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Off-resonance unenhanced total internal reflection (TIR) Raman Spectroscopy was explored to investigate supported single lipid bilayers with incorporated membrane peptides/proteins at water/solid interface. A model membrane was formed on a planar supported lipid layer (pslb) by the fusion of the reconstituted small unilamellar vesicles (SUVs), and the intensity of bilayer was confirmed by a comparison of Raman spectral intensity in the C-H stretching modes with C₁₆TAB. With prominent Raman sensitivity attained, we studied the 2-D phase transition of DMPC and DPPC pslbs and the temperature-dependent polarised spectra revealed a broad transition range of ca. 10 °C commencing at the calorimetric phase transition temperature. We applied polarised TIR-Raman Spectroscopy to pslbs formed by DMPC SUVs reconstituted with a model membrane-spanning peptide gramicidin D. A preferential channel structure formed by dissolution of trifluoroethanol could be probed by polarised Raman Spectroscopy qualitatively showing an antiparallel β-sheet conformation (different from "standard" one) and our Raman spectra by correlation with NMR and CD data confirmed single-stranded π^6.3 β-helical channel structure in the single bilayer. We also studied the membrane-penetrating peptide indolicidin in the presence of DMPC pslb over the chain melting temperature and a β-turn structure was dominantly observed concomitant with membrane perturbation. Dynamic adsorption of DPPC to form pslb from a micellar solution of n-dodecyl-β- _D-maltoside could be examined with high sensitivity of every 1-min acquisition. Finally we used polarised TIR-Raman scattering to porcine pancreatic phospholipase A₂ hydrolytic activity on DPPC pslbs and revealed lipid-active conformation different from that of the enzyme alone.

In the last few decades, an increasing number of physicists specialized in soft matter, including polymers, have turned their attention to biologically relevant materials. The properties of various molecules and fibres, such as DNA, RNA, proteins, and filaments of all sorts, are studied to better understand their behaviours and functions. Self-assembled biological membranes, or lipid bilayers, are also the focus of much attention as many life processes depend on these. Small lipid bilayers vesicles dubbed liposomes are also frequently used in the pharmaceutical and cosmetic industries. In this thesis, work is presented on both the elastic properties of polymers and the response of lipid bilayer vesicles to extrusion in narrow-channels. These two areas of research may seem disconnected but they both concern deformed soft materials. The thesis contains four articles: the first presenting a fundamental study of the entropic elasticity of circular chains; the second, a simple universal description of the effect of sequence on the elasticity of linear polymers such as DNA; the third, a model of the symmetric thermophoretic stretch of a nano-confined polymer; the fourth, a model that predicts the final sizes of vesicles obtained by pressure extrusion. These articles are preceded by an extensive introduction that covers all of the essential concepts and theories necessary to understand the work that has been done.

Lycopene has recently received interest as an antioxidant in human tissues. These same antioxidant properties present challenges in preventing oxidative degradation within food products. In this research, degradation of lycopene in model emulsion systems was examined to better understand the chemical stability of this potential functional food ingredient.

Endotoxin recognition by the innate immune receptor CD14 is a critical part of the innate immune system’s early detection and activation of the inflammatory response during microbial invasion. The differential recognition and high affinity binding of endotoxins from gram-negative and gram-positive bacteria is performed by the innate immune receptor CD14. Upon endotoxin binding, CD14 transfers the specific endotoxins to a Toll-like receptor signaling complex, which is responsible for initiating the intracellular signaling cascade. In the presence of overwhelming infection, the effects of CD14 lead to the over-activation of the inflammatory response, which results in the life threatening condition known as sepsis. Preparation of a 15N isotopically labeled truncated version of soluble CD14, using Pichia pastoris, allowed direct structural observation of the binding interaction between CD14 and two endotoxin ligands, lipopolysaccharide (LPS) and lipoteichoic acid (LTA), from gram-negative and gram-positive bacteria, respectively using solution NMR spectroscopy. These studies revealed that CD14 uses both a common set of residues, and endotoxin specific subsets of residues, to bind LPS and LTA. To further investigate the structural features of each endotoxin recognized by CD14, 13C 15N isotopically labeled Kdo2–Lipid A, a fully active chemically defined gram-negative endotoxin, and LTA lipid anchor, the minimal unit of LTA, were produced. This allowed detailed NMR spectral mapping of these agonist ligands bound to sCD14 which identified, for the first time, structural regions and features in each that are strongly affected during complex formation with sCD14. Additionally, the presence of differential dynamic behavior was seen in both CD14 and the ligands upon complexation. This behavior suggests a likely role for dynamics in the mechanism of pattern recognition by CD14, which uses the dynamic ability of specific residue combinations to differentially affect endotoxin binding. Using NMR, the dynamic behavior of CD14 was further investigated using temperature and pH-dependence studies of isotopically labeled CD14. These studies clearly demonstrated the presence of multiple conformations for several residues, and may provide a possible explanation for the broad specificity of ligand binding by CD14. In addition, the spin-labeling of isotopically labeled lipid A enabled the collection of intermolecular distances on CD14 bound lipid A.

Regular soy consumption has been shown to reduce cardiovascular (CV) risk through plasma cholesterol reduction. According to the current health claim, this benefit is attributed to soy protein (SP). Dietary intervention trials indicate that isoflavones (ISO), weak phytoestrogens in soy, may also contribute by offering additional vascular and metabolic protection. Equol, a metabolite of the ISO daidzein (DAZ) with greater estrogenic potency, may be an important mediator of such effects. This thesis examines effects of soy, in particular, ISO consumption on CV risk factors and the potential for ISOs to enhance cognition, possibly through improvements of circulatory function. Two crossover design intervention trials were undertaken: a food-based intervention, investigating differential effects of SP and ISO on plasma lipids and other risk factors for CVD, and an ISO supplementation trial, examining effects on cognition and vascular function. Both addressed whether benefits were dependent on equol production. In the first trial, 91 subjects with untreated mild hypercholesterolemia were randomised to consume each of the following three diets in random order for sequential 6 week periods: (S) soy foods containing 24 g of SP and 75-90 mg ISO per day, (SD) soy/dairy foods containing 12 g SP, 12 g dairy protein (DP) and 75-90 mg ISO per day or (D) dairy foods containing 24 g DP only per day. At the end of each diet period, blood lipids, flow-mediated dilatation (FMD) of the brachial artery, blood pressure, arterial compliance and anthropometric measures were assessed. Compared with the control diet (D), there was a small but significant reduction in total cholesterol on the S diet only (2.8 + 1.1%, P<0.05), which could be accounted for by a decrease in saturated fat intake. FMD was found to be significantly improved when SD and S diet data were nested (P=0.03). Plasma triglycerides (TG) improved on both the SD and S diets compared with D (P<0.01). Other lipid, metabolic and vascular parameters did not differ between diets. There were no differences in outcomes between equol (n=30) and non equol producers (n=61). In a subsequent 12 week double-blind supplementation trial, 34 healthy males were randomised to take 4 capsules providing 120mg ISO per day or a matching placebo for 6 weeks, after which they crossed over to the alternate supplement. FMD and cognitive assessments relating to measures of memory and executive function were performed at the beginning and end of each treatment phase. Spatial working memory, a test in which females consistently perform better than males, was significantly improved by ISO supplementation (P<0.02). However, other measures of cognition and FMD were unaffected and there were no differences between equol (n=8) and non-equol producers (n=26). These interventions indicate that ISOs offer specific health benefits, independent of equol production. ISO supplementation can enhance specific cognitive processes which appear dependent on estrogen activation. Additionally, soy foods containing ISOs improved FMD and TG but were unable to improve LDL cholesterol, even in equol producers. Thus dietary ISOs may reduce CV risk but the validity of the current health claim for SP is questioned. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1345614 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

Nuclear Hormone Receptors (NRs) are hormone dependent DNA binding proteins that translate physiological signals into gene expression. Gene products have been identified that belong to the NR superfamily on the basis of homology. However, the endogenous and /or synthetic ligands that regulate their activity remain unknown, consequently, this subgroup of proteins are designated as orphans). Retinoic acid receptor related orphan receptors alpha and gamma(RORα and γ) are orphan NRs, and are preferentially expressed in skeletal muscle a major metabolic tissue and other tissues including pancreas, thymus, prostate, liver, adipose and testis. Surprisingly, the specific roles of ROR α and γ in skeletal muscle, a peripheral tissue, have not been examined. Muscle is one of the most energy demanding tissues which accounts for ~40% of the total body mass and energy expenditure, ~75% of glucose disposal and relies heavily on β-oxidation of fatty acids. We hypothesize that ROR α and γ regulates metabolism in this major mass lean tissue. Initially, this hypothesis was examined by “gain and loss” of function studies in an in-vitro mouse skeletal muscle cell culture model. Previous in vitro studies analyzed the role of RORα in the regulation of lipid homeostasis in skeletal muscle cells. We similarly conducted in vitro RORγ gain and loss of function studies in skeletal muscle cells to understand the role of this isoform in metabolism. We utilized stable ectopic over-expression of VP16-RORγ (gain of function), native RORγ and RORγΔH12 (loss of function) vectors to modulate RORγ mRNA expression and function. Candidate driven expression profiling of lines that ectopically express the native and variant forms of RORγ suggested that this orphan NR has a function in regulating the expression of genes that control lipid homeostasis (fatty acid-binding protein 4), CD36 (fatty acid translocase), lipoprotein lipase and uncoupling protein 3), carbohydrate metabolism (GLUT5 (fructose transporter), adiponectin receptor 2 and interleukin 15 (IL-15)) and muscle mass (including myostatin and IL-15). Interestingly, our study revealed a function for RORγ in the pathway that regulates production of reactive oxygen species which was also correlated with increased expression of UCP3 mRNA. Subsequently, we conducted in vivo studies with mouse models displaying global and muscle specific perturbation in RORα expression and function to elucidate the physiological role of this orphan NR in the context of metabolism.Along these lines, we characterized homozygous staggerer mice (sg/sg) in the context of lipid, carbohydrate and energy homeostasis. Staggerer mice were characterized by decreased and dysfunctional retinoic acid receptor-related orphan receptor alpha (RORα) expression. We observed decreases in serum (and liver) triglycerides and total and high density lipoprotein serum cholesterol in staggerer mice. Moreover, the staggerer mice were associated with reduced adiposity, decreased fat pad mass and adipocyte size. Candidate-based expression profiling demonstrated that the dyslipidemia in staggerer mice was associated with decreased hepatic expression of SREBP-1c, and the reverse cholesterol transporters, ABCA1 and ABCG1. This was consistent with the reduced serum lipids. Furthermore, the lean phenotype in staggerer mice was also characterized by significantly increased expression of PGC-1α, PGC-1β, and lipin1mRNAin liver and white and brown adipose tissue from staggerer mice. In addition, we observed a significant 4-fold increase in β2-adrenergic receptor mRNA in brown adipose tissue. Finally, dysfunctional RORα expression protects against diet-induced obesity. Following a 10-week high fat diet, wild-type (but not sg/sg) mice exhibited a ~20% weight gain, increased hepatic triglycerides, and notable white and brown adipose tissue accumulation. In summary, these changes in gene expression (that modulate lipid homeostasis) in metabolic tissues were involved in decreased adiposity and resistance to diet induced obesity in the sg/sg mice, despite hyperphagia. Finally, we specifically modulated RORα signaling in skeletal muscle by the targeted over-expression of truncated RORαΔDE (lacking the ligand binding domain) driven by a myogenic specific promoter, to investigate the contribution of this peripheral tissue to the RORα phenotype. Interestingly, transgenic heterozygous animals exhibit increased fasting blood glucose levels and mild glucose intolerance. Expression profiling (and western analysis) identified perturbations in the insulin signaling cascade. For example, we observed attenuation of p85alpha (PI3K) and Akt2 (mRNA and protein) expression; and insulin dependent induction of phospho-Akt2. In concordance, significantly increased levels of active phospho-AMPK were detected in the muscle of transgenic mice (relative to wt littermates). The increase in phospho-AMPK correlated with: (i) the suppression of lipogenic gene expression; and (ii) increased phospho-ACC and activation of genes involved in fatty acid oxidation in the skeletal muscle of transgenic animals. In conclusion, we suggest these orphan nuclear receptors (RORα and γ) are key modulators of fat and carbohydrate homeostasis in skeletal muscle tissue. Specifically, we propose that, RORα plays vital role in fat accumulation in adipose tissue and insulin mediated glucose homeostasis in skeletal muscle. Therefore we suggest that selective muscle specific RORα modulators may have utility in the treatment of type2 diabetes and obesity.

The first step in the synthesis of steroid hormones occurs in the mitochondria where cholesterol is converted to pregnenolone by cytochrome P450scc (CYP11A1). Cholesterol is insoluble in water and is supplied to the CYP11A1 directly from the inner mitochondrial membrane to which the enzyme is bound. The aim of this study was to characterise the interaction of bovine CYP11A1 with the phospholipid membrane. The effect of osmotic stress provided by glycerol on the spin-state, activity and degree of hydration of CYP11A1 was also investigated. Multiple sequence alignment of mitochondrial P450s revealed that there are 46 absolutely conserved residues, with the highest conservation in the heme-binding region at the C-terminal. The greatest variablility between subfamilies is in the regions believed to be involved in substrate binding (SRSs), as defined for the CYP2B family. The secondary structure prediction for CYP11A1 suggests that there is strong similarity in secondary structure to P450s of known structure. A model structure of CYP11A1 was built from primary sequence alignment to template P450 structures using the SwissModel automated server. From the model and other bioinformatic analyses, the distal face of the P450 which includes the A’ helix, F-G loop and beta sheet 1 regions, were predicted to interact with the membrane. Tryptic digests of CYP11A1 were performed with the aim of identifying membrane bound peptides that may be protected from protease activity. HPLC tryptic maps were similar in profile between soluble and vesicle-bound P450 which suggests that there is not a large region of CYP11A1 protected from protease digestion when the enzyme is attached to a membrane. Mass spectrometric analysis of peptides resulting from tryptic digestion revealed a number of peptides in the soluble digest that were not present in the digest of vesicle-bound P450. These peptides were located at the N-terminal and the J to J’ helix and interestingly, there was an absence of C-terminal peptides for both digests. This C-terminal peptide could be detected in digests of vesicle-bound P450 but not in digests of soluble P450 by tricine SDS polyacrylamide gel electrophoresis, Western transfer and N-terminal sequence analysis. Based upon the bioinfomatic and tryptic digestion data, a set of N- and C-terminal deletion mutants of CYP11A1 were expressed in E. coli and fractionated based on their association with the soluble or membrane fraction of the cells. The N-terminal deletion of the A’ helix resulted in an increase in the proportion of CYP11A1 in the soluble fraction while the C-terminal deletion did not alter membrane localisation. There are eight tryptophan residues in mature CYP11A1. The accessibility of these tryptophans to a water-soluble fluorescence quencher was determined for soluble and vesicle-bound enzyme. When CYP11A1 was associated with the vesicle membrane an average of 2 tryptophan residues were protected from quenching compared to soluble CYP11A1. This suggests that these tryptophan residues become buried within the membrane following association of CYP11A1 with the vesicles and are no longer accessible to quencher. The only free cysteine (C265S) of bovine CYP11A1 was removed by site directed mutagenesis and new cysteine residues introduced at selected sites based upon earlier results and the modelled CYP11A1 structure. The cysteine mutants were expressed, purified and labelled with the environmentally sensitive fluorescent probe, N-(7-nitrobenz-2-oxal-3-diazol-4-yl)ethylenediamine (NBD). There was an increase in the hydrophobicity of the NBD environment following the association of CYP11A1 with vesicles for the labeled mutants V212C and L219C. This indicates that these residues which are in the F-G loop, become localized to a more hydrophobic environment following membrane binding. Labeled cysteine residues introduced into the A’, B’ and G helices and β4-2 did not show major changes in hydrophobicity following membrane integration of CYP11A1. Osmotic stress of CYP11A1 induced by glycerol resulted in a low-spin spectral response and inhibition of activity. The change to low spin correlated with the dissociation of five or six water molecules from CYP11A1 and the inhibition of activity with cholesterol as substrate correlated with the dissociation of two molecules of water. In conclusion, this study shows that CYP11A1 is held to the membrane, at least in part, by the F-G loop region, and that the removal of water from the active site of CYP11A1 by osmotic stress causes a low spin spectral response and inhibition of activity.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.