Genome-wide RNA interference screen for the identification of crucial host cell factors in Chlamydia trachomatis infection:
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| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Berlin
Mensch und Buch Verl.
2013
|
| Schriftenreihe: | Biology
|
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | X, 112 S. Ill., graph. Darst. |
| ISBN: | 9783863874148 |
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| 245 | 1 | 0 | |a Genome-wide RNA interference screen for the identification of crucial host cell factors in Chlamydia trachomatis infection |c von Erik González Martínez |
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Datensatz im Suchindex
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| adam_text | TABLE OF CONTENTS
TABLE OF CONTENTS
ABSTRACT
ZUSAMMENGFASSUNG
1. INTRODUCTION 1
1.1 CHLAMYDIALES 1
1.1.1 DISCOVERY AND CLASSIFICATION OF THE CHLAMYDIALES 1
1.1.2 THE HUMAN PATHOGEN C. TRACHOMATIS 3
1.1.2.1 EPIDEMIOLOGY AND CLINICAL MANIFESTATION 3
1.1.3 TREATMENT OF CHLAMYDIA INFECTIONS 4
1.2 THE BIOLOGY OF CHLAMYDIA 5
1.2.1 THE CHLAMYDIAL DEVELOPMENTAL CYCLE AND HOST-CHLAMYDIA INTERACTIONS
5
1.2.2 ATTACHMENT AND INVASION OF THE HOST CELL BY CHLAMYDIA 7
1.2.3 INTRACELLULAR EVENTS AND EXPANSION OF CHLAMYDIA 9
1.2.4 PERSISTENCE OF CHLAMYDIA 11
1.2.5 HOST CELL EXIT OF CHLAMYDIA 11
1.2.6 NUTRIENT ACQUISITION BY CHLAMYDIA 12
1.2.7 NUCLEOTIDE METABOLISM OF CHLAMYDIA 15
1.3 THE HOST CELL PURINE METABOLISM 16
1.3.1 THE PURINE SYNTHESIS 16
1.3.2 REGULATION OF GUANOSINE MONOPHOSPHATE SYNTHASE (GMPS) (EC 6.3.5.2)
17
1.4 RNA INTERFERENCE 19
1.4.1 MECHANISM OF RNA INTERFERENCE 19
1.4.2 RNA INTERFERENCE SCREENS 20
2. GOAL OF THE STUDY 24
3. RESULTS 25
3.1 ASSAY DEVELOPMENT 25
3.1.1 OVERVIEW OF THE GENOME-WIDE RNAI SCREEN 25
3.1.2 ESTABLISHMENT OF THE MICROSCOPIC READ-OUT 27
3.1.3 ESTABLISHMENT OF THE KNOCKDOWN CONDITIONS 29
3.1.4 ESTABLISHMENT OF SIRNA CONTROLS FOR THE SCREEN 30
3.1.5 DETERMINATION OF CYTOTOXIC SIRNA EFFECTS WITH LACTATE
DEHYDROGENASE (LDH) ASSAY 32
3.2 GENOME-WIDE SIRNA SCREEN 36
3.2.1 QUALITY CONTROL AND CORRELATION ANALYSIS OF SCREENING DATA 37
3.2.2 DATA NORMALIZATION 37
3.2.3 STATISTICAL ANALYSIS AND HIT CLASSIFICATION OF THE PRIMARY SCREEN
39
3.2.4 HIT VALIDATION AND CLASSIFICATION OF THE VALIDATED HITS 42
3.3 NUCLEOTIDE METABOLISM IN C. TRACHOMATIS 12 INFECTION 48
3.3.1 CHARACTERIZATION OF THE ROLE OF GUANOSINE MONOPHOSPHATE SYNTHASE
(GMPS) IN C. TRACHOMATIS 12 INFECTION
48
3.3.2 EXPRESSION OF GMPS IN HELA CELLS INFECTED WITH C. TRACHOMATIS 12
50
3.3.3 GMPS INHIBITOR ACIVICIN BLOCKS CHLAMYDIA REPLICATION AND
PRODUCTION OF PROGENY 51
3.3.4 RECOVERY OF INFECTION BY EXTERNAL ADDITION OF GUANOSINE
TRIPHOSPHATE (GTP) OR GUANOSINE MONOPHOSPHATE
(GMP) IN CELLS TREATED WITH ACIVICIN 53
3.3.5 GMPS IS RECRUITED TO THE C. TRACHOMATIS L2 INCLUSION 55
SEITE | I
HTTP://D-NB.INFO/1046691635
TABLE OF CONTENTS
4. DISCUSSION 57
4.1 PREFACE 57
4.2 ESTABLISHMENT OF THE ASSAY 59
4.3 DATA ANALYSIS 61
4.4 ESSENTIAL CELLULAR FACTORS IDENTIFIED IN THE GENOME-WIDE RNAI SCREEN
FOR CHLAMYDIA INFECTION 62
4.5 NUCLEOTIDE METABOLISM IN CHLAMYDIA AND IN HUMAN CELLS 65
4.6 GMPS REGULATION IN HELA CELLS DURING CHLAMYDIA INFECTION 70
4.7 GMPS-MORE THAN AN ENZYME INVOLVED IN DE NOVO NUCLEOTIDE SYNTHESIS 72
5. OUTLOOK 74
6. MATERIALS AND METHODS 75
6.1 MATERIALS 75
6.1.1 BACTERIAL STRAIN 75
6.1.2 CELL LINE 75
6.1.3 CELL CULTURE MEDIA 75
6.1.4 GENE SPECIFIC PRIMERS 76
6.1.5 CHEMICALS 76
6.1.6 ANTIBIOTICS 77
6.1.7 GMPS INHIBITORS 77
6.1.8 COMMERCIALS KITS 77
6.1.9 BUFFERS AND SOLUTIONS 78
6.1.10 ANTIBODIES 80
6.1.10.1 PRIMARY ANTIBODIES 80
6.1.10.2 SECONDARY ANTIBODIES 81
6.1.11 HUMAN WHOLE GENOME SIRNA SET 81
6.1.12 LABORATORY EQUIPMENT 81
6.1.13 COMPUTER SOFTWARE 82
6.2 METHODS 82
6.2.1 CELL CULTURE 82
6.2.2 PROPAGATION AND PREPARATION OF CHLAMYDIA TRACHOMATIS L2 STOCK 82
6.2.3 SIRNA TRANSFECTION OF HELA CELLS 83
6.2.4 INFECTION OF TRANSFECTED HELA CELLS WITH CHLAMYDIA 84
6.2.5 INFECTIVITY ASSAY 84
6.2.6 IMMUNOSTAINING PROCEDURES 84
6.2.7 DETERMINATION OF CELL NUMBERS BY LDH ASSAY 85
6.2.8 AUTOMATED MICROSCOPY AND IMAGE ANALYSIS 85
6.2.9 TREATMENT OF INFECTED HELA CELLS WITH GMPS INHIBITOR ACIVICIN 86
6.2.10 RECOVERY OF INFECTION BY EXTERNAL ADDITION GMP OR GTP 86
6.2.11 PROTEIN TECHNIQUES 86
6.2.11.1 SAMPLES PREPARATIONS 86
6.2.11.2 SDS-PAGE 86
6.2.11.3 PROTEIN TRANSFER AND IMMUNOBLOTTING 87
6.2.12 RNA TECHNIQUES 87
6.2.12.1 ISOLATION OF RNA 87
6.2.12.2 REVERSE TRANSCRIPTION AND RELATIVE QUANTIFICATION OF MRNA BY
REAL-TIME (RT-)PCR 88
6.2.13 STATISTICAL ANALYSIS 88
SEITE | II
TABLE OF CONTENTS
7. REFERENCES 90
8. APPENDIX 105
8.1 ABBREVIATIONS 105
8.2 LIST OF FIGURES 108
8.3 ACKNOWLEDGEMENTS 109
8.4 CURRICULUM VITAE 110
8.5 PUBLICATION LIST ILL
8.6 SELBSTANDIGKEITSERKIARUNG 112
SEITE | III
|
| any_adam_object | 1 |
| author | González, Erik 1975- |
| author_GND | (DE-588)1046754203 |
| author_facet | González, Erik 1975- |
| author_role | aut |
| author_sort | González, Erik 1975- |
| author_variant | e g eg |
| building | Verbundindex |
| bvnumber | BV041588677 |
| classification_rvk | WG 1900 |
| ctrlnum | (OCoLC)915391041 (DE-599)BVBBV041588677 |
| discipline | Biologie |
| format | Thesis Book |
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| spelling | González, Erik 1975- Verfasser (DE-588)1046754203 aut Genome-wide RNA interference screen for the identification of crucial host cell factors in Chlamydia trachomatis infection von Erik González Martínez Berlin Mensch und Buch Verl. 2013 X, 112 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Biology Zugl.: Berlin, Humboldt-Univ., Diss., 2013 Wirtszelle (DE-588)4232591-2 gnd rswk-swf Chlamydia-trachomatis-Infektion (DE-588)4192859-3 gnd rswk-swf RNS-Interferenz (DE-588)4802706-6 gnd rswk-swf Screening (DE-588)4054045-5 gnd rswk-swf Chlamydia trachomatis (DE-588)4192858-1 gnd rswk-swf Genanalyse (DE-588)4200230-8 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Chlamydia-trachomatis-Infektion (DE-588)4192859-3 s Chlamydia trachomatis (DE-588)4192858-1 s Wirtszelle (DE-588)4232591-2 s Genanalyse (DE-588)4200230-8 s RNS-Interferenz (DE-588)4802706-6 s Screening (DE-588)4054045-5 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027033792&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | González, Erik 1975- Genome-wide RNA interference screen for the identification of crucial host cell factors in Chlamydia trachomatis infection Wirtszelle (DE-588)4232591-2 gnd Chlamydia-trachomatis-Infektion (DE-588)4192859-3 gnd RNS-Interferenz (DE-588)4802706-6 gnd Screening (DE-588)4054045-5 gnd Chlamydia trachomatis (DE-588)4192858-1 gnd Genanalyse (DE-588)4200230-8 gnd |
| subject_GND | (DE-588)4232591-2 (DE-588)4192859-3 (DE-588)4802706-6 (DE-588)4054045-5 (DE-588)4192858-1 (DE-588)4200230-8 (DE-588)4113937-9 |
| title | Genome-wide RNA interference screen for the identification of crucial host cell factors in Chlamydia trachomatis infection |
| title_auth | Genome-wide RNA interference screen for the identification of crucial host cell factors in Chlamydia trachomatis infection |
| title_exact_search | Genome-wide RNA interference screen for the identification of crucial host cell factors in Chlamydia trachomatis infection |
| title_full | Genome-wide RNA interference screen for the identification of crucial host cell factors in Chlamydia trachomatis infection von Erik González Martínez |
| title_fullStr | Genome-wide RNA interference screen for the identification of crucial host cell factors in Chlamydia trachomatis infection von Erik González Martínez |
| title_full_unstemmed | Genome-wide RNA interference screen for the identification of crucial host cell factors in Chlamydia trachomatis infection von Erik González Martínez |
| title_short | Genome-wide RNA interference screen for the identification of crucial host cell factors in Chlamydia trachomatis infection |
| title_sort | genome wide rna interference screen for the identification of crucial host cell factors in chlamydia trachomatis infection |
| topic | Wirtszelle (DE-588)4232591-2 gnd Chlamydia-trachomatis-Infektion (DE-588)4192859-3 gnd RNS-Interferenz (DE-588)4802706-6 gnd Screening (DE-588)4054045-5 gnd Chlamydia trachomatis (DE-588)4192858-1 gnd Genanalyse (DE-588)4200230-8 gnd |
| topic_facet | Wirtszelle Chlamydia-trachomatis-Infektion RNS-Interferenz Screening Chlamydia trachomatis Genanalyse Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027033792&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT gonzalezerik genomewidernainterferencescreenfortheidentificationofcrucialhostcellfactorsinchlamydiatrachomatisinfection |