Thesis (Ph. D.)--University of Lund, 1983. / Includes bibliographical references.

Thesis (Ph.D.)--McMaster University, 2004. / Adviser: Bernardo Trigatti. Includes bibliographical references (leaves 153-178). Also available online.

The PLFA profiling method was adopted and used to determine changes in microbial community structure and abundance along natural and human-induced environmental gradients. The presented studies were based on field sampling campaigns combined with targeted laboratory experiments. According to the aims of particular studies, microbial PLFA fingerprinting was combined with the auxiliary below- and aboveground ecosystem characteristics to identify the drivers of microbial responses to environmental changes or with 13C-labelling and metagenomics to obtain more complex information about running processes and involved microorganisms.

The use of fatty acid (FA) analysis has become an important tool in recent years to investigate a broad spectrum of questions in fisheries and aquatic ecology. One of these applications has been the use of FA profiles as biomarkers for indicating diet and recent feeding history of fishes and other aquatic consumers. Differences in forage FA profiles and spatial differences in food web structure within aquatic systems may lead to corresponding spatial patterns of FA profiles in consumers, thereby facilitating the potential applicability of FA biomarkers as indicators of habitat use and origin for mobile consumers such as fishes. However, little information is available regarding differences in FA profiles among habitat types in river-floodplain ecosystems and the time-scale over which fish FA profiles change when fish move between habitats that differ in the FA profiles of prey resources. The objectives of this study were to test whether the tissue FA profiles of channel catfish (Ictalurus punctatus) differed among three reaches of the lower Kaskaskia River and its floodplain lakes, to determine the time-course of tissue FA profile turnover when fish are relocated to a new habitat type, and to compare FA profiles among muscle, liver, and adipose fin tissues in channel catfish. Fatty acid profiles of channel catfish were significantly different among sites, especially between upper and lower river sites, and between river channel and oxbow lake sites, suggesting differing energy sources among habitats and river reaches in the Kaskaskia system. More specifically, there was a significant increase in the essential FAs, 18:2n-6 and 18:3n-3, in channel catfish with increasing distance downstream, which could reflect an increase in river-floodplain connectivity at downstream sites. Channel catfish transplanted from the Kaskaskia River to ponds at Southern Illinois University Carbondale showed a significant linear decrease in n-3 and medium-chain polyunsaturated FAs (MC-PUFA), and a significant linear increase in monounsaturates (MUFA) with longer residence time in a lentic environment. Liver tissue exhibited the fastest turnover rate of the three tissue types (< 2 weeks), with muscle and adipose fin tissue both displaying similar, longer turnover times (approximately 10 weeks). Results of this study support the use of FA profiles as indicators of energy sources for fishes in large river-floodplain ecosystems, including the potential for indentifying habitat-specific (river channel vs. floodplain lake) or river reach-specific energy sources. Additionally, results of this study provide a timeline for FA turnover in channel catfish tissues when fish move among habitats with distinct FA signatures, which is crucial for detecting temporal shifts in use of habitat-specific energy sources by channel catfish. Results also suggest that adipose fin tissue samples may be a less invasive alternative to muscle tissue for analysis of FA profiles in channel catfish. Improved understanding of which habitats or river reaches provide trophic support for riverine fishes through use of FA profile analysis has potentially important implications for habitat conservation and rehabilitation in river-floodplain ecosystem.

Aquaculture is currently the fastest growing sector of protein production, and is expected to overtake the harvest of wild fisheries. Limitations in nutrition, specifically fatty acid nutrition, are preventing even more dramatic growth of many species of commercial importance. Currently, much of the research involving fatty acids examines requirements as being correlated to thermal guilds (warm vs. cool water) or salinity tolerance (marine vs. freshwater). However, recent studies have revealed the potential for trophic level to be as much, if not more, influential in determining fatty acid requirements of a species. As such, two feeding trials were conducted to determine the requirements of two species of different trophic levels (Oreochromis niloticus and Trachinotus carolinus) based on C18 PUFA vs. LC-PUFA. Nile Tilapia, O. niloticus, exhibited similar growth regardless of the inclusion of C18 PUFA or LC-PUFA, however, tissue fatty acid profiles were influenced per the diet provided. As such, it was concluded that Nile Tilapia exhibit the capacity to effectively synthesize LC-PUFA from C18 PUFA as is seen in many species that occupy low trophic levels. Florida Pompano, T. carolinus, did not exhibit any significant differences in growth regardless of the diet provided, but numerical differences indicated benefits towards inclusion of dietary LC-PUFAs. Similar to O. niloticus, tissue fatty acid profiles were significantly affected by dietary treatment. Based on numerical differences in growth performance and significant differences in tissue fatty acids, it was concluded that Florida Pompano show a typical carnivorous requirement for LC-PUFA.

Abstract In eukaryotic cells, fatty acids are mainly synthesized in the cytoplasm, but recently, in yeast and in humans, the ability to synthesize fatty acids has been characterized in mitochondria. This mitochondrial pathway is similar to bacterial type II fatty acid synthesis (FAS). The main feature of mitochondrial FAS in yeast is the respiratory deficient phenotype and loss of cytochromes when any of genes encoding enzymes for mitochondrial FAS is deleted. Mitochondrial FAS has been demonstrated to have an important role in lipoic acid production, namely it synthesizes octanoyl-ACP, which is used as a precursor for lipoic acid. However, the role and function of mitochondrial FAS is not yet fully understood. Many components of the mitochondrial FAS pathway in yeast have been identified according to their similarity to bacterial counterparts, but 3-hydroxyacyl-ACP dehydratase does not show any easily recognizable similarity to bacterial dehydratases and thus remained unidentified. In this study 3-hydroxyacyl-ACP dehydratases of mitochondrial FAS were characterized from the yeast Saccharomyces cerevisiae, humans, and the human pathogen Trypanosoma brucei. The yeast 3-hydroxyacyl-ACP dehydratase (Htd2p) was identified by using a genetic screen, and this protein was shown to be encoded by open reading frame (ORF) YHR067w. The product of this gene shows mitochondrial localization and exhibits hydratase 2 activity. The deletion of HTD2 leads to a respiratory deficient phenotype, loss of cytochromes, reduced lipoic acids levels and changes in mitochondrial morphology. The ORF encoding human 3-hydroxyacyl-ACP hydratase (HsHTD2) was identified by functional complementation of the respiratory deficient phenotype of the yeast htd2 mutant with a human cDNA library. The complementing cDNA was previously identified as the RPP14 transcript encoding the 14 kDa subunit of the human RNase P complex. It was found that this transcript contains another 3' ORF, which encodes a protein that displays hydratase 2 activity and has mitochondrial localization. The bicistronic nature of the transcript is conserved in vertebrates and indicates a genetic link between mitochondrial FAS and RNA processing. The mitochondrial 3-hydroxyacyl-ACP hydratase in T. brucei is homologous to human HTD2, can complement the yeast respiratory deficient phenotype, exhibits hydratase 2 activity and localizes to the T. brucei mitochondrion.

Abstract Fatty acid degradation in living organisms occurs mainly via the β-oxidation pathway. When this work was started, it was known that the hydration and dehydrogenation reactions in mammalian peroxisomal β-oxidation were catalyzed by only multifunctional enzyme type 1 (MFE-1; Δ2-Δ3-enoyl-CoA isomerase/2-enoyl-CoA hydratase 1/(3S)-hydroxyacyl-CoA dehydrogenase) via the S-specific pathway, whereas in the yeast peroxisomes via the R-specific pathway by multifunctional enzyme type 2 (MFE-2; 2-enoyl-CoA hydratase 2/(3R)-hydroxyacyl-CoA dehydrogenase). The work started with the molecular cloning of the rat 2-enoy-CoA hydratase 2 (hydratase 2). The isolated cDNA (2205 bp) encodes a polypeptide with a predicted molecular mass of 79.3 kDa, which contains a potential peroxisomal targeting signal (AKL) in the carboxyl terminus. The hydratase 2 is an integral part of the cloned polypeptide, which is assigned to be a novel mammalian peroxisomal MFE-2. The physiological role of the mammalian hydratase 2 was investigated with the recombinant hydratase 2 domain derived from rat MFE-2. The protein hydrates a physiological intermediate (24E)-3α, 7α, 12α-trihydroxy-5β-cholest-24-enoyl-CoA to (24R, 25R)-3α, 7α, 12α, 24-tetrahydroxy-5β-cholestanoyl-CoA in bile acid synthesis. The sequence alignment of human MFE-2 with MFE-2(s) of different species reveals 12 conserved protic amino acid residues, which are potential candidates for catalysis of the hydratase 2. Each of these residues was replaced by alanine. Complementation of Saccharomyces cerevisiae fox-2 (devoid of endogenous MFE-2) with human MFE-2 provided a model system for examing the in vivo function of the variants. Two protic residues, Glu366 and Asp510, of the hydratase 2 domain of human MFE-2 have been identified and are proposed to act as a base and an acid in catalysis. Mammalian MFE-2 has a (3R)-hydroxyacyl-CoA dehydrogenase domain, whereas the yeast MFE-2 has two dehydrogenase domains, A and B. The present work, applying site-directed mutagenesis to dissect the two domains, shows that the growth rates of fox-2 cells expressing a single functional domain are lower than those of cells expressing S. cerevisiae MFE-2. Kinetic experiments with the purified proteins demonstrate that domain A is more active than domain B in catalysis of medium- and long-chain (3R)-hydroxyacyl-CoA, whereas domain B is solely responsible for metabolism of short-chain substrates. Both domains are required when yeast cells utilize fatty acids as the carbon source.

This study investigated the effect of nutritional energy levels on meat quality characteristics, through effects on the fatty acid profiles of the subcutaneous fat in sheep. Two rations containing 11. 76MJ ME/kg DM and 10 .18MJ ME/kg DM were fed to Dorper and SA Mutton Merino wethers from respectively 20.51 ± 2.51kg and 22.30 ± 3.99kg to 25, 31, 37 and 43kg live mass. M.longissimus lumborum samples were removed and a trained taste panel evaluated sensory parameters on a lOcm unstructured scale. Subcutaneous fat samples and plasma samples were collected, and various carcass measurements were taken. It was found that high energy nutrition significantly increased the concentration of unsaturated fatty acids in the subcutaneous adipose tissue, with subsequent effects on the fat quality and sensory properties of lamb. Both the aroma and incipient juiciness of taste samples from wethers on the high energy treatment were noticeably improved, while the increased amount of fat, coupled with its poor consistency significantly impaired the overall acceptability of taste samples. / Dissertation (MSc Agric)--University of Pretoria, 1992. / gm2014 / Animal and Wildlife Sciences / Unrestricted

This study (Wassell & Young 2007; Wassell et al., 2010a) shows that behenic (C22:0) fatty acid rich Monoacylglycerol (MAG), or its significant inclusion, has a pronounced effect on crystallisation (Wassell et al., 2010b; 2012; Young et al., 2008) and interfacial kinetics (3.0; 4.0). New interfacial measurements demonstrate an unusual surface-interactive relationship of long chain MAG compositions, with and without Polyglycerol Polyricinoleate (PGPR). A novel MAG synthesised from Moringa oleifera Triacylglycerol (TAG) influenced textural behaviour of water-in-oil (W/O) emulsions and anhydrous TAG systems (4.0: 5.0; 6.0). Emulsifier mixtures of PGPR and MAG rich in C18:1 / 18:2 and C16:0 / C18:0 do not decrease interfacial tension compared with PGPR alone. Only those containing MAG with significant proportion of C22:0 impacted interfacial behaviour. A mixture of C22:0 based MAG and PGPR results with decreasing tension from ~20°C and is initially dominated by PGPR, then through rearrangement, the surface is rapidly dominated by C22:0 fatty acids. A Moringa oleifera based MAG showed unusual decreased interfacial behaviour not dissimilar to PGPR. All other tested MAG (excluding a C22:0 based MAG), irrespective of fatty acid composition resulted with high interfacial tension values across the measured temperature spectrum (50°C to 5°C). A relative decrease of interfacial tension, with decreased temperature, was greater, the longer the chain length (Krog & Larsson 1992). Moreover, results from bulk and interfacial rheology showed that the presence of C22:0 based MAG has a pronounced effect on both elastic modulus (G’) and viscous modulus (G’’). Through a multidisciplinary approach, results were verified in relevant product applications. By means of ultrasonic velocity profiling with pressure difference (UVP-PD) technique, it was possible to examine the effect of a C22:0 based MAG in an anhydrous TAG system whilst in a dynamic non-isothermal condition (3.0). The non-invasive UVP-PD technique conclusively validated structural events. The application of a Moringa oleifera based MAG in low TAG (35% - 41%), W/O emulsions, results in high emulsion stability without a co-surfactant (PGPR). The bi-functional behaviour of Moringa oleifera based MAG is probably attributed to miscibility (Ueno et al., 1994) of its fatty acids, ranging ~30% of saturated fatty acids (SAFA), with ~70% of C18:1 (5.0). It is concluded that the surface-interactive behaviour of Moringa oleifera based MAG, is attributed to approximately 10% of its SAFA commencing from C20:0. When examined separately and compared, results showed that physical effect of a Moringa oleifera based MAG was not dissimilar to PGPR, influencing the crystallisation kinetics of the particular anhydrous TAG system. When either was combined with a C22:0 rich MAG, enhanced gelation onset and strong propensity to form dendrite structure occurred (5.0). Macrobeam and synchrotron radiation microbeam small angle x-ray diffraction (SR-μ-SAXD) was utilized (6.0) to assess behavior of C22:0 rich MAG, with and without PGPR (Wassell et al., 2012). The C22:0 based MAG combined with PGPR promoted TAG crystallisation as observed by differential scanning calorimetry (DSC). Polarised optical microscopy (POM) observations indicated that C22:0 based MAG eliminates formation of large crystal aggregates, resulting in the likely formation of tiny Pickering TAG / MAG crystals (6.0). It is concluded that the presence and interactive behaviour of Pickering surface-active MAG, is strongly linked to increased fatty acid chain length, which induce increased textural resilience owing to viscoelasticity (4.0; 5.0). A multidisciplinary approach was able to verify structuring behaviour (4.0; 5.0), using multiple analyses (Wassell et al., 2010b; 2012; Young et al., 2008). Novel structuring solutions in reduced TAG based systems have been provided (4.0; 5.0). This study both enhances current understanding of structuring in low TAG W/O emulsions and has led to novel MAG compositions, which address emulsification, structuring and texture in TAG based food systems (Wassell et al., 2010a; 2012a; 2012b; 2012c; 2012d; 2012e; Bech et al., 2013).

The industrial uses of cottonseed oil are limited by its fatty acid composition. Genetic modification of cotton lipid profiles using seed-specific promoters could allow cotton growers to produce valuable new oils in the seed without adverse effects on fiber quality and yield, therefore making this crop more commercially profitable. Transgenic cotton callus harboring a diverged fatty acid desaturase gene (FADX) from Momordica charantia was characterized for production of alpha-eleostearic acid (conjugated double bonds: 18:3 D9 cis, 11 trans, 13 trans), not normally found in cotton. Gas chromatography (GC) in conjunction with mass spectrometry (MS) confirmed production of alpha-eleostearic acid in the transgenic cotton tissues. A second series of transformation experiments introduced the cotton fatty acid thioesterase B (FATB) cDNA, fused to the seed-specific oleosin promoter into cotton to promote the over-expression of FATB, to generate cotton with increased palmitate in the cottonseed. PCR amplification, as well as fatty acid analysis by gas chromatography, confirmed introduction of the FATB cDNA in transgenic tissues. Collectively, these results demonstrate the feasibility of manipulating the fatty acid composition in cotton via transgenic approaches and form the basis for continued efforts to create novel oils in cottonseed.