Yes / Platinum drugs are widely used for cancer treatment. Other precious metals are promising, but their clinical progress depends on achieving different mechanisms of action to overcome Pt-resistance. Here, we evaluate 13 organo-Os complexes: 16-electron sulfonyl-diamine catalysts [(η6-arene)Os(N,N′)], and 18-electron phenylazopyridine complexes [(η6-arene)Os(N,N’)Cl/I]+ (arene = p-cymene, biphenyl, or terphenyl). Their antiproliferative activity does not depend on p21 or p53 status, unlike cisplatin, and their selective potency toward cancer cells involves the generation of reactive oxygen species. Evidence of such a mechanism of action has been found both in vitro and in vivo. This work appears to provide the first study of osmium complexes in the zebrafish model, which has been shown to closely model toxicity in humans. A fluorescent osmium complex, derived from a lead compound, was employed to confirm internalization of the complex, visualize in vivo distribution, and confirm colocalization with reactive oxygen species generated in zebrafish. / Wellcome Trust (grant no. 107691/Z/15/Z), ERC (grant nos. 247450, 324594), Science City (AWM and ERDF), WCPRS and Bruker Daltonics (Studentship for JPCC), Mike and Enfys Bagguley, and EPSRC (Studentship for HEB, and grant no. EP/F034210/1)

Arsenical-induced carcinogenesis in human bladder has been established through epidemiological evidence, but unfortunately, no mode of action had been determined for this phenomenon. UROtsa cells, a normal, immortalized cell culture model of human urothelium does not form tumors when injected into immuno-compromised mice nor does it have anchorage-independent growth. UROtsa cells were shown to be malignantly transformed following low-level exposure to both arsenite [As(III)] and its more toxic metabolite, monomethylarsonous acid [MMA(III)] providing additional models for studying arsenical-induced carcinogenesis of the bladder. These transformed cell lines allow researchers the ability to investigate the process of urothelial tumorigenesis at multiple time points of arsenical exposure. In the studies discussed here in, environmentally relevant levels of As(III) and MMA(III) were chosen. UROtsa cells were exposed to As(III) and MMA(III) both acutely and chronically to begin investigations into signaling pathway alterations that can lead to carcinogenesis in the human bladder upon exposure to arsenicals. In acute studies, it was shown that As(III) and MMA(III) generate oxidative stress response in UROtsa at low, environmentally relevant levels. The ROS generated by MMA(III) led to an increased 8-oxo-dG formation after 30 min, supporting the importance of MMA(III) in damage caused in the bladder by arsenicals. Because ROS has been linked to MAPK signaling, it was shown that 50 nM MMA(III) and 1 µM As(III) induce MAPK signaling following acute exposures and this increase is dependent on the production of ROS.Next, it was necessary to begin to look at changes that occur during transformation of UROtsa with MMA(III). Chronic exposure to 50 nM MMA(III) constitutively increases the amounts of EGFR, activated Ras, and COX-2 protein in MSC cells. Chronic upregulation of COX-2 in MSC52 cells is due to increased levels of ROS. Phenotypic changes seen in MSC52 cells (hyperproliferation and anchorage independent growth) are dependent on the secondary generation of excess ROS in MSC52 cells. These data clearly present evidence supporting a role for ROS in both acute and chronic toxicities associated with low-level arsenical exposure, and gives evidence that ROS are important in cellular transformation following MMA(III) exposure.

Parkinson’s disease (PD) is the most common movement neurodegenerative disease affecting approximately 1% of the population over 60. Though originally thought to be sporadic in nature, a genetic component is increasingly being linked to the disease. Of these genes, mutations in DJ-1 (PARK7) cause early onset autosomal recessive PD. Initial workup of the DJ-1 protein has suggested that it may act in the cell by combatting oxidative stress though the mechanism by which it does so is unclear. Thus, though much work has attempted to elucidate a function at the biochemical, cellular and organismal level, the overt physiological role of DJ-1 remains elusive. In this dissertation, we explore the mechanisms through which DJ-1 confers neuroprotection, particularly in the case of oxidative stress insult. We demonstrate that DJ-1 acts through the pro-survival protein AKT to accomplish its neuroprotective function. Moreover, we note that DJ-1 likely serves its role as an antioxidant through the NRF2 master antioxidant regulator pathway a pathway that is, itself, likely to be regulated by AKT. Together, our results demonstrate that neuroprotection by DJ-1 is done through a signaling pathway involving both AKT and NRF2 and that disruption of the former in PD likely results in abolishing this signaling pathway. Finally, to generate a better animal model of PD, we demonstrate that backcrossing DJ-1 null mice - which originally did not demonstrate any gross histopathological or behavioral phenotypes – display unilateral dopaminergic degeneration that progresses to bilateral degeneration with aging, a feature reminiscent of classical PD progression. Collectively, this thesis takes a two-sided approach to address the biochemical and physiological functions of DJ-1 within the cell and the mouse in hopes of elucidating mechanisms of neuronal death to devise better translational therapies.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS), saccharide (i.e. monosaccharide, disaccharide and polysaccharide), are continuously generated, transformed and consumed in the living systems. As a consequence of their significant value towards human health in aerobic life, it is very important and has drawn much attention in the chemical and biological sensing of the species. It is our long-standing interest in the recognition of monosaccharide (e.g. glucose) through exploration of various boronate-based fluorescence probes, thus, based on the previous work, we started on the design, synthesis and evaluation of novel fluorescent chemosensors for breakthrough discoveries in the detection of saccharide and ROS selectively and specifically, which are made up of different receptors and diverse singaling fluorophores, e.g. anthracene, coumarin, fluorescein, naphthalimine. Firstly, “integrated” and “insulated” boronate-based fluorescent probes (2-naphthylboronic acid and N-Methyl-o-(aminomethyl)phenylboronic acid) have been evaluated for the detection of hydrogen peroxide in the presence of saccharides (i.e. D-fructose). In the presence of D-fructose the initial fluorescence intensity of the “insulated” system is much higher and produces a blue visible fluorescence. Based on the experimental observation above in the boronate-based systems (i.e. B-N bond protection), a new water-soluble boronate-based fluorescent probe was designed and evaluated for the detection of peroxynitrite (much stronger oxidant) in the presence of D-fructose. The enhanced fluorescence of probe when bound with D-fructose was switched off in the presence of peroxynitrite. While, other reactive oxygen/nitrogen species led to only slight fluorescence decreases due to protection by the internal N-B interaction. The interaction of probe with D-fructose not only strengthens the fluorescence signal, but also protects the boronic acid to oxidation by other ROS/RNS. Therefore, under conditions generating various ROS/RNS, the boronate-based sugar complex preferentially reacts with peroxynitrite (ONOO−). The sensor displays good “on-off” response towards peroxynitrite both in RAW 264.7 cells and HeLa cells. A new ICT (internal charge transfer) sensing system was developed for the detection of hydrogen peroxide and peroxynitrite. The probe displayed an enhanced fluorescence change when bound with D-fructose due to the prolonged N-B distance. The fluorescence intensity of the probe dropped down both in the detection of H2O2 and ONOO− which was attributed to the oxidation of arylboronic acid even though in the presence of D-fructose. Using the self-assembly of aromatic boronic acids with Alizarin Red S (ARS), we developed a new chemo/biosensor for the selective detection of peroxynitrite. Phenylboronic acid, benzoboroxole and 2-(N, N-dimethylaminomethyl) phenylboronic acid were employed to bind with ARS to form the complex probes. In particular the ARS-NBA system with a high binding affinity can preferably react with peroxynitrite over hydrogen peroxide and hypochlorite due to the protection of the boron via the solvent-insertion B-N interaction. Our simple system produces a visible naked-eye colorimetric change and on-off fluorescence response towards peroxynitrite. By coupling a chemical reaction that leads to an indicator displacement, we have developed a new sensing strategy, referred to herein as RIA (Reaction-based Indicator displacement Assay). Next, we developed a novel class of simple materials for sensing monosaccharides by the functionalization of graphene oxide (GO) with boronate-based fluorescence probes. The composite materials were characterized by atomic force microscopy, Raman spectroscopy, and UV-vis/fluorescence spectroscopy. The strong fluorescence of the fluorescence probes is quenched in the presence of GO through fluorescence resonance energy transfer (FRET). The BA@GO composite sensors formed provide a useful platform for fluorogenic detection of monosaccharides based on the strong affinity between the boronic acid receptor and monosaccharides. The BA@GO composite sensor displayed a “turn-on” fluorescence response with a good linear relationship towards fructose over a range of other saccharides. Next, new water-soluble copper (II) complex fluorescence probes were developed and evaluated for the detection of nitric oxide and nitroxyl in a physiological condition. A significant fluorescence “off-on” response displayed by using the copper (II) complex for the detection of NO and HNO (Na2N2O3 as a donor). Under pathological conditions generating various ROS/RNS, the copper (II) complex fluorescent probe preferentially reacts with NO/HNO over other reactive oxygen species. The dual-analyte recognitions of the simple, sensitive probe were further applied in living cell for the exogenous NO/HNO. In the following work, we synthesised a phosphorous-based compound for the detection of HNO which derived from Angeli’s salt in a biological condition. Significantly, it displayed a high sensitivity and selectivity toward HNO over other various ROS species, especially NO since they have a similar chemical property. The underlying mechanism was attributed to the cleavage of C-O bond induced by Staudinger Ligation.

The migration of keratinocytes across wound beds is a key step in dermal wound healing. In aged human skin, wound healing rates decrease, and reactive oxygen species damage accumulation increases, but it is unclear if these factors relate specifically to migration of human skin keratinocytes (HSKs). In this study, two concentrations of oxygen (4% and 21%) were used to model low and high oxidative stress to produce varying levels of reactive oxygen species. When migration of HSKs from young and old primary skin were compared by scratch assay, those from old skin migrated faster in high oxygen tension than did young HSKs, which was an opposite trend from that seen in young skin. An intense increase in reactive oxygen species at margins immediately after scratching was seen in both young and old HSKs, but reactive oxygen species disappeared from young skin at 21% oxygen most quickly. These cells also had the slowest migration. These findings suggest that old and young keratinocytes respond differently to oxidative stress, and that migration of keratinocytes--a key step in re-epithelialization of wounds, is effected by the efficacy of reactive oxygen species removal.

It has been shown that reactive oxygen species (ROS) are released in response to articular cartilage injury. The excessive release of ROS has been shown to be mitochondrial in nature and leads to chondrocyte death which in turn can lead to post-traumatic osteoarthritis (PTOA). Evidence suggests that mitochondria are attached to chondrocytes' cytoskeleton. Upon tissue level deformation, it is believed that mitochondria also experience deformation in response to cytoskeletal strain, releasing ROS. Therefore, it was hypothesized that inhibition of chondrocytes' cytoskeleton would prevent mitochondrial distortion rendering them unable to release ROS in response to the applied strain, saving chondrocytes. Osteochondral explants treated with cytoskeletal inhibitors were found to reduce mitochondrial ROS production directly after impact and increase chondrocyte viability 24 hours after impact. The release of mitochondrial ROS is an important mechanotranduction pathway in the initiation of PTOA.

Polychlorniated Biphenyls (PCBs), a group of 209 individual congeners, are ubiquitous environmental pollutants and classified as probable human carcinogens. Hallmarks of aging and carcinogenesis are changes in telomerase activity and telomere length. I hypothesize that PCBs modulate telomerase activity and telomeres via interference in gene regulation and generation of reactive oxygen species (ROS) resulting in the dysregulation of cell growth. To explore this possibility, I exposed human skin keratinocytes (HaCaT) to a synthetic airborne PCB mixture (CAM) and individual congeners, i.e. PCB28, PCB52, PCB126 and PCB153. To mimic the chronic human exposure to PCBs and the slow process of carcinogenesis, a long term exposure period of 48 days and beyond was employed. All PCB congeners and CAM reduced telomerase activity, telomere length and cell growth. Among all PCBs, PCB126 had the most pronounced effect with reduction in telomerase activity, telomere length, hTERT and hTR gene expression and cell growth, while increasing TRF1 & TRF2 gene expression. PCB126 elicited an increase in CYP1A1 mRNA, CYP1A1 activity, DHE and DCFH oxidation levels from days 6 to 48, suggesting that increased ROS might be a causative factor for the reduction in telomerase activity and telomere length. However, transduction with hTERT and hTR subunits partly rescued telomerase activity, while treatment with PEG-catalase did not rescue telomerase activity suggesting that telomerase subunits play an important role on PCB126 induced effects on telomerase activity and telomere length. Since cells with shortened telomeres may escape crisis through telomerase reactivation, PCB126 treatment was continued until day 90. A change in growth behavior was observed from day 54 to 90, with cells recovering the proliferation rate, and increasing c-Myc, hTERT, and hTR gene expression level, re-activating telomerase activity and re-elongating telomere length. TRF1 & 2 gene expression started to decrease after day 66. From day 78, no increase in CYP1A1mRNA and its activity as well as CYP1B1, ALDH3A1, UGT1A1 and AhRRmRNA was observed suggesting that the AhR response pathway may have been altered. This study shows for the first time that PCBs initially reduce telomerase activity, telomere length, and cell growth, and can later lead to telomerase re-activation, telomere lengthening and increased cell growth with modulation of the AhR receptor pathway. This observation has broad implications for chronic PCB exposure scenarios.

Telomere attrition is a natural process that occurs due to inadequate telomere maintenance. Once at a critically short threshold, telomeres signal the cell to cease division and enter a cell fate termed senescence. Telomeres can be elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Mutations in the telomerase complex or telomere-related genes give rise to the premature aging disorder Dyskeratosis Congenita (DC). DC provides a unique model system to study human aging in relation to telomerase insufficiency and the subsequent accelerated telomere attrition. In this thesis, skin fibroblasts as well as keratinocytes and T-cells were analyzed from patients with Autosomal Dominant Dyskeratosis Congenita (AD DC) caused by a single allele mutation in the telomerase RNA component (TERC) that leads to telomerase haploinsufficiency. These cells were determined to have a severe proliferative defect and extremely short telomeres. It is demonstrated that the short telomeres in AD DC cells initiate a DNA damage response transduced by the p53/p21WAF/CIP pathway which mediate an elevation in steady-state levels of mitochondrially-derived superoxide and oxidative stress. Exogenous expression of the catalytic reverse transcriptase component of telomerase (TERT) activated telomerase in DC fibroblasts but resulted in reduced activity (~50% compared to control fibroblasts); however telomeres were successfully maintained, albeit at a short length. Simultaneous expression of both TERT and TERC led to robust telomerase activity and elongation of telomeres, indicating that TERC haploinsufficiency is a rate-limiting step in telomere maintenance in DC cells. Reconstitution of telomerase activity in AD DC cells ameliorated the proliferative defects, reduced the p53/p21WAF/CIP response and decreased oxidative stress. Increased superoxide and slow proliferation found in DC cells could also be mitigated by inhibiting p21WAF/CIP or by decreasing the oxygen tension to which the cells are exposed. Together, these results support the hypothesis that the insufficient telomerase leads to critically short telomeres which signal the activation of p21WAF/CIP, leading to increased steady-state levels of mitochondrial superoxide and metabolic oxidative stress as a means to engage senescence. These studies provide insight into mechanisms whereby shortened telomeres lead to premature aging in a humans and point to potential strategies to reduce the effects of tissue dysfunction in DC patients.

Friedreich’s ataxia (FRDA) is an autosomal recessive inherited disorder affecting approximately 1 every 40,000 individuals in Western Europe, is characterized by progressive gait and limb ataxia, dysarthria, areflexia, loss of vibratory and position sense, and a progressive weakness of central origin. Additional features particularly include an hypertrophic cardiomyopathy that can cause premature death. A large GAA repeat expansion in the first intron of the FXN gene is the most common mutation underlying FRDA. Patients show severely reduced levels of the FXN-encoded mitochondrial protein frataxin. Frataxin function is not yet completely elucidated. In frataxin deficiency conditions abnormalities of iron metabolism occur: decreased activities of iron-sulfur cluster (ISC) containing proteins, accumulation of iron in mitochondria and depletion in the cytosol, enhanced cellular iron uptake, and, in some models, reduced heme synthesis. Evidence of oxidative stress has also been found in most though not all models of frataxin deficiency. Accordingly, yfh1-deficient yeast and cells from FRDA patients are highly sensitive to oxidants. Respiratory chain dysfunction further aggravate oxidative stress by increasing leakage of electrons and the formation of superoxide. Frataxin deficient cells not only generate more free radicals, but, they also show a reduced ability to mobilize antioxidant defenses, in particular to induce superoxide dismutase 2 (SOD2). Peroxisome proliferator-activated receptor (PPAR) isoform-gamma play a key role in numerous cellular functions and is a key regulator of mitochondrial biogenesis and of the ROS metabolism. Recruitment of the PPAR coactivator-1a (PGC-1a) mediates many effects of the PPAR-γ activation. In a first work we assessed the potential beneficial effects of a potent PPAR-gamma agonist on frataxin expression in primary fibroblasts from healthy controls and FRDA patients, and Neuroblastoma cells. We used the APAF molecule (1-0-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocoline; C33H66NO9P). Our results show that this compound is able to increase frataxin amount both at transcriptional and post-transcriptional level. At a dose of 20µM frataxin mRNA significantly increases in both controls (p=0.03) and FRDA patients (p=0.002) fibroblasts (1). The finding was confirmed in Neuroblastoma cells (p=0.042). According to previous publications APAF, as others PPAR-gamma agonists is able to up-regulate PGC-1a transcription. In a second part of the study we investigate the role of the PPAR-gamma/PGC-1a pathway in the pathogenesis of FRDA. We performed a microarray analysis of heart and skeletal muscle in a mouse model of frataxin deficiency and we found molecular evidence of increased lipogenesis in skeletal muscle and alteration of fiber-type composition in heart, consistent with insulin resistance and cardiomyopathy, respectively. Since the PPAR-gamma pathway is known to regulate both processes, we hypothesized that dysregulation of this pathway could play a key role in frataxin deficiency. We confirmed this by showing a coordinate dysregulation of Pgc1a and the transcription factor Srebp1 in cellular and animal models of frataxin deficiency, and in cells from FRDA patients, who have marked insulin resistance. Particularly, PGC-1a was found significantly reduced (2) in primary fibroblasts and lymphocytes from FRDA patients (p<0.05). Furthermore, PGC-1a mRNA levels strongly correlate with frataxin relative mRNA levels (r2=0.9, p<0.001). According to this observation, in C2C12 myoblasts, PGC-1a and a reporter gene under the control of the PGC-1a promoter are rapidly down-regulated (p<0.05) when frataxin expression is inhibited by an shRNA in vitro. To further investigate this relation, we then generate PGC-1a deficient fibroblasts cells using a specific siRNA; at 72 hours of transfection frataxin was found down-regulate (p<0.05) in control cells. Taken together those data indicate that some mechanism directly links an early effect of frataxin deficiency with reduced PGC-1a transcription in this cell type, and presumably in other cells that also down-regulate PGC-1α when frataxin levels are low. Finally, since PGC-1a has also emerged as a key factor in the induction of many antioxidant programs in response to oxidative stress, both in vivo and in vitro, in particular in neurons, we tested whether the PGC-1a down-regulation occurring in FRDA cells could be in part responsible for the blunted antioxidant response observed in frataxin deficiency. Using primary fibroblasts from FRDA patients we found reduced SOD2 levels (p<0.05), according to PGC1 and frataxin reduced levels. Our finding confirm previous publications showing that PGC-1a directly regulate SOD2 levels in vitro and in vivo. We then tested the response to oxidative stress induced by the addition of hydrogen peroxide (H2O2) at different time and doses. Our data show that H2O2 directly increase PGC-1a and SOD2 levels (p<0.01 and p<0.05) in control cells; no effect was observed in FRDA cells, suggesting a lack in the activation of this response. Moreover, PGC-1α direct silencing, using a specific siRNA, in control fibroblasts led to a similar loss of SOD2 response (p<0.001) to oxidative stress as observed in FRDA fibroblasts, confirming its crucial role in this response (3). We then measured the same parameters after pharmacological manipulations of PGC-1a. PGC-1a activation with the PPAR agonist (Pioglitazone) or with a cAMP-dependent protein kinase (AMPK) agonist (AICAR) restored normal SOD2 induction (4) in FRDA cells (p<0.01). In vivo treatment of the KIKO mice (35-40% of wiled-type frataxin) with Pioglitazone significantly up-regulate SOD2 (5) in cerebellum (p<0.01) and spinal cord (p<0.05), two primary affected tissues in patients. The search for experimental drugs increasing the amount of frataxin is a very active and timely area of investigation. In cellular and in animal model systems, the replacement of frataxin function seems to alleviate the symptoms or completely reverts the phenotype. Therefore, drugs that are able to increase directly the amount of frataxin, at least up to the level of an asymptomatic carrier, are attractive candidates for new approaches to the therapy of FRDA. Our findings show (1) that a potent PPAR-gamma agonists can increase frataxin expression. We do also show a regulatory loop between frataxin and PGC-1a. Thus, we suggest that this loop could play a critical role in the pathogenesis of the disease and breaking this loop could help to slow down the pathological phenotype observed in FRDA patients. Particularly, PGC-1α down-regulation (3) is likely to contribute to the blunted antioxidant response observed in cells from FRDA patients. This response can be restored by AMPK and PPAR agonists in vitro (4) and in vivo, as shown by Pioglitazone treatment (5) in a mouse model for the disease. To conclude, our study provide evidences that PPAR-gamma agonists are a potential treatment for Friedreich’s ataxia, consisting with their action on both mitochondrial biogenesis and oxidative stress defenses.

Parkinson’s disease (PD) is the most common movement neurodegenerative disease affecting approximately 1% of the population over 60. Though originally thought to be sporadic in nature, a genetic component is increasingly being linked to the disease. Of these genes, mutations in DJ-1 (PARK7) cause early onset autosomal recessive PD. Initial workup of the DJ-1 protein has suggested that it may act in the cell by combatting oxidative stress though the mechanism by which it does so is unclear. Thus, though much work has attempted to elucidate a function at the biochemical, cellular and organismal level, the overt physiological role of DJ-1 remains elusive. In this dissertation, we explore the mechanisms through which DJ-1 confers neuroprotection, particularly in the case of oxidative stress insult. We demonstrate that DJ-1 acts through the pro-survival protein AKT to accomplish its neuroprotective function. Moreover, we note that DJ-1 likely serves its role as an antioxidant through the NRF2 master antioxidant regulator pathway a pathway that is, itself, likely to be regulated by AKT. Together, our results demonstrate that neuroprotection by DJ-1 is done through a signaling pathway involving both AKT and NRF2 and that disruption of the former in PD likely results in abolishing this signaling pathway. Finally, to generate a better animal model of PD, we demonstrate that backcrossing DJ-1 null mice - which originally did not demonstrate any gross histopathological or behavioral phenotypes – display unilateral dopaminergic degeneration that progresses to bilateral degeneration with aging, a feature reminiscent of classical PD progression. Collectively, this thesis takes a two-sided approach to address the biochemical and physiological functions of DJ-1 within the cell and the mouse in hopes of elucidating mechanisms of neuronal death to devise better translational therapies.