Characterization of signaling pathways activated by the HCMV encoded GPCR homolog pUS28:
Gespeichert in:
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| Format: | Abschlussarbeit Elektronisch Software E-Book |
| Sprache: | English |
| Veröffentlicht: |
2004
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Titel von interner Quelle |
| Beschreibung: | 1 CD-R 12 cm |
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Datensatz im Suchindex
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| adam_text | Table of contents
1 Introduction 1
1.1 Signal transduction 1
1.2 G protein coupled receptors 3
1.2.1 Hetrotrimeric G proteins 4
1.2.2 G protein a and Py subunits 5
1.2.3 Regulation of G protein coupled receptors 6
1.2.4 Chemokines and chemokine receptors 7
1.3 Small GTP binding proteins 12
1.4 Phospholipase C 13
1.5 Human cytomegalovirus (HCMV) 14
1.6 Aim of the study 15
2 Materials 17
2.1 Chemicals 17
2.2 Radiolabelled chemicals 18
2.3 Enzymes, kits and nucleic acids 18
2.4 Materials 19
2.5 Prokaryotic and eukaryotic cell lines 19
2.6 Viral strains 20
2.7 Plasmids 20
2.8 Oligonucleotides 21
2.8.1 Plasmid specific oligonucleotides 21
2.8.2 US28 specific oligonucleotides 21
2.9 Recombinant plasmids 22
2.10 Peptides 22
2.11 Antibodies 23
2.12 Special instruments 23
2.13 Softwares 23
3 Methods 25
3.1 Microbiological methods 25
3.1.1 Preparation of Luria Bertani (LB) medium 25
3.1.2 Preparation of LB ampicillin plates 25
3.1.3 Preparation of competent bacteria 26
3.1.4 Transformation of competent bacteria 26
3.1.5 Selection of transformed bacteria 26
3.1.6 Storage of E.coli 27
3.2 Molecolar biological methods 27
3.2.1 Preparation of plasmid DNA 27
3.2.2 Spectroscopic measurement of DNA concentration 28
3.2.3 Phenol chloroform extraction of precipitation of DNA 28
3.2.4 Agarose gel electrophoresis 29
3.2.5 Extraction of DNA from agarose gel 30
3.2.6 Restriction digestion of DNA 30
3.2.7 Dephosphorylation of plasmid vectos 30
3.2.8 Filling in reaction 31
3.2.9 Ligation of DNA fragments into plasmid vectors 31
3.2.10 Polymerase chain reaction 32
3.2.11 DNA sequencing 33
3.2.12 Generation of mutated receptor cDNA 33
3.3 Eukaryotic cell culture 35
3.3.1 Human foreskin fibroblast (HFF) and astrcytoma cell line culture 35
3.3.2 COS 7 cell culture 36
3.4 Viral infection 36
3.4.1 Viral titering 36
3.4.2 Infection of HFF 37
3.4.3 UV light treatment 37
3.5 Protein expression in mammalian cells 38
3.5.1 Transfection on COS 7 cells and astrocytoma cell lines 38
3.5.2 Membrane preparation 38
3.6 Protein biochemistry 39
3.6.1 Estimation of protein concentration 39
3.7 Methods to determine receptor activity 39
3.7.1 Radioligand binding 39
3.7.2 [35S]GTP[S] binding 40
3.7.3 Inositol phosphate formation in HFF cells 41
3.7.4 Inositol phosphate formation in COS 7 and astrocytoma cells 42
3.7.5 Luciferase assay 42
3.7.6 Measurement of intracellular Ca2+ release 43
4 Results 45
4.1 Binding of chemokines to HCMV infected HFF cells 45
4.2 Measurement of intracellular Ca2+ concentration in HFF cells infected with
HCMV AD 169 or HCMV Towne 49
4.3 Receptor mediated stimulation of [35S]GTP[S] binding in membranes of
HCMV infected cells 50
4.4 Inositol phosphate formation in HCMV infected HFF cells 52
4.4.1 Effect of chemokines 52
4.4.2 Inositol phosphate formation in HFF cells inoculated with UV
light irradiated HCMV virus 55
4.4.3 Dependence of inositol phosphate formation in HCMV infected
HFF cells on pUS28 57
4.4.4 Effect of pertussis toxin pretreatment on the formation of inositol
phosphate in HCMV HFF infected cells 58
4.4.5 Dependence of inositol phosphate formation on time of infection
and multiplicity of infection 59
4.4.6 Effect of RANTES on early time infection and low MOI 60
4.4.7 Inositol phosphate formation of HCMV laboratory strains and
a clinical HCMV isolate at increasing MOI 62
4.4.8 Effect of the incubation medium on enhanced inositol phosphate
formation in HCMV infected HFF cells 64
4.5 Cloning of US28Adi69DNA in expression vector 66
4.6 Binding of chemokines to COS 7 cells expressing US28adi69 70
4.7 Measurement of intracellular Ca2+ in COS 7 cells expressing US28adi69 V1
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| author_GND | (DE-588)129671231 |
| author_facet | Tulone, Calogero 1971- |
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| spelling | Tulone, Calogero 1971- Verfasser (DE-588)129671231 aut Characterization of signaling pathways activated by the HCMV encoded GPCR homolog pUS28 presented by Calogero Tulone 2004 1 CD-R 12 cm txt rdacontent c rdamedia cd rdacarrier Titel von interner Quelle Ulm, Univ., Diss., 2004 (DE-588)4113937-9 Hochschulschrift gnd-content HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=013206583&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Tulone, Calogero 1971- Characterization of signaling pathways activated by the HCMV encoded GPCR homolog pUS28 |
| subject_GND | (DE-588)4113937-9 |
| title | Characterization of signaling pathways activated by the HCMV encoded GPCR homolog pUS28 |
| title_auth | Characterization of signaling pathways activated by the HCMV encoded GPCR homolog pUS28 |
| title_exact_search | Characterization of signaling pathways activated by the HCMV encoded GPCR homolog pUS28 |
| title_full | Characterization of signaling pathways activated by the HCMV encoded GPCR homolog pUS28 presented by Calogero Tulone |
| title_fullStr | Characterization of signaling pathways activated by the HCMV encoded GPCR homolog pUS28 presented by Calogero Tulone |
| title_full_unstemmed | Characterization of signaling pathways activated by the HCMV encoded GPCR homolog pUS28 presented by Calogero Tulone |
| title_short | Characterization of signaling pathways activated by the HCMV encoded GPCR homolog pUS28 |
| title_sort | characterization of signaling pathways activated by the hcmv encoded gpcr homolog pus28 |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=013206583&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT tulonecalogero characterizationofsignalingpathwaysactivatedbythehcmvencodedgpcrhomologpus28 |