The DNA glycosylases function in the first step of the base excision repair (BER) process, that is responsible for removing base lesions resulting from oxidation, alkylation or deamination. The DNA glycosylases that recognize oxidative base damage fall into two general families: the Fpg/Nei family and the Nth superfamily. Based on protein sequence alignments, we identified four putative Fpg/Nei family members as well as a putative Nth protein in Mycobacterium tuberculosis H37Rv, the causative agent of tuberculosis. While Fpg proteins are widely distributed among the bacteria and plants, Nei homologs are sparsely distributed across phyla, and are only found in γ-proteobacteria, actinobacteria and metazoans. Interestingly, M. tuberculosis H37Rv harbors two proteins (Rv2464c and Rv3297) from the Nei clade and two (Rv2924c and Rv0944) from the Fpg clade. All four Fpg/Nei proteins were successfully overexpressed by using a novel bicistronic vector, which theoretically prevented stable mRNA secondary structure(s) surrounding the translation initiation region (TIR) thereby improving translation efficiency. Additionally, MtuNth (Rv3674c) was also overexpressed in soluble form. The substrate specificities of the purified enzymes were characterized in vitro with oligonucleotide substrates containing single lesions. Some were further characterized by gas chromatography/mass spectrometry (GC/MS) analysis of products released from γ-irradiated DNA. MtuFpg1 (Rv2924c) has a substrate specificity similar to that of EcoFpg and recognizes oxidized purines. Both EcoFpg and MtuFpg1 are more efficient at removing spiroiminodihydantoin (Sp) than 7,8-dihydro-8-oxoguanine (8-oxoG); however, MtuFpg1 has a substantially increased opposite base discrimination compared to EcoFpg. The Rv0944 gene encodes MtuFpg2, which contains only the C-terminal domain of an Fpg protein and has no detectable DNA binding activity or DNA glycosylase/lyase activity and thus appears to be a pseudogene. MtuNei1 (Rv2464c) recognizes oxidized pyrimidines not only on doublestranded DNA but also on single-stranded DNA. It also exhibits uracil DNA glycosylase activity as well as weak activity on FapyA and FapyG. MtuNth recognizes a variety of oxidized bases, such as urea, 5,6-dihydrouracil (DHU), 5-hydroxyuracil (5- OHU), 5-hydroxycytosine (5-OHC) and methylhydantoin (MeHyd) as well as FapyA, FapyG and 8-oxoadenine (8-oxoA). Both MtuNei1 and MtuNth excise thymine glycol (Tg); however, MtuNei1 strongly prefers the (5R) isomers of Tg, whereas MtuNth recognizes only the (5S) isomers. The other Nei paralog, MtuNei2 (Rv3297), did not demonstrate activity in vitro as a recombinant protein, but when expressed in Escherichia coli, the protein decreased the spontaneous mutation frequency of both the fpg mutY nei triple and nei nth double mutants, suggesting that MtuNei2 is functionally active in vivo recognizing both guanine and cytosine oxidation products. The kinetic parameters of the MtuFpg1, MtuNei1 and MtuNth proteins on selected substrates were also determined and compared to those of their E. coli homologs. Since pathogenic bacteria are often exposed to an oxidative environment, such as in macrophages, our data, together with previous observations, support the idea that the BER pathway is of importance in protecting M. tuberculosis against oxidative stress, as has been observed with other pathogens .

Oxidative stress is caused when there are more reactive oxygen species (ROS), than antioxidants to scavenge them, resulting in damage to cellular components. It has been implicated as a major player at multiple points in the disease process of Parkinson’s disease (PD) and many other conditions. For example, evidence suggests oxidative damage to the α-synuclein protein may affect its aggregation propensity. In addition, α-synuclein may increase ROS production. However, how this oxidative stress relates to neurodegeneration is not known. Therefore, there is a need for models of α-synucleinopathies and tools to assess the involvement of oxidative stress in the disease process. In order to model α-synucleinopathies, overexpression of the α-synuclein protein was used. A BacMam viral expression system containing human α-synuclein was generated and used to assess toxicity. α-Synuclein overexpression in undifferentiated or differentiated SH-SY5Y cells failed to show toxicity. However, the stability of α-synuclein protein expression and the cell line used may have influenced in the lack of toxicity. The current work provides important guidance for future experimental design. Flavonoids are found in plants and have antioxidant capability. AO-1-530 is a synthetic compound with a flavonoid head group and a long hydrocarbon tail. It is highly cell permeable and localises to the mitochondria. In order to investigate its protective properties, toxin-induced oxidative stress cell assays were established. AO-1-530, in the low micromolar range, was protective against high doses of tert-butyl hydroperoxide (tBHP), whereas natural antioxidants, such as myricetin and quercetin, showed limited protection or required at least 10-fold higher concentrations to achieve similar protection. The ability of AO-1-530 to directly scavenge radicals was assessed cell-free in solution and in a cell-based assay. In solution the mechanism of action was investigated by electron paramagnetic resonance (EPR) spectroscopy. AO-1-530 had similar scavenging ability to myricetin, but was a slightly stronger scavenger than quercetin. The intracellular scavenging ability was quantified by CellROX® Deep Red live imaging. Although the compounds had similar cell-free scavenging abilities, AO-1-530 significantly out-performed both myricetin and quercetin in the intracellular assay, suggesting the mitochondrial localisation is critical to its highly protective properties. AO-1-530 is a powerful, novel tool to study the involvement of oxidative stress in diverse disease models.

Oxidative stress can result in changes to many biomolecules and also affect their activities. We are interested in protein carbonylation, a type of unnatural oxidation which has been associated with numerous degenerative disease states and is also a consequence of the natural aging process. Protein carbonyls are stable species, but countless analytical barriers exist in terms of their identification. Thus, the main goal of this work was to develop and optimize analytical methods that could be used to help us better understand which, where, and how proteins are being carbonylated. Initial studies involved method validation for carbonylating, tagging, and enriching the model protein human serum albumin (HSA). We have developed a reproducible method of producing carbonylated protein in vitro in which HSA is treated with acrolein to carbonylate cysteines, histidines, and lysines. Protein carbonyls are compatible with various affinity labels and enrichment techniques. We strived to learn more about the efficiencies of various biotin affinity labels and avidin enrichment techniques using quantitative assays and mass spectrometry. Results showed a preference for different affinity labels based on their chemical properties and suggested that monomeric columns are selective for particular peptides. Most recently, method development and validation work was done involving a cleavable biotin tag that enables both enrichment and identification of protein carbonylation modification sites. This affinity tag offered the highest labeling efficiency of all tags tested in the past and greater coverage of modification sites than biotin hydrazide reagents. We applied our analytical methods to two sets of human blood samples. The first sample set was plasma taken from chronic kidney disease (CKD) patients. No carbonylation patterns were elucidated, but this project marked the beginning of blood analyses in which existing protocols were adapted to blood samples. The second sample set was serum/plasma taken from patients with traumatic injuries. We effectively applied our analytical methods to these sample sets and were able to visualize and quantitate temporal protein carbonylation patterns via Western blotting and iTRAQ-based mass spectrometry experiments. ProteoMiner experiments proved successful in that we were able to identify a larger and more diverse amount of carbonylated proteins via mass spectrometry.

Periodontal diseases are bacterially induced, inflammatory diseases which are responsible for loss of alveolar bone and connective tissue supporting the teeth which results in loss of teeth. Gram negative anaerobic bacteria are highly associated with these diseases. One of them is Porphyromonas gingivalis belonging to the phylum Bacteroidetes. Infection by P. gingivalis is recurrent after physical removal of the bacteria from the oral cavity and even after antibiotic treatment as development of resistance is not rare. Hence complete understanding the biology of this bacterium is of significance. This gram negative obligate anaerobe, being aerotolerant, manages to survive inside the oral cavity, where oxidative stress is ubiquitous. Genome sequence of P. gingivalis shows the presence of a transcriptional regulator OxyR which is a homologue of OxyR present in E. coli. P. gingivalis OxyR induces the expression of antioxidant defense genes like sod, ahpC-F, dps to protect the bacteria from oxidative stress. Expression of P. gingivalis OxyR regulon is not very well understood. Microarray studies carried out in our lab using P. gingivalis W83 to study gene regulation by OxyR, indicated that several genes in P. gingivalis are co-regulated by iron-and OxyR. Literature also supports that in iron deplete conditions genes involved in oxidative stress are down-regulated. These studies formed the basis of our hypothesis that OxyR might regulate the genes in P. gingivalis in an iron dependent manner. To study the mechanism of regulation by P. gingivalis OxyR and to determine whether OxyR regulation is iron dependent, two approaches were applied - in vitro characterization of binding and in vivo characterization. First step of in vitro characterization was to perform CHIP-chip assay to determine OxyR-binding sites present on the genomic DNA of P. gingivalis. As this assay was performed under completely anaerobic conditions, the target fragments to which OxyR was found to bind during this assay were not same as reported in literature. These and the fragments reported in literature were used for EMSA. EMSAs carried out using crude cell lysates and in vitro OxyR protein preparations showed expected results but the results were not reproducible. In vivo expressed and purified P. gingivalis OxyR never bound to the target fragments used. Preparation of a stable protein preparation and improvement in the parameters of EMSA is very important to further investigate the binding in vitro. The second approach is based on in vivo characterization of binding. This requires tagging the P. gingivalis OxyR at its C-terminus with fluorescent protein to observe its binding to the target DNA sequences. Fluorescently tagged OxyR, is expected to emit fluorescence from a highly localized area to produce sharp fluorescent spots when it is bound to its target sequences. Unbound OxyR is expected to emit a fluorescent signal which is spread over the entire area of the cell. This technique will help to determine the conditions under which OxyR binds to its target DNA sequences. This provides a means to confirm the results obtained from in vitro characterization instead of just extrapolating them.

It is well established that free radical mediated oxidative stress plays a critical role in aging and age-related diseases. Among the post-translational protein modifications, carbonylation has attracted a great deal of attention due to its irreversible and irreparable nature. Despite the fact that protein carbonylation is associated with a series of physiological and pathological processes, there are still issues to be clarified such as why certain proteins are more vulnerable to modification, what are the locations of the protein modifications, and how does the nature of the oxidant affect the preferred site of modification. In this study, we will seek an answer to these questions and examine the global effect of oxidative stress on protein abundance. The study embraces three distinct specific aims. In the first, methods are developed for identifying sites of protein carbonylation. In the second specific aim, these methods are used to identify carbonylaytion sites in model proteins subjected to chemical oxidants. In the third aim, the focus is on a model organism, C. elegans, subjected to paraquat-induced oxidative stress. This is exploratory work and mass spectrometry is used to assess the impact of oxidative stress on the mitochondrial proteome.

The aim of this study was to observe levels of oxidative DNA damage in patients with multiple injuries in correlation with the nutritional support that the patients have received during their hospital stay. Oxidative DNA damage was evaluated in two periods of time, first evaluation was performed during standard nutritional support according to the ESPEN guidelines. Second evaluation was performed after a change in nutrition according to individual parameters of metabolism and utilization of nutritional components based on indirect calorimetric measurements. This study included 6 patients with multiple injuries hospitalized in the Intensive Care Unit 1 at the Department of Surgery, University Hospital in Hradec Kralove. In this experiment DNA isolated from peripheral lymphocytes was used to evaluate oxidative DNA damage. This DNA was analyzed using the Comet Assay method. The enzymatic version of the Comet Assay was used to determine the oxidative damage of purines and pyrimidines, and the alkaline version was used for detection of single strand breaks. Mann-Whitney test was used for statistic evaluation the difference between both measuremetns, correlation analysis for relations between Comet Assay results and clinical parameters. Significant correlations between a total amount of nutrients given...

The aim of this work was to study the expression of proteins involved in reactions in which harmful free radicals are degraded in an organism. was observed difference between the expression of selected myocardial proteins in non-influenced animals, animals who were treated with low dosage of morphine (0.1 mg/kg/day or 1 mg/kg/day), and animals administered high dosage of morphine (10 mg/kg/day). Low dosages were administered for 28 days and high dosage for 10 days. In addition, the effect of abstinence lasting one week was assessed after cessation of morphine administration (1 mg/kg/day). Morphine at low dosage (0.1 mg/kg/day) increased levels of glutathion peroxidase-1/2, which may be considered as one of the possible consequences of the ongoing oxidative stress. There were no significant differences in glutathion peroxidase-6 expression. Next aim of this work was to study the expression of antioxidant enzymes. These experiments were carried out on myocardial preparations from the animals treated with a constant dosage of morphine (10 mg/kg/day) for 10 days. Samples from these animals were used for measuring the total antioxidant capacity of the left and right ventricles. These samples were also used for determination of concentration of the oxidative stress marker 8-isoprostane. We also aimed to...

Four new synthetic methods employing an oxidative dearomatization - rearomatization strategy are presented. In Chapter 2, a new oxidative dearomatization - radical cyclization - rearomatization approach to form fused oxygen-containing heterocycles is presented. Origins, design, reaction, and optimizations are discussed. In Chapter 3, meta-selective alkylation of catechol mono-ethers is described employing an oxidative dearomatization - radical addition - rearomatization approach using trialkylboranes as source of alkyl radicals. In Chapter 4, a metal-free method to synthesize fluorinated indoles from aniline starting materials is described. Chapter 5 lays the groundwork for para-selective functionalization of catechol mono-ethers. Chapters 6 and 7 highlight the work related to pharmaceutical drug analyses. Chapter 6 presents the FDA approved drugs organized in Disease Focused Posters. Chapter 7.1 and 7.2 present the drug analysis of Sulfur- and Fluorine-Containing Drugs, and Nitrogen-Heterocycle Containing Drugs, respectively.

Renal ischemia/reperfusion (I/R) is a main cause of acute kidney injury (AKI) and delayed graft function after renal transplantation. Previous studies in human and experimental models have identified that inflammation and oxidative stress are two key players in renal I/R injury. However, the underlying mechanisms remain speculative. The overall objective of the study was to investigate the biochemical and molecular mechanisms of I/R-induced renal injury and the effect of tyrosol supplementation on I/R-induced kidney oxidative stress damage. In the present study, renal I/R was induced in Sprague-Dawley rats and in a human kidney proximal tubular cell line. A significantly elevated expression of pro-inflammatory cytokine expression (MCP-1, IL-6) was observed. There was a significant decrease in mRNA and protein levels of two hydrogen sulphide (H2S)-producing enzymes, CBS and CSE, with a concomitant reduction of glutathione and H2S production. In the cell culture model, hypoxia–reoxygenation of proximal tubular cells led to a decrease in CBS and CSE expression and an increase in pro-inflammatory cytokine expression. Supplementation of glutathione or H2S donor (NaHS) effectively abolished cytokine expression in tubular cells. Experiments were conducted to detect oxidative stress markers. It was demonstrated that there was a significant increase in peroxynitrite formation and lipid peroxidation in the kidney after I/R insult, which might be caused by the elevation in nitric oxide (NO) metabolites and inducible nitric oxide synthase (iNOS). Administration of tyrosol, a natural phenolic compound, reduced peroxynitrite formation, lipid peroxidation and the level of NO metabolites via inhibiting NF-B activation and iNOS expression. Tyrosol treatment improved kidney function and had a protective effect against I/R-induced AKI. The present study has clearly demonstrated that (1) there is a reduction of H2S production via inhibition of CBS and CSE expression, which contributes to increased pro-inflammatory cytokine expression in the kidney and in tubular cells upon I/R insult; (2) restoration of endogenous H2S production would be of therapeutic value in regulating inflammatory response in I/R-induced kidney injury; (3) tyrosol treatment has a beneficial effect against renal I/R-induced oxidative stress, in part, through its inhibition on NF-B activation and iNOS-mediated NO production. / May 2016

The cannabinoid partial agonist BAY 59-3704 has been identified as an attractive target to explore structure-activity relationships at cannabinoid receptors for the development of a therapeutic agent for psychostimulant addiction. This thesis will describe the studies associated with the optimization of a palladium-catalyzed oxidative ring closure reaction for the synthesisof dibenzofuran analogues from substituted diaryl ethers. These dibenzofurans are viewed as rigid analogues of BAY 59-3704 and will provide useful information about molecular interactions at cannabinoid receptors. The scope and limitations of the palladium-catalyzed oxidative ring closure reaction as it relates to the synthesis of the target dibenzofuran analogues will be presented.