Study of surface treated historical materials

Import 05/08/2014Diplomová práce se zabývá obecným studiem povrchových úprav technikou plátkového zlacení na historických předmětech, zejména obrazových rámech. V teoretické části práce pojednává o historii a principech zhotovování technologií zlacení povrchu různých materiálů od minulosti k současnosti. Zřetel je kladen na dřevěné obrazové rámy a jejich části. Cílem práce je průzkum složení povrchových úprav a to jak složení slitiny použitého plátkového kovu, tak složení pojiv a podkladové vrstvy. Experimentální část je zaměřena na průzkum povrchu historických předmětů pomocí skenovací elektronové mikroskopie a analýzu typu syntetických pryskyřic rámů pomocí spektroskopických a chromatografických metod.This thesis deals with the general study of finishes leaf gilding technique on historical subjects, especially picture frames. The theoretical part deals with the history and principles of making technology gilding the surface of various materials from past to present. Consideration shall be given to the wooden picture frames and parts. The aim of the work is investigation of the finishes in both the composition of the alloy used leaf metal and binder composition and the underlying layer. The experimental part will focus on exploration of historical objects surface using scanning electron microscopy and analysis of the type of synthetic resin frames using chromatographic methods.636 - Katedra materiálového inženýrstvívýborn

Radial Glial Neural Progenitors Regulate Nascent Brain Vascular Network Stabilization Via Inhibition of Wnt Signaling

<div><p>The cerebral cortex performs complex cognitive functions at the expense of tremendous energy consumption. Blood vessels in the brain are known to form stereotypic patterns that facilitate efficient oxygen and nutrient delivery. Yet little is known about how vessel development in the brain is normally regulated. Radial glial neural progenitors are well known for their central role in orchestrating brain neurogenesis. Here we show that, in the late embryonic cortex, radial glial neural progenitors also play a key role in brain angiogenesis, by interacting with nascent blood vessels and regulating vessel stabilization via modulation of canonical Wnt signaling. We find that ablation of radial glia results in vessel regression, concomitant with ectopic activation of Wnt signaling in endothelial cells. Direct activation of Wnt signaling also results in similar vessel regression, while attenuation of Wnt signaling substantially suppresses regression. Radial glial ablation and ectopic Wnt pathway activation leads to elevated endothelial expression of matrix metalloproteinases, while inhibition of metalloproteinase activity significantly suppresses vessel regression. These results thus reveal a previously unrecognized role of radial glial progenitors in stabilizing nascent brain vascular network and provide novel insights into the molecular cascades through which target neural tissues regulate vessel stabilization and patterning during development and throughout life.</p> </div

Activation of canonical Wnt signaling induces, while attenuation substantially suppresses, vessel regression.

<p>(A–D) Effects of LiCl-mediated Wnt pathway activation on vessel development. LiCl treatment at E16.5–17.5 induces hemorrhage throughout E18.5 brains (A). Ter119 staining confirmed microhemorrhage throughout LiCl-treated brains (insets in A). Quantification showed significant decreases in vessel density in the cortex (<i>p</i> = 0.0057, <i>n</i> = 7), but not in the striatum (<i>p</i> = 0.88, <i>n</i> = 6) or the heart (<i>p</i> = 0.45, <i>n</i> = 3) following LiCl treatment (B). Vessel morphology in NaCl- and LiCl-treated E18.5 cortices is shown in (C and D). (E–F) Effects of SB216763-mediated Wnt pathway activation on vessel development. SB216763 treatment at E15.5–17.5 induces vessel loss in E18.5 brains (E). Quantification showed significant decreases in vessel density in the cortex (<i>p</i> = 3.4×10⁻⁵, <i>n</i> = 4) (F). (G–H) Effects of <i>wnt7b</i> mutation on Glut-1 expression in <i>orc3</i> mutants. Introduction of <i>wnt7b</i> into <i>orc3</i> mutant background suppresses increases in Glut-1 expression at E16.5 (G). Quantification of Glut-1 expression and analysis by ANOVA followed by Tukey's post hoc test shows that <i>wnt7b</i> mutation alone has no significant effects but suppresses Glut-1 expression in <i>orc3</i> mutants (** <i>p</i><0.01, <i>n</i> = 13) (H). (I–L) Effects of <i>wnt7b</i> mutation on vessel regression in <i>orc3</i> mutants at P0. Introduction of <i>wnt7b</i> into <i>orc3</i> mutant background suppresses brain hemorrhage (I). Quantification of vessel density and analysis by ANOVA followed by Tukey's post hoc test shows that <i>wnt7b</i> mutation alone has no significant effects on vessel density but suppresses vessel regression in <i>orc3</i> mutants (**<i>p</i><0.01, <i>n</i> = 4) (J). Cortical vessel morphology in <i>orc3</i> single and <i>orc3/wnt7b</i> double mutants is shown in (K and L). Scale bar (in J): 200 µm for (C–E), 100 µm for (G), and 500 µm for (K–L).</p

Ablation of neural progenitors results in defective cortical angiogenesis.

<p>(A–B) Neonatal brain hemorrhage in <i>orc3</i> mutants. Immunoglobulin reactivity further confirmed patterns of hemorrhage (B). (C–F) Vessel morphology at P0 along the anterior-posterior axis. IB4 (in green) and laminin (LN, in red) staining revealed severe loss of vessels in mutants in both the anterior (C and D) and posterior (E and F) cortex. (G–H) Quantitative analysis of cortical plate vessel density and branching frequency. Dramatic reductions in both vessel density (G) and branch point frequency (H) were observed (<i>p</i> = 1.76×10⁻¹⁴ and <i>p</i> = 1.01×10⁻⁸, respectively; <i>n</i> = 9). (I–J) Correlation between radial glial density and vessel density (I) as well as between radial glial density and vessel branching frequency (J) in the medial cortex of control, <i>orc3/nestin-cre</i>, and <i>orc3/hGFAP-cre</i> mutant neonates. The correlation coefficient was 0.97 for the former (I) and 0.98 for the latter (J). Scale bar (in F): 500 µm for (B–F).</p

Ectopic Wnt signaling may destabilize cortical vessels in part through up-regulation of MMP-2.

<p>(A–B) Effects of radial glial ablation on vessel basement membrane. Staining with highly diluted anti-laminin (LN) antibodies (in red) revealed even labeling along control vessels (A), but frequent bright puncta in mutants (B). (C–D) MMP-2 expression along vessels at E16.5. Antibody staining (in brown) revealed low levels of MMP-2 along control vessels (C), which appear elevated in mutants (D). (E–F) Gelatin zymography. Full-length pro-MMP-2 activity appears substantially up-regulated in mutants at E16.5. Cleaved MMP-2 was also detected in mutants but not in controls. Quantification confirmed an over 170% increase in pro-MMP-2 levels (<i>p</i> = 0.006; <i>n</i> = 3) (F). (G–H) MMP-2 expression in brains treated with LiCl at E17.5. LiCl dramatically elevated MMP-2 expression along cortical vessels. (I–L) Effects of <i>Timp2</i> mutation on vessel regression in <i>orc3</i> mutants. Introduction of <i>Timp2</i> mutation into <i>orc3</i> mutant background substantially suppressed brain hemorrhage (I) and restored cortical vessel network (K and J). Analysis by ANOVA followed by Tukey's post hoc test shows that <i>Timp2</i> mutation alone has no significant effects on vessel density, but suppresses vessel regression in <i>orc3</i> mutants (** <i>p</i><0.01, <i>n</i> = 6) (L). Scale bar (in K): 70 µm for (A–B) and 200 µm for (C–D, G–H, and J–K).</p

Blockade of cell cycle progression reduces cortical neural progenitors without affecting cell fate.

<p>(A–D) Effects of <i>orc3</i> deletion on radial glial division. BrdU labeling (in red) revealed no obvious defects in ventricular zone division at E13.5, but substantial reductions at E15.5, especially at the ventricular surface (arrowheads in C and D). (E–H) Effects of <i>orc3</i> deletion on radial glial density. RC2 staining (in green) revealed substantial reductions in radial glial density at E15.5 and near complete loss at P0. Boxed areas in (E–H) are shown in (E′–H′). Also notice more severe loss of radial glia in the mutant medial cortex (right side in H). (I–J″) Effects of <i>orc3</i> deletion on radial glial fate. Although reduced in number, mutant radial glia express normal levels of Pax6 (in red) (I and J) at E16.5. PH3 (in green) staining also revealed a reduced number of mitotic cells at the ventricular surface (I′ and J′). (K–L) Quantification of Pax6⁺ cell density and expression level. Significant differences were observed in Pax6⁺ cell density (<i>p</i> = 0.0003, <i>n</i> = 4) but not Pax6 staining intensity (<i>p</i> = 0.32, <i>n</i> = 20). Note cells are less densely packed in mutants. (M–N) Effects of <i>orc3</i> deletion on intermediate progenitors. Tbr2 staining (red) revealed severe reduction in intermediate progenitors in mutants at E16.5. (O–P″) Cell cycle exit analysis at E15.5. BrdU was administered at E14.5 followed by staining for BrdU (red in O, P, O″, and P″) and Ki67 (green in O′, P′, O″, and P″) at E15.5. (Q) Quantification of Tbr2⁺ cells in the subventricular zone at E16.5. Significant reductions were observed in mutants at E16.5 (<i>p</i> = 3×10⁻¹⁵, <i>n</i> = 9). (R) Cell cycle exit indices at E15.5 and E16.5. Significant increases were observed in mutants at E15.5 (<i>p</i> = 0.0001, <i>n</i> = 5), but not at E16.5 (<i>p</i> = 0.39, <i>n</i> = 5). Scale bar (in D): 200 µm for (A–D), 50 for µm for (E–H), and 60 µm for (I–J″) and (M–P″).</p

Ablation of neural progenitors results in cortical vessel regression independent of defects in pericyte recruitment.

<p>(A–H) Vessel morphology from E15.5 to E18.5. IB4 staining (in green) revealed no obvious defects at E15.5 (A and B) but significant defects at E16.5 (C and D), E17.5 (E and F), and E18.5 (G and H). (I–J) Quantification of vessel density and branching frequency from E15.5–17.5. Significant decreases (*) in both parameters were observed at E16.5 (<i>p</i> = 0.03 and 0.0003, <i>n</i> = 3) and E17.5 (<i>p</i> = 0.7×10⁻⁵ and 0.007, <i>n</i> = 5), but not at E15.5 (<i>p</i> = 0.52 and 0.25, <i>n</i> = 4). (K–N) Pericyte investment of vessels. Desmin staining (in red) surrounds CD31⁺ (in green) vessels similarly in mutants (L), as in controls (K). Boxed areas in (K and L) are shown in two right panels. PDGFRβ⁺ (in green) cells (some highlighted by arrowheads) were also associated with IB4⁺ (in red) vessels similarly in mutants (N), as in controls (M). PDGFRβ staining in boxed areas are shown as insets. (O) Quantification of PDGFRβ⁺ pericyte density. Pericytes along all vessels were quantified. No significant differences were observed (<i>p</i> = 0.62, <i>n</i> = 4). (P–Q′) Collagen IV staining. Collagen IV⁺ (in red) empty sleeves (arrowheads in Q′) were frequently observed in mutants (Q–Q′), in contrast to controls (P–P′). (R–S) Vessel perfusion. Neonates were perfused trans-cardially using 1% FITC-dextran (in green) followed by IB4 (in red) staining. Many vessels in mutant brains (S) were not well perfused (arrowhead in S), in contrast to those in controls (R). Scale bar (in D): 200 µm for (A–H), (K–N), and (R–S), 80 µm for (P–Q′).</p