Phenolic residues which accumulate in the environment as a result of agro-industrial practices has resulted in the need to find and use Eco-Friendly techniques, rather than the traditional methods of burning or burying this kind of waste. Bioremediation and bioconversion are attractive alternatives using whole cell or enzyme-based systems. The aims of this project were to isolate and uses thermophilic Actinomycetes, which produce thermo-tolerant oxidoreductase enzymes, which can be used to bioconvert a model industrial phenolic waste commonly genersated in the wine-making industry of South Africa. Current research in bioconversion and bioremediation focuses on mesophilic microbes in that their enzymes can catalyse reactions at higher temperatures without affecting its activity and lower contamination levels. Three novel Actinomycete isolates were isolated (RU-A0l , RU-A03 and RU-A06) from a compost site and characterized using a combination of conventional identification techniques and 16S rDNA methodology to identity the three isolates. All three isolates belong to the Streptomyces clade. In addition, five known Actinomycetes were selected from an internation culture collection and also screened for oxidoreductase activity in comparision to the three novel isolates. Although the five isolates were selected based on their ability to produce oxidoreductase enzymes, unexpectedly, no activity was detected. Screening assays for peroxidase, polyphenol oxidase and laccase on RU-AO 1, RU-A03 and RU-A06, showed that all three isolated produced peroxidases and peroxidases but no laccase. Substrate specificity studies revealed that the most suitable substrates to determine peroxidase and polyphenol oxidase activity on these isolates were catechol for polyphenol oxidase, 2,4-dichlorophenol for peroxidases and veratryl alcohol for lignin peroxidases. Previous studies have indicated that peroxidases and polyphenol oxidases are produced in Actinomycetes during the primary stage of growth. This was the case with RU-AOI , RU-A03 and RU-A06. Growth rates were higher that other Actinomycetes, with maxImum biomass being reached at 36 hours for the isolates RU-AOI and RU-A06 and 48 hours for isolate RUA03. pH studies showed that the three isolates were adaptable and could grow over a broad pH range. Catabolism studies of phenolic model compounds showed that the three isolates were capable of catabolizing the model phenolic compounds within a period of 24 hours. Further studies were carried out to determine the effect of these microbes and their enzymes in whole cell and enzyme-based systems on a model phenolic waste, graoe waste consisting of compressed grape skins, pips and stalks. Whole cell studies showed that the isolates were capable of bioconverting the waste at a maximum concentration of 30% grape waste (vol:vol). Peroxidase and polyphenol oxidase activity increased indicating induction of these enzymes in the presence of phenolic compounds, with a maximum increase of up to 15.9 fold increase in extracellular lignin peroxidase activity in RU-AO1. HPLC and phenolic determination assays indicated that bioconversion of the phenolic grape waste had occurred in the presence of the three isolates. Attempts were made to isolate and identify a peroxidase or phenol oxidase gene from one the isolates. As bacteria, Actinomycetes are amendable to gene manipulation making them suitable candidates for methods such as site directed evolution in comparison to fungi. Two clones were selected for sequencing based on positive activity results when assayed for peroxidase activity. However the resultant sequences did not identify a functional gene sequence. Southern Blotting was then carried out to determine the nature of the peroxidase gene. Previous studies have been focused on the catalase-peroxidase gene (CalC gene) found Actinomycetes and other bacteria. A probe was developed from the CalC gene. No hybridization occurred with any of the enzyme restricted DNA from the three isolates. The implications of these results are that the peroxidase genets in the three isolates are in fact lignin peroxidase in nature. This project has the potential in the bioconversion of phenolic wastes and is the first description of the use of thermophilic Actinomycetes in the bioconversion of an industrial phenolic waste.

<div lang="en" class="abs"> Abstract The increasing awareness of the causes and consequences of climate chance has led to actions to reduce the dependency on oil and other finite energy and raw material sources. Plant biomass is used in increasing amounts as a resource for biofuel, biochemical and fiber production. Carbohydrate enzymology has provided new ways to utilize and modify renewable carbon sources, especially the lignocellulolytic systems of fungi. Cellulolytic enzymes work in a synergistic manner on recalcitrant structure of cellulose, hydrolyzing it into soluble oligosaccharides, and eventually, glucose. Lytic polysaccharide monooxygenases (LPMOs) contribute to this system by oxidizing either C1- or C4-carbon from the carbohydrate chain on a crystalline cellulose with the help of copper-core induced radicals, thus creating available substrates for the other cellulolytic enzymes. Since their discovery in 2010, the research on their activities and specificities have increased rapidly, but the analytical methods to investigate this diverse group of enzymes is mostly limited to short and soluble products, which are only a fraction of the oxidation products. In addition, most of the methods require special equipment, wide range of standards and expertise to interpret the results. In this study, HPLC and HPAEC-PAD were tested, unsuccessfully, to quantify soluble products from LPMO-catalysis. A TTC-method, in which 2,3,5-triphenyl-2H-tetrazolium chloride is reduced into red and spectrophotometrically quantifiable formazan by reducing ends from insoluble LPMO-products, was successfully optimized and downscaled, and used to optimize reaction conditions for a type 3 LPMO from Trichoderma reesei, TrAA9A, with Whatman filter paper 1 as a substrate. Experiments were conducted to investigate the effects of pH, temperature, donor, time and the presence/absence of H₂O₂ to the accumulation of reducing ends. The results did not show any substantial differences in the accumulation of aldehydes in different reaction conditions. This study showed that cellulose degrades in the presence of TrAA9A and an electron donor. The greatest effects were observed with longer reaction times and the addition of H₂O₂, both increasing the amount of measured aldehydes in the insoluble products. The highest yield was recorded from the reactions with gallic acid as a donor at pH 6, and in the presence of 0.7 mM H₂O₂. The results from this study could lead to understanding the rate-limiting factors of the LPMOs and further improve the utilization of this enzyme in the degradation of lignocellulosic biomass.

Many microorganisms, and in particular fungi, are able to grow over a wide pH range. Thus, these microorganisms must possess some regulatory mechanism or system that senses the environmental pH signal and ensures that gene expression of certain molecules is tailored to the pH of the environment (Penalva and Arst, 2002). In Aspergillus species and several other fungi, pH regulation is mediated by seven genes viz. palA, palB, palC, palF, palH, palI and the global pH regulatory gene, pacC (MacAbe et al, 1996; Negrete-Urtasun, 1999; Denison, 2000). The activated form of the PacC protein activates genes that are required at alkaline pH, e.g. genes coding for alkaline phosphatases, and represses certain genes that are functional at acidic pH, e.g. genes encoding acid phosphatases (Negrete-Urtasun, 1999). PacC (and its homologues) also positively regulates genes involved in penicillin biosynthesis, e.g. the isopenicillin N synthase gene, ipnA, in A. nidulans (Penalva and Arst, 2002). It has also been hypothesised that pacC may negatively regulate aflatoxin biosynthesis, a carcinogenic secondary metabolite in several species of Aspergillus (Keller et al, 1997). To elucidate the role of pacC a novel method of post-transcriptional gene silencing known as RNA interference was utilized. This method involved the cloning of a partial pacC gene fragment first in the forward and then the reverse orientations in a fungal expression cassette to create an RNA interference (RNAi) vector. The unique structure of this vector would allow the cloned fragments to be expressed and the resulting RNA to immediately form a double stranded stem-loop structure or short hairpin RNA (shRNA; McDonald et al, 2005). The formation of this shRNA, in turn, would be responsible for activating the endogenous RNA degradation complexes that would lead to mRNA degradation and subsequent gene silencing (Liu et al, 2003; Kadotoni et al, 2003; McDonald et al, 2005). The results presented here have shown that confirmed pacC RNAi mutants produced aflatoxins irrespective of environmental pH (i.e. the mutants produce aflatoxins under acidic and alkaline conditions). Thus, pacC is essential for pH regulation of aflatoxin production in A. flavus. There are numerous other biological (e.g. presence of oxylipins, lipooxygenases) and non-biological factors (pH, carbon source etc.) which affect maize colonisation and aflatoxin production by A. flavus (Burrow et al, 1996; Wilson et al, 2001; Calvo et al; 2002; Tsitsigiannis et al, 2006). However, all the genetic mechanisms involved have as yet not been identified. It has been shown by Caracuel et al (2003) that pacC acts as a negative virulence regulator in plants and these workers have hypothesised that PacC prevents expression of genes that are important for infection and virulence of the pathogen. Therefore the physiological effects that pacC silencing had on the growth, conidiation and pathogenicity of A. flavus mutants were also investigated. The results of this study showed that pacC does not play a significant role in primary growth and development but does affect conidial production. SEM results showed that mutants have many “open ended” phialides and poorly developed conidiophores. This would suggest that pacC activation of conidial production genes is also required. Furthermore, pacC RNAi silencing severely impaired the ability of the A. flavus mutants to infect and cause damage on maize. The results obtained here are similar to that of pacC null mutants in A. nidulans, C. albicans and F. oxysporum which also exhibited low pathogenicity (Davis et al, 2000; Fonzi, W.A, 2002; Caracuel et al, 2003; Bignell et al, 2005 and Cornet et al, 2005). This study indicates that pathogenicity of A. flavus on maize is directly related to the structural integrity of conidia, which in turn is greatly influenced by PacC. This gene is a global transcriptional regulator and may either repress or activate one or many genes in each of the above pathways (Penalva and Arst, 2002). Studies on the genetic mechanisms of pacC regulation on these pathways are needed to elucidate the mechanisms of activation or repression of these genes.

A three part study was carried out to examine rheological, colloidal and floe microstructural aspects of industrial brewing yeast strains. Following a review of the literature, the rheological properties of four yeast strains (two flocculent ale and lager types and their non-flocculent variants) were examined. In related colloidal studies, orthokinetic flocculation rates of these strains as well as their cell surface charge were determined. Floc microstructure was characterized using both light and scanning electron microscopy. In a summary chapter, the cell floc model (a modification of Hunter's elastic floc model) was used to the explain the rheological and colloidal behaviour of brewing yeast suspensions. Flow behaviour studies of the commercial yeast suspensions suspended in a calcium-containing sodium acetate buffer revealed that yeast flocculent characteristics had an important influence on their suspension flow behaviour. As cell concentrations increased, suspension flow properties become increasingly non-Newtonian and could be described by the Casson model at low rates of shear and the Bingham model at shear rates above 100 s⁻¹. The cell floc model was proposed to explain the Bingham flow behaviour of these csuspensions. The Bingham yield stress in these suspensions was believed to be a function of the orthokinetic capture coefficient, cell volume and the energy to break up doublet cells. Increasing temperature tended to lower the Bingham yield stress in lager strains and increase the yield stress in ale strains. A semi-empirical explanation for the viscosity increase of deflocculated cell suspensions and the estimation of pseudo-capture coefficients was presented. Furthermore, studies of the flow behaviour of yeast strains suspended in decarbonated ale and lager beer revealed that: 1) suspensions of flocculent strains show higher yield stress values than their non-flocculent variants, 2) ale strain suspensions tended to have higher yield values than the lager strains and 3) yeast dispersed in beer had higher yield stress values than when suspended in buffered calcium suspensions. This last observation was believed to reflect the influence of ethanol on the cell binding process which has important implications for future measurements of yeast flocculation. Colloidal studies revealed for the first time, that the orthokinetic rate of flocculation of brewing yeast cells could be modelled by a first order equation, as predicted by fundamental colloid theory. While subject to considerable variation, measured rate constants led to the calculation of orthokinetic capture coefficients. Yeast cell zeta potential values generally agreed with literature data but could not be employed in the DLVO model of colloid flocculation to explain measured orthokinetic capture coefficient values. Examination of the cell zeta potential data indicated that the data had non-normal distributions. SEM examination of the four industrial yeast strains suggested that a number of distinct structures mediated cell-to-cell interaction and that intra-strain differences occurred. These findings, along with the observation of non-normal surface charge distributions, indicated that these industrially pure strains had undergone substantial variation. Treatment of the flocculent cells with pronase tended to reduce cell-to-cell contacts. In the summary chapter the cell floe model was employed to describe the rheological behaviour of the yeast suspensions. Estimation of the force needed to separate doublet yeast cells were made using critical shear rate data (i.e., the point at which Bingham flow begins). This estimate was similar to that reported for single antibody bonds and may be due to the presence of lectin-like structures on the yeast cell wall. / Land and Food Systems, Faculty of / Graduate

In the last decades, the prevalence of obesity increased dramatically worldwide. According to the WHO, 2.1 billion people (30% of the population) around the world are obese or overweight. Effective ways to decrease food intake are needed. Evidence from emerging studies indicates an association between feeding behavior and the IL-6 expression in the central nervous system (CNS). The study aimed to investigate the effect of IL-6 modulation on feeding behavior and learning and memory process in the ventral hippocampus (vHPC). To implement this aim two groups of rats were used, the first group was exposed to the reduction of the IL-6 expression (knockdown), whereas the other one to microinjections of exogenous IL-6 (EX IL-6). Both experimental groups were subjected to a set of behavioral and molecular tests specific for investigating memory process, emotional/affective behavior and food intakes like novel object recognition, Morris water maze, and social interaction test. The results for IL-6 knockdown (KD) showed improvement in the short term memory, but did not affect the food intake. On the other hand, EX IL-6 caused an increase in the locomotor’s activity and the food intake during the 24 hours, but at the same time caused impairment in the spatial and learning memory. Taken together, these results provide new insight on the role of IL-6 outside of inflammation highlighting its ability to modulate hippocampus-dependent mnemonic process, and affective and feeding behaviors in the vHPC, however several questions still remain not addressed and the study require further investigation.

No description available.

Cerebral stroke has become one of the leading causes of death and disability worldwide. During an ischemic stroke, oxygen and nutrient deprivation occurs, which combined lead to cell starvation, anoxia, and eventually cell death. However, when blood flow is restored, reperfusion damage occurs resulting in increased cell death through several mechanisms. One of the main reasons behind ischemia/reperfusion damage is oxidative stress due to elevated production of reactive oxygen species (ROS) during reperfusion. There are several proteins and processes that are thought to be involved in elevated oxidative stress and the formation of ROS during reperfusion, among which the NADPH oxidase (Nox) family is suggested to be the main contributor of ROS.To examine this hypothesis, in the present work, we inhibited activity of the Nox2 and Nox4 enzymes during ischemia/reperfusion with the Glucox Biotech AB (Sweden) inhibitors M4, M107, and M114 to evaluate whether reducing Nox activity could reduce the ischemia/reperfusion-induced cell death, hence be used as a potential stroke treatment, the cell viability was measured with MTS after ischemia/reperfusion induction and treatment with the Nox substances. We also examined the gene expression levels of the Nox enzymes Nox2 and Nox4 with qPCR after induced ischemia/reperfusion in the neuroblastoma cell line NB69.Our results showed a decrease in Nox4 gene expression after 1h ischemia/8h reperfusion and an increased expression after 1h ischemia/24h reperfusion in NB69 cells. Treatment with M114 resulted in increased cell viability after 2h ischemia/72h reperfusion. However, the toxic effect of ischemia/reperfusion-induced response was found to be inadequate, as indicated by extensive proliferation and lack of cell death. This unfavorable outcome is suggested to be excess of oxygen in medium, metabolization of L-glutamine, and effects of growth factors in the serum used in cell culture medium during the ischemic phase. Therefore, the cell culture protocol was modified to the use of PBS instead of glucose-free medium under serum-free condition during the ischemia. The altered ischemic conditions resulted in continuous reduction in cell viability at increasing ischemic time points with total cell death at 2h ischemia, suggesting applicable conditions for ischemia/reperfusion studies. Even though a conclusion could not be made about the inhibitors M4, M107, and M114 as the cell viability assay was performed under insufficient conditions; the Nox inhibitors shows high potential as future ischemic stroke treatments, which may help save lives and improve life quality for affected patients.

Affibody® molecules are small affinity proteins with great opportunities in the application of biotechnological areas. Their popularity as biomolecules in pharmaceutical applications and research entails that the purity of Affibody® molecules is essential for their use as safe medical drugs. Hydrophobic Interaction Chromatography (HIC) is a purification technique that separates molecules based on their inherent difference in hydrophobicity, and the application as a purification strategy for Affibody® molecules may be possible. This thesis investigates the implementation of HIC on Affibody® molecules by screening for differences in binding, recovery, and purity influenced by different parameters, such as resin, pH, salt type, and salt concentration, have on binding, recovery, and purity. HIC presents as a viable purification method where an approximate 97% reduction in Host Cell Proteins was obtained. The yield of the purified product also presented as promising with a recovery of approximately 82%. These results indicate that further investigation and optimization of this technique may benefit the downstream process of the investigated Affibody® molecule.

No description available.

Molecular electronics is an active research area for the future information technology. The fabrication of basic electronic elements with molecules as the core-operators has been made experimentally in the laboratory in recent years. However, the underlying electron or charge transport mechanisms for most devices are still under debate, Theoretical modelling based on the first-principles methods are expected to play an important role in this field. A generalized quantum chemical approach based on Green's function scattering theory has been developed and applied to two- and three-terminal molecular devices. It allows to study both elastic and inelastic electron scattering at hybrid density functional theory levels. It can treat molecular devices where the metal electrodes and the molecule are either chemically or physically bonded on equal footing. As one of the applications, we have studied the length dependence of electron transport in gold-oligophenylene-gold junctions. We have shown that the experimental results for molecular junctions of oligophenylene with di erent lengths can be well reproduced by hybrid density functional theory calculations. It is also found that the current-voltage characteristics of the junctions depend strongly on the metal-molecule bonding distances. With the help of the calculations, the possible gold-molecule bonding distances in the experimental devices are identi ed. The central focus of this thesis is to study the three-terminal molecular devices, namely the eld e ect transistor (FET). An extension of our quantum chemical approach to FET devices has been made and successfully applied to different FET devices constructed with polymer, small and middle sized conjugated molecules. The experimentally observed conductance oscillation in polymer FET and three orders of magnitude enhancement of the current in electrochemical gated molecular FET have been verified by the calculations. The electron transport mechanisms of these devices are revealed. / QC 20101129