In vitro spheroids and metastatic epithelial ovarian cancer

This thesis investigated the role of cellular aggregation in the spread of epithelial ovarian cancer. Cell aggregation was studied in vitro in order to identify markers of disease progression and cell-cell adhesion. Proteomics was then used to identify additional proteins associated with cell aggregation and survival.

Mechanical characterizations of thick laminated composites before and after bonded repair

One set of composite laminates was manufactured from bi-directional carbon fibre woven cloth pre-impregnated with epoxy resin and used to establish experimental techniques. Another, similar set was used for extensive data collection. One other set of laminates, manufactured from uni-directional carbon fibre, was also subjected to extensive tests to represent a different material. The results give the pre and post characterizations of repaired composite materials; outlining at each stage the losses and gains of structural strength and stiffness and discusses the difficulties experienced.

Epithelial cell folate is an accurate marker when compared with whole tissue biopsy folate for examining the role of folate status in colorectal cancer

Background – Epidemiological studies have shown low folate status is associated with colorectal cancer. Colonic tissue folate levels at different stages of cancer development should give important information, but different methodologies to extract the colonic tissue folates have been used. This has hampered progress in defining the relationship between systemic and tissue folate levels.
Objective – To evaluate two methods of colonic tissue preparation for estimation of total folate content.
Design – Whole tissue punch biopsy samples were obtained from the descending colon of 31individuals following a normal colonoscopy. Blood samples were obtained for the determination of plasma homocysteine (Hcy), red cell folate (RCF), methylenetetrahydrofolate reductase 677C>T genotype, and serum vitamin B12 and folate. Colonic tissue folate was measured both in washed whole tissue biopsies and in epithelial cells isolated from tissue biopsies.
Outcomes - Whole biopsy and epithelial cell folate concentrations were significantly correlated (R=.375; P=.038). Hcy was inversely correlated with both measures (R=-.365; P=.043 and R=-.364; P=.044 respectively). RCF was significantly correlated with isolated epithelial cell folate (R=.477; P=.007) but not with whole tissue biopsy folate (R=.264; P=.151). There were no significant associations between serum and colonic folate in this study.
Conclusion - Both methods are useful for comparing systemic and localised tissue folate status but epithelial cells may provide more reliable data.

Influence of age on expression of DNA repair genes in Arabidopsis

Numerous studies suggest that ageing in mammals may be associated with a reduction in DNA repair, whereas little is known about the DNA repair capacity of plants as they age. In this study we examined the effects of ageing on the expression of genes thought to be involved in nucleotide excision repair (AtERCC1, AtGTF2H2, AtGTF2H5, AtXPB1, AtXPD, AtXPF) or translesion replication (AtPOLH, AtREV1, AtREV3, AtUBC2) of UV photoproducts in Arabidopsis (Arabidopsis thaliana). Two- or four-week old plants were unirradiated or treated with 254 nm ultraviolet (UV) radiation (0.75 or 1.5 kJm^-2), incubated for 3 or 9 hr, and gene expression was analysed via quantitative PCR. With the exception of AtPOLH, transcript levels for all genes investigated were at least four-fold greater in unirradiated four-week old plants than unirradiated two-week old plants. Compared to unirradiated age-matched plants, two-week old plants generally showed no consistent change in transcript levels for either UV dose or post-irradiation incubation period. On the other hand, transcript levels in four-week old plants were increased over those in two-week old plants for the majority of genes by 9 hr post-irradiation with 0.75 or 1.5 kJm^-2 UV. No other consistent responses were observed for UV treatment. Collectively, our results are consistent with the possibility that ageing may be associated with increased DNA repair capacity in plants.

Age and exposure to arsenic alter base excision repair transcript levels in mice

Arsenic (As) induces DNA-damaging reactive oxygen species. Most oxidative DNA damage is countered by base excision repair (BER), the capacity for which may be reduced in older animals. We examined whether age and consumption of As in lactational milk or drinking water influences BER gene transcript levels in mice. Lactating mothers and 24-week-old mice were exposed (24 h or 2 weeks) to As (2 or 50 p.p.m.) in drinking water. Lung tissue was harvested from adults, neonates (initially 1 week old) feeding from lactating mothers and untreated animals 1– 26 weeks old. Transcripts encoding BER proteins were quantified. BER transcript levels decreased precipitously with age in untreated mice but increased in neonates whose mothers were exposed to 50 p.p.m. As for 24 h or 2 weeks. Treatment of 24-week-old mice with 2 or 50 p.p.m. As for 2 weeks decreased all transcript levels measured. Exposure to As attenuated the age-related transcript level decline for only one BER gene. We conclude that aging is associated with a rapid reduction of BER transcript levels in mice, which may contribute to decreased BER activity in older animals. Levels of As that can alter gene expression are transmitted to neonatal mice in lactational milk produced by mothers drinking water containing As, raising concerns about breastfeeding in countries having As-contaminated groundwater. Reduction of BER transcript levels in 24- week-old mice exposed to As for 2 weeks suggests As may potentiate sensitivity to itself in older animals.

Moral repair with offenders : ethical issues arising From victimization experiences

In this article, the authors examine the relevance of the concept of moral repair for sex offenders who have been victims of sexual or physical abuse. First, they briefly review the literature on victimization rates and effects in sexual offenders. Second, the notion of moral repair and its constituent tasks is examined with particular emphasis given to Margaret Walker's recent analysis of the concept. Third, the concept of moral repair is applied to offenders and its implications and possible constraints discussed. Fourth, the authors outline a normative framework for addressing victimization issues with sexual offenders, drawing on the resources of human rights theory and strength-based treatment approaches. Finally, they conclude with a brief consideration of the ethical and clinical implications of their normative model.

Antioxidant enzyme activities of iron-saturated bovine lactoferrin (Fe-bLf) in human gut epithelial cells under oxidative stress

Chemoprevention by dietary constituents in the form of functional food has emerged as a novel approach to control inflammatory diseases and cancers. Recently we reported for the first time that iron content is a critical determinant in the anti-tumour activity of bovine milk lactoferrin (bLf). We therefore wanted to evaluate the chemo-preventative efficacy of Apo-bLF and 100% iron-saturated bLF (Fe-bLF) on hydrogen peroxide (H2O 2)-induced colon carcinogenesis, and their influence on antioxidant enzyme activities within colon carcinogenesis. This was undertaken through observing how oxidative stress induced by H2O2 alters antioxidant enzyme activity within HT29 colon cancer cells, and then observing changes in this activity by treatments with the different antioxidants ascorbic acid (AA), Apo-bLF and Fe-bLF. All antioxidant enzymes (catalase, glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-s-transferase (GsT) and superoxide dismutase (SOD)) appeared to be increased within HT29 cells, even prior to H2O2 exposure, and all enzymes showed significant decreased activity when cells were treated with the antioxidants AA, Apo-bLF or Fe-bLF, with or without H2O2 exposure. The results indicate that all three antioxidants have the ability to scavenge ROS, lower antioxidant enzyme activities within already excited states, and possibly allow colon cancer cells to be overcome by oxidative stress that would normally be prevented, perhaps leading to damage and potential apoptosis of the cancer cells. In conclusion, the anti-oxidative effects of Apo-bLF and Fe-bLf studied for the first time, show dynamic changes that may allow for necessary protection from imbalanced oxidative conditions, and potential at reducing the ability of cancer cells to protect themselves from oxidative stress states.

c-Cbl facilitates endocytosis and lysosomal degradation of cystic fibrosis transmembrane conductance regulator in human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl− channel expressed in the apical membrane of fluid-transporting epithelia. The apical membrane density of CFTR channels is determined, in part, by endocytosis and the postendocytic sorting of CFTR for lysosomal degradation or recycling to the plasma membrane. Although previous studies suggested that ubiquitination plays a role in the postendocytic sorting of CFTR, the specific ubiquitin ligases are unknown. c-Cbl is a multifunctional molecule with ubiquitin ligase activity and a protein adaptor function. c-Cbl co-immunoprecipitated with CFTR in primary differentiated human bronchial epithelial cells and in cultured human airway cells. Small interfering RNA-mediated silencing of c-Cbl increased CFTR expression in the plasma membrane by inhibiting CFTR endocytosis and increased CFTR-mediated Cl− currents. Silencing c-Cbl did not change the expression of the ubiquitinated fraction of plasma membrane CFTR. Moreover, the c-Cbl mutant with impaired ubiquitin ligase activity (FLAG-70Z-Cbl) did not affect the plasma membrane expression or the endocytosis of CFTR. In contrast, the c-Cbl mutant with the truncated C-terminal region (FLAG-Cbl-480), responsible for protein adaptor function, had a dominant interfering effect on the endocytosis and plasma membrane expression of CFTR. Moreover, CFTR and c-Cbl co-localized and co-immunoprecipitated in early endosomes, and silencing c-Cbl reduced the amount of ubiquitinated CFTR in early endosomes. In summary, our data demonstrate that in human airway epithelial cells, c-Cbl regulates CFTR by two mechanisms: first by acting as an adaptor protein and facilitating CFTR endocytosis by a ubiquitin-independent mechanism, and second by ubiquitinating CFTR in early endosomes and thereby facilitating the lysosomal degradation of CFTR.

Apical localization of zinc transporter ZnT4 in human airway epithelial cells and its loss in a murine model of allergic airway inflammation

The apical cytoplasm of airway epithelium (AE) contains abundant labile zinc (Zn) ions that are involved in the protection of AE from oxidants and inhaled noxious substances. A major question is how dietary Zn traffics to this compartment. In rat airways, in vivo selenite autometallographic (Se-AMG)-electron microscopy revealed labile Zn-selenium nanocrystals in structures resembling secretory vesicles in the apical cytoplasm. This observation was consistent with the starry-sky Zinquin fluorescence staining of labile Zn ions confined to the same region. The vesicular Zn transporter ZnT4 was likewise prominent in both the apical and basal parts of the epithelium both in rodent and human AE, although the apical pools were more obvious. Expression of ZnT4 mRNA was unaffected by changes in the extracellular Zn concentration. However, levels increased 3-fold during growth of cells in air liquid interface cultures and decreased sharply in the presence of retinoic acid. When comparing nasal versus bronchial human AE cells, there were significant positive correlations between levels of ZnT4 from the same subject, suggesting that nasal brushings may allow monitoring of airway Zn transporter expression. Finally, there were marked losses of both basally-located ZnT4 protein and labile Zn in the bronchial epithelium of mice with allergic airway inflammation. This study is the first to describe co-localization of zinc vesicles with the specific zinc transporter ZnT4 in airway epithelium and loss of ZnT4 protein in inflamed airways. Direct evidence that ZnT4 regulates Zn levels in the epithelium still needs to be provided. We speculate that ZnT4 is an important regulator of zinc ion accumulation in secretory apical vesicles and that the loss of labile Zn and ZnT4 in airway inflammation contributes to AE vulnerability in diseases such as asthma.

A potential copper-regulatory role for cytosolic expression of the DNA repair protein XRCC5

Copper (Cu) has a critical role in the generation of oxidative stress during neurodegeneration and cancer. Reactive oxygen species generated through abnormal elevation or deficiency of Cu can lead to lipid, protein, and DNA damage. Oxidation of DNA can induce strand breaks and is associated with altered cell fate including transformation or death. DNA repair is mediated through the action of the multimeric DNA-PK repair complex. The components of this complex are the Ku autoantigens, XRCC5 and XRCC6 (Ku80 and Ku70, respectively). How this repair complex responds to perturbed Cu homeostasis and Cu-mediated oxidative stress has not been investigated. We previously reported that XRCC5 expression is altered in response to cellular Cu levels, with low Cu inhibiting XRCC5 expression and high Cu levels enhancing expression. In this study we further investigated the interaction between XRCC5 and Cu. We report that cytosolic XRCC5 is increased in response to Cu, but not zinc, iron, or nickel, and the level of cytosolic XRCC5 correlates with protection against oxidative damage to DNA. These observations were made in both HeLa cells and fibroblasts. Cytosolic XRCC5 interacted with the Cu chaperone and detoxification protein human Atox1 homologue (HAH), and down regulation of XRCC5 expression using siRNA led to enhanced HAH expression when cells were exposed to Cu. XRCC5 could also be purified from cytosolic extracts using a Cu-loaded column. These findings provide further evidence that cytosolic XRCC5 has a key role in protection against DNA oxidation from Cu, through either direct sequestration or signaling through other Cu-detoxification molecules. Our findings have important implications for the development of therapeutic treatments targeting Cu in neurodegeneration and/or cancer.

Effect of selenium-saturated bovine lactoferrin (Se-bLF) on antioxidant enzyme activities in human gut epithelial cells under oxidative stress

Cancer and many chronic inflammatory diseases are associated with increased amounts of reactive oxygen species (ROS). The potential cellular and tissue damage created by ROS has significant impact on many disease and cancer states and natural therapeutics are becoming essential in regulating altered redox states. We have shown recently that iron content is a critical determinant in the antitumour activity of bovine milk lactoferrin (bLF). We found that 100% iron-saturated bLF (Fe-bLF) acts as a potent natural adjuvant and fortifying agent for augmenting cancer chemotherapy and thus has a broad utility in the treatment of cancer. Furthermore, we also studied the effects of iron saturated bLF's ability as an antioxidant in the human epithelial colon cancer cell line HT29, giving insights into the potential of bLF in its different states. Thus, metal saturated bLF could be implemented as anti-cancer neutraceutical. In this regard, we have recently been able to prepare a selenium (Se) saturated form of bLF, being up to 98% saturated. Therefore, the objectives of this study were to determine how oxidative stress induced by hydrogen peroxide (H₂O₂) alters antioxidant enzyme activity within HT29 epithelial colon cancer cells, and observe changes in this activity by treatments with different antioxidants ascorbic acid (AA), Apo (iron free)-bLF and selenium (Se)-bLF. The states of all antioxidant enzymes (glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-s-transferase (GsT), catalase and superoxide dismutase (SOD)) demonstrated high levels within untreated HT29 cells compared to the majority of other treatments being used, even prior to H₂O₂ exposure. All enzymes showed significant alterations in activity when cells were treated with antioxidants AA, Apo-bLF or Se-bLF, with and/or without H₂O₂ exposure. Obvious indications that the Se content of the bLF potentially interacted with the glutathione (GSH)/GPx/GR/GsT associated redox system could be observed immediately, showing capability of Se-bLF being highly beneficial in helping to maintain a balance between the oxidant/antioxidant systems within cells and tissues, especially in selenium deficient systems. In conclusion, the antioxidative defence activity of Se-bLf, investigated in this study for the first time, shows dynamic adaptations that may allow for essential protection from the imbalanced oxidative conditions. Because of its lack of toxicity and the availability of both selenium and bLF in whole milk, Se-bLF offers a promise for a prospective natural dietary supplement, in addition to being an immune system enhancement, or a potential chemopreventive agent for cancers.