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The G protein-coupled estrogen receptor GPER/GPER1, also known as GPR30, was originally cloned as an orphan receptor and later shown to be specifically activated by 17-ß-estradiol. This has led to its classification as an estrogen receptor and expanded the perspective on the mechanisms underlying the rapid estrogenic effects reported over the years. GPER is strongly expressed in the central nervous system and peripheral tissues and appears to be involved in a wide variety of physiological and pathological processes. Estrogens are known to alter the processing of nociceptive sensory information and analgesic responses in the central nervous system. Both analgesic and pro-nociceptive effects of estrogens have been reported. Some pro-algesic estrogenic responses have a short latency, suggesting a non-genomic mechanism of action. Immunohistochemical studies in rodents prove the existence of GPER in pain-relevant areas of the nervous system such as dorsal root ganglia, superficial dorsal horn of the spinal cord, periaqueductal gray (PAG), amygdala, trigeminal sensory nucleus and thalamus. In the periphery, activation of GPER results in pro-nociceptive effects. However, GPER involvement in pain processing at central levels is largely unexplored. Thus, the work presented in this thesis was aimed at investigating whether GPER modulates nociception at spinal and supraspinal sites. The behavioral response to GPER activation in the spinal cord and PAG was evaluated in an acute grooming test (scratching, biting and licking behavior) and in the hot plate test, respectively. Intrathecal challenge of mice with the GPER agonist G-1 (0.1-1 nmol) induced a dose-dependent increase in pain-related behaviors, that was abolished by pre-treatment with the GPER antagonist G15 (1-10 nmol), confirming GPER specificity of the response. Likewise, intra-PAG microinjection of G-1 (10-100 pmol) to rats reduced the nociceptive threshold in the hot plate test, an effect that was G15 sensitive. To obtain further insight on the mechanisms involved in the behavioral effects observed in whole animals, we tested the effect of GPER ligands on neuronal membrane potential, intracellular calcium concentration ([Ca2+]i) and reactive oxygen species (ROS) accumulation. The membrane depolarization and the increases in [Ca2+]i and ROS levels are markers of neuronal activation, underlying pain sensitization in the spinal cord and pain facilitation in the PAG. Electrophysiological recordings from superficial dorsal horn and lateral PAG neurons indicate neuronal depolarization upon G-1 application, an effect that was fully prevented by G15 pre-treatment. Both cultured spinal neurons and cultured PAG neurons responded to G-1 administration by elevating [Ca2+]i and mitochondrial and cytosolic ROS levels. In the presence of G15, G-1 did not elicit the calcium and ROS responses. Collectively, these results demonstrate that GPER modulates both the ascending and descending pain pathways to increase nociception via cytosolic calcium elevation and ROS accumulation in spinal and PAG neurons, respectively. These findings broaden the current knowledge on GPER involvement in physiology and pathophysiology, providing the first evidence of its pro-nociceptive effects at central levels and characterizing some of the mechanisms involved. Moreover, we show for the first time ROS accumulation downstream of GPER activation, extending the current understanding of GPER signaling. / Pharmacology

Bud dormancy is an essential characteristic of deciduous woody perennials, including apple, to cope with the low temperatures during winter. The release from dormancy and subsequent budburst in apple can only occur after fulfillment of chilling and heat requirements. In the Mid-Atlantic region, dormancy release and bud break of apple often coincide with late-spring freezes that cause severe damages to flowers, and small fruitlets. Therefore, the present study aimed to better understand mechanisms underlying bud dormancy in apple, with an ultimate goal of exploring chemical and/or genetic approaches for bloom-time modulation to avoid spring frost. Using two apple cultivars, 'Cripps Pink' and 'Honeycrisp,' representing early- and late-blooming cultivars, respectively, the present study specifically investigated the accumulation kinetics of plant hormones, carbohydrates, and reactive oxygen species (ROS) throughout the dormancy-regrowth cycle. Our results indicated that both cultivars required 1000 chilling hours for endodormancy release, but 'Honeycrisp' required 1000 growing degree hours (GDHs) more than 'Cripps Pink' for ecodormancy release and budburst. Among plant hormones, abscisic acid (ABA) showed remarkably elevated levels in the dormant buds of both cultivars during endodormancy, but its levels were significantly higher in 'Honeycrisp'. The decline of the ABA level at bud burst was combined with increased levels of cytokinin (CK). The ABA accumulation pattern during dormancy paralleled with an upregulation and downregulation of ABA biosynthetic and catabolic genes, respectively. On the other hand, the levels of hydrogen peroxide (H2O2) and superoxide (O2.-) were significantly higher in 'Cripps Pink' than 'Honeycrisp', particularly by the time of endodormancy and ecodormancy release, respectively. Our findings also showed a gradual decline in starch levels with the dormancy progression and increased levels of total soluble sugars (TSS) that were generally higher in the early-blooming cultivars. Transcriptomic profiling and module-trait relationship identified two modules that contrast between two cultivars mainly during eco-dormancy. Gene ontology (GO) analysis indicated that these DEGs were mostly involved in pathways related to hormones and signaling and co-expressed with H2O2 whereas, during ecodormancy pathways related to glutathione metabolism, auxin biosynthesis, carbohydrate metabolism and reproductive development were co-expressed with O2.-. Together, our results suggest that the contrasting bloom dates between 'Cripps Pink' and 'Honeycrisp' can be explained, at least partially, by the differential accumulation levels of ABA, ROS, antioxidants, and their associated genes in the buds of these cultivars throughout the dormancy cycle. / Doctor of Philosophy / Spring frosts represent a significant threat to apple production in many fruit-producing states of the United States including Virginia. The risk of frost damage is rising due to global climate change, and there is a high demand for effective measures to reduce frost damage. Exogenous applications of plant growth regulators (PGRs) to delay bloom has been suggested as an effective frost avoidance strategy, but with limited success. Therefore, the present study aimed to investigate molecular and biochemical pathways regulating bud dormancy and bloom time in apple, which can ultimate lead to novel approaches for bloom delay and frost mitigation. To this end, the accumulation patterns of major plant hormones (e.g. abscisic acid, ABA, cytokinin, CK and jasmonic acid, JA), reactive oxygen species (ROS) and carbohydrates (e.g. starch, sucrose, glucose and fructose) were thoroughly monitored throughout the dormancy-regrowth cycle in two apple cultivars, 'Cripps Pink' and 'Honeycrisp,' representing early- and late-blooming cultivars, respectively. Both these cultivars had similar chilling requirements (1000 chilling hours) but differed in their heat requirements; with 'Honeycrisp' requiring 1000 growing degree hours more than 'Cripps Pink'. Among plant hormones, ABA increased with the progression of dormancy and decreased with dormancy release in both cultivars. However, ABA levels were significantly higher in 'Honeycrisp' compared to 'Cripps Pink'. On the contrary, during dormancy release, the growth-promoting hormone, CK, increased earlier in 'Cripps Pink'. The levels of ROS, e.g., hydrogen peroxide (H2O2), and superoxide (O2.-), were also higher in 'Cripps Pink' than 'Honeycrisp', particularly by the time of endodormancy and ecodormancy release, respectively. Our data showed that starch levels generally declined during dormancy, whereas soluble sugars increased. However, there was no significant alternations in the carbohydrate accumulation profiles between the two cultivars that could account for the differences in their bloom dates. These results were verified further at the transcriptomic level. Using the RNA-sequencing technology, identified two modules that contrast between two cultivars mainly during eco-dormancy. Gene ontology (GO) analysis indicated that these genes were mostly involved in pathways related to hormones and signaling and co-expressed with H2O2 whereas during ecodormancy pathways related to glutathione metabolism, auxin biosynthesis, carbohydrate metabolism and reproductive development were co-expressed with O2.-. Overall, our results suggest that ABA, cytokinin, H2O2, and O2.- may, at least partially, explain the differences in the bloom time between the two apple cultivars. Further analysis of these molecules and their associated genes in other apple cultivars with contrasting bloom dates is necessary for better understanding of bloom time regulation in apple and developing strategies against frost damage.

Deficits in skeletal muscle mitochondrial metabolism are associated with a wide variety of chronic skeletal muscle and metabolic-related diseases, including diabetes and sarcopenia. Even in patients with advanced skeletal muscle-related diseases, exercise is a well-established method to improve skeletal muscle mitochondrial metabolism, culminating in enhanced whole-body metabolism and decreased disease severity. In response to exercise, there is an increase in reactive oxygen species (ROS) production. Historically, ROS were solely considered to drive disease development. However, ROS are also required for physiological adaptation and many questions still remain regarding their downstream pathways. One significant producer of skeletal muscle ROS with exercise is Nadph oxidase 4 (Nox4). Nox4 is unique compared to other Nox members as it predominantly produces hydrogen peroxide (H2O2), an effective signaling molecule. Here we demonstrate an essential role for Nox4 in mediating the beneficial effects of exercise. This work will contribute to our understanding of physiological ROS and their downstream targets by identifying a novel role for Nox4 in exercise adaptation. Further defining the molecular events that promote exercise adaptation will be essential for formulating new treatment strategies for patients with chronic metabolic diseases. / Doctor of Philosophy / Exercise is a widely effective tool for both preventing and reversing disease. Even patients with advanced skeletal muscle and metabolic-related diseases can benefit from continual and repeated exercise training. While decades of work have supported the effectiveness of exercise as a therapeutic intervention, the mechanistic understanding of what occurs at the cellular level remains incomplete. Here, we elucidate a novel pathway mediating important metabolic adaptations to exercise. In response to exercise stress, reactive oxygen species (ROS) are produced in skeletal muscle. ROS facilitate metabolic adaptations to meet the body's need for increased energy. One significant source of ROS comes from Nadph oxidase 4 (Nox4) which plays an essential role in metabolic regulation. The skeletal muscle metabolic response to stress is largely dependent on adaptations that include changes in gene expression, substrate oxidation, and mitochondrial metabolic adaptations. These mitochondrial adaptations include mitochondrial recycling after exercise in skeletal muscle (referred to as mitophagy). We have shown that Nox4 increases the expression of a subset of metabolic genes, is required for substrate oxidation after exercise, and is important for exercise-induced mitophagy.

Indiscriminate use of pesticides appears to alter immune response in non-target organisms such as humans and other animals. Thus, immune modulation is considered as one of the potential risks and consequences following exposure to these chemicals. Because of the widespread usage, exposure to mixtures of pesticides during the lifetime of individuals is unavoidable and can result in potentiation of the toxic effects. Because immune cells are more susceptible to toxic insults at a lower dose than most other cell types, the effects of pesticides and their mixtures on murine splenocytes were evaluated. C57BL/6 male mouse splenocytes, in vitro, were exposed to permethrin and endosulfan, individually and in-combination (25-200 µM). The immunotoxic potential of these pesticides was monitored using a flow cytometric technique in combination with 7-Amino Actinomycin D (7-AAD) staining. Endosulfan exposures (25-150 µM) resulted in time- and dose-dependent increase in apoptotic and necrotic cell death in murine splenocytes, in vitro. Permethrin exposure (50-200 µM) resulted in neither a time-dependent/dose-dependent loss of splenocyte viability nor induction of apoptosis in splenocytes. With mixtures of permethrin and endosulfan, depressed viability and enhanced early apoptosis and late apoptosis/necrosis were observed. Exposure to mixtures of 50 µM endosulfan with 50 or 100 µM permethrin increased late apoptosis/necrosis compared to exposure to either chemical alone. DNA fragmentation, a hall mark of apoptosis was observed by DNA ladder technique, confirming the occurrence of apoptosis. Morphological observation using cytospun slides was also carried out to further confirm the presence of apoptosis and necrosis. These findings suggest that the immunotoxicity of endosulfan both individually and in mixtures with permethrin is associated with the occurrence of apoptotic and necrotic processes. Further, the ability of these pesticides to alter the oxidative status of the cells, via reactive oxygen species (ROS) generation and modulation of intracellular antioxidant enzymes levels, was investigated. We monitored the generation of ROS such as hydrogen peroxide (H₂O₂) with 2´, 7´- dichlorofluorescin diacetate (DCFH-DA) assay and superoxide anion (O₂⁻) with hydroethidine (HE) assay in combination with flow cytometry. Spectrophotometric techniques were used to measure antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and glutathione peroxidase (GPX). Results of the analyses revealed that individual pesticides increased the production of H₂O₂ in a time and dose-dependent manner. Both time and dose-dependent increases in O₂⁻ production were caused by permethrin; whereas endosulfan exposure resulted in only a dose-dependent increase. However, exposure to mixtures of these pesticides had little or no effect on the generation of H₂O₂ and O₂⁻ radicals as compared to individual pesticides. The levels of SOD and GPX in pesticide-treated splenocytes were found to be not different from solvent control. An increase in GR and CAT levels in cells was noticed with permethrin (100 µM) exposure. These findings suggest that permethrin and endosulfan have the ability to affect the cellular oxidative status and can cause toxicity in immune cells, in vitro. / Master of Science

Yes / Platinum complexes are the most widely used anticancer drugs; however, new generations of agents are needed. The organoiridium(III) complex [(η5-Cpxbiph)Ir(phpy)(Cl)] (1-Cl), which contains π-bonded biphenyltetramethylcyclopentadienyl (Cpxbiph) and C^N-chelated phenylpyridine (phpy) ligands, undergoes rapid hydrolysis of the chlorido ligand. In contrast, the pyridine complex [(η5-Cpxbiph)Ir(phpy)(py)]+ (1-py) aquates slowly, and is more potent (in nanomolar amounts) than both 1-Cl and cisplatin towards a wide range of cancer cells. The pyridine ligand protects 1-py from rapid reaction with intracellular glutathione. The high potency of 1-py correlates with its ability to increase substantially the level of reactive oxygen species (ROS) in cancer cells. The unprecedented ability of these iridium complexes to generate H2O2 by catalytic hydride transfer from the coenzyme NADH to oxygen is demonstrated. Such organoiridium complexes are promising as a new generation of anticancer drugs for effective oxidant therapy. / We thank the ERC (247450), SNSF (PA00P2_145308 for N.P.E.B.), IAS (for I.R.C.), BBSRC (for J.M.H.), Science City (AWM and ERDF), and the EPSRC for support, and Prof. Timothy Bugg and members of EC COST Action CM1105 for stimulating discussions. We also thank Professor Pat Unwin, Mike Snowden, and Rob Lazenby for their help with the electrochemical experiments and the National Cancer Institute for NCI-60 human tumor cell panel screening.

No / Accumulation of large-scale mitochondrial DNA (mtDNA) deletions and chronic, subclinical inflammation are concomitant during skin aging, thus raising the question of a causal link. To approach this, we generated mice expressing a mutant mitochondrial helicase (K320E-TWINKLE) in the epidermis to accelerate the accumulation of mtDNA deletions in this skin compartment. Mice displayed low amounts of large-scale deletions and a dramatic depletion of mtDNA in the epidermis and showed macroscopic signs of severe skin inflammation. The mtDNA alterations led to an imbalanced stoichiometry of mitochondrial respiratory chain complexes, inducing a unique combination of cytokine expression, causing a severe inflammatory phenotype, with massive immune cell infiltrates already before birth. Altogether, these data unraveled a previously unknown link between an imbalanced stoichiometry of the mitochondrial respiratory chain complexes and skin inflammation and suggest that severe respiratory chain dysfunction, as observed in few cells leading to a mosaic in aged tissues, might be involved in the development of chronic subclinical inflammation. / Deutsche Forschungsgemeinschaft (Wi 889/6-3 to RJW, SFB 829 A14 to RJW, Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases–CECAD to RJW, BR2304/9-1 to BB, and SFB 829 A1, A5, and Z2 to CMN) and the Center of Molecular Medicine Cologne of the Medical Faculty (CMMC, to RJW)

Yes / Boron dipyrromethene derivates (BODIPYs) are promising photosensitisers (PSs) for cancer treatment using photodynamic therapy (PDT). This study investigates the functionalisation of graphene oxide (GO) with a BODIPY derivate for glutathione (GSH) depletion and PDT. The functionalisation of GO with a 3,5-dichloro-8-(4-boronophenyl) BODIPY via a diol derivatisation with the phenyl boronic acid moiety at the meso position of the BODIPY core, allowed to preserve the intrinsic properties of GO. We demonstrated that both chlorine atoms were substituted by GSH in the presence of glutathione transferase (GST), inducing a relevant bathochromic shift in the absorption/emission features and thus generating the active PS. Ex vitro assessment using cell lysates containing cytoplasmatic GST revealed the intracellular catalytic mechanism for the nucleophilic substitution of the GO-BODIPY adduct with GSH. Confocal microscopy studies showed important differences in the cellular uptake of free BODIPY and GO-BODIPY and revealed the coexistence of GO-BODIPY, GO-BODIPY-GS, and GO-BODIPY-GS2 species inside vesicles and in the cytoplasm of the cells after 24 h of incubation. In vitro biocompatibility and safety of GO and GO-BODIPY were evaluated in 2D and 3D models of prostate adenocarcinoma cells (PC-3), where no toxicity was observed up to 100 µg ml−1 of GO/GO-BODIPY in all treated groups 24 h post-treatment (cell viability > 90%). Only a slight decrease to 80% at 100 µg ml−1 was observed after 48 h of incubation. We demonstrated the efficacy of a GO adduct containing an α-chlorine-substituted BODIPY for the simultaneous depletion of intracellular GSH and the photogeneration of reactive oxygen species using a halogen white light source (5.4 mW cm−2) with a maximum in the range of 500–800 nm, which significantly reduced cell viability (<50%) after irradiation. Our study provides a new vision on how to apply BODIPY derivates and potentiate the toxicity of PDT in prostate and other types of cancer.

Master of Science / Department of Kinesiology / Timothy I. Musch / During exercise aged individuals exhibit endothelial dysfunction and decreased levels of whole-limb blood flow (BF), both of which may be linked mechanistically to age-related increases in reactive oxygen species (ROS). Ascorbic acid (AA) reduces levels of ROS and has been shown to alleviate vascular and hyperemic dysfunction at rest (Jablonski et al., 2007) and during small muscle mass exercise in humans (Kirby et al., 2009). However, the effect of AA on vascular function and BF to individual muscles during whole-body exercise is not known. PURPOSE: To test the hypothesis that a single high-dose infusion of AA would increase BF to the hindlimb musculature of old rats at rest and during treadmill running. METHODS: 18 old (~28 months) Fischer 344 x Brown Norway rats were randomized into rest (n=9) and exercise (n=9) groups. BF to the total hindlimb and individual muscles (28 individual muscles and muscle parts) was evaluated via radiolabeled microspheres before and after intra-arterial AA administration (76 mg/kg in 3 ml heparinized saline, 30 minute infusion) at rest and during submaximal treadmill running (20m/min, 5% grade). Total antioxidant capacity (TAC) and thiobarbituric acid reactive species (TBARS) were measured before and after AA to determine the ability of this specific dose of AA to increase levels of plasma antioxidants and decrease levels of ROS, respectively. RESULTS: At rest: AA increased TAC (~37%, P<0.05) but did not change TBARS (Pre: 6.8±0.7 vs Post: 7.0±1.0 µM, P>0.05). AA decreased total hindlimb BF (Pre: 25±3 vs Post: 16±2 ml/min/100g, P<0.05) and BF to 8 of the 28 muscles that were evaluated. During exercise: TAC was increased (~35%, P<0.05) and TBARS were decreased (Pre: 9.8±2.0 vs Post: 7.0±1.0 µM, P<0.05). However, there was no effect on either total hindlimb BF (Pre: 154±14 vs Post: 162±13, P>0.05) or BF to any of the individual muscles evaluated. CONCLUSIONS: Increased TAC via AA infusion reduces hindlimb muscle BF at rest but had no effect on BF during whole-body dynamic exercise. Thus, even though TBARS decreased, there was no evidence that AA supplementation increases blood flow to the locomotor muscles of old rats during whole-body exercise.