Characterisation of the drug transport properties of the Plasmodium falciparum chloroquine resistance transporter through expression in Xenopus laevis oocytes [Elektronische Ressource] / presented by Anurag Dave

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DISSERTATION Submitted to the combined faculties for Natural Sciences and Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences Presented by Anurag Dave born in Ahmedabad, India Date of oral examination : ___________________ Characterisation of the drug transport properties of the Plasmodium falciparum chloroquine resistance transporter through expression in Xenopus laevis oocytes ^ Referees: Prof. Dr. Michael Lanzer Prof. Dr. Christine Clayton Ich erkläre hiermit, dass ich die vorliegende Doktorarbeit selbstständig unter Anleitung verfasst und keine anderen als die angegebenen Quellen und Hilfsmittel benutzt 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 Department für Infektiologie, Abteilung Parasitologie des Universitätsklinikum Heidelberg in der Zeit von Oktober 2006 bis März 2011 unter der Leitung von Prof. Dr. Michael Lanzer ausgeführt. ……… …………………… Datum Anurag Dave Acknowledgements Acknowledgements I would like take this opportunity to first of all thank Prof. Dr.

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DISSERTATION

Submitted to
the combined faculties for Natural Sciences and
Mathematics of the Ruperto-Carola University of
Heidelberg, Germany for the degree of Doctor of Natural
Sciences







Presented by
Anurag Dave
born in Ahmedabad, India



Date of oral examination : ___________________








Characterisation
of the drug transport properties of the
Plasmodium falciparum
chloroquine resistance transporter
through expression
in Xenopus laevis oocytes













^ Referees: Prof. Dr. Michael Lanzer
Prof. Dr. Christine Clayton

Ich erkläre hiermit, dass ich die vorliegende Doktorarbeit selbstständig unter Anleitung
verfasst und keine anderen als die angegebenen Quellen und Hilfsmittel benutzt 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 Department für Infektiologie, Abteilung Parasitologie des
Universitätsklinikum Heidelberg in der Zeit von Oktober 2006 bis März 2011 unter der
Leitung von Prof. Dr. Michael Lanzer ausgeführt.





……… ……………………
Datum Anurag Dave





Acknowledgements
Acknowledgements

I would like take this opportunity to first of all thank Prof. Dr. Michael Lanzer for offering me
the possibility to pursue this project in his laboratory and under his supervision, as well as for
his support and trust. I am thankful to Prof. Dr. Christine Clayton for agreeing to be the
second examiner of my thesis.

I am grateful to Dr. Gabrielle Planelles for her participation in the thesis advisory committee
meetings as well as for her words of encouragement. I would also like to thank
P. D. Dr. Barbara Kappes and Dr. Ann-Kristin Müller for participating in the thesis defence
committee.

Amongst the laboratory members, I would like to thank Dr. Cecilia Sanchez for her help and
guidance. Working in a research group entails team work, and a big thanks to my colleagues
in the laboratory who made this team work an enjoyable experience. So a big thanks to all the
members of the lab who part of the group during the course of my PhD project - Sebastiano,
Elizabeth, Marina, Stefan, Yulin , Sybille, Bianca, Gabriel, Astuti, Yvonne, Tim, Theodora,
Philipp, Nicole, Dorothee, Carolin, Carine, Kathrin, Anne, Christian, Parkash, Ina, Ines and
Dominik. I would also like to thank Miriam Griesheimer for having helped me out with all the
paperwork I have had to deal with, and Sebastiano for his help with all issues related to MS
Word and Excel. A very special note of thanks to Dr. Alexander Rotmann for having
supervised me in the lab, as well as for imparting training to deal with the Xenopus laevis
oocyte system.

I am grateful to the MalParTraining PhD Programme for giving me the opportunity to work as
part of the Marie Curie Early stage researcher training programme, as well as for the financial
support extended and the courses organized. I especially thank Prof. Henri Vial and Nathalie
Modjeska in Montpellier in this regard.

And lastly I would like to express my gratitude to my sisters Dr. Anoushka Dave,
Dr. Anuja Dave, my father Dr. Rajhans Dave and my mother Dr. Jyoti Dave, without whose
support and affection I could not have made it this far.

IAbbreviations
Abbreviations

3D 3 Dimesional
A Adenine or Alanine
Å Armstrong
AG Aktiengesellschaft
Amp Ampicillin
AmpR β-Lactamase gene for Ampicillin Resistance Apicoplast
APS Ammoniumpersulphate
AQ odiaquine
ATP Adenosine Triphosphate
BCE Before common era
Bp Base pairs
BSA Bovine Serum Albumin
C Cytosine or Cysteine
CaCl Calcium Chloride 2
cDNA compleentary DNA
cm Centimeter
C-terminus Carboxy terminus
CQ Chloroquine
CQR resistance
CQS sensitive
D Aspartic aid a Dlon
dd HO double distilled water 2
DEPC Diethylpyrocarbonate
DMSO Dimethylsulfoxide
DNA Deoxyribonucleic acid
DNAse Deoxyribonuclease
dNTP Deoxyribonucleoside triphosphate
dsDNA stranded DNA
DV Digestive vacuole
E Glutamic acid
E. coli Escherichia coli
ECL Enhanced chemiluminescence
EDTA Ethylene Diaminotetraacetate
EM Electrone microscopy
EMP1 Erythrocyte membrane protein1
ER Endoplasmic reticulum
EtBr Ethidium bromide
Fig. Figure
FV Food vacuole
FP Forward primer
G Glycine or Guanine
g Gram
GmbH eslchaft mit beschränkter Haftung
GNP Gross National Product
GTP Guanidine triphosphate
IIAbbreviations
h hour
H Histidine
HO Water 2
HEPES N-(2-Hydroxylethyl)piperacin-N´-(2-ethylsulphonacid)
I Isoleucine
IC Half of maximal inhibitory concentration 50
k Kilo
K Lysne
KAHRP Knob Associated Histidine Rich Protein
Kb Kilobasepair
KCl Potassium Chloride
KOH Hydroxide
L eucine
l Liter
LB Luria Bertani
LSC Liquid scintillation counting
m Milli or Meter
M Molar or Methionine
MC Maure’sCleft
MgCl Magnesium Chloride 2
min inute
MnCl Manganese Chloride 2
mRNA mesnger RNA
MSP-1 Merozoite surface protein-1
n Nano
N Asparigine
NaAc Sodium Acetate
NaCl Chloride
NaOH Hydroxide
nm Nanometrs
NPP New Permeation Pathway
Nu ucleus
O Oxygen 2
°C degre Clsiu
O.D. Optical density
OR Ooocyte Ringer solution 2
P Proline
pasmd
P. Plasmodium
PAGE Polyacrylamide Gel Electrophoresis
PBS Phosphate Buffered Saline
PBST buffered supplemented
with 0.1% Tween-20
PCR Polymerase Chain Reaction
Pf Plasmodium falciparum
PfiRBC Plasmodium falciparum infected red blood cells
pfcrt Plasmodium falciparum chloroquine resistance
Transporter coding sequence

PfCRT Plasmodium falciparum chloroquine resistance
IIIAbbreviations
Transporte
pfmdr1 Plasmodium falciparum multidrug resistance protein 1
coding sequence
Pfmdr1 Plasmodium falciparumsistance protein 1
pH Potential hydrogenii
PIPES Piperazine-N,N ′-bis(2-ethanesulfonic acid)
pmol picomles
POD Peroxidase
PPM Parasite plasma membrane
PV Parasitophorous vacuole
PVDF Polyvinyldifluoride
PVM vacuolar membrane
Q Glutamine
QD Quinidine
QN Quine
R Arginine
RP Reverse primer
RIFIN Repetitive Intersped family
RNA Ribonucleic acid
RNAse Ribonuclease
rpm revoluations per minute
RT Room Temperature
S Serin
SAP Shrimp Alkaline Phosphatase
SDS Sodiumdodecyl sulphate
sec Second
SEM Standard error of measurement
SP Signal peptide
T Thymine or Threonine
T Bacteriphage T4 4
TAE Triacetate/EDTA
Taq Thermus aquaticus
TE Tris/EDTA
TEMED triethylmethylethyldiamine
TMD Transmembrane domain
Tris tris (hydroxymethyl)-aminomethane
U Units SedStates
UTR Untranslated region
UV Ultraviolet
V Volt or Valine
v/v volume to volume
vol
W Tryptophan
w/v weight to volume
WHO World Health Organization
x times
X any amino acid
X-ray Roentgen ray
X .laevis Xenopus laevis
IVAbbreviations
Y Tyrosine
α Ant
µ Micro
φ Hydrophobic amino acid

VTable of Contents

Contents
Acknowledgements..............................................................................................................................I
Abbreviations......................................................................................................................................II
Table of Contents..............................................................................................................................VI
Summary...............1
Zusammenfassung2
Contents
1 Introduction.....................................................................................................................3
1.1 Malaria ...........................................................................................................................3
1.1.1 Global impact .................................................................................................................3
1.1.2 Origin and history...........................................................................................................4
1.1.3 Clinical manifestations...................................................................................................5
1.1.4 Life cycle........................................................................................................................6
1.1.5 Remodelling of P. falciparum infected erythrocytes ...................................................10
1.2 Chemotherapy for malaria............................................................................................13
1.2.1 Antifolates....................................................................................................................13
1.2.2 Antibiotics.......14
1.2.3 Inhibitors of the respiratory chain ................................................................................14
1.2.4 Artemisinin derivatives ................................................................................................15
1.2.5 Aminoquinolines and arylaminoalcohols.....................................................................16
1.2.6 New antimalarials undergoing development................................................................17
1.3 Quinolines - mechanism of action and resistance ........................................................18
1.3.1 Haemoglobin degradation ............................................................................................18
1.3.2 Mechanism of action of quinoline antimalarials..........................................................19
1.3.3 Chloroquine & quinine resistance in P. falciparum.....................................................20
1.3.4 PfCRT...........................................................................................................................20
1.4 Use of Xenopus laevis oocytes to study membrane proteins .......................................22
1.5 Aim of the study...........................................................................................................26
2 Materials and methods..................................................................................................28
2.1 Materials.......................................................................................................................28
2.1.1 Equipments...................................................................................................................28
2.1.2 Disposables...................................................................................................................29
2.1.3 Chemicals.....................................................................................................................30
2.1.3.1 Non-radioactive chemicals...........................................................................................30
2.1.3.2 Radioactive chemicals..................................................................................................30
2.1.4 Kits...............................................................................................................................31
2.1.5 Biological materials......................................................................................................31
2.1.5.1 Size Markers and loading buffer ..................................................................................31
2.1.5.2 Enzymes........31
2.1.5.3 Plasmids.......................................................................................................................31
2.1.5.4 Oligonucleotides...........................................................................................................32
2.1.5.5 Bacteria...........33
2.1.5.6 Antibodies....................................................................................................................33
2.1.5.7 Xenopus laevis frogs.....................................................................................................33
2.1.6 Buffers, media and solutions........................................................................................34
2.2 Methods........................................................................................................................37