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Alpha-1 antitrypsin deficiency (AATD) in the liver results from misfolding and intracellular aggregation of Alpha-1 antitrypsin (AAT) proteins. A system capable of tracking intracellular AAT levels would be highly useful as a means of identifying the effects of potential therapeutic compounds or genetic modulations. The objective of this study was to apply fusion protein constructs comprising AAT and fluorescent reporters to visualize and quantify intracellular trafficking and levels of wild-type (M) and mutant (Z) AAT in the liver. Initially, fusion protein constructs were synthesized using Gibson Assembly. To study the relevance to hepatic biology lentiviruses expressing these constructs were then produced and used to transduce the construct into HepG2, a liver cell line. After sorting and isolating a mEmerald+ve HepG2 cell population, the cells were characterized through Enzyme Linked Immunosorbent Assay, Fluorescence-activated cell sorting and Immunofluorescence image analyses. Fusion protein constructs represent an approach that will allow real-time quantification of AAT levels in live cells. Future directions will include the application of the constructs generated in this project to evaluate the effects of potential therapeutic compounds and studying the disease risk modifying genes using high throughput screening on levels of intracellular mutant ZAAT proteins.

Terminal uridylation of RNA 3ꞌ ends has recently been recognised as an important biological regulatory mechanism. The human terminal uridyl transferase ZCCHC11 was shown previously to uridylate and thereby regulate replication-dependent histone mRNAs and several miRNA precursors and mature miRNAs. Studies in cancer cell lines, mouse models and patient samples suggest that ZCCHC11 is a potential oncogene. However, very little is known about regulation of its expression and subcellular localisation. In this study, the regulation of the expression of ZCCHC11 was investigated, especially in relation to the roles played by the untranslated regions of its mRNA. The second aim of this study was to investigate the subcellular localisation of ZCCHC11, both under physiological conditions and under cellular stress. The results presented show that ZCCHC11 expression is regulated negatively through its 5ꞌ and 3ꞌUTRs. Upstream open reading frames in the 5ꞌUTR decrease its translation, while regulation through the 3ꞌUTR involves the combined activity of positive and negative factors. In this study it was shown that HuR positively regulates ZCCHC11 expression, whereas TTP contributes to its negative regulation. An homologous enzyme, ZCCHC6, negatively regulates ZCCHC11 expression. Regulation through the 3ꞌUTR of ZCCHC11 is particularly marked following S-phase arrest. ZCCHC11 expression increases under cellular stress and both UTRs take part in this regulation. Regulation under cellular stress also involves an alteration in sub-cellular localisation. ZCCHC11 is predominantly cytoplasmic in unstressed cells, but re-localises to p-bodies and stress granules under cellular stress. Mutating one of three zinc knuckle motifs within ZCCHC11 significantly decreased differential co-localisation with stress granule marker TIA-1 under oxidative stress. Taken together, the data presented provide insights into the post-transcriptional regulation of this post-transcriptional regulatory protein and its subcellular localisation, especially under cellular stress.

Persistent accumulation of free radicals in cells leads to oxidative stress, which plays a causative role in the induction and progression of various chronic diseases. Therapeutic focus has therefore shifted towards the use of antioxidants, with recent interest in those of plant origin. This study investigated radical scavenging and cytoprotective activities of phytochemicals (quercetin, curcumin, sulforaphane, rosmarinic acid, caffeic acid, danshensu (3,4-dihydroxyphenyllactic acid), ferulic acid and m-coumaric acid) against DPPH free radical in a non-cellular assay, and oxidative damage in hepatic (HepG2) and pancreatic (1.1B4) cells, elicited by an organic hydroperoxide (tert-butylhydroperoxide - tBHP) and a more physiologically relevant stressor (palmitate). Direct and indirect cytoprotective activities were assessed by neutral red viability assay after 5 h co-exposure and 20 h pre-exposure conditions, respectively. Radical scavenging activities of three well-known phytochemicals - quercetin, curcumin and sulforaphane - were initially validated against DPPH (non-cellular assay), where quercetin was shown to be more potent than curcumin; sulforaphane was without effect. With quercetin as positive control, radical scavenging activities of rosmarinic acid and three of its principal metabolites (caffeic acid, danshensu and ferulic acid) were comparable, while m-coumaric acid lacked antiradical activity against DPPH radical. Subsequently in HepG2 hepatoma cells, quercetin and curcumin were confirmed to possess direct and indirect cytoprotective acitivities against 0.5 mM tBHP while, sulforaphane only had indirect cytoprotective acitivities. Additionally, co-treatment of HepG2 cells with low concentrations of quercetin and curcumin (used together) exhibited direct cytoprotective activities against tBHP. However, direct cytoprotective potencies of rosmarinic acid and caffeic acid were less than quercetin. Similar pattern was observed for indirect cytoprotective activities; with danshensu, ferulic acid and m-coumaric acid lacking hepatoprotective activity in co-exposure and pre-exposure conditions. These results highlight the discrepancy between non-cellular and cellular antioxidant activities, which could be accounted to the poor lipophilicity profiles of rosmarinic acid and its principal metabolites. Cytotoxicity assay in 1.1B4 human pancreatic β-cells revealed that these cells were more vulnerable to tBHP-induced oxidative damage than HepG2 cells. An investigation of selected phytochemicals in 1.1B4 cells produced novel findings, with quercetin exhibiting direct and indirect cytoprotective activities against tBHP (0.125 mM and 0.5 mM). Curcumin and caffeic acid were also cytoprotective against 0.125mM tBHP but only exhibited direct cytoprotection against 0.5mM tBHP. Sulforaphane lacked both direct and indirect cytoprotective activities in 1.1B4 cells, exhibiting marked cytotoxic effects in both conditions. Further analysis in both HepG2 and 1.1B4 cells proved that indirect cytoprotective activities of selected phytochemicals were not dependent on pro-proliferative activities of quercetin, curcumin, caffeic acid and sulforaphane. Moreover, it was observed that high concentrations of curcumin and sulforaphane caused necrosis in both cell types, rather than apoptosis; caffeic acid also produced necrotic effect in 1.1B4 cells. Whilst prolonged exposure of HepG2 and 1.1B4 cells to high glucose concentrations failed to elicit any evidence of glucotoxicity, sodium palmitate caused concentration-dependent cytotoxicity after short-term (5 h) and long-term (20 h) exposure to both cell types. Overall, selected phytochemicals caused additive cytotoxicity in the presence of palmitate, although quercetin demonstrated direct cytoprotection alone in HepG2 cells. Using Western blot, curcumin, caffeic acid and sulforaphane did not upregulate NQO1, but 20 h exposure to 0.1 mM quercetin resulted in upregulation in HepG2 cells, amidst high basal levels of NQO1 in this cell type. However, both basal and inductive expression of NQO1 has not been observed in 1.1B4 cells. Thus, although rosmarinic acid, danshensu, caffeic acid and ferulic acid may possess good intrinsic antioxidant properties, their physicochemical properties may limit pharmacological activities at the cellular level. Moreover, the additive cytotoxicity resulting from treatment with selected phytochemicals and sodium palmitate highlights a discrepancy between mechanisms of cytotoxicities by tBHP and palmitate.

<p>Sun-damaged skin is a relevant target tissue for studying the development of skin cancer. The aim of the present study was to investigate the epidermal response to ultraviolet radiation (UVR) in human skin<i> in vivo</i> and <i>in vitro</i> and to explore the mutagenic effect of UVA. The prevalence and the genetic background of epidermal p53 clones were furthermore analysed.</p><p>Large inter- and intraindividual differences were observed in the epidermal response to UVR. Repair of UV-induced DNA damage appeared more efficient in chronically sun-exposed skin than in non-sun-exposed skin. Irradiation with UVA1 induced p53 mutations in keratinocytes. The pattern of mutations was indicative of oxidative damage, consistent with UVA acting as a mutagen.</p><p>The prevalence of p53 clones in skin adjacent to basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and benign melanocytic nevus in patients of different age groups was analysed. An age-dependent increase in number and size of p53 clones was observed. Epidermal p53 clones were significantly larger and more frequent in skin adjancent to SCC than adjacent to BCC or melanocytic nevus. Mutation analysis of the entire coding region of the p53 gene showed that 57% of p53 clones in normal skin surrounding BCC and SCC have a mutated p53 gene. </p><p>In conclusion, this study has increased our knowledge of the effects of UVR in chronically sun-exposed skin. The mutation spectra observed in epidermal p53 clones resembled that of non-melanoma skin cancer. The increased prevalence of epidermal p53 clones adjacent to SCC indicates that epidermal p53 clones may represent a step prior to actinic keratosis in the development of SCC.</p>

Sun-damaged skin is a relevant target tissue for studying the development of skin cancer. The aim of the present study was to investigate the epidermal response to ultraviolet radiation (UVR) in human skin in vivo and in vitro and to explore the mutagenic effect of UVA. The prevalence and the genetic background of epidermal p53 clones were furthermore analysed. Large inter- and intraindividual differences were observed in the epidermal response to UVR. Repair of UV-induced DNA damage appeared more efficient in chronically sun-exposed skin than in non-sun-exposed skin. Irradiation with UVA1 induced p53 mutations in keratinocytes. The pattern of mutations was indicative of oxidative damage, consistent with UVA acting as a mutagen. The prevalence of p53 clones in skin adjacent to basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and benign melanocytic nevus in patients of different age groups was analysed. An age-dependent increase in number and size of p53 clones was observed. Epidermal p53 clones were significantly larger and more frequent in skin adjancent to SCC than adjacent to BCC or melanocytic nevus. Mutation analysis of the entire coding region of the p53 gene showed that 57% of p53 clones in normal skin surrounding BCC and SCC have a mutated p53 gene. In conclusion, this study has increased our knowledge of the effects of UVR in chronically sun-exposed skin. The mutation spectra observed in epidermal p53 clones resembled that of non-melanoma skin cancer. The increased prevalence of epidermal p53 clones adjacent to SCC indicates that epidermal p53 clones may represent a step prior to actinic keratosis in the development of SCC.

The purpose of this study was to gather normative data regarding the phonological system of bilingual Creole-English children ages three and five and to compare performance to norms for English speaking children. The forty participants lived in Miami and represented low socio-economic groups. Participants were assessed using the Goldman-Fristoe Test of Articulation-2 and a Haitian Creole Picture Naming Assessment. The results indicated that the percentage of correct phonemes in Creole (M=91.6) were not significantly different when compared to the correct production of the same phonemes in English (M=92.8). Further analysis revealed that the accuracy of all phonemes was higher for the five-year (M= 90.8) as compared to the three-year-olds (M= 85) in Creole. In English, the five-year-olds performed better than the three-year-olds participants. These findings revealed patterns of phonological development in bilingual Creole/English Children similar to patterns reported in other bilingual children. This information is essential in the evaluation and treatment of this population.

In the Northeast, powdery mildew (PM), caused by Uncinula necator (Schewein.) Burrill is one of the most important grape diseases in terms of economic loss. It has been established that cultural practices, including proper sanitation, are the first step in preventing disease, and fungicide sprays are regularly applied to manage the disease. Currently, fungicides that successfully control PM have a strong potential to develop pathogen resistance, and alternatives with low risk of initiating resistance are not as effective in disease control. Our approach to this emerging resistance dilemma is to combine a systemic acquired resistance inducer (salicylic acid and potassium phosphate) with a topical fungicide, potassium bicarbonate. To determine each treatment’s level of efficacy, multiple aspects of infection and defense were quantified and qualified, including germination rate, lethality, lignin formation, callose formation, and vine and leaf growth. The first trial showed potassium bicarbonate and the standard fungicide (Pristine) inhibited the most germination and was most lethal against PM conidia. Potassium phosphate had little effect on germination and conidia death, and when combined with bicarbonate there was no different than the water control. In the second trial, the biorational mixture was able to reduce the level of powdery mildew infection significantly more than the other compounds, including the commercial standard. The salicylic acid and potassium bicarbonate mix may be successful enough to use in the vineyard to determine if the compound can tolerate field conditions with the same level of efficacy.