αSNAP Alpha-soluble NSF attachment protein AG Arabinogalactan AIDS Acquired immune deficiency syndrome Amp Ampicillin
AraLAM Arabinose Lipoarabinomanan
ATCC American type culture collection
ATP Adenosine tri phosphate
ATPase Adenosine tri phosphatase
BCG Bacillus Calmette-Guèrin
BSA Bovine serum albumin
Ca2+ Calcium
CaMKII Calmodulin complex II
cAMP Cyclic adenosine mono phosphate CFP-10 10 kilo dalton Culture filtrate protein CFU Colony forming units
CPM Counts per minute
CuSO4 Copper Sulphate
DAG Diacylglycerol
DC Dendritic cells
DeADMAn Designer arrays for defined mutant analysis DFI Differential fluorescence induction
DHFRS Dihydrofolate reductase
DIG Digoxigenin DMEM Dulbecos minimum essential medium DNA Deoxy ribo nucleic acid
DTT Dithiothreitol E.coli Escherichia coli
ECL Enhanced chemiluminescence (?)
EDTA Ethylene diamine tetraacetic acid
EE Early endosomes EEA1 Early endosomal antigen 1
EGTA Ethylene glycol tetraacetic acid
EMBL European Molecular Biology Laboratory
ER Endoplasmic Reticulum
ES/MS Electrospray mass spectrometry ESAT-6 Early secreted antigen
ESI-TOF Electro spray inonisation – time of flight FACS Fluorescence activated cell sorter
FBS Foetal bovine serum
FITC Fluorescein isothiocyanate
GAPDH Glycerinaldehyde-3-phosphate- dehydrogenase
GC Guanosine Cytosine
GDI Guanine nucleotide dissociation inhibitor GFP Green Fluorescent protein
Gly-Gly-Ala Glycine-Glycine-alanine Gly-Gly-Asn Glycine-glycine-asparagine GPLs Glycopeptidolipidis GTP Guanosine tri phosphate
GTPase Guanosine tri phosphatase
HCl Hydrochloric acid
HEK Human embryonic kidney
His Histidine HMDM Human Monocyte Derived Macrophages HPLC High performance liquid chromatographic HPTLC High performance thin layer chromatography
Hrs Hepatocyte growth factor-regulated tyrosine kinase substrate
H2SO4 Slulphuric acid
Hyg Hygromycin
ICL Isocitrate lyase
IFN-γ Interferon gamma
IgG Immunoglobulin G
IL Interleukin IPTG Isopropyl β-D-1-thiogalactopyranoside Kana Kanamycin
Kb Kilo bases
KCI Potassium chloride
kDa Kilo dalton
KOD Thermococcus kodakaraensispolymerase
KOH Potassium hydroxide
Kv Kilo volt
LAM Lipoarbinomanan LAMP Lysosome asscociated membrane protein LB Luria-Bertani LBPA Lysobisphosphatidi c acid
LE Late endosome
LOS Lipooligosaccharides
M. avium Mycobacterium avium
MACS Magnetic cell sorting
MALDI Matrix assisted laser desorption/ionization
ManLAM Mannose Lipoarabinomannan
MCP-1 Macrophage chemotactic protein-1 MFP Membrane fusion protein
Mg Magnesium
MgCI Magnesium Chloride
MHC Major Histocompatibility complex
MmpLs Mycobaterial membrane protein large mmpS Mycobacterial membrane protein small MOI Multiplicity of Infection
MP Mycobacterial phagosome
mRNA Messenger RNA
MS Mass spectrometry
Mtb Mycobacterium tuberculosis
NaCl Sodium Chloride
NADPH Nicotinamide adenine dinucleotide phosphate NaHCO3 Sodium bi carbonate
NaHPO4 Sodium hydrogen phosphate
NaOH Sodium hydroxide
Ni-NTA Nickel-nitrilotriacetic acid
NK Natural killer
NMR Nuclear Magnetic Resonance
NSF N-ethylmaleimide-sensitive fusion protein OADC Oleic acid albumin dextrose complex
OD Optical density
OmpA Outer membrane protein A
ORF Open reading frame
PI3K Phosphoinositol-3-kinase PI3P Phosphoinositol-3-phosphate PBS Phosphate buffer saline
PCR Polymerase chain reaction PDIM Phthiocerol di mycocerosate
PE Proline-Glutamine
PE-PGRS Proline-glutamine – polymorphic GC-rich repetitive sequence
PGL Phenolic Glycolipid
PGRS Polymorphic GC-rich-repetitive sequence (?) p-HBAD P-hydroxybenzoic acid derivatives
PI3P Phosphatidylinositol 3-phosphate
PIM Phosphoinositol mannoside
PIPES Piperazine-N,N´-bis ethansulfonic acid (?)
PknA Protein kinaseA
pks Polyketide synthase PL Phagolsosome
PM Phagosome maturation
pmiA Phagosome maturation inhibition A
PMSF Phenylmethylsulphonyl fluoride
PPE Proline-Proline-Glutamine ppm Parts per million
PtdIns(4,5)P Phosphoinositol-4,5-bisphosphate
PVDF Polyvinylidene difluoride
RF Retention factor
RGD Arginine-glycine-aspargine RILP Rab7-interacting lysosomal protein
RNA Ribo nucleic acid
RND Resistance, nodulation, and cell division proteins rpm Rotations per minute
RPMI Roswell Park Memorial Institute medium
RT Room temperature
SapM Secreted mycobacterial lipid phosphatase SCOTS Selective capture of trancribed sequence SDS Sodium dodecyl sulphate
SDS-PAGE Sodium dodecyl sulphate Poly acrylamide gel electrophoresis
SE Sorting endosomes
SFM Serum free medium
SMART Simple Modular Architecture Research Tool STM Signature tagged mutagenesis
TB Tuberculosis
TC Tissue culture
TDM Trehalose dimycolate
TE Tris EDTA
Tf Transferrin
Tfr Transferrin receptor TGN Trans Golgi network
TLC Thinlayer chromatography
TLR Toll like receptor
TMD Transmembrane domains
Tn Transposon TNF-α Tumor necrosis factor alpha
TraSH Transposon site hybridization
TRITC Tetramethylrhodamine-5-isothiocyanate
UV Ultra violet
WHO World health organisation
WT Wild-type WTHK Wild-type heat killed
YFP Yellow fluorescent protein
Declaration
I hereby declare that the work in this thesis is original and has been carried out by me at the Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Universität Köln, under the supervision of Priv. Doz. Dr. med. Georg Plum and in partial fulfilment of the requirements of the Doctor of Philosophy degree of the Universität Bonn. I further declare that this work has not been the basis for the awarding of any degree, diploma, fellowship, associateship or similar title of any university or institution.
Nirmal Robinson May 2007
Acknowledgement
“No one who achieves success does so withot acknowledging the help of others. The wise and confident acknowledge this help with gratitude”
- Alfred North Whitehead
The writing of this thesis has been one of the most significant academic challenges I have ever faced. Without the guidance, support, encouragement and patience of the following people, this study would not have been completed. It is to them I owe my deepest gratitude.
First I express my sincere gratitude to Dr. Georg Plum my mentor for his excellent guidance, valuable discussions and for all the support rendered throughout the study.
Most of all I thank him for giving me an opportunity to carry out this study and grooming my research capabilities.
Working in the lab would not have been easier for me if not for the charm and support of Mrs. Martina Wolke. I am grateful to her for putting up with me and for all the assistance with the experiments.
I thank Dr. Thomas Kolter who was instrumental in the characterisation of the lipid. I also thank him for the helpful discussions.
I acknowledge with gratitutde the support and encouragement given by Prof. Martin Krönke throughout the study.
I would like to thank Dr. Olaf Utermöhlen and his team for the help rendered in conducting experiments with mice, Dr. Karen Ernestus for her help with histopathology, Dr. Wiltrud Kalkamoll and Ms. Sonja Meemboor for their assistance in the analysis of GFP expression by flowcytometry.
I am grateful to Mrs. Anne Abrams for critical reading of the thesis and the manuscript and her valuable suggestions.
Besides my supervisor Dr. Plum, I would like to thank the rest of my doctoral thesis committee members Prof. Wiedemann, Prof. König and Dr. Kolter for reviewing my thesis.
Special thanks go to my friends Sabu, Tom, Paul, Giri, Poornima and Jorly who have lent their helping hands whenever needed.
I thank my family- my parents Mrs and Mr. Robinson for educating me, for their unconditional support and encouragement to pursue my interests and for their boundless love – Mahizh my sister and Frederick my brother-in-law for encouraging me throughout my academic career. “A hundred men may make an encampment but it takes a woman to make a home”. Last but not the least, I am thankful to Jini my wife for her love, support, endurance and patience all throughout and most of all lending her shoulders during rough and happy moments and my beloved son Bryan who eases me out with his charm and love.
This work was supported by grant PL 268/2-1 from Deutsche Forschungsgemeinschaft and by grant 135/2005 from the Koeln Fortune Program, Facult of Medicine, University of Cologne.
CURRICULUM VITAE
Family Name First Name Date of Birth
Robinson Nirmal 31.07.1976 Nationality : Indian
Present Position: PhD student
Institution : Institute for Medical Microbiology and Immunology, University of Cologne, Germany
Education:
Year Degree University (Country) Field of Study 1997
1999
B.Pharm
M.Pharm
Tamil Nadu Dr. MGR Medical University, Chennai, India
Tamil Nadu Dr. MGR Medical University, Chennai, India
Pharmacy
Pharmaceutical Biotechnology Research Experience
Period Institution Position Discipline
1999 – 2002
2002 – 2003
2003
All India Institute of Medical Sciences (AIIMS) All India Institute of Medical Sciences (AIIMS) Institute for Medical Microbiology and
Immunology
Research Fellow
Research Fellow
PhD student, Researcher
Dept of Biotechnology
Dept of Neurology
Molecular and Medical Microbiology