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Neuroblastoma is the most common malignant disease of infancy, and amplification of the MYCN oncogene is closely associated with poor prognosis. Recently, expression of MYCN was shown to be inversely correlated with aryl hydrocarbon receptor (AHR) expression in neuroblastoma, and overexpression of AHR downregulated MYCN expression, promoting cell differentiation. Therefore, we further investigated the potential of AHR to serve as a prognostic indicator or a therapeutic target in neuroblastoma. First, the clinical significance of AHR in neuroblastoma was examined. Positive AHR immunostaining strongly correlated with differentiated histology of neuroblastoma and predicted better survival for patients. The mouse xenograft model showed that overexpression of AHR significantly suppressed neuroblastoma tumor growth. In addition, activation of AHR by the endogenous ligand kynurenine inhibited cell proliferation and promoted cell differentiation in vitro and in vivo. kynurenine treatment also upregulated the expression of KISS1, a tumor metastasis suppressor, and attenuated metastasis in the xenograft model. Finally, analysis of KISS1 levels in neuroblastoma patient tumors using the R2: Genomics Analysis and Visualization Platform revealed that KISS1 expression positively correlated with AHR, and high KISS1 expression predicted better survival for patients. In conclusion, our results indicate that AHR is a novel prognostic biomarker for neuroblastoma, and that overexpression or activation of AHR offers a new therapeutic possibility for patients with neuroblastoma. Significance: These findings show that AHR may function as a tumor suppressor in childhood neuroblastoma, potentially influencing the aetiologic and therapeutic targeting of the disease.

Aryl hydrocarbon receptor (AHR) signaling has been suggested to play roles in various physiological functions independent of its xenobiotic activity, including cell cycle regulation, immune response, and embryonic development. Several endogenous ligands were also identified by high-throughput screening techniques. However, the mechanism by which these molecules mediate AHR signaling in certain functions is still elusive. In this study, we investigated the possible pathway through which AHR and its endogenous ligands regulate neural development. We first identified two neuroactive steroids, 3α,5α-tetrahydrocorticosterone and 3α,5β-tetrahydrocorticosterone (5α- and 5β-THB), as novel AHR endogenous ligands through the use of an ultrasensitive dioxin-like compound bioassay and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS). We then treated zebrafish embryos with 5α- and 5β-THB, which enhance the expression of neurogenesis marker HuC. Furthermore, 5α- and 5β-THB both enhanced the expression of myelinating glial cell markers, sex determining region Y-box 10 (Sox10), and myelin-associated proteins myelin basic protein (Mbp) and improved the mobility of zebrafish larvae via the Ahr2 pathway. These results indicated that AHR mediates zebrafish neurogenesis and gliogenesis, especially the differentiation of oligodendrocyte or Schwann cells. Additionally, we showed that these molecules may induce neuroblastoma (NB) cell differentiation suggesting therapeutic potential of 5α- and 5β-THB in NB treatment. In summary, our results reveal that 5α- and 5β-THB are endogenous ligands of AHR and have therapeutic potential for NB treatment. By the interaction with THB, AHR signaling regulates various aspects of neural development.

The electrochemical behavior of selected phenolic benzotriazoles (BZTs), namely 2‐(2H‐benzotriazol‐2‐yl)‐4,6‐bis(1‐methyl‐1‐phenylethyl)phenol and 2,4‐di‐tert‐butyl‐6‐(5‐chlorobenzotriazol‐2‐yl)phenol (commercial names UV234 and UV327, respectively) were examined with cyclic voltammetry (CV) and controlled potential electrolysis (CPE) in acetonitrile and dichloromethane solutions. CV indicated that both phenolic BZTs undergo a chemically irreversible oxidation process at approximately Ep°x=+1.0 V vs. Fc/Fc+ (where Ep°x is the anodic peak potential and Fc=ferrocene) to form compounds that cannot be electrochemically converted back to the starting material on the voltammetric timescale. In basic conditions, cyclic voltammetry experiments indicated that the corresponding phenolates (prepared by reacting the phenols with equiv. mols of n‐Bu4NOH) were oxidized at Ep°x∼−0.2 V vs. Fc/Fc+ via a one‐electron diffusion controlled process with anodic (ip°x) to cathodic (ipred) peak current ratios (ip°x/ipred)≫1, suggesting that the produced phenoxyl radicals decomposed rapidly via a chemical step. However, electron paramagnetic resonance (EPR) experiments performed on the bulk electrolyzed solutions of the phenolates after one‐electron bulk oxidation indicated long lifetimes of the UV234. and UV327. phenoxyl radicals. Therefore, the long timescale CPE and spectroscopic (UV‐vis and EPR) studies provided good evidence of a reversible dimerization mechanism between the phenoxyl radicals, which explained the apparent discrepancy with the short timescale CV experiments. MOE (Min. of Education, S’pore)

Electrochemical oxidation studies were performed in aprotic organic solvents on two types of artificial hydroxyl containing emerging contaminants; namely bisphenol A and phenolic benzotriazoles (UV234 and UV327) as well as on the natural hydroxyl containing vitamin D compounds (vitamins D3 and D2). The techniques utilized to study the compounds included CV, CPE as well as combining spectroscopic methods such as UV-vis and EPR with electrochemistry and chemical oxidation. All 3 types of hydroxyl containing compounds were found to undergo chemically irreversible oxidation processes on the short voltammetric timescale, indicating that the initial heterogeneous electron transfer steps produced oxidized intermediates that were not long-lived. Further investigations enabled the determination of the exact electron transfer and chemical steps involved in the oxidation process. In this thesis, the oxidative mechanism for the compounds are proposed along with the identity of the intermediate species and final oxidized products which can provide useful insights into the reactions that can occur in lipophilic environments during oxidative metabolism as well as highlight the possible role of the intermediate species as reactive metabolites in oxidative degradation pathways. The electrochemical oxidation of the two artificial phenolic classes of compounds occurred at the phenolic moiety in a mechanism that is similar to phenols in general, while the electrochemical oxidation of the natural hydroxyl containing vitamin D most likely occurred at the triene moiety. Bisphenol A is chemically oxidized via a fourelectron/two-proton process (‒4e‒/‒2H+) in a 2 × ECE mechanism to form a relatively unstable dication which reacts quickly in the presence of water in acetonitrile. UV234 andUV327 displayed similar behavior and can be electrochemically oxidized via a twoelectron/one-protonprocess (‒2e‒/‒1H+) in an ECE mechanism under neutral and acidic conditions. In basic conditions, the phenolic benzotriazole is electrochemically oxidized in a CECdim process to form neutral benzotriazolyl-substituted phenoxyl radicals that immediately undergo a reversible dimerization (Cdim-step) to form a dimer which exists in equilibrium with the radicals. Vitamin D is electrochemically oxidized in an ECCE mechanism initially via one-electron to form a cation radical, which reacts homogeneously in two chemical steps [where one chemical step is fast (< 1 s) and one is relatively slow (> 1 s)]. On the longer electrolysis timescale, the oxidized product undergoes a second one-electron transfer at a less positive potential. Surface coverage of drop cast nanomaterial coatings formed on electrodes were assessed after investigations at a MWCNT-coated GC electrode suggested that electrode modification with nanomaterials was not useful in resolving the electrode fouling problem caused by adsorption of oxidized products during oxidation. It was demonstrated that the drop casting procedure is unable to prevent solution phase ions from diffusing through the nanomaterial coating to the base electrode and the contribution from the base electrode on the current-potential trace must be taken into account to fully understand the current response. DOCTOR OF PHILOSOPHY (SPMS)

Burning of joss paper and incense is still a very common traditional custom in countries with a majority Chinese population. The Hungry Ghost Festival which is celebrated in the 7 month of the Chinese calendar is one of the events where joss paper and incense are burned as offerings. This study investigates the impact of the Ghost Month Festival (open burning event) on air quality by analysis of the chemical composition of particulate matter (PM) and rainwater samples collected during this event, compared with data collected throughout the year, as well as bottom ash samples from burning the original joss paper and incense. The results showed that the change in the chemical composition of the rainwater and PM2.5 (PM ≤ 2.5 μm) atmospheric samples could be correlated directly with burning events during this festival, with many elements increasing between 18% and 60% during August and September compared to the yearly mean concentrations. The order of percentage increase in elemental composition (in rain water and PM2.5) during the Hungry Ghost Festival is as follows: Zn > Ca > K > Mg > Fe > Al > Na ∼ Mn ∼ Ti ∼ V > Cu > As > Ni > Co > Cd > Cr > Pb. The chemical composition of the original source materials (joss paper and incense for combustion) and their associated bottom ash were analysed to explain the impact of burning on air quality. MOE (Min. of Education, S’pore) Published version

The electrochemical behavior of vitamins D 2 and D 3 were examined by performing cyclic voltammetry (CV), rotating disk electrode voltammetry, controlled potential electrolysis and chemical oxidation in aprotic organic solvents. Both vitamins were electrochemically oxidized in dichloromethane and acetonitrile ( E p ox ∼ +0.8 vs. (Fc/Fc + )/V, where E p ox is the anodic peak potential and Fc = ferrocene) via a one-electron chemically irreversible process on the short voltammetric time scale (≤ seconds). Varying the scan rate (0.1 V s −1 to 20 V s −1 ) and temperature (233 K to 293 K) did not strongly affect the voltammetric response recorded on platinum and glassy carbon electrode surfaces with the oxidation process remaining chemically irreversible over the range of scan rates and temperatures tested, indicating that the initially formed cation radical was not long-lived. Repetitive CV experiments indicated that the oxidized product partially adsorbed onto the electrode surface, resulting in diminishing peak currents with multiple scans. Bulk controlled potential electrolysis of the vitamin D compounds performed by alternating several cycles of oxidative electrolysis and reductive pulsed stripping proved to be effective in stripping the adsorbed species off the electrode surfaces. Longer time scale bulk electrolysis experiments led to the detection of a new oxidation peak appearing at less positive potentials as the electrolysis progressed, suggesting that the compounds underwent oxidation on long time scales (minutes to hours) via a two electron process. The vitamins were most likely initially oxidized via one-electron (E-step) to form a cation radical, which reacts homogeneously in two chemical steps (C-steps) where one chemical step is fast ( 1 s). On the electrolysis timescale, the oxidized product then undergoes a second electron transfer (E) at less positive potentials, so the overall mechanism is an ECCE process. Chemical oxidation of vitamin D 3 with 2 mol equiv of the one-electron oxidant, NO + SbF 6 − in acetonitrile/dichloromethane 1:4 ratio (v/v) resulted in the complete oxidation of the starting material in an overall two-electron process, with NMR analysis of the reaction mixture indicating that the triene moiety is absent from the products.

The electrochemical behavior of bisphenol A (BPA) was examined using cyclic voltammetry, bulk electrolysis and chemical oxidation in aprotic organic solvents. It was found that BPA undergoes a chemically irreversible voltammetric oxidation process to form compounds that cannot be electrochemically converted back to the starting materials on the voltammetric timescale. To overcome the effects of electrode fouling during controlled potential electrolysis experiments, NO + was used as a one-electron chemical oxidant. A new product, hydroxylated bisdienone was isolated from the chemical oxidation of BPA with 4 mol equiv of NO + SbF 6 − in low water content CH 3 CN. The structure of the cation intermediate species was deduced and it was proposed that BPA is oxidized in a four-electron/two-proton process to form a relatively unstable dication which reacts quickly in the presence of water in acetonitrile (in a mechanism that is similar to phenols in general). However, as the water content of the solvent increased it was found that the chemical oxidation mechanism produced a nitration product in high yield. The findings from this study provide useful insights into the reactions that can occur during oxidative metabolism of BPA and highlight the possibility of the role of a bisdienone cation as a reactive metabolite in biological systems.

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