Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: A laboratory-scale model was used to simulate biological sulfate removal. The focus of the research was microbial community response, such as the relative abundance of functional groups to changes in influent medium composition. Specific oligonucleotide probes were obtained that recognised sulfate reducing bacteria (SRB) within the biofilm community. Terminal restriction fragment length polymorphism (T-RFLP) and BIOLOG™ Ecoplate analyses were used to study the SRB community when provided with sodium lactate, sucrose or ethanol as carbon sources in complex Postgate C broth. These two analyses, as well as conventional methods, were applied to follow succession patterns in the laboratory scale reactors, and to determine the possible presence and relative abundance of microorganisms other than bacteria under sulfate reducing conditions. T-RFLP and BIOLOG™ Ecoplate analyses indicated a few dominant organisms in the community and a slight decline after a shift to another carbon source. Fluorescent hybridization showed higher numbers of SRB relative to the total microbial community than conventional culturing techniques. Furthermore, microscopic observations showed that not only SRB and other bacteria, but also yeast and filamentous fungi were integrated in a biofilm under sulfate reducing conditions. These microscopic observations were verified with fluorescent in situ hybridization (FISH) and yeast Live I Dead viability probes. / AFRIKAANSE OPSOMMING: Biologiese sulfaat-verwydering is met behulp van "n laboratoriumskaalmodel gesimuleer. Die doel van die navorsing was om die respons van "n mikrobiese gemeenskap met byvoorbeeld die relatiewe hoeveelheid van funksionele groepe op veranderinge in invloeiende medium samestelling te bestudeer. Spesifieke oligonukleotiedpeilers wat sulfaatreduserende bakterieë (SRB) in "n biofilmgemeenskap kan opspoor is gebruik. Die SRB gemeenskap is bestudeer met behulp van terminale-restriksiefragmentlengtepolimorfisme (TRFLP) en BIOLOGTM Ecoplate analise waar natriumlaktaat, sukrose of etanol as koolstofbronne toegevoeg is. Hierdie twee tipes analise en konvensionele metodes is aangewend om suksessiepatrone in die laboratoriumskaalreaktor te volg en die moontlike teenwoordigheid en relatiewe hoeveelheid van organismes, uitsluitende bakterieë, onder sulfaatreduserende kondisies te bepaal. Analise van T-RFLP en BIOLOGTM Ecoplate het aangedui dat In paar dominante organismes in die gemeenskap teenwoordig was, wat effens afgeneem het na verskuiwing na 'n ander koolstofbron. Fluoresserende hibridisasie het hoër getalle van SRB relatief tot die totale mikrobiese gemeenskap aangedui as konvensionele kultuur tegnieke. Mikroskopiese analises het verder getoon dat benewens SRB en ander bakterieë ook giste en filamentagtige swamme onder sulfaatreduserende kondisies in "n biofilm geïntegreer was. Hierdie mikroskopiese waarneminge is bevestig deur fluoresserende in situ hibridisasie (FISH) en gis Lewe / Dood lewensvatbaarheid peilers.

Development of the visual system involves the precise orchestration of neural connections between the retina of the eye, the thalamus (dorsal lateral geniculate nucleus; dLGN) and the superior colliculus (SC). During early development, receptor molecules on the growth cones of retinal ganglion cell (RGC) axons sense molecular guidance cues in the extra cellular matrix (ECM) that define their route and branching behaviour within the visual system. Heparan sulphate proteoglycans (HSPGs) are ECM molecules composed of a core protein and a variable number of disaccharide residues that have been implicated in mediating axon guidance. HSPGs are modified by a number of enzymes that contribute to their structural diversity. Based on this structural diversity; the “heparan sulphate code” hypothesis of Bulow and Hobert (2004) postulated that different HSPG modifications confer different axon navigation responses as the growth cones traverse the local environment. To investigate the roles played by specific modifications of HSPG molecules in the guidance of axons, we examined two lines of mutant mice harbouring mutations in the genes encoding HSPG modifying enzymes, Heparan sulphate-6-O-sulphotransferase-1 (Hs6st1) and Heparan sulphate-2-O-sulphotransferase (Hs2st). These two mutant lines were generated through the use of gene trapping. Previous observations of RGC axon development in the two mutant lines revealed distinct axon guidance errors at the optic chiasm. Loss of Hs6st1 sulphation resulted in RGC axons navigating ectopically into the contralateral eye. Loss of Hs2st sulphation resulted in RGC axons navigating outside the normal boundary of the optic chiasm. Early observations suggested that both Hs2st sulphation and Hs6st1 sulphation have distinct, non-overlapping actions and thus, influence different axon guidance signalling pathways at the optic chiasm. Based on our findings and previous work describing the expression patterns and functions of the chemo-repellent axon guidance molecules, Slit1 and Slit2 at the optic chiasm and their Robo2 in the retina, we formulated the hypothesis of an HSPG sulphation code where Hs2st sulphation is specifically required for Slit1-Robo2 signalling and Hs6st1 sulphation is specifically required for Slit2-Robo2 signalling at the optic chiasm. To further our understanding of the roles Hs2st sulphation and Hs6st1 sulphation have on axon guidance, we looked at a number of key choice points that navigating axons encounter and are known to be influenced by Slit signalling. Further observations of RGC axons at the optic chiasm of Hs2st-/- mutants and Hs6st1-/- mutants showed distinct axon guidance phenotypes, both resulting in statistically significant increases in the width of the optic chiasm at the midline. While Hs6st1 sulphation had no effect on RGC axon navigation within the eye (possibly due to 6-O-sulphation compensation by Hs6st3); the loss of Hs6st1 sulphation at the dLGN resulted in a significant increase in the defasciculation of the optic tract. Observations of other axonal tracts influenced by Slit signalling revealed the importance of Hs2st and Hs6st1 sulphation in aiding callosal axons to successfully traverse the midline in corpus callosum development. Observations of the thalamocortical (TCA)/corticothalamic (CTA) tracts revealed that neither Hs2st sulphation nor Hs6st1 sulphation was required for the development of the mouse TCA tract (the latter may be explained by 6-O-sulphation compensation by Hs6st2). To test whether Hs2st and Hs6st1 enzymes have redundant functions in optic chiasm development, we attempted to create Hs2st-/-/Hs6st1-/- double mutants. A PCR genotyping strategy was developed for the identification of Hs6st1 animals and showed that Hs6st1-/- mutants had high postnatal lethality with only 3% of the offspring surviving to weaning while Hs2st-/-/Hs6st1-/- double mutants all died very early during embryonic development. Observations of Hs2st-/-/Hs6st1+/- mutants and Hs2st+/-/Hs6st1-/- mutants that lacked three of the four Hst alleles showed no differences when compared to single Hst knockouts. Finally, we showed that altered Slit expression at the optic chiasm and Robo expression in the retina could not explain the mutant phenotypes observed in Hs2st-/- mutants and Hs6st1-/- mutants, and therefore we hypothesized that Hs2st sulphation and Hs6st1 sulphation regulate distinct aspects of Slit-Robo signalling at the surface of the navigating axon growth cone.

No description available.

No description available.

Osteopontin (OPN) is a matricellular cytokine found in most tissues and body fluids. It is involved in a variety of cell processes by binding to integrins and CD44 receptors, and it modulates immune responses. To investigate the functions of OPN during colitis the DSS acute colitis model in OPN-/- and WT control mice was utilized. OPN-/- mice were more susceptible to DSS-induced colitis than the DSS-treated WT control mice. The increased susceptibility of the OPN-/- mice was characterized by greater intestinal crypt destruction; high myeloperoxidase activity of infiltrating neutrophils; lack of differentiation of inflammatory cells such as lymphocytes subsets (CD4+, CD8+) and macrophages (F4/80); reduced production of certain cytokines, especially TNF-alpha; and non-programmed cell death of enterocytes. It is postulated that the hyperactivity of neutrophils may explain the increased tissue destruction during experimental colitis in the absence of OPN. Analysis of OPN’s impact on neutrophil function showed that while OPN may be important for the recruitment and migration of neutrophils, the expression of OPN by neutrophils is not required for manifestation of their destructive capabilities. This would suggest that OPN administration may protect the intestines from the adverse effects of colitis. Exogenous bovine milk OPN (20 μg/ml), administered for 8 days dissolved in the drinking water, ameliorated DSS-induced colitis. It diminished signs of disease, with a greater impact in the WT than the OPN-/- mice. It reduced the levels of neutrophils, macrophages, and pro-inflammatory mediators in the colon tissue. Recombinant OPN failed to reproduce the beneficial effects of milk OPN, which suggests that post-translational modifications of OPN are crucial to ameliorate experimental colitis. Collectively, these studies demonstrate that OPN has a protective effect during experimental colitis and that the oral administration of bovine milk OPN (20 μg/ml) ameliorates acute DSS-induced colitis. The results of this study also imply that the protective effect probably depends on a post-translationally modified form of OPN, and may require intracellular-OPN as a cofactor for significant attenuation of colitis. Future research could concentrate on more detailed investigation of these latter findings to determine OPN’s mechanism of action.

No description available.

Heparan sulphate proteoglycans (HSPGs) are a family of molecules that are found on the surface of cells or in the extracellular matrix, where they are involved in regulating key signalling events required for normal mammalian brain development. It is thought that specificity of HSPGs for particular signalling processes is encoded by their heparan sulphate (HS) sugar side chains, which can be modified post-translationally to yield huge variation in HS structure. Different sulphation patterns are generated by the action of the heparan sulphate sulfotransferases (HSTs) and sulfatase enzymes, which add or remove sulphate groups to specific positions on residues of the HS side chains. Depending on the expression of these enzymes and the resulting heparan sulphate ‘code’, it is proposed that cells are then able to regulate signals they receive and send in the ligand rich extracellular environment of the developing forebrain. Hs6st1 and Hs2st catalyse 6-O and 2-O HS sulphation, respectively. Following loss of either of these two HSTs, commissural tracts including the corpus callosum fail to develop normally during late mouse embryogenesis. The telencephalic midline environment is perturbed, with a striking mis-positioning of glial cell populations that normally act to guide axons towards the contralateral hemisphere. Too many radial glial cells at the glial wedge (GW) migrate towards the indusium griseum (IG) in mutant embryos. The running hypothesis to explain this phenotype is a change in critical signalling pathways required to set up the correct midline glia environment, such as Fgf8/ERK signalling which has already been identified as up-regulated at the Hs6st1-/- corticoseptal boundary (CSB). In order to further study what changes are occurring at the developing midline of HST-/- embryos compared to WT, we took a hypothesis free approach using RNA-sequencing analysis. RNA extracted from dissected midline regions of WT, Hs2st-/- and Hs6st1-/- mouse embryos at E16.5 was sent for sequencing, and a list of differentially expressed genes obtained. Overall we find few differentially expressed genes at the Hs6st1-/- midline compared to WT. At the Hs2st- /- midline there are a larger number of differentially expressed genes. Following validation studies, we find a significant and specific increase in Fgf17 protein distribution at the CSB of Hs2st-/- embryos compared to WT at E14.5. The results suggest the hypothesis that Hs2st’s normal role is to regulate Fgf17 protein distribution to limit exposure of GW radial glia cells to this translocation signal. When 2-O HS sulphation is lost then in Hs2st-/- embryos, ectopic Fgf17 signalling induces aberrant glia migration which ultimately prevents callosal axons from crossing the telencephalic midline to form the corpus callosum. To test this hypothesis, we used ex vivo slice culture experiments and showed ectopic Fgf17 protein expression is sufficient to trigger precocious translocation of midline glia in WT CSB, phenocopying the glia behaviour of Hs2st-/- embryos. Also consistent with the hypothesis, the Hs2st-/- glia phenotype can be rescued by addition of an FgfR1 inhibitor which reduces number of translocated glia cells. From these results we find for the first time that 2-O sulphated HS plays a remarkably specific role in regulating Fgf17-mediated translocation of midline glia cells at the developing mammalian telencephalic midline.

<p>This thesis is focused on the heparan sulphate (HS) structure from blind mole rat liver. HS is a glycosaminoglycan that is produced as a proteoglycan, in which linear polysaccharide chains are attached covalently to a protein core. Proteoglycans are widespread molecules in the body and have many important physiological functions. HS is synthesized as a polymer of alternating glucuronic acid and N-acetylglucosamine units. Parts of the polymer are subsequently modified by N-deacetylation /N-sulphation of the glucosamine units, C-5 epimerization of glucuronic acid to iduronic acid and O-sulphation at various positions.</p><p>The mole rats are from Israel and are of the Spalax ehrenbergi superspecies. Spalax Judaei (S60) has 60 chromosomes and Spalax Galili (S52) has 52 chromosomes. They are both completely blind and spend their entire life underground in hypoxic conditions. Spalax Galili (S52) inhabits the cool-humid Upper Galilee Mountains and Spalax Judaei (S60) inhabits the warm-dry southern regions. There is no current information about the heparan sulphate structure of these animals.</p><p>The two blind mole rats (S52 and S60) were metabolically labelled with [3H] Glucosamine. The animals were sacrificed and the organs were taken and frozen. The liver was chosen for the purpose of my project.</p><p>The HS structure was studied using various chromatographic methods such as ion-exchange and gel filtration. Structural analysis of HS indicated that the size of HS from the liver was the same in both species. However, the domain structure differed between the two animals, particularly with regard to sample S52(1) which had obvious differences. This leads to the study of the heparanase cleavage sites. Disaccharide composition analysis identified varying proportions of disaccharide species in S52 and also the possibility of an unknown disaccharide species.</p>

Most metal ions have negative impacts on pulp mill operations. The concentrations of metal ions on pulp fibers and in washwaters rise significantly with increased wastewater recycling. The development of technology to remove these metal ions requires an understanding of how metal ions are bound to pulp components. It is also desirable to predict distribution of metal ions between the pulp fibers and the washwaters. The adsorption isotherms for eight metal ions (Ca, Ba, Mn, Zn, Pb, Cd, Ni, Na) were measured on bleached and unbleached (brownstock) kraft pulps at neutral pH and temperatures ranging from 25 to 75��C. On bleached pulps, the metal ion adsorption increased rapidly with increasing metal ion concentration in solution and then leveled off. At neutral pH, the adsorption on bleached pulp was stoichiometric to the carboxylate sites, whereas the adsorption on unbleached pulp was not, especially at high metal ion concentration in solution and low temperature. The pH isotherms specify the adsorption isotherms of sodium and calcium on wood pulps as pH ranging from 2.5 to 11.0. The pH isotherms on bleached pulp with only COOH functional groups (pK[subscript a] of 3.77) were saturated at pH 4 and above, whereas those on brownstock pulp with both COOH and PhOH (pK[subscript a] of 10) functional groups increased in two steps, at pH 4 and 8. The brownstock pulp is heterogeneous material. Therefore, only the empirical Freundlich model was applied to the data. To predict the metal ion adsorption on bleached pulps, two fundamental equilibrium models were developed: the multi-component ion exchange and the Donnan equilibrium models. The ion-exchange model better predicts the metal adsorption at neutral pH, whereas the Donnan equilibrium model more accurately predicts the pH isotherms. The adsorption kinetics of Ba����� and Ni����� were measured on wood pulps as a function of mixing speed, initial metal ion concentration, and temperature. The adsorption of metal ions reached equilibrium rapidly. The intraparticle diffusion model, based on first principle with a linear relationship assumption between adsorbed and free metal ion concentration, satisfactorily predicted the adsorption kinetics at low metal ion concentration in solution. / Graduation date: 2001

Laminar entrained flow reactor (LEFR) was used to determine the effect of temperature and residence time on the distribution of carbon, sulfur and nitrogen between gaseous and condensed phase products from low temperature pyrolysis of kraft black liquor. The operating furnace temperatures were between 400��C-600��C where the effect of condensable organic and organic sulfur compounds may be important. The residence times ranged from 0.3 to 2.0 seconds. In the evolution of carbon as gases, an oxidizer was used to convert all oxidizable components in LEFR effluent gas to carbon dioxide which was detected by an infrared carbon dioxide meter. With this, measurement of total carbon in the gas phase, the fine particles, and the char residue were made. The carbon yield in the gas phase increased as residence time increased. The higher the temperature, the higher the carbon yield as gases phase at each residence time. The carbon yield in the fine particles differed very little with temperature at residence time below 1.1 seconds. At higher temperature, the carbon yield in the fine particles is about the same at 500��C and 600��C, but lower at 400��C. The carbon yield in the char residue decreased as residence time increased. The carbon yield in the char residue at 500��C and residence time above 1.1 seconds was a little lower than at temperature 600��C, due to an apparent loss of char at 500��C. The char yield at 500��C was lower than expected based on the 400��C and 600��C data because of accumulation of larger, more highly swollen char particles at the tip of collector at this temperature. The average of the sum of carbon recovered as char residue, gases, and fine particles was 96.2% at 600��C, 88.1% at 500��C, and 95.7% at 400��C. The main reason for the poorer carbon recovery at 500��C was the loss of char particles which accumulated on the tip of the collector. When the char yield at 500��C is increased so that the carbon balance closed to 96%, the char yield, carbon yield, and sulfur yield at 500��C fell between the values at 400��C and 600��C. The sulfur yield in the char residue decreased as residence time increased. The higher the temperature, the lower the sulfur yield in the char residue. The nitrogen yield in the char residue also decreased as residence time increased. / Graduation date: 1997