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Chemical-induced nephrotoxicity is a major cause of acute kidney injury. My dissertation reveals that all-trans-retinoic acid (ATRA) affords cytoprotection against renal cell injury. Pretreatment with ATRA (25 μM, 24 hr) affords selective cytoprotection against p-aminophenol (PAP), iodoacetamide (IDAM), and 2-(glutathion-S-yl)-hydroquinone-induced necrosis. In contrast, pretreatment of cells with ATRA provides no protection against cisplatin-induced apoptosis. Inhibition of protein synthesis blunts ATRA-mediated cytoprotection, suggesting that critical cell survival signaling pathways are activated prior to toxicant exposure. Oxidative stress is a major contributor to cellular damage. To investigate the mechanism(s) by which ATRA affords cytoprotection, we determined its effects on ROS generation using a DCFDA assay. ATRA did not alter PAP or MGHQ-induced ROS levels. Moreover, ATRA had no effect on GSH levels nor Nrf2 expression, suggesting that other cytoprotective mechanisms are engaged by ATRA. Elevated ROS disrupt endoplasmic reticulum protein folding guided by the molecular chaperone Grp78. During ATRA-mediated pretreatment, the ER stress proteins Grp78 and p-eIF2α were induced (2-fold) in a time-dependent manner (24 and 4 hr respectively). In addition to influencing organelle stress proteins in the ER, ATRA rapidly (15 min) induced levels of the cellular stress kinases p-ERK and p-AKT with maximum levels achieved at 30 min. Moreover, induction of these stress kinases was observed at concentrations of ATRA (10 and 25 μM) required for cytoprotection. Inhibition of p-ERK with PD98059 reduced the ability of ATRA to provide protection against PAP toxicity, implying a role for p-ERK and downstream target genes in the protective effects of ATRA. Gene ontology analysis of a microarray experiment of cells treated with ATRA revealed that ATRA rapidly (0.5, 1 hr) induced growth factors and genes involved in cell proliferation, with subsequent (4, 8, 12 hr) induction of genes involved in ribosome biogenesis, DNA replication and repair, and cell cycle regulation. Complementary data from a cell stress protein array and western blot analyses indicated that ATRA induced HIF1α 3-fold at 8 hr. Furthermore, the microarray data indicated the HIF1α target gene BHLHE40 (which encodes a basic-helix-loop-helix protein involved in cell differentiation) was increased 3-fold. As ATRA induced genes that were associated with cell proliferation, related assays were employed. ATRA had a small effect on cell cycle distribution demonstrated by an increase in the population of cells in the S and G2 phases between 8 and 24 hr. In addition, ATRA markedly increased total DNA content and cell number at 24 hr suggesting that mitogenic/proliferative effects contribute to ATRA cytoprotection. The present studies indicate that a signaling cascade of proteins downstream of p-ERK associated with mitogenesis work cooperatively to afford ATRA protection against renal cell injury. Understanding the mechanism of ATRA-mediated cytoprotection will provide insights into the development of novel therapeutic strategies for renal pathological conditions.

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The thesis reports the use of electrospun nanofibres as a platform for the development of colorimetric probes. Three colorimetric probes in the form of electrospun nanofibre test strips were developed for the selective detection of ascorbic acid and dopamine because they are crucial biomolecules for physiological processes in human metabolism and usually coexist in biological samples. The simultaneous detection of the biomolecules is very important as their abnormal concentration levels would lead to diseases such as Parkinson's and schizophrenia. Different methods of incorporating detector agents into the nanofibre were exploited for the detection of the biomolecules. The methods included physical incorporation of nanoparticles, covalent bonding of ligand/dyes through surface modification of the fibres. The first colorimetric test strip for ascorbic acid was based on copper-gold alloy nanoparticles prepared in-situ and hosted in nylon6. The test strip showed selectivity in detecting ascorbic acid in the pH range 2 – 7. The suitability of fibres in hosting copper-gold alloy nanoparticles for the colorimetric detection of ascorbic acid was investigated using nylon6, poly(vinyl benzyl chloride)-styrene and cellulose acetate based test strips. All the test strips exhibited leaching and the nylon6 based test strip was found to be thermally stable up to 60 ˚C. The colorimetric performance of the test strips was maintained and neither was colour decay exhibited after 10 months of storage in a shelf. The test strip achieved an eye-ball limit of detection of 1.76 x10-2 mg L-1 and its suitability was demonstrated by the determination of ascorbic acid in fruit juices, urine, serum, and vitamin C tablets. The second colorimetric test strip for ascorbic acid and dopamine employed prussian blue synthesised in-situ in nylon6. Ascorbic acid turned the deep blue test strip to light blue at pH 3, and a faded navy blue colour at a pH range of 6 - 7 while dopamine changed the strip to purple at the same pH range. The versatility of the test strip was demonstrated by detecting ascorbic acid in commercial fruit juices as well as by detecting ascorbic acid as well as dopamine in fortified urine. The eye-ball detection limit of the Prussian blue test strip for ascorbic acid and dopamine was 17.6 mg L-1 and 18.9 mg L-1, respectively. The third method involved a covalent approach, where poly(vinylbenzyl chloride) nanofibers were post functionalised with 2-(2′-pyridyl)-imidazole and iron(III) for the selective detection of ascorbic acid and dopamine. The eye-ball detection limit for ascorbic acid and dopamine was 17.6 mg L-1 and 18.9 mg L-1, respectively. The test strip was selective for dopamine, but the detection of ascorbic acid suffered from interference by glutathione. The application of the test strips was nevertheless demonstrated by the detection of ascorbic acid in fruit juices and dopamine in fortified urine. The developed test strips employing the three approaches were applied without sample pre-treatment and use of supporting equipment.

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The fortification of skim milk powder with vitamin A has been found to be ineffective with available methods. The purpose of this study was to assess new methods and materials for their effectiveness in providing stability to vitamin A in fortified skim milk powder. The first phase of the project involved trials in Pilot Plants which evaluated 14 different treatments for vitamin A stability during storage periods of twelve months at 22°C and six months at 37°C. The second and third phases of the experiment consisted of primary and instant powder trials in commercial plants using the most stable methods from the Pilot Plant trials. In the latter phases of the project, eight treatments were tested for primary powder and ten for instant type of powder. Results show that levels of antioxidants were important to control the oxidative degradation of vitamin A in the milk powder. The vitamin A concentrate containing BHA (5 mg), BHT (55 mg) and ⍺-tocopherol (12.5 mg) antioxidants produced the best results for primary powder. Ascorbyl palmitate-⍺-tocopherol combination of antioxidants was found to be more effective than the BHA-BHT-⍺-tocopherol blend for instant powder. The level of hydrogenated coconut oil (HCO) used as the vitamin carrier was also found to be important for stability, 0.2% being slightly better than 0.1% in primary powder. A 12% emulsion injected at such a rate as to add 0.027% oil in milk solids was the best treatment of the instant powder trials. Hay-like flavour in reconstituted skim milk powder was correlated with vitamin A destruction. / Land and Food Systems, Faculty of / Graduate