^ƵŵĂƌ

hD&͞ĂƌŽůĂǀŝůĂ͕͟ƵĐƵƌĞƕƚŝ
&ĂĐƵůƚĂƚĞĂĚĞDŽĂƕĞƕŝƐŝƐƚĞŶƜĉDĞĚŝĐĂůĉ
1. DetecƜŝĂ ƕŝĂŶĂůŝnjĂE

Curs 04: 2. ŶĂůŝnjĂĞƉŝŐĞŶŽŵƵůƵŝ

3. ŶĂůŝnjĂĞdžƉƌĞƐŝĞŝƕŝĨƵŶĐƜŝĞŝŐĞŶĞůŽƌ
NoƜŝƵŶŝ de ŐĞŶŽŵŝĐĉ, ƉƌŽƚĞŽŵŝĐĉ
ƕŝŝŶŐŝŶĞƌŝĞŐĞŶĞƚŝĐĉ
Titular curs: Conf. univ. dr. Mocanu Maria-Magdalena
e-mail: mocanu.umfcd@gmail.com

dĞŚŶŽůŽŐŝĂEƌĞĐŽŵďŝŶĂŶƚ
= manipularea ADN cu precizie in vitro sau in vivo
Principalele tehnici:
1. ^ĐŝŶĚĂƌĞĂEĐƵĞŶnjŝŵĞĚĞƌĞƐƚƌŝĐƜŝĞ
2. >ŝŐĂƌĞĂ E
ϭ͘ĞƚĞĐƜŝĂƕŝĂŶĂůŝnjĂE 3. ůŽŶĂƌĞĂE
• Vectori de clonare
• ZĞĂĐƜŝĂŠŶůĂŶƜĂpolimerazei
LP 4-6
4. ,ŝďƌŝĚŝnjĂƌĞĂE
5. ^ŝŶƚĞnjĂE
6. ^ĞĐǀĞŶƜŝĞƌĞĂ E

3 4
dĞŚŶŽůŽŐŝĂEƌĞĐŽŵďŝŶĂŶƚ
Avantaje:
• ^ĞƉŽƚŝĚĞŶƚŝĨŝĐĂƐĞĐǀĞŶƜĞŠŶƚƌĞŐŝĚĞŶƵĐůĞŽƚŝĚĞ – ex: bacterii,
ĐŝƵƉĞƌĐŝŠŶĐąƚĞǀĂŽƌĞ͖gemom ƵŵĂŶŠŶŵĂŝƉƵƜŝŶĚĞϭnjŝ
• ^ĞƉŽƚŝnjŽůĂŐĞŶĞĐƵƵƕƵƌŝŶƜĉ
• Se pot sintetiza in vitro ĐĂŶƚŝƚĉƜŝŵĂƌŝĚĞƉƌŽƚĞŝŶĞcodificate de
genele izolate prin tehnici de laborator – ex: insulina, interferonul Ϯ͘ŶĂůŝnjĂĞƉŝŐĞŶŽŵƵůƵŝ
• ^ĞƉŽƚƚƌĂŶƐĨĞƌĂŐĞŶĞde la un organism la altul – ex: organisme
transgenice utilizate pentru studiul unor boli

5 6

ŶĂůŝnjĂĞƉŝŐĞŶŽŵƵůƵŝ

• ŶĂůŝnjĂŵŽĚŝĨŝĐĉƌŝůŽƌE

• ŶĂůŝnjĂƐƚƌƵĐƚƵƌŝŝƕŝĚŝŶĂŵŝĐŝŝĐƌŽŵĂƚŝŶĞŝ
Ϯ͘ϭ͘ŶĂůŝnjĂŵŽĚŝĨŝĐĉƌŝůŽƌE

7 8
 ŶĂůŝnjĂŵŽĚŝĨŝĐĉƌŝůŽƌE ŶĂůŝnjĂŵŽĚŝĨŝĐĉƌŝůŽƌE
‰ŝŐĞƐƚŝĂEŐĞŶŽŵŝĐ ĐƵĂũƵƚŽƌƵůƵŶŽƌĞŶnjŝŵĞĚĞƌĞƐƚƌŝĐƜŝĞ
• ĞĂŵĂŝĚŝŶĂŵŝĐĉŶƵĐůĞŽƚŝĚĉ – ĐŝƚŽnjŝŶĂ
ƐĞŶƐŝďŝůĞůĂƉƌĞnjĞŶƜĂϱŵ

• Cca 50-60% din cele ϮϴŵŝůŝŽĂŶĞĚĞĐŝƚŽnjŝŶĞ de tip CpG sunt ŵĞƚŝůĂƚĞ • ^ĞƵƚŝůŝnjĞĂnjĉĞŶnjŝŵĞĚĞƌĞƐƚƌŝĐƜŝĞ
la nivelul atomului de ĐĂƌďŽŶĚŝŶƉŽnjŝƜŝĂϱ (5mC) (endonucleaze care ƐĐŝŶĚĞĂnjĉEůĂŽŶŝǀĞůƵůƵŶĞŝ
ĂŶƵŵŝƚĞƐĞĐǀĞŶƜĞnucleotidice)

• Prin ƐĞĐǀĞŶƜŝĞƌĞ ƐĞŝĚĞŶƚŝĨŝĐĉƕŝƐĞ

ĐŽŵƉĂƌĉŵĞƚŝůĂƌĞĂcitozinei
(ƉƌŽĐĞƐƵůŠŶĐĂƌĞƐĞĚĞƚĞƌŵŝŶĉŽƌĚŝŶĞĂƉƌĞĐŝƐĉĂ
nucleotidelor de la nivelul genelor)

9 ϭϬ
ϮϬϬϰ͕Head et al., Biotechniques

ŶĂůŝnjĂŵŽĚŝĨŝĐĉƌŝůŽƌE ŶĂůŝnjĂŵŽĚŝĨŝĐĉƌŝůŽƌE
‰/ŵƵŶŽƉƌĞĐŝƉŝƚĂƌĞĂ EŵĞƚŝůĂƚ ĐƵĂũƵƚŽƌƵůĂŶƚŝĐŽƌƉŝůŽƌĂŶƚŝ-ϱŵ ‰ ŽŶǀĞƌƐŝĂĐŚŝŵŝĐĉĂϱŵĐƵĂũƵƚŽƌƵůďŝƐƵůĨŝƚƵůƵŝĚĞƐŽĚŝƵ

• ŶƚŝĐŽƌƉŝŝrecunosc citozinele • Tratamentul cu ďŝƐƵůĨŝƚƵůĚĞ

metilate Æ izolarea ƕŝ
ĐŽŶĐĞŶƚƌĂƌĞĂƐĞĐǀĞŶƜĞůŽƌE
metilat
• Imunoprecipitarea ĞƐƚĞƵƌŵĂƚĉ
de ƐĞĐǀĞŶƜŝĞƌĞĂ nucleotidelor
ϭϭ
ϮϬϬϰ͕Head et al., Biotechniques
ƐŽĚŝƵÆ ĐŽŶǀĞƌƐŝĂ ĐŝƚŽnjŝŶĞůŽƌ
ŶĞŵĞƚŝůĂƚĞ ŠŶƵƌĂĐŝů
• ŝƚŽnjŝŶĞůĞ ŵĞƚŝůĂƚĞ sunt
ƌĞnjŝƐƚĞŶƚĞ la modificare
• Analiza citozinei metilate prin
tehnici de ƐĞĐǀĞŶƜŝĞƌĞ
ϭϮ
ϮϬϬϰ͕Head et al., Biotechniques

Ϯ͘Ϯ͘ŶĂůŝnjĂƐƚƌƵĐƚƵƌŝŝƕŝĚŝŶĂŵŝĐŝŝ
ĐƌŽŵĂƚŝŶĞŝ
ŶĂůŝnjĂĐƌŽŵĂƚŝŶĞŝŝŶƚĞƌŶƵĐůĞŽnjŽŵĂůĞ
EƵĐůĞŽnjŽŵŝŝ, proteinele asociate
ĐƌŽŵĂƚŝŶĞŝďůŽĐŚĞĂnjĉƐĐŝŶĚĂƌĞĂ
ĞŶnjŝŵĂƚŝĐĉĂEÆ ĐƌĞĞĂnjĉ
ĂŵƉƌĞŶƚĞŐĞŶĞƚŝĐĞ(lb. engl.
footprints) care corespund
ŝŶƚĞƌĂĐƜŝƵŶŝůŽƌĚŝŶƚƌĞƐĞĐǀĞŶƜĞůĞ
EƕŝƉƌŽƚĞŝŶĞ
Accesul la cromatina
ŝŶƚĞƌŶƵĐůĞŽnjŽŵĂůĉ:
• Enzimatic
• Chimic
• Epigenetic
2014, Zentner & Henikoff, NATURE REVIEWS | GENETICS
ŶĂůŝnjĂƐƚƌƵĐƚƵƌŝŝƕŝĚŝŶĂŵŝĐŝŝĐƌŽŵĂƚŝŶĞŝ
WƌŽĐĞƐĞŝŵƉůŝĐĂƚĞŠŶƌĞŐůĂƌĞĂĞdžƉƌĞƐŝĞŝŐĞŶŝĐĞ:
• Cromatina este extrem de ĚŝŶĂŵŝĐĉ Æ nucleozomii se pot deplasa de-a lungul
ĨŝďƌĞŝĚĞĐƌŽŵĂƚŝŶĉ͕ƉŽƚĨŝĞdžĐůƵƕŝƐĂƵŠŶůŽĐƵŝƜŝ
• ƌŽŵĂƚŝŶĂĨŽƌŵĞĂnjĉďƵĐůĞ ce permit ŝŶƚĞƌĂĐƜŝƵŶĞĂƵŶŽƌůŽĐŝ ƐŝƚƵĂƜŝůĂĚŝƐƚĂŶƜĉ
ƵŶƵůĚĞĂůƚƵůƉĞĂĐĞůĂƕŝĐƌŽŵŽnjŽŵƐĂƵƉĞĐƌŽŵŽnjŽŵŝĚŝĨĞƌŝƜŝ
dĞŚŶŝĐŝĚĞĂŶĂůŝnjĉ:
• ŶĂůŝnjĂĐƌŽŵĂƚŝŶĞŝŝŶƚĞƌŶƵĐůĞŽnjŽŵĂůĞ
• /ŵƵŶŽƉƌĞĐŝƉŝƚĂƌĞĂ ĐƌŽŵĂƚŝŶĞŝĐƵĂŶƚŝĐŽƌƉŝƐƉĞĐŝĨŝĐŝ
ϭϯ ϭϰ
ŶĂůŝnjĂĐƌŽŵĂƚŝŶĞŝŝŶƚĞƌŶƵĐůĞŽnjŽŵĂůĞ
EƵĐůĞŽnjŽŵŝŝ, proteinele asociate
ĐƌŽŵĂƚŝŶĞŝďůŽĐŚĞĂnjĉƐĐŝŶĚĂƌĞĂ
ĞŶnjŝŵĂƚŝĐĉĂEÆ ĐƌĞĞĂnjĉ
ĂŵƉƌĞŶƚĞŐĞŶĞƚŝĐĞ(lb. engl. Nucleozomii, proteinele asociate cromatinei
footprints) care corespund
ŝŶƚĞƌĂĐƜŝƵŶŝůŽƌĚŝŶƚƌĞƐĞĐǀĞŶƜĞůĞ ďůŽĐŚĞĂnjĉƐĐŝŶĚĂƌĞĂĞŶnjŝŵĂƚŝĐĉĂEÆ
EƕŝƉƌŽƚĞŝŶĞ ĐƌĞĞĂnjĉamprente genetice (lb. engl.
footprints) care corespund ŝŶƚĞƌĂĐƜŝƵŶŝůŽƌ
Accesul la cromatina ŝŶƚĞƌŶƵĐůĞŽnjŽŵĂůĉ: ĚŝŶƚƌĞƐĞĐǀĞŶƜĞůĞEƕŝƉƌŽƚĞŝŶĞ
Accesul la cromatina • Enzimatic
ŝŶƚĞƌŶƵĐůĞŽnjŽŵĂůĉ:
• Enzimatic • Chimic
• Chimic • Epigenetic
• Epigenetic
ϮϬϭϬ͕ůďĞƌƚƐĞƚĂů͕ƐƐĞŶƚŝĂůĞůůŝŽůŽŐLJ
ϭϱ ϭϲ
ϮϬϭϰ͕Zentner & Henikoff, EdhZZs/t^| 'Ed/^
 ^ĐŝŶĚĂƌĞĂĞŶnjŝŵĂƚŝĐĉĂĐƌŽŵĂƚŝŶĞŝ ^ĐŝŶĚĂƌĞĂĞŶnjŝŵĂƚŝĐĉĂĐƌŽŵĂƚŝŶĞŝ
EƵĐůĞĂnjĞ • ŝŐĞƐƚŝĂĐƌŽŵĂƚŝŶĞŝĐƵĂũƵƚŽƌƵůDEĂnjĞŝ
• EƵĐůĞĂnjĂ ŵŝĐƌŽĐŽĐĂůĉ (DEĂƐĞ) (MNase-seq)
• ĞnjŽdžŝƌŝďŽŶƵĐůĞĂnjĂ I (EĂƐĞ I)
• ŝŐĞƐƚŝĂĐƌŽŵĂƚŝŶĞŝĐƵĂũƵƚŽƌƵůEĂnjĞŝ I
• dŶϱƚƌĂŶƐƉŽnjĂnjĂ ;dŶϱĂƐĞͿ
(DNase-seq)

• ŶĂůŝnjĂĐƌŽŵĂƚŝŶĞŝĐƵĂũƵƚŽƌƵůdŶϱĂnjĞŝ
(lb. engl. tagmentation/ assay for
transposase-accessible chromatin using
sequencing/ ATAC-seq)

ϭϳ ϭϴ
2014, Zentner & Henikoff, NATURE REVIEWS | GENETICS

^ĐŝŶĚĂƌĞĂĐŚŝŵŝĐĉĂĐƌŽŵĂƚŝŶĞŝ DĞƚŝůĂƌĞĂEŝŶƚĞƌŶƵĐůĞŽnjŽŵĂů

• ^ĞŝnjŽůĞĂnjĉŶƵĐůĞŝŝ
• ^ĞŵĂƌĐŚĞĂnjĉŶƵĐůĞŽnjŽŵŝŝ (ex:
phenantrolina ƌĞĐƵŶŽĂƕƚĞ,ϰ-S47C)
• ^ĞƚƌĂƚĞĂnjĉĐƵŵĞƚŝůƚƌĂƐĨĞƌĂnjĉ Æ sunt
ŵĞƚŝůĂƚĞ ĚŝŶƵĐůĞŽƚŝĚĞůĞ GpC
• ^ĞĂĚĂƵŐĉƵ2+ ƕŝ,2O2 Æ ƐĐŝŶĚĂƌĞĂ E
• Eeste fragmentat, purificat

• /njŽůĂƌĞĂ ƕŝƐĞĐǀĞŶƜŝĞƌĞĂ E • ^ĞƚƌĂƚĞĂnjĉĐƵďŝƐƵůĨŝƚ Æ citozinele

metilate sunt rezistente la conversia
ĐŝƚŽnjŝŶĉ-uracil

• Se ƐĞĐǀĞŶƜŝĂnjĉ E

ϭϵ ϮϬ
2014, Zentner & Henikoff, NATURE REVIEWS | GENETICS 2014, Zentner & Henikoff, NATURE REVIEWS | GENETICS
 /ŵƵŶŽƉƌĞĐŝƉŝƚĂƌĞĂ ĐƌŽŵĂƚŝŶĞŝĐƵĂŶƚŝĐŽƌƉŝ
/ŵƵŶŽƉƌĞĐŝƉŝƚĂƌĞĂ ĐƵĂŶƚŝĐŽƌƉŝƐƉĞĐŝĨŝĐŝ;ChIP)
ƐƉĞĐŝĨŝĐŝ;ChIP)
(lb. engl. chromatin immunoprecipitation – ChIP)
• Ś/W ĐƵĐƌŽŵĂƚŝŶĉĨŝdžĂƚĉ(lb. engl. cross-linking ChIP/ XChIP) – ƵƚŝůŝnjĂƚĉ
Scop: pentru studierea histonelor ƐĂƵĨĂĐƚŽƌŝůŽƌĚĞƚƌĂŶƐĐƌŝƉƜŝĞ͖ƚĞŚŶŝĐĂ
ƉƌĞƐƵƉƵŶĞĨŝdžĂƌĞĂĐƌŽŵĂƚŝŶĞŝĐƵĨŽƌŵĂůĚĞŚŝĚĉ;ĨĂĐŝůŝƚĞĂnjĉĂƐŽĐŝĞƌĞĂ
• ^ĞĂŶĂůŝnjĞĂnjĉŝŶƚĞƌĂĐƜŝƵŶĞĂĚŝŶƚƌĞƉƌŽƚĞŝŶĞƕŝE ƉƌŽƚĞŝŶĞůŽƌůĂEͿ

DĞƚŽĚĂĐŽŵďŝŶĉŝŵƵŶŽƉƌĞĐŝƉŝƚĂƌĞĂ ĐƌŽŵĂƚŝŶĞŝƕŝƐĞĐǀĞŶƜŝĞƌĞĂ E • Ś/W ŶĂƚŝǀĉ(lb. engl. native ChIP/ NChIP) – ƵƚŝůŝnjĂƚĉƉĞŶƚƌƵƐƚƵĚŝƵů
Æ identificĉ situsurile de legare a proteinelor ůĂE ŵŽĚŝĨŝĐĉƌŝůŽƌƉŽƐƚƌĂŶƐůĂƜŝŽŶĂůĞ ale histonelor

Ϯϭ 22

/ŵƵŶŽƉƌĞĐŝƉŝƚĂƌĞĂ ĐƵĐƌŽŵĂƚŝŶĉĨŝdžĂƚĉ;XChIP) /ŵƵŶŽƉƌĞĐŝƉŝƚĂƌĞĂ ĐƌŽŵĂƚŝŶĞŝŶĂƚŝǀĞ;NChIP)

MNase, ŶƵĐůĞĂnjĉ ŵŝĐƌŽĐŽĐĂůĉ (lb engl micrococcal nuclease)

23 24
2014, Zentner & Henikoff, NATURE REVIEWS | GENETICS
S 2014, Zentner & Henikoff, NATURE REVIEWS | GENETICS
ǀĂŶƚĂũĞƕŝĚĞnjĂǀĂŶƚĂũĞNChIP ǀƐ XChIP
ĂƌĂĐƚĞƌŝƐƚŝĐŝ yŚ/W EŚ/W
ƵƌĂƚĉ 5 ore ϭ-2 zile

Fixare Da (celulele ŶƵƐƵŶƚŠŶƐƚĂƌĞ

ŶĂƚŝǀĉ– fixarea poate masca unii
EƵ;ĐĞůƵůĞ ŠŶƐƚĂƌĞŶĂƚŝǀĉͿ
ϯ͘ŶĂůŝnjĂĞdžƉƌĞƐŝĞŝƕŝĨƵŶĐƜŝĞŝ
epitopiͬŶƵŵĂŝƐƵŶƚƌĞĐƵŶŽƐĐƵƜŝ
de anticorpi) ŐĞŶĞůŽƌ
Identificarea ĞƚĞĐƜŝĂŵŽĚŝĨŝĐĉƌŝůŽƌ histonice ĞƚĞĐƜŝĂŵŽůĞĐƵůĞůŽƌ de interes
ŵŽĚŝĨŝĐĉƌŝůŽƌ ƕŝĂĨĂĐƚŽƌŝůŽƌĚĞƚƌĂŶƐĐƌŝƉƜŝĞ ĞdžƉƌŝŵĂƚĞŠŶĐĂŶƚŝƚĂƚĞŵĂƌĞ
post- hŶŝŝĨĂĐƚŽƌŝĚĞƚƌĂŶƐĐƌŝƉƜŝĞĐƵ
ƚƌĂŶƐůĂƜŝŽŶĂůĞ ĞdžƉƌĞƐŝĞƌĞĚƵƐĉŶƵƉŽƚĨŝ
ĚĞƚĞĐƚĂƜŝ
25 26

ŶĂůŝnjĂĞdžƉƌĞƐŝĞŝƕŝĨƵŶĐƜŝĞŝŐĞŶĞůŽƌ

‰ŶĂůŝnjĂĞdžƉƌĞƐŝĞŝŐĞŶĞůŽƌůĂŶŝǀĞůZE

‰ŶĂůŝnjĂĞdžƉƌĞƐŝĞŝŐĞŶĞůŽƌůĂŶŝǀĞůĚĞƉƌŽƚĞŝŶĞ
ϯ͘ϭ͘ŶĂůŝnjĂĞdžƉƌĞƐŝĞŝŐĞŶĞůŽƌůĂ
ŶŝǀĞůĚĞZE

27 28
 'ĞŶĞƚŝĐĂŝŶǀĞƌƐĉ 'ĞŶĞƚŝĐĂŝŶǀĞƌƐĉ– ƚĞŚŶŝĐŝ
'ĞŶĞƚŝĐĉ • dĞŚŶŝĐĂ͟ŐĞŶĞŬŶŽĐŬŽƵƚ͟
• Organism mutant Æ identificarea genei Æ identificarea proteinei • DŽĚŝĨŝĐĂƌĞĂĞdžƉƌĞƐŝĞŝŐĞŶŝĐĞ
• KƌŐĂŶŝƐŵĞƚƌĂŶƐŐĞŶŝĐĞ
'ĞŶĞƚŝĐĉ ŝŶǀĞƌƐĉ • dĞŚŶŽůŽŐŝŝďĂnjĂƚĞƉĞŝŶƚĞƌĨĞƌĞŶƜĂZE;ZEŝ)
• 'ĞŶĞƌĂƉŽƌƚŽĂƌĞ
• Modificarea genei de interes Æ generarea de organisme mutante
• ,ŝďƌŝĚŝnjĂƌĞĂin ƐŝƚƵ ĂZE
• ZĞĂĐƜŝĂ ƉŽůŝŵĞƌĂnjĞŝ ŠŶůĂŶƜƉƌŝŶƚƌĂŶƐĐƌŝƉƜŝĞŝŶǀĞƌƐĉ;Zd-WZͿ
• DŝĐƌŽƌĞƜĞůĞ E
• /ŵƵŶŽƉƌĞĐŝƉŝƚĂƌĞĂ ĐƌŽŵĂƚŝŶĞŝ
ϮϬϭϱ͕Alberts et al, Molecular ŝŽůŽŐLJ of the Cell
29 ϯϬ
ϮϬϭϳ͕Covic et al, Genetica Medicala

dĞŚŶŝĐĂ͟ŐĞŶĞŬŶŽĐŬŽƵƚ͟ DŽĚŝĨŝĐĂƌĞĂĞdžƉƌĞƐŝĞŝŐĞŶĞŝĚĞŝŶƚĞƌĞƐ
= ĚĞůĞƜŝĂ unei gene pe cei doi cromozomi omologi Æ se observĉ сŵŽĚŝĨŝĐĉƌŝĐĞƉĞƌŵŝƚŽƉƌŝƌĞĂͬƉŽƌŶŝƌĞĂƚƌĂŶƐĐƌŝƉƜŝĞŝŐĞŶĞůŽƌ
ĞĨĞĐƚĞůĞŠŶůŝƉƐĂŐĞŶĞŝ • ^ŝƐƚĞŵdĞƚ-KEͬdĞƚ-OFF
• La modele animale ĚŽdžŝĐŝĐůŝŶĂ ƐĞĂĚĂƵŐĉŠŶĂƉĂĚĞďĉƵƚ
• ĂĐĉrepresorul Tet este construit cu ƐƉĞĐŝĨŝĐŝƚĂƚĞƉĞŶƚƌƵƵŶĂŶƵŵŝƚƜĞƐƵƚ,
KďƐĞƌǀĂƜŝĞ͗ ŐĞŶĂƐĞĞdžƉƌŝŵĉĚŽĂƌŠŶĂĐĞůƜĞƐƵƚ
• /ŵƉŽƌƚĂŶƚĞƐƚĞĐĂŐĞŶĂƐĉŶƵĨŝĞĞƐĞŶƜŝĂůĉƉĞŶƚƌƵƐƵƉƌĂǀŝĞƜƵŝƌĞ

ϯϭ 32
 DŽĚŝĨŝĐĂƌĞĂĞdžƉƌĞƐŝĞŝŐĞŶĞŝĚĞŝŶƚĞƌĞƐ DŽĚŝĨŝĐĂƌĞĂĞdžƉƌĞƐŝĞŝŐĞŶĞŝĚĞŝŶƚĞƌĞƐ

‰ ^ŝƐƚĞŵdĞƚ-KE ‰ ^ŝƐƚĞŵdĞƚ-OFF
• &ƵnjŝƵŶĞ • &ƵnjŝƵŶĞ
• Domeniu ĂĐƚŝǀĂƚŽƌ
• Domeniu ĂĐƚŝǀĂƚŽƌ
• Domeniu ƌĞƉƌĞƐŽƌ ĐĞƌĞĐƵŶŽĂƕƚĞƌĞŐŝƵŶĞĂ
ĐĂƌĞƌĞŐůĞĂnjĉƚƌĂŶƐĐƌŝƉƜŝĂŐĞŶĞŝy • Domeniu ƌĞƉƌĞƐŽƌ ĐĞƌĞĐƵŶŽĂƕƚĞƌĞŐŝƵŶĞĂ
ĐĂƌĞƌĞŐůĞĂnjĉƚƌĂŶƐĐƌŝƉƜŝĂŐĞŶĞŝy

• ĂƚŽƌŝƚĉƉƌĞnjĞŶƜĞŝĂĐƚŝǀĂƚŽƌƵůƵŝ – este • 2ŶƉƌĞnjĞŶƜĂĚŽdžŝĐŝĐůŝŶĞŝ ƚƌĂŶƐĐƌŝƉƜŝĂŐĞŶĞŝ

ĂŵƉůŝĨŝĐĂƚŶŝǀĞůƵůƚƌĂŶƐĐƌŝƉƜŝĞŝŐĞŶĞy este ŽƉƌŝƚĉ, deoarece complexul represor-
ĂĐƚŝǀĂƚŽƌŶƵŵĂŝŝŶƚĞƌĂĐƜŝŽŶĞĂnjĉĐƵE
• GĞŶĂ ĞƐƚĞ ĞdžƉƌŝŵĂƚĉ ^ĞĐǀĞŶƜĂƌĞŐůĂƚŽĂƌĞcis
;ŠŶĂďƐĞŶƜĂdoxiciclinei͕ŽǀĞƌƐŝƵŶĞƐƚĂďŝůĉĂ ƉĞŶƚƌƵŐĞŶĂy
tetraciclinei, Tet)

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WƌŽĐĞĚƵƌĂĚĞŽďƜŝŶĞƌĞĂĂŶŝŵĂůĞůŽƌ
KƌŐĂŶŝƐŵĞƚƌĂŶƐŐĞŶŝĐĞ
ƚƌĂŶƐŐĞŶŝĐĞ
= organisme modificate genetic prin ĚĞůĞƜŝĂ sau înlocuirea unei gene
^͕ĐĞůƵůĞƐƚĞŵĞŵďƌŝŽŶĂƌĞ
= celule ƉůƵƌŝƉŽƚĞŶƚĞ derivate
din blastocist (un stadiu de
ĂĐĉŽŵŽůĞĐƵůĉEĐĂƌĞƚƌĂŶƐƉŽƌƚĉŽŐĞŶĉŵƵƚĂŶƚĉĞƐƚĞƚƌĂŶƐĨĞƌĂƚĉ dezvoltare a embrionului,
unei celule murine: ŠŶĂŝŶƚĞĚĞŝŵƉůĂŶƚĂƌĞͿ

• IŶƐĞƌƜŝĞĂůĞĂƚŽƌŝĞ
ĞůƵůĞƉůƵƌŝƉŽƚĞŶƚĞ
• 2ŶůŽĐƵŝĞƕƚĞŐĞŶĂŶŽƌŵĂůĉ сĐĞůƵůĞĐĂƌĞĂƵƉŽƚĞŶƜŝĂůƵů
ĚĞĂƐĞĚŝĨĞƌĞŶƜŝĂŠŶŽƌŝĐĂƌĞ
dintre cele trei straturi
embrionare (endoderm,
mesoderm, ectoderm)

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 ŶŝŵĂůĞƚƌĂŶƐŐĞŶŝĐĞ
cŽĂƌĞĐĞŶŽƌŵĂů cŽĂƌĞĐĞtransgenic ĐĂƌĞĞdžƉƌŝŵĉŽŚĞůŝĐĂnjĉ E
ŵƵƚĂŶƚĉ͖helicaza ĞƐƚĞŝŵƉůŝĐĂƚĉŠŶƚƌĂŶƐĐƌŝƉƜŝĂEŠŶ
ZEƕŝŠŶƌĞƉĂƌĂƌĞĂE
Fenotip͗ŠŵďĉƚƌąŶŝƌĞƉƌĞŵĂƚƵƌĉ͕ŽƐƚĞŽƉŽƌŽnjĉ͕ĂůďŝƌĞĂ
ƉƌĞŵĂƚƵƌĉĂďůĉŶŝŝ͕ŝŶĨĞƌƚŝůŝƚĂƚĞ͕ĚƵƌĂƚĂƌĞĚƵƐĉĂ
ƐƵƉƌĂǀŝĞƜƵŝƌŝŝ 37
ϮϬϭϱ͕Alberts et al, Molecular ŝŽůŽŐLJ of the Cell
dĞŚŶŽůŽŐŝŝďĂnjĂƚĞƉĞŝŶƚĞƌĨĞƌĞŶƜĂZE;ZEŝ)
• WůĂĐŝĐƵϵϲĚĞŐŽĚĞƵƌŝĐĂƌĞĐŽŶƜŝŶE. coli cu o ŐĞŶĉ
ƉĞŶƚƌƵĨĞƌƚŝůŝƚĂƚĞŵŽĚŝĨŝĐĂƚĉ
• 2ŶĨŝĞĐĂƌĞŐŽĚĞƵƐƵŶƚĂĚĉƵŐĂƜŝϭϬC. elegans care vor
ingera bacteriile Æ ĨĞŶŽƚŝƉƵů acestora ƐĞva ŵŽĚŝĨŝĐĂ
• Examinare la ŵŝĐƌŽƐĐŽƉ a plĉĐŝŝ cu 96 de godeuri Æ
nematode normale vs nematode cu abilitate redusĉ
de a se reproduce
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dĞŚŶŽůŽŐŝŝďĂnjĂƚĞƉĞŝŶƚĞƌĨĞƌĞŶƜĂZE;ZEŝ)
сƵƚŝůŝnjĞĂnjĉƉƌŽƉƌŝĞƚĂƚĞĂŽƌŐĂŶŝƐŵĞůŽƌǀŝŝĚĞĂƐĞ
proteja împotriva virusurilor sau elementelor
genetice mobile
(B) Embrion normal de la C. elegans ambii pro-nucleii
(ƃ,ƂͿƐĞĂĨůĉŠŶƌĞŐŝƵŶĞĂƉŽƐƚĞƌŝŽĂƌĉĂĞŵďƌŝŽŶƵůƵŝ͕ůĂ
ƐĐƵƌƚƚŝŵƉĚƵƉĉĨĞƌƚŝůŝnjĂƌĞ
(C) Embrion mutant ĂĐĉƌƵŝŐĞŶĉĚĞŝŶƚĞƌĞƐĂĨŽƐƚ
ŝŶĂĐƚŝǀĂƚĉ prin ƚĞŚŶŽůŽŐŝĂďĂnjĂƚĉƉĞŝŶƚĞƌĨĞƌĞŶƜĂZEÆ
cei 2 pro-nuclei nu au migrat ŠŵƉƌĞƵŶĉŠŶƌĞŐŝƵŶĞĂ
ƉŽƐƚĞƌŝŽĂƌĉĂĞŵďƌŝŽŶƵůƵŝ
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'ĞŶĞƌĂƉŽƌƚŽĂƌĞ
• ^ĞĐǀĞŶƜĂƌĞŐůĂƚŽĂƌĞcis Æ ĐŽŶƚƌŽůĞĂnjĉ
ƚƌĂŶƐĐƌŝƉƜŝĂ
• Se poate introduce o ŐĞŶĉƌĂƉŽƌƚŽĂƌĞ
= o gene ĂƐŽĐŝĂƚĉ ƐĞĐǀĞŶƜĞŝƌĞŐůĂƚŽĂƌĞĂ
unei gene de interes; caracteristicile genei
raportoare ƐĞƉŽƚŵĉƐƵƌĂĐƵƵƕƵƌŝŶƜĉ
;ŵŽůĞĐƵůĉĨůƵŽƌĞƐĐĞŶƚĉ͕ĞŶnjŝŵĉͿ
• În celulele EK cele mai comune proteine
raportoare sunt:
• ĞŶnjŝŵĂɴ-ŐĂůĂĐƚŽnjŝĚĂnjĂ
• ƉƌŽƚĞŝŶĂĨůƵŽƌĞƐĐĞŶƚĉǀĞƌĚĞ
ϰϬ
ϮϬϭϱ͕Alberts et al, Molecular ŝŽůŽŐLJ of the Cell
 'ĞŶĞƌĂƉŽƌƚŽĂƌĞ– ƉƌŽƚĞŝŶĂĨůƵŽƌĞƐĐĞŶƚĉ
'ĞŶĞƌĂƉŽƌƚŽĂƌĞ– ĞŶnjŝŵĂɴ-ŐĂůĂĐƚŽnjŝĚĂnjĂ
ǀĞƌĚĞ;ŐƌĞĞŶ ĨůƵŽƌĞƐĐĞŶƚƉƌŽƚĞŝŶ, GFP)
D. melanogaster – embrion
'&WĞƐƚĞŝnjŽůĂƚĉĚŝŶ
Aequorea victoria ;ŵĞĚƵnjĉͿ

^ƚƌƵĐƚƵƌĉĨŽƌŵĂƚĉĚŝŶϭϭĨŽŝ
ɴ-ƉůŝĂƚĞƕŝƵŶĐƌŽŵŽĨŽƌ
plasat central

D. melanogaster – embrion
Promoter ĂĐƚŝǀĚŽĂƌŠŶŶĞƵƌŽŶŝ
– fuzionat cu GFP Æ
vizualizeazĉΕϮϬneuroni
localizaƜŝůĂƐƵƉƌĂĨĂƜĂ
embrionului (recunosc stimulii
Stripe͕ďĂŶĚĉ din mediul extern)
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ZĞĂĐƜŝĂ ƉŽůŝŵĞƌĂnjĞŝ ŠŶůĂŶƜƉƌŝŶƚƌĂŶƐĐƌŝƉƜŝĞ

,ŝďƌŝĚŝnjĂƌĞĂin situ ĂZE
ŝŶǀĞƌƐĉ;Zd-WZ)
• WŽĂƚĞĨŝƵƚŝůŝnjĂƚĉƉĞŶƚƌƵŐĞŶĞƌĂƌĞĂƵŶƵŝ (lb. engl. reverse transcription-polymerase chain reaction)
ĂƚůĂƐĂůĞdžƉƌĞƐŝĞŝŐĞŶĞůŽƌŠŶĞŶĐĞĨĂůƵů
ŵƵƌŝŶ
• ^ĞŵĉƐŽĂƌĉŶŝǀĞůƵůĞdžƉƌĞƐŝĞŝZEŵ
• Sunt prezentate zonele asociate ŠŶǀĉƜĉƌŝŝ
ƕŝŵĞŵŽƌŝnjĉƌŝŝ
• &ůƵŽƌĞƐĐĞŶƜĂ ĞƐƚĞŵĉƐƵƌĂƚĉĚŽĂƌĂƚƵŶĐŝĐąŶĚ
ŵŽůĞĐƵůĂĨůƵŽƌĞƐĐĞŶƚĉƐĞĨŝdžĞĂnjĉůĂEĚĐ
• fĞƐƵƚƵůĞƐƚĞĨŝdžĂƚƉĞŶƚƌƵĂƌĞƜŝŶĞZEŵ
Æ expunerea ZEŵ Æ ŚŝďƌŝĚŝnjĂƌĞĂ cu
ŽůŝŐŽŶƵĐůĞŽƚŝĚĞDEͬZE • WƌŽďĂŠŶƌŽƕƵ(ƐƚąŶŐĂͿĂƌĞŽĐŽŶĐĞŶƚƌĂƜŝĞŵĂƌĞĚĞ
ZEŵ Æ ŶĞĐĞƐŝƚĉŵĂŝƉƵƜŝŶĞĐŝĐůƵƌŝWZpentru a
• Avantaj: ŶƵŶĞĐĞƐŝƚĉƵƚŝůŝnjĂƌĞĂƚĞŚŶŝĐŝůŽƌ ĂũƵŶŐĞůĂĂĐĞĞĂƕŝĐŽŶĐĞŶƚƌĂƜŝĞŵĂdžŝŵĉÆ se poate
ĚĞŝŶŐŝŶĞƌŝĞŐĞŶĞƚŝĐĉ determina ƉƌĞĐŝƐcantitatea de ZEŵ

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 DŝĐƌŽƌĞƜĞůĞ E;ůď͘ĞŶŐů͘microarray) DŝĐƌŽƌĞƜĞůĞ E
• Analiza ĂƐƵƚĞĚĞŵŽůĞĐƵůĞĚĞŵZE
ŠŶƚƌ-ƵŶƐŝŶŐƵƌĞdžƉĞƌŝŵĞŶƚ (ŵŝĐƌŽƌĞƜĞůĞ ŽĚĐƵůŽĂƌĞ džƉƌĞƐŝĞŐĞŶĞ
ĐŽŶƜŝŶnjĞĐŝ– ƐƵƚĞĚĞŽůŝŐŽŶƵĐůĞŽƚŝĚĞ
ĚŝĨĞƌŝƚĞ fixate pe un suport solid) ZŽƕƵ 'ĞŶĞůĞĚŝŶƉƌŽďĂϭƐƵŶƚ
exprimate la un nivel mai ridicat
• Planificare experiment: ĐŽŵƉĂƌĂƜŝĞ ĚĞĐąƚŐĞŶĞůĞĚŝŶƉƌŽďĂϮ
ĚŝƌĞĐƚĉ ŠŶƚƌĞĐĞůƵůĞƚƌĂƚĂƚĞĐƵŚŽƌŵŽŶŝƕŝ
ĐĞůƵůĞĨĉƌĉƚƌĂƚĂŵĞŶƚ Verde Genele din proba 2 sunt
exprimate la un nivel mai ridicat
• Etape: ĐŽŶǀĞƌƐŝĂZEŵ ŠŶEĚĐ Æ ĚĞĐąƚŐĞŶĞůĞĚŝŶƉƌŽďĂϭ
ĚĞŶĂƚƵƌĂƌĞEƕŝŵĂƌĐĂƌĞ
ĨůƵŽƌĞƐĐĞŶƚĉ (roƕƵƉĞŶƚƌƵŽƉƌŽďĉƕŝ
Galben Genele au un nivel al expresiei
ǀĞƌĚĞƉĞŶƚƌƵĂĐĞĂůĂůƚĉͿÆ probele ĂƐĞŵĉŶĉƚŽƌŠŶĂŵďĞůĞƉƌŽďĞ
marcate ƐĞ ĐŽŵďŝŶĉƕŝƐĞ ƉůĂƐĞĂnjĉ pe EĞŐƌƵ EƵĞdžŝƐƚĉ ĞdžƉƌĞƐŝĞŐĞŶŝĐĉ
ƐƵƉƌĂĨĂƜĂŵŝĐƌŽƌĞƜĞůĞŝ Æ ŚŝďƌŝĚŝnjĂƌĞ Æ
(26x26) ƐƉĉůĂƌĞ Æ ƐĐĂŶĂƌĞĂ ĨůƵŽƌĞƐĐĞŶƜĞŝ
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/ŵƵŶŽƉƌĞĐŝƉŝƚĂƌĞĂ ĐƌŽŵĂƚŝŶĞŝůĂŶŝǀĞůƵů /ŵƵŶŽƉƌĞĐŝƉŝƚĂƌĞĂ ĐƌŽŵĂƚŝŶĞŝůĂŶŝǀĞůƵů

ŠŶƚƌĞŐƵůƵŝŐĞŶŽŵ
• Metoda permite ŝĚĞŶƚŝĨŝĐĂƌĞĂƚƵƚƵƌŽƌ
ƐŝƚƵƐƵƌŝůŽƌ ĚŝŶŐĞŶŽŵƉĞĐĂƌĞƵŶƌĞŐůĂƚŽƌĂů
ƚƌĂŶƐĐƌŝƉƜŝĞŝůĞŽĐƵƉĉin ǀŝǀŽ
• Tehnica: În celule, proteinele sunt ĨŝdžĂƚĞ de
EƉƌŝŶƵƚŝůŝnjĂƌĞĂĚĞĨŽƌŵĂůĚĞŚŝĚĉÆ
celulele sunt ůŝnjĂƚĞ Æ Eeste ĨƌĂŐŵĞŶƚĂƚ
ŵĞĐĂŶŝĐ Æ ƐĞĂĚĂƵŐĉĐĂŶƚŝ-ĨĂĐƚŽƌŝĚĞ
ƚƌĂŶƐĐƌŝƉƜŝĞ Æ ƐĞƐĞůĞĐƚĞĂnjĉĨƌĂŐŵĞŶƚĞůĞE
ĂƐŽĐŝĂƚĞĨĂĐƚŽƌŝůŽƌĚĞƚƌĂŶƐĐƌŝƉƜŝĞƌĞĐƵŶŽƐĐƵƜŝ
de Ac Æ se ƐĞĐǀĞŶƜŝĂnjĉ E Æ se identificĉ
ůŽĐĂůŝnjĂƌĞĂĨŝĞĐĉƌƵŝĨƌĂŐŵĞŶƚEprin
ĐŽŵƉĂƌĂƌĞĂƐĞĐǀĞŶƜĞŝEĐƵƐĞĐǀĞŶƜĞůĞE
de la nivelul genomului
ŠŶƚƌĞŐƵůƵŝŐĞŶŽŵ
&ĂĐƚŽƌŝĚĞƚƌĂŶƐĐƌŝƉƜŝĞ
Oct4, octamer-binding transcription factor 4 – ĞdžƉƌŝŵĂƚŠŶĐĞůƵůĞůĞ
ĞŵďƌŝŽŶĂƌĞ͖ŝŵƉůŝĐĂƚŠŶĚŝĨĞƌĞŶƜŝĞƌĞĂĐĞůƵůĞůŽƌƐƚĞŵpluripotente
Sox2, (sex determining region Y)-box 2 – ĞdžƉƌŝŵĂƚŠŶĐĞůƵůĞůĞƐƚĞŵ
ĞŵďƌŝŽŶĂƌĞ͖ŝŵƉůŝĐĂƚŠŶĚŝĨĞƌĞŶƜŝĞƌĞĂĐĞůƵůĞůŽƌƐƚĞŵŶĞƵƌŽŶĂůĞ
EĂŶŽŐ, ”Tir na nog” – ŵŝƚŽůŽŐŝĂĐĞůƚŝĐĉ͟ƜĂƌĂĐĞůŽƌƚŝŶĞƌŝ͟– ĞdžƉƌŝŵĂƚŠŶ
ĐĞůƵůĞƐƚĞŵĞŵďƌŝŽŶĂƌĞ͖ŝŵƉůŝĐĂƚŠŶƌĞŐůĂƌĞĂĚŝĨĞƌĞŶƜŝĞƌŝŝĐĞůƵůĞůŽƌƐƚĞŵ
pluripotente
Localizarea Oct4, Sox2, EĂŶŽŐ ŠŶŐĞŶŽŵÆ ƌĞŐůĞĂnjĉĞdžƉƌĞƐŝĂŐĞŶŝĐĉĂ
Oct4
,ŝƐƚŽŶĞ
H3K4 – ƉŽnjŝƜŝĂhistonei ŵŽĚŝĨŝĐĂƚĞĞƐƚĞĂƐŽĐŝĂƚĉĐƵŐĞŶĂŠŶƐƚĂƌĞĂĐƚŝǀĂƚĉ
DŽůĞĐƵůĞZE
Permit identificarea exonilor ƕŝĂintronilor

47 48
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 ŶĂůŝnjĂĞdžƉƌĞƐŝĞŝŐĞŶĞůŽƌůĂŶŝǀĞůĚĞƉƌŽƚĞŝŶĞ
WƵƌŝĨŝĐĂƌĞĂƉƌŽƚĞŝŶĞůŽƌ
• ^ĞƉĂƌĂƌĞĂĨƌĂĐƜŝŝůŽƌĐĞůƵůĂƌĞ
• Cromatografia
ϯ͘Ϯ͘ŶĂůŝnjĂĞdžƉƌĞƐŝĞŝŐĞŶĞůŽƌůĂ
ŶĂůŝnjĂƉƌŽƚĞŝŶĞůŽƌ
ŶŝǀĞůĚĞƉƌŽƚĞŝŶĞ • tĞƐƚĞƌŶblot
• Imunohistochimia
• ŶĂůŝnjĂƉƌŽƚĞŝŶĞůŽƌƉƌŝŶƐƉĞĐƚƌŽŵĞƚƌŝĞĚĞŵĂƐĉ
• DŝĐƌŽƌĞƜĞůĞ proteice

49 ϱϬ

^ĞƉĂƌĂƌĞĂĨƌĂĐƜŝŝůŽƌĐĞůƵůĂƌĞ ^ĞƉĂƌĂƌĞĂĨƌĂĐƜŝŝůŽƌĐĞůƵůĂƌĞ
ƵůƚƌĂĐĞŶƚƌŝĨƵŐĂƌĞ

low speed: ϭϬϬϬƚŝŵĞƐŐƌĂǀŝƚLJĨŽƌϭϬŵŝŶƵƚĞƐ

medium speed: ϮϬ͕ϬϬϬƚŝŵĞƐŐƌĂǀŝƚLJĨŽƌϮϬŵŝŶƵƚĞƐ
high speed: ϴϬ͕ϬϬϬƚŝŵĞƐŐƌĂǀŝƚLJĨŽƌϭŚŽƵƌ
very high speed: ϭϱϬ͕ϬϬϬƚŝŵĞƐŐƌĂǀŝƚLJĨŽƌϯhours

ϱϭ 52
ϮϬϭϱ͕Alberts et al, Molecular ŝŽůŽŐLJ of the Cell ϮϬϭϱ͕Alberts et al, Molecular ŝŽůŽŐLJ of the Cell
 ^ĞƉĂƌĂƌĞĂĨƌĂĐƜŝŝůŽƌĐĞůƵůĂƌĞ–
ƉƌŝŶǀŝƚĞnjĂĚĞƐĞĚŝŵĞŶƚĂƌĞ
• Componentele subcelulare –
ƐĞĚŝŵĞŶƚĞĂnjĉůĂǀŝƚĞnjĞĚŝĨĞƌŝƚĞŠŶ
ĨƵŶĐƜŝĞĚĞĚŝŵĞŶƐŝƵŶĞƕŝĚĞŶƐŝƚĂƚĞ
ĂƚƵŶĐŝĐąŶĚƐƵŶƚĐĞŶƚƌŝĨƵŐĂƚĞ
deasupra unui ŐƌĂĚŝĞŶƚĚĞŐůƵĐŽnjĉ
(5-ϮϬйͿ
• ƵƉĉĐĞŶƚƌŝĨƵŐĂƌĞĚŝĨĞƌŝƚĞůĞ
componente pot fi colectate
ŝŶĚŝǀŝĚƵĂů
53
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^ĞƉĂƌĂƌĞĂĨƌĂĐƜŝŝůŽƌĐĞůƵůĂƌĞ–
ƉƌŝŶĐƌŽŵĂƚŽŐƌĂĨŝĞ*
dŝƉƵƌŝĚĞŵĂƚƌŝĐĞƵƚŝůŝnjĂƚĞŠŶĐƌŽŵĂƚŽŐƌĂĨŝĞ
55
ϮϬϭϱ͕Alberts et al, Molecular ŝŽůŽŐLJ of the Cell
^ĞƉĂƌĂƌĞĂĨƌĂĐƜŝŝůŽƌĐĞůƵůĂƌĞ–
ƉƌŝŶĐƌŽŵĂƚŽŐƌĂĨŝĞ
• WƌŽďĂ ƐĞƉůĂƐĞĂnjĉŠŶƚƌ-o ĐŽůŽĂŶĉ
deasupra unei ŵĂƚƌŝĐĞƐŽůŝĚĞƉĞƌŵĞĂďŝůĞ
– ex: celuloza
• ^ĞĂĚĂƵŐĉůĞŶƚ ƐŽůǀĞŶƚƵůŠŶĐĂŶƚŝƚĂƚĞ
ŵĂƌĞ
• 2ŶĨƵŶĐƜŝĞĚĞĚŝĨĞƌŝƚĞĐĂƌĂĐƚĞƌŝƐƚŝĐŝ;ƐƚĂƌĞ
ŝŽŶŝĐĉ͕ĚŝŵĞŶƐŝƵŶĞ͕ŝŶƚĞƌĂĐƜŝƵŶŝĞŶnjŝŵĞ-
ƐƵďƐƚƌĂƚͿŵŽůĞĐƵůĞůĞŝŶƉƌŽďĉŵŝŐƌĞĂnjĉ
prin matrice
• &ƌĂĐƜŝŝůĞŵŽůĞĐƵůĂƌĞƐĞĐŽůĞĐƚĞĂnjĉŠŶ
ƚƵďƵƌŝƐĞƉĂƌĂƚĞ
54
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tĞƐƚĞƌŶďůŽƚ
;ŝŵƵŶŽďůŽƚƚŝŶŐ, ŝŵƵŶŽĂŵƉƌĞŶƚĉ)
1. ůĞĐƚƌŽĨŽƌĞnjĂƉƌŽƚĞŝŶĞůŽƌ(lb. engl. sodium ĚŽĚĞĐLJů sulfate –
ƉŽůLJĂĐƌLJůĂŵŝĚĞ gel electrophoresis, SDS-PAGE)
2. dƌĂŶƐĨĞƌƵůƉƌŽƚĞŝŶĞůŽƌ
3. DetecƜŝĂ ƕŝǀŝnjƵĂůŝnjĂƌĞĂ ƉƌŽƚĞŝŶĞůŽƌ
сƚĞŚŶŝĐĉĚĞůĂďŽƌĂƚŽƌďĂnjĂƚĉƉĞƉƌŝŶĐŝƉŝƵůƌĞĂĐƜŝĞŝĂŶƚŝŐĞŶ-anticorp pentru detectarea
ƐƉĞĐŝĨŝĐĉĂƉƌŽƚĞŝŶĞůŽƌ
56
 tĞƐƚĞƌŶďůŽƚ tĞƐƚĞƌŶďůŽƚ

ϭ͘ůĞĐƚƌŽĨŽƌĞnjĂƉƌŽƚĞŝŶĞůŽƌ
сƐĞƉĂƌĂƌĞĂƉƌŽƚĞŝŶĞůŽƌŠŶĨƵŶĐƜŝĞĚĞ
ŵĂƐĂůŽƌŵŽůĞĐƵůĂƌĉƉƌŝŶ
electroforeza cu SDS în gel de
ƉŽůŝĂĐƌŝůĂŵŝĚĉ

• SDS, sodiul dodecilsulfat –

detergent – ĐŽŶĨĞƌĉƉƌŽƚĞŝŶĞůŽƌ
caracter electronegativ
• ɴ-mercaptoetanol – agent
ƌĞĚƵĐĉƚŽƌĐĂƌĞƐĐŝŶĚĞĂnjĉůĞŐĉƚƵƌŝůĞ
S-S

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tĞƐƚĞƌŶďůŽƚ tĞƐƚĞƌŶďůŽƚ

• ŽůŽĂŶĂϭ– markeri moleculari

standard

• Coloana 2 – proteina cu masa

ŵŽůĞĐƵůĂƌĉŶĞĐƵŶŽƐĐƵƚĉ

59 ϲϬ
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 tĞƐƚĞƌŶďůŽƚ tĞƐƚĞƌŶďůŽƚ
• Colorare Coomasie a gelului
Ϯ͘dƌĂŶƐĨĞƌƵůƉƌŽƚĞŝŶĞůŽƌ ƚĂƉĞƉƌŝŶĐŝƉĂůĞ

• ŽůŽĂŶĂϭсĂŵĞƐƚĞĐĚĞƉƌŽƚĞŝŶĞ • ^ĞƵƚŝůŝnjĞĂnjĉŽŵĞŵďƌĂŶĉĚĞ
• Coloanele 2-ϱсĨƌĂĐƜŝŝůĞƉƌŽƚĞŝĐĞŽďƜŝŶƵƚĞ ŶŝƚƌŽĐĞůƵůŽnjĉ
prin cromatografie
• Se ĨŽůŽƐĞƕƚĞŽƐƵƌƐĉĚĞĐƵƌĞŶƚĞůĞĐƚƌŝĐ
• ĂŶƚŝƚĂƚĞĂĚĞƉƌŽƚĞŝŶĞƵƚŝůŝnjĂƚĉͬĐŽůŽĂŶĉс
10 ʅŐ • Se ƌĞĂůŝnjĞĂnjĉƵŶsandwich –
ŵĞŵďƌĂŶĂĚĞŶŝƚƌŽĐĞůƵůŽnjĉƕŝŐĞůƵů
ƐƵŶƚƉůĂƐĂƚĞŠŶƚƌĞŚąƌƚŝŝĚĞĨŝůƚƌƵÆ
curentul electric va facilita transferul
proteinelor din gel pe membranĉ

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tĞƐƚĞƌŶďůŽƚ tĞƐƚĞƌŶďůŽƚ

ϯ͘ĞƚĞĐƜŝĂƕŝǀŝnjƵĂůŝnjĂƌĞĂƉƌŽƚĞŝŶĞůŽƌ ƚĂƉĞƉƌŝŶĐŝƉĂůĞ͗ ϯ͘ĞƚĞĐƜŝĂƕŝǀŝnjƵĂůŝnjĂƌĞĂƉƌŽƚĞŝŶĞůŽƌ ƚĂƉĞƉƌŝŶĐŝƉĂůĞ͗

• ůŽĐĂƌĞĂůĞŐĉƌŝŝŶĞƐƉĞĐŝĨŝĐĞĂ • /ŶĐƵďĂƌĞĂĐƵƐƵďƐƚƌĂƚƵů – enzima: HRP

(lb. engl. horseradish peroxidase),
ĂŶƚŝĐŽƌƉŝůŽƌ– BSA (bovine serum ĚĞƚĞƌŵŝŶĉconversia substratului (peroxid
albumine) ĚĞŽdžŝŐĞŶƕŝluminol) ŠŶƚƌ-un semnal
vizibil
• ŶƚŝĐŽƌƉƵůƉƌŝŵĂƌ – ĚĞƚĞĐƜŝĂƉƌŽƚĞŝŶĞŝ
de interes (complex Ag-Ac)
• ĞƚĞĐƜŝĂƉƌŝŶĐŚŝŵŝŽůƵŵŝŶŝƐĐĞŶƜĉ=
• ŶƚŝĐŽƌƉƵůƐĞĐƵŶĚĂƌ– ƌĞĐƵŶŽĂƕƚĞĐ emisia de unde electromanetice ŠŶ
ƉƌŝŵĂƌƕŝĞƐƚĞĐŽŶũƵŐĂƚĐƵŽĞŶnjŝŵĉ ĚŽŵĞŶŝƵůs/^͕ĨĞŶŽŵĞŶĐĞƉŽĂƚĞƐĉĂƉĂƌĉ
ĐĂƌĞnjƵůƚĂƚĂůƵŶŽƌƌĞĂĐƜŝŝĐŚŝŵŝĐĞ

63 64
ϮϬϭϭ͕Roitt͛ƐĞƐƐĞŶƚŝĂůŝŵŵƵŶŽůŽŐLJ ŚƚƚƉ͗ͬͬǁǁǁ͘ŽŶůŝŶĞďŝŽůŽŐLJŶŽƚĞƐ͘ĐŽŵ
 ^ƵƌƐĞĚĞǀĂƌŝĂďŝůŝƚĂƚĞŐĞŶĞƚŝĐĉ
hD&͞ĂƌŽůĂǀŝůĂ͕͟ƵĐƵƌĞƕƚŝ
&ĂĐƵůƚĂƚĞĂĚĞDŽĂƕĞƕŝƐŝƐƚĞŶƜĉDĞĚŝĐĂůĉ
1. ZĞĐŽŵďŝŶĉƌŝŐĞŶĞƚŝĐĞ
2. DŝŐƌĂƜŝŝ
3. DƵƚĂƜŝŝ
Curs 05:
sĂƌŝĂďŝůŝƚĂƚĞĂŐĞŶĞƚŝĐĉ
Titular curs: Conf. univ. dr. Mocanu Maria-Magdalena
e-mail: mocanu.umfcd@gmail.com

ZĞĐŽŵďŝŶĉƌŝůĞŐĞŶĞƚŝĐĞ
= ĐŽŵďŝŶĂƜŝŝŐĞŶĞƚŝĐĞŶŽŝprin rearanjarea materialului genetic la
nivelul a ĚŽƵĉƵŶŝƚĉƜŝŐĞŶĞƚŝĐĞĚŝĨĞƌŝƚĞ
dŝƉƵƌŝĚĞƌĞĐŽŵďŝŶĂƌĞŐĞŶĞƚŝĐĉ– ŠŶĨƵŶĐƜŝĞĚĞŵĉƌŝŵĞĂƵŶŝƚĉƜŝůŽƌŝŵƉůŝĐĂƚĞ͗

1. ZĞĐŽŵďŝŶĂƌĞŐĞŶŽŵŝĐĉ
ϭ͘ZĞĐŽŵďŝŶĉƌŝůĞŐĞŶĞƚŝĐĞ 2. ZĞĐŽŵďŝŶĂƌĞĐƌŽŵŽnjŽŵŝĂůĉ
3. ZĞĐŽŵďŝŶĂƌĞŐĞŶŝĐĉ

3 4
 Recombinarea ŐĞŶŽŵŝĐĉ ZĞĐŽŵďŝŶĂƌĞĂĐƌŽŵŽnjŽŵŝĂůĉ
= are loc prin recombinarea ŐĞŶŽŵƵůƵŝŐĂŵĞƜŝůŽƌcare vor forma = are loc în cursul ŐĂŵĞƚŽŐĞŶĞnjĞŝîntre cromozomi omologi (cu origine
zigotul ĚŝĨĞƌŝƚĉ͕ŵĂƚĞƌŶĉƐĂƵƉĂƚĞƌŶĉͿƉƌŝŶĨĞŶŽŵĞŶĞƐƉĞĐŝĨŝĐĞŵĞŝŽnjĞŝƉƌŝŵĂƌĞ
Rol:
• ƐŝŐƵƌĉĐŽŶƚŝŶƵŝƚĂƚĞĂƕŝƉĉƐƚƌĂƌĞĂŐĞŶĞůŽƌĚĞůĂƉĉƌŝŶƜŝ dŝƉƵƌŝĚĞƌĞĐŽŵďŝŶĂƌĞĐƌŽŵŽnjŽŵŝĂůĉ:
• ƐŝŐƵƌĉĐƌĞƕƚĞƌĞĂǀĂƌŝĂďŝůŝƚĉƜŝŝƵƌŵĂƕŝůŽƌ a) ZĞĐŽŵďŝŶĂƌĞĂŝŶƚƌĂ-ĐƌŽŵŽnjŽŵŝĂůĉ;ŽŵŽůŽĂŐĉͿ
ZĞĐŽŵďŝŶĂƌĞĂŐĞŶĞƚŝĐĉĂĚŽŝŐĂŵĞƜŝƉƌŽǀĞŶŝƜŝĚĞůĂ͗ b) ZĞĐŽŵďŝŶĂƌĞĂŝŶƚĞƌ-ĐƌŽŵŽnjŽŵŝĂůĉ
a) /ŶĚŝǀŝnjŝĚŝĨĞƌŝƜŝ;ŶĞŠŶƌƵĚŝƜŝŐĞŶĞƚŝĐͿÆ ŚĞƚĞƌŽnjŝƐ = vitalitate sporitĉ͕ĂĚĂƉƚĂƌĞ
ŵĂŝďƵŶĉůĂŵĞĚŝƵ͕ĨĞƌƚŝůŝƚĂƚĞĐƌĞƐĐƵƚĉ
b) /ŶĚŝǀŝnjŝĂƐĞŵĉŶĉƚŽƌŝ;ŠŶƌƵĚŝƜŝŐĞŶĞƚŝĐͿÆ ĐŽŶƐĂŶŐǀŝŶŝnjĂƌĞ = variabilitatea
ŐĞŶĞƚŝĐĉƐĞƌĞĚƵĐĞ
5 6

ZĞĐŽŵďŝŶĂƌĞĂĐƌŽŵŽnjŽŵŝĂůĉ ZĞĐŽŵďŝŶĂƌĞĂĐƌŽŵŽnjŽŵŝĂůĉ
‰ ZĞĐŽŵďŝŶĂƌĞĂŝŶƚƌĂ-ĐƌŽŵŽnjŽŵŝĂůĉ ‰ ZĞĐŽŵďŝŶĂƌĞĂŝŶƚƌĂ-ĐƌŽŵŽnjŽŵŝĂůĉ
= schimbul reciproc de segmente cromozomiale între cromozomii omologi = ĐƌŽƐƐŝŶŐ-ŽǀĞƌ

ƌŽŵŽnjŽŵ ƌŽŵŽnjŽŵ
ƉĂƚĞƌŶƌĞƉůŝĐĂƚ ŵĂƚĞƌŶƌĞƉůŝĐĂƚ WƌŽĨĂnjĂĚŝŶ
ŵĞŝŽnjĂ
ĐĞŶƚƌŽŵĞƌ
ƉƌŝŵĂƌĉ

ĐƌŽŵĂƚŝĚĞƐƵƌŽƌŝ

cƌŽŵŽnjŽŵ ďŝǀĂůĞŶƚ ĐŚŝĂƐŵĂ ĐƌŽƐƐŝŶŐ-ŽǀĞƌ

7 8
Figure 19-8 Essential Cell Biology (© Garland Science 2010) 2012, Brooker, Genetics
 ZĞĐŽŵďŝŶĂƌĞĂĐƌŽŵŽnjŽŵŝĂůĉ Recombinarea ŐĞŶŝĐĉ
‰ ZĞĐŽŵďŝŶĂƌĞĂŝŶƚĞƌ-ĐƌŽŵŽnjŽŵŝĂůĉ = ƌĞĐŽŵďŝŶĂƌĞĂŝŶƚƌĂŐĞŶŝĐĉ; se produce un crossing-over egal între
• ĂƌĂĐƚĞƌŝƐƚŝĐĉĂŶĂĨĂnjĞŝĚŝŶƉƌŽĨĂnjĂ ĚŽƵĉŐĞŶĞĂůĞůĞ͕ĐĞĞĂĐĞƉƌŽĚƵĐĞŽŐĞŶĉŚŝďƌŝĚĉͬĨƵnjŝƵŶĞŐĞŶŝĐĉce
ƉƌŝŵĂƌĉ ĐŽŶƜŝŶĞƵŶĨƌĂŐŵĞŶƚƚĞƌŵŝŶĂůĚŝŶƚƌ-ŽĂůĞůĉƕŝƌĞƐƚƵůĚĞƐĞĐǀĞŶƜĉĚŝŶ
• Perechile de cromozomi omologi (matern ĐĞĂůĂůƚĉĂůĞůĉ
ƕŝƉĂƚĞƌŶͿƐĞƐĞƉĂƌĉĚŝŶďŝǀĂůĞŶƚƕŝ
ŵŝŐƌĞĂnjĉƐƉƌĞĐĞůƵůĞůĞĨŝŝĐĞсĂƐŽƌƚĂƌĞ
ŝŶĚĞƉĞŶĚĞŶƚĉ ƌŽƐƐŝŶŐ-ŽǀĞƌ
• 223 tipuri (8,4+ ŵŝůŝŽĂŶĞͿĚĞŐĂŵĞƜŝ
ĚŝĨĞƌŝƜŝ͕feminini sau masculini Æ pot
rezulta teoretic 246 ĐŽŵďŝŶĂƜŝŝpentru
njŝŐŽƚ
&ƵnjŝƵŶĞŐĞŶŝĐĉ
9 10
2012, Brooker, Genetics ĚĂƉƚĂƌĞĚƵƉĉϮϬϭϳ͕Covic ĞƚĂů͕͘'ĞŶĞƚŝĐĂDĞĚŝĐĂůĉ

DŝŐƌĂƜŝŝ
= transferul de alele prin ĚĞƉůĂƐĂƌĞĂ ƕŝŠŶĐƌƵĐŝƕĂƌĞĂ unui grup de
indivizi dintr-ŽƉŽƉƵůĂƜŝĞĐƵŽĂůƚĉƉŽƉƵůĂƜŝĞĐƵŽĐŽŵƉŽnjŝƜŝĞĂůĞůŝĐĉ
ĚŝĨĞƌŝƚĉ
ĨĞĐƚĞůĞŵŝŐƌĂƜŝĞŝ͗
• ƌĞƕƚĞƌĞĂ ŶƵŵĉƌƵůƵŝĚĞŝŶĚŝǀŝnjŝŚĞƚĞƌŽnjŝŐŽƜŝ
Ϯ͘DŝŐƌĂƜŝŝ • Sunt introduse ĂůĞůĞŶŽŝŠŶƉŽƉƵůĂƜŝĂŐĂnjĚĉÆ Æ ƉŽƉƵůĂƜŝŝŚŝďƌŝĚĞ(ex: în Hawaii –
ĐĉƐĉƚŽƌŝĂƵŶŽƌŝŶĚŝǀŝnjŝĚŝŶƉŽƉƵůĂƜŝŝĚŝĨĞƌŝƚĞ– formate din polinezienei, orientali,
ĐĂƵĐĂnjŝĞŶŝͿ
DŝŐƌĂƜŝĂ– ƉŽƉƵůĂƜŝĂƵŵĂŶĉ
• /ŵŝŐƌĂƜŝĂ– dpdv ĂůůŽĐĂůŝƚĉƜŝŝĚĞĚĞƐƚŝŶĂƜŝĞ
• ŵŝŐƌĂƜŝĞ – dpdv ĂůůŽĐĂůŝƚĉƜŝŝĚĞƉůĞĐĂƌĞ
11 12
Flux genic
сĚŝĨƵnjŝĞůĞŶƚĉĂŐĞŶĞůŽƌĚŝŶƚƌ-ŽƉŽƉƵůĂƜŝĞŠŶƚƌ-ŽĂůƚĉƉŽƉƵůĂƜŝĞ

Efectele fluxului genic:

• 2ŵďŽŐĉƜŝƌĞĂĐŽŶƐƚĞůĂƜŝĞŝŐĞŶŝĐĞĂƉŽƉƵůĂƜŝĞŝŐĂnjĚĉ
• ZĞĚƵĐĞƌĞĂĐŽŶƐĂŶŐǀŝŶŝnjĉƌŝŝ;ƐĐĉĚĞƌĞĂŚŽŵŽnjŝŐŽƜŝĞŝ Æ reducerea frecvenƜĞŝ
bolilor recesive
• ƉĂƜŝĂ ĞĨĞĐƚƵůƵŝĚĞŚĞƚĞƌŽnjŝƐ;ĐƌĞƕƚĞǀŝŐŽĂƌĞĂŚŝďƌŝĚƵůƵŝ– ĐƌĞƕƚĞƌĞĂǀŝĂďŝůŝƚĉƜŝŝƕŝ
ϯ͘DƵƚĂƜŝŝůĞ
ĂĨĞƌƚŝůŝƚĉƜŝŝͿ

DŝŐƌĂƜŝĂĞƐƚĞĐŽŶƐŝĚĞƌĂƚĉŽƐƵƌƐĉŝŵƉŽƌƚĂŶƚĉĂǀĂƌŝĂďŝůŝƚĉƜŝŝŐĞŶĞƚŝĐĞ

13 14

DƵƚĂƜŝŝŐĞŶĞƚŝĐĞ
‰EŽƜŝƵŶŝŐĞŶĞƌĂůĞĚĞƐƉƌĞŵƵƚĂƜŝŝ
‰DŽĚŝĨŝĐĉƌŝĂůĞŶƵŵĉƌƵůƵŝƕŝƐƚƌƵĐƚƵƌŝŝĐƌŽŵŽnjŽŵŝůŽƌ
‰DƵƚĂƜŝŝŐĞŶŝĐĞ

ϯ͘ϭ͘EŽƜŝƵŶŝŐĞŶĞƌĂůĞ

15 16
DƵƚĂƜŝĞŐĞŶŝĐĉсǀĂƌŝĂƜŝĞĚĞƐĞĐǀĞŶƜĉ ůĂƐŝĨŝĐĉƌŝĂůĞŵƵƚĂƜŝŝůŽƌ
сŽƌŝĐĞŵŽĚŝĨŝĐĂƌĞŠŶƐĞĐǀĞŶƜĂnucleotidelor ĚŝŶEƐĂƵŠŶŶƵŵĉƌƵůĚĞ ‰DŽĚĂůŝƚĂƚĞĂĚĞĂƉĂƌŝƜŝĞ
ĐŽƉŝŝĂůĞƵŶĞŝƐĞĐǀĞŶƜĞƐƉĞĐŝĨŝĐĞĚĞE 9 ŶĂƚƵƌĂůĞ;ƐƉŽŶƚĂŶĞͿ
9 ĂƌƚŝĨŝĐŝĂůĞ;ŝŶĚƵƐĞͿ
‰dŝƉƵůĚĞĐĞůƵůĞĂĨĞĐƚĂƚĞ
9 ƐŽŵĂƚŝĐĞ;ĚŽďąŶĚŝƚĞͿ– ƐĞǀĂĨŽƌŵĂŽĐůŽŶĉĐĞůƵůĂƌĉĂŶŽƌŵĂůĉсŵŽnjĂŝĐŝƐŵ
• A introdus termenul de ŵƵƚĂƜŝĞ ƐŽŵĂƚŝĐ
• 1901, a elaborat dĞŽƌŝĂŵƵƚĂƜŝŽŶĂůĉ 9 ŐĂŵĞƚŝĐĞ ;ŵŽƕƚĞŶŝƚĞͿ– se mai numesc germinale; sunt ereditare
(lb. engl. mutation theoryͿ ‰DŽĚƵůĚĞĞdžƉƌŝŵĂƌĞĨĞŶŽƚŝƉŝĐĉ
9 ĚŽŵŝŶĂŶƚĞ
9 ƌĞĐĞƐŝǀĞ
,ƵŐŽDĂƌŝĞ de sƌŝĞƐ 9 co-ĚŽŵŝŶĂŶƚĞ
ŽƚĂŶŝƐƚƕŝŐĞŶĞƚŝĐŝĂŶŽůĂŶĚĞnj
;ϭϴϰϴ-1935)
17 18

ůĂƐŝĨŝĐĉƌŝĂůĞŵƵƚĂƜŝŝůŽƌ
‰dŝƉƵůŐĞŶŽŵƵůƵŝĂĨĞĐƚĂƚ
9 ŶƵĐůĞĂƌ
9 ŵŝƚŽĐŽŶĚƌŝĂů
‰ĨĞĐƚƵůĂƐƵƉƌĂǀŝĂďŝůŝƚĉƜŝŝŽƌŐĂŶŝƐŵĞůŽƌͬĐŽŶƐĞĐŝŶƜĞůĞĨĞŶŽƚŝƉŝĐĞ
9 ďĞŶĞĨŝĐĞ;ƉƌŽŐƌĞƐŝǀĞͿ– ĂĚĂƉƚĂƌĞјůĂŵĞĚŝƵ
ϯ͘Ϯ͘DŽĚŝĨŝĐĉƌŝĂůĞŶƵŵĉƌƵůƵŝƕŝ
9 ĚĉƵŶĉƚŽĂƌĞ;ƌĞŐƌĞƐŝǀĞͿ– ĂƉĂƌĂĨĞĐƜŝƵŶŝŐĞŶĞƚŝĐĞ ; 8-10% din genom; ϭ͕ϮйĞdžŽŶŝ
9 ŶĞƵƚƌĞ– peste ϵϬйdin genom
structuri cromozomilor
9 letale – ŝŶĐŽŵƉĂƚŝďŝůĞĐƵǀŝĂƜĂ
‰DĉƌŝŵĞĂƐĞŐŵĞŶƚƵůƵŝĚĞEŝŵƉůŝĐĂƚ
9 ĐƌŽŵŽnjŽŵŝĂůĞ
9 ŐĞŶŝĐĞ
19 20
 Cariotipul normal Localizarea centromerului
= aranjarea cromozomilor dintr-ŽĐĞůƵůĉŠŶƚŝŵƉƵůĚŝǀŝnjŝƵŶŝŝĐĞůƵůĂƌĞ͕ƌĞƐƉĞĐƚąŶĚ
ŐƌĂĚŝĞŶƚƵůĚĞŵĉƌŝŵĞ͕ĨŽƌŵĉ͕ƉŽnjŝƜŝĂĐĞŶƚƌŽŵĞƌƵůƵŝƐĂƵĂůƚĐĂƌĂĐƚĞƌƌĞƉƌĞnjĞŶƚĂƚŝǀ
pentru individ sau specie

46 ϴ 20

21 22
2012, Brooker, Genetics – Analysis and Principles 2012, Brooker, Genetics – Analysis and Principles

Cariotipul uman normal Clasificarea anomaliilor cromozomiale

ƌŝƚĞƌŝƵ dŝƉĚĞŵŽĚŝĨŝĐĂƌĞ
DŽŵĞŶƚƵůĂƉĂƌŝƜŝĞŝ ŽŶƐƚŝƚƵƜŝŽŶĂůĞ
ŽďąŶĚŝƚĞ
Modul de afectare a materialului genetic EƵŵĞƌŝĐĞ ^ƚƌƵĐƚƵƌĂůĞ
• WŽůŝƉůŽŝĚŝŝ • ĐŚŝůŝďƌĂƚĞ – fenotip normal
• ŶĞƵƉůŽŝĚŝŝ • EĞĞĐŚŝůŝďƌĂƚĞ– fenotip anormal
EƵŵĉƌƵůĚĞĐĞůƵůĞĂĨĞĐƚĂƚĞ KŵŽŐĞŶĞ – în toate celulele individului afectat
DŽnjĂŝĐ – ĚŽƵĉƐĂƵŵĂŝŵƵůƚĞůŝŶŝŝĐĞůƵůĂƌĞĐĞĚĞƌŝǀĉĚŝŶĂĐĞůĂƕŝnjŝŐŽƚ͕ĐĂƌĞĚŝĨĞƌĉƉƌŝŶ
ŶƵŵĉƌƵůƐĂƵƐƚƌƵĐƚƵƌĂĐƌŽŵŽnjŽŵŝůŽƌ
Tipul de cromozom afectat ƵƚŽnjŽŵĂůĞ
,ĞƚĞƌŽnjŽŵĂůĞ
DŝdžƚĞ
Tipul de celule afectate ^ŽŵĂƚŝĐĞ
'ĞƌŵŝŶĂůĞ

23 24
2012, Brooker, Genetics – Analysis and Principles 2017, Covic et al͕͘'ĞŶĞƚŝĐĂDĞĚŝĐĂůĉ
 Anomalii cromozomiale numerice
сĂŶŽŵĂůŝŝŶƵŵĞƌŝĐĞĐĂƌĞĐŽŶƐƚĂƵŠŶŵŽĚŝĨŝĐĂƌĞĂŶƵŵĉƌƵůƵŝŶŽƌŵĂůĚĞ
cromozomi din complementul cromozomial caracteristic speciei

3.2.1. Anomalii cromozomiale Clasificarea anomaliilor cromozomiale numerice:

numerice a) WŽůŝƉůŽŝĚŝŝ– ƉƌĞnjĞŶƜĂƐƵƉůŝŵĞŶƚĂƌĉĂƵŶƵŝĂƐĂƵŵĂŝŵƵůƚŽƌƐĞƚƵƌŝŚĂƉůŽŝĚĞĚĞ

ĐƌŽŵŽnjŽŵŝ ĨĂƜĉĚĞŶƵŵĉƌƵůŶŽƌŵĂůĚĞƐĞƚƵƌŝĚĞĐƌŽŵŽnjŽŵŝ

b) ŶĞƵƉůŽŝĚŝŝ – ǀĂƌŝĂƜŝĂŠŶƉůƵƐͬŠŶŵŝŶƵƐĂƵŶƵŝĐƌŽŵŽnjŽŵͬĂƵŶŽƌƐĞƚƵƌŝĚĞ
ĐƌŽŵŽnjŽŵŝ

25 26

Poliploidii Aneuploidii
dŝƉĚĞĂŶĞƵƉůŽŝĚŝĞ Eƌ͘ ĐƌƐ. ĂƌĂĐƚĞƌŝƐƚŝĐŝ
EƵůŝƐŽŵŝĞ ϮŶ-2 ďƐĞŶƜĂĂŵďŝůŽƌĐƌŽŵŽnjŽŵŝĚŝŶƚƌ-o pereche; incompatibil cu
dŝƉŽƌŐĂŶŝƐŵ Eƌ͘ĐƌƐ. KďƐĞƌǀĂƜŝŝ ǀŝĂƜĂ
Homo sapiens DŽŶŽƐŽŵŝĞ ϮŶ-1 Lipsa unui cromozom
ŝƉůŽŝĚ 2n = 46 EƵŵĉƌŶŽƌŵĂůĚĞƐĞƚƵƌŝĚĞĐƌŽŵŽnjŽŵŝ • ƵƚŽnjŽŵĂůĞ M. autozomale sunt letale
• ,ĞƚĞƌŽnjŽŵĂůĞ M. ŚĞƚĞƌŽnjŽŵĂůĉ – sindrom Turner ;yϬͿ
dƌŝƉůŽŝĚ 2n = 69 Incompatibil ĐƵǀŝĂƜĂ
dƌŝƐŽŵŝĞ ϮŶнϭ WƌĞnjĞŶƜĂƐƵƉůŝŵĞŶƚĂƌĉĂƵŶƵŝĐƌŽŵŽnjŽŵ– letale, pierderi
dĞƚƌĂƉůŽŝĚ 2n = 92 WƌŽĚƵƐĚĞĐŽŶĐĞƉƜŝĞƉŝĞƌĚƵƚƚŝŵƉƵƌŝƵ • ƵƚŽnjŽŵĂůĞ ƐƉŽŶƚĂŶĞĚĞƐĂƌĐŝŶĉ
• ,ĞƚĞƌŽnjŽŵĂůĞ T. autozomale – perechile de crs. 21, 18, 13
T. heterozomale – XXX, XXY, XYY
dĞƚƌĂƐŽŵŝĞͬWĞŶƚĂƐŽŵŝŝ ϮŶнϮ 48, XXXX; 48, XXYY
• ,ĞƚĞƌŽnjŽŵĂůĞ 49, XXXXX; 49, XXXXY

27 28
 Aneuploidii

dŝƉĚĞĂŶĞƵƉůŽŝĚŝĞ ĂƌĂĐƚĞƌŝƐƚŝĐŝ
ŶĞƵƉůŽŝĚŝŝ ŽŵŽŐĞŶĞ dŽĂƚĞĐĞůƵůĞůĞƉƌĞnjŝŶƚĉĂŶŽŵĂůŝĂ
Se produc în timpul meiozei prin:
ĂͿ Non-ĚŝƐũƵŶĐƜŝĂĐƌŽŵŽnjŽŵŝůŽƌŠŶĂŶĂĨĂnjĂŵĞŝŽnjĞŝƉƌŝŵĂƌĞƐĂƵŠŶ
anafaza meiozei secundare
3.2.2. Anomalii cromozomiale
ŶĞƵƉůŽŝĚŝŝ ŠŶŵŽnjĂŝĐ
ďͿ Pierderea ĂŶĂĨĂnjŝĐĉ
Co-ĞdžŝƐƚĉůŝŶŝŝĐĞůƵůĂƌĞĚŝĨĞƌŝƚĞ͗ƵŶĂŶŽƌŵĂůĉƕŝƵŶĂŵŽĚŝĨŝĐĂƚĉ
Se produc în timpul meiozei prin:
structurale
ĂͿ Non-ĚŝƐũƵŶĐƜŝĂĐƌŽŵŽnjŽŵŝůŽƌŠŶĂŶĂĨĂnjĂŵĞŝŽnjĞŝƐĞĐƵŶĚĂƌĞ
ďͿ Pierderea ĂŶĂĨĂnjŝĐĉ

29 30

Anomalii cromozomiale structurale Anomalii cromozomiale structurale

Mecanisme de producere: Mecanisme de producere:
1. ZƵƉĞƌĞĂĐƌŽŵŽnjŽŵŝůŽƌ 2. ƌŽƌŝĚĞƌĞĐŽŵďŝŶĂƌĞŵĞŝŽƚŝĐĉ
• În ĨĂnjĂ'ϭ;ŶƵĞƐƚĞƌĞƉĂƌĂƚĉŠŶĨĂnjĂ^Ϳ • Se produc în ĐĞůƵůĞůĞŐĞƌŵŝŶĂůĞ
• Se produc ƌĞŐŝƵŶŝƚĞƌŵŝŶĂůĞŝŶƐƚĂďŝůĞ(lipsesc telomereleͿ • Erori la formarea complexelor sinaptomenale, urmate de ĐƌŽƐƐŝŶŐ-ŽǀĞƌŝŶĞŐĂů
• ^ĞƵŶĞƐĐŝŶĐŽƌĞĐƚĐĂƉĞƚĞůĞĐƌŽŵŽnjŽŵŝůŽƌƌƵƉƜŝÆ ĐƌŽŵŽnjŽŵŝ ĚĞƌŝǀĂƜŝ • Se produc: ĚĞůĞƜŝĂ, ĚƵƉůŝĐĂƜŝĂ, ŝŶǀĞƌƐŝĂ sau ƚƌĂŶƐůŽĐĂƜŝĂ unor segmente
(acentrici, dicentriciͿÆ se pierd în timpul diviziunii cromozomiale
• Se produc: ĚĞůĞƜŝŝ, ŝŶǀĞƌƐŝƵŶŝ, ĐƌŽŵŽnjŽŵŝŝŶĞůĂƌŝ, ƚƌĂŶƐůŽĐĂƜŝŝ

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Anomalii cromozomiale structurale ĞůĞƜŝŝůĞ (del)
‰ ŠŶĨƵŶĐƜŝĞĚĞůŽĐĂůŝnjĂƌĞĂƐĞŐŵĞŶƚƵůƵŝƉŝĞƌĚƵƚ
ŶŽŵĂůŝŝĐƌŽŵŽnjŽŵŝĂůĞĐĂƌĞŝŵƉůŝĐĉƵŶƐŝŶŐƵƌĐƌŽŵŽnjŽŵ
ĞůĞƜŝĞ ƚĞƌŵŝŶĂůĉ ĞůĞƜŝĞ ŝŶƚĞƌƐƚŝƜŝĂůĉ
• ĞůĞƜŝŝ ĞůĞƜŝŝ ƚĞƌŵŝŶĂůĞ

• ƵƉůŝĐĂƜŝŝ - Ruperea unui cromozom ŠŶƚƌ-ƵŶƉƵŶĐƚ

ƵƌŵĂƚĉĚĞƉŝĞƌĚĞƌĞĂĨƌĂŐŵĞŶƚƵůƵŝacentric ƕŝ
• /ŶǀĞƌƐŝŝ ĨŽƌŵĂƌĞĂƵŶƵŝďƌĂƜĂůĐƌŽŵŽnjŽŵƵůƵŝĨĉƌĉ
telomer
• ƌŽŵŽnjŽŵŝŝŶĞůĂƌŝ
• /njŽĐƌŽŵŽnjŽŵŝ ĞůĞƜŝŝ ŝŶƚĞƌƐƚŝƜŝĂůĞ
- Ruperea unui cromozom ŠŶĚŽƵĉƉƵŶĐƚĞ,
• ƌŽŵŽnjŽŵŝŵĂƌŬĞƌ ƐŝƚƵĂƚĞƉĞĂĐĞůĂƕŝďƌĂƜ͕ƵƌŵĂƚĉĚĞƉŝĞƌĚĞƌĞĂ
ĨƌĂŐŵĞŶƚƵůƵŝŝŶƚĞƌƐƚŝƜŝĂůƕŝƌĞƵŶŝƌĞĂ
ISCN, International System for Human fragmentelor restante
Cytogenetic Nomenclature
33 34

ĞůĞƜŝŝůĞ ĞůĞƜŝŝůĞ
‰ ĚŝŶƉƵŶĐƚĚĞǀĞĚĞƌĞĂůĚŝŵĞŶƐŝƵŶŝŝ
^ŝŶĚƌŽŵƵůĐƌŝ-du-chat
ĞůĞƜŝŝ ŵŝĐƌŽƐĐŽƉŝĐĞ • 2ŶĐŽƉŝůĉƌŝĞƉůąŶƐĂƐĞŵĉŶĉƚŽƌƜŝƉĉƚƵůƵŝ
• •5Mb unei pisici
• Vizibile prin marcaj cromozomial • ^ƚĂƚƵƌĉƌĞĚƵƐĉ
• ƐƉĞĐƚĨĂĐŝĂůĐĂƌĂĐƚĞƌŝƐƚŝĐ;ĨĂƜĂ
ĞůĞƜŝŝ ƐƵďŵŝĐƌŽƐĐŽƉŝĐĞсŵŝĐƌŽĚĞůĞƜŝŝ ƚƌŝƵŶŐŚŝƵůĂƌĉ͕ŽĐŚŝŵŝŐĚĂůĂƜŝͿ
• sŝnjŝďŝůĞƉƌŝŶƚĞŚŶŝĐŝĚĞĐŝƚŽŐĞŶĞƚŝĐĉ • Microencefalie
ŵŽůĞĐƵůĂƌĉ͗&/^,͕ŵŝĐƌŽƌĞƜĞůĞ

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 Cromozomii inelari (r, lb. engl. ring) Inversii (inv)
Mecanismul de producere:
• Cromozomi circulari /ŶǀĞƌƐŝĞƉĂƌĂĐĞŶƚƌŝĐĉ /ŶǀĞƌƐŝĞƉĞƌŝĐĞŶƚƌŝĐĉ • Ruperea cromozomului în ĚŽƵĉƉƵŶĐƚĞÆ
ƌŽƚŝƌĞĂ fragmentului intermediar cu 180° Æ
ƌĞƵŶŝƌĞĂ fragmentelor
a a
Mecanismul de producere: a a
b d Clasificare:
• ruperea unui cromozom ŠŶĚŽƵĉƉƵŶĐƚĞ
/ŶǀĞƌƐŝŝƉĂƌĂĐĞŶƚƌŝĐĞ
localizate pe ďƌĂƜĞĚŝĨĞƌŝƚĞ Æ ƵƌŵĂƚĉĚĞ b b
pierderea fragmentelor acentrice ƚĞƌŵŝŶĂůĞƕŝ c c • Fragmentul inversat ŶƵĐŽŶƜŝŶĞĐĞŶƚƌŽŵĞƌƵů
unirea capetelor fragmentului centric = ĚƵďůĉ c d • ĞůĞϮƉƵŶĐƚĞĚĞƌƵƉƚƵƌĉƐƵŶƚƐŝƚƵĂƚĞƉĞ
d b ĂĐĞůĂƕŝďƌĂƜal cromozomului
ĚĞůĞƜŝĞͬmonosomie ƉĂƌƜŝĂůĉĂƌĞŐŝƵŶŝůŽƌ d c
telomerice e e e e
/ŶǀĞƌƐŝŝƉĞƌŝĐĞŶƚƌŝĐĞ
• Fragmentul inversat ĐŽŶƜŝŶĞĐĞŶƚƌŽŵĞƌƵů
• ĞůĞϮƉƵŶĐƚĞĚĞƌƵƉƚƵƌĉ sunt situate pe ďƌĂƜĞ
ĚŝĨĞƌŝƚĞ

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Izocromozomii (i) Cromozomi marker (mar)

• WƌĞnjĞŶƜĂŠŶĚƵďůƵĞdžĞŵƉůĂƌĂƵŶƵŝĂĚŝŶƚƌĞďƌĂƜĞс
ƚƌŝƐŽŵŝĞƉĂƌƜŝĂůĉ • Cromozomi de ĚŝŵĞŶƐŝƵŶĞ ĨŽĂƌƚĞ ŵŝĐĉ
p
q sau
• AďƐĞŶƜĂ = ŵŽŶŽƐŽŵŝĞ ƉĂƌƜŝĂůĉƵŶƵŝĂĚŝŶƚƌĞďƌĂƜĞ
• Cromozomi ƐƵƉƌĂŶƵŵĞƌĂƌŝĨĂƜĉĚĞcariotipul normal
p
Clasificare
/njŽĐƌŽŵŽnjŽŵŝ ĚĞďƌĂƜůƵŶŐ Tipuri de cromozomi markeri
q
q
p
/njŽĐƌŽŵŽnjŽŵŝ ĚĞďƌĂƜƐĐƵƌƚ • ƌŽŵŽnjŽŵŝĚŝĐĞŶƚƌŝĐŝ ĐƵƐĂƚĞůŝƜŝ
Mecanism de formare: • ƌŽŵŽnjŽŵŝŵŝĐŝŝŶĞůĂƌŝ
V1: ůŝǀĂƌĞĂƚƌĂŶƐǀĞƌƐĂůĉĂĐĞŶƚƌŽŵĞƌƵůƵŝÆ formarea
unui cromozom alcĉƚƵŝƚ ĚŽĂƌĚŝŶďƌĂƜĞƐĐƵƌƚĞͬůƵŶŐŝ
V2: Schimb inegal de material genetic între
cromatidele surori Æ cromozom dicentric

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 Anomalii cromozomiale dƌĂŶƐůŽĐĂƜŝŝƌĞĐŝƉƌŽĐĞ;t)
ŶŽŵĂůŝŝĐƌŽŵŽnjŽŵŝĂůĞĐĂƌĞŝŵƉůŝĐĉĚŽŝĐƌŽŵŽnjŽŵŝ • Se produc în timpul ŝŶƚĞƌĨĂnjĞŝprin
ruperea a doi cromozomi neomologi
• dƌĂŶƐůŽĐĂƜŝŝ;ƌĞĐŝƉƌŽĐĞ͕ƌŽďĞƌƚƐŽŶŝĞŶĞ) Æ ƐĐŚŝŵb ƌĞĐŝƉƌŽĐ al ĨƌĂŐŵĞŶƚĞůŽƌ
• /ŶƐĞƌƜŝŝ ĂĐĞŶƚƌŝĐĞ Æ formarea a ĚŽŝ
ĐƌŽŵŽnjŽŵŝĚĞƌŝǀĂƜŝ
• ƌŽŵŽnjŽŵŝĚŝĐĞŶƚƌŝĐŝ
• /ŶĐŝĚĞŶƜĂ ϭ͗ϰϬϬindivizi

==
>ĞƵĐĞŵŝĂŵŝĞůŽŝĚĉ ĐƌŽŶŝĐĉ
• WŽnjŝƚŝǀĉƉĞŶƚƌƵƚ;ϵ͖ϮϮͿ;Ƌϯϰ͖ƋϭϭͿсĐƌŽŵŽnjŽŵƵů
ƚ;ϵ͖ϮϮͿ;Ƌϯϰ͖ƋϭϭͿ WŚŝůĂĚĞůƉŚŝĂ(PhͿ

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dƌĂŶƐůŽĐĂƜŝŝrobertsoniene (rob) dƌĂŶƐůŽĐĂƜŝŝrobertsoniene (rob)

• /ŵƉůŝĐĉĚŽŝĐƌŽŵŽnjŽŵŝĂĐƌŽĐĞŶƚƌŝĐŝ,
ŽŵŽůŽŐŝ;ϱйͿƐĂƵneomologi ;ϵϱйͿ
Æ ƐĞƌƵƉůĂŶŝǀĞůƵůĐĞŶƚƌŽŵĞƌĞůŽƌ
ƐĂƵůĂŶŝǀĞůƵůďƌĂƜĞůŽƌƐĐƵƌƚĞ͕ĨŽĂƌƚĞ
aproape de centromer Æ
fuzioneazĉÆ se formeazĉƵŶ
ĐƌŽŵŽnjŽŵĚĞƌŝǀĂƚƉƐĞƵĚŽĚŝĐĞŶƚƌŝĐ
;ƵŶƐŝŶŐƵƌĐĞŶƚƌŽŵĞƌĨƵŶĐƜŝŽŶĂůͿĐĞ
ĐŽŶƜŝŶĞďƌĂƜĞůĞůƵŶŐŝ

• ĂƌĂĐƚĞƌŝƐƚŝĐĉƉĞŶƚƌƵĐƌŽŵŽnjŽŵŝŝ
ĂĐƌŽĐĞŶƚƌŝĐŝ͗ϭϯ͕ϭϰ͕ϭϱ͕Ϯϭ͕ϮϮ
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/ŶƐĞƌƜŝŝůĞ;ins) Cromozomi dicentrici (dic)
• Sunt ƚƌĂŶƐůŽĐĂƜŝŝŶĞƌĞĐŝƉƌŽĐĞ • dƌĂŶƐůŽĐĂƜŝĞŶĞĞĐŚŝůŝďƌĂƚĉ
• /ŵƉůŝĐĉƚƌĂŶƐĨĞƌƵůƵŶƵŝĨƌĂŐŵĞŶƚ • ZƵƉĞƌĞĂŠŶĐąƚĞƵŶƉƵŶĐƚĂĚŽŝ
ĐƌŽŵŽnjŽŵŝĂůde pe un cromozom, pe cromozomi Æ ƵŶŝƌĞĂ ĨƌĂŐŵĞŶƚĞůŽƌ
un alt cromozom neomologͬĂĐĞůĂƕŝ ĐĞŶƚƌŝĐĞ într-un ĐƌŽŵŽnjŽŵĚĞƌŝǀĂƚƕŝ
cromozom
ƉŝĞƌĚĞƌĞĂĨƌĂŐŵĞŶƚĞůŽƌĂĐĞŶƚƌŝĐĞ
• EƵŵĉƌƵůĚĞĐƌŽŵŽnjŽŵŝƌĞĚƵĐĞĚĞůĂ
DĞĐĂŶŝƐŵƵůĚĞĂƉĂƌŝƜŝĞ͗ 46 la 45
• EĞĐĞƐŝƚĉƚƌĞŝƐŝƚƵƐƵƌŝ de ĐůŝǀĂƌĞ – 2
situsuri de clivare ŐĞŶĞƌĞĂnjĉƵŶ
ĨƌĂŐŵĞŶƚĐĞƐĞŝŶƐĞƌĞĂnjĉ;ŠŶƉŽnjŝƜŝĞ
ŶŽƌŵĂůĉƐĂƵŝŶǀĞƌƐĂƚĉͿŠŶĂůƚ situs de
clivare

45 46

DƵƚĂƜŝŝŐĞŶĞƚŝĐĞ
‰dŝƉƵƌŝĚĞŵƵƚĂƜŝŝŐĞŶŝĐĞ
‰ĂƵnjĞůĞŵƵƚĂƜŝŝůŽƌ
‰DĞĐĂŶŝƐŵĞůĞĚĞƌĞƉĂƌĂƌĞĂůĞnjŝƵŶŝůŽƌE

ϯ͘ϯ͘DƵƚĂƜŝŝŐĞŶŝĐĞ

47 48
 dŝƉƵƌŝĚĞŵƵƚĂƜŝŝŐĞŶŝĐĞ
• ^ƵďƐƚŝƚƵƜŝŝŶƵĐůĞŽƚŝĚŝĐĞ ;ϲϬйͿ
• ĞůĞƜŝŝ ƕŝŝŶƐĞƌƜŝŝŵŝĐŝ(sub 20 nucleotideͿ
• ZĞŵĂŶŝĞƌŝŐĞŶŝĐĞĂďĞƌĂŶƚĞÆ deleƜŝŝƕŝŝŶƐĞƌƜŝŝŵĂƌŝ;ƉĞƐƚĞϮϬ
nucleotideͿ͕ĚƵƉůŝĐĂƜŝŝ͕ŝŶǀĞƌƐŝŝƐĂƵƚƌĂŶƐůŽĐĂƜŝŝ

ϯ͘ϯ͘ϭ͘dŝƉƵƌŝĚĞŵƵƚĂƜŝŝŐĞŶŝĐĞ • ŵƉůŝĨŝĐĂƌĞĂƵŶŽƌƌĞƉĞƚŝƜŝŝŶƵĐůĞŽƚŝĚŝĐĞ

49 50

Bazele azotate ^ƵďƐƚŝƚƵƜŝĂƵŶƵŝnucleotid

 Transversie dŝƉƵƌŝĚĞƐƵďƐƚŝƚƵƜŝŝnucleotidice:
dƌĂŶnjŝƜŝĞ
• dƌĂŶnjŝƜŝŝ – înlocuirea unei baza
C T pirimidinice (d͕ͿƐĂƵpurinice (͕'Ϳ
ĐƵŽďĂnjĉĚĞĂĐĞůĂƕŝƚŝƉ
• dƌĂŶƐǀĞƌƐŝŝ – ŽďĂnjĉƉƵƌŝŶŝĐĉ este
G
ŠŶůŽĐƵŝƚĉĚĞŽďĂnjĉƉŝƌŝŵŝĚŝŶŝĐĉ

ƚƌĂŶƐǀĞƌƐŝĞ

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 ĨĞĐƚĞůĞƐƵďƐƚŝƚƵƜŝŝůŽƌ ĨĞĐƚĞůĞƐƵďƐƚŝƚƵƜŝŝůŽƌ

DƵƚĂƜŝŝƐŝůĞŶƜŝŽĂƐĞ

DƵƚĂƜŝŝĐƵƐĞŶƐŐƌĞƕŝƚ

DƵƚĂƜŝŝŶŽŶƐĞŶƐ

DƵƚĂƜŝŝƉƌŝŶĐĂƌĞƐĞ
ĚĞƉůĂƐĞĂnjĉĐĂĚƌƵůĚĞ
ĐŝƚŝƌĞ

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ĞůĞƜŝŝ ƕŝŝŶƐĞƌƜŝŝŵŝĐŝ
ZĞƉůŝĐĂƌĞŶŽƌŵĂůĉ

Catena sens
Catena anti-sens
Apar prin ŵĞĐĂŶŝƐŵĞĚĞ
ŐůŝƐĂƌĞͬĂůƵŶĞĐĂƌĞ
(lb. engl. slippageͿ
3.3.2. Mecanismele de reparare a
Disocierea ADN de catena sens produce ŝŶƐĞƌƜŝŝ Disocierea ADN de catena anti-sens produce ĚĞůĞƜŝŝ

Catena sens
leziunilor ADN
Catena anti-sens
Catena sens
Catena anti-sens

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 ĂƵnjĞůĞĂƉĂƌŝƜŝĞi ŵƵƚĂƜŝŝůŽƌ ĨĞĐƚĞĂůĞĂĐƜŝƵŶŝŝĨĂĐƚŽƌŝůŽƌgenotoxici
• &ĂĐƚŽƌŝĨŝnjŝĐŝ DŽůĞĐƵůĂĚĞEĞƐƚĞƐƵƐĐĞƉƚŝďŝůĉůĂ͗
• ZĂĚŝĂƜŝŝůĞŝŽŶŝnjĂŶƚĞ– ƌĂĚŝĂƜŝŝȖ ‫܈‬L; • ƚĂĐŚŝĚƌŽůŝƚŝĐ
• ZĂĚŝĂƜŝŝůĞƵůƚƌĂǀŝŽůĞƚĞ– UV-C (Ȝ = 260 nmͿ͕ • ĞƉƵƌŝŶĂƌĞ – cca 5000 ͬ'ƐĞƉŝĞƌĚ njŝůŶŝĐ
UV-B (Ȝ = 280-315 nmͿÆ ĚŝŵĞƌŝ de ƚŝŵŝŶĉ • ĞĂŵŝŶĂƌĞ – conversia C Æ T cca 100
• &ĂĐƚŽƌŝŝĐŚŝŵŝĐŝ bpͬnjŝůŶŝĐ
• Hidrocarburile aromatice policiclice ĐƜŝƵŶĞŐĞŶŽƚŽdžŝĐĉ • ^ƚƌĞƐŽdžŝĚĂƚŝǀ
;ĨƵŵƵůĚĞƜŝŐĂƌĉͿ • Anionii superoxid – atacĉƐƚƌƵĐƚƵƌŝůĞ
• Aflatoxine ;ĂƌĂŚŝĚĞůĞŵƵĐĞŐĉŝƚĞͿ ciclice ale bazelor azotate
• Chimioterapicele • DĞƚŝůĉƌŝĐĂƌĞŶƵƐƵŶƚ ƉƌŽĚƵƐĞ de 5-
• &ĂĐƚŽƌŝďŝŽůŽŐŝĐŝ ŵĞƚŝůĐŝƚŽnjŝŶĂ
• Virusurile • Prin S-adenosyl ŵĞƚŚŝŽŶŝŶĉ Æ ďĂnjĞ
ĂnjŽƚĂƚĞ ĐŝƚŽƚŽdžŝĐĞ

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ĨĞĐƚĞĂůĞĂĐƜŝƵŶŝŝĨĂĐƚŽƌŝůŽƌgenotoxici Principalele mecanisme de reparare a ADN

ĚĞƉƵƌŝŶĂƌĞ

‰ZĞƉĂƌĂƌĞĚŝƌĞĐƚĉĂE
dŝŵĞƌŝ de ƚŝŵŝŶĉ
‰ZĞƉĂƌĂƌĞĂƉƌŝŶĞdžĐŝnjŝĂďĂnjĞůŽƌĂnjŽƚĂƚĞ
‰ZĞƉĂƌĂƌĞĂƉƌŝŶĞdžĐŝnjŝĂŶƵĐůĞŽƚŝĚĞůŽƌ
‰ZĞƉĂƌĂƌĞĂůĞnjŝƵŶŝůŽƌEĚĐ
ĚĞĂŵŝŶĂƌĞ

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 ZĞƉĂƌĂƌĞĂĚŝƌĞĐƚĉĂE Repararea prin excizia bazei azotate

• cca ϮϬϬϬϬĚĞďĂnjĞĂnjŽƚĂƚĞƐƵŶƚŵŽĚŝĨŝĐĂƚĞͬnjŝůŶŝĐŠŶŽƌŐĂŶŝƐŵƵůƵŵĂŶ
• 'ůŝĐŽnjŝůĂnjĂ – ŠŶĚĞƉĉƌƚĞĂnjĉŵŽĚŝĨŝĐĂƚĉ
• ŶĚŽŶƵĐůĞĂnjĂ͕ĨŽƐĨŽĚŝĞƐƚĞƌĂnjĂ ĐůŝǀĞĂnjĉEƕŝŠŶĚĞƉĉƌƚĞĂnjĉƵŶƌĞnjŝĚƵƵŐůƵĐŝĚŝĐ
• ^ƉĂƜŝƵůƌĉŵĂƐůŝďĞƌĞƐƚĞĐŽŵƉůĞƚĂƚƉƌŝŶƌĞ-ƐŝŶƚĞnjĂEƌĞĂůŝnjĂƚĉĚĞEƉŽůŝŵĞƌĂnjĂ

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2012, Brooker, Genetics – Analysis and Principles 2011, Strachan & Read, Human Molecular Genetics

Repararea prin excizia nucleotidelor Repararea leziunilor ADNdc

ZĞĐŽŵďŝŶĂƌĞŽŵŽůŽĂŐĉ ZĞĐŽŵďŝŶĂƌĞĂŶĞ-ŽŵŽůŽĂŐĉ

Este
Nu este întotdeauna
întotdeauna ƉŽƐŝďŝůĉ͕ĚĂƌƐĞ
ƉŽƐŝďŝůĉ ƌĞĂůŝnjĞĂnjĉĐƵ
ĚĞůĞƜŝĂ unei
ŵŝĐŝƐĞĐǀĞŶƜĞ
ADN

• Scop: ŠŶĚĞƉĉƌƚĂƌĞĂĚŝŵĞƌŝůŽƌĚĞd
• ^ĞŠŶĚĞƉĉƌƚĞĂnjĉŽƌĞŐŝƵŶĞŵĂŝŵĂƌĞĚŝŶĐĂƚĞŶĂE
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2011, Strachan & Read, Human Molecular Genetics 2011, Strachan & Read, Human Molecular Genetics
 ^ƵŵĂƌ
hD&͞ĂƌŽůĂǀŝůĂ͕͟ƵĐƵƌĞƕƚŝ
&ĂĐƵůƚĂƚĞĂĚĞDŽĂƕĞƕŝƐŝƐƚĞŶƜĉDĞĚŝĐĂůĉ

ϭ͘ ƐƉĞĐƚĞŐĞŶĞƚŝĐĞŠŶŽŶĐŽŐĞŶĞnjĉ

ƵƌƐϬϳ͗ Ϯ͘ ůĞŵĞŶƚĞ ĚĞƚĞƌĂƚŽŐĞŶĞnjĂ

EŽƜŝƵŶŝĚĞŐĞŶĞƚŝĐĉƵŵĂŶĉ
ϯ͘ ůĞŵĞŶƚĞĚĞƉƌŽĨŝůĂdžŝĞĂďŽůŝůŽƌŐĞŶĞƚŝĐĞ
dŝƚƵůĂƌĐƵƌƐ͗ŽŶĨ͘ƵŶŝǀ͘Ěƌ͘DŽĐĂŶƵDĂƌŝĂͲDĂŐĚĂůĞŶĂ
ĞͲŵĂŝů͗ŵŽĐĂŶƵ͘ƵŵĨĐĚΛŐŵĂŝů͘ĐŽŵ

ϭ Ϯ

^ƵŵĂƌ
‡ EŽƜŝƵŶŝŝŶƚƌŽĚƵĐƚŝǀĞ
‡ sŝƌƵƐƵƌŝůĞƕŝƚƌĂŶƐĨŽƌŵĂƌĞĂŵĂůŝŐŶĉ
‡ KŶĐŽŐĞŶĞ
ϭ͘ƐƉĞĐƚĞŐĞŶĞƚŝĐĞŠŶ ‡ 'ĞŶĞƐƵƉƌĞƐŽĂƌĞƚƵŵŽƌĂůĞ
ŽŶĐŽŐĞŶĞnjĉ ‡ DĂůŝŐŶŝƚĉƜŝĞƌĞĚŝƚĂƌĞ

ϯ ϰ
 ĞĨŝŶŝƜŝĞ

EŽƜŝƵŶŝŝŶƚƌŽĚƵĐƚŝǀĞ
Žůŝ ŵĂůŝŐŶĞ
сĐĂƚĞŐŽƌŝĞĚĞĂĨĞĐƜŝƵŶŝĐĂƵnjĂƚĉĚĞŽƉƌŽůŝĨĞƌĂƌĞĐĞůƵůĂƌĉĂŶĂƌŚŝĐĉ͕ŶĞĐŽŶƚƌŽůĂƚĉƕŝ
ŶĞŠŶƚƌĞƌƵƉƚĉ͕ĐĂƌĂĐƚĞƌŝnjĂƚĉƉƌŝŶĂůƚĞƌĂƌĞĂƉƌŽĐĞƐĞůŽƌĚĞĐƌĞƕƚĞƌĞƕŝƉƌŽůŝĨĞƌĂƌĞ
ĐĞůƵůĂƌĉ͘ ϮϬϭϰ͕ŝĐƜŝŽŶĂƌĚĞŐĞŶĞƚŝĐĉ
ϱ ϲ
ϮϬϭϮ͕ƌŽŽŬĞƌ͕'ĞŶĞƚŝĐƐ

džƉĞƌŝŵĞŶƚʹ /ƉŽƚĞnjĂĚĞůƵĐƌƵ džƉĞƌŝŵĞŶƚʹ ƚĞƐƚĂƌĞĂŝƉŽƚĞnjĞŝĚĞůƵĐƌƵ

ZŽůƵůŐĞŶĞůŽƌͬEŠŶĚĞĐůĂŶƕĂƌĞĂƚƌĂŶƐĨŽƌŵĉƌŝŝŵĂůŝŐŶĞ

EĐĞůƵůĂƌŝnjŽůĂƚĚŝŶĐĞůƵůĞůĞŵĂůŝŐŶĞ
;ůŝŶŝŝĐĞůƵůĂƌĞƚƌĂŶƐĨŽƌŵĂƚĞĐƵĂŐĞŶƜŝ
ĨŝnjŝĐŝƕŝĐŚŝŵŝĐŝ͕ŶƵƉƌŝŶŝŶĨĞĐƜŝĞĐƵ
ǀŝƌƵƐƵƌŝͿǀĂƚƌĂŶƐĨŽƌŵĂĨŝďƌŽďůĂƐƚĞůĞ
ŶŽƌŵĂůĞŠŶĨŝďƌŽďůĂƐƚĞ ŵĂůŝŐŶĞ

ĞůƵůĞůĞŶŽƌŵĂůĞĐƌĞƐĐŠŶŵŽŶŽƐƚƌĂƚ͕ŠŶƚŝŵƉĐĞ
ĐĞůƵůĞůĞŵĂůŝŐŶĞĐƌĞƐĐƐƵďĨŽƌŵĉĚĞ͟ŐƌĉŵĞnjŝ͟
ϳ ϴ
ϮϬϭϮ͕ƌŽŽŬĞƌ͕'ĞŶĞƚŝĐƐ ϮϬϭϮ͕ƌŽŽŬĞƌ͕'ĞŶĞƚŝĐƐ
 džƉĞƌŝŵĞŶƚʹ ƚĞƐƚĂƌĞĂŝƉŽƚĞnjĞŝĚĞůƵĐƌƵ džƉĞƌŝŵĞŶƚʹ ƌĞnjƵůƚĂƚĞƕŝŝŶƚĞƌƉƌĞƚĂƌĞ

ZĞnjƵůƚĂƚĞ /ŶƚĞƌƉƌĞƚĂƌĞĂƌĞnjƵůƚĂƚĞůŽƌ

‡ ĚŽĂƌŽƉĂƌƚĞĚŝŶEĞdžƚƌĂƐĚŝŶĐĞůƵůĞůĞ
ŵĂůŝŐŶĞ ƉŽĂƚĞŝŶĚƵĐĞƚƌĂŶƐĨŽƌŵĂƌĞĂŵĂůŝŐŶĉ
ĂĨŝďƌŽďůĂƐƚĞůŽƌ
‡ ƉŽƐŝďŝůĞĞdžƉůŝĐĂƜŝŝ͗
‡ ƵŶĞůĞŐĞŶĞĐĂƌĞŝŶĚƵĐƚƌĂŶƐĨŽƌŵĂƌĞĂƐƵŶƚŽŶĐŽŐĞŶĞ
ĚŽŵŝŶĂŶƚĞ͕ŝĂƌĂůƚĞůĞƌĞĐĞƐŝǀĞ
‡ ŝŶĐĞůƵůĞůĞŐĂnjĚĉĞdžŝƐƚĉŐĞŶĞƐƵƉƌĞƐŽĂƌĞƚƵŵŽƌĂůĞ
ĐĂƌĞĂƵŝŶŚŝďĂƚŵŽĚŝĨŝĐĉƌŝůĞŝŶĚƵƐĞĚĞEĞdžŽŐĞŶ

‡ EĞdžƚƌĂƐĚŝŶĐĞůƵůĞůĞŶŽƌŵĂůĞŶƵĂŝŶĚƵƐ
ƚƌĂŶƐĨŽƌŵĂƌĞĂŵĂůŝŐŶĉ

ϵ ϭϬ
ϮϬϭϮ͕ƌŽŽŬĞƌ͕'ĞŶĞƚŝĐƐ ϮϬϭϮ͕ƌŽŽŬĞƌ͕'ĞŶĞƚŝĐƐ

ĂƌĂĐƚĞƌŝƐƚŝĐŝĐŽŵƵŶĞĂůĞĐĞůƵůĞůŽƌŵĂůŝŐŶĞ KŶĐŽŐĞŶĞnjĂ ĞƐƚĞƵŶƉƌŽĐĞƐŵƵůƚŝƐƚĂĚŝĂů

‡ KƌŝŐŝŶĞĂĐůŽŶĂůĉ͘ĞůĞŵĂŝŵƵůƚĞƚƵŵŽƌŝŵĂůŝŐŶĞŠƕŝĂƵŽƌŝŐŝŶĞĂŠŶƚƌͲŽ
ƐŝŶŐƵƌĉĐĞůƵůĉŵĂůŝŐŶĉ͕ĐĂƌĞƵůƚĞƌŝŽƌƐĞǀĂĚŝǀŝĚĞƕŝǀĂĚĂŶĂƕƚĞƌĞƵŶĞŝ
ĐůŽŶĞ

‡ ĂƌĐŝŶŽŐĞŶŝ͘WƌŽĐĞƐƵůŵĂůŝŐŶƉŽƌŶĞƕƚĞĚĞůĂŽŵŽĚŝĨŝĐĂƌĞďĞŶŝŐŶĉ͕ĐĂƌĞ
ŠŶƉƌĞnjĞŶƜĂƵŶŽƌĨĂĐƚŽƌŝĚŝŶŵĞĚŝƵůĞdžƚĞƌŝŽƌ;ƌĂĚŝĂƜŝŝhs͕ƐƵďƐƚĂŶƜĞ
ĐŚŝŵŝĐĞͿƐĞƚƌĂŶƐĨŽƌŵĉŠŶƚƌͲŽĂĨĞĐƜŝƵŶĞŵĂůŝŐŶĉ;ϴϬйĐĂnjƵƌŝͿ

W͕ĂĚĞŶŽŵĂƚŽƵƐ ƉŽůLJƉŽƐŝƐ ĐŽůŝ

Z^͕ƌĂƚ ƐĂƌĐŽŵĂ
͕ĚĞůĞƚĞĚ ŝŶĐŽůŽƌĞĐƚĂů ĐĂƌĐŝŶŽŵĂ
ϭϭ ĞnjǀŽůƚĂƌĞĂŵƵůƚŝƐƚĂĚŝĂůĉĂĐĂŶĐĞƌƵůƵŝĚĞĐŽůŽŶ ϭϮ
ϮϬϭϮ͕ƌŽŽŬĞƌ͕'ĞŶĞƚŝĐƐ
 ,ĞƚĞƌŽŐĞŶŝĐŝƚĂƚĞĂ ƚƵŵŽƌĂůĉʹ ŝĚĞŶƚŝĨŝĐĂƌĞĂ
/ŶƐƚĂďŝůŝƚĂƚĞĂŐĞŶĞƚŝĐĉŠŶďŽůŝůĞŵĂůŝŐŶĞ
ƉƌŽĨŝůƵůƵŝŵŽůĞĐƵůĂƌŠŶďŽůŝůĞŵĂůŝŐŶĞ

džĞŵƉůĞĚĞĨĞŶŽƚŝƉ ŠŶ
ĐĂŶĐĞƌƵů ŵĂŵĂƌ

ϭ͘ Zн ΀ƚĞƌĂƉŝĞ͗ƚĂŵŽdžŝĨĞŶ΁
Ϯ͘ ,ZϮн΀ƚĞƌĂƉŝĞ͗ƚƌĂƐƚƵnjƵŵĂď΁
ϯ͘ dE;ZͲ͕,ZϮͲ͕'&ZнͿ
΀ƚĞƌĂƉŝĞ͗ĐĞƚƵdžŝŵĂď΁
ĞůƵůĉŶŽƌŵĂůĉ ĞůƵůĉŵĂůŝŐŶĉ
ϭϯ ϭϰ
ϮϬϭϮ͕ƌŽŽŬĞƌ͕'ĞŶĞƚŝĐƐ ϮϬϭϬ͕ŝŽƐŝŵŽ ΘĂƐĞůŐĂ͕EĂƚƵƌĞ ZĞǀŝĞǁƐͮůŝŶŝĐĂůKŶĐŽůŽŐLJ

sŝƌƵƐƵƌŝůĞƕŝƚƌĂŶƐĨŽƌŵĂƌĞĂŵĂůŝŐŶĉ
‡ ĞůĞŵĂŝŵƵůƚĞǀŝƌƵƐƵƌŝŶƵƐƵŶƚĐĂƉĂďŝůĞƐĉŝŶĚƵĐĉ
ƚƌĂŶƐĨŽƌŵĂƌĞĂŵĂůŝŐŶĉ͖ĚŽĂƌĐĐĂϰϬǀŝƌƵƐƵƌŝƉŽƚŝŶĚƵĐĞ
ƚƌĂŶƐĨŽƌŵĂƌĞĂŵĂůŝŐŶĉ

‡ ϭϵϭϭ͕ǀŝƌƵƐŽůŽŐƵůĂŵĞƌŝĐĂŶWĞLJƚŽŶ ZŽƵƐ ĂŝnjŽůĂƚǀŝƌƵƐƵů

sŝƌƵƐƵƌŝůĞƕŝƚƌĂŶƐĨŽƌŵĂƌĞĂ ƐĂƌĐŽŵƵůƵŝZŽƵƐ

ŵĂůŝŐŶĉ ‡ ĂƵĨŽƐƚŝŶĨĞĐƚĂƚĞĨŝďƌŽďůĂƐƚĞ ĂǀŝĞŶĞ ĐƵǀŝƌƵƐƵů

ƐĂƌĐŽŵƵůƵŝZŽƵƐ Æ ƉƌŽůŝĨĞƌĂƌĞ ĐĞůƵůĂƌĉ

‡ ŐĞŶĂƌĞƐƉŽŶƐĂďŝůĉĚĞƉƌŽůŝĨĞƌĂƌĞĂĐĞůƵůĂƌĉ͕ ǀͲƐƌĐ
ƌĞƉƌĞnjŝŶƚĉƉƌŝŵƵůĞdžĞŵƉůƵĚĞŽŶĐŽŐĞŶĉ

‡ ϭϵϲϲ͕ĂƉƌŝŵŝƚWƌĞŵŝƵůEŽďĞůƉĞŶƚƌƵĚĞƐĐŽƉĞƌŝƌĞĂ
ǀŝƌƵƐƵƌŝůŽƌĐĂƌĞŝŶĚƵĐĂƉĂƌŝƜŝĂƚƵŵŽƌŝůŽƌŵĂůŝŐŶĞ

ϭϱ ϭϲ
ǁǁǁ͘ŶŽďĞůƉƌŝnjĞ͘ŽƌŐ
 sŝƌƵƐƵƌŝůĞƕŝƚƌĂŶƐĨŽƌŵĂƌĞĂŵĂůŝŐŶĉ
‡ 2ŶĐĞůƵůĞůĞŶŽƌŵĂůĞĞdžŝƐƚĉŐĞŶĂ
ĐͲƐƌĐ ʹ ĐĂƌĞŶƵ ƉƌŽĚƵĐĞ
ƚƌĂŶƐĨŽƌŵĂƌĞĂŵĂůŝŐŶĉ
‡ /ŶƚĞŐƌĂƌĞĂŐĞŶĞŝĐͲƐƌĐ ŠŶƚƌͲƵŶ
ŐĞŶŽŵǀŝƌĂůƉŽĂƚĞĐŽŶĚƵĐĞůĂ
ƚƌĂŶƐĨŽƌŵĂƌĞŵĂůŝŐŶĉ
sŝƌƵƐƵƌŝůĞƕŝƚƌĂŶƐĨŽƌŵĂƌĞĂŵĂůŝŐŶĉ
DĞĐĂŶŝƐŵĞĚĞƚƌĂŶƐĨŽƌŵĂƌĞ͗ ‰ ZĞƚƌŽǀŝƌƵƐƵƌŝ
ϭ͘ 2ŶƚŝŵƉƵůƌĞƉůŝĐĉƌŝŝǀŝƌĂůĞƐĞ
ƉƌŽĚƵĐŵƵůƚĞĐŽƉŝŝĂůĞŐĞŶĞŝĐͲƐƌĐ
Æ ĂŵƉůŝĨŝĐĂƌĞ ŐĞŶŝĐĉ sŝƌƵƐ ĞƐĐƌŝĞƌĞ
Ϯ͘ 2ŶĐŽƌƉŽƌĂƌĞĂŐĞŶĞŝĐͲƐƌĐ ůąŶŐĉ sŝƌƵƐƵů ƐĂƌĐŽŵƵůƵŝZŽƵƐ WƌŽĚƵĐĞ ƐĂƌĐŽŵĂǀŝĂŶ
ƉƌŽŵŽƚŽƌƵůǀŝƌĂůÆ sŝƌƵƐƵů ƐĂƌĐŽŵƵůƵŝƐŝŵŝĂŶ WƌŽĚƵĐĞƐĂƌĐŽŵůĂ ŵĂŝŵƵƜĞ
ƐƵƉƌĂͲĞdžƉƌŝŵĂƌĞĂ ƉƌŽƚĞŝŶĞŝ
sŝƌƵƐƵů ůĞƵĐĞŵŝĞŝďĞůƐŽŶ WƌŽĚƵĐĞƐĂƌĐŽŵ ŵƵƌŝŶ
ĐŽĚŝĨŝĐĂƚĉĚĞŐĞŶĂĐͲƐƌĐ
ϯ͘ 'ĞŶĂĐͲƐƌĐ ƉŽĂƚĞƐƵĨĞƌŝŵƵƚĂƜŝŝ ƕŝ
ƐĞƚƌĂŶƐĨŽƌŵĉŠŶǀͲƐƌĐ
ƐĂƌĐŽŵ͕ƚƵŵŽĂƌĞŵĂůŝŐŶĉĐĞĂƉĂƌĞŠŶƜĞƐƵƚƵůĐŽŶũƵĐƚŝǀ

ϭϳ ϭϴ
ϮϬϭϮ͕ƌŽŽŬĞƌ͕'ĞŶĞƚŝĐƐ

sŝƌƵƐƵƌŝůĞƕŝƚƌĂŶƐĨŽƌŵĂƌĞĂŵĂůŝŐŶĉ
‰ ĚĞŶŽǀŝƌƵƐƵƌŝ
sŝƌƵƐ ĞƐĐƌŝĞƌĞ
sŝƌƵƐƵůŚĞƉĂƚŝƚĞŝ͕ ǀŝƌƵƐƵů WŽƚƉƌŽĚƵĐĞƚƵŵŽƌŝŵĂůŝŐŶĞ ŚĞƉĂƚŝĐĞ
ƐŝŵŝĂŶϰϬ;^sϰϬͿ͕ǀŝƌƵƐƵů
ƉŽůŝŽŵŝĞůŝƚŝĐ
sŝƌƵƐƵů ƉĂƉŝůŽŵĂ ;,WsͿ WŽĂƚĞƉƌŽĚƵĐĞƚƵŵŽƌŝďĞŶŝŐŶĞƐĂƵŵĂůŝŐŶĞůĂŽĂŵĞŶŝ͘WŽĂƚĞ
ƉƌŽĚƵĐĞĐĂŶĐĞƌĐĞƌǀŝĐĂů KŶĐŽŐĞŶĞ
sŝƌƵƐƵƌŝŚĞƌƉĞƚŝĐĞ WŽƚŝŶĚƵĐĞƚƌĂŶƐĨŽƌŵĂƌĞĂŵĂůŝŐŶĉĂůŝŵĨŽĐŝƚĞůŽƌĂǀŝĞŶĞ͘

>Ă ŽĂŵĞŶŝ͕ǀŝƌƵƐƵůƉƐƚĞŝŶͲĂƌƌ;ǀ͘ŚĞƌƉĞƚŝĐƵŵĂŶϰͿƉƌŽĚƵĐĞ
ůŝŵĨŽŵƵƌŬŝƚƚ ;ŠŶƐƉĞĐŝĂůůĂƉĞƌƐŽĂŶĞůĞŝŶĨĞĐƚĂƚĞĐƵ,/sͿ

ϭϵ ϮϬ
ϮϬϭϮ͕ƌŽŽŬĞƌ͕'ĞŶĞƚŝĐƐ
 DW<͕ŵŝƚŽŐĞŶͲĂĐƚŝǀĂƚĞĚ ƉƌŽƚĞŝŶŬŝŶĂƐĞƐ
'&͕ĞƉŝĚĞƌŵĂů ŐƌŽǁƚŚ ĨĂĐƚŽƌ D<͕DW<ͬZ<ŬŝŶĂƐĞ
KŶĐŽŐĞŶĉ͕ ^ŽƐ͕ƐŽŶŽĨƐĞǀĞŶůĞƐƐ ZĂĨͲϭ͕ǀŝƌƵƐŝŶĚƵĐĞĚ ƌĂƉŝĚĞůLJ ĂĐĐĞůĞƌĂƚĞĚ ĨŝďƌŽƐĂƌĐŽŵĂ

ŐĞŶĉ͟ĐąƕƚŝŐͲĚĞͲĨƵŶĐƜŝĞ͟;ůď͘ĞŶŐů͘ŐĂŝŶͲŽĨͲĨƵŶĐƚŝŽŶͿ
KŶĐŽŐĞŶĞ 'ƌďϮ͕ŐƌŽǁƚŚ ĨĂĐƚŽƌƌĞĐĞƉƚŽƌͲďŽƵŶĚ ƉƌŽƚĞŝŶ Ϯ
ZĂƐ͕ƌĂƚ ƐĂƌĐŽŵĂ ǀŝƌƵƐ
'Wͬ'dW͕ŐƵĂŶŽnjŝŶĞ ĚŝͲͬƚƌŝͲƉŚŽƐƉŚĂƚĞ
DLJĐ͕ĂǀŝĂŶ ŵLJĞůŽĐLJƚŽŵĂƚŽƐŝƐ ǀŝƌƵƐ
:ƵŶ͕ĂǀŝĂŶ ƐĂƌĐŽŵĂ ǀŝƌƵƐϭϳ
&ŽƐ͕&ŝŶŬĞůͲŝƐŬŝƐͲ:ŝŶŬŝŶƐ ŵƵƌŝŶĞ ŽƐƚĞŽŐĞŶŝĐƐĂƌĐŽŵĂ ǀŝƌƵƐ

сŐĞŶĉĐĂƉĂďŝůĉĚĞĂĐĂƵnjĂƚƌĂŶƐĨŽƌŵĂƌĞĂĐĞůƵůĞůŽƌŶŽƌŵĂůĞŠŶĐĞůƵůĞ
ŵĂůŝŐŶĞ͕ƐƚŝŵƵůąŶĚƉƌŽůŝĨĞƌĂƌĞĂƕŝĐƌĞƕƚĞƌĞĂĐĞůƵůĂƌĉ
2ŶĐĞůƵůĞůĞŵĂůŝŐŶĞ
ĐĂůĞĂĚĞ
ƐĞŵŶĂůŝnjĂƌĞĂ'&Z
KŐĞŶĉŶŽƌŵĂůĉĐĂƌĞĂƌĞƉŽƚĞŶƜŝĂůƐĉĚĞǀŝŶĉŽŶĐŽŐĞŶĉсƉƌŽƚŽͲŽŶĐŽŐĞŶĉ ĞƐƚĞĂĐƚŝǀĂƚĉ
ĐŽŶƐƚŝƚƵƚŝǀ͖Ž
ƉŽƐŝďŝůĉĐĂƵnjĉ͗
WƌŝŶĐŝƉĂůĞůĞĞĨĞĐƚĞĂůĞƚƌĂŶƐĨŽƌŵĉƌŝŝƉƌŽƚŽͲŽŶĐŽŐĞŶĞůŽƌÆ ŽŶĐŽŐĞŶĞ͗ ƚƌĂŶƐĨŽƌŵĂƌĞĂ
ϭ͘ WƌŽƚĞŝŶĂĞƐƚĞĞdžƉƌŝŵĂƚĉŠŶĐĂŶƚŝƚĉƜŝŵĂƌŝ ƉƌŽƚŽͲŽŶĐŽŐĞŶĞŝ
ĐͲĞƌďď ŠŶ ŽŶĐŽŐĞŶĂ
Ϯ͘ ƉĂƌŵŽĚŝĨŝĐĉƌŝ ŠŶƐƚƌƵĐƚƵƌĂƉƌŽƚĞŝŶĞůŽƌĐĂƌĞůĞŵĞŶƜŝŶŠŶƐƚĂƌĞĂĐƚŝǀĂƚĉ ǀͲĞƌďď
ϯ͘ WƌŽƚĞŝŶĞůĞƐƵŶƚĞdžƉƌŝŵĂƚĞŠŶƜĞƐƵƚƵƌŝŠŶĐĂƌĞŶƵƐĞĞdžƉƌŝŵĉŠŶŵŽĚ
ŶŽƌŵĂů
ĹŝǀŝnjŝƵŶĞĐĞůƵůĂƌĉ

Ϯϭ ϮϮ
ϮϬϭϮ͕ƌŽŽŬĞƌ͕'ĞŶĞƚŝĐƐ ϮϬϭϮ͕ƌŽŽŬĞƌ͕'ĞŶĞƚŝĐƐ

KŶĐŽŐĞŶĞ ʹ ĞdžĞŵƉůĞ KŶĐŽŐĞŶĞ ʹ ĞdžĞŵƉůĞ

WƌŽƚŽͲŽŶĐŽŐĞŶĂ >ŽĐĂůŝnjĂƌĞĐƌƐ͘ KŶĐŽŐĞŶĂ &ƵŶĐƜŝĂƉƌŽƚĞŝŶĞŝ ĨĞĐƜŝƵŶĞǀŝƌĂůĉ WƌŽƚŽͲŽŶĐŽŐĞŶĂ >ŽĐĂůŝnjĂƌĞĐƌƐ͘ KŶĐŽŐĞŶĂ &ƵŶĐƜŝĂƉƌŽƚĞŝŶĞŝ ĨĞĐƜŝƵŶĞǀŝƌĂůĉ
ǀŝƌĂůĉ ǀŝƌĂůĉ
ĐͲƐŝƐ ϮϮƋϭϯ͘ϭ ǀͲƐŝƐ ^ƵďƵŶŝƚĂƚĞĂĂĨĂĐƚŽƌƵůƵŝĚĞĐƌĞƕƚĞƌĞ ^ĂƌĐŽŵƐŝŵŝĂŶ ĐͲũƵŶ ϭƉϯϮ͘ϭ ǀͲũƵŶ &ĂĐƚŽƌĚĞƚƌĂŶƐĐƌŝƉƜŝĞ ^ĂƌĐŽŵ ĂǀŝĂŶ ϭϳ
ĚĞƌŝǀĂƚĚŝŶƉůĂĐŚĞƚĞůĞ ƐĂŶŐƵŝŶĞ ĐͲŵLJĐ ϴƋϮϰ͘Ϯϭ ǀͲŵLJĐ &ĂĐƚŽƌĚĞƚƌĂŶƐĐƌŝƉƜŝĞ DŝĞůŽĐŝƚŽŵĂƚŽnjĉ ĂǀŝĂŶĉ
;W'&Ϳ
ĐͲĨŽƐ ϭϰƋϮϰ͘ϯ ǀͲĨŽƐ &ĂĐƚŽƌĚĞƚƌĂŶƐĐƌŝƉƜŝĞ KƐƚĞŽƐĂƌĐŽŵŵƵƌŝŶ
ĐͲĞƌďď ϳƉϭϭ͘Ϯ ǀͲĞƌďď ZĞĐĞƉƚŽƌƵůĨĂĐƚŽƌƵůƵŝĚĞĐƌĞƕƚĞƌĞ ƌŝƚƌŽůĞƵĐĞŵŝĞ
ĞƉŝĚĞƌŵĂů ;'&ZͿ ĂǀŝĂŶĉ
ĐͲƌĂƐ ϭϭƉϭϱ͘ϱ ǀͲƌĂƐ dŝƌŽnjŝŶͲŬŝŶĂnjĉ ŝŶƚƌĂĐĞůƵůĂƌĉ ;ZĂƐͿ ^ĂƌĐŽŵŵƵƌŝŶ
,ĂƌǀĞLJ

ĐͲĂďůϭ ϵƋϯϭ͘ϭ ǀͲĂďů dŝƌŽnjŝŶͲŬŝŶĂnjĉ ŝŶƚƌĂĐĞůƵůĂƌĉ >ĞƵĐĞŵŝĂŵƵƌŝŶĉ

ďĞůƐŽŶ

Ϯϯ Ϯϰ
ϮϬϭϭ͕^ƚƌĂĐŚĂŶ͕,ƵŵĂŶ DŽůĞĐƵůĂƌ'ĞŶĞƚŝĐƐ ϮϬϭϭ͕^ƚƌĂĐŚĂŶ͕,ƵŵĂŶ DŽůĞĐƵůĂƌ'ĞŶĞƚŝĐƐ
 DĞĐĂŶŝƐŵĞĚĞƚƌĂŶƐĨŽƌŵĂƌĞĂ DĞĐĂŶŝƐŵĞĚĞƚƌĂŶƐĨŽƌŵĂƌĞĂ
ƉƌŽƚŽͲŽŶĐŽŐĞŶĞůŽƌŠŶŽŶĐŽŐĞŶĞ ƉƌŽƚŽͲŽŶĐŽŐĞŶĞůŽƌŠŶŽŶĐŽŐĞŶĞ
‰ ĂŵƉůŝĨŝĐĂƌĞŐĞŶŝĐĉ ‰ ŵƵƚĂƜŝĞƉƵŶĐƚŝĨŽƌŵĉ

ŵƉůŝĨŝĐĂƌĞĂ ŽŶĐŽŐĞŶĞŝ EDzŠŶŶĞƵƌŽďůĂƐƚŽŵ

Ϯϱ Ϯϲ
ϮϬϭϭ͕^ƚƌĂĐŚĂŶ͕,ƵŵĂŶ DŽůĞĐƵůĂƌ'ĞŶĞƚŝĐƐ ϮϬϭϮ͕ƌŽŽŬĞƌ͕'ĞŶĞƚŝĐƐ

DĞĐĂŶŝƐŵĞĚĞƚƌĂŶƐĨŽƌŵĂƌĞĂ DĞĐĂŶŝƐŵĞĚĞƚƌĂŶƐĨŽƌŵĂƌĞĂ
ƉƌŽƚŽͲŽŶĐŽŐĞŶĞůŽƌŠŶŽŶĐŽŐĞŶĞ ƉƌŽƚŽͲŽŶĐŽŐĞŶĞůŽƌŠŶŽŶĐŽŐĞŶĞ
‰ ƚƌĂŶƐůŽĐĂƜŝĞ
DĞĐĂŶŝƐŵ KŶĐŽŐĞŶĉ ĨĞĐƜŝƵŶĞ ŵĂůŝŐŶĉ
ŵƉůŝĨŝĐĂƌĞŐĞŶŝĐĉ ZϮ;,ZϮͿ ĂŶĐĞƌŵĂŵĂƌ͕ŐĂƐƚƌŝĐ͕ƉƵůŵŽŶĂƌ͕ĐŽůŽƌĞĐƚĂů
EDz EĞƵƌŽďůĂƐƚŽŵ
DƵƚĂƜŝĞƉƵŶĐƚŝĨŽƌŵĉ ,Z^ ĂŶĐĞƌǀĞnjŝĐĂů͕ƉƵůŵŽŶĂƌ͕ĐŽůŽƌĞĐƚĂů͖ ŵĞůĂŶŽŵ
dƌĂŶƐůŽĐĂƜŝŝ ZͲ>ϭ >ĞƵĐĞŵŝĞ ŵŝĞůŽŝĚĉ ĐƌŽŶŝĐĉ
Dz >ŝŵĨŽŵ ƵƌŬŝƚƚ

Z͕ďƌĞĂŬƉŽŝŶƚ ĐůƵƐƚĞƌƌĞŐŝŽŶ
>ĞƵĐĞŵŝĞŵŝĞůŽŝĚĉ ĐƌŽŶŝĐĉ >ŝŵĨŽŵƵƌŬŝƚƚ
ƚ;ϵ͖ϮϮͿ ƚ;ϴ͖ϭϰͿ
Ϯϳ Ϯϴ
ϮϬϭϮ͕ƌŽŽŬĞƌ͕'ĞŶĞƚŝĐƐ ϮϬϭϭ͕^ƚƌĂĐŚĂŶ͕,ƵŵĂŶ DŽůĞĐƵůĂƌ'ĞŶĞƚŝĐƐ ϮϬϭϭ͕^ƚƌĂĐŚĂŶ͕,ƵŵĂŶ DŽůĞĐƵůĂƌ'ĞŶĞƚŝĐƐ

'ĞŶĞƐƵƉƌĞƐŽĂƌĞƚƵŵŽƌĂůĞ
'ĞŶĞƐƵƉƌĞƐŽĂƌĞƚƵŵŽƌĂůĞ
ĻŝǀŝnjŝƵŶĞĐĞůƵůĂƌĉ
‰ 'ĞŶĂZ
‡ &ĂĐƚŽƌƵůĚĞƚƌĂŶƐĐƌŝƉƜŝĞϮ&
ĨĂǀŽƌŝnjĞĂnjĉŝŶƚƌĂƌĞĂĐĞůƵůĞůŽƌŠŶĐŝĐůƵů
ĚĞĚŝǀŝnjŝƵŶĞ
‡ >ĞŐĂƌĞĂƉƌŽƚĞŝŶĞŝZďůĂϮ&ďůŽĐŚĞĂnjĉ
ĂĐƚŝǀŝƚĂƚĞĂƚƌĂŶƐĐƌŝƉƜŝŽŶĂůĉ ĂϮ&
Ϯ&͕ϮĨĂĐƚŽƌ
ĹŝǀŝnjŝƵŶĞĐĞůƵůĂƌĉ
ϮϬϭϮ͕ƌŽŽŬĞƌ͕'ĞŶĞƚŝĐƐ
'ĞŶĞƐƵƉƌĞƐŽĂƌĞƚƵŵŽƌĂůĞͬĂŶƚŝͲŽŶĐŽŐĞŶĞ
сĂƵƌŽůƵůĚĞĂƉƌĞǀĞŶŝƉƌŽůŝĨĞƌĂƌĞĂĐĞůƵůĞůŽƌŵĂůŝŐŶĞ
‰ 'ĞŶĂZ
‡ /ĚĞŶƚŝĨŝĐĂƚĉƉĞŶƚƌƵƉƌŝŵĂĚĂƚĉŠŶƚƌͲŽƚƵŵŽƌĉƌĞƚŝŶŝĂŶĉůĂ
ĐŽƉŝŝ;ƌĞƚŝŶŽďůĂƐƚŽŵͿ
‡ ŽĂůĂĞƐƚĞĞƌĞĚŝƚĂƌĉĚĂĐĉƐƵŶƚĂĨĞĐƚĂƜŝĂŵďŝŝŽĐŚŝ͕ƕŝ
ƐƉŽƌĂĚŝĐĉĚĂĐĉĞƐƚĞĂĨĞĐƚĂƚĚŽĂƌƵŶƵůĚŝŶƚƌĞŽĐŚŝ
‡ ϭϵϳϭ͕ŐĞŶĞƚŝĐŝĂŶƵůĂŵĞƌŝĐĂŶůĨƌĞĚ<ŶƵĚƐŽŶ ĂƉƌŽƉƵƐ
ŝƉŽƚĞnjĂ͟ĚŽƵĉͲůŽǀŝƚƵƌŝ͟;ůď ĞŶŐů ƚǁŽͲŚŝƚŚLJƉŽƚŚĞƐŝƐͿ͕ĐĨ
ĐĉƌĞŝĂƐƵŶƚŶĞĐĞƐĂƌĞϮŵƵƚĂƜŝŝƉƚ ĐĂďŽĂůĂƐĉƐĞŵĂŶŝĨĞƐƚĞ
Ϯϵ ϯϬ
ϮϬϭϭ͕^ƚƌĂĐŚĂŶ͕,ƵŵĂŶ DŽůĞĐƵůĂƌ'ĞŶĞƚŝĐƐ
'ĞŶĞƐƵƉƌĞƐŽĂƌĞƚƵŵŽƌĂůĞ
‰ 'ĞŶĂdWϱϯ
‡ ĐŽĚŝĨŝĐĉƉƌŽƚĞŝŶĂƉϱϯ
‡ ĂĐƚŝǀĞĂnjĉŐĞŶĞĐĂƌĞ͗
ϭͿ /ŶĚƵĐƌĞƉĂƌĂƌĞĂE
ϮͿ /ŶĚƵĐďůŽĐĂƌĞĂƉƌŽŐƌĞƐŝĞŝ
ĐŝĐůƵůƵŝĐĞůƵůĂƌ
ϯͿ WƌŽŵŽǀĞĂnjĉĂƉŽƉƚŽnjĂ
dD͕ ĂƚĂdžŝĂͲƚĞůĂŶŐŝĞĐƚĂƐŝĂŵƵƚĂƚĞĚ
dZ͕dDͲ ĂŶĚZĂĚϯͲZĞůĂƚĞĚ
DĚŵϮ͕DŽƵƐĞĚŽƵďůĞŵŝŶƵƚĞϮŚŽŵŽůŽŐ
ŚŬϭ͕ŚĞĐŬƉŽŝŶƚ ŬŝŶĂƐĞ ϭ
ϱϬйĚŝŶĐĂŶĐĞƌĞůĞƵŵĂŶĞƉƌĞnjŝŶƚĉ
ŵƵƚĂƜŝŝůĂŶŝǀĞůƵůŐĞŶĞŝƉϱϯ
ϯϭ ϯϮ
ϮϬϭϱ͕ůďĞƌƚƐ ĞƚĂů͕͘DŽůĞĐƵůĂƌŝŽůŽŐLJ ŽĨƚŚĞ Ğůů
 'ĞŶĞƐƵƉƌĞƐŽĂƌĞƚƵŵŽƌĂůĞ
љWƌŽůŝĨĞƌĂƌĞĐĞůƵůĂƌĉ
ϮϬϭϱ͕ůďĞƌƚƐ ĞƚĂů͕͘DŽůĞĐƵůĂƌŝŽůŽŐLJ ŽĨƚŚĞ Ğůů
'ĞŶĞƐƵƉƌĞƐŽĂƌĞƚƵŵŽƌĂůĞ
‰ 'ĞŶĂW ‰ 'ĞŶĞůĞZͲϭ͕ZͲϮ
ZͲϭ
‡ >ď͘ĞŶŐů͘ĂĚĞŶŽŵĂƚŽƵƐ ƉŽůLJƉŽƐŝƐ ĐŽůŝ ‡ >ď͘ĞŶŐů͘ďƌĞĂƐƚ ĐĂŶĐĞƌƐƵƐĐĞƉƚŝďŝůŝƚLJ
‡ ŽĚŝĨŝĐĉƉƌŽƚĞŝŶĂW͕ĐĂƌĞĞƐƚĞ ŐĞŶĞƐ
ƌĞŐůĂƚŽƌŶĞŐĂƚŝǀ ĂůĐĉŝŝĚĞƐĞŵŶĂůŝnjĂƌĞ &ŽƐĨŽƌŝůĞĂnjĉ ŬŝŶĂnjĞůĞ ‡ /ŵƉůŝĐĂƚĞŠŶƌĞƉĂƌĂƌĞĂE
tŶƚ͕ƌĞƐƉĞĐƚŝǀĂůɴͲĐĂƚĞŶŝŶĞŝ͕ĐĂƌĞŠŶ dDͬdZ ‡ &ƵŶĐƜŝŽŶĞĂnjĉĐĂƉƌŽƚĞŝŶĞŝŵƉůŝĐĂƚĞŠŶ
ůŽĐĂƌĞĂ
ŵŽĚŶŽƌŵĂůƉƌŽŵŽǀĞĂnjĉĚŝǀŝnjŝƵŶĞĂ ĐŝĐůƵůƵŝ ĐĞůƵůĂƌ ƉƵŶĐƚĞůĞĚĞĐŽŶƚƌŽůĂůĞĐŝĐůƵůƵŝ
ĐĞůƵůĂƌĉ ĐĞůƵůĂƌ
Ļ dƌĂŶƐĐƌŝƉƜŝĂ ƕŝ
‡ DƵƚĂƜŝŝĂůĞŐĞŶĞŝW ƉŽƚĐŽŶĚƵĐĞůĂ
ƚƌĂŶƐĨŽƌŵĂƌĞĂ ƉŽƉƚŽnjĉ
ĂƉĂƌŝƜŝĂĐĂŶĐĞƌƵůƵŝĐŽůŽƌĞĐƚĂů ŵĂůŝŐŶĉ dD͕ĂƚĂdžŝĂͲƚĞůĂŶŐŝĞĐƚĂƐŝĂŵƵƚĂƚĞĚ
ZĞƉĂƌĂƌĞ ůŽĐĂƌĞĂ dZ͕dDͲ ĂŶĚZĂĚϯͲZĞůĂƚĞĚͬ
E ĐŝĐůƵůƵŝ ĐĞůƵůĂƌ ƐĞƌŝŶͬƚƌĞŽŶŝŶ ƉƌŽƚĞŝŶ ŬŝŶĂƐĞ
tŶƚ͕tŝŶŐůĞƐƐͲƌĞůĂƚĞĚŝŶƚĞŐƌĂƚŝŽŶƐŝƚĞ >&ϭ͕>LJŵƉŚŽŝĚ ŶŚĂŶĐĞƌ ŝŶĚŝŶŐ &ĂĐƚŽƌϭ
>ZW͕ůŝƉŽƉƌŽƚĞŝŶ ƌĞĐĞƉƚŽƌͲƌĞůĂƚĞĚ ƉƌŽƚĞŝŶ d&͕dͲĐĞůů ĨĂĐƚŽƌ ZĂĚϯ͕ŚĞůŝĐĂƐĞ
'^<ϯ͕'ůLJĐŽŐĞŶƐLJŶƚŚĂƐĞŬŝŶĂƐĞϯ &ƌŝnjnjůĞĚ͕'ͲƉƌŽƚĞŝŶ ĐŽƵƉůĞĚ ƌĞĐĞƉƚŽƌ
<͕ƌĞĂƚŝŶŬŝŶĂnjĂ ϮϬϭϮ͕^ůĂƌŬĞƚĂů͕͘ŽŵƉƵƚĂƚŝŽŶĂů ĂŶĚ ^ƚƌƵĐƚ ŝŽƚĞĐŚŶŽů :͕sŽůƵŵĞEŽ͗ϭ͕/ƐƐƵĞ͗ϭ͕ĞϮϬϭϮϬϰϬϬϱ͕
ϯϯ ϯϰ
ϮϬϭϯ͕DĂƌĞĐŚĂů ĞƚĂů͕͘ ŽůĚ^ƉƌŝŶŐ,Ăƌď WĞƌƐƉĞĐƚ ŝŽů ϮϬϭϯ͖ϱ͗ĂϬϭϮϳϭϲ

'ĞŶĞƐƵƉƌĞƐŽĂƌĞƚƵŵŽƌĂůĞ
DĞĐĂŶŝƐŵĞĚĞŝŶĂĐƚŝǀĂƌĞͬ͟ůŽƐƐͲŽĨͲĨƵŶĐƚŝŽŶ͟

ϭ͘ DƵƚĂƜŝŝ ůĂŶŝǀĞůƵůŐĞŶĞůŽƌƐƵƉƌĞƐŽĂƌĞƚƵŵŽƌĂůĞÆ ƉƌĞǀŝŶƐŝŶƚĞnjĂ

ƵŶŽƌƉƌŽƚĞŝŶĞĨƵŶĐƜŝŽŶĂůĞ

Ϯ͘ DĞƚŝůĂƌĞĂEʹ ĚĞƌĞŐƵůĉŠŶĐĞůƵůĞůĞĞƵĐĂƌŝŽƚĞŵĞƚŝůĂƌĞĂE
;ŝŶƐƵůĞůĞƉ' ĚŝŶĂƉƌŽƉŝĞƌĞĂƌĞŐŝƵŶŝŝƉƌŽŵŽƚŽƌͿŝŶŚŝďĉƚƌĂŶƐĐƌŝƉƜŝĂ DĂůŝŐŶŝƚĉƜŝĞƌĞĚŝƚĂƌĞ
ϯ͘ ŶĞƵƉůŽŝĚŝĂʹ ƉŝĞƌĚĞƌĞĂƵŶŽƌĐƌŽŵŽnjŽŵŝƉŽĂƚĞĐŽŶĚƵĐĞůĂ
ƉŝĞƌĚĞƌĞĂŐĞŶĞůŽƌƐƵƉƌĞƐŽĂƌĞƚƵŵŽƌĂůĞ

ϯϱ ϯϲ
 DĂůŝŐŶŝƚĉƜŝ ĞƌĞĚŝƚĂƌĞ
'ĞŶĞƐƵƉƌĞƐŽĂƌĞƚƵŵŽƌĂůĞ
W ;ĂĚĞŶŽŵĂƚŽƵƐƉŽůLJƉŽƐŝƐĐŽůŝͿ
Z ;ƌĞƚŝŶŽďůĂƐƚŽŵĂͿ
dWϱϯ
WdE ;ƉŚŽƐƉŚĂƚĂƐĞĂŶĚƚĞŶƐŝŶ ŚŽŵŽůŽŐͿ
ZͲϭ͕ZͲϮ ;ďƌĞĂƐƚ ĐĂŶĐĞƌ
ƐƵƐĐĞƉƚŝďŝůŝƚLJŐĞŶĞͿ
&ĂĐƚŽƌŝĚĞƌŝƐĐŝŶĐĂŶĐĞƌƵůŵĂŵĂƌΎ
dƌĂŶƐŵŝƚĞƌĞĂ ĞƌĞĚŝƚĂƌĉĂĐĂŶĐĞƌƵůƵŝŵĂŵĂƌ
WŝĞƌĚĞƌĞĂŚĞƚĞƌŽnjŝŐŽnjŝƚĉƜŝŝ
DĂůŝŐŶŝƚĂƚĞ ;ůď͘ĞŶŐů͘ůŽƐƐ ŽĨŚĞƚĞƌŽnjLJŐŽƐŝƚLJͿ
WŽůŝƉŽnjĉ ĂĚĞŶŽŵĂƚŽĂƐĉ ĨĂŵŝůŝĂůĉƕŝĐĂŶĐĞƌĚĞĐŽůŽŶĨĂŵŝůŝĂů
ZĞƚŝŶŽďůĂƐƚŽŵ
^ŝŶĚƌŽŵƵů>ŝͲ&ƌĂƵŵĞŶŝ ʹ ĚĞnjǀŽůƚĂƌĞĂĂŵĂŝŵƵůƚŽƌƚƵŵŽƌŝ͗
ƐĂƌĐŽĂŵĞ͕ƚƵŵŽƌŝĐĞƌĞďƌĂůĞ͕ĐĂŶĐĞƌŵĂŵĂƌ
ZŝƐĐ ĐƌĞƐĐƵƚƉĞŶƚƌƵĐĂƌĐŝŶŽĂŵĞŵĂŵĂƌĞƕŝƚŝƌŽŝĚŝĞŶĞ
ĂŶĐĞƌŵĂŵĂƌĨĂŵŝůŝĂů
ϯϳ ϯϴ
ϮϬϭϮ͕ƌŽŽŬĞƌ͕'ĞŶĞƚŝĐƐ
ŽŶĐĞƉƚĞ ĨƵŶĚĂŵĞŶƚĂůĞ ;ϭͿ
‡ ĂŶĐĞƌƵůĂƌĞŽŽƌŝŐŝŶĞĐůŽŶĂůĉ ƕŝĞƐƚĞƵŶƉƌŽĐĞƐŵƵůƚŝƐƚĂĚŝĂůĐĂƌĞ
ŶĞĐĞƐŝƚĉŵĂŝŵƵůƚĞŵƵƚĂƜŝŝƉĞŶƚƌƵĐĂŽĨŽƌŵĂƜŝƵŶĞďĞŶŝŐŶĉƐĉƐĞ
ƚƌĂŶƐĨŽƌŵĞŠŶƚƌͲŽƚƵŵŽƌĉŵĂůŝŐŶĉ͕ŝĂƌĂƉŽŝƐĉĚĞǀŝŶĉŝŶǀĂnjŝǀĉƕŝƐĉ
ŵĞƚĂƐƚĂnjĞnjĞ
‡ /ŶƐƚĂďŝůŝƚĂƚĞĂŐĞŶĞƚŝĐĉĞƐƚĞƵŶĨĞŶŽŵĞŶĐŽŵƵŶŠŶďŽůŝůĞŵĂůŝŐŶĞ
‡ /ĚĞŶƚŝĨŝĐĂƌĞĂƉƌŽĨŝůƵůƵŝŵŽůĞĐƵůĂƌĞƐƚĞŶĞĐĞƐĂƌĉƉĞŶƚƌƵĂƉůŝĐĂƌĞĂƵŶĞŝ
ƚĞƌĂƉŝŝĐŽƌĞƐƉƵŶnjĉƚŽĂƌĞ͕ĚĞŽĂƌĞĐĞďŽůŝůĞŵĂůŝŐŶĞƉƌĞnjŝŶƚĉƵŶŐƌĂĚ
ŵĂƌĞĚĞŚĞƚĞƌŽŐĞŶŝĐŝƚĂƚĞ
ϯϵ ϰϬ
&OHPRQV 0 3*RVV1(QJO -0HG9RO1R-DQXDU\ ,)
ŽŶĐĞƉƚĞ ĨƵŶĚĂŵĞŶƚĂůĞ ;ϮͿ ŽŶĐĞƉƚĞ ĨƵŶĚĂŵĞŶƚĂůĞ ;ϯͿ
‡ hŶĞůĞǀŝƌƵƐƵƌŝƚƌĂŶƐƉŽƌƚĉŽŶĐŽŐĞŶĞ ‡ DĞĐĂŶŝƐŵĞůĞĐĂƌĞƚƌĂŶƐĨŽƌŵĉƉƌŽƚŽͲŽŶĐŽŐĞŶĞůĞŠŶŽŶĐŽŐĞŶĞƐƵŶƚ͗
ŵƵƚĂƜŝŝůĞƉƵŶĐƚŝĨŽƌŵĞ͕ĂŵƉůŝĨŝĐĂƌĞĂŐĞŶŝĐĉƕŝƚƌĂŶƐůŽĐĂƜŝŝůĞĐƌŽŵŽnjŽŵŝĂůĞ

‡ KŶĐŽŐĞŶĞůĞ ŠŶŵŽĚĨƌĞĐǀĞŶƚĂĨĞĐƚĞĂnjĉƉƌŽůŝĨĞƌĂƌĞĂƉƌŝŶƉƌŽƚĞŝŶĞ
ŝŵƉůŝĐĂƚĞŠŶĚŝǀŝnjŝƵŶĞĂĐĞůƵůĂƌĉ
‡ KŶĐŽŐĞŶĞůĞƐĞĨŽƌŵĞĂnjĉƉƌŝŶĨĞŶŽŵĞůĞ ĚĞƚŝƉ ͟ĐąƕƚŝŐͲĚĞͲĨƵŶĐƜŝĞ͟Ăů
ƉƌŽƚŽͲŽŶĐŽŐĞŶĞůŽƌ
‡ 'ĞŶĂƐƵƉƌĞƐŽĂƌĞƚƵŵŽƌĂůĉdWϱϯ ĞƐƚĞŝŵƉůŝĐĂƚĉŠŶƌĞƉĂƌĂƌĞĂůĞnjŝƵŶŝůŽƌE
ƉƌŝŶŵĞĐĂŶŝƐŵĞĚĞďůŽĐĂƌĞĂĐŝĐůƵůƵŝĐĞůƵůĂƌ͕ŵĞĐĂŶŝƐŵĞĚĞƐŝŶƚĞnjĉĂ
ĞŶnjŝŵĞůŽƌŝŵƉůŝĐĂƚĞŠŶƌĞƉĂƌĂƌĞĂE͕ŵĞĐĂŶŝƐŵĞĂƉŽƉƚŽƚŝĐĞ
‡ 'ĞŶĞůĞƐƵƉƌĞƐŽĂƌĞƚƵŵŽƌĂůĞƉŽƚĨŝŝŶĂĐƚŝǀĂƚĞ ƉƌŝŶŵƵƚĂƜŝŝ;ůŽƐƐͲŽĨͲ
ĨƵŶĐƚŝŽŶͿ͕ƉŝĞƌĚĞƌĞĂĐƌŽŵŽnjŽŵŝůŽƌƕŝŵĞƚŝůĂƌĞĂE

ϰϭ ϰϮ

&ĂĐƚŽƌŝƚĞƌĂƚŽŐĞŶŝ
сĂŐĞŶƜŝĚŝŶŵĞĚŝƵůŠŶĐŽŶũƵƌĉƚŽƌĐĂƌĞŝŶƚĞƌĨĞƌĞĂnjĉĐƵĚĞnjǀŽůƚĂƌĞĂ
ŝŶƚƌĂƵƚĞƌŝŶĉĂƉƌŽĚƵƕŝůŽƌĚĞĐŽŶĐĞƉƜŝĞƕŝŝŶĚƵĐĂŶŽŵĂůŝŝĚĞĚĞnjǀŽůƚĂƌĞůĂ
ŶĂƕƚĞƌĞ
ĨĞĐƚĞůĞĨĂĐƚŽƌŝůŽƌƚĞƌĂƚŽŐĞŶŝĚĞƉŝŶĚĚĞ͗

Ϯ͘ůĞŵĞŶƚĞ ĚĞƚĞƌĂƚŽŐĞŶĞnjĂ ‡ dŝƉƵůƚĞƌĂƚŽŐĞŶƵůƵŝ

‡ WĞƌŝŽĂĚĂŠŶĐĂƌĞĂĐĞƐƚĂĂĐƜŝŽŶĞĂnjĉĂƐƵƉƌĂƉƌŽĚƵƐƵůƵŝĚĞĐŽŶĐĞƉƜŝĞ;ƌŝƐĐŵĂdžŝŵŠŶƉƌŝŵƵůƚƌŝŵĞƐƚƌƵ
ĚĞƐĂƌĐŝŶĉͿ
‡ ŽnjĂĂĐĞƐƚƵŝĂ
‡ ƵƌĂƚĂĞdžƉƵŶĞƌŝŝůĂĂŐĞŶƚƵůƚĞƌĂƚŽŐĞŶ

ϰϯ ϰϰ
&ĂĐƚŽƌŝƚĞƌĂƚŽŐĞŶŝ &ĂĐƚŽƌŝƚĞƌĂƚŽŐĞŶŝ
ŶŽŵĂůŝŝůĞĐŽŶŐĞŶŝƚĂůĞ͗ ‡ ŚŝŵŝĐŝ
,ĞŵĂŶŐŝŽŵ͕ĂŐůŽŵĞƌĂƌĞĚĞ
ϭ͘ ŵďƌŝŽƉĂƚŝŝ ʹ ƉƌŝŵĞůĞϯůƵŶŝĚĞƐĂƌĐŝŶĉ ǀĂƐĞĐĂƉŝůĂƌĞŶŽƵĨŽƌŵĂƚĞĐĂƌĞ ‡ &ŝnjŝĐŝ
Ϯ͘ &ĞƚŽƉĂƚŝŝ ʹ ĂĐƜŝƵŶĞĂƚĞƌĂƚŽŐĞŶƵůƵŝƉĞƉĂƌĐƵƌƐƵůůƵŶŝůŽƌϰͲϵĚĞƐĂƌĐŝŶĉ ƐĞƉƌĞnjŝŶƚĉĐĂŽƉĂƚĉƌŽƕŝĞͲ
ǀŝŽůĂĐĞĞ͖ƚƵŵŽƌĉďĞŶŝŐŶĉĂ ‡ /ŶĨĞĐƜŝŽƕŝ
ǀĂƐĞůŽƌƐĂŶŐƵŝŶĞ ‡ DĞƚĂďŽůŝĐŝ
ĨĞĐƚĞůĞĐůŝŶŝĐĞ͗
ϭ͘ DŝŶŽƌĞ ʹ ŚĞŵĂŶŐŝŽĂŵĞ͕ƐŝŶĚĂĐƚŝůŝĞ ^ŝŶĚĂĐƚŝůŝĞ͕ŵĂůĨŽƌŵĂƜŝĞ
Ϯ͘ DĂũŽƌĞ ʹ ĂĨĞĐƜŝƵŶŝĂůĞ^E͕s͕'/͕ƵƌŽͲŐĞŶŝƚĂů͕ŵĞŵďƌĞůŽƌ ĐŽŶŐĞŶŝƚĂůĉĂŵąŝŶŝŝƐĂƵ
ƉŝĐŝŽƌƵůƵŝ͕ĐĂƌĞĐŽŶƐƚĉŠŶůŝƉŝƌĞĂ
ĂĚŽƵĉƐĂƵŵĂŝŵƵůƚŽƌĚĞŐĞƚĞ
ŠŶƚƌĞĞůĞƉƌŝŶƐƵĚĂƌĞĂƉĉƌƜŝůŽƌ
WƌŽĨŝůĂdžŝĂĞƐƚĞŠŶƚŽƚĚĞĂƵŶĂŝŵƉŽƌƚĂŶƚĉ ŵŽŝ͕ŽĂƐĞůĞƌĉŵąŶąŶĚ
ƐĞƉĂƌĂƚĞ

ϰϱ ϰϲ

dĞƌĂƚŽŐĞŶŝĐŚŝŵŝĐŝ dĞƌĂƚŽŐĞŶŝĐŚŝŵŝĐŝ
ůĐŽŽůƵůÆ ƐŝŶĚƌŽŵƵů ĂůĐŽŽůŝĐ ĨĞƚĂů tĂƌĨĂƌŝŶĂ
‡ /ŶĐŝĚĞŶƜĉ ϭ͗ϯϬϬůĂϭ͗ϮϬϬϬEE
‡ ŶƚŝĐŽĂŐƵůĂŶƚ
‡ ^ĞƌĞĐŽŵĂŶĚĉĞǀŝƚĂƌĞĂĐŽŶƐƵŵƵůƵŝĚĞĂůĐŽŽůƉĞƉĞƌŝŽĂĚĂƐĂƌĐŝŶŝŝ
‡ ƌĞƕƚĞŶŝǀĞůƵůĂĐĞƚĂůĚĞŚŝĚĞŝŵŝƚŽĐŽŶĚƌŝĂůĞÆ ĞĨĞĐƚĐŝƚŽƚŽdžŝĐ͕ŵƵƚĂŐĞŶ͕ƚĞƌĂƚŽŐĞŶĂƐƵƉƌĂ ‡ ^ƵƐĐĞƉƚŝďŝůŝƚĂƚĞĂƚąƚŠŶƉĞƌŝŽĂĚĂĞŵďƌŝŽŶĂƌĉ͕ĐąƚƕŝĨĞƚĂůĉ
ƉƌŽĚƵƐƵůƵŝĚĞĐŽŶĐĞƉƜŝĞ ‡ ƌĞƕƚĞĨƌĞĐǀĞŶƜĂƉŝĞƌĚĞƌŝůŽƌĚĞƐĂƌĐŝŶĉƐƉŽŶƚĂŶĞ
‡ ŽƉŝŝĂĨĞĐƚĂƜŝƉƌĞnjŝŶƚĉƌĞƚĂƌĚŵŝŶƚĂů;/YсϲϯͲϴϯͿ͕ŚŝƉĞƌĂĐƚŝǀŝƚĂƚĞ͕ƵŶĞŽƌŝŝŵƉƵůƐŝǀŝƚĂƚĞ ‡ ŽƉŝŝĂĨĞĐƚĂƜŝƉƌĞnjŝŶƚĉŚŝƉŽƚŽŶŝĞŵƵƐĐƵůĂƌĉ͕ƐĐŽůŝŽnjĉ͕ƌĞƚĂƌĚŵĞŶƚĂů͕ĂŶŽŵĂůŝŝĐĂƌĚŝĂĐĞ

dŚĂůŝŽŵŝĚĂ
‡ DĞĚŝĐĂŵĞŶƚƐĞĚĂƚŝǀ͕ƵƚŝůŝnjĂƚŠŶƚƌĂƚĂŵĞŶƚƵůĚŝƐŐƌĂǀŝĚŝĞŝ;ǀĉƌƐĉƚƵƌŝƌĞƉĞƚĂƚĞ͕ŝŶƚŽůĞƌĂŶƜĉĂůŝŵĞŶƚĂƌĉ DĞĚŝĐĂŵĞŶƚĞĂŶƚŝĐŽŶǀƵůƐŝǀĞ͗ŚŝĚĂŶƚŽŝŶĂ ;ϮϱйƌŝƐĐͿ͕ƚƌŝŵĞƚĂĚŝŽŶĂ
ĐĂƌĞĐŽŶĚƵĐůĂĚĞnjĞĐŚŝůŝďƌĞŚŝĚƌŽͲĞůĞĐƚƌŽůŝƚŝĐĞͿ
;ϴϬйƌŝƐĐͿ
‡ ^ƵƐĐĞƉƚŝďŝůŝƚĂƚĞŵĂdžŝŵĉŠŶƉƌŝŵĞůĞϱͲϲƐĉƉƚĉŵąŶŝĚĞƐĂƌĐŝŶĉ
‡ ŽƉŝŝĂĨĞĐƚĂƜŝƉƌĞnjŝŶƚĉĂŶŽŵĂůŝŝĐĂƌĚŝĂĐĞ͕ŽƐŽĂƐĞ͕ƉƵůŵŽŶĂƌĞ͕ĚŝŐĞƐƚŝǀĞ͕ƌĞŶĂůĞ͕ŚŝĚƌŽĐĞĨĂůŝĞ ‡ WŽƚƉƌŽĚƵĐĞƌĞƚĂƌĚŠŶĐƌĞƕƚĞƌĞĂƕŝĚĞnjǀŽůƚĂƌĞĂĞŶĐĞĨĂůƵůƵŝ͕ĨŽŶƚĂŶĞůĂĂŶƚĞƌŝŽĂƌĉĚĞĚŝŵĞŶƐŝƵŶŝ
ŵĂƌŝ͕ĂŶŽŵĂůŝŝĂůĞŵĞŵďƌĞůŽƌ͕ůƵdžĂƜŝĞĐŽŶŐĞŶŝƚĂůĉĚĞƕŽůĚ͕ĂŶŽŵĂůŝŝsƕŝƵƌŽŐĞŶŝƚĂůĞ

ϰϳ ϰϴ
dĞƌĂƚŽŐĞŶŝĐŚŝŵŝĐŝ dĞƌĂƚŽŐĞŶŝĐŚŝŵŝĐŝ
ŵŝƉƚĞƌŝŶĂ ƕŝŵĞƚŚŽƚƌĞdžĂƚƵů ;ĚĞƌŝǀĂƚĂůĂŵŝŶŽƉƚĞƌŝŶĞŝͿ DĞƚŝůͲŵĞƌĐƵƌƵů
‡ ŶƚĂŐŽŶŝƐƚĂůĂĐŝĚƵůƵŝĨŽůŝĐ ‡ &ŽůŽƐŝƚĐĂĨƵŶŐŝĐŝĚŠŶĂŐƌŝĐƵůƚƵƌĉ͖ŝŶƚƌŽĚƵƐŠŶŽƌŐĂŶŝƐŵƉƌŝŶĐŽŶƐƵŵƵůĚĞĐĞƌĞĂůĞƚƌĂƚĂƚĞƐĂƵƉĞƕƚĞ
‡ 2ŶƉƌŝŵĞůĞϱƐĉƉƚĉŵąŶŝÆ ƉŝĞƌĚĞƌĞĂ ƐĂƌĐŝŶŝŝ ĚŝŶĂƉĞĐŽŶƚĂŵŝŶĂƚĞ
‡ WĞƌŝŽĂĚĂ ϱƐĉƉƚĉŵąŶŝ ʹ ϯůƵŶŝÆ ĞĨĞĐƚĞƚĞƌĂƚŽŐĞŶĞ͗ƌĞƚĂƌĚĚĞĐƌĞƕƚĞƌĞ͕ƌĞƚĂƌĚŵĞŶƚĂů͕ĞdžŽĨƚůĂŵŝĞ͕ ‡ ZŝƐĐŵĂdžŝŵŠŶƉƌŝŵĞůĞϮͲϱƐĉƉƚĉŵąŶŝ
ĂŶŽŵĂůŝŝĚĞĚĞnjǀŽůƚĂƌĞĂŵĞŵďƌĞůŽƌ
‡ ĨĞĐƚĞƚĞƌĂƚŽŐĞŶĞ͗ƌĞƚĂƌĚƉƐŝŚŽͲŵŽƚŽƌƐĞǀĞƌ͕ƚƵůďƵƌĉƌŝŶĞƵƌŽůŽŐŝĐĞŐƌĂǀĞ͕ŵŝĐƌŽĐĞĨĂůŝĞ

sŝƚĂŵŝŶĂƕŝĂĐŝĚƵůϭϯͲĐŝƐ ƌĞƚŝŶŽŝĐ
‡ hƚŝůŝnjĂƚĞŠŶƚƌĂƚĂŵĞŶƚƵůĂĐŶĞĞŝ
ŽĐĂŝŶĂ;ƌŝƐĐϯϯйͿ
‡ ĨĞĐƚƚĞƌĂƚŽŐĞŶ͗ƌŝƐĐĐƌĞƐĐƵƚĚĞƉŝĞƌĚĞƌĞƐƉŽŶƚĂŶĉĂƐĂƌĐŝŶŝŝƕŝĞŵďƌŝŽƉĂƚŝŝ ‡ ZŝƐĐŵĂdžŝŵŠŶƉƌŝŵƵůƚƌŝŵĞƐƚƌƵĚĞƐĂƌĐŝŶĉ
‡ ŽƉŝŝĂĨĞĐƚĂƜŝ͗ĨƌƵŶƚĞŠŶŐƵƐƚĉ͕ŶĂƐƚƵƌƚŝƚ͕ĂŶŽŵĂůŝŝĐĂƌĚŝŽǀĂƐĐƵůĂƌĞ͕ƉĂǀŝůŝŽĂŶĞĂƵƌŝĐƵůĂƌĞŵŝĐŝƐĂƵ ‡ WŝĞƌĚĞƌŝƐƉŽŶƚĂŶĞĚĞƐĂƌĐŝŶĉ͕ŶĂƕƚĞƌŝƉƌĞŵĂƚƵƌĞ͕ĚĞnjůŝƉŝƌĞĚĞƉůĂĐĞŶƚĉ͕ƚƵďƵƌĉƌŝ ŶĞƵƌŽůŽŐŝĐĞ͕
ĐŚŝĂƌĂďƐĞŶƚĞ͕ĂŶŽŵĂůŝŝs͕ĂŶŽŵĂůŝŝĂůĞ^E ĂŶŽŵĂůŝŝĚŝŐĞƐƚŝǀĞ͕ĂŶŽŵĂůŝŝŐĞŶŝƚŽͲƵƌŝŶĂƌĞ

ϰϵ ϱϬ

dĞƌĂƚŽŐĞŶŝĨŝnjŝĐŝ dĞƌĂƚŽŐĞŶŝĨŝnjŝĐŝ
ZĂĚŝĂƜŝŝůĞŝŽŶŝnjĂŶƚĞ &ĞƚŽƉĂƚŝĂ ĂŵŶŝŽƚŝĐĉ
‡ ƚąƚƌĂĚŝĂƜŝŝůĞŠŶƐĐŽƉĚŝĂŐŶŽƐƚŝĐ͕ĐąƚƕŝƚĞƌĂƉĞƵƚŝĐʹ ŶƵƐƵŶƚŝŶĚŝĐĂƚĞĨĞŵĞŝůŽƌŐƌĂǀŝĚĞ ‡ ĂƵnjĞ͗ƉƌĞƐŝƵŶĞĐƌĞƐĐƵƚĉƐĂƵƕŽĐƵƌŝŵĞĐĂŶŝĐĞĞdžĞƌĐŝƚĂƚĞĂƐƵƉƌĂƉƌŽĚƵƐƵůƵŝĚĞĐŽŶĐĞƉƜŝĞ͖
‡ /ƌĂĚŝĞƌĞĂƚĞƌĂƉĞƵƚŝĐĉʹ ŝŶĚŝĐĉŠŶƚƌĞƌƵƉĞƌĞĂƐĂƌĐŝŶŝŝ ƉƌĞƐŝƵŶĞĂŝŶƚƌĂƵƚĞƌŝŶĉƉŽĂƚĞĐƌĞƕƚĞĚŝŶĐĂƵnjĂƐĂƌĐŝŶŝŝŐĞŵĞůĂƌĞ͕ŵĂůƉŽnjŝƜŝĞŝĨĞƚĂůĞ͕ƚƵŵŽƌŝ
ŝŶƚƌĂĂďĚŽŵŝŶĂůĞ͕ĐŽŵƉƌĞƐŝƵŶĞĞdžƚĞƌŶĉ;ŚĂŝŶĞĞdžĐĞƐŝǀĚĞƐƚƌąŵƚĞͿ
‡ WŽƚƉƌŽĚƵĐĞƉŝĞƌĚĞƌŝƐƉŽŶƚĂŶĞĚĞƐĂƌĐŝŶĉ͕ƌĞƚĂƌĚŵĞŶƚĂů͕ŵŝĐƌŽĐĞĨĂůŝĞ͕ĂŶŽŵĂůŝŝǀŝƐĐĞƌĂůĞ
‡ ŽŶƐĞĐŝŶƜĞ͗ĂŵƉƵƚĂƜŝŝĂůĞŵĞŵďƌĞůŽƌ͕ƐŝŶĚĂĐƚŝůŝĞ͕ƐƚƌĂŶŐƵůĂƌĞĂƵŶƵŝĂŶƵŵŝƚƐĞŐŵĞŶƚĚĞĐŽƌƉ

,ŝƉĞƌƚĞƌŵŝĂŵĂƚĞƌŶĉ;јϯϵΣͿ
‡ WĞƉĞƌŝŽĂĚĞůƵŶŐŝĚĞƚŝŵƉʹ ŠŶƉƌŝŵĞůĞϭϰƐĉƉƚĉŵąŶŝĂƌĞĞĨĞĐƚƚĞƌĂƚŽŐĞŶ
‡ WŽĂƚĞĐŽŶĚƵĐĞůĂƌĞƚĂƌĚĚĞĐƌĞƕƚĞƌĞƉƌĞŶĂƚĂů͕ŵŝĐƌŽĐĞĨĂůŝĞ͕ƚƵůďƵƌĉƌŝŶĞƵƌŽůŽŐŝĐĞŐƌĂǀĞ͕ƌĞƚĂƌĚ
ŵĞŶƚĂů

ϱϭ ϱϮ
dĞƌĂƚŽŐĞŶŝŝŶĨĞĐƜŝŽƕŝ dĞƌĂƚŽŐĞŶŝŝŶĨĞĐƜŝŽƕŝ
dŽdžŽƉůĂƐŵĂŐŽŶĚŝŝ sĂƌŝĐĞůĂ
‡ /ŶĐŝĚĞŶƜĉϭ͗ϱϬϬϬEE ‡ ŽŶƐĞĐŝŶƜĞ͗ƌĞƚĂƌĚƐŽŵĂƚŝĐ͕ĂƉĂƌŝƜŝĂĚĞĐŝĐĂƚƌŝĐŝĐŚĞůŽŝĚĞ͕ƚƵůďƵƌĉƌŝŶĞƵƌŽůŽŐŝĐĞ͕ƉĂƌĂůŝnjŝŝ͕ĐŽŶǀƵůƐŝŝ͕
ĂƚƌŽĨŝĂŶĞƌǀƵůƵŝŽƉƚŝĐ͕ƌĞƚĂƌĚŵĞŶƚĂů
‡ ^ƵƌƐĂĚĞŝŶĨĞĐƜŝĞ͗ĂŶŝŵĂůĞůĞĚĞĐĂƐĉ;ĐąŝŶŝ͕ƉŝƐŝĐŝ͕ĂŶŝŵĂůĞĚĞĐƵƌƚĞͿƐĂƵĐŽŶƐƵŵƵůĚĞĐĂƌŶĞĚĞ
ƉŽƌĐŝŶĨĞƐƚĂƚĉƕŝƚƌĂƚĂƚĉŝŶƐƵĨŝĐŝĞŶƚƚĞƌŵŝĐ
‡ ŽŶƐĞĐŝŶƜĞ͗ƉŝĞƌĚĞƌĞĂƐƉŽŶƚĂŶĉĂƐĂƌĐŝŶŝŝ͕ŚŝĚƌŽĐĞĨĂůŝĞ͕ƚƵůďƵƌĉƌŝŶĞƵƌŽůŽŐŝĐĞ͕ŐůĂƵĐŽŵ͕ĐĂƚĂƌĂĐƚĉ͕ sŝƌƵƐƵůĐŝƚŽŵĞŐĂůŝĐ
ŝĐƚĞƌ͕ŚĞƉĂƚŽͲƐƉůĞŶŽŵĞŐĂůŝĞ͕ƌĞƚĂƌĚƉƐŝŚŽͲŵŽƚŽƌ͕ƐĞƉƚŝĐĞŵŝĞ ‡ ZŝƐĐϰϬй
‡ ŽŶƐĞĐŝŶƜĞ͗ƉŝĞƌĚĞƌĞĂƐĂƌĐŝŶŝŝ͕ĐĂƌĚŝŽƉĂƚŝŝ͕ĂĨĞĐƜŝƵŶŝŽĐƵůĂƌĞ͕ƚƵůďƵƌĉƌŝŶĞƵƌŽůŽŐŝĐĞ
‡ /ŶĨĞĐƚĂƌĞĂŠŶƚŝŵƉƵůŶĂƕƚĞƌŝŝƉŽĂƚĞĐŽŶĚƵĐĞůĂ͗ƐĞƉƚŝĐĞŵŝĞ͕ƉŶĞƵŵŽŶŝĞ͕ĞŶĐĞĨĂůŝƚĉ
ZƵďĞŽůĂ
‡ ZŝƐĐϴϱйŠŶƉƌŝŵĞůĞϮůƵŶŝĚĞƐĂƌĐŝŶĉ
dƌĞƉŽŶĞŵĂƉĂůůŝĚƵŵ ;ůƵĞƐĐŽŶŐĞŶŝƚĂůͿ
‡ ŽŶƐĞĐŝŶƜĞ͗ƌĞƚĂƌĚŵĞŶƚĂů͕ĂŶŽŵĂůŝŝs͕ŐůĂƵĐŽŵ͕ƐƵƌĚŝƚĂƚĞ͕ŚĞƉĂƚŽͲƐƉůĞŶŽŵĞŐĂůŝĞ͕ĂŶŽŵĂůŝŝ
ŽƐŽĂƐĞ ‡ /ĐƚĞƌ͕ŚĞƉĂƚŝƚĉ͕ĂĨĞĐƚĂƌĞŚĞŵĂƚŽůŽŐŝĐĉ͕ƌĞŶĂůĉ͕ŶĞƵƌŽůŽŐŝĐĉ͕ŽĐƵůĂƌĉ͕ĐƵƚĂŶĂƚĉ;ƐŝĨŝůŝĚĞĐƵƐĞĐƌĞƜŝĞ
ƉƵƌƵůĞŶƚĉͿ͕ƐƵƌĚŝƚĂƚĞ͕ĂƚƌŽĨŝĞŽƉƚŝĐĉ
‡ WƌŽĨŝůĂdžŝĞ͗ǀĂĐĐŝŶĂƌĞĂŶƚŝͲƌƵďĞŽůŝĐĉ

ϱϯ ϱϰ

&ĂĐƚŽƌŝŵĞƚĂďŽůŝĐŝ
ŝĂďĞƚƵůŵĂƚĞƌŶ
‡ ĂƵnjĂ͗ŐůŝĐĞŵŝĂŝŶĐŽƌĞĐƚĐŽŶƚƌŽůĂƚĉ
‡ ŽŶƐĞĐŝŶƜĞ͗ĂŶŽŵĂůŝŝĐŽŶŐĞŶŝƚĂůĞ͕ƉƌŽĚƵƐƵůĚĞĐŽŶĐĞƉƜŝĞĞƐƚĞƐƵƉƌĂƉŽŶĚĞƌĂůĚĞůĂŶĂƕƚĞƌĞ͕
ƉŝĞƌĚĞƌĞƐƉŽŶƚĂŶĉĂƐĂƌĐŝŶŝŝ͕ĂŶŽŵĂůŝŝĐĞƌĞďƌĂůĞ͕ĐĂƌĚŝĂĐĞ͕ŐĞŶŝƚĂůĞ͕ŽƐŽĂƐĞ͕ŵĞƚĂďŽůŝĐĞ
‡ WƌŽĨŝůĂdžŝĂ͗ĐŽŶƚƌŽůƵůƐƚƌŝĐƚĂůŐůŝĐĞŵŝĞŝƕŝĐŽƌĞĐƚĂƌĞĂƚƵůďƵƌĉƌŝůŽƌŵĞƚĂďŽůŝĐĞƉĞƉĞƌŝŽĂĚĂƐĂƌĐŝŶŝŝ
ŽŶĐĞƉƚĞĨƵŶĚĂŵĞŶƚĂůĞ;ϭͿ
‡ &ĂĐƚŽƌŝŝƚĞƌĂƚŽŐĞŶŝƐƵŶƚĂŐĞŶƜŝĚŝŶŵĞĚŝƵůŠŶĐŽŶũƵƌĉƚŽƌĐĂƌĞŝŶƚĞƌĨĞƌĞĂnjĉ
ĐƵĚĞnjǀŽůƚĂƌĞĂŝŶƚƌĂƵƚĞƌŝŶĉĂƉƌŽĚƵƕŝůŽƌĚĞĐŽŶĐĞƉƜŝĞ ƕŝŝŶĚƵĐĂŶŽŵĂůŝŝ ĚĞ
ĚĞnjǀŽůƚĂƌĞůĂŶĂƕƚĞƌĞ
‡ WƌŽĨŝůĂdžŝĂ ĞƐƚĞŝŵƉŽƌƚĂŶƚĉŠŶĞǀŝƚĂƌĞĂĨĂĐƚŽƌŝůŽƌƚĞƌĂƚŽŐĞŶŝ

&ĞŶŝůĐĞƚŽŶƵƌŝĂ ŵĂƚĞƌŶĉ ‡ &ĂĐƚŽƌŝŝƚĞƌĂƚŽŐĞŶŝƉŽƚĨŝĐŚŝŵŝĐŝ͕ĨŝnjŝĐŝ͕ŝŶĨĞĐƜŝŽƕŝƕŝŵĞƚĂďŽůŝĐŝ

‡ ĂƵnjĂ͗ŶŝǀĞůĐƌĞƐĐƵƚĚĞĨĞŶŝůĂůĂŶŝŶĉ ŠŶƐąŶŐĞůĞŵĂƚĞƌŶ &ĞŶŝůĐĞƚŽŶƵƌŝĂ͕ĞƐƚĞŽďŽĂůĉŐĞŶĞƚŝĐĉ
ŐƌĂǀĉĚĞƚĞƌŵŝŶĂƚĉĚĞŽƚƵůďƵƌĂƌĞĂ
‡ ŽŶƐĞĐŝŶƜĞ͗ƉŝĞƌĚĞƌĞƐƉŽŶƚĂŶĉĂƐĂƌĐŝŶŝŝ͕ƉƌĞŵĂƚƵƌŝƚĂƚĞ͕
ƌĞƚĂƌĚŵĞŶƚĂů͕ŚŝƉĞƌĂĐƚŝǀŝƚĂƚĞ͕ƌĞƚĂƌĚĚĞĐƌĞƕƚĞƌĞ
ŵĞƚĂďŽůŝƐŵƵůƵŝĨĞŶŝůĂůĂŶŝŶĞŝ͖ĨĉƌĉƵŶ ‡ džĞŵƉůĞĚĞĨĂĐƚŽƌŝƚĞƌĂƚŽŐĞŶŝĐŚŝŵŝĐŝƐƵŶƚ͗ĂůĐŽŽůƵů͕ĂŶƚŝĐŽŶǀƵůƐŝǀĞůĞ
ƚƌĂƚĂŵĞŶƚĂĚĞĐǀĂƚĐŽŶĚƵĐĞůĂĂƉĂƌŝƜŝĂ
;ƚĞƌĂƉŝĂĞƉŝůĞƉƐŝĞŝͿ͕ĂŶƚŝĐŽĂŐƵůĂŶƚĞůĞ;ǁĂƌĨĂƌŝŶĂͿ͕ǀŝƚĂŵŝŶĂ͕ĂůĐŽŽůƵů͕
‡ WƌŽĨŝůĂdžŝĂ͗ƌĞĚƵĐĞƌĞĂĨĞŶŝůĂůĂŶŝŶĞŝ ƉƌŝŶĚŝĞƚĉ ĚĞŠŶƚąƌnjŝĞƌŝŵŝŶƚĂůĞ;ŽůŝŐŽĨƌĞŶŝĞ
ĐŽĐĂŝŶĂ
ĨĞŶŝůƉŝƌƵǀŝĐĉͿ
ϱϱ ϱϲ
ŽŶĐĞƉƚĞĨƵŶĚĂŵĞŶƚĂůĞ;ϮͿ
‡ džĞŵƉůĞĚĞĨĂĐƚŽƌŝƚĞƌĂƚŽŐĞŶŝĨŝnjŝĐŝƐƵŶƚ͗ƌĂĚŝĂƜŝŝůĞŝŽŶŝnjĂŶƚĞ͕
ƉƌĞƐŝƵŶĞĂŝŶƚƌĂƵƚĞƌŝŶĉĐƌĞƐĐƵƚĉ

‡ džĞŵƉůĞĚĞĨĂĐƚŽƌŝƚĞƌĂƚŽŐĞŶŝŝŶĨĞĐƜŝŽƕŝ͗ǀŝƌƵƐƵůƌƵďĞŽůĞŝ͕ǀŝƌƐƵůƵŝ ϯ͘ůĞŵĞŶƚĞĚĞƉƌŽĨŝůĂdžŝĞĂďŽůŝůŽƌ
ǀĂƌŝĐĞůĞŝ͕dƌĞƉŽŶĞŵĂƉĂůůŝĚƵŵ͕dŽdžŽƉůĂƐŵĂŐŽŶĚŝŝ
ŐĞŶĞƚŝĐĞ
‡ džĞŵƉůĞĚĞĨĂĐƚŽƌŝŵĞƚĂďŽůŝĐŝ͗ĚŝĂďĞƚƵůnjĂŚĂƌĂƚŵĂƚĞƌŶ͕
ĨĞŶŝůĐĞƚŽŶƵƌŝĂ ŵĂƚĞƌŶĉ

ϱϳ ϱϴ

ůĞŵĞŶƚĞĚĞƉƌŽĨŝůĂdžŝĞĂďŽůŝůŽƌĞƌĞĚŝƚĂƌĞ dŝƉƵƌŝĚĞƉƌŽĨŝůĂdžŝĞ
‡ dŝƉƵƌŝĚĞƉƌŽĨŝůĂdžŝĞ ‰WƌŽĨŝůĂdžŝĂƉƌŝŵĂƌĉʹ ƉƌĞǀĞŶŝƌĞĂĂƉĂƌŝƜŝĞŝďŽůŝŝůĂƵŶŽƌŐĂŶŝƐŵĐĂƌĞŶƵ
‡ WƌŝŵĂƌĉ ĂĨŽƐƚĂĨĞĐƚĂƚĚĞďŽĂůĉ
‡ ^ĞĐƵŶĚĂƌĉ ‡ ƉƌĞǀĞŶŝƌĞĂĂƉĂƌŝƜŝĞŝŵƵƚĂƜŝŝůŽƌ
‡ ĞǀŝƚĂƌĞĂĨĂĐƚŽƌŝůŽƌŵƵƚĂŐĞŶŝ
‡ ƌĞĚƵĐĞƌĞĂǀąƌƐƚĞŝĚĞƌĞƉƌŽĚƵĐĞƌĞ
‡ ƉƌĞǀĞŶŝƌĞĂƚƌĂŶƐŵŝƚĞƌŝŝŵƵƚĂƜŝŝůŽƌ
‡ ^ĐƌĞĞŶŝŶŐͲƵů ‡ ĚĞƉŝƐƚĂƌĞĂŚĞƚĞƌŽnjŝŐŽƜŝůŽƌ
‡ WŽƉƵůĂƜŝŽŶĂů ‡ ƌĞĚƵĐĞƌĞĂĐŽŶƐĂŶŐǀŝŶŝnjĉƌŝŝ
‡ &ĂŵŝůŝĂů ‡ ƐĨĂƚŐĞŶĞƚŝĐ
‡ ƉƌĞǀĞŶŝƌĞĂĂƉĂƌŝƜŝĞŝďŽůŝŝ
‡ ŝĚĞŶƚŝĨŝĐĂƌĞĂŝŶĚŝǀŝnjŝůŽƌĐƵƉƌĞĚŝƐƉŽnjŝƜŝĞŐĞŶĞƚŝĐĉ
‡ ŠŶĚĞƉĉƌƚĂƌĞĂĨĂĐƚŽƌŝůŽƌĐĂƌĞƉŽƚƚƌĂŶƐĨŽƌŵĂƉƌĞĚŝƐƉŽnjŝƜŝĂŠŶďŽĂůĉ

ϱϵ ϲϬ
dŝƉƵƌŝĚĞƉƌŽĨŝůĂdžŝĞ
‰WƌŽĨŝůĂdžŝĂƐĞĐƵŶĚĂƌĉʹ ƉƌĞǀĞŶŝƌĞĂƌĞĐƵƌĞŶƜĞŝůĂƵŶŽƌŐĂŶŝƐŵĂĨĞĐƚĂƚ
ĂŶƚĞƌŝŽƌĚĞďŽĂůĉ
‡ ĚĞƉŝƐƚĂƌĞĂƉƌĞĐŽĐĞĂďŽůŝŝƕŝƉƌĞǀĞŶŝƌĞĂĐŽŵƉůŝĐĂƜŝŝůŽƌ
‡ ƐĐƌĞĞŶŝŶŐƉƌĞŶĂƚĂů
‡ ƉƌĞǀĞŶŝƌĞĂŵĂŶŝĨĞƐƚĉƌŝŝďŽůŝůŽƌŐĞŶĞƚŝĐĞůĂĐŽƉŝŝĐƵďŽůŝŐĞŶĞƚŝĐĞ
‡ ƐĐƌĞĞŶŝŶŐŶĞŽŶĂƚĂů
dŝƉƵƌŝĚĞƐĐƌĞĞŶŝŶŐ
‡ WŽƉƵůĂƜŝŽŶĂů
‡ WƌĞŶĂƚĂů ʹ ƉƌĞǀĞŶŝƌĞĂŶĂƕƚĞƌŝŝƵŶƵŝ
ŝŶĚŝǀŝĚĐƵŐĞŶŽƚŝƉĂŶŽƌŵĂů
‡ EĞŽŶĂƚĂů ʹ ƉƌĞǀĞŶŝƌĞĂŵĂŶŝĨĞƐƚĉƌŝůŽƌ
ďŽůŝůŽƌŐĞŶĞƚŝĐĞƐĂƵĂůĞĐŽŵƉůŝĐĂƜŝŝůŽƌ
;Ğdž͗ĨĞŶŝůĐĞƚŽŶƵƌŝĞ͕ŚŝƉŽƚŝƌŽŝĚŝƐŵͿ
‡ ůŚĞƚĞƌŽnjŝŐŽƜŝůŽƌ ʹ ŝĚĞŶƚŝĨŝĐĂƌĞĂ
ƉƵƌƚĉƚŽƌŝůŽƌĚĞŐĞŶĞŵƵƚĂŶƚĞ;Ğdž͗
ƐŝĐůĞŵŝĞ͕ƚĂůĂƐĞŵŝĞ͕ĨŝďƌŽnjĂĐŚŝƐƚŝĐĉͿ
‡ &ĂŵŝůŝĂů ʹ ƐĞĂĚƌĞƐĞĂnjĉĨĂŵŝůŝŝůŽƌĐƵŽ
ďŽĂůĉŐĞŶĞƚŝĐĉƐĂƵĐƵŽƉƌĞĚŝƐƉŽnjŝƜŝĞ
ĐĉƚƌĞŽďŽĂůĉŐĞŶĞƚŝĐĉ
ϲϭ
ǁǁǁ͘ŐŽǀ͘ƵŬ
&ŝďƌŽnjĂĐŚŝƐƚŝĐĉ;ŵƵĐŽǀŝƐĐŝĚŽnjĂͿ͕ĞƐƚĞ
ƉƌŽǀŽĐĂƚĉĚĞŵƵƚĂƜŝĂŐĞŶĞŝ&dZ;ĐLJƐƚŝĐ
ĨŝďƌŽƐŝƐ ƚƌĂŶƐŵĞŵďƌĂŶĞ ĐŽŶĚƵĐƚĂŶĐĞ
ƌĞŐƵůĂƚŽƌLJͿĐĞĐŽĚŝĨŝĐĉƉƌŽƚĞŝŶĞĂůĞ
ĐĂŶĂůĞůŽƌĚĞĐůŽƌĚŝŶŵĞŵďƌĂŶĂ
ĐĞůƵůĞůŽƌĞƉŝƚĞůŝĂůĞ͘ƌĞůŽĐŠŶŐƌŽƕĂƌĞĂ
ƐĞĐƌĞƜŝŝůŽƌĂƐŽĐŝĂƚĉĐƵŝŶĨůĂŵĂƜŝĞĐƌŽŶŝĐĉ
ƕŝŝŶĨĞĐƜŝŝƌĞƉĞƚĂƚĞ͘WŽĂƚĞĐŽŶĚƵĐĞůĂ
ĚŝƐƚƌƵŐĞƌĞĂƜĞƐƵƚƵůƵŝƉƵůŵŽŶĂƌƕŝ
ŝŶƐƵĨŝĐŝĞŶƜĉƉƵůŵŽŶĂƌĉƵƌŵĂƚĉĚĞ
ĚĞĐĞƐ͘
dĂůĂƐĞŵŝĞ͕ĂŶĞŵŝĞĞƌĞĚŝƚĂƌĉŐƌĂǀĉ
ĐĂƵnjĂƚĉĚĞƉƌĞnjĞŶƜĂŚĞŵŽŐůŽďŝŶĞŝĨĞƚĂůĞ
ŠŶƐąŶŐĞ
ϲϯ
^ĐƌĞĞŶŝŶŐ
сŝĚĞŶƚŝĨŝĐĂƌĞĂƉƌĞĐŽĐĞĂƵŶƵŝŐĞŶŽƚŝƉĂŶŽƌŵĂůƐĂƵĂƵŶĞŝďŽůŝŐĞŶĞƚŝĐĞ͕
ĂƐƚĨĞůŠŶĐąƚƉƌŽĐĞƐĞůĞƉĂƚŽůŽŐŝĐĞƐĉƉŽĂƚĉĨŝƉƌĞǀĞŶŝƚĞ͕ŝĂƌƉĞƌƐŽĂŶĞůĞ
ŝŵƉůŝĐĂƚĞƐĉƉŽĂƚĉůƵĂŽĚĞĐŝnjŝĞŝŶĨŽƌŵĂƚĉ
ϲϮ
^ĐƌĞĞŶŝŶŐƉƌĞŶĂƚĂůʹ ƉƌŝŵƵůƚƌŝŵĞƐƚƌƵĚĞ
ƐĂƌĐŝŶĉ
‡ 2ŶƚƌĞƐĉƉƚĉŵąŶŝůĞϵƕŝϭϯ
‡ ŝƚĞƐƚƵůͬƚĞƐƚƵůĚƵďůƵʹ ŵĂƌŬĞƌŝ ďŝŽĐŚŝŵŝĐŝ͗
‡ WƌŽƚĞŝŶĂƉůĂƐŵĂƚŝĐĉ;WWWͲͬƉƌĞŐŶĂŶĐLJ
ĂƐƐŽĐŝĂƚĞ ƉůĂƐŵĂƉƌŽƚĞŝŶ ͿĂƐŽĐŝĂƚĉƐĂƌĐŝŶŝŝ dĞƐƚƵůĐŽŵďŝŶĂƚ
‡ 'ŽŶĂƚŽĚƌŽƉŝŶĂ ĐŽƌŝŽŶŝĐĉ ƵŵĂŶĉ;ŚŽƌŵŽŶ
ƉƌŽĚƵƐĚĞƉůĂĐĞŶƚĉĚƵƉĉŝŵƉůĂŶƚĂƌĞͿ
‡ hůƚƌĂƐŽŶŽŐƌĂĨŝĞ
ϲϰ
 ^ĐƌĞĞŶŝŶŐƉƌĞŶĂƚĂůʹ ĂůĚŽŝůĞĂƚƌŝŵĞƐƚƌƵĚĞ
/ŶƚĞƌƉƌĞƚĂƌĞƌĞnjƵůƚĂƚĞ
ƐĂƌĐŝŶĉ
‡ 2ŶƚƌĞƐĉƉƚĉŵąŶŝůĞϭϰƕŝϮϭ;ϭϲƕŝϭϴʹ ŽƉƚŝŵͿ
dĞƐƚƵůĐŽŵďŝŶĂƚ
‡ dƌŝƉůƵůƚĞƐƚ
WWWͲ DĂůĂĚŝŝĂƐŽĐŝĂƚĞ ‡ ɲͲ&ĞƚŽƉƌŽƚĞŝŶĂ ;&W͕ƉƌŽƚĞŝŶĉĨĞƚĂůĉĚĞƚĞĐƚĂƚĉŠŶ
^ĐĉnjƵƚ ^ŝŶĚƌŽŵŽǁŶ ;ƚƌŝƐŽŵŝĂϮϭͿ ƐąŶŐĞůĞŵĂƚĞƌŶͿ
нƵůƚƌĂƐŽŶŽŐƌĂĨŝĞ
dĞƐƚƵůŝŶƚĞŐƌĂƚ
;ϭͲϭϰ ƐĉƉƚͿ ^ŝŶĚƌŽŵ ĚǁĂƌĚ ;ƚƌŝƐŽŵŝĂϭϴͿ ‡ ƐƚƌŝŽů;ϯͿŶĞĐŽŶũƵŐĂƚ
‡ 'ŽŶĂĚŽƚƌŽƉŝŶĂ ĐŽƌŝŽŶŝĐĉ ƵŵĂŶĉ
‡ YƵĂĚƌƵƉůƵůƚĞƐƚ
Ś' DĂůĂĚŝŝĂƐŽĐŝĂƚĞ ‡ ɲͲ&ĞƚŽƉƌŽƚĞŝŶĂ
ƌĞƐĐƵƚ ^ŝŶĚƌŽŵŽǁŶ ;ƚƌŝƐŽŵŝĂϮϭͿ ‡ ƐƚƌŝŽů;ϯͿŶĞĐŽŶũƵŐĂƚ
‡ 'ŽŶĂĚŽƚƌŽƉŝŶĂ ĐŽƌŝŽŶŝĐĉ ƵŵĂŶĉ
‡ /ŶŚŝďŝŶĂ ĚŝŵĞƌŝĐĉ ;ŚŽƌŵŽŶƐĞĐƌĞƚĂƚĚĞĐŽƌƉƵů
ŐĂůďĞŶƕŝƉůĂĐĞŶƚĉŠŶƚŝŵƉƵůƐĂƌĐŝŶŝŝͿ
ϲϱ ϲϲ
ϮϬϭϯ͕^^ŚŝĞĨĂ͕/ŶĚ :ůŝŶ ŝŽĐŚĞŵ ;:ĂŶͲDĂƌϮϬϭϯͿϮϴ;ϭͿ͗ϯʹϭϮ

/ŶƚĞƌƉƌĞƚĂƌĞĂƌĞnjƵůƚĂƚĞůŽƌ ŽŶĐĞƉƚĞĨƵŶĚĂŵĞŶƚĂůĞ;ϭͿ
dƌŝƉůƵůƚĞƐƚ ‡ WƌŽĨŝůĂdžŝĂƉƌŝŵĂƌĉĂďŽůŝůŽƌŐĞŶĞƚŝĐĞĐŽŶƐƚĉŠŶ͗
&W ϯŶĞĐŽŶũƵŐĂƚ Ś' DĂůĂĚŝŝĂƐŽĐŝĂƚĞ 9ƉƌĞǀĞŶŝƌĞĂĂƉĂƌŝƜŝĞŝŵƵƚĂƜŝŝůŽƌ
^ĐĉnjƵƚ ^ĐĉnjƵƚ ƌĞƐĐƵƚ ^ŝŶĚƌŽŵŽǁŶ ;ƚƌŝƐŽŵŝĂϮϭͿ 9ƉƌĞǀĞŶŝƌĞĂƚƌĂŶƐŵŝƚĞƌŝŝŵƵƚĂƜŝŝůŽƌ
^ĐĉnjƵƚ ^ĐĉnjƵƚ ^ĐĉnjƵƚ ^ŝŶĚƌŽŵĚƵĂƌĚ;ƚƌŝƐŽŵŝĂϭϴͿ 9ƉƌĞǀĞŶŝƌĞĂĂƉĂƌŝƜŝĞŝďŽůŝůŽƌĞƌĞĚŝƚĂƌĞ
ƌĞƐĐƵƚ E E ĞĨĞĐƚĞĂůƚĞƚƵďƵůƵŝŶĞƵƌĂů

‡ WƌŽĨŝůĂdžŝĂƐĞĐƵŶĚĂƌĉĂďŽůŝůŽƌŐĞŶĞƚŝĐĞĐŽŶƐƚĉŠŶ͗
YƵĂĚƌƵƉůƵůƚĞƐƚ 9ĚĞƉŝƐƚĂƌĞĂƉƌĞĐŽĐĞĂďŽůŝŝƕŝƉƌĞǀĞŶŝƌĞĂĐŽŵƉůŝĐĂƜŝŝůŽƌ
/ŶŚŝďŝŶĂ  DĂůĂĚŝŝĂƐŽĐŝĂƚĞ 9ƉƌĞǀĞŶŝƌĞĂŵĂŶŝĨĞƐƚĉƌŝŝďŽůŝŝ
ƌĞƐĐƵƚ ^ŝŶĚƌŽŵŽǁŶ ;ƚƌŝƐŽŵŝĂϮϭͿ
^ĐĉnjƵƚ ^ŝŶĚƌŽŵĚƵĂƌĚ;ƚƌŝƐŽŵŝĂϭϴͿ

ϲϳ ϲϴ
ŽŶĐĞƉƚĞĨƵŶĚĂŵĞŶƚĂůĞ;ϮͿ
‡ ^ĐƌĞĞŶŝŶŐƵů ƌĞƉƌĞnjŝŶƚĉ ŝĚĞŶƚŝĨŝĐĂƌĞĂƉƌĞĐŽĐĞĂƵŶƵŝŐĞŶŽƚŝƉĂŶŽƌŵĂů
ƐĂƵĂƵŶĞŝďŽůŝŐĞŶĞƚŝĐĞ͕ĂƐƚĨĞůŠŶĐąƚƉƌŽĐĞƐĞůĞƉĂƚŽůŽŐŝĐĞƐĉƉŽĂƚĞĨŝ
ƉƌĞǀĞŶŝƚĞ͕ŝĂƌƉĞƌƐŽĂŶĞůĞŝŵƉůŝĐĂƚĞƐĉƉŽĂƚĉůƵĂŽĚĞĐŝnjŝĞŝŶĨŽƌŵĂƚĉ
‡ ^ĐƌĞĞŶŝŶŐƉŽƉƵůĂƜŝŽŶĂůƉŽĂƚĞĨŝƉƌĞŶĂƚĂů͕ŶĞŽŶĂƚĂůƐĂƵĂů
ŚĞƚĞƌŽnjŝŐŽƜŝůŽƌ
‡ ^ĐƌĞĞŶŝŶŐĨĂŵŝůŝĂůƐĞĂĚƌĞƐĞĂnjĉĨĂŵŝůŝŝůŽƌĐƵďŽůŝŐĞŶĞƚŝĐĞ
ϲϵ
ŽŶĐĞƉƚĞĨƵŶĚĂŵĞŶƚĂůĞ;ϯͿ
‡ ^ĐƌĞĞŶŝŶŐƉƌĞŶĂƚĂůŠŶƉƌŝŵƵůƐĞŵĞƐƚƌƵĚĞƐĂƌĐŝŶĉĞƐƚĞƌĞƉƌĞnjĞŶƚĂƚĚĞ
ƚĞƐƚƵůĐŽŵďŝŶĂƚͬĚƵďůƵůƚĞƐƚ͗WWWͲ͕Ś'͖ŶŝǀĞůƵůƐĐĉnjƵƚĚĞWWWͲƕŝ
ŶŝǀĞůƵůĐƌĞƐĐƵƚĚĞŚ' ƐƵŶƚĂƐŽĐŝĂƚĞĐƵƚƌŝƐŽŵŝĂϮϭ
‡ ^ĐƌĞĞŶŝŶŐƉƌĞŶĂƚĂůŠŶĂůĚŽŝůĞĂƚƌŝŵĞƐƚƌƵĚĞƐĂƌĐŝŶĉĞƐƚĞƌĞƉƌĞnjĞŶƚĂƚ
ĚĞ͗
‡ dĞƐƚƵůŝŶƚĞŐƌĂƚͬ ƚƌŝƉƵů ƚĞƐƚ͗&W͕ϯŶĞĐŽŶũƵŐĂƚ͕Ś'
‡ YƵĂĚƌƵƉůƵůƚĞƐƚ͗ƐĞĂĚĂƵŐĉŝŶŚŝďŝŶĂ 
ϳϬ