The aim of this in vitro study was to investigate the feasibility of the transdermal delivery of isoniazid (INH) and rifampicin (RMP) by means of the novel PheroidTM technology system. 'The application of the latter is being investigated in combination with various actives such as peptides (insulin, human growth hormone), anti-malarial drugs (chloroquine), anti-fungals (ketoconazole), local anaesthetics (lidocaine, prilocaine) as well as tuberculostatics (ethambutol, pyrazinamide etc.) via different administration routes at the North- West University. PheroidTM, a stable skin-friendly carrier, comprises of a submicron (200 nm - 2 m) emulsion type formulation for which previous studies have confirmed the ability to penetrate keratinised tissue, skin, intestinal linings, the vascular system, fungi, bacteria and even parasites. Studies involving an oral PheroidTM formulation containing the current approved regime of four anti-tuberculosis drugs showed improved efficacy results whilst an in vitro analysis of bacterial growth indicated a reduction in drug resistance in multidrug resistant tuberculosis (MDR-TB) strains. Therefore we thought it prudent to ascertain whether or not the PheroidTM system would be able to improve the transdermal delivery of a combination of INH and RMP as a possible treatment against cutaneous tuberculosis (tuberculosis involving the skin). The latter refers to pathological lesions of the skin caused by any one of the following: Mycobacterium tuberculosis, Mycobacterium bovis or the bacilli Calmette- Guerin (BCG) vaccine. Demonstration of M. tuberculosis within the infected tissues by traditional acid-fast bacilli (AFB) staining, culture or polymerase chain reaction (PCR) confirms the diagnosis. CTB lesions are associated with various degrees of one or more of the following ulceration, plaque formation, hyperkeratosis or the presence of necrotic matter. Seeing as C-TB is mostly associated with systemic involvement, current treatment comprises of the standard three/four drug regimens used for pulmonary 'TB in general. Cases of CTB usually show improvement within 1 month of therapy with anti-TB drugs, but complete resolution is only attained after 4 - 6 months. 'The major drawback to current therapy is that patients not only remain a source of infection (viable organisms can still be demonstrated in the lesions), but they also suffer from constant embarrassment due to the disfiguring nature of CTB until these lesions have healed completely. No evidence of an already existing topical formulation of this kind could be found. Therefore in vitro permeation studies were conducted using vertical Franz diffusion cells and female abdominal skin as permeation membrane over a period of 12 hours. Concentrations of 5 mg/ml and 10 mg/ml for isoniazid( INH) and rifampicin (RMP) respectively, were applied to the donor phase suspended in either phosphate buffered saline (PBS) or entrapped in PheroidTM. Permeation studies were conducted at pH 5.5. In vitro penetration of INH and RMP were assayed directly by HPLC. Particle size distribution for rifampicin and entrapment of actives within the PheroidTM carrier system was determined by polarized light and laser scanning microscopy (CLSM) respectively and revealed definite entrapment. Permeation profiles obtained for INH in PheroidTM indicated a biphasic character, whilst that obtained for RMP in PheroidTM showed a triphasic character. The PheroidTM delivery system proved more efficacious for delivery of both anti-tubercular drugs and resulted in greater percentage yield as well as flux values than that for a PBS solution. Furthermore, the PheroidTM formulation was able to deliver, the entrapped INH and RMP in concentrations sufficient to exceed their respective minimum inhibitory concentrations (MIC). / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2008.

The aim of this in vitro study was to investigate the feasibility of the transdermal delivery of isoniazid (INH) and rifampicin (RMP) by means of the novel PheroidTM technology system. 'The application of the latter is being investigated in combination with various actives such as peptides (insulin, human growth hormone), anti-malarial drugs (chloroquine), anti-fungals (ketoconazole), local anaesthetics (lidocaine, prilocaine) as well as tuberculostatics (ethambutol, pyrazinamide etc.) via different administration routes at the North- West University. PheroidTM, a stable skin-friendly carrier, comprises of a submicron (200 nm - 2 m) emulsion type formulation for which previous studies have confirmed the ability to penetrate keratinised tissue, skin, intestinal linings, the vascular system, fungi, bacteria and even parasites. Studies involving an oral PheroidTM formulation containing the current approved regime of four anti-tuberculosis drugs showed improved efficacy results whilst an in vitro analysis of bacterial growth indicated a reduction in drug resistance in multidrug resistant tuberculosis (MDR-TB) strains. Therefore we thought it prudent to ascertain whether or not the PheroidTM system would be able to improve the transdermal delivery of a combination of INH and RMP as a possible treatment against cutaneous tuberculosis (tuberculosis involving the skin). The latter refers to pathological lesions of the skin caused by any one of the following: Mycobacterium tuberculosis, Mycobacterium bovis or the bacilli Calmette- Guerin (BCG) vaccine. Demonstration of M. tuberculosis within the infected tissues by traditional acid-fast bacilli (AFB) staining, culture or polymerase chain reaction (PCR) confirms the diagnosis. CTB lesions are associated with various degrees of one or more of the following ulceration, plaque formation, hyperkeratosis or the presence of necrotic matter. Seeing as C-TB is mostly associated with systemic involvement, current treatment comprises of the standard three/four drug regimens used for pulmonary 'TB in general. Cases of CTB usually show improvement within 1 month of therapy with anti-TB drugs, but complete resolution is only attained after 4 - 6 months. 'The major drawback to current therapy is that patients not only remain a source of infection (viable organisms can still be demonstrated in the lesions), but they also suffer from constant embarrassment due to the disfiguring nature of CTB until these lesions have healed completely. No evidence of an already existing topical formulation of this kind could be found. Therefore in vitro permeation studies were conducted using vertical Franz diffusion cells and female abdominal skin as permeation membrane over a period of 12 hours. Concentrations of 5 mg/ml and 10 mg/ml for isoniazid( INH) and rifampicin (RMP) respectively, were applied to the donor phase suspended in either phosphate buffered saline (PBS) or entrapped in PheroidTM. Permeation studies were conducted at pH 5.5. In vitro penetration of INH and RMP were assayed directly by HPLC. Particle size distribution for rifampicin and entrapment of actives within the PheroidTM carrier system was determined by polarized light and laser scanning microscopy (CLSM) respectively and revealed definite entrapment. Permeation profiles obtained for INH in PheroidTM indicated a biphasic character, whilst that obtained for RMP in PheroidTM showed a triphasic character. The PheroidTM delivery system proved more efficacious for delivery of both anti-tubercular drugs and resulted in greater percentage yield as well as flux values than that for a PBS solution. Furthermore, the PheroidTM formulation was able to deliver, the entrapped INH and RMP in concentrations sufficient to exceed their respective minimum inhibitory concentrations (MIC). / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2008.

The membrane aerated bioreactor (MABR) is a unique technological innovation where a gas permeable membrane is applied to biological processes. In an MABR, oxygen and other substrates diffuse from the opposite directions into a biofilm, and thus simultaneous chemical oxygen demand (COD) and nitrogen removal can be achieved. However, controlling biofilm thickness, stability, and attachment is challenging. The objectives of this research were to study the effect of oxygen partial pressure on process performance with respect to nitrogen removal and examine the biomass properties in MABRs at different oxygen partial pressures and COD loadings. The conditions within the bioreactors were based on a low hydrodynamic condition (average fluid velocity 22 cm/min along the membrane surface), with the intention of minimizing the impact of the hydrodynamic shear on biomass properties. Simultaneous nitrification and denitrification were achieved in the reactors, and increasing oxygen partial pressure enhanced the total nitrogen removal. The biomass at the membrane-biofilm interface was more porous at a loading of 11.3 kg COD/1000 m2/day (areal porosity about 0.9) as compared with a loading of 22.6 kg COD/1000 m2/day (areal porosity about 0.7), indicating carbon substrate was limiting near the membrane. Long-term (over 30 days) experimental results showed that at the loading of 11.3 kg COD/1000 m2/day, the oxygen partial pressures of 0.59 atm and 0.88 atm caused over 80% of the biomass to become suspended in the bulk phase while at 0.25 atm and 0.41 atm oxygen over 97% of the biomass was immobilized on the membrane. There is a critical oxygen partial pressure that can sustain the biofilm, which increases with an increasing COD loading. The nitrifying population in the reactors was examined by applying fluorescence in situ hybridization (FISH). At the loading of 22.6 kg COD/1000 m2/day, there were 12% beta-proteobacterial ammonia oxidizing bacteria (AOB) and 17%Nitrobacter in homogenized biofilm biomass at 0.59 atm oxygen while there were 7% beta-proteobacterial AOB and 4% Nitrobacter at 0.25 atm oxygen. The ratio of protein to carbohydrate in extracellular polymeric substances (EPS) of the homogenized biomass in the reactor decreased with increasing oxygen partial pressure. Surface characterization of the biomass revealed that the higher the oxygen partial pressure, the lower the biomass hydrophobicity and surface charge. The ratio of EPS protein to carbohydrate in a membrane aerated biofilm decreased when approaching the membrane-biofilm interface. The distribution of nitrifiers and dissolved oxygen profiles inside the biofilm suggested that dual substrate limitations exist, and it was concluded that the membrane aerated biofilm had an aerobic region in the inner layer and an anoxic region in the outer layer. It is proposed that the loss of EPS due to secondary substrate consumption, especially the loss of EPS proteins, at the bottom of the biofilm was responsible for biofilm detachment subjected to a critical oxygen partial pressure.

No description available.

For the first time, a novel processing method of co-refining wood and polyolefin (PO) by steam-explosion was scientifically explored for wood-thermoplastic composites without a coupling agent. Traditional studies have addressed the improvement of adhesion between components of wood thermoplastic composites through the use of coupling agents such as maleated PO. The objective of this study was to increase adhesion between wood and PO through reactive processing conditions of steam-explosion. PO characteristics, such as type (polyethylene or polypropylene), form (pellet, fiber, or powder) and melt viscosity were studied along with oxygen gas content of the steam-explosion reactor vessel. Modification of co-processed wood fiber was characterized in four studies: microscopy analysis of dispersion of PO with wood fiber, sorption properties of co-processed material, chemical analysis of fractionated components, and morphological investigation of co-processed material. Two additional studies are listed in the appendices that relate to adsorption of amphiphilic polymers to the cellulose fiber surface, which is one hypothesis of fiber surface modification by co-steam-explosion. Microscopy studies revealed that PO melt viscosity was found to influence the degree of dispersion and uniformity of the steam-exploded material. The hygroscopic nature of the co-processed fiber declined as shown by sorption isotherm data. Furthermore, a water vapor kinetics study found that all co-refined material had increased initial diffusion coefficients compared to the control fiber. Chemical changes in fractionated components were PO-type dependent. Lignin extracted from co-processed wood and polyethylene showed PO enrichment determined from an increase of methylene stretching in the Fourier Transform infrared subtraction spectra, while lignin from co-processed wood and polypropylene did not. Additionally, extracted PO showed indirect signs of oxidation as reflected by fluorescence studies. Solid state nuclear magnetic resonance spectroscopy revealed a number of differences in the co-processed materials such as increased cellulose crystallinity, new covalent linkages and an alternative distribution of components on the nanoscale reflected in the T1Ï relaxation parameter. Steam-explosion was shown to modify wood fiber through the addition of "non-reactive" polyolefins without the need for coupling agents. In light of these findings, co-refining by steam-explosion should be viewed as a new reactive processing method for wood thermoplastic composites. / Ph. D.

Background: Syllids are a species rich annelid family possessing remarkable regenerative ability, which is not only the response after traumatic injury, but also a key step during the life cycle of several syllid taxa. In these animals the posterior part of the body becomes an epitoke and is later detached as a distinct unit named stolon. Such a sexual reproductive mode is named schizogamy or stolonization. The prostomium and the proventricle, a modified foregut structure, have been proposed to have a control function during this process, though the concrete mechanisms behind it have never been elucidated. Results: By using different experimental set-ups, histology and immunohistochemistry combined with subsequent cLSM analyzes, we investigate and document the regeneration and stolonization in specimens of Typosyllis antoni that were amputated at different levels throughout the antero-posterior body axis. The removal of the anterior end including the proventricle implies an incomplete anterior regeneration as well as severe deviations from the usual reproductive pattern, i.e. accelerated stolonization, masculinization and the occurrence of aberrant stolons. The detailed anatomy of aberrant stolons is described. A histological study of the proventricle revealed no signs of glandular or secretory structures. The ventricle and the caeca are composed of glandular tissue but they are not involved in the reproductive and regenerative processes. Conclusions: As in other investigated syllids, the proventricle region has a significant role during stolonization and reproduction processes in Typosyllis antoni. When the proventricle region is absent, anterior and posterior regeneration are considerably deviated from the general patterns. However, proventricle ultrastructure does not show any glandular component, thereby questioning a direct involvement of this organ itself in the control of reproduction and regeneration. Our findings offer a comprehensive starting point for further studies of regeneration and reproductive control in syllids as well as annelids in general.

Escapin is an L-amino acid oxidase that produces antimicrobial metabolites collectively called “Escapin Intermediate Products” (EIP-K). EIP-K and H2O2 together were previously shown to be bactericidal towards diverse planktonic bacteria. The present work investigates the ability of EIP-K and H2O2 to antagonize bacterial biofilms, using Pseudomonas aeruginosa as a model. The project had three aims: 1) determine the most effective concentrations of EIP-K and H2O2 necessary to break down existing P. aeruginosa biofilms, using a crystal violet assay; 2) examine the ability of EIP-K + H2O2 to inhibit biofilm formation, using triphenyl tetrazolium chloride dye; and 3) determine the effect of EIP-K + H2O2 on the viability, biomass and structure of biofilms cultivated in flow cells using confocal laser scanning microscopy (CLSM). Results showed that EIP-K + H2O2 significantly reduced biofilm biomass relative to controls and that the compounds are effective at nanomolar concentrations.

Les biofilms sont des communautés de microorganismes adhérant à une surface et encastrées dans une substance polymérique produite par les cellules du système, dite matrice extracellulaire. Du fait de leur nature ubiquitaire, les biofilms colonisent de nombreux environnements et causent souvent de sérieux problèmes dans les secteurs de la santé et de l’industrie. La dispersion par ajout d’agent chimique est l’une des stratégies de lutte contre les biofilms. Un acide gras, l’acide cis-2-décénoique (CDA), semble être prometteur pour ce faire, grâce à l’étendue de son action dispersante sur les espèces et règnes du vivant. L’objectif de ce travail de thèse est d’investiguer les mécanismes de dispersion des biofilms de l’espèce bactérienne Escherichia coli (E. coli) par la molécule modèle CDA. Le CDA modifie-t-il les structures du biofilm ou induit-il une réponse génétique des bactéries lors de la dispersion ? Pour répondre à ces questions, la dispersion des biofilms d’E. coli a été étudiée in situ dans des chambres microfluidiques par microscopie confocale à balayage laser (CLSM). Des souches bactériennes spécifiques ont été construites par clonage de promoteurs d’intérêt en fusion transcriptionnelle avec un gène codant pour une protéine fluorescente verte. Les résultats confirment l’activité dispersante du CDA avec une réduction significative de la biomasse, de l’épaisseur moyenne et de l’aire de recouvrement par couche du biofilm. Un outil innovant d’analyse d’images CLSM a été développé en collaboration dans le but de déterminer les propriétés structurales du biofilm et l’intensité de fluorescence in situ du rapporteur étudié. Les résultats indiquent une augmentation de l’intensité moyenne de fluorescence des biofilms après dispersion avec le CDA, au niveau global en considérant tout le biofilm et au niveau local en considérant une segmentation du biofilm en microcolonies, ainsi qu’en profondeur. Ces résultats évoquent un changement d’expression génique des bactéries en présence de CDA. Par ailleurs, les résultats montrent que le CDA ne semble pas avoir d’effet en culture planctonique, ni sur la croissance bactérienne ni sur l’activité des promoteurs sélectionnés. Ceci suggère que les effets du CDA sont biofilm-dépendants. / Biofilms are microbial communities adhering to a surface and embedded in a self-produced polymeric substance, called extracellular matrix. By being ubiquitous in nature, biofilms colonize numerous environments, and they often cause serious problems for both health and industry sectors. Dispersion is one of the strategies for fighting biofilms. A fatty acid, cis-2-decenoic acid (CDA), seems to be promising for dispersing biofilms by the extent of its action on different species of microbes. The aim of this thesis work is to investigate the mechanisms of biofilm dispersion of the bacterial species Escherichia coli (E. coli) by the model molecule CDA. Does CDA modify the biofilm structures or does it induce a genetic response from bacteria during dispersion? To answer these questions, E. coli biofilm dispersal has been studied in situ in microfluidic chambers by confocal laser scanning microscopy (CLSM). Specific bacterial strains have been developed by cloning promoters of interest in transcriptional fusion with a gene encoding for a green fluorescent protein. The results confirm the dispersing activity of CDA with a significant decrease of biomass, biofilm average thickness and area over biofilm depth. A novel tool for analyzing CLSM images has been developed in collaboration in order to measure the biofilm structural properties as a function of in situ fluorescence intensity of the studied reporter. The results indicate an increase in the mean fluorescence intensity of the biofilms after dispersion with CDA, at a global level for the whole biofilm and at a local scale by considering a biofilm segmentation into microcolonies. These results evoke a change in gene expression by bacteria in the presence of CDA. Furthermore, the results show that CDA does not seem to have an effect on planktonic bacteria, neither on the bacterial growth nor on the activity of the selected promoters. This suggests that the CDA effects are biofilm-dependent.

No description available.

4 Abstract The present thesis focuses on testate amoebae (TA) and their relationship to their natural environment, as well as on relevant microscopic imaging methods. The bulk of the data has been published in original scientific papers and is compiled into three separate chapters (Pt I, Pt II and Pt III), each annotated by a brief introduction. (Pt I) The methods section is devoted to specialized microscopic techniques employed to broaden the scope of the ecological analyses. In particular, precise discrimination between live and dead individuals, biomass determination inside individual tests and a multi-modal visualization of the cytoplasm and organelles enhance the data. Laser scanning confocal microscopy and two-photon microscopy are the main imaging modalities employed to study TA morphology in detail. The data have implications for taxonomy and ecophysiology, including the use of TA as bioindicators of pollution. (Pt II) An actuoecological analysis focuses on the seasonal variability of TA species composition in a freshwater ecosystem, namely the Komo any ponds in Prague, during the course of the year. The species composition variation is correlated to simultaneously recorded limnological parameters such as temperature, pH, contamination by (heavy) metals (As, Cd, Mn, Ni, Fe, Pb), polycyclic aromatic...