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The presented diploma thesis deals with the extraction and storage stability of lipophilic pigments produced by selected strains of yeasts and algae. In this thesis, there was studied the influence of the selected solvents on the efficiency of extraction, as well as the effect of ambient temperature on the stability of the pigments during storage. The work is divided into two main parts, theoretical and practical part. In the theoretical part knowledge about algae, yeasts and their cultivation parameters is summarized. Furthermore, some information regarding the selected lipid metabolites, their properties and possibilities of application in various branches of industry is mentioned. The practical part deals with the preparation of extracts and stability tests. Extracts were prepared from selected biomass samples in solvents suitable for applications in food industry or cosmetics (ethanol and hexane). Subsequently, the long-term stability tests lasting 4 months and short-term stability tests lasting a total of 28 days were performed on these extracts. The pigments were determined by HPLC and spectrophotometrically, the fatty acid content was determined by GC. For the extraction of pigments from biomass, in the most cases ethanol appears to be the optimal solvent. However, for lipid extraction from biomass, hexane appears to be the optimal solvent for a significant number of samples. In most samples, storage in the freezer showed the most favourable effect on pigment stability, but some samples showed comparable stability even when stored in the refrigerator.

Formulation of local anaesthetics in different dosage forms, including those for oral, parenteral, and topical application have being widely investigated. All of these formulations include local anaesthetics in their salt forms. The lipophilic nature of the bases of local anaesthetics may influence the rate of the pharmacological effect. There has been very little research done towards this aspect of local anaesthetics. Prilocaine base and lignocaine base possess greater lipophilicity than their salts. The salt forms undergo dissociation in the body. To maximise the absorption rate lipophilicity plays an important role. The aim of the present study is to evaluate the potential of using prilocaine and lignocaine individually and in combination as bases for parenteral formulations using cyclodextrins as complexing agents. Cyclodextrins are widely used as complexing agents to increase the solubility of poorly soluble drugs. Hydroxypropyl-β-cyclodextrin (HPβCD) was the first choice amongst the different cyclodextrins to be evaluated as a solubility enhancer as it does not show nephrotoxicity and is more bio-available compared to other cyclodextrins. / Method: Prilocaine base was prepared from its salt and lignocaine base was obtained from Sigma Pharmaceuticals. Solubilities were examined individually and in combination by the phase solubility method and complex formation investigated. The mobile phase used was methanol:water (55:45) with phosphate buffer at pH 5.5. An AL type solubility isotherm was obtained for the influence of HPβCD on the solubilities of prilocaine and lignocaine. Complexation was investigated for both prilocaine and lignocaine to HPβCD by NMR. Results: The measured solubilities of prilocaine and lignocaine individually at 30% HPβCD from 25°C to 42°C were 1.96-7.91 moles/L and 1.69-4.55 moles/L respectively. The solubilities in combination were 0.91-3.68 moles/L for prilocaine and 1.03-8.35 moles/L for lignocaine respectively. The NMR data suggested that complexation involves the aromatic ring for both prilocaine and lignocaine apart from methene and methyl groups for prilocaine and ethyl amide and aromatic methyl groups for lignocaine.

Ph.D. / The main objective of this study was to investigate the presence, variations and the fate of lipophilic extractives during all the stages in the dissolving pulp production process. Lipophilic wood extractives which include fatty acids, sterols, hydrocarbons, steroid hydrocarbons and ketones are organic compounds present in the wood and dissolving pulp which are soluble in organic solvents. Their presence in wood and pulp has negative effects on both the pulping process and the quality of the produced dissolving pulp. Comparison of two Eucalyptus species (E. grandis and E. dunnii) at three different sites (Richmond, Umvoti and Ixopo in KwaZulu-Natal Province, South Africa) revealed that lipophilic extractives were higher in E. dunnii than in E. grandis in all the sampled sites and that the woods at the Ixopo site were found to have the highest amount of lipophilic extractives. The quantitative variation of lipophilic extractives in wood was found to be significantly affected by respective sites and tree species. The principal component analysis (PCA) model, which was used to correlate soil parameters and amount of lipophilic extractives in woods per each site, revealed that high amounts of lipophilic extractives were found in trees grown at sites with a high composition of clay soil and organic matter. Thus if the industry is to avoid the high levels of lipophilic extractives in wood, sandy sites should be the choice to grow the trees. Partial least squares (PLS) modelling revealed that sterols and saturated fatty acids significantly influence the quality parameters (Kappa number, viscosity, copper number, cellulose yield and retained carbohydrates) of dissolving pulp, whereas unsaturated fatty acids and unsaturated sterols in particular were found to positively correlate with the Kappa number, thus affecting its measurement.The main objective of this study was to investigate the presence, variations and the fate of lipophilic extractives during all the stages in the dissolving pulp production process. Lipophilic wood extractives which include fatty acids, sterols, hydrocarbons, steroid hydrocarbons and ketones are organic compounds present in the wood and dissolving pulp which are soluble in organic solvents. Their presence in wood and pulp has negative effects on both the pulping process and the quality of the produced dissolving pulp. Comparison of two Eucalyptus species (E. grandis and E. dunnii) at three different sites (Richmond, Umvoti and Ixopo in KwaZulu-Natal Province, South Africa) revealed that lipophilic extractives were higher in E. dunnii than in E. grandis in all the sampled sites and that the woods at the Ixopo site were found to have the highest amount of lipophilic extractives. The quantitative variation of lipophilic extractives in wood was found to be significantly affected by respective sites and tree species. The principal component analysis (PCA) model, which was used to correlate soil parameters and amount of lipophilic extractives in woods per each site, revealed that high amounts of lipophilic extractives were found in trees grown at sites with a high composition of clay soil and organic matter. Thus if the industry is to avoid the high levels of lipophilic extractives in wood, sandy sites should be the choice to grow the trees. Partial least squares (PLS) modelling revealed that sterols and saturated fatty acids significantly influence the quality parameters (Kappa number, viscosity, copper number, cellulose yield and retained carbohydrates) of dissolving pulp, whereas unsaturated fatty acids and unsaturated sterols in particular were found to positively correlate with the Kappa number, thus affecting its measurement. Viscosity of pulp which is an important quality parameter was also found to be positively influenced by sterol residues, whereas the reducing properties of pulp were found to be affected by sterols and saturated fatty acids with a carbon chain length of less than C20.

Espécies de Malassezia fazem parte da microbiota de humanos e de animais. Essas leveduras lipofílicas são microrganismos oportunistas que estão associados com muitas doenças superficiais como: pitiriase versicolor, dermatite seborréica, foliculite, dermatite atópica e algumas infecções sistêmicas. As espécies de Malassezia têm sido identificadas através de procedimentos morfológicos e bioquímicos, no entanto, pequenas semelhanças entre algumas espécies estão presentes em algumas regiões do genoma. Foi avaliado o PCR-RFLP, método molecular para genotipar espécies de Malassezia obtidas de 55 amostras clinicas isoladas. Foram analisados 23 pacientes com dermatite seborréica, pitiriase versicolor e com a síndrome de Gourgerot- Carteaud. Encontramos quatro espécies diferentes: M. furfur, M. globosa, M. sympodialis and M. slooffiae. A identificação fisiológica e o PCR-RFPL foram compatíveis em 83% das amostras. M. furfur esteve presente em 52% dos casos, o que sugere que é o agente causador da pitiriase versicolor e da dermatite seborréica. Os resultados sugerem que a utilização do PCR-RFLP em amostras clínicas é importante no diagnóstico de algumas micoses causadas por esta levedura. / Malassezia species are part of the microbiota of humans and other animals. These lipophilic yeasts are opportunistic microorganisms that are associated with some dermatoses including pityriasis versicolor, seborrheic dermatitis, folliculitis ptirospórica, atopic dermatitis, some systemic infections among others. The species of Malassezia have been identified by morphological and biochemical and molecular techniques currently. We identify the species of Malassezia obtained from 55 clinical samples and 15 samples that formed the control group, by conventional techniques using a medium with not described in the literature, the mixture of media and Dixon Kimming, which was higher than each when used separately. Used the molecular method of PCR-RFLP to genotype 23 of the 55 clinical samples of Malassezia spp. from patients with seborrheic dermatitis, pityriasis versicolor and Gourgerot - Carteaud syndrome. Could the isolation of four species: M. furfur, M. globosa, M. Sympodialis and M. slooffiae. The physiological identification obtained with the PCRRFLP was consistent in 83% of the samples. M. furfur was present in 52% of cases, suggesting that is the main causative agent of pityriasis versicolor and perhaps the agent most frequently found in seborrheic dermatitis. The results suggest that the use of PCR-RFLP method in clinical samples is of great use for the identification of Malassezia species associated with diseases in humans.

Espécies de Malassezia fazem parte da microbiota de humanos e de animais. Essas leveduras lipofílicas são microrganismos oportunistas que estão associados com muitas doenças superficiais como: pitiriase versicolor, dermatite seborréica, foliculite, dermatite atópica e algumas infecções sistêmicas. As espécies de Malassezia têm sido identificadas através de procedimentos morfológicos e bioquímicos, no entanto, pequenas semelhanças entre algumas espécies estão presentes em algumas regiões do genoma. Foi avaliado o PCR-RFLP, método molecular para genotipar espécies de Malassezia obtidas de 55 amostras clinicas isoladas. Foram analisados 23 pacientes com dermatite seborréica, pitiriase versicolor e com a síndrome de Gourgerot- Carteaud. Encontramos quatro espécies diferentes: M. furfur, M. globosa, M. sympodialis and M. slooffiae. A identificação fisiológica e o PCR-RFPL foram compatíveis em 83% das amostras. M. furfur esteve presente em 52% dos casos, o que sugere que é o agente causador da pitiriase versicolor e da dermatite seborréica. Os resultados sugerem que a utilização do PCR-RFLP em amostras clínicas é importante no diagnóstico de algumas micoses causadas por esta levedura. / Malassezia species are part of the microbiota of humans and other animals. These lipophilic yeasts are opportunistic microorganisms that are associated with some dermatoses including pityriasis versicolor, seborrheic dermatitis, folliculitis ptirospórica, atopic dermatitis, some systemic infections among others. The species of Malassezia have been identified by morphological and biochemical and molecular techniques currently. We identify the species of Malassezia obtained from 55 clinical samples and 15 samples that formed the control group, by conventional techniques using a medium with not described in the literature, the mixture of media and Dixon Kimming, which was higher than each when used separately. Used the molecular method of PCR-RFLP to genotype 23 of the 55 clinical samples of Malassezia spp. from patients with seborrheic dermatitis, pityriasis versicolor and Gourgerot - Carteaud syndrome. Could the isolation of four species: M. furfur, M. globosa, M. Sympodialis and M. slooffiae. The physiological identification obtained with the PCRRFLP was consistent in 83% of the samples. M. furfur was present in 52% of cases, suggesting that is the main causative agent of pityriasis versicolor and perhaps the agent most frequently found in seborrheic dermatitis. The results suggest that the use of PCR-RFLP method in clinical samples is of great use for the identification of Malassezia species associated with diseases in humans.

The work presented in this thesis details the synthetic modification of the clinically important aminoglycoside antibiotics, neomycin B, paromomycin and tobramycin. We sought to modify aminoglycosides by attaching lipophilic groups, including fatty acids and steroids, with a view to improving the bacterial membrane permeability of these species, and ultimately their efficacy in the treatment of tuberculosis. Our initial synthetic strategy involved direct and specific functionalization of the singular primary hydroxyl group of the aminoglycoside antibiotic neomycin B, with lipophilic groups containing carboxylic acid functions via Mitsunobu esterification. Although, direct and selective Mitsunobu acylation of the primary hydroxyl group proved unsuccessful in the case of the pseudo tetrasaccharide neomycin B, the Mitsunobu reaction did however result in selective chemistry elsewhere in the molecule and this has been exploited for modification of the ido (ring IV) and streptamine (ring II) ring systems. Under carefully controlled conditions, the Mitsunobu reaction has been used for the selective dehydration of the ido ring, to give the talo epoxide, and, under more forcing Mitsunobu dehydration conditions, an aziridine function has been introduced into the streptamine moiety. Both the epoxide and the epoxide-aziridine neomycin building blocks were utilized as synthons in subsequent chemical transformations. Seventeen novel neomycin derivatives featuring modification of ring IV and/or ring II were obtained using this approach. Explicit structural elucidation of all the synthetic intermediates and the final products was achieved using high temperature NMR spectroscopy. Direct and specific functionalization of the singular primary hydroxyl group at the C5 position of the ribose ring (ring III) of neomycin B was achieved, via a procedure based in part on selective tripsylation of the C5III primary hydroxyl group of neomycin B reported previously, followed by subsequent displacement of the tripsyl group by azide. Terminal alkyne containing lipophilic esters were then successfully attached to the ribose residue of neomycin B via Cu(I)-mediated azide-alkyne coupling reaction. In addition to the isolation of two fortuitous, new and versatile synthons i.e. monoanhydro neomycin and bis-anhydro neomycin for modification of ring IV and ring II of neomycin, a third synthon based on neomycin framework, allowing stepwise modification of ring III and ring IV was designed and synthesized. This synthon features an epoxide function in the ido ring, and a protected amine function at the C5 position of the ribose ring. Examples of the stepwise use of this synthon for further synthetic modification of the neomycin framework were demonstrated. Fourteen novel neomycin derivatives featuring modification of ring III and /or ring IV were obtained and characterized. Regioselective Mitsunobu esterification of the single primary hydroxyl group of the pseudo trisaccharide tobramycin was utilized successfully to link a variety of hydrophobic esters with tobramycin. Nine lipophilic tobramycin derivatives with significant structural diversity were synthesised and characterized. In a preliminary study, the applicability of the Mitsunobu dehydration reaction for the regioselective formation of an epoxide ring in the ido moiety of the pseudo tetrasaccharide aminoglycoside antibiotic paromomycin system was confirmed. The regioselective ring-opening of the derived epoxide with azide at C3IV of paromomycin was also successfully demonstrated. In total, forty-two new potential aminoglycoside antibiotics have been synthesized and characterized.

Interest in microemulsions as potential platforms for polymerization stems from the wide range of phase behaviour dependant morphologies and domain sizes that can be generated in a low viscosity environment. By introducing polymerizable components into the oil and aqueous phases of a microemulsion, we may essentially create a low viscosity, low interfacial tension, bicontinuous template with nanostructured morphologies and narrow domain size distributions analogous to those generated through conventional interpenetrating polymer network (IPN) synthesis and spinodal decomposition. The main objective of this dissertation is to test the application of bicontinuous microemulsion templates to the formulation and polymerization of a silicone-hydrogel IPN. In addition, the project expands on the classical definition of IPNs to a scale of entanglement at the level of groups of polymer chains, as opposed to molecular or chain-level entanglement. This study is divided into two main parts. In the first part, silicone microemulsions were developed and characterized according to the Hydrophilic-Lipophilic Difference (HLD) framework. The hydrophobicity of silicone oils, the characteristic curvature of silicone surfactants and the co-surfactant contribution of methacrylic acid (MAA) and hydroxyethyl methacrylate (HEMA) were quantified. These findings led to the successful formulation of bicontinuous microemulsions (μEs) containing silicone oil, silicone alkyl polyether and reactive monomers in aqueous solution. Ternary phase diagrams of these systems revealed the potential for silicone-containing polymer composites with bicontinuous morphologies. In the second part of this study, the formulation and simultaneous polymerization of polydimethylsiloxane-poly(methacrylic acid – hydroxyethyl methacrylate), (PDMS-P(MAA-HEMA) IPNs from bicontinuous microemulsions was demonstrated. Laser scanning confocal microscopy (LSCM) on swollen polymers highlights aqueous pathways, and indicates the formation of bicontinuous morphologies with domain sizes at equilibrium swelling ranging from ~100 nm to 1 μm. Incorporating polymerizable surfactants into the microemulsion aided in stabilizing the initial microemulsion structure during polymerization. The process developed demonstrates a simple, single-step polymerization approach to forming IPNs from low viscosity microemulsion templates, and could potentially be extended to a variety of hydrophilic and hydrophobic monomers.

Interest in microemulsions as potential platforms for polymerization stems from the wide range of phase behaviour dependant morphologies and domain sizes that can be generated in a low viscosity environment. By introducing polymerizable components into the oil and aqueous phases of a microemulsion, we may essentially create a low viscosity, low interfacial tension, bicontinuous template with nanostructured morphologies and narrow domain size distributions analogous to those generated through conventional interpenetrating polymer network (IPN) synthesis and spinodal decomposition. The main objective of this dissertation is to test the application of bicontinuous microemulsion templates to the formulation and polymerization of a silicone-hydrogel IPN. In addition, the project expands on the classical definition of IPNs to a scale of entanglement at the level of groups of polymer chains, as opposed to molecular or chain-level entanglement. This study is divided into two main parts. In the first part, silicone microemulsions were developed and characterized according to the Hydrophilic-Lipophilic Difference (HLD) framework. The hydrophobicity of silicone oils, the characteristic curvature of silicone surfactants and the co-surfactant contribution of methacrylic acid (MAA) and hydroxyethyl methacrylate (HEMA) were quantified. These findings led to the successful formulation of bicontinuous microemulsions (μEs) containing silicone oil, silicone alkyl polyether and reactive monomers in aqueous solution. Ternary phase diagrams of these systems revealed the potential for silicone-containing polymer composites with bicontinuous morphologies. In the second part of this study, the formulation and simultaneous polymerization of polydimethylsiloxane-poly(methacrylic acid – hydroxyethyl methacrylate), (PDMS-P(MAA-HEMA) IPNs from bicontinuous microemulsions was demonstrated. Laser scanning confocal microscopy (LSCM) on swollen polymers highlights aqueous pathways, and indicates the formation of bicontinuous morphologies with domain sizes at equilibrium swelling ranging from ~100 nm to 1 μm. Incorporating polymerizable surfactants into the microemulsion aided in stabilizing the initial microemulsion structure during polymerization. The process developed demonstrates a simple, single-step polymerization approach to forming IPNs from low viscosity microemulsion templates, and could potentially be extended to a variety of hydrophilic and hydrophobic monomers.

Cyanobacteria (“blue-green algae”) are known to produce a diverse repertoire of biologically active secondary metabolites. When associated with so-called “harmful algal blooms”, particularly in freshwater systems, a number of these metabolites have been associated - as “toxins”, or commonly “cyanotoxins” - with human and animal health concerns. In addition to the known water-soluble toxins from these genera (i.e. microcystins, cylindrospermopsin, and saxitoxins), our studies have shown that there are metabolites within the lipophilic extracts of these strains that inhibit vertebrate development in zebrafish embryos. Following these studies, the zebrafish embryo model was implemented in the bioassay-guided purification of four isolates of cyanobacterial harmful algal blooms, namely Aphanizomenon, two isolates of Cylindrospermopsis, and Microcystis, in order to identify and chemically characterize the bioactive lipophilic metabolites in these isolates. We have recently isolated a group of polymethoxy-1-alkenes (PMAs), as potential toxins, based on the bioactivity observed in the zebrafish embryos. Although PMAs have been previously isolated from diverse cyanobacteria, they have not previously been associated with relevant toxicity. These compounds seem to be widespread across the different genera of cyanobacteria, and, according to our studies, suggested to be derived from the polyketide biosynthetic pathway which is a common synthetic route for cyanobacterial and other algal toxins. Thus, it can be argued that these metabolites are perhaps important contributors to the toxicity of cyanobacterial blooms. In addition to the PMAs, a set of bioactive glycosidic carotenoids were also isolated because of their inhibition of zebrafish embryonic development. These pigmented organic molecules are found in many photosynthetic organisms, including cyanobacteria, and they have been largely associated with the prevention of photooxidative damage. This is the first indication of these compounds as toxic metabolites and the hypothesized mode of action is via their biotransformation to retinoids, some of which are known to be teratogenic. Additional fractions within all four isolates have been shown to contain other uncharacterized lipophilic toxic metabolites. This apparent repertoire of lipophilic compounds may contribute to the toxicity of these cyanobacterial harmful algal blooms, which were previously attributed primarily to the presence of the known water-soluble toxins.