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A genetic approach towards identification of factors affecting quinine accumulation in P. falciparum [Elektronische Ressource] / presented by Astutiati Nurhasanah

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108 Pages
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Dissertation Submitted to the Combined Faculties for the Natural Sciences and Mathematics the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences A Genetic Approach towards Identification of Factors Affecting Quinine Responses in P. falciparum Presented by Astutiati Nurhasanah born in Jakarta, Indonesia Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdiesntes (DAAD) Dissertation Submitted to the Combined Faculties for the Natural Sciences and Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences Presented by Astutiati Nurhasanah born in Jakarta, Indonesia Oral examination: ...................................... Genetic Approach towards Identification of Factors Affecting Quinine Responses in P. falciparum Referees: Prof. Dr. Michael Lanzer Prof. Dr. Christine Clayton Ich erkläre hiermit, dass ich die vorgelegte Dissertation selbst verfasst und mich dabei keiner anderen als der von mir ausdrücklich bezeichneten Quellen und Hilfen bedient habe. Ich erkläre hiermit, dass ich an keiner anderen Stelle ein Prüfungsverfahren beantragt bzw.

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Published 01 January 2009
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Dissertation
Submitted to the
Combined Faculties for the Natural Sciences and Mathematics
the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences







A Genetic Approach towards Identification of Factors
Affecting Quinine Responses in P. falciparum









Presented by
Astutiati Nurhasanah
born in Jakarta, Indonesia




































Gedruckt mit Unterstützung des Deutschen Akademischen
Austauschdiesntes (DAAD)
Dissertation
Submitted to the
Combined Faculties for the Natural Sciences and Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences
















Presented by
Astutiati Nurhasanah
born in Jakarta, Indonesia





Oral examination: ......................................






Genetic Approach towards Identification of Factors
Affecting Quinine Responses in P. falciparum
















Referees: Prof. Dr. Michael Lanzer
Prof. Dr. Christine Clayton
Ich erkläre hiermit, dass ich die vorgelegte Dissertation selbst verfasst und mich
dabei keiner anderen als der von mir ausdrücklich bezeichneten Quellen und Hilfen
bedient habe.

Ich erkläre hiermit, dass ich an keiner anderen Stelle ein Prüfungsverfahren
beantragt bzw. die Dissertation in dieser oder anderer Form bereits anderweitig als
Prüfungsarbeit verwendet oder einer anderen Fakultät als Dissertation vorgelegt
habe.

Die vorliegende Arbeit wurde am Hygieneinstitut, Abteilung Parasitologie der
Universität Heidelberg in der Zeit vom Oktober 2005 bis April 2009 unter der Leitung
von Prof. Dr. Michael Lanzer durchgeführt.


















Publications resulting from this work:

§ §Sybille Mayer , Astutiati Nurhasanah , Cecilia P. Sanchez, Wilfred D. Stein, and
§ Michael Lanzer ( Both authors contribute equally) “Genetic linkage analysis of
quinine responses reveals a novel locus synergistically associated with pfcrt in
Plasmodium falciparum”. Submitted to PNAS.
Acknowledgment
Acknowledgment

First of all I would like to thank Prof. Dr. Michael Lanzer for the opportunity he had
given me in working in his lab, his trust and his constant support during the years of
my PhD.

I also would like to thank Prof. Wilfred Stein for the collaboration, support and his
priceless input to the work.

I am very grateful to the Deutscher Akademischer Austauschdienst (DAAD) for the
scholarship they gave me, without which, it would have been hardly possible for me
to finish this work.

My heartfelt appreciation goes to Sybille Mayer, for the collaboration and (of course!)
also the friendship, we had during the years I spent in Lanzer’s lab.

And to the other members of Lanzer’s lab, the past and the current, big big thanks to
you (Anurag, Yvonne, Alex, Sebastiano, Tim, Theodora, Phillipp, Doro, Nicole and
our ‘super TAs’ Marina and Eli, and all the others whom I failed to mention here) for
the good time I had all these years. All of you had really made my stay in Germany
truly memorable.

Last but not least I would like to thank my family and friends, for their love and
support through these years. Especially to my Indonesian friends (who are as dear to
me as my own family), thank you for making my staying away from home felt easier ☺
(Terimakasih untuk semuanya ya..). Last but not least, to my husband and dearest
friend, Tomi, for everything.
I Abbreviations
Abbreviations

3H Tritium, tritiated
ABC ATP Binding Cassette
BLAST Basic Local Alignment Search Tool
BSA Bovine Serum Albumin
CaCl Calcium Chloride 2
Chrom. Chromosome
Ci Curie
cM centiMorgan
CQ Chloroquine
CQR Resistant
CQS Sensitive
CSA Chondroitin sulphate A
D Dd2
DHFR Dihydrofolate reductase
DHPS Dihydropteroate synthase
DNA Deoxyribonucleic Acid
dNTP Deoxyribonucleoside triphosphate
DPAP1 dipeptidyl aminopeptidase 1
EBA Erythrocyte binding antigen
EDTA Ethylene Diaminotetraacetate
et al et alii (Latin) – and others
EtBr Ethidium Bromide
f Femto
F regression variance
fL femtoLiter
FV Food vacuole
FVM Food vacuolar membrane
fwd Forward
gDNA Genomic DNA
Glu Glutamine
Gly Glycine
GmbH Gesellschaft mit beschränkter Haftung
H Hydrogen or HB3
HO Water 2
H O Hydrogen peroxide 2 2
HAP histoaspartic protease
HCl Hydrochloric Acid
HEPES N-(2-Hydroxylethyl)piperacin-N´-(2-ethylsulphonacid)
His Histidine
HRP2 Histidine rich protein 2
IC 90% Inhibition Concentration 90
ICAM1 Intracellular adhesion molecule 1
iRBC Infected red blood cell
K Lycine
k Kilo
K & D Kyte and Doolittle
K76T Threonine substitution to Lycine at amino acid 76
Kb Kilobases
KCl Potassium chloride
KCl Potassium Chloride
II Abbreviations
L Liter
LOD Logarithm of odds
m Milli or meter
M Molar
MACS Magnet Activated Cell Sorter
Met Methionine
MgCl Magnesium Chloride 2
MgSOMagnesiumsulphate 4
min Minute
ml Milliliter
2mm Millimeter square
3mmcubic
MR4 Malaria Research and Reference Reagent Resource Center
MS Micro satellites
MSD multiple membrane spanning domain
MSP Merozoite surface protein
n Nano
N Nitrogen 2
NaCl Sodium chloride
NaOH hydroxide
NBD nucleotide binding domain
NCBI National Center for Biotechnology Information
O Oxygen 2
P. Plasmodium
PBS Phosphate Buffered Saline
PCR Polymerase Chain Reaction
Pf Plasmodium falciparum
PfCRT Plasmodium falciparum chloroquine resistant transporter (protein
name)
pfcrt Plasmodiume resistant transporter (gene
name name)
PfEMP1 Plasmodium falciparum erythrocyte membrane protein 1 (protein
name)
pfemp1 Plasmodium erythrocyte membrane protein 1 (gene
name name)
pfmdr1 Plasmodium falciparum multidrugresistant gene
PfNHE odium Sodium Hydrogen Exchanger (protein
name)
pfnhe Plasmodium falciparum Sodium Hydrogen Exchanger (gene name)
P-gp P-glycoprotein
pH Potential hydrogenii
pLDH Plasmodium lactose dehydrogenase
Plm plasmepsin
PV Parasitophorous Vacuole
PVM rous Vacuolar Membrane
QN Quinine
QN IC 90% inhibition concentration of quinine 90
QN-VP aine in the presence of verapamil
IC 90
QTL Quantitative Trait Loci
R or r Pearson regression
RBC Red Blood Cell
RDT Rapid diagnostic test
rev Reverse
III Abbreviations
RFLP Restriction fragment length polymorphisms
rpm rotations per minute
RPMI Rosewell Park Memorial Institute
RT Room Temperature
SDS Sodium Dodecyl Sulphate
sec Second
Ser Serine
STS Sequence Tagged Site
T Threonine
TAE Tris/acetic acid/EDTA
Taq Thermus aquaticus
TE Tris/ EDTA
TKM Tris-KCl-Magnesium
TMD Transmembrane domain
TMHMM rane domain Hidden Markov Model
Tris tris (hydroxymethyl)-aminomethane
TVN Tubulovesicular Network
U Units
US United States
UV Ultra Violet
V Volt
v/v volume to volume
vol Volume
VP Verapamil
w/v weight to volume
WHO World Health Organization
x Times
θ recombination frequency
µ Micro
IV Table of Contents
Table of Contents

Acknowledgment...........................................................................................................I
Abbreviations ...............................................................................................................II
Table of Contents........................................................................................................ V
Table of Figures ....................................................................................................... VIII
List of Tables X
Summary......................................................................................................................1
Zusammenfassung.......................................................................................................2
1. Introduction..........................................................................................................3
1.1. Malaria.........................................................................................................3
1.1.1. Malaria; the Origin and History.............................................................3
1.1.2. Malaria; the Global Impact....................................................................4
1.1.3. Clinical Manifestations..........................................................................6
1.1.4. Life Cycle..............................................................................................7
1.1.5. Chemotherapy....................................................................................10
1.2. Quinine......................................................................................................13
1.2.1. Quinoline drugs – The Discovery and History ....................................13
1.2.2. Haemoglobin Degradation..................................................................15
1.2.3. Chloroquine and Quinine – the Mechanism of Action ........................16
1.2.4. Chloroquine and Quinine – the Resistance........................................17
1.3. Quantitative Trait Loci (QTL) Analysis....................................................... 21
1.3.1. QTL Mapping of P. falciparum............................................................22
1.3.1.1. P. falciparum Genetics System...................................................23
1.3.1.2. More Than One Gene Involved in Quinoline Drug Responses...24
1.3.2. Mapping of Quantitative Trait Loci (QTL) By Linear Regression
Method ............................................................................................................25
1.4. Aim of the Study ........................................................................................ 26
2. Materials and Methods.......................................................................................28
2.1. Materials....................................................................................................28
2.1.1. Equipments.........................................................................................28
2.1.2. Disposables29
2.1.3. Chemicals...........................................................................................30
2.1.3.1. Non Radioactive Chemicals........................................................30
2.1.3.2. Radioactive Chemicals...............................................................30
2.1.4. Biological Materials.............................................................................31
V