Indiana University-Purdue University Indianapolis (IUPUI) / Tobacco smoking is considered a major modifiable risk factor for periodontal disease. Tobacco contains about 6700 compounds and almost 4000 compounds of these have been identified in tobacco smoke. Nicotine is the addictive ingredient in tobacco and has been shown to affect multiple cellular processes. Cigarette smoke condensate (CSC) is the particulate matter of smoke. It is believed to be a powerful inducer of inflammatory responses. Neutrophils are the first line of host defense and are critical cells in the maintenance of periodontal health through their role in the control of bacteria, but they can also contribute to the progression of periodontal disease by the production and release of reactive oxygen species (ROS). Virulence factors from periodontal pathogens, such as Porphyromonas gingivalis (P. gingivalis), stimulate the respiratory burst of neutrophils. In this dissertation, three studies aimed at understanding the oxidative activity of neutrophils when stimulated with either nicotine, cigarette smoke condensate (CSC) or four other components of tobacco smoke (2-naphthylamine, hydroquinone, acrolein, and acetaldehyde) with or without P. gingivalis supernatant. The release of matrix metalloproteinase-9 (MMP-9) was also examined. ROS production increased significantly when the neutrophils were stimulated with nicotine. P. gingivalis induced the maximum ROS production when compared to all the other components examined. The combination of nicotine and P. gingivalis did not have an additive effect on ROS production. Nicotine significantly increased the MMP-9 release from the neutrophils. On the contrary, CSC inhibited ROS production at all the concentrations examined. The combination of CSC and P. gingivalis resulted in the inhibition of ROS production. MMP-9 release was also increased from the CSC-treated neutrophils. The four other tobacco smoke components examined affected ROS production and MMP-9 release differently. These projects demonstrated that CSC inhibited the ROS production from neutrophils, which can be attributed to several components in tobacco smoke that may include acrolein and hydroquinone. More research is needed to determine the mechanisms of inhibition and if other tobacco components are involved in ROS inhibition

Das Rett-Syndrom ist eine postnatale neurologische Entwicklungsstörung, der eine Mutation im Methyl-CPG-bindenden Protein 2 (MECP2) zugrunde liegt. Es betrifft überwiegend Mädchen und geht mit kognitiven Beeinträchtigungen, motorischen Stereotypien und Atmungsstörungen einher. Es existieren vielfältige Hinweise dafür, dass die Pathogenese des Rett-Syndroms im Zusammenhang mit einer beeinträchtigten Mitochondrienfunktion steht. Genetische Untersuchungen des Rett-Genoms zeigten, dass eine Untereinheit des Komplex III der Atmungskette dysreguliert ist und die innere Mitochondrienmembran ein Protonenleck aufweist. Weiterhin fanden sich Hinweise für erhöhten oxidativen Stress in Blut- und Liquoruntersuchungen. Um den intrazellulären Redox-Status zu quantifzieren, wurde die genetisch kodierte optische Sonde roGFP1 verwendet, die semiquantitative Messungen reaktiver Sauerstoffspezies ermöglichte. Es zeigte sich, dass Mecp2(-/y)-Hirnschnitte bereits unter Ruhebedingungen erhöhtem oxidativen Stress ausgesetzt sind. Auf der Suche nach der Ursache wurden die intrazellulären antioxidativen Schutzenzyme Superoxid-Dismutase und Katalase sowie das Glutathionsystem überprüft. Alle drei Enzymsysteme zeigten Funktionsstörungen und waren nicht in der Lage, extern applizierten oxidativen Stress im gleichen Umfang zu kompensieren wie die Enzyme der Wildtyp-Vergleichsgruppe. Um die zytosolischen Redox-Verhältnisse zu beeinflussen, wurden Untersuchungen mit den Antioxidantien Ascorbat, Trolox und Melatonin vorgenommen. Dabei zeigte sich, dass Antioxidantien eine potentielle pharmakologische Maßnahme darstellen, um die zu oxidativen Verhältnissen verschobene Redox-Homöostase in Mecp2(-/y)-Hippokampi zu senken und folglich zu normalisieren. Vor allem das Vitamin E-Derivat Trolox stellte sich als wirkungsvoller Radikalfänger heraus und bietet sich für weitere detaillierte Untersuchungen hinsichtlich einer therapeutischen Option des Rett-Syndroms an. Die externe Störung der mitochondrialen Funktion durch die Induktion einer transienten Hypoxie sowie die gezielte Inhibition verschiedener Atmungskettenkomplexe zeigte eine deutlich erhöhte Hypoxieempfindlichkeit der Mecp2(-/y)-Hippokampi und war mit einer erhöhten ROS-Produktion verbunden. In der Arbeit gelang es erstmals, die bereits mehrfach postulierte Störung der Redox-Homöostase im Rett-Syndrom direkt auf zellulärer Ebene nachzuweisen. Die erhobenen Befunde liefern mögliche mechanistische Erklärungsansätze für die Störung der synaptischen Plastizität im Rett-Syndrom, da es klare Verbindungen zwischen dem zellulären Redox-Status und dem Kalziumhaushalt gibt, der durch redoxsensitive Proteine mitreguliert wird. Somit konnte eine zentrale Dysregulation der Erkrankung identifziert werden, die unter Umständen auch neue pharmakologische Angriffspunkt aufzeigt, um die Symptomatik des Rett-Syndroms zu mildern.

Systemic acquired resistance (SAR) is a form of inducible defense response triggered upon localized infection that confers broad-spectrum disease resistance against secondary infections. Several factors are known to regulate SAR and these include phenolic phytohormone salicylic acid (SA), phosphorylated sugar glycerol-3-phosphate (G3P), and dicarboxylic acid azelaic acid (AzA). This study evaluated a role for free radicals nitric oxide (NO) and reactive oxygen species (ROS) in SAR. Normal accumulation of both NO and ROS was required for normal SAR and mutations preventing NO/ROS accumulation and/or biosynthesis compromised SAR. A role for NO and ROS was further established using pharmacological approaches. Notably, both NO and ROS conferred SAR in a concentration dependent manner. This was further established using genetic mutants that accumulated high levels of NO. NO/ROS acted upstream of G3P and in parallel to SA. Collectively, these results suggest that NO and ROS are essential components of the SAR pathway.

In many chronic inflammatory diseases, patients suffer from skeletal muscle weakness, exacerbating their symptoms. Serum levels of tumor necrosis factor-alpha (TNF) and sphingomyelinase are increased, suggesting their possible role in the progression of this weakness. This dissertation focuses on the role that reactive oxygen species (ROS) and nitric oxide (NO) play in mediating TNF-induced skeletal muscle weakness and to what extent sphingolipid signaling mediates cellular response to TNF. The first aim of this work was to identify which endogenous oxidant species stimulated by TNF contributes to skeletal muscle weakness. In C57BL/6 mice (n=38), intraperitoneal injection of TNF elicited a 25% depression of diaphragm contractile function. In separate experiments, diaphragm fiber bundles harvested from mice (n=39) and treated with TNF ex vivo showed a 38% depression of contractile function compared to untreated controls. Using ROS and NO-sensitive fluorescence microscopy in parallel with a genetic knockout animal model, TNF-induced contractile dysfunction was found to be mediated by NO generated by a specific isoform of nitric oxide synthase (NOS), nNOS. Basal levels of ROS were necessary co-mediators, but were not sufficient to elicit TNF-induced diaphragm weakness. The second aim of this dissertation was to investigate the extent to which sphingolipids could serve as a signaling cascade post-TNF stimulus leading to the generation of NO in skeletal muscle. The effects of TNF exposure in C2C12 skeletal muscle cells were studied in vitro using mass spectroscopy to measure sphingolipid metabolism and fluorescent microscopy to quantify oxidant production. TNF exposure was associated with significant mean increases in sphingosine (+52%), general oxidant activity (+33%), and NO production (+14%). These increases were due to specific modulation of nNOS as demonstrated by siRNA knockdown of neutral ceramidase and nNOS, and confirmed by pharmacologic inhibition using N-Oleoylethanolamine and di-methylsphingosine. In summary, these findings confirm NO as a major causative oxidant contributing to TNF’s deleterious phenotype in skeletal muscle. Moreover, the work suggests a new role for sphingosine in skeletal muscle and warrants further study of the enzymatic regulation of sphingosine to advance the discovery of new therapies for patients suffering from chronic inflammation.

Tyrosyl phosphorylation plays an important role in many fundamental cellular processes, including cell growth, differentiation and proliferation. The levels of phosphotyrosine (pY) are regulated by the opposing actions of protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). A limitation to understanding the roles of PTPs in physiological and pathological cell signaling has been the absence of global proteomic approaches that enable the systematic and comprehensive analysis of PTP expression, regulation and function. This dissertation describes the development and application of novel proteomic methodologies that permit the global analysis of PTP expression (qPTPome), regulation (by oxidation and nitrosylation; q-oxPTPome) and substrates/binding proteins. These methods provide a workflow to begin assessing PTP function at a systems level, rather than its current targeted format. Application of these techniques will provide invaluable information to begin bridging the gap in our understanding of PTP and PTK function in normal and malignant cell signaling.

Chemotherapy, employing single-molecule or multidrug concoctions inspired by the diverse repository of plant chemicals, has been the mainstay of cancer treatment for years. However, isolating single molecules has proven to be expensive along with limited therapeutic window and toxicity. On the other hand, whole foods, while preserving the natural complex balance between their constituent phytochemicals and being non-toxic, have proven to impart better disease-fighting efficacies, thus leading to an increased focus on dietary interventions to both treat and prevent cancer. Owing to the complex interactions between their constituent phytochemicals, several dietary agents have been investigated for their therapeutic and preventive efficacies. However, due to lack of emphasis on confounding factors like bioavailability, absorption, metabolism, and excretion, essentially driven by phytocomplexity, incorporation of whole foods in therapeutic regimen has not been successful. This thesis exemplifies the need to investigate factors associated with the limitations in the current approach with respect to dietary agents. Bioactivity-guided fractionation of sweet potato greens extract (SPGE) led to the identification of ~100-fold more potent fraction in vitro. However, this efficacy could not be translated in vivo. We also studied whole ginger extract (GE) for its in vitro and in vivo prostate tumor growth-inhibitory and apoptosis-inducing effects. In addition, GE proved to be more efficacious as compared to its individual most-active constituents owing to the differences in their pharmacokinetic (PK) and bioavailability measurements. Hence, these studies emphasize the crucial role of synergistic/additive interactions among the constituents of whole foods in successful translation of their therapeutic benefits. Another factor that seeks further attention is the unique cellular mechanisms engaged by these phytochemicals to confer their remarkable effects. Phenolic compounds, the most-abundant of all phytochemicals, are well known for their antioxidant properties and act via reactive oxygen species (ROS)-mediated mechanisms. We however assert the underappreciated xenohormetic prooxidant role of phenolics, where cancer cell death is caused by induction of intolerable levels of ROS. We demonstrated that a Piper betel constituent, hydroxychavicol (HC), mediates cytotoxicity via ROS-induced DNA-damage. This thesis thus provides compelling grounds for future preclinical studies to validate their potential usefulness for cancer management.

This study presents the first attempt to combine the fields of ultraviolet (UV) photobiology, plant cell wall biochemistry, aerobic methane production and reactive oxygen species (ROS) mechanisms to investigate the effect of UV radiation on vegetation foliage. Following reports of a 17% increase in decomposition rates in oak (Quercus robur) due to increased UV, which were later ascribed to changes in cell wall carbohydrate extractability, this study investigated the effects of decreased UV levels on ash (Fraxinus excelsior), a fast-growing deciduous tree species. A field experiment was set up in Surrey, UK, with ash seedlings growing under polytunnels made of plastics chosen for the selective transmission of either all UV wavelengths, UV-A only, or no UV. In a subsequent field decomposition experiment on end-of-season leaves, a significant increase of 10% in decomposition rate was found after one year due to removal of UV-B. However, no significant changes in cell wall composition were found, and a sequential extraction of carbohydrate with different extractants suggested no effects of the UV treatments on cell wall structure. Meanwhile, the first observations of aerobic production of methane from vegetation were reported. Pectin, a key cell wall polysaccharide, was identified as a putative source of methane, but no mechanism was suggested for this production. This study therefore tested the effect of UV irradiation on methane emissions from pectin. A linear response of methane emissions against UV irradiation was found. UV-irradiation of de-esterified pectin produced no methane, demonstrating esters (probably methyl esters) to be the source of the observed methane. Addition of ROS-scavengers significantly decreased emissions from pectin, while addition of ROS without UV produced large quantities of methane. Therefore, this study proposes that UV light is generating ROS which are then attacking methyl esters to create methane. The study also demonstrates that this mechanism has the potential to generate several types of methyl halides. These findings may have implications for the global methane budget. In an attempt to demonstrate ROS generation in vivo by UV irradiation, radio-labelling techniques were developed to detect the presence of oxo groups, a product of carbohydrate attack by ROS. Using NaB3H4, the polysaccharides of ash leaflets from the field experiment were radio-labelled, but did not show any significant decrease in oxo groups due to UV treatments. However, UV-irradiation of lettuce leaves showed a significant increase in radio-labelling, suggesting increased UV irradiation caused an increase in the production of ROS. The study shows that the use of this radio-labelling technique has the potential to detect changes in ROS production due to changes in UV levels and could be used to demonstrate a link between ROS levels and methane emissions.

The biological control agent Pseudomonas chlororaphis PA23 is effective at protecting Brassica napus (canola) from the necrotrophic fungus Sclerotinia sclerotiorum via direct antagonism. Despite the growing importance of biocontrol bacteria in protecting crop plants from fungal pathogens, little is known about how the host plant responds to bacterial priming on the leaf surface and certainly nothing about global changes in gene activity in the presence and absence of S. sclerotiorum. PA23 priming of mature canola plants reduced the number of lesion-forming petals by 90%. Global RNA sequencing of canola tissue at the host-pathogen interface showed a 16-fold reduction in the number of genes uniquely upregulated in response to S. sclerotiorum when pretreated with PA23. Upstream defense-related gene patterns suggest MAMP-triggered immunity via surface receptors detecting PA23 flagellin and peptidoglycans. Although systemic acquired resistance (SAR) was induced in all treatment groups, a response centered around a glycerol-3-phosphate (G3P)-mediated pathway was exclusively observed in canola plants treated with PA23 alone. Activation of these defense mechanisms by PA23 involved production of reactive oxygen species as well as pronounced thylakoid membrane structures and plastoglobule formation in leaf chloroplasts. PA23 therefore primes defense responses in the plant through the induction of unique local and systemic regulatory networks. / October 2016

Morphine alkaloids and their derivatives are pharmaceutically important substances. Huge production and consumption of these compounds predetermines them to be significant pollutants in the environment. Some of them have been detected in surface waters. The aim of this study was to characterize effects of morphine alkaloids on the physiology of three model organisms: Agrobacterium sp. R89-1, Escherichia coli XL-1 (Blue), and Raoultella sp. kDF8, and elucidation of the mechanisms leading to toxicity. The biotransformation potential and utilization ability were characterized for model organisms. It was demonstrated that the microorganism Agrobacterium sp. R89-1 is capable of rapid biotransformation of codeine to its 14-OH derivatives. The manifestation of morphine compounds toxic effects for the strain R89-1 is the highest. In contrast, microorganism Raoultella sp. KDF8 is able to utilize codeine as a carbon and energy source. The accumulation of 14-OH-derivatives was not observed. Escherichia coli XL-1 (Blue) is not able to biotransform or utilize codeine. Α, β-unsaturated ketones (morphinone, codeinone, 14-OH-morphinone and 14-OH-codeinone) were found as a most toxic intermediates of codeine metabolism. Bacterial cell growth (strains R89-1 and KDF8) in the presence of codeine is characteristic with...

O dinoflagelado unicelular marinho fotossintetizante Gonyaulax polyedra tem sido utilizado como modelo para o estudo de relógios biológicos. Neste organismo já foram descritos os ritmos de: migração vertical, divisão celular, atividade de superóxido dismutase e nitrato redutase, bioluminescência e capacidade fotossintética. Investigamos a variação circadiana dos pigmentos carotenóides e de RuBisCo II e PCP, as quais estão intimamente ligadas ao processo fotossintético. Experimentos de supressão de espécies reativas de oxigênio (EROs) por carotenóides foram preparados e mostraram que extratos de carotenóides de G. polyedra são capazes de suprimir o O2(1Δg) (oxigênio singlete) in vitro confirmando o importante papel destes no controle das EROs nestas algas. Os extratos metanólicos apresentaram vários pigmentos, tais como clorofila a, β-caroteno e peridinina em diferentes concentrações. A peridinina representa 80 % do total de carotenóides enquanto que o β-caroteno somente 4%. As análises dos cromatogramas de HPLC mostraram que a razão peridinina/clorofila a não varia ao longo de 24 h porém, por outro lado, o β-caroteno apresenta uma variação significativa na sua quantidade, com níveis duas vezes maiores no meio do dia em comparação com os níveis no meio da noite. Esta variação é conservada mesmo quando as células são mantidas em condições de luz constante. A curva de dose-resposta para a síntese de β-caroteno induzida pela luz mostra uma resposta linear com 45 minutos de exposição a luz branca. A indução é máxima quando utilizamos as células do meio período da noite (CT 18) que após esta exposição apresentam níveis de β-caroteno semelhantes as células do meio do dia. Esta alteração de fase no CT 18 sugere que este pigmento pode ser um dos compostos-captadores de luz envolvidos no mecanismo de ajuste de fase por luz em G. polyedra. Culturas de G. polyedra do meio da noite foram expostas à diferentes irradiações (azul, vermelha e verde) e os seus pigmentos extraídos e analisados. Em outra série de experimentos, as células foram mantidas durante o período de claro (12: 12 h) sob diferentes irradiações (vermelha, verde e azul) por 36 horas e os seus pigmentos analisados. Os resultados sugerem que a síntese foto-induzida e a oscilação circadiana do β-caroteno estão ligadas a um fotorreceptor de luz azul/ verde. Nas condições utilizadas não foram observadas variações significativas no conteúdo protéico da RuBisCo II e da PCP ao longo do dia. As análises de RNA total da RuBisCo II mostram que não há variação nos seus níveis quando as células são coletadas no meio do dia e no meio da noite. Quando expostas a condições adversas, G. polyedra apresenta a capacidade de encistar. Embora se conheça bem este mecanismo de defesa, existem poucas informações sobre o estado fisiológico destas células. Células encistadas induzidas por dias curtos apresentam uma alteração na composição de pigmentos com diminuição nas quantidades de β-caroteno e de clorofila a e aumento da quantidade de peridinina, indicando um rearranjo do aparato fotossintético nesta situação, com a peridinina desempenhando um papel mais estrutural. Em consequência, embora o conteúdo protéico de RuBisCo permaneça inalterado, os níveis protéicos de PCP se encontram diminuídos nas células encistadas. / Gonyaulax polyedra, a marine dinoflagellate which has been used as a model to study the biological clock, displays numerous circadian processes, such as bioluminescence, cell aggregation, cell division, superoxide dismutase and nitrate reductase activities and photosynthesis. In this alga, the photosynthesis is maximal in the middle of the day and minimal in the middle of the night. We investigated the pigments content and the amounts of two proteins related to the photosynthesis: ribulose- 1,5- bisphosphate carboxylase/ oxygenase form II (RuBisCo II) and peridinin: chlorophyll a: protein (PCP) in a 24 h cycle. Using the thermal decomposition of 1,4-dimethylnaphtalene endoperoxide, it was shown that the carotenoids could act as effective quenchers of synglet oxygen in G. polyedra. G. polyedra pigments were extracted every three hours over 24 hours. The amounts of peridinin and chlorophyll a remain constant over the day while the levels of β-carotene oscillate, being two times higher at the day than at the night phase. This variation persists when the cells were kept under constant dim light. The dose-response curve for light-induced β-carotene synthesis showed a linear response up to 45 minutes of light exposure, after which night-phase cells contained the same levels of β-carotene as day-phase cells. Cells exposed to light pulses at different times displays the highest β-carotene induction in the middle of the night. This may suggest that β-carotene may be one of the light-harvesting compounds involved in the light induced phase-shift in Gonyaulax polyedra. To identify which was the photoreceptor involved in β-carotene synthesis, cell of the middle of the night-phase (CT 18) were exposed for 45 minutes to different irradiations (red, blue and green) and their pigments extracted and analysed. Also, cells were grown under red, blue and green light during the light phase (12 h light: 12 h dark ) for 36 hours and their pigments analysed. The results suggested that the circadian oscillation and the photoinduced response synthesis of β-carotene, are related to a blue light receptor. The amounts of RuBisCo II and PCP do not change over the circadian cycle when the cultures were grown under constant dim light. The levels of these proteins also remain constant when cells were kept under ither white light or different light qualities (red, blue and green ) in light: dark (12: 12 h) regime. The G. polyedra RuBisCo form II transcrits levels are the same in middle-day and middle-night cells, suggesting a post-translational control for this enzyme in this organism. Adverse environmental conditions elicit the encystment of G. polyedra. Our results showed an alteration in pigment composition of cysts. An increase in peridinin levels and a decrease in β-carotene and chlorophyll a content were observed. Although RuBisCo form II protein levels remained constant, there was a reduction in the amounts of PCP in cysts. This suggests an important role in thylakoids structure stabilizer for free peridinin.