Mini Re iew
Volume 3 Issue 4 - Janua y 2018
DOI: 10.19080/NAPDD.2018.03.555617
No App o D ug Des De
Copy igh © All igh s a e ese ed by Ca lo a Saldanha
E y h ocy e Ace ylcholines e ase is a Signaling
Recep o
Ca lo a Saldanha* and Ana Sil a He dade
Ins i u e o Biochemis y, Ins i u e o Molecula Medicine, Facul y o Medicine, Uni e si y o Lisbon, Po ugal
Recei ed Da e: Janua y 12, 2018; Published Da e: Janua y 23, 2018
*Co esponding au ho : Ca lo a Saldanha, Ins i u e o Biochemis y, Ins i u e o Molecula Medicine, Facul y o Medicine, Uni e si y o Lisbon,
Po ugal, Email:
Mini Re iew
The need o cha ac e ize he composi ion, s uc u e and
unc ion o he ed blood cells (RBCs) memb ane, he p esence
o he ace ylcholines e ase (AChE) enzyme, which kine ically
esembled he b ain es e ase, bu ma kedly di e en om
he cholines e ase ound in se um was e idenced [1]. Human
e y h ocy e AChE enzyme ac i i y is he highes one in he
mammalian scale [2,3]. The p esence o AChE a away o he
neu onal sys em aises ques ions abou i s unc ion beyond he
enzyme ac ion. He zand Kaplan in hei e ision pape poin ed
ha a e in i o hemolysis, ace ylcholines e ase ac i i y can be
eco e ed in he e y h ocy e memb ane [4]. Also he endency o
couple ion anspo mechanisms ac oss e y h ocy e memb ane
wi h AChE was explo ed wi h ailu e [4]. La e was epo ed
AChE enzyme ac i i y as a bioma ke o human ed blood
cell aging and o RBC memb ane in eg i y [5,6]. The blood o
heal hy humans p esen s a wide a ie y o a g adien scale wi h
di e en aged classes om olde o younge e y h ocy es each
one showing a iable amoun o RBCs esul ing om i s na u al
li e span o 120 days. Du ing his pe iod, exo esicles en iched
wi h AChE a e eleased om e y h ocy e memb ane o blood
ci cula ion es ing he olde e y h ocy e wi h lowe enzyme
ac i i y [5,7,8]. Conside ing gende he human emale has highe
AChE enzyme ac i i y han he men ma ched in age [9].
Highe human e y h ocy e AChE enzyme ac i i y we e
e i ied in pa ien s wi h glaucoma, essen ial hype ension, and
ALS [10]. These pa hologies a e in lamma o y ascula diseases
cha ac e ized by high concen a ion in blood o in lamma o y
molecules, eac i e oxygen species, and eac i e ni ogen species,
[10]. The RBCs AChE enzyme ac i i y da a ob ained in hose
abo e men ioned ascula diseases ein o ce he Das`s a emen
o e y h ocy e AChE be conside ed a bioma ke o in lamma ion
[11]. Das e i ied also, ha in blood samples o in lamma o y
diseases, wi h high enzyme ac i i y o e y h ocy e AChE, lowe
plasma ace ylcholine (ACh) le els we e ob ained [11].
Ace ylcholine molecule ac ing in he pa asympa he ic
sys em and in he neu on muscula junc ion, was conside ed
as a neu o ansmi e du ing se e al decades. Fu he was
also ecognized also as an an i-in lamma o y agen h ough he
in e play be ween immune and neu onal sys ems, he nomina ed
“choline gic an i-in lamma o y pa hway” [12-14]. This consis s
in he ac i a ion o ad ene gic neu ons in he spleen ha libe a e
no - epineph ine nea he T cell capable o sec e e ace ylcholine.
This non-neu onal ACh ac s on α7 subuni –con aining nico inic
ace ylcholine ecep o s exp essed on mac ophages which a e
binding induce supp ession on he syn hesis and on he sec e ion
o in lamma o y cy okines. Mac ophages ac as an in e ace
be ween he b ain and he immune sys em [15]. The ac i a ion
o a e en agus ne e by endo oxin o p o-in lamma o y
cy okines s imula es hypo halamic-pi ui a y-ad enal an i-
in lamma o y esponses conduc ed by he e e en agus ne e.
No App o D ug Des De 3(4): NAPDD.MS.ID.555617 (2018) 001
Abs ac
The ace ylcholines e ase (AChE), is an enzyme loca ed in he e y h ocy e memb ane ha mo i a e con inually ques ions, abou i s
physiological unc ion. The aim o his mini e iew is highligh he ecep o beha io o AChE in human ed blood cells o chemical signals
associa ed wi h ni ic oxide mobiliza ion and e lux om e y h ocy es. Da a om he expe imen al models used a e p esen ed. Consequen ly, key
pa icipa i e p o eins in he s eps o he signal ansduc ion pa hways, in dependence o AChE ecep o , a e sugges ed o be u he conside ed
as po en ial he apeu ic a ge s.
Keywo ds: E y h ocy e; Ace ylcholines e ase; signaling ; Ace ylcholine; Recep o
Abb e ia ions: RBCs: Red Blood Cells; AChE: Ace ylcholines e ase; ACh: Ace ylcholine; AC: Adenylyl cyclase; cAMP: cyclic Adenosine
Monophospha e (cAMP)
How o ci e his a icle: Ca lo a Saldanha and Ana Sil a He dades. E y h ocy e Ace ylcholines e ase is a Signaling Recep o . No App o D ug Des De
2018; 3(4) : 555617 DOI: 10.19080/NAPDD.2018.03.555617.
002
No el App oaches in D ug Designing & De elopmen
Back o e y h ocy e memb ane enzyme, AChE has he
pa icula i y o be inhibi ed by high concen a ions o ACh
meaning by i s own na u al subs a e, [10,16]. So, di e en ypes
o enzyme complexes may be p esen ed namely, ac i e, inac i e
and less ac i e ones acco ding he amoun o ACh exis en in
he expe imen al medium [16,17]. The unusual AChE kine ic
beha io a non-neu onal en i onmen and he disco e o ni ic
oxide (NO) p oduced in he lumen o abbi ao ic endo helium
cells unde he p esence ACh, aised us he ques ion i he e is
NO inside e y h ocy es [18]. NO was obse ed, in e y h ocy es
wi h added ACh, by luo escence mic oscopy [19]. Human
e y h ocy e suspensions, in p esence o ACh, we e loaded wi h
he pe meable non luo escen p obe diamino luo esceine-2
diace a e (DAF-2Da). In a e y h ocy e luo escence in ensi y
o iazolo luo escein (DAF-2T) was isualized as a esul om
he eac ion be ween NO and he 4,5- diamino luo escein. We
concluded ha ACh, in a dose-dependen way, is able o induce
NO mobiliza ion inside he e y h ocy e [19].
Ace ylcholine is known o be p esen in human blood
ci cula ion being p oduced by T lymphocy es and endo helial
cells [20]. The ci cula ing ACh induces asodila ion o
asocons ic ion in dependence o in eg i y o he endo helium,
ia he NO syn hesized and eleased o smoo h muscle [21].
Also he NO eleased om endo helial cells can mo e o
he lumen o he essels whe e in he blood ci cula ion is
cap u ed by ee hemoglobin o sca enged by e y h ocy e
p esen [22]. The e y h ocy e NO-heme-hemoglobin adduc o
ni osylhemoglobin is o med a high issues oxygen ension,
o example, when deoxygena ed blood en e s in o he ascula
bed o pulmona y ci cula ion [23]. A e , NO is ans e ed o he
hiol g oup o cys eine β93 o Hb o ming S- ni osohemoglobin
(SNOHb) conside ed as a NO ese oi molecule [23]. Low issue
oxygen ension is pe cei ed by e y h ocy es wi h occu ence
o s uc u al allos e ic ansi ions in SNOHb which a o he
ans e o i s NO o he hiol g oup o band 3 p o ein, allowing
he NO e lux om e y h ocy es o he capilla ies sp eading
hose issues [24].
In in i o s udies wi h e y h ocy es ob ained om heal hy
dono s unde he p esence o ACh i was e idenced inc eased
o NO e lux, e y h ocy e de o mabili y (EE) and dec eased
e y h ocy e agg ega ion and hemoglobin oxygen a ini y [25].
O he in i o s udy wi h blood samples aken om pa ien s wi h
ascula diseases such as hype ension, hype choles e olemia
and kidney ansplan , showing lowe EE han heal hy pe sons
ha was amelio a e by he p esence o ACh [24]. A nega i e
associa ion be ween EE alues and NO e lux om e y h ocy es
was showed in blood samples o hose pa ien s [26].
The NO e lux measu emen s om e y h ocy es a e based
on he ACh signal ansduc ion pa hway h ough he AChE–
ACh ac i e enzyme complex ( ac i e con o ma ion), associa ed
wi h Gαip o ein, adenylyl cyclase (AC) inhibi ion , band 3
p o ein phospho yla ion by p o ein y osine kinase (PTK),
p o ein kinase C (PKC) ac i a ion, phosphodies e ase-3 (PDE3)
ac i a ion, and low le el o adenosine iphospha e (cAMP)
molecules [17,27-31]. Lowe NO e lux om e y h ocy es we e
ob ained in p esence o elnac ine and imolol, which a e s ong
and mode a e AChE inhibi o s espec i ely, [27,31,32].
E y h ocy e S-ni osoglu a hione (GSNO) is o he NO
ese oi molecule exis ing inside e y h ocy e bu able o
be sec e ed by he binding o ACh o imolol o AChE o ming
ac i e and less ac i e enzyme complex espec i ely [33]. A
a iance GSNO e lux om e y h ocy e is null in absence o AChE
subs a e o inhibi o [33]. Meanwhile, inside he e y h ocy es
he p esence o he inac i e complex AChE-VM showed highe
GSNO concen a ion in ela ion o he ac i e enzyme complex
AChE-ACh [27,31]. A opposi e GSNO le els inside e y h ocy es
we e lowe in p esence o he less ac i e enzyme complex AChE–
imolol han in he case o he ac i e enzyme complex AChE-ACh
[33,34]. F om all he da a ob ained in he abo e desc ibed s udies
we e idenced ha e y h ocy e AChE beyond i s enzyme ac i i y
unc ion as a ecep o o signal molecules able o induce escue
o NO e lux om human e y h ocy es and i s mobiliza ion om
ese oi molecules. The NO de i a i e molecules inside hem
as ni i e, ni a e and pe oxyni i e we e also quan i ied in all
men ioned s udies.
The key p o ein molecules pa icipan s in he chemical
signal ansduc ion mechanism o NO unde AChE ecep o
once iden i ied we e submi ed o inhibi o s o ac i a o s in in
i o models o hype ib inogenemia [35,36]. The acu e phase
p o ein ib inogen binds o RBCs memb ane CD47p o ein
pa ne o he Rh mac o-complex which is connec ed o he
cy oskele on ia p o ein 4.2, and in e ac s also wi h he majo
RBC mac o- -complex band 3 p o ein [37]. The p esence o
AChE-ACh and AChE- imolol complexes in RBCs induced he
e u n o no mal alues he highe NO e lux ob ained unde
high ib inogen concen a ion [35,38-40]. Consequen ly his NO
sca enged a oids he inc ease concen a ions o he eac i e
ni ogen and oxygen species p esen in in lamma o y condi ions.
Remembe ing ha he in lamma o y esponse is sys emically
associa ed wi h a iable deg ee o endo helium dys unc ion,
high plasma ACh and ib inogen concen a ions, he abo e da a
a e is acco dance wi h o he s ha conside ace ylcholine as an i-
in lamma o y agen . Ou s udies pe o med in i o wi h animal
models o in lamma ion demons a ed he an i-in lamma o y
unc ion o ACh [41].
The e y h ocy es ob ained om blood samples o pa ien s
wi h open glaucoma e idenced ex- i o highe NO e lux alues,
in e nal GSNO le els and AChE enzyme ac i i y alues, han hose
assessed in e y h ocy es o heal hy pe sons [34,42]. Wi h hese
da a we can unde s and he he apeu ic e icacy o o skolin
in glaucoma pa ien s [43]. Fo skolin is AC ac i a o , meaning
syn hesis o cAMP le els inside RBCs, ha e e ed he highe
NO e lux om e y h ocy es model o hype ib inogenemia [44].
Fib inogen binding o RBCs augmen ed he NO e lux unde lowe
le els o cAMP [36]. I will be necessa y o con i m in glaucoma
pa ien s unde o skolin he apy whe he no maliza ion o NO
How o ci e his a icle: Ca lo a Saldanha and Ana Sil a He dades. E y h ocy e Ace ylcholines e ase is a Signaling Recep o . No App o D ug Des De
2018; 3(4) : 555617 DOI: 10.19080/NAPDD.2018.03.555617.
003
No el App oaches in D ug Designing & De elopmen
e lux and lowe ni ogen eac i e species occu [34,44]. Also
NO dono s like ni oglyce in ac s in ea men and p e en ion o
endo helium dys unc ion [45].
Conclusion
In conclusion he p o eins he apeu ic a ge s o con ol he
RBCs bioa ailabili y in NO (e lux o sca enge) in in lamma o y
ascula disease a e AChE, AC, Gi , PTP , PTK, PKC, PI3K and
PD3 . Some ac i a o s and inhibi o s o hose p o eins a e
al eady disposal o examples ace ylcholine, elnac ine, imolol,
ad enaline, o skolin, alsa an, wo mannin de i a e and
ilop os .
Acknowledgmen
This wo k was unded by Fundação pa a a Ciência e Tecnologia:
LISBOA-01-0145-FEDER-007391, p ojec co undedby FEDER,
h ough POR Lisboa 2020 - P og ama Ope acional Regional de
Lisboa, Po ugal 2020. The au ho is also g a e ul o Emilia Al es
o ype-w i ing he e e ences.
Re e ences
1. Alles GA, Hawes RC (1940) Cholines e ases in he blood o man. J Biol
Chem 133: 375-390.
2. Zajicek J, Da a N (1953) In es iga ion o ace ylcholines e ase ac i i y
o e y h ocy es, pla ele s and plasma in di e en animal species.
Ac aHaema ol 9: 115-123.
3. Callahan JF, K uckenbe g SM (1967) E y h ocy e ace ylcholines e ase
ac i i y o domes ic and labo a o y animals: no mal le els o some
species. Am J Ve Res 28: 1509-1512.
4. F i z H, Kaplan E (1973) A Re iew: Human E y h ocy e
Ace ylcholines e ase Pedia . Res 7: 204-214.
5. P all YG, Gambhi KK, Ampy FR (1998) Ace ylcholines e ase: An
enzyma ic ma ke o human ed blood cell aging. Li e Sci 63: 177-184.
6. Aloni B, Li ne A (1974) Ace ycholines e ase as a p obe o e y h ocy e-
memb ane in ac ness. Biochim BiophysAc a 339: 359-366.
7. Bü iko e P, B odbeck U, O P (1987) Modula ion o e y h ocy e
esicula ion by amphiphilic d ugs. Biochim Biophys Ac a 901: 291-
295.
8. Saldanha C, San os NC, Ma ins e Sil a J (2004) A colo ime icp ocess
o isualize e y h ocy e exo esicles agg ega es. Biochem Mol Biol Educ
32: 250 -253.
9. Hilá io S, Saldanha C, Ma ins e Sil a J (2003) An in i o s udy o
ad enaline e ec on human e y h ocy e p ope ies in bo h gende . Clin
Hemo heol Mic oci c 28: 89-98.
10. Saldanha C (2017) Human E y h ocy e Ace ylcholines e ase in Heal h
and Disease. Molecules 22(9). pii: E1499.
11. Das UN (2007) Ace ylcholines e ase and bu y ylcholines e ase as
possible ma ke s o low-g ade sys emic in lamma ion. Med Sci Moni
13(12): 214-221.
12. Bo o iko a LV, I ano a S, Zhang M, Yang H, Bo chkina GI (2000) Vagus
ne e s imula ion a enua es he sys emic in lamma o y esponse o
endo oxin. Na u e 405: 458-462.
13. T acey KJ, Czu a CJ, I ano a S (2001) Mind o e immuni y. FASEB J.
15: 1575-1576.
14. T acey KJ (2002) The in lamma o y e lex. Na u e 420 (6917): 853-
859.
15. de Jonge WJ, an de Zanden EP, The FO, Bijlsma MF, an Wes e loo
DJ, e al. (2005) S imula ion o he agus ne e a enua es mac ophage
ac i a ion by ac i a ing he Jak2-STAT3 signaling pa hway. Na
Immunol 6(8): 844-851.
16. Saldanha C (1986) Ace ilcolines e ase-Con ibuição pa a o es udo
ciné ico da enzima e i oci á ia de homem. Ph.D. Thesis, Uni e sidade
No a de Lisboa, Lisboa, Po ugal.
17. Teixei a P, Du o N, Napoleão P, Saldanha C (2015) Ace ylcholines e ase
con o ma ional s a es in luence ni ic oxide mobiliza ion in
hee y h ocy e. J Memb. Biol 248(2): 349-354.
18. Igna o LJ, By ns RE, Buga GM, Wood KS (1987) Endo helium-
de i ed elaxing ac o om pulmona y a e y and ein possesses
pha macologic and chemical p ope ies iden ical o hose o ni ic
oxide adical. Ci c Res 61(6): 866-879.
19. Mesqui a R, Saldanha C, Ma ins-Sil a J (2000) Ace ylcholine induces
ni ic oxide p oduc ion by e y h ocy es in i o. Mee ing Repo :
Fi s In e na ional Con e ence Ni ic Oxide: Biology, Chemis y and
The apeu ic Applica ions o Ni ic Oxide, 3-7 June 2000. In: Ni ic
Oxide Biology and Chemis y 4: 313-314.
20. Fujii T, Yamada S, Yamaguchi N, Fujimo o K, Suzuki T, Kawashima K
(1995) Species di e ences in he concen a ion o ace ylcholine, a
neu o ansmi e , in whole blood and plasma. Neu osci Le 201(3):
207-210.
21. Fu chgo RF, Vanhou e PM (1989) Endo helium-de i ed elaxing and
con ac ing ac o s. FASEB J 3(9): 2007-2018.
22. Vaughn MW, Huang KT, Kuo L, Liao JC (2000) E y h ocy es possess
an in insic ba ie o ni ic oxide consump ion. J Biol Chem 275(4):
2342-2348.
23. Son eaux P, Lobyshe a II, Fe on O, McMahon TJ (2007) T anspo and
pe iphe al bioac i i ies o ni ogen oxides ca ied by ed blood cell
hemoglobin: ole in oxygen deli e y. Physiology (Be hesda) 22: 97-
112.
24. Jia L, Bona en u a C, Bona en u a J, S amle JS (1996)
Sni osohaemoglobin: a dynamic ac i i y o blood in ol ed in ascula
con ol. Na u e 380(6571): 221-226.
25. Mesqui a R, Pi es I, Saldanha C, Ma ins-Sil a J (2001) E ec s o
ace ylcholine and spe mineNONOa e on e y h ocy e hemo heologic
and oxygen ca ying p ope ies. Clin Hemo heol Mic oci 25(3-4):
153-163.
26. Ca alho FA, Ma ia AV, B az Noguei a JM, Gue a J, Ma ins-Sil a J,
e al. (2006) The ela ion be ween he e y h ocy e ni ic oxide and
hemo heological pa ame e s. Clin Hemo heol Mic oci c 35(1-2): 341-
347.
27. Ca alho FA, Lopes de Almeida JP, F ei as-San os T, Saldanha C (2009)
Modula ion o e y h ocy es ace ylcholines e ase ac i i y and i s
associa ion wi h G p o ein Band 3 in e ac ions. J Memb Biol 228(2):
89-97.
28. Almeida JP, Ca alho FA, Ma ins-Sil a J, Saldanha C (2008)
The modula ion o cyclic nucleo ide le els and PKC ac i i y by
ace ylcholines e ase e ec o s in human e y h ocy es. Ac aBioquímica
9: 111-114.
29. Low PS, Allen DP, Zioncheck TF, Cha i V, Willa dson BM, e al. (1987)
Ty osine phospho yla ion o band 3 inhibi s pe iphe al p o ein
binding. J Biol Chem 262(10): 4592-4596.
30. Shaku Y, Hols LS, Lands om TR, Mo sesian M, Dege man E,
e al. (2001) Regula ion and unc ion o he cyclic nucleo ide
phosphodies e ase (PDE3) gene amily. P og Nucleic Acid Res Mol Biol
66: 241-277.
31. Ca alho FA, Almeida JP, Fe nandes IO, F ei as-San os T, Saldanha
C (2008) Non-neu onal choline gic sys em and signal ansduc ion
How o ci e his a icle: Ca lo a Saldanha and Ana Sil a He dades. E y h ocy e Ace ylcholines e ase is a Signaling Recep o . No App o D ug Des De
2018; 3(4) : 555617 DOI: 10.19080/NAPDD.2018.03.555617.
004
No el App oaches in D ug Designing & De elopmen
pa hways media ed by band 3 in ed blood cells. Clin Hemo heol
Mic oci c 40(3): 207-227.
32. Saldanha C, Teixei a P, San os-F ei as T, Napoleão P (2013) Timolol
modula es e y h ocy e ni ic oxide bioa ailabili y. J Clin Exp Op almol
4:285.
33. Teixei a P, Napoleão P, Saldanha C (2015) S-ni osoglu a hione e lux in
he e y h ocy e. Clin Hemo heol Mic oci c 60(4):397-404.
34. Es e es R, F ei as T, Teixei a P, Napoleão P, Ne es C, e al. (2016)
E y h ocy eni ic oxide in glaucoma pa ien s - ex i o s udy. Clin
Hemo heol Mic oci c 64(4): 989-994
35. Lopes de Almeida JP, F ei as-San os T, Saldanha C (2011) E idence ha
he deg ee o band 3 phospho yla ion modula es human e y h ocy es
ni ic oxide e lux-in i o model o hype ib inogenemia. Clin Hemo h
Mic oc 49(1-4): 407-416.
36. Sil a-He dade AS, F ei as T, Almeida JP, Saldanha C (2016) Fib inogen
Signaling in E y h ocy e Ni ic oxide Mobiliza ion in P esence o PI3-K
and Adenylyl Ciclase Inhibi o s. Eu J Biom Pha m Science 3(4): 28-34.
37. De Oli ei a S, Vi o ino de Almeida V, Calado A, Saldanha C (2012)
In eg in- associa ed p o ein (CD47) is a pu a i e media o o soluble
ib inogen in e ac ion wi h human ed blood cells memb ane, Biochem
Biophys Ac a 1818(3): 481-490.
38. Saldanha C, F ei as T, Almeida JP (2012) Fib inogen e ec s on
e y h ocy e ni ic oxide mobiliza ion in p esence o ace ylcholine. Li e
Sci 91(21-22): 1017-1022.
39. Saldanha C, Sil a-He dade AS (2017) Physiological p ope ies o
human e y h ocy es in in lamma ion. J Cell Bioc en 3: 15-20.
40. Saldanha C, He dade AS (2017) E y h ocy e as a he apeu ic a ge .
No App o D ug des De 2 (3): 1-3.
41. Saldanha C, Sil a-He dade AS. (2014) The Ubiqui y Na u e o
Ace ylcholine. Clinical & Expe imen al Pha macology 4: 3.
42. Zabala L, Saldanha C, Ma ins e Sil a J, Souza-Ramalho P (1999) Red
blood cell memb ane in eg i y in p ima y open angle glaucoma: ex i o
and in i o s udies. Eye 13: 101-103.
43. Mu olo MG, Albanese G, Rusciano D, Pescosolido N (2016) O al
Adminis a ion o Fo skolin, Homo au ine, Ca nosine, and Folic Acid
in Pa ien s wi h P ima y Open Angle Glaucoma: Changes in In aocula
P essu e, Pa e n Elec o e inog am Ampli ude, and Fo eal Sensi i i y.
J Ocul Pha macol The 32(3): 178-183.
44. Galassi F, Renie i G, Sodi A, Ucci F, Vannozzi L, e al. (2004) Ni ic
oxide p oxies and ocula pe usion p essu e in p ima y open angle
glaucoma. B J Oph halmol 88: 757-760.
45. Saldanha C, Lopes de Almeida JP, Sil a-He dade AS (2014) Applica ion
o ni ic oxide senso in biomedicine. Biosenso s 4(1): 1-17.
You nex submission wi h Junipe Publishe s
will each you he below asse s
• Quali y Edi o ial se ice
• Swi Pee Re iew
• Rep in s a ailabili y
• E-p in s Se ice
• Manusc ip Podcas o con enien unde s anding
• Global a ainmen o you esea ch
• Manusc ip accessibili y in di e en o ma s
( Pd , E-pub, Full Tex , Audio)
• Unceasing cus ome se ice
T ack he below URL o one-s ep submission
h ps://junipe publishe s.com/online-submission.php
This wo k is licensed unde C ea i e
Commons A ibu ion 4.0 License
DOI:
10.19080/NAPDD.2018.03.555617