Molecular analysis of annexin-mediated immunosuppression in tumour development:
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| 100 | 1 | |a Hein, Tobias |d 1989- |e Verfasser |0 (DE-588)120887697X |4 aut | |
| 245 | 1 | 0 | |a Molecular analysis of annexin-mediated immunosuppression in tumour development |c Tobias Hein |
| 264 | 1 | |a Heidelberg |c 2020 | |
| 300 | |a XVI, 116 Seiten |b Illustrationen, Diagramme |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Ruperto Carola University of Heidelberg, Germany |d 2020 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 856 | 4 | 2 | |m B:DE-101 |q application/pdf |u https://d-nb.info/1212193911/04 |3 Inhaltsverzeichnis |
| 856 | 4 | 2 | |m DNB Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032320334&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 999 | |a oai:aleph.bib-bvb.de:BVB01-032320334 | ||
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| _version_ | 1804181791262638080 |
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| adam_text | CONTENTS
ABSTRACT
....................................................................................................................................................
IX
ZUSAMMENFASSUNG
.................................................................................................................................
XI
1
INTRODUCTION
.....................................................................................................................................
1
1.1
THE
IMMUNE
SYSTEM
................................................................................................................
1
1.1.1
INNATE
IMMUNITY
.............................................................................................................
1
1.1.2
ADAPTIVE
IMMUNITY
........................................................................................................
2
1.2
TOLERANCE
.................................................................................................................................
4
1.2.1
CENTRAL
TOLERANCE
...........................................................................................................
4
1.2.2
PERIPHERAL
TOLERANCE
......................................................................................................
5
1.2.3
DENDRITIC
CELLS
IN
THE
CENTRE
OF
IMMUNITY
...................................................................6
1.3
APOPTOSIS
.................................................................................................................................8
1.3.1
APOPTOTIC
CELL
CLEARANCE
................................................................................................
9
1.3.2
IMMUNE
TOLERANCE
INDUCTION
BY
APOPTOTIC
CELL
CLEARANCE
......................................
10
1.4
APOPTOSIS
IN
CANCER...............................................................................................................12
1.4.1
THERAPY-INDUCED
CELL
DEATH
IN
CANCER........................................................................
14
1.5
ANNEXINS
................................................................................................................................
15
1.5.1
EXTERNALISATION
AND
CLEAVAGE
......................................................................................
16
1.5.2
FUNCTIONS.......................................................................................................................
17
1.5.3
THE
IMMUNOSUPPRESSIVE
EFFECTS
OF
ANXS
TOWARDS
APCS
.........................................
18
1.5.4
RECEPTORS
......................................................................................................................
19
1.6
AIM
OF
THIS
WORK
...................................................................................................................22
2
MATERIALS
........................................................................................................................................23
2.1
CHEMICALS
AND
REAGENTS
.......................................................................................................23
2.1.1
CHEMICALS
......................................................................................................................
23
2.1.2
REAGENTS
........................................................................................................................
23
2.1.3
COMMERCIAL
KITS
...........................................................................................................25
2.2
BUFFERS
AND
SOLUTIONS
...........................................................................................................25
2.3
CONSUMABLES
.........................................................................................................................27
2.4
CULTURE
MEDIA
AND
SUPPLEMENTS
.........................................................................................28
2.4.1
BACTERIAL
CULTURE
MEDIA
...............................................................................................28
2.4.2
MEDIA
FOR
EUKARYOTIC
CELL
CULTURE
...............................................................................
28
2.5
BIOLOGICAL
MATERIAL
.................................................................................................................29
2.5.1
EUKARYOTIC
CELL
LINES
......................................................................................................29
2.5.2
MOUSE
STRAIN
..................................................................................................................30
2.5.3
BACTERIAL
STRAIN
..............................................................................................................
30
2.6
ANTIBODIES
..............................................................................................................................
31
2.6.1
PRIMARY
WESTERN
BLOT
ANTIBODIES
...............................................................................31
2.6.2
SECONDARY
WESTERN
BLOT
ANTIBODIES
...........................................................................
32
2.6.3
ANTIBODIES
FOR
FLOW
CYTOMETRY
....................................................................................32
2.6.4
PROTEINS
AND
PEPTIDES
.................................................................................................33
2.7
MATERIALS
FOR
MOLECULAR
BIOLOGY
.........................................................................................33
2.7.1
PRIMERS
FOR
RT-QPCR
.....................................................................................................
33
2.7.2
PRIMERS
FOR
CLONING
.......................................................................................................35
2.7.3
PRIMERS
FOR
SEQUENCING
...............................................................................................35
2.7.4
RESTRICTION
ENZYMES
.....................................................................................................35
2.7.5
VECTORS
...........................................................................................................................35
2.8
INSTRUMENTS
............................................................................................................................
36
2.9
SOFTWARE
L
DATABASES
..........................................................................................................37
2.9.1
SOFTWARE
.........................................................................................................................
37
2.9.2
DATABASE
.......................................................................................................................
37
3
METHODS
..........................................................................................................................................
39
3.1
CELL
BIOLOGY
.............................................................................................................................
39
3.1.1
GENERAL
CULTURE
CONDITIONS
.........................................................................................
39
3.1.2
DETERMINATION
OF
CELL
DENSITY
.....................................................................................
39
3.1.3
THAWING
AND
FREEZING
OF
CELLS
......................................................................................
39
3.1.4
CELL
LYSIS
..........................................................................................................................
39
3.1.5
APOPTOSIS
INDUCTION
.....................................................................................................
40
3.1.6
EDTA
WASH
OF
APOPTOTIC
SURFACE
MOLECULES
.............................................................
40
XIV
3.1.7
CELL
DEATH
ANALYSIS
.......................................................................................................
40
3.1.8
PREPARATION
OF
DONE
MARROW-DERIVED
DC
(BMDC)
...................................................
41
3.1.9
BMDC
SUPPRESSION
ASSAY
.............................................................................................
41
3.1.10
DETERMINATION
OF
REACTIVE
OXYGEN
SPECIES
(ROS)
PRODUCTION
...............................
41
3.1.11
TRANSIENT
TRANSFECTION
OF
CHO-S
CELLS
........................................................................
42
3.1.12
ISOLATION
OF
HUMAN
NEUTROPHILS
.................................................................................
42
3.1.13
IN
VIVO
STUDY
.................................................................................................................
42
3.1.14
RESTIMULATION
OF
SPLENOCYTES
.....................................................................................
42
3.2
MOLECULAR
BIOLOGY
................................................................................................................
43
3.2.1
POLYMERASE
CHAIN
REACTION
(PCR)
...............................................................................
43
3.2.2
AGAROSE
GEL
ELECTROPHORESIS
AND
DNA
EXTRACTION
....................................................
43
3.2.3
RESTRICTION
ENZYME
DIGESTION
AND
LIGATION
................................................................
44
3.2.4
SITE
DIRECTED
MUTAGENESIS
BY
PCR
..............................................................................
44
3.2.5
ISOLATION
OF
RNA
...........................................................................................................
45
3.2.6
REVERSE
TRANSCRIPTION
(RT)
OF
RNA
INTO
CDNA
..........................................................
45
3.2.7
QUANTITATIVE
PCR
(QPCR)
............................................................................................
46
3.2.8
BACTERIAL
TRANSFORMATION
............................................................................................
46
3.2.9
PLASMID
PURIFICATION
.....................................................................................................
46
3.2.10
DNA
SEQUENCING
...........................................................................................................
46
3.3
BIOCHEMICAL
METHODS
..........................................................................................................
47
3.3.1
SDS-PAGE
AND
WESTERN
BLOT
.......................................................................................
47
3.3.2
COOMASSIE
STAINING
AND
SILVER
STAINING
.....................................................................
47
3.3.3
FLOW
CYTOMETRY
............................................................................................................
47
3.3.4
ENZYME
LINKED
IMMUNOSORBENT
ASSAY
(ELISA)
........................................................
48
3.3.5
LRP1
BINDING
ELISA
.......................................................................................................
48
3.3.6
LIPID-BINDING
ELISA
.......................................................................................................
49
3.3.7
ENZYME
LINKED
IMMUNOSPOT
ASSAY
(ELISPOT)
...........................................................
49
3.3.8
LRPLC4-FC
AFFINITY
PURIFICATION
...................................................................................
50
3.3.9
RECOMBINANT
ANNEXINS
................................................................................................
50
XV
4
RESULTS
.............................................................................................................................................51
4.1
EXPRESSION
PROFILES
OF
THE
ANX
FAMILY
MEMBERS
................................................................
51
4.2
ANX
EXPRESSION
LEVELS
IN
CANCER
AND
HEALTHY
TISSUE..........................................................
53
4.3
DETECTION
OF
ANNEXINS
ON
THE
SURFACE
OF
APOPTOTIC
CANCER
CELLS
....................................
54
4.4
LRP1
CLUSTER
IV
FC
CHIMERA
BINDS
SPECIFICALLY
TO
THE
ANX
CORE
DOMAIN
..........................
56
4.5
EFFECTS
OF
LRPLC4-FC
ON
ANX-MEDIATED
ROS
PRODUCTION
..................................................
58
4.6
EFFECTS
OF
LRPLC4-FC
ON
ANX-MEDIATED
DC
INHIBITION
.......................................................
60
4.7
PRODUCTION
OF
LRPLC4-FC
IN
CHO
CELLS
.................................................................................62
4.8
EFFECTS
OF
LRPLC4-MUTFC
ON
IN
VIVO
TUMOUR
GROWTH
AND
T
CELL
RESPONSE
......................65
4.8.1
TUMOUR
GROWTH
AND
SURVIVAL
......................................................................................65
4.8.2
ANTI-TUMOUR
T
CELL
RESPONSE
.......................................................................................66
4.8.3
IN
VITRO
RESTIMULATION
OF
SPLENOCYTES.........................................................................
68
5
DISCUSSION.......................................................................................................................................69
5.1
CANCER
CELL
LINES
HAVE
STABLE
ANX
EXPRESSION
PROFILES
.....................................................69
5.2
ANX
EXPRESSION
AFTER
MALIGNANT
TRANSFORMATION
VARIES
AMONG
CANCER
ENTITIES
............71
5.3
ALL
EXPRESSED
ANXS
ARE
TRANSLOCATED
TO
THE
CELL
SURFACE
DURING
APOPTOSIS
IN
CANCER
CELL
LINES
.........................................................................................................................................73
5.4
LRP1
CLUSTER
IV-FC
DISPLAYS
STRONG
AND
SPECIFIC
BINDING
TO
THE
ANX
CORE
DOMAIN
........73
5.5
LRPLC4-FC
SHOWS
VARIABLE
RESULTS
IN
BLOCKING
ANX-MEDIATED
EFFECTS
.............................75
5.6
APG
LRPLC4-FC
PREPARATIONS
DIFFER
FROM
THE
COMMERCIAL
PROTEIN
.................................76
5.7
LRPLC4-MUTFC
HAD
LITTLE
INFLUENCE
ON
TUMOUR
PROGRESSION
OR
IMMUNE
RESPONSE
........78
5.8
CONCLUSION
..............................................................................................................................
82
REFERENCES...............................................................................................................................................
85
APPENDIX
...............................................................................................................................................
107
I.
SUPPLEMENTARY
RESULTS
........................................................................................................
107
II.
LIST
OF
FIGURES
.......................................................................................................................
112
III.
LIST
OF
TABLES
.........................................................................................................................
113
IV.
ABBREVIATIONS
.......................................................................................................................
114
XVI
|
| adam_txt |
CONTENTS
ABSTRACT
.
IX
ZUSAMMENFASSUNG
.
XI
1
INTRODUCTION
.
1
1.1
THE
IMMUNE
SYSTEM
.
1
1.1.1
INNATE
IMMUNITY
.
1
1.1.2
ADAPTIVE
IMMUNITY
.
2
1.2
TOLERANCE
.
4
1.2.1
CENTRAL
TOLERANCE
.
4
1.2.2
PERIPHERAL
TOLERANCE
.
5
1.2.3
DENDRITIC
CELLS
IN
THE
CENTRE
OF
IMMUNITY
.6
1.3
APOPTOSIS
.8
1.3.1
APOPTOTIC
CELL
CLEARANCE
.
9
1.3.2
IMMUNE
TOLERANCE
INDUCTION
BY
APOPTOTIC
CELL
CLEARANCE
.
10
1.4
APOPTOSIS
IN
CANCER.12
1.4.1
THERAPY-INDUCED
CELL
DEATH
IN
CANCER.
14
1.5
ANNEXINS
.
15
1.5.1
EXTERNALISATION
AND
CLEAVAGE
.
16
1.5.2
FUNCTIONS.
17
1.5.3
THE
IMMUNOSUPPRESSIVE
EFFECTS
OF
ANXS
TOWARDS
APCS
.
18
1.5.4
RECEPTORS
.
19
1.6
AIM
OF
THIS
WORK
.22
2
MATERIALS
.23
2.1
CHEMICALS
AND
REAGENTS
.23
2.1.1
CHEMICALS
.
23
2.1.2
REAGENTS
.
23
2.1.3
COMMERCIAL
KITS
.25
2.2
BUFFERS
AND
SOLUTIONS
.25
2.3
CONSUMABLES
.27
2.4
CULTURE
MEDIA
AND
SUPPLEMENTS
.28
2.4.1
BACTERIAL
CULTURE
MEDIA
.28
2.4.2
MEDIA
FOR
EUKARYOTIC
CELL
CULTURE
.
28
2.5
BIOLOGICAL
MATERIAL
.29
2.5.1
EUKARYOTIC
CELL
LINES
.29
2.5.2
MOUSE
STRAIN
.30
2.5.3
BACTERIAL
STRAIN
.
30
2.6
ANTIBODIES
.
31
2.6.1
PRIMARY
WESTERN
BLOT
ANTIBODIES
.31
2.6.2
SECONDARY
WESTERN
BLOT
ANTIBODIES
.
32
2.6.3
ANTIBODIES
FOR
FLOW
CYTOMETRY
.32
2.6.4
PROTEINS
AND
PEPTIDES
.33
2.7
MATERIALS
FOR
MOLECULAR
BIOLOGY
.33
2.7.1
PRIMERS
FOR
RT-QPCR
.
33
2.7.2
PRIMERS
FOR
CLONING
.35
2.7.3
PRIMERS
FOR
SEQUENCING
.35
2.7.4
RESTRICTION
ENZYMES
.35
2.7.5
VECTORS
.35
2.8
INSTRUMENTS
.
36
2.9
SOFTWARE
L
DATABASES
.37
2.9.1
SOFTWARE
.
37
2.9.2
DATABASE
.
37
3
METHODS
.
39
3.1
CELL
BIOLOGY
.
39
3.1.1
GENERAL
CULTURE
CONDITIONS
.
39
3.1.2
DETERMINATION
OF
CELL
DENSITY
.
39
3.1.3
THAWING
AND
FREEZING
OF
CELLS
.
39
3.1.4
CELL
LYSIS
.
39
3.1.5
APOPTOSIS
INDUCTION
.
40
3.1.6
EDTA
WASH
OF
APOPTOTIC
SURFACE
MOLECULES
.
40
XIV
3.1.7
CELL
DEATH
ANALYSIS
.
40
3.1.8
PREPARATION
OF
DONE
MARROW-DERIVED
DC
(BMDC)
.
41
3.1.9
BMDC
SUPPRESSION
ASSAY
.
41
3.1.10
DETERMINATION
OF
REACTIVE
OXYGEN
SPECIES
(ROS)
PRODUCTION
.
41
3.1.11
TRANSIENT
TRANSFECTION
OF
CHO-S
CELLS
.
42
3.1.12
ISOLATION
OF
HUMAN
NEUTROPHILS
.
42
3.1.13
IN
VIVO
STUDY
.
42
3.1.14
RESTIMULATION
OF
SPLENOCYTES
.
42
3.2
MOLECULAR
BIOLOGY
.
43
3.2.1
POLYMERASE
CHAIN
REACTION
(PCR)
.
43
3.2.2
AGAROSE
GEL
ELECTROPHORESIS
AND
DNA
EXTRACTION
.
43
3.2.3
RESTRICTION
ENZYME
DIGESTION
AND
LIGATION
.
44
3.2.4
SITE
DIRECTED
MUTAGENESIS
BY
PCR
.
44
3.2.5
ISOLATION
OF
RNA
.
45
3.2.6
REVERSE
TRANSCRIPTION
(RT)
OF
RNA
INTO
CDNA
.
45
3.2.7
QUANTITATIVE
PCR
(QPCR)
.
46
3.2.8
BACTERIAL
TRANSFORMATION
.
46
3.2.9
PLASMID
PURIFICATION
.
46
3.2.10
DNA
SEQUENCING
.
46
3.3
BIOCHEMICAL
METHODS
.
47
3.3.1
SDS-PAGE
AND
WESTERN
BLOT
.
47
3.3.2
COOMASSIE
STAINING
AND
SILVER
STAINING
.
47
3.3.3
FLOW
CYTOMETRY
.
47
3.3.4
ENZYME
LINKED
IMMUNOSORBENT
ASSAY
(ELISA)
.
48
3.3.5
LRP1
BINDING
ELISA
.
48
3.3.6
LIPID-BINDING
ELISA
.
49
3.3.7
ENZYME
LINKED
IMMUNOSPOT
ASSAY
(ELISPOT)
.
49
3.3.8
LRPLC4-FC
AFFINITY
PURIFICATION
.
50
3.3.9
RECOMBINANT
ANNEXINS
.
50
XV
4
RESULTS
.51
4.1
EXPRESSION
PROFILES
OF
THE
ANX
FAMILY
MEMBERS
.
51
4.2
ANX
EXPRESSION
LEVELS
IN
CANCER
AND
HEALTHY
TISSUE.
53
4.3
DETECTION
OF
ANNEXINS
ON
THE
SURFACE
OF
APOPTOTIC
CANCER
CELLS
.
54
4.4
LRP1
CLUSTER
IV
FC
CHIMERA
BINDS
SPECIFICALLY
TO
THE
ANX
CORE
DOMAIN
.
56
4.5
EFFECTS
OF
LRPLC4-FC
ON
ANX-MEDIATED
ROS
PRODUCTION
.
58
4.6
EFFECTS
OF
LRPLC4-FC
ON
ANX-MEDIATED
DC
INHIBITION
.
60
4.7
PRODUCTION
OF
LRPLC4-FC
IN
CHO
CELLS
.62
4.8
EFFECTS
OF
LRPLC4-MUTFC
ON
IN
VIVO
TUMOUR
GROWTH
AND
T
CELL
RESPONSE
.65
4.8.1
TUMOUR
GROWTH
AND
SURVIVAL
.65
4.8.2
ANTI-TUMOUR
T
CELL
RESPONSE
.66
4.8.3
IN
VITRO
RESTIMULATION
OF
SPLENOCYTES.
68
5
DISCUSSION.69
5.1
CANCER
CELL
LINES
HAVE
STABLE
ANX
EXPRESSION
PROFILES
.69
5.2
ANX
EXPRESSION
AFTER
MALIGNANT
TRANSFORMATION
VARIES
AMONG
CANCER
ENTITIES
.71
5.3
ALL
EXPRESSED
ANXS
ARE
TRANSLOCATED
TO
THE
CELL
SURFACE
DURING
APOPTOSIS
IN
CANCER
CELL
LINES
.73
5.4
LRP1
CLUSTER
IV-FC
DISPLAYS
STRONG
AND
SPECIFIC
BINDING
TO
THE
ANX
CORE
DOMAIN
.73
5.5
LRPLC4-FC
SHOWS
VARIABLE
RESULTS
IN
BLOCKING
ANX-MEDIATED
EFFECTS
.75
5.6
APG
LRPLC4-FC
PREPARATIONS
DIFFER
FROM
THE
COMMERCIAL
PROTEIN
.76
5.7
LRPLC4-MUTFC
HAD
LITTLE
INFLUENCE
ON
TUMOUR
PROGRESSION
OR
IMMUNE
RESPONSE
.78
5.8
CONCLUSION
.
82
REFERENCES.
85
APPENDIX
.
107
I.
SUPPLEMENTARY
RESULTS
.
107
II.
LIST
OF
FIGURES
.
112
III.
LIST
OF
TABLES
.
113
IV.
ABBREVIATIONS
.
114
XVI |
| any_adam_object | 1 |
| any_adam_object_boolean | 1 |
| author | Hein, Tobias 1989- |
| author_GND | (DE-588)120887697X |
| author_facet | Hein, Tobias 1989- |
| author_role | aut |
| author_sort | Hein, Tobias 1989- |
| author_variant | t h th |
| building | Verbundindex |
| bvnumber | BV046910880 |
| ctrlnum | (OCoLC)1189754868 (DE-599)DNB1212193911 |
| discipline | Biologie Medizin |
| discipline_str_mv | Biologie Medizin |
| format | Thesis Book |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV046910880 |
| illustrated | Illustrated |
| index_date | 2024-07-03T15:28:10Z |
| indexdate | 2024-07-10T08:57:16Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-032320334 |
| oclc_num | 1189754868 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR |
| owner_facet | DE-355 DE-BY-UBR |
| physical | XVI, 116 Seiten Illustrationen, Diagramme 30 cm |
| publishDate | 2020 |
| publishDateSearch | 2020 |
| publishDateSort | 2020 |
| record_format | marc |
| spelling | Hein, Tobias 1989- Verfasser (DE-588)120887697X aut Molecular analysis of annexin-mediated immunosuppression in tumour development Tobias Hein Heidelberg 2020 XVI, 116 Seiten Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Ruperto Carola University of Heidelberg, Germany 2020 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf https://d-nb.info/1212193911/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032320334&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Hein, Tobias 1989- Molecular analysis of annexin-mediated immunosuppression in tumour development |
| subject_GND | (DE-588)4113937-9 |
| title | Molecular analysis of annexin-mediated immunosuppression in tumour development |
| title_auth | Molecular analysis of annexin-mediated immunosuppression in tumour development |
| title_exact_search | Molecular analysis of annexin-mediated immunosuppression in tumour development |
| title_exact_search_txtP | Molecular analysis of annexin-mediated immunosuppression in tumour development |
| title_full | Molecular analysis of annexin-mediated immunosuppression in tumour development Tobias Hein |
| title_fullStr | Molecular analysis of annexin-mediated immunosuppression in tumour development Tobias Hein |
| title_full_unstemmed | Molecular analysis of annexin-mediated immunosuppression in tumour development Tobias Hein |
| title_short | Molecular analysis of annexin-mediated immunosuppression in tumour development |
| title_sort | molecular analysis of annexin mediated immunosuppression in tumour development |
| topic_facet | Hochschulschrift |
| url | https://d-nb.info/1212193911/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032320334&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT heintobias molecularanalysisofannexinmediatedimmunosuppressionintumourdevelopment |
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