Recognition of Occluded Object Using Wavelets

Ph.DDOCTOR OF PHILOSOPH

Effects of Hydrogen Peroxide on Wound Healing in Mice in Relation to Oxidative Damage

10.1371/journal.pone.0049215PLoS ONE711

Ectodermal Influx and Cell Hypertrophy Provide Early Growth for All Murine Mammary Rudiments, and Are Differentially Regulated among Them by Gli3

Mammary gland development starts in utero with one or several pairs of mammary rudiments (MRs) budding from the surface ectodermal component of the mammalian embryonic skin. Mice develop five pairs, numbered MR1 to MR5 from pectoral to inguinal position. We have previously shown that Gli3Xt-J/Xt-J mutant embryos, which lack the transcription factor Gli3, do not form MR3 and MR5. We show here that two days after the MRs emerge, Gli3Xt-J/Xt-J MR1 is 20% smaller, and Gli3Xt-J/Xt-J MR2 and MR4 are 50% smaller than their wild type (wt) counterparts. Moreover, while wt MRs sink into the underlying dermis, Gli3Xt-J/Xt-J MR4 and MR2 protrude outwardly, to different extents. To understand why each of these five pairs of functionally identical organs has its own, distinct response to the absence of Gli3, we determined which cellular mechanisms regulate growth of the individual MRs, and whether and how Gli3 regulates these mechanisms. We found a 5.5 to 10.7-fold lower cell proliferation rate in wt MRs compared to their adjacent surface ectoderm, indicating that MRs do not emerge or grow via locally enhanced cell proliferation. Cell-tracing experiments showed that surface ectodermal cells are recruited toward the positions where MRs emerge, and contribute to MR growth during at least two days. During the second day of MR development, peripheral cells within the MRs undergo hypertrophy, which also contributes to MR growth. Limited apoptotic cell death counterbalances MR growth. The relative contribution of each of these processes varies among the five MRs. Furthermore, each of these processes is impaired in the absence of Gli3, but to different extents in each MR. This differential involvement of Gli3 explains the variation in phenotype among Gli3Xt-J/Xt-J MRs, and may help to understand the variation in numbers and positions of mammary glands among mammals

Engineering a scaffold-free 3D tumor model for in vitro drug penetration studies

10.1016/j.biomaterials.2009.10.049BIOMATERIALS3161180-1190UNITED KINGDO

High concentrations of H₂O₂ increase levels of MMP-8 in wounds.

<p>Western blot analysis of wound tissues lysate collected 6 days after wounding. Each lane represents a sample from a different animal. (A) Representative blot of MMP-8. (B) Densitometry analysis of MMP-8 normalized against α-tubulin re-probed from their respective blot. Results are mean ± S.E.M. (n = 4) and were analyzed using 1-way ANOVA followed by Tukey multiple comparison among all the columns. ** p<0.01 (C) Representative blot of MMP-9. (D) Densitometry analysis of MMP-9 normalized against α-tubulin re-probed from their respective blot. Results are mean ± S.E.M. (n = 4), p values for 1-way ANOVA is p = 0.13.</p

Low concentrations of H₂O₂ increased wound angiogenesis.

<p>Paraffin sections from day 6 wounds were stained for CD31 using an immunohistochemical method. Representative photomicrograph of (A) control, (B) 166 mM H₂O₂ and (C) 10 mM H₂O₂ treated wounds are shown. (D) The number of brown lumen-like structures within the neodermis was counted in a single blinded fashion and analyzed using 1-way ANOVA followed by Dunnett’s multiple comparison test with control. Graph shown is the mean ± S.E.M, n = 6–7, *** p<0.001.</p

Wounding increases ERK1/2 and p38 phosphorylation which can be further increased by 166 mM H₂O₂ treatment.

<p>(A) Representative blots of wound tissues lysate collected 30 min after wounding. Skin denotes skin from non-wounded animals while control refers to wounds treated with PBS. (B) The density of phosphorylated ERK and pan-ERK and (C) phosphorylated p38 and pan p38 were normalized against α-tubulin re-probed from their respective blot. Results shown are mean ± S.E.M. (n = 4). Densitometry results were analyzed by 1-way ANOVA and test of significance between all column was determined using Tukey’s post hoc test. Only the comparison between 166 mM treated wounds and skin was statistically significant for both B and C. ** p<0.01.</p

Wounding increased lipid peroxidation and nitrative damage but not protein carbonylation.

<p>Levels of F₂-isoprostanes levels in skin and wounds were compared by normalizing against arachidonic acid (A) or tissue weight (B). Results shown are mean ± S.E.M, n = 5. Wounds were compared to skin using 1-way ANOVA with Dunnett’s post-hoc test. Asterisks denote level of significance when compared to skin. Control and H₂O₂ wounds were also compared against each other using 2-way ANOVA but the differences was not statistically significant. (C) Levels of arachidonic acid in skin and wound tissues. Results shown are mean ± S.E.M, n = 5. Wounds were compared to skin using 1-way ANOVA with Dunnett’s post-hoc test. Asterisks denote level of significance when compared to skin. Control and H₂O₂ wounds were also compared against each other using 2-way ANOVA but the differences was not statistically significant. (D) Levels of protein carbonyls in wounds were compared to intact skin and expressed as fold change. The results shown are the mean fold change ± S.E.M. No difference in the levels of protein carbonyl was observed in control wounds and 166 mM H₂O₂ treated wounds. (E) Comparison of 3-nitrotyrosine level in skin and wounds. Results shown are mean ± S.E.M., n = 5. The 3-nitrotyrosine levels of skin were compared to control wounds or H₂O₂ treated wounds and analyzed with 1-way ANOVA followed by Dunnett’s post-hoc test. Levels of 3-nitrotyrosine were significantly higher at day 6 after wounding. Levels of 3-nitrotyrosine in control and 166 mM H₂O₂ treated wounds were also compared using 2-way ANOVA and the differences were not statistically significant. *p<0.05, ** p<0.01, ***p<0.001.</p

High concentrations of H₂O₂ retard connective tissue formation.

<p>Paraffin sections from day 6 wounds were stained with the Masson-Goldner trichrome stain as described in material and method. Connective tissues are stained green. Fibrin, eschar and cytoplasm are stained red. Nuclei are stained dark brown. Representative images for control (A,D) 10 mM (B, E) and 166 mM (C, F) treated wounds are shown. Images A-C are at 100X magnification while D-F are at 200X magnification. (G) Quantification of the fraction of pixels that are stained green. The number of pixels stained green within the neodermis at 100X magnification was quantified using a custom software. The area quantified is outlined with the dashed line. Results were analyzed using 1-way ANOVA followed by Dunnett’s multiple comparison test with control. Graph shown is the mean ± S.E.M. n = 6–7, *** p<0.001.</p