Aspects of the quantitative separation and estimation of thiamine and its phosphate esters : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

Methods for the separation and estimation of thiamine, thiamine monophosphate and thiamine diphosphate which would be applicable to biological extracts were investigated. Two methods for the estimation of thiamine were compared, the acid dye method and the thiochrome method. The thiochrome method was preferred as the acid dye method was more difficult to perform and some interference by certain amino acids was indicated. As both methods only estimate free thiamine, the optimum conditions for hydrolysis of thiamine phosphate esters by wheat germ acid phosphatase were also investigated. High phosphatase concentrations in the digestion mixture interfered with the extraction of thiochrome, by isobutanol, after oxidation of the free thiamine produced. Variation of the buffer in which the digestion was performed also affected the recoveries obtained. The inclusion of magnesium ions in the digestion mixture increased the activity of the enzyme so that it was possible to use an amount of phosphatase which was low enough to avoid interference with the extraction of thiochrome but which was sufficient to completely hydrolyse thiamine phosphate esters. The presence of magnesium ions also prevented the interference observed when formate rather than acetate buffers were used in the digestion mixture. A variety of separation techniques were investigated. Compared to paper and thin layer chromatography, high voltage paper electrophoresis (at 3kV in pH 3.5 buffer) gave the best and quickest separations. However only a 60% recovery was obtained after samples were eluted from the paper with 0.1M hydrochloric acid. Separation was achieved by elution of the thiochrome derivatives of thiamine, TMP and TDP from Sephadex G10 gel. Recoveries, estimated spectrophotometrically, indicated that this method could be used for the quantitative separation of thiamine and its phosphate esters. However since the method does not allow concentration of samples, it would be unsuitable for the estimation of biological extracts. Separation of thiamine and its esters using three ion exchange resins was also investigated. Partial separation of thiamine and its phosphate esters was obtained with Amberlite GC50 resin, the separation being determined by the form of the resin used. The hydrogen form of the resin allowed separation between TDP and thiamine-TMP while the sodium form separated thiamine from TMP-TDP. Neither form of the resin bound TDP firmly even when water was used as the eluent, so that separation of TDP and TTP would not be possible. Separation was attempted by eluting samples from Dowex 1-X8 resin with formate buffers of increasing ionic strength or pH. While the separation of thiamine, TMP and TDP appeared to be complete, by the elution profile, it was found that sample breakdown occurred. Electrophoresis of the eluted samples showed that the only peak which contained a single component was that corresponding to thiamine. Sample break-down was further indicated by a low recovery obtained when a sample containing only TDP was eluted. Identification of the peak contaminants was attempted using high voltage electrophoresis but proved difficult due to salt retardation affecting the positions of the peak components after electrophoresis. With Dowex 50 resin TDP and TMP were easily separated and eluted with ammonium acetate buffer of varying pH and ionic strength but the elution of thiamine required high pH or ionic strength solutions. Sample breakdown also appeared to occur on elution of samples from the resin. When TMP and TDP were eluted, separation appeared to be complete but a recovery of greater than 100% was obtained for TMP and both eluted compounds exhibited a progressive breakdown after elution. Sample breakdown was particularly notable when thiamine alone was eluted as 2 peaks were eluted and, after oxidation, yellow fluorescent material as well as the usual blue (characteristic of thiochrome) was observed. Characterisation of the yellow fluorescent compound(s) was attempted using electrophoresis, ultra-violet spectra and fluorescent spectra and it was found to be similar, but not identical, to thiamine

On Saturated kk-Sperner Systems

Given a set $X$, a collection $\mathcal{F}\subseteq\mathcal{P}(X)$ is said to be $k$-Sperner if it does not contain a chain of length $k+1$ under set inclusion and it is saturated if it is maximal with respect to this property. Gerbner et al. conjectured that, if $|X|$ is sufficiently large with respect to $k$, then the minimum size of a saturated $k$-Sperner system $\mathcal{F}\subseteq\mathcal{P}(X)$ is $2^{k-1}$. We disprove this conjecture by showing that there exists $\varepsilon>0$ such that for every $k$ and $|X| \geq n_0(k)$ there exists a saturated $k$-Sperner system $\mathcal{F}\subseteq\mathcal{P}(X)$ with cardinality at most $2^{(1-\varepsilon)k}$. A collection $\mathcal{F}\subseteq \mathcal{P}(X)$ is said to be an oversaturated $k$-Sperner system if, for every $S\in\mathcal{P}(X)\setminus\mathcal{F}$, $\mathcal{F}\cup\{S\}$ contains more chains of length $k+1$ than $\mathcal{F}$. Gerbner et al. proved that, if $|X|\geq k$, then the smallest such collection contains between $2^{k/2-1}$ and $O\left(\frac{\log{k}}{k}2^k\right)$ elements. We show that if $|X|\geq k^2+k$, then the lower bound is best possible, up to a polynomial factor.Comment: 17 page

Saturation in the Hypercube and Bootstrap Percolation

Let $Q_d$ denote the hypercube of dimension $d$. Given $d\geq m$, a spanning subgraph $G$ of $Q_d$ is said to be $(Q_d,Q_m)$-saturated if it does not contain $Q_m$ as a subgraph but adding any edge of $E(Q_d)\setminus E(G)$ creates a copy of $Q_m$ in $G$. Answering a question of Johnson and Pinto, we show that for every fixed $m\geq2$ the minimum number of edges in a $(Q_d,Q_m)$-saturated graph is $\Theta(2^d)$. We also study weak saturation, which is a form of bootstrap percolation. A spanning subgraph of $Q_d$ is said to be weakly $(Q_d,Q_m)$-saturated if the edges of $E(Q_d)\setminus E(G)$ can be added to $G$ one at a time so that each added edge creates a new copy of $Q_m$. Answering another question of Johnson and Pinto, we determine the minimum number of edges in a weakly $(Q_d,Q_m)$-saturated graph for all $d\geq m\geq1$. More generally, we determine the minimum number of edges in a subgraph of the $d$-dimensional grid $P_k^d$ which is weakly saturated with respect to `axis aligned' copies of a smaller grid $P_r^m$. We also study weak saturation of cycles in the grid.Comment: 21 pages, 2 figures. To appear in Combinatorics, Probability and Computin

Varying the Abundance of O Antigen in \u3cem\u3eRhizobium etli\u3c/em\u3e and Its Effect on Symbiosis with \u3cem\u3ePhaseolus vulgaris\u3c/em\u3e

Judged by migration of its lipopolysaccharide (LPS) in gel electrophoresis, the O antigen of Rhizobium etli mutant strain CE166 was apparently of normal size. However, its LPS sugar composition and staining of the LPS bands after electrophoresis indicated that the proportion of its LPS molecules that possessed O antigen was only 40% of the wild-type value. Its LPS also differed from the wild type by lacking quinovosamine (2-amino-2,6-dideoxyglucose). Both of these defects were due to a single genetic locus carrying a Tn5 insertion. The deficiency in O-antigen amount, but not the absence of quinovosamine, was suppressed by transferring into this strain recombinant plasmids that shared a 7.8-kb stretch of the R. etli CE3 lps genetic region α, even though this suppressing DNA did not carry the genetic region mutated in strain CE166. Strain CE166 gave rise to pseudonodules on legume host Phaseolus vulgaris, whereas the mutant suppressed by DNA from lps region α elicited nitrogen-fixing nodules. However, the nodules in the latter case developed slowly and were widely dispersed. Two other R. etli mutants that had one-half or less of the normal amount of O antigen also gave rise to pseudonodules on P. vulgaris. The latter strains were mutated in lps region α and could be restored to normal LPS content and normal symbiosis by complementation with wild-type DNA from this region. Hence, the symbiotic role of LPS requires near-normal abundance of O antigen and may require a structural feature conferred by quinovosamin

Dimensions of Power and Collaboration in the Context of Destination Branding: A Theoretical Framework

This paper aims to present a theoretical framework for the study of power and collaboration in the process of branding a tourism destination. The study has the objective to discover how stakeholder power influences the destination branding process that has generally been discussed as a collaborative process. This research draws upon sociological theory and within it focuses on power and collaboration theories. The process of destination branding provides a context for the research along with the related concepts of tourism destination, destination branding and stakeholder theory. A theoretical framework that integrates these concepts is provided together with three research questions. The theoretical framework is useful in improving the efficiency of collaborative stakeholder based processes such as destination branding

Whale Watching: The Roles of Small Firms in the Evolution of a New Australian Niche Market

This chapter provides two comparative case studies focusing on the roles of small businesses in the development of a new niche market and traces their interaction with small communities. The cases are set within the context of the development of commercial leisure viewing of humpback whales (Megaptera novaeangliae) in two coastal communities in Australia. Small businesses have been able to successfully develop a new niche market in one destination, but failed to in the other. These different outcomes are related to the economic developments and benefits in one destination while in the other region, social values appear to have dictated limits to the development of whale watching. The cases also provide insight into the dynamics of entrepreneurial development, the formation of supplier networks and the consequent development of standardized products

Yield applied to destination management: an inefficient analogy

This paper reports on a research project that examines the use of yield as a performance indicator for destination management. It reviews the history, definitions and use of yield and yield management in hospitality and transport businesses and then examines how these ideas have been transferred to the literature of tourism destinations. A series of recommendations on usage of the term 'yield' are provided

Genetic Locus and Structural Characterization of the Biochemical Defect in the O-Antigenic Polysaccharide of the Symbiotically Deficient \u3cem\u3eRhizobium etli\u3c/em\u3e Mutant, CE166

The O-antigen polysaccharide (OPS) of Rhizobium etli CE3 lipopolysaccharide (LPS) is linked to the core oligosaccharide via an N-acetylquinovosaminosyl (QuiNAc) residue. A mutant of CE3, CE166, produces LPS with reduced amounts of OPS, and a suppressed mutant, CE166α, produces LPS with nearly normal OPS levels. Both mutants are deficient in QuiNAc production. Characterization of OPS from CE166 and CE166α showed that QuiNAc was replaced by its 4-keto derivative, 2-acetamido-2,6-dideoxyhexosyl-4-ulose. The identity of this residue was determined by NMR and mass spectrometry, and by gas chromatography-mass spectrometry analysis of its 2-acetamido-4-deutero-2,6-dideoxyhexosyl derivatives produced by reduction of the 4-keto group using borodeuteride. Mass spectrometric and methylation analyses showed that the 2-acetamido-2,6-dideoxyhexosyl-4-ulosyl residue was 3-linked and attached to the core-region external Kdo III residue of the LPS, the same position as that of QuiNAc in the CE3 LPS. DNA sequencing revealed that the transposon insertion in strain CE166 was located in an open reading frame whose predicted translation product, LpsQ, falls within a large family of predicted open reading frames, which includes biochemically characterized members that are sugar epimerases and/or reductases. A hypothesis to be tested in future work is that lpsQ encodes UDP-2-acetamido-2,6-dideoxyhexosyl-4-ulose reductase, the second step in the synthesis of UDP-QuiNAc from UDP-GlcNAc

Muslim world and its tourisms

The study of tourism in the Muslim world can be about religious topics such as hajj and pilgrimage, but it actually means and involves much more. Because religious life and secular life in Islam are closely intertwined, study of its tourism is also partly about its worldview and culture as well as a means of reflecting on Western concepts of travel and hedonistic tourism. This review article introduces selected aspects of Islam to non-Muslims and reviews the tourism literature to identify themes and areas for further research. In addition to scholarly goals, an understanding of the patterns and requirements of the growing numbers of Muslim travellers is of practical importance for the tourism industry. Significantly, the Muslim world provides opportunities for studying differences in policy and development decisions that can offer new insights and inform tourism by providing alternative perspectives