S udies on he gene ics o a emisinin
esis ance in mala ia
Ana Júlia Pin o Fonseca Sieu e A onso Rod igues
Uni e sidade No a de Lisboa
Ins i u o de Higiene e Medicina T opical
Cen o de Malá ia e ou as Doenças T opicais – Labo a ó io Associado
2007
2
3
Ana Júlia Pin o Fonseca Sieu e A onso Rod igues
S udies on he gene ics o a emisinin esis ance in mala ia
Disse ação de candida u a ao g au de Dou o em Pa asi ologia Médica
subme ida ao Ins i u o de Higiene e Medicina T opical
Uni e sidade No a de Lisboa
O ien ado es – P o esso Dou o Vi gílio Es ólio do Rosá io
P o esso Dou o Ped o Ví o Lemos C a o
4
5
Ana Júlia Pin o Fonseca Sieu e A onso Rod igues oi bolsei a
da Fundação pa a a Ciência e Tecnologia do Minis é io da
Ciência Tecnologia e Ensino Supe io
SFRH/BD/8913/2002

6
7
I decla e ha unless s a ed o he wise, he wo k p esen ed he e is my own.
A numbe o igu es and ables ha e been kindly made a ailable by o he scien is s o
de eloped in pa ne ship wi h o he scien is s.
Ana Júlia Pin o Fonseca Sieu e A onso Rod igues
8
9
Í aca
Se um dia pa i es umo a Í aca,
eza pa a que o caminho seja longo,
cheio de a en u a e de conhecimen o.
Não emas mons os como os Ciclopes ou o zangado Poseidon:
Nunca os encon a ás no eu caminho
enquan o man i e es o eu espí i o ele ado,
enquan o uma a a exci ação agi a o eu espí i o e o eu co po.
Nunca encon a ás os Ciclopes ou ou os mons os
a não se que os agas con igo den o da ua alma,
a não se que a ua alma os c ie em en e a i.
Deseja que o caminho seja bem longo
pa a que haja mui as manhãs de Ve ão em que,
com quan o p aze , com an a aleg ia,
en es em po os que ês pela p imei a ez;
Pa a que possas pa a em pos os de comé cio enícios
aí comp a coisas inas, mad epé ola, co al e âmba ,
e pe umes sensuais de odos os ipos
an os quan os pude es encon a ;
e pa a que possas isi a mui as cidades egípcias
e ap ende e con inua semp e a ap ende com os seus escola es.
Tem semp e Í aca na ua men e.
Chega lá é o eu des ino.
Mas não e ap esses na iagem.
Se á melho que ela du e mui os anos
pa a que sejas elho quando chega es à ilha,
ico com udo o que encon as e no caminho,
sem espe a es que Í aca e aga iquezas.
Í aca deu- e a ua bela iagem.
Sem ela não e ias seque pa ido.
Não em mais nada a da - e.
E, sábio como e e ás o nado,
ão cheio de sabedo ia e expe iência,
já e ás pe cebido, à chegada, o que signi ica uma Í aca.
Kons an inos Ka á is (1863-1933)
.
16

17
TABLE OF CONTENTS
18
19
Page
Acknowledgemen s / Ag adecimen os
11
Table o Con en s
17
Lis o Figu es
23
Lis o Tables
27
Lis o Abb e ia ions
31
Abs ac
35
Resumo
39
Chap e I – In oduc ion
43
1.1 Mala ia: gene al ea u es
45
1.2 Mala ia oday
47
1.3 The pa asi e and i s li e
-
cycle
50
1.4 The gene ics o mala ia pa asi es
53
1.5 An imala ial d ugs and a ge s
57
1.6 A emisinin and i s de i a i es
58
1.6.1 A emisinin mode o ac ion
63
1.7 D ug Resis ance
67
1.7.1 Resis ance o a emisinin
71
1.8
Roden mala ia pa asi es
7
3
1.8
.1 The gene i
cs o oden mala ia pa asi es
78
1.9 Linkage G oup Selec ion
80
1.9.1. Ampli ied F agmen Leng h Polymo phism
83
1.10 Aims o he p ojec
8
4
Chap e II – Ma e ials and Me hods
8
5
2.1. Mice
87
2.2. Mosqui oes
87
2.3. Pa asi es
87
2.4 P epa a ion o
10
7
s anda d pa asi e inocula
88
2.5 Cloning
88
2.6 P epa a ion and adminis a ion o a emisinin and a esuna e
88
2.7 D ug es s
89
2.8 A emisinin and a esuna e selec ion expe imen s: gene al p ocedu e
89
2.9 Tes s o e alua e he s abili y o d ug- esis ance
90
2.10. T ansmission h ough Anopheles s ephensi
90
2.11. DNA ex ac ion
91
2.12. Iden i ica ion o he
P. chabaudi
c p
and
a p6
genes
9
1
2.13. Ampli ica ion and sequencing o he md 1, cg10, c p and a p6 genes o P. chabaudi
92
2.14. Es ima ion o copy numbe s o he pcmd 1, pc c p and pca p6 genes
92
2.15 P oduc ion o c oss p ogeny - O e iew o p ocedu e
93
2.15.1 Mosqui o eeds
9
4
2.15.2 In ec ion o mice om spo ozoi es
95
2.15.3 P epa a ion o a emisinin and a esuna e
95
2.16 Linkage G oup Selec ion
9
5
2.16.1 Selec ion o c oss p ogeny
9
5
2.16.2 Ampli ied F agmen Leng h Polymo phism (AFLP) analysis
96
2.16.2.1 P epa a ion o pa asi e DNA om expe imen al g oups
96
2.16.3 AFLP analysis
9
7
2.16.4 Measu emen and c
ompa ison o he in ensi y o AFLP ma ke s
10
1
2.16.5 Assignmen o AFLP ma ke s o loca ions in a P. chabaudi gene ic linkage map.
102
2.16.6 Sequencing o AFLP ma ke s, and hei loca ion on he Plasmodium alcipa um
genome 103
2.16.7 AFLP band sequen
cing and pu i ica ion o PCR p oduc s
10
3
2.17 Expe imen s wi h gene ic c osses o AS-ART and AJ o AS-ATN and AJ
105
2.17.1 Blood collec ion o P opo ional sequencing analyses
105
2.17.2 Ex ac ion o pa asi e DNA om blood o spo ozoi es
10
6
2.17.
3 De e mina ion o he p opo ions o clones in gene ic c osses by p opo ional
sequencing
10
6
2.17.3.1 P inciple o p opo ional sequencing
106
2.17.3.2 P opo ional sequencing
-
PCR eagen s and eac ion condi ions
10
9
20
2.17.3.3 P opo ional sequencing
–
Sequencing and pu i ica ion o PCR
p oduc s
1
10
2.17.3.4 P opo ional sequencing – Analyses o sequencing esul s
110
2.18. Ampli ica ion and sequencing o he ubp-1 gene o P. chabaudi
112
Resul s
11
3
Chap e III – Expe imen s o d ug selec ion o a emisinin and
a esuna e esis ance
11
7
3.1 In oduc ion
119
3.2 A emisinin d ug selec ion
120
3.
3
A esuna e d ug selec ion
12
4
3.
4
Cloning o esis an pa asi es
1
2
8
3.5 D ug esis ance s abili y es s
130
3.5.1. Resis ance s abili y a e liquid ni ogen p ese a ion (deep- eezing)
130
3.5.2. Resis ance s abili y a e blood passages in he absence o d ug p essu e
131
3.
5
.3. Resis ance s abili y a e cyclical ansmission h ough mosqui oes
1
31
3.6 Tes o c oss- esis ance be ween a emisinin and a esuna e clones
133
3.7 Discussion
134
Chap e IV – Analysis o he pu a i e gene ic modula o s o a emisinin
and a esuna e esis ance
137
4.
1
.
Sequencing o
P. chabaudi md 1
,
cg10, c p
and
a p6
genes,
in he selec ed mu an
clones.
1
3
9
4.1.1. Isola ion o he md 1, cg10, c p and a p6 P.chabaudi o hologues
139
4.2.2. Sequnece compa ions o P. chabaudi md 1, cg10, c p and a p6 in he
selec ed mu an clones and hei sensi i e p ogeni o s. 143
4.2. Es ima ion o gene copy numbe s o he pcmd 1, pc c p and pca p6 genes, in he
selec ed mu an clones. 144
4.3. Discussion
146
4.3.1. Summa y
14
6
4.3.2. Gene al discussion 147
Chap e V – Expe imen s using Linkage G oup Selec ion as an a emp o
iden i y he locus o loci in ol ed in a emisinin and a esuna e esis ance
149
5.1. In oduc ion
151
5.2
P oduc ion o c oss p ogeny
1
52
5.3 Selec ion o c oss
p ogeny
1
53
5.3.1 Gene al p ocedu e
153
5.3.2 Resul s
154
5.3.2.1 Selec ion o he AS
-
ART x AJ c oss
1
55
5.3.2.2 Selec ion o
he AS
-
ATN x AJ c oss
1
58
5.4 AFLP analysis
160
5.4.1 Gene al p ocedu e
160
5.4. 2 Resul s
1
61
5.4.2.1 AFLP analysis o he AS
-
ART x AJ
1
62
5.4.2.2 AFLP analysis o he AS-ATN x AJ
165
5.4.2.3 AFLP analysis compa ison be ween pooled c osses AS-ART x AJ
and AS-ATN x AJ 169
5.5 Sequencing and mapping o AFLP bands unde selec ion
17
0
5.5.1 Gene al p ocedu e
170
5.5.2 Resul s
171
5.6 Fu he in es iga ion o ma ke s unde selec ion on ch omosome 2
1
73
5.6.1 Iden i ica ion o
ubp
-
1
gene mu a ion o
n
P. chabaudi
ch omosome 2
1
75
5.7 Discussion
178
Chap e VI – Gene al conclusions
181
Supplemen
185
Appendix 1 -
Solu ions and bu e s
1
87
Appendix 2 –
Gene and p ime sequence o he genes pcmd 1, cg10, pc c p and pca p6
189
Appendix 3 –
P ime sequence and PCR ampli ica ion condi ions o de e mining gene
copy numbe o pcmd 1, pc c p and pca p6 genes
193
Appendix 4 –
Gene and p ime sequence o he gene ubp-1 194
Appendix 5 –
The ela i e in ensi y and compa a i e in ensi ies o all AFLP ma ke s
analysed in he LGS expe imen s desc ibed in Chap e V
202
21
Glossa y
2
21
Re e ences
2
25
Online e e ences
2
39
Publica ions ha a ose om his p ojec
243
Hun
P,
A onso
A
, C easey
A, Culle on
R, Sidhu A, Logan
J, Valde amos
S, McNae
I, Cheesman
S, do
Rosa io
V, Ca e
R, Fidock D and C a o
P. Gene encoding a de-ubiqui ina ing enzyme is mu a ed in
a emisinin- and chlo oquine- esis an oden mala ia pa asi es. In p ess a he Molecula Mic obiology
Cheesman S, C easey A, Degnan K, Kooij T,
A onso A
, C a o P, Ca e R, Hun P. Valida ion o
Py osequencing o accu a e and high h oughpu es ima ion o allele equencies in mala ia pa asi es Mol
Biochem Pa asi ol. 2007 Ap ; 152 (2): 213-219.
C a o P, Culle on R,
A onso A
, Fe ei a ID, do Rosá io VE Mechanisms o D ug Resis ance in Mala ia:
Cu en and New Challenges. An i-In ec i e Agen s in Medical Chemis y 2006. 5: 63-73.
A onso A
, Hun P, Cheesman S, Al es AC, Cunha CV, do Rosá io V, C a o P Mala ia pa asi es can de elop
s able esis ance o a emisinin bu lack mu a ions in candida e genes a p6 (se ca), c p, md 1 and cg10. An i-
Mic obial Agen s Chemo he apy 2006. 50 (2): 480-489.

22
23
LIST OF FIGURES
24
25
Page
Co e
-
A emisia annua
lowe s and lea es.
Sou ce: The Uni e si y o Yo k (www.yo k.ac.uk)
Figu e 1
-
Illus a ion d awn by La e an o a ious s ages o mala ia pa asi es as seen on esh blood. 46
Figu e 2
–
Wo ld geog aphic dis ibu ion o mala ia, da a om 2003.
49
Figu e 3
–
Mala ia a
nsmission a eas and
P. alcipa um
d ug esis ance dis ibu ion da a om Wo ld
Hea h O ganiza ion da a om 2004
49
Figu e 4
-
Li e cycle o mala ia pa asi e. 52
Figu e 5
–
C ossing and ch omosomal e en s in
Plasmodium.
55
Figu e
6
-
Schema ic ep esen
a ion o
A emisia annua
L.
58
Figu e
7
–
A - Chemical s uc u e o (1) Qinghaosu o a emisinin and some o i s de i a i es (2)
dihyd oa emisinin, (3) a eme he ; (4) a ee he and (5) a esunic acid o a esuna e. 60
Figu e
8
The geog aphic o igins o
he oden mala ia pa asi es.
7
3
Figu e
9
Plasmodium chabaudi
pa asi es ( ophozoi e s age) in mouse pe iphe al blood.
7
5
Figu e 10
–
Roden mala ia genomes syn eny map o P. alcipa um.
79
Figu e 11
– Schema ic ep esen a ion o he Linkage G oup Selec ion p o ocol. 82
Figu e 12
-
An example o an AFLP gel.
10
0
Figu e 13
-
The ou polymo phisms on he
pcdh
ps
gene ha allowed
di e en ia ing
be ween s ains
AS and AJ.
10
8
Figu e 14
–
Example o an elec ophe og am o p opo ional sequence analysis o wo
polymo phisms on he pcdhps gene. 111
Figu e 15
–
A schema ic ep esen a ion o he a emisinin
selec ion p ocedu e. AS
-
ART* is uncloned.
1
20
Figu e 16
– The inc ease o he a emisinin dose (mg/kg/day), ole a ed by AS-30CQ clone, du ing 15
blood passages. 121
Figu e 17
–
The mean day o ec udescence on each passage unde selec ion and a emisinin dose
(mg/kg/day).
12
2
Figu e18
– A schema ic ep esen a ion o he a esuna e selec ion p ocedu e. AS-ATN* is uncloned. 124
Figu e 19
– The inc ease o he a esuna e dose (mg/kg/day), ole a ed by AS-15CQ clone, du ing 14
blood passages. 125
Figu e 20
–
The mean day o ec udescence unde selec ion and a esuna e dose (mg/kg/day).
1
2
6
Figu e 21
– A schema ic ep esen a ion o he clones and pa asi e lines o Plasmodium chabaudi used
in his p ojec . 128
Figu e 22
–
A schema ic ep esen a ion o he a emisinin and a esuna e selec ion p ocedu e.
13
2
Figu e 23
–
Ex en o sequence analysed o he gene
pcmd 1
o he clones AS
-
15CQ, AS
-
30CQ,
AS-ART and AS-ATN.
1
41
Figu e 24
–
Ex en o sequence analysed o he gene pccg10 o he clones AS-15CQ, AS-30CQ, AS-
ART and AS-ATN. 141
Figu e 2
5
–
Ex en o sequence analysed o he gene
p
c c p
o he clones AS
-
15CQ, AS
-
30CQ, AS
-
ART and AS-ATN.
14
2
Figu e 2
6
–
Ex en o sequence analysed o he gene pca p6 o he clones AS-15CQ, AS-30CQ, AS-
ART and AS-ATN 142
Figu e 27
–
Rela i e di e ences (N
-
old) in gene copy numbe be ween a emisinin (AS
-
ART) and
a esuna e (AS-ATN) esis an pa asi es and hei sensi i e p ogeni o s, AS (30CQ) and AS (15CQ)
espec i ely.
1
4
5
Figu e 28
-
Schema ic ep esen a ion o he selec ion o he pooled c oss p ogeny (LGS) expe imen
using p e iously gene a ed indi idual c osses be ween AJ and AS-ART
1
53
Figu e 2
9
-
Pa asi aemia cu es o he “un ea ed” and “a emisinin ea ed” uncloned c oss p ogeny
o he AS-ART x AJ c oss. 155
Figu e 30
–
De ail o he
pcdhps
gene sequence wi h indica ion o polymo phisms 1
-
4 be ween AS
and AJ. Fo en i e sequence o he gene please check igu e 13.
1
56
Figu e 31
-
Pa asi aemia cu es o he “un ea ed” and “a esuna e ea ed” uncloned c oss p ogeny
o he AS-ATN x AJ c oss. 158
Figu e 32
-
Compa a i e In ensi ies (CI) o AJ (sensi i e pa en ) speci ic AFLP ma ke s in
he ART
ea ed g oup compa ed o he un ea ed g oup, o he AS-ART x AJ c oss.
1
63
Figu e 33
-
Compa a i e In ensi ies (CI) o AS ( esis an pa en ) speci ic AFLP ma ke s in he ART
ea ed g oup compa ed o he un ea ed g oup, o he AS-ART x AJ c oss 164
Figu e 34
-
Compa a i e In ensi ies (CI) o AJ (sensi i e pa en ) speci ic AFLP ma ke s in he ATN
ea ed g oup compa ed o he un ea ed g oup, o he AS-ATN x AJ c oss.
1
67
Figu e 35
-
Compa a i e In ensi ies (CI) o AS ( esis an pa en ) speci ic AFLP ma ke s in he ATN
ea ed g oup compa ed o he un ea ed g oup, o he AS-ATN x AJ c oss. 168
Figu e 36
-
P. chabaudi
ch omosome 2 wi h syn enic blocks ep esen ed,
P. alcipa um
ch omosome
1
74
32

33
3D7
P. alcipa um
cul u e, sensi i e
6-PGD - 6-Phosphoglucona e dehyd ogenase
A - Adenine (in DNA con ex )
A
Alanine (Ala) (in p o ein con ex )
a.a. -
Amino acid
ACT - A emisinin combina ion he apy
AFLP - Ampli ied agmen leng h polymo phism
ART
-
A emisin
in
AJ -
Plasmodium chabaudi
clone, o ally d ug sensi i e. Gene ically di e en om AS
line
.
AS-ART - Plasmodium chabaudi clone, py ime hamine-, high chlo oquine- esis an , ob ained om
AS-30CQ by a emisinin d ug p essu e (clone ob ained du ing his p ojec )
AS-ART* -
Plasmodium chabaudi
pa asi e line, py ime hamine
-
, high chlo oquine
-
esis an , ob ained
om AS-30CQ by a emisinin d ug p essu e (pa asi e line ob ained du ing his p ojec ),
his pa asi e line was hen cloned by limi ing dilu ion
AS-ATN - Plasmodium chabaudi clone, py ime hamine-, in e media e chlo oquine- esis an , ob ained
om AS-15CQ by a esuna e d ug p essu e (clone ob ained du ing his p ojec )
AS-ATN* -
Plasmodium chabaudi
pa asi e line, py ime hamine
-
, in e media e c
hlo oquine
-
esis an ,
ob ained om AS-15CQ by a esuna e d ug p essu e (pa asi e line ob ained du ing his
p ojec ), his pa asi e line was hen cloned by limi ing dilu ion
AS-SENS -
Plasmodium chabaudi
clone, o ally d ug sensi i e. Gene ically di e
en om AJ.
AS-PYR -
Plasmodium chabaudi
clone, py ime hamine
-
esis an ob ained om AS
-
SENS by
py ime hamine d ug p essu e
AS-3CQ - Plasmodium chabaudi clone, py ime hamine and low chlo oquine esis an ob ained om
AS-PYR by chlo oquine d ug p essu e
AS-15CQ -
Plasmodium chabaudi
pa asi e isola e, py ime hamine and in e media e chlo oquine
esis an , ob ained om he AS-3CQ by clo oquine d ug p essu e
AS-15MEF - Plasmodium chabaudi pa asi e clone, py ime hamine, in e media e chlo oquine esis an
and me loquine esis an , ob ained om he AS-15CQ by me loquine d ug p essu e
AS-30CQ -
Plasmodium chabaudi
pa asi e clone, py ime hamine and high chlo oquine esis an ,
ob ained om he AS-15CQ by clo oquine d ug p essu e
ARMD - Accele a ed esis an o mul iple d ugs pheno ype
ama-1 -
Apical
m
emb ane
a
n igen
-
1
ATN -
A esuna e
a p6 - Encoding he Sa coplasmic and Endoplasmic Re iculum Ca
2+
a pase gene
C - Cys eine (Cys)
CDC -
Na ional Cen e o Disease Con ol
cDNA -
Complemen a y DNA
cg10 - Gene coding o a pu a i e p o ein anspo e , P. chabaudi o hologue o he P. alcipa um
c .
CI -
Compa a i e
i
n ensi y
CQ
-
Chlo oquine
c - Chlo oquine esis ance anspo e gene
CSP - Ci cumspo ozoi e p o ein
D -
Aspa ic acid
(Asp)
Dd2
P. alcipa um
cul u e,
om Indochina,
py ime hamine
, me loquine and chlo oquine
esis an
DDT - Dichlo o-diphenyl- ichlo oe hane
DHA -
Dihyd oa emisinin
DHFR
Dihyd o ola e
-
educ ase enzyme
DMSO -
Dime hyl sul oxide
DNA - Deoxy ibonucleic acid
F -
Phen
ylalanine
(Phe)
gDNA -
Genomic DNA
HB3 - P. alcipa um cul u e, om Hondu as, py ime hamine, me loquine and chlo oquine
esis an
IC
50
-
The d ug dose necessa y o elimina e 50% o he pa asi es
II
In ensi y index
i. p.
-
In ape i oneally
iRBC - In ec ed ed blood cells (e y h ocy es)
K
i
Biochemis y ca aly ic cons an
34
L
Leucine (Leu)
LDH - Lac a e dehyd ogenase
LGS
- Linkage g oup selec ion
Mb - Mega base
MCD -
Minimum
c
u a i e
d
ose
md 1 -
Mul i
d
ug
esis ance 1 gene
msp-1 Me ozoi e su ace p o ein-1
msp-2 -
Me ozoi e su ace p o ein-2
N -
Aspa agine
(Asn)
NADH -
Hyd ogen
n
ico inamide
a
denine
d
inucleo ide
NCBI/NIH - Na ional Ins i u e o Heal h
nM - Nanomola
PABA -
Pa aminobenzoic
a
cid
pcdhps - Gene o Plasmodium chabaudi codi ying o enzyme dihyd op e oa e syn he ase
PCR - Polyme ase chain eac ion
Phe - Phenylalanine
PYR -
Py ime hamine
RBC -
Red blood
c
ells (e y h ocy es)
RIIs - Rela i e in ensi y indices
RMP - Roden mala ia pa asi e
RNA -
Ribonucleic
a
cid
RNA -
Ribosomal RNA
RTQ
-
PCR
-
Real ime quan i a i e – PCR
S - Se ine (Se )
s.
c
.
-
Subcu aneous
SDS -
Sodium dodecyl sul a e
sRNA - Small RNA
SP
-
Sul adoxine-py ime hamine
T -
Tymine
c p - T ansla ionally con olled umou p o ein gene
RNA - T ans e RNA
Ty - Ty osine
ubp-1 -
De
-
ubiqui ina ing enzyme,
u
biqui in ca boxyl
-
e minal hyd olase, pu a i e, 1
ubp-1
De
-
ubiqui ina ing enzyme,
u
biqui in ca boxyl
-
e minal hyd olase, pu a i e, 1 gene
V
-
Valine (Val)
WHO - Wo ld Hea h O ganiza ion
Y -
Ty osine (Ty )
6-PGD - 6-Phosphoglucona e dehyd ogenase
35
ABSTRACT
36
37
Resis ance o Plasmodium alcipa um o mul iple d ugs including chlo oquine (CQ) and
sul adoxine-py ime hamine (SP) is a majo p oblem in mala ia con ol. New d ugs, such as
a emisinin (ART) de i a i es, pa icula ly in combina ion wi h o he d ugs, a e hus
inc easingly used o ea mala ia. Al hough s able esis ance o ART has ye o be epo ed
om labo a o y o ield s udies, i s eme gence would be disas ous because o he lack o
al e na i e ea men s.
The wo k p esen ed in his hesis desc ibes he selec ion o pa asi es wi h s able esis ance o
ART and a esuna e (ATN), and hei gene ic analysis. This wo k was ca ied ou using he
oden mala ia pa asi e Plasmodium chabaudi chabaudi (Plasmodium chabaudi).
Two di e en oden mala ia pa asi e lines AS-15CQ and AS-30CQ we e con inually
passaged in he p esence o inc easing concen a ions o ATN o ART, espec i ely. A e
selec ion, hese lines, named AS-ATN and AS-ART, showed 6- old and 15- old inc eased
esis ance o ATN and ART espec i ely. Resis ance emained s able a e cloning,
eeze/ hawing, blood passage in he absence o d ug p essu e and ansmission h ough
mosqui oes. The nucleo ide sequences and he gene copy numbe o he possible gene ic
modula o s o ART esis ance md 1, cg10, c p and a p6; we e compa ed be ween sensi i e
and esis an pa asi es. No mu a ions o changes in he gene copy numbe o hese genes we e
ound.
Linkage G oup Selec ion (LGS) was used o in es iga e he gene ic basis o ART esis ance.
Gene ic c osses be ween AS-ART o AS-ATN and he ART-sensi i e clone AJ we e analysed
be o e and a e d ug ea men . Using quan i a i e ma ke s, a gene ic locus on ch omosome 2
was ound o be unde s ong selec ion. Loci on ch omosomes 1, 8 and 14 o P. chabaudi also
appea o be unde selec ion. On ch omosome 2, wo di e en mu a ions V739F and V770F in
a de-ubiqui ina ing enzyme (ubp-1) we e iden i ied in AS-ATN and AS-ART espec i ely,
ela i e o hei sensi i e p ogeni o s. The implica ions o hese esul s a e discussed.

38
39
RESUMO
40
41
A exis ência de es i pes do pa asi a, Plasmodium alcipa um esis en es a mul iplos á macos
ais como; clo oquina (CQ) e sul adoxina-pi ime amina (SP) é um dos p oblemas mais g a es
no con olo da malá ia.
No os á macos, como a a emisinina (ART) e seus de i ados, pa icula men e em
combinação com ou os á macos, são cada ez mais u ilizados no a amen o da mala ia.
Embo a a é ao momen o a á maco- esis ência es á el à ART que in i o que in i o não
enha sido egis ada, o seu su gimen o se ia desas oso de ido á al a de al e na i as.
O abalho ap esen ado nes a ese desc e e a selecção de esis ência es á el a ART e ao
a esuna o (ATN). Es e abalho oi ealizado usando o modelo oedo de malá ia Plasmodium
chabaudi chabaudi (Plasmodium chabaudi).
Duas linhas pa asi á icas di e en es, AS-15CQ e AS-30CQ, o am ei as c esce na p esença
de concen ações c escen es de ATN e ART, e que no inal ap esen a am uma esis ência de 6
e 15 ezes supe io ao ATN e à ART, espec i amen e (es as no as linhas ob idas o am
nomeadas AS-ATN e AS-ART).
A esis ência é es á el mesmo após clonagem, congelamen o/descongelamen o, passagem
sanguínea na ausência de p essão de á maco e ansmissão a a és do mosqui o ec o .
A sequência nucleo ídica e o núme o de cópias dos genes desc i os como modulado es
pu a i os de esis ência à ART: md 1, cg10, c p e a p6; oi compa ada en e pa asi as
esis en es e sensí eis. Não endo sido encon adas al e ações na sequência ou no núme o de
cópias des es genes.
Numa en a i a de iden i ica os genes encol idos na esis ância à ART e ao ATN a écnica de
Linkage G oup Selec ion (LGS) oi u ilizada e dois c uzamen os gené icos en e os clones
á maco- esis en es; AS-ART e AS-ATN e o clone gene icamen e dis in o dos an e io es e
sensí el aos á macos em es udos; AJ; o am ealizados. Fo am encon ados sob e selecção
em ambos os c uzamen os gené icos qua o loci; c omossomas de P. chabaudi 1, 2, 6 e 8.
A endendo a que, a selecção no c omossoma 2 e a mais o e, es e locus oi subme ido a
análises gené icas subsequen es. Tendo sido encon adas duas mu ações di e en es (V739F e
V770F) num gene que codi ica pa a um enzima de desubiqui inação (ubp-1). As implicações
des es esul ados se ão discu idas.
48
P. alcipa um has become esis an o almos all d ug classes excep he a emisinin
de i a i es. Nowadays chlo oquine- esis an P. alcipa um occu s ac oss all mala ia endemic
a eas. The e ec i eness o sul adoxine-py ime hamine has apidly declined in all egions
whe e i has been in oduced due o esis ance, and mul id ug esis ance is now es ablished in
Sou heas Asia, Sou h Ame ica and A ica (See Figu e 3) [Collins WJ e al. 2006; G een MD
2006; Kshi saga NA 2006; Lina es GE e al. 2007].
D ug esis ance is mos likely o eme ge when backg ound immuni y is weak, pa asi e
numbe s in an indi idual a e high, ansmission is low and d ug p essu e is in ense o e y
in ense [Has ings IM e al. 2000].
Wi h an inc ease in insec icide and an imala ial-d ug esis ance, he de elopmen o a mala ia
accine and abo e all new d ugs o new d ug combina ions, using d ugs al eady in use,
ca ies huge expec a ions [Cha e jee S e al. 2006; Gi a d MP e al. 2007; G eenwood B e al.
2007; Ma uschewski K 2006, Shanks GD 2006; S epniewska K e al. 2006].

49
Figu e 2 – Wo ld geog aphic dis ibu ion o mala ia, da a om 2003.
Sou ce: Na ional Cen e o Disease Con ol (CDC) wi h kind pe mission o CDC.
Figu e 3 – Mala ia ansmission a eas and P. alcipa um d ug esis ance dis ibu ion da a om Wo ld Hea h
O ganiza ion da a om 2004.
Sou ce: Wo ld Hea h O ganiza ion (WHO) wi h kind pe mission o WHO.
50
1.3 The pa asi e and i s li e-cycle
In na u e, mala ia pa asi es sp ead by in ec ing successi ely wo ypes o hos s: humans and
emale Anopheles mosqui oes.
Mala ia is ansmi ed among humans by emale mosqui oes o he genus Anopheles. Female
mosqui oes equi e blood meals in o de o ca y ou egg p oduc ion, and such blood meals
a e he link be ween he human and he mosqui o hos s in he pa asi e li e cycle. O he
app oxima ely 430 known species o Anopheles, only 30-50 ansmi mala ia in na u e
(“ ec o s”). The success ul de elopmen o he mala ia pa asi e in he mosqui o ( om he
“game ocy e” s age o he “spo ozoi e” s age – See Figu e 4) depends on se e al ac o s. The
mos impo an is ambien empe a u e and humidi y and whe he he Anopheles su i es long
enough o allow he pa asi e o comple e i s cycle in he mosqui o hos (“spo ogonic” o
“ex insic” cycle, du a ion 10 o 18 days). Mala ia’s li e cycle is comp ised o bo h he sexual
and asexual o ms (See Figu e 4). The sexual cycle occu s mainly in he mosqui o; while he
asexual cycle akes place in he human hos a e he pa asi es ha e en e ed he hos ’s blood
s eam when he mosqui o bi es o a blood meal. Du ing a blood meal, a mala ia in ec ed
emale Anopheles mosqui o inocula es spo ozoi es in o he human hos . Though he sali a y
glands o an in ec ed mosqui o con ain housands o spo ozoi es, less han 100 o hese a e
ansmi ed in any one bi e [Rosenbe g R e al. 1990]. Wi hin 30-45 minu es o he pa asi e’s
spo ozoi es en e ing he bloods eam, hey en e pa enchymal cells o he li e ; his is
achie ed by he binding o he h ombospondin domains o he ci cumspo ozoi e and
h ombospondin- ela ed adhesi e p o eins (csp and ap espec i ely) o he hepa in sulpha e
p o eoglygan on he hepa ocy es [F e e U e al. 1993]. This phase is called he p e-
e y h ocy ic s age las ing 5-15 days in which he pa asi e unde goes asexual ep oduc ion
(schizogony): he end p oduc s o his a e he me ozoi es. In P. i ax and P. o ale a do man
s age called hypnozoi es, can pe sis in he li e and can cause elapses by in ading
e y h ocy es weeks o yea s la e [Du an e Mangoni E e al. 2003].
Hepa ocy es up u e o elease me ozoi es ha en e ed blood cells. In asion o e y h ocy es
by me ozoi es in ol e an ini ial low a ini y in e ac ion be ween p o eins on he su ace coa
o he me ozoi e (me ozoi e su ace p o ein-1 (msp-1), and apical memb ane an igen-1 (ama-
1)) and he su ace o he e y h ocy e. Once inside he e y h ocy e he me ozoi e ini ia es he
eeding p ocess o ming he in acellula pa asi e, he ophozoi e (e y h ocy ic schizogony
s age). Mi o ic di isions (asexual ep oduc i e s age) occu in he cells gi ing ise o
schizon s, which con ain up o abou hi y haploid me ozoi es. This ed cell up u es,
eleasing mo e ma u e me ozoi es, which in ade mo e ed blood cells, hence main aining his
51
“asexual cycle”. Some pa asi es unde go game ocy ogenesis wi hin he e y h ocy e, p oducing
male o emale mic o and mac ogame ocy es espec i ely. These emain in he blood
ci cula ion whe e hey a e a ailable o inges ion by a eeding mosqui o. The asexual
ep oduc i e s age, occu ing in he blood o he e eb a e hos (human, p ima e o oden ), is
he a ge o mos an imala ial d ugs, including a emisinin and i s de i a i es. Inside he
mosqui o mid-gu , emale and male game ocy es unde go game ogenesis, in which he emale
mac ogame ocy e escapes om he e y h ocy e memb ane and he male mic ogame ocy e
unde goes he p ocess o ex lagella ion which p oduces 8 mo ile mic ogame es. The mic o
and mac o-game es use o o m a zygo e ha in u n becomes an ookine e, ( he only diploid
s age o he pa asi e). The ookine e c osses he gu wall and encys s on he ou e wall o he
gu benea h he basal lamella o ming an oocys o spo ocys . Di ision and mul iplica ion o
he spo ocys akes place o p oduce many haploid spo ozoi es. When he spo ocys bu s s he
spo ozoi es a e eleased and hen mig a e o he sali a y gland, wai ing o e-in ec again once
he mosqui o akes ano he blood-meal [Ba nwell JW e al. 1998; Beie JC e al. 1998; Sinden
RE 1997].
See Figu e 4 o de ails on he pa asi e li e cycle.
52
Figu e 4 - Li e cycle o mala ia pa asi e.
Mos o he biological wo k p esen ed he e occu s in he e y h ocy ic cycle o he pa asi e (p esen ed in he
Figu e as B). The d ug esis ance selec ion p ocess occu s on his pa o he pa asi e li e cycle.
B ie ly: he mala ia pa asi e li e cycle in ol es wo hos s. Du ing a blood meal, a mala ia-in ec ed emale
Anopheles mosqui o inocula es spo ozoi es in o he human hos . Spo ozoi es in ec li e cells and ma u e
in o schizon s , which up u e and elease me ozoi es . A e his ini ial eplica ion in he li e (exo-
e y h ocy ic schizogony ), he pa asi es unde go asexual mul iplica ion in he e y h ocy es (e y h ocy ic
schizogony ). Me ozoi es in ec ed blood cells . The ing s age ophozoi es ma u e in o schizon s, which
up u e eleasing me ozoi es . Some pa asi es di e en ia e in o sexual e y h ocy ic s ages (game ocy es) .
Blood s age pa asi es a e esponsible o he clinical mani es a ions o he disease. The game ocy es, male
(mic ogame ocy es) and emale (mac ogame ocy es), a e inges ed by an Anopheles mosqui o du ing a blood
meal . The pa asi es’ mul iplica ion in he mosqui o is known as he spo ogonic cycle . While in he
mosqui o's s omach, he mic ogame es pene a e he mac ogame es gene a ing zygo es . The zygo es in u n
become mo ile and elonga ed (ookine es) which in ade he midgu wall o he mosqui o whe e hey de elop
in o oocys s . The oocys s g ow, up u e, and elease spo ozoi es , which make hei way o he mosqui o's
sali a y glands. Inocula ion o he spo ozoi es in o a new human hos pe pe ua es he mala ia li e cycle.
Sou ce: Na ional Cen e o Disease Con ol (CDC) wi h kind pe mission o CDC.
53
1.4 The gene ics o mala ia pa asi es
Mala ia pa asi es, as all membe s o he phylum Apicomplexa, a e haploid o almos hei
en i e li e cycle (exo-e y h ocy ic and e y h ocy ic blood s ages, spo ogony and
mic ogame ogenesis), and in he haploid phase o he pa asi e li e cycle hey mul iply by
mi osis.
The only phase o he pa asi e li e cycle whe e he pa asi e genome is diploid is he zygo e
s age (ookine es), p io o he meio ic di ision ha esul s in he p oduc ion o spo ozoi es.
Mala ia pa asi es ha e h ee indi idual genomes; an ex a-ch omosomal mi ochond ial
genome, a 35kb ci cula genome and a la ge nuclea genome.
The mi ochond ial genome, also known as he 6kb elemen con ains genes encoding wo
unca ed ibosomal sRNA and h ee p o eins componen s in ol ed in he elec on anspo
sys em; cy och ome c oxidase subuni s I and II and cy och ome b [Funes S e al 2004]. The
inhe i ance o he 6 kb elemen appea s o ollow he same pa e n as o he mi ochond ial
genomes in euka yo es meaning i is inhe i ed om he emale pa en only [C easey AM e al.
1993].
The 35kb ci cula genome associa ed wi h he apicoplas encodes 30 p o eins, mainly RNA,
RNA, which a e p ima ily in ol ed in gene exp ession [Funes S e al. 2004, Ga dne MJ e
al. 2002]. The exac ole o he apicoplas emains unclea , bu i is known o be in ol ed in
he anabolic syn hesis o a y acids, isop enoids and haem [Ga dne MJ e al. 2002].
The haploid nuclea genome o P. alcipa um is whe e mos pa asi e genes eside. I consis s
o 14 ch omosomes and encodes app oxima ely 5,300 genes wi h a o al genome size o 22.8
Mb. The pa asi e ch omosomes ha e a cen al domain ha con ain conse ed coding egions
and ch omosome ends ha consis o elome ic epea sequences and sub elome ic epea
egions, con aining polymo phic gene amilies ( o example p emp1, s e o s and i ins)
[Lanze M e al. 1994].
The P. alcipa um nuclea genome is e y (A+T)- ich, wi h an o e all (A+T) con en o 81%,
ising o 90% in in onic and in e genic egions [Ga dne MJ e al. 2002]. The e is
conside able ch omosomal size polymo phism be ween s ains o pa asi es [Co co an LM e
al. 1986], which could be due o unequal c ossing-o e o homologous ch omosomes du ing
meiosis, o non-meio ic ch omosome b eaking and healing e en s [Babike HA e al. 1994,
Ga dne MJ e al. 2002, He nandez-Ri as R e al. 1996, Sche A e al. 1992].
Va ious gene ic polymo phisms can be obse ed when compa ing di e en s ains e en
wi hin he same Plasmodium species. This di e si y obse ed o he geno ype le el has i s
o igin on ei he spon aneous gene ic mu a ion, occu ing a any s age o he pa asi e

54
de elopmen and also h ough gene ic ecombina ion occu ing in he mosqui o ec o s age.
Gene ic ecombina ion which he pa asi es unde go in he mosqui o midgu can hen esul in
independen asso men o genes on di e en ch omosomes (See Figu e 5) [Wallike D e al.
1998].
When a mosqui o eeds on an in ec ed hos con aining wo gene ically dis inc pa asi es,
game ocy es om he wo pa asi es a e aken up, and may e ilise, ecombining in o
he e ozygous zygo es, by meiosis, p oducing ou gene ically dis inc haploid daugh e cells,
which a e called he ecombinan p ogeny (See Figu e 5). In he case whe e he e a e equal
numbe s o male and emale game es p esen om each pa en al s ain in he mosqui o, hen
sel ing will occu 50% o he ime, esul ing in 25% o he zygo es being gene ically iden ical
o one pa en al s ain, and 25% iden ical o he o he . The emaining 50% will be hyb id
be ween he wo pa en als.Wha is mean is ha i he ecombinan p ogeny was unde going a
no mal Mendelian seg ega ion pa e n meaning wi hou any kind o sel ing/c ossing bias, hus
in he p esence o a andom seg ega ion and i he e a e equal numbe s o male and emale
game es p esen om each pa en al s ain in he mosqui o, hen sel ing ( ecombina ion wi hin
he same s ain) will occu 50% o he ime, esul ing in 25% o he zygo es being gene ically
iden ical o one pa en al s ain, and 25% iden ical o he o he . The emaining 50% will be
hyb id be ween he wo pa en als.
55
A B C D
Figu e 5 – C ossing and ch omosomal e en s in Plasmodium.
Clone 1 and clone 2 a e game es (haploid s age) om wo gene ically dis inc pa asi es, a e he blood meal
zygo es a e o med. The o ma ion o hese zygo es can esul om sel ing (equal o he p ogeni o game es) o
om c ossing (c oss be ween di e en clones and a e he e o e he e ozygous). Th ough meiosis ou gene ically
dis inc haploid daugh e cells a e p oduced, which a e called he ecombinan p ogeny. A and D a e he esul o
sel ing on he o he hand B and C a e he p oduc o ecombina ion.
F om: Wallike D 2000, wi h kind pe mission o P o esso Da id Wallike .
Recombina ion be ween mala ia pa asi es was i s p o en wi h gene ic c ossing expe imen s
done wi h he oden pa asi es Plasmodium yoelii [Wallike D e al. 1971], and Plasmodium
chabaudi [Wallike D e al. 1975]. In he P. chabaudi expe imen s, wo cloned pa asi es
56
which di e ed in hei esponse o he an i-mala ial d ug py ime hamine and in he
elec opho e ic pa e ns o wo enzymes (6-phosphoglucona e dehyd ogenase (6-PGD) and
lac a e dehyd ogenase (LDH)) we e mixed in mosqui oes and he esul ing p ogeny we e
cloned and cha ac e ized o hei enzyme ype and hei pheno ypic esponse o
py ime hamine. I was ound ha no only had he wo enzyme iso o ms ecombined, bu ha
py ime hamine suscep ibili y seg ega ed independen ly [Wallike D e al. 1975], which
showed ha ecombina ion be ween he pa en al cha ac e s had occu ed.
In P. alcipa um he p oduc ion o he e ozygo es (in he oocys ) be ween wo he e ologous
mala ia pa asi es has also been demons a ed expe imen ally, by dissec ing indi idual oocys s
om mosqui oes ha had ed on a mixed P. alcipa um blood in ec ion o clones 3D7 and
HB3. A e pe o ming gene ic yping o alleles o msp-1 and msp-2 genes, i was ound ha
some oocys s con ained alleles exclusi ely om HB3, some con ained alleles only om 3D7,
and he emainde o he oocys s con ained alleles om bo h pa en s, and we e he e o e
hyb ids, meaning, he p oduc s o e iliza ion be ween he wo di e en pa en al s ains. The
p opo ion o he homozygous and he e ozygous o ms was consis en wi h andom
e iliza ion be ween pa en s [Ran o d-Ca w igh L e al. 1993].
Wi h he objec i e o sequencing he genome o he human mala ia pa asi e Plasmodium
alcipa um (clone 3D7), an In e na ional Mala ia Genome Sequencing Conso ium was
o med in 1996. The genome was sequenced by h ee g oups: The Ins i u e o Genomic
Resea ch and he Mala ia P og am o he Na al Medical Resea ch Cen e (ch omosomes 2,
10, 11 and 14), The Wellcome T us Sange Ins i u e (ch omosomes 1, 3-9, 13) and S an o d
Uni e si y (ch omosome 12).
In 2002, he comple e genome o Plasmodium alcipa um was published, igge ing he “pos -
genomic” age o mala iology [Ga dne MJ e al. 2002].
57
1.5 An imala ial d ugs and a ge s
An imala ial d ugs a e one o he mos impo an measu es o con ol he disease. The d ug o
choice depends on he pa asi e species and local condi ions, d ug esis ance p e alence and
speci ici y. T adi ionally, an imala ial agen s a e classi ied as blood schizon ocides, issue
schizon icides, game ocides and spo on ocides, depending on he s ages o he mala ia li e
cycle which a e a ge ed by he d ug [T acey J e al. 1996]. Fo de ails see Table 1.
Blood schizon ocides a e d ugs ac ing on asexual in ae y h ocy ic s ages o mala ial
pa asi es. They supp ess he p oli e a ion o plasmodia in he e y h ocy es.
Tissue schizon ocides p e en he de elopmen o hepa ic schizon s. They a e causally
p ophylac ic because hey a ec he ea ly de elopmen al s ages o he p o ozoa and p e en
he in asion o he e y h ocy es.
A hypnozoi icide ac s on pe sis en in ahepa ic s ages o P. i ax and P. o ale in he li e .
Game ocides des oy he in ae y h ocy ic sexual o ms (game es) o he p o ozoa and he
p e en ansmission om human o ano he mosqui o. An imala ials a e a ely used clinically
jus o hei game ocidal ac ion
Table 1 – Classi ica ion o an imala ial agen s acco ding o hei s age o ac ion.
S age o Ac ion An imala ial
Tissue
schizon ocides P imaquine, py ime hamine, sul onamides (and o he 8-
aminoquinolines and o he ola e inhibi o s)
Hypnozoi icides P imaquine, a enoquine
Blood schizon ocides
Type 1, quick onse
: Chlo oquine, me loquine, quinine,
halo an ine, a emisinin
Type 2, slow onse : Py ime hamine, sul onamides, sul ones, o he
an ibio ics, a o aquone
Game ocides P imaquine o P. alcipa um
Quinine o P. i ax, P. mala iae and P. o ale
Spo on ocides P imaquine, chlo oquine
64
ha e also shown ha he b eakdown o a emisinin esul s in ee adicals [Meshnick SR e al
1996] du ing his a emisinin b eakdown p ocess e yl ions (Fe[IV]=O) appea o be o med
[Kape anaki S e al 2000]. The e a e o he elec ochemical s udies ha ha e shown ha
heme/i on can ca alyse he i e e sible b eakdown o a emisinin de i a i es [Zhang F e
al.1992] and also s uc u e–ac i i y ela ionship s udies ha e shown a high co ela ion
be ween an imala ial ac i i ies and heme-binding [Meshnick SR e al 1996] and be ween
an imala ial ac i i y and p o ein alkyla ion [Meshnick SR e al 1996]. Also, he p edic ed
pha macopho e o d ug ecep o om se e al s uc u e–ac i i y ela ionship s udies seems o
esemble heme [Meshnick SR e al 1996] and also he e a e a lo o o he heo e ical s udies
ha ha e shown ha a emisinin could bind o and eac wi h heme i sel [Gu HM e al. 1984]
u he mo e, he p esence o he heme polyme , hemozoin, is associa ed wi h sensi i i y o
a emisinins o example a emisinins a e inac i e agains he RC s ain o Plasmodium
be ghei [Pe e s W e al. 1986], and he ela ed in ae h ocy ic apicomplexan pa asi e,
Babesia mic o i [Wi ne M e al. 1996],.which bo h lack hemozoin, ye a e mos ac i e
agains schis osomes which also p oduce hemozoin [U zinge J e al.2001].
Though all hese e idences one could no say ha a emisinins ac like he ypical oxida ing
d ugs which cause p omiscuous damage o p o ein, nucleic acids and lipid i s ly because,
unlike mos o he oxidan d ugs (and oxidizing agen s pe si), a emisinin canno be cyclically
oxidised and educed causing he cascade e ec ypical om ee adical eac ions [Zhang F
e al.1992] in a way ha only one ee adical can esul om one d ug molecule, secondly, all
o he oxidan end p oduc s obse ed expe imen ally we e only obse ed a e y high d ug
concen a ions [Be man PA e al. 1997], bu he d ug is e ec i e a much lowe
concen a ions. This is a e y s ong indica ion ha , a emisinin de i a i es mus ha e a mo e
selec i e oxic e ec . One sugges ion o he selec i e oxici y o a emisinins may be he
o ma ion o co alen adduc s wi h pa asi e componen s, which will hen se e as media o s
o ee adical in e media es. One impo an alkyla ion a ge is heme i sel . A emisinin–
heme adduc s ha e been demons a ed in pa asi e cul u es ea ed wi h he apeu ic
concen a ions o a emisinin de i a i es [Hong YL e al. 1994] his means ha heme is bo h
an ac i a o and a ge o he a emisinin de i a i es.
The modi ica ion ha occu s in heme ia i s liga ion o a emisinin could kill he pa asi e in
se e al ways, i s ly, a emisinin o i s heme adduc migh be able o inhibi hemozoin
biosyn hesis o cause hemozoin deg ada ion, o example Pande and co-wo ke s p o ed ha
a mic omola concen a ions, a emisinin inhibi s hemoglobin diges ion by mala ia pa asi es
and inhibi s hemozoin o ma ion [Pandey AV e al. 1999] hough his obse a ion has only
been demons a ed in cell- ee condi ions, a emisinin ea men o li ing in ae y hocy ic P.

65
alcipa um in cul u e caused no change in hemozoin con en [Asawamahasakda W e al.
1994], sugges ing ha heme me abolism migh no be he majo in acellula a ge . Bu heme
is no he only p o ein ha a emisinin o ms adduc s wi h, a emisinins also o m co alen
adduc s wi h o he p o ein, bu no wi h DNA [Yang YZ e al 1993, Yang YZ e al. 1994].
The alkyla ion o speci ic mala ia p o eins by a emisinins has been demons a ed
[Asawamahasakda W e al. 1994] and his could media e he killing ac ion o a emisinin
de i a i es since i occu ed a he apeu ic concen a ions o d ug. One o he majo alkyla ion
a ge s desc ibed in he li e a u e is he mala ial ansla ionally con olled umou p o ein ( c p
p o ein), a p o ein ha binds heme [Bhisu hibhan J e al. 1998], also p e ious mic oscopic
s udies had p o en ha some o he mala ial c p p o ein is p esen in he ood acuole
memb anes, whe e i is in p oximi y o he heme- ich ood acuole [Bhisu hibhan J e al.
1998]. Thus, i is likely ha he eac ion be ween a emisinin and c p p o ein occu s because
o an associa ion be ween c p p o ein and heme, howe e , he e is a lack o e idences o c p
o any o he mala ial p o ein in associa ion wi h a emisinin is di ec ly causing he pa asi e
elimina ion hough a a ie y o ul as uc u al s udies ha e been ca ied ou on in ec ed ed
cells ea ed wi h a emisinin de i a i es and om hose s udies i was desc ibed ha
memb ane-con aining s uc u es, such as he plasma memb ane, endoplasmic e iculum,
nuclea en elope, ood acuola memb ane and mi ochond ia appea o be mos sensi i e o
he ac ion o a emisinins [Maeno Y e al.1993] hough om hese obse a ions a a ie y o
mechanisms o ac ion migh be sugges ed. Ano he e y in e es ing obse a ion is ha when
adiolabelled a emisinin de i a i es a e ed o mala ia in ec ed cells, he d ug has been ound
o accumula e in hemozoin and in he memb anes o he ood acuole and mi ochond ia
[Maeno Y e al.1993]. These obse a ions a e consis en wi h he ole o heme in he
mechanism o ac ion.
Though he e a e as desc ibed be o e many heo ies o explain a emisinin mode o ac ion
one canno e en say wha is he cellula a ge o a emisinin. Tc p p o ein o he easons
s a ed be o e was one o he sugges ed a ge s and mo e ecen ly K ishna and co-wo ke s ha e
indica ed ha a emisinins migh inhibi he sa co–endoplasmic e iculum Ca
2+
-ATPase
(SERCA) o P. alcipa um (P a p6) and, he e o e, also SERCAs o o he Plasmodium
species because hapsiga gin a chemical componen ha is a po en and selec i e inhibi o o
SERCAs and sha e some chemical simila i ies wi h a emisinins [Ecks ein-Ludwig U e al.
2003]. A emisinins, bu no o he an imala ial d ugs, inhibi ed P a p6 p o ein ac i i y when
P a p6 was exp essed and assayed in Xenopus oocy es [Ecks ein-Ludwig U e al. 2003].
O he s ha e ca ied ou docking simula ion s udies o a emisinin de i a i es o models o he
hapsiga gin binding si e in P a p6 p o ein [Uhlemann AC e al. 2005]. Se e al po en ial
66
hyd ophobic in e ac ions be ween side chains o a emisinin de i a i es and amino acids o
P a p6 p o ein ha e been iden i ied including Leu263 ha seems o modula e a emisinin
suscep ibili y when examined using mu agenesis expe imen s o mala ial SERCAs [Jung M e
al. 2005, Uhlemann AC e al. 2005]. Taken oge he , he e a e independen lines o e idence
ha ha e been ob ained om a ange o expe imen al echniques o sugges ha P a p6
p o ein migh be he p ima y a ge o a emisinins. Howe e , i is sugges ed ha gene ic
s udies a e equi ed o suppo his hypo hesis.
Mo e ecen ly, Li and co-wo ke s ha e sugges ed ha he elec on anspo chain o P.
alcipa um migh be a a ge o a emisinins [Li W e al. 2005]. In suppo o his idea, when
yeas was g own in non- e men able media (making i dependen on mi ochond ial
espi a ion), sensi i i y o a emisinin inc eases, because o e exp ession o some
mi ochond ial- anspo p o eins seems o inc ease sensi i i y o a emisinins, i has been
sugges ed ha he elec on anspo chain s imula es he ac i i y o a emisinins, and ha
hese ac i a ed a emisinins impede mi ochond ial unc ion by depola izing mi ochond ial
memb ane po en ial [Li W e al. 2005]. The mechanism o his inhibi ion is unclea bu i
migh be ela ed o he p esence o an i on g oup in he cy och ome cen e ha induces he
o ma ion o adicals [Haynes RK e al. 2006].
67
1.7 D ug Resis ance
The eme gence and sp ead o pa asi e esis ance o an i-mala ial d ugs has p esen ed one o
he la ges obs acles hinde ing he e ec i e ea men and con ol o mala ia.
The WHO’s o icial de ini ion o mala ia pa asi e esis ance da es om 1973; “ he abili y o a
pa asi e s ain o su i e and/o mul iply despi e he adminis a ion and abso p ion o a d ug
in doses equal o o highe han hose usually ecommended bu wi hin he limi s o ole ance
o he subjec ” [Wo ld Heal h O ganisa ion. Chemo he apy o Mala ia and Resis ance o
An imala ials: Repo o a WHO Scien i ic G oup].
The WHO 1973 de ini ion o he le el o pa asi e d ug esis ance emains in use;
A - Sensi i e (S): The asexual pa asi e coun educes o <25% o he p e- ea men
le el in 48 hou s a e s a ing he ea men , and comple e clea ance a e 7 days, wi hou
subsequen ec udescence - Comple e Reco e y.
B - RI Delayed Rec udescence: The asexual pa asi aemia educes o < 25% o p e-
ea men le el in 48 hou s, bu eappea s be ween 2-4 weeks.
C - RI Ea ly Rec udescence: The asexual pa asi aemia educes o < 25% o p e-
ea men le el in 48 hou s, bu eappea s wi hin 2 weeks.
D - RII Resis ance: Ma ked educ ion in asexual pa asi aemia (dec ease >25% bu
<75%) in 48 hou s, wi hou comple e clea ance in 7 days.
E - RIII Resis ance: Minimal educ ion in asexual pa asi aemia, (dec ease <25%) o
an inc ease in pa asi aemia a e 48 hou s.
Though i is impo an o cla i y ha clinical ea men ailu e o inc eased in i o IC
50
alues
alone a e no su icien o p o e d ug esis ance. A pa asi e isola e should be classi ied as
esis an only a e analysis o ea men esponse pa ame e s (pa asi e and e e clea ance)
and ea men success (de e mina ion o possible e-in ec ion, in he case o appa en
ec udescence) in co ela ion wi h he in i o d ug sensi i i y. Inc eased IC
50
alues
combined wi h p olonged pa asi e clea ance and ea men ailu e de ine a case o con i med
d ug esis ance [Noedl H 2005]. One migh sugges ha he i s s ep in he de elopmen o
clinical esis ance may be a dec ease in pa asi e in i o suscep ibili y, associa ed wi h a key
mu a ion in a a ge enzyme which ul ima ely esul s in clinical ailu e. Though i is also e y
impo an o no ice ha he pa asi e esponse and esis ance is no only dependen on he
pa asi e geno ype bu also in ol es he gene al heal h s a e and immune s a us o he pa ien .
A numbe o ac o s in luence he likelihood o esis ance occu ing and he speed wi h which
i sp eads. Fo ins ance, he mechanism by which he d ug wo ks agains he pa asi e is
68
impo an ; simple modes o ac ion such as simple enzyme inhibi ion a e likely o lead o apid
e olu ion o esis ance, as he numbe o gene ic mu a ions equi ed o al e enzyme s uc u e
is low. This is he case wi h py ime hamine esis ance, which e ol ed e y quickly a e he
in oduc ion o he d ug, in con as o he pa e n seen wi h he eme gence o chlo oquine
esis ance, which ook much longe o e ol e, as mos s udies desc ibed chlo oquine mode o
ac ion has being much mo e complex han py ime hamine.
The pha macokine ic dynamics o d ugs a e also impo an in de e mining he selec ion
p essu e o d ug esis ance. Wa kins and Mosobo [Wa kins WM e al. 1993], o example,
showed ha he long hal -li e o sul adoxine-py ime hamine was a conside able ac o in he
selec ion p essu e o esis an mu an s, as he d ug was p esen in pa ien s a sub- he apeu ic
le els o long pe iods o ime. D ugs wi h high e iciencies o pa asi e killing, apid
achie emen o le els abo e he minimal inhibi o y concen a ions and sho hal -li es, will
be he mos e ec i e a minimizing he selec ion p essu e o esis an mu an s [Wins anley
PA e al. 2002].
Resis ance has been eco ded o e e y an i-mala ial cu en ly in use, wi h he excep ion o
a emisinin and i s de i a i es. Quinine, he i s d ug used speci ically o ea mala ia was
used ex ensi ely. The i s epo s o esis ance o he d ug occu ed a he beginning o he
20
h
cen u y, when Cou o (1908) and la e Noch and We ne (1910) epo ed he ea men o
pa ien s who did no espond o quinine ea men [Pe e s W 1987]. Despi e he appea ance o
quinine esis ance, he d ug emains ema kably use ul oday, especially as a i s line d ug o
ea ing complica ed ce eb al mala ia. In ac , quinine esis ance is su p isingly uncommon,
and in he ew ins ances i has eme ged, i is o en associa ed wi h pa asi es ha a e al eady
esis an o o he d ugs such as chlo oquine and me loquine [Looa eesuwan S e al.1990;
Meshnick SR 1997; Pe e s W 1987; Puk i ayakamee S e al. 1994]. Chlo oquine, i sel based
on he s uc u e o quinine, was de eloped in Ge many in he ea ly 1940s, esis ance o
chlo oquine was a mo e o hcoming han wi h quinine, and he i s epo s o pa asi es
ailing o espond o he d ug eme ged independen ly om Sou h Ame ica and Sou h Eas
Asia in he la e 1950s [Moo e DV e al. 1961; Young MD e al. 1961]. The sp ead o
esis ance om hese pionee a eas was ela i ely apid, and chlo oquine esis ance is now a
majo p oblem h oughou he mala ia a ec ed a eas o he wo ld.
One o he p oposed mechanisms o he eme gence o d ug esis ance is h ough he p esence
o a d ug a sub- he apeu ic le els wi hin a popula ion. The e can be no doub ha he
eme gence o chlo oquine esis ance in Sou h Ame ica was acili a ed by he policy o
dis ibu ing chlo oquina ed sal o he a ea as pa o a well-in en ioned con ol p oblem. This
esul ed in a la ge p opo ion o he popula ion being exposed o he d ug a sub-cu a i e
69
doses, hus conside ably enhancing he chances o selec ion o chlo oquine esis an pa asi es
[Payne D 1988]. The p oli e a ion o chlo oquine esis ance esul ed in he de elopmen o
me loquine, a d ug ha was e ec i e agains chlo oquine esis an pa asi es. Ini ial
indica ions ha me loquine esis ance was likely o eme ge came, howe e , in 1977, when
esis ance was expe imen ally induced in a oden mala ia pa asi e [Pe e s W e al. 1977].
E o s o educe he possibili y o he eme gence o esis an pa asi es in he ield by using
me loquine in combina ion wi h o he d ugs (especially py ime hamine) me wi h ailu e,
howe e , and epo s o me loquine esis an pa asi es eme ged h oughou he 1980s [Pe e s
W 1998]. In oduced as a i s line ea men o Thailand in 1984, subs an ial esis ance had
de eloped wi hin 6 yea s [P ice RN e al. 2004].
Conce ning he gene ic o d ug esis ance, wo main genes ha e been implica ed in
chlo oquine esis ance; he p md 1 (P. alcipa um mul i d ug esis ance 1 gene) and he p c
(P. alcipa um chlo oquine esis ance anspo e gene).
P md 1 p o ein is a memb ane p o ein, belonging o he sub g oup o ABC- ype mul id ug
anspo sys em. P. alcipa um gene md 1 is localized on ch omosome 5 and acco ding o he
P. chabaudi syn eny map he pcmd 1 (gene homologous on P. chabaudi o he p md 1) gene
is localized on ch omosome 12.
P c p o ein is a diges i e acuole ansmemb ane p o ein, associa ed o chlo oquine
esis ance. P. alcipa um gene c is localized on ch omosome 7 and acco ding o he P.
chabaudi syn eny map he p c gene e e ed o as cg10 gene is localized on ch omosome 6.
I has been shown ha some poin polymo phisms in he p md 1 gene can be co ela ed wi h
chlo oquine esis ance in some ield isola es [Basco LK e al. 1995, Cox-Singh J e al. 1995,
Du aisingh MT e al. 1997, Du aisingh MT e al. 2000]. Simila ly, ans ec ion wo k has
sugges ed ha he gene p md 1 can modula e he sensi i i y o chlo oquine [Reed MB e al.
2000]. Howe e he e a e o he s udies ound in he li e a u e wi h pa asi es collec ed om he
ield whe e no associa ion be ween poin polymo phisms in he gene p md 1 and chlo oquine
esis ance was ound [Chaiya oj SC e al. 1999, C eme G e al. 1995, Po oa MM e al.
1998]. In addi ion, he analysis o a gene ic c oss be ween a P. alcipa um chlo oquine-
esis an clone; Dd2 and a chlo oquine sensi i e one, HB3, showed ha mu a ions in he gene
p md 1 did no gene ically seg ega e wi h chlo oquine esis ance [Wellems TE e al. 1990].
La e de ailed linkage analysis and ine ch omosome mapping o he p ogeny clones o he P.
alcipa um Dd2 x HB3 gene ic c oss allowed he iden i ica ion o ano he gene, he p c
gene, in which a pa icula mu a ion a he amino acid posi ion 76 (K76T) a lysine o a
h eonine change, ha appea s o co ela e comple ely wi h chlo oquine esis ance among

70
ield isola es o P. alcipa um [Fidock DA e al. 2000]. Babike HA and colleagues desc ibed
ha he combina ion o mu a ion in bo h p md 1 and p c gene loci con e highe chlo oquine
esis ance pheno ypes in ield popula ions o P. alcipa um [Babike HA e al. 2001].
In ela ion o he gene ic o me loquine esis ance, in i o s udies on P. alcipa um ha e
shown ha gene ic ampli ica ion o he p md 1 gene may co ela e wi h bo h me loquine and
quinine esis ance [Cowman AF e al. 1994, Peel SA e al. 1994]. Howe e , ield s udies on
he associa ion be ween he p md 1 gene and he pa asi e esponse o me loquine ha e no
p o ided unanimous esul s, while some appea o ha e shown an associa ion be ween he
ampli ica ion o he p md 1 gene and me loquine esis ance [P ice RN e al. 1997, P ice RN e
al. 1999, Wilson CM e al. 1993] o he s as Chaiya oj SC and co-wo ke s ha e no ound any
co ela ion be ween he wo e en s [Chaiya oj SC e al. 1999], one possible explana ion
comes om he ac ha he geno ype-pheno ype associa ions depends om he pa asi e
geog aphical o igin. In addi ion and as wi h chlo oquine i has also been demons a ed
h ough gene ic c ossing and ans ec ion expe imen s ha poin mu a ions in he p md 1 gene
may modula e he sensi i i y o bo h me loquine and quinine in P. alcipa um [Du aisingh
MT e al. 2000, Reed MB e al. 2000]. In P. chabaudi C a o PV and colleagues [C a o PV e
al. 2003] shown ha ampli ica ion o he pcmd 1 gene (gene homologous o he P. alcipa um
md 1 gene) is an impo an e en in he gene a ion o me loquine esis ance, pa alleling he
si ua ion obse ed by Cowman AF and co-wo ke s and Peel SA and colleagues [Cowman AF
e al. 1994, Peel SA e al. 1994] hough o he genes a e sugges ed o be also in ol ed.
71
1.7.1 Resis ance o a emisinin
Clinical pa asi e esis ance o a emisinin d ugs has no ye been obse ed, al hough a ia ions
in sensi i i y ha e been desc ibed [ an Ag mael MA e al. 1999] o example a ious isola es
o P. alcipa um om Vie nam and Thailand ha e been ound o a y in hei sensi i i y o
a emisinins in i o [B ockman A e al. 2000; Woi sch B e al. 2004; Wongs ichanalai C e
al. 1997; Wongs ichanalai C. e al. 1999]. Di e ences in he sensi i i y le el o P. alcipa um
isola es o a emisinins can be due o gene ic al e a ions o he pa asi e ha con e di e en ial
sensi i i y o hese d ugs o simply can be due o he na u al gene ic a ia ion o he pa asi e
in ha pa icula pa o he wo ld.
To his s age, se e al p o eins, including a Ca
2+
-depending SERCA ype ATPase p o ein ha
in P. alcipa um is codi ied by he p a p6 gene ha is localized in P. alcipa um ch omosome
1, he P. alcipa um chlo oquine esis ance anspo e p o ein codi ied by he p c gene, he
P. alcipa um mul id ug esis ance p o ein-1 codi ied by he gene p md 1, and he
ansla ionally con olled umo p o ein ( c p) ha is codi ied by he gene p c p in P.
alcipa um ( his gene being localized in P. alcipa um ch omosome 5), ha e been implica ed
in modula ion o pa asi e suscep ibili y o a emisinin d ugs.
Mu a ions in p c gene we e associa ed wi h inc eased suscep ibili y o P. alcipa um isola es
ga he ed in Asia, A ica and Sou h Ame ica [Sidhu AB e al. 2002] o a emisinin.
Measu emen o he p md 1 gene copy by eal- ime PCR on iled isola es ha we e
signi ican ly mo e esis an o me loquine, quinine, a emisinin and a esuna e and mo e
sensi i e o chlo oquine showed ha his isola es had 3 copies o p md 1 gene (in no mal
condi ions p md 1 is a single copy gene), hus, educed in i o sensi i i y o a emisinin and
a esuna e was co ela ed o an inc eased gene copy numbe s o he gene p md 1 [Picka d AL
e al. 2003, P ice RN e al. 2004]. P ice RN and colleagues also associa ed polymo phisms in
he gene p md 1 wi h an inc eased a emisinin suscep ibili y in isola es wi h a single copy o
he gene p md 1, in his s udy he N86Y mu a ion was associa ed wi h lowe IC
50
s o
me loquine han in hose isola es wi h a wild- ype p md 1 gene. By con as , he p esence o
ei he he S1034C mu a ion o he N1042D mu a ion in isola es wi h single copies o he gene
p md 1 was associa ed wi h a highe a esuna e IC
50
s han was he wild- ype, a bo h hese
loci. On he o he hand o he poin polymo phisms o he gene p md 1 ha e been associa ed
wi h inc eased sensi i i y o a emisinin [Du aisingh MT e al. 2000]. Likewise, he iple
mu a ion in he gene p md 1, S1034C/N1042D/D1246Y, highly p e alen in Sou h Ame ica,
was ound o enhance pa asi e suscep ibili y o me loquine, halo an ine and a emisinin
[Sidhu AB e al. 2005]. F om all hese s udies he gene p md 1 appea s o be one impo an
72
modula o o he pa asi e suscep ibili y o a emisinin d ugs. Highe copy numbe s o his
gene p edic ea men ailu e e en o chemo he apy wi h he highly e ec i e combina ion o
me loquine and 3 days a esuna e [P ice RN e al. 2004]. I seems ha he e is a co ela ion
be ween he esis ance o a emisinins and o o he an imala ials. Since he gene p md 1 has a
gene al impo ance in an imala ial d ug esis ance, induc ion o esis ance o one d ug may be
ollowed by esis ance o o he d ugs ha a e no ac i e by he same mechanism [Ande son TJ
e al. 2005; Du aisingh MT e al. 2000; Fe e -Rod iguez I e al 2004; Ngo T e al. 2003;
Picka d AL e al. 2003; P ice RN e al. 2004; Reed MB e al. 2000; Sidhu AB e al. 2005].
Resis ance o a emisinin in P. yoelii had been p e iously been selec ed by d ug p essu e and
was co ela ed o a high p o ein exp ession le el o pa asi e c p p o ein (T ansla ionally
Con olled Tumo P o ein Homolog) [Walke DJ e al. 2000], which has been shown o bind
a emisinin [Bhisu hibhan J e al. 1998], howe e , he esis an pa asi es eadily los
esis ance once d ug-selec ion p essu e was wi hd awn [Pe e s W e al. 1999], so no clea
associa ion was ac ually made be ween a emisinin esis ance and he p o ein c p, as being a
ansien pheno ype, he gene ic in ol ed on his esis ance pheno ype is no possible, ha is
why one o he mains objec i es o his p ojec was o selec a emisinin esis ance o s able
pheno ype.
So in conclusion we can say ha we hink ha he gene ic mechanism esponsible o
a emisinin and i s de i a i es esis ance need cla i ica ion. To s a he p ocess o
cla i ica ion i is be e o cla i y ha al hough s udies made di ec ly on P. alcipa um may
p o ide mo e incisi e in o ma ion, his p esen s se e al limi a ions s a ing by he ac ha in
he a emisinin case he e is no a emisinin esis ance ye epo ed, so he wo k in animal
models can ci cum en ed all he human mala ia pa asi e limi a ions. The e a e se e al oden
mala ia models a ailable; hose will be p esen ed in he ollowing chap e .
73
1.8 Roden mala ia pa asi es
The hos speci ici y o human mala ia pa asi es ep esen s a majo cons ain on he s udy o
mala ia as, unlike he o he majo opical diseases such as ypanosomiasis and leishmaniasis,
he ac ual causa i e o ganisms canno be main ained in con enien small labo a o y animals.
The need o sui able labo a o y models has esul ed in he use o a ian and simian pa asi es
and un il 1948 hese models we e he only ones a ailable.
In 1948 he si ua ion changed wi h he disco e y and isola ion o a mala ia pa asi e ha was
capable o in ec ing labo a o y a s and mice [Vincke IH e al. 1948]. This pa asi e,
Plasmodium be ghei, soon became he mos in ensi ely s udied mala ia pa asi e.
All expe imen s desc ibed wi hin his PhD hesis we e ca ied ou using he oden mala ia
pa asi e, Plasmodium chabaudi. P. chabaudi belongs o a g oup o ou Plasmodium species
ha in ec mu ine oden s om Cen al A ica he o he species being Plasmodium inckei,
Plasmodium yoelii and Plasmodium be ghei. A map o he loca ions om which he a ious
oden pa asi es we e isola ed is shown in Figu e 8.
Figu e 8 The geog aphic o igins o he oden mala ia pa asi es.
F om Ca l on JM e al.2001 wi h kind pe mission o D a. Jane Ca l on.
80
1.9 Linkage G oup Selec ion
As any o he li ing o ganism, in Plasmodium he iden i ica ion and ully unde s anding o he
gene ic mechanisms in ol ed in impo an pheno ypes such as d ug esis ance is o g ea
impo ance. In he pa icula case o d ug esis ance he knowledge o he genes in ol ed in
his pheno ype allows epidemiological s udies and o he long e m s udies o example as he
ones moni o ing he esis ance o a pa icula an imala ial in he ield. In he case o mala ia
pa asi es he e a e nowadays mainly wo gene ic me hods ha can be used o y o loca e
genes con olling any gene ic ai such as d ug esis ance o example; hose a e linkage
analysis and linkage g oup selec ion (LGS).
When using classical linkage analysis o iden i y he gene ic loci in ol ed in d ug esis ance,
he esis an mu an s need o be c ossed wi h gene ically dis inguishable pa asi es (like AS
and AJ s ains o example), his gene ically dis inguishable pa asi es mus di e in a numbe
o gene ic ma ke s. These ma ke s can be es ic ion leng h polymo phism (RFLPs) o
mic osa eli es o example, and will dis inguish he sensi i e om he esis an pa asi es.
When using classical linkage analysis, he p ogeny o he gene ic c oss ob ained needs o be
cloned and he esul ing cloned p ogeny will be analysed o linkage o he pheno ype, le s
conside o he momen d ug esis ance, wi h he inhe i ance o any pa en al ma ke s ei he
esis ance o sensi i e. Classical linkage analyses b ing o no ice g oups o ma ke s whose
inhe i ance is linked o he inhe ence o d ug esis ance. In he e en ual case o ha ing a
numbe o ma ke s conside ed su icien a linkage map can be cons uc ed [ e ised by Ca e
R e al 2007]. Classical linkage analysis has been used be o e wi h success o iden i y egions
o he pa asi e genome ha a e impo an o d ug esis ance as o example he case o
Wellems TE and co-wo ke s, Ca l on JM and co-wo ke s and Hun P and collabo a o s
[Ca l on JM e al. 1998, Hun P e al. 2004b, Wellems TE e al. 1991] bu only once has i
lead o he ac ually iden i ica ion o a gene esponsible o a pa icula pheno ype, in his case,
i was he iden i ica ion o p c has he gene in ol ed in chlo oquine esis ance [Fidock DA
e al. 2000, Su X e al. 1997]. Due o he ac ha o using classical linkage analysis a la ge
numbe o gene ic ma ke s is indeed necessa y, i is also necessa y o know he posi ion o
hese ma ke s in he pa asi e genome and hen in ol es he cloning o a la ge numbe o
clones om he ecombinan p ogeny and also he gene ic cha ac e iza ion o each one, his
echnique is e y labo ious and ime consuming.
The o he echnique ha enables he disco e y o genes con olling biological p ope ies in
mala ia pa asi es as s a ed be o e is linkage g oup selec ion (LGS). LGS is a no el app oach
de eloped o mala ia pa asi es in o de o iden i y genes esponsible o selec able

81
pheno ypes, as o example d ug esis ance. LGS was p e ious alida ed o inding genes
in ol ed in d ug esis ance by Culle on R and co-wo ke s [Culle on R e al. 2005]. LGS can
be applied o he gene ic analysis o any mala ia pa asi e as long as he e a e expe imen al
means o in ec ing mosqui oes wi h game ocy es om pa asi es and also means o passaging
he gene ic p ogeny o a c oss h ough he li e s age o de elopmen and in o he blood. LGS
has in common o he classical linkage analysis he ac ha i is an app oach ha uses a
gene ic c oss be ween wo un ela ed pa asi es om he same species, one o which is sensi i e
and he o he one is esis an o a pa icula cha ac e is ic ha is going o be applied as
selec i e p essu e [Culle on R e al. 2005], bu di e s om he adi ional app oach by
a oiding cloning e y la ge numbe s o cloned lines om he p ogeny o a gene ic c oss
[Ca e R e al. 2007]. In LGS he uncloned p ogeny o a gene ic c oss, be ween a sensi i e
and a esis an pa asi e o a pa icula cha ac e is ic and ha a e gene ically dis inguishable by
a la ge numbe o gene ic ma ke s such as AFLPs ma ke s o example, is placed unde a
selec ion p essu e ep esen ing he biological p ope y o use, in he case o ou p ojec ;
a emisinin and a esuna e esis ance. The DNA ob ained om he su i ing p ogeny is hen
sc eened om he p esence o a la ge numbe o molecula ma ke s dis ibu ed h oughou he
pa asi e genome. P io o he de eloped o he LGS echnique an a las o a la ge numbe o
molecula gene ic ma ke s dis inguishing o example he wo di e en s ains AS and AJ was
de eloped [Ma inelli A e al 2005]. The gene ic ma ke s om he sensi i e p ogeni o ha
a e linked o he gene o in e es ( o example in ou case he gene con e ing a emisinin
esis ance) will be unde - ep esen ed o e en elimina ed om he p ogeny o he gene ic c oss
a e he d ug selec ion. Finding he gene ic posi ion o he ma ke s unde selec ion would
allow us an a ea in he genome whe e he gene o in e es migh be loca ed. I is impo an o
no iced ha he in ensi y o educ ion o any pa icula ma ke is di ec ly p opo ional o he
dis ance o he gene o in e es hus allowing he cons uc ion o a selec ion alley a ound he
gene ic a ea o in e es , whe e he a ge gene in supposedly loca ed a he alley base [Ca e
R e al. 2007]. See Figu e 11 o he esume o he LGS p o ocol.
82
Figu e 11 – Schema ic ep esen a ion o he Linkage G oup Selec ion p o ocol.
P
Pa
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83
1.9.1. Ampli ied F agmen Leng h Polymo phism
LGS equi es ha he wo pa en al clones a e dis inguished by a la ge enough numbe o
gene ic ma ke s o ensu e ha some will be linked o he genes o in e es . LGS was
op imized using ampli ied agmen leng h polymo phism (AFLP) a PCR-based me hod o
ampli ying DNA agmen s om gene ically dis inc cloned lines o pa asi es, o example AS
and AJ. AFLP is a echnique in which la ge numbe s o ma ke s a e gene a ed ac oss a
genome [Masiga DK e al. 2000]. Ha ing a high densi y o hese ma ke s in he genome
means ha he e is a high p obabili y ha some o hem will be linked o he gene o in e es .
I has been p e iously shown ha AFLP mee s he equi emen s o LGS bo h as ega ds
numbe s o ma ke s gene a ed in di e en s ains o P. chabaudi [G ech K e al. 2000] and
hei quan isa ion in a mix u e o he s ains [Ma inelli A e al. 2004].
The AFLP echnique allows he isualiza ion o es ic ion agmen s o DNA. This enables
he de ec ion o he a ia ion in DNA be ween wo s ains wi hou p io knowledge o he
nucleo ide sequence. Depending upon he polymo phism be ween he wo s ains, la ge
numbe s o gene ic ma ke s can be p oduced in a ela i ely sho ime [Vos P e al. 1995].
AFLP in ol es cu ing genomic DNA in o a la ge numbe o DNA agmen s wi h wo
di e en es ic ion enzymes (an enzyme called a equen cu e and ano he called a a e
cu e ), hus gene a ing op imal size agmen s o isualiza ion on polyac ylamide gels.
Using adia ion o label one o he p ime s allows isualiza ion o he p oduc s. Gene ic
di e ences be ween s ains occu ing a a cu ing si e esul in agmen s o di e en sizes
which can hen be iden i ied on a gel as being p esen in one s ain and absen in he o he
(See Figu e 12 o an example o an AFLP gel).
Polymo phic bands can be p oduced as a esul o polymo phisms be ween s ains a he
enzyme ecogni ion si es, polymo phisms be ween s ains a he selec i e bases used in each
PCR, o as a esul o inse ion/dele ion polymo phisms wi hin agmen s.
84
1.10 Aims o he p ojec
This p ojec in ol es iden i ying and cha ac e izing genes in ol ed in esis ance o
a emisinin using he oden mala ia specie P. chabaudi, s a ing by selec ing s able
a emisinin d ug esis ance.
To selec a emisinin and a esuna e mu an s using he oden model, P. chabaudi, hough
p olonged exposu e o d ug-sensi i e lines o low and inc easing le els o he d ug in mice.
A emp s o selec ing a emisinin and a esuna e mu an s in P. chabaudi would be ca ied ou
hough p olonged exposu e o d ug-sensi i e lines o low and inc easing le els o he d ug in
mice; so ha he su i ing pa asi es o one lowe dose o a esuna e and a emisinin would
ecei e an inc easing dose o he same d ug. These esul s will be p esen ed on
chap e III
.
A e selec ing mu an clones esis an o a emisinin and a esuna e he objec i e would be, as
a i s app oach, o s udy he in ol emen o P. chabaudi gene o hologues p md 1, p c ,
p c p and p a p6, in he selec ed mu an clones, p e iously desc ibed as being pu a i e gene ic
modula o s o a emisinin. These esul s will be p esen ed on
chap e IV
.
The las objec i e was o pe o m gene ic c osses in mosqui oes wi h he p e iously selec ed
and cloned a emisinin and a esuna e mu an s in P. chabaudi (AS-ART and AS-ATN
espec i ely) and he gene ic dis inc i e sensi i e pa asi e line o P. chabaudi AJ, and o
pe o m linkage g oup selec ion (LGS) on he gene ic c osses using a emisinin and
a esuna e; hese esul s will be p esen ed on
chap e V
.
85
CHAPTER II
MATERIALS AND METHODS

86
87
2.1. Mice
Inb ed emale CD1 (Mus musculus) om Ha lan-Tekld Ibe ica we e used o d ug es s and
selec ion expe imen s. Inb ed emale CBA/CA and C57B1/6J mice (Mus musculus) om he
Uni e si y o Edinbu gh we e used in he es o he expe imen s desc ibed in his wo k. All
mice used we e 4-6 weeks old a he s a o he expe imen s. They we e housed in
polyp opylene cages wi h sawdus as bedding, and we e p o ided wi h 41B a and mouse
main enance die (Ha lan-Tekld) ad libidum. D inking wa e was supplemen ed wi h 0.05%
pa aminobenzoic acid (PABA), an essen ial elemen o he pa asi e g ow h, and gi en ad
libidum. The cages we e kep in a oom whe e empe a u e was main ained a a cons an 25º C
± 3 º C, on a 12-hou ligh /da k cycle.
2.2. Mosqui oes
Anopheles s epehensi mosqui oes we e used o all expe imen s. Insec a ies we e main ained
on a 12-hou ligh /da k cycle a 25-27º C empe a u e and 75-86 % humidi y. La ae we e ed
on Liqui y™ un il 2
nd
ins a and he ea e on g ound Te amin™ ish ood, Adul
mosqui oes we e kep on 10% glucose and 2% PABA-supplemen ed wa e solu ion. S ock
adul s ecei ed weekly a -blood eeds, which a e essen ial o he p oduc ion o eggs.
2.3. Pa asi es
All pa asi es used in he expe imen s desc ibed he e we e Plasmodium chabaudi chabaudi
( e e ed o as P. chabaudi he ea e ). See Table 5 and also Table 6.
Table 6 - Pa asi e clones and lines used in he p esen wo k.
CLONE DRUG RESPONSE
AJ D ug sensi i e, gene ically di e en and dis inguishable om AS line
AS-PYR De i ed om AS-SENS; py ime hamine- esis an
AS-15CQ De i ed om AS-3CQ; esis an o 6 daily doses o chlo oquine a
5mg/kg mouse body weigh
AS-30CQ De i ed om AS-15CQ; esis an o 6 daily doses o chlo oquine a
30 mg/kg mouse body weigh
88
2.4 P epa a ion o 10
7
s anda d pa asi e inocula
10
7
iRBC was es ablished as he s anda d pa asi e numbe o be in ec ed in o indi idual mice
a he ime o he d ug es s, du ing selec ion expe imen s and du ing p epa a ion o inocula
o he p oduc ion o mixed in ec ions.
The p epa a ion o he inocula o d ug es s and o he selec ion expe imen s was as ollows:
he pa asi aemia and ed blood cell densi y (RBC/ml) we e calcula ed in he dono mouse,
a e which he equi ed amoun o blood was collec ed om he mouse ail by calib a ed
capilla y pipe e and dilu ed o a inal concen a ion o 10
7
iRBCs/0.1 ml in a solu ion o
hepa inised 1:1 cal se um/mammalian Ringe ’s solu ion (Appendix 1). P epa a ions we e
kep on ice a all imes and an aliquo o 0.1 ml was inocula ed in o mice.
Fo he p epa a ion o inocula o he p oduc ion o mixed in ec ions con aining equal
p opo ions o wo pa asi e clones, in ec ions o bo h clones in o a single mouse we e induced
as desc ibed abo e.
2.5 Cloning
Dilu ions o cloning we e p epa ed as in 2.4 excep ha each mouse was in ec ed ei he wi h
a mean o 0.5 o 1 pa asi e. G oups o 50 mice we e inocula ed in hese expe imen s. I
app oxima ely 30% o mice became in ec ed, i could be p edic ed ha 75% o he in ec ions
had esul ed om a single pa asi e; a lowe pe cen age o in ec ed mice would indica e highe
p opo ion o pu e clones. Cloning was done once d ug esis ance s abili y was e i ied.
2.6 P epa a ion and adminis a ion o a emisinin and a esuna e
A emisinin powde was ob ained as a gi om A ican A emisia.
A esuna e powde was ob ained as a gi om Daph a Pha ma.
In he ini ial es s a emisinin was dissol ed in dime hyl sulphoxide (DMSO) and co n oil, and
a esuna e was dissol ed in DMSO, Na
2
CO
3
o co n oil, bu o u he analysis DMSO was
always used.
Bo h a emisinin and a esuna e we e eshly dilu ed daily in dime hyl sulphoxide (DMSO)
and we e kep a oom empe a u e p o ec ed om he ligh . Bo h d ugs we e adminis e ed o
mice by ga age using a lub ica ed ca he e adap ed o a 1 ml sy inge. D ug doses we e
exp essed as millig ams o d ug pe kilog am (Kg) o mouse body weigh pe day. Bo h d ugs
89
we e dilu ed o a concen a ion such ha he amoun o d ug co esponding o he desi ed dose
was p esen in 0.1 ml when gi en o a mouse weighing 20 g ams. A he ime o d ugging,
mice we e indi idually weighed so ha he amoun o d ug gi en could be adjus ed.
2.7 D ug es s
Ini ially, a emisinin and a esuna e d ug es ials we e ca ied ou on ou P. chabaudi
clones (AS-PYR, AS-15CQ and AS-30CQ), o es ablish he app op ia e d ug egimen and a
s anda d es o dis inguish be ween esis an and sensi i e pa asi es as well as pa asi es wi h
in e media e le els o esis ance.
Mice ecei ed 10
7
iRBC each by in a-pe i oneal (i. p.) injec ion.
G oups o i e mice we e p epa ed; one g oup was un ea ed and used as a con ol o he
in ec ion, while he emaining g oup was d ugged h ee hou s a e injec ion, o allow
pa asi es o each he blood s eam. Depending on he pa icula d ug egimen he d ug dose
was epea ed e e y 24 hou s o he desi ed numbe o days.
Blood smea s om con ol and a emisinin o a esuna e ea ed mice in he single dose, in he
h ee-day supp essi e es and in he i e-day supp essi e es , we e aken on day i e pos
in ec ion and e e y day he ea e , un il he in ec ion peaked o i was clea ha no pa asi es
we e going o appea . The pa asi aemias and numbe o days o ec udescence o he
di e en pa asi e lines we e compa ed.
2.8 A emisinin and a esuna e selec ion expe imen s: gene al p ocedu e
Two g oups o i e (4-6 week old) CD1 mice we e inocula ed wi h 10
7
pa asi es o he clone
o be used o selec ion o inc eased a emisinin o a esuna e esis ance.
Th ee hou s a e inocula ion one o he g oups we e ea ed o ally wi h he equi ed doses o
a emisinin o a esuna e o a o al o i e days. The emaining g oup was le un ea ed and
se ed as a con ol. Bo h con ols and pa asi es ha su i ed d ug ea men we e passaged
weekly om he mouse exhibi ing he highes pa asi aemia in o unin ec ed mice and he
ea men epea ed. The d ug doses we e inc eased acco ding o he pa asi e esponse o
ea men in he p e ious passage. To add ess he possibili y ha po en ial inc eases in d ug
ole ance could be o due o inc eased i ulence a ibu ed o mul iple sub-inocula ions, an
un ea ed and unselec ed pa asi e line was main ained in pa allel and passaged in un ea ed
mice he same numbe o imes as he d ug selec ed line. Following hese passages in he
96
Fo he AS-ATN x AJ c oss, pa asi es we e pooled and sub-inocula ed om he “non-
passaged” g oup in o wo u he g oups o mice, one o which was ea ed wi h a esuna e
(“ATN ea ed” g oup), and he o he le un ea ed (“un ea ed” g oup). These wo g oups
p o ided he ma e ial o he compa ison o ma ke s be ween d ug- ea ed and un ea ed
pa asi e popula ions. A emisinin was adminis e ed o ally a a dose o 25 mg/kg o mouse
body weigh daily a 24-h in e als o 5 days, s a ing 3h a e pa asi e challenge. A esuna e
was adminis e ed o ally a a dose o 5 mg/kg o mouse body weigh daily a 24-h in e als o
5 days, s a ing 3h a e pa asi e challenge. Bo h he a emisinin- ea ed and a esuna e- ea ed
and he un ea ed blood-s age c oss p ogeny we e allowed o g ow o peak pa asi aemia
(30%–40% o un ea ed, and 20%–30% o ea ed), a which poin he blood was ha es ed.
Two samples o pa asi e DNA we e p epa ed o AFLP and o he molecula analyses by
pooling sepa a ely he blood om he ea ed and un ea ed mice.
2.16.2 Ampli ied F agmen Leng h Polymo phism (AFLP) analysis
2.16.2.1 P epa a ion o pa asi e DNA om expe imen al g oups
When in ec ions eached peak pa asi aemia, blood was ex ac ed om all mice in each g oup
by se e ance o he b achial a e y, pooled, and p epa ed o DNA ex ac ion. Blood was
il e ed in o de o emo e any mouse lymphocy es o o he nuclea ed cells, by passing i
wice h ough a 5 ml column o powde ed cellulose (Sigma) washed wi h ci a e saline. Blood
was hen il e ed h ough Plasmodipu ™ il e s (Eu o-Diagnos ica) wice. The il a e was
cen i uged o 5 mins a 3000 pm and he supe na an emo ed, lea ing a pelle o packed
cells. 0.15% saponin in Phospha e Bu e ed Saline (PBS) was added o p omo e cell lysis
cells. A e lysis o e y h ocy es occu ed (associa ed wi h a change in colou o he solu ion
om b igh ed o bu gundy colou ), PBS was added in excess o p e en pa asi e lysis. This
solu ion was hen cen i uged again a 4000 pm o 5 minu es and washed wice in PBS.
Supe na an was disca ded and pelle s s o ed a -70°C. Th ee hick blood smea s we e aken,
one p io o il a ion, one a e cellulose il a ion and one a e Plasmodipu ™ il a ion in
o de o de e mine he e iciency o hos cell emo al a each s age.
The ozen pelle was e-suspended in 0.4 ml bu e A (Appendix 1), and 10 µl o 10% SDS
and 50 µg P o einase K (Sigma) we e added. The pelle was le a 37°C o e nigh a e which
an equal olume o 1:1 phenol/chlo o o m mix u e was added, mixed o 3 min, and
cen i uged a 5,000 g o 1-2 minu es. The uppe aqueous laye was ans e ed o a esh

97
ube. The s ep was epea ed 2-3 imes. Then an equal olume o chlo o o m was added, and
he ube was cen i uged as be o e o 1-2 minu es. The uppe aqueous laye was emo ed o a
esh ube. This p ocedu e was epea ed once be o e an equal olume o e he was added, he
solu ion cen i uged as be o e o 1-2 minu es and he uppe laye emo ed. The emaining
e he was le o d y in he ai . Th ee olumes o absolu e e hanol (0º C) and 1/10
h
olume o
3M sodium ace a e (pH 5.2) we e added o he d ied pelle , and he ube was mixed and
placed on ice o 15-45 minu es o p ecipi a e he DNA. The ube was hen cen i uged a
10,000 g in a Speed Vac (Sa an ) o 10 minu es and he e hanol mix u e emo ed. The ube
was again cen i uged a 10,000 g o 5-10 minu es o emo e inal aces o e hanol and he
pelle was esuspended in 100-200 µl o TE bu e (pH 8.0) (see Appendix 1) and le a 37°C
o 10 minu es be o e s o age a -20º C.
2.16.3 AFLP analysis
0.5µg o pa asi e genomic DNA was cu using wo enzymes. Fi s ly, 10 U o EcoRI (MBI
Fe men as, ecogni ion sequence: G↓AATTC) we e added and DNA incuba ed a 37°C o 1
h, hen 5 U o T uI/MseI (MBI Fe men as, ecogni ion sequence: T↓TAA) we e added and
DNA incuba ed o u he 3 h a 65°C.
The diges ion s age was pe o med in a 40 µl solu ion con aining 2X Y+/ Tango bu e
(P omega). The agmen s we e hen liga ed wi h adap e s ma ching he cu ends p oduced by
he enzymes.
All p ime s used we e p o ided by MWG-Bio ech UK L d. Adap e s dis up ed he cu ing si e
ecognised by he enzymes in o de o p e en cu ing o he adap e s om he DNA
agmen .
These adap e s also p o ided a ecogni ion si e o p ime s.
-MseI adap e s: MeI.a1 and MeI.a2
-MeI.a1: 5’-GACGATGAGTCCTGAG-3’
-MeI.a2: 3’-TACTCAGGACTCAT-5’
-EcoRI adap e s: EoI.a1 and EoI.a2
-EoI.a1: 5’-CTCGTAGACTGCGTACC-3’
-EoI.a2: 3’-CATCTGACGCATGGTTAA-5’
98
Bold le e s indica e base subs i u ion o dis up enzyme cu ing si e.
Adap e s we e p epa ed by adding equimola amoun s o bo h s ands and hen pe o ming he
ollowing p ocedu e:
- Hea adap e s a 94°C o 5 mins, hen cool o 21°C o 5 mins
- Hea adap e s a 72°C o 60 s., hen cool o 21°C o 5 mins
- Hea adap e s a 65°C o 60 s., hen cool o 21°C o 5 mins.
Adap e s we e hen dilu ed o 50 pmol/µl. 10 µl o liga ion mix u e we e hen added o 40 µl
o diges ed DNA and his incuba ed a 37°C o 3h, hen o e nigh a 15°C. Liga ed ma e ial
was dilu ed 1:10 in TE bu e (pH 8.0) (Appendix 1) and s o ed a -20°C.
A p elimina y PCR ampli ica ion was pe o med in ol ing he use o “non-selec i e” p ime s
(meaning p ime s wi h no ex a nucleo ides added a hei 3’ ends ex ending beyond he
adap e s sequence) ma ching he adap e s:
- Non-selec i e EcoRI p ime : 5’-GACTGCGTACCAATTC-3’
- Non-selec i e MseI p ime : 5’-GATGAGTCCTGAGTAA-3’
A 20µl PCR solu ion con aining 0.32 µM o each o he non-selec i e p ime s, 1 µl empla e
DNA, 0.4 U Taq polyme ase (P omega), 1X Mg- ee PCR Bu e (P omega), 1.5 mM MgCl2,
and 0.2mM o all 4 dNTPs was se up. The ollowing cycles we e pe o med:
- 94°C o 60 s., hen
- 94°C o 30 s.
- 56°C o 60 s.
- 65°C o 60 s.
epea he h ee s eps o 20 cycles
The esul ing PCR ma e ial was dilu ed 50- old in TE bu e (pH 8.0) and s o ed a - 20°C.
Selec i e ampli ica ion was pe o med using adiolabelled p ime (EcoRI-p ime ) and
selec i e p ime s (meaning p ime s wi h a selec i e ex ension a he 3’-end, in o de o educe
he numbe o agmen s ampli ied). Fo adiolabeling, 2.5x Kinase bu e (P omega), 20U T4
polynucleo ide Kinase (P omega), 100 µCi [γ-
33
P] ATP / [γ-
32
P] ATP (ICN) and 500 ng o
oligonucleo ide p ime we e incuba ed in a 20 µl solu ion a 37°C o 60 min. The eac ion
99
was s opped by adding 1 µl o 0.1 M EDTA pH 8.0 and hea ing a 70°C o 10 min. The
mix u e was hen made up o a olume o 50 µl by adding s e ile, dis illed wa e .
P ime pu i ica ion was pe o med using TE Mic o Selec -D, G-25 mic ocen i uge spin
columns p oduced by Eppendo -5 P ime Inc.
Ho PCR wi h he adiolabelled p ime was pe o med as ollows: 0.32 µM o he selec i e
MseI p ime , 0.05 µM o he labelled selec i e EcoRI p ime , 1 µl empla e DNA, 0.4 U Taq
polyme ase (P omega), 1X Mg- ee PCR Bu e (P omega), 2.5 mM MgCl2, and 0.2mM o
all 4 dNTP’s we e added o a 20µl o al olume PCR solu ion.
PCR condi ions o his s age we e:
- 94°C o 60 s., hen
- 94°C o 30 s.
- 65°C o 60 s., annealing empe a u e is educed a each cycle by 0.7°C o he nex 12
cycles, hen emained a 56°C o he emaining 23 cycles
- 65°C o 60 s.
epea he p eceding h ee s eps o 35 cycles
The PCR p oduc s we e mixed wi h 20 µl o loading dye speci ic o poly-ac ylamide
sequencing gels (Anachem), hen hea ed a 99°C o 3 min and immedia ely cooled on ice.
5µl o each sample was loaded on o a 5% dena u ing polyac ylamide gel (5% ac ylamide,
0.25% me hylene bisac yl, 7.5 M u ea in 50mM T is/50mM Bo ic acid/1mM EDTA). 500 µl
o a 10% Ammonium Pe sul a e solu ion (APS) and 100µl o TEMED (Sigma) we e added o
100 ml o gel solu ion and he gel cas using a SequiGen 38x50 cm gel appa a us (BioRad).
Elec opho esis was pe o med a 110 W o 2 h in 1x TBE bu e . Gels we e hen d ied in a
acuum gel d ie (model 583, Bio Rad) and exposed o e nigh in phospho image sc eens
(Fuji) a -70°C. Resul s we e isualised on an au o adiog aphy ilm (Kodak XAR-5). They
we e de eloped in an au oma ic au o adiog aphe de elope (Exog aph).
As an example o an AFLP gel and he band nomencla u e see Figu e 12.
Thus, and jus as an example, he ma ke AJAG02CA deno es he second la ges ( ha is
whe e he 02 cames om) AJ-speci ic band ( hus beginning wi h AJ) ob ained using EcoRI
p ime s wi h AG ( he i s pai o nucleo ide le e s, like he o wa d p ime ) as addi ional
“selec i e” 3’-nucleo ides, and CA as “selec i e” nucleo ides on he MseI p ime s ( he second
pai o nucleo ide le e s, like he e e se p ime ).
100
Figu e 12 - An example o an AFLP gel.
In his pa icula gel we can see he esul s o bands gene a ed wi h Mse1 (CA selec i e bases, so called o wa d
p ime ) + EcoR1 (AG selec i e bases, so called e e se p ime ). One o he ma ke s o he sensi i e pa en
(AJAG02CA) was absen in he ea ed popula ion and is hen p esumably closely linked o he locus con e ing
esis ance.
Each one o he lanes is labelled as ollowed;
AS: deno es DNA ex ac ed om single AS-SENS clone in ec ion (in ou p ojec he AS alleles a e he
esis an ones).
AJ: deno es DNA ex ac ed om single AJ clone in ec ion (in ou p ojec he AJ alleles a e he
sensi i e ones).
1: deno es DNA ex ac ed om he unpassaged pooled g oup.
2: deno es DNA ex ac ed om he ea ed pooled g oup (in he case o ou p ojec he AS-ART x AJ
c oss was ea ed wi h ART and he AS-ATN x AJ c oss was ea ed wi h ATN).
3: deno es DNA ex ac ed om he un ea ed pooled g oup.
Each lane ep esen s a DNA sample ob ained om he pooled DNA o all mice on each expe imen al g oup.
Polymo phic ma ke s a e bands ha a e di e en o AS o AJ. AS speci ic ma ke s ( hus esis an ) a e ma ked
wi h a b oken a ow (), and AJ speci ic ma ke s ( hus sensi i e) wi h an open a ow (), bands ma ks wi h an
a ow () ep esen a non-polymo phic ma ke ( he e a e no di e ences be ween AS and AJ wi hin his ma ke ).
Adap ed om Culle on R e al. 2005 wi h kind pe mission o D Richa d Culle on.
101
2.16.4 Measu emen and compa ison o he in ensi y o AFLP
ma ke s
This echnique makes uses o a la ge numbe o molecula gene ic ma ke s dis inguishing wo
s ains o mala ia pa asi es, he AFLP ma ke s. These ma ke s can be isualised as bands on a
polyac ilamide gel. Mos o he bands ha will appea on a gel will be p esen in bo h he
pa asi es and a e called non-polymo phic bands and some a e unique o one o he clones in
ou case ei he AS - esis an o AJ - sensi i e. Once he selec ion p essu e is applied in he
gene ic p ogeny o a c oss, in he case o his p ojec , a emisinin o a esuna e p essu e, he
pa asi es ha ca y he allele ha is sensi i e (in his case he AJ allele) will be emo ed, and
om an expe imen al poin o iew his disappea ance is isible by he dec ease in in ensi y
o comple e disappea ance o an AFLP band speci ic o he sensi i e allele (AJ band). Thus
he p ogeny a e selec ion is hen sc eened o ma ke s. Specially hose om he sensi i e
pa en ha would ei he be signi ican ly educed o absen because hese ma ke s should be
linked o he locus o loci unde selec ion, in ou case unde a emisinin o a esuna e
selec ion. Polymo phic ma ke s (be ween AS-ART and AJ and be ween AS-ATN and AJ,
he e simpli ied o AS and AJ), meaning hose ha a e di e en be ween AS and AJ, we e
named and placed in he AFLP map, o deno e speci ici y o he polymo phic band meaning
he size o he band ( ela i e o o he polymo phic bands in he same gel lane) and he
selec i e bases used, his would make i e y s aigh o wa d he iden i ica ion o a pa icula
band o he AFLP map al eady de eloped by Ma inelli A and collegues [Ma inelli A e al.
2005]. As s a ed be o e we we e looking o he disappea ing o AJ sensi i e ma ke s on he
ea ed g oup (meaning a emisinin o a esuna e ea ed g oup) in compa ison o he
un ea ed g oup, each one o hese ma ke s dec eased in in ensi y o disappea ed is p edic ed
o lay in wha we call a selec ion alley ha can hen be associa ed o a pa icula gene
unde ling he esis an pheno ype o a leas esponsible o gi en so ype o selec i e
ad an age o e he sensi i e pa asi es. An example o an AFLP gel is ep esen ed in Ma e ials
and Me hods Figu e 12.
As desc ibed in he ma e ials and me hods chap e , each ma ke band in ensi y was measu ed
wi h Phospho Image and IMAGEQUANT so wa e (Molecula Dynamics). Fo each ma ke
o in e es , ei he AS o AJ, an in ensi y index (II) was calcula ed by aking he in ensi y o a
polymo phic ma ke (ei he AS o AJ) and compa e i o a no polymo phic ma ke , making
su e ha his is done using he same PCR ma e ial applied in he same polyac ilamide gel.
Each II is hen con e ed o a ela i e in ensi y index (RII) ei he unselec ed RII
u
o selec ed
RII
s.
The RII
u
is calcula ed by making a a ion be ween he II ob ained o he unselec ed

102
ma e ial and he II ob ained o he pa en al (ei he AS i we a e looking a an AS speci ic
ma ke s o AJ is we a e looking o an AJ ma ke ). The RII
s
is calcula ed by making he a io
be ween he II ob ained o he selec ed ma e ial and he II ob ained o he pa en al (ei he
AS o AJ) [Ma inelli A e al. 2004].
To compa e be ween di e en ma ke s a compa a i e In ensi y (CI) o he polymo phic
ma ke s was calcula ed. This compa a i e in ensi ies a e de ined as he RII o an AFLP
ma ke in he c oss p ogeny selec ed in “ ea ed” mice (RII
), di ided by he RII o he ma ke
o he c oss p ogeny g own in a pa allel “un ea ed” g oup o mice (RII
u
), and exp essed as a
pe cen age:
CI = (RII
/RII
u
) x 100.
Keeping in mind ha he objec i e is o iden i y he genomic loci unde d ug selec ion ei he
by a emisinin o a esuna e ha would co espond o a selec ion alley associa ed o he
esis an pheno ype, AFLP ma ke s wi h low CIs we e iden i ied and hei posi ion acco ding
o he p e iously de ine gene ic linkage map [Ma inelli A e al. 2005] we e no ed.
2.16.5 Assignmen o AFLP ma ke s o loca ions in a P. chabaudi
gene ic linkage map.
Ma ke s had p e iously been o de ed on a gene ic linkage map o P. chabaudi [Ma inelli A
e al. 2004, Ma inelli A e al.2005] ob ained om p e iously gene a ed c osses be ween AS-
de i ed clones and AJ s ains o P. chabaudi [Ca l on JM e al. 1998; Rosa io VE 1976;
Wallike D e al. 1975].
A o al o 674 AFLP ma ke s we e yped o each uncloned p ogeny analysed in his wo k,
and we e subsequen ly assigned o linkage g oups using he p e iously gene a ed map
[Ma inelli A e al. 2005].
A o al o 674 AFLP ma ke s we e yped o each o 28 c oss-p ogeny clones, and we e
subsequen ly assigned o linkage g oups using he Map Manage QTX so wa e [Manly KF e
al. 2001]. A o al o 44 RFLP ma ke s cha ac e ized in a p e ious s udy [Ca l on JM e al.
1998] we e used as gene ic ancho s o allow he assignmen o he a ious linkage g oups o
ch omosomes. In o al, 11 ch omosomes could be iden i ied, while 12 linkage g oups o as ye
unknown assignmen emain, which include he h ee emaining ch omosomes (ch omosomes
2, 4, and 14).
103
2.16.6 Sequencing o AFLP ma ke s, and hei loca ion on he
Plasmodium alcipa um genome
AFLP bands ha appea ed o be unde selec ion we e excised om ac ylamide gels using a
s e ile scalpel. The gel agmen s we e hen soaked in an Eppendo ube in 50 µl o
au ocla ed, dis illed wa e (sdH
2
O) o e nigh .
Gel slices we e cen i uged a 12000g p io o emo al o he liquid phase con aining he
DNA. DNA was p ecipi a ed using 1/10 olume o 3M sodium ace a e (pH 5.2) and 3
olumes o ice-cold absolu e e hanol. The solu ion was placed a -20°C o a leas 1 h.
The ea e he ubes we e spun o 30 minu es a high speed. The liquid phase was emo ed
and he DNA pelle washed wice in 70% e hanol, be o e ai d ying. The pelle was hen
dissol ed in 50 µl TE bu e (pH 8.0).
The ex ac ed DNA agmen was ampli ied by PCR, using he same selec i e AFLP p ime s
and PCR condi ions ha p oduced i .
Sequencing eac ions we e ca ied ou using he p o ocol desc ibed, below in sec ion 2.16.7.
The sequences o ma ke s we e hen physically mapped in he P. alcipa um genome
(sequence da a o P. alcipa um we e ob ained om he Sange Cen e websi e, which can be
accessed a www.sange .ac.uk_P ojec s_P_ alcipa um), using BLAST sea ches.
2.16.7 AFLP band sequencing and pu i ica ion o PCR p oduc s
Sequencing PCR eac ions we e se up using he ABI PRISM Big DyeTM Te mina o Cycle
Sequencing Ready Reac ion Ki , (PE Applied Biosys ems). PCR eac ions we e p epa ed o a
inal olume o 10µl, con aining he ollowing eagen s: 4µl empla e DNA (100-250ng), 4µl
Te mina o Ready Reac ion Mix, 1µl p ime (3.2pmol), 1µl s e ile dis illed wa e
Sequencing PCR condi ions we e as ollows;
Dena u ing 95°C o 30 seconds
Annealing 50°C o 20 seconds
Ex ension 60°C o 4 minu es
Following he sequencing PCR, he p oduc s we e pu i ied by p ecipi a ion wi h sodium
ace a e and 95% e hanol, and washed in 70% e hanol.
x 25 cycles
104
Fo sequencing o pu i ied PCR p oduc s, in e nal p ime s co e ing he ex eme 5’ and 3’
ends o he agmen s we e used. All agmen s we e sequenced in opposi e di ec ions.
Sequencing esul s we e analysed using he SeqED V 1.0.3 so wa e (Applied Biosys ems
Inc., 1992). The p og amme allows he isualisa ion o ch oma og ams o he sequenced
DNA.
So as a summa y: adi ional linkage analysis o indi idual c oss p ogeny clones, is labou -
in ensi e and expensi e, as i in ol es he geno ypic and pheno ypic cha ac e iza ion o
indi idual clones om a cloned p ogeny o a gene ic c oss. Wi hou he gene a ion o an
ex emely la ge numbe o ecombinan clones i has a poo esolu ion, which makes he
ac ual iden i ica ion o he unde lying genes ex emely di icul unless s ong candida es a e
al eady suspec ed. The e is an in e se ela ionship be ween he size o he locus wi hin which
possible a ge genes may be loca ed and he numbe o ecombinan clones ha mus be
gene a ed [Wellems TE e al. 1991].
LGS, used in his p ojec , has enabled he disco e y o genes con olling biological p ope ies
in mala ia pa asi es o be g ea ly accele a ed [Culle on R e al. 2005; Ma inelli A e al. 2005].
LGS di e s om he adi ional app oach o gene ic analysis o mala ia pa asi es by no
equi ing indi idually cha ac e isa ion o e y la ge numbe s o clones om he p ogeny o
he c oss [Ca e R e al. 2007]. LGS cha ac e ises he uncloned p ogeny o a gene ic c oss
(be ween a esis an and sensi i e pa asi e o he pa icula pa asi e cha ac e is ic in s udy) by
measu ing he p opo ion o pa en al polymo phic ma ke s a genome-wide loci (AFLP),
be o e and a e d ug ea men . Ma ke s om he sensi i e pa en ha a e linked o he gene
unde lying he esis ance pheno ype will be unde - ep esen ed o elimina ed a e d ug
ea men , o ming a “selec ion alley”. In he case o his p ojec , he AJ ma ke s co espond
o he ma ke s om he sensi i e pa en ; hose ma ke s will be unde ep esen ed o
elimina ed a e a emisinin and a esuna e ea men .
As Ca e R and colleagues so clea ly summa ise [Ca e R e al. 2007], LGS analysis
depends upon se e al dis inc expe imen al componen s.
A) The c ossing o wo gene ically dis inc lines o mala ia pa asi e by p epa ing a mixed
in ec ion o game ocy e-p oducing blood-s age pa asi es in a mouse, allowing
mosqui oes o eed upon he mix u e, wi h consequen pa asi e de elopmen and
in asion o spo ozoi es o sali a y glands;
105
B) The subjec ion o he p ogeny o he c oss o a selec ion p essu e (in he case o his
esea ch, ART o ATN p essu e). This d ug selec ion is applied o he blood s age
pa asi es in mice in ec ed wi h he c oss p ogeny spo ozoi es.
C) The sc eening o he uncloned, selec ed c oss p ogeny wi h quan i a i e ma ke s, such
as quan i a i e eal- ime PCR, quan i a i e AFLP and p opo ional sequencing. So
be o e he LGS echnique could be applied he de elopmen o quan i a i e ma ke s
co e ing as much he pa asi e genome as possible is necessa y. P io o his p ojec
quan i a i e AFLP we e op imized and de eloped o being use wi h AS and AJ
pa asi e lines om P. chabaudi [Culle on R e al. 2005; Ma inelli A e al. 2005].
The loca ion o he gene ic ma ke s in a Plasmodium genome da abase wi h he expec a ion
ha ma ke s linked o genes con olling he a ge o d ug selec ion will o m a selec ion
alley con aining he locus o he selec ed genes. A gene ic linkage map, in he case o his
p ojec al eady p e ious de eloped by Ma inelli A and co-wo ke s [Ma inelli A e al. 2005],
a genome sequence da abase, in he case o ou model P. chabaudi sequence hough no ully
comple e wi h a big co e age and a comple e syn enic map de eloped p io o his p ojec
[Kooij TW e al. 2006] allowed us o map genes and ma ke s gene ically and physically, hus
allowing iden i ica ion o loci unde selec ion. Then mu a ions in suspec ed candida e genes
wi hin he locus can be iden i ied by compa a i e sequencing o genes om bo h he esis an
mu an and i s sensi i e p ogeni o [Ca e R e al. 2007].
2.17 Expe imen s wi h gene ic c osses o AS-ART and AJ o AS-ATN and
AJ
To analyse he e icacy and composi ion o each o he gene ic c osses and he p og ession o
he d ug selec ion p opo ional sequencing was used. P opo ional sequencing was used wi h
DNA samples om spo ozoi es and mice blood samples be o e and a e d ug selec ion.
2.17.1 Blood collec ion o P opo ional sequencing analyses
5 µl o blood we e emo ed om each in ec ed mice daily using a glass capilla y. The blood
was placed in an Eppendo ube which con ains 2 d ops o ci a e saline solu ion. The
samples we e hen spun in a mic ocen i uge a 10,000 pm o 2 minu es, and he supe na en
emo ed. The esul ing pelle was ozen a -70°C.
112
2.18. Ampli ica ion and sequencing o he ubp-1 gene o P. chabaudi
The ampli ica ion and sequencing o ubp-1 gene has done using he same p o ocol as he one
desc ibed o md 1, cg10, c p and a p6 genes, and his p o ocol is al eady desc ibed in 2.13.
Genomic DNA was used as empla e in 50µl PCR eac ions, con aining 0.2µM o each
oligonucleo ide p ime , 1x PCR bu e (P omega), 2.5 mM MgCl
2
, 0.2mM dNTPs and
0.025U/µl o Taq DNA polyme ase. These we e used in PCR ampli ica ions o AS-15CQ,
AS-30CQ, AS-ATN and AS-ART. Nega i e con ols we e also p epa ed, which con ained 1µl
o s e ile dis illed wa e in place o empla e DNA. Posi i e con ols we e p epa ed using a
p e iously ampli ied DNA empla e.
The oligonucleo ide p ime s sequence is p esen ed in Appendix 4.
All PCR eac ions we e ca ied ou using a UNO-The moblock machine (Biome a).
PCR p oduc s we e un on a 2% aga ose gel in TBE solu ion and isualized unde UV.
P oduc s we e pu i ied using he QIAquick PCR Pu i ica ion Ki om QIAGEN and
sequenced using BigDye chain e mina ion 3.1 (Applied Biosys ems). The sequencing
eac ions we e analysed by Mac ogen®. The p ime s used in sequencing eac ions we e hose
used o he ini ial ampli ica ion o he agmen s.
Gene and p edic ed amino-acid sequences we e manually compiled, and hen compa ed
be ween d ug selec ed and unselec ed clones using an in e ne -based in e ace deno ed
Mul iple Sequence Alignmen wi h hie a chical clus e ing [Co pe F e al. 1998], using
de aul alignmen pa ame e s (h p://p odes. oulouse.in a. /mul alin/ mul alin.h ml).

113
RESULTS
114
115
The esul s o he p esen wo k a e di ided in o h ee chap e s:
• Chap e III desc ibes he esul s o expe imen s designed o selec a emisinin and
a esuna e esis an pa asi es. These expe imen s also in ol ed e alua ion o ce ain
esis ance ea u es such as s abili y.
• Chap e IV desc ibes he esul s o expe imen s designed o analyse he in ol emen o
p e iously desc ibed pu a i e gene ic modula o o a emisinin and a esuna e esis ance,
he P. chabaudi genes md 1, cg10, c p and a p6.
• Chap e V is conce ned wi h he esul s o expe imen s on gene ic c osses be ween an
a emisinin esis an (AS-ART) and a sensi i e (AJ) cloned s ain o P. chabaudi and an
a esuna e esis an (AS-ATN) and a sensi i e (AJ) cloned s ain o P. chabaudi.
P esen ed he e a e also he esul s o LGS expe imen s conduc ed wi h he uncloned
ecombinan p ogenies o he gene ic c osses.
116
117
RESULTS
CHAPTER III
EXPERIMENTS FOR DRUG SELECTION OF ARTEMISININ
AND ARTESUNATE RESISTANCE

118
119
This chap e p esen s he esul s o expe imen s o selec a emisinin and a esuna e esis ance
in P. chabaudi, o s able pheno ype, o cloning he pa asi e lines ob ained and in he
cha ac e iza ion o he esis ance ob ained, including i s s abili y.
3.1. In oduc ion
The objec i e o his pa o he p ojec was o selec a emisinin and a esuna e mu an s in he
oden mala ia P. chabaudi, h ough p olonged exposu e o d ug-sensi i e lines o low and
inc easing le els o he d ug, adminis e ed o mice.
Table 7 depic s he maximum doses ole a ed by he p ogeni o pa asi e lines as es ablished in
p elimina y wo k.
Table 7 – Maximum doses ole a ed by he p ogeni o pa asi e lines used o selec o esis ance.
P. chabaudi Maximum dose o
a emisinin (mg/kg/day)
Maximum dose o a esuna e
(mg/kg/day)
AS-15CQ 1.2 2
AS-30CQ 4 1.6
120
3.2 A emisinin d ug selec ion
P. chabaudi clone AS-30CQ was exposed o g adually inc easing concen a ions o
a emisinin, du ing se e al consecu i e passages in mice, s a ing wi h a d ug dose o 4
mg/kg/day, p e iously de e mined o be subcu a i e o hese pa asi es (da a no shown). See
Table 7.
The d ug selec ion p ocedu e is desc ibed in mo e de ail in he Ma e ials and Me hods
Chap e (see Poin 2.8) and summa ized in Figu e 15.
Figu e 15 – A schema ic ep esen a ion o he a emisinin selec ion p ocedu e. AS-ART* is uncloned.
AS
-
30CQ
pa asi e
clone
Sensi i e o
a emisinin
4
mg/kg/day
15 blood passages
60
mg/kg/day
D ug
dose
AS
-
ART
*
pa asi e line
Resis an o
a emisinin
121
Figu e 16 ep esen s he s epwise inc ease in d ug dose du ing he a emisinin selec ion
p ocedu e.
Blood passage numbe and a emisinin dose
0
10
20
30
40
50
60
70
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Numbe o sucessi e blood passage
A emisinin dose
(mg/kg/day)
D ug dose (mg/kg/day)
Figu e 16 – The inc ease o he a emisinin dose (mg/kg/day), ole a ed by AS-30CQ clone, du ing 15 blood
passages.
Figu e 16 shows he s epwise inc emen on he a emisinin dose du ing selec ion, s a ing by a
4 mg/kg/day dose o a emisinin, his dose being he maximum dose ole a ed by he ini ial
pa asi es, AS-30CQ.
The d ug esis ance selec ion p ocess las ed o app oxima ely 27 weeks (app oxima ely 7
mon hs); meaning ha du ing his pe iod he P. chabaudi pa asi e clone AS-30CQ was kep
unde inc easing d ug p essu e un il a concen a ion o 60 mg/kg/day, a he end o which
esis ance seemed o ha e been selec ed. Due o ime cons ains he selec ion p ocess was
inished a his s age whe e s abili y o esis ance was assayed.
In he i s ou passages unde d ug p essu e, he o al inc emen on he a emisinin dose was
o only 6 mg ( om i s passage o passage numbe 10) only om passage numbe i e
onwa ds we we e able o inc ease 5mg o ART on each passage.
128
3.4 Cloning o esis an pa asi es
A he end o he selec ion p ocedu e, i was conside ed ha a signi ican le el o esis ance o
bo h o a emisinin and a esuna e had been ob ained. The N- old le el o esis ance was
calcula ed. The a emisinin esis an pa asi e line AS-ART* was 15 old mo e esis an han
he ini ial clone AS-30CQ; he a esuna e esis an pa asi e line AS-ATN* was 6 old mo e
esis an o ATN han he ini ial clone AS-15CQ.
Bo h pa asi e popula ions we e cloned by he limi ing dilu ion me hod. (See Sec ion 2.5 o
de ails). The cloning p ocedu e is ex ensi ely desc ibed a Rosa io V 1976
.
F om he cloning p ocedu e o he pa asi e line AS-ART*, om he 50 mice inocula ed, i e
mice de eloped pa en pa asi emias (10 % in ec ion a e ) om which pa asi es we e
ha es ed and ozen in liquid ni ogen
F om he cloning p ocedu e o he pa asi e line AS-ATN*, om he 50 mice inocula ed, se en
mice de eloped pa en pa asi emias (14% in ec ion a e) om which pa asi es we e ha es ed
all ozen in liquid ni ogen.
O hese, one clone om each line was chosen on he basis o hei as e g ow h a e, o
u he s udies, including s abili y assays and gene ic c osses and analysis. The a emisinin
and a esuna e esis an clones we e designa ed P. chabaudi AS-ART and AS-ATN
espec i ely. See Figu e 21 o de ails.
Figu e 21 – A schema ic ep esen a ion o he clones and pa asi e lines o Plasmodium chabaudi used in his
p ojec .
AS
-
SENS
AS-PYR
AS-3CQ
AS-15CQ
AS-30CQ
PYR esis an , CQ sensi i e
CQ esis an (low)
CQ esis an (in e media e)
CQ esis an (high)
AS-ATN
AS-ART
A esuna e d ug
selec ion and cloning
A emisinin d ug
selec ion and cloning

129
AS-ART and AS-ATN we e es ed o hei esponse o a emisinin and a esuna e
espec i ely, immedia ely a e cloning. AS-ART and AS-ATN clones we e inocula ed in o
naï e CD1 mice and hen e- es ed o hei suscep ibili y o a emisinin and a esuna e, by
compa ing hei esponse o each d ug wi h ha o non- ea ed and passaged he same numbe
o imes as he pa asi es unde selec ion, con ol pa asi es, AS-30CQ and AS-15CQ
espec i ely.
AS-ART and AS-ATN we e shown o e ain he same pheno ype as ha o he d ug- esis an
popula ion om which hey had been de i ed, unde he same d ug p essu e o espec i ely
60 mg/kg/day ea men o 5 days wi h a emisinin, o a 12 mg/kg/day ea men o 5 days
wi h a esuna e. These pa asi es we e hen used in subsequen s udies o in es iga e u he
whe he he obse ed d ug esis ance was s able. These esul s will be p esen ed on Sec ion
3.5.
130
3.5 D ug esis ance s abili y es s
Once esis ance o a emisinin and a esuna e we e selec ed and cloned s abili y o he
esis an pheno ype, unde absence o d ug p essu e was assayed.
D ug- esis an pa asi e clones, AS-ART and AS-ATN we e e- es ed o hei d ug esponses
a e each o h ee di e en p ocedu es:
i) F eeze- hawing cycles in liquid ni ogen,
ii) 12 con inuous sub inocula ions in mice in he absence o d ug ea men and
iii) T ansmission h ough Anopheles s ephensi mosqui oes in o new mice.
N- old esis ance index was calcula ed as i has been p e iously desc ibed.
3.5.1. Resis ance s abili y a e liquid ni ogen p ese a ion (deep-
eezing)
To check o esis ance s abili y a e liquid ni ogen p ese a ion, esis an clones we e
ozen down in liquid ni ogen and hen hawed, and e- es ed o esis ance.
B ie ly in ec ed blood is d awn, when he majo i y o asexual pa asi es a e a he ing s age o
de elopmen , in o hepa inised sy inges. The ed blood cells a e pelle ed by cen i uga ion
(2000 pm o 5 minu es) and he majo i y o he plasma is emo ed, wo olumes o deep-
eeze solu ion (Appendix 1) a e added o one olume o he packed ed blood cells wi h
cons an mixing in a d op wise ashion. Such ma e ial, inocula ed i. p. in o a mouse, a e any
pe iod o s o age, induced an in ec ion, which became pa en om 7-14 days a e
inocula ion. The pa asi es g owing in his ashion we e e alua ed o hei esponse o
a emisinin o a esuna e in compa ison wi h unselec ed con ol pa asi es, he same way as
du ing he d ug esis ance selec ion.
AS-ART and AS-ATN pa asi e clones pheno ypes d ug esis ance emained unchanged in
bo h cases. The wo cloned lines e ained a 15 and 6 imes N- old esis an pheno ype
espec i ely. The pa asi es esponse o he d ug was he same a e eezing and hawing.
S abili y o esis ance was he e o e, con i med.
131
3.5.2. Resis ance s abili y a e blood passages in he absence o d ug
p essu e
AS-ART and AS-ATN cloned pa asi es we e subjec ed o 12 passages in un ea ed mice, a e
which hey we e es ed o hei d ug esponses.
B ie ly, 10
7
in ec ed ed blood cells we e es ablished as he s anda d pa asi e numbe o be
in ec ed in o indi idual mice. In ec ions in he absence o d ug p essu e we e main ained by
passaging esis an pa asi es wel e imes in CD1 mice i. p. ou e. T ea men was 60
mg/kg/day o 5 days o ART and as 12 mg/kg/day o 5 days o ATN.
Once d ug es s we e p e o med a e he wel e passages in he absence o d ug ea men , i
was e i ied ha he esis an pheno ype main ained because esis an pa asi es ec udesced as
expec ed while sensi i e pa asi es did no .
Resis an clones e ained esis ance o he co esponding d ug.
3.5.3. Resis ance s abili y a e cyclical ansmission h ough
mosqui oes
A e con i ming he s abili y o he esis ance pheno ype a e cloning, eezing and hawing
o hese clones, in he absence o d ug p essu e, i was necessa y o demons a e ha his
gene ic ai was ansmissible h ough mosqui oes.
The p ocedu e o cyclical ansmission h ough mosqui oes was p e iously desc ibed by
Landau and colleagues [Landau I e al. 1966].B ie ly, in ec ed splenec omised a s, in which
game ocy es we e p esen , we e exposed o mosqui oes, which had been s a ed o 24-48
hou s. The in ec ed oden s we e exposed o mosqui oes o a a ied leng h o ime (1-2
hou s) depending on he eeding pe o mance o he mosqui oes. Se en o en days la e a
small numbe o mosqui oes we e dissec ed in o de o coun he numbe o oocys s, which
had de eloped on each midgu . Fi een and, e en ually, se en een days a e he blood meal,
when spo ozoi es we e p esen in he sali a y glands, an unin ec ed mouse was exposed o he
mosqui oes o ansmission o he mala ia in ec ion. Pa en blood in ec ions could be
de ec ed a e 4-8 days in blood smea s om he mice. Resis an clones success ully
ansmi ed in his ashion we e es ed o hei esponse o a emisinin o a esuna e in
compa ison wi h unselec ed con ol pa asi es.
Mice in ec ed wi h AS-ART o AS-ATN we e used o eed A. s ephensi mosqui oes. AS-ART
was success ully ansmi ed h ough mosqui oes on wo sepa a e occasions, and he esul ing
132
blood o ms ha de eloped in mice we e enamed AS-ARTA and AS-ARTB. The N- old
esis ance index o AS-ARTB we e hen assessed in pa allel o he, un ea ed bu passaged
con ol line, AS-30CQ ( his line was also ansmi ed h ough mosqui oes a he same ime, as
a con ol). AS-ARTB showed an N- old esis ance index o 15- old o a emisinin ela i e o
AS-30CQ. Thus, a emisinin esis ance emained s able a e ansmission o he esis an
pa asi es h ough mosqui oes. AS-ARTA was no es ed and he pa asi es whe e deep- ozen.
Fo simplici y and cla i y on u he analysis du ing his p ojec , AS-ARTB clone was
enamed AS-ART.
In a simila ashion, AS-ATN was also subjec ed o mosqui o ansmission and showed a 6-
old inc ease in he N- old esis ance o a esuna e ela i e o sensi i e con ol; he e o e we
also conside a esuna e esis ance o be s able a e mosqui o ansmission. See Figu e 22 o
a summa y o he d ug selec ion / s abili y es s p ocedu e.
Figu e 22 – A schema ic ep esen a ion o he a emisinin and a esuna e selec ion p ocedu e.
Clone AS-30CQ and pa asi e line AS-15CQ we e passaged in he absence and p esence o g adually inc easing
doses o d ug (a emisinin and a esuna e, espec i ely). Ini ial d ug sensi i i ies (4 mg/kg/day o 2 mg/kg/day)
dec eased ( o 60 o 12 mg/kg/day) a e 15 o 14 passages, espec i ely. The d ug esponses a e cloning,
passage in he absence o d ug (“un ea ed”) and ansmission h ough mosqui oes emained unchanged. Con ol
selec ion p ocedu es in he absence o d ug a e also shown.
Adap ed om: A onso A e al. 2006.
The a emisinin and a esuna e esis ance pheno ypes we e unal e ed a e passaging in he
absence o d ug p essu e, a e eezing and hawing and a e ansmission h ough labo a o y
mosqui oes (Figu e 22). Thus a emisinin and a esuna e esis ance ob ained in ou oden
model P. chabaudi is s able, indica ing ha esis ance is likely o be gene ically encoded.
AS-30CQ
AS-15CQ
4 mg
2 mg
60mg
12mg
CLONING
[
D ug
]
sensi i e
sensi i e
AS
-ART
AS
-ATN
14
passages
15
passages
esis an
esis an
12
passages
12
passages
Un ea ed
esis an
esis an
AS
-
ARTA
No
es ed
AS
-
ARTB
Resis an
o 60mg
AS
-
ATN
Resis an
o 12mg
AS
-
30CQ
sensi i e
AS
-
15CQ
sensi i e
133
3.6 Tes o c oss- esis ance be ween a emisinin and a esuna e clones
In o de o e alua e whe he he mechanisms o esis ance o a emisinin and o a esuna e
sha e simila ea u es, he esponses o AS-ART o a esuna e and AS-ATN o a emisinin
we e es ed, esis an clones we e c oss es ed wi h each o he ’s selec i e d ugs. A emisinin
a chosen dose was gi en o he a esuna e esis an clone (AS-ATN) and a esuna e a chosen
dose was gi en o he a emisinin esis an clone (AS-ART).
Fo hese es s se e al d ug doses we e ini ially es ed o bo h esis an clones, he maximum
dose o a emisinin ole a ed by he a esuna e esis an clone (AS-ATN) is 32 mg/kg/day o
i e days and he maximum dose o a esuna e ole a ed by he a emisinin esis an clone
(AS-ART) is 8 mg/kg/day (da a no shown). AS-ART showed a i e- old inc ease in he
MCD o a esuna e ela i e o AS-30CQ while AS-ATN showed a g ea e han en- old
inc ease in he MCD o a emisinin, ela i e o AS-15CQ. These es s he e o e e ealed ha
bo h clones showed c oss- esis ance and ha in bo h pa asi es he e we e g ea e inc eases in
a emisinin esis ance (15–26 old) han o a esuna e esis ance (5–6 old). The
demons a ed c oss- esis ance sugges s some sha ing o gene ic o pa hways ea u es in he
pa asi es clones desc ibed he e. Though he exis ence o c oss- esis ance sugges s ha
esis ance o a emisinin and a esuna e do sha e simila ea u es, a leas in he pa asi es
clones desc ibed in his p ojec .
Table 10 - N- old esis ance o P. chabaudi AS-ATN and AS-ART. The absolu e and ela i e (N- old) d ug
sensi i i ies o AS-ATN and AS-ART a e blood passage in he absence o ea men , eeze/ haw and mosqui o
ansmission a e gi en. MCD – minimum cu a i e dose.
F om: A onso A e al. 2006.
P. chabaudi MCD ATN
(mg/kg/day)
MCD ART
(mg/kg/day)
N- old ATN N- old ART
AS-15CQ 2 1.2 - -
AS-ATN 12 32 6 26
AS-30CQ 1.6 4 - -
AS-ART 8 60 5 15

134
3.7 Discussion
D ug esis ance gene ic basis can be s udy using a ious me hodologies. A co ec and
comple e me hod o s udy d ug esis ance gene ics depends on i s ly selec ing d ug esis ance
mu an s o s able pheno ype, because only he p esence o a s able pheno ype gua an ees ha
esis ance is gene ically encoded and no a physiological adap a ion o he cons an p esence
o d ug p essu e. D ug esis ance can be selec ed in i o using o example chemical
mu agenesis o in i o by d ug p essu e ei he using a single e y high dose like he me hod
used o selec PYR esis ance o by p og essi ely inc easing he d ug concen a ion. The la e
si ua ion is he one ha mos closely mimics he ac ually scena io aking place in na u al
pa asi e popula ions. The e o e, an app op ia e me hod ha has been used o selec ing and
unde s anding d ug esis ance is he u iliza ion o gene ically s able esis an mu an s selec ed
h ough d ug p essu e, o igina ed om cloned sensi i e pa asi e lines. Bo h he o iginal d ug-
sensi i e and he selec ed d ug- esis an pa asi es should be gene ically iden ical, (o
isogenic), excep o any mu a ions in ol ed in esis ance; such mu a ions can hen be
pinpoin ed using di e en app oaches.
This p ojec ep esen s he i s s udy whe e mala ia pa asi es wi h gene ically s able and
ansmissible esis ance o he an imala ial d ugs a emisinin and a esuna e we e selec ed and
used in gene ic c osses.
The easons o ob aining s able esis ance whe e o he s ha e p e iously ied unsuccess ully
a e no clea . One eason could be he use o he oden model P. chabaudi, which had been
used be o e o selec o py ime hamine, chlo oquine and me loquine esis ance. The
exis ence o pa asi e clones wi h an accumula ion o d ug esis ance gene ic ma ke s may
c ea e a a ou able backg ound o he selec ion o a emisinin and a esuna e esis ance.
Two pa asi e lines we e selec ed and cloned: AS-ART is 15 imes mo e esis an o
a emisinin han i s p ogeni o pa asi e line, AS-30CQ and AS-ATN which is 6 imes mo e
esis an o a emisinin han i s p ogeni o pa asi e line, AS-15CQ.
In his wo k he u iliza ion o app oxima ely he same numbe o blood passages, du ing a
simila ime pe iod unde d ug p essu e, p oduced an N- old esis ance o a esuna e in e io
o he N- old esis ance o a emisinin and his is no explainable.
Me aboliza ion di e ences be ween he wo d ugs may ha e had an in insic di e ence in he
selec i e p essu e wi hin he hos , bu d ug concen a ion s udies we e no ca ied ou .
A e y in e es ing inding du ing his pa o he p ojec was he c oss esis ance be ween he
selec ed esis an clones, gi ing a e y s ong indica ion ha esis ance o ART and ATN
sha e simila ea u es.
135
The success ul selec ion o hese pa asi es and he ac ha hey can be ansmi ed h ough
Anopheles sp. mosqui oes is a signi ican achie emen o wo main easons: a) hese
obse a ions demons a e ha mala ia pa asi es a e gene ically and biologically capable o
sus aining s able esis ance o a emisinins which had been selec ed h ough d ug p essu e.
Consequen ly, i is also concei able ha , in human mala ia, a emisinin esis ance may appea
in he u u e due o ex ensi e and/o inapp op ia e d ug usage; once i does, i may sp ead in
he pa asi e popula ion and become es ablished; b) he esis an P. chabaudi pa asi es epo ed
he e could hen be used o in es iga e he gene ic de e minan s o esis ance o hese d ugs.
I was possible o gene a e ART o ATN esis ance om wo di e en pa asi e clones, AS-
30CQ and AS-15CQ which we e al eady esis an o chlo oquine and py ime hamine. E o s
o gene a e ART o ATN esis ance om he e y o iginal chlo oquine sensi i e clone (AS-
PYR), we e abandoned, because his clone showed, unde he same d ug p essu e
me hodology, high suscep ibili y o he ea men s (da a no shown). As s a ed be o e, his
may be a esul o a gene ic abili y, i no po en ia ion o he pa asi e abili y o gene a e
mu a ions in esponse o d ug ea men (called he “Accele a ed Resis ance o Mul iple
D ugs” (ARMD) pheno ype) [Ra hod PK e al. 1997] which migh ha e occu ed du ing he
gene a ion o p e ious d ug selec i e me hods, including chlo oquine.
Al e na i ely, i is possible ha he ART esis ance pheno ype is only exp essed in
chlo oquine esis an clones, almos as esis ance o CQ is equi ed o he selec ion o ART
and ATN.
This sugges s he p esence o unc ional in e ac ions be ween he pa hways unde lying
chlo oquine and a emisinin esis ance o ha esis ance o clo oquine gene ically c ea es
gene ic condi ions which acili a e he appea ance o ART and ATN esis ance. These
ques ions ha e signi ican ele ance o he p ac ical li e ime o a d ug in a eas whe e
esis ance o o he d ugs (o o chlo oquine speci ically) is p e alen .
The inspec ion o mu a ion o mu a ions unde lying esis ance o ART and ATN, will be
desc ibed in Chap e IV (known o suspec ed mu a ions) and in Chap e V using high-
h oughpu compa a i e genomic s udies based on genome-wide app oaches, Linkage G oup
Selec ion (LGS) (new mu a ions) [Culle on R e al. 2005].
136
137
RESULTS
CHAPTER IV
ANALYSIS OF THE PUTATIVE GENETIC MODULATORS
FOR ARTEMISININ AND ARTESUNATE RESISTANCE
144
4.2. Es ima ion o gene copy numbe s o he pcmd 1, pc c p and pca p6
genes, in he selec ed mu an clones.
Besides gene mu a ions, changes in copy numbe s may also be esponsible o d ug
esis ance. The e o e, gene copy numbe o pcmd 1, pc c p and pca p6 on he p e iously
selec ed and cloned a emisinin and a esuna e mu an s in P. chabaudi (AS-ART and AS-
ATN espec i ely) we e s udied. A his s age he gene pccg10 was no analysed o gene
copy numbe change due o he ac ha he e we e no e e ences ound in he li e a u e ha
accoun o changes in he gene copy numbe o ei he p c o i s P. chabaudi homologue
pccg10.
The gene copy numbe can be analysed by a ious me hods, he me hod o using eal- ime
quan i a i e PCR (RTQ-PCR) has been ex ensi ely alida ed o he use in mala ia pa asi es.
We ound ha he mos obus me hod o copy numbe de e mina ion by eal- ime PCR is he
compa a i e C (2
-∆∆C
) me hod [Li ak KJ e al. 2001]. While equi ing an endogenous con ol
and a calib a o , meaning a gene om which p e ious knowledge is needed o he exac gene
numbe ( o example msp-1 gene ha is known o su e o be a single copy gene in P.
chabaudi) and a sample ha is use as a con ol o ha gene, i di e s om ela i e s anda d
me hod by elying on equal PCR e iciencies wi h he a ge and he endogenous con ol
genes. The 2
-∆∆C
me hod is desc ibed in de ail by Li ak and colleagues [Li ak KJ e al. 2001].
B ie ly o he ∆∆C calcula ion o be alid, he e iciency o he ampli ica ion o a ge and
e e ence gene mus be app oxima ely equal. The con ol gene was msp-1 and he a ge genes
we e md 1, c p and a p6 genes. The a e age C was calcula ed o bo h con ol and a ge
genes and he ∆C (C
a ge gene
– C
msp-1
) was de e mined. Di e en plo s o he log DNA
dilu ion e sus ∆C we e made and whene e he slope was close o ze o he e iciencies o
he a ge and e e ence genes we e simila , he ∆∆C was calcula ed o he ela i e
quan i ica ion o he a ge gene; ∆∆C = (C
a ge gene
- C
msp-1
)
α
- (C
a ge gene
- C
msp-1
)
β
, whe e
α = ART o ATN esis an sample and β = AS-30CQ o AS-15CQ samples espec i ely.
A e he me hod was alida ed he esul s o each sample we e exp essed in N- old numbe s
in α gene copy numbe wi h no maliza ion o he msp-1gene copy numbe acco ding o he
equa ion: gene numbe o a ge = 2
-∆∆C
[Li ak KJ e al. 2001].
A e op imisa ion o all he RTQ-PCR condi ions we aimed o in es iga e i ART and ATN
esis ance could be ela ed o changes in he gene copy numbe o md 1, c p and a p6 genes.
Fo his he N- old gene numbe was e alua ed and md 1, c p and a p6 genes we e
no malised o msp-1 gene, in a emisinin esis an pa asi es P. chabaudi AS-ART ela i e o

145
a emisinin sensi i e P. chabaudi AS-30CQ om which his pa asi e line was ob ained and
a esuna e esis an P. chabaudi AS-ATN ela i e o a esuna e sensi i e P. chabaudi
AS15CQ om which his pa asi e line was ob ained. Th ee independen expe imen s we e
ca ied ou ; he mean N- old alues o he expe imen s a e p esen ed in Figu e 27. The e we e
no changes in he gene copy numbe o md 1, c p and a p6 genes de ec ed (Figu e 27),
meaning ha du ing he selec ion o a emisinin and a esuna e esis ance he genes pcmd 1,
pc c p and pca p6 did no su e any gene copy numbe change.
Figu e 27 – Rela i e di e ences (N- old) in gene copy numbe be ween a emisinin (AS-ART) and a esuna e
(AS-ATN) esis an pa asi es and hei sensi i e p ogeni o s, AS (30CQ) and AS (15CQ) espec i ely.
The y-axis ep esen s he mean N- old o gene numbe (g ey ba s) and s anda d de ia ions ( e ical lines) a 95%
con idence in e al o each o he genes unde s udy no malised agains msp-1, gene a ed a e h ee independen
assays.
F om: A onso A e al 2006.
0
0,5
1
1,5
2
AS-ART AS-ATN AS-ART AS-ATN AS-ART AS-ATN
md 1 c p a p6
0
0,5
1
1,5
2
0
0,5
1
1,5
2
AS-ART AS-ATN AS-ART AS-ATN AS-ART AS-ATN
md 1 c p a p6
146
4.3. Discussion
4.3.1. Summa y
The p e iously desc ibed pu a i e gene ic modula o s o a emisinin and a esuna e d ug
esis ance, pcmd 1, pcg10, pc c p and pca p6 ha a e P. chabaudi gene homologues o P.
alcipa um md 1, c , c p and a p6, espec i ely, we e ully isola ed and analysed o hei
coding egions. In he case o he gene a p6 gene an a ea o 4kb ups eam and 1 kb
downs eam was also analysed, in bo h he esis an clone lines, AS-ART and AS-ATN in
ela ion o hei sensi i e p ogeni o s AS-30CQ and AS-15CQ espec i ely and ound o be
no mu a ed.
Collec i ely, he abo e da a allowed us o conclude ha in ou model P. chabaudi he
pcmd 1, pcg10, pc c p and pca p6 genes a e no in ol ed in a emisinin o a esuna e
esis ance by gene poin mu a ion.
Besides looking o poin mu a ions, he genes pcmd 1, pc c p and pca p6 we e also
in es iga ed o hei in ol emen in a emisinin and a esuna e esis ance by inspec ing
pu a i e changes in gene copy numbe . This allowed us o conclude ha he pheno ype o
a emisinin and a esuna e esis ance in ou model, P. chabaudi, is also no associa ed wi h an
inc ease in he gene copy numbe o he same genes.
147
4.3.2. Gene al Discussion
This s udy has shown ha no changes in nucleo ide sequence o copy numbe o pcmd 1,
cg10, c p o a p6 genes we e ound in he a emisinin o a esuna e esis an pa asi es when
compa ed o hei sensi i e p ogeni o s.
We conside , howe e , ha o he gene ic mechanisms such as p o ein u no e and/o pos -
ansla ional modi ica ions o he gene p oduc s may accoun o he in ol emen o hese
genes in he esis ance pheno ype. Howe e he in es iga ion o hese pu a i e mechanisms
was no con empla ed in his s udy.
In mala ia, he gene ic mechanisms unde lying d ug esis ance ha e been ex ensi ely s udied
o mos an imala ials, bu a e no ully unde s ood, excep o esis ance o py ime hamine.
Resis ance o his d ug has been o shown o be con e ed by cumula i e single nucleo ide
mu a ions in he dh gene [Si awa apo n W e al. 1997] in P. alcipa um and in all oden
mala ia models analyzed. A emisinin has been used in he ield o cen u ies wi hou
esis ance e e being egis e ed, so one migh expec a complex mechanism o esis ance, no
as simple as ha o py ime hamine.
In ou s udy in a oden mala ia model, we selec ed o high le els o d ug esis ance, which
sugges s he in ol emen o mo e han one gene.
The e idences suppo ing he ole o p a pase6 p o ein in a emisinin sensi i i y in P.
alcipa um a ise om he in e ac ion o a emisinin in an ex i o he e ologous sys em
(Xenopus oocy es) [Ecks ein-Ludwig U e al. 2003] which may e lec a his ological,
physiological and biochemis y poin o iew which may be di icul o in e p e in ligh o he
human mala ia pa asi e. E idence o he in ol emen o his gene in modula ing a emisinin
suscep ibili y also came om he ac ha ecen e idence indica es ha mu a ions in he
p a pase6 gene may co ela e wi h a ying deg ees o in i o esponses o P. alcipa um o
a emisinin de i a i es [Jambou R e al. 2005]. In his case he p a pase6 p o ein S769N,
A623E and E431K polymo phisms we e associa ed wi h an inc eased mean in he IC50 o
a emisinins [Jambou R. e al. 2005]. The impo ance o hese indings is unclea howe e ,
since: i) he co espondence be ween educed sensi i i y o he ield isola es o a emisinins
and he p esence o he di e en polymo phisms was incomple e and ii) he e was a egion-
speci ic associa ion o hese polymo phisms wi h a ying deg eees o suscep ibili y
co ela ing highly (bu no comple ely) wi h he S769N mu a ion in F ench Guiana bu no in
Senegal o Cambodia. In addi ion, la e s udies ound no associa ion be ween mu a ions in he
p a pase6 gene and he sensi i i y o ield isola es o a emisinins om Tanzania [Mugi u K
e al. 2007] and São Tomé and P incipe [Fe ei a ID e al. 2007].
148
In e es ingly, i may be possible ha ano he gene closely link o he a p6 gene may be
in ol ed in he esis ance o his d ug, which could explain he s ong bu incomple e
associa ion obse ed in F ench Guiana [Jambou R. e al. 2005]. In ac ual ac , a simila
scena io has al eady been desc ibed be o e wi h he chlo oquine esis ance de e minan in P.
alcipa um which had been mapped by linkage analysis o a segmen on ch omosome 7 whe e
wo candida e genes cg1 and cg2 wi h complex polymo phisms ini ially linked o he
chlo oquine esis ance pheno ype whe e ound [Du aisingh MT e al. 2000a, Fidock DA e al.
2000a]. Mo e de ailed analysis wi hin ha egion la e e ealed a di e en , bu closely linked
gene, p c , o be he majo de e minan o chlo oquine esis ance.
Finally, i is ele an o men ion ha , i p a pase6 u ns ou o be he majo modula o o
a emisinin esponses in P. alcipa um, i is concei able ha he oden mala ia P. chabaudi
may e eal al e na i e mechanisms o esis ance o hose o P. alcipa um, as is he case wi h
chlo oquine esis ance [Hun P e al. 2004].
149
RESULTS
CHAPTER V
EXPERIMENTS USING LINKAGE GROUP SELECTION AS
AN ATTEMPT TO IDENTIFY THE LOCUS OR LOCI
INVOLVED IN ARTEMISININ AND ARTESUNATE
RESISTANCE

150
151
5.1 In oduc ion
As desc ibed in he p e ious chap e (Resul s – Chap e IV) sequence analysis o genes wi h
po en ial in ol emen in modula ing pa asi e esponses o a emisinin de i a i es, was ca ied
ou o he P. chabaudi o hologues o p a p6 [Ecks ein-Ludwig U e al. 2003; Jambou R e
al. 2005; Uhlemann AC e al. 2005], p c [Sidhu AB e al. 2002], p md 1 [Fe e -Rod íguez I
e al. 2004; P ice RN e al. 2004; Reed MB e al. 2000; Sidhu AB e al. 2005], and p c p
[Bhisu hibhan J e al. 1998; Walke DJ e al. 2000]. Sequencing o AS-ART and AS-ATN
and hei p ogeni o s showed ha he e we e no mu a ions o copy numbe changes in hese
genes (da a published A onso A e al. 2006).
This chap e desc ibes he applica ion o Linkage G oup Selec ion (LGS) as an app oach o
iden i y he gene ic locus o loci in ol ed in a emisinin and a esuna e esis ance in P.
chabaudi.
Some o he esul s desc ibed below ha e been accep ed o publica ion [Hun P e al. in p ess
a he Molecula Mic obiology].
152
5.2 P oduc ion o c oss p ogeny
The p ocedu e o ob ain a c oss p ogeny ha e been al eady desc ibed (See Chap e II -
Ma e ials and Me hods).
Th ee independen gene ic c osses we e ob ained using AS-ART and AJ and wo independen
gene ic c osses we e ob ained using AS-ATN and AJ.
One o he condi ions o a co ec and use ul u iliza ion o LGS o iden i ying gene ic locus
in ol ed in a pa icula pheno ypic ai , is ha la ge numbe s o ecombinan p ogeny clones
a e p esen bo h in he p ogeny p io o he selec ion, and pos selec ion. Fo ha eason and in
o de o inc ease he numbe o ecombinan s, he uncloned p ogeny om hese c osses
(called “unpassaged”) we e pooled in equal p opo ions (equal numbe o pa asi es) and
passaged h ough he wo ea ed and un ea ed g oups. See Figu e 28 o de ails on he
expe imen al g oups.
Fo u he analysis pooled c osses we e used. Those pooled c osses we e named supe
c osses. The h ee gene ic c osses ob ained be ween AS-ART and AJ we e pooled oge he in
a supe c oss ha o cla i y was named AS-ART x AJ. The wo gene ic c osses ob ained
be ween AS-ATN and AJ we e polled oge he in a supe c oss ha o cla i y was named AS-
ATN x AJ.
Each c oss was no analysed indi idually du ing his p ojec .
153
5.3 Selec ion o c oss p ogeny
5.3.1 Gene al p ocedu e
When he spo ozoi es-induced in ec ions (so called “unpassaged”) eached pa asi aemias o
be ween 10%–15%, he pa asi es we e ha es ed o AFLP analysis (p o iding a e e ence
poin o ma ke s analysed in he subsequen ea men g oups), pooled, and inocula ed (each
mouse in each g oup ecei ed 1 x 10
7
pa asi es) in o wo g oups o mice, one d ug ea ed
wi h ei he a emisinin o a esuna e (so called “ ea ed”) and he o he le un ea ed (so
called “un ea ed”). See Figu e 28 o de ails.
Figu e 28 - Schema ic ep esen a ion o he selec ion o he pooled c oss p ogeny (LGS) expe imen using
p e iously gene a ed indi idual c osses be ween AJ and AS-ART
Fo he pooled c oss AS-ATN x AJ only wo indi idual c osses we e used.
Adap ed om Culle on R, 2005 wi h kind pe mission om D . Richa d Culle on.
Gene ic c oss 1
Gene ic c oss 2
Gene ic c oss 3
Unpassaged
Supe c oss pooled in equal
p opo ion o pa asi e numbe s
om indi idual c osses
T ea ed Un ea ed