The role of scavenger receptor BI in hepatitis - eTheses Repository ...

More documents

Recommendations

Info

Figure 2-8 Titration of sE2. 92 Serial dilutions of JFH-1 (A) and JFH-1 G451R (B) sE2 were captured in a GNA ELISA, bound glycoprotein was detected with 1µg/ml anti-tag mAb 10/76B. The relative concentration of each sE2 was assessed by calculation of OD units; three readings were taken from a linear section of each curve (at 1/750,1250 and 1750). OD units were calculated by multiplying the absorbance by the reciprocal dilution for each reading. Comparison of the mean OD units for either glycoprotein suggests that JFH-1 sE2 is ~8% more concentrated than JFH-1 G451R sE2, therefore JFH-1 sE2 was diluted appropriately before use in the assays presented in the results section.
93 3 Results: Investigations using CHO cell expressed SR-BI. The work documented in this thesis is reliant on a number of techniques and reagents, this chapter covers the validation of these tools along with studies using a panel of SR-BI mutants and the splice variant SR-BII. 3.1 Expression of SR-BI in CHO cells. To investigate the interaction of HCV glycoproteins with SR-BI it was necessary to identify a cell background lacking functioning viral receptors. Hamster derived CHO cells do not bind HCV sE2 (274) and it has been shown that SR-BI expressed in a CHO cell is functional with regards to lipid uptake and trafficking (239, 259, 349). We therefore began work with a CHO cell line stably expressing human SR-BI. We assessed SR-BI expression in CHO cells using a panel of anti-SR-BI antibodies by flow cytometry and immunofluorescent microscopy (Figure 3-1). Figure 3-1 A-C show live cell staining of CHO-SRBI and represent plasma membrane SR-BI expression.
Page 1 and 2:
The role of scavenger receptor B-I
Page 3 and 4:
i Abstract. With 170 million infect
Page 5 and 6:
iii Acknowledgements. I would like
Page 7 and 8:
v 5 Results: Identification of a re
Page 9 and 10:
vii Figure 5-5 Analysis of JFH-1 wt
Page 11 and 12:
1 Introduction 1.1 The disease. 1 D
Page 13 and 14:
3 predominantly immuno-pathogenic i
Page 15 and 16:
5 Aside from considerations of the
Page 17 and 18:
7 Current attempts to design a HCV
Page 19 and 20:
1.2 An elusive pathogen. 9 The emer
Page 21 and 22:
11 PCR). This technique is highly s
Page 23 and 24:
13 Within six years of the descript
Page 25 and 26:
1.3 Basic virology. 15 Like other m
Page 27 and 28:
17 Figure 1-2 HCV genome and polypr
Page 29 and 30:
19 241). E1 and E2 are anchored to
Page 31 and 32:
21 The characterisation of HCV geno
Page 33 and 34:
23 in the E1 and E2 glycoprotein ge
Page 35 and 36:
25 The solution reached by a great
Page 37 and 38:
27 binding to mouse cells. sE2 inte
Page 39 and 40:
29 density lipoprotein (HDL), allow
Page 41 and 42:
31 blood brain barrier is permeable
Page 43 and 44:
33 act as receptors for viral entry
Page 45 and 46:
1.4.2 Attachment factors 35 The tru
Page 47 and 48:
1.4.3 Endocytosis and fusion 37 Aft
Page 49 and 50:
39 1.4.4 Co-receptor interplay and
Page 51 and 52: 41 inhibition of protein kinase A (
Page 53 and 54: 1.5 Lipoproteins 43 Since 1992 ther
Page 55 and 56: 45 Lipoproteins exist in a dynamic
Page 57 and 58: 47 and delivers its cargo via the w
Page 59 and 60: 49 uptake of cholesterol from LDL (
Page 61 and 62: Figure 1-4 SR-BI-lipoprotein intera
Page 63 and 64: 53 In summary the class B scavenger
Page 65 and 66: 55 In 1992 Thomssen et. al. demonst
Page 67 and 68: 57 lipoprotein association promotes
Page 69 and 70: 59 Figure 1-5 Lipo-viro-particle as
Page 71 and 72: 61 In vitro culture medium, into wh
Page 73 and 74: 2 Materials and methods. 2.1 Cell l
Page 75 and 76: Preparation of rat anti-E2 mAbs. 65
Page 77 and 78: 2.4 Basic techniques. Flow cytometr
Page 79 and 80: 69 6. Finally, the cells were washe
Page 81 and 82: 71 (Promega, WI, USA) kits accordin
Page 83 and 84: 73 proteins and are incapable of fu
Page 85 and 86: 75 spectrophotometer (Amersham), we
Page 87 and 88: 77 Figure 2-2 Electroporated Huh-7.
Page 89 and 90: 79 5. Data is expressed as percenta
Page 91 and 92: 81 (Invitrogen). The samples to be
Page 93 and 94: 83 1. The SR-BII coding region was
Page 95 and 96: 2.7 Cloning and synthesis of JFH-1
Page 97 and 98: The sequence encoding amino acids 3
Page 99 and 100: Figure 2-6 sE2 sequencing. 89 JFH-1
Page 101: 91 7. Large scale transfections wer
Page 105 and 106: 95 3.2 CHO cells expressing human S
Page 107 and 108: Figure 3-3 Ability of anti-E2 mAb t
Page 109 and 110: 99 3.3 The interaction of sE2 with
Page 111 and 112: 101 Figure 3-5 Position of SR-BI mu
Page 113 and 114: 103 Table 3-2 Investigation of cell
Page 115 and 116: 105 3.4 Expression of SR-BII in CHO
Page 117 and 118: 3.5 Discussion. 107 We have demonst
Page 119 and 120: 109 may offer a tool to study the i
Page 121 and 122: 111 Figure 4-1A displays endogenous
Page 123 and 124: 113 4.2 Exogenous expression of SR-
Page 125 and 126: 115 4.3 Evidence of enhanced JFH-1
Page 127 and 128: 117 4.4 SR-BI expression levels lim
Page 129 and 130: 119 4.5 Murine SR-BI does not enhan
Page 131 and 132: 121 4.6 SR-BI/II expression levels
Page 133 and 134: 123 Figure 4-5 Over expression of S
Page 135 and 136: 125 Figure 4-6 Anti-SR-BI serum inh
Page 137 and 138: 127 Figure 4-7 Infection of Huh-7.5
Page 139 and 140: 129 cells is largely manifested in
Page 141 and 142: 131 5 Results: Identification of a
Page 143 and 144: 133 Figure 5-1 JFH-1 G451R has an a
Page 145 and 146: 135 Figure 5-2 CD81 dependence of J
Page 147 and 148: 137 G451R sE2 with hCD81 LEL we com
Page 149 and 150: 139 Figure 5-4 JFH-1 wt and G451R s
Page 151 and 152: 141 demonstrated a lower dependence
Page 153 and 154:
143 Figure 5-6 Relationship between
Page 155 and 156:
145 flow cytometry (data not shown)
Page 157 and 158:
147 5.6 Low density JFH-1 particles
Page 159 and 160:
5.7 Discussion. 149 We have demonst
Page 161 and 162:
151 Numerous reports have used dens
Page 163 and 164:
153 demonstrate that low density JF
Page 165 and 166:
155 to over express SR-BI we were a
Page 167 and 168:
157 5-1), better able to engage CD8
Page 169 and 170:
159 Figure 6-1 An overview of antib
Page 171 and 172:
161 it is believed that ligation of
Page 173 and 174:
163 Given our and other’s finding
Page 175 and 176:
6.3 HCV lipo-viro-particles. 165 As
Page 177 and 178:
167 represent the hyper-variable re
Page 179 and 180:
169 The G451R mutation disrupts the
Page 181 and 182:
7 Bibliography 171 1. Acton, S., D.
Page 183 and 184:
173 include the CD81 tetraspanin an
Page 185 and 186:
175 receptor class B type I and inh
Page 187 and 188:
177 83. Drummer, H. E., K. A. Wilso
Page 189 and 190:
179 KewalRamani, D. R. Littman, C.
Page 191 and 192:
181 King. 1996. Efficient infection
Page 193 and 194:
183 hepatitis C virus envelope glyc
Page 195 and 196:
185 recovered chimpanzees exhibit r
Page 197 and 198:
187 218. Monazahian, M., I. Bohme,
Page 199 and 200:
189 243. Perez-Berna, A. J., J. Gui
Page 201 and 202:
191 single-cycle production assay t
Page 203 and 204:
193 298. Strickland, G. T., S. S. E
Page 205 and 206:
195 Remaley, G. Csako, T. L. Eggerm
Page 207:
197 2007. Scavenger receptor class
show all

The role of scavenger receptor BI in hepatitis - eTheses Repository ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?