<p>Arsenic (As) exposure via groundwater consumption is a global health problem affecting millions. Monitoring exposure is a key step in understanding and predicating future health outcomes. This thesis explores the relationships between arsenic concentrations in toenails and arsenic in water. Three case studies were investigated, with residents from: North Carolina, USA (n=103); the Rift Valley, Ethiopia (n=60); and the Mekong Delta, Vietnam (n=65). Arsenic concentrations above the WHO's recommended 10ppb limit were found in groundwater from the three research sites. </p><p>Arsenic in toenails was analyzed by inductively coupled plasma mass spectrometry (ICP-MS). </p><p>In the Rift Valley of Ethiopia, 53% of the tested drinking wells (n=34) had As above the WHO's limit. Arsenic concentrations in toenails (n=60) were significantly correlated to As concentrations in groundwater (r=0.72; p<0.001), reflecting the direct exposure of rural communities to As in well water, which is their principle water source. Male minors (<18 years old) were found to have greater nail-As concentrations compared with adults consuming equal amounts of As (p<0.05). Estimated As dose specifically from drinking water sources was also associated with nail concentrations (p<0.01). </p><p>In the Mekong Delta of Vietnam (Dong Thap Province), 36 out of the 68 tested wells had As content above the WHO's recommended limit of 10ppb, with levels as high as 981 ppb. Arsenic contents in nails collected from local residents (n=62) were significantly correlated to As in drinking water (r=0.49, p<0.001). Demographic and survey data show that the ratio of As in nail to As in water varies among residents that reflects differential As accumulation in the exposed population. The data show that water filtration and diet, particularly increased consumption of animal protein and dairy and reduced consumption of seafood, were associated with lower ratios of As in nail to As in water and thus could play important roles in mitigating As exposure.</p><p>Sixty-one wells were tested from Union County, North Carolina, with 15 out of 61 wells exceeded the WHO's 10 ppb limit. Arsenic values ranged from below the limit of detection (0.07) to 130ppb, with a mean of 11ppb (median=1.5ppb). Nails were collected from county residents (n=103) and were statistically correlated with As-water concentrations (r=0.48, p<0.001). </p><p>Integration of the data from the three cases studies across different populations and ethnicities show high correlation between As concentrations in groundwater and As in nails in all the three locations (r(Union County)= 0.48, p<0.001; r(Ethiopia)=0.72 p<0.001; r(Vietnam)=0.49, p<0.001). For As-nail to As-water pairs in which As in water was above 1ppb, these three locations are statistically indistinguishable from one another (r=0.62, p<0.001, n=176). These results support the hypothesis that nails can be used as a biomarker of exposure regardless of geographic or ethnic differences in populations considered. Nutrition (meat, seafood, and milk consumption) rather than gender, ethnicity, or dose is suggested to be the major confounding issue affecting the magnitude of As exposure in the human body.</p> / Dissertation

Previous research in our laboratory found that inhibiting expression of vimentin, a marker of epithelial-to mesenchymal transition, inhibited cell growth and motility in vitro and in vivo. Tumors derived from vimentin knockdown cells showed features of epithelial redifferentiation and increased expression of differentiation-specific keratins. It is unknown what causes re-expression of keratins when vimentin is inhibited. Although, canonical Wnt signaling may activate NF-κB and repress of keratin and/or induce vimentin expression through β-catenin. We hypothesize that downregulation of differentiation-specific keratins contributes to tumor progression, mediated directly or indirectly by expression of vimentin. Vimentin-negative HN4 cells were transfected with plasmids encoding wild-type, PKCε-phosphomimetic, or unphosphorylatable versions of vimentin. Expression of vimentin was confirmed by western blot and immunofluorescence. Effects on cell growth and motility were determined using MTT, cell proliferation, and wound-closure assays. These results indicate that mutation of vimentin PKCε-phosphorylation sites cause changes in proliferation and filament assembly. Treatment of cells with an NF-κB inhibitor or 5-Aza-C, which allows re-expression of the Wnt inhibitor DKK3, led to a decrease in proliferation. These results suggest that inhibiting Wnt signaling removes the inhibition on GSK-3β and prevents activation of NF-κB, which decreases proliferation.

Bibliography: leaves [127-137]

<p>Epidermolytic hyperkeratosis is a rare inherited disease of the skin caused by a dominant-negative mutation in keratin 1 (K1) or 10 (K10). Keratins are the major structural protein in epidermis and mutations causes instability of intermediate filament and keratinocyte fragility. No curative treatment is available, but some patients benefit from retinoid therapy. More knowledge is needed about the genotype/phenotype correlation in epidermolytic hyperkeratosis and the mechanism of action of retinoids including regulation of keratin expression. </p><p>Fifteen patients were identified in Scandinavia, 13 with a generalised disease and 2 with localised lesions. Different types of mutation were identified such as point, splice site, deletion, and deletion-insertion mutations. An association was found between mutation in K1 and the appearance of palmoplantar keratoderma. Only the patients with K10 mutation benefited from the treatment, although no differences in the mRNA levels for K1 and K10 were detected. However, retinoids caused a pronounce down-regulation of K2e in upper epidermis and upregulation of K4 not normally present in the skin. This was further investigated in normal healthy skin and in keratinocytes grown in a reconstructed skin model. By adding retinoids with different affinity for the nuclear receptors RAR and RXR to the culture, the most potent retinoids were found to be RARα agonist, the effect of which could be inhibited by addition of a pan RAR antagonist. </p><p>In conclusion, several novel keratin mutations have been shown to cause epidermolytic hyperkeratosis, and genotype/phenotype correlations have been found. Treatment with retinoids is only useful for patients with a K10 mutation, possibly because they are less vulnerable too the pronounce down-regulation of K2e also seen in normal skin. This effect and the upregulation of K4 seem to be mediated through RARα , expressed in the keratinocytes.</p>

Epidermolytic hyperkeratosis is a rare inherited disease of the skin caused by a dominant-negative mutation in keratin 1 (K1) or 10 (K10). Keratins are the major structural protein in epidermis and mutations causes instability of intermediate filament and keratinocyte fragility. No curative treatment is available, but some patients benefit from retinoid therapy. More knowledge is needed about the genotype/phenotype correlation in epidermolytic hyperkeratosis and the mechanism of action of retinoids including regulation of keratin expression. Fifteen patients were identified in Scandinavia, 13 with a generalised disease and 2 with localised lesions. Different types of mutation were identified such as point, splice site, deletion, and deletion-insertion mutations. An association was found between mutation in K1 and the appearance of palmoplantar keratoderma. Only the patients with K10 mutation benefited from the treatment, although no differences in the mRNA levels for K1 and K10 were detected. However, retinoids caused a pronounce down-regulation of K2e in upper epidermis and upregulation of K4 not normally present in the skin. This was further investigated in normal healthy skin and in keratinocytes grown in a reconstructed skin model. By adding retinoids with different affinity for the nuclear receptors RAR and RXR to the culture, the most potent retinoids were found to be RARα agonist, the effect of which could be inhibited by addition of a pan RAR antagonist. In conclusion, several novel keratin mutations have been shown to cause epidermolytic hyperkeratosis, and genotype/phenotype correlations have been found. Treatment with retinoids is only useful for patients with a K10 mutation, possibly because they are less vulnerable too the pronounce down-regulation of K2e also seen in normal skin. This effect and the upregulation of K4 seem to be mediated through RARα , expressed in the keratinocytes.

Natural diseases caused by keratin mutations are rare and have only been reported in humans. We have recently identified a heritable skin disorder in Norfolk terriers caused by a mutation in KRT10. Affected dogs have a tendency to form shallow erosions or blisters following mild trauma, which is first noted after the birthing process. As the dogs age, they display generalized hyperpigmentation and scaling that is most severe in the axillary and inguinal regions. The main histologic and ultrastructural features include: marked hyperkeratosis, epidermal hyperplasia, prominent vacuolation of the upper suprabasal layers, eosinophilic intracytoplasmic aggregates (keratin bundles), numerous and frequently enlarged keratohyaline granules, and epidermal hyperplasia. Analysis of an extended pedigree through seven generations confirmed an autosomal recessive mode of inheritance. The keratin 10 mutation was defined as a G-T point mutation in intron 5 that affected splicing at the boundary of exon 4 and intron 5. The primary outcome of the mutation was a 35 bp deletion in exon 4 caused by use of a cryptic splice site. Real-time PCR quantitation of KRT10 confirmed that this mutation led to premature mRNA decay and an average 35-fold decrease in KRT10 message. Organotypic cell culture techniques were used to establish in vitro models for normal and affected Norfolk terriers. After 21 days of culture, normal epidermis was cornified with a compact and multifocally parakeratotic stratum corneum. Affected epidermis largely reproduced the expected morphologic alterations. Immunoblotting and immunohistochemistry for keratin 10 protein and real-time PCR quantitation of KRT10 message showed significantly less keratin expression in vitro than in vivo suggesting that the differentiation program in vitro underwent significant alterations. A diagnostic PCR assay was established for detection of the carrier state. Global analysis of gene expression between normal, carrier and affected dogs was performed with DermArray cDNA microarrays. Affected and carrier dogs showed differential regulation of 320 and 298 genes, respectively, between normal dogs. In affected dogs, 217 were upregulated and 103 were downregulated. In carrier dogs, 222 were upregulated and 76 were downregulated. 72 genes (65 upregulated, 7 downregulated) were altered in both affected and heterozygous dogs.

There has been an increase in rates of chromosomal abnormalities in newborns as a result of reproductive aging. For the past decades, a lot of effort has been placed on identifying pregnancies at risk of genetic defects. Conventional prenatal genetic diagnosis is achieved by invasive procedures that have been associated with an increased risk of pregnancy loss. This has led the researchers to explore the use of non-/minimally invasive techniques for prenatal diagnosis. Trophoblasts are known to be shed from regressing chorionic villi into the lower uterine pole of pregnant women during the first trimester. These cells are trapped within cervical mucus, which can be retrieved with a cytobrush. By using human leukocyte antigen G (HLA-G) and cytokeratin-7 (CK7) as trophoblast markers, this study aims to investigate the possibility of isolating individual fetal trophoblast from transcervical samples for genetic diagnosis. 195 healthy pregnant women requesting for legal termination of pregnancy (TOP) were recruited in this study. Transcervical cells were collected from them with the use of a cytobrush before TOP. HLA-G+ or CK7+ cells were then isolated by a combination of mucolytic action, fluorescent immunohistochemistry, and micromanipulation. The origin of these cells was subsequently investigated by either fluorescent in situ hybridization (FISH) or allelic profiling by quantitative fluorescent polymerase chain reaction (QF-PCR) based on chromosome 16, chromosome X, amelogenin gene and sex determining region Y (SRY) gene. This study first demonstrated the presence of fetal cells in transcervical samples based on the detection of chromosome Y signal by ordinary PCR. Cells expressing HLA-G and CK7 were also identified among transcervical cells. Immunopositive cells were isolated by micromanipulation under fluorescent microscopy. One isolated cell expressing CK7 was shown to inherit paternal allele at a locus on chromosome 16, suggesting the possible fetal origin of this cell. However, this study was still hampered by a number of technical factors. Further optimization of the protocol is required before transcervical trophoblasts can be retrieved in a reliable manner. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

This thesis investigates the use of an enzyme immunoassay to measure keratin glucocorticoid concentrations in mammalian hair, bird feathers and reptilian shed skins. Keratin glucocorticoid concentrations were compared to fecal glucocorticoid concentrations produced during the period of keratin growth in the African Elephant (Loxodonta africana), the Western Lowland Gorilla (Gorilla gorilla gorilla), the Sumatran Orangutan (Pongo abelii), the Domestic Chicken (Gallus gallus domesticus), the Eastern Loggerhead Shrike (Lanius ludovicianus migrans), the African House Snake (Lamprophis fuliginosus) and the Eastern Massasauga Rattlesnake (Sistrurus catenatus catenatus). Complete biochemical validation was performed for the feather and shed skin corticosterone and fecal corticosterone enzyme immunoassays in the Domestic Chicken and the African House Snake. Biological validation was performed in the Domestic Chicken. Biological and physiological validation were attempted in the African House Snake. African Elephant, Western Lowland Gorilla and Sumatran Orangutan hair cortisol concentrations ranged from 2.10 - 312.70 ng/g. African Elephant hair corticosterone concentrations ranged from 2.68 - 20.70 ng/g. Domestic Chicken and Eastern Loggerhead Shrike feather corticosterone concentrations ranged from 1.31 - 8.09 pg/mm and from 1.09 - 6.59 pg/mm, respectively. African House Snake and Massasauga Rattlesnake shed skin corticosterone concentrations ranged from 4.42 - 124.35 ng/g and 3.82 - 22.85 ng/g, respectively. In the majority of cases, a statistically significant association was not found between summary statistical measures of fecal and keratin glucocorticoid concentrations. A statistically significant positive association was detected between hair cortisol and the coefficient of variation of fecal corticosterone in the African Elephants. A statistically significant negative association was detected between feather corticosterone and the 75th percentile and coefficient of variation of fecal corticosterone in the Eastern Loggerhead Shrikes. A statistically significant positive association was also detected between shed skin corticosterone and the mean fecal corticosterone from 3 weeks before to 1 week after the previous ecdysis in the African House Snake. Feather corticosterone concentrations were significantly higher in feathers from Domestic Chickens that were socially housed with roosters than in feathers from chickens housed individually in laying cages. A statistically significant difference was not detected between the shed skin corticosterone concentrations of the minimally handled control and the weekly handled experimental African House Snakes. Adrenocorticotropic hormone stimulation did not result in the physiological validation anticipated for shed skin corticosterone concentrations in the African House Snake. / Toronto Zoological Foundation and Ontario Ministry of Agriculture, Food and Rural Affairs

Bibliography: leaves [127-137] / 126 [78] leaves, [26] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1985

vii, 136 leaves : ill., graphs, tables, photos ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1979) from the Dept. of Biochemistry, University of Adelaide