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CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Estudos sobre o desenvolvimento da composiÃÃo salivar no primeiro ano de vida podem promover informaÃÃes a cerca da maturaÃÃo imunolÃgica em crianÃas. A saliva à o fluido humano mais disponÃvel e de fÃcil acesso, o que faz dela uma das ferramentas mais pesquisadas no campo de diagnÃstico e biomarcadores. Nesse contexto, essa tese, constituÃda de 1 artigo objetivou caracterizar proteÃnas salivares nos primeiros meses de vida, utilizando eletroforese unidimensional. Nesse estudo, saliva total estimulada e foram obtidas de 79 bebÃs de ambos os gÃneros no primeiro e terceiro meses de vida. Os sobrenadantes foram analisados, o fluxo salivar foi calculado (mL/min) para cada crianÃa e a concentraÃÃo de proteÃnas totais foi determinada pelo MÃtodo do Ãcido BicinconÃnico (BCA). ProteÃnas foram caracterizadas de acordo com o peso molecular atravÃs de eletroforese unidimensional. Os resultados demonstraram fluxos salivares significativamente diferentes entre o primeiro e terceiro meses de vida (p = 0,000), sendo o fluxo salivar no terceiro mÃs maior (Mediana = 0,10; Min-Max = 0,02-0,20 mL/ min) do que no primeiro mÃs (Mediana = 0,02; Min-Max = 0,02-0,06 mL/min).O peso em kg mostrou diferenÃa significante (p = 0,000) entre o primeiro mÃs (Mediana: 3,32; Min-Max: 2,02-4,03) e o terceiro (Mediana: 6,26; Min-Max: 4,49-9,30). O presente trabalho tambÃm demonstrou diferenÃa estatisticamente significante entre as concentraÃÃes de proteÃnas totais do primeiro e terceiro meses de vida (p < 0,001), onde a concentraÃÃo de proteÃnas totais foi maior na saliva total do primeiro mÃs (Mediana = 1,29; Min-Max = 0,43-3,93 &#956;g/mL) que no terceiro (Mediana: 1,293; Min-Max: 0,427-3,931 &#956;g/mL). Bandas com 150 (p=0,004), 100 (p=0,000), 42 (p=0,001), 30 (p=0,039), 25 (p=0,001), 20 (p=0,009), 15 (p=0,011) e 13 kDa (p=0,002) apresentaram maior intensidade na saliva total durante o primeiro mÃs. A banda com 218 kDa foi observada exclusivamente durante o terceiro mÃs de vida. O Apgar no primeiro minuto se correlacionou positivamente com o nÃmero total de bandas expressas no primeiro (p = 0,019) e terceiro mÃs (p = 0,014). Em conclusÃo, a concentraÃÃo de proteÃnas totais salivares reduz entre o primeiro e o terceiro meses de vida, e a expressÃo de proteÃnas salivares muda em funÃÃo da idade, onde a expressÃo de determinadas bandas proteicas à especÃfica a determinados perÃodos de desenvolvimento durante os primeiros 3 meses de vida. / Studies on the development of salivary composition in the first year of life can promote the information about the immune maturation in children. Saliva is the most easily available and accessible body fluid, which makes it one of the most sought after tools in diagnostic and biomarks. In this context, this thesis, constituted by 01 article aimed to characterize salivary proteins in the first months of life using unidimensional electrophoresis. On this study, stimulated whole saliva was obtained from 79 babies, both genders at first and third months of life. Supernatants were analyzed, salivary flow rate (mL/min) was calculated for each child and total protein concentration determined by the Bicinchoninic Acid Protein (BCA) method. Proteins were characterized according to their molecular weights within the unidimensional electrophoresis. The results showed salivary flow rates significantly different (p = 0.000) between the first and third months of life, being the flow rate of whole saliva at third month (Median: 0.10, min-max: 0.02-0.2 mL/ min) higher than at first month (Median: 0.02, min-max: 0.02-0.06mL/min). The weight in Kg showed significant differences (p = 0.000) between the first month (Median: 3.32, Min-Max: 2.02-4.03) and third months (Median: 6.26, Min-Max: 4.49-9.30).This study also showed difference concerning to total protein concentration between saliva of the first and third months (p = 0.000), being higher concentrations found in first (Median: 1.293, min-max: 0.427-3.931 &#956;g/mL) than in third (Median: 0.720, min-max: 0.164-2.244 &#956;g/mL). Bands with 150 (p=0.004), 100 (p=0.000), 42 (p=0.001), 30 (p=0.039), 25 (p=0.001), 20 (p=0.009), 15 (p=0.011) and 13 kDa (p=0.002) presented higher intensity in whole saliva of the first month. A band with 218 kDa was expressed exclusively during the third month of life. Apgar at 1 min was positively correlated with the total number of bands expressed in the first (p = 0.019) and third months (p = 0.014). In conclusion, the concentration of total salivary proteins decreases between the first and third months, and the expression of the salivary proteins changes depending on the age, where the expression of specific protein bands is specific to certain developmental stages during the first 3 months.

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M.Tech.

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The kinetics of the coordination reaction between the carboxyl group of the acetate radical and the trivalent chromium ion has been examined by independent methods at a temperature selected to yield reasonably accurate rate data. The study has been extended to an examination of the rate of chromium "fixation" by the carboxyl groups of hide collagen in a series of miniature tannages carried out under similar conditions for comparative purposes. The effect of olation of the chromium ions in solution on the kinetics of t he coordination reaction has also been examined. The rate of coordination of the acetate radical was followed by solvent extraction and spectrophotometric techniques over a range of concentration and pH levels. The rate data revealed a reaction having typical mass-action characteristics, the rate of reaction depending on the concentration levels of the chromium ion and the ionised acetate radical. An attempt made to distinguish a differential reaction rate in the case of a parallel reaction series using boiled and aged chromium nitrate reactant solutions, failed to reveal any significant differences between the series. The reactant solutions gave absorption spectra characteristic of the trivalent chromium ion with maxima in the 420 mµ and 570 mµ wavelength regions. The changes in optical density occurring in the vicinity of the maxima were followed spectrophotometrically, the height of the 570 mµ being found to increase during the course of reaction, while the height of the 420 mµ peak decreased. The variation in optical density at the 570 mµ peak was found to be directly related to the increase in concentration of the product complex in solution in accordance with the Baer-Bougher law, while the decrease in the height of the 420 mµ peak was related to the properties of unolated OH groups associated with the chromium complex as governed by pH and olation changes during the course of reaction. Boiled and aged chromium nitrate solutions gave reactant solutions having initial optical densities greater than those of the correspending fresh reactant solutions at the 570 mµ peak, and less than those of the fresh solutions at the 420 mµ peak. During the course of reaction, however, the two series converged to the same values. The equilibrium reaction solutions were subjected to paper electrophoretic study which indicated that all tho chromium was present in the form of cationic species. This finding was in accordance with stoicheiometric indications in the solvent extraction studies where coordination in a l : 1 ratio was reflected, giving rise to positively charged complex ions only. Making due allowance for band spreading the numbers of species predicted from the kinetic studies correspond with the electrophoretic patterns found. Application of the classical second order kinetic expression to the solvent extraction rate data, yielded plots having two distinctly linear sections, apparently indicating consecutive second order processes occurring in solution, contrary to the findings of previous workers who assign a first order mechanism to the reaction. This finding was consistent with the view that in the case of both the fresh and aged series, reaction consisted of successive coordination of an acetate radical to each chromium atom of a diol complex. Second order rate "constants" were calculated for each step and their dependency on pH level demonstrated. The findings of Hamm et al that these were first order reactions independent of acetate co'ncentration are believed to be due to their use of a large excess of acetate in their experiments. The kinetic study was further extended to an examination of the rate of "fixation"of trivalent chromium by the carboxyl groups of hide collagen under comparable conditions in a series of miniaturv tanning experiments in which the tannage substrate was provided in each of two physical forms:- (a) As hide powder where surface development was large, and (b) as prepared pelt pieces in which the fibrous weave pattern was retained. In view of the heterogeneity of the tannage systems, remarkable similarity was observed in the reaction course when compared with that of the acetate coordination studies, particularly in the case of the hide powder tannages. The dependency of the tannage rate data upon concentration and pH conditions was also found to be the same as in the case of the pure chemical system. The exact correlation between the rate data for the tannage and pure chemical systems was demonstrated by means of correlation plots. Close correlation was revealed in the case of hide powder tannage while the smaller degree of correlation observed in the case of the pelt tannage systems was attributed to the modifying effects of diffusion, particularly on the initial reaction velocity. The effect of using boiled and aged chromium solutions in the tannages was to increase the initial rate of chroniill!l "fixation" apparently due to tho coordination of a dial species at each coordination site. After the initial reaction period, the two series showed a tendency to converge as in the case of the spectrophotometric studies on the acetate reaction. The convergence trend was regarded as indicative of the tendency for the chromium in fresh solutions to under go rapid olation to the ame level as in the boiled and aged solutions. The experimental observations made on the various systems lead to the following conclusions:- (a) The mechanism of coordination of the acetate radical ttrivalent chromium appears to involve the successive coordination of ligand to each of the chromium atoms of a diol complex, both coordination steps proceeding by second order reaction mechanisms. (b) At the pH levels at which the reaction is carried out, the formation of elated bodios is rapid so that reaction in the case of both fresh and aged solutions essentially occurs between an elated species and the ionised acetate radical. (c) Modificati0ns in the absorption spectrum of the reactant solution at the 570 mµ peak are directly related to coordination, while changes at the 420 mµ peak are related to the formation of loosely-bound hydroxo chromium compounds, the concentrations of which depend on the pH level. (d) The mechanism of chromium fixation in hide is essentially similar to that operating in the case of coordination of the acetate radical to chromium, involving attachment of chromium to the side chain acid r sidues of collagen. (e ) The effect of olation is to enhance the rate of chromium fixation by hide protein during the initial reaction stages and to render possible bridge formation between adjacent side chains by secondary coordination during the latter reaction stages. (f) Where pelt pieces are used instead of hide powder, the initial rate of chromium fixation is dominated by the rate of diffusion of chromium into the fibrous structure. It should be stressed that the observations made on the various reaction systems cannot be regarded as exhaustive and the conclusions are subject to further confirmation. Consequently, the present study is essentially of a preliminary nature, but it is felt that the need for further investigation along similar lines has been demonstrated.

The presence of harmful algal blooms (HAB) is a growing concern in aquatic environments. Among HAB organisms, cyanobacteria are of special concern because they have been reported worldwide to cause environmental and human health problem through contamination of drinking water. Although several analytical approaches have been applied to monitoring cyanobacteria toxins, conventional methods are costly and time-consuming so that analyses take weeks for field sampling and subsequent lab analysis. Capillary electrophoresis (CE) becomes a particularly suitable analytical separation method that can couple very small samples and rapid separations to a wide range of selective and sensitive detection techniques. This paper demonstrates a method for rapid separation and identification of four microcystin variants commonly found in aquatic environments. CE coupled to UV and electrospray ionization time-of-flight mass spectrometry (ESI-TOF) procedures were developed. All four analytes were separated within 6 minutes. The ESI-TOF experiment provides accurate molecular information, which further identifies analytes.

Bioanalysis is a subdivision of analytical chemistry and deals with biological analytes such as metabolites, proteins, nucleic acids, small molecules, virus particles and entire cells. The rationale of my thesis was to achieve two goals: (i) develop a set of ready to use methods (ii) which are capable providing exact concentrations of analytes as well as kinetic and thermodynamic parameters of their interactions. To investigate interactions between biomolecules special conditions are required which do not interfere with the course if biomolecule interactions. Establishing these conditions and optimization of separation and detection parameters can be tedious and can take longer than actual analysis of samples. I developed a variety of Capillary Electrophoresis – Mass Spectrometry (CE-MS) methods suitable for bionalalysis. CE-MS establishes a new paradigm that separation methods together with MS detection can be used as comprehensive kinetic tools. Most previous attempts to use chromatography and electrophoresis for studying nucleic acid interactions were restricted to assuming slow or no equilibrium between reactants. Kinetic CE (KCE) shows that non-zero kinetics and structural dynamics must be taken into account when separation happens. KCE-MS could be a valuable supplement to IM-MS due to the separation of ions in solution according to their size-to-charge ratio. These methods allowed to reveal new facts about biomolecules and added novel data to the bank of the mankind knowledge. For the best of my knowledge, kinetic parameters for TG2 and thrombin G-quadruplex folding were reported for the first time. I developed a homogeneous method to determine kon, koff and Kd of fast and weak noncovalent interactions between multiple unlabeled ligands (small molecule drugs) and an oligosaccharide (α- or β-cyclodextrin) simultaneously in one capillary microreactor. It has been shown for the first time that KCE can be used to separate and detect the slowly interconverting open and closed conformations of human TG2. It allowed the first direct measurement of the Kd value for calcium binding. Sixteen new substrates were discovered for three aminotransferases (AAT, BCAT, and DAAT). In addition, Viral qCE showed a feasibility to analyse both the count of intact viral particles and sample nucleic acid contamination.

This thesis examines the use of capillary electrophoresis for the study of several biomolecules and their interactions and viruses. The first two experimental chapters focus on its utility for thermodynamic and kinetic analysis of molecules. Chapter one focuses on the use of non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), to calculate the dissociation constant for the interaction between double stranded microRNA-122 and protein p19. NECEEM was used to calculate the rate constants (koff = 0.059 ± 0.013 s-1, kon = 0.0022 ± 0.0008 s-1M-1) and the dissociation constant between miR-122 and wild type p19 (Kd = 27 ± 9 nM). A new method was developed to calculate the rate constant koff, by using multiple electric fields; which resulted in a koff value of 0.072 ± 0.022 s-1. In chapter two, the dissociation constant, Kd, was determined between HIV trans-activation response element and nuclear protein TOE1. It was demonstrated that TOE1, more specifically peptides ER19 and ED35, were binding to TAR with Kd values of 4.08 ± 0.19 µM for ER19 and 7.43 ± 1.60 µM for ED35. The discovery of the peptides’ inhibitory action of viral replication at the transcription level is a significant step towards further elucidating mechanisms for host response to HIV-1 infection. The third chapter focuses on the use of capillary electrophoresis for studying vesicular stomatitis virus (VSV) and vaccinia virus (VV). A new method was developed for quantification of VSV, using dithiothreitol. Furthermore, CE was used to study the preservation of VSV by a previously selected aptamer construct (quadramer) during freeze-thaw cycles. It was found that the infectivity of quadramer and aptamer pool-protected virus was higher than pure virus after 60 freeze−thaw cycles. It was also found that adding quadramers to the virus without freezing (cycle 0) increased the virus infectivity by 30%. We also investigated the potency of a carbohydrate-based ice recrystallization inhibitor, N-octyl-D-gluconamide (NOGlc) for its ability to eliminate the cold chain and stabilize the potency of VV. Viral potency after storage at room temperature demonstrated that NOGlc conserved the infectivity of VV, during 40 days.

Please read the abstract (summary) in the section 00front of this document. / Thesis (PhD)--University of Pretoria, 2011. / Food Science / unrestricted