The Role of Helium on Cavity Growth and Swelling at High Damage Levels in Ferritic-Martensitic Steels

The desire for cleaner energy sources for baseload power generation drive an interest in new nuclear power plant designs. The development of radiation-tolerant materials for new nuclear power plants requires extensive research programs. Traditionally, radiation effects studies have been conducted using materials test reactors followed by expensive and time-consuming post-irradiation examination due to the neutron induced radioactivity. Additionally, the damage rate, temperature and helium generation rate within a reactor are all correlated with each other and dependent on the flux of neutrons and gamma rays in a reactor making the analysis for the underlying mechanisms of cavity growth and swelling difficult. Ion irradiation experiments allow for the separation of single variable dependencies to uncover the processes and understand the mechanisms underlying cavity growth and swelling with orders of magnitude higher damage rates compared to reactor irradiations and no induced radioactivity. The objective of this thesis is to understand the process by which cavity growth and swelling changes with helium content across a wide range of damage levels. Alloys HT9, heat 84425, and T91, heat 30176, were irradiated with 5.0 MeV Fe2+ ions with 2.03-2.85 MeV He2+ ions at helium co-injection rates of 0 to 4 appm He/dpa at 460°C (HT9) and 445°C (T91) at damage levels from 17 to 650 dpa in the Michigan Ion Beam Laboratory. The swelling and dislocation evolution for all irradiation conditions were characterized. Scanning transmission electron microscopy (STEM) was used to characterize the cavities greater than 5 nm in diameter and dislocations. Transmission electron microscopy (TEM) was used to characterize the cavities less than 5 nm in diameter. The peak in swelling was observed at the highest examined helium-to-dpa ratio at the lowest damage level. The location of the peak was demonstrated to be due to the enhanced nucleation of cavities at higher helium co-injection rates. As the damage level was increased, cavity growth started to dominate swelling, so the increased bubble density and resulting increase in sink strength caused a shift in the peak swelling toward lower helium-to-dpa ratios. The results of the shift were that at an intermediate damage level of 50 dpa, the maximum swelling was observed to occur at an intermediate helium co-injection rate of 0.2 appm He/dpa. At damage levels greater than 150 dpa, the peak swelling was observed to occur without the co-injection of helium. Examining helium effects in existing literature, the mode of helium injection was demonstrated to not have an influence on the effect of helium. Specifically, at low damage levels, swelling is driven by the nucleation of cavities. As the damage level increases, higher helium levels correspond to higher cavity densities and higher sink strengths which retard swelling and shift the maximum swelling location to lower helium concentrations. At high damage levels, the peak in swelling as a function of helium concentration is observed to occur at 0 appm He. This work provides substantial insight into the impact of helium on the evolution of cavities.PHDNuclear Engineering & Radiological SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/162904/1/dawoodle_1.pd

The 105-kDa Basement Membrane Autoantigen p105 Is N-Terminally Homologous to a Tumor-Associated Antigen

Certain constitutive skin basement membrane components, such as bullous pemphigoid antigens and epidermolysis bullosa acquisita antigen, were discovered because they were targeted by an autoimmune reaction. We aimed to purify and characterize a 105-kDa skin basement membrane protein termed p105 recognized by autoantibodies (anti-p105) from patients with a unique immune-mediated subepidermal blistering skin disease. A simian virus 40-transformed human fibroblast cell line that synthesizes and secretes p105 was utilized as the protein source. p105 was partially purified by salt-gradient fractionation of serum-free conditioned medium through a Mono Q anion-exchange column and by examining each fraction with protein staining and immunoblotting against anti-p105. p105 was isolated from polyacrylamide gel electrophoresis gels, blotted onto polyvinylidene difluoride membrane, and subjected to protein microsequencing. The 20 microsequenced N-terminal amino acids exhibited no homology to known basement membrane proteins but exhibited a 70% homology to a 90-kDa tumor-associated antigen. Antibodies raised against a peptide generated from these amino acid sequences reacted to a 105-kDa western-blotted keratinocyte and fibroblast protein and a basement membrane component. p105 resisted digestion by glycosidases chondroitinase ABC, neuraminidase, and N-glycosidase F but was cleaved by protease V8 to antigenic fragments of 22kDa and 14kDa. The synthesis of p105 was inhibited by cycloheximide. We conclude that p105 is a unique basement membrane component produced by both keratinocytes and fibroblasts

Spreading and Enhanced Motility of Human Keratinocytes on Fibronectin

Soluble human plasma fibronectin or collagen types I or IV, when preincubated with tissue culture plastic dishes, were effective spreading agents for cultured human keratinocytes and increased spreading in a time and concentration-dependent manner. Spreading on fibronectin, but not on type IV collagen, was inhibited by antifibroneetin; therefore, the contribution of fibronectin to the spreading activity of the natural matrix produced by keratinocytes could not be determined using antifibronectin. Fibroneetin mediated spreading at both high (1.1 mM) and low (0.1 mM) Ca++ concentrations, and spreading was not altered by cycloheximide. Insoluble fibronectin deposited by keratinocytes correlated with phagokinetic tracks on particulate gold salts, and added fibronectin, as well as type I collagen and type IV collagen, enhanced motility of keratinocytes. These studies show that production of fibronectin and responsiveness to it are similar in fibroblasts and keratinocytes and demonstrate that fibronectin can act as a matrix factor for keratinocytes

Production of Soluble and Cell-Associated Fibronectin by Cultured Keratinocytes

Fibronectin has been demonstrated in epithelial cell types in culture, but published studies of keratinocytes have shown patterns of fibronectin produced by cells grown in medium with serum, which contains fibronectin. Since plasma fibronectin can bind to cells in vitro, cells grown in serum-supplemented media could show artifactual patterns of cell-associated fibronectin. To study insoluble fibronectin produced by keratinocytes, we plated cells in the absence of feeder layers in medium lacking fibronectin. Medium conditioned by metabolically labeled keratinocytes was studied by immunoprecipitation and by extraction with gelatin-Sepharose. Cells grown in fibronectin-free medium were labeled using affinity-purified anti-fibronectin antibody and fluorescein-conjugated antirabbit IgG. Keratinocytes produced soluble fibronectin, since both immunoprecipitation and adsorption to gelatin-Sepharose detected 35S-methionine-labeled material which comigrated with human plasma fibronectin on sodium dodecyl sulfate polyacrylamide gels. Demonstration of insoluble, cell-associated fibronectin was enhanced in Triton X-100-extracted cells and was seen in subcellular fibrillar arrays at both physiologic and reduced Ca++ concentrations, but in intracellular locations only at physiologic Ca++ concentrations. When cells grown in 1.1 mM Ca++ were removed with Triton X-100, diffusely distributed fibrillar fibronectin remained on the surface of the coverslip. Asymmetric "tracks" of fibronectin left by sparsely plated cells suggested movement. Fibronectin is deposited by keratinocytes on the culture surface and may be modulated by culture conditions

Effects of Timber Harvesting and Plantation Development on Cavity-nesting Birds in New Brunswick

We studied the abundance of cavity-nesting birds in forestry-related habitats in a region of Acadian forest in New Brunswick. We examined five reference stands of natural forest, a chronosequence of conifer plantations up to 19 years old (the oldest in the study area), two selectively harvested stands, and a 30-year-old naturally regenerated clear-cut. The species richness and abundance of cavity-nesting birds were higher in reference forest (average 10.0 species per stand; 5.3 territories per 10 ha) than in plantations (2.3/stand; 1.0/10 ha), selectively harvested stands (7.0/stand; 3.8/10 ha), or the naturally regenerated clear-cut (5.0/stand; 2.5/10 ha). A cluster analysis segregated the “community” of cavity-nesting birds of natural forest from those of other treatments. Of the various harvested stands and plantations, five with a relatively large number of residual snags clustered similarly in the cluster analysis, while those with no or very few snags also clustered together. We used arrays of nest boxes (12 per stand) to examine whether the availability of cavities was limiting the use of habitats otherwise suitable for foraging by cavity-dependent species. Nest-box use for nesting and roosting was much higher in the seven plantations examined (average 4.0/10 ha for nesting and 2.9/10 ha for roosting) than in three reference stands (each 0.3/10 ha), suggesting that the plantations were deficient in this critical-habitat element. Our results suggest that certain mitigations, such as leaving residual snags and living cavity-trees, would help maintain populations of some cavity-dependent birds in clear-cuts and plantations. However, some cavity-dependent species might not be accommodated by these mitigations and are potentially at risk in intensively managed areas, unless landscape-scale management plans ensure the survival of sufficient areas of older mixed-wood forest

Adult Human Keratinocytes Migrating over Nonviable Dermal Collagen Produce Collagenolytic Enzymes That Degrade Type I and Type IV Collagen

Human adult keratinocytes migrating on a nonviable dermal substrate in cultures without fibroblasts induce thinning and degradation of the collagen substrate beneath the migrating epithelium. Further, unconcentrated conditioned medium from the cultures exhibit collagenolytic activity against both type I and type IV collagen which is inhibited by EDTA but not by phenylmethylsulfonyl fluoride or N-ethylmaleimide. Since the migrating epithelium and dermal substrate do not contain fibroblasts, this study shows that migratory keratinocytes in contact with interstitial collagen are capable of producing collagenases against type I and type IV collagen. Moreover, migratory keratinocytes appear to be similar to highly metastatic cells in their ability to degrade basement membrane collagen

Characterization of “Neo-Dermis” Formation Beneath Cultured Human Epidermal Autografts Transplanted on Muscle Fascia

Cultured human keratinocyte autografts were transplanted to burn wounds that had been completely excised down to muscle fascia such that all cutaneous elements were removed from the wounds. Healing autografts were biopsied from days 6-153 in five patients, and the "neo-dermis" beneath the autografts was examined by immunofluorescent staining using antibody probes to connective tissue molecules, by histochemical staining for elastin fibers, and by electron microscopy. We found that the neo-dermis contained most of the major connective tissue elements early in the post-transplantation period. However, regardless of the time examined, there was a paucity of elastin fibers and poor organization of linkin (microthread-like fibers) in the neo-dermis beneath autografts. The perturbations of these connective tissue components in the neo-dermis may play a role in the poor recoil and elastic properties of burn wounds treated with autografts

A Mouse Monoclonal Antibody Against a Newly Discovered Basement Membrane Component, the Epidermolysis Bullosa Acquisita Antigen

A mouse monoclonal antibody, H3a, directed against the newly described epidermolysis bullosa acquisita (EBA) antigen was obtained using hybridoma techniques. The distribution of the monoclonal antibody is identical to that of the polyclonal serum antibody of patients with EBA. By immunofluorescence, a linear band is seen at the dermal-epidermal junction and, by immunoelectron microscopy, immune reaction products are present in the lamina densa and sublamina densa regions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western immunoblot analysis shows that the monoclonal antibody recognizes 290 and 145 kilodalton proteins present in the immunizing junctional extract, identical with the newly discovered EBA antigen. This monoclonal antibody should be useful in the further isolation and characterization of the EBA antigen

Dibutyryl Cyclic AMP Modulates Keratinocyte Migration Without Alteration on Integrin Expression

Cyclic adenosine monophosphate (cAMP) has long been regarded as a second messenger and a regulator of human keratinocyte proliferation. It has been demonstrated that cAMP inhibits keratinocyte proliferation when used at high concentrations. Nevertheless, new recent reports have demonstrated that cAMP may stimulate or inhibit keratinocyte growth depending upon the concentration used. Studies to examine the influence of cAMP upon the migration of other cell types have been contradictory. To determine the direct effect of dibutyryl cAMP (DBcAMP) upon human keratinocyte migration, we used a quantitative locomotion assay using a wide range of DBcAMP concentrations. We found a bi-phasic effect of DBcAMP on keratinocyte migration across connective tissue matrices. Keratinocyte locomotion on the matrices was promoted at 10-5 M and 10-6 M of DBcAMP, but not at higher or lower concentrations. Timecourse experiments demonstrated that the effect of DBcAMP on keratinocyte locomotion and proliferation occurred independently. Fluorescence-activated cell sorter analysis demonstrated that the effect of DBcAMP on the migration of human keratinocytes was independent from the modulation of integrin receptors. Although the cellular mechanisms by which DBcAMP promotes keratinocyte migration is unclear, the addition of DBcAMP or TPA to keratinocyte cultures enhanced the synthesis of a 92-kDa metalloproteinase in association with enhanced cellular migration. These observations suggest a possible link between metalloproteinase expression and cellular migration

Neonatal Foreskin Substrate Has Limitations for the Immunofluorescent Screening of Monoclonal Antibodies

Two monoclonal antibodies to type IV collagen showed a marked decrease in the labeling of the dermal-epidermal junction of neonatal foreskin while the basement membrane around dermal blood vessels was brightly stained. In contrast, these antibodies labeled the junction and dermal blood vessels with approximately equal intensity when adult skin of nonforeskin site was used as substrate. Other antibodies to matrix molecules (bullous pemphigoid antigen, epidermolysis bullosa acquisita antigen, and laminin) showed excellent staining of both the dermal-epidermal junction and dermal blood vessels in both neonatal foreskin and adult skin. Further, the ultrastructural appearance of the substrates appeared identical. The implication is that neonatal foreskin is not a good substrate to use for the routine screening of monoclonal antibodies to matrix components by indirect immunofluorescence since a "false negative" evaluation may occur