Selenium is a required micronutrient in mammalian cells. It is incorporated in the form of selenocysteine into selenoenzymes such as glutathione peroxidase and thioredoxin reductase, and is absolutely required for activity. Thioredoxin reductase is necessary for reduction of oxidized thioredoxin and therefore plays a major role in maintaining the redox status of the cell. Glutathione peroxidase is responsible for reducing peroxides into their corresponding alcohols and water. Together, these selenoenzymes constitute a significant part of the cell's arsenal to defend itself against oxidative stress. Exogenous sources of oxidative stress, such as UV radiation, are capable of generating reactive oxygen species (ROS). Elevated levels of ROS can lead to covalent modifications of lipids, nucleic acids, and proteins within a cell. This damage has been implicated in the development of cancer and degenerative diseases. As the skin is the first level of defense for UV radiation, skin cancer is an obvious concern. Previous studies have demonstrated a protective effect against UV-induced cytotoxicity when selenium compounds were administered to skin cells in cell culture models. Topical selenium application to mice has also been shown to reduce UV damage to skin. Although a variety of chemical forms of selenium are available in nutritional supplements, the efficiency by which they are used for selenoprotein synthesis varies greatly. It is debated within the selenium research community which form is best for use as a supplement. In this study, we have focused on a selenotrisulfide derivative of alpha-lipoic acid (LASe). We have examined its utilization for selenoprotein synthesis through radiolabeling studies (75Se) in a human keratinocyte cell line (HaCaT). We have determined that is incorporated into selenoproteins with nearly the same efficiency as selenite and L-selenocysteine. We have also determined that LASe is far more efficient as a supplement in cell culture than selenate or L-selenomethionine, two forms of selenium commonly used as supplements. LASe was also found to protect HaCaT keratinocytes from UV- induced cytotoxicity. Cells pretreated with LASe and exposed to 500J/m2 and 750J/m2 of broadband (UVA/UVB) UV radiation showed greater survival than untreated controls in a dose –dependent manner. Cells pre-treated either with lipoic acid or selenium in the form of selenite alone also observed protection. Nonetheless, these finding are significant given that LASe was previously shown to penetrate the skin better than other forms of selenium. These results indicate that LASe has the potential for use as a topical antioxidant upon further testing in animal studies.

Arsenic exposure, through various routes, is associated with the development of cancer of the skin, lung, liver, kidney, and bladder. Treatment of cells in culture with trivalent arsenicals has been shown to increase reactive oxygen species (ROS). In particular, monomethylarsonous acid (MMAIII), a trivalent metabolite of arsenite, is highly cytotoxic and possibly carcinogenic. Three trivalent arsenicals; arsenite, arsenic trioxide (ATO), and MMAIII, are also known inhibitors of the selenoprotein thioredoxin reductase (TrxR). Selenium, an essential micronutrient in mammals, is needed in the form of selenocysteine for activity of this enzyme and other selenoproteins. TrxR is part of a key component of the cell's ability to defend against ROS. It has been speculated that TrxR is also involved directly in selenium metabolism, but this has yet to be demonstrated in vivo. The promoter region of the gene encoding the cytosolic TrxR (TrxR1) also contains an antioxidant responsive element (ARE). The ARE is activated by the transcription factor, Nrf2, which is governed by the Nrf2/Keap1 response, and can be triggered by certain oxidants. ATO and arsenite both inhibited incorporation of selenium into selenoproteins. Auranofin, a gold chemotherapeutic inhibitor of TrxR1, also inhibited selenoprotein synthesis. These results seem to support the hypothesis that TrxR1 is needed for selenoprotein synthesis. However, siRNA mediated reduction of TrxR1 did not block incorporation of selenium into selenoproteins. It is likely that ATO and auranofin are forming As-Se and Au-Se complexes, respectively. We also found that exposure of primary lung fibroblasts (WI-38) to MMAIII led to increased synthesis of TrxR1. This increase was dependent on the activation of transcription of the TrxR1 gene, specifically mediated through the ARE element. These results indicate exposure to MMAIII induces the Nrf2 response. The results obtained in these studies aid in both our understanding of the carcinogenic potential of arsenic as well as give new insight into the mechanism of action of emerging cancer drugs.

Selenoproteins are unique proteins in which selenocysteine is inserted into the polypeptide chain by highly specialized translational machinery. They exist within all three kingdoms of life. The functions of these proteins in biology are still being defined. In particular, the importance of selenoproteins in pathogenic microorganisms has received little attention. We first established that a nosocomial pathogen, Clostridium difficile, utilizes a selenoenzyme dependent pathway for energy metabolism. Following this initial characterization, we demonstrate that this pathway is linked to production of toxins by this organism. Finally, we show that interruption of selenium metabolism is a viable pathway for development of antimicrobials against this, and other selenoprotein dependent pathogens. We investigated whether Stickland reactions (paired amino acid fermentation) might be at the heart of C. difficile's bioenergetic pathways. Growth of C. difficile on Stickland pairs yielded large increases in cell density in a limiting basal medium, demonstrating these reactions are tied to ATP production. Selenium supplementation was required for this increase in cell yield. Analysis of genome sequence data reveals genes encoding the protein components of two key selenoenzyme reductases; glycine and D-proline reductase. These selenoenzymes were expressed upon addition of the corresponding Stickland acceptor (glycine, proline or hydroxyproline). Purification of the selenoenzyme D-proline reductase revealed a mixed complex of PrdA and PrdB (SeCys containing) proteins. D-proline reductase utilized only D-proline but not L-hydroxyproline, even in the presence of an expressed and purified proline racemase. The enzyme was found to be independent of divalent cations, and zinc was a potent inhibitor. These results show that Stickland reactions are key to the growth of C. difficile and that the mechanism of D-proline reductase may differ significantly from similar enzymes from non-pathogenic species. C. difficile pathogenesis is due to the production of toxins, A and B, members of the large clostridial cytotoxin family. Previous studies have shown that toxin production by this organism is influenced by the composition of the growth medium. We examined the impact of Stickland acceptor amino acids (Stickland acceptors; glycine, proline and hydroxyproline) on growth kinetics and yield, protein synthesis, toxin production and gene expression. Although addition of Stickland acceptors moderately increases growth yield and total protein synthesis, there does not appear to be a clear impact on entry into stationary phase. Glycine dramatically increases the amount of toxin released into the growth medium. Conversely, the addition of hydroxyproline suppresses toxin production. We examine possible mechanisms of regulation and demonstrate that CodY, a regulator of toxin gene transcription does not appear to mediate this effect. Given the importance of selenium dependent Stickland reactions to C. difficile growth and toxin production we aimed to examine the efficacy of blocking such pathways as a means of antimicrobial development. Selenide is the only known substrate for selenophosphate synthetase, the first enzyme involved in the specific incorporation of selenium into selenoproteins. We have identified a stable complex formed upon reaction of auranofin (a gold containing drug) with selenide in vitro. Auranofin potently inhibits the growth of C. difficile but does not similarly affect other clostridia that do not utilize selenoproteins to obtain energy. Moreover, auranofin inhibits the incorporation of radioisotope selenium (75Se) in selenoproteins in both E. coli, the prokaryotic model for selenoprotein synthesis, and C. difficile without impacting total protein synthesis. Auranofin blocks the uptake of selenium and results in the accumulation of the auranofin-selenide adduct in the culture medium. Addition of selenium in the form of selenite or L-selenocysteine to the growth media significantly reduces the inhibitory action of auranofin on the growth of C. difficile. Based on these results, we propose that formation of this complex and the subsequent deficiency in available selenium for selenoprotein synthesis is the mechanism by which auranofin inhibits C. difficile growth. The antimicrobial potential of blocking selenium metabolism is further demonstrated in the dental pathogen Treponema denticola. We show that auranofin blocks the growth this organism which also participates in Stickland fermentation. In addition, we provide evidence that the antimicrobial action of stannous salts against T. denticola is also mediated through inhibition of the metabolism of selenium. These studies clearly show that, at least in a subset of microbes that use selenium for the synthesis of selenoproteins, the need for this metalloid can be a useful target for future antimicrobial development.

Selenium (Se), an essential trace element, plays an important role in the normal function of a number of Se-dependent biological processes. Many studies have demonstrated that selenium deficiency in the body may contribute to an increased risk for certain neoplastic diseases (including colonic carcinoma, gastric carcinoma, pulmonary carcinoma and prostate carcinoma), as well as diseases of the cardiovascular, osseous, nervous systems and retardation of bone formation. However, at higher concentrations Se is cytotoxic. For these reasons it is desirable to have a means of monitoring selenium concentration in humans. The feasibility of measuring selenium in humans using the in vivo neutron activation analysis (IVNAA) technique was studied. For this purpose human hand tissue equivalent phantoms were prepared with varying amounts of selenium and irradiated by a low energy neutron beam produced by the 7Li(p,n)7Be reaction by employing the high beam current Tandetron accelerator. The counting data saved using the 4π NaI(TI) detection system in anticoincidence, coincidence and singles modes of detection were analyzed. The selenium was detected via the neutron capture reaction, 76Se(n,γ)77mSe, whereas calcium was detected through the 48Ca(n,γ)49Ca reaction. The peak areas of Se and Ca were computed and the Se concentrations were normalized to the Ca concentrations for various time segments of detection. The calibration lines were drawn between Se/Ca concentration and Se/Ca counts ratio. The minimum detection limits (MDL) were obtained and the inverse variance weighted mean value of MDL was finally calculated for three time segments. During the analysis of counting data it was also found that 18O is activated in water phantoms and becomes short lived radioactive 19O having T1/2=26.9 s. To the author’s best knowledge, this study for the first time presents the MDL value in terms of Se/Ca concentration for the human hand bone equivalent phantom obtained from in vivo neutron activation analysis and these results will provide a good basis for future investigations. / Thesis / Master of Science (MSc)

<p> A 19F nmr study has shown that SeO2F2, SeOF2 and SeOCl2 behave as bases (B) towards SbF5 forming the adducts, (SbF5)n•B where n = 1-5 and in which they are bonded to antimony through an oxygen. Structural information about these adducts was also obtained. Solutions of SbF5 in SeOF2 and SeOCl2 were also shown to contain the SbF-6 and cis and trans [SbF4 (B)2]+ ions. The order of basicity towards SbF5 for the following bases is, SeOCl2 > SeOF2 > SbF-6 > SOF2 > SeO2F2 > SO2ClF.</p> <p> A 77Se nmr study of the SeOCl2 solvent system has shown that the order of Lewis acidity for the following acids is, SbF5 ~ SO3 > SbCl5 > SnCl4 > SbCl3 > AsCl3.</p> <p> A new selenium oxyfluoride, SeOF4 has been identified and is shown to form an ionic adduct SeOF+3SbF-6 with SbF5.</p> <p> Polyselenium oxyfluorides, F(SeO3)nSeO2F, where n=1-3, were prepared and are found to have acyclic structures. The 77Se spectra of Se4^2+ and Se8^2+ were studied. </p> <p> Redistribution reactions between selenium and phosphorus halides and oxyhalides were studied using the nmr resonances of 19F, 31P and 77Se.</p> / Thesis / Doctor of Philosophy (PhD)

Background. Speed is getting more and more critical in modern Software Engineering to be able to respond to users’ expectations of product development. One practice that takes a significant amount of time in the process of releasing software to the customers is testing. It is a clear trend that organizations are increasing the amount of test automation compared to manual testing. However, manual testing is still prominent in GUI-based testing due to challenges interacting with a GUI from test scripts. One of the most prominent challenges is synchronizing test script execution with the system under test. Objectives. This research aims to compare static and dynamic synchronization of GUI-based tests. This comparison will be conducted by replicating an existing Selenium test suite using static synchronization to Playwright, which will use dynamic synchronization and run the test suites against a web-based application. These test suites will then be used to compare the two types of synchronization from the perspective of test execution efficiency, test output correctness, and maintenance cost.Methods. The research methodology we chose is experiment. We have chosen Selenium to represent static synchronization and Playwright to represent dynamic synchronization. We used an existing test suite in Selenium that was translated into Playwright. There are a total of 81 tests in the test suite. The test suites were used to compare test scripts that use static and dynamic synchronization from the perspective of test execution efficiency, test output correctness, and maintenance cost.Results. The data collected from the experiments shows that execution efficiency for test scripts using dynamic synchronization is significantly faster than static synchronization. A mean difference between the test suites showed a decrease of 87%. For defect identification, ten defects where used and both test suites managed to identify all of them, resulting in no difference could be found. Test maintenance cost was found that test scripts using dynamic synchronization had a positive effect with an average of 60% less time spent on maintenance.Conclusions. Based on the result, we found that test scripts using dynamic synchronization improved execution efficiency and maintenance costs without sacrificing the test output correctness.

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