Thesis (Ph. D.)--University of Nevada, Reno, 2008. / "May 2008." Includes bibliographical references. Online version available on the World Wide Web.

Universiẗat, Diss., 2004--München.

Thiamine (vitamin B1) is involved in several basal metabolic processes. It is an essential compound for many organisms and in aquatic systems it is mainly produced by phytoplankton and prokaryotes and transferred to higher trophic levels through grazing and predation. The occurrence of thiamine deficiency in top predators has been reported from several aquatic systems. In the Baltic populations of the Atlantic salmon (Salmo salar) this has been observed since 1974 and recently thiamine deficiency has also been reported for Baltic sea birds. This thesis aims at investigating what processes that governs the flow of thiamine from the primary producers to top predators via zooplankton grazers and planktivoric fish. Paper I showed that abiotic stress factors such as salinity, temperature and light conditions can alter the thiamine content of phytoplankton. Paper II showed that abiotic factors indirectly can affect the stress resistance of zooplankton grazers by changing the nutritional quality of their food. In Paper III we found that the in situ thiamine content of zooplankton grazers was directly affected by that of the phytoplankton diet. In Paper IV we found a similar connection between the thiamine contents of Baltic salmon and herring, one of the major salmon prey species. In Paper V we looked at the thiamine content of the pelagic food web of the Baltic Sea as a whole and found a pattern of trophic dilution; the higher the trophic level of an organism (i.e. the further away from the source of thiamine in the food web), the lower was its thiamine content. In all, the results of this thesis suggests a bottom up effect on the thiamine status of the higher trophic levels of  the Baltic Sea and that external factors, both natural and man-made, have the capability to affect the thiamine status of the plankton communities and thereby the whole Baltic ecosystem. Thiamine and other micronutrients are not something generally considered in the environmental management of aquatic systems but the results of this thesis suggest that ecological disturbances indirectly can have negative effects on top predators via a disrupted supply of essential substances. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript. Paper 4: Manuscript. Paper 5: Manuscript.</p>

The level of gamma-aminobutyric acid in the brains of white rats suffering from thiamine deficiency symptoms resulting from pyrithiamine or oxythiamine antagonism of thiamine or from thiamine deprivation was measured. The level of this amino acid was 17.5% lower in the brains of pyrithiamine-treated rats and 20.0% lower in the brains of thiamine-deprived rats than in the control rats. Daily injections of ten times the required amounts of vitamin B6 in rats treated with pyrithiamine or oxythiamine or by thiamine deprivation had no effect on the level of brain gamma-aminobutyric acid or on the psiological behavior of these rats. The activity of glutamic decarboxylase was assayed and found to be 29.0% lower in the brains of thiamine-deprived rats than in the controls. This enzyme was not significantly altered in pyrithiamine-treated or in oxythiamine-treated rats. The activity of glutamic-gamma-aminobutyric transaminase with succinic semialdehyde dehydrogenase in the brains of any of these groups was not significantly different than the activity in the control animals.

The enzyme ATP:thiamine pyrophosphotransferase (thiamine pyrophosphokinaseJ was purified from acetone powders which were prepared from rat brains up to the chromatography on alumina C_γ step by a modified procedure that Mano (Y. Mano, J. Biochem., 42, 283 (1960)] developed for the purification of the rat liver thiamine pyrophosphokinase. The enzyme was purified 12 fold over that in the original extract of the acetone powder. A spectrophotometric assay for the brain thiamine pyrophosphokinase was developed utilizing brewer's yeast apotransketolase. The transketolase assay was able to detect less than 10^-11 moles of thiamine diphosphate, which was synthesized by the kinase, in the presence of ATP and thiamine. Contaminating yeast thiamine pyrophosphokinase was removed from the transketolase by chromatography of the transketolase on DEAE-cellulose at pH 6.0 with 0.01 M phosphate buffer. The thiamine pyrophosphokinase from 2.0 g of acetone powder (equivalent to 8 rat brains> could synthesize 276 mumoles of thiamine diphosphate per hour at 37°. The K_m 's for ATP and thiamine were found to be 0.02 M and 1.2 x 10-7 M, respectively. The pH optimum was found to be pH 7.8 with glycylglycine buffer. Pyrithiamine and oxythiamine were found to be competitive Inhibitors of thiamine pyrophosphorylation by the kinase. Pyrithiamine and oxythiamine have K_i 's of 2 x 10^-7 M and 2 x I0^-4 M, respectively. An oxythiamine phosphate compound was synthesized by brain thiamine pyrophosphokinase from ATP and oxythiamine which in turn inhibited the formation of transketolase from apotransketolase and thiamine diphosphate.

<p>U okviru ove doktorske disertacije razvijene su elektroanalitičke metode za određivanje pojedinih vitamina B grupe. Za određivanje vitamina B₁ i B₃ primenjena je adsorpciona hronopotenciometrijska striping analiza (AdHSA) na tankoslojnoj živinoj elektrodi kao radnoj elektrodi, dok je za određivanje vitamina B2 primenjena hronopotenciometrijska analiza (HA) na dvema geometrijski različitim elektrodama od staklastog ugljenika: planarnoj disk elektrodi i elektrodi u vidu procesne posude. U cilju optimizacije metoda ispitan je uticaj najznačajnijih eksperimentalnih faktora. Optimalni eksperimentalni uslovi za određivanje vitamina B₁ su podrazumevali primenu 0,2 mol/l citratnog pufera vrednosti pH 6 kao pomoćnog elektrolita, potencijala i vremena akumulacije od -1,313 V i 50 s, redom, i struje rastvaranja depozita od 1,9 &ndash; 6,1 &mu;A. Odgovarajući eksperimentalni faktori za određivanje vitamina B₂ su bili: 0,025 mol/l HCl kao pomoćni elektrolit, inicijalni potencijal od 0,023 V i struja redukcije od 0,8 &ndash; 4,2 &mu;A, dok su optimalni radni uslovi za određivanje vitamina B₃ obuhvatali primenu 0,05 mol/l citratnog pufera pH 6, potencijala akumulacije od -1,405 V pri vremenu akumulacije od 15 s, i struji rastvaranja u intervalu od 1,4 &ndash; 15,1 &mu;A. U slučaju određivanja vitamina B₂ primenom radne elektrode u vidu procesne posude ispitan je i uticaj aktivne povr&scaron;ine radne elektrode na analitički signal vitamina B₂. Optimalna vrednost aktivne povr&scaron;ine radne elektrode iznosila je 13,4 cm². Pod optimalnim eksperimentalnim uslovima, dolazilo je do elektrooksidacije molekula vitamina B₁ i B₃ na tankoslojnoj živinoj elektrodi u analitičkom koraku, dok se vitamin B₂ redukovao na elektrodama od staklastog ugljenika. U okviru validacije metoda definisani su opsezi linearnosti, određene su vrednosti granice detekcije i granice kvantitativnog određivanja, ocenjena je preciznost i ispitane su interferencije. Uz odgovarajuće uslove rada, dobijena je dobra linearnost analitičkog signala od sadržaja za sva tri ispitivana vitamina. Ostvarene su granice detekcije od 1,64 mg/l za vitamin B₁, 0,076 mg/l za vitamin B₂ uz primenu planarne disk elektrode i 0,018 mg/l (vitamin B₂) uz primenu procesne posude od staklastog ugljenika kao radne elektrode. Ostvarena granica detekcije za vitamin B₃ je iznosila 2,20 mg/l. Nakon optimizacije i validacije, razvijene metode HA i AdHSA primenjene su za određivanje vitamina B₁, B₂ i B₃ u komercijalnim multivitaminskim dodacima ishrani i multivitaminskim instant napicima. Tačnost razvijenih metoda je potvrđena paralelnim analizama izvedenim primenom visokopritisne tečne hromatografije.</p> / <p>Within the scope of this doctoral dissertation, electroanalytical methods for the determination of several vitamins of the B-complex are developed. For the determination of vitamin B1 and B3 adsorptive chronopotentiometric stripping analysis was applied, with mercury film electrode as the working electrode. For vitamin B2 determination, the chronopotentiometric analysis was performed on two geometrically different glassy carbon working electrodes: the planar disc electrode and the process vessel electrode. The most important experimental parameters of the analysis were investigated and optimized. For vitamin B₁ determination, the optimized experimental conditions were: 0,2 mol/l citrate buffer pH 6 as the supporting electrolyte, accumulation potential of -1,313 V, accumulation time of 15 s and the oxidation current between 1,9 &mu;A and 6,1 &mu;A. The appropriate experimental factors for vitamin B₂ determination included 0,025 mol/l HCl solution (supporting electrolyte), initial potential of 0,023 V and reduction current in the range from 0,8 &ndash; 4,2 &mu;A, whereas the optimal working parameters for vitamin B3 determination were as follows:0,05 mol/l citrate buffer pH 6, accumulation potential of -1,405 V, accumulation time of 15 s and dissolution current from 1.4 &ndash; 15.1 &mu;A. When the process vessel was used as the working electrode, the optimal volume of the analyzed solution i.e. the active surface area of the electrode was optimized. The optimal value of the active surface area was 13,4 cm2. As well, under the optimal experimental conditions, vitamin B₁ and vitamin B3 underwent electrooxidation process in the analytical step, whereas vitamin B₂ was electrochemically reduced on glassy carbon electrodes. A validation procedure of the optimized methods was performed by evaluation of the following parameters: linearity, the limit of detection (LOD), the limit of quantitation (LOQ), precision, selectivity, and accuracy. Under optimal working conditions, the linearity of the proposed methods was very good. The achieved limits of detection were 1.64 mg/l for vitamin B₁, 0,076 mg/l for vitamin B₂ (planar disc electrode) and 0,018 mg/l (process vessel electrode) and 2,2 mg/l for vitamin B₃.<br />After optimization and validation procedures, the developed methods were applied for vitamin B₁, B₂, and vitamin B₃ determination in commercially available multivitamin supplements and instant multivitamin beverages. The accuracy of the proposed methods was tested by parallel HPLC analyses of the same samples.</p>

Call number: LD2668 .T4 1985 K44 / Master of Science

Nesta dissertação, foi caracterizada a via de biossíntese de tiamina (Vitamina B1) nas três formas intraeritrocitárias de P. falciparum. Foram realizadas marcações metabólicas, utilizando diferentes precursores radioativos envolvidos na biossíntese de tiamina, já descritos para outros organismos. A utilização do precursor [1-14C] acetato de sódio demonstrou que a via de biossíntese de tiamina encontra-se ativa em todos os estágios intraeritrocitários de P. falciparum. Investigamos os precursores que poderiam estar envolvidos na biossíntese do intermediário tiazol, e nossos dados sugerem que a cistéina é a doadora do enxofre presente na molécula de tiamina; que o aminoácido tirosina pode ser o precursor da biossíntese de tiamina, e nicotinamida não é utilizada como precursor em P. falciparum. Também se avaliou o efeito da fosmidomicina e 3ClDHP e foi demonstrado que ambos propiciaram uma inibição no crescimento dos parasitas. Estes dados sugerem que a via de biossíntese de tiamina, pode ser explorada como alvo para drogas antimaláricas, devido ausência em humanos. / In the present work we have demonstrated the biosynthesis of thiamin (vitamin B1) in the intraerytrocytic stages of P. falciparum. We have demonstrated active biosynthesis of thiamine in the three parasite stages metabolically labeled with [1-14C] sodium acetate. We also investigated which precursors could be involved in the biosynthesis of the thiazole intermediate, by metabolic labelling with different precursors. Our data suggest that the sulphur present in the thiamine molecule is formed from cysteine white that tyrosine can be the precursor of thiamine biosynthesis. Nicotinamide is not utilized as a precursor in P.falciparum. We also investigated the effect of fosmidomycin (an inhibitor of the DOXP reductoisomerase in the MEP pathway) and 3CIDHP (an analogue of bacimethrin) in vitro cultures and both showed an inhibitory effect on parasite growth. These data suggest that the biosynthesis of thiamine can be an attractive target for the development of antimalarial drugs since this pathway is absent in humans.

Thiamin transport in Escherichia coli is a model system to establish the tolerance of derivatives for transport into the cell. Since little is known about what types of thiamin derivatives may be successfully taken into the cell through the transport system, a series of thiamin derivatives are synthesized. A thiamin amino analog is synthesized and tested to determine the use of the analog as an alternate source of thiamin for growth of an E. coli thiamin mutant. Formate, acetate, and benzoate thiamin esters are synthesized and tested as alternate sources for growth of an E. coli thiamin mutant. Thiamin esters or amides may provide a scaffold for attaching other small molecules of interest to be imported into the cell by thiamin transport system. Thiamin containing formate, acetate, and benzoate esters were synthesized and tested as alternative growth source for thiamin using an E. coli mutant strain incapable of synthesizing thiamin. All three synthesized ester thiamin forms gave a zone of growth determined by disk-assay study. Also, an amino thiamin is synthesized to determine uptake through thiamin transport system by growth study using an E. coli mutant incapable of synthesizing thiamin. The growth curves resulting show concentration-dependent growth in the absence of natural thiamin, indicating amino thiamin is taken up by thiamin transport system as an alternate source of thiamin for growth. More characterization of the thiamin transport system is desired in order to develop thiamin conjugates of interest such as a photoaffinity probe for isolating thiamin-utilizing enzymes.

Thesis (Ph. D.)--State University of New York at Binghamton, Department of Psychology, Behavioral Neuroscience, 2008. / Includes bibliographical references.