Consideration is given to the use of the time differential perturbed directional correlation (TDPDC) technique in biomedical applications such as structural studies of the selenoproteins. Mathematical models of the perturbed directional correlations show that the application of the TDPDC technique to the study of the electric quadrupole interactions experienced by an ensemble of probe nuclei bound to a protein is capable of yielding the fraction of probe nuclei occupying each of a number of distinct types of binding site in the protein, the strength, symmetry and inhomogeneity of the nuclear quadrupole interaction occurring at each different type of site and, if the environment of the protein is a viscous solution, the diffusion constant for the protein in the solution, from which the associated correlation time and hence the radius of the protein may be derived. A time spectrometer employing a pair of barium fluoride (BaF2) scintillation counters was designed and constructed to allow TDPDC experiments to be performed. The optimum time resolution of the spectrometer was found to be 354+/-10 ps for the 1.173 and 1.332 MeV gamma-rays emitted in the decay of 60Co. The characteristics of the spectrometer were inferior to those of similar systems which are described elsewhere. A number of improvements to the spectrometer that was used in this work have been suggested. The manner in which various deviations from the ideal experimental arrangement affect both the observed perturbed directional correlation and the optimum methods of conducting TDPDC experiments has been carefully considered. Of particular importance was the effect of source decentring, since the employed apparatus does not allow the radioactive source to be precisely centred. It was found that the implications of source decentring are that the values of the parameters describing the perturbation of the directional correlation must be derived from a single TDPDC spectrum, as must those of a number of additional parameters which are of little interest. The viability of this procedure was investigated by carrying out a TDPDC study of the 356-81 keV gamma-gamma cascade in 133Cs, taking account of the interference from sum-coincidence effects and competing cascades. From the TDPDC spectrum that was obtained, the smearing effects of the finite time resolution of the spectrometer were partially removed by deconvolution, resulting in an unfolded time spectrum to which attempts were made to fit a suitable model. Although it was not possible to achieve a satisfactory fit, a number of potential remedies for the poor fit have been proposed. A TDPDC study of the 121-280 keV gamma-gamma cascade in 75As was performed for the purpose of assessing the feasibility of using the TDPDC technique in future investigations employing 75Se-labelled selenoproteins. Account was again taken of the interference from sum-coincidence effects and competing cascades. A bid to deconvolve the smeared TDPDC spectrum that was acquired was not successful, and so attempts were made to fit to the smeared spectrum a model which allowed for the finite time resolution of the spectrometer. The fitting proved to be problematic, indicating that it would be difficult to apply the TDPDC technique to an investigation of 75Se-labelled selenoproteins. However, this conclusion is perhaps unduly pessimistic because some of the problems that arose in the feasibility study will not be present in practice when a 75Se-labelled selenoprotein is used. The available evidence suggests that 73Se offers advantages over 75Se as a radiolabel in TDPDC studies of the selenoproteins.

Graduation date: 1997

Graduation date: 2004

Thesis (M.S. in animal sciences)--Washington State University, May 2008. / Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

Rat selenoprotein W (SeW) promoter activity was investigated using different concentrations of cadmium, copper, and zinc. Two fragments (404bp and 1265bp) of the SeW promoter, containing a single metal response element (MRE), were ligated into the multiple cloning site of a pGL3-Basic reporter plasmid. The constructs were transfected into cultured rat C6 (glial) and L8 (myoblast) cells and promoter activity measured by means of luciferase reporter gene fused to the SeW promoter fragments in the reporter plasmid. With post-transfection exposure of these cell lines to these metals, copper and zinc, but not cadmium, significantly increased promoter activity of the unmutated 1265bp (not 404bp) construct (p<0.05) only in the C6 cells. Mutation of the MRE sequence abolished promoter response to metal exposure but did not eliminate promoter activity. The results suggest that SeW expression in glial cells can be increased on exposure to copper and zinc and that this response is dependent on the MRE sequence present in the SeW promoter. To understand transcriptional regulation of the SeW gene, we used in vitro binding assays to identify transcription factors that may be involved in the transcriptional regulation of the SeW gene. Using protein from rat C6 (glial) cell nuclear extracts, oligonucleotides containing putative regulatory elements in the SeW promoter, and antibodies, we were able to show that the specificity protein 1(Sp1) transcription factor binds to the Sp1 consensus sequence in the SeW promoter as well as the MRE. However, the MRE, GRE, AP-1 and LF-A1 did not yield any specific binding. Although, competition analysis showed specific binding at the TFII-1 site, super-shift analysis using anti-TFII-1 antibody did not yield any super-shifted band. Therefore the SeW gene may be a target for Sp1 whose interaction with the SeW promoter may activate or repress the transcription of SeW. / Graduation date: 2003

Proteins containing selenocysteine (selenoproteins) have been found to exist in organisms from all domains of life. Selenoproteins are important for many in vivo processes such as the removal of reactive oxygen containing species (ROS), redox disulfide shuffling reactions, and pro-hormone activation. Structurally and functionally analogous to cysteine, selenocysteine's lower pKa appears to be the defining chemical difference between these two amino acids. Using a single-molecule electrical recording technique, rate constants for the reaction of selenocysteine with small molecule disulfides were obtained over a pH range of 6 - 10. Analogous single molecule ~riments carried out ~ .. - using cysteine, revealed that, after correcting for the ratio of selenolate to selenol and thiolate to thiol based on the pKa of each amino acid, the nuc1eophilicity of selenocysteine was comparable to that of cysteine. The selenium atom of the selenylsulfide bond was found to be substantially more electrophilic than a sui fur atom of the analogous disulfide bond and the leaving group ability of the selenolate of selenocysteine compared to the thiolate of cysteine were found to be comparable. Another biologically relavant interaction that occurs in vivo is the reaction between selenocysteine and organoarsenic (Ill) molecules. It is known that arsenic (Ill) compounds are toxic to organisms, and that this toxicity stems from the ability to coordinate to the thiol and selenol groups of the cysteine and selenocysteine residues within proteins. The reaction of selenocysteine with an organoarsenic species was investigated at the single molecule level over the pH range 6.5 - 8.5. By carrying out an analogous reaction between cysteine and the organoarsenic (Ill) species, it was found that selenocysteine and cysteine exhibit similar reaction rates. The organoarsenic reagent could exist in a range of different protonation states in solution and it was concluded that the rate of reaction was governed by the equilibrium of the arsenic molecule, where only some of the forms were reactive towards the selenocysteine and cysteine groups.

No description available.

O aminoácido selenocisteína (Sec) representa a principal forma biológica de selênio sendo requerida uma complexa maquinaria molecular para sua síntese e incorporação co-traducional em selenoproteínas. A Seril-tRNA sintetase (SerRS) inicia essa via, aminoacilando o Ser-tRNASec (SelC) com uma serina e também aminoacila os tRNAsSer. Sendo assim, um dos focos deste trabalho foi estudar a interação da SerRS de Trypanosoma brucei (T. brucei) com os tRNAsSer e o SelC utilizando a técnica de anisotropia de fluorescência para determinar suas constantes de dissociação. Em Kinetoplastidae, além da via de síntese de selenocisteína, há três selenoproteínas: SelT, SelK e SelTryp. No entanto, pouco se sabe a respeito das mesmas, sendo o estudo destas selenoproteínas o outro foco deste trabalho. Os fragmentos de DNA que codificam estas selenoproteínas foram subclonados em vetor de expressão pET 28a e 29a para posterior uso em células de Escherichia coli (E. coli). Para as proteínas SelK e SelTryp os ensaios de expressão apresentaram resultados insuficientes para dar continuidade aos experimentos planejados, pois o rendimento foi baixo e a purificação não foi possível. Já com a proteína SelT, devido à grande dificuldade encontrada para tornà-la solúvel, descobriu-se, no decorrer do trabalho, que tratava-se de uma proteína de membrana, ocasionando mudanças de alguns objetivos previamente propostos e consequentemente busca por novas estratégias. Conseguiu-se expressá-la na de forma solúvel e purificá-la por cromatografias. Ensaios realizados no SEC-MALLS mostraram uma estabilidade do complexo proteína-detergente. Com a TbSerRS é possível concluir que a organização de especificidade de ligação da enzima com seus ligantes se dá crescentemente: SelC>tRNASer7>tRNASer3a>tRNASer3b. E com as selenoproteínas do T. brucei faz-se necessários novas contruções para SelK e SelTryp e dar continuidade aos experimentos com a SelT tentando cristalizá-la, já que prototolo para a obtenção do complexo proteína-detergente está montado e estabilizado. / Selenocysteine (Sec) amino acid is the major biological form of selenium and requires a complex molecular machinery for its synthesis and co-translational incorporation into selenoproteins. The Seryl-tRNA synthetase (SerRS) starts this biosynthesis and matches the tRNASec (SELC) with a serine and the tRNAsSer, therefore the focus of this study is on SerRS of Trypanosoma brucei (T. brucei) and tRNAsSer and SELC interactions, with fluorescence anisotropy techinic to determinat dissociation constants. Three selenoproteins, namely SelT, SelK and SelTryp, besides the route of selenocysteine synthesis there be in Kinetoplastidae. DNA fragments that coding for these selenoproteins were subcloned in 28a and 29a to use into Escherichia coli (E. coli) cells. For Selk and SelTryp proteins, the expression protocol did not show an unsatisfactory result to continue the experiments. Many difficulties were encountered in studies with Selt protein, mainly in attempts to make it soluble. Our analyses revealed SelT was a membrane protein, therefore it could cause changes in some objectives and search for new strategies. It could be expressed and purified in cromatographis. SEC-MALLS assays showed a stability of the protein detergent complex. With TbSerRS is possible to conclude that the organization of binding specificity of the enzyme with its ligands occurs increasingly: SelC>tRNASer7>tRNASer3a>tRNASer3b. And selenoproteins in T. brucei, it is necessary for new constructions to SelK and SelTryp to continue the experiments trying to crystallizes SelT, since prototolo for obtaining the protein-detergent complex is assembled and stabilized.

The objective of this study was to further determine the distribution of selenoprotein W (SeW) in tissues from rats and sheep fed different selenium levels and to search for the possible functions of this protein. In the rat study a total of 28 rat tissues were examined and SeW was found in all of the tissues except for liver, thyroid, pancreas, pituitary and eyes regardless of the level of Se fed. SeW was not detected in heart, lungs, prostate, esophagus, small intestine, tongue, skin diaphragm and skeletal muscle from selenium deficient rats, but was present in these tissues when the two higher levels of selenium (0.1 and 4.0 mg/kg) were fed. SeW has the highest expression in muscle, brain, testis and spleen when selenium is adequate. Interestingly, selenium deficiency resulted in undetectable SeW levels in heart and muscle from deficient sheep and rats, but the content in brain was unaffected by selenium status. Second generation selenium depleted and repleted rats indicated that the expression of SeW in cortex and cerebellum was not significantly affected by selenium, but selenium increased its levels in thalamus. Cortex had the highest SeW expression among the three parts of the rat brain. SeW levels in muscle, spleen, skin and testis were undetectable in weanling rats, but became detectable after 6 weeks of selenium repletion. Studies with various brain cell cultures indicated that Se appears to be metabolized differently by different brain cell types. As demonstrated in neuroblastoma and glial cells, glutathione peroxidase (GPX) activity decreased at a faster rate than SeW with neuroblastoma cells whereas SeW decreased at a faster rate than GPX activity in glial cells when selenium was removed from the media. Since other work showed that glutathione was bound to SeW, it was speculated that it has antioxidant function similar to other selenoproteins. SeW overexpressed and underexpressed cell lines were established by DNA recombinant techniques. There was a greater survival rate of overexpressed cells when incubated with 2,2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) than control cells, suggesting SeW possibly has an antioxidant function. / Graduation date: 1999