Identification of host genes facilitating Plasmodium liver infection:
Saved in:
| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
Berlin
Mensch und Buch Verl.
2013
|
| Series: | Biological science
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | X, 164 S. Ill., graph. Darst. |
| ISBN: | 9783863872687 |
Staff View
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Record in the Search Index
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| adam_text | CONTENTS
CONTENTS IV
LIST OF FIGURES VIII
LIST OF TABLES X
1 INTRODUCTION 1
1.1 MALARIA GENERAL CONSIDERATIONS . . . . 1
1.2 PREVENTION AND CONTROL 2
1.3 THE PLASMODIUM LIFE CYCLE 4
1.4 THE PRE-ERYTHROCYTIC STAGE 5
1.4.1 THE PLASMODIUM SPOROZOITE . 5
1 4.2 HEPATOCYTE INVASION AND TRANSVERSAL 9
14 2.1 CELL TRANSVERSAL . 9
1.4.2.2 HEPATOCYTE INVASION 10
1.4 3 THE SPOROZOITE S JOURNEY AND METAMORPHOSIS 10
1.5 WHY STUDY HOST-PARASITE INTERACTIONS DURING THE LIVER STAGE? ... 14
1.6 RNA INTERFERENCE OVERVIEW 15
1 7 HOST FACTORS AND SPOROZOITE LIVER INFECTIVITY ... 16
1.7.1 HEPARAN SULFATE PROTEOGLYCANS . . 18
1.7.2 LOW DENSITY LIPOPROTEIN RECEPTOR RELATED-PROTEIN 18
1.7.3 CLUSTER OF DIFFERENTIATION 81 18
1.7.4 SCAVENGER RECEPTOR CLASS B TYPE I 19
1 7.5 HEPATOCYTE GROWTH FACTOR .... 20
17 6 HEME OXYGENASE 1 (HO-1) 21
IV
HTTP://D-NB.INFO/106633143X
CONTENTS
1.7.7 FETUIN A 22
1.7.8 LIVER-FATTY ACID BINDING PROTEIN . .22
1.7.9 PROTEIN KINASES . . . . 22
1.7.10 F-ACTIN 23
1.8 AIMS . . . ... . 24
2 MATERIALS AND METHODS 25
2.1 CELLS AND PARASITES .... . .... 25
2 2 ANTIBODIES 25
2 3 OLIGONUCLEOTIDE SEQUENCES ... 26
2.4 SOFTWARE ... 27
2.5 SIRNA LIBRARY AND SCREENING CONTROLS 27
2.6 KITS, ENZYMES AND OTHER REAGENTS 28
2.7 MEDIA, BUFFERS AND SOLUTIONS 29
2.8 PARASITOLOGY METHODS 30
2.8.1 PARASITE MAINTENANCE . 30
2.8.2 ASSESSMENT OF PARASITAEMIA 30
2.8.3 TERMINAL REMOVAL OF BLOOD FROM MICE 30
2 8 4 PURIFICATION OF PARASITE BLOOD STAGES 30
2.8.5 SPOROZOITE PRODUCTION . 31
2.8.6 HEPATOCYTE INFECTION 32
2.8.7 ASSESSMENT OF SPOROZOITES TO LIVER SCHIZONT CONVERSION . . 32
2.8.8 IN VITRO CULTURE AND PURIFICATION OF SCHIZONTS 33
2 8 9 SCHIZONT TRANSFECTION . 34
2.8.10 SELECTION OF PYRIMETHAMINE RESISTANT PARASITES 34
2 8 11 CLONING BY LIMITING DILUTION 34
2.8.12 ASSESSMENT OF GFP EXPRESSION 35
2 8 13 ASSESSMENT OF HEPATIC MEROZOITE MFECTIVITY 35
2.9 CELL AND MOLECULAR BIOLOGY METHODS . . 35
2.9.1 P. BERGHEI GENOMIC DNA PURIFICATION 35
2.9.2 POLYMERASE CHAIN REACTION (PCR) 36
2 9.3 CLONING OF PCR PRODUCTS 36
2.9.4 RESTRICTION DIGEST . . . .... 36
V
CONTENTS
2.9.5 DNA DEPHOSPHORYLATION 37
2.9 6 DNA LIGATION . . ... ... 37
2.9.7 E COLL TRANSFORMATION 37
2.9.8 DNA PURIFICATION AND GEL ELUTION . . ... . ... 37
2.9.9 PLASMID CONSTRUCTION 37
2.9.10 DNA SEQUENCING . 38
2 9 11 DIAGNOSTIC PCR 39
2.9.12 RADIOACTIVE LABELLING OF DNA PROBES ... 39
2.9.13 SOUTHERN BLOTTING ... 39
2.9.14 LUCIFERASE ASSAY . ... 40
2.9.15 HEPG2C3A TRANSFECTION OPTIMIZATION . . 40
2 9.16 HIGH-THROUGHPUT SIRNA SCREENING . 41
2.9.17 QUANTITATIVE REVERSE TRANSCRIPTASE-PCR ... . ... 41
2 9 18 STAINING OF SCREENING PLATES 42
2.9.19 IMAGE ACQUISITION AND ANALYSIS 42
2.9.20 DATA ANALYSIS 43
2.9.21 INTERACTIONS NETWORK 44
2.9.22 INDIRECT IMMUNOFLUORESCENCE 44
2.9.23 LYSOTRACKER RED-UPTAKE. . ... 45
2.9.24 TRANSIENT TRANSFECTION OF MAMMALIAN CELLS .... ... 45
2.9.25 LIVE-CELL MICROSCOPY . 45
2.9.26 INTRA-EXTRACELLULAR PARASITE ASSAY . . 46
3 RESULTS 47
3.1 ASSAY DEVELOPMENT . . . . 47
3.1.1 ESTABLISHMENT AND OPTIMIZATION OF HEPATOCYTE INFECTION 47
3.1.2 OPTIMIZATION OF SPOROZOITE PRODUCTION . . 51
3.1.3 READ-OUT SELECTION 52
3.1 3.1 GENERATION OF TRANSGENIC P. BERGHEI EXPRESSING GFP-
LUCIFERASE FUSION PROTEIN 52
3.1.3.2 ANALYSIS OF PYRIMETHAMINE-RESISTANT PARASITES . . 53
3.1.3.3 TRANSGENIC P. BERGHEI PARASITES DEVELOP AND COM
PLETE NORMAL LIFE CYCLE 55
VI
CONTENTS
3.2 CHARACTERIZATION OF GFP AND LUCIFERASE EXPRESSION IN TRANSGENIC P.
BERGHEI 56
3 3 HEPATOCYTE SIRNA TRANSFECTION OPTIMIZATION 59
3.4 PRIMARY SCREEN AND SELECTION OF CANDIDATE GENES . . 64
3.5 SECONDARY SCREEN AND HIT SELECTION . . . 69
3.6 DISSECTING PUTATIVE MECHANISMS 73
4 DISCUSSION 89
4.1 RNAI SCREENING DESIGN . . . 89
4.2 NOVEL HOST-FACTORS FOR PLASMODIUM LIVER INFECTION 92
4.3 RNA INTERFERENCE SCREENS AND THEIR LIMITATIONS 99
4.4 CONCLUDING REMARKS 100
REFERENCES 102
APPENDIX 1. ABBREVIATIONS 134
APPENDIX 2. PL0018 VECTOR MAP 136
APPENDIX 3. PLATE LAYOUT 137
APPENDIX 4. Z-SCORE LIST 139
APPENDIX 5. SECONDARY SCREEN DATA 152
APPENDIX 6. KNOCKDOWN QUANTIFICATION 157
APPENDIX 7. CURRICULUM VITAE 159
APPENDIX 8. PUBLICATIONS 160
APPENDIX 9. ACKNOWLEDGEMENTS 161
APPENDIXLO. SELBSTSTANDIGKEITSERKLARUNG 162
APPENDIX 11. VIDEO MATERIAL 163
VII
|
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| discipline | Medizin |
| format | Thesis Book |
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| isbn | 9783863872687 |
| language | English |
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| publishDate | 2013 |
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| spelling | Montes Vergara, Beatriz Alicia 1976- Verfasser (DE-588)1033672076 aut Identification of host genes facilitating Plasmodium liver infection von Beatriz Alicia Montes Vergara Berlin Mensch und Buch Verl. 2013 X, 164 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Biological science Zugl.: Berlin Humboldt-Univ., Diss., 2013 In vitro (DE-588)4250701-7 gnd rswk-swf Pathogenese (DE-588)4115512-9 gnd rswk-swf Infektionskrankheit (DE-588)4026879-2 gnd rswk-swf Plasmodium (DE-588)4248576-9 gnd rswk-swf RNS-Interferenz (DE-588)4802706-6 gnd rswk-swf Leberepithelzelle (DE-588)4120659-9 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Plasmodium (DE-588)4248576-9 s Infektionskrankheit (DE-588)4026879-2 s Leberepithelzelle (DE-588)4120659-9 s Pathogenese (DE-588)4115512-9 s RNS-Interferenz (DE-588)4802706-6 s In vitro (DE-588)4250701-7 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025932891&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Montes Vergara, Beatriz Alicia 1976- Identification of host genes facilitating Plasmodium liver infection In vitro (DE-588)4250701-7 gnd Pathogenese (DE-588)4115512-9 gnd Infektionskrankheit (DE-588)4026879-2 gnd Plasmodium (DE-588)4248576-9 gnd RNS-Interferenz (DE-588)4802706-6 gnd Leberepithelzelle (DE-588)4120659-9 gnd |
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| title | Identification of host genes facilitating Plasmodium liver infection |
| title_auth | Identification of host genes facilitating Plasmodium liver infection |
| title_exact_search | Identification of host genes facilitating Plasmodium liver infection |
| title_full | Identification of host genes facilitating Plasmodium liver infection von Beatriz Alicia Montes Vergara |
| title_fullStr | Identification of host genes facilitating Plasmodium liver infection von Beatriz Alicia Montes Vergara |
| title_full_unstemmed | Identification of host genes facilitating Plasmodium liver infection von Beatriz Alicia Montes Vergara |
| title_short | Identification of host genes facilitating Plasmodium liver infection |
| title_sort | identification of host genes facilitating plasmodium liver infection |
| topic | In vitro (DE-588)4250701-7 gnd Pathogenese (DE-588)4115512-9 gnd Infektionskrankheit (DE-588)4026879-2 gnd Plasmodium (DE-588)4248576-9 gnd RNS-Interferenz (DE-588)4802706-6 gnd Leberepithelzelle (DE-588)4120659-9 gnd |
| topic_facet | In vitro Pathogenese Infektionskrankheit Plasmodium RNS-Interferenz Leberepithelzelle Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025932891&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT montesvergarabeatrizalicia identificationofhostgenesfacilitatingplasmodiumliverinfection |