LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

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Figure 4.3.22: 14-3-3γ loss does not alter the localization of KIF5B to to microtubules. HCT116 derived vector control or 14-3-3γ knockdown clones were fixed and immuno stained with the indicated antibodies (KIF5B and α-tubulin). Please note in the zoom images that KIF5B localizes to microtubules in both cell types (Original magnification x 630 with 2X optical zoom). 4.3.12 DSC2/3 recruitment to cell border is microtubule dependent. Recently a report has shown that the desmosomal cadherins are recruited to the cell border in a microtubule dependent manner (260). The cell line used in the above experiment was SCC9 cells which is cells of stratified squamous epithelia (298) and is different from the HCT116 cells which are derived from simple epithelia (299). So we asked whether desmocollin 2/3 (DSC2/3) recruitment to cell border during desmosome biogenesis is dependent on microtubule based processes in HCT116 cells. A calcium switch assay was performed as previously described (19) in the presence or absence of nocodazole. The HCT116 cells were grown in low calcium medium for 20 hrs and then shifted to medium with standard calcium levels in presence or absence of nocodazole. The cells were fixed for immunofluorescence experiment at 30 min and 60 min time intervals and stained with antibodies to DSC2/3 and α-tubulin. A diffused cytoplasmic staining was observed for DSC2/3 in the cells cultured in low calcium medium (Figure 144
4.3.23 (A and B)). These proteins gradually accumulated at the cell-cell border at 60 min for HCT116 cells treated with DMSO and showed typical membrane localization. However at 60 min DSC2/3 protein failed to localize to the cell border in the HCT116 cells treated with nocodazole upon addition of calcium. As shown in the (Figure 4.3.23 (A and B)) the nocodazole used in the experiment was effective enough to depolymerize microtubules. These results suggest that both calcium dependent and calcium independent desmosomal proteins may be recruited to the cell border in a microtubule dependent manner. However the transport of calcium independent desmosomal proteins (plakoglobin) is followed by the recruitment of calcium dependent desmosomal proteins (desmocollin 2/3) to the cell border in a microtubule dependent manner. Figure 4.3.23: Desmocollin2/3 recruitment to the cell border is microtubule dependent. (A) HCT116 cells were grown in low calcium medium for 20 hrs and then shifted to 145
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Generation of knockdown mice that l
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STATEMENT BY AUTHOR This dissertati
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CERTIFICATE I certify that the thes
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I am thankful to Mr Uday Dandekar f
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3.16 Isolation of Genomic DNA (gDNA
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A synopsis of thesis to be submitte
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esults in loss of 14-3-3 binding an
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and NheI (NEB) to remove tetO-CMV,
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Hanging drop and wound healing assa
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360). To generate transgenic animal
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asement of the seminiferous tubules
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normal female, pups were screened f
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14-3-3ε only in the presence of HP
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proteins are expressed in testis (3
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5. Lalit Sehgal, Srikanth B., Khyat
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Presented a poster at 33rd All Indi
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shRNA siRNA TBS-T U.V WT C IF DSG D
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List of Figures: Page No 1. Figure
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37. Figure 4.3.20: 14-3-3γ interac
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CHAPTER 1 INTRODUCTION 39
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1.1.1 Cyclin Cdk complexes Eukaryot
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1.1.1.c Cyclin A cdk complexes The
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of which are associated with acquis
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1.2.2 The G1/S DNA Damage checkpoin
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complexes. DNA damage induced degra
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cytoplasmic accumulation of cdc25 p
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14-3-3 binding site. 14-3-3 binding
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G2 arrest and induced premature chr
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emoval of S216 phosphorylation as i
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embryogenesis, a loss of cdc25A can
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dimer was essential for Raf activat
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of enhanced apoptosis of peripheral
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the generation of transgenic mice a
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CHAPTER 2 AIMS AND OBJECTIVES 67
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CHAPTER 3 MATERIALS AND METHODS 69
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Name of oligonucleotide EF1α Forwa
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cleavage sites GAAGGTATATTGCTGTTGAC
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R E1 A GAACGAATAGTGAAGCCACAGATGTA s
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100mm 25ug 225ul 250ul 500 1000ul T
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for the transgene were considered a
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solution in TBS-T containing 0.02%
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mounting agent. Confocal images wer
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3.26 Antibodies used for Western bl
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each plate was added cold 500ml of
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3.32 Hanging drop assay to determin
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3.37 Biodistribution of 18 F -FDG u
Page 93 and 94: 2.5M NaCl 150mM 60ml Tween-20 (Sigm
Page 95 and 96: CHAPTER 4 RESULTS 95
Page 97 and 98: Figure 4.1.1 Generation of lentivir
Page 99 and 100: 4.1.2 Generation of lentiviral vect
Page 101 and 102: transduction with the virus. Untran
Page 103 and 104: Figure 4.1.6: Application of bi-cis
Page 105 and 106: Figure 4.1.7: Application of bi-cis
Page 107 and 108: Figure 4.2.1: Infection of morulae
Page 109 and 110: Figure 4.2.2: Generation of transge
Page 111 and 112: Figure 4.2.3: EGFP-f expression in
Page 113 and 114: sequenced and the distribution amon
Page 115 and 116: 4.3 Generation of 14-3-3γ knockdow
Page 117 and 118: numbering). Alignment was done usin
Page 119 and 120: presence of EGFP-f by performing po
Page 121 and 122: the mouse tested infected with vect
Page 123 and 124: Figure 4.3.7: Staging of 14-3-3γ k
Page 125 and 126: containing spermatozoa is on the Y-
Page 127 and 128: Figure 4.3.9: 14-3-3γ knockdown le
Page 129 and 130: Figure 4.3.11: 14-3-3γ knockdown i
Page 131 and 132: intercellular space (12, 110, 115,
Page 133 and 134: Figure 4.3.13: 14-3-3γ loss leads
Page 135 and 136: Figure 4.3.16: Localization of PKP3
Page 137 and 138: Figure 4.3.18: Reintroduction of sh
Page 139 and 140: A B 139
Page 141 and 142: from our laboratory demonstrated th
Page 143: Figure 4.3.21: Plakoglobin recruitm
Page 147 and 148: the desmosomal proteins recruitment
Page 149 and 150: Figure 4.3.25: Generation of stable
Page 151 and 152: KIF5B, however its reduction does n
Page 153 and 154: compared to vector control cells. (
Page 155 and 156: 4.4 Generation of 14-3-3ε knockdow
Page 157 and 158: into the interstitial spaces of the
Page 159 and 160: Figure 4.4.3: 14-3-3ε knockdown in
Page 161 and 162: Figure 4.4.4: Patchy hair loss in 1
Page 163 and 164: follicle formation marked by black
Page 165 and 166: Figure 4.4.7: Phenotypes in 14-3-3
Page 167 and 168: compared to WT lungs (indicated by
Page 169 and 170: compared to wild type (Figure 4.4.1
Page 171 and 172: and CD4 positive lyumphocyte number
Page 173 and 174: Figure 4.4.13: Bone marrow cells fr
Page 175 and 176: Figure 4.4.15: Generation of stable
Page 177 and 178: Figure 4.4.16: NIH3T3 cells lacking
Page 179 and 180: Figure 4.4.18: Generation of induci
Page 181 and 182: CHAPTER 5 DISCUSSION 181
Page 183 and 184: to generate transgenic animals expr
Page 185 and 186: transgenic research and the use of
Page 187 and 188: (reviewed in (152, 374)). Kinesins
Page 189 and 190: subtypes known to date. Type IV col
Page 191 and 192: Figure 5.1 Schematic representation
Page 193 and 194: loss of 14-3-3ε the T cell activat
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BIBLIOGRAPHY: 1. Abraham, R. T. 200
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29. Blomer, U., L. Naldini, T. Kafr
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58. Cheng, C. Y., and D. D. Mruk. 2
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90. Elia, A. E., P. Rellos, L. F. H
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118. Girard, F., U. Strausfeld, J.
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150. Herrmann, H., M. Haner, M. Bre
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and activation of a cyclin E-cdk2 c
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213. Liu, D., J. Bienkowska, C. Pet
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242. Moll, R., W. W. Franke, D. L.
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274. Pagano, M., R. Pepperkok, F. V
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305. Russell, L. D. 1978. The blood
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inhibitor of metalloproteases-1 in
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364. Tsunekawa, N., M. Matsumoto, S
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397. Yashiro, M., N. Nishioka, and
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Research: Biology to Prevention to
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LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

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