Eimeria bovis-mediated modulation of the host cell cholesterol metabolism
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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Gießen
VVB Laufersweiler Verlag
2014
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| Ausgabe: | 1. Aufl. |
| Schriftenreihe: | Edition Scientifique
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| 245 | 1 | 0 | |a Eimeria bovis-mediated modulation of the host cell cholesterol metabolism |c eingereicht von Penny Humaidah Hamid |
| 250 | |a 1. Aufl. | ||
| 264 | 1 | |a Gießen |b VVB Laufersweiler Verlag |c 2014 | |
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| 650 | 7 | |a E. bovis |2 cabt | |
| 650 | 7 | |a cholesterol metabolism |2 cabt | |
| 650 | 7 | |a bovine serum albumin |2 cabt | |
| 650 | 0 | 7 | |a Cholesterinstoffwechsel |0 (DE-588)4147860-5 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Eimeria bovis |0 (DE-588)4453723-2 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Rind |0 (DE-588)4050061-5 |2 gnd |9 rswk-swf |
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| 689 | 0 | 0 | |a Rind |0 (DE-588)4050061-5 |D s |
| 689 | 0 | 1 | |a Eimeria bovis |0 (DE-588)4453723-2 |D s |
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Datensatz im Suchindex
| _version_ | 1819624571050917888 |
|---|---|
| adam_text | TABLE OF CONTENTS
1 INTRODUCTION 1
2 LITERATURE REVIEW 3
2.1 EIMERIABOVIS 3
2.1.1 GENERAL INTRODUCTION 3
2.1.2 BOVINE COCCIDIOSIS AND E. BOVIS LIFE CYCLE 5
2.1.3 MODULATION OF THE HOST CELL BY E. BOVIS INFECTIONS 8
2.2 CHOLESTEROL 1
2.2.1 CELLULAR CHOLESTEROL SOURCES 12
2.2.2 CYTOSOLIC LIPID DROPLETS (LDS) 16
2.2.3 INTRACELLULAR CHOLESTEROL TRANSPORT AND REGULATION OF
HOMEOSTASIS 17
2.3 MODULATION OF HOST CELL CHOLESTEROL METABOLISM BY PROTOZOAN
PARASITES 20
3 MATERIALS AND METHODS 25
3.1 CELL CULTURE 25
3.1.1 PRIMARY ENDOTHELIAL CELL ISOLATION AND CULTIVATION 25
3.1.2 ENDOTHELIAL CELL SUBCULTIVATION AND CRYOPRESERVATION 26
3.2 PARASITE PREPARATIONS 27
3.2.1 EXPERIMENTAL ANIMALS 27
3.2.2 ANIMAL INFECTIONS WITH E. BOVIS 27
3.2.3 OOCYST ISOLATION FROM THE FAECES 28
3.2.4 OOCYST EXCYSTATION 28
3.2.5 E. BOVIS IN VITRO INFECTION 29
3.3 CHOLESTEROL-RELATED ASSAYS 30
3.3.1 CHOLESTEROL STAINING 30
3.3.2 CHOLESTEROL QUANTIFICATION 31
3.3.2.1 TOTAL LIPID EXTRACTION 31
3.3.2.2 TOTAL CHOLESTEROL QUANTIFICATION 32
3.3.3 STEROLS ENRICHMENT 32
3.3.4 CHOLESTEROL PULSE-LABELLING 33
3.3.5 CHOLESTEROL DEPLETION PRIOR TO INFECTION 34
3.4 LIPID DROPLET-RELATED ASSAYS 35
3.4.1 LIPID DROPLET STAINING 35
3.4.1.1 NILE RED STAINING 35
3.4.1.2 BODIPY 493/503 STAINING 35
3.4.1.3 OSMIUM TETROXIDE STAINING 36
3.4.2 LIPID DROPLET QUANTIFICATION 36
3.4.2.1 FLOW CYTOMETRY ANALYSIS 36
3.4.2.2 SEMIQUANTITATIVE ASSAY OF LIPID ACCUMULATION 37
3.4.2.3 OLEIC ACID ENRICHMENT 38
3.5 LOW DENSITY LIPOPROTEIN-RELATED ASSAYS 39
VII
HTTP://D-NB.INFO/1060269430
3.5.1 QUANTIFICATION OF SURFACE LDL RECEPTOR EXPRESSION 39
3.5.2 LOW DENSITY LIPOPROTEIN (LDL) BINDING ASSAY 40
3.5.3 LOW DENSITY LIPOPROTEIN (LDL) ENRICHMENT 40
3.6 INHIBITION ASSAYS 41
3.6.1 TOXICITY ASSAY 41
3.6.2 INHIBITION OF E. BOVIS PROLIFERATION IN VITRO 42
3.7 QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (QPCR) 43
3.7.1 GENE TRANSCRIPTION OF CHOLESTEROL METABOLISM-RELATED
MOLECULES 43
3.7.1.1 TARGET GENES, PRIMERS AND PROBES DESIGN 43
3.7.1.2 GENERATION OF QPCR STANDARDS 45
3.7.1.3 ANALYSIS OF QPCR EFFICIENCIES 47
3.7.1.4 RNA PREPARATION AND CDNA SYNTHESIS 48
3.7.1.5 TOTAL RNA ISOLATION AND DNA DIGESTION 48
3.7.1.6 CDNA SYNTHESIS 49
3.7.1.7 QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION
(QPCR) ASSAY 49
3.7.1.8 DATA ANALYSIS 50
3.7.2 QPCR-BASED E. BOVIS MEROZOITE 1 QUANTIFICATION 50
3.8 IMMUNOB LOTTING 51
3.9 STATISTICAL ANALYSIS 54
4 RESULTS 55
4.1 CHOLESTEROL AND LIPID DROPLET LOCALIZATION IN FREE PARASITE STAGES
AND
INFECTED CELLS 55
4.1.1 CHOLESTEROL LOCALIZATION IN E. BOVIS STAGES 55
4.1.2 DANSYL-CHOLESTEROL INCORPORATION INTO E. BOVIS STAGES 58
4.1.3 LIPID DROPLET (LD) FORMATION IN E. BOVIS STAGES 60
4.2 QUANTIFICATION OF CHOLESTEROL AND LIPID DROPLET (LD) CONTENTS IN .
6OVU-INFECTED HOST CELLS 65
4.2.1 CHOLESTEROL ACCUMULATION IN E. TOWS-INFECTED HOST CELLS 65
4.2.2 LIPID DROPLET (LD) ACCUMULATION IN E. FCOVIS-INFECTED HOST
CELLS 67
4.3 INFLUENCE OF CHOLESTEROL AND LIPID DROPLET (LD) ENRICHMENT ON E.
BOVIS DEVELOPMENT IN VITRO 68
4.3.1 EFFECTS OF CHOLESTEROL SUPPLEMENTATION ON MACROMERONT
DEVELOPMENT 68
4.3.2 EFFECTS OF CHOLESTEROL DEPLETION ON E. BOVIS DEVELOPMENT IN
VITRO 69
4.3.3 EFFECTS OF INCREASED HOST CELLULAR LIPID DROPLET DISPOSABILITY
ON E. BOVIS MEROZOITE I PRODUCTION 70
4.4 INVOLVEMENT OF LDL IN E. BOVIS MACROMERONT DEVELOPMENT IN VITRO 74
4.4.1 BINDING OF LDL AND ACETYLATED LDL (ACLDL) ON PARASITE-
INFECTED HOST CELLS 74
4.4.2 SURFACE LDL RECEPTOR (LDLR) EXPRESSION ON E. BOVIS-
INFECTED HOST CELLS 78
VIII
4.4.3 EFFECTS OF LDL ENRICHMENT ON E. BOVIS IN VITRO DEVELOPMENT 78
4.5 GENE TRANSCRIPTION AND PROTEIN EXPRESSION OF CHOLESTEROL METABOLISM-
RELATED MOLECULES IN E. IOVIS-INFECTED HOST CELLS 80
4.5.1 ESTABLISHMENT AND VALIDATION OF QPCR SYSTEMS 80
4.5.2 TRANSCRIPTIONAL PROFILING OF DIFFERENT MOLECULES RELEVANT FOR
HOST CHOLESTEROL METABOLISM IN E. IOV/J-INFECTED BUVEC 82
4.5.3 PROTEIN EXPRESSION OF ACAT1, CH25H, OLR1 AND SOAT1 IN
E. TOVW-INFECTED BUVEC 87
4.6 INHIBITION OF E. BOVIS IN VITRO DEVELOPMENT BY INTERFERENCE WITH THE
MEVALONATE BIOSYNTHESIS PATHWAYS AND FATTY ACID SYNTHESIS 88
4.6.1 EVALUATION OF ADEQUATE INHIBITOR CONCENTRATIONS 88
4.6.2 ESTABLISHMENT OF EBMIC4-BASED QPCR FOR MEROZOITES I
QUANTIFICATION 89
4.6.3 INHIBITION OF HMGCOA REDUCTASE 91
4.6.4 INHIBITION OF SQUALENE SYNTHASE 93
4.6.5 INHIBITION OF ACYL-COA CHOLESTEROL ACYLTRANSFERASE 95
4.6.6 INHIBITION OF FATTY ACID SYNTHASE 98
5 DISCUSSION 101
5.1 FREE CHOLESTEROL AND LIPID DROPLETS ACCUMULATE IN E. IOVI S-INFECTED
HOST CELLS AND REVEAL AS KEY FACTORS OF PARASITE REPLICATION 101
5.1.1 FREE CHOLESTEROL ACCUMULATION IN PARASITE STAGES 101
5.1.2 LIPID DROPLET FORMATION IN PARASITE STAGES AND INFECTED HOST
CELLS 106
5.2 E. BOVIS UP-REGULATES BOTH HOST CELL CHOLESTEROL DE NOVO SYNTHESIS
AND
LDL-MEDIATED UPTAKE 112
5.2.1 UP-REGULATION OF THE MEVALONATE BIOSYNTHESIS PATHWAY AND OF
HOST CELLULAR CHOLESTEROL PROCESSING BY E. BOVIS INFECTIONS 112
5.2.2 INHIBITION OF HOST CELLULAR CHOLESTEROL DE NOVO SYNTHESIS AND
ESTERIFICATION BLOCKS PARASITE GROWTH 115
5.2.3 KEY ROLE OF HOST CELLULAR LDL UP-TAKE IN E. BOVIS
DEVELOPMENT 119
6 SUMMARY 123
7 ZUSAMMENFASSUNG 125
8 REFERENCES 127
9 APPENDIX 144
9.1 STATISTICAL ANALYSIS 144
ACKNOWLEDGMENT 147
DECLARATION 148
IX
|
| any_adam_object | 1 |
| author | Hamid, Penny Humaidah |
| author_facet | Hamid, Penny Humaidah |
| author_role | aut |
| author_sort | Hamid, Penny Humaidah |
| author_variant | p h h ph phh |
| building | Verbundindex |
| bvnumber | BV042368221 |
| ctrlnum | (OCoLC)915574673 (DE-599)DNB1060269430 |
| dewey-full | 636.2089693601 |
| dewey-hundreds | 600 - Technology (Applied sciences) |
| dewey-ones | 636 - Animal husbandry |
| dewey-raw | 636.2089693601 |
| dewey-search | 636.2089693601 |
| dewey-sort | 3636.2089693601 |
| dewey-tens | 630 - Agriculture and related technologies |
| discipline | Agrar-/Forst-/Ernährungs-/Haushaltswissenschaft / Gartenbau |
| edition | 1. Aufl. |
| format | Thesis Book |
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| series2 | Edition Scientifique |
| spellingShingle | Hamid, Penny Humaidah Eimeria bovis-mediated modulation of the host cell cholesterol metabolism E. bovis cabt cholesterol metabolism cabt bovine serum albumin cabt Cholesterinstoffwechsel (DE-588)4147860-5 gnd Eimeria bovis (DE-588)4453723-2 gnd Rind (DE-588)4050061-5 gnd |
| subject_GND | (DE-588)4147860-5 (DE-588)4453723-2 (DE-588)4050061-5 (DE-588)4113937-9 |
| title | Eimeria bovis-mediated modulation of the host cell cholesterol metabolism |
| title_auth | Eimeria bovis-mediated modulation of the host cell cholesterol metabolism |
| title_exact_search | Eimeria bovis-mediated modulation of the host cell cholesterol metabolism |
| title_full | Eimeria bovis-mediated modulation of the host cell cholesterol metabolism eingereicht von Penny Humaidah Hamid |
| title_fullStr | Eimeria bovis-mediated modulation of the host cell cholesterol metabolism eingereicht von Penny Humaidah Hamid |
| title_full_unstemmed | Eimeria bovis-mediated modulation of the host cell cholesterol metabolism eingereicht von Penny Humaidah Hamid |
| title_short | Eimeria bovis-mediated modulation of the host cell cholesterol metabolism |
| title_sort | eimeria bovis mediated modulation of the host cell cholesterol metabolism |
| topic | E. bovis cabt cholesterol metabolism cabt bovine serum albumin cabt Cholesterinstoffwechsel (DE-588)4147860-5 gnd Eimeria bovis (DE-588)4453723-2 gnd Rind (DE-588)4050061-5 gnd |
| topic_facet | E. bovis cholesterol metabolism bovine serum albumin Cholesterinstoffwechsel Eimeria bovis Rind Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027804526&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT hamidpennyhumaidah eimeriabovismediatedmodulationofthehostcellcholesterolmetabolism |