The prominence of insect cell culture has grown rapidly due to its ability to produce baculovirus biopesticides and recombinant proteins using the Baculovirus Expression Vector System. A critical problem in the mass production of these products is CO2 accumulation to inhibitory levels within the bioreactor. The current research investigated the effect of elevated CO2 concentrations on insect cell growth and metabolism and the roles of oxidative stress and intracellular pH (pHi) in CO2 inhibition. Spodoptera frugiperda Sf-9 insect cells were cultured in a 3 L bioreactor (1.2 L working volume) controlled at 20% air saturation, 27oC and a pH of 6.2. The cells were exposed to a constant CO2 concentration by purging the medium with CO2 and the headspace with air. The experiments were repeated for different CO2 concentrations and samples were taken every 24 h to determine cell density, viability, metabolism and oxidative stress. The population doubling time (PDT) of Sf-9 cells increased with increasing CO2 concentration. Specifically, the PDT for 0-37, 73, 147, 183 and 220 mm Hg CO2 concentrations were 23.2 ± 6.7, 32.4 ± 7.2, 38.1 ± 13.3, 42.9 ± 5.4 and 69.3 ± 35.9 h (n = 3 or 4; 95% confidence level), respectively. An 80 mL working volume shaker flask was maintained as a control and had an average PDT of 24.9 ± 3.1 h (n = 7; 95% confidence level). The viability of cells in all experiments was above 90%. The osmolality for all bioreactor experiments was observed to be 300 - 360 mOsm/kg, a range that is known to have a negligible effect on insect cell culture. Elevated CO2 concentration did not alter the cell specific glucose consumption rate (2.5 to 3.2 x 10-17 mol/cell-s), but slightly increased the specific lactate production rate from -3.0 x 10-19 mol/cell-s to 10.2 x 10-19mol/cell-s. Oxidative stress did not contribute to CO2 inhibition in uninfected Sf-9 cells as no significant increase in the levels of lipid hydroperoxide and protein carbonyl concentrations was discovered at elevated CO2 concentration. The experiments conducted to determine the effect of CO2 on pHi were not successful and different experimental methods tested were well documented.

Oxidative stress, the imbalance of free radical generation with antioxidant defenses, leads to cellular inflammation, apoptosis and cell death. This compromised environment results in debilitating diseases, such as oral mucositis (OM), atherosclerosis, and ischemia/reperfusion injury. Antioxidant therapeutics has been a proposed strategy to ameliorate these imbalances and maintain homeostatic environments. However, the success of these approaches, specifically curcumin, has been limited due to characteristics such as hydrophobicity and high reactivity when released as bolus doses to contest to oxidative stress induced diseases. The development of a controlled release system to aid in protection of the antioxidant capacity of curcumin, as well as a tunable system to aid in proper rate of release for disease can overcome these limitations. Previously, the use of a poly(beta-amino ester) (PBAE) chemistry has been developed in Dziubla and Hilt laboratories to provide desirable properties. The dynamic mechanical analysis and efficacy in cellular protection has been studied, yet the sensitivity and responsiveness of these polymers to abnormal environments found within oxidative stress compromised environments are unknown. In this work, a series of networks were comprised of different molar ratios of modified acrylated curcumin, poly(ethylene glycol) diacrylate, and a primary diamine crosslinker to create tunable hydrolytically degradable crosslinked hydrogels. I hypothesized a consumption rate difference of free curcumin and curcumin as a released product from the crosslinked network in the presence of a free radical generating system. After the consumption profiles of each were reported differently, the experimental data was translated into a kinetic rate model to identify quantitative consumption rate parameters of curcumin and active film degradation products. The effect on the released products arose the question of curcumin consumption in other oxidizing environments. These networks were then investigated in low concentrations of a hydrogen peroxide insult, and interestingly showed sensitivity to hydrolysis by recovering significantly more curcumin at an accelerated rate of release. Identifying the sensitivity of these tunable networks to environmental stimuli, they were then presented to a series of low pH environments, which significantly reduced the degradation time, finding a dependence of rate of release on the weight loading of curcumin present within the film. To translate these responsive materials to an application-based system, the curcumin conjugated PBAE polymers were investigated as an oral rinse drug delivery system for the treatment of radiation-induced OM in a hamster model. Radiation-induced OM onset and severity was reduced with a 20 wt% microparticle loaded mucoadhesive system that releases curcumin over 24 hours, providing promising results of a therapeutic effect from curcumin when incorporated in to a controlled release delivery system. Overall, curcumin conjugated PBAE polymers show selectivity of hydrolysis in abnormal environments related to oxidative stress. This information is beneficial to the proper design and loading of antioxidant therapeutics within crosslinked polymers, giving the ability to tune release to treat and deliver based on the environment’s insult. This can advance the potential use for antioxidant therapeutics in pharmaceutical applications in the future.

Axonal pathology is a key contributor to long-term disability in multiple sclerosis (MS), an inflammatory demyelinating disease of the central nervous system (CNS), but the mechanisms that underlie axonal insults remain unclear. While most axonal pathologies characterized in MS are a direct consequence of myelin loss, we propose that axonal pathologies also occur independent of demyelination. In support of this idea, we recently reported that mice that develop experimental autoimmune encephalomyelitis (EAE), a model commonly used to mimic the pathogenesis of MS, exhibit a structural and functional disruption of the axon initial segment (AIS), a subdomain of the axon that acts as the trigger-zone for action potential generation. Importantly, this disruption is independent of myelin loss. Although the mechanism responsible for AIS disruption remains unclear, we observed an attenuation of the AIS insult following treatment with a known scavenger of oxygen free radicals. To further investigate the role of oxidative stress in modulating AIS stability, we employed an in vitro model in which neurons were exposed to a spontaneous reactive oxygen and nitrogen species generator. Through this approach, we demonstrated that oxidative stress is capable of AIS modulation acting through induction of cytosolic calcium (Ca2+) influx from both extracellular and intracellular sources, resulting in calpain protease activation. Furthermore, because rises in intracellular Ca2+ are central to these and other mechanisms of AIS disruption, we next investigated the cisternal organelle (CO), an AIS-localized Ca2+-regulating structure. Although this organelle could prove to be central to AIS modulation, very little is known about the mechanisms regulating its stability. Through this line of investigation, we provide the first evidence of pathological alteration to the CO in a disease state. This disruption precedes loss of AIS protein clustering and axo-axonic GABAergic input in both EAE and MS postmortem tissue. Overall, these studies reveal a primary axonal insult, independent of myelin loss, in a disease classically characterized as a white-matter pathology. Instead, this insult is most likely driven by oxidative stress through local Ca2+ dysregulation at the AIS, providing novel therapeutic targets for MS.

Overexpression of the enzyme iNOS induces apoptotic cellular death by increasing indices of pro-inflammation and oxidative stress. Aerobic physical activity has been known to have anti- inflammatory benefits and reduce oxidative stress. Purpose: Therefore, this study aimed to examine the impact of aerobic fitness on LPS-induced iNOS mRNA expression and the relationship of this expression with indices of oxidative stress, pro-inflammation and apoptosis in isolated leukocytes. Methods: Whole blood samples from aerobically fit and unfit males were stimulated with and without LPS. Thereafter, iNOS mRNA expression and MDA, TNF-α and p53 concentrations were analyzed. Results: iNOS mRNA expression levels following LPS stimulation were not increased in both groups, and correlational analyses were not consistent with mechanistic predictions. Discussion: Numerous factors including timing of sample quantification, the high level of health of the subject population, and alternative intracellular mechanisms impacting biomarkers analyzed, may have influenced leukocyte iNOS mRNA expression levels.

COPD is characterized by tissue inflammation and impaired remodeling that suggests fibroblast maintenance of structural homeostasis is dysregulated. Thus, we performed in vitro wound healing experiments on normal and diseased human lung fibroblasts and developed an ABM of fibroblasts closing a scratched monolayer using NetLogo to evaluate differences due to COPD or cigarette smoke condensate exposure. This ABM consists of a rule-set governing the healing response, accounting for cell migration, proliferation, death, activation and senescence rates; along with the effects of heterogeneous activation, phenotypic changes, serum deprivation and exposure to cigarette smoke condensate or bFGF. Simulations were performed to calibrate parameter-sets for each cell type using in vitro data of scratch-induced migration, viability, senescence-associated beta-galactosidase and alpha-smooth muscle actin expression. Parameter sensitivities around each calibrated parameter-set were analyzed. This model represents the prototype of a tool designed to explore fibroblast functions in the pathogenesis of COPD and evaluate potential therapies.

Orientador: Antonio de Queiroz Neto / Banca: José Correa de Lacerda Neto / Banca: Wilson Roberto Fernandes / Banca: Flávio Desessards de La Cortê / Banca: Thiago Luiz de Salles Gomes / Abstract: The aim of this study was to register the physiologic and metabolic alterations and investigate the exercise induced oxidative stress that Arabians horses undergo during long distance endurance exercises, under tropical climate. Five endurance rides were followed through 2004 state championship. Blood samples were collected from jugular vein 2 to 5 hours before the beginning of each ride and during rides, after the veterinary check-point. During the recovery period, venous samples were collected 24, 48 and 72 hours after the rides, at each horses’ stables. Hemoconcentration (elevated erythrocytes count, hemoglobin concentration and hematocrit), dehydration (elevated total plasmatic proteins concentration and weight losses) and, possibly, decreased renal perfusion (elevated seric urea and creatinin concentration) were revealed by horses in this study. All data returned to basal values during the recovery period except serum urea concentration. Hormonal changes were also monitored and data revealed an important elevation in plasma cortisol concentration during the ride, directly related to the duration of it. Insulin response was decreased by catecholamines suppressing action during exercise. The endurance effort, evaluated in this experiment, could induce muscular alterations by an increase in muscular enzyme activities during the rides, with different periods of return to basal values in the recovery period. An interesting result was the exercise-induced oxidative stress measured by cyclic voltammetry to determine the total antioxidant capacity of plasma during endurance exercise. The results of this study can contribute to a better understanding of what really occurs in the horses’ body to maintain homeostasis while submitted to long distance endurance efforts under tropical climate. / Doutor

While mechanical loading is essential for articular cartilage homeostasis, it also plays a central role in the etiology of osteoarthritis. The mechanotransduction events underlying these dual effects, however, remain unclear. Previously, we have shown that lethal amounts of reactive oxygen species (ROS) were liberated from mitochondrial complex 1 in response to a mechanical insult. The sensitivity of this response to an actin polymerase inhibitor, cytochalasin B, indicated a link between ROS release and cytoskeletal distortion caused by excessive compressive strain. It did not, however, rule out the possibility that ROS may also mediate the beneficial effects of normal stresses that induce lower tissue strains required for proper homeostasis. If this possibility is true, one would expect the amount of ROS released in loaded cartilage to be positively correlated with the level of strain, and ROS should only reach lethal levels under super-physiological deformations. To test this hypothesis, full cartilage tissue strains were measured in cartilage explants subjected to static normal stresses of 0, 0.1, 0.25, 0.5, and1.0 MPa. After compression, the percentage of ROS-producing cells was measured using the oxidation-sensitive fluorescent probe, dihydroethidium, and confocal microscopy. In support of our theory, the percentage of fluorescing cells increased linearly with increasing strains (0-75%, r2 = 0.8, p < 0.05). Additionally, hydrostatic stress, which causes minimal tissue strain, induced minimal ROS release. In terms of cell viability, cartilage explants compressed with strains >40% experienced substantial cell death, while explants with strains

Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants that are known to elicit adverse health effects including skin toxicity and cancer to animals and humans. 4-Monochlorobiphenyl (PCB3), a low-chlorinated airborne PCB conger is present in human blood and the environment. 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ), a quinone metabolite of PCB3, has been shown to induce oxidative stress and toxicity in human mammary and prostate epithelial cells. These studies were designed to investigate and characterize the cellular responses to 4-ClBQ in HaCaT human skin keratinocytes. We found that 4-ClBQ treatment increased cellular reactive oxygen species (ROS) production, inhibited cell proliferation, and induced toxicity in HaCaT cells. Results from a Human Antioxidant Mechanism PCR array and quantitative RT-PCR assay showed that the mRNA levels of antioxidant gene selenoprotein P (sepp1) and catalase were significantly downregulated by the treatment, which correlated with evident decreases in their protein levels and catalase enzymatic activity. Pharmacological (sodium selenite supplementation) and molecular (sepp1overexpression) manipulation of SEPP1 expression significantly suppressed 4-ClBQ induced oxidative stress and toxicity. Additional results demonstrated that decreased catalase expression was associated with an inhibition in transcriptional coactivator peroxisome proliferator activated receptor Γ coactivator 1α (PGC-1α) expression. Overexpression of pgc-1α restored catalase expression and activity and consequently protected HaCaT cells from 4-ClBQ induced oxidative stress and toxicity. Furthermore, results from metabolic flux analysis using Seahorse XF96 Analyzer showed that 4-ClBQ treatment increased extracellular acidification rate, proton production rate, and oxygen consumption rate, which were associated with increases in glucose uptake and in the expression of glucose metabolism regulatory gene hexokinase 2, pyruvate kinase M2, and glucose-6-phosphate dehydrogenase (G6PD). G6PD is the rate-limiting enzyme of the pentose phosphate pathway. The enhanced expression of G6PD correlated with an increase in cellular glutathione content; and inhibition of G6PD activity sensitized HaCaT cells to 4-ClBQ induced toxicity, suggesting that the protective function of the pentose phosphate pathway is active in 4-ClBQ treated cells. Interestingly, we also found that 4-ClBQ selectively and significantly decreased mitochondrial complex II subunits C (sdhc) and D (sdhd) mRNA expression and subsequently reduced complex II activity leading to metabolic oxidative stress and toxicity, which were significantly suppressed by overexpressing sdhc and sdhd in HaCaT cells. Taken together, findings from this project demonstrate that 4-ClBQ treatment increases ROS production through perturbing cellular metabolism and mitochondrial function and decreases antioxidant capacity by inhibiting SEPP1 and catalase expression in HaCaT cells. This imbalance due to increased mitochondrial prooxidant production and decreased antioxidant capacity leads to oxidative stress and toxicity. Importantly, antioxidant supplementation could abrogate 4-ClBQ induced toxicity, suggesting that antioxidants, especially nutrient-based manipulation of selenoproteins could be promising countermeasures for PCB induced adverse health effects in humans.

The brain renin-angiotensin system (RAS), of which angiotensin II (AngII) is the primary effector peptide, plays a critical role in the neurohumoral regulation of cardiovascular and body fluid homeostasis by modulating blood pressure, secretion of hypothalamic and pituitary hormones, and water intake. AngII produced locally in the brain or in the systemic circulation can act on brain regions called circumventricular organs (CVO), which lack the blood-brain-barrier. Dysregulation of central AngII signaling is implicated in the pathogenesis of hypertension; therefore, understanding the mechanisms of AngII in the CNS is an important area of investigation. Recently, a novel signaling mechanism for AngII in the periphery has been shown to involve NAD(P)H oxidase-derived reactive oxygen species (ROS). Although ROS are now known to be involved in numerous AngII-regulated processes in peripheral tissues, and are increasingly implicated in CNS neurodegenerative diseases, the role of ROS in central regulation of AngII-induced cardiovascular function remains unexplored. The hypothesis that ROS are critically involved in central AngII signaling and in AngII-dependent blood pressure and drinking behavior was tested by harnessing the power of an array of selective genetic tools, in combination with novel technologies for analysis of cardiovascular function in conscious mice. More specifically, central injections of adenoviruses encoding ROS-modulating molecules were used to examine the redox mechanisms in central AngII-mediated cardiovascular responses in vivo. Neuronal cell cultures were also used to investigate the involvement of NAD(P)H oxidase-derived ROS in AngII signaling, as well as to examine a link between calcium and ROS in intra-neuronal AngII signaling. Finally, in order to better understand the potential role of ROS in the brain in the pathogenesis of AngII-dependent hypertension, a mouse model that recapitulates the characteristics of human hypertension was employed in conjunction with genetic modulation of the redox state of the brain. These studies provide new evidence that ROS are involved in the intracellular signaling mechanism of AngII in the brain under normal and pathological conditions and offer new insight to how dysregulation of redox mechanisms in the brain may lead to the pathogenesis of AngII-dependent hypertension.

Mitochondrial superoxide production during the early and late radiation response is increasingly recognized as a critical driver of oxidative damage and injury processes in mammalian cells. The role of Sirtuin 3, a key mitochondrial regulatory deacetylase, in preventing mitochondrial superoxide generation in conditions of nutrient and oxidative stress may be critical during the radiation response in mammalian liver. Because several tumor types express lower than normal levels of Sirtuin 3, the involvement of Sirtuin 3 in the radiation response may also provide clues to improving cancer radiation therapy and understanding the process of carcinogenesis. Studies of how the SIRT3 loss impacts the hepatic radiation response may also provide insight into the role of superoxide in normal liver physiology as well as in conditions of pathology. Increased superoxide production has largely been associated with disease, but oftentimes without clear demonstration of mechanism or even clear descriptions of pathogenesis. Here we identify a target of Sirtuin 3, the mitochondrial antioxidant enzyme manganese superoxide dismutase, and delineate the role that Sirtuin 3-mediated increases in manganese superoxide dismutase may be playing in the prevention of injury following biologically relevant doses of low linear energy transfer and high linear energy transfer radiation types including Cs-137 and Fe and Si particle radiation. Loss of Sirtuin 3 appears to correlate with decreases in hepatocellular carcinoma 16 months after 0.1 and 1 Gy doses of particle radiation known to increase hepatocellular carcinoma rates. These results indicate that Sirtuin 3 is a critical regulator of superoxide metabolism in the liver following whole body irradiation.