• ‘˜‡”‡† „› ‡–ƒ„‘Ž‹  ’”‘ˆ‹Ž‡ ƒˆ–‡” ’‘•‹–‹˜‡‘ˆ†ƒ—‰Š–‡”Ǥ‘†‘ —‡–‡† †‡”ƒ‰‡‡–•ƒ• Š‹Ž†Ǥ ›’–ǣ•›’–‘ƒ–‹ ǡ•›’–ǣ•›’–‘ƒ–‹ Ǥƒ–‡‰‘”‹œƒ–‹‘‘ˆ•›’–‘ƒ–‘Ž‘‰›ƒ ‘”†‹‰–‘ȏͳͲȐ.ǣƒŽ‡ǡ ǣˆ‡ƒŽ‡Ǥ Ǧ ƒ”ǣǦ ƒ”‹–‹‡Ǥ–ǣƒ–‹‡–Ǥ  Š‡ Ǥͻͺͷε  ACADM —–ƒ–‹‘ ƒ—•‡• ‹’ƒ‹”‡– ‘ˆ –Š‡  ’”‘–‡‹ ˆ‘Ž†‹‰ ȏʹͷȐ ƒ† ’”‘–‡‹‹•–ƒ„‹Ž‹–›ȏʹ͸ǡʹ͹Ȑ”‡•—Ž–‹‰‹†‡‰”ƒ†ƒ–‹‘‘ˆ–Š‡—–ƒ–‡†’”‘–‡‹ƒ†Žƒ ‘ˆƒ –‹˜‹–› ȏʹͺȐǤ‡•’‡ ‹ˆ‹ ƒŽŽ›•–—†‹‡†ˆ‹„”‘„Žƒ•–•–Šƒ–‘”‹‰‹ƒ–‡†ˆ”‘’ƒ–‹‡–•ȋͶ›’–‘ƒ–‹  ƒ†͸ƒ•›’–‘ƒ–‹ ‹†‹˜‹†—ƒŽ•ˆ”‘͹ˆƒ‹Ž‹‡•Ȍ™Š‘™‡”‡ Ž‹‹ ƒŽŽ›ƒ• ‡”–ƒ‹‡†„‡ˆ‘”‡–Š‡

ʹͲͻ Chapter 6

‹–”‘†— –‹‘‘ˆ–Š‡‡™„‘”• ”‡‡‹‰ȋȌ’”‘‰”ƒƒ†‘ˆ™Š‘–Š‡•›’–‘ƒ–‘Ž‘‰›Šƒ† „‡‡’”‡˜‹‘—•Ž›†‡• ”‹„‡†ȏͳͲȐǤ –•Š‘—Ž†„‡‘–‡†–Šƒ–•‘‡‘ˆ–Š‡‹†‹˜‹†—ƒŽ•–Šƒ–™‡”‡ ƒ–‡‰‘”‹œ‡† ƒ• ƒ•›’–‘ƒ–‹ ǡ ”‡ ‡‹˜‡† –”‡ƒ–‡– „‡ˆ‘”‡ –Š‡ ƒ˜‡”ƒ‰‡ ƒ‰‡ ‘ˆ †‹•‡ƒ•‡ ’”‡•‡–ƒ–‹‘ ȋƒ„Ž‡ ͳȌǤ Š‡•‡ Š‹Ž†”‡ ™‡”‡ †‹ƒ‰‘•‡† ƒˆ–‡” ƒ •‹„Ž‹‰ ’”‡•‡–‡† ™‹–Š •›’–‘•Ǥ ˆƒ –ǡ–Š‡‡†‹ƒȋ”ƒ‰‡Ȍƒ‰‡‘ˆ‘•‡–‹•›’–‘ƒ–‹ ‰”‘—’™ƒ•ʹȋͳǦʹȌ›‡ƒ”•ǡ ™Š‹Ž‡ –Š‡ ‡†‹ƒ ƒ‰‡ ‘ˆ †‹ƒ‰‘•‹• ‹ –Š‡ ƒ•›’–‘ƒ–‹  ‰”‘—’ ™ƒ• ͳǤʹͷ ȋͲǦ͵ͲȌ ›‡ƒ”•Ǥ • –”‡ƒ–‡– ƒ ‹–‡”ˆ‡”‡ ™‹–Š –Š‡ ƒ–—”ƒŽ ‘—”•‡ ‘ˆ †‹•‡ƒ•‡ †‡˜‡Ž‘’‡–ǡ ‹– ‹• ‘– ‡”–ƒ‹ ™Š‡–Š‡” •‘‡ ‘ˆ –Š‡  ‹†‹˜‹†—ƒŽ• ƒ–‡‰‘”‹œ‡† ƒ• ƒ•›’–‘ƒ–‹  ƒ”‡ –”—Ž› ƒ•›’–‘ƒ–‹ ǤŠ‡”‡ˆ‘”‡ǡ‹–‡”’”‡–ƒ–‹‘‘ˆ”‡•—Ž–•‘„–ƒ‹‡†‹ˆ‹„”‘„Žƒ•–•‘ˆƒ•›’–‘ƒ–‹  ‹†‹˜‹†—ƒŽ••Š‘—Ž†„‡ ‘•‹†‡”‡†™‹–Š ƒ—–‹‘Ǥ ’‘”–ƒ–Ž›ǡ’ƒ–‹‡–ͳͲȋ–ͳͲȌ™ƒ• –Š‡ ‡Ž†‡•–  ‹†‹˜‹†—ƒŽ ‹ –Š‡ •–—†‹‡† ‘Š‘”–Ǥ –ͳͲ Šƒ• ”‡ƒ‹‡† ƒ•›’–‘ƒ–‹  –Š”‘—‰Š‘—–Ž‹ˆ‡™‹–Š‘—––”‡ƒ–‡–ǤŠ‡™ƒ•‹†‡–‹ˆ‹‡†ƒ–ƒ‰‡͵Ͳ„‡ ƒ—•‡‘ˆƒ’‘•‹–‹˜‡”‡•—Ž–‹ –Š‡ ‡™„‘” • ”‡‡‹‰ ‘ˆ Š‡” ‡™„‘” Š‹Ž†Ǥ –ͳͲ ƒ –Š—• „‡ ‘•‹†‡”‡† –Š‡ ‘•– ƒ• ‡”–ƒ‹‡† ƒ•›’–‘ƒ–‹   ‹†‹˜‹†—ƒŽǤ ‘” –Š‹• ”‡ƒ•‘ǡ –Š‡ ”‡•—Ž–• ‘ˆ –ͳͲ ™‹ŽŽ „‡ ’”‡•‡–‡† ƒ† †‹• —••‡† •‡’ƒ”ƒ–‡Ž›Ǥ Šƒ”ƒ –‡”‹•–‹ • ‘ˆ –Š‡ ‘–”‘Ž ‘Š‘”– ƒ”‡ ‰‹˜‡ ‹ —’’Ž‡‡–ƒ”›ƒ„Ž‡ͳǤ  Acyl-carnitines ‹”•–ǡ ™‡ ’‡”ˆ‘”‡† ƒ ‡š’Ž‘”ƒ–‹˜‡ •–—†› ‘ ƒ •‹‰Ž‡ •ƒ’Ž‡ ‘ˆ ‡ƒ Š ‘ˆ –Š‡ —Ž–—”‡† •‹ ˆ‹„”‘„Žƒ•–• ‘ˆ ƒŽŽ ‹†‹˜‹†—ƒŽ•Ǥ Š‡ ‘–‹˜ƒ–‹‘ ˆ‘” –Š‹• ƒ’’”‘ƒ Š ™ƒ• –Šƒ– ™‡ ‡š’‡ –‡† †‹ˆˆ‡”‡ ‡• „‡–™‡‡ –Š‡ ‰”‘—’• ”ƒ–Š‡” –Šƒ „‡–™‡‡ –Š‡ ‹†‹˜‹†—ƒŽ•Ǥ ‡ ‡ƒ•—”‡† ƒ ›ŽǦ ƒ”‹–‹‡ ’”‘ˆ‹Ž‡• ƒ† –Š‡ –ƒ”‰‡–‡† ’”‘–‡‘‡ ‘ˆ ‹–‘ Š‘†”‹ƒŽ ‡‡”‰› ‡–ƒ„‘Ž‹•Ǥ Š‡ ‘ ‡–”ƒ–‹‘•‘ˆ͸ǦǡͺǦƒ†ͳͲǣͳǦƒ ›ŽǦ ƒ”‹–‹‡ǡ„—–‘–‘ˆͳͲǦƒ ›ŽǦ ƒ”‹–‹‡ǡ™‡”‡Š‹‰Š‡” ‹ –Š‡ —Ž–—”‡ ‡†‹— ‘ˆ  ˆ‹„”‘„Žƒ•–• –Šƒ ‹ –Šƒ– ‘ˆ ‘–”‘Ž ˆ‹„”‘„Žƒ•–• ȋ—’’Ž‡‡–ƒ”› ‹‰—”‡ͳȌǤ•–Š‡ˆ”‡•Š‡†‹—ƒŽ”‡ƒ†› ‘–ƒ‹‡†ƒ ›ŽǦ ƒ”‹–‹‡•ǡ™‡ƒŽ•‘ ƒŽ —Žƒ–‡†–Š‡ Šƒ‰‡‹ƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘•‹–Š‡‡†‹—†—”‹‰ —Ž–‹˜ƒ–‹‘ƒ† ‘”ƒŽ‹œ‡†–Š‡”‡•—Ž––‘ ‡ŽŽ—Žƒ”’”‘–‡‹ ‘–‡–ǡ–‘ƒ ‘—–ˆ‘”†‹ˆˆ‡”‡ ‡•‹ ‘ˆŽ—‡ ‡‘ˆ –Š‡ ‡ŽŽ•Ǥ Š‡  ˆ‹„”‘„Žƒ•–• •‡ ”‡–‡† ‘”‡ ͸Ǧ ƒ† ͺǦƒ ›ŽǦ ƒ”‹–‹‡• ‘’ƒ”‡† –Š‡ ‘–”‘Ž ˆ‹„”‘„Žƒ•–•Ǥ  ƒ††‹–‹‘ǡ –Š‡ ˆ‹„”‘„Žƒ•–• ˆ”‘ –ͳͲ •‡ ”‡–‡† ‘”‡ ͸ǦͳͲǦƒ ›ŽǦ ƒ”‹–‹‡–Šƒ–Š‡‘–Š‡”ˆ‹„”‘„Žƒ•–•ȋ ‹‰—”‡ͳǦȌǤ‡ƒŽ•‘‡ƒ•—”‡†‹–”ƒ ‡ŽŽ—Žƒ” ƒ ›ŽǦ ƒ”‹–‹‡Ž‡˜‡Ž•ǤŠ‹••Š‘™‡†–Šƒ–•›’–‘ƒ–‹ ƒ†ƒ•›’–‘ƒ–‹ ˆ‹„”‘„Žƒ•–• Šƒ† •‹‰‹ˆ‹ ƒ–Ž› Š‹‰Š‡” ‹–”ƒ ‡ŽŽ—Žƒ” ͸Ǧǡ ͺǦ „—– •‹‹Žƒ” ͳͲǣͳǦ ƒ† ͳͲǦƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘• ‘’ƒ”‡† –‘ ‘–”‘Ž ˆ‹„”‘„Žƒ•–• ȋ ‹‰—”‡ ͳǦ ȌǤ —”–Š‡”‘”‡ǡ ™‡ †‹† ‘– ‘„•‡”˜‡†‹ˆˆ‡”‡ ‡•„‡–™‡‡–Š‡ ‹–”ƒ ‡ŽŽ—Žƒ” ‘ ‡–”ƒ–‹‘•‘ˆƒ ›ŽǦ ƒ”‹–‹‡•‹–Š‡•‹ ˆ‹„”‘„Žƒ•–‘ˆ–ͳͲƒ†–Š‘•‡‘ˆ–Š‡•›’–‘ƒ–‹ ƒ†‘–Š‡”ƒ•›’–‘ƒ–‹ ˆ‹„”‘„Žƒ•–•ȋ ‹‰—”‡ ͳǦ ȌǤ–Š‡”ƒ ›ŽǦ ƒ”‹–‹‡•™‡”‡‘–†‹ˆˆ‡”‡–„‡–™‡‡ƒ›‘ˆ–Š‡‰”‘—’•ȋ†ƒ–ƒ‘–•Š‘™ȌǤ ‹ ‡–ͳͲ†‹ˆˆ‡”‡†ˆ”‘ƒŽŽ‘–Š‡”‹†‹˜‹†—ƒŽ•ǡ™‡•‡–‘—––‘˜ƒŽ‹†ƒ–‡–Š‡‘„•‡”˜ƒ–‹‘•™‹–Š —Ž–‹’Ž‡ ”‡’Ž‹ ƒ–‡ ˆ‹„”‘„Žƒ•– —Ž–—”‡• ’‡” ‹†‹˜‹†—ƒŽǤ Š‡ ‘ ‡–”ƒ–‹‘• ‘ˆ ͸Ǧǡ ͺǦǡ ƒ† ͳͲǣͳǦƒ ›ŽǦ ƒ”‹–‹‡‹–Š‡‡†‹—‘ˆƒŽŽ„—–‘‡’ƒ–‹‡– ‡ŽŽŽ‹‡ȋ–͹Ȍ™‡”‡Š‹‰Š‡”–Šƒ–Š‘•‡ ‘ˆ–Š‡ ‘–”‘Žˆ‹„”‘„Žƒ•–•ȋ—’’Ž‡‡–ƒ”› ‹‰—”‡ʹǦȌǤ‡˜‡”ƒŽ’ƒ–‹‡–ˆ‹„”‘„Žƒ•– —Ž–—”‡• Šƒ†ƒŽ•‘Š‹‰Š‡”ͳͲǦƒ ›ŽǦ ƒ”‹–‹‡Ž‡˜‡Ž•‹–Š‡‡†‹—ȋ—’’Ž‡‡–ƒ”› ‹‰—”‡ʹȌǤ

ʹͳͲ In vitro metabolic adaptations that explain symptomatology in MCADD individuals

A B 1.5 15 ## ##,*

1.0 10

0.5 5 medium C8 medium C6 mol/) mol/g mol/g protein) mol/g §§ ' ' P P §,¥ ( (

0.0 0 Sympt Asympt (ex. Pt10) Pt10 Ctrl Sympt Asympt (ex. Pt10) Pt10 Ctrl

C D 2.5 2.0 # 2.0 1.5

1.5 1.0 1.0 6 medium C10 mol/g protein) mol/g mol/g protein) mol/g 0.5 medium C10:1 medium ' P

P 0.5 ( ( '

0.0 0.0 Sympt Asympt (ex. Pt10) Pt10 Ctrl Sympt Asympt (ex. Pt10) Pt10 Ctrl

EF 1.5 0.25

0.20 1.0 0.15

0.10 0.5 0.05 intracellular C8 intracellular C6

§§ protein) (nmol/g §§ (nmol/g protein) (nmol/g

0.0 0.00 Sympt Asympt (ex. Pt10) Pt10 Ctrl Sympt Asympt (ex. Pt10) Pt10 Ctrl

GH 1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2 (nmol/g protein) (nmol/g (nmol/g protein) (nmol/g intracellular C10 intracellular C10:1 0.0 0.0 Sympt Asympt (ex. Pt10) Pt10 Ctrl Sympt Asympt (ex. Pt10) Pt10 Ctrl  Figure 1Ǥ ͸ǦͳͲǦƒ ›ŽǦ ƒ”‹–‹‡ •‡ ”‡–‹‘ ‹–‘ ‹ –Š‡ ‡†‹— ȋǦȌ ƒ† ‹–”ƒ ‡ŽŽ—Žƒ” ͸Ǧ ƒ† ͺǦƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘•ȋǦ ȌǤ‡ ”‡–‹‘‘ˆƒ ›ŽǦ ƒ”‹–‹‡•‹••Š‘™ƒ•–Š‡†‹ˆˆ‡”‡ ‡‹ ‘ ‡–”ƒ–‹‘‘ˆ ƒ ›Ž ƒ”‹–‹‡•‹–Š‡‡†‹—„‡ˆ‘”‡ƒ†ƒˆ–‡” —Ž–—”‡‘ˆˆ‹„”‘„Žƒ•–•ǡ‘”ƒŽ‹œ‡†ˆ‘”–Š‡ ‡ŽŽ—Žƒ”’”‘–‡‹ ‘ ‡–”ƒ–‹‘ǤŠ‡”‡•—Ž–’‡”–ƒ‹–‘–Š‡‡š’Ž‘”ƒ–‹˜‡’ƒ”–‘ˆ–Š‡•–—†›ǤαͶǡͷǡƒ†Ͷ†‹ˆˆ‡”‡–‹†‹˜‹†—ƒŽ•ǡˆ‘” •›’–‘ƒ–‹ ǡƒ•›’–‘ƒ–‹ ƒ† ‘–”‘Žˆ‹„”‘„Žƒ•–•ǡ”‡•’‡ –‹˜‡Ž›ǤŠ‡ ‘–”‘Žˆ‹„”‘„Žƒ•–•™‡”‡ͳǡ͵ǡͶǡ ͷƒ†͸Ǥȋ—’’Ž‡‡–ƒ”›ƒ„Ž‡ͳȌǤȚǣ•‹‰‹ˆ‹ ƒ–†‹ˆˆ‡”‡ ‡ ‘’ƒ”‡†–‘ˆ‹„”‘„Žƒ•–•ȋ‹ Ž—†‹‰ –ͳͲȌǡ͓ǣ•‹‰‹ˆ‹ ƒ–†‹ˆˆ‡”‡ ‡ ‘’ƒ”‡†–‘ˆ‹„”‘„Žƒ•–•ȋ‡š Ž—†‹‰–ͳͲȌǡ͎ǣ•‹‰‹ˆ‹ ƒ–†‹ˆˆ‡”‡ ‡ ‘’ƒ”‡† –‘ –ͳͲǡ șǣ •‹‰‹ˆ‹ ƒ– †‹ˆˆ‡”‡ ‡ ‘’ƒ”‡† –‘ •›’–‘ƒ–‹   ˆ‹„”‘„Žƒ•–•ǡ Șǣ •‹‰‹ˆ‹ ƒ– †‹ˆˆ‡”‡ ‡ ‘’ƒ”‡†–‘ƒ•›’–‘ƒ–‹ ˆ‹„”‘„Žƒ•–•ǡƬǣ‹‰‹ˆ‹ ƒ– ‘’ƒ”‡†–‘–Š‡‘–Š‡”ˆ‹„”‘„Žƒ•–•Ǥ ȗǣ•‹‰‹ˆ‹ ƒ–†‹ˆˆ‡”‡ ‡ ‘’ƒ”‡†–‘ ‘–”‘Žˆ‹„”‘„Žƒ•–•Ǥ‡•›„‘Ž†‡‘–‡•’δͲǤͲͷǡ–™‘•›„‘Ž•†‡‘–‡• ’δͲǤͲͳǤ  ‘•‡“—‡–Ž›ǡ„‘–Š•›’–‘ƒ–‹ ƒ†ƒ•›’–‘ƒ–‹ ‰”‘—’•Šƒ†•‹‰‹ˆ‹ ƒ–Ž›Š‹‰Š‡” ͸ǦͳͲǦƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘•‹–Š‡‡†‹—–Šƒ–Š‡ ‘–”‘Ž‰”‘—’ȋ—’’Ž‡‡–ƒ”› ‹‰—”‡ʹǦ ȌǤ ƒ††‹–‹‘ǡ–Š‡ ‘–”‘Žˆ‹„”‘„Žƒ•–• ‘•—‡†͸ǦͳͲǦƒ ›ŽǦ ƒ”‹–‹‡•ǡ™Š‹Ž‡ ˆ‹„”‘„Žƒ•–•†‹†‘–ȋ—’’Ž‡‡–ƒ”›ˆ‹‰—”‡͵ƒ† ‹‰—”‡ʹǦȌǤ

ʹͳͳ Chapter 6

A B 0.6 ‚‚ 6

0.4 4

0.2 ††ÁÁ‚‚–– 2 0.0 ††ÁÁ‚––

Sympt Asympt (ex. Pt10) Pt10 Ctrl C8 medium medium C6 mol/g protein) mol/g 0 mol/g protein) mol/g ' ' P

P -0.2

( Sympt Asympt (ex. Pt10) Pt10 Ctrl (

-0.4 -2

C D 0.3 0.3

0.2 0.2

0.1 ††ÁÁ‚–– 0.1 0.0 Sympt Asympt (ex. Pt10) Pt10 Ctrl ††Á–– medium C10 mol/g protein) mol/g mol/g protein) mol/g 0.0 medium C10:1 ' P P -0.1

( Sympt Asympt (ex. Pt10) Pt10 Ctrl ( '

-0.2 -0.1

EF 0.5 0.20

0.4 0.15

0.3 0.10 0.2 0.05 ††ÁÁ‚‚–– 0.1 intracellular C6 intracellular C8 (nmol/g protein) (nmol/g

††ÁÁ‚‚–– protein) (nmol/g

0.0 0.00 Sympt Asympt (ex. Pt10) Pt10 Ctrl Sympt Asympt (ex. Pt10) Pt10 Ctrl

GH 0.08 0.04

0.06 0.03

0.04 0.02

0.02 0.01 (nmol/g protein) (nmol/g (nmol/g protein) (nmol/g intracellular C10 intracellular C10:1 0.00 0.00 Sympt Asympt (ex. Pt10) Pt10 Ctrl Sympt Asympt (ex. Pt10) Pt10 Ctrl  Figure 2Ǥ͸ǦͳͲǦƒ ›ŽǦ ƒ”‹–‹‡•‡ ”‡–‹‘‹–‘‹–Š‡‡†‹—ȋǦȌƒ†‹–”ƒ ‡ŽŽ—Žƒ”͸ǦͳͲǦƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘• ȋǦ ȌǤ ‡ ”‡–‹‘ ‘ˆ ƒ ›ŽǦ ƒ”‹–‹‡• ‹• •Š‘™ ƒ• –Š‡ †‹ˆˆ‡”‡ ‡ ‹ ‘ ‡–”ƒ–‹‘ ‘ˆ ƒ ›ŽǦ ƒ”‹–‹‡• ‹ –Š‡ ‡†‹— „‡ˆ‘”‡ ƒ† ƒˆ–‡” —Ž–—”‡ ‘ˆ ˆ‹„”‘„Žƒ•–•ǡ ‘”ƒŽ‹œ‡† ˆ‘” –Š‡ ‡ŽŽ—Žƒ” ’”‘–‡‹ ‘ ‡–”ƒ–‹‘ǤŠ‡”‡•—Ž–•’‡”–ƒ‹–‘–Š‡˜ƒŽ‹†ƒ–‹‘’Šƒ•‡‘ˆ–Š‡•–—†›Ǥαͳˆ‘”ƒ–‹‡– ͓͹ƒ† ‘–”‘Ž ˆ‹„”‘„Žƒ•–͓ͷǡαʹˆ‘”ƒ–‹‡– ͓ͳǦ͸ǡͺǦͻƒ† ‘–”‘Žˆ‹„”‘„Žƒ•–•͓͵ǡͶǡ͸ƒ†α͵ˆ‘”ƒ–‹‡– ͓ͳͲƒ† ‘–”‘Žˆ‹„”‘„Žƒ•–͓͸ǤǤ‹‰‹ˆ‹ ƒ ‡˜‹•—ƒŽ‹œƒ–‹‘‹•ƒ ‘”†‹‰–‘ ‹‰—”‡ͳǤ  Ž‹‡™‹–Š–Š‡‡š’Ž‘”ƒ–‹˜‡•–—†›ǡ–Š‡ˆ‹„”‘„Žƒ•–•‘ˆ–ͳͲ•‡ ”‡–‡†•‹‰‹ˆ‹ ƒ–Ž›‘”‡͸ǦͳͲǦ ƒ ›ŽǦ ƒ”‹–‹‡–Šƒ–Š‡ˆ‹„”‘„Žƒ•–•‘ˆƒŽŽ‘–Š‡”‹†‹˜‹†—ƒŽ• ‘„‹‡†ȋ—’’Ž‡‡–ƒ”› ˆ‹‰—”‡͵ƒ† ‹‰—”‡ʹǦȌǤ ‹ƒŽŽ›ǡ–Š‡ˆ‹„”‘„Žƒ•–•‘ˆ•›’–‘ƒ–‹ ƒ†ƒ•›’–‘ƒ–‹  ‹†‹˜‹†—ƒŽ•Šƒ†•‹‰‹ˆ‹ ƒ–Ž›Š‹‰Š‡”‹–”ƒ ‡ŽŽ—Žƒ”͸Ǧƒ†ͺǦǡ„—–•‹‹Žƒ”ͳͲǣͳǦƒ†ͳͲǦƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘• ‘’ƒ”‡† –‘ ‘–”‘Ž ˆ‹„”‘„Žƒ•–• ȋ—’’Ž‡‡–ƒ”› ˆ‹‰—”‡ Ͷ ƒ† ‹‰—”‡ʹǦ ȌǤŠ‡”‡™ƒ•‘†‹ˆˆ‡”‡ ‡„‡–™‡‡–Š‡‹–”ƒ ‡ŽŽ—Žƒ”ƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘• ‘ˆ–Š‡ˆ‹„”‘„Žƒ•–•‘ˆ–ͳͲƒ†–Šƒ–‘ˆ‘–Š‡”•›’–‘ƒ–‹ ‘”ƒ•›’–‘ƒ–‹ ‹†‹˜‹†—ƒŽ•

ʹͳʹ In vitro metabolic adaptations that explain symptomatology in MCADD individuals

ȋ—’’Ž‡‡–ƒ”›ˆ‹‰—”‡Ͷƒ† ‹‰—”‡ʹǦ ȌǤ˜‡”ƒŽŽǡ–Š‡•‡”‡•—Ž–••—‰‰‡•––Šƒ– ‡ŽŽ•‘ˆ–ͳͲǡ –Š‡‡Ž†‡•–ƒ•›’–‘ƒ–‹ ‹†‹˜‹†—ƒŽǡ•‡ ”‡–‡‘”‡͸ǦͳͲǦƒ ›ŽǦ ƒ”‹–‹‡•Ǥ  Targeted proteomics Š‡‡ƒ•—”‡†‹–‘ Š‘†”‹ƒŽ’”‘–‡‹• ‘’”‹•‡‡œ›‡•‹˜‘Ž˜‡†‹ ƒ† › Ž‡ǡ ‘”‡ •—„—‹–• ‘ˆ ‡ƒ Š ‘ˆ –Š‡ ”‡•’‹”ƒ–‘”›Ǧ Šƒ‹ ‘’Ž‡š‡•ǡ ƒ† –”ƒ•’‘”–‡”• ”‡“—‹”‡† –‘ –”ƒ•’‘”–‡–ƒ„‘Ž‹–‡•‹–‘ƒ†‘—–‘ˆ–Š‡‹–‘ Š‘†”‹ƒȏʹͶȐǤ ƒŽŽ ‘–”‘Ž•ƒ’Ž‡•™ƒ• †‡–‡ –‡†ǡ ™Š‹Ž‡ ‹– ™ƒ• Ž‘™ ‹ –Š‡ ’ƒ–‹‡– •ƒ’Ž‡• ȋ ‹‰—”‡ ͵ȌǤ –Š‡”™‹•‡ –Š‡”‡ ™‡”‡ ‘ •‹‰‹ˆ‹ ƒ–†‹ˆˆ‡”‡ ‡•„‡–™‡‡ƒ† ‘–”‘Žˆ‹„”‘„Žƒ•–•‹–Š‡‡š’Ž‘”ƒ–‹˜‡’Šƒ•‡‘ˆ–Š‡ •–—†›Ǥ A B 1.5 20 §§ && g) P g)

P 15 6 1.0

10

0.5

Superoxide 5 MCAD (fmol/ l.o.d. (fmol/ dismutase 0.0 0 Sympt Asympt (ex.Pt10) Pt10 Ctrl Sympt Asympt (ex.Pt10) Pt10 Ctrl

C D 0.8 && 0.6 && g) g)

0.6 P P 0.4 0.4 l.o.d. 0.2 0.2 ANT1 (fmol/ ETFQO (fmol/

0.0 0.0 Sympt Asympt (ex.Pt10) Pt10 Ctrl Sympt Asympt (ex.Pt10) Pt10 Ctrl

E && 0.5

g) 0.4 P l.o.d. 0.3

0.2

0.1 SCAD (fmol/

0.0 Sympt Asympt (ex.Pt10) Pt10 Ctrl  Figure 3Ǥ‡Ž‡ –‹‘‘ˆ’”‘–‡‹•‘ˆ–Š‡‡š’Ž‘”ƒ–‘”›’Šƒ•‡‘ˆ–Š‡•–—†›Ǥ‡’‹ –‡†ƒ”‡–Š‡’”‘–‡‹•™Š‡”‡–ͳͲ ˆ‹„”‘„Žƒ•–••Š‘™‡†–Š‡Žƒ”‰‡•–†‹ˆˆ‡”‡ ‡‹ ‘ ‡–”ƒ–‹‘ ‘’ƒ”‡†–‘–Š‡ˆ‹„”‘„Žƒ•–•‘ˆ–Š‡‘–Š‡” ƒ† ‘–”‘Ž‹†‹˜‹†—ƒŽ•ƒ†Ǥ‹‰‹ˆ‹ ƒ ‡˜‹•—ƒŽ‹œƒ–‹‘‹•ƒ ‘”†‹‰–‘ ‹‰—”‡ͳǤ  –Š‡‡š’Ž‘”ƒ–‘”›’Šƒ•‡‘ˆ–Š‡•–—†›ǡ‹–ƒ’’‡ƒ”‡†–Šƒ––Š‡’”‘–‡‘‡‘ˆ–Š‡ˆ‹„”‘„Žƒ•–•‘ˆ–ͳͲ ™ƒ•“—‹–‡†‹ˆˆ‡”‡–ǡ•Š‘™‹‰Š‹‰Š‡”Ž‡˜‡Ž•‘ˆ•‡˜‡”ƒŽ’”‘–‡‹•–Šƒ–Š‡‘–Š‡”ˆ‹„”‘„Žƒ•–•ȋ–‘’ ͵ƒ†•Š‘™‹ ‹‰—”‡͵ǦȌǤŠ‡’”‘–‡‹ ‘ ‡–”ƒ–‹‘•–Šƒ–™‡”‡‘•–‡Ž‡˜ƒ–‡†‹ –ͳͲ ‡ŽŽ• ‘’ƒ”‡† –‘ –Š‡ ‘–Š‡” ˆ‹„”‘„Žƒ•–•ǡ ™‡”‡ –Š‘•‡ ‘ˆ ‹–‘ Š‘†”‹ƒŽ •—’‡”‘š‹†‡ †‹•—–ƒ•‡ʹȋ ‘†‡†„›ʹȌǡȀ–”ƒ•Ž‘ ƒ•‡ͳȋͳǡ ‘†‡†„›ʹͷͶȌƒ†  †‡Š›†”‘‰‡ƒ•‡ȋ Ǧ—„‹“—‹‘‡‘š‹†‘”‡†— –ƒ•‡ǡ ǡ ‘†‡†„›  Ȍȋ ‹‰—”‡͵ǦȌǤ  ƒ††‹–‹‘ǡ–ͳͲ•Š‘™‡†Š‹‰Š‡”Ž‡˜‡Ž•‘ˆ ‘’ƒ”‡†–‘–Š‡‘–Š‡”ˆ‹„”‘„Žƒ•–•ȋ ‹‰—”‡͵ȌǤ

ʹͳ͵ Chapter 6

–Š‡˜ƒŽ‹†ƒ–‹‘’Šƒ•‡‘ˆ–Š‡•–—†›™‡ ‘ˆ‹”‡†–Š‡Ž‘™ ‘ ‡–”ƒ–‹‘•‹ ‘’ƒ”‡†–‘ ‘–”‘Žˆ‹„”‘„Žƒ•–•ȋ ‹‰—”‡Ͷƒ†—’’Ž‡‡–ƒ”› ‹‰—”‡ͷȌǤŠ‡ˆ‹„”‘„Žƒ•–• ‘ˆ –Š‡  ‹†‹˜‹†—ƒŽ• •Š‘™‡† •Ž‹‰Š– „—– •‹‰‹ˆ‹ ƒ–Ž› Š‹‰Š‡” Ž‡˜‡Ž• ‘ˆ –Š‡ ƒ–‹‘š‹†ƒ– ’”‘–‡‹‡”‘š‹”‡†‘š‹Ǧ͸ȋ ‘†‡†„›”†š͸Ȍȋ ‹‰—”‡Ͷƒ†—’’Ž‡‡–ƒ”› ‹‰—”‡ͷȌǤŠ‹•‹• ‹Ž‹‡™‹–Š’”‘–‡‹‘š‹†ƒ–‹˜‡†ƒƒ‰‡ƒ†ƒŽ–‡”‡†ƒ–‹‘š‹†ƒ–†‡ˆ‡•‡‘„•‡”˜‡†‹ ‹†‹˜‹†—ƒŽ• ȏʹͻȐǤ  ƒ††‹–‹‘ǡ –Š‡ ˆ‹„”‘„Žƒ•–• –ͳͲ †‹† ‘– •Š‘™ •‹‰‹ˆ‹ ƒ– †‹ˆˆ‡”‡ ‡• ‹ ’”‘–‡‘‡ ‘’ƒ”‡†–‘ƒ›‘ˆ–Š‡‰”‘—’•ǡ‡š ‡’–ˆ‘”•‹‰‹ˆ‹ ƒ–Ž›Š‹‰Š‡” ‘ ‡–”ƒ–‹‘•‘ˆ ‹ˆ‹„”‘„Žƒ•–‘ˆ–ͳͲ ‘’ƒ”‡†–‘–Š‡‘–Š‡”ƒ•›’–‘ƒ–‹ ˆ‹„”‘„Žƒ•–•ȋ ‹‰—”‡Ͷƒ† —’’Ž‡‡–ƒ”› ‹‰—”‡ͷȌǤ

A B

§§ g) 20

4 P

g) 15

P 3 §§ 2 10

1 5 MCAD (fmol/ l.o.d. 0 0 Sympt Asympt (ex.Pt10) Pt10 Ctrl (fmol/ -6 Sympt Asympt (ex.Pt10) Pt10 Ctrl

C ‚ 0.5

g) 0.4 P 0.3

0.2

0.1 SCAD (fmol/

0.0 Sympt Asympt (ex.Pt10) Pt10 Ctrl Figure 4Ǥ ‡Ž‡ –‹‘ ‘ˆ ’”‘–‡‹• ‘ˆ –Š‡ ˜ƒŽ‹†ƒ–‹‘ ’ƒ”– ‘ˆ –Š‡ •–—†›Ǥ αʹ ˆ‘” ƒ–‹‡–  ͓ͳǦͻ ƒ† ‘–”‘Ž ˆ‹„”‘„Žƒ•–• ͓͵ǡͶǡ͸Ǥ ƒ† ͸Ǥ ƒ† α͵ ˆ‘” ƒ–‹‡–  ͓ͳͲ ‹‰‹ˆ‹ ƒ ‡ ˜‹•—ƒŽ‹œƒ–‹‘ ‹• ƒ ‘”†‹‰ –‘ ‹‰—”‡ͳǤ  Discussion  –Š‹• •–—†› ™‡ ‹˜‡•–‹‰ƒ–‡† ™Š‡–Š‡” –Š‡”‡ ƒ”‡ ‡–ƒ„‘Ž‹  ƒ†ƒ’–ƒ–‹‘• ‹ —Ž–—”‡† •‹ ˆ‹„”‘„Žƒ•–• ‘ˆ  ‹†‹˜‹†—ƒŽ• –Šƒ– •‡’ƒ”ƒ–‡ •›’–‘ƒ–‹  ˆ”‘ ƒ•›’–‘ƒ–‹   ‹†‹˜‹†—ƒŽ•Ǥ ‡ †‹† ‘– ‘„•‡”˜‡ ‘•‹•–‡– †‹ˆˆ‡”‡ ‡• ‹ ƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘• „‡–™‡‡ˆ‹„”‘„Žƒ•–‘ˆ•›’–‘ƒ–‹ ƒ†ƒ•›’–‘ƒ–‹ ‹†‹˜‹†—ƒŽ•ǡ‡š ‡’–ˆ‘”–ͳͲǡ –Š‡ ‡Ž†‡•–  ‹†‹˜‹†—ƒŽ ™‹–Š ƒ Ž‹ˆ‡Ž‘‰ ƒ„•‡ ‡ ‘ˆ •›’–‘•Ǥ – ƒ’’‡ƒ”•ǡ ˆ”‘ „‘–Š ’Šƒ•‡•‘ˆ–Š‡•–—†›–Šƒ–ˆ‹„”‘„Žƒ•–‘ˆ–ͳͲ•‡ ”‡–‡‹–”ƒ ‡ŽŽ—Žƒ”ƒ ——Žƒ–‡†ƒ ›ŽǦ ƒ”‹–‹‡• ‘”‡‡ˆˆ‹ ‹‡–Ž›Ǥ‡˜‡”–Š‡Ž‡••ǡ–Š‡‹–”ƒ ‡ŽŽ—Žƒ” ‘ ‡–”ƒ–‹‘•‘ˆƒ ›ŽǦ ƒ”‹–‹‡•”‡ƒ‹‡† Š‹‰Š ‘’ƒ”‡†–‘ ‘–”‘Žˆ‹„”‘„Žƒ•–•ǤŠ‡“—‡•–‹‘ƒ”‹•‡•ǡ™Šƒ– ƒ—•‡•–Š‡Š‹‰Š‡””ƒ–‡‘ˆƒ ›ŽǦ ƒ”‹–‹‡ •‡ ”‡–‹‘Ǥ – ‘—Ž† „‡ ƒ ”‡•—Ž– ‘ˆ Š‹‰Š‡” ”ƒ–‡• ‘ˆ   ™‹–Š ‘ ‘‹–ƒ– ƒ ›ŽǦ ƒ”‹–‹‡’”‘†— –‹‘‹ˆ‹„”‘„Žƒ•–•‘ˆ–ͳͲ ‘’ƒ”‡†–‘–Š‡ˆ‹„”‘„Žƒ•–•‘ˆ–Š‡‘–Š‡” ‹†‹˜‹†—ƒŽ•ǤŽ–‡”ƒ–‹˜‡Ž›ǡ–Š‡’”‘–‡‹•‹˜‘Ž˜‡†‹–Š‡•‡ ”‡–‹‘‘ˆƒ ›ŽǦ ƒ”‹–‹‡• ‘—Ž†„‡ —’”‡‰—Žƒ–‡† ‹ –ͳͲǤ Š‹• ™ƒ• ‘– –‡•–‡†ǡ •‹ ‡ –Š‡ –ƒ”‰‡–‡† ‡–Š‘† †‹† ‘– ‹ Ž—†‡ ƒ› –”ƒ•’‘”–‡”•‘—–•‹†‡–Š‡‹–‘ Š‘†”‹ƒǤ”‘–‡‘‹ ••Š‘™‡†Šƒ”†Ž›ƒ›ƒ†ƒ’–ƒ–‹‘•‹ ‡ŽŽ• ‘’ƒ”‡†–‘ ‘–”‘Ž•ǤŠ‹•‹• ‘•‹•–‡–™‹–Š‡ƒ”Ž‹‡”ˆ‹†‹‰•‹Ǧ‘—•‡Ž‹˜‡” ȋChapter 3ƒ†ȏʹͶȐȌǤ‡‘„•‡”˜‡†ƒ†ƒ’–ƒ–‹‘•‹–Š‡’”‘–‡‘‡‘ˆ–ͳͲǡ„—––Š‡›™‡”‡Ž‡••

ʹͳͶ In vitro metabolic adaptations that explain symptomatology in MCADD individuals

’”‘‘— ‡†‹–Š‡˜ƒŽ‹†ƒ–‹‘’Šƒ•‡–Šƒ‹–Š‡‡š’Ž‘”ƒ–‘”›’Šƒ•‡‘ˆ–Š‡•–—†›ǤŠ‡”‡ˆ‘”‡ǡ™‡ ‘—Ž† ‘– —ƒ„‹‰—‘—•Ž› ‡•–ƒ„Ž‹•Š ‹ˆ –Š‡› ‘–”‹„—–‡ –‘ –Š‡ Žƒ  ‘ˆ •›’–‘• ‹ –ͳͲǤ ’”‡‰—Žƒ–‹‘‘ˆǡƒ• ‘•‹•–‡–Ž›‘„•‡”˜‡†‹–Š‡‡š’Ž‘”ƒ–‘”›ƒ†–Š‡˜ƒŽ‹†ƒ–‹‘’Šƒ•‡ǡ ‘—Ž†„‡ƒˆ— –‹‘ƒŽƒ†ƒ’–ƒ–‹‘ǤŠ‹•‡œ›‡ ƒ–ƒŽ›œ‡•–Š‡•ƒ‡”‡ƒ –‹‘ƒ•ǡ™‹–Šƒ ‘˜‡”Žƒ’’‹‰•‡–‘ˆ•—„•–”ƒ–‡•ǡƒ† ƒ–Š‡”‡ˆ‘”‡’ƒ”–Ž› ‘’‡•ƒ–‡ˆ‘”–Š‡Ž‘••‘ˆǤ †‡”–Š‡ —Ž–—”‡ ‘†‹–‹‘•–‡•–‡†‹–Š‹••–—†›ǡ™‹–Š͸Ǥͳ‘ˆ‰Ž— ‘•‡ǡ–Š‡ ‡ŽŽ•ƒ›‘– †‡’‡† •–”‘‰Ž› ‘ ˆƒ––›Ǧƒ ‹† ‘š‹†ƒ–‹‘Ǥ ‘ ‡Ž‹ ‹– Žƒ”‰‡” ƒ† ‘•‹•–‡– ƒ†ƒ’–ƒ–‹‘•ǡ ‘”‡ •–”‹‰‡– ‘†‹–‹‘• ƒ› „‡ ”‡“—‹”‡†ǡ •— Š ƒ• Ž‘™‡” ‰Ž— ‘•‡ Ž‡˜‡Ž• ƒ† Š‹‰Š‡” ˆƒ––›Ǧƒ ‹† ‘ ‡–”ƒ–‹‘•Ǥ  Materials and Methods Patients and controls ”‹ƒ”› Š—ƒ •‹ ˆ‹„”‘„Žƒ•–• ‘”‹‰‹ƒ–‡† ˆ”‘ ’ƒ–‹‡–• Š‘‘œ›‰‘—• ˆ‘” –Š‡ Ǥͻͺͷε  ACADM 6 —–ƒ–‹‘ǡ ™Š‘ ™‡”‡ Ž‹‹ ƒŽŽ› ƒ• ‡”–ƒ‹‡† „‡ˆ‘”‡ ʹͲͲ͹ ™‡”‡ •‡Ž‡ –‡†Ǥ ›’–‘ƒ–‘Ž‘‰› ™ƒ• ƒ–‡‰‘”‹œ‡†ƒ ‘”†‹‰–‘ȏͳͲȐǤ •Š‘”–ǡ–Š‡•›’–‘ƒ–‹ ’Š‡‘–›’‡™ƒ•†‡ˆ‹‡†„›–Š‡‘ —””‡ ‡‘ˆ ƒ–Ž‡ƒ•–‘‡ƒ —–‡‡–ƒ„‘Ž‹  ”‹•‹•™‹–Š†‘ —‡–‡†Š›’‘‰Ž› ‡‹ƒȀ ‘ƒȀ‡ ‡’ŠƒŽ‘’ƒ–Š›‘”•‡‹œ—”‡•Ǥ ‘–”‘Ž ˆ‹„”‘„Žƒ•–• ™‡”‡ ˆ”‘ ’ƒ–‹‡–• ™‹–Š‘—– ƒ †‘ —‡–‡† ‡–ƒ„‘Ž‹  †‹•‡ƒ•‡ ™‡”‡ —•‡† ȋ—’’Ž‡‡–ƒ”›–ƒ„Ž‡ͳȌǤŠ‡ˆ‹„”‘„Žƒ•–•™‡”‡‘„–ƒ‹‡†ˆ”‘–Š‡‡’ƒ”–‡–‘ˆŽ‹‹ ƒŽ ‡‡–‹ •‘ˆ –Š‡‹˜‡”•‹–›‡†‹ ƒŽ‡–‡” ”‘‹‰‡ǤŠ‡‡†‹ ƒŽ–Š‹ ƒŽ‘‹––‡‡‘ˆ–Š‡‹˜‡”•‹–›‡†‹ ƒŽ ‡–‡” ”‘‹‰‡ ‘ˆ‹”‡† –Šƒ– –Š‡ ‡†‹ ƒŽ ‡•‡ƒ” Š ˜‘Ž˜‹‰ —ƒ —„Œ‡ –•  – ™ƒ• ‘– ƒ’’Ž‹ ƒ„Ž‡ƒ†–Šƒ–‘ˆˆ‹ ‹ƒŽ•–—†›ƒ’’”‘˜ƒŽ„›–Š‡‡†‹ ƒŽ–Š‹ ƒŽ‘‹––‡‡™ƒ•‘–”‡“—‹”‡†ȋ  ʹͲͳ͸ȀͷͻͲȌǤ †‡”–Š‡•‡ ‹” —•–ƒ ‡• ƒ†™Š‡ †ƒ–ƒ ƒ”‡—•‡† ƒ‘›‘—•Ž›ǡ‹†‹˜‹†—ƒŽ•—„Œ‡ –̵• ‹ˆ‘”‡† ‘•‡–‹•‘–”‡“—‹”‡†ǡƒ•’‡”‹•–‹–—–‹‘ƒŽƒ†ƒ–‹‘ƒŽ”—Ž‡•Ǥȋ ʹͲͳ͸ȀͷͻͲȌǤ  Cell culture and harvest

Š‡ ‡ŽŽ•™‡”‡ —Ž–—”‡†ƒ–͵͹鋐ͷΨʹ‹ ƒ̵• ǦͳͲ—–”‹‡–‹šȋŠ‡”‘ ‹•Š‡” ‹‡–‹ˆ‹ Ȍǡ •—’’Ž‡‡–‡† ™‹–Š ͳͲΨ ˆ‡–ƒŽ ƒŽˆ •‡”— ȋ ǡ ‹„ ‘Ȍ ƒ† ͳΨ ’‡‹ ‹ŽŽ‹Ȁ•–”‡’–‘› ‹ ȋ‡–”‡’ǡ ‹„ ‘ȌǤ ‘” –Š‡ ‡š’‡”‹‡–•ǡ –Š‡ —Ž–—”‡ ‡†‹— ™ƒ• •—’’Ž‡‡–‡† ™‹–Š ͶͲͲ Ɋ Ǧ ƒ”‹–‹‡ ȋ‡š’‡”‹‡–ƒŽ‡†‹—ȌǤ ‘”–ƒ”‰‡–‡†’”‘–‡‘‹ •ǡ ‡ŽŽ•™‡”‡ˆ‹”•–•‡‡†‡†ƒ–̱͹ͷΨ ‘ˆŽ—‡ ›‹͹ͷ —Ž–—”‡ˆŽƒ••ȋ‘”‹‰Ž‹ˆ‡• ‹‡ ‡•ȌǤˆ–‡”ʹͶŠ‘—”•ǡ–Š‡ ‡ŽŽ•™‡”‡‹ —„ƒ–‡†‹‡š’‡”‹‡–ƒŽ‡†‹—Ǥ ˆ–‡”͹ʹŠ‘—”•ǡ–Š‡ ‡ŽŽ•™‡”‡™ƒ•Š‡†™‹–Šȋǡ ‹„ ‘Ȍǡ–”›’•‹‹œ‡†™‹–ŠͲǤʹͷΨ–”›’•‹ ȋ ‹„ ‘Ȍƒ†”‡•—•’‡†‡†‹ˆ”‡•Š‡†‹—ǤŠ‡ ‡ŽŽ•™‡”‡•’—†‘™ȋͷǯƒ–ͳǡͲͲͲ”’Ȍƒ†™ƒ•Š‡† ™‹–ŠͳͲŽ–Š”‡‡–‹‡•Ǥ ‹ƒŽŽ›ǡ–Š‡ ‡ŽŽ•™‡”‡ƒŽ‹“—‘–‡†ȋʹȉͳͲͷǦͳȉͳͲ͸ ‡ŽŽ•’‡”ƒŽ‹“—‘–Ȍ‹ͳǤͷ ”‘–‡‹ ‘‹† –—„‡• ȋ’’‡†‘”ˆȌǡ ’‡ŽŽ‡–‡† ȋͷǯ ƒ– ͳǡͷͲͲ ”’Ȍ ƒ† •–‘”‡† ƒ– ǦͺͲι —–‹Ž ˆ—”–Š‡” ’”‘ ‡••‹‰ƒ†ƒƒŽ›•‹•Ǥ‡ŽŽ’‡ŽŽ‡–•™‡”‡Ž›•‡†‹ʹͷρ‹ ‡Ǧ ‘Ž†ǦͶͲŽ›•‹•„—ˆˆ‡”ȋͲǤͳΨǦͶͲǡͲǤͶ ƒŽǡͳͲ”‹•’ ͺǤͲƒ†ͳ’ ͺǤͲȌˆ‘”ʹͲ‹—–‡•ǡ˜‘”–‡š‡†•Š‘”–Ž›ǡ•’—‘ƒ”‘–ƒ–‘” ƒ–ͶͲ”’ƒ†Ͷιˆ‘”ͳͷ‹—–‡•ƒ†•’—ˆ‘”ͳͲ‹—–‡•ƒ–ͳʹǡͲͲͲ‰ƒ†ͶιǤʹͲρ‘ˆ–Š‡•—’‡”ƒ–ƒ– ™ƒ•–”ƒ•ˆ‡””‡†–‘ƒ•‡ ‘†‘‹†–—„‡ƒ†ͷρ™ƒ•—•‡†ˆ‘”’”‘–‡‹†‡–‡”‹ƒ–‹‘ȋ‹‡” ‡̹ ”‘–‡‹••ƒ›‹–ǡŠ‡”‘ ‹‡–‹ˆ‹ ȌǤ‡”–‹‡’‘‹–͵™‡ŽŽ•‘ˆƒ͸Ǧ™‡ŽŽ’Žƒ–‡ȋ ”‡‹‡”‹‘Ǧ‡ǡͲͷͳ͸ͲȌ ™‡”‡ •‡‡†‡† ƒ– ά͹ͷΨ ‘ˆŽ—‡ ›Ǥ ʹͶ Š‘—”• ƒˆ–‡” •‡‡†‹‰ǡ ‡ŽŽ• ™‡”‡ ‹ —„ƒ–‡† ™‹–Š ͳ Ž ‘ˆ ‡š’‡”‹‡–ƒŽ‡†‹—Ǥ ‘”–Š‡‡–ƒ„‘Ž‘‹ •ǡ•’‡–‡†‹—™ƒ• ‘ŽŽ‡ –‡†ƒ† ‡ŽŽ•™‡”‡Šƒ”˜‡•–‡† ‘‹ ‡Ǥ‡ŽŽ•ˆ”‘–™‘͸Ǧ ™‡ŽŽ•™‡”‡™ƒ•Š‡†–™‹ ‡™‹–Š‹ ‡Ǧ ‘Ž†ǡ• ”ƒ’‡†™‹–ŠͷͲͲρ‡–Šƒ‘Ž ȋǦʹͲιȌǡ ƒŽ‹“—‘–‡† ȋͳͲͲͲ ρŽ ’‡” ƒŽ‹“—‘–Ȍ ƒ† •–‘”‡† ƒ– ǦͺͲι —–‹Ž ˆ—”–Š‡” ƒƒŽ›•‹•Ǥ ‘” ’”‘–‡‹ †‡–‡”‹ƒ–‹‘ǡ ‡ŽŽ•‹‘‡™‡ŽŽ™‡”‡• ”ƒ’‡†™‹–ŠʹͲͲρ‹ ‡Ǧ ‘Ž†ǡƒŽ‹“—‘–‡†ƒ†•–‘”‡†ƒ–ǦͺͲιǤ  Targeted quantitative proteomics of mitochondrial proteins  •‡– ‘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 –‹˜‹–›™‡”‡

ʹͳͷ Chapter 6

“—ƒ–‹ˆ‹‡†‹–Š‡’”‡’”‘ ‡••‡† ‡ŽŽ•ƒ’Ž‡•—•‹‰‹•‘–‘’‹ ƒŽŽ›Žƒ„‡ŽŽ‡†•–ƒ†ƒ”†•ȋͳ͵ǦŽƒ„‡Ž‡†Ž›•‹‡• ƒ†ƒ”‰‹‹‡•Ȍ†‡”‹˜‡†ˆ”‘•›–Š‡–‹ ’”‘–‡‹ ‘ ƒ–‡‡”•ȋ ‘Ȍȋ‘Ž›—ƒ– „ ǡƒ†„„ƒ Šǡ ‡”ƒ›Ȍƒ ‘”†‹‰–‘‘Ž–‡”•et al.ȏʹͶȐǤ  Acyl-carnitine levels  ›ŽǦ ƒ”‹–‹‡Ž‡˜‡Ž•™‡”‡‡ƒ•—”‡†ƒ ‘”†‹‰–‘‡”•et al.ȏͳʹȐǤ  Statistical analysis Š‡•‹‰‹ˆ‹ ƒ ‡‘ˆ†‹ˆˆ‡”‡ ‡•ǡ™‡”‡ƒ••‡••‡†—•‹‰’ƒ”ƒ‡–”‹ ȋ‘‡Ǧ™ƒ›ƒ†•—„•‡“—‡– ‘”—‡–ǯ•’‘•–Š‘ –‡•–Ȍ‘”‘’ƒ”ƒ‡–”‹ –‡•–•ȋ”—•ƒŽǦƒŽŽ‹•—ǯ•’‘•–Š‘ –‡•–Ȍ †‡’‡†‹‰‘–Š‡‹”†‹•–”‹„—–‹‘Ǥ–ƒ’˜ƒŽ—‡‘ˆδͲǤͲͷ–Š‡—ŽŽŠ›’‘–Š‡•‹•™ƒ• ‘•‹†‡”‡†–‘„‡ ”‡ˆ—–‡†ǤƮ–ƒ–‹•–‹ ƒŽƒƒŽ›•‡•™‡”‡’‡”ˆ‘”‡†—•‹‰ȋ˜ʹʹȌƒ†–Š‡†ƒ–ƒ™ƒ•˜‹•—ƒŽ‹œ‡†—•‹‰ ”ƒ’Šƒ†”‹••‘ˆ–™ƒ”‡ȋ ”ƒ’Šƒ†‘ˆ–™ƒ”‡  Ǥǡ˜‡”•‹‘ͷǤͲͲǡʹͲͲ͹ȌǤ

References ͳǤ Žƒƒ” ƒ Ǥƒ•••’‡ –”‘‡–”›‹ Ž‹‹ ƒŽ Š‡‹•–”›ǣŠ‡ ƒ•‡‘ˆ‡™„‘”• ”‡‡‹‰Ǥ Šƒ” ‹‘‡†ƒŽǤʹͲͳͶǢͳͲͳǣͳ͹ͶȂͺʹǤ ʹǤ ƒ’Š‡”ǡ”—‡––‹Ǧ‹‡””‹ǡ‡‡Ǥ „‘”‡””‘”•‘ˆ‡–ƒ„‘Ž‹•ǣ–Š‡ˆŽ—šˆ”‘‡†‡Ž‹ƒ–‘ ‘’Ž‡š†‹•‡ƒ•‡•Ǥƒ–‡˜ ‡‡–ǤʹͲͲ͸ —Ǣ͹ȋ͸ȌǣͶͶͻȂ͸ͲǤ ͵Ǥ ƒŽ†‘ƒ†‘ ǡ ƒŠƒ ǡ ƒŠƒ ǡ ƒŠƒ Ǥ ›•–‡• ‹‘Ž‘‰› ’’”‘ƒ Š‡• ‘™ƒ”† †‡”•–ƒ†‹‰”‹ƒ”›‹–‘ Š‘†”‹ƒŽ‹•‡ƒ•‡•Ǥ ”‘– ‡‡–ǤʹͲͳͻǢͳͲȋ ‡„”—ƒ”›ȌǣͳͻǤ ͶǤ ”‰ƒǡ ‘—–‡ǡŠ— ǡ Šƒ†–Ǥ‡š– ‡‡”ƒ–‹‘—Ž–‹• ƒŽ‡‹‡™‘ˆ „‘”””‘”• ‘ˆ‡–ƒ„‘Ž‹•Ǥ‡ŽŽ‡–ƒ„ǤʹͲͳ͸Ǣʹ͵ȋͳȌǣͳ͵Ȃʹ͸Ǥ ͷǤ ‹Ž ‡ǡ ƒƒ•ǡ ‘›ǡ‹Ž‡›ǡŠƒ’Ž‹ǡŽƒ ǡ Ž‡– Š‡” ǡ ‹ŽŽ ǡ‘‡ŠǤ—– ‘‡‘ˆ ‡‘ƒ–ƒŽ• ”‡‡‹‰ˆ‘”‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›‹—•–”ƒŽ‹ƒǣƒ ‘Š‘”– •–—†›Ǥƒ ‡–ǤʹͲͲ͹Ǣ͵͸ͻȋͻͷͷͷȌǣ͵͹ȂͶʹǤ ͸Ǥ ‘ŠŽ—ŽŽ‡” ǡ ƒ›ƒ–‡’‡ ǡ  Š—Žœ‡ ǡ ‘ˆˆƒ ǡ ‹†‡” ǡ Ž‰‡‘ŽŽ‡” Ǥ š’ƒ†‡† ‡™„‘” ”‡‡‹‰ˆ‘” „‘”””‘”•‘ˆ‡–ƒ„‘Ž‹•„›Ž‡ –”‘•’”ƒ› ‘‹œƒ–‹‘Ǧƒ†‡ƒ•• ’‡ –”‘‡–”›ǣ‡•—Ž–•ǡ—– ‘‡ǡƒ† ’Ž‹ ƒ–‹‘•Ǥ‡†‹ƒ–”‹ •ǤʹͲͲͶǢͳͳͳȋ͸Ȍǣͳ͵ͻͻȂͶͲ͸Ǥ ͹Ǥ ƒ‰‡”ǡ—‹Œ’‡”•ǡ‘• Šǡ—Ž†‡” ǡ—„‹‘Ǧ ‘œƒŽ„‘ǡ‹••‡” ǡ†‡”‹‡•ǡ‹ŽŽ‹ƒ• ǡ ƒ–‡”Šƒ ǡ ˜ƒ ’”‘•‡ ǡ  Š‹‡Ž‡  ǡ ‡”•  Ǥ  ƒ–‹‘™‹†‡ ‡–”‘•’‡ –‹˜‡ „•‡”˜ƒ–‹‘ƒŽ –—†› ˆ ‘’—Žƒ–‹‘ ‡™„‘”  ”‡‡‹‰ ‘” ‡†‹—ǦŠƒ‹  ›ŽǦ‘ ‡Š›†”‘‰‡ƒ•‡ȋȌ‡ˆ‹ ‹‡ › Š‡‡–Š‡”Žƒ†•Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͻ’”ʹ͵Ǣ͵ͳȋͲȌǤ ͺǤ  Šƒ–œǡ•‡ƒ—‡”ǤŠ‡ Ž‹‹ ƒŽƒ‹ˆ‡•–ƒ–‹‘‘ˆ†‡ˆ‹ ‹‡ ›ǣŠƒŽŽ‡‰‡•–‘™ƒ”†• ƒ†—Ž–Š‘‘†‹–Š‡• ”‡‡‡†’‘’—Žƒ–‹‘Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͲǢ͵͵ȋͷȌǣͷͳ͵ȂʹͲǤ ͻǤ ‡–Ž‡”ǡŠƒ‹ǡŽ•„‡ ‡”ǡ”‘Ž† ǡ—”–‘ǡ‘ Ž‡› ǡƒ‡”‘ǡ ‹‡” ǡ†‹   ǡ‡””›ǡŠ‘ƒ• ǡ‘†‰‡ǡ‹‰Šǡƒ•Šƒǡ‘ƒŽ‡›ǡ–‡„”‹†‰‡ǡ—••‹ǡ Š‹ŽŽ‹’•ǡ—”–‘ǡ†ƒ‘ǡŠ”‡•–Šƒǡ ‘‰ƒ•‘ ǡ™›‡”ǡ ƒ‹Ž‹‡ǡ‘‹‡ǡ ƒ‹Ž‹‡ǡ •ƒ‘ƒŠ ǡ ‘‘†‹ ǡ ƒŒƒƒ”—ƒ ǡ ƒ •‘ ǡ ƒ‘•Š ǡ ‡”‘ ǡ ‹–Š ǡ Šƒ† ǡ ‹’‹•‹ǡ ‡Ž†ƒ ǡ‡””›ǡŽ•„‡ ‡”ǡ‡–Ž‡”ǡ ‘–Ǧ‘–‰‘‡”›ǡ‡ ǡ‡ƒǡ ‘‡„‡”Žǡ ‹ƒ‰Š—‹ǡ‹•Šƒ‹ǡ‹œœ‘ǡƒ™•‘ǡ„”‘•‡ǡ‡˜›ǡ”‘ǡ ‘‰ –‘ǡǯ ‘ǡ ƒ”–ǡ”„‡ǡƒ‘•ǡ‡•Ž‹‡ǡ‘™‡”•ǡƒ”–Š‘Ž‘‡™ǡ ‘ˆˆǡ˜ƒƒŽ ƒ”ǡ ƒ•‡ ǡ”‘Ž† ǡ‘ Ž‡› ǡƒŽ•ŠǦ‘ Ž‡›ǡŠ‡ƒ†ǡ‹‘ ǡ‰‡Ž‹‰ǡ‹”†ǡ™ƒ ǡ Š™‘‡”‡” ǡ ‡”›ǡƒ”—ƒ Š‹ǡ —‡Žǡ‹Ž‹• ǡƒ˜‹•Ǧ‡’’‡ǡ–‡‹ǡ‘ƒ ǡ ƒ‡”‘ ǡ †‹   ǡ ‹‡”  ǡ —•–‹ ‡ ǡ Šƒ‹ Ǥ ʹʹͳ ‡™„‘”Ǧ• ”‡‡‡† ‡‘ƒ–‡• ™‹–Š ‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ ‘‡œ›‡†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›ǣ ‹†‹‰•ˆ”‘–Š‡ „‘”””‘”•‘ˆ ‡–ƒ„‘Ž‹•‘ŽŽƒ„‘”ƒ–‹˜‡Ǥ‘Ž ‡‡–‡–ƒ„ǤʹͲͳ͸ǢͳͳͻȋͳȂʹȌǣ͹ͷȂͺʹǤ ͳͲǤ ‡”• 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 ͳͳǤ ‡”• ǡƒ’”‘•‡ ǡƒ‡ ǡƒ‡” ‹Ž•–ǡ’ƒǡ‹– Ǥ ƒˆ‡ƒ†—•ƒˆ‡ †—”ƒ–‹‘‘ˆˆƒ•–‹‰ˆ‘” Š‹Ž†”‡™‹–Š†‡ˆ‹ ‹‡ ›Ǥ—” ‡†‹ƒ–”ǤʹͲͲ͹Ǣͳ͸͸ȋͳȌǣͷȂͳͳǤ ͳʹǤ ‡”• ǡ‘‡”ǡ••‡ǡ‘•ǡ—‹–‡” ǡƒ–‡”Šƒ ǡ‹‡œ‡Ǧ‘‹‰ǡƒ†‡”• ǡ ‘†‡‡Ž ǡ‘‡„‡” ǡ‡ƒ–‡ǡ‹– ǡ‡‹Œ‰‘—† Ǥ‡‘ƒ–ƒŽ• ”‡‡‹‰ˆ‘”‡†‹—Ǧ

ʹͳ͸ In vitro metabolic adaptations that explain symptomatology in MCADD individuals

Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋȌ †‡ˆ‹ ‹‡ › ‹ Š‡ ‡–Š‡”Žƒ†•ǣ Š‡ ‹’‘”–ƒ ‡ ‘ˆ ‡œ›‡ƒƒŽ›•‹•–‘ƒ• ‡”–ƒ‹–”—‡†‡ˆ‹ ‹‡ ›Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͲͺǢ͵ͳȋͳȌǣͺͺȂͻ͸Ǥ ͳ͵Ǥ ‘—™ǡ‹– ǡ‹‡œ‡Ǧ‘‹‰ǡ‘•‰”ƒƒˆǦ†‡‘‡”ǡ ‡”†‹‰ǡ‡‹Œ‰‘—†Ǧ ǡ‡”•  Ǥ  ˜‹–”‘ ƒ† ‹ ˜‹˜‘ ‘•‡“—‡ ‡• ‘ˆ ˜ƒ”‹ƒ– ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ‰‡‘–›’‡•Ǥ”’Šƒ‡– ƒ”‡‹•ǤʹͲͳ͵ǢͺȋͳȌǣͶ͵Ǥ ͳͶǤ ‘—™ǡ‹– ǡ†‡”‹‡•ǡ†‡Ž‡” ǡ‘• Šǡ‹••‡” ǡ—Ž†‡” ǡ—„‹‘Ǧ ‘œƒŽ„‘ ǡŽ˜‡”•ǡ‹‡œ‡Ǧ‘‹‰ǡƒ†‡”• ǡƒ–‡”Šƒ ǡ‡‹Œ‰‘—†Ǧ ǡ‡”• Ǥ‹• •–”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 ͳͷǤ ’‹‡‡”‘‡––‡” ǡ ƒ•–‹ ǡ ‹ŽŽ‹‰Šƒ ǡ ‘””‹• ǡ ‹Œ„—”‰ ǡ ‹Ž ‡ Ǥ —””‡– ‹••—‡• ”‡‰ƒ”†‹‰ –”‡ƒ–‡– ‘ˆ ‹–‘ Š‘†”‹ƒŽ ˆƒ––› ƒ ‹† ‘š‹†ƒ–‹‘ †‹•‘”†‡”•Ǥ  Š‡”‹– ‡–ƒ„ ‹•Ǥ ʹͲͳͲǢ͵͵ȋͷȌǣͷͷͷȂ͸ͳǤ ͳ͸Ǥ ”‘‡–›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 ͳ͹Ǥ ‡ŽŽ›ǡŠ‡Žƒ ǡ‰†‡ǡŽ’‡”•ǡŠƒ‰ ǡ‡ŽŽ—• ǡ ”‡‰‡”•‡ǡ‘”Žƒ†ǡ–”ƒ—•• 6 Ǥ‘Ž‡ —Žƒ” Šƒ”ƒ –‡”‹œƒ–‹‘‘ˆ‹Š‡”‹–‡†‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›Ǥ ”‘ ƒ–Ž ƒ† ‹ǤͳͻͻͲǢͺ͹ȋʹ͵Ȍǣͻʹ͵͸ȂͶͲǤ ͳͺǤ —”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 ͳͻǤ  Šƒ–œƒǤǡ•‡ƒ—‡”ǤŠ‡ Ž‹‹ ƒŽƒ‹ˆ‡•–ƒ–‹‘‘ˆ†‡ˆ‹ ‹‡ ›ǣŠƒŽŽ‡‰‡•–‘™ƒ”†• ƒ†—Ž–Š‘‘†‹–Š‡• ”‡‡‡†’‘’—Žƒ–‹‘Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͲǢ͵͵ȋͷȌǣͷͳ͵ȂʹͲǤ ʹͲǤ ‘—–‡ ǡ ‹‘Žƒ–‡ ǡ ‡–—”ƒ  Ǥǡ ƒ†‡”•  Ǥ Š‡ ‹‘ Š‡‹•–”› ƒ† Š›•‹‘Ž‘‰› ‘ˆ ‹–‘ Š‘†”‹ƒŽ ƒ––›  ‹† ȾǦš‹†ƒ–‹‘ ƒ† –• ‡‡–‹  ‹•‘”†‡”•Ǥ — ‡˜ Š›•‹‘ŽǤ ʹͲͳ͸Ǣ͹ͺȋͳȌǣʹ͵ȂͶͶǤ ʹͳǤ ƒ‰ ǡ ‘Š•‡ ǡ ‡ Š‹ ǡ  Š”‡‹„‡” ǡ ƒŽƒ•—„”ƒƒ‹ ǡ ƒ› ǡ ‹ Šƒ‡Ž ƒ”ƒ†ƒ ǡ ‘ Ž‡› Ǥ ‘’Ž‡š Šƒ‰‡• ‹ –Š‡ Ž‹˜‡” ‹–‘ Š‘†”‹ƒŽ ’”‘–‡‘‡ ‘ˆ •Š‘”– Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡–‹ ‡Ǥ‘Ž ‡‡–‡–ƒ„ǤʹͲͳͶǢͳͳʹȋͳȌǣ͵ͲȂͻǤ ʹʹǤ ‘ Šƒ ǡ ‡””‡‹”ƒǡƒ”˜ƒŽŠ‘ ǡ‹–‘”‹‘ǡƒ–ƒǡ‘’‡•ǡ ”‡‰‡”•‡ǡ‹Žƒ”‹Š‘ǡƒ†‘ Ǥ Šƒ”ƒ –‡”‹œƒ–‹‘ ‘ˆ ‹–‘ Š‘†”‹ƒŽ ’”‘–‡‘‡ ‹ ƒ •‡˜‡”‡ ƒ•‡ ‘ˆ  Ǧ †‡ˆ‹ ‹‡ ›Ǥ  ”‘–‡‘‹ •ǤʹͲͳͳǢ͹ͷȋͳȌǣʹʹͳȂͺǤ ʹ͵Ǥ †Šƒ‰‡”Ǥǡ–‡„”‘‡ǡ‹‡Ž•‡ǡ”‘••ǡŽ•‡ ǡ ”‡‰‡”•‡ǡƒŽˆ‡Ž†– Ǥ”‘–‡‘‹  ‹˜‡•–‹‰ƒ–‹‘‘ˆ —Ž–‹˜ƒ–‡†ˆ‹„”‘„Žƒ•–•ˆ”‘’ƒ–‹‡–•™‹–Š‹–‘ Š‘†”‹ƒŽ•Š‘”–Ǧ Šƒ‹ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›Ǥ‘Ž ‡‡–‡–ƒ„ǤʹͲͳͶǢͳͳͳȋ͵Ȍǣ͵͸ͲȂͺǤ ʹͶǤ ‘Ž–‡”• ǡ ‹ƒ’ƒ‹–‡ ǡ ˜ƒ —‡ ǡ ‹‡œ‡Ǧ‘‹‰ ǡ ƒ––‘ ǡ ‘”–‡  ǡ ‘”˜ƒ–‘˜‹ Š ǡ ƒ‡” ǡ ‹• Š‘ˆˆ ǡ ‡”‡–‹‡” Ǥ ”ƒ•Žƒ–‹‘ƒŽ –ƒ”‰‡–‡† ’”‘–‡‘‹ • ’”‘ˆ‹Ž‹‰ ‘ˆ ‹–‘ Š‘†”‹ƒŽ ‡‡”‰› ‡–ƒ„‘Ž‹  ’ƒ–Š™ƒ›• ‹ ‘—•‡ ƒ† Š—ƒ •ƒ’Ž‡•Ǥ —„‹–– –‘ —„ŽǤ ʹͲͳ͸Ǣ ʹͷǤ ƒ‹Œ‘•ǡ‹ŽŽ‹ƒ ǡƒƒƒŠǤ –”ƒ‹–‘ Š‘†”‹ƒŽ ‘Ž†‹‰ƒ†••‡„Ž›‘ˆ‡†‹—Ǧ Šƒ‹ ›ŽǦ ‘‡Š›†”‘‰‡ƒ•‡ȋȌǤͳͻͻͶǢʹ͸ͻȋ͸ȌǣͶͶͲͳȂͺǤ ʹ͸Ǥ ”‘••ǡ ‡•’‡”•‡ǡ ‡•‡ ǡ†”‡•‡ǡ”‹•–‡•‡ ǡ‹–‡”ǡƒ†›ǡ”ƒ—–Ž‡ ǡ Š‹•Žƒǡ‘Ž—†ǡ‹ ǡ ”‡‰‡”•‡Ǥˆˆ‡ –•‘ˆ–™‘—–ƒ–‹‘•†‡–‡ –‡†‹‡†‹— Šƒ‹  ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋȌǦ†‡ˆ‹ ‹‡– ’ƒ–‹‡–• ‘ ˆ‘Ž†‹‰ǡ ‘Ž‹‰‘‡” ƒ••‡„Ž›ǡ ƒ† •–ƒ„‹Ž‹–›‘ˆ‡œ›‡Ǥ‘ŽǤʹ͹Ͳǡ ‘—”ƒŽ‘ˆ‹‘Ž‘‰‹ ƒŽŠ‡‹•–”›ǤͳͻͻͷǤ’ǤͳͲʹͺͶȂͻͲǤ ʹ͹Ǥ ƒ ǡƒ‹‡”ǡ‡‹„ǡ ƒ•Ž„‡ ǡ‡–‡” ǡ”—‰‡”ǡ‘‡”Š‘ˆˆǡ ‡”†‹ƒ†—••‡ ǡ ƒ†‡”•  ǡ ‡”•–‹‰ ǡ —–ƒ— Ǥ Š‡ †‘ƒ‹Ǧ•’‡ ‹ˆ‹  ƒ† –‡’‡”ƒ–—”‡Ǧ†‡’‡†‡– ’”‘–‡‹‹•ˆ‘Ž†‹‰ ’Š‡‘–›’‡ ‘ˆ ˜ƒ”‹ƒ– ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡Ǥ ‘ ‡Ǥ ʹͲͳͶǢͻȋͶȌǤ ʹͺǤ —‹–‡„‡‡ǡ‘‡Ž• ǡ—„—ŽŽǡ ƒ”ƒ˜ƒ‰Ž‹ƒǡŠƒŽ‡”•ǡƒ›Ž‘”Ǥ‘•‡–‡†‹— Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›ǤͳͻͻͷǢʹͲͻȂͳͶǤ ʹͻǤ ‡”• ǡ‘—™ǡ‹„ƒ• ǡ‹ƒ ‹‹ ǡƒœ‹ǡ‡‰”‡––‘ ǡ‡• ƒǡ‡‹Œ‰‘—† ǡ ‹– ǡ ƒŒ‡” ǡ ƒ”‰ƒ• Ǥ š’‡”‹‡–ƒŽ ‡˜‹†‡ ‡ ˆ‘” ’”‘–‡‹ ‘š‹†ƒ–‹˜‡ †ƒƒ‰‡ ƒ† ƒŽ–‡”‡†ƒ–‹‘š‹†ƒ–†‡ˆ‡•‡‹’ƒ–‹‡–•™‹–Š‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͶǢ͵͹ȋͷȌǣ͹ͺ͵ȂͻǤ 

ʹͳ͹ Chapter 6



ʹͳͺ In vitro metabolic adaptations that explain symptomatology in MCADD individuals

Supplementary Figures and tables  Supplementary table ST1: Characteristics of the individuals from which the control fibroblasts originated. ID Age (years) Indication Outcome C1 Ͷʹ —††‡†‡ƒ–Š  —–‡ ƒ”†‹ƒ †‡ƒ–Š C2 ͹ʹ —††‡†‡ƒ–Š ƒ”†‹ƒ ƒ””‡•–ǡ‡—”‘†‡‰‡‡”ƒ–‹˜‡†‹•‡ƒ•‡ C3 ͷ͸ ‹‰”ƒ‹‡ ‘‰‡‡–‹ ‘”‹‰‹‘ˆ†‹•‡ƒ•‡ C4 ͳ —††‡†‡ƒ–Š ‡’–‹ •Š‘  C5 Ͳ —††‡†‡ƒ–Š Š‡•—•ƒ–ƒ‰‘‹• C6.K ”‡•—ƒ„Ž›ʹͲǯ•  ‹‡–‹ˆ‹ ‡•‡ƒ” Š Ǧ C6.W Ͳ ‘–ƒ˜ƒ‹Žƒ„Ž‡ ‘–ƒ˜ƒ‹Žƒ„Ž‡ Ǯ‰‡ǯ ”‡ˆ‡”• –‘ –Š‡ ƒ‰‡ ƒ– ™Š‹ Š ˆ‹„”‘„Žƒ•–• ™‡”‡ –ƒ‡Ǥ Ǯ †‹ ƒ–‹‘ǯ †‡• ”‹„‡• –Š‡ ”‡ƒ•‘ ˆ‘” „‹‘’•›Ǥ Ǯ—– ‘‡ǯ†‡• ”‹„‡•†‹•‡ƒ•‡‘—– ‘‡Ǥ‘‡–ƒ„‘Ž‹ ‘”‹‰‹‘ˆ†‹•‡ƒ•‡™ƒ•‹†‡–‹ˆ‹‡†Ǥ  6 A B 0.15 0.8

M) M) 0.6 P P * 0.10

0.4

0.05 ††Á‚ 0.2 ††ÁÁ‚– medium C6 ( C6 medium ( C8 medium

0.00 0.0 Sympt Asympt (ex. Pt10) Pt10 Ctrl Sympt Asympt (ex. Pt10) Pt10 Ctrl

CD 0.15 0.08 #,* M) M) P

P 0.06 0.10

0.04

0.05 ††Á 0.02

medium C10 ( C10 medium ¥ medium C10:1 ( C10:1 medium 0.00 0.00 Sympt Asympt (ex. Pt10) Pt10 Ctrl Sympt Asympt (ex. Pt10) Pt10 Ctrl  Supplementary figure S1Ǥ ‘ ‡–”ƒ–‹‘ ‘ˆ ƒ ›ŽǦ ƒ”‹–‹‡• ‹ –Š‡ ‡†‹—Ǥ Š‡ ”‡•—Ž–• ’‡”–ƒ‹ –‘ –Š‡ ‡š’Ž‘”ƒ–‘”›’Šƒ•‡‘ˆ–Š‡•–—†›Ǥ‡–ƒ‹Ž•ƒ•‹ ‹‰—”‡ͳǤ‹‰‹ˆ‹ ƒ ‡˜‹•—ƒŽ‹œƒ–‹‘‹•ƒ ‘”†‹‰–‘ ‹‰—”‡ͳǤ 

ʹͳͻ Chapter 6

A B 0.15 1.5 M) M) P P 0.10 1.0

0.05 0.5 medium C8 ( medium medium C6 (

0.00 0.0 Pt1 Pt5 Pt7 Pt8 Pt2 Pt3 Pt4 Pt6 Pt9 Pt10 C3 C4 C5 C6.KC6.W Pt1 Pt5 Pt7 Pt8 Pt2 Pt3 Pt4 Pt6 Pt9 Pt10 C3 C4 C5 C6.KC6.W

C D 0.08 0.08 M) M) P

0.06 P 0.06

0.04 0.04

0.02 0.02 medium C10 ( C10 medium medium C10:1 ( C10:1 medium 0.00 0.00 Pt1 Pt5 Pt7 Pt8 Pt2 Pt3 Pt4 Pt6 Pt9 Pt10 C3 C4 C5 C6.KC6.W Pt1 Pt5 Pt7 Pt8 Pt2 Pt3 Pt4 Pt6 Pt9 Pt10 C3 C4 C5 C6.KC6.W

EF 0.15 1.5 M) M) P 0.10 P 1.0

0.05 0.5 ††Á‚–– medium C6 ( C6 medium medium C8 ( medium ††ÁÁ‚–– 0.00 0.0 Sympt Asympt (ex. Pt10) Pt10 Ctrl Sympt Asympt (ex. Pt10) Pt10 Ctrl

GH

0.08 0.08 M) M) P

0.06 P 0.06

0.04 0.04

0.02 ††Á‚– 0.02 ¥ medium C10 ( medium medium C10:1 ( C10:1 medium 0.00 0.00 Sympt Asympt (ex. Pt10) Pt10 Ctrl Sympt Asympt (ex. Pt10) Pt10 Ctrl

Supplementary figure S2Ǥ‘ ‡–”ƒ–‹‘‘ˆƒ ›ŽǦ ƒ”‹–‹‡•‹–Š‡‡†‹—ǡ†‡’‹ –‡†ˆ‘”–Š‡ˆ‹„”‘„Žƒ•–• •‡’ƒ”ƒ–‡Ž› ȋǦȌ ƒ† †‡’‹ –‡† ƒ• •›’–‘ƒ–‹ ǡ ƒ•›’–‘ƒ–‹  ƒ† ‘–”‘Ž ˆ‹„”‘„Žƒ•– ‰”‘—’• ȋǦ ȌǤ Š‡ ”‡•—Ž–• ’‡”–ƒ‹ –‘ –Š‡ ˜ƒŽ‹†ƒ–‹‘ ’Šƒ•‡ ‘ˆ –Š‡ •–—†›Ǥ ‡–ƒ‹Ž• ƒ• ‹ ‹‰—”‡ ʹǤ ‹‰‹ˆ‹ ƒ ‡ ˜‹•—ƒŽ‹œƒ–‹‘ ‹• ƒ ‘”†‹‰–‘ ‹‰—”‡ͳǤ 

ʹʹͲ In vitro metabolic adaptations that explain symptomatology in MCADD individuals

A B 0.6 6

0.4 4 0.2 2 0.0 Pt1 Pt5 Pt7 Pt8 Pt2 Pt3 Pt4 Pt6 Pt9 Pt10 C3 C4 C5 C6.KC6.W medium C6 medium C8 medium

mol/g protein) mol/g protein) mol/g 0 ' '

P -0.2 P Pt1 Pt5 Pt7 Pt8 Pt2 Pt3 Pt4 Pt6 Pt9 Pt10 C3 C4 C5 C6.KC6.W ( (

-0.4 -2

CD 0.3 0.3

0.2 0.2 0.1 0.1 0.0 Pt1 Pt5 Pt7 Pt8 Pt2 Pt3 Pt4 Pt6 Pt9 Pt10 C3 C4 C5 C6.KC6.W medium C10 medium

mol/g protein) mol/g protein) mol/g 6 0.0 medium C10:1 medium ' P -0.1 P Pt1 Pt5 Pt7 Pt8 Pt2 Pt3 Pt4 Pt6 Pt9 Pt10 C3 C4 C5 C6.KC6.W ( ( '

-0.2 -0.1 Supplementary figure S3Ǥ ›ŽǦ ƒ”‹–‹‡•‡ ”‡–‹‘‹–‘‹–Š‡‡†‹—ǡ†‡’‹ –‡†’‡”ˆ‹„”‘„Žƒ•– ‡ŽŽŽ‹‡Ǥ Š‡”‡•—Ž–•’‡”–ƒ‹–Š‡˜ƒŽ‹†ƒ–‹‘’Šƒ•‡‘ˆ–Š‡•–—†›Ǥ‡–ƒ‹Ž•ƒ•‹ ‹‰—”‡ʹǤ 

A B 0.5 0.20

0.4 0.15

0.3 0.10 0.2

intracellular intracellular 0.05 0.1 C6 (nmol/g. protein) C8 (nmol/g. protein) 0.0 0.00 Pt1 Pt5 Pt7 Pt8 Pt2 Pt3 Pt4 Pt6 Pt9 Pt10 C3 C4 C5 C6.KC6.W Pt1 Pt5 Pt7 Pt8 Pt2 Pt3 Pt4 Pt6 Pt9 Pt10 C3 C4 C5 C6.KC6.W

CD 0.08 0.04

0.06 0.03

0.04 0.02

intracellular 0.02 intracellular 0.01 C10 (nmol/g. protein)

C10:1 (nmol/g. protein) 0.00 0.00 Pt1 Pt5 Pt7 Pt8 Pt2 Pt3 Pt4 Pt6 Pt9 Pt10 C3 C4 C5 C6.KC6.W Pt1 Pt5 Pt7 Pt8 Pt2 Pt3 Pt4 Pt6 Pt9 Pt10 C3 C4 C5 C6.KC6.W  Supplementary figure S4Ǥ –”ƒ ‡ŽŽ—Žƒ”ƒ ›ŽǦ ƒ”‹–‹‡ ‘ ‡–”ƒ–‹‘•ǡ†‡’‹ –‡†’‡”ˆ‹„”‘„Žƒ•– ‡ŽŽŽ‹‡ǤŠ‡ ”‡•—Ž–•’‡”–ƒ‹–‘–Š‡˜ƒŽ‹†ƒ–‹‘’Šƒ•‡‘ˆ–Š‡•–—†›Ǥ‡–ƒ‹Ž•ƒ•‹ ‹‰—”‡ʹǤ  

ʹʹͳ Chapter 6

A B

4 g) 20 P g)

P 3 15

2 10

1 5 MCAD (fmol/ l.o.d. 0 0

Pt1 Pt5 Pt7 Pt8 Pt2 Pt3 Pt4 Pt6 Pt9 Pt10 C3 C4 C5 C6.KC6.W Peroxiredoxin-6 (fmol/ Pt1 Pt5 Pt7 Pt8 Pt2 Pt3 Pt4 Pt6 Pt9 Pt10 C3 C4 C5 C6.KC6.W

C 0.5

g) 0.4 P 0.3

0.2

0.1 SCAD (fmol/

0.0 Pt1 Pt5 Pt7 Pt8 Pt2 Pt3 Pt4 Pt6 Pt9 Pt10 C3 C4 C5 C6.KC6.W Supplementary figure S5Ǥ‡Ž‡ –‹‘‘ˆ’”‘–‡‘‹ ”‡•—Ž–•‘ˆ–Š‡˜ƒŽ‹†ƒ–‹‘’Šƒ•‡‘ˆ–Š‡•–—†›ǡ†‡’‹ –‡†’‡” ˆ‹„”‘„Žƒ•– ‡ŽŽŽ‹‡Ǥ‡–ƒ‹Ž•ƒ•‹ ‹‰—”‡ͶǤ‹‰‹ˆ‹ ƒ ‡˜‹•—ƒŽ‹œƒ–‹‘‹•ƒ ‘”†‹‰–‘ ‹‰—”‡ͳǤ 

ʹʹʹ 

Chapter 7

General discussion

                      

‡‡”ƒŽ†‹• —••‹‘

 –Š‹• –Š‡•‹•ǡ  –‘‘ ƒ ›•–‡• ‡†‹ ‹‡ ƒ’’”‘ƒ Š –‘ ƒ††”‡•• –Š‡ “—‡•–‹‘ ™Šƒ– ‡Ž‹ ‹–• Š›’‘‡–‘–‹ Š›’‘‰Ž› ‡‹ƒ‹•‘‡†‡ˆ‹ ‹‡–•—„Œ‡ –•ƒ†™Šƒ–’”‘–‡ –•‘–Š‡”•ƒ‰ƒ‹•– –Š‹•Ǥ ‘”†‡”–‘†‘•‘ ‡˜ƒŽ—ƒ–‡††‡ˆ‹ ‹‡ ›‹ƒ ‘’—–ƒ–‹‘ƒŽ‘†‡Žǡ‹‹ ‡‹˜‹˜‘ ƒ†‹Š—ƒ•‹˜‹–”‘Ǥ –Š‹• Šƒ’–‡” ™‹ŽŽˆ‹”•–†‹• —••–Š‡’”‡•‡–‡†’”‘Œ‡ –•ǡ–Š‡”‡•—Ž–•ǡ ‹’Ž‹ ƒ–‹‘•ǡˆ‘ŽŽ‘™Ǧ—’”‡ ‘‡†ƒ–‹‘•ƒ†ˆ—–—”‡’”‘•’‡ –•ǡˆ”‘ ‘’—–ƒ–‹‘ƒŽ‘†‡Ž–‘ ‹ ‡–‘ƒǤ ‹ƒŽŽ›ǡ ™‹ŽŽ‡†™‹–Š•‘‡‘˜‡”ƒ” Š‹‰ ‘ Ž—•‹‘•ˆ”‘–Š‹•–Š‡•‹•Ǥ  Navigating from computational metabolic model to mice to man

Computational modeling of mFAO and beyond Chapter 2ǡ ‹˜‡•–‹‰ƒ–‡†–Š‡‘”‹‰‹‘ˆ–Š‡•–‡ƒ†›Ǧ•–ƒ–‡ˆŽ—šǦ†‡ Ž‹‡’Š‡‘‡‘‘„•‡”˜‡† ‹ –Š‡ ‘’—–ƒ–‹‘ƒŽ ‘†‡Ž ‘ˆ –Š‡ ‹•‘Žƒ–‡†   ’ƒ–Š™ƒ› ‹ ”ƒ– Ž‹˜‡”Ǥ  ƒ††‹–‹‘ǡ  ‹˜‡•–‹‰ƒ–‡† –Š‡ ”‘Ž‡ ‘ˆ ‡œ›‡ ’”‘‹• —‹–› ‹ ‹–Ǥ  ‹†‡–‹ˆ‹‡† –Šƒ– ‡œ›‡ ’”‘‹• —‹–› ‡•’‡ ‹ƒŽŽ›–Šƒ–‘ˆǡ’Žƒ›•ƒ‹’‘”–ƒ–”‘Ž‡‹–Š‡ˆŽ—š†‡ Ž‹‡Ǥœ›‡’”‘‹• —‹–› Ž‡ƒ†•–‘‹Š‹„‹–‹‘‘ˆ‡–ƒ„‘Ž‹ ”‡ƒ –‹‘•„› ‘’‡–‹‰•—„•–”ƒ–‡•ǤŠ‹•‹Š‹„‹–‹‘ƒ’’‡ƒ”•–‘ 7 ”‡•—Ž–‹ƒ•–”‘‰‹Š‹„‹–‹‘‘ˆ–Š‡ ˆŽ—šƒ– ›–‘•‘Ž‹ ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘•Š‹‰Š‡” –Šƒƒ ‡”–ƒ‹–Š”‡•Š‘Ž†˜ƒŽ—‡Ǥ ’‘”–ƒ–Ž›ǡ ‘’‡–‹–‹˜‡‹Š‹„‹–‹‘ƒ†‡œ›‡’”‘‹• —‹–› ƒ”‡‹–”‹ ƒ–‡’”‘’‡”–‹‡•‘ˆƒ–—”ƒŽ„‹‘Ž‘‰‹ ƒŽ’”‘ ‡••‡•Ǥ –‹•–Š—•‹–‡”‡•–‹‰–‘‘–‹ ‡–Šƒ– ”‡ƒ –‹‘• ƒ”‡ƒ Š•—„•–ƒ–‹ƒŽŽ›Š‹‰Š‡””ƒ–‡•‹‹•‘Žƒ–‹‘ǡ™Š‡ƒŠ‘‘‰‡‡‘—••—„•–”ƒ–‡ ‹š–—”‡‹•’”‡•‡–‡†–‘–Š‡‡œ›‡ǡ–Šƒ™Š‡–Š‡‡œ›‡ǯ•’”‘’‡”–‹‡•ƒ”‡ Šƒ‰‡††—‡–‘ –Š‡ ’”‡•‡ ‡ ‘ˆ ‘’‡–‹‰ •—„•–”ƒ–‡• ‘” ‘–Š‡” ‘†‹ˆ‹‡”•Ǥ ‡”•‹•–‡– ‹„ƒŽƒ ‡• ‹ ”ƒ–‡• ™‹–Š‹ ƒ ‡–™‘” ‘—Ž† „‡ ‘‡ †‡–”‹‡–ƒŽ –‘ ‡–ƒ„‘Ž‹  Š‘‡‘•–ƒ•‹• ƒ† ƒ—•‡ –Š‡ †‡˜‡Ž‘’‡–‘ˆƒ’ƒ–Š‘Ž‘‰‹ •–ƒ–‡Ǥ ƒŽ•‘‹˜‡•–‹‰ƒ–‡†–Š‡•‡“—‡ ‡‘ˆ‡˜‡–•Ž‡ƒ†‹‰—’–‘–Š‡ Ž‘™ˆŽ—šƒ–Š‹‰Š’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘•Ǥ •Š‘™‡†–Šƒ–ƒ–Š‹‰Š ›–‘•‘Ž‹ ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘•͸•—„•–”ƒ–‡‘ˆ•–ƒ”–‡†ƒ ——Žƒ–‹‰•—„•–ƒ–‹ƒŽŽ›„‡ˆ‘”‡–Š‡‘–Š‡” ‹–‡”‡†‹ƒ–‡• ‘ˆ –Š‡ ’ƒ–Š™ƒ›Ǥ ’’ƒ”‡–Ž›ǡ  †‹† ‘– ‡‡– –Š‡ Š‹‰Š ”‡ƒ –‹‘ ”ƒ–‡• †‡ƒ†‡† „› –Š‡ Žƒ”‰‡ •—„•–”ƒ–‡ •—’’Ž›ǡ ’ƒ”–‹ —Žƒ”Ž› ™Š‡ ‹– ™ƒ• ‹Š‹„‹–‡† „› ƒŽŽ –Š‡ ‹–‡”‡†‹ƒ–‡ƒ ›ŽǦ‘‡•–‡”•Ǥ‘•‡“—‡–Ž›ǡƒ˜‹ ‹‘—• › Ž‡‘ˆ‹Š‹„‹–‹‘„›‹–•‘™•—„•–”ƒ–‡• ƒ†’”‘†— –•‘ —””‡†ǡ”‡•—Ž–‹‰‹‘ †‡’Ž‡–‹‘ƒ†Ž‘™‡”•–‡ƒ†›Ǧ•–ƒ–‡ˆŽ—š‡•ƒ–Š‹‰Š ›–‘•‘Ž‹ ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘•Ǥ –‡”‡•–‹‰Ž›ǡ‰ƒ‹‡†’‘•‹–‹˜‡ˆŽ—š ‘–”‘Žƒ† ͳ‰ƒ‹‡†‡‰ƒ–‹˜‡ˆŽ—š ‘–”‘Žƒ–Š‹‰Š’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘•ȏͳȐǤ›’‹ ƒŽŽ›ǡ–Š‡ˆ‹”•– ‡œ›‡‹ƒ’ƒ–Š™ƒ›Ž‘•‡•ˆŽ—š ‘–”‘Ž™Š‡‹–••—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘‹ ”‡ƒ•‡•ƒ†–Š‡ ‡œ›‡ •–ƒ”–• –‘ •‡•‡ ‹Š‹„‹–‹‘ „› ‹–• ‘™ ’”‘†— –• ‘” †‘™•–”‡ƒ ‡–ƒ„‘Ž‹–‡•Ǥ Ž—š ‘–”‘Ž–Š‡•Š‹ˆ–•–‘‡œ›‡•ˆ—”–Š‡”†‘™•–”‡ƒȏʹȐǤŠ‡‡‰ƒ–‹˜‡ˆŽ—š ‘–”‘Ž‡š‡”–‡†„› ͳƒ–Š‹‰Š•—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘•†‹•–‹‰—‹•Š‡†–Š‡ ‘†‡Žˆ”‘‘–Š‡”’ƒ–Š™ƒ› •–”— –—”‡•Ǥ –Š‡‡š–•‡ –‹‘ ™‹ŽŽ‡˜ƒŽ—ƒ–‡–Š‡‡ˆˆ‡ –•‘ˆ–ƒ ‹–ƒ••—’–‹‘•‹–Š‡‘†‡Žǡ –Š‡‹” ‘•‡“—‡ ‡•ˆ‘”–Š‡‘†‡Žƒ†ƒ’‘••‹„Ž‡™ƒ›–‘ ‹” —˜‡––Š‡Ǥ  Assumptions •’”‡˜‹‘—•Ž›‹†‹ ƒ–‡†ǡ–Š‡’—„Ž‹•Š‡†‘†‡Ž• ‘–ƒ‹•‡˜‡”ƒŽ‹’Ž‹‡†ƒ••—’–‹‘•–Šƒ–ƒ› „‡”‡Ž‡˜ƒ––‘’‘‹–‘—–‹–Š‡Ž‹‰Š–‘ˆ—•‹‰–Š‡•‡‘†‡Ž•–‘ƒ‡’”‡†‹ –‹‘•ƒ„‘—–Š‡ƒŽ–Š ƒ††‹•‡ƒ•‡Ǥ ‹”•–ǡ ™‹ŽŽ†‹• —••ƒ••—’–‹‘•‘–Š‡‹‡–‹ •‘ˆ–Š‡‡œ›ƒ–‹ ”‡ƒ –‹‘•ǡƒˆ–‡” ™Š‹ Š ™‹ŽŽ†‡ƒŽ™‹–Šƒ••—’–‹‘•™‹–Š”‡‰ƒ”†–‘–Š‡‡–ƒ„‘Ž‹–‡•Ǥ

ʹʹ͹ Šƒ’–‡”ͽ

œ›ƒ–‹  ”‡ƒ –‹‘•’”‘ ‡‡†‹ ƒ ‡”–ƒ‹ †‹”‡ –‹‘ ™Š‡ –Š‡ ȟ  ‹••ƒŽŽ‡” –Šƒœ‡”‘Ǥ ‘”

‡œ›ƒ–‹ ”‡ƒ –‹‘•–Š‡ ƒŽ†ƒ‡”‡Žƒ–‹‘•Š‹’†‡• ”‹„‡•–Š‡”‡Žƒ–‹‘•Š‹’„‡–™‡‡‡“‘ˆ–Š‡ ”‡ƒ –‹‘ƒ†–Š‡”‡ƒ –‹‘Ǧ‡ Šƒ‹•Ǧ•’‡ ‹ˆ‹ ‹‡–‹ ’ƒ”ƒ‡–‡”•Ǥƒ Š”‡ƒ –‹‘‡ Šƒ‹• ™‹–Š–™‘‘”‘”‡•—„•–”ƒ–‡•ƒ†’”‘†— –•ǡƒ›Šƒ˜‡–™‘‘”‘”‡ ƒŽ†ƒ‡”‡Žƒ–‹‘•ȏ͵ȐǤŠ‡   ‘†‡Ž ‹ ‹–• —””‡– ˆ‘” ‹• Žƒ”‰‡Ž› „ƒ•‡† ‘ ‘˜‡‹‡ ‡ ‹‡–‹ • ȏͶȐǤ Š‹• ™ƒ• ƒ ’”ƒ‰ƒ–‹  Š‘‹ ‡ǡ•‹ ‡‹‡–‹ ’ƒ”ƒ‡–‡”•–‘†‡• ”‹„‡‘”‡ ‘’Ž‡š‹‡–‹ ‡ Šƒ‹••ƒ”‡ • ƒ” ‡Ǥ ‘” ‡šƒ’Ž‡ǡ  ‡ƒ ‡– ƒŽǤ ‡š’‡”‹‡–ƒŽŽ› †‡–‡”‹‡† –Šƒ– –Š‡ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡Ǧ ƒ–ƒŽ›œ‡† ”‡ƒ –‹‘ ’”‘ ‡‡†• ƒ ‘”†‹‰ –‘ Dz”†‡”‡†Ǧ‹Ǧ‹dz ‡ Šƒ‹• ȏͷȐǤ ‡‘ˆ–Š‡ ‘””‡•’‘†‹‰ ƒŽ†ƒ‡”‡Žƒ–‹‘•Š‹’•ȏ͵Ȑ‹•

ி஺஽ு ିοீᇱ ௘௡௢௬௟ି஼௢஺ మ బ ܸ௙ ܭ௠ ܭ ݁ ோ் ൌܭ ൌ ή ή ௜  ௘௤ ܸ ௔௖௬௟ି஼௢஺ ܭி஺஽ ௥ ܭ௜ ௠

 –Š‡   ‘†‡Ž ‹– ™ƒ• ƒ••—‡† –Šƒ– –Š‡ ‡“ ‘ˆ –Š‡ †‡Š›†”‘‰‡ƒ•‡ ”‡ƒ –‹‘ ™ƒ• ‹†‡’‡†‡– ‘ˆ Šƒ‹ǦŽ‡‰–ŠǤ ‘”‡‘˜‡”ǡ ‹ –Š‡ ‘˜‡‹‡ ‡ ‹‡–‹ • ܭ݅ܨܣܦܪʹ ƒ† ‰‹™‘ŽŽ‘ˆ ‡Š– ‹ ‰‹–Ž—•‡” ǡ‘†ܣ݋ܥݕ݈Ϋܿܽ݉ܭ †ƒ ʹܪܦܣܨ݉ܭ  –—„ ǡ”— ‘ –‘ ‘† ܣ݋ܥݕ݈Ϋܿܽ݅ܭ ƒŽ†ƒ‡”‡Žƒ–‹‘ǣ

௘௡௢௬௟ି஼௢஺ ி஺஽ுమ ܸ௙ ܭ ܭ ܭ ൌ ή ௠ ή ௠  ௘௤ ܸ ௔௖௬௟ି஼௢஺ ி஺஽ ௥ ܭ௠ ܭ௠ Š‡“—‡•–‹‘ƒ”‹•‡•–‘™Šƒ–‡š–‡––Š‡•‡ƒ••—’–‹‘• Šƒ‰‡–Š‡ ‘ Ž—•‹‘•†”ƒ™ˆ”‘–Š‡

‘†‡ŽǤŠ‡ˆ‹”•–ƒ••—’–‹‘–Šƒ––Š‡‡“‹• Šƒ‹ǦŽ‡‰–Š‹†‡’‡†‡–‹‰Š–„‡“—‡•–‹‘ƒ„Ž‡Ǥ

•‹‰–Š‡‘Ž‹‡ ƒŽ —Žƒ–‘”‘ˆȟ Ͳǯƒ†‡“˜ƒŽ—‡•ȋ‡—‹Ž‹„”ƒ–‘”Ȍȏ͸Ȑ  ƒŽ —Žƒ–‡†–Šƒ––Š‡”ƒ–‹‘

‘ˆ‡“˜ƒŽ—‡•‘ˆͳ͸Ǧƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ–‹‘‘˜‡”ͶǦƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ–‹‘™ƒ•Ž‡•• –ŠƒͲǤͲ͵͵Ǥ•›‡–ǡ‹–‹•‘– Ž‡ƒ”Š‘™™‡ŽŽƒ†™‹–Š™Šƒ–‡””‘”–Š‡’”‡†‹ –‹‘•™‡”‡ƒ†‡Ǥ

Š‡ ‘””‡ –ǡŠ‘™‡˜‡”ǡ™‹–Š–Š‡•ƒ‡˜ƒŽ—‡•ˆ‘”–Š‡ǯ•–Š‡ˆ‘”™ƒ”†ƒš™‘—Ž†„‡Š‹‰Š‡”

”‡Žƒ–‹˜‡–‘–Š‡”‡˜‡”•‡ƒš™Š‡–Š‡ƒ ›ŽǦ‘ Šƒ‹‹••Š‘”–‡”Ǥ‘”‡‘˜‡”ǡ•‘‡†ƒ–ƒ™‡”‡

ƒ˜ƒ‹Žƒ„Ž‡ˆ‘”–Š‡‹ǯ•ȏͷȐǤ ‘”ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡ǡˆ‘”‹•–ƒ ‡ǡ–Š‡›†‹ˆˆ‡”‡†„›ƒˆƒ –‘”

‘ˆ Ͷ ˆ”‘ –Š‡ ‘””‡•’‘†‹‰ ǯ• ȏͷȐǤ Š‡  Œ—•– ”‡’Žƒ ‡ –Š‡ ‹ǯ• „› –Š‡ ǯ•  ™‘—Ž† ‡••‡–‹ƒŽŽ›‡‡†–‘‹–”‘†— ‡ƒ ‘””‡ –‹‘ˆƒ –‘”ȋȽȌ‹–Š‡ ƒŽ†ƒ‡‡“—ƒ–‹‘ǣ

௘௡௢௬௟ି஼௢஺ ி஺஽ுమ ͳ ܸ௙ ܭ ܭ ܭ ൌ ή ή ௠ ή ௠  ௘௤ ߙ ܸ ௔௖௬௟ି஼௢஺ ி஺஽ ௥ ܭ௠ ܭ௠ ௏ ‘”‡ •’‡ ‹ˆ‹ ƒŽŽ›ǡ ™‹–Š‘—– –Š‡ ‘””‡ –‹‘ ˆƒ –‘”ǡ –Š‡ ”ƒ–‹‘ ೑ ‹• ͲǤ͵ͺ ˆ‘” –Š‡ ͶǦƒ ›ŽǦ‘ ௏ೝ †‡Š›†”‘‰‡ƒ–‹‘”‡ƒ –‹‘ǡ‹ ‘–”ƒ•––‘ƒ˜ƒŽ—‡‘ˆͳǤͷʹ™Š‡–Š‡ ‘””‡ –‹‘ˆƒ –‘”‘ˆͶ™ƒ•  Ž—†‡†Ǥ ‘–Š‡”™‘”†•ǡ–Š‡‡•–‹ƒ–‹‘‘ˆ–Š‡”ƒ–‡ƒ–™Š‹ Š–Š‡”‡˜‡”•‡”‡ƒ –‹‘”—•‹‰Š– „‡ˆƒ”Ž‡••‹ ‘’ƒ”‹•‘™‹–Š–Š‡ˆ‘”™ƒ”†”‡ƒ –‹‘–Šƒ—•‡†‹–Š‡‘”‹‰‹ƒŽ ‘†‡ŽǤ‘ ‡˜ƒŽ—ƒ–‡ –Š‹• ’”‘’‡”Ž›ǡ Š‘™‡˜‡”ǡ –Š‡ ™Š‘Ž‡ Ǯ”†‡”‡† ‹ ‹ ‡“—ƒ–‹‘ǯ •Š‘—Ž† „‡ —•‡†Ǥ Š‡

†‡‘‹ƒ–‘”‘ˆ–Š‡Dz”†‡”‡†‹‹dz”ƒ–‡‡“—ƒ–‹‘ƒŽ•‘ ‘–ƒ‹•‹’ƒ”ƒ‡–‡”•ǤŠ‹•‡ƒ•

–Šƒ––Š‡‹αƒ••—’–‹‘ƒˆˆ‡ –•–Š‡‘†‡Ž‡†”‡ƒ –‹‘”ƒ–‡‹‘”‡–Šƒ‘‡™ƒ›ȏ͵ǡ͹ȐǤ  –Š‡ ’—„Ž‹•Š‡†   ‘†‡Ž•ǡ ›–‘•‘Ž‹  ’ƒŽ‹–‘›ŽǦ‘ ™ƒ• –Š‡ •‘Ž‡ •—„•–”ƒ–‡ ƒ† ͳ ‘†‡Ž‡†ƒ•ƒ•–”‹ –Ž›ͳ͸Ǧ•’‡ ‹ˆ‹ ‡œ›‡ȏͺǡͻȐǤŠ‡™ƒ•’‡”ˆ‘”‡†‘–Š‡’—„Ž‹•Š‡† ”ƒ– ‘†‡Ž ‹– •Š‘™‡† –Šƒ– ͳ Šƒ† •—„•–ƒ–‹ƒŽ ˆŽ—š ‘–”‘Ž ‘˜‡” –Š‡ ‘’Ž‡–‡ ”ƒ‰‡ ‘ˆ ›–‘•‘Ž‹ ’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘•ȏͳȐǤŠ‡–Š‡ͳ͸Ǧ•’‡ ‹ˆ‹ ͳ™ƒ•”‡’Žƒ ‡†„›ƒ ͳ™‹–Šƒ„”‘ƒ†Ǧ•’‡ –”— Šƒ‹ǦŽ‡‰–Š•’‡ ‹ˆ‹ ‹–›™Š‹Ž‡‡‡’‹‰ƒŽŽ‘–Š‡”’ƒ”ƒ‡–‡”•–Š‡

ʹʹͺ ‡‡”ƒŽ†‹• —••‹‘

•ƒ‡ǡ–Š‡•–”‘‰ˆŽ—š†‡ Ž‹‡ƒ–Š‹‰Š’ƒŽ‹–‘›ŽǦ‘ ‘ ‡–”ƒ–‹‘•™ƒ•‘–’”‡•‡–ǡ‡˜‡™‹–Š

ƒ–™‹ ‡ƒ•Š‹‰Šͳƒšȋ ‹‰—”‡ͳȌǤ 14 Published rat model

) 12 Rat model w. sens. CPT1 -1 Rat model w. sens. CPT1, 2x Vcpt1 10

8 .gProtein -1 6

4

2 J (μmol.min

0 0 50 100 [Palmitoyl-CoA] CYT ;ʅDͿ Figure 1. Computational rat dynamic model of isolated mFAO with C16-specific CPT1 (solid lines), with C16-C4 sensitive CPT1 (dashed lines) and with C16-C4 sensitive CPT1 at twice larger Vmax (short dashed lines).  Ž–Š‘—‰Š–Š‡”‡ƒ”‡‘ͶǦͳͶƒ ›ŽǦ‘•—„•–”ƒ–‡•’”‡•‡–‹–Š‹••‹—Žƒ–‹‘ǡ–Š‡ͳͶǦͳͶ ƒ ›ŽǦ ƒ”‹–‹‡’”‘†— –• ƒ„‡ˆ‘”‡†‹–Š‡‹–‘ Š‘†”‹ƒŽƒ–”‹šƒ†–”ƒ•’‘”–‡†‘—–„› Ǥ –Š‹•™ƒ›–Š‡•—„•–”ƒ–‡ƒ†’”‘†— –•’‡ ‹ˆ‹ ‹–›‘ˆͳ ƒ’Žƒ›ƒ”‘Ž‡‡˜‡‹–Š‡ 7 ƒ„•‡ ‡‘ˆͶǦͳͶ•—„•–”ƒ–‡•ǤŠ‹•‹†‹ ƒ–‡•–Šƒ––Š‡ Šƒ‹ǦŽ‡‰–Š•’‡ ‹ˆ‹ ‹–›‘ˆͳŠƒ•ƒ •–”‘‰‡ˆˆ‡ –‘–Š‡ ˆŽ—šǡƒ†–Š‡”‡ˆ‘”‡™ƒ””ƒ–•ˆ—”–Š‡”•–—†›Ǥ ‘”‹•–ƒ ‡ǡ‹–Š‡ ‡š–‡†‡†Š—ƒ‹œ‡†‘†‡Žˆ‘”†‡ˆ‹ ‹‡ ›ȋChapter 5Ȍƒ ——Žƒ–‹‰‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ƒ”‡–”ƒ•’‘”–‡†‘—–‘ˆ–Š‡‹–‘ Š‘†”‹ƒƒ•ˆ”‡‡ƒ ‹†•ǤŠ‡–Š‡•‡ƒ”‡”‡ƒ –‹˜ƒ–‡†–‘ ƒ ›ŽǦ‘ ‹ –Š‡ ›–‘•‘Ž ƒ† –”ƒ•‡•–‡”‹ˆ‹‡† –‘ ƒ ›ŽǦ ƒ”‹–‹‡ „› ͳǡ ‹–• Šƒ‹ǦŽ‡‰–Š •’‡ ‹ˆ‹ ‹–›„‡ ‘‡•‹’‘”–ƒ–Ǥ••Š‘™‹Chapter 5ǡƒ•–”‹‹‰†‹ˆˆ‡”‡ ‡™ƒ•‘„•‡”˜‡†‹ –Š‡ ”‘„—•–‡•• ‘ˆ –Š‡   ™Š‡ –Š‡ Šƒ‹ǦŽ‡‰–Š •’‡ ‹ˆ‹ ‹–› ‘ˆ ͳ ™ƒ• ”‡•–”‹ –‡† –‘ ’ƒŽ‹–‘›ŽǦ‘Ǥ  ••—’–‹‘• ™‡”‡ ƒŽ•‘ ƒ†‡ ƒ„‘—– –Š‡ „‘—†ƒ”› ‡–ƒ„‘Ž‹–‡• ‹ –Š‡ ‘†‡ŽǤ ‡‘˜ƒŽǡ ”‡‰‡‡”ƒ–‹‘ ‘” ”‡‘š‹†ƒ–‹‘ ‘ˆ •‡˜‡”ƒŽ ‘ˆ –Š‡•‡ ‘’‘—†• ™‡”‡ ”‡’”‡•‡–‡† „› ƒ Dz•‹dz ”‡ƒ –‹‘ǤŠ‡•‹”‡ƒ –‹‘•™‡”‡‹–”‘†— ‡†•— Š–Šƒ––Š‡›†‘‘–‡š‡”–ƒ›ˆŽ—š ‘–”‘ŽǤ Š‡•‡ƒ”‡–Š‡”‡‘š‹†ƒ–‹‘‘ˆ ǡ ‘˜‡”•‹‘‘ˆƒ ‡–›ŽǦ‘„ƒ ‹–‘‘ ƒ†”‡‘š‹†ƒ–‹‘

‘ˆ  ʹ –Šƒ– ™‡”‡ ‹ Ž—†‡† ƒ• ƒ†Š•‹ǡ ƒ ‡•‹ ƒ† ˆƒ†Š•‹ǡ ”‡•’‡ –‹˜‡Ž›Ǥ ‘™‡˜‡”ǡ ‘–”‘Ž‘ˆ–Š‡ˆŽ—š–Š”‘—‰Š–Š‡ „›‡š–‡”ƒŽ‡ˆˆ‡ –‘”•‹˜‹˜‘‹•‘–ƒ„•‡–ǤŠ‡–Š‡•‡ •‹ ”‡ƒ –‹‘• ™‘—Ž† „‡ ‘†‡ŽŽ‡† ‡š’Ž‹ ‹–Ž›ǡ –Š‡› ‹‰Š– •Šƒ”‡ ‹ –Š‡ –‘–ƒŽ ˆŽ—š ‘–”‘Ž †‡’‡†‹‰‘–Š‡‹”‹‡–‹ ’ƒ”ƒ‡–‡”•ƒ† ƒ–ƒŽ›–‹  ƒ’ƒ ‹–›ǤŠ‹•‹•ƒŽ•‘‹Ž‹‡™‹–Š–Š‡ˆŽ—š ‘–”‘Ž‡š‡”–‡†„› ‹Š—ƒ‘†‡ŽȋChapter 5ȌǤŠ—•ǡ–Š‡”‡•—Ž–•‹Chapter 2•Š‘—Ž† „‡‹–‡”’”‡–‡†ƒ•–Š‡‡œ›‡•ǯ•Šƒ”‡‘ˆ ‘–”‘Žˆ‘”–Š‡’ƒ”–‘ˆ–Š‡–‘–ƒŽˆŽ—š ‘–”‘Ž–Šƒ–”‡•‹†‡• ‹–Š‡ ’ƒ–Š™ƒ›‹–•‡ŽˆǤ  ›ŽǦ ƒ”‹–‹‡•™‡”‡‘–‡š’‘”–‡†ˆ”‘–Š‡ ‡ŽŽ ‹–Š‡‘†‡Ž ˜‡”•‹‘ —•‡† ‹ Chapter 2 ƒ† ƒ”‡ –Š‡”‡ˆ‘”‡ –‡ Š‹ ƒŽŽ› †‡ƒ†Ǧ‡† ‡–ƒ„‘Ž‹–‡•Ǥ Š‹• ”‡’”‡•‡–•–Š‡‡š’‡”‹‡–ƒŽ ‘†‹–‹‘•‘ˆ‹•‘Žƒ–‡†‹–‘ Š‘†”‹ƒ‹ƒ•ƒŽŽ”‡ƒ –‹‘˜‡••‡Žǡ ˆ‘”™Š‹ Š–Š‡‘†‡ŽŠƒ†„‡‡˜ƒŽ‹†ƒ–‡†ȏͳͲȐǤ ˜‹˜‘ǡŠ‘™‡˜‡”ǡƒ ›ŽǦ ƒ”‹–‹‡• ƒ„‡‡š’‘”–‡† ‘—– ‘ˆ –Š‡ Š‡’ƒ–‘ ›–‡Ǥ š’‘”– ‘ˆ ƒ ——Žƒ–‡† ƒ ›Ž ‡“—‹˜ƒŽ‡–• ‘—Ž† ”‡Ž‹‡˜‡ –Š‡ ‘†‡ŽŽ‡† •›•–‡ ˆ”‘ –Š‡ ‘•‡“—‡ ‡• ‘ˆ ‡š ‡••‹˜‡ •—„•–”ƒ–‡ •—’’Ž›Ǥ †‡‡†ǡ ‹–”‘†— –‹‘ ‘ˆ –Š‡ Ǧ͸ ‡š’‘”–‘ˆƒ ›ŽǦ ƒ”‹–‹‡•ǡ„›‡ƒ•‘ˆƒ††‹‰•‹’Ž‡ƒ••ƒ –‹‘‹‡–‹ •ȋ˜αͳͲ ȗȏǦƒ ›ŽǦ

ƒ”‹–‹‡ȐȌǡŽƒ”‰‡Ž›”‡•‘Ž˜‡†–Š‡ˆŽ—šǦ†‡ Ž‹‡’Š‡‘‡‘ȋ ‹‰—”‡ʹȌǤ

ʹʹͻ Šƒ’–‡”ͽ

14 Published rat model

) 12 model w. ac-carn export -1

10

8 .gProtein -1 6

4

2 J (μmol.min

0 0 50 100 [Palmitoyl-CoA] CYT ;ʅDͿ Figure 2. Computational rat dynamic model of isolated mFAO with (dashed lines) and without (solid lines) cytosolic acyl-carnitine export.

‹ƒŽŽ›ǡ‹–Š‡‘†‡Ž–Š‡–ƒ ‹–ƒ••—’–‹‘‹•ƒ†‡–Šƒ–Ǧ ƒ”‹–‹‡ ƒ‡˜‡”‰‡–†‡’Ž‡–‡†Ǥ ‘™‡”’Žƒ•ƒǦ ƒ”‹–‹‡Ž‡˜‡Ž•Šƒ˜‡Š‘™‡˜‡”„‡‡”‡’‘”–‡†‹‹†‹˜‹†—ƒŽ•ȏͳͳȐǤŠ‹• ‹‰Š–„”‹‰ƒ„‘—–Ž‘™‡” ›–‘•‘Ž‹ Ǧ ƒ”‹–‹‡Ž‡˜‡Ž•Ǥ‹ ‡ ƒ”‹–‹‡‹•‹˜‘Ž˜‡†‹„‘–Š–Š‡ ͳƒ†ʹ ƒ–ƒŽ›œ‡†”‡ƒ –‹‘•ǡ Šƒ‰‡•‹ ƒ”‹–‹‡Ž‡˜‡Ž•‹‰Š–„‡Žƒ”‰‡‡‘—‰Š–‘Šƒ˜‡ ƒ‡ˆˆ‡ –‘–Š‡Ǧ ƒ–ƒŽ›œ‡†”‡ƒ –‹‘•Ǥ †‡‡†ǡƒ••Š‘™‹Chapter 5ǡŽ‘™‡”Ǧ ƒ”‹–‹‡ Ž‡˜‡Ž• Ž‹‹– –Š‡   ˆŽ—š •‡˜‡”‡Ž›Ǥ Š—• ‹ ”‡ƒŽ‹–›ǡ ›–‘•‘Ž‹  Ǧ ƒ”‹–‹‡ Ž‡˜‡Ž• ‘—Ž† „‡ ‘•‹†‡”‡†ƒ•’ƒ”–‘ˆƒ”‡‰—Žƒ–‘”›‡ Šƒ‹•–Šƒ–‡‡’•ͳǦ‡†‹ƒ–‡†‹’‘”–‘ˆ•—„•–”ƒ–‡ ‹–‘–Š‡‹–‘ Š‘†”‹ƒƒ†‘š‹†ƒ–‹‘‘ˆ–Š‹••—„•–”ƒ–‡‹„ƒŽƒ ‡ǤŠ‡–Š‡‹–‘ Š‘†”‹ƒŽ ‘š‹†ƒ–‹‘‹•‘—–‘ˆ„ƒŽƒ ‡™‹–Š–Š‡•—„•–”ƒ–‡‹’‘”–ǡ‹–‡”‡†‹ƒ–‡‡–ƒ„‘Ž‹–‡•ƒ ——Žƒ–‡ ƒ† ƒ „‡ ‡š ”‡–‡† ‹ –Š‡ —”‹‡ ƒ• –Š‡‹” ƒ”‹–‹‡ ‘—–‡”’ƒ”–•Ǥ ‘•‡“—‡–Ž›ǡ ƒ”‹–‹‡ ‘‹‡–‹‡•ƒ”‡Ž‘•–ˆ”‘–Š‡ ‡ŽŽǤ ˆ†‡‘˜‘•›–Š‡•‹•‘ˆǦ ƒ”‹–‹‡ ƒ‘–‡‡’—’™‹–Š–Š‡ ƒ‘—– ‘ˆ ƒ”‹–‹‡ǡ Ǧ ƒ”‹–‹‡ Ž‡˜‡Ž• ™‹ŽŽ †‡ ”‡ƒ•‡Ǥ ‘™‡” ›–‘•‘Ž‹  ƒ ›ŽǦ ƒ”‹–‹‡ Ž‡˜‡Ž• •—„•‡“—‡–Ž›‹Š‹„‹––Š‡”ƒ–‡‘ˆ–Š‡ͳǦ ƒ–ƒŽ›œ‡†”‡ƒ –‹‘ƒ†•—„•‡“—‡–Ž›–‡’‘”ƒ”‹Ž› †‡ ”‡ƒ•‡–Š‡•—’’Ž›‘ˆ•—„•–”ƒ–‡–‘–Š‡ ǤŠ‡ ’ƒ–Š™ƒ› ƒ‹–—”‡‡’—’™‹–Š –Š‡ •—„•–”ƒ–‡ •—’’Ž› ‘ˆ ͳǤ Š‡ “—‡•–‹‘ –‘ „‡ ƒ††”‡••‡† ‹• ‹ˆ –Š‹• ‡ Šƒ‹• ‹• “—ƒ–‹–ƒ–‹˜‡Ž› ƒ’ƒ„Ž‡‘ˆ’”‡˜‡–‹‰–Š‡ˆŽ—š†‡ Ž‹‡ǤƒŽ–‡”ƒ–‹˜‡• ‡ƒ”‹‘™‘—Ž†„‡–Šƒ– –Š‡ˆŽ—š†‡ Ž‹‡Šƒ’’‡•ǡ‘–„‡ ƒ—•‡‘ˆ‘ †‡’Ž‡–‹‘„—–„‡ ƒ—•‡ ƒ”‹–‹‡Ž‡˜‡Ž•ƒ”‡•‘ Ž‘™–Šƒ–ͳ‹••‡˜‡”‡Ž›•Ž‘™‡††‘™ǤŠ—•ǡ“—ƒ–‹–ƒ–‹˜‡ƒƒŽ›•‹•‘ˆ–Š‡ ƒ”‹–‹‡‡ ‘‘› ƒ›Šƒ˜‡ Ž‹‹ ƒŽ‹’‘”–ƒ ‡Ǥ   Chapter ͷ ‡š–‡†‡†–Š‡‘†‡Ž™‹–Šƒ•‡–‘ˆ‡œ›ƒ–‹ ”‡ƒ –‹‘™Š‹ Š ƒ’Žƒ›ƒ”‘Ž‡‹ •–ƒ„‹Ž‹œ‹‰–Š‡ Ǥ †‹†‘–›‡–•–—†›–Š‡‡ˆˆ‡ –•‘ˆ’‘•–Ǧ–”ƒ•Žƒ–‹‘ƒŽ‘†‹ˆ‹ ƒ–‹‘•ȋ•Ȍ ‘ ‡œ›‡ ’ƒ”ƒ‡–‡”•Ǥ ‘Ǧ‡œ›ƒ–‹  ƒ ›Žƒ–‹‘ ‘ˆ   ‡œ›‡• ‘ —”• ƒ• –Š‡ ‘•‡“—‡ ‡‘ˆ–Š‡˜‡”›Š‹‰Š ‘ ‡–”ƒ–‹‘•‘ˆƒ ›ŽǦ‘‡•–‡”•‹‹–‘ Š‘†”‹ƒ’ƒ”–‹ —Žƒ”Ž› †—”‹‰ †‡ˆ‹ ‹‡ ›Ǥ‡’‡†‹‰‘–Š‡‡–ƒ„‘Ž‹ †‡ˆ‡ –‡œ›‡•™‡”‡’ƒŽ‹–‘›Žƒ–‡†ȏͳʹȐǡ ƒ ‡–›Žƒ–‡†ǡƒŽ‘›Žƒ–‡†ǡ•— ‹›Žƒ–‡†ǡ‰Ž—–ƒ”›Žƒ–‡†ǡ‘”„—–›”›Žƒ–‡†ȏͳ͵ǡͳͶȐǤŠ‡‡ˆˆ‡ –•‘ ‡œ›‡‹‡–‹ •Šƒ•‘–›‡–„‡‡•–—†‹‡†‹†‡–ƒ‹ŽǤ ‘™‡˜‡”ǡ”‡ ‡–Ž›ƒ ›Žƒ–‹‘†ƒ–ƒ„ƒ•‡• ȏͳʹǡͳ͵ȐŠƒ˜‡„‡‡‰‡‡”ƒ–‡†ƒ†–Š‡‘‰‘‹‰†‡˜‡Ž‘’‡–‘ˆ–‘‘Ž•–‘’”‡†‹ ––Š‡‡ˆˆ‡ –‘ˆ –Š‡•‡•‘‹‡–‹ ’”‘’‡”–‹‡•ȏͳͷȐǡ”‡•—Ž–‡†˜‡”›”‡ ‡–Ž›‹–Š‡‰‡‡”ƒ–‹‘‘ˆƒ†ƒ–ƒ„ƒ•‡ ™‹–Š†‹•‡ƒ•‡Ǧƒ••‘ ‹ƒ–‡†•ȏͳ͸ȐǤŠ‡•‡†‡˜‡Ž‘’‡–•ƒ›‹–Š‡‡ƒ”ˆ—–—”‡‘ˆˆ‡”‘”‡ ‹•‹‰Š–‹–‘–Š‡‡ˆˆ‡ –•‘ˆ•‘‡œ›‡‹‡–‹ ’”‘’‡”–‹‡•‹Š‡ƒŽ–Šƒ††‹•‡ƒ•‡Ǥ Šƒ˜‡ƒŽ•‘ ‘–›‡– ‘•‹†‡”‡†–Š‡”‘Ž‡‘ˆ Šƒ‡Ž‹‰ƒ†•—„‹–‘ Š‘†”‹ƒŽŽ‘ ƒŽ‹œƒ–‹‘‹–Š‡‘†‡ŽǤ• ‹†‹ ƒ–‡†‹Chapter 1 and 2ǡƒŽŽ–Š‡ ‡œ›‡•Šƒ˜‡„‡‡•Š‘™–‘ƒ••‘ ‹ƒ–‡–‘  •—’‡” ‘’Ž‡š‡•ƒ†Ȁ‘”‘–Š‡”‡œ›‡•ȋ‹ Ž—†‹‰‘’Ž‡š ǡ  ʹǡ ‹–”ƒ–‡•›–Šƒ•‡ǡ  ƒ†  Ȍ –Šƒ– ƒ ˆƒ ‹Ž‹–ƒ–‡ –Š‡ ”‡ƒ –‹‘• ‘ˆ –Š‡ ‘””‡•’‘†‹‰   ‡œ›‡•Ǥ ’‘”–ƒ–Ž›ǡ ‹– Šƒ• ƒŽ•‘ „‡‡ ”‡’‘”–‡† –Šƒ– ‡˜‡ ‹ ͷ͹Ȁ͸ ‹ ‡ǡ –Šƒ– Šƒ˜‡ ƒ —•–ƒ„Ž‡

ʹ͵Ͳ ‡‡”ƒŽ†‹• —••‹‘

•—’‡” ‘’Ž‡šƒ••‡„Ž›ˆƒ –‘”ͳȋ ȌǡƒŽŽ•—’‡” ‘’Ž‡š‡•ƒ”‡ƒ–‡ƒ”‘”ƒŽŽ‡˜‡Ž• ƒ†ƒŽ•‘–Š‡”‡•’‹”ƒ•‘‡ȋ‹Ǥ‡ ‘’Ž‡–‡ƒ••‡„Ž›‘ˆ‘’Ž‡š ǡ ǡ ƒ†ȌŠƒ•„‡‡ˆ‘—†–‘ „‡ƒ–‘”ƒŽŽ‡˜‡Ž•‹–Š‡Ž‹˜‡”ǡƒŽ–Š‘—‰Š–Š‡”‡’‘”–‡†”‡•—Ž–•ƒ”‡‘– ‘•‹•–‡–ȏͳ͹Ȃʹ͵ȐǤŠ‹• •—‰‰‡•–• ‘†‹–‹‘Ǧ†‡’‡†‡†•—’‡” ‘’Ž‡šˆ‘”ƒ–‹‘ƒ† –Š‹••Š‘—Ž†„‡‹˜‡•–‹‰ƒ–‡†‹ Ǧ‹ ‡Ǥ  So, what can be done to improve our understanding of the model’s behavior and to improve the accuracy of our dynamic modeling predictions? Š‡ ƒ„‘˜‡ †‹• —••‹‘ •Š‘™‡† –Šƒ– –Š‡ ƒ••—’–‹‘• ƒˆˆ‡ – –Š‡ ’”‡†‹ –‹‘• ƒ†‡ ™‹–Š –Š‡ ‘†‡ŽǤ –‹• Ž‡ƒ”–Šƒ–‡š’‡”‹‡–ƒŽ†ƒ–ƒ‘‡œ›‡’ƒ”ƒ‡–‡”•ǡ™Š‡”‡ƒŽŽ–Š‡ƒ –—ƒŽ‹• ‹ Ž—†‡†ǡ ƒ† ‡–ƒ„‘Ž‹–‡ ‘ ‡–”ƒ–‹‘• ƒ”‡ ‡‡†‡†Ǥ ‡‡”ƒŽŽ›ǡ Ǧ‹ • –‡ Š‘Ž‘‰›ǡ ‹ Ž—†‹‰’”‘–‡‘‹ •ǡ‡–ƒ„‘Ž‘‹ •ƒ†Ž‹’‹†‘‹ •ƒ”‡’”‘‹•‹‰–‡ Š‘Ž‘‰‹‡•™Š‹ Š‹‰Š– Š‡Ž’ ”‡•‘Ž˜‡ •‘‡ ‘ˆ –Š‡ Žƒ  ‘ˆ ‡š’‡”‹‡–ƒŽ †ƒ–ƒǤ ‘” –Š‡ Š—ƒ‹œ‡† ‘†‡Žǡ Š—ƒ ’”‡ ‹•‹‘Ǧ —–Ž‹˜‡”•Ž‹ ‡•ƒ”‡ƒ’”‘‹•‹‰‡š’‡”‹‡–ƒŽ•›•–‡–‘‹‹ Š—ƒŽ‹˜‡”ȏʹͶȐǤ ‘ŽŽƒ„‘”ƒ–‹‘™‹–Š–Š‡Ž‹˜‡”–”ƒ•’Žƒ–ƒ–‹‘–‡ƒ‹–Š‡ ‘ˆˆ‡”•ƒ—‹“—‡‘’’‘”–—‹–›–‘ 7 ‘„–ƒ‹–Š‡•‡†ƒ–ƒǤ –Š‹••‡ –‹‘ ™‹ŽŽˆ—”–Š‡”‡š’Ž‹ ‹–Ž›‰‹˜‡•‘‡Ž‡••‘„˜‹‘—•‡šƒ’Ž‡•‘ˆ ™Šƒ– ƒ„‡†‘‡–‘‹’”‘˜‡‘—”—†‡”•–ƒ†‹‰‘ˆ–Š‡‘†‡Žǯ•„‡Šƒ˜‹‘”ƒ†–Š‡ƒ —”ƒ ›‘ˆ –Š‡‘†‡Ž’”‡†‹ –‹‘•Ǥ ˆ–Š‡‡š’‡”‹‡–ƒŽŽ›Ǧ†‡–‡”‹‡†”‡ƒ –‹‘‡ Šƒ‹•‘ˆƒ”‡ƒ –‹‘‹• Š‘•‡ˆ‘”—•‡‹–Š‡†›ƒ‹ ‘†‡Žǡ–Š‡–Š‡ ‘””‡•’‘†‹‰ ƒŽ†ƒ‡”‡Žƒ–‹‘•Š‹’ ƒ„‡

—•‡†–‘‡•–‹ƒ–‡‹••‹‰’”‘†— –ǯ•ƒ†‹ǯ•Ǥ ‘”‡šƒ’Ž‡ǡˆ‘”–Š‡ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡

”‡ƒ –‹‘ǡ–Š‡ˆȀ””ƒ–‹‘‹•‡š’‡ –‡†–‘„‡͵Ǥͷƒ†͹Ǥͷȏ͹ǡʹͷȐƒ†–Š‡ Šƒ‹ǦŽ‡‰–ŠǦ•’‡ ‹ˆ‹ ‡“

„‡–™‡‡͸ƒ†ͳͺͲȏ͸ǡ͹ǡʹͷȐǤŠ‡–Š‡‹ƒ†˜ƒŽ—‡•ˆ‘” ƒ†  ʹƒ”‡‡’––Š‡•ƒ‡

ƒ•‹–Š‡‘”‹‰‹ƒŽ‘†‡Žȋ˜ƒŽ—‡•‘”‹‰‹ƒ–‹‰ˆ”‘ȏ͹ǡʹ͸ȐȌǡ–Š‡‡‘›ŽǦ‘ǯ• ƒ„‡‡•–‹ƒ–‡†Ǥ – ƒ•—„•‡“—‡–Ž›„‡†‡–‡”‹‡†™Š‡–Š‡”—•‹‰–Š‡ ƒŽ†ƒ‡”‡Žƒ–‹‘•Š‹’‹–Š‹•™ƒ›ˆ‘”–Š‡ ‡œ›‡•‹–Š‡‘†‡ŽŠƒ•ƒ‡ˆˆ‡ –‘–Š‡‘†‡Ž„‡Šƒ˜‹‘”Ǥ ‹ƒŽŽ›ǡ’‡”–ƒ‹‹‰–‘‡š’‡”‹‡–ƒŽ˜ƒŽ‹†ƒ–‹‘‘ˆ†›ƒ‹ ‡–ƒ„‘Ž‹ ‘†‡Ž‹‰ǡ’”‡ˆ‡”‡ ‡ ƒ „‡‰‹˜‡–‘”‡•—Ž–•‹–Š‡ˆ‹”•–‹—–‡•‘”Š‘—”‘ˆ–Š‡•‹—Žƒ–‹‘Ǥ‡›‘†–Š‹•ǡ‡•’‡ ‹ƒŽŽ›‹ ‡–ƒ„‘Ž‹•ǡ–Š‡‘„–ƒ‹‡†•‹—Žƒ–‹‘”‡•—Ž–•‹‰Š–„‡ ‘‡‘”‡ƒ†‘”‡—”‡Ž‹ƒ„Ž‡ǤŠ‹•‹• „‡ ƒ—•‡‹‡–ƒ„‘Ž‹•ǡ’”‘ ‡••‡•Ȃ™‹–Š‡š ‡’–‹‘•Ȃ ƒŠƒ”†Ž›„‡ ‘•‹†‡”‡†–‘ƒ””‹˜‡ƒ–ƒ •–‡ƒ†› •–ƒ–‡Ǥ ‡–ƒ„‘Ž‹  ’ƒ–Š™ƒ›• ƒ”‡ ‹–”‹ ƒ–‡Ž› ‹–‡” ‘‡ –‡†ǡ ‡š– –‘ –Š‡ •™‹ˆ– ƒ† •—„•–ƒ–‹ƒŽ ‡ˆˆ‡ – –Šƒ– ‡˜‹”‘‡–ƒŽ ‘†‹ˆ‹‡”• ‘—Ž† Šƒ˜‡ ‘ –Š‡ ˆ— –‹‘‹‰ ‘ˆ –Š‡•‡ ’ƒ–Š™ƒ›•Ǥ –‹• ‘•‡“—‡–Ž›—†‡”•–ƒ†ƒ„Ž‡–Šƒ–‹‡š’‡”‹‡–•’‡”–ƒ‹‹‰–‘‡–ƒ„‘Ž‹• “—‹ ƒ –‹‘‹•–ƒ‡–‘“—‡ Š”‡ƒ –‹‘‹š–—”‡•‘”ˆ”‡‡œ‡Ǧ Žƒ’‘”‰ƒ•–‘Ž‘ ƒ‡–ƒ„‘Ž‹  •–ƒ–‡—–‹Ž•ƒ’Ž‡ƒƒŽ›•‹•ǤŠ‡‘†‡Ž‹‰ƒ‡–ƒ„‘Ž‹ ’ƒ–Š™ƒ›‹ƒ†›ƒ‹ ‘†‡Žǡ™Š‡”‡ –Š‹• ’ƒ–Š™ƒ› ‹• ‘ˆ–‡ Žƒ”‰‡Ž› ‹•‘Žƒ–‡†ǡ ‹– ƒ –ƒ‡ Š‘—”• „‡ˆ‘”‡ ”‡ƒ Š‹‰ ƒ •–‡ƒ†›Ǧ•–ƒ–‡Ǥ ƒŽ‹†ƒ–‹‘‘ˆ•–‡ƒ†›•–ƒ–‡•‘ˆ•— Šƒ‘†‡Ž‹‰Š–ƒŽ•‘„‡‡••‡–‹ƒŽŽ›—ƒ––ƒ‹ƒ„Ž‡Ǥ—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘• ƒ† –Šƒ– ‘ˆ ‘–Š‡” ‡–ƒ„‘Ž‹–‡• ƒ† ’ƒ”ƒ‡–‡”• –Šƒ– Šƒ˜‡ „‡‡ ˆ‹š‡† ‹ –Š‡ ‘†‡Ž ™‘—Ž† ƒŽ•‘ Šƒ˜‡ –‘ „‡ ƒ‹–ƒ‹‡† ‘•–ƒ– ‹ ˜‹–”‘‘”‹ ˜‹˜‘Ǥ ‰‹‡‡”‹‰ ‘ˆ ‡š’‡”‹‡–ƒŽ•‡–—’•™‘—Ž†„‡”‡“—‹”‡†ǡŽ‹‡—•‹‰ ‘–‹—‘—••–‹””‡†Ǧ–ƒ”‡ƒ –‘”•ȋ •Ȍ ƒ†ƒŽƒ”‰‡ƒ‘—–‘ˆŽ‹˜‹‰ƒ–‡”‹ƒŽˆ‘”ƒŽƒ”‰‡”ƒ‰‡‘ˆ ‘†‹–‹‘•™‘—Ž†„‡‡‡†‡†ˆ‘”–Š‡ ‹–‡†‡† ˜ƒŽ‹†ƒ–‹‘Ǥ  ƒ•‡ ‘ˆ ‹•‘Žƒ–‡† ‹–‘ Š‘†”‹ƒ ˆ‘” ‹•–ƒ ‡ǡ •—„•–ƒ–‹ƒŽ ˆ— –‹‘ƒŽ †‡–‡”‹‘”ƒ–‹‘ ƒ„‡‡š’‡ –‡†„‡ˆ‘”‡–Š‡”‡“—‹”‡†–‹‡’‡”‹‘†‡ ‡••ƒ”›–‘ƒ‡˜ƒŽ‹†ƒ–‹‘ ‘ˆ •–‡ƒ†›Ǧ•–ƒ–‡ ”‡•—Ž–• ’‘••‹„Ž‡Ǥ ‘ ˜ƒŽ‹†ƒ–‡ •’‡ ‹ˆ‹  •–‡ƒ†›Ǧ•–ƒ–‡ ‘†‡Ž „‡Šƒ˜‹‘”•ǡ ƒ Žƒ”‰‡ —„‡”‘ˆ•—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘•‡‡†–‘„‡–‡•–‡†‹ƒŽ‹˜‹‰•›•–‡ǡ‘–‘Ž›–‘‘„–ƒ‹ –Š‡‡ ‡••ƒ”›ƒ‘—–‘ˆ ‘†‹–‹‘•–‘ƒ‡‹–’‘••‹„Ž‡˜ƒŽ‹†ƒ–‡•–‡ƒ†›Ǧ•–ƒ–‡‹•‹Ž‹ ‘”‡•—Ž–•ǡ

ʹ͵ͳ Šƒ’–‡”ͽ

„—–ƒŽ•‘†—‡–‘— ‡”–ƒ‹–‹‡•‹‡š’‡”‹‡–•ƒ†‹•‹Ž‹ ‘ȋƒ•‹†‹ ƒ–‡†ƒ„‘˜‡ȌǤŠ‡”‡ˆ‘”‡ǡ‹– ‘—Ž†„‡‘”‡”‡ƒŽ‹•–‹ –‘•‹—Žƒ–‡ ‘†‹–‹‘•–Šƒ–ƒ”‡’‘••‹„Ž‡‘” Ž‘•‡–‘’‘••‹„Ž‡‹Ž‹˜‹‰ •›•–‡•Ǥ ‡”‡‹ǡ‹–•Š‘—Ž†ƒŽ•‘„‡ ‘•‹†‡”‡†™Šƒ–‡ˆˆ‡ –•‡š–‡”ƒŽ’ƒ”ƒ‡–‡”•Šƒ˜‡‘–Š‡ ”‡ƒ†‘—–•–Šƒ–ƒ”‡‹–‡†‡†–‘˜ƒŽ‹†ƒ–‡–Š‡‹Ǧ•‹Ž‹ ‘”‡•—Ž–•Ǥ

Mouse models as tools to understand symptomatology in MCADD Chapter 3ǡ ‹˜‡•–‹‰ƒ–‡†™Š‹ Š‡–ƒ„‘Ž‹ ’ƒ–Š™ƒ›• ‘–”‹„—–‡–‘–Š‡”‘„—•–‡••‘ˆǦ  ‹ ‡ —†‡” ˆ‡† ƒ† ˆƒ•–‹‰ ‘†‹–‹‘• ‘ ƒ Š‹‰ŠǦ ‘” Ž‘™Ǧˆƒ– †‹‡–Ǥ ‘—” –”ƒ• ”‹’–• ‘ˆ ‡–ƒ„‘Ž‹  ƒ –‹˜‡ ‡œ›‡• ™‡”‡ †‹ˆˆ‡”‡–‹ƒŽŽ› ‡š’”‡••‡†Ǥ ‘–ƒ„Ž›ǡ –™‘ ‘ˆ –Š‡ Ͷ –”ƒ• ”‹’–• ™‡”‡ˆ”‘‰‡‡•‡ ‘†‹‰ˆ‘”‡œ›‡•–Šƒ– ƒ–ƒŽ›œ‡‘ǦƒȋȌȋ ȌǦ‹˜‘Ž˜‹‰”‡ƒ –‹‘•Ǥ  ƒ††‹–‹‘ǡ „› ‡ƒ• ‘ˆ ‰‡‡Ǧ•‡–Ǧ‡”‹ Š‡– ƒƒŽ›•‡•ǡ  •Š‘™‡† –Šƒ– ‰‡‡• ‘†‹‰ ˆ‘” ‡œ›‡• –Šƒ– ƒ–ƒŽ›œ‡ ‘Ǧ ƒ ȋȌȋ ȌǦ‹˜‘Ž˜‹‰ ”‡ƒ –‹‘• ƒ”‡ ƒŽ–‡”‡† ‹ ‡š’”‡••‹‘ „‡–™‡‡  ƒ†  ‹ ‡Ǥ Š‹•‹• ‹ Ž‹‡ ™‹–Š ƒ ‹’‘”–ƒ– ”‘Ž‡ ‘ˆ „‘–Š ‘ˆƒ –‘”• ‹ –Š‡ ‘’—–ƒ–‹‘ƒŽ‘†‡Ž‘ˆ‹•‘Žƒ–‡†”ƒ– ȋChapter 2Ȍƒ†‘ˆ‘‹–Š‡ǦŠ—ƒ ‘’—–ƒ–‹‘ƒŽ ‘†‡Ž ‘–ƒ‹‹‰ –Š‡ ‘”‡ ͳ͸Ǧ•’‡ ‹ˆ‹  ͳ ‡œ›‡ ȋChapter 5ȌǤ —” ‡š’‡”‹‡–ƒŽ”‡•—Ž–ˆ”‘–Š‹••–—†›ƒ”‡”‡Ž‡˜ƒ–‹–Š‡ ‘–‡š–‘ˆ–Š‡DzdzŠ›’‘–Š‡•‹•„› ‹– Š‡ŽŽ ‡– ƒŽǤ ȏʹ͹ǡʹͺȐǤ Š‹• Š›’‘–Š‡•‹• ’”‡†‹ –• –Šƒ– ‘  •‡“—‡•–”ƒ–‹‘ǡ –‘š‹ ‹–› ‘” ”‡†‹•–”‹„—–‹‘ȋŠ‡ ‡Ȍ™‘—Ž†‘ —”‹ƒ›‘ˆ‹’ƒ‹”‡–•‹‘‡•–‡”†‡‰”ƒ†ƒ–‹‘ ’ƒ–Š™ƒ›•ǡ‹ Ž—†‹‰ †‡ˆ‹ ‹‡ ‹‡•Ǥ –Šƒ•„‡‡’”‘’‘•‡†ȏʹ͹ǡʹͺȐ–Šƒ–ƒ›‘ˆ–Š‡–›’‹ ƒŽ •›’–‘• ’”‡•‡–‹‰ ‹ –Š‡•‡ ƒ•‡•ǡ •— Š ƒ• Š›’‘‰Ž› ‡‹ƒǡ ƒ”‡ ƒ ”‡•—Ž– ‘ˆ –Š‹•  ’Š‡‘‡‘Ǥ  ƒŽ•‘ ‘„•‡”˜‡† ƒ †‘™”‡‰—Žƒ–‹‘ ‹ ‰‡‡• ‹˜‘Ž˜‡† ‹ †‡–‘š‹ˆ‹ ƒ–‹‘ ‡ Šƒ‹••Ǥ  Ž‹‡ ™‹–Š –Š‹•ǡ Š‡’ƒ–‘Š‹•–‘Ž‘‰‹ ƒŽ ƒƒŽ›•‹• •Š‘™‡† ƒ –”‡† –‘™ƒ”†• ‹ˆŽƒƒ–‹‘‹–Š‡Ž‹˜‡”‘ˆǦ‹ ‡Ǥ –‡”‡•–‹‰Ž›ǡ‹Ǧ‘—•‡™‹–Š–Š‡‘•– •‡˜‡”‡‡–ƒ„‘Ž‹ ’Š‡‘–›’‡ƒˆ–‡” ‘Ž†‡š’‘•—”‡ȋChapter 4ȌǡƒŽ•‘•‡˜‡”‡•–‡ƒ–‘Š‡’ƒ–‹–‹•™ƒ• ‘„•‡”˜‡†ǤŠ‹•‹‰Š–’‘‹––‘’”‘‰”‡••‹‘‘ˆ–Š‡Ǧ’Š‡‘–›’‡—’‘•™‹– Š‹‰ˆ”‘ ‡š’‘•—”‡ –‘ ƒ ‹Ž† ‘†‹–‹‘ –‘ ƒ ‘†‹–‹‘ ‘ˆ Š‹‰ŠǦ‡‡”‰› †‡ƒ†Ǥ Š‡ ‘„•‡”˜‡† Ž‹˜‡” ‹ˆŽƒƒ–‹‘‹‰Š–„‡ƒ••‘ ‹ƒ–‡†™‹–Šƒ ——Žƒ–‡†ǡƒ•™ƒ•”‡’‘”–‡†‹Š—ƒ –‹••—‡•ȏʹͻǡ͵ͲȐǤ –‹•ƒŽ•‘’‘••‹„Ž‡–Šƒ––Š‡”‘„—•–‡••‹Ǧ‹ ‡‹•’ƒ”–Ž›‡†‹ƒ–‡†„› ƒƒ†ƒ’–ƒ–‹‘‹Š‡’ƒ–‹ ‹–‘ Š‘†”‹ƒŽ ‘–‡–ǤƒŽ›•‹•‘ˆŽ‹’‹†Ǧƒ†’”‘–‡‹‘š‹†ƒ–‹‘ƒ† ’‘–‡–‹ƒŽƒ†ƒ’–ƒ–‹‘„›‹ ”‡ƒ•‡†‹–‘ Š‘†”‹ƒŽ ‘–‡–‹–Š‡ͳ͸ǦŠ‘—”ˆƒ•–‡†‹ ‡ȋChapter 3Ȍƒ”‡—†‡”™ƒ›Ǥ Chapter 4ǡ™Š‡”‡Ǧ‹ ‡™‡”‡ˆƒ•–‡†ƒ†•—„•‡“—‡–Ž› ‘Ž†Ǧ‡š’‘•‡†ǡ ‘„–ƒ‹‡† –Š”‡‡‘–Š‡”‹–‡”‡•–‹‰”‡•—Ž–•Ǥ ‹”•–Ž›ǡ •Š‘™‡†ˆ‘”–Š‡ˆ‹”•––‹‡–Šƒ–Ǧ‹ ‡Šƒ† •‹‰‹ˆ‹ ƒ–Ž›Ž‘™‡”„Ž‘‘†‰Ž— ‘•‡Ž‡˜‡Ž•ƒ––‡”‹ƒ–‹‘–Šƒ‹ ‡Ǥ ƒ††‹–‹‘ǡ–Š‡‘—•‡ ™‹–Š –Š‡ ‘•– •‡˜‡”‡ ’Š‡‘–›’‡ǡ •Š‘™‡† „‘–Š •‡˜‡”‡ •–‡ƒ–‘Š‡’ƒ–‹–‹• ƒ† „Ž‘‘† ‰Ž— ‘•‡ †‡ ”‡ƒ•‡†—”‹‰ ‘Ž†‡š’‘•—”‡ǡƒ•‘’’‘•‡†–‘–Š‡‰‡‡”ƒŽ„Ž‘‘†‰Ž— ‘•‡‹ ”‡ƒ•‡‘„•‡”˜‡†‹ „‘–Šƒ†‹ ‡†—”‹‰ ‘Ž†‡š’‘•—”‡Ǥ‹–Š–Š‹• Šƒ˜‡ ‘ˆ‹”‡†–Šƒ––Š‡Ǧ ‘—•‡ ‘†‡Žǡ —†‡” •’‡ ‹ˆ‹  ‘†‹–‹‘•ǡ ‘—Ž† „‡ ‘•‹†‡”‡† ƒ ‰‘‘† ‘†‡Ž ˆ‘”  †‡ˆ‹ ‹‡ ›‹Š—ƒ•Ǥ‡ ‘†Ž›ǡ ‘„•‡”˜‡†ƒ ‘„‹ƒ–‹‘‘ˆ•‹‰‹ˆ‹ ƒ–Ž›Š‹‰Š‡”„Ž‘‘†ƒ ›ŽǦ ƒ”‹–‹‡Ž‡˜‡Ž•ǡ•‹‹Žƒ”Š‡’ƒ–‹ ƒ ›ŽǦ ƒ”‹–‹‡Ž‡˜‡Ž•ƒ†Š‹‰Š‡”Š‡’ƒ–‹ Ž‡˜‡Ž•‘ˆ‡†‹—Ǧ Šƒ‹ Ž‡˜‡Ž•‹Ǧ ‘’ƒ”‡†–‘‹ ‡ǤŠ‡ˆ‹†‹‰–Šƒ– ‘Ž†Ǧ‡š’‘•—”‡Ǧ‹†— ‡• ƒ ›ŽǦ ƒ”‹–‹‡ ‡š’‘”– ‘—– ‘ˆ –Š‡ Ž‹˜‡” ȏ͵ͳǡ͵ʹȐ –‘‰‡–Š‡” ™‹–Š ‹ ‘”’‘”ƒ–‹‘ ‘ˆ ƒ ——Žƒ–‡† ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘‡•–‡”•‹–‘  • ‹‰Š– Š‡Ž’ ’”‘–‡ – Ǧ ‹ ‡ ƒ‰ƒ‹•– •‡˜‡”‡ Ž‹˜‡”†ƒƒ‰‡ǤŠ‹”†Ž›ǡ ‘„•‡”˜‡†Ž‘™‡”Š‡’ƒ–‹ ƒ‹‘ƒ ‹†Ž‡˜‡Ž•ǡ„—–•‹‹Žƒ”„Ž‘‘†ƒ‹‘ ƒ ‹†Ž‡˜‡Ž•‹Ǧ ‘’ƒ”‡†–‘‹ ‡Ǥ•‹†‹ ƒ–‡†‹Chapter 4ǡ–Š‡Š‡’ƒ–‹ ƒ‹‘

ʹ͵ʹ ‡‡”ƒŽ†‹• —••‹‘

ƒ ‹†•‹‰Š–Šƒ˜‡„‡‡—•‡†ƒ•ƒŽ–‡”ƒ–‹˜‡ˆ—‡Ž„›–Š‡Ž‹˜‡”ǡ„—–‘––‘ƒ‡š–‡––Šƒ–‹–™‘—Ž† ƒˆˆ‡ –„Ž‘‘†Ž‡˜‡Ž•Ǥ‡ƒ›™‘†‡”ǡŠ‘™‡˜‡”ǡ™Š‡–Š‡”‘–Š‡”‘”‰ƒ•–Šƒ–†‡’‡†‘ ǡ ™‘—Ž†ƒŽ•‘—•‡‘”‡ƒ‹‘ƒ ‹†•ƒ†™Š›–Š‹•†‘‡•‘–ƒˆˆ‡ ––Š‡„Ž‘‘† ‘ ‡–”ƒ–‹‘•Ǥ – ™‘—Ž†–Š‡”‡ˆ‘”‡ „‡ ‹–‡”‡•–‹‰ –‘ ‡ƒ•—”‡ ƒ‹‘ ƒ ‹†• ‹ ‘–Š‡” –‹••—‡• ƒ† ’‘••‹„Ž› ƒŽ•‘ •–—†›–Š‡–—”‘˜‡”‘ˆƒ‹‘ƒ ‹†•™‹–Š•–ƒ„Ž‡‹•‘–‘’‡•ȏ͵͵ȐǤŠ‹•‹‰Š–‰‹˜‡ƒ‹†‹ ƒ–‹‘‹ˆ –Š‡•›•–‡‹ ƒ‹‘Ǧƒ ‹†Š‘‡‘•–ƒ•‹•†‡’‡†•‘Š‹††‡”‡‰—Žƒ–‹‘Ǥ  /ŶǀŝƚƌŽ cell models of human MCADD Chapter 6ǡ ’”‡•‡–‡†ƒ•Š‘”–•–—†›ǡ™Š‡”‡–Š‡Ž‡˜‡Ž•‘ˆƒ ›ŽǦ ƒ”‹–‹‡ƒ†•‡Ž‡ ––ƒ”‰‡–‡† ‹–‘ Š‘†”‹ƒŽ ’”‘–‡‹• ‘ˆ –Š‡ ‡‡”‰› ‡–ƒ„‘Ž‹• ™‡”‡ †‡–‡”‹‡† ‹ —Ž–—”‡† •‹ ˆ‹„”‘„Žƒ•–•‘ˆ•›’–‘ƒ–‹ ƒ†ƒ•›–‘ƒ–‹ ‹†‹˜‹†—ƒŽ• ‘–”‘Ž•ǤŠ‡ˆ‹„”‘„Žƒ•–•‘ˆ –Š‡‡Ž†‡•–ǡƒ•›’–‘ƒ–‹ ‹†‹˜‹†—ƒŽ•Š‘™‡††‹ˆˆ‡”‡ ‡•™‹–Š–Š‡‘–Š‡”ƒ† Š‡ƒŽ–Š› ‘–”‘Žˆ‹„”‘„Žƒ•–ǡ•—‰‰‡•–‹‰‡–ƒ„‘Ž‹ ƒ†ƒ’–ƒ–‹‘•–Šƒ–ƒ›”‡Žƒ–‡–‘–Š‡ƒ„•‡ ‡‘ˆ •›’–‘•Ǥ‡š––‘–Š‹•ǡ‘–Š‡” ‡ŽŽ–›’‡• ‘—Ž†„‡—•‡†–‘•–—†›‡ Šƒ‹••—†‡”Ž›‹‰–Š‡ ˜ƒ”‹‘—• ’Š‡‘–›’‡• ‹  ‹†‹˜‹†—ƒŽ•Ǥ ”‘‹•‹‰ ‡ŽŽ –›’‡• ‹ Ž—†‡ ’ƒ–‹‡–Ǧ†‡”‹˜‡† 7 ‹†— ‡†’Ž—”‹’‘–‡–•–‡ȋ‹Ȍ ‡ŽŽ•™Š‹ Š ƒ„‡†‹ˆˆ‡”‡–‹ƒ–‡†‹–‘Š‡’ƒ–‘ ›–‡•ȏ͵ͶȂ͵ͻȐǤ – ™‹ŽŽ„‡ ŠƒŽŽ‡‰‹‰–‘†‡˜‡Ž‘’–Š‹•‹–‘ƒ•›•–‡–Šƒ–•Š‘™•ƒ’ƒ–‹‡–Ǧ•’‡ ‹ˆ‹ Ž‹˜‡”’Š‡‘–›’‡Ǥ ˆ•— ‡••ˆ—ŽǡŠ‘™‡˜‡”ǡ‘–‘Ž›ƒ„‹‘ Š‡‹ ƒŽ Šƒ”ƒ –‡”‹œƒ–‹‘‘ˆ–Š‡ ‘””‡•’‘†‹‰ ‡ŽŽ• ƒ „‡’‡”ˆ‘”‡†ǡ„—–ƒŽ•‘–Š‡› ƒƒŽ•‘„‡—•‡†–‘–‡•––Š‡‡ˆˆ‡ –‘ˆ’‘–‡–‹ƒŽ–”‡ƒ–‡–•–”ƒ–‡‰‹‡• ‘Ž‹˜‡”‡–ƒ„‘Ž‹•Ǥšƒ’Ž‡•‘ˆ’‘–‡–‹ƒŽ–”‡ƒ–‡–•‹ Ž—†‡–Š‡—•‡‘ˆ–Š‡’Š‡›Ž„—–›”ƒ–‡ǡ ƒ Ǧ•’‡ ‹ˆ‹  š‡‘„‹‘–‹  •—„•–ƒ ‡ ƒ† †”—‰ ȋƒ˜‹ –‹Ȍǡ –‘ •–ƒ„‹Ž‹œ‡ –Š‡ ͻͺͷε  —–ƒ–‡†’”‘–‡‹ȏͶͲȂͶʹȐǡ–ƒ”‰‡–‹‰ ‡ƒ–Š‘ ”‘–‡‹ͳͲȋ •’ͳͲȌ–‘‹ ”‡ƒ•‡’”‘–‡‹ ˆ‘Ž†‹‰‡ˆˆ‹ ‹‡ ›ȏͶ͵Ȑǡƒ†•—’’Ž‡‡–ƒ–‹‘‘ˆǦ ƒ”‹–‹‡ȏͶͶȂͶ͹Ȑƒ†‘ ’”‡ —”•‘”• ȏͶͺȂͷʹȐǤ‡ Š‹“—‡•–Šƒ– ƒ„‡—•‡†‘–Š‡‘„–ƒ‹‡†’ƒ–‹‡–ƒ–‡”‹ƒŽ•–‘„‡––‡”—†‡”•–ƒ† –Š‡ Šƒ”ƒ –‡”‹•–‹ •‘ˆ•›’–‘ƒ–‹ ƒ†ƒ•›’–‘ƒ–‹ ‹†‹˜‹†—ƒŽ•ƒ† Šƒ”ƒ –‡”‹•–‹ • ‘ˆ •’‡ ‹ˆ‹   ‹†‹˜‹†—ƒŽ• ‹ Ž—†‡ ™Š‘Ž‡Ǧ‡š‘‡ •‡“—‡ ‹‰ ȋ–‘ ”‡˜‡ƒŽ ‰‡‡–‹  „ƒ ‰”‘—† ƒ† ’‘–‡–‹ƒŽ ‰‡‡–‹  ‘†‹ˆ‹‡”•Ȍǡ  •‡“—‡ ‹‰ ȋ–‘ ”‡˜‡ƒŽ ‰‡‡Ǧ”‡‰—Žƒ–‹‘Ǧ ”‡Žƒ–‡† ƒ†ƒ’–ƒ–‹‘•Ȍǡ –ƒ”‰‡–‡† ƒ† •Š‘–‰— ’”‘–‡‘‹ • ȋ–‘ ”‡˜‡ƒŽ •‘‡ ‘ˆ –Š‡ ˆ— –‹‘ƒŽ ƒ†ƒ’–ƒ–‹‘•Ȍǡ —–ƒ”‰‡–‡† ‡–ƒ„‘Ž‘‹ • ƒ† ˆŽ—š‘‹ • ȋ–Š‡ ”‡˜‡ƒŽ –Š‡ ‘˜‡”ƒŽŽ ‡Ž—Žƒ” ‡–ƒ„‘Ž‹ ’Š‡‘–›’‡ȌǤŠ‡•‡–‡ Š‹“—‡•‹ ‘„‹ƒ–‹‘™‹–Š†‹•‡ƒ•‡Ǧ”‡Ž‡˜ƒ–‡š’‡”‹‡–ƒŽ ‘†‹–‹‘•‹˜‹–”‘‘”‡š˜‹˜‘‹‰Š–”‡˜‡ƒŽ•›’–‘–‘Ž‘‰›Ǧ”‡Žƒ–‡†‰‡‡–‹ ƒ†‡˜‹”‘‡–ƒŽ ‘†‹ˆ‹‡”•ǤŠ‡•ƒŽŽ—„‡”•‘ˆ’ƒ–‹‡–•‹ ‘„‹ƒ–‹‘™‹–Š–Š‡Žƒ”‰‡—„‡”‘ˆ‰‡‡–‹  †‹ˆˆ‡”‡ ‡• –Šƒ– ƒ”‡ —•—ƒŽŽ› ˆ‘—†ǡ ™‹ŽŽ ‡ ‡••‹–ƒ–‡ –‘ ‘„‹‡ –Š‡•‡ Žƒ••‹ ƒŽ Ǯ‘‹ • –‡ Š‘Ž‘‰‹‡•™‹–Š‡ Šƒ‹•–‹ ‘†‡Ž‹‰Ǥ ‡š––‘—•‹‰’ƒ–‹‡–Ǧ†‡”‹˜‡†ƒ–‡”‹ƒŽ•ǡŠ—ƒŠ‡’ƒ–‹  ‡ŽŽŽ‹‡• ‘—Ž†„‡—•‡†–‘—†‡”•–ƒ† ‰‡‡”ƒŽ‡ Šƒ‹••–Šƒ–ƒˆˆ‡ – ™Š‡‹•†‡ˆ‹ ‹‡–Ǥ‘–Š‹•‡†ǡ™‡Šƒ˜‡‰‡‡”ƒ–‡† ƒǦ˜‡”•‹‘‘ˆ–Š‡Š—ƒŠ‡’ƒ–‘ ƒ”‹‘ƒ ‡ŽŽŽ‹‡ ‡’ ʹǡ„› Ȁƒ•ͻ‰‡‡ ‡†‹–‹‰ –‡ Š‹“—‡ ƒ ‘”†‹‰ –‘ ȏͷ͵Ȑ ȋƒ”–‹‡•ǡ ‹‡‹”ƒ ƒ”ƒǡ —‹Œƒǡ ƒ †‡ Ž—‹• —’—„Ž‹•Š‡†ȌǤŠ‡ ‡’ ʹ ‡ŽŽŽ‹‡™ƒ• Š‘•‡‘—–‘ˆˆ‘—”Š‡’ƒ–‘ ƒ” ‹‘ƒ ‡ŽŽŽ‹‡•ǡ•‹ ‡‹– Šƒ† –Š‡ Š‹‰Š‡•– ‘–‡– ‘ˆ ’”‘–‡‹• ‹˜‘Ž˜‡† ‹ –Š‡   ƒ• †‡–‡”‹‡† „› –ƒ”‰‡–‡† ’”‘–‡‘‹ •ȋ ‹‰—”‡͵ȌǤ 

ʹ͵͵ Šƒ’–‡”ͽ

A HepG2 IHH HUH-7 Hep3B CPT1 liver isoform

CPT1 muscle isoform

CPT2

VLCAD

LCAD

MCAD

SCAD

MTPa

MTPb

CROT

MSCHAD

MCKAT 

B

150 * Control 100 100 100 +HoPan

100

50 50 50 *** *** 50

Relative cell number after number cell Relative 72h (% untreated control) 0 0 0 0 HepG2 IHH HUH-7 Hep3B

C  KO KO KO KO KO KO KO MCAD

GAPDH  Figure 3. Generation of an MCAD-KO hepatocyte cell line. Aǣ ‡˜‡Ž• ‘ˆ   ’”‘–‡‹• ‹ –Š‡ Š‡’ƒ–‘ ƒ” ‹‘ƒ ‡ŽŽŽ‹‡ ‡’ ʹǡ–Š‡‹‘”–ƒŽ‹œ‡†Š—ƒŠ‡’ƒ–‘ ›–‡ȋ Ȍƒ†–Š‡Š‡’ƒ–‘ ƒ” ‹‘ƒ ‡ŽŽ Ž‹‡•  Ǧ͹ƒ† ‡’͵ȋαͳȌǡ†‡–‡”‹‡†ƒ ‘”†‹‰–‘ȏͷͶȐǤŠ‡Š‡ƒ–ƒ’™ƒ•‰‡‡”ƒ–‡†™‹–Š–Š‡‘Ž‹‡ –‘‘Ž ‡–ƒ„‘ƒƒŽ›•– ȏͷͷȐǤ Bǣ  ‡Žƒ–‹˜‡ ‡ŽŽ —„‡” ƒˆ–‡” ͹ʹŠ –”‡ƒ–‡– ™‹–Š ‘ƒ ˜‹–ƒ‹ ͷǦŽƒ ‹‰ ‡†‹—ǤŠ‡ ‡ŽŽ•™‡”‡ —Ž–—”‡†‹–Š‡š‹‰‡ ‡•›•–‡ȋ‘ Š‡’’Ž‹‡† ‹‡ ‡ǡ‡œ„‡”‰ǡ ‡”ƒ›Ǣ ‘ŽŽƒ„‘”ƒ–‹‘™‹–Š”‘ˆǤǤ‘Ž‰ƒȌƒ† ‡ŽŽ‹†‡š˜ƒŽ—‡•™‡”‡ ƒŽ —Žƒ–‡†ƒ ‘”†‹‰–‘ȏͷ͸ȐǤŠ‡ ‡ŽŽ‹†‡š ˜ƒŽ—‡•™‡”‡ ‘•‹†‡”‡†–‘„‡’”‘’‘”–‹‘ƒŽ–‘–Š‡ ‡ŽŽ—„‡”ƒ†—•‡†–Š‹•–‘‡•–‹ƒ–‡–Š‡’‡” ‡–ƒ‰‡‘ˆ ”‡ƒ‹‹‰ ‡ŽŽ•ƒˆ–‡” ‘ƒ–”‡ƒ–‡–Ǥ ‘ƒ™ƒ•ƒ‹†‰‹ˆ–ˆ”‘”‘ˆǤǤ‹„‘Ǥ‡•—Ž–•ƒ”‡•Š‘™ƒ•‹ „ƒ”’Ž‘–•ƒ•‡ƒάǡα͵Ǧ͸’‡”‰”‘—’ǡȗ’δͲǤͲͷǡȗȗȗ’δͲǤͲͲͲͳ—•‹‰—’ƒ‹”‡†–Ǧ–‡•–ǤC: ‡•–‡”Ž‘– ƒƒŽ›•‹•’”‘–‡‹Ž‡˜‡Ž•‹ƒ•‡Ž‡ –‹‘‘ˆ ‡’ ʹ Ž‘‡•Ǥ Ž› ‡”ƒŽ†‡Š›†‡͵Ǧ’Š‘•’Šƒ–‡†‡Š›†”‘‰‡ƒ•‡ ȋ  Ȍ™ƒ•—•‡†ƒ•–Š‡Š‘—•‡Š‘Ž†‰‡‡Ǥ‡’‹ –‡†Š‡”‡‹•–Š‡„Ž‘– ‘–ƒ‹‹‰–Š‡ Š‘•‡ ‡’ ʹ ȋŽƒ‡ͷȌǤ  Š‡ ™‹Ž†Ǧ–›’‡ ‡’ ʹ ‡ŽŽ Ž‹‡ ™ƒ• ƒŽ•‘ •‡•‹–‹˜‡ –‘ –Š‡ ‘  •›–Š‡•‹• ‹Š‹„‹–‘” ‘’ƒ–‡ƒ–‡ȋ ‘ƒȌ‹‡†‹—™‹–Š‘—––Š‡‘ ’”‡ —”•‘”‹–ƒ‹ͷȋ ‹‰—”‡͵ȌǤŠ‹• ™‹ŽŽ„‡”‡Ž‡˜ƒ––‘•–—†›–Š‡‹–‡”’Žƒ›„‡–™‡‡‘ ƒ˜ƒ‹Žƒ„‹Ž‹–›ƒ† Ǥ ’”‘ ‡‡†‡† ™‹–Š–Š‡Ǧ ‡’ ʹ Ž‘‡™‹–Š–Š‡ Ž‡ƒ‡•–•‡“—‡ ‡ȋŽƒ‡ͷ‹ ‹‰—”‡͵ȌǤŠ‡ ‡’ ʹ ‡ŽŽ•ƒ”‡ —Ž–—”‡†‹ʹΨŠ—ƒ•‡”—ǡ–Š‡›”‡•‡„Ž‡•’”‹ƒ”›Š—ƒŠ‡’ƒ–‘ ›–‡ ‘”‡ –Šƒ ™Š‡ –Š‡› ƒ”‡ —Ž–—”‡† ‹  ͳͲΨ ˆ‡–ƒŽ ƒŽˆ •‡”— ȏͷ͹ȂͷͻȐǤ †‡‡†ǡ —’‘ ˜‹•—ƒŽ ‹•’‡ –‹‘  ‘•‹•–‡–Ž›‘–‹ ‡†–Šƒ––Š‡‘”’Š‘Ž‘‰›‘ˆ„‘–Š–Š‡ƒ†Ǧ ‡’ ʹ

ʹ͵Ͷ ‡‡”ƒŽ†‹• —••‹‘

‡ŽŽ• •–ƒ”–‡† ”‡•‡„Ž‹‰ –Šƒ– ‘ˆ ’”‹ƒ”› Š‡’ƒ–‘ ›–‡• ‹ –Š‡ ʹΨ Š—ƒ •‡”—Ǥ ‘”‡‘˜‡”ǡ Ǧ —Ž–—”‡••Š‘™‡†Ž‡••†‹˜‹†‹‰ ‡ŽŽ• ‘’ƒ”‡†–‘–Š‡ ‡ŽŽ•ȋ†ƒ–ƒ‘–•Š‘™ȌǤŠ‡ ‡š– •–‡’ ™‹ŽŽ  „‡ –‘ ƒƒŽ›•‡ „‹‘ Š‡‹ ƒŽŽ› –‘ ™Šƒ– ‡š–‡– –Š‡•‡ ‡ŽŽ• ‡šŠ‹„‹– ƒ Ž‹˜‡”ǦŽ‹‡ ‡–ƒ„‘Ž‹  ’Š‡‘–›’‡ ȋ ˆǤȏ͸ͲȐȌǤ Š‡ –Š‡ Ǧ ‡’ ʹ ‡ŽŽ Ž‹‡ ™‹ŽŽ ’”‘˜‹†‡ ƒ ˜‡”› ’”ƒ –‹ ƒŽ‹˜‹–”‘•›•–‡–‘‹˜‡•–‹‰ƒ–‡–Š‡‹–‡”’Žƒ›‘ˆ–Š‡ ’ƒ–Š™ƒ›™‹–Š•—””‘—†‹‰ ’ƒ–Š™ƒ›• ƒ† –‘ ˜ƒŽ‹†ƒ–‡ ‘’—–‡” •‹—Žƒ–‹‘•Ǥ šƒ’Ž‡• ‹ Ž—†‡ –Š‡ ‡ˆˆ‡ – ‘ˆ ‘  †‡’Ž‡–‹‘ƒ†Ž‘™Ǧ ƒ”‹–‹‡‘ ˆŽ—šǡ‰Ž— ‘•‡’”‘†— –‹‘ǡ‡–‘‡„‘†›’”‘†— –‹‘ƒ† ‡ŽŽ˜‹ƒ„‹Ž‹–›‹–Š‡Ǧ ‘’ƒ”‡†–‘–Š‡ ‡’ ʹ ‡ŽŽ•Ǥ  Conclusion Š‹• –Š‡•‹• ”‡’”‡•‡–• ƒ ˆ‹”•– ‡š’Ž‘”ƒ–‹‘ ‹–‘  †‡ˆ‹ ‹‡ › —•‹‰ ƒ ‘„‹ƒ–‹‘ ‘ˆ ›•–‡• ‡†‹ ‹‡ –‘‘Ž•Ǥ Š‡ ™‘” ’”‘˜‹†‡† ‹•‹‰Š– ‹–‘ –Š‡ Ž‹‹–ƒ–‹‘•ǡ ƒ†˜ƒ–ƒ‰‡• ƒ† †‹•ƒ†˜ƒ–ƒ‰‡• ‘ˆ ‘’—–ƒ–‹‘ƒŽ ‘†‡Ž‹‰ǡ ‹ ˜‹˜‘ ‡š’‡”‹‡–• ‹ ‹ ‡ ƒ† ‹ ˜‹–”‘ ‡š’‡”‹‡–•‹Š—ƒƒ–‡”‹ƒŽǤ‡‘ˆ›‡›‘˜‡”ƒ” Š‹‰ˆ‹†‹‰•ǡ„‘–Š‹•‹Ž‹ ‘ƒ†‹˜‹˜‘ǡ ‹•–Š‡”‘Ž‡‘ˆ‘‡œ›‡ƒ†ȋȌȋ Ȍ‹ ˆ— –‹‘ƒ††‡ˆ‹ ‹‡ ›ǤŠ‡Š—ƒ 7 ˜‡”•‹‘‘ˆ–Š‡ ‘’—–ƒ–‹‘ƒŽ‘†‡ŽȋChapter 5Ȍ‘ˆˆ‡”•–Š‡‘’’‘”–—‹–›–‘‹–‡‰”ƒ–‡Š—ƒ †ƒ–ƒǡ‡Ǥ‰Ǥˆ”‘‹˜‹–”‘‡š’‡”‹‡–•™‹–Š’ƒ–‹‡–Ǧ•’‡ ‹ˆ‹  ‡ŽŽ•‘”–Š‡‡™Ǧ ‡’ ʹ ‡ŽŽŽ‹‡ǤŠ—•ǡ Š‘’‡–Šƒ––Š‡ ‘’”‡Š‡•‹˜‡‰”‘—†™‘”ƒ††‹• —••‹‘•Žƒ‹†‘—–‹ –Š‹• –Š‡•‹• •Š‘™ ’”‘‹•‡ ˆ‘” ˆ—–—”‡ ˆ‘ŽŽ‘™Ǧ—’ •–—†‹‡• –‘ —†‡”•–ƒ† –Š‡ ‡ Šƒ‹•• —†‡”Ž›‹‰ –Š‡ Š‡–‡”‘‰‡‡‘—• ’Š‡‘–›’‡• ‹  †‡ˆ‹ ‹‡ ›Ǥ Š‹• •Š‘—Ž† ‘’‡ —’ ‡™ ƒ˜‡—‡• ˆ‘” ‘”‡ ’‡”•‘ƒŽ‹œ‡† †‹ƒ‰‘•‡•ǡ ’”‘‰‘•‡• ƒ† –”‡ƒ–‡– •–”ƒ–‡‰‹‡• ˆ‘”  †‡ˆ‹ ‹‡ ›Ǥ  References ͳǤ ƒ”–‹‡•Ǧ ǡ˜ƒ—‡ǡ‡‹Œ‰‘—†Ǧ ǡƒ‡”ǤŠ‡’”‘‹• —‘—•‡œ›‡‡†‹—Ǧ Šƒ‹ ͵Ǧ‡–‘Ǧƒ ›ŽǦ‘ –Š‹‘Žƒ•‡ –”‹‰‰‡”• ƒ ˜‹ ‹‘—• › Ž‡ ‹ ˆƒ––›Ǧƒ ‹† „‡–ƒǦ‘š‹†ƒ–‹‘Ǥ  ‘’—–‹‘ŽǤʹͲͳ͹Ǣͳ͵ȋͶȌǣͳȂʹʹǤ ʹǤ ƒ—”‘ Ǥ‘””‡ –‹‘–‘Dz‘–”‘Žƒ†”‡‰—Žƒ–‹‘‘ˆ’ƒ–Š™ƒ›•˜‹ƒ‡‰ƒ–‹˜‡ˆ‡‡†„ƒ dzǤ ‘  –‡”ˆƒ ‡ǤʹͲͳ͹ǢͳͶȋͳʹͻȌǤ ͵Ǥ Ǥ Š‡ ‹‡–‹ • ‘ˆ ‡œ›‡Ǧ ƒ–ƒŽ›œ‡† ”‡ƒ –‹‘• ™‹–Š –™‘ ‘” ‘”‡ •—„•–”ƒ–‡•‘” ’”‘†— –•Ǥ Ǥ‘‡ Žƒ–—”‡ƒ†”ƒ–‡‡“—ƒ–‹‘•Ǥ‹‘ Š‹‹‘’Š›• –ƒǤͳͻ͸͵ ƒͺǢ͸͹ȋʹȌǣͳͲͶȂ͵͹Ǥ ͶǤ ‹‡„‡”‡‹•–‡” ǡ Ž‹’’ Ǥ ”‹‰‹‰ ‡–ƒ„‘Ž‹  ‡–™‘”• –‘ Ž‹ˆ‡ǣ ‘˜‡‹‡ ‡ ”ƒ–‡ Žƒ™ ƒ† –Š‡”‘†›ƒ‹  ‘•–”ƒ‹–•ǤŠ‡‘”‹‘Ž‡†‘†‡ŽǤʹͲͲ͸Ǣ͵Ǥ ͷǤ  ‡ƒǡ ”‡”ƒ ǡ‹‡Ž‡Ǥ ‡‡”ƒŽƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡ˆ”‘’‹‰Ž‹˜‡”Ǥ‹‡–‹  ƒ†„‹†‹‰•–—†‹‡•Ǥ ‹‘ŽŠ‡Ǥͳͻ͹ͻ’”ʹͷǢʹͷͶȋͺȌǣʹ͹͵ͲȂͷǤ ͸Ǥ ŽƒŠ‘Žœ ǡ ‘‘” ǡ ƒ”Ǧ˜‡ ǡ ‹Ž‘ Ǥ —‹Ž‹„”ƒ–‘” Ǧ Š‡ „‹‘ Š‡‹ ƒŽ –Š‡”‘†›ƒ‹ • ƒŽ —Žƒ–‘”Ǥ— Ž‡‹  ‹†•‡•ǤʹͲͳʹǢͶͲȋͳȌǣ͹͹ͲȂͷǤ ͹Ǥ —‰‹ Ǥ ›ƒ‹  ‹‡–‹  ‘†‡Ž‹‰ ‘ˆ ‹–‘ Š‘†”‹ƒŽ ‡”‰› ‡–ƒ„‘Ž‹•Ǥ Ǧ‡ŽŽ ›•– ƒ•‹  ‘ ‡’–•’’ŽǤʹͲͳ͵ǢͳͲͷȂͶʹǤ ͺǤ ˜ƒ—‡ǡ‹‘• ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ†‡‡•–‡ ǡ‘—™ǡ ‘—–‡ǡ ”‘‡ ǡ”ƒ„ǡ‡‹Œ‰‘—† ǡƒ‡”Ǥ‹‘ Š‡‹ ƒŽ‘’‡–‹–‹‘ƒ‡• ƒ––›Ǧ ‹†ȾǦš‹†ƒ–‹‘ —Ž‡”ƒ„Ž‡–‘—„•–”ƒ–‡˜‡”Ž‘ƒ†Ǥ‘‘’—–‹‘ŽǤʹͲͳ͵ǢͻȋͺȌǣʹȂͻǤ ͻǤ ˜ƒ—‡ǡ‘Ž‡”Ǧ‘—™ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ‘Ž–‡”• ǡ˜ƒ‹Œ– ǡƒ”–‹‡• ǡ ‹‡œ‡Ǧ‘‹‰ǡ ‡‹‡”ǡ‡”‡–‹‡” ǡ ”‘‡ǡ‡‹Œ‰‘—† ǡ‡”• ǡƒ‡”Ǥ ‹˜‹‰‘–Š‡‡†‰‡ǣ—„•–”ƒ–‡ ‘’‡–‹–‹‘‡š’Žƒ‹•Ž‘••‘ˆ”‘„—•–‡••‹‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧ ƒ ‹†‘š‹†ƒ–‹‘†‹•‘”†‡”•Ǥ‹‘ŽǤʹͲͳ͸ǢͳͶȋͳȌǣͳȂͳͷǤ ͳͲǤ ˜ƒ—‡ǡ‹‘• ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ†‡‡•–‡ ǡ‘—™ǡ ‘—–‡ǡ ”‘‡ ǡ”ƒ„ǡ‡‹Œ‰‘—† ǡƒ‡”Ǥ‹‘ Š‡‹ ƒŽ‘’‡–‹–‹‘ƒ‡• ƒ––›Ǧ ‹†ȾǦš‹†ƒ–‹‘ —Ž‡”ƒ„Ž‡–‘—„•–”ƒ–‡˜‡”Ž‘ƒ†Ǥ‘‘’—–‹‘ŽǤʹͲͳ͵ǢͻȋͺȌǣʹȂͻǤ ͳͳǤ –ƒŽ‡›ǡ ƒŽ‡ǡ‘ƒ–‡•ǡ ƒŽŽǡ‘”‡›ǡƒ‰ǡ‡ŽŽ‡› ǡ ‘œƒŽ‡•ǡ‹ŽŽ‹ƒ•‘

ʹ͵ͷ Šƒ’–‡”ͽ

ǡ ƒ‡” Ǥ ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ †‡ˆ‹ ‹‡ › ‹ Š‹Ž†”‡ ™‹–Š ‘Ǧ‡–‘–‹  Š›’‘‰Ž› ‡‹ƒƒ†Ž‘™ ƒ”‹–‹‡Ž‡˜‡Ž•Ǥ‡†‹ƒ–”‡•Ǥͳͻͺ͵Ǣͳ͹ȋͳͳȌǣͺ͹͹ȂͺͶǤ ͳʹǤ Žƒ ǡƒ˜‹† ǡ„”ƒ‹ǡ‹‰Ž‹‘œœ‹ǡ”ƒ† ǡò”‰‹ ǡ˜ƒ†‡” ‘‘– Ǥ™‹••ƒŽǣ”‘–‡‹ ƒŽ‹–‘›Žƒ–‹‘†ƒ–ƒ„ƒ•‡Ǥ ͳͲͲͲ‡•‡ƒ” ŠǤʹͲͳͷǢͶȋͲȌǣʹ͸ͳǤ ͳ͵Ǥ — ǡŠ‘— ǡ‹ǡ‡‰ǡŠƒ‰ǡ—‡Ǥǣ—’†ƒ–‡††ƒ–ƒ”‡•‘—” ‡‘ˆ’”‘–‡‹Ž›•‹‡ ‘†‹ˆ‹ ƒ–‹‘•Ǥ  ‡‡– ‡‘‹ •ǤʹͲͳ͹ƒ›ʹͲǢͶͶȋͷȌǣʹͶ͵ȂͷͲǤ ͳͶǤ ‘—‰‘˜‹ƒǡ–‡”‹‡ ǡƒ†‡”• ǡ ‘—–‡ǡ†‡‘‡” Ǥ„‡””ƒ–’”‘–‡‹ƒ ›Žƒ–‹‘‹• ƒ ‘‘ ‘„•‡”˜ƒ–‹‘ ‹ ‹„‘” ‡””‘”• ‘ˆ ƒ ›ŽǦ‘ ‡–ƒ„‘Ž‹•Ǥ  Š‡”‹– ‡–ƒ„ ‹•Ǥ ʹͲͳͶǢ͵͹ȋͷȌǣ͹ͲͻȂͳͶǤ ͳͷǤ —†ƒ‰‘––‘ǡƒŽ‡”ƒ”‘Ǥ”‘–‡‹’‘•–Ǧ–”ƒ•Žƒ–‹‘ƒŽ‘†‹ˆ‹ ƒ–‹‘•ǣ •‹Ž‹ ‘’”‡†‹ –‹‘–‘‘Ž• ƒ†‘Ž‡ —Žƒ”‘†‡Ž‹‰Ǥ‘’—––”— –‹‘–‡ Š‘Ž ǤʹͲͳ͹Ǣͳͷǣ͵Ͳ͹ȂͳͻǤ ͳ͸Ǥ — ǡ ƒ‰ ǡ‹ ǡ ‡‰ ǡ ‡‰ ǡ —‹ ǡ —‡ Ǥǣ  ƒ–ƒ„ƒ•‡ ‘ˆ —ƒ ‹•‡ƒ•‡Ǧ ƒ••‘ ‹ƒ–‡† ‘•–Ǧ–”ƒ•Žƒ–‹‘ƒŽ ‘†‹ˆ‹ ƒ–‹‘•Ǥ ‡‘‹ • ”‘–‡‘‹ • ‹‘‹ˆ‘”ƒ–‹ •Ǥ ʹͲͳͺǢͳ͸ȋͶȌǣʹͶͶȂͷͳǤ ͳ͹Ǥ ƒ’—‡–‡Ǧ”— ǡ ‘”‡‘Ǧ‘•Š—‡”–‘• ǡ  ‹Ǧ‡”‡œ ǡ ƒ–‘””‡Ǧ‡ŽŽ‹ ‡” ǡ ‘Žƒ• ǡ ƒŽ•ƒ ǡ ‡”ƒŽ‡•ǦŽ‡‡–‡ǡ—‹”‘•ǡƒŽ˜‘ǡ‘†”‹‰—‡œǦ ‡”ƒ†‡œǡƒ˜ƒ•ǡ”—œǡƒ””ƒ ‡†‘ ǡ ‘’‡œǦ–‹ ǡ ‡”‡œǦƒ”–‘• ǡ ‡”ƒ†‡œǦ‹Ž˜ƒ ǡ ‡”ƒ†‡œǦ‹œƒ””ƒ ǡ ”‹“—‡œ Ǥ —’‡” ‘’Ž‡š ••‡„Ž› ‡–‡”‹‡• Ž‡ –”‘ Ž—š ‹ –Š‡ ‹–‘ Š‘†”‹ƒŽ Ž‡ –”‘ ”ƒ•’‘”– Šƒ‹Ǥ ‹‡ ‡ȋͺͲǦȌǤʹͲͳ͵Ǣ͵ͶͲȋ͸ͳͶͲȌǣͳͷ͸͹Ȃ͹ͲǤ ͳͺǤ Šƒǡƒ‰ǡ—™‡”š ǤƒŽ›•‹•‘ˆ‹–‘ Š‘†”‹ƒŽ‡•’‹”ƒ–‘”›Šƒ‹—’‡” ‘’Ž‡š‡••‹‰ Ž—‡ ƒ–‹˜‡ ‘Ž›ƒ ”›Žƒ‹†‡ ‡Ž Ž‡ –”‘’Š‘”‡•‹• ȋǦ ȌǤ —”” ”‘–‘  ‘—•‡ ‹‘ŽǤ ʹͲͳ͸Ǣ͸ȋͳȌǣͳȂͳͶǤ ͳͻǤ ‘‰Ž‹ƒ–‹ ǡ ƒŽ˜‘ ǡ ‘—”‡‹”‘ ǡ —ƒ”ƒ• ǡ ‹‡–‘Ǧ”‡ŽŽƒ‘ ǡ ƒ” ‹ƒǦ‘›ƒ–‘• ǡ œ—”†‹ƒ ǡ ‡” ƒ†‡”ǡžœ“—‡œ ǡ”‹“—‡œ Ǥ‡ Šƒ‹•‘ˆ•—’‡”Ǧƒ••‡„Ž›‘ˆ”‡•’‹”ƒ–‘”› ‘’Ž‡š‡•  ƒ† Ǥƒ–—”‡ǤʹͲͳ͸Ǣͷ͵ͻȋ͹͸͵ͲȌǣͷ͹ͻȂͺʹǤ ʹͲǤ ‘—”‹‡” ǡ ƒ–‹  ǡ —œœ‡‡–‡ ǡ ƒ”••‘ Ǧ ǡ ‹Ž‡‘˜‹  Ǥ Š‡ ”‡•’‹”ƒ–‘”› Šƒ‹ •—’‡” ‘’Ž‡š ‘”‰ƒ‹œƒ–‹‘ ‹• ‹†‡’‡†‡– ‘ˆ ͹ƒʹŽ ‹•‘ˆ‘”•Ǥ ‡ŽŽ ‡–ƒ„Ǥ ʹͲͳͶ ‡  ʹǢʹͲȋ͸ȌǣͳͲ͸ͻȂ͹ͷǤ ʹͳǤ ‹ŽŽ‹ƒ• ǡ—ǡ Šƒǡ—„—‹•ǡŽƒ––ƒǡ”‰ƒǡ ‘—–‡ǡƒ”‹—–ƒǡ‘Ž•‹ ǡ ƒ„‘‹ ǡ ‡„‡”•‘Ž† ǡ —™‡”š Ǥ ›•–‡• ’”‘–‡‘‹ • ‘ˆ Ž‹˜‡” ‹–‘ Š‘†”‹ƒ ˆ— –‹‘Ǥ  ‹‡ ‡ǤʹͲͳ͸ —ͳͲǢ͵ͷʹȋ͸ʹͻͳȌǣƒƒ†ͲͳͺͻǤ ʹʹǤ Š‹„ƒ ǡ ‡†ƒ ǡ ‘”‹‡Ǧ ‘—‡ ǡ ƒƒ›ƒƒ ǡ ƒƒƒ ǡ ‘—‡ Ǥ ‡ˆ‹ ‹‡ › ‘ˆ ͹ǡ ƒ ‹–‘ Š‘†”‹ƒŽ•—’‡” ‘’Ž‡šƒ••‡„Ž›’”‘‘–‹‰ˆƒ –‘”ǡŽ‘™‡”•„Ž‘‘†‰Ž— ‘•‡Ž‡˜‡Ž‹‹ ‡Ǥ ‹ ‡’ǤʹͲͳ͹Ǣ͹ȋͳȌǣ͹͸Ͳ͸Ǥ ʹ͵Ǥ ‹Ž‡‘˜‹ ǡŽƒœƒ ǡƒ”••‘ ǡ ‹”•– ǤŠ‡‹‰ƒ‘ˆ–Š‡‡•’‹”ƒ–‘”›Šƒ‹—’‡” ‘’Ž‡šǤ ‡ŽŽ‡–ƒ„ǤʹͲͳ͹ǢʹͷȋͶȌǣ͹͸ͷȂ͹͸Ǥ ʹͶǤ ƒ–ƒ—–‹ǡŽ‹‰ƒǡ‡‹‰• ǡ ”‘‘–Š—‹• ǤƒŽ‹†ƒ–‹‘‘ˆ’”‡ ‹•‹‘Ǧ —–Ž‹˜‡”•Ž‹ ‡•–‘ •–—†›†”—‰Ǧ‹†— ‡† Š‘Ž‡•–ƒ•‹•ǣƒ–”ƒ• ”‹’–‘‹ •ƒ’’”‘ƒ ŠǤ” Š‘š‹ ‘ŽǤʹͲͳ͹Ǣͻͳȋ͵ȌǣͳͶͲͳȂ ͳʹǤ ʹͷǤ ‘Šǡ ƒ”ˆ‹‡ŽǤ‘’—–‡”•‹—Žƒ–‹‘‘ˆ‡–ƒ„‘Ž‹•‹’ƒŽ‹–ƒ–‡Ǧ’‡”ˆ—•‡†”ƒ–Š‡ƒ”–Ǥ Ǥ ƒŽ‹–ƒ–‡‘š‹†ƒ–‹‘Ǥ‹‘‡†‰Ǥͳͻͺ͵ǢͳͳȋͷȌǣ͵͸ͳȂͺͶǤ ʹ͸Ǥ —‰‹ ǡ ‘‹–ƒ Ǥ  ‰‡‡”ƒŽ ‘’—–ƒ–‹‘ƒŽ ‘†‡Ž ‘ˆ ‹–‘ Š‘†”‹ƒŽ ‡–ƒ„‘Ž‹• ‹ ƒ ™Š‘Ž‡ ‘”‰ƒ‡ŽŽ‡• ƒŽ‡Ǥ‹‘‹ˆ‘”ƒ–‹ •ǤʹͲͲͶǢʹͲȋͳͳȌǣͳ͹ͻͷȂ͸Ǥ ʹ͹Ǥ ‹– Š‡ŽŽ ǡ ƒ—–Š‹‡”ǡ‡•‹’Ž‡ǡƒ‰ǡƒ‡”ǡ—”‡•Š‹ Ǥ ‡”‡†‹–ƒ”›ƒ†ƒ “—‹”‡† †‹•‡ƒ•‡•‘ˆƒ ›ŽǦ ‘‡œ›‡‡–ƒ„‘Ž‹•Ǥ‘Ž ‡‡–‡–ƒ„ǤʹͲͲͺǢͻͶȋͳȌǣͶȂͳͷǤ ʹͺǤ ƒ‰ ǡŠƒ‘ǡƒ‰ǡƒ‰ǡ‹– Š‡ŽŽ Ǥ ‡”‡†‹–ƒ”›†‹•‡ƒ•‡•‘ˆ ‘‡œ›‡–Š‹‘‡•–‡” ‡–ƒ„‘Ž‹•Ǥ‹‘ Š‡‘ ”ƒ•ǤʹͲͳͻ ‡„ʹͺǢͶ͹ȋͳȌǣͳͶͻȂͷͷǤ ʹͻǤ ‹ǡƒŒ‹ƒǡŠ‹—”ƒǡƒ”‡›ǡ–”‘—†ǡ—”ƒ›ƒƒǡŠ–ƒ‡ǡ ‡œ‹‡Ǥ‘••‘ˆ –Š‡ ‹–‘ Š‘†”‹ƒŽ ƒ––›  ‹† ȾǦš‹†ƒ–‹‘ ”‘–‡‹ ‡†‹—ǦŠƒ‹  ›ŽǦ‘‡œ›‡  ‡Š›†”‘‰‡ƒ•‡‹•”—’–•š‹†ƒ–‹˜‡Š‘•’Š‘”›Žƒ–‹‘”‘–‡‹‘’Ž‡š–ƒ„‹Ž‹–›ƒ† — –‹‘Ǥ  ‹‡’ǤʹͲͳͺǢͺȋͳȌǣͳȂͳ͹Ǥ ͵ͲǤ ‡”• ǡ‘—™ǡ‹„ƒ• ǡ‹ƒ ‹‹ ǡƒœ‹ǡ‡‰”‡––‘ ǡ‡• ƒǡ‡‹Œ‰‘—† ǡ ‹– ǡ ƒŒ‡” ǡ ƒ”‰ƒ• Ǥ š’‡”‹‡–ƒŽ ‡˜‹†‡ ‡ ˆ‘” ’”‘–‡‹ ‘š‹†ƒ–‹˜‡ †ƒƒ‰‡ ƒ† ƒŽ–‡”‡†ƒ–‹‘š‹†ƒ–†‡ˆ‡•‡‹’ƒ–‹‡–•™‹–Š‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡†‡ˆ‹ ‹‡ ›Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳͶǢ͵͹ȋͷȌǣ͹ͺ͵ȂͻǤ ͵ͳǤ ‹ ‘š ǡ ‡‘‰Š‡‰ƒ ǡƒ• Š‡ ǡ‡•ƒ”†ǡƒ•“—ƒŽ‹ǡ‹ƒ‘ǡ‡‡ǡ ‹ƒ‰ǡ —  ǡ

ʹ͵͸ ‡‡”ƒŽ†‹• —••‹‘

‡”•Šƒ™ǡ‘ƒ–‘ ǡ’–‡ǡ‘‰‘ǡ—––‡” ǡ Š”‡‹„‡”ǡ‡ Š‡”ǡ‘š ǡ‹ŽŽƒ—‡˜ƒ Ǥ Ž‘„ƒŽƒŽ›•‹•‘ˆŽƒ•ƒ‹’‹†• †‡–‹ˆ‹‡•‹˜‡”Ǧ‡”‹˜‡† ›Ž ƒ”‹–‹‡•ƒ•ƒ —‡Ž‘—” ‡ˆ‘” ”‘™ ƒ–Š‡”‘‰‡‡•‹•Ǥ‡ŽŽ‡–ƒ„ǤʹͲͳ͹Ǣʹ͸ȋ͵ȌǣͷͲͻȂͷʹʹǤ‡͸Ǥ ͵ʹǤ „—”ƒ†ǤŠ‡‹˜‡”ƒ•ƒ —„‹Š‡”‘‰‡‡•‹•Ǥ‡ŽŽ‡–ƒ„ǤʹͲͳ͹Ǣʹ͸ȋ͵ȌǣͶͷͶȂͷǤ ͵͵Ǥ —‹ ǡ Š‡”‰—”‘˜‹ Š ǡ ‘”• Š‡”  ǡ ƒ‰ ǡ ‡‰ ǡ — ǡ •’ƒ”œƒ ǡ ‡›ƒ ǡ ‡ Šƒǡ ƒš‹ƒ‰ —‘ ǡŠ‹–‡ǡƒ„‹‘™‹–œ Ǥ Ž— ‘•‡ˆ‡‡†•–Š‡ › Ž‡˜‹ƒ ‹” —Žƒ–‹‰Žƒ –ƒ–‡Ǥ ƒ–—”‡ǤʹͲͳ͹Ǣͷͷͳȋ͹͸͹ͺȌǣͳͳͷȂͺǤ ͵ͶǤ —ƒ”ǡŽƒ‰‡”‘ ǡ—””ƒ Ǥ †— ‡†’Ž—”‹’‘–‡–•–‡ ‡ŽŽ•‹†‹•‡ƒ•‡‘†‡Ž‹‰ƒ†‰‡‡ ‹†‡–‹ˆ‹ ƒ–‹‘Ǥ‡–Š‘†•‘Ž‹‘ŽǤʹͲͳͺǢͳ͹Ͳ͸ǣͳ͹Ȃ͵ͺǤ ͵ͷǤ —––ƒǡ ‡‘ ǡŽ‡˜‡”• Ǥ‹•‡ƒ•‡‘†‡Ž‹‰‹–‡‡ŽŽǦ‡”‹˜‡†͵”‰ƒ‘‹†›•–‡•Ǥ”‡†• ‘Ž‡†ǤʹͲͳ͹Ǣʹ͵ȋͷȌǣ͵ͻ͵ȂͶͳͲǤ ͵͸Ǥ — Šǡ‘„Ž‹ Š ǡ—–‘Žˆǡƒ”–‹‡œǦ”‹ƒ•ǤŠ‡Š‘’‡ƒ†–Š‡Š›’‡‘ˆ‘”‰ƒ‘‹†”‡•‡ƒ” ŠǤ ‡˜‡Ž‘’‡–ǤʹͲͳ͹ǢͳͶͶȋ͸Ȍǣͻ͵ͺȂͶͳǤ ͵͹Ǥ ƒ‰ ǡ—ǡ‹—ǡƒ‘Ǥƒ–‹‡–Ǧ†‡”‹˜‡†‘”‰ƒ‘‹†•ǣƒ’”‘‹•‹‰‘†‡Žˆ‘”’‡”•‘ƒŽ‹œ‡† ƒ ‡”–”‡ƒ–‡–Ǥ ƒ•–”‘‡–‡”‘Ž‡’ǤʹͲͳͺǢ͸ȋͶȌǣʹͶ͵ȂͷǤ ͵ͺǤ ƒ††”‹ ǡ‹’•‘Ǥ”‰ƒǦ‘ǦƒǦ Š‹’–‡ Š‘Ž‘‰›ǣ–—”‹‰–‹–•’‘–‡–‹ƒŽˆ‘” Ž‹‹ ƒŽ„‡‡ˆ‹– ‹–‘”‡ƒŽ‹–›Ǥ”—‰‹• ‘˜‘†ƒ›ǤʹͲͳͻǢͲͲȋͲͲȌǤ ͵ͻǤ ‡ ™‹–– ǡŽƒ”ǡŠ‡‡Ž‡”ǡƒ›Ž‘”ǡ–‘Žœǡ ”‹ˆˆ‹–Šǡ‡ŽŽ•Ǥ‹˜‡”Ǯ‘”‰ƒ‘ƒ Š‹’Ǥǯ š’‡ŽŽ‡•ǤʹͲͳͺǢ͵͸͵ȋͳȌǣͳͷȂʹͷǤ 7 ͶͲǤ ƒ‰ Ǥ š’Ž‘”‹‰ –Š‡”ƒ’‡—–‹  ƒ’’”‘ƒ Š‡• ˆ‘” –”‡ƒ–‡– ‘ˆ ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ȋȌ†‡ˆ‹ ‹‡ ›ǤʹͲͳͶǤ ͶͳǤ ‘ Ž‡› Ǥ •‡ ‘ˆ ƒ˜‹ –‹ ‹ ƒ–‹‡–• ‹–Š  ‡ˆ‹ ‹‡ › ‹–Š –Š‡ ͻͺͷε  ȋ͵ͲͶȌ —–ƒ–‹‘ ȏ –‡”‡–ȐǤ ʹͲͳ͹ ȏ ‹–‡† ʹͲͳͻ ’” ʹͳȐǤ ˜ƒ‹Žƒ„Ž‡ ˆ”‘ǣ Š––’•ǣȀȀ Ž‹‹ ƒŽ–”‹ƒŽ•Ǥ‰‘˜Ȁ –ʹȀ•Š‘™Ȁ•–—†›ȀͲͳͺͺͳͻͺͶ ͶʹǤ ƒŽ‹” ǡ —‹–‡” ǡ ƒ†‡”•  ǡ ‘—–‘‘’‡”  Ǥ †‡–‹ˆ‹ ƒ–‹‘ ‘ˆ ‡œ›‡• ‹˜‘Ž˜‡† ‹ ‘š‹†ƒ–‹‘‘ˆ’Š‡›Ž„—–›”ƒ–‡Ǥ ‹’‹†‡•ǤʹͲͳ͹ǢͷͺȋͷȌǣͻͷͷȂ͸ͳǤ Ͷ͵Ǥ —ǡŠ‡ǡ’‘–‡ǡƒ––ƒ‰Ž‹ƒǡ — ‡ǡƒ Ǥ”‘Ž‘‰‡†ˆƒ•–‹‰‹†‡–‹ˆ‹‡•Š‡ƒ–•Š‘  ’”‘–‡‹ͳͲƒ•ƒ•‹”–—‹͵•—„•–”ƒ–‡ǣŽ— ‹†ƒ–‹‰ƒ‡™‡ Šƒ‹•Ž‹‹‰‹–‘ Š‘†”‹ƒŽ’”‘–‡‹ ƒ ‡–›Žƒ–‹‘–‘ˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘‡œ›‡ˆ‘Ž†‹‰ƒ†ˆ— –‹‘Ǥ ‹‘ŽŠ‡ǤʹͲͳͷǢʹͻͲȋͶȌǣʹͶ͸͸Ȃ ͹͸Ǥ ͶͶǤ ƒ‡”ƒ•  ǡ ƒ ‡‡‰Š‡Ž ǡ ‡‹• ǡ ‹ ‘Žƒ› ǡ ”‘’‡”• ǡ ‘—–‡ Ǥ ƒ”‹–‹‡ •—’’Ž‡‡–ƒ–‹‘ ƒ––‡—ƒ–‡• ›‘ ƒ”†‹ƒŽ Ž‹’‹† ƒ ——Žƒ–‹‘ ‹ Ž‘‰Ǧ Šƒ‹ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡‘ ‘—–‹ ‡Ǥ  Š‡”‹–‡–ƒ„‹•ǤʹͲͳ͵Ǣ͵͸ȋ͸Ȍǣͻ͹͵ȂͺͳǤ ͶͷǤ ‡‡ ǡ ƒ†œ•‹‡˜ ǡ ‡Ž‡‰ŠǤ ‘Ž‡ ‘ˆ ƒ”‹–‹‡ ƒ† ‹–• †‡”‹˜ƒ–‹˜‡• ‹ –Š‡ †‡˜‡Ž‘’‡– ƒ† ƒƒ‰‡‡–‘ˆ–›’‡ʹ†‹ƒ„‡–‡•Ǥ—–”‹ƒ„‡–‡•ǤʹͲͳͺǢͺȋͳȌǣͳȂͳͲǤ Ͷ͸Ǥ ’‹‡‡”‘‡––‡” ǡ ƒ•–‹ ǡ ‹ŽŽ‹‰Šƒ ǡ ‘””‹• ǡ ‹Œ„—”‰ ǡ ‹Ž ‡ Ǥ —””‡– ‹••—‡• ”‡‰ƒ”†‹‰ –”‡ƒ–‡– ‘ˆ ‹–‘ Š‘†”‹ƒŽ ˆƒ––› ƒ ‹† ‘š‹†ƒ–‹‘ †‹•‘”†‡”•Ǥ  Š‡”‹– ‡–ƒ„ ‹•Ǥ ʹͲͳͲǢ͵͵ȋͷȌǣͷͷͷȂ͸ͳǤ Ͷ͹Ǥ ƒŽ–‡” ǤǦƒ”‹–‹‡‹‹„‘”‡””‘”•‘ˆ‡–ƒ„‘Ž‹•ǣŠƒ–‹•–Š‡‡˜‹†‡ ‡ǫ  Š‡”‹–‡–ƒ„ ‹•ǤʹͲͲ͵Ǣʹ͸ȋʹȂ͵ȌǣͳͺͳȂͺǤ ͶͺǤ ”‹‹˜ƒ•ƒǡƒ”ƒ–ƒ•Š˜‹Ž‹ǡ™ƒƒ‰ƒ‡”ǡƒ‘ǡ‘Ž‘„‡ŽŽ‹ǡ Šƒƒ’ǡ‘ŽŽ‡ǡ ‘†‰‘”懍ǡ‘•‡  ǡ‡–‘˜‹ ǡ ƒ›ˆŽ‹ Ǥš–”ƒ ‡ŽŽ—Žƒ”ͶǯǦŠ‘•’Š‘’ƒ–‡–Š‡‹‡‹•ƒ•‘—” ‡ ˆ‘”‹–”ƒ ‡ŽŽ—Žƒ”‘‡œ›‡•›–Š‡•‹•Ǥƒ–Š‡‹‘ŽǤʹͲͳͷǢͳͳȋ—‰—•–ȌǣͳȂʹ͸Ǥ ͶͻǤ Šƒ”ƒǡ—„”ƒƒ‹ƒǡ—ǡ ”ƒǡŠ‹–‡ǡ‘ ǡ‡‡ǡ ƒ ‘™•‹Ǥ –Š‡”ƒ’‡—–‹  ƒ’’”‘ƒ Š –‘ ’ƒ–‘–Š‡ƒ–‡ ‹ƒ•‡ ƒ••‘ ‹ƒ–‡† ‡—”‘†‡‰‡‡”ƒ–‹‘Ǥ ƒ– ‘—Ǥ ʹͲͳͺǢͻȋͳȌǤ ͷͲǤ ‹‡‘ ǡ‘Ž‘„‡ŽŽ‹ǡ”‹‹˜ƒ•ƒǡ†‡‹ŽŽ‹‡”•ǡ ƒƒ†ƒ ǡ ‡‘‰ǡ ‘šǡ‘Ž–Œ‡”ǡ‡’’‡”  ǡƒŠƒ›‡ǡ‹œœ‡––‘ǡ ƒ””• ǡ†‡‘‡”ǡ˜ƒ†‡”™ƒƒ‰ǡ ‡‘ǡ,—•ƒǡƒŠ‘” ǡ ‘•‡  ǡ ”œ‡• Š‹ǡ ƒ›ˆŽ‹  ǡ‹”ƒ–‹ǡ‹„‘Ǥ ‡–›ŽǦͶǯǦ’Š‘•’Š‘’ƒ–‡–Š‡‹‡‹••–ƒ„Ž‡ ‹•‡”—ƒ†’”‡˜‡–•’Š‡‘–›’‡•‹†— ‡†„›’ƒ–‘–Š‡ƒ–‡‹ƒ•‡†‡ˆ‹ ‹‡ ›Ǥ ‹‡’ǤʹͲͳ͹ ‡’ͳʹǢ͹ȋͳȌǣͳͳʹ͸ͲǤ ͷͳǤ ‹– Š‡ŽŽ ǡ ƒ—–Š‹‡”ǡ‡•‹’Ž‡ǡƒ‰ǡƒ‡”ǡ—”‡•Š‹ Ǥ ‡”‡†‹–ƒ”›ƒ†ƒ “—‹”‡† †‹•‡ƒ•‡•‘ˆƒ ›ŽǦ ‘‡œ›‡‡–ƒ„‘Ž‹•Ǥ‘Ž ‡‡–‡–ƒ„ǤʹͲͲͺǢͻͶȋͳȌǣͶȂͳͷǤ ͷʹǤ ƒ‰ ǡŠƒ‘ǡƒ‰ǡƒ‰ǡ‹– Š‡ŽŽ Ǥ ‡”‡†‹–ƒ”›†‹•‡ƒ•‡•‘ˆ ‘‡œ›‡–Š‹‘‡•–‡” ‡–ƒ„‘Ž‹•Ǥ‹‘ Š‡‘ ”ƒ•ǤʹͲͳͻǢͶ͹ȋͳȌǣͳͶͻȂͷͷǤ ͷ͵Ǥ ƒ ǡ •—ǡ”‹‰Š– ǡ‰ƒ”™ƒŽƒǡ ‘––ǡŠƒ‰ Ǥ ‡‘‡‡‰‹‡‡”‹‰—•‹‰–Š‡ Ǧ

ʹ͵͹ Šƒ’–‡”ͽ

ƒ•ͻ•›•–‡Ǥƒ–”‘–‘ ǤʹͲͳ͵ǢͺȋͳͳȌǣʹʹͺͳȂ͵ͲͺǤ ͷͶǤ ‘Ž–‡”• ǡ ‹ƒ’ƒ‹–‡ ǡ ƒ —‡ ǡ ‹‡œ‡Ǧ‘‹‰ ǡ ƒ––‘ ǡ ‘”–‡  ǡ ‘”˜ƒ–‘˜‹ Š ǡ ƒ‡” ǡ ‹• Š‘ˆˆ ǡ ‡”‡–‹‡” Ǥ ”ƒ•Žƒ–‹‘ƒŽ ƒ”‰‡–‡† ”‘–‡‘‹ • ”‘ˆ‹Ž‹‰ ‘ˆ ‹–‘ Š‘†”‹ƒŽ ‡”‰› ‡–ƒ„‘Ž‹  ƒ–Š™ƒ›• ‹ ‘—•‡ ƒ† —ƒ ƒ’Ž‡•Ǥ  ”‘–‡‘‡ ‡•Ǥ ʹͲͳ͸ǢͳͷȋͻȌǣ͵ʹͲͶȂͳ͵Ǥ ͷͷǤ ‹ƒ ǡ•› Š‘‰‹‘•ǡ‘—‰ǡ‹•Šƒ”–Ǥ‡–ƒ„‘ƒŽ›•–᩿ǣƒ™‡„•‡”˜‡”ˆ‘”‡–ƒ„‘Ž‘‹ †ƒ–ƒ ƒƒŽ›•‹•ƒ†‹–‡”’”‡–ƒ–‹‘ǤʹͲͲͻǢ͵͹ȋƒ›Ȍǣ͸ͷʹȂ͸ͲǤ ͷ͸Ǥ ‹‡‡”– ǡ ‘Ž‰ƒ ǡ ‘„ƒ„‡ ǡ ”‘Š ǡ ˆ‡‹ˆ‡” ǡ ‡šŽ‡” ǡ —Ž•‡‡ Ǥ ’‡†ƒ ‡ ‡ƒ•—”‡‡– ˆ‘” ”‡ƒŽ –‹‡ †‡–‡ –‹‘ ‘ˆ ‡—”‘ƒŽ ‡ŽŽ †‡ƒ–ŠǤ  ‡—”‘• ‹ ‡–Š‘†•Ǥ ʹͲͳʹ ƒ ͳͷǢʹͲ͵ȋͳȌǣ͸ͻȂ͹͹Ǥ ͷ͹Ǥ — ǡ ”‡‡ ǡ”ƒˆƒŽǡ ‘†•‘Ǥ ˜‹–”‘ ‡ŽŽ—Žƒ”‘†‡Ž•‘ˆŠ—ƒŠ‡’ƒ–‹ ˆƒ––›ƒ ‹† ‡–ƒ„‘Ž‹•ǣ ‹ˆˆ‡”‡ ‡• „‡–™‡‡ —Š͹ ƒ† ‡’ ʹ ‡ŽŽ Ž‹‡• ‹ Š—ƒ ƒ† ˆ‡–ƒŽ „‘˜‹‡ —Ž–—”‹‰•‡”—ǤŠ›•‹‘Ž‡’ǤʹͲͳ͹ǢͷȋʹͶȌǣͳȂͳʹǤ ͷͺǤ –‡‡„‡”‰‡ǡ–‹ǡ‡” ‘”•–ǡƒ–ǡ‡—ˆ‡Ž†–ǡ‡Ž‘˜‘†•‹›ǡŠ—ƒǡŠ‘ ǡ ‘› ‡ǡ —–‹ŽŠ ǡ ›””‡ŽŽ Ǥ •–ƒ„Ž‹•Š‹‰ ‘”ƒŽ ‡–ƒ„‘Ž‹•ƒ† †‹ˆˆ‡”‡–‹ƒ–‹‘ ‹ Š‡’ƒ–‘ ‡ŽŽ—Žƒ” ƒ” ‹‘ƒ ‡ŽŽ•„› —Ž–—”‹‰‹ƒ†—Ž–Š—ƒ•‡”—Ǥ ‹‡’ǤʹͲͳͺ—‰͵ǢͺȋͳȌǣͳͳ͸ͺͷǤ ͷͻǤ ”ƒˆƒŽǡƒ”••‘ǡ ¡”†ˆ‡Ž†– ǡ”‹••‘ǡƒ”‹‹Ǥ—Ž–—”‹‰‘ˆ ‡’ ʹ ‡ŽŽ•™‹–ŠŠ—ƒ •‡”— ‹’”‘˜‡ –Š‡‹” ˆ— –‹‘ƒŽ‹–› ƒ† •—‹–ƒ„‹Ž‹–› ‹ •–—†‹‡• ‘ˆ Ž‹’‹† ‡–ƒ„‘Ž‹•Ǥ ‹‘ Š‹ ‹‘’Š›• –ƒǦ‘Ž‡ŽŽ‹‘Ž‹’‹†•ǤʹͲͳ͸Ǣͳͺ͸ͳȋͳȌǣͷͳȂͻǤ ͸ͲǤ ƒƒ‡œ ǡƒ”‘ǡ”‹ƒ†ǡ‡Š‘†– ǡ—’Žƒ ǡ—‹’‡”• ǡ ‡—›‡”ǡŽƒ˜‡›ǡ–ƒ‡Ž•Ǥ Š‡ Š—ƒ Š‡’ƒ–‘ ›–‡ ‡ŽŽ Ž‹‡•  ƒ† ‡’ƒ ǣ ‘†‡Ž• –‘ •–—†› ‰Ž— ‘•‡ǡ Ž‹’‹† ƒ† Ž‹’‘’”‘–‡‹‡–ƒ„‘Ž‹•Ǥ” ŠŠ›•‹‘Ž‹‘ Š‡ǤʹͲͳʹǢͳͳͺȋƒ” ŠȌǣͳͲʹȂͳͳǤ

ʹ͵ͺ

Appendices



Summary

 

Summary

‡†‹—Ǧ Šƒ‹ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡ȋȌ†‡ˆ‹ ‹‡ ›ȋ ʹͲͳͶͷͲȌǡƒ†‡ˆ‹ ‹‡ ›‘ˆ ƒ‹•‘‡œ›‡‘ˆ–Š‡‹–‘ Š‘†”‹ƒŽˆƒ––›ƒ ‹†‘š‹†ƒ–‹‘ȋ Ȍ’ƒ–Š™ƒ›ǡ‹•‘‡‘ˆ–Š‡‘•– ’”‡˜ƒŽ‡–‹„‘”‡””‘”•‘ˆ‡–ƒ„‘Ž‹•ȋ ȌǤ†‡ˆ‹ ‹‡ ›‹•ƒŽ•‘–Š‡‘•– ‘‘  ‘ˆ –Š‡   ’ƒ–Š™ƒ›Ǥ Š‡  ’”‡˜ƒŽ‡ ‡ ƒ– „‹”–Š ™ƒ• ͳȀͺ͵ͲͲ ‹ –Š‡ ‡–Š‡”Žƒ†• „‡–™‡‡ʹͲͲ͹ƒ†ʹͲͳͷȋŠƒ’–‡”ͳȌǤ ’Žƒ›•ƒ‹’‘”–ƒ–”‘Ž‡‹‡‡”‰›Š‘‡‘•–ƒ•‹• †—”‹‰ˆƒ•–‹‰ƒ† ‘†‹–‹‘•‘ˆŠ‹‰ŠǦ‡‡”‰›†‡ƒ†Ǥ†‡”–Š‡•‡ ‘†‹–‹‘•ǡ–Š‡Ž‹˜‡”—•‡• –Š‡ ’ƒ–Š™ƒ›–‘‰‡‡”ƒ–‡‡–‘‡„‘†‹‡•ƒ†–Š‡–Šƒ–‹•”‡“—‹”‡†ˆ‘”de novo ‰Ž— ‘•‡ ’”‘†— –‹‘Ǥ Š‡”‡„› –Š‡   ‹• ‘ˆ ”‹–‹ ƒŽ ‹’‘”–ƒ ‡ ˆ‘” ˆ—‡Ž •—’’Ž› –‘ –Š‡ ’‡”‹’Š‡”ƒŽ –‹••—‡•Ǥ–”‡ƒ–‡†Ǧ†‡ˆ‹ ‹‡–ȋȌ Š‹Ž†”‡”—ƒ•‡˜‡”‡”‹•‘ˆŽ‹ˆ‡Ǧ–Š”‡ƒ–‡‹‰Ž‘™ „Ž‘‘†‰Ž— ‘•‡ƒ†Ž‘™‡–‘‡„‘†›Ž‡˜‡Ž•ȋŠ›’‘‡–‘–‹ Š›’‘‰Ž› ‡‹ƒȌǤ‡˜‡”–Š‡Ž‡••ǡ ‹†‹˜‹†—ƒŽ•Šƒ˜‡„‡‡”‡’‘”–‡†–‘–‘Ž‡”ƒ–‡ͳͺǦʹͶŠ‘—”•‘ˆˆƒ•–‹‰Ǥ’‡ ‹ˆ‹ ƒŽŽ›ǡƒ ‘„‹ƒ–‹‘ ‘ˆ’”‘Ž‘‰‡†ˆƒ•–‹‰–‘‰‡–Š‡”™‹–Šƒƒ††‹–‹‘ƒŽ–”‹‰‰‡”ǡ•— Šƒ•ƒ‹”™ƒ›‘”‰ƒ•–”‘‹–‡•–‹ƒŽ ‹ˆ‡ –‹‘•‘” ‘Ž†‡š’‘•—”‡ǡ ƒ’—–—–”‡ƒ–‡†‹†‹˜‹†—ƒŽ•ƒ–”‹•Ǥ˜‡™‹–Š‹–Š‡ •—„’‘’—Žƒ–‹‘‘ˆ‹†‹˜‹†—ƒŽ•™‹–ŠƒŠ‘‘œ›‰‘—•Ž‘••Ǧ‘ˆǦˆ— –‹‘ Ǥͻͺͷε —–ƒ–‹‘‹–Š‡ Ǧ‡ ‘†‹‰ ACADM ‰‡‡ǡ •›’–‘• ƒ ”ƒ‰‡ ˆ”‘ ˆƒ–ƒŽ Š›’‘‡–‘–‹  Š›’‘‰Ž› ‡‹ƒ ‡ƒ”Ž› ‹ Ž‹ˆ‡ –‘ ƒ ‘’Ž‡–‡ Žƒ  ‘ˆ •›’–‘• –Š”‘—‰Š‘—– Ž‹ˆ‡Ǥ ‡‡–‹  ƒ† ‡˜‹”‘‡–ƒŽ ‘†‹ˆ‹‡”• ‘—Ž† ’Žƒ› ƒ ”‘Ž‡ ‹ –Š‡ Š‡–‡”‘‰‡‡‹–› ‹ ˜—Ž‡”ƒ„‹Ž‹–› –‘ ‘” ”‘„—•–‡•• ƒ‰ƒ‹•– ‡–ƒ„‘Ž‹ •–”‡••Ǥ ‘™–Š‹•˜—Ž‡”ƒ„‹Ž‹–›‘””‘„—•–‡••ƒ”‹•‡•‹•ƒ•›‡–— Ž‡ƒ”Ǥ ›–Š‡•‹• Šƒ˜‡–Š‡”‡ˆ‘”‡–ƒ‡ƒ•›•–‡•‡†‹ ‹‡ƒ’’”‘ƒ Š–‘‰‡–ƒ„‡––‡”‹•‹‰Š–‹–Š‡ ‘’Ž‡š ‡–‹‘Ž‘‰› ‘ˆ Ǥ › ‘˜‡”ƒ” Š‹‰ “—‡•–‹‘ Šƒ• „‡‡ ™Šƒ– ‡Ž‹ ‹–• Š›’‘‡–‘–‹  Š›’‘‰Ž› ‡‹ƒ ‹ •‘‡  ’ƒ–‹‡–• ƒ† ™Šƒ– ’”‘–‡ –• ‘–Š‡”•Ǥ  ‹˜‡•–‹‰ƒ–‡† ™Š‹ Š ‘†‹ˆ›‹‰ˆƒ –‘”•™‹–Š‹ƒ†‘—–•‹†‡–Š‡’ƒ–Š™ƒ›ƒ›’—•Š‡–ƒ„‘Ž‹•ˆ”‘ƒƒ’’ƒ”‡–Ž› Š‡ƒŽ–Š› •–ƒ–‡ ‹–‘ –‘™ƒ”†• Š›’‘‡–‘–‹  Š›’‘‰Ž› ‡‹ƒ ƒ† ™Š‹ Š ˆƒ –‘”• ƒ› ‡‡’ –Š‡ ‡–ƒ„‘Ž‹ ‡–™‘”•ƒˆ‡Ž›ƒ™ƒ›ˆ”‘–Š‹•†ƒ‰‡”‘—••–ƒ–‡Ǥ ‘”†‡”–‘ƒ•™‡”–Š‹•“—‡•–‹‘ǡ  ‡˜ƒŽ—ƒ–‡††‡ˆ‹ ‹‡ ›‹ƒ ‘’—–ƒ–‹‘ƒŽ‘†‡Žȋi.e. in silicoȌǡ‹‹ ‡in vivo ƒ†‹ Š—ƒ ‡ŽŽ•in vitroǤ ˆ‹”•–‹˜‡•–‹‰ƒ–‡–Š‡„‡Šƒ˜‹‘”‘ˆƒ’—„Ž‹•Š‡† ‘’—–ƒ–‹‘ƒŽ‘†‡Ž‘ˆ‹•‘Žƒ–‡† ‹”ƒ– Ž‹˜‡”ȋŠƒ’–‡”ʹȌǡ•‹ ‡ ‹–‡†‡†–‘—•‡–Š‹•‘†‡Žƒ•ƒ„ƒ•‹•–‘ ‘•–”— –ƒŠ—ƒ‘†‡Ž ˜‡”•‹‘Žƒ–‡”Ǥ —”ƒ˜‡Ž‡†–Š‡‘”‹‰‹‘ˆ–Š‡•Šƒ”’†‡ Ž‹‡‘ˆ–Š‡ ˆŽ—š–Šƒ–‘ —””‡†‹–Š‡ ‘†‡ŽŠ‹‰Š•—„•–”ƒ–‡ ‘ ‡–”ƒ–‹‘•ǤŠ‹•ˆŽ—š†‡ Ž‹‡™ƒ• Ž‡ƒ”Ž›”‡Žƒ–‡†–‘–Š‡’”‘‹• —‹–› ‘ˆ–Š‡ ‡œ›‡•ǡi.e.–Š‡ˆƒ ––Šƒ––Š‡›ƒ ‡’–—Ž–‹’Ž‡Š‘‘Ž‘‰‘—••—„•–”ƒ–‡•ǡ™Š‹ Š ‘’‡–‡ˆ‘”„‹†‹‰–‘–Š‡ƒ –‹˜‡•‹–‡‘ˆ–Š‡‡œ›‡Ǥ ‹†‡–‹ˆ‹‡†ƒ˜‹ ‹‘—• › Ž‡ǡ–Šƒ–•–ƒ”–‡† ƒ– ‡†‹—Ǧ Šƒ‹ ‡–‘ƒ ›ŽǦ‘ –Š‹‘Žƒ•‡ ȋȌ ƒ† ’”‘’ƒ‰ƒ–‡† –Š”‘—‰Š‘—– –Š‡ •›•–‡ǡ Ž‡ƒ†‹‰–‘Ž‘™ ˆŽ—šǤ ‡”‡‹ǡ’”‘‹• —‹–›‘ˆ™ƒ•„‘–Š‡ ‡••ƒ”›ƒ†•—ˆˆ‹ ‹‡––‘ ‡Ž‹ ‹––Š‡ˆŽ—š†‡ Ž‹‡ǤŠƒ†‹•—ˆˆ‹ ‹‡– ƒ’ƒ ‹–›–‘ ‘’‡™‹–ŠŠ‹‰Š•—„•–”ƒ–‡‹ˆŽ—šƒ† ‘•‡“—‡–Ž›ǡ ‹–ǯ• ‡–‘ƒ ›ŽǦ‘ •—„•–”ƒ–‡• •–ƒ”–‡† –‘ ƒ ——Žƒ–‡Ǥ Š‹• ƒ ——Žƒ–‹‘ ™ƒ• ƒ’Ž‹ˆ‹‡† „› –Š‡ ’”‡ ‡†‹‰ ‡œ›‡ǡ Ž‡ƒ†‹‰ –‘ ƒ ——Žƒ–‹‘ ‘ˆ —’•–”‡ƒ ‘ ‡•–‡”•ǡ ‹ Ž—†‹‰ƒ ›ŽǦ‘‡•–‡”•ǡƒ†•‡“—‡•–”ƒ–‹‘‘ˆˆ”‡‡‘ȋ‘ ȌǤ‹ ‡‘ ‹•ƒ‡••‡–‹ƒŽ ‘ˆƒ –‘”‘ˆƒ†ƒ ›ŽǦ‘‡•–‡”•ƒ”‡’”‘†— –•ǡ‹–•ƒ –‹˜‹–›™ƒ•ˆ—”–Š‡”‹Š‹„‹–‡†Ǥ ƒ††‹–‹‘ǡ‘ ‹•ƒŽ•‘ƒ•—„•–”ƒ–‡‘ˆ‘–Š‡”‡œ›‡•‹–Š‡ ’ƒ–Š™ƒ›ǡƒ†–Š‡”‡„›‹–• •‡“—‡•–”ƒ–‹‘ ‡ˆˆ‹ ‹‡–Ž› ‘—‹ ƒ–‡† –Š‡ Ǯ–”ƒˆˆ‹  Œƒǯ ƒ–  –‘ –Š‡ ‡–‹”‡ ’ƒ–Š™ƒ›ǡ —Ž–‹ƒ–‡Ž›”‡•—Ž–‹‰‹Ž‘™ ˆŽ—šǤ ‹ƒŽŽ›ǡ ˆ‘—†–Šƒ––Š‡˜‹ ‹‘—• › Ž‡™ƒ•ƒ‰‰”ƒ˜ƒ–‡† „›ƒŠ‹‰Š  ‘ ‡–”ƒ–‹‘ǤŠ‡Žƒ––‡” ƒ—•‡•’”‘†— –‹Š‹„‹–‹‘‘ˆ–Š‡ ƒ†–Š‡”‡„› ‡Šƒ ‡•–Š‡ƒ ——Žƒ–‹‘‘ˆ‘‡•–‡”•ƒ†•‡“—‡•–”ƒ–‹‘‘ˆ‘ Ǥ

ʹͶͷ Appendices

Šƒ’–‡”͵ǡ ‹˜‡•–‹‰ƒ–‡†™Š‹ Š‡–ƒ„‘Ž‹ ’ƒ–Š™ƒ›• ‘–”‹„—–‡–‘–Š‡”‘„—•–‡••‘ˆˆƒ•–‡† Ǧ‹ ‡ǡ•‹ ‡‹–Š‡•‡‹ ‡Ž‘™„Ž‘‘†‰Ž— ‘•‡‘”‡–‘‡„‘†› ‘ ‡–”ƒ–‹‘•Šƒ† ‡˜‡”„‡‡‘„•‡”˜‡†ǤŠ‡‘ –ƒ‘›ŽǦ‘‘š‹†ƒ–‹‘ ƒ’ƒ ‹–›‘ˆŽ‹˜‡”‹–‘ Š‘†”‹ƒˆ”‘Ǧ ‹ ‡™ƒ•Ž‘™‡”–Šƒ–Šƒ–‘ˆ‹ ‡ǤŠ‹•Ž‘™‡” ƒ’ƒ ‹–›™ƒ•‘– ‘’‡•ƒ–‡†ˆ‘”„› Š‹‰Š‡”’”‘–‡‹Ž‡˜‡Ž•‘ˆ–Š‡•Š‘”–Ǧƒ†Ž‘‰Ǧ Šƒ‹‹•‘‡œ›‡•ƒ†‹–Š‡Ǧ ‹ ‡Ǥ‡†‹†Š‘™‡˜‡”‘„•‡”˜‡•—„–Ž‡ƒ†ƒ’–ƒ–‹‘•‹–Š‡‡š’”‡••‹‘‘ˆ‰‡‡•‡ ‘†‹‰ ‡œ›‡•–Šƒ––ƒ‡‘ ǡȋȌΪ‘”ȋȌ ƒ•ƒ•—„•–”ƒ–‡‘”’”‘†— –ǡƒ•™‡ŽŽƒ•‡œ›‡• ‹˜‘Ž˜‡†‹†‡–‘š‹ˆ‹ ƒ–‹‘ǤŠ‡•‡ƒ†ƒ’–ƒ–‹‘•ƒ› ‘–”‹„—–‡–‘–Š‡”‘„—•–‡••‘ˆ–Š‡Ǧ ‹ ‡ƒ†™ƒ””ƒ–ˆ—”–Š‡”‹˜‡•–‹‰ƒ–‹‘ǤŠ‡•‡”‡•—Ž–• ‘””‘„‘”ƒ–‡–Š‡‹’‘”–ƒ–”‘Ž‡‘ˆ ‘ ƒ† ˆ‘” ˆ— –‹‘•Š‘™‹Šƒ’–‡”ʹǤ Šƒ’–‡”Ͷǡ ‹˜‡•–‹‰ƒ–‡†–Š‡Š‡’ƒ–‹ ‡–ƒ„‘Ž‹•‘ˆ ‘Ž†Ǧ‡š’‘•‡†ǡˆƒ•–‡†Ǧ‹ ‡‹ ”‡•’‘•‡–‘•‡˜‡”‡‡‡”‰‡–‹ •–”‡••Ǥ†‡”–Š‡•‡ ‘†‹–‹‘•–Š‡Ǧ‹ ‡•Š‘™‡†ˆ‘” –Š‡ˆ‹”•––‹‡ǡŽ‘™‡”’Žƒ•ƒ‰Ž— ‘•‡Ž‡˜‡Ž•–Šƒ–Š‡Ǥ ƒ††‹–‹‘ǡ–Š‡Ǧ‹ ‡Šƒ† Ž‘™‡” Š‡’ƒ–‹  ƒ‹‘ ƒ ‹† Ž‡˜‡Ž• ƒ† Š‹‰Š‡” Ž‹˜‡” ™‡‹‰Š–•ǡ Ž‹˜‡” –‘–ƒŽ –”‹‰Ž› ‡”‹†‡•ǡ Ž‹˜‡” ‡†‹—Ǧ Šƒ‹ –”‹‰Ž› ‡”‹†‡• ƒ† Š‹‰Š‡” ’Žƒ•ƒ ‡†‹—Ǧ Šƒ‹ ƒ ›ŽǦ ƒ”‹–‹‡ Ž‡˜‡Ž•Ǥ  –Š‡ ‘•–•‡˜‡”‡Ž›ƒˆˆ‡ –‡†Ǧ‹ ‡ǡ™‡ƒŽ•‘‘„•‡”˜‡†Š‡’ƒ–‹ ‹ˆŽƒƒ–‹‘ǡ™Š‹ Š‹•‹ Ž‹‡™‹–Š–Š‡–”‡†–‘™ƒ”†•Ž‹˜‡”‹ˆŽƒƒ–‹‘‘„•‡”˜‡†‹ˆƒ•–‡†Ǧ‹ ‡‹Šƒ’–‡” ͵ǤŠ‡”‡•—Ž–••—‰‰‡•–ƒ‹’‘”–ƒ–”‘Ž‡‘ˆŽ‹˜‡” ‹‰Ž— ‘•‡Š‘‡‘•–ƒ•‹•†—”‹‰ ‘Ž† •–”‡••‹ ‘„‹ƒ–‹‘™‹–Šˆƒ•–‹‰ǤǦ‹ ‡ƒ’’‡ƒ”‡†–‘—•‡ƒ‹‘ƒ ‹†•ƒƒŽ–‡”ƒ–‹˜‡ ˆ—‡Ž –‘ ‘’‡•ƒ–‡ ˆ‘” –Š‡ †‡ˆ‹ ‹‡–   ‹ –Š‡ ‰‡‡”ƒ–‹‘ ‘ˆ ǡ ‰Ž— ‘•‡ǡ ƒ† ‡–‘‡ „‘†‹‡•ǡ ™Š‹Ž‡ ‡š ‡•• ‡†‹—Ǧ Šƒ‹ ˆƒ––›Ǧƒ ‹† ‹–‡”‡†‹ƒ–‡• ™‡”‡ ‡š’‘”–‡† ƒ† ”‡”‘—–‡† †‹”‡ –Ž›‘”‹†‹”‡ –Ž›„› Šƒ‹‡Ž‘‰ƒ–‹‘‹–‘–”‹‰Ž› ‡”‹†‡•Ǥ Šƒ’–‡”ͷǡ ‹˜‡•–‹‰ƒ–‡†Š‘™’ƒ–Š™ƒ›••—””‘—†‹‰–Š‡ ǡ‘–ƒ„Ž›–Š‘•‡–Šƒ–ƒˆˆ‡ – ‘ ǡ‘†—Žƒ–‡–Š‡‡–ƒ„‘Ž‹ ’Š‡‘–›’‡™Š‡‘”‘–Š‡”ƒ ›ŽǦ‘†‡Š›†”‘‰‡ƒ•‡• ƒ”‡†‡ˆ‹ ‹‡–ǤŠ‹••Š‘—Ž†‰‹˜‡‹•‹‰Š–•‹‘†‹ˆ›‹‰ˆƒ –‘”•–Šƒ–ƒ›’Žƒ›ƒ”‘Ž‡‹–Š‡†‹ˆˆ‡”‡– †‹•‡ƒ•‡’”‡•‡–ƒ–‹‘‘ˆ‹†‹˜‹†—ƒŽ’ƒ–‹‡–•Ǥ ˆ‹”•–ƒ†ƒ’–‡†–Š‡ ‘’—–ƒ–‹‘ƒŽ‘†‡Ž–Šƒ–™ƒ• —•‡† ‹ Šƒ’–‡” ʹ „› ‹ ‘”’‘”ƒ–‹‰ –Š‡ ƒ˜ƒ‹Žƒ„Ž‡ Š—ƒ ‹‡–‹  ’ƒ”ƒ‡–‡”•Ǥ ‘”‡‘˜‡”ǡ  ƒ††‡†ƒ—„‡”‘ˆ’ƒ–Š™ƒ›•‹˜‘Ž˜‡†‹ƒ ›ŽǦ‘‡–ƒ„‘Ž‹•ǤŠ‡•‡‘†‹ˆ‹ ƒ–‹‘••–ƒ„‹Ž‹œ‡† –Š‡   ƒ† ’”‡˜‡–‡† –Š‡ ˆŽ—š †‡ Ž‹‡ ‘„•‡”˜‡† ‹ Šƒ’–‡” ʹǤ ‹—Žƒ–‡† ˆŽ—š‡• ƒ† ‡–ƒ„‘Ž‹–‡ ’”‘ˆ‹Ž‡• ”‡ˆŽ‡ –‡† –Š‡ ”‡Žƒ–‹˜‡ †‹•‡ƒ•‡ •‡˜‡”‹–‹‡• ‘ˆ ǡ ǡ ƒ†  †‡ˆ‹ ‹‡ ›‹Š—ƒ•Ǥ‡’”‡••‹‘‘ˆ–Š‡ˆ—–‹Ž‡ › Ž‡‘ˆƒ ›ŽǦ‘Š›†”‘Ž›•‹•ƒ†”‡ƒ –‹˜ƒ–‹‘ǡ „›‘†—Žƒ–‹‰–Š‡•—„•–”ƒ–‡•’‡ ‹ˆ‹ ‹–›‘ˆͳǡ‹’ƒ‹”‡†–Š‡ ˆŽ—š•’‡ ‹ˆ‹ ƒŽŽ›‹–Š‡ †‡ˆ‹ ‹‡–‘†‡ŽǤŠ‡”‡•—Ž–•‰‹˜‡ƒˆ‹”•–‹†‹ ƒ–‹‘Š‘™‰‡‡–‹ ‘†‹ˆ‹‡”•”‡Žƒ–‡†–‘–Š‡ •—””‘—†‹‰’ƒ–Š™ƒ›• ‘—Ž†ƒˆˆ‡ – ˆ— –‹‘‹’ƒ–‹‡–•Ǥ  Šƒ’–‡” ͸ǡ  ‹˜‡•–‹‰ƒ–‡† ™Š‡–Š‡” –Š‡”‡ ƒ”‡ ‡–ƒ„‘Ž‹  ƒ†ƒ’–ƒ–‹‘• ‹ —Ž–—”‡† •‹ ˆ‹„”‘„Žƒ•–•‘ˆ‹†‹˜‹†—ƒŽ•–Šƒ–†‹•–‹‰—‹•Š•›’–‘ƒ–‹ ˆ”‘ƒ•›’–‘ƒ–‹  ‹†‹˜‹†—ƒŽ•ǤŠ‡ˆ‹„”‘„Žƒ•–•‘ˆ‘•–‘ˆ–Š‡ƒ•›’–‘ƒ–‹ ‹†‹˜‹†—ƒŽ•”‡•‡„Ž‡†–Š‘•‡ ‘ˆ –Š‡‹” •›’–‘ƒ–‹  ‘—–‡”’ƒ”–•Ǥ ‘‡ ‘ˆ –Š‡ ƒ•›’–‘ƒ–‹  Š‹Ž†”‡ ™‡”‡ Š‘™‡˜‡” †‹ƒ‰‘•‡†ƒ–ƒ›‘—‰ƒ‰‡ƒ†‘‡‹‰Š–™‘†‡”‹ˆ–Š‡›™‡”‡ƒ•›’–‘ƒ–‹ „‡ ƒ—•‡‘ˆ–Š‡ –”‡ƒ–‡– ‘” „‡ ƒ—•‡ ‘ˆ –Š‡‹” ‰‡‡–‹  ƒ‡Ǧ—’Ǥ ‡  ‹†‹˜‹†—ƒŽǡ Š‘™‡˜‡”ǡ ™ƒ• †‹ƒ‰‘•‡† ƒ– –Š‡ ƒ‰‡ ‘ˆ͵Ͳ ƒ† ™ƒ• ‰‡—‹‡Ž› ƒ•›’–‘ƒ–‹  –Š”‘—‰Š‘—– Ž‹ˆ‡ǡ ™‹–Š‘—– ƒ› –”‡ƒ–‡–ǤŠ‡ˆ‹„”‘„Žƒ•–•‘ˆ–Š‹•’‡”•‘•‡ ”‡–‡†ƒ ›ŽǦ ƒ”‹–‹‡•ƒ–ƒŠ‹‰Š‡””ƒ–‡ƒ†Šƒ† Š‹‰Š‡” ‘ ‡–”ƒ–‹‘‘ˆǤ–Š‡”†‹ˆˆ‡”‡ ‡•„‡–™‡‡–Š‡’”‘–‡‘‡‘ˆ–Š‹•’‡”•‘ƒ†ƒŽŽ ‘–Š‡”•ǡ™‡”‡‘–”‡’”‘†— ‹„Ž‡Ǥ •—‰‰‡•––Šƒ––Š‡†‹ˆˆ‡”‡ ‡•ƒ›„‡Žƒ”‰‡”™Š‡–Š‡ ‡ŽŽ• †‡’‡†‘”‡‘–Š‡ ǡ‡Ǥ‰Ǥ™Š‡–Š‡‰Ž— ‘•‡ ‘ ‡–”ƒ–‹‘‹•Ž‘™‡”‡†ˆ—”–Š‡”Ǥ

ʹͶ͸ Summary

Š‹•–Š‡•‹•Šƒ•ƒ††‡†–‘–Š‡—†‡”•–ƒ†‹‰‘ˆ†‡ˆ‹ ‹‡ ›„›—•‹‰ƒ ‘„‹ƒ–‹‘‘ˆ ›•–‡•‡†‹ ‹‡–‘‘Ž•ǤŠ‡”‡•—Ž–•‘„–ƒ‹‡†„› ‘’—–ƒ–‹‘ƒŽ‘†‡Ž‹‰ƒ†‡š’‡”‹‡–• ™‹–Š‹ ‡ƒ†’ƒ–‹‡–Ǧ†‡”‹˜‡† ‡ŽŽ•ǡ•—‰‰‡•–ƒ‹’‘”–ƒ––Š‡”‘Ž‡‘ˆ‘ ǡ‹–• ‘Œ—‰ƒ–‡† –Š‹‘‡•–‡”•ƒ†ȋȌȋ Ȍ‹ ˆ— –‹‘ƒ††‡ˆ‹ ‹‡ ›ǤŠ‡Š—ƒ˜‡”•‹‘‘ˆ–Š‡ ‘’—–ƒ–‹‘ƒŽ‘†‡ŽȋŠƒ’–‡”ͷȌ™‹ŽŽ„‡ƒ‹’‘”–ƒ––‘‘Ž–‘‹–‡‰”ƒ–‡‡š’‡”‹‡–ƒŽ†ƒ–ƒ ˆ”‘Š—ƒ–‹••—‡ƒ†‘„–ƒ‹ƒ„‡––‡”—†‡”•–ƒ†‹‰‘ˆ†‡ˆ‹ ‹‡ ›ǤŠ‡‰”‘—†™‘” ƒ††‹• —••‹‘•Žƒ‹†‘—–‹–Š‹•–Š‡•‹•’”‘˜‹†‡‘’’‘”–—‹–‹‡•ˆ‘”ˆ‘ŽŽ‘™Ǧ—’•–—†‹‡•–‘—”ƒ˜‡Ž –Š‡‡–‹‘Ž‘‰›‘ˆŠ‡–‡”‘‰‡‡‘—•’Š‡‘–›’‡•‹†‡ˆ‹ ‹‡ ›Ǥ Š‘’‡–Šƒ–‹–Š‡ˆ—–—”‡ǡ–Š‡ ‘„–ƒ‹‡†”‡•—Ž–™‹ŽŽ‡ƒ„Ž‡‘”‡’‡”•‘ƒŽ‹œ‡††‹ƒ‰‘•‡•ǡ’”‘‰‘•‡•ƒ†–”‡ƒ–‡–•–”ƒ–‡‰‹‡• ˆ‘”†‡ˆ‹ ‹‡ ›Ǥ   

ʹͶ͹

  ^ĂŵĞŶǀĂƚƚŝŶŐ     





























 

































 Samenvatting

‹††‡Ǧ‡–‡ ƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡ ȋȌ †‡ˆ‹ ‹´–‹‡ ȋ  ʹͲͳͶͷͲȌ ‹• ‡‡ ‡œ›†‡ˆ‡ – ‹ †‡ ‹–‘ Š‘†”‹´Ž‡ ˜‡–œ——”‘š‹†ƒ–‹‡”‘—–‡Ǥ Ǧ†‡ˆ‹ ‹´–‹‡ ‹• ‡‡ ˜ƒ †‡ ‡‡•–˜‘‘”‘‡†‡ƒƒ‰‡„‘”‡•–‘ˆ™‹••‡Ž‹‰•œ‹‡–‡•Ǥ ‡–‹•‘‘Š‡–‡‡•–˜‘‘”‘‡†‡ ƒƒ‰‡„‘”‡ †‡ˆ‡ – ‹ †‡ ˜‡–œ——”‘š‹†ƒ–‹‡Ǥ   ‡†‡”Žƒ† ™ƒ• †‡ ’”‡˜ƒŽ‡–‹‡ „‹Œ ‰‡„‘‘”–‡ ͳȀͺ͵ͲͲ–—••‡ʹͲͲ͹‡ʹͲͳͷȋ ‘‘ˆ†•–—ͳȌǤ‡‹–‘ Š‘†”‹´Ž‡˜‡–œ——”‘š‹†ƒ–‹‡•’‡‡Ž–‡‡ „‡Žƒ‰”‹Œ‡”‘Ž‹‘œ‡‡‡”‰‹‡Š—‹•Š‘—†‹‰ǡ‡–ƒ‡–‹Œ†‡•˜ƒ•–‡‘ˆƒŽ•™‡˜‡‡Ž‡‡”‰‹‡ ˜‡”„”—‹‡Ǥ†‡”†‡œ‡‘•–ƒ†‹‰Š‡†‡œ‘”‰–˜‡–œ——”‘š‹†ƒ–‹‡‹†‡Ž‡˜‡”˜‘‘”†‡’”‘†— –‹‡ ˜ƒ‡–‘Ž‹ Šƒ‡‡˜‘‘”†‡†‹‡‘†‹‰‹•‘‹‡—™‡‰Ž— ‘•‡ –‡ƒ‡Ǥ ‹‡”‡‡‹•†‹– ’”‘ ‡•˜ƒ‡••‡–‹‡‡Ž„‡Žƒ‰˜‘‘”†‡„”ƒ†•–‘ˆ˜‘‘”œ‹‡‹‰˜ƒ†‡‘”‰ƒ‡‹Š‡–Ž‹ ŠƒƒǤ Ž•Ǧ†‡ˆ‹ ‹´–‡ȋȌ‹†‡”‡‹‡–™‘”†‡„‡Šƒ†‡Ž†ǡŽ‘’‡œ‡‡‡‡”•–‹‰”‹•‹ ‘ ‘’ Ž‡˜‡•„‡†”‡‹‰‡†‡ Žƒ‰‡ „Ž‘‡†•—‹‡”™ƒƒ”†‡ ‡ Žƒ‰‡ ‘ ‡–”ƒ–‹‡• ˜ƒ ‡–‘Ž‹ Šƒ‡Ǥ ‹–™‘”†–Š›’‘‡–‘–‹• Š‡Š›’‘‰Ž› ‡‹‡‰‡‘‡†Ǥ‘ Š‹•„‡• Š”‡˜‡†ƒ–Ǧ’ƒ–‹´–‡ ͳͺ –‘– ʹͶ ——” —‡ ˜ƒ•–‡Ǥ ‡– ‹• †‡ ‘„‹ƒ–‹‡ ˜ƒ Žƒ‰†—”‹‰ ˜ƒ•–‡ ‡– ‡‡ ‡š–”ƒ ”‹•‹ ‘ˆƒ –‘”ǡ œ‘ƒŽ•‡‡ Ž— Š–™‡‰Ǧ ‘ˆ †ƒ”‹ˆ‡ –‹‡ ‘ˆ „Ž‘‘–•–‡ŽŽ‹‰ ƒƒ ‘—ǡ †‹‡ ‰‡˜ƒƒ”Ž‹Œ ‹• ˜‘‘” Œ‘‰‡ Ǧ’ƒ–‹´–‡Ǥ ” ‹• ‡ Š–‡” ‡‡ ‰”‘–‡ Ž‹‹• Š‡ Š‡–‡”‘‰‡‹–‡‹– ‹ †‡œ‡ ’ƒ–‹´–‡‰”‘‡’Ǥ‡Žˆ•„‹‡†‡•—„’‘’—Žƒ–‹‡˜ƒ’ƒ–‹´–‡‡–†‡œ‡Žˆ†‡ ͻͺͷε —–ƒ–‹‡ǡ †‹‡ Š‡– ‡œ› ˜‘ŽŽ‡†‹‰ ‹ƒ –‹˜‡‡”–ǡ —‡ †‡ •›’–‘‡ ˜ƒ”‹´”‡ ˜ƒ ‡‡ ˆƒ–ƒŽ‡ Š›’‘‡–‘–‹• Š‡Š›’‘‰Ž› ‡‹‡–‘–‡‡Ž‡˜‡•Žƒ‰‘–„”‡‡˜ƒ‡‹‰‡œ‹‡–‡˜‡”• Š‹Œ•‡Ž‡ †‹‡ ‡–  ‹ ˜‡”„ƒ† —‡ ™‘”†‡ ‰‡„”ƒ Š–Ǥ ‡‡–‹• Š‡ ‡ ‘‰‡˜‹‰•ˆƒ –‘”‡ —‡‡‡”‘Ž•’‡Ž‡‹†‡Š‡–‡”‘‰‡‹–‡‹–˜ƒ†‡œ‡’ƒ–‹´–‡‰”‘‡’Ǥƒ–†‡‰‡˜‘‡Ž‹‰Š‡‹† ˜ƒ‹†‹˜‹†—‡Ž‡’ƒ–‹´–‡’”‡ ‹‡•„‡’ƒƒŽ–ǡ‹•‡ Š–‡”‹‡–†—‹†‡Ž‹ŒǤ ‹Œ’”‘‡ˆ• Š”‹ˆ–Š‡„‹ †ƒƒ”‘†‡ƒƒ’ƒ˜ƒ†‡Ǯ•›•–‡‡‰‡‡‡•—†‡ǯ‰‡˜‘Ž‰†‘‡‡„‡–‡”‹œ‹ Š––‡”‹Œ‰‡‹ Š‡–‘–•–ƒƒ˜ƒ•›’–‘‡„‹ŒǤ‹Œ‘˜‡”‘‡’‡Ž‡†‡˜”ƒƒ‰‹•™ƒ–‡”˜‘‘”œ‘”‰–†ƒ– „‹Œ •‘‹‰‡ ’ƒ–‹´–‡ ‡‡ Š›’‘‡–‘–‹• Š‡ Š›’‘‰Ž› ‡‹‡ ‘’–”‡‡†–ǡ –‡”™‹ŒŽ ƒ†‡”‡ Š‹‡”–‡‰‡ „‡• Š‡”† Ž‹Œ‡Ǥ  Š‡„ ‘†‡”œ‘ Š– ™‡Ž‡ ‘†—Žƒ–‹‡• „‹‡ ‡ „—‹–‡ †‡ ˜‡–œ——”‘š‹†ƒ–‹‡ǡ †‡ •–‘ˆ™‹••‡Ž‹‰ ˜ƒ—‹– ‡‡ • Š‹Œ„ƒƒ” ‰‡œ‘†‡ –‘‡•–ƒ† ƒƒ” ‡‡ Š›’‘‡–‘–‹• Š‡ Š›’‘‰Ž› ‡‹‡ —‡ †—™‡ǡ ƒƒ” ‘‘ ™‡Ž‡ ˆƒ –‘”‡ Œ—‹•– „‡• Š‡”‹‰ „‹‡†‡ –‡‰‡ †‡œ‡ ‰‡˜ƒƒ”Ž‹Œ‡ –‘‡•–ƒ†Ǥ  †‡œ‡ ˜”ƒ‰‡ –‡ —‡ „‡ƒ–™‘‘”†‡ Š‡„ ‹ Ǧ†‡ˆ‹ ‹´–‹‡ ‘†‡”œ‘ Š– †‘‘” ‹††‡Ž ˜ƒ ‘’—–‡”•‹—Žƒ–‹‡• ‡ ‡š’‡”‹‡–‡ ‡– —‹œ‡‡‡•‡Ž‹Œ‡ ‡ŽŽ‡Ǥ  „‡ „‡‰‘‡ Š‡– ‰‡†”ƒ‰ ˜ƒ ‡‡ ”‡‡†• „‡•–ƒƒ† ‘’—–‡”‘†‡Ž ˜ƒ †‡ ‰‡Ã•‘Ž‡‡”†‡ ˜‡–œ——”‘š‹†ƒ–‹‡ ‹ ”ƒ––‡Ž‡˜‡” –‡ ‘†‡”œ‘‡‡ ȋ ‘‘ˆ†•–— ʹȌǤ ‹– ‘†‡Ž œ‘— ‹ Žƒ–‡” ‹ ‘‘ˆ†•–— ͷ ‘†‹‰ Š‡„„‡ ƒŽ• „ƒ•‹• ˜‘‘” ‡‡ ‘†‡Ž ˜ƒ †‡ ‡•‡Ž‹Œ‡ ˜‡–œ——”‘š‹†ƒ–‹‡Ǥ ‘’—–‡”•‹—Žƒ–‹‡• „”ƒ Š–‡ ‡‡ • Š‡”’‡ †ƒŽ‹‰ ˜ƒ †‡ •‡ŽŠ‡‹† ˜ƒ ˜‡–œ——”‘š‹†ƒ–‹‡ „‹Œ Š‘‰‡ •—„•–”ƒƒ– ‘ ‡–”ƒ–‹‡ ƒƒ Š‡– Ž‹ Š–Ǥ  Š‡„ Š‡– ‡ Šƒ‹•‡ †ƒ– Š‹‡”˜‘‘” ˜‡”ƒ–™‘‘”†‡Ž‹Œ ‹•ǡ ‘–”ƒˆ‡Ž†Ǥ ‡ †ƒŽ‹‰ ˜ƒ †‡ •‡ŽŠ‡‹† Šƒ† †—‹†‡Ž‹Œ –‡ ƒ‡ ‡– †‡ ’”‘‹• —Ö‡‹–˜ƒ†‡„‡–”‘‡‡œ›‡Ǥ ‹‡”‡‡„‡†‘‡Ž‹†ƒ–†‡‡œ›‡˜‡”• Š‹ŽŽ‡†‡ǡ ‘’‡Žƒƒ”Ž‹Œ‡†‡•—„•–”ƒ–‡ƒ ‡’–‡”‡ǡ™‡Ž‡‘’Š—„‡—”– ‘ —””‡”‡˜‘‘”†‡„‹†‹‰ ƒƒŠ‡–ƒ–ƒŽ›–‹• Š‡ ‡–”—˜ƒŠ‡–‡œ›Ǥ ™ƒ‡‡˜‹ ‹‡—œ‡ ‹”‡Ž‘’Š‡–•’‘‘”†‹‡ œ‹Œ‘‘”•’”‘‰Šƒ†‹Š‡–‡œ›‹††‡‡–‡‡–‘ƒ ›ŽǦ–Š‹‘Žƒ•‡ȋȌǡœ‹ Š˜‘‘”–’Žƒ––‡ †‘‘” Š‡– ‰‡Š‡Ž‡ •›•–‡‡ ‡ —‹–‡‹†‡Ž‹Œ Ž‡‹††‡ –‘– Š‡– ‹•–‘”–‡ ˜ƒ †‡ ˜‡–œ——”‘š‹†ƒ–‹‡•‡ŽŠ‡‹†Ǥ‡’”‘‹• —Ö‡‹–˜ƒ™ƒ•‡‡‘‘†œƒ‡Ž‹Œ‡‡˜‘Ž†‘‡†‡ ˜‘‘”™ƒƒ”†‡˜‘‘”†‹–˜‡”• Š‹Œ•‡ŽǤŠƒ†–‡™‡‹‹‰ƒ–ƒŽ›–‹• Š‡ ƒ’ƒ ‹–‡‹–‘‡‡Š‘‰‡ ‹•–”‘‘˜ƒ˜‡–œ—”‡–‡—‡˜‡”™‡”‡Ǥƒƒ”†‘‘”„‡‰‘‡‡–‘Ǧƒ ›ŽǦ‘‘Ž‡ —Ž‡ǡ †‡ •—„•–”ƒ–‡ ˜ƒ ǡ ‘’ –‡ Š‘’‡Ǥ ‡œ‡ ‡–ƒ„‘Ž‹‡–•–ƒ’‡Ž‹‰ ™‡”† ˜‡”•–‡”– †‘‘”

ʹͷͳ Appendices

’”‘†— –”‡‹‰ ˜ƒ Š‡– ˜‘‘”‰ƒƒ†‡ ‡œ›ǡ œ‘†ƒ– ‘‘ ƒ ›ŽǦ‘ ‡–ƒ„‘Ž‹‡–‡ •–”‘‘‘’™ƒƒ”–•„‡‰‘‡‘’–‡Š‘’‡Ǥ†ƒ–†‹–ƒŽŽ‡ƒƒŽ–Š‹‘‡•–‡”•˜ƒ ‘‡œ›œ‹Œǡ Ž‡‹††‡ †‹– –‘– ‡‡ –‡‘”– ƒƒ „‡• Š‹„ƒƒ” ‘‡œ› Ǥ — œ‹Œ †‡ ƒ ›ŽǦ‘ ‡–ƒ„‘Ž‹‡–‡ –‡˜‡•’”‘†— –‡˜ƒ‡‹•˜”‹Œ ‘‡œ›‡‡•—„•–”ƒƒ–˜ƒ†‹–‡œ›Ǥ ‡––‡‘”– ƒƒ ‘‡œ›‡†‡‘˜‡”ƒƒ–˜ƒƒ ›ŽǦ‘Šƒ††ƒƒ”†‘‘”‡‡•–‡””‡‡†‡™‡”‹‰‘’ Ǥ‘‡œ›‹•„‘˜‡†‹‡‡‡‡••‡–‹´Ž‡ ‘ˆƒ –‘”˜‘‘”˜‡‡Žƒ†‡”‡‡œ›‡‹†‡ ˜‡–œ——”‘š‹†ƒ–‹‡Ǥ ‡– –‡‘”– ƒƒ ‘‡œ›  ‘—‹ ‡‡”†‡ †‡ ‘–•–ƒ‡ ‘’•–‘’’‹‰ „‹Œ †ƒƒ”†‘‘”•‡Žƒƒ”†‡”‡•–˜ƒŠ‡–•›•–‡‡Ǥ‹–”‡•—Ž–‡‡”†‡—‹–‡‹†‡Ž‹Œ‹‡‡•–‡” ˜‡”Žƒƒ‰†‡ ˜‡–œ——”‘š‹†ƒ–‹‡•‡ŽŠ‡‹†Ǥ ‡ •Ž‘––‡ ™ƒ ‹ ‡”ƒ Š–‡” †ƒ– †‡ ˜‹ ‹‡—œ‡ ‹”‡Ž ˜‡”•–‡”–™‡”††‘‘”‡‡Š‘‰‡  ‘ ‡–”ƒ–‹‡Ǥ œ‘”‰–˜‘‘”’”‘†— –”‡‹‰˜ƒ†‡ ˜‡–œ——”‘š‹†ƒ–‹‡‡˜‡”‡”‰‡”–†‡‘’Š‘’‹‰˜ƒ‡•–‡”•˜ƒ ‘‡œ› ‡†‡—‹–’—––‹‰˜ƒ ˜”‹Œ„‡• Š‹„ƒƒ” ‘‡œ›Ǥ  ‘‘ˆ†•–—͵Š‡„‹‘†‡”œ‘ Š–™‡Ž‡•–‘ˆ™‹••‡Ž‹‰•’”‘ ‡••‡—‡„‹Œ†”ƒ‰‡ƒƒ†‡ ”‘„——•–Š‡‹†˜ƒ—‹œ‡™ƒƒ”‹Š‡–‰‡†ƒ– ‘†‡‡”–˜‘‘”ǡ™ƒ•—‹–‰‡• Šƒ‡Ž††‘‘” ‰‡‡–‹• Š‡‘†‹ˆ‹ ƒ–‹‡Ǥ †‡œ‡—‹œ‡™ƒ”‡‘‰‹‡–‡‡”†‡”Žƒ‰‡„Ž‘‡†•—‹‡”™ƒƒ”†‡•‘ˆ ‘ ‡–”ƒ–‹‡•˜ƒ‡–‘Ž‹ Šƒ‡ƒƒ‰‡–‘‘†–‹Œ†‡•˜ƒ•–‡Ǥ‡Ž‡˜‡”•˜ƒ—‹œ‡œ‘†‡” Šƒ††‡‡‡Žƒ‰‡”‡ƒ–ƒŽ›–‹• Š‡ ƒ’ƒ ‹–‡‹–˜‘‘”†‡‘š‹†ƒ–‹‡˜ƒ‘ –ƒ‘›ŽǦ‘†ƒ†‡ ‹‡–Ǧ‰‡‘†‹ˆ‹ ‡‡”†‡ —‹œ‡Ǥ ‡œ‡ Žƒ‰‡”‡ ƒ’ƒ ‹–‡‹– ™‡”† ‹‡– ‰‡ ‘’‡•‡‡”† †‘‘” ˜‡”Š‘‘‰†‡ ‡‹™‹–‹˜‡ƒ—• ˜ƒ †‡ ‹•‘‡œ›‡ ‘”–‡Ǧ‡–‡Ǧ ‡ Žƒ‰‡Ǧ‡–‡Ǧƒ ›ŽǦ‘ †‡Š›†”‘‰‡ƒ•‡•ǡ”‡•’‡ –‹‡˜‡Ž‹Œ‡Ǥ‡Žƒ‹•—„–‹‡Ž‡ƒƒ’ƒ••‹‰‡™ƒƒ”‹ †‡‡š’”‡••‹‡˜ƒ‰‡‡†‹‡ ‘†‡”‡˜‘‘”‡œ›‡†‹‡ ‘‡œ›ǡȋȌΪ‘ˆȋȌ ƒŽ• •—„•–”ƒƒ–‘ˆ’”‘†— –Š‡„„‡ǡƒŽ•‡†‡‹‡œ›‡†‹‡„‡–”‘‡œ‹Œ„‹Œ‘–‰‹ˆ–‹‰•”‡ƒ –‹‡•Ǥ ‡œ‡ ƒƒ’ƒ••‹‰‡ —‡ ‘‰‡Ž‹Œ‡”™‹Œ• „‹Œ†”ƒ‰‡ ƒƒ †‡ ”‘„——•–Š‡‹† ˜ƒ †‡ —‹œ‡ œ‘†‡”‡œ‹Œ†‡‘‡‹–‡™ƒƒ”†‘˜‡”†‡”–‡‘†‡”œ‘‡‡Ǥ‡”‡•—Ž–ƒ–‡˜‡”•–‡”‡ ‘‘‹Œ‡‡”†‡”‡„‡˜‹†‹‰‡‹ ‘‘ˆ†•–—ʹ†ƒ– ‘‡œ›‡ „‡Žƒ‰”‹Œœ‹Œ˜‘‘” Š‡–ˆ— –‹‘‡”‡˜ƒ†‡˜‡–œ——”‘š‹†ƒ–‹‡Ǥ  ‘‘ˆ†•–— Ͷ Š‡„ ‹ †‡ Ž‡˜‡”•–‘ˆ™‹••‡Ž‹‰ ‘†‡”œ‘ Š– ™ƒ‡‡” —‹œ‡ œ‘†‡”  ™‡”†‡„Ž‘‘–‰‡•–‡Ž†ƒƒ‡‡ ‘„‹ƒ–‹‡˜ƒ‘—†‡‡˜ƒ•–‡ǡ™ƒ–‡‡‰”‘–‡‡‡”‰‡–‹• Š‡ —‹–†ƒ‰‹‰ –‡™‡‡‰„”‡‰–Ǥ †‡” †‡œ‡ ‘•–ƒ†‹‰Š‡†‡ ˜‘† ‹ ˜‘‘” Š‡– ‡‡”•– Žƒ‰‡”‡ „Ž‘‡†‰Ž— ‘•‡™ƒƒ”†‡ ‹ —‹œ‡ œ‘†‡”  †ƒ ‹ †‡ ‹‡–Ǧ‰‡‘†‹ˆ‹ ‡‡”†‡ —‹œ‡Ǥ ‘˜‡†‹‡Šƒ††‡†‡—‹œ‡œ‘†‡”Žƒ‰‡”‡ƒ‹‘œ——” ‘ ‡–”ƒ–‹‡•‹†‡Ž‡˜‡”Ǥ‡ Šƒ††‡‘‘‡‡œ™ƒƒ”†‡”‡Ž‡˜‡”‡‡‡Š‘‰‡”‡ ‘ ‡–”ƒ–‹‡˜ƒ–”‹‰Ž› ‡”‹†‡•‹†‡Ž‡˜‡” ȋœ‘™‡Ž †‡ –‘–ƒŽ‡ ‘ ‡–”ƒ–‹‡ ƒŽ• †‹‡ ˜ƒ †‡ ‹††‡Ǧ‡–‡ –”‹‰Ž› ‡”‹†‡•Ȍǡ ‡ Š‘‰‡”‡ ‘ ‡–”ƒ–‹‡•˜ƒ‹††‡Ǧ‡–‡ƒ ›ŽǦ ƒ”‹–‹‡•‹Š‡–„Ž‘‡†Ǥ †‡—‹•†‹‡Š‡–•–‡”•–™ƒ• ƒƒ‰‡†ƒƒ†‘‘”†‡ƒˆ™‡œ‹‰Š‡‹†˜ƒǡƒ‹‘‘‡‡‘–•–‡‹‰˜ƒ†‡Ž‡˜‡”™ƒƒ”Ǥ‹– ’ƒ•–„‹Œ‹Œ™ƒƒ”‡‹‰‹ ‘‘ˆ†•–—͵†ƒ–†‡—‹œ‡œ‘†‡”ƒŽ–‹Œ†‡•‰‡™‘‘ ˜ƒ•–‡ ‡‹‰‡ ‡‹‰‹‰ –‘– ‘–•–‡‹‰ ˜ƒ †‡ Ž‡˜‡” Š‡„„‡Ǥ  ‡ ”‡•—Ž–ƒ–‡ •—‰‰‡”‡”‡ †ƒ– ˜‡–œ——”‘š‹†ƒ–‹‡‹†‡Ž‡˜‡”‡‡„‡Žƒ‰”‹Œ‡”‘Ž•’‡‡Ž–‹†‡Š‘‡‘•–ƒ•‡˜ƒ‰Ž— ‘•‡–‹Œ†‡• ˜ƒ•–‡ ‹ ‘„‹ƒ–‹‡ ‡– ‘—Ǥ —‹œ‡ œ‘†‡”  Ž‡‡ ƒ‹‘œ—”‡ –‡ ‰‡„”—‹‡ ƒŽ• ƒŽ–‡”ƒ–‹‡˜‡„”ƒ†•–‘ˆ ‘ –‡ ‘’‡•‡”‡ ˜‘‘” †‡ †‡ˆ‡ –‡ ˜‡–œ——”‘š‹†ƒ–‹‡ǡ œ‘†ƒ– œ‡ –‘ Š ‡‰Ž— ‘•‡‘†‡ƒ‡Ǥ‡‘˜‡”ƒƒ–˜ƒ‹††‡Ǧ‡–‡˜‡–œ—”‡™‡”††‘‘”†‡Ž‡˜‡” ‰‡´š’‘”–‡‡”†‹†‡˜‘”˜ƒƒ ›ŽǦ ƒ”‹–‹‡•‘ˆ‹‰‡„‘—™†‹–”‹‰Ž› ‡”‹†‡•ǡƒŽ†ƒ‹‡–ƒ ˜‡”Ž‡‰‹‰˜ƒ†‡˜‡–œ——”‡–‡•Ǥ  ‘‘ˆ†•–— ͷ Š‡„ ‹ †‡ ‹˜Ž‘‡† ‘†‡”œ‘ Š– ˜ƒ †‡ •–‘ˆ™‹••‡Ž‹‰•”‘—–‡• †‹‡ ‹ ‘–ƒ – •–ƒƒ‡–†‡˜‡–œ——”‘š‹†ƒ–‹‡ǡ‡–ƒ‡†‡‰‡‡™ƒƒ”‹ ‘‡œ›„‡–”‘‡‹•Ǥ Š‡„Š‡–

ʹͷʹ Samenvatting

‡ˆˆ‡ – ˜ƒ †‡œ‡ ’”‘ ‡••‡ ‘’ Š‡–  ˆ‡‘–›’‡ „‡•–—†‡‡”† †‘‘” ‹††‡Ž ˜ƒ ‘’—–‡”•‹—Žƒ–‹‡•˜ƒ†‡Ž‡˜‡”•–‘ˆ™‹••‡Ž‹‰Ǥ‹–ƒ‹œ‹ Š–‰‡˜‡‹™‡Ž‡ˆƒ –‘”‡‡‡ ”‘Ž•’‡Ž‡‹Š‡–œ‹‡–‡„‡‡Ž†˜ƒ‹†‹˜‹†—‡Ž‡’ƒ–‹´–‡Ǥ‡”•–Š‡„‹Š‡– ‘’—–‡”‘†‡Ž—‹– ‘‘ˆ†•–— ʹ ƒƒ‰‡’ƒ•– †‘‘” ‡” †‡ „‡• Š‹„ƒ”‡ ‹‡–‹• Š‡ ’ƒ”ƒ‡–‡”• ˜‘‘” ‡•‡Ž‹Œ‡ ‡œ›‡‹–‡˜‘‡‰‡Ǥ‡”†‡”Š‡„‹‡‡ƒƒ–ƒŽ’”‘ ‡••‡–‘‡‰‡˜‘‡‰††‹‡„‡–”‘‡œ‹Œ„‹Œ †‡‘œ‡––‹‰‡˜ƒƒ ›ŽǦ‘Ǥ‡œ‡ƒƒ’ƒ••‹‰‡ƒƒ–‡†‡˜‡–œ——”‘š‹†ƒ–‹‡•–ƒ„‹‡Ž‡”‡ ˜‘‘”™ƒ‡ †‡ Š‡– ‹•–‘”–‡ ˜ƒ †‡ ˜‡–œ——”‘š‹†ƒ–‹‡ˆŽ—š †‹‡ ‹ ‹ ‘‘ˆ†•–— ʹ Šƒ† „‡•–—†‡‡”†Ǥ ‡ ‰‡•‹—Ž‡‡”†‡ •‡ŽŠ‡†‡ ‡ ‡–ƒ„‘Ž‹‡–’”‘ˆ‹‡Ž‡ ™‡‡”•’‹‡‰‡Ž†‡ †‡ ‡”•– ˜ƒǦǡǦ‡Ǧ†‡ˆ‹ ‹´–‹‡‹‡•‡Ǥ‘‡‹†‡‹‡–Ǧ’”‘†— –‹‡˜‡ › Ž—•˜ƒ ƒ ›ŽǦ‘Š›†”‘Ž›•‡‡”‡ƒ –‹˜‡”‹‰‘†‡”†”—–‡†‘‘”†‡•—„•–”ƒƒ–•’‡ ‹ˆ‹ ‹–‡‹–˜ƒͳ–‡ ˜‡”ƒ†‡”‡ǡ „Ž‡‡ †‹– †‡ ˜‡–œ——”‘š‹†ƒ–‹‡ ‡– ƒ‡ ‹ Š‡–  ‘†‡Ž –‡ ”‡‡Ǥ ‡ ”‡•—Ž–ƒ–‡ ‰‡˜‡ ‡‡ ‡‡”•–‡ ‹†”— Š‘‡ ‰‡‡–‹• Š‡ ˜‡”• Š‹ŽŽ‡ ‡– „‡–”‡‹‰ –‘– †‡ ’”‘ ‡••‡ ”‘†‘ †‡ ˜‡–œ——”‘š‹†ƒ–‹‡ —‹–‡‹†‡Ž‹Œ †‡ ˜‡–œ——”‘š‹†ƒ–‹‡ œ‡Žˆ —‡ „‡Ã˜Ž‘‡†‡‹Ǧ’ƒ–‹´–‡Ǥ  ‘‘ˆ†•–—͸Š‡„‹–‡•Ž‘––‡‰‡œ‘ Š–ƒƒ”ƒƒ’ƒ••‹‰‡‹†‡•–‘ˆ™‹••‡Ž‹‰™ƒƒ”‡‡™‡ ‘†‡”• Š‡‹†—‡ƒ‡–—••‡Ǧ’ƒ–‹´–‡‡–‡œ‘†‡”•›’–‘‡Ǥ Š‡„†‹– ‘†‡”œ‘ Š– ‹ ‰‡™‡‡–‡ ˆ‹„”‘„Žƒ•–‡ ˜ƒ †‡ Š—‹† †‹‡ ™ƒ”‡ ƒˆ‰‡‘‡ ˜ƒ ‹†‹˜‹†—‡Ž‡ Ǧ’ƒ–‹´–‡Ǥ ‡ ˆ‹„”‘„Žƒ•–‡ ˜ƒ •›’–‘ƒ–‹• Š‡ ‡ ƒ•›’–‘ƒ–‹• Š‡ ’ƒ–‹´–‡ Ž‡‡•–‡”‘’‡Žƒƒ”Ǥ‘‹‰‡ƒ•›’–‘ƒ–‹• Š‡’ƒ–‹´–‡Šƒ††‡Š—†‹ƒ‰‘•‡‡ Š–‡”‘’ Œ‘‰‡ Ž‡‡ˆ–‹Œ† ‰‡”‡‰‡ ‡ Œ‡ —– Œ‡ ƒˆ˜”ƒ‰‡ ‘ˆ œ‡ ƒ•›’–‘ƒ–‹• Š ™ƒ”‡ ˜ƒ™‡‰‡ Š— „‡Šƒ†‡Ž‹‰ ‘ˆ ˜ƒ™‡‰‡ Š— ‰‡‡–‹• Š‡ ƒ Š–‡”‰”‘†Ǥ ± Ǧ’ƒ–‹´– ™ƒ• ‡ Š–‡” ’ƒ• ‰‡†‹ƒ‰‘•–‹ ‡‡”† ‘’ ͵ͲǦŒƒ”‹‰‡ Ž‡‡ˆ–‹Œ† ‡ ™ƒ• ™‡”‡Ž‹Œ Ž‡˜‡•Žƒ‰ ƒ•›’–‘ƒ–‹• Š ‰‡„Ž‡˜‡ǡ œ‘†‡” ‡‹‰‡ „‡Šƒ†‡Ž‹‰Ǥ ‡ ˆ‹„”‘„Žƒ•–‡ ˜ƒ †‡œ‡ ’‡”•‘‘ ˜‡”–‘‘†‡ ‡‡ Š‘‰‡”‡ —‹–• Š‡‹†‹‰ ˜ƒ ƒ ›ŽǦ ƒ”‹–‹‡• ‡‡ Š‘‰‡”‡ ‘ ‡–”ƒ–‹‡˜ƒǤ†‡”‡ ˜‡”• Š‹ŽŽ‡ –—••‡ Š‡– ’”‘–‡‘‘ ˜ƒ †‡ ˆ‹„”‘„Žƒ•–‡ ˜ƒ †‡œ‡ ’‡”•‘‘ ‡ †ƒ– ˜ƒ ƒŽŽ‡ ƒ†‡”‡™ƒ”‡‹‡–Š‡”ŠƒƒŽ„ƒƒ”Ǥ •–‡Ž˜‘‘”†ƒ–†‡˜‡”• Š‹ŽŽ‡‘‰‡Ž‹Œ‰”‘–‡”œ—ŽŽ‡œ‹Œ ƒŽ•†‡ ‡ŽŽ‡‡‡”ƒˆŠƒ‡Ž‹Œ™‘”†‡‰‡ƒƒ–˜ƒ˜‡–œ——”‘š‹†ƒ–‹‡ǡ„‹Œ˜‘‘”„‡‡Ž††‘‘”†‡ ‰Ž— ‘•‡ ‘ ‡–”ƒ–‹‡˜‡”†‡”‘Žƒƒ‰–‡„”‡‰‡Ǥ ‡– ‹Œ ’”‘‡ˆ• Š”‹ˆ– Š‘‘’ ‹ –‡ Š‡„„‡ „‹Œ‰‡†”ƒ‰‡ ƒƒ Š‡– „‡‰”‹’ ˜ƒ  †‘‘” ‰‡„”—‹ –‡ ƒ‡ ˜ƒ ‡–Š‘†‡• —‹– †‡ •›•–‡‡‰‡‡‡•—†‡Ǥ ‡ ”‡•—Ž–ƒ–‡ †‹‡ ‹ Š‡„ ˜‡””‡‰‡†‘‘”‹††‡Ž˜ƒ ‘’—–‡”•‹—Žƒ–‹‡•‡‡š’‡”‹‡–‡‡–—‹œ‡‡ ‡ŽŽ‡˜ƒ ’ƒ–‹´–‡ǡ•—‰‰‡”‡”‡†ƒ– ‘‡œ›ǡ‡•–‡”•˜ƒ ‘‡œ›‡ȋȌ ‡‡„‡Žƒ‰”‹Œ‡”‘Ž •’‡Ž‡‹Š‡–ˆ— –‹‘‡”‡˜ƒ†‡˜‡–œ——”‘š‹†ƒ–‹‡™ƒ‡‡”ƒˆ™‡œ‹‰‘ˆ‹ƒ –‹‡ˆ‹•Ǥ‡ ‡•‡Ž‹Œ‡ ˜‡”•‹‡ ˜ƒ Š‡– ‘’—–‡”‘†‡Ž ȋ ‘‘ˆ†•–— ͷȌ ƒ ‡‡ „‡Žƒ‰”‹Œ Š—Ž’‹††‡Ž œ‹Œ ‘ ‡š’‡”‹‡–‡Ž‡ ‰‡‰‡˜‡• —‹– ‡•‡Ž‹Œ‡ ™‡‡ˆ•‡Ž• –‡ ‹–‡‰”‡”‡ ‡ ‘ ‡‡ „‡–‡” „‡‰”‹’ –‡ ”‹Œ‰‡ ˜ƒ ͵Ǥ ‡ „ƒ•‹• †‹‡ ‹ ‹ †‹– ’”‘‡ˆ• Š”‹ˆ– Š‡„ ‰‡Ž‡‰†ǡ „‹‡†– ‘‰‡Ž‹ŒŠ‡†‡ ˜‘‘” ˜‡”˜‘Ž‰•–—†‹‡• ‘ Š‡– Š‡–‡”‘‰‡‡ œ‹‡–‡„‡‡Ž† ˜ƒ  ˜‡”†‡” –‡ ‘–”ƒˆ‡Ž‡Ǥ Š‘‘’†ƒ–†‡”‡•—Ž–ƒ–‡‹†‡–‘‡‘•–’‡”•‘‘Ž‹Œ‡”‡†‹ƒ‰‘•‡•ǡ’”‘‰‘•‡•‡ „‡Šƒ†‡Ž‹‰‡˜‘‘”Ǧ’ƒ–‹´–‡‘‰‡Ž‹Œœ—ŽŽ‡ƒ‡Ǥ   

ʹͷ͵

Acknowledgements

 

Acknowledgements

Š‡›‡ƒ”•‘ˆ›Š™‡”‡ƒ„‹‰ƒ†˜‡–—”‡ǡˆ—ŽŽŠƒ”†™‘”ǡ‹–‡”‡•–‹‰‡˜‡–•ǡˆ”—•–”ƒ–‹‘•ǡ •— ‡••‡•ǡˆƒ‹Ž—”‡•ǡƒ†‡™†‹• ‘˜‡”‹‡•Ǥ—”‹‰›Šǡ ‹–‡”ƒ –‡†™‹–ŠƒŽ‘–‘ˆ’‡‘’Ž‡ǡ‡ƒ Š ‘ˆ™Š‘ ‘–”‹„—–‡†‹–Š‡‹”‘™™ƒ›–‘›ŠǤ –Š‹••‡ –‹‘ ™‘—Ž†Ž‹‡–‘–ƒ‡–Š‡ ‘’’‘”–—‹–›–‘‡š’”‡••›‰”ƒ–‹–—†‡Ǥ  ƒ”„ƒ”ƒƒ†‹”Ǧ ƒǡƒ•ƒŠ ƒ†‹†ƒ–‡™‹–Š›‘—ƒ•’”‘‘–‘”•ǡ Šƒ˜‡Ž‡ƒ”‡†ƒŽ‘–ǡ‘‹••—‡• ™‹–Š‹ ƒ†‘—–•‹†‡”‡•‡ƒ” ŠǤ Šƒ ›‘— ˆ‘” ›‘—” ’‡”•’‡ –‹˜‡• ‘ Š‘™ –‘ Šƒ†Ž‡ ’”‘Œ‡ –•ǡ ’‡‘’Ž‡ƒ†Š‹‰‡‡”ƒŽǤƒ”„ƒ”ƒǡ†—”‹‰›Š’‡”‹‘†‹›‘—”‰”‘—’›‘—‰ƒ˜‡‡–Š‡ ‘’’‘”–—‹–›–‘‡š’Ž‘”‡•›•–‡•„‹‘Ž‘‰›ǡ•›•–‡•‡†‹ ‹‡ǡ„‹‘‹ˆ‘”ƒ–‹ •ǡ‘Ž‡ —Žƒ”„‹‘Ž‘‰› ƒ† •‡˜‡”ƒŽ ‘–Š‡” –‡ Š‹“—‡• –‘ „‡––‡” —†‡”•–ƒ† „‹‘ Š‡‹•–”›ǡ ‡–ƒ„‘Ž‹• ƒ† ‹„‘” ‡””‘”•‘ˆ‡–ƒ„‘Ž‹•ȋ ȌǤ ™ƒ•ƒŽ•‘ƒ„Ž‡–‘™‘”™‹–Š Ž‹‹ ‹ƒ•–‘„‡‡ˆ‹–’ƒ–‹‡–•™‹–Šƒ ǤŠƒ›‘—ǤŠƒ›‘—ˆ‘”‡ ‘—”ƒ‰‹‰‡–‘‰‘‘‹–‡”‡•–‹‰ƒ†‹•’‹”‹‰ ‘‰”‡••‡•Ǥ Š‡ ʹͲͳͶ‹‡Ž„‘—”‡ǡ—•–”ƒŽ‹ƒǡƒˆ–‡””‡ ‡‹˜‹‰ƒ”‡‰‹•–”ƒ–‹‘’”‹ ‡ƒ†ƒŽ•‘ƒ–”ƒ˜‡Ž ‰”ƒ–ǡ‡˜‡–Š‘—‰Š–Š‡”‡™‡”‡ƒ„— Š‘ˆ‰”‡ƒ–‡˜‡–•ƒˆ–‡”™ƒ”†•ǡ™ƒ•’ƒ”–‹ —Žƒ”Ž›•’‡ ‹ƒŽǤ ‘—ƒŽ•‘†‹†›‘—”„‡•––‘ƒ ‘‘†ƒ–‡ƒŽŽ›‘—”†‹˜‡”•‡”‡•‡ƒ” Š‰”‘—’‡„‡”•ƒ†  ‘—Ž† ˜‘‹ ‡›•— ‡••‡•ƒ†ˆ”—•–”ƒ–‹‘•–‘›‘—ǤŠ”‘—‰Š”‡‰—Žƒ”†‹• —••‹‘•™‡ƒ†‡•‡˜‡”ƒŽ †‹• ‘˜‡”‹‡•ƒ†ƒ”‡‘‘—”™ƒ›–‘’—„Ž‹•Šƒ›‘ˆ–Š‡Ǥ ‹ƒŽŽ›ǡ–Šƒ›‘—ˆ‘”„”‹‰‹‰•‘‡ ‰”‡ƒ– ƒ† Ž‘˜‡Ž› ”‡•‡ƒ” Š‡”• ‹–‘ –Š‡ ‰”‘—’Ǥ ‹”Ǧ ƒǡ ›‘—” ‡–Š—•‹ƒ• ƒ†‘™Ž‡†‰‡ ‹ „‹‘ Š‡‹•–”›ǡ Ž‹‹ ƒŽ Š‡‹•–”›ƒ†‡–ƒ„‘Ž‹ †‹•‡ƒ•‡‹•˜‡”›ƒ†‹”ƒ„Ž‡ǤŠƒ–ˆ‹–•˜‡”›™‡ŽŽ ™‹–Š›‘™‡–Š—•‹ƒ•ƒ†‹–‡”‡•–•‹–Š‡•‡ˆ‹‡Ž†•Ǥ‡Šƒ†˜‡”›‹–‡”‡•–‹‰ǡ‡ ‘—”ƒ‰‹‰ǡ ‹˜ƒŽ—ƒ„Ž‡†‹• —••‹‘•ƒ„‘—–‘–‘Ž›–Š‡•‡–‘’‹ •ǡ›’”‘Œ‡ –•‹’ƒ”–‹ —Žƒ”ǡ„—–ƒŽ•‘ƒ„‘—– ›’‘–‡–‹ƒŽˆ—–—”‡Š‡”‡‹ǤŽ•‘–Šƒ›‘—ˆ‘”Ž‡––‹‰‡’‘—”›Š‡ƒ”–‘—––‘›‘—‹ƒ› ‘ ƒ•‹‘•ǤŠƒ›‘—Ǥ  ™‘—Ž†ƒŽ•‘Ž‹‡–‘–Šƒ›ƒ••‡••‡– ‘‹––‡‡ǡ’”‘ˆǤ ”ƒ”—‰‰‡ƒǡ’”‘ˆǤ‡”– ”‘‡ ƒ†’”‘ˆǤ ”ƒ Œƒ˜ƒ’”‘•‡ǡˆ‘”–ƒ‹‰–Š‡–‹‡–‘”‡ƒ†ƒ†ƒ’’”‘˜‡›–Š‡•‹•Ǥ ”ƒǡ  ƒ†‹”‡ ›‘—” ’ƒ••‹‘ ƒ† ‡š–‡•‹˜‡ ‘™Ž‡†‰‡ ‹ •›•–‡• „‹‘Ž‘‰›ǡ ‡œ›‡ ‹‡–‹ • ƒ† –Š‡”‘†›ƒ‹ •Ǥ‡”–ǡ›‘—”„”‘ƒ†‘™Ž‡†‰‡ǡ›‘—”œ‡•–ˆ‘”Ž‹ˆ‡ƒ†ˆ”‹‡†Ž‹‡••Šƒ˜‡ƒŽ™ƒ›• ‹’”‡••‡†‡Ǥ ”ƒ Œƒǡ ƒ†‹”‡›‘—”‹–‡”‡•–ƒ•ƒ Ž‹‹ ‹ƒ‹•›•–‡•„‹‘Ž‘‰›Ǥ –‰‹˜‡•‡ ‰”‡ƒ–Œ‘›–‘•‡‡ Ž‹‹ ‹ƒ•‹–‡”‡•–‡†‹‹–‡‰”ƒ–‹‰–Š‡•‡–›’‡•‘ˆƒ’’”‘ƒ Š‡•–‘—†‡”•–ƒ† ƒ†„‡‡ˆ‹––Š‡Š‡ƒŽ–Š‘ˆ’ƒ–‹‡–•ǤŠƒ›‘—ƒŽŽǤ  ™‘—Ž†Ž‹‡–‘–ŠƒƒŽŽ–Š‡’”‡•‡–ƒ†ˆ‘”‡”‡„‡”•‘ˆ–Š‡ƒ”„ƒ”ƒǯ•‰”‘—’ǤŽ„‡”–ǡ›‘— Šƒ˜‡ˆ”‘–Š‡•–ƒ”–„‡‡ƒ‰”‡ƒ–Š‡Ž’ƒ†• ‹‡–‹ˆ‹ ƒ†‡‘–‹‘ƒŽ•—’’‘”–ˆ‘”‡–Š”‘—‰Š‘—– –Š‡›‡ƒ”•Ǥ Šƒ˜‡‰”‡ƒ–‰”ƒ–‹–—†‡ˆ‘”™Šƒ–›‘—†‘ƒ†Š‘™›‘—Šƒ†Ž‡ƒŽŽ‘ˆ—•Ǥ ˆ‡‡ŽŽ‹‡›‘— ƒ”‡–Š‡‰Ž—‡–Šƒ–‡‡’•—•ƒŽŽ–‘‰‡–Š‡”ƒ†‘–‹˜ƒ–‡†–‘‰‘‘‹–Š‡Šƒ”†ŠŽ‹ˆ‡Ǥˆ ‘—”•‡ǡ ‹–ǯ•–‘‘— Š–‘‡–‹‘™Šƒ–›‘—†‹†ˆ‘”ƒ†™‹–Š‡Ǥ ”‘•ƒ’Ž‡’”‡’ǡ•ƒ’Ž‡ƒƒŽ›•‹•ǡ

ʹ ‘•—’–‹‘•ǡ ‘—•‡ ‡š’‡”‹‡–•ǡ ƒ –‹˜‡ ’ƒ”–‹ ‹’ƒ–‹‘ ‹ ‡‡–‹‰• ƒ† ƒ†˜‹ ‡ ƒ„‘—– ‡š’‡”‹‡–•ƒ††ƒ–ƒ‹–‡”’”‡–ƒ–‹‘ƒ†‘—–•‹†‡–Š‡Žƒ„ƒ‰”‡ƒ–ƒ†‡’ƒ–Š‹ ’‡”•‘–‘–ƒŽ –‘ƒ†ƒ‡š ‡ŽŽ‡– ‘‘ǨŠƒ›‘—˜‡”›— ŠǨ ‹Œ•ǡ–Šƒ›‘—ˆ‘”ƒŽ™ƒ›•„‡‹‰™‹ŽŽ‹‰–‘ Š‡Ž’–Š”‘—‰Š‘—–›Šǡ‡˜‡™ƒ›ƒˆ–‡”›‘—Ž‡ˆ–‘—”‰”‘—’ǡ›‘—ƒ”‡ƒŽ™ƒ›•“—‹ –‘ƒ•™‡” ƒ›“—‡•–‹‘• Šƒ˜‡Ǥƒ”‡ǡ–Šƒ›‘—ˆ‘”†‘‹‰–Š‡‰”‘—†™‘”ˆ‘”–Š‡‘†‡Ž‹‰–Šƒ– †‹† ‹›ŠǤ‘—”‹’—–™ƒ•‹’‘”–ƒ–‹›•–ƒ”–‹‰—’‹‘†‡Ž‹‰ƒ†‹–Š‡Žƒ„ǤŽ•‘–Šƒ ›‘—ˆ‘””‡˜‹‡™‹‰›™‘”‹–Š‡„‡‰‹‹‰ƒ†ˆ‘” Š‡ ‹‰›”ƒ–‘†‡ŽƒƒŽ›•‹•”‡•—Ž–•Ǥ —”‰‡ǡ–Šƒ›‘—ˆ‘”–ƒ‹‰ƒ‹–‡”‡•–‹›™‘”ƒ†Šƒ˜‹‰‰”‡ƒ–“—‡•–‹‘•Ȁ ‘‡–•

ʹͷ͹ Appendices

†—”‹‰‘—”‡‡–‹‰•Ǥ ‘Ž‹–ƒǡ–Šƒ›‘—ˆ‘”–‡ƒ Š‹‰‡Š‘™–‘’”‡’ƒ”‡ˆ‘”ƒ†ƒƒŽ›œ‡†ƒ–ƒ ˆ”‘”‘„‘”‘•ǡ’”‘–‡‘‹ •ƒ†‘—•‡‡š’‡”‹‡–•Ǥ ‹‘ƒǡ ‡”–Œƒȋƒ† ‘Ž‹–ƒȌǡ–Šƒ›‘—ˆ‘” ‰‹˜‹‰‡–Š‡‘’’‘”–—‹–›–‘Ž‡ƒ”ƒ†’”ƒ –‹ ‡–‡”‹ƒ–‹‘•ƒ––Š‡‡”‹‡ˆƒ ‹Ž‹–›ǤŠ‘†Šƒ ƒ‘ǡ Šƒ†ƒ‰‘‘† ‘‡ –‹‘™‹–Š›‘—ˆ”‘–Š‡•–ƒ”–Ǥ‡Šƒ†•— Šƒ‰”‡ƒ––‹‡ƒ––Š‡  ʹͲͳ͵Ǥš’Ž‘”‹‰–Š‡ ‹–›ǡ Ž‹„‹‰–Š‡–‘™‡”ƒ†Œ—•–„‡‹‰–Š‡”‡ˆ‘”‡ƒ Š‘–Š‡”‹–Š‡‰”‘—’ ƒ†„‡‹‰ƒ•Š‘—Ž†‡”–‘ ”›‘ǤŠƒ›‘—ˆ‘”•Šƒ”‹‰›‘—”ƒ–Š‡ƒ–‹ ••‹ŽŽ•ƒ†™‹•‡ƒ† Š‘‡•–™‘”†•™‹–Š‡ǡˆ‘”ƒŽŽ‘™‹‰‡‹–‘›‘—”Š‘‡ǡ’Žƒ›‹‰’‹‰’‘‰ƒ†„‡‹‰ƒ‰‘‘† ˆ”‹‡†Ǥƒ›—ǡ–Šƒ›‘—ˆ‘”Š‡Ž’‹–”›‹‰–‘ˆ‹‰—”‡‘—––Š‡„‡Šƒ˜‹‘”•‘ˆ–Š‡”ƒ– ‘’—–ƒ–‹‘ƒŽ  ‘†‡ŽǤŠ”‹•ǡ–Šƒ›‘—ˆ‘”‘—”‰‡œ‡ŽŽ‹‰‡–ƒŽ•‹–Š‡Žƒ„ǡƒ†›‘—”’ƒ–‹‡ ‡™Š‡ ™ƒ• –ƒ‹‰˜‡”›Ž‘‰‹–Š‡Š‘‘†Ǥ‡Žƒ›ǡ–Šƒ›‘—ˆ‘”›‘—”Š‡Ž’‘–Š‡ ‡’ ʹ’”‘Œ‡ –ƒ†ˆ‘” ƒŽ™ƒ›•„‡‹‰•‘‹ ‡ƒ†ˆ—›Ǩ‡”ƒ”†ǡ –Š‹›‘—ǯ”‡ƒ‰”‡ƒ–‰—›™‡Šƒ˜‡†‘‡“—‹–‡•‘‡ •Šƒ”‹‰–Š”‘—‰Š‘—––Š‡›‡ƒ”•ƒ† ƒŽ™ƒ›•ˆ‹†‹–ƒ„”‡‡œ‡ƒ†–Š‡”ƒ’‡—–‹ ™Š‡ –ƒŽ–‘›‘—Ǥ ‘— ƒƒŽ™ƒ›• ‘‡–‘‡™‹–Š“—‡•–‹‘•‘”Œ—•––‘•Šƒ”‡Š‘™–Š‹‰•ƒ”‡‰‘‹‰Ǥ ‡–ƒ™ǡ –Šƒ›‘—„‡‹‰ƒƒ††‹–‹‘ƒŽƒ••‡–‹‘†‡Ž‹‰‹‘—”‰”‘—’Ǥ –Š‹›‘—ƒ”‡†‘‹‰‹’‘”–ƒ– ™‘”–‘—†‡”•–ƒ†™Šƒ–™‡ƒ”‡‘†‡Ž‹‰‹–Š‡‰”‘—’Ǥ‘–•‘ˆ–Š‘•‡–Š‹‰• ™‘—Ž†ǯ–Šƒ˜‡ „‡‡ƒ„Ž‡–‘†‘–Š‡ƒ•™‡ŽŽƒ•›‘—ǨŠ”‹•–‘ˆˆ†‡†ƒƒŽǡ–Šƒ›‘—ˆ‘”„‡‹‰‹–‡”‡•–‡†‹ ‘–‹—‹‰›™‘”ƒ†ƒŽ”‡ƒ†›‡–‹ —Ž‘—•Ž›”‡˜‹‡™‹‰›Žƒ–‡•– ‘’—–ƒ–‹‘ƒŽ‘†‡ŽǤ  –Š‹›‘—™‹ŽŽ„‡ƒ‹‰•‘‡‹–‡”‡•–‹‰†‹• ‘˜‡”‹‡•”‡Žƒ–‡†–‘†‡ˆ‹ ‹‡ ›ǤƒŽ‹‡ǡ –Šƒ›‘—ˆ‘”„‡‹‰›•‡ ‘†‹–‡”Ǥ Ž‡ƒ”‡†ƒŽ‘–ƒ•›‘—”•—’‡”˜‹•‘”ƒ†Š‘’‡–‘ƒŽ•‘Šƒ˜‡ ’ƒ••‡† ‘ •‘‡ ‘ˆ › ‡š’‡”‹‡ ‡ –‘ ›‘—Ǥ Šƒ ›‘— ˆ‘” ‘–‹—‹‰ –‘ ”‡•‡ƒ” Š  †‡ˆ‹ ‹‡ ›‹‘—”‰”‘—’Ǥƒ”ƒŠƒ†‰‹‡•œƒǡ›ˆ‡ŽŽ‘™–™‹•ǡ–Š‘—‰Š™‡†‹††‹ˆˆ‡”‡–™‘” –Š”‘—‰Š‘—–‘—”Šǡ™‡™‡”‡ƒ„Ž‡–‘‡š’‡”‹‡ ‡ƒŽ‘––‘‰‡–Š‡”ƒ†•—’’‘”–‡ƒ Š‘–Š‡”‹ ƒ›™ƒ›•Ǥ ƒ‰”ƒ–‡ˆ—Ž–‘Šƒ˜‡‡–ƒ††‹†›Š™‹–Š„‘–Š‘ˆ›‘—ǤŠƒ›‘—ˆ‘”‘—” †‹• —••‹‘•ƒ„‘—–™‘”ƒ†•—’’‘”–‹‰‡–Š”‘—‰Š‘—–ƒŽŽ–Š‡—’•ƒ††‘™•Ǥ–—†›‹‰‹–Š‡ ‡–Š‡”Žƒ†•Ȁ—”‘’‡ǡ‹•„›ˆƒ”‘––Š‡•ƒ‡ƒ•‰”‘™‹‰—’Š‡”‡ǤŠƒ•ˆ‘”‘’‡‹‰›‘—” ‹†• ƒ† Š‡ƒ”–• •Šƒ”‹‰ ›‘—” —”‘’‡ƒ ‘™Ž‡†‰‡ ƒ† ‡š’‡”‹‡ ‡ ™‹–Š ‡Ǥ ‰‹‡•œƒǡ –Šƒ›‘—ˆ‘”›‘—”Š‡Ž’‹•‹†‡ƒ†‘—–•‹†‡–Š‡Žƒ„ǡ–Š”‘—‰Š›‘—”‡š ‡ŽŽ‡–™‡–ƒ††”›Žƒ„ •‹ŽŽ•ǡ–Šƒ•ˆ‘”›‘—”’Žƒ›ˆ—Ž‡••ǡ›‘—”‰ƒ‡‹‰Š–•ǡ›‘—”ˆ—›‡”†›Œ‘‡•ǡ–‡ƒ Š‹‰‡ ƒ„‘—–—”‘’‡ƒŠ‹•–‘”›ƒ†‘Ž‹•Š —Ž–—”‡ǡ‹–”‘†— ‹‰‡–‘–Š‡Š†ƒ› ‘‹––‡‡ƒ†–‘ ‹’”‘˜ǡ‰—ƒ”†‹‰‡‘–Š‡‹††›•Ž‘’‡†—”‹‰›ˆ‹”•– •‹‹‰–”‹’ȋǣǦȌƒ†‘—”’‡”•‘ƒŽ ‘˜‡”•ƒ–‹‘•Ǥ Š”‘—‰Š ›‘—” Šƒ”† ƒ† ‹’”‡••‹˜‡ ™‘” ‹ ƒ† ‘—–•‹†‡ ‘ˆ –Š‡ ‘ˆˆ‹ ‡ǡ ‹– ‹• ‡˜‹†‡––Šƒ–›‘—ƒ”‡ƒ’‡”•‘–‘„‡ƒ†‹”‡†Ǥƒ”ƒŠǡ•‹‹Žƒ”Ž›™‡•Šƒ”‡†•‡˜‡”ƒŽ‡š’‡”‹‡ ‡• ‹•‹†‡ƒ†‘—–•‹†‡–Š‡‘ˆˆ‹ ‡Ǥ†‘ˆ ‘—”•‡ǡˆ‘”ƒŽŽ›‘—”Š‡Ž’™‹–Š‡“†ƒ–ƒƒƒŽ›•‹•‹ ƒ†‘–Š‡”—•‡ˆ—Ž–‘‘Ž•Ǥ ™ƒ•ƒŽ•‘ƒ„Ž‡–‘Šƒ˜‡‰”‡ƒ– ‘˜‡”•ƒ–‹‘•™‹–Š›‘—ǡ‡˜‡–ƒŽ•ƒ„‘—– Ž‘˜‡ƒ† ‘—‹ ƒ–‹‘ǡ™Š‹ Š ”‡ƒŽŽ›Ž‹‡Ǥ ™‘—Ž†Ž‹‡–‘–Šƒ„‘–Š›‘—ƒ†‰‹‡•œƒˆ‘” ‘—”˜‡”›‹ ‡†‹‡”•ƒ†‘˜‹‡‹‰Š–•–‘‰‡–Š‡”™‹–Šƒƒ”–‡ǡ‹‘ǡƒ‹‡Žƒƒ†‘–Š‡”•ǡ‘—” Šƒ‹”‹‰Š–ƒ†ˆ—ƒ– ‘ˆ‡”‡ ‡•‡– Ǥ‘™  ƒƒŽ•‘ˆ‹ƒŽŽ›–ƒ‡ƒ‰‘‘†Ž‹•–‡–‘DzŠ‡”–‘ˆ ‘˜‹‰dzƒ—†‹‘„‘‘›‘—”‡ ‘‡†‡†‡-Ǥƒ” ‡Žǡ›‘— ƒ‡‹–‘›Ž‹ˆ‡‹–Š‡‡†‘ˆʹͲͳͷ ƒ†›Ž‹ˆ‡Šƒ•‡˜‡”„‡‡–Š‡•ƒ‡ǣǦǤ‡ Ž‹ ‡†ˆ”‘–Š‡‰‡–Ǧ‰‘ƒ†›‘—†‹†‘–‘Ž› „‡ ‘‡ƒ‡š ‡ŽŽ‡–‹–‡”ǤŠ”‘—‰ŠŠƒ”†™‘”™‡ƒ†‡ƒǦ ‡ŽŽŽ‹‡ǡ‡š’ƒ†‡†ƒ ‘†‡Žǡ ‘‘”†‹ƒ–‡†‡š’‡”‹‡–•–‘‰‡–Š‡”ǡ•Šƒ”‡†•‘‡Š‹Žƒ”‹‘—•Œ‘‡•ǡ™‡–‘ƒ™‘”•Š‘’ –‘‰‡–Š‡”‹ƒ”•‡‹ŽŽ‡ǡ•Šƒ”‡†„‡ƒ—–‹ˆ—Ž• ‡‡”‹‡•‹ƒ” ‡Ž‘ƒƒ†ƒ”•‡‹ŽŽ‡ȋ‘–—‰‡–ǨȌǡ ™‡––‘‡‘”ƒ„Ž‡‡˜‡–•‘—–•‹†‡–Š‡Žƒ„ǡ‹–”‘†— ‡†‡–‘•‘‡ˆƒ–ƒ•–‹ ’‡‘’Ž‡ȋŽ‹‡‡†”‘ ƒ†ƒ’Šƒ‡ŽȌƒ†™‡•—’’‘”–‡†‡ƒ Š‘–Š‡”–Š”‘—‰ŠŽƒ„ƒ†’‡”•‘ƒŽ—’•ƒ††‘™•Ǥ‘—” ˆ”‹‡†•Š‹’‡ƒ•ƒŽ‘––‘‡ǡƒ† ™‹•Š›‘—ƒ‰”‡ƒ–ƒ‘—–‘ˆŽ‘˜‡ƒ†•— ‡••‹›‘—”Ž‹ˆ‡Ǩ

ʹͷͺ Acknowledgements

 ‘ƒŽŽ‘ˆ–Š‡ ‘ƒ—–Š‘”•ǡ ‘ŽŽƒ„‘”ƒ–‘”•ǡƒ†‹’‘”–ƒ– ‘–”‹„—–‘”• Šƒ˜‡‘–‡–‹‘‡†›‡–ǡ –Šƒ›‘—ˆ‘”–Š‡‹’‘”–ƒ–”‘Ž‡•›‘—’Žƒ›‡†‹›’”‘Œ‡ –•Ǥ‡””›ǡ–Šƒ›‘—ˆ‘”„‡‹‰ƒ‹† ’‡†‹ƒ–”‹ ‹ƒǡ ‹˜‘Ž˜‡† ‹ •‡˜‡”ƒŽ ‘ˆ › ’”‘Œ‡ –• –‘ ƒ• Ž‹‹ ƒŽŽ›Ǧ”‡Ž‡˜ƒ– “—‡•–‹‘•ǡ ‰‹˜‡ Ž‹‹ ƒŽ‹•‹‰Š–ǡƒ† ‘•–ƒ–Ž›™‘”‹‰‘ƒ‹–ƒ‹‹‰ƒ ‘—‹ ƒ–‹‘ƒ† ‘ŽŽƒ„‘”ƒ–‹˜‡ Ž‹• „‡–™‡‡ –Š‡ Ž‹‹  ƒ† „ƒ•‹  ”‡•‡ƒ” ŠǤ ‘„ ‡‹‰ǡ  ‡š’‡”‹‡ ‡† ›‘— ƒ• •— Š ƒ ’Ž‡ƒ•ƒ–ǡ‘’‡ƒ†‹•’‹”‹‰’”‘ˆ‡••‘”Ǥ‘ǡ‘ˆ ‘—”•‡ǡ Šƒ˜‡•‡‡›‘—”–ƒŽƒ†™ƒ•–Š‡”‡ ƒ–›‘—”Ž‡ –—”‡†—”‹‰–Š‡Š†ƒ›‘ˆʹͲͳͷǤ ™‘—Ž†Ž‹‡–‘•‘‘ˆ‘ŽŽ‘™Ǧ—’‘—” ‘˜‡”•ƒ–‹‘ ‘ –Š‡ ‘Ž†Ǧ‡š’‘•—”‡ ‡š’‡”‹‡– ”‡•—Ž–•Ǥ ƒƒ‹‡ Ǥǡ  ™ƒ• Šƒ’’› –‘ ™‘” ™‹–Š ›‘— ƒ•  ‡š’‡”‹‡ ‡†›‘—ƒ••— Šƒƒ‹ ƒ„Ž‡ǡ„—„„Ž›ǡ‡–Š—•‹ƒ•–‹ ’‡”•‘Ǥ –™ƒ•‰”‡ƒ––‘™‘”™‹–Š ›‘—‘› ‘Ž†Ǧ‡š’‘•—”‡‡š’‡”‹‡–•ƒ†™‡Šƒ†•‘‡ ƒ†‹†ǡ‹•’‹”‹‰–ƒŽ•‹–Š‡Ǥ Šƒ ›‘—Ǩ ‡š—ƒǡ –Šƒ ›‘— ˆ‘” ™‘”‹‰ ™‹–Š ‡ ‘ –Š‡ ‘Ž†Ǧ‡š’‘•—”‡ ’”‘Œ‡ – ƒ† ™‘”‹‰ Ž‘•‡™‹–ŠƒŽ‹‡ƒ† ‘–Š‡ˆ‹„”‘„Žƒ•–’”‘Œ‡ –Ǥ ‡š’‡”‹‡ ‡›‘—ƒ•ƒ ’‡”•‘–Šƒ–‹•˜‡”›‘’‡ǡƒ‰”‡ƒ–‡š’‡”–‹Ž‹’‹†‘‹ •ǡƒ†‹ ‡–‘™‘”™‹–ŠǤ‡™‹ŽŽ–Š‡”‡ˆ‘”‡ —†‘—„–‡†Ž›’—„Ž‹•Š‰”‡ƒ–’ƒ’‡”•–‘‰‡–Š‡”ǤŠƒ›‘—ǤŽƒ‹ƒ†ƒ—”ƒǡ–Šƒ›‘—ˆ‘”›‘—” ‘–”‹„—–‹‘•–‘›’ƒ’‡”•–Š”‘—‰Š›‘—”Š‹•–‘Ž‘‰›™‘”Ǥ ‹Ž†‡ Ǥǡ–Šƒ›‘—ˆ‘”›‘—” ‹ ”‘ƒ””ƒ›†ƒ–ƒƒ†›‘—”Š‡Ž’‹›“—‡•––‘—†‡”•–ƒ†–Š‡Ǧ‘—•‡Ǥƒ‹‡”ǡ–Šƒ ›‘—ˆ‘””‡˜‹‡™‹‰› ‘Ž†Ǧ‡š’‘•—”‡ƒ—• ”‹’– ƒ”‡ˆ—ŽŽ›ƒ† ‘–”‹„—–‹‰–‘‘‹ •‡–Š‘†• ‹‰‡‡”ƒŽǤŽƒƒ•”ƒ„ǡ–Šƒ›‘—ˆ‘”•Šƒ”‹‰›‘—”‹•‹‰Š–‹–‘Š‘™–‘ƒƒŽ›œ‡–Š‡„‡Šƒ˜‹‘”‘ˆ –Š‡”ƒ– ‘’—–ƒ–‹‘ƒŽ‘†‡ŽǤ ƒ†‡‰‘‘†—•‡‘ˆ›‘—” ƒ—••Ǧ ‘”†ƒŽ‹‹ƒ–‹‘š ‡Žˆ‹Ž‡›‘— ’”‘˜‹†‡†Ǥ ƒ•‡•–‡”Š‘ˆˆǡ–Šƒ›‘—ˆ‘”›‘—”‹–‡”‡•–‹›‘†‡Ž‹‰™‘”ƒ†ƒŽŽ‘ˆ–Š‡ ‰”‘—†™‘” ›‘— Žƒ‹† ‘—– ˆ‘” › Š ™‹–Š ›‘—” ‹’‘”–ƒ– ƒ† ‹’ƒ –‹‰ ’—„Ž‹ ƒ–‹‘•‘ ‹‡–‹ •ǡ ‘–”‘ŽƒƒŽ›•‹•ƒ†•›•–‡•„‹‘Ž‘‰›Ǥ‘—ƒ”‡ƒŽ•‘‹•’‹”‹‰„ƒ•‡†‘›‘—”„ƒ•‹ Ǧ ”‡•‡ƒ” ŠǦ–”ƒ• ‡†‹‰ ƒ –‹˜‹–‹‡• ƒ† ’ƒ••‹‘ ‹ ƒ’’Ž›‹‰ •›•–‡• „‹‘Ž‘‰› ƒ† •›•–‡• ‡†‹ ‹‡‹Š‡ƒŽ–Š ƒ”‡ǡƒŽ•‘ƒ‡‡‹–‡”‡•–‘ˆ‹‡Ǥ‰‡Ž‹ƒǡ–Šƒ›‘—ˆ‘”Š‡Ž’‹‰‡™‹–Š ‘—•‡–‡”‹ƒ–‹‘•ǡŠ‹•–‘Ž‘‰›ƒ†‘–Š‡”‹ ”‘• ‘’›–‡ Š‹“—‡•Ǥ ‹”ŒƒǤǡ–Šƒ›‘—ˆ‘” ›‘—”‹–‡”‡•–‹›Š’”‘‰”‡••ǡƒ†‘ˆ ‘—”•‡–Šƒ›‘—ˆ‘”ƒŽŽ–Š‡Š‹•–‘Ž‘‰›™‘”›‘—†‹† ˆ‘” ‡Ǥ  ™‘—Ž† Ž‹‡ –‘ –Šƒ –Š‡ ‡‡–‹ • †‡’ƒ”–‡– ˆ‘” –Š‡‹”  ‡“—‡ ‹‰ ƒ† „‹‘‹ˆ‘”ƒ–‹ •™‘”‘›ˆƒ•–‹‰’”‘Œ‡ –ȋ•’‡ ‹ƒŽ–Šƒ•–‘‹‡–‡”Ǥƒ† ‡”„‡Ȍƒ†‘ ‹†Ž›’”‘˜‹†‹‰ƒ† ‘–”‘Žˆ‹„”‘„Žƒ•–•Ǥƒ”‹‡‡ǡ‹–™ƒ•˜‡”›‹ ‡™‘”‹‰™‹–Š›‘— ‘–Š‡ ‘Ž†Ǧ‡š’‘•—”‡‡š’‡”‹‡–•™Š‡Ž„‡”–™ƒ•‘––Š‡”‡Ǥ‘—‡˜‡‰ƒ˜‡‡•‘‡˜‡”› —•‡ˆ—Ž –‹’•Ǥ Šƒ ›‘—Ǥ ‹ ‘Ž‡––‡ǡ –Šƒ ›‘— ˆ‘” „‡‹‰ Š‡Ž’ˆ—Ž ‹ ƒ› ™ƒ›• ‹ –Š‡ Žƒ„Ǥ  ƒ’’”‡ ‹ƒ–‡†›‘—”Š‘‡•–›ƒ†›‘—”‰‡—‹‡‹–‡”‡•–‹–Š‡Š—ƒƒ•’‡ –‘ˆŠŽ‹ˆ‡Ǥ† –Šƒ ›‘— ˆ‘” ›‘—” ƒ –‹˜‡ ‹˜‘Ž˜‡‡–ǡ –‘‰‡–Š‡” ™‹–Š –Š‡ ‘–Š‡” –‡ Š‹ ‹ƒ•ǡ ‹ ƒ‹‰ ‡˜‡”›‘‡ˆ‡‡Žƒ’’”‡ ‹ƒ–‡†™Š‡–Š‡›ƒ””‹˜‡ƒ†™Š‡–Š‡›Ž‡ƒ˜‡–Š‡Žƒ„Ǥ†‘ˆ ‘—”•‡ǡƒ› –Šƒ• ˆ‘” ›‘—” Š‡Ž’ ƒ† ’ƒ–‹‡ ‡ ™‹–Š –Š‡ ‰‡‡”ƒ–‹‘ ‘ˆ –Š‡ Ǧ ‡’ ʹ ‡ŽŽ•ǡ ƒ ‹’‘”–ƒ–‹Ž‡•–‘‡‹‘—”‡ˆˆ‘”–•–‘—†‡”•–ƒ††‡ˆ‹ ‹‡ ›ǤƒŽ‹ƒƒ†‹”‰‹–ǡ–Šƒ• ˆ‘” ‹†Ž› ’”‘˜‹†‹‰ ›‘—” š‹‰‡ ‡ ‡“—‹’‡– ƒ† Š‡Ž’‹‰ ™‹–Š › ‡ŽŽ ‰”‘™–Š ‡š’‡”‹‡–•Ǥ†›‹„‘ƒ†ƒ”‹ƒ‡˜ƒ†‡”™ƒƒ‰ǡ–Šƒ›‘—ˆ‘”›‘—” ‘ŽŽƒ„‘”ƒ–‹‘‘‘—” ’—”•—‹––‘—†‡”•–ƒ†–Š‡”‘Ž‡‘ˆ‘‡œ›‡‹†‡ˆ‹ ‹‡ ›Ǥ  ™‘—Ž†ƒŽ•‘Ž‹‡–‘–Šƒ–Š‡‡†‹ƒ–”‹ ••–ƒˆˆǤƒƒ‹‡Ǥǡ–Šƒ›‘—ˆ‘”›‘—” ‘–”‹„—–‹‘•ƒ– ‡‡–‹‰•ǡ›‘—”‰‡‡”ƒŽƒ†• ‹‡–‹ˆ‹ ƒ†˜‹ ‡–Š”‘—‰Š‘—–›Šǡƒ„‘—–™Šƒ––‘†‘™‹–Š› Ǧ‹ ‡ƒ†Š‘™›‘—Š‡Ž’‡†‡•–ƒ”–—’› ‘Ž†Ǧ‡š’‘•—”‡‡š’‡”‹‡–•Ǥ‡„‡ ƒǡ –Šƒ›‘—ˆ‘”‘—”–ƒŽ•ƒ„‘—– Ž‹‹ ƒŽ Š‡‹•–”›ƒ†‘—”‡‡–‹‰•–‘•–—†›†‡ˆ‹ ‹‡ ›ǡ

ʹͷͻ Appendices

Žƒ”›ǡ–Šƒ›‘—ˆ‘”‘—” ‘˜‡”•ƒ–‹‘•ƒ†–Šƒ›‘—ˆ‘”’”‘˜‹†‹‰—•‡ˆ—Ž‹ˆ‘”ƒ–‹‘ˆ‘”› ’”‘Œ‡ –•Ǥƒ”‹ǡ–Šƒ›‘—ˆ‘”ƒŽŽ›‘—”’”‘–‡‘‹ •™‘”‹›’”‘Œ‡ –•Ǥƒ”–ǡ–Šƒ›‘—ˆ‘” ’”‘˜‹†‹‰—•™‹–Š›‘—”‡š’‡”–‹•‡†—”‹‰‘—”‡†‡ƒ˜‘”•–‘ƒ‡–Š‡Ǧ ‡’ ʹ ‡ŽŽŽ‹‡ǡ ƒ†ˆ‘”„‡‹‰ƒ‹ ‡’‡”•‘–‘–ƒŽ–‘Ǥ ‡Œƒǡ–Šƒ›‘—ˆ‘”›‘—”ˆƒ• ‹ƒ–‹‰Ž›‹–‡”‡•–‹‰ “—‡•–‹‘•ǡ–Š‡›”‡ƒŽŽ›ƒ‡‡–Š‹Ǥ ƒŽ•‘ˆ‘—†›‘—”–‡ƒ Š‹‰•‡‹ƒ”‘Š‘™–‘”‡ƒ† ’ƒ’‡”•˜‡”›‹•‹‰Š–ˆ—ŽǤ‡„„›ǡ–Šƒ›‘—ˆ‘”›‘—”–ƒŽ•ƒ„‘—–ŠŽ‹ˆ‡ƒ†Š•—’‡”˜‹•‹‘ ƒ†Š‘™›‘— ƒƒƒ‰‡Ž‹ˆ‡ƒ•ƒ• ‹‡–‹•–‹‰‡‡”ƒŽǤ ‘Ž‡”–ǡ ‡š’‡”‹‡ ‡†›‘—ƒ•ƒ˜‡”›‹† ƒ†ƒ’’”‘ƒ Šƒ„Ž‡Ž‡ƒ†‡”ǡ™‹–Š‡‡‹–‡”‡•–‹–Š‡”‡•‡ƒ” Š–‘’‹ •‘ˆ–Š‡Š•–—†‡–•ǤŠƒ ›‘—ˆ‘”–Šƒ–ǤŽ•‘ǡ–‘ƒŽŽ‘–Š‡”•–ƒˆˆ‡„‡”•ǡƒ”–‡ȋ‹‡‘”‹ƒȌǡƒ–Š”‹ǡƒ”‹–ǡ ‹‰›—ƒǡ ƒ• Ǥǡ ƒ‹‡ǡ—‹ˆǡ–Šƒ›‘—ˆ‘”›‘—”‹•‹‰Š–ˆ—Ž ‘–”‹„—–‹‘•ƒ––Š‡†‡’ƒ”–‡–‡‡–‹‰•Ǥ  ‘Žˆǡƒ”‹Œ‡ǡ ‹Ž†‡ǡƒ—Žƒǡ˜‡Ž‹‡ǡ–Šƒ›‘—˜‡”›— Šˆ‘”ƒŽŽ–Š‡‘”‰ƒ‹œƒ–‹‘ƒŽ–Š‹‰•›‘— ƒ””ƒ‰‡ˆ‘”—•Š ƒ†‹†ƒ–‡•„‡Š‹†–Š‡• ‡‡•ǤŠƒ›‘—ˆ‘”ƒŽ•‘„‡‹‰•‘ƒ’’”‘ƒ Šƒ„Ž‡ ƒ†Šƒ’’›–‘Ž‹•–‡–‘—•Ǥ‡„‡ ƒǡ–Šƒ›‘—ˆ‘”„‡‹‰‹˜‘Ž˜‡†‹Ǧ†‡ˆ‹ ‹‡ ›Ǧ”‡Žƒ–‡† ”‡•‡ƒ” Šƒ†„‡‹‰‹–‡”‡•–‡†‹ •‡––‹‰—’ ”‡Žƒ–‡†’”‘Œ‡ –•Ǥ ƒ††‹–‹‘ǡ–Šƒ›‘—ˆ‘”‘—” ‘˜‡”•ƒ–‹‘• ƒ„‘—– Ž‹‹ ƒŽ Š‡‹•–”›Ǥ ‹‡‡ǡ –Šƒ ›‘— ˆ‘” ›‘—” ‹–”‘†— –‹‘ –‘  †‡ˆ‹ ‹‡ › ƒ† ›‘—” ‹’‘”–ƒ– ™‘” ‹ †‡˜‡Ž‘’‹‰ ƒ† Šƒ”ƒ –‡”‹œ‹‰ –Š‡ ˆ‹”•– ’—”‡Ǧ „ƒ ‰”‘—†Ǧ‘—•‡‘†‡ŽǤ  Šƒ˜‡‡š’‡”‹‡ ‡†‘—”ȋŽƒ„Ȍ–‡ Š‹ ‹ƒ•ƒ•ƒ‹’‘”–ƒ–„ƒ „‘‡‹–Š‡™‘”™‡†‘‹‘—” ŠǤƒ›–Šƒ•–‘ƒŽŽ–Š‡–‡ Š‹ ‹ƒ•™Š‘Šƒ˜‡‹†Ž›Š‡Ž’‡†™‹–Šƒ›“—‡•–‹‘•ƒ†Žƒ„ ‹••—‡• Šƒ†ƒ†ˆ‘”ƒŽ•‘ƒ –‹˜‡Ž›’‡”ˆ‘”‹‰ƒŽ‘–‘ˆ•ƒ’Ž‡’”‡’ƒ†‡ƒ•—”‡‡–•ˆ‘”› ’”‘Œ‡ –•Ǥ‹ ‡–ǡ–Šƒ›‘—ˆ‘”›‘—”‹˜‘Ž˜‡‡–‹”‡˜‹‡™‹‰›ƒ—• ”‹’–•ǡ•‡†‹‰‡ ‹–‡”‡•–‹‰’—„Ž‹ ƒ–‹‘•ƒ†‰‹˜‹‰•–ƒ–‹•–‹ •ƒ†˜‹ ‡ƒ†„‡‹‰ƒŽ™ƒ›•‘’‡–‘Š‡Ž’Ǥ‹ ǡ–Šƒ ›‘— ˆ‘” ›‘—” ‹†‡•• ƒ† ›‘—” Š‡Ž’ ƒ– –Š‡ „‡‰‹‹‰ ‘ˆ –Š‡ ‘Ž†Ǧ‡š’‘•—”‡ ’”‘Œ‡ – ƒ† ’”‘„ƒ„Ž› ƒ› ‘”‡ ‡š’‡”‹‡–ƒŽ ƒ† ’”‘Œ‡ –Ǧ”‡Žƒ–‡† –Š‹‰•ǡ ƒ† ‘– –‘ ˆ‘”‰‡– –Š‡ „‹Ž‡ ƒ—Žƒ–‹‘•™‡†‹†ˆ‘”ƒ”‡†Ǩ ˆ‡Ž–Ž‹‡ƒ–”—‡‹ ”‘•—”‰‡‘-Ǥ‡ƒŽ•‘Šƒ†•‘‡‘–‹˜ƒ–‹‰ ‘˜‡”•ƒ–‹‘•ƒ„‘—–›ŠǤŠƒ›‘—˜‡”›— ŠǤƒ‘ǡ–Šƒ›‘—ˆ‘”‹–”‘†— ‹‰‡–‘ –Š‡š‹‰‡ ‡‡–Š‘†ƒ†ˆ‘”ƒŽ™ƒ›•„‡‹‰™‹ŽŽ‹‰–‘Š‡Ž’Ǥ‹”‹ƒǤǦǤǡ–Šƒ›‘—ˆ‘” Š‡Ž’‹‰–‘ƒ‡ͶŽƒ„”—Ž‹‡ƒ•‘‘–Šƒ Š‹‡ƒ†ˆ‘”„‡‹‰˜‡”›ƒ’’”‘ƒ Šƒ„Ž‡Ǥ‹‡Ž•ǡ –Šƒ›‘—ˆ‘”›‘—”‡–Š—•‹ƒ•ƒ–™‘”Ǩ –™ƒ•ƒŽ™ƒ›• ‘–ƒ‰‹‘—•–‘Š‡ƒ”›‘—Žƒ—‰Šˆ”‘–Š‡ ‘ˆˆ‡‡”‘‘ƒŽŽ–Š‡™ƒ›–‘›†‡•‹–Š‡‘ˆˆ‹ ‡Ǥ†–Šƒ›‘—ˆ‘”ƒŽ™ƒ›•„‡‹‰ƒ„Ž‡–‘Š‡Ž’ ‡™‹–Š›“—‡•–‹‘•ƒ„‘—–Žƒ„–‡ Š‹“—‡•‘”ƒ›‘–Š‡”‹••—‡•‹–Š‡Žƒ„Ǥ ƒŽ•‘–Š‘—‰Š–‹–™ƒ• ”‡ƒŽŽ› ‘‘Ž™Š‡  ‘—Ž†Š‡Ž’›‘—™‹–Š›‘—”Š‹‰ŠǦ•’‡‡†ǡŠ‹‰ŠǦ–Š”‘—‰Š’—–‘—•‡‹•‡‹ƒ–‹‘Ǥ †‘ˆ ‘—”•‡ǡƒŽ•‘–Šƒ›‘—ˆ‘”Š‡Ž’‹‰‡™‹–Š›‹••—‡•™‹–Š›Ž‘‰‰‡”Ǧ‹’Žƒ–‡†‹ ‡ ™Š‡ƒƒ‹‡ Ǥ™ƒ•‘––Š‡”‡Ǥ› Šƒǡ–Šƒ›‘—ˆ‘”„‡‹‰Š‡Ž’ˆ—Ž™‹–Šƒ›“—‡•–‹‘• Šƒ†Ǥ Šƒ•–‘ƒŽŽ‘–Š‡”–‡ Š‹ ‹ƒ•‹‘—”†‡’ƒ”–‡–™Š‘Šƒ˜‡Š‡Ž’‡†‡‹‘‡™ƒ›‘”ƒ‘–Š‡”ǡ ‹ Ž—†‹‰ǡŽ‹‡ǡ ‡ǡƒ” ‡ŽǤǡƒ”–‹Œǡ‹‡Ž•Ǥǡ‡œ‡ǡŠ‡‘˜ƒ‹ŒǡŒƒ••‘ǡƒ††™‹‡Ǥ  ƒŽ•‘™‘—Ž†Ž‹‡–‘–Šƒ–Š‡™Š‘Ž‡‹ƒ‹‰›‘—•‡‡š’‡”‹‡–•–Š‡”‡’‘••‹„Ž‡Ǥ  ƒŽ•‘‡•’‡ ‹ƒŽŽ›™‘—Ž†Ž‹‡–‘–Šƒ”ƒ†ƒ–ƒ• Šƒˆ‘”–ƒ‹‰•— Š‰‘‘† ƒ”‡‘ˆ–Š‡‹ ‡ƒ† ƒŽ™ƒ›• „‡‹‰ •‘ ‹ ‡ –‘ –ƒŽ –‘ ƒ„‘—– ƒ›–Š‹‰Ǥ ‹ Š‡Ž ‡‹Œǡ –Šƒ ›‘— ˆ‘” ‘—” ’Ž‡ƒ•ƒ– ‹–‡”ƒ –‹‘• ƒ† ›‘—” Š‡Ž’ ™‹–Š –Š‡ ˜ ’”‘–‘ ‘Ž•Ǥ ‘— ƒŽ•‘ ‰ƒ˜‡ ‡†‡”‹ ‘ ƒ†  –Š‡ ‘’’‘”–—‹–›–‘’”ƒ –‹ ‡‘—”Š‡ƒ”–’— –—”‡–‡ Š‹“—‡•ǡ–Šƒ˜‡”›— Šˆ‘”–Šƒ–Ǥ ——Žƒ†

ʹ͸Ͳ Acknowledgements

‹”‹ƒ˜Ǥ†ǤǦ Ǥǡ–Šƒ›‘—ˆ‘”›‘—”Š‡Ž’‹–Š‡’”‡’ƒ”ƒ–‹‘ˆ‘”–Š‡‡š’‡”‹‡–•Ǥ†ƒŽ•‘ǡ –Šƒ•‹ ‡Ǩ  Šƒ›‘—–‘‡˜‡”›‘‡‹ƒ–Š”‹ǯ•‰”‘—’ǡ‹ Ž—†‹‰Ž‡šǡƒ–”‹ ‹ƒǡƒ”–‹ǡ ‡‡ƒ†Ž”‹‡ǡˆ‘” ›‘—”Š‡Ž’™‹–Š™‡•–‡”„Ž‘––‹‰ǡ‹ ”‘• ‘’›ƒ†ƒ›–Š‹‰‡Ž•‡ ‡‡†‡†‘” ‘—Ž†ǯ–ˆ‹†Ǥ‘— ‰—›• ƒ”‡ •— Š ƒ†‹”ƒ„Ž‡ Šƒ”† ™‘”‡”• ƒ† ’—„Ž‹•Š‡”•Ǥ Šƒ ›‘— –‘ ‡˜‡”›‘‡ ˆ”‘ –Š‡ ‘Ž‡ —Žƒ” ‡‡–‹ • ‰”‘—’ǡ ‹ Ž—†‹‰ –‘‹‡ǡ ‹Ž‹‡ǡ ‘—ǡ ‹„‹ǡ ”‡†ƒǡ ‡Ž‡ǡ ƒ”‡‡„ƒǡ ƒ” ‘ǡ‡Ž‹†‡ǡƒ–ƒŽ‹ƒǡƒ†‡‡–‹ƒǤŠƒ›‘—ˆ‘”‘—” ƒ†‹†ǡ‘–‹˜ƒ–‹‰–ƒŽ•ƒ†ƒŽŽ›‘—” Š‡Ž’‹ƒ†‘—–•‹†‡‘ˆ–Š‡Žƒ„Ǥƒ”‡†ǡ‹–™ƒ•ƒ’Ž‡ƒ•—”‡Š‡Ž’‹‰›‘—™‹–Š›‘—”‡š’‡”‹‡–•ƒ† ƒŽ•‘•Šƒ”‹‰‘—”‹••—‡•ǡˆ”—•–”ƒ–‹‘•ƒ†•— ‡••‡•™‹–Š‡ƒ Š‘–Š‡”Ǥ‹ƒ‰Ǥ™‡Šƒ†ƒ•Šƒ”‡† ’ƒ•–†—‡–‘‘—”‹–‡”•Š‹’•ƒ–ǡ™Š‹ ŠŠ‡Ž’‡†—• ‘‡ –Ǥ ƒŽ•‘ƒ†‹”‡†›‘—ˆ‘”›‘—” ’ƒ––‡” ”‡ ‘‰‹–‹‘ǡ •–ƒ–‹•–‹ ƒŽ ƒ† ’”‘‰”ƒ‹‰ •‹ŽŽ•ǡ „—– ‡˜‡ ‘”‡ ‹’‘”–ƒ–Ž› ›‘—” ‹†‡••ǡ Ž‹ˆ‡ ’‡”•’‡ –‹˜‡• ƒ† ‘’‡‡•• –‘ Š‡ƒ”‹‰ ƒ† •—’’‘”–‹‰ ‡ ƒ† ‘–Š‡”• ‹ ƒ›–Š‹‰Ǥ  ™ƒ• ƒ† ƒ ƒŽ™ƒ›• ˜‡”› Šƒ’’› –‘ Š‡Ž’ ›‘— ™‹–Š ƒ›–Š‹‰ ›‘— ‡‡†Ǥ  •–‹ŽŽ ”‡‡„‡”–Š‡†‡Ž‹ ‹‘—•–Š‡ ƒ‡›‘—”™‹ˆ‡ƒ†‡ˆ‘”‡ǨŠƒ›‘—Ǩ ‡†‡”‹ ‘ǡ‘‡‘ˆ–Š‡‘•– ’ƒ••‹‘ƒ–‡Š”‡•‡ƒ” Š‡”• ǯ˜‡‡–Ǥ‘—Šƒ†ƒ•–”‘‰™‘”‡–Š‹ ƒ†™‡”‡˜‡”›†‹• ‹’Ž‹‡†Ǥ –‘’‘ˆ–Šƒ–›‘—™‡”‡˜‡”›‹†ƒ†Šƒ†ƒ‹•ƒ–‹ƒ„Ž‡‡–Š—•‹ƒ•ƒ†‘’‡‡••–‘Š‡Ž’‹ –Š‡ Žƒ„ǡ ‘—” ‘—•‡ –‡”‹ƒ–‹‘• –‘‰‡–Š‡” „‘–Š ›‘—”• ƒ† ‹‡ǡ ™‘”‹‰ ‘—– ‘—” ‹‹–‹ƒŽ ‡“‹••—‡•–‘‰‡–Š‡”ǡ Š‡Ž’‡™‹–Šƒ’”‡•‡–ƒ–‹‘ǡ –ƒŽƒ„‘—–ŠŽ‹ˆ‡ǡ‹••—‡•‹‹–ƒ† ƒ†˜‹ ‡ǤŠƒ›‘—ˆ‘”„‡‹‰–Š‡”‡ˆ‘”‡ƒ†•‘ƒ›‘–Š‡”•Ǥ  Šƒ›‘—–‘‡˜‡”›‘‡‡Ž•‡ˆ”‘–Šƒ– ‡–‹ ǡ‹ Ž—†‹‰ƒǡ‹ ›ǡ ƒ ”‡ƒ”ǡ ”‡‡ǡ ƒ”Ž‡‡Ǥǡ‘”‹ǡ‹”ŒƒǤǡ‡ǡ‹ƒǡƒ†”ƒǡ‡”ƒǡ‹‘Ž‡–ƒǡ‹ǡ‡‹Ž‹ǡƒ†ƒ‹ Ǥ—” ‹–‡”ƒ –‹‘•™‡”‡ƒŽ™ƒ›•’Ž‡ƒ•ƒ––‘‡ǤŽ„‡”–‘Ǥ ǯŽŽ‡˜‡”ˆ‘”‰‘–Š‘™›‘—ƒ†‡–Š‡•–ƒ”–‘ˆ ›ŠŽ‹ˆ‡Š‡”‡•‘— Šˆ—ǡ•–ƒ”–‹‰™‹–Šƒƒ™‡•‘‡Š‹–”‘†— –‹‘ƒ–ŽŽ‡”•ƒ„‘”‰Ǩ ƒǯ–™ƒ‹–ˆ‘”—•–‘ ƒ– Š—’•‘‘Ǩ  ™‘—Ž†ƒŽ•‘Ž‹‡–‘–Šƒ–‘•‘‡‘ˆ›’”‡˜‹‘—•‹•’‹”‹‰–‡ƒ Š‡”•ƒ†•—’‡”˜‹•‘”•™Š‘ ’”‘ˆ‘—†Ž›•Šƒ’‡†‡‘–Š‡”‘ƒ†–‘™ƒ”†•›ŠǤ”‘ˆ•Ǥ ‡˜ƒ†‡‡”‰ƒ†‘—‹•˜ƒ †‡” ƒǡ–Šƒ›‘—ˆ‘”„‡‹‰–Š‡”‡‹› Š‡‹ ƒŽ‡‰‹‡‡”‹‰•–—†›ƒ†ˆ‘”„‡‹‰•— Š ‹•’‹”‹‰ǡ™‡Ž ‘‹‰ǡ‘–‹˜ƒ–‹‰ƒ†•—’’‘”–‹˜‡–‡ƒ Š‡”•ƒ†•—’‡”˜‹•‘”•Ǥ”‘ˆǤ Ǥ Ǥ Ǥȋ ‘ŠƒȌ ‰„‡”•‡ǡ –Šƒ ›‘— ˆ‘” „‡Ž‹‡˜‹‰ ‹ ‡ ™Š‡  ™ƒ–‡† –‘ ‡š’ƒ† › „‹‘ Š‡‹•–”› ‘™Ž‡†‰‡ƒ††‘ƒ•‡ŽˆǦ ‘•–”— –‡†„‹‘ Š‡‹•–”›ƒ––Š‡ƒ†„‘—†‹˜‡”•‹–›ƒŽŽ–Š‡™ƒ›‹ ‹Œ‡‰‡Ǥ‹ Šƒ”†ǡ‡–‡”ƒ† ƒǡ–Šƒ›‘—ˆ‘”–ƒ‹‰ƒ Šƒ ‡‘‡ǡƒˆ”‡•ŠŽ›‹–‡†  Š‡‹ ƒŽ‡‰‹‡‡”‹‰‰”ƒ†—ƒ–‡ȋi.e.ƒ”‘‘‹‡‹„‹‘ Š‡‹•–”›ƒ†„ƒ•‹ ‘Ž‡ —Žƒ”Ž‹ˆ‡• ‹‡ ‡ ”‡•‡ƒ” Šƒ†–‡ Š‹“—‡•Ȍƒ†–‡ƒ Š‹‰‡•‘— Š‹–Š‹•ˆ‹‡Ž†‹ƒ•Š‘”–’‡”‹‘†‘ˆ–‹‡Ǥ ƒǡ ƒŽ•‘”‡ƒŽŽ›ƒ’’”‡ ‹ƒ–‡†–Šƒ–›‘—•Š‘™‡†–Š‡ Ž‹‹ ƒŽ’ƒ”–‘ˆ‘’Ž‡š †‡ˆ‹ ‹‡ ›Ǥ ƒŽ•‘”‡ƒŽŽ› ƒ’’”‡ ‹ƒ–‡†‘—”–ƒŽ•ƒ†Šƒ˜‡•‘— Šƒ†‹”ƒ–‹‘ˆ‘”–Š‡ ƒ”‡›‘—–ƒ‡‹›‘—”™‘”ƒ† –Š‡’ƒ••‹‘›‘—Šƒ˜‡‹‹–Ǥ ——•ǡ ƒ”‘Ž†ƒ†‘”‹‡ǡ–Šƒ›‘—ˆ‘”™‡Ž ‘‹‰‡™‹–Š‘’‡ ƒ”•‹›‘—”‰”‘—’ǡƒ ‘‘†ƒ–‹‰›‹–‡”‡•–‹†‘‹‰ Ž‹‹ ƒŽǦ Š‡‹•–”›Ǧ”‡Žƒ–‡†”‡•‡ƒ” Š ƒ†‹ ”‡ƒ•‹‰› Ž‹‹ ƒŽ Š‡‹•–”›‘™Ž‡†‰‡Ǥ ™‹ŽŽƒŽ•‘ƒŽ™ƒ›• Š‡”‹•Š–Šƒ– ™ƒ•ƒ„Ž‡–‘ ’—„Ž‹•Šƒ„‡ƒ—–‹ˆ—Ž”‡˜‹‡™™‹–Š›‘—ƒ†™”‹–‡‰”ƒ–’”‘’‘•ƒŽ•™‹–Š›‘—Ǥ ‡š’‡”‹‡ ‡†›‘—” •—’‡”˜‹•‹‘ƒ†‹–‡”ƒ –‹‘•‹‰‡‡”ƒŽƒ•ƒ˜‡”›’Ž‡ƒ•ƒ–ƒ†‹•’‹”‹‰ƒ† Ž‡ƒ”‡†ƒŽ‘–Ǥ Šƒ›‘—Ǥ 

ʹ͸ͳ Appendices

†—‹ƒ†ƒ–ƒ•ŠƒǦǤǨŠƒ›‘—˜‡”›— Šˆ‘”ƒ‰”‡‡‹‰–‘„‡›’ƒ”ƒ›’Š•Ǥˆ–‡”‘– „‡‹‰ƒ„Ž‡–‘„‡ƒ–›™‡††‹‰ Œ—•–Šƒ†–‘‹˜‹–‡› Ž‘•‡•–ˆ”‹‡†Ȁˆƒ‹Ž›‡„‡”Ǥ Šƒ˜‡ •‘— ŠŽ‘˜‡ƒ†ƒ†‹”ƒ–‹‘ˆ‘”›‘—„‘–ŠǨŠ”‘—‰Š‘—–›Š›‘—’ƒ–‹‡–Ž›Š‡ƒ”†ƒŽŽ› –”‹ƒŽ•ƒ†–”‹„—Žƒ–‹‘•ǡ„‡‹‰ƒ„Ž‡–‘•—’’‘”–‡ƒ†‘–‹˜ƒ–‡‡–Š”‘—‰Š‹–ƒŽŽǤŠƒ›‘—Ǩ  ƒ›–Šƒ•–‘ƒŽŽ›ˆ”‹‡†•ǡˆ‘”–Š‡›—™ƒ˜‡”‹‰•—’’‘”––Š”‘—‰Š‘—–›ŠŒ‘—”‡›Ǥ Šƒ›‘— ˆǡŒƒ›ǡƒ•‹–ƒǡ ‡ƒ†‘‘ǡƒ—Žƒ† ‰‡ǡ‡–‡”Ǥƒ†Š”‹•–‹ƒǡ‡”–Ǥƒ† ƒ”‰‘ǤŠƒ›‘—ˆ‘”ƒŽŽ–Š‡Ž‹‰Š–ǡˆ—ǡ†‡‡’ƒ†’Š‹Ž‘•‘’Š‹ ƒŽ ‘˜‡”•ƒ–‹‘•™‡Šƒ†ǡ›‘—” Ž‘˜‡ǡ ›‘—” •—’’‘”–ǡ ›‘—” ‡ ‘—”ƒ‰‡‡–ǡ –Š‡ Ž‘˜‡Ž› ‡‘”‹‡• ™‡ ”‡ƒ–‡† –‘‰‡–Š‡” ‡˜‡ –Š‘—‰Š ™ƒ••‘„—•›Ǥ Š‘’‡–‘„‡ƒ„Ž‡–‘ƒ‡ƒ›ƒ›‘”‡‡™‡‘”‹‡•™‹–Š›‘— ‘™–Šƒ–›Š‹•ˆ‹ƒŽ‹œ‡†Ǥ  ›ˆƒ‹Ž›ǡ–Šƒ›‘—•‘— ŠǨŠƒ›‘—–‘›ƒ”–‹‡•ˆƒ‹Ž›ȋ‹‘Š‡ Š‘ǡ‹‘‹Ž‘ǡƒ Š‹ ‹Ž†”‡†ǡƒ Š‹‹–‹ǡƒ Š‹ Š‹ǡƒ Š‹°ǡ‡›‘Ž†ǡƒ Š‹‡Ž‹ƒǡ ”ƒǡƒ Š‹֒‹ǡ‘›ǡ ƒ Š‹‹‹ǡ–Š‘›ǡƒ Š‹‹ Š‹ǡƒ›‘ǡƒ Š‹ƒŽƒǡ —›ǡ‘™ǡƒ‹ƒǡ‡––›ǡ —”‹ǡƒ”›ǡ ‹‡–Œ‡ǡ ”ƒ ‹•ǡ‘‡•Œ‡ǡŠ—†ƒ”‹ǡ‡””›ǡ ‹‘ǡ‹ Šƒ‡Žǡ— ‹ŽŽ‡ǡ•‡”ƒŽ†ƒǡŠƒŠƒ‹Žƒǡ‡—‰‡‡ǡ Š‘—”‡ŽŽƒǡ™‡•ƒǡ‡”ŽƒǡŽ‰‹ǡ—‡‡›ǡƒ˜‡›ǡƒ–Šƒ‹‡Žǡƒ›•Šƒǡ—‹ǡ—•‹ǡŠƒ‹ƒǡ –‘ǡ ‘Ž‹ǡ‘–Œ‡ǡ —†‡••ƒ‹–—”‹ƒ Š–—’”‹—‹ƒ Š–—’”‹ƒƒȌǡ›‹”‹†‘‰‘ˆƒ‹Ž›ȋ‹‘ǡ™‹†ƒǡ ‡›ǡ †‡Ž‹‡‡ǡƒ‹ǡŠƒ”Ž–‘ǡƒ”Ž›•‘ǡƒŽŽ›ǡƒ–”‹ ǡƒ•Œ‡ǡ ‡””‹–ǡƒ”ƒ‹ƒ’Š‡›Ȍǡ ›‹ǦŽƒ™ˆƒ‹Ž›ȋ‹ Ž—†‹‰ ƒ”‘Ž†”ǡ ƒ‹ ‡ǡ —‹ǡ—Ž‹ǡƒ–ƒ•ŠƒǤǡ”†‹–Šǡ ‡›ǡ‹–ƒǡƒ†› ‹Ž‹ˆˆ‘”†Ȍˆ‘”–Š‡‹”Ž‘˜‡ƒ†•—’’‘”––Š”‘—‰Š‘—––Š‡›‡ƒ”•Ǩ   Š—‘‰‘—• –Šƒ• ‰‘‡• –‘ › ’ƒ”‡–•ǡ ‰”‹† ƒ† †™‹ǡ ˆ‘” –Š‡‹” Ž‘˜‡ǡ •—’’‘”– ƒ† ‡ ‘—”ƒ‰‡‡– –Š”‘—‰Š‘—– –Š‡ ›‡ƒ”•ǡ ‹•–‹ŽŽ‹‰ ‹ ‡ ˆ”‘ –Š‡ –ƒ –‹ ƒŽǡ ‡†— ƒ–‹‘ƒŽǡ ”‡ ”‡ƒ–‹‘ƒŽǡ–Š‡‡‘–‹‘ƒŽǡ‡–ƒŽǡ•’‹”‹–—ƒŽƒ†‘”‡-Ǥ›•‹•–‡”ǡ”‹• ‡ŽŽƒǡ–Šƒ›‘—ˆ‘” ›‘—”Ž‘˜‡ǡ•—’’‘”–ǡ„‡‹‰’”‘—†‘ˆ‡ƒ† ‘ˆ‹†‹‰‹‡Ǥ ƒ’’”‡ ‹ƒ–‡‘—”‹–‡”ƒ –‹‘ƒ†‹– Šƒ•–ƒ—‰Š–‡ƒŽ‘–‘ˆ–Š‹‰•‹Ž‹ˆ‡Ǥ  ƒ•–„—–‘–Ž‡ƒ•–ǡ ƒ—–‘•–‰”ƒ–‡ˆ—Ž–‘›Š—•„ƒ†ǡƒ”– Š‘‘’Ǥ‹–ŠƒŽŽ›Š‡ƒ”–ǡ  ™‘—Ž†Ž‹‡–‘ƒˆˆ‹”–Šƒ–  ‘—Ž†ǯ–†‘‹–™‹–Š‘—–›‘—Ǥ ”‘Š‡Ž’‹‰‡‘˜‡ǡ ‘‹‰ˆ”‘ˆƒ” ƒ™ƒ›–‘ƒ ‘’ƒ›‡‘” ‘‡‰‡–‡Žƒ–‡ˆ”‘™‘”ǡ™ƒ‹–‹‰’ƒ–‹‡–Ž›ˆ‘”‡–‘ˆ‹‹•Š ™‘”‹‰ǡŽ‘‘™‹–Šƒˆ”‡•Š‡›‡ƒ–›™‘”ƒ†Š‡ƒ”‹‰›‹••—‡•ƒ†ˆ”—•–”ƒ–‹‘•™Š‡ ˆ‡Ž– Ž‘•–ǡ’—•Š‹‰‡–‘ ‡Ž‡„”ƒ–‡›•— ‡••‡•‘ƒ––‡”Š‘™Ž‹––Ž‡ –Š‘—‰Š––Š‡›™‡”‡ǡ–‡ŽŽ‡ –‘‰‘–‘•Ž‡‡’™Š‡ ™ƒ•–‹”‡†ǡ–ƒ‹‰ ƒ”‡‘ˆ‡™Š‡ ™ƒ••‹ ƒ†ƒ••—”‹‰‡ ‡˜‡”›–Š‹‰ ™‘—Ž†„‡‘‡˜‡–Š‘—‰Š›‘—™‡”‡‘–•—”‡‹–™‘—Ž†Ǥ‡ŽŽ‡›‘—Ž‘˜‡‡‘˜‡”ƒ†‘˜‡”ƒ† ƒ‹‰‡ˆ‡‡ŽŠƒ’’›‹•‘ƒ›†‹ˆˆ‡”‡–™ƒ›•Ǥ‘˜‡›‘—ᇡǡ–Šƒ›‘—Ǩ   

ʹ͸ʹ  

  ŝŽŐƌĂƉŚLJ  

  



























 



































 ‹‘‰”ƒ’Š›

‡ǦŽƒ‹”‡ƒ”–‹‡•™ƒ•„‘”‘ –‘„‡”ͳͲ–Šͳͻͺͳ‹‹ŽŽ‡•–ƒ†ǡ—”ƒ­ƒ‘ǡƒ†‰”‡™—’‹ ƒ†‘”‡–ǡ ™‹–Š Š‡” ’ƒ”‡–• ƒ† •‹•–‡”Ǥ  ͳͻͻͻǡ •Š‡ ‰”ƒ†—ƒ–‡† ƒ”‹ƒ ƒ —Žƒ–ƒ › ‡— ȋȌ‹‹ŽŽ‡•–ƒ†Ǥ—‡–‘Š‡”‡‡‹–‡”‡•–‹–‡ Š‹ ƒŽǡ„‹‘Ž‘‰‹ ƒŽƒ†‡†‹ ƒŽƒ•’‡ –•‘ˆ Š‡‹•–”›ǡ•Š‡†‡ ‹†‡†–‘ˆ‘ŽŽ‘™ƒ’ƒ–Š–‘‡†— ƒ–‡Š‡”•‡Žˆƒ†‰ƒ‹‡š’‡”‹‡ ‡‹–Š‡•‡ˆ‹‡Ž†• ƒ†Š‘’‡ˆ—ŽŽ›‹–Š‹•™ƒ› ƒ‡–Š‡ ™‘”Ž†ƒ„‡––‡”’Žƒ ‡ ƒ†Š—ƒŠ‡ƒŽ–Šƒ•‘’–‹ƒŽƒ• ’‘••‹„Ž‡Ǥ Š‡”‡ˆ‘”‡ǡ ƒˆ–‡” Š‡” •‡ ‘†ƒ”› ‡†— ƒ–‹‘ǡ •Š‡ •–ƒ”–‡† Š‡” Š‡‹ ƒŽ ‡ Š‘Ž‘‰› •–—†› ƒ– –Š‡ ‹˜‡”•‹–› ‘ˆ ™‡–‡Ǥ Š‡ ˆ‘ŽŽ‘™‡† –Š‡ ”‘ ‡•• ‡ Š‘Ž‘‰› –”ƒ  ™‹–Š –Š‡ •—„†‹• ‹’Ž‹‡”‘ ‡••ƒ†Žƒ–†‡•‹‰Ǥ—”‹‰Š‡”•–—†‹‡••Š‡–‘‘–‹‡–‘ƒ‘‰•–‘–Š‡”• •–ƒ”– ƒ •–—†‡–Ǧˆ‘ —•‡† ‘’—–‡” ‘’ƒ› ȋ”‘ ›‘ ‘’—–‡”•ǡ ”Š‡Ȍ –‘ ’ƒ”–‹ —Žƒ”Ž› Š‡Ž’‡™•–—†‡–•ˆŽ›‹‰‹ˆ”‘—”ƒ­ƒ‘ƒ†‘–Š‡”—– Š–‹ŽŽ‡•‹•Žƒ†•™‹–Šƒ ‘’—–‡”Ǥ Š‹•‹•„‡ ƒ—•‡ǡƒ•ƒ––Šƒ––‹‡ǡ†—‡–‘•‘‡–‡ Š‘Ž‘‰‹ ƒŽŽ‹‹–ƒ–‹‘• ‘’ƒ”‡†–‘‘™ǡ‹•™ƒ• ‘–‡ƒ•›ˆ‘”–Š‡‡™•–—†‡–•–‘‹–‡‰”ƒ–‡‹–‘–Š‡ —Ž–—”‡ǡˆ‹‰Š–Š‘‡•‹ ‡••ƒ†ˆ‘ —•‘ –Š‡‹” •–—†‹‡•Ǥ ‡ ‡‹˜‹‰ ƒ ‘’—–‡” ™‹–Š ‹–‡”‡– ƒ ‡•• —’‘ ƒ””‹˜ƒŽ ™ƒ• –Š‡”‡ˆ‘”‡ ˜‡”› Š‡Ž’ˆ—ŽǤ ˆ–‡” –Š‹•ǡ –‘ ‰ƒ‹ ™‘” ‡š’‡”‹‡ ‡ •Š‡ —†‡”–‘‘ ƒ ͶǦ‘–Š ‹–‡”•Š‹’ ƒ– ‡‹Œ‹ ”ƒ‹†ǡ”Š‡ǡ™Š‡”‡•Š‡†‹†’”‘ ‡••’Žƒ–‘’–‹‹œƒ–‹‘ƒ††‡•‹‰ˆ‘” › Ž‘Š‡š›Žƒ‹‡ ’”‘†— –‹‘ǡ—†‡”–Š‡•—’‡”˜‹•‹‘‘ˆ”‹ ‡šǡ’”‘ˆǤ‘—‹•˜ƒ†‡” ƒǤ—”‹‰Š‡”•–—†‹‡•ǡ •Š‡ƒŽ•‘ˆ‘ŽŽ‘™‡†ƒ•‡ŽˆǦ†‡˜‹•‡†‹‘”‹‹‘ Š‡‹•–”›ƒ––Š‡ƒ†„‘—†‹˜‡”•‹–›‹‹Œ‡‰‡Ǥ —„•‡“—‡–Ž›ǡˆ‹‹•Š‡†Š‡”•–—†›ȋ Ȍ™‹–Šƒ‘‡Ǧ›‡ƒ”‹–‡”•Š‹’ƒ––‹Ǧ ˆ‡ –‹˜‡•ǡ ‡Žˆ–ǡ—†‡”–Š‡•—’‡”˜‹•‹‘‘ˆƒƒ”–‡˜ƒ†‡ ”ƒƒˆǡŽ• Š—Ž–‡ǡ’”‘ˆǤ‹ ‡–‹‡”•–”ƒ•œǡ ’”‘ˆǤ ‘—‹• ˜ƒ †‡” ƒ ƒ† ’”‘ˆǤ ‡ ˜ƒ †‡ ‡”‰Ǥ Š‡”‡ •Š‡ †‡•‹‰‡† ƒ „‹‘ƒ••Ǧ •‡’ƒ”ƒ–‹‘ ’”‘ ‡•• ˆ‘” –Š‡ •—•–ƒ‹ƒ„Ž‡ Žƒ”‰‡Ǧ• ƒŽ‡ ’”‘†— –‹‘ ‘ˆ •— ‹‹  ƒ ‹† —•‹‰ ‹ ”‘‘”‰ƒ‹••Ǥ‘ ‘–‹—‡‘Š‡”Œ‘—”‡›ǡ•Š‡ˆ‘ŽŽ‘™‡†ƒ•‡ ‘†ƒ•–‡”•–—†›‹‡†‹ ƒŽ ‹‘ Š‡‹•–”›ƒ––Š‡ƒ†„‘—†‹˜‡”•‹–›‹‹Œ‡‰‡ǤŠ‡”‡ǡ•Š‡ˆ‘ŽŽ‘™‡†Š‡”ͳǦ›‡ƒ”ƒ•–‡” –Š‡•‹•‹–‡”•Š‹’‹‡†‹ ƒŽ‹‘ Š‡‹•–”›—†‡”–Š‡•—’‡”˜‹•‹‘‘ˆ‹ Šƒ”†‘†‡„—”‰ǡ‡–‡” ‹ŽŽ‡•ƒ†’‡†‹ƒ–”‹ ‹ƒ’”‘ˆǤ ƒ‡‹–‹‹–Š‡‡†‹ƒ–”‹ •†‡’ƒ”–‡–ƒ†–Š‡ƒ†„‘—† •–‹–—–‡ˆ‘”‘Ž‡ —Žƒ”‹ˆ‡ ‹‡ ‡•‘ˆ–Š‡ƒ†„‘—†— Ǥ—”‹‰–Š‹•‹–‡”•Š‹’ǡ•Š‡•–—†‹‡† –Š‡ ‡ˆˆ‡ – ‘ˆ –Š‡ ƒ–‹‘š‹†ƒ– ͳ ‘ ‡ŽŽǦ„‹‘Ž‘‰‹ ƒŽ ‘•‡“—‡ ‡• ‘ˆ ‘’Ž‡š Ǧ†‡ˆ‹ ‹‡– ’ƒ–‹‡– ‡ŽŽŽ‹‡•ǤŠ‡ƒŽ•‘ˆ‘ŽŽ‘™‡†ƒ͸Ǧ‘–Š‹‘””‡•‡ƒ” Š‹–‡”•Š‹’‹ƒ†„‘—†— ǯ• š’‡”‹‡–ƒŽŽ‹‹ ƒŽŠ‡‹•–”›”‡•‡ƒ” Š‰”‘—’ǡ—†‡”–Š‡•—’‡”˜‹•‹‘‘ˆ ——•‘”–ƒƒ† ‰”‘—’ǦŽ‡ƒ†‡”’”‘ˆǤ‘”‹‡™‹‡Ž•ǡƒ†‰ƒ‹‡†‘”‡‘™Ž‡†‰‡‹Ž‹‹ ƒŽŠ‡‹•–”›Ǥ—”‹‰ ƒ†ƒˆ–‡”Š‡”‹–‡”•Š‹’ǡ•Š‡•–—†‹‡†–Š‡‡ˆˆ‡ –‘ˆ‹”‘Ǧ”‹ Šƒ†‹”‘Ǧ†‡ˆ‹ ‹‡–‡–‡”‹ „ƒ –‡”‹ƒ ‘  ‡š’”‡••‹‘ ƒ† ’”‘–‡‹ Ž‡˜‡Ž• ‘ˆ –Š‡ ‹”‘Ǧ„‹†‹‰ ’”‘–‡‹  ǡ ‹ ‹–‡•–‹ƒŽ ‡’‹–Š‡Ž‹ƒŽ ‡ŽŽ• ‹ ˜‹–”‘Ǥ  –Š‡ Ž‹‹ ƒŽ Š‡‹•–”› ˆ‹‡Ž†ǡ   ™ƒ• ‘•–Ž› ‘™ ˆ‘” ‹–• ƒ••‘ ‹ƒ–‹‘™‹–Š‹†‡›‹Œ—”›Ǥ‘‰‡–Š‡”™‹–ŠŠ‡”Ž‹–‡”ƒ–—”‡–Š‡•‹•†‹• —••‹‰–Š‡ȋ’‘–‡–‹ƒŽȌ ˆ— –‹‘‘ˆ ƒ†’‘–‡–‹ƒŽ™ƒ›•–Š‡—•‡‘ˆ ‹†‹ƒ‰‘•–‹ •ƒ†–Š‡”ƒ’›ǡƒ‘˜‡Ž ”‡•‡ƒ” ŠŽ‹‡‹–Š‹•ƒ”‡ƒ™ƒ••–ƒ”–‡†‹‘”‹‡ǯ•‰”‘—’ǤŠ‘”–Ž›ƒˆ–‡”ǡ•Š‡™‡–‘–‘™”‹–‡ –™‘‰”ƒ–’”‘’‘•ƒŽ•™‹–Š‘”‹‡ǡ‘ˆ™Š‹ Š‘‡™ƒ•‰”ƒ–‡†ǡƒ†’—„Ž‹•Šƒ”‡˜‹‡™‹‹†‡› ‹”‘‡–ƒ„‘Ž‹•‹ƒ–Ǥ‡˜Ǥ‡’Š”Ǥǡ—†‡”‘”‹‡Ǥ ‡”†‹• ‘˜‡”›†—”‹‰Š‡”•–—†‹‡•–Šƒ––Š‡ •›•–‡•„‹‘Ž‘‰›ƒ†•›•–‡•‡†‹ ‹‡ƒ’’”‘ƒ ŠŠƒ˜‡–Š‡’‘–‡–‹ƒŽ–‘•‹‰‹ˆ‹ ƒ–Ž›‹’”‘˜‡ †‹ƒ‰‘•–‹ • ƒ† ’”‘‰‘•–‹ • ‹ ’ƒ–‹‡–• ™‹–Š ƒ ‹„‘” †‡ˆ‹ ‹‡ › ƒ† ‹’”‘˜‡ ‘—” —†‡”•–ƒ†‹‰‘ˆŠ—ƒŠ‡ƒŽ–Š‹‰‡‡”ƒŽǡ•’ƒ”‡†Š‡”‹–‡”‡•–‹–Š‡•‡ˆ‹‡Ž†•ƒ†–Š‡Š ”‡•‡ƒ” Š•Š‡’”‡•‡–‡†‹–Š‹•–Š‡•‹•Ǥ ‡”Š™ƒ• ƒ””‹‡†‘—–—†‡”–Š‡•—’‡”˜‹•‹‘‘ˆ’”‘ˆǤ ƒ”„ƒ”ƒ ƒ‡” ƒ† ’”‘ˆǤ ‹”Ǧ ƒ ‡‹Œ‰‘—†ǡ ‹ ‘ŽŽƒ„‘”ƒ–‹‘ •‡˜‡”ƒŽ ‘–Š‡”  ǯ• ƒ† ”‡•‡ƒ” Š‡”• ƒ† ™‹–Š Ž‹‹ ‹ƒ• ‘ˆ –Š‡ ‡†‹ƒ–”‹ • †‡’ƒ”–‡–Ǥ ‡”‡‹ǡ ƒ• –Š‡ –Š‡•‹• –‹–Ž‡ ‹†‹ ƒ–‡•ǡ •Š‡ ‡š’Ž‘”‡†–Š‡ ‡ Šƒ‹•• —†‡”Ž›‹‰ –Š‡ ’Š‡‘–›’‡ ‘ˆ  †‡ˆ‹ ‹‡ › „› —•‹‰ƒ•›•–‡•‡†‹ ‹‡ƒ’’”‘ƒ ŠǤ—”‹‰Š‡”Š•Š‡’”‡•‡–‡†ƒ–•‡˜‡”ƒŽƒ–‹‘ƒŽƒ† ‹–‡”ƒ–‹‘ƒŽ ‘ˆ‡”‡ ‡•ƒ†‘„–ƒ‹‡†ƒ–”ƒ˜‡Ž‰”ƒ–ˆ‘”ƒŽƒ”‰‡•›•–‡•„‹‘Ž‘‰› ‘ˆ‡”‡ ‡ ‹‡Ž„‘—”‡ǡ—•–”ƒŽ‹ƒǡƒ†ƒ„‡•–Ǧ‘”ƒŽǦ’”‡•‡–ƒ–‹‘’”‹ ‡ƒ––Š‡‘‡œ›‡™‘”•Š‘’‹ ƒ”•‡‹ŽŽ‡ǡ ”ƒ ‡Ǥ ˆ–‡” Š‡” Šǡ •Š‡ ™‹ŽŽ –ƒ‡ ƒ ™‡ŽŽǦ†‡•‡”˜‡† •Š‘”– •ƒ„„ƒ–‹ ƒŽ ƒ† ™‹ŽŽ ˆ—”–Š‡”’—”•—‡Š‡”‹–‡”‡•–•‹‹’”‘˜‹‰Š—ƒŠ‡ƒŽ–Š„›ƒ’’Ž›‹‰–Š‡•›•–‡•‡†‹ ‹‡ ƒ’’”‘ƒ Šƒ†™‘”‹‰ Ž‘•‡Ž›™‹–Š”‡•‡ƒ” Š‡”•ǡ Ž‹‹ ‹ƒǯ•ƒ†Š‡ƒŽ–Š ƒ”‡’”‘˜‹†‡”•Ǥ

ʹ͸͹

  WŽƌƚĨŽůŝŽ  

  



























 



































 ‘”–ˆ‘Ž‹‘

‘”Ž‘ƒ† ‡• ”‹’–‹‘ ‡ƒ” ȋȌ ‡ƒ Š‹‰‘Ž‡ —Žƒ”‡ŽŽŠ›•‹‘Ž‘‰› ‘—”•‡ʹͲͳ͹ ʹͲͳ͹ ͲǤͷ ‹ˆ‡ ‹‡ ‡•™‹–Š †—•–”›™‘”•Š‘’ ʹͲͳ͹ ͳǤͶ —’‡”˜‹•‘”‘ˆ‹–‡”ƒŽ‹‡ ƒ‰‡”ȋ͸Ǧ‘–Š‹–‡”•Š‹’Ȍ ʹͲͳ͹ ͶǤͲ Šƒ›ʹͲͳ͹‘”‰ƒ‹œƒ–‹‘ ‘‹––‡‡Ǧ†˜‹•‘”‹„‘ƒ”†‘ˆ ‘‹––‡‡ ʹͲͳ͸ ͺǤͲ  ʹͲͳ͸ ‘ˆ‡”‡ ‡ǡ ‡ƒǡ ‡”ƒ› ʹͲͳ͸ ͲǤͷ ƒ–Š‡ƒ–‹ ƒǦƒ•‹ ’”‹ ‹’Ž‡•  ʹͲͳ͸ ͲǤ͵ –”‡’”‡‡—”•Š‹’ƒ†ƒŽ‘”‹œƒ–‹‘ˆ‘”‡†‹ ƒŽ• ‹‡ ‡• ʹͲͳ͸ ͷǤͲ —”‘’‡ƒ–—†‡–‘— ‹Ž›’‘•‹—ʹͲͳ͸ ʹͲͳ͸ ͲǤͷ ”‹œ‡ˆ‘”„‡•–‘”ƒŽ’”‡•‡–ƒ–‹‘ƒ–‘‡œ›‡™‘”•Š‘’‹ ”ƒ ‡ ʹͲͳ͸ ͲǤͷ ‘‡œ›‡™‘”•Š‘’ǡƒ”•‡‹ŽŽ‡ǡ ”ƒ ‡ ʹͲͳ͸ ͳǤͲ •‹Ž‹ ‘Ž‹ˆ‡ǣ‘•–”ƒ‹–Ǧ„ƒ•‡†‘†‡ŽŽ‹‰ƒ–‰‡‘‡• ƒŽ‡ ʹͲͳ͸ ͲǤͻ Šƒ›ʹͲͳ͸‘”‰ƒ‹œƒ–‹‘ ‘‹––‡‡Ǧ’‘•‘”•Š‹’–‡ƒ‡„‡” ʹͲͳ͸ ͶǤͲ  ʹͲͳ͸ ‘ˆ‡”‡ ‡ ʹͲͳ͸ ͲǤͷ ‡ƒ Š‹‰‘Ž‡ —Žƒ”‡ŽŽŠ›•‹‘Ž‘‰› ‘—”•‡ʹͲͳ͸ ʹͲͳ͸ ͳǤͲ ‡ƒ Š‹‰ˆ‘”Š•–—†‡–• ʹͲͳ͸ ʹǤͲ —’‡”˜‹•‘”‘ˆ‹–‡”ƒ” ‡Ž‹‡‹”ƒƒ”ƒȋ͸Ǧ‘–Š‹–‡”•Š‹’Ȍ ʹͲͳ͸ ͶǤͲ ›–Š‘ ‘—”•‡ˆ‘”‹ˆ‡ ‹‡–‹•–• ʹͲͳͷ ͲǤͻ –Š‹ •‹‡•‡ƒ” Šƒ† ‹‡–‹ˆ‹  –‡‰”‹–›ȋ ʹͳͷͳȌ ʹͲͳͷ ʹǤͲ Šƒ›ʹͲͳͷȋƒ––‡†‡†ƒ•’ƒ”–‹ ‹’ƒ–Ȍ ʹͲͳͷ ͳǤͲ –”‘†— –‹‘‹–‘ ʹͲͳͷ ͳǤͶ ‹‘ ‘—”•‡ǣǦ•‡“†ƒ–ƒƒƒŽ›•‹• ʹͲͳͷ ͲǤͻ ‡”•‘ƒŽ‡ˆˆ‡ –‹˜‡‡•• ʹͲͳͷ ͲǤͷ –ƒ–‹•–‹ •‘—”•‡ʹͲͳͷ ʹͲͳͷ ʹǤͲ  ʹͲͳͷ ‘ˆ‡”‡ ‡ ʹͲͳͷ ͲǤͷ ‹‘ ‘—”•‡ǣ‹ •–ƒ”– ʹͲͳͷ ͲǤ͵ ‡ƒ Š‹‰‘Ž‡ —Žƒ”‡ŽŽŠ›•‹‘Ž‘‰› ‘—”•‡ʹͲͳͷ ʹͲͳͷ ͲǤͷ ‹‘ ‘—”•‡ǣƒ––‡”‡ ‘‰‹–‹‘ ʹͲͳͷ ͵ǤͲ ̷ʹͲͳͶ ‘ˆ‡”‡ ‡ ʹͲͳͶ ͲǤͷ ‹ ”‘„‹‘Ž‘‰‹ ƒŽ•ƒˆ‡–› ʹͲͳͶ ͳǤͲ ʹͲͳͶ ‘ˆ‡”‡ ‡ǡ‡Ž„‘—”‡ǡ—•–”ƒŽ‹ƒ ʹͲͳͶ ͲǤͷ  ʹͲͳͶ ‘ˆ‡”‡ ‡ǡ—”Šƒǡ ʹͲͳͶ ͲǤͷ ‡ƒ Š‹‰‘Ž‡ —Žƒ”‡ŽŽŠ›•‹‘Ž‘‰› ‘—”•‡ʹͲͳͶ ʹͲͳͶ ͲǤͷ †˜ƒ ‡†‡ –—”‡‘—”•‡‘›•–‡•‹‘Ž‘‰›ȋ›•„‹‘ʹͲͳͶȌǡ •„”— ǡ—•–”‹ƒ ʹͲͳͶ ʹǤͲ ̷ʹͲͳ͵ ‘ˆ‡”‡ ‡ ʹͲͳ͵ ͲǤͷ ”‡•‡–ƒ–‹‘‹ŽŽ• ʹͲͳ͵ ͲǤͷ ™‘Ǧ†ƒ› ‘—”•‡‘ƒ••’‡ –”‘‡–”› ʹͲͳ͵ ͲǤ͸ ”‘Œ‡ –ƒƒ‰‡‡–ƒ†  –”‘†— –‹‘ ʹͲͳ͵ ʹǤͲ ‘‘Ž•ƒ†ƒ’’”‘ƒ Š‡•‘ˆ•›•–‡•„‹‘Ž‘‰› ʹͲͳ͵ ͵ǤͲ –‡”ƒ–‹‘ƒŽ‘ˆ‡”‡ ‡‘›•–‡•‹‘Ž‘‰›ʹͲͳ͵ȋ ʹͲͳ͵Ȍǡ‘’‡Šƒ‰‡ǡ ‡ƒ” ʹͲͳ͵ ͲǤͷ ƒ–Š‡ƒ–‹ ƒǦƒ•‹ ’”‹ ‹’Ž‡•  ʹͲͳ͵ ͲǤ͵    

ʹ͹ͳ

  >ŝƐƚŽĨƉƵďůŝĐĂƚŝŽŶƐ  

  



























 



































 —„Ž‹ ƒ–‹‘•

 ƒ”–‹‡•  ǡ ‡–œ‡Ž• ǡ ™‹‡Ž• Ǥ Œœ‡” ‡ ‹‡”• Šƒ†‡Ǥ ƒ’‹–ƒ ‡Ž‡ –ƒ ‡ˆ”‘Ž‘‰‹‡ǡ —‡”ʹǡʹͲͳʹ  ƒ”–‹‡•  ǡ ƒ•‡”‡‡—™ ǡ ŒƒŽ•ƒ ǡ ‘‡†‡”‘’ ǡ ‡–œ‡Ž• ǡ ™‹‡Ž• Ǥ ”‘ ‡–ƒ„‘Ž‹• ‹ –Š‡ ’ƒ–Š‘‰‡‡•‹• ‘ˆ ‹”‘Ǧ‹†— ‡† ‹†‡› ‹Œ—”›Ǥ ƒ– ‡˜ ‡’Š”‘ŽǤ ʹͲͳ͵ —ŽǢͻȋ͹Ȍǣ͵ͺͷȂͻͺ  ˜ƒ—‡ǡ‘Ž‡”Ǧ‘—™ǡ ‡”†‹‰ǡŽ‡‡‡”ǡ‘Ž–‡”• ǡ˜ƒ‹Œ– ǡƒ”–‹‡• ǡ ‹‡œ‡Ǧ‘‹‰ǡ ‡‹‡”ǡ‡”‡–‹‡” ǡ ”‘‡ǡ‡‹Œ‰‘—† ǡ‡”• ǡƒ‡”Ǥ ‹˜‹‰‘–Š‡‡†‰‡ǣ—„•–”ƒ–‡ ‘’‡–‹–‹‘‡š’Žƒ‹•Ž‘••‘ˆ”‘„—•–‡••‹‹–‘ Š‘†”‹ƒŽˆƒ––›Ǧ ƒ ‹†‘š‹†ƒ–‹‘†‹•‘”†‡”•Ǥ‹‘ŽǤʹͲͳ͸ǢͳͶȋͳȌǣͳȂͳͷ  ƒ”–‹‡•Ǧ ǡ˜ƒ—‡ǡ‡‹Œ‰‘—†Ǧ ǡƒ‡”ǤŠ‡’”‘‹• —‘—•‡œ›‡‡†‹—Ǧ Šƒ‹ ͵Ǧ‡–‘Ǧƒ ›ŽǦ‘ –Š‹‘Žƒ•‡ –”‹‰‰‡”• ƒ ˜‹ ‹‘—• › Ž‡ ‹ ˆƒ––›Ǧƒ ‹† „‡–ƒǦ‘š‹†ƒ–‹‘Ǥ  ‘’—–‹‘ŽǤʹͲͳ͹Ǣͳ͵ȋͶȌǣͳȂʹʹǤ   

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