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Doctor of Philosophy / In this thesis, a set of wheat varieties (Triticum aestivum L.) produced by the Value Added Wheat Cooperative Research Centre with lower swelling power as compared to commercial Australian wheat varieties were studied to enhance our understanding of the role of amylose in starch functionality. These starches originated from a heterogeneous genetic background and had a narrow range of elevated amylose content (35 to 43%) linked with diverse functional properties. Small-angle X-ray scattering together with complementary techniques of differential scanning calorimetry and X-ray diffraction have been employed to investigate the features of starch granular structure at the nanometer scale. Starch chemical structure was characterized in terms of amylose content and amylopectin chain length distribution. Starch functionality was studied by a series of swelling, pasting and enzymic digestion methods. This study showed that swelling power of flour is a simple test that reflects a number of industrially relevant characteristics of starch, and therefore can be used as an indicator of amylose content and pasting properties of starch. In contrast to waxy starches and starches with normal amylose content, wheat starches with increased amylose content displayed characteristic pasting properties that featured decreasing peak, breakdown and final viscosities with increasing amylose contents. Existence of a threshold value in amylose content, above which final viscosity of starch paste does not further increase with increasing amylose content, was proposed. Variability in amylopectin chain length distribution was shown to have an additional effect on the swelling and pasting properties of the starches. On the molecular level, increased amylose content was correlated with increased repeat spacing of the lamellae present in the semicrystalline growth rings. In agreement with current understanding of starch synthesis, amylose was shown to accumulate in both crystalline and amorphous parts of the lamella. Using waxy starch as a distinctive comparison with the other samples confirmed general trend of increasing amylose content being linked with the accumulation of defects within crystalline lamellae. Amylose content was shown to directly influence the architecture of semicrystalline lamellae, whereas thermodynamic and functional properties were proposed to be brought about by the interplay of amylose content and amylopectin architecture. Subjecting starch granules with varying amylose content to pancreatic α-amylase showed differences in their digestion patterns. Pancreatic α-amylase preferentially attacked amorphous regions of waxy starch granules, whereas these regions for initial preferential hydrolysis gradually diminished with increasing amylose content. Observed variations in the extent of enzymic digestion were concluded to be primarily determined by the level of swelling of amorphous growth rings, which can also explain observed morphologies of partly digested granules with varying amylose content. It was confirmed that access to the granular components is not a function of the extent of crystallinity but rather the spatial positioning of the crystalline regions within the granule. Digestion kinetics is governed by factors intrinsic to starch granules, whereas influence of enzyme type was shown to be critical in determining the absolute rate of hydrolysis. Wheat starches with increased amylose content offer the potential to be used as slow digestible starch, mostly in their granular form or when complexed with lipids. Differences among varieties largely diminished when starches were gelatinized or allowed to retrograde demonstrating the importance of granular structure on starch hydrolysis. Wheat varieties used in this study displayed widely differing pasting properties in a Rapid Visco Analyser (RVA) and textural characteristics of the respective retrograded starch gels. Varietal differences in starch chemical composition among wheat varieties were shown to have significant effect on the extent of the response of starch viscoelastic characteristics to the addition of monopalmitin. Amylose content was positively correlated with the increase in final viscosity, which was attributed to the presence of more amylose in non-aggregated state contributing to higher apparent viscosity of the starch paste. Comparison of stored gels obtained from amylose-rich starches with gel prepared from waxy wheat varieties confirmed the critical role of amylose on the formation of starch network and thus providing the strength of the gel. Lack of correlation between textural properties of stored gels with amylose content or rheological characteristics measured by the RVA indicated that subtle differences in starch structure may have far-reaching consequences in relation to the strength of the gels, although these differences may have only limited effect on pasting properties in the RVA Viscoelastic properties of starch paste prepared from commercial wheat starch were significantly altered depending on the chain length and saturation of the fatty acid of the monoglyceride added during repeated heating and cooling in the Rapid Visco Analyser. Varying effects of different monoglycerides on the paste viscosity were attributed to different complexation abilities of these lipids with starch. It was proposed that stability and structure of the starch-lipid complexes formed affect the viscosity trace of the paste subjected to multiple heating and cooling. Our study indicated that differing monoglycerides in combination with the number of heat-cool cycles can be used to induce form I or form II starch-lipid complexes and thus manipulate paste rheology, gel structure and resistant starch content.

Includes bibliographical references (leaves 169-184) / xiv, 184 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines the concept of membrane homeostatis, which implies that biological membranes tend to maintain a constant level of lipid fluidity in the face of potential exogenous and endogenous pertubations. Manipulations of dietary cholesterol and/or saturated (coconut oil) v/s unsaturated (sunflower seed oil) fatty acids have been used to study the relationship between membrane lipid composition, membrane lipid fluidity and membrane-bound enzymes of lipid metabolism; and, to see whether these enzymes act co-ordinately for the maintenance of a membrane homeostatis under these dietary conditions. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Sciences, 1986

N-linked glycosylation often imparts important properties to protein therapeutics. An essential step in this intracellular process is the transfer of oligosaccharide from dolichol monophosphate (Dol-P) to a potential glycosylation site. Variability in the success rate of this reaction affects the extent of protein glycosylation. The critical role of Dol-P suggests that its availability may influence the extent of glycosylation by limiting the pool of lipid-linked oligosaccharides (LLOs), the glycosyl donor. To test this hypothesis, the impact of Dol-P supplementation on protein glycosylation in Chinese hamster ovary (CHO) cells was investigated. Although exogenous Dol-P was incorporated by CHO cells and processed into LLOs in a dose-dependent manner, Dol-P supplementation had no marked effects on LLO or overall cellular glycosylation levels. While concentrations of exogenous Dol-P exceeding 100 µg/ml were detrimental to CHO cell viability, maximum non-toxic supplemental doses of Dol-P had no significant impact on the glycosylation of recombinant interferon-γ produced by batch cultures of CHO cells. These results show that glycosylation in CHO cells cannot be readily enhanced by Dol-P feeding under normal culture conditions. / Singapore-MIT Alliance (SMA)

Thesis (M.Sc.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 86-96.

Triacylglycerols are the predominant storage form of energy in eukaryotes. As obesity has become a worldwide problem and excessive accumulation of triacylglycerols in adipose tissue causes obesity, enzymes catalyzing the synthesis of triacylglycerols are of great interest. Acyl CoA:diacylglycerol acyltransferase (DGAT), including the isoforms DGAT1 and DGAT2, catalyze the final and committed step in triacylglycerol synthesis. Proteins that physically interact with DGAT1 may provide information regarding the metabolic role of DGAT1. We chose HEK-293T cell line to express DGAT1 and used mass spectrometry to identify proteins that co-immunoprecipitated with DGAT1. We confirmed that DGAT2 and ACAT1 did interact with DGAT1. The interaction of DGAT1 with DGAT2 appeared to interrupt the synthesis of triacylglycerol since the co-expression of DGAT1 and DGAT2 was expected to increase triacylglycerol synthesis. This implied that DGAT1 and DGAT2 might serve different functional roles. On the other hand, DGAT1 overexpression may increase the synthesis of cholesterol esters that was the product of ACAT1. Additionally, ACAT1 overexpression did increase triacylglycerol synthesis and ACAT1 disruption by siRNA did decrease triacylglycerol synthesis. Our findings indicated that DGAT1 and ACAT1 might be involved in the same lipid-synthesizing protein complex.

ABSTRACT SANDRA C. METCALF Associations of Youth Weight Status Categories and Cholesterol Levels: Analysis of Data from the National Health and Nutrition Examination Survey (NHANES) (Under the direction of RODNEY LYN, PhD, Assistant Professor) Obesity is recognized as a risk factor for dyslipidemia, however studies specific to overweight youth and dyslipidemia are scarce. Nonetheless, expert bodies have established BMI at the 85th percentile or greater as the threshold for “at risk” in youth and advocate for lipid screening. This study analyzed associations of weight categories and occurrence of dyslipidemia among a multi-racial sample of youth 6 to 18 years of age. In this NHANES cohort, overweight youth were at significant increased risk of dyslipidemias of total cholesterol, high-density lipoprotein cholesterol and non-high-density lipoprotein cholesterol. Obese children and adolescents demonstrated even higher risk. This study supports the hypothesis that overweight youth are at increased risk for dyslipidemia. Despite the increased risk, test agreement for overweight and dyslipidemia was poor. BMI at the 85th percentile did not provide good discrimination in detecting children with dyslipidemia and use of this threshold warrants further review.

Alzheimer's Disease (AD) is a neurodegenerative disorder that is prevalent among the elderly population. Aß protein has been heavily implicated in the pathogenesis of AD. This protein in its fibrillar form is a major component in the senile plaques that form on neuronal cellular membranes during the course of AD. Despite substantial efforts the exact mechanism of Aß toxicity towards a cell membrane is not well-understood. The determination of this mechanism, however, is of utmost importance, since the membrane presents the first site of Aß interaction with neurons, which in turn maybe the origin of Aß neurotoxicity. The purpose of this study was to find a lipid composition that can be used as a model of neuronal membrane for subsequent studies of the role of topographical heterogeneity (domain formation) on Aß-membrane interaction as related to AD. The lipids used in the study were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), cholesterol (Chol), sphingomyelin (SM) and ganglioside GM1 (GM1). These lipids were combined in different proportions and deposited on a mica substrate to form supported monolayers. They were then imaged with an atomic force microscope (AFM) to determine if any of them exhibited domain formation. Three of the studied samples: POPC/POPG/SM 40:40:20 +5%Chol, POPC/SM/Chol 75:20:5 and POPC/SM/GM1/Chol 74:2:1:23 were found to possess interesting topography, rich in structural features: pores and domains. The average height difference between the domain features for each sample was found to be 0.58±015 nm, 0.61±0.12 nm and 0.27±0:07 nm.

Coral reefs ecosystems are some of the most productive and diverse in the world. The symbiotic association between cnidarians and their symbiotic microalgae is of great importance in coral reef ecosystems; however, its underlying molecular mechanism remains unclear even after decades of research. Rab small GTP binding proteins are critical regulators of vesicle trafficking. Here we present the experimental evidence supporting a possible association of ApRab37 with the surface of lipid droplets in the endosymbiosis between the sea anemone, Aiptasia pulchella and the symbiotic dinoflagellate (commonly known as zooxanthellae). ApRab37, a protein of 215 amino acids, displays strong homology with human Rab37. In transfected COS7 cells, EGFP-ApRab37 localized to lipid droplets and clustered in the peri-nuclear region, which stained positive for the ER (endoplasmic reticulum) marker. Immunostaining analysis found ApRab37 associated with symbiosomes and lipid droplets, which was also confirmed by Western blot analysis of in the enrich lipid droplet fraction. Phagocytosis assay showed that ApRab37 involved in late phase of phagocytosis. DCMU treatment indicates symbiosome association of ApRab37 is mediated by zooxanthellae. I propose that ApRab37 plays a pivotal role in the regulation of lipid trafficking from the symbiosomes to the host cell during the Aiptasia-zooxanthellal endosymbiosis.

Rotavirus (RV) nonstructural protein 4 (NSP4) is a multifunctional glycoprotein that induces secretory diarrhea in mouse pups in the absence of other viral proteins. The intracellular transport route(s) and functional mechanism(s) of NSP4 are poorly understood; however, the recent association of the enterotoxin with cellular caveolin-1 may provide a link between NSP4 transport and function. To determine if NSP4 traffics to a specific subset of lipid rafts at the plasma membrane (PM), we isolated caveolae from a PM-enriched fraction with a new method that yielded endoplasmic reticulum (ER)-free caveolae membranes with a unique membrane structure and composition. Comparison of these caveolae with other detergent- and non-detergent-extracted membranes revealed that each caveolae/raft fraction contained caveolae markers; however, only our PM caveolae fraction mimicked the membrane structure and sterol exchange dynamics of intact PM without ER or non-raft PM contaminants. When these PM caveolae were isolated from RV-infected cells, full-length, high-mannose glycosylated NSP4 was present. Confocal imaging showed association of NSP4 with caveolin-1 moving from perinuclear and cytoplasmic sites toward the PM as the infection progressed. Fluorescent imaging also indicated exposure of the NSP4 Cterminus at the exofacial PM surface without transport of the enterotoxin through the Golgi apparatus. Surface-specific biotinylation was used to confirm NSP4 exposure at the surface of infected MDCK cells and to determine that the exposed protein was fulllength and high-mannose glycosylated. This study presents an ER contaminant-free PM caveolae isolation methodology, identifies the presence of full-length, high-mannose glycosylated NSP4 in both PM caveolae and exposed at the cell surface, and confirms the Golgi-bypassing nature of NSP4 ER to PM transport in RV-infected MDCK cells.