The IE1 protein of human cytomegalovirus as an antagonist of intrinsic cellular defense mechanisms: = Antagonisierung zellulärer Abwehrmechanismen durch das IE1-Protein des Humanen Cytomegalovirus
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| Format: | Thesis/Dissertation Book |
| Language: | English |
| Published: |
2013
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| Subjects: | |
| Links: | https://open.fau.de/handle/openfau/3436 https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-46890 http://d-nb.info/1036774929/34 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026735882&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| Physical Description: | 118 S. Ill., graph. Darst. 21 cm |
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| 245 | 1 | 0 | |a The IE1 protein of human cytomegalovirus as an antagonist of intrinsic cellular defense mechanisms |b = Antagonisierung zellulärer Abwehrmechanismen durch das IE1-Protein des Humanen Cytomegalovirus |c vorgelegt von Myriam Scherer |
| 246 | 1 | 1 | |a Antagonisierung zellulärer Abwehrmechanismen durch das IE1-Protein des Humanen Cytomegalovirus |
| 264 | 1 | |c 2013 | |
| 300 | |a 118 S. |b Ill., graph. Darst. |c 21 cm | ||
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Record in the Search Index
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| adam_text | IMAGE 1
TABLE OF CONTENTS
A. SUMMARY .... ...... 1
A. ZUSAMMENFASSUNG 2
B. INTRODUCTION ......... ....... 3
C. OBJECTIVES - 13
D. MATERIALS AND METHODS 14
1. BIOLOGICAL MATERIALS 14
1.1. BACTERIA 14
1.2. EUKARYOTIC CELL CULTURES 14
1.3. VIRUS STRAINS 14
1.4. ANTIBODIES IS
1.4.1. MONOCLONAL ANTIBODIES 15
1.4.2. POLYCLONAL ANTIBODIES 16
1.4.3. SECONDARY ANTIBODIES 17
2. NUCLEIC ACIDS 17
2.1. OLIGONUCLEOTIDES 17
2.2. VECTORS AND PLASMIDS 20
2.2.1. VECTORS AND VECTOR SYSTEMS 20
2.2.2. PLASMIDS CONTAINING SYNTHETIC GENES 21
2.2.3. READY-TO-USE CONSTRUCTS 21
2.2.4. NEWLY GENERATED PLASMIDS 22
3. ENZYMES, CHEMICALS, AND MEDIA 25
3.1. ENZYMES 25
3.2. MEDIA 26
3.2.1. BACTERIAL MEDIA 26
3.2.2. CELL CULTURE MEDIA 26
3.3. CHEMICALS 27
3.4. STANDARD BUFFERS AND SOLUTIONS 27
4. STANDARD MOLECULAR BIOLOGY TECHNIQUES 28
4.1. IN VITRO MUTAGENESIS 29
4.2. GATEWAY CLONING 29
5. STANDARD CELL CULTURE TECHNIQUES 29
5.1. MAINTENANCE OF CELL CULTURES 29
5.2. TRANSFECTION AND INFECTION OF MAMMALIAN CELLS 30
I
HTTP://D-NB.INFO/1038246296
IMAGE 2
6. INDIRECT IMMUNOFLUORESCENCE ANALYSIS 30
7. COIMMUNOPRECIPITATION 31
8. GENERATION OF LENTH/IRALLY TRANSDUCED CELL LINES 31
8.1. GENERATION OF INFECTIOUS LENTIVIRAL PARTICLES 31
8.2. LENTIVIRAL TRANSDUCTION AND SELECTION OF STABLY TRANSDUCED CELLS 32
9. INFECTION EXPERIMENTS OF PRIMARY HUMAN FORESKIN FIBROBLASTS 32
9.1. PREPARATION OF VIRUS STOCKS 32
9.2. VIRUS TITRATION 33
9.3. DETERMINATION OF VIRUS RELEASE 33
9.4. GFP REPORTER ASSAY 33
9.5. PLAQUE ASSAY 34
9.6. DETERMINATION OF THE VIRAL LOAD IN INFECTED HFFS 34
9.7. CHARACTERIZATION OF RECOMBINANT HCMV BY MULTISTEP GROWTH-CURVE
ANALYSIS 35
10. PROTEIN EXPRESSION AND PURIFICATION 35
10.1. COOMASSIE BLUE STAINING 35
10.2. SMALL-SCALE INDUCTION OF PROTEIN EXPRESSION 35
10.3. LARGE-SCALE INDUCTION OF PROTEIN EXPRESSION 36
10.4. PURIFICATION OF GST-TAGGED PROTEINS BY AFFINITY CHROMATOGRAPHY 36
10.5. REMOVAL OF THE GST-TAG FROM GST-IE1 FUSION PROTEINS 37
10.6. GEL FILTRATION 37
10.7. LIMITED PROTEOLYSIS, CD SPECTROSCOPY, AND CRYSTALLIZATION
EXPERIMENTS 37
E. RESULTS 38
1. INTERPLAY BETWEEN HCMV AND THE CELLULAR SUMO CONJUGATION PATHWAY 38
1.1. CHARACTERIZATION OF LEL-INDUCED CHANGES OF PROTEIN SUMOYIATION 38
1.1.1. GENERATION OF PRIMARY HUMAN FIBROBLASTS WITH A STABLE EXPRESSION
OF FLAG-TAGGED SUMO-1 OR SUMO-3 39
1.1.2. CHARACTERIZATION OF HFFS STABLY EXPRESSING F-SUMO-1 OR F -SUMO-3
40
1.1.3. INVESTIGATION OF HCMV-INDUCED CHANGES OF THE SUMOYIATION PATTERN
43
1.1.4. INVESTIGATION OF LEL-INDUCED CHANGES OF THE SUMOYIATION PATTERN
44
1.1.5. IDENTIFICATION OF SPLOO AS DIRECT TARGET OF LEL-MEDIATED
DE-SUMOYLATION 45
1.2. ROLE OF SUMO PROTEINS DURING HCMV REPLICATION 46
1.2.1. LOCALIZATION OF F-SUMO PROTEINS DURING THE HCMV REPLICATION CYCLE
46
1.2.2. ANALYSIS OF THE ROLE OF IE2 IN THE RECRUITMENT OF F-SUMO-1 AND
F-SUMO-3 INTO VIRAL REPLICATION COMPARTMENTS 48
1.2.3. ANALYSIS OF THE EFFECT OF F-SUMO-1 AND F-SUMO-3 EXPRESSION ON
HCMV REPLICATION 49
1.2.4. PREDICTION OF SUMOYIATION SITES WITHIN HCMV PROTEINS 52
IMAGE 3
2. INSIGHTS INTO THE MECHANISM AND STRUCTURAL DETERMINANTS OF ND10
DISRUPTION BY IE1 53
2.1. STRUCTURE DETERMINATION OF A SUBDOMAIN OF PRIMATE CYTOMEGALOVIRUS
IE1 53
2.1.1. EXPRESSION OF FULL LENGTH IE1 INF. COLI 53
2.1.2. PURIFICATION AND CRYSTALLIZATION TRIALS OF FULL LENGTH IE1 54
2.1.3. IDENTIFICATION OF A STABLE SUBDOMAIN WITHIN IE1 56
2.1.4. PURIFICATION AND CRYSTALLIZATION OF TRUNCATED IE1 VARIANTS 57
2.1.5. PURIFICATION AND CRYSTALLIZATION OF A SUBDOMAIN OF HOMOLOGOUS
PRIMATE IE1 PROTEINS 59
2.1.6. STRUCTURE DETERMINATION OF THE SUBDOMAIN OF RHESUS MACAQUE IE1 62
2.2. FUNCTIONAL CHARACTERIZATION OF THE IE1 PROTEIN OF RHESUS MACAQUE
CYTOMEGALOVIRUS 63
2.2.1. INVESTIGATION OF THE INFLUENCE OF RHCMV INFECTION ON ND10 DOMAINS
63
2.2.2. INVESTIGATION OF THE INFLUENCE OF RHIEL EXPRESSION ON ND10
DOMAINS 64
2.3. FUNCTIONAL CHARACTERIZATION OF THE IE1 SUBDOMAIN OF HUMAN
CYTOMEGALOVIRUS 65
2.3.1. LOCALIZATION OF THE IE1 SUBDOMAIN IN TRANSFECTED HFF CELLS 66
2.3.2. LOCALIZATION OF THE IE1 SUBDOMAIN 1-382 IN STABLY TRANSDUCED HFF
CELLS 68
2.3.3. INTERACTION ANALYSIS OF THE IE1 SUBDOMAIN WITH PML VI 69
2.3.4. INTERACTION ANALYSIS OF THE IE1 SUBDOMAIN WITH PML ISOFORMS I TO
VI 71
2.3.5. CHARACTERIZATION OF A SELF-INTERACTION OF IE1 72
2.3.6. INFLUENCE OF THE IE1 SUBDOMAIN ON THE SUMOYLATION STATUS OF PML
AND SPLOO 73
2.3.7. EFFECT OF THE IE1 SUBDOMAIN ON THE LOCALIZATION OF ND10 MAIN
COMPONENTS 75
2.4. CHARACTERIZATION OF A RECOMBINANT HCMV ENCODING THE IE1 SUBDOMAIN
1-382 77
2.4.1. EFFECT OF AD169/IE11-382 ON THE SUBIIUDEAR LOCALIZATION OF ND10
PROTEINS 77
2.4.2. EFFECT OF AD169/IE11-382 ON THE SUMOYLATION STATUS OF ND10
PROTEINS 80
2.4.3. ANALYSIS OF PROTEIN EXPRESSION KINETICS OF AD169/IE11-382
COMPARED TO WT AD169 80
2.4.4. COMPARATIVE GROWTH ANALYSIS OF WT AD169 AND AD169/IE11-382 82
2.5. CHARACTERIZATION OF IE1 MUTANTS DESIGNED ON BASIS OF THE CRYSTAL
STRUCTURE 84
2.5.1. ANALYSIS OF THE SUBNUDEAR LOCALIZATION OF THE STRUCTURE-DERIVED
IE1 MUTANTS 86
2.5.2. ANALYSIS OF THE DE-SUMOYLATION ACTIVITY OF THE STRUCTURE-DERIVED
IE1 MUTANTS 88
F. DISCUSSION 91
G. ABBREVIATIONS ...... 105
H. REFERENCES 106
|
| any_adam_object | 1 |
| author | Scherer, Myriam |
| author_GND | (DE-588)103824577X |
| author_facet | Scherer, Myriam |
| author_role | aut |
| author_sort | Scherer, Myriam |
| author_variant | m s ms |
| building | Verbundindex |
| bvnumber | BV041286814 |
| collection | ebook |
| ctrlnum | (OCoLC)858928621 (DE-599)DNB1038246296 |
| dewey-full | 570 610 |
| dewey-hundreds | 500 - Natural sciences and mathematics 600 - Technology (Applied sciences) |
| dewey-ones | 570 - Biology 610 - Medicine and health |
| dewey-raw | 570 610 |
| dewey-search | 570 610 |
| dewey-sort | 3570 |
| dewey-tens | 570 - Biology 610 - Medicine and health |
| discipline | Biologie Medizin |
| format | Thesis Book |
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| indexdate | 2024-12-20T16:44:11Z |
| institution | BVB |
| language | English |
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| oclc_num | 858928621 |
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| spellingShingle | Scherer, Myriam The IE1 protein of human cytomegalovirus as an antagonist of intrinsic cellular defense mechanisms = Antagonisierung zellulärer Abwehrmechanismen durch das IE1-Protein des Humanen Cytomegalovirus Immunität Medizin (DE-588)4120476-1 gnd Cytomegalie-Virus (DE-588)4238363-8 gnd |
| subject_GND | (DE-588)4120476-1 (DE-588)4238363-8 (DE-588)4113937-9 |
| title | The IE1 protein of human cytomegalovirus as an antagonist of intrinsic cellular defense mechanisms = Antagonisierung zellulärer Abwehrmechanismen durch das IE1-Protein des Humanen Cytomegalovirus |
| title_alt | Antagonisierung zellulärer Abwehrmechanismen durch das IE1-Protein des Humanen Cytomegalovirus |
| title_auth | The IE1 protein of human cytomegalovirus as an antagonist of intrinsic cellular defense mechanisms = Antagonisierung zellulärer Abwehrmechanismen durch das IE1-Protein des Humanen Cytomegalovirus |
| title_exact_search | The IE1 protein of human cytomegalovirus as an antagonist of intrinsic cellular defense mechanisms = Antagonisierung zellulärer Abwehrmechanismen durch das IE1-Protein des Humanen Cytomegalovirus |
| title_full | The IE1 protein of human cytomegalovirus as an antagonist of intrinsic cellular defense mechanisms = Antagonisierung zellulärer Abwehrmechanismen durch das IE1-Protein des Humanen Cytomegalovirus vorgelegt von Myriam Scherer |
| title_fullStr | The IE1 protein of human cytomegalovirus as an antagonist of intrinsic cellular defense mechanisms = Antagonisierung zellulärer Abwehrmechanismen durch das IE1-Protein des Humanen Cytomegalovirus vorgelegt von Myriam Scherer |
| title_full_unstemmed | The IE1 protein of human cytomegalovirus as an antagonist of intrinsic cellular defense mechanisms = Antagonisierung zellulärer Abwehrmechanismen durch das IE1-Protein des Humanen Cytomegalovirus vorgelegt von Myriam Scherer |
| title_short | The IE1 protein of human cytomegalovirus as an antagonist of intrinsic cellular defense mechanisms |
| title_sort | the ie1 protein of human cytomegalovirus as an antagonist of intrinsic cellular defense mechanisms antagonisierung zellularer abwehrmechanismen durch das ie1 protein des humanen cytomegalovirus |
| title_sub | = Antagonisierung zellulärer Abwehrmechanismen durch das IE1-Protein des Humanen Cytomegalovirus |
| topic | Immunität Medizin (DE-588)4120476-1 gnd Cytomegalie-Virus (DE-588)4238363-8 gnd |
| topic_facet | Immunität Medizin Cytomegalie-Virus Hochschulschrift |
| url | https://open.fau.de/handle/openfau/3436 https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-46890 http://d-nb.info/1036774929/34 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026735882&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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