Interaction of Human Polyomavirus JC with cells of the hematopoietic system in the periphery:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | Volltext Inhaltsverzeichnis |
| Beschreibung: | IV, 145 S. Ill. |
Internformat
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| 100 | 1 | |a Sbiera, Silviu |d 1976- |e Verfasser |0 (DE-588)1029725225 |4 aut | |
| 245 | 1 | 0 | |a Interaction of Human Polyomavirus JC with cells of the hematopoietic system in the periphery |c von Silviu Sbiera |
| 246 | 1 | 3 | |a Interaktion zwischen Human Polyomavirus JC mit Zellen des haematopoietischen Systems in der Peripherie |
| 264 | 1 | |c 2012 | |
| 300 | |a IV, 145 S. |b Ill. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
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| 502 | |a Würzburg, Univ., Diss., 2012 | ||
| 650 | 0 | 7 | |a Polyomaviren |0 (DE-588)7757390-0 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Blutbildendes Gewebe |0 (DE-588)4239650-5 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Persistenz |0 (DE-588)4136610-4 |2 gnd |9 rswk-swf |
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
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| 689 | 0 | 1 | |a Persistenz |0 (DE-588)4136610-4 |D s |
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Datensatz im Suchindex
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|---|---|
| adam_text | IMAGE 1
TABLE O F CONTENTS
1. INTRODUCTION 1
2. MATERIALS AND METHODS 6
2.1. MATERIALS 6
2.1.1. BLOOD MATERIALS: 6
2.1.2 CELL-LINES 6
2.1.3. GROWTH MEDIA FOR BACTERIA AND CELL CULTURES 7
2.1.4. RECOMBINANT DNA AND DNA LADDERS 8
2.1.4.1. CLONED VIRUS DNA 8
2.1.4.2. MARKERS FOR ELECTROPHORETIC SEPARATION 9
2.1.5. BUFFERS 9
2.1.6. CHEMICALS AND ENZYMES: 12
2.1.7. ANTIBODIES 13
2.1.8. KITS 14
2.1.9. EQUIPMENT 14
2.1.10. MISCELLANEOUS 15
2.2. METHODS 17
22. 1. ESCHERICHIA COLI BACTERIAL CULTURE FOR VECTOR DNA EXPRESSION 17
2.2.1.0. PREPARATION O F AGAR PLATES 17
2.2.1.1. GROWTH ON AGAR-PLATES 17
2.2.1.2. GROWTH IN LIQUID LB-MEDIUM 17
2.2.1.3. LONG TERM STORAGE O F BACTERIA 18
2.2.2. CELL CULTURES AS CONTROLS FOR THE VIRUS DETECTION 18
2.2.2.1. SPLITTING ADHERENT MAMMAL CELL CULTURES 19
2.2.2.2. SPLITTING SUSPENSION CELL CULTURES 19
2.2.2.3. SPLITTING ADHERENT INSECT CELLS IN CULTURE 19
2.2.2.4. PREPARATION O F VIRUS INFECTED CELLS FOR SINGLE CELL DETECTION
O F VIRAL PRODUCTS 19
2.2.2.5. RECOMBINANT JC VIRUS INFECTION IN SF-9 CELLS 20
2.2.3. DNA ISOLATION AND CHARACTERIZATION 20
2.2.3.1. PREPARATION O F PLASMID DNA 20
2.2.3.2. LEUKOCYTE DNA ISOLATION 21
2.2.3.3. DNA ETHANOL PRECIPITATION 22
I
HTTP://D-NB.INFO/1033485314
IMAGE 2
2.2.3.4. PREPARATION O F CARRIER DNA SOLUTION 22
2.2.3.5. AGAROSE GEL ELECTROPHORESIS O F NUCLEIC ACIDS 23
2.2.3.6. PURIFICATION O F RADIOACTIVE PHOSPHOR 32- AND
DIGOXIGENIN-LABELED PCR PRODUCTS BY
SEPHADEX G50 COLUMN CHROMATOGRAPHY 23
2.2.4. ISOLATION O F BLOOD CELLS AND SEPARATION O F HAEMATOPOIETIC
SUBPOPULATIONS. 24
2.2.4.1. ISOLATION O F PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) BY
FICOLL DENSITY
CENTRIFUGATION 24
2.2.4.2. ISOLATION O F GRANULOCYTES AND PBMCS FROM PERIPHERAL BLOOD BY A
TWO-STEP PERCOLL
GRADIENT CENTRIFUGATION 24
2.2.4.3. ERYTHROLYSIS 25
2.2.4.4. VIABILITY AND CELL-COUNT DETERMINATION BY TRYPAN-BLUE EXCLUSION
26
2.2.4.5. SEPARATION O F PBMC SUBPOPULATIONS BY MAGNETIC BEAD ISOLATION
26
2.2.4.5.1. MAGNETIC ISOLATION O F PERIPHERAL BLOOD B-CELLS, T-CELLS AND
MONOCYTES BY POSITIVE
SELECTION 26
2.2.4.6. DETERMINATION O F THE PURITY O F SEPARATED SUBPOPULATIONS BY
FACS ANALYSIS 27
2.2.5. DETECTION O F VIRUS SPECIFIC DNA IN PERIPHERAL BLOOD CELLS BY
POLYMERASE CHAIN REACTION
(PCR) 28
2.2.6. SPECIFICITY CHARACTERIZATION O F PCR AMPLIFIED DNA FRAGMENTS
USING HYBRIDIZATION
TECHNIQUES 28
2.2.6.1. TRANSFER O F PCR AMPLIFIED DNA FRAGMENTS ON NITROCELLULOSE
MEMBRANE FOR FURTHER
ANALYSIS USING SOUTHERN BLOT 28
2.2.6.2. DETERMINING THE SPECIFICITY O F THE PCR AMPLIFIED PRODUCTS
USING RADIOACTIVE
HYBRIDIZATION O F SOUTHERN BLOTS 29
2.2.6.2.1. RADIOACTIVE LABELING O F SPECIFIC OLIGONUCLEOTIDES AS
HYBRIDIZATION PROBES 29
2.2.6.2.2. HYBRIDIZATION O F MEMBRANE BOUND DNA WITH RADIOACTIVE LABELED
OLIGONUCLEOTIDE
PROBES 31
2.2.6.3. DETECTION O F VIRUS DNA IN SITU USING FLUORESCENCE IN SITU
HYBRIDIZATION (FISH) 32
2.2.6.3.1. FIXATION AND PERMEABILIZATION O F CELLS ON SLIDES 32
2.2.6.3.2. PREPARATION O F DIGOXIGENIN-LABELLED DNA PROBES FOR FISH 32
2.2.6.3.3. FLUORESCENCE IN SITU HYBRIDIZATION WITH CHEMICAL
AMPLIFICATION O F THE SIGNAL 36
2.2.6.3.4. FLUORESCENCE IN SITU HYBRIDIZATION WITHOUT CHEMICAL
AMPLIFICATION 38
2.2.6.4. ESTIMATION O F DIGOXIGENIN LABELED DNA PROBE SPECIFICITY BY
DOT-BLOT HYBRIDIZATION 38
2.2.6.4.1. IMMUNOSTAINING DETECTION O F DOT-BLOT HYBRIDIZATION PRODUCTS
38
2.2.8. VISUALIZATION O F IN SITU FLUORESCENT SIGNALS USING MICROSCOPY 39
II
IMAGE 3
2.2.8.1. ASSESSING THE FISH RESULTS USING CLASSICAL FLUORESCENCE
MICROSCOPY 39
2.2.8.2. VISUALIZATION AND LOCALIZATION O F FLUORESCENT SIGNALS USING
LASER SCANNING MICROSCOPY
(LSM) 40
3. RESULTS 41
3.1. AIM OF THE STUDY 41
3.2. STUDY GROUPS FOR ANALYSIS O F JCV INFECTION IN PERIPHERAL BLOOD
CELLS 41
3.3. MATERIAL COLLECTION STRATEGY 42
3.3.1. ISOLATION O F PERIPHERAL BLOOD CELL POPULATIONS 42
3.3.1.1. ISOLATION O F PBMCS AND GRANULOCYTES BY DENSITY GRADIENT
CENTRIFUGATION 42
3.3.1.2. ENRICHMENT O F PBMC SUBPOPULATIONS BY MAGNETIC CELL SEPARATION
SYSTEM 44
3.3.1.3. PURITY O F HEMATOPOIETIC CELL SUBPOPULATIONS 44
3.3.1.3.1. PURITY O F DENSITY GRADIENT PURIFIED PBMCS AND GRANULOCYTES
44
3.3.1.3.2. PURITY O F MACS ISOLATED LEUKOCYTE SUBPOPULATIONS DETERMINED
BY FACS ANALYSIS
45
3.4. DETECTION OF JCV DNA IN PERIPHERAL BLOOD CELLS 50
3.4.1. SENSITIVITY AND SPECIFICITY O F THE POLYMERASE CHAIN REACTION
(PCR) FOR THE DETECTION O F JCV
DNA IN PERIPHERAL BLOOD CELLS 50
3.4.2. PRESENCE O F JCV DNA IN PERIPHERAL BLOOD CELL PREPARATIONS 51
3.4.2.1. DETECTION O F JCV DNA IN PERIPHERAL BLOOD CELLS O F HEALTHY
BLOOD DONORS 51
3.4.2.2. DETECTION O F JCV DNA IN PERIPHERAL BLOOD CELLS O F IMMUNE
IMPAIRED PATIENTS 56
3.5. CHARACTERIZATION OF JCV-PERIPHERAL BLOOD CELLS ASSOCIATION 60
3.5.1. ESTABLISHMENT O F THE FLUORESCENCE IN SITU HYBRIDIZATION
TECHNIQUE FOR SUB-CELLULAR
LOCALIZATION O F JCV DNA IN PERIPHERAL BLOOD CELLS 60
3.5.1.1. FIXATION O F PRIMARY PERIPHERAL BLOOD CELLS FOR FISH 61
3.5.1.2. DEVELOPMENT O F A HIGHLY SENSITIVE JCV SPECIFIC FLUORESCENCE
PROBE FOR IN SITU
HYBRIDIZATION 68
3.5.1.3. DETECTION CONDITIONS FOR DIGOXIGENIN-LABELED HYBRIDIZATION
PROBES IN PRIMARY BLOOD
CELLS 73
3.5.1.4. JCV SPECIFICITY O F THE FISH HYBRIDIZATION IN SINGLE CELLS 79
3.5.2.1. IMPROVEMENT O F THE IN-SITU VISUALIZATION BY DECONVOLUTION
SOFTWARE 81
3.5.2.2. JCV LOCALIZATION AT SINGLE CELL LEVEL BY CONFOCAL MICROSCOPY 83
III
IMAGE 4
3.5.2.3. SOFTWARE BASED LOCALIZATION O F JCV SIGNALS AT SUBCELLULAR
LEVEL 83
3.5.3.1. JCV LOCALIZATION IN PERIPHERAL BLOOD CELLS 85
3.5.3.2. RATE O F PERIPHERAL BLOOD CELLS ASSOCIATED WITH THE JCV DNA 86
3.5.3.3. INTRACELLULAR QUANTIFICATION O F VIRAL GENOMES IN PBCS 92
3.5.3.4. INFLUENCE O F SHORT TIME IMMUNE SYSTEM ACTIVATION ON JCV
ASSOCIATION WITH PERIPHERAL
BLOOD CELLS 96
4. SUMMARY AND DISCUSSIONS 101
5. BIBLIOGRAPHY 116
6. ABBREVIATIONS 132
7. ENGLISH ABSTRACT 134
8. GERMAN ABSTRACT 136
9. CURRICULLUM VITAE 138
I V
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| indexdate | 2024-07-10T00:28:47Z |
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| language | English |
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| physical | IV, 145 S. Ill. |
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| spelling | Sbiera, Silviu 1976- Verfasser (DE-588)1029725225 aut Interaction of Human Polyomavirus JC with cells of the hematopoietic system in the periphery von Silviu Sbiera Interaktion zwischen Human Polyomavirus JC mit Zellen des haematopoietischen Systems in der Peripherie 2012 IV, 145 S. Ill. txt rdacontent n rdamedia nc rdacarrier Würzburg, Univ., Diss., 2012 Polyomaviren (DE-588)7757390-0 gnd rswk-swf Blutbildendes Gewebe (DE-588)4239650-5 gnd rswk-swf Persistenz (DE-588)4136610-4 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Polyomaviren (DE-588)7757390-0 s Persistenz (DE-588)4136610-4 s Blutbildendes Gewebe (DE-588)4239650-5 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-74183 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-74183 Resolving-System kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025498853&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Sbiera, Silviu 1976- Interaction of Human Polyomavirus JC with cells of the hematopoietic system in the periphery Polyomaviren (DE-588)7757390-0 gnd Blutbildendes Gewebe (DE-588)4239650-5 gnd Persistenz (DE-588)4136610-4 gnd |
| subject_GND | (DE-588)7757390-0 (DE-588)4239650-5 (DE-588)4136610-4 (DE-588)4113937-9 |
| title | Interaction of Human Polyomavirus JC with cells of the hematopoietic system in the periphery |
| title_alt | Interaktion zwischen Human Polyomavirus JC mit Zellen des haematopoietischen Systems in der Peripherie |
| title_auth | Interaction of Human Polyomavirus JC with cells of the hematopoietic system in the periphery |
| title_exact_search | Interaction of Human Polyomavirus JC with cells of the hematopoietic system in the periphery |
| title_full | Interaction of Human Polyomavirus JC with cells of the hematopoietic system in the periphery von Silviu Sbiera |
| title_fullStr | Interaction of Human Polyomavirus JC with cells of the hematopoietic system in the periphery von Silviu Sbiera |
| title_full_unstemmed | Interaction of Human Polyomavirus JC with cells of the hematopoietic system in the periphery von Silviu Sbiera |
| title_short | Interaction of Human Polyomavirus JC with cells of the hematopoietic system in the periphery |
| title_sort | interaction of human polyomavirus jc with cells of the hematopoietic system in the periphery |
| topic | Polyomaviren (DE-588)7757390-0 gnd Blutbildendes Gewebe (DE-588)4239650-5 gnd Persistenz (DE-588)4136610-4 gnd |
| topic_facet | Polyomaviren Blutbildendes Gewebe Persistenz Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-74183 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025498853&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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