The studies presented in this thesis focus on the synthesis of air-stable solid supported lipid bilayers by anhydrobiotic mechanisms. Supported lipid bilayers (SLBs) serve as platforms that mimic cellular membrane surfaces in appearance and behavior. One of the most attractive aspects of the SLB is that it exhibits two-dimensional fluidity that allows for individual components to rearrange as they would in actual cellular membranes. The one thing that would allow the SLB to become an ideal biosensor is the ability to remain stable in the absence of bulk water. As it stands now, unprotected SLBs are unstable in the presence of air causing the membrane to rearrange and delaminate from the surface. Several biological organisms utilize the process of anhydrobiosis to persevere in severe dehydrated states. Anhydrobiosis occurs when organisms employ large amounts of sugars, particularly disaccharides, to protect their cell membranes. The sugars, often released as a stress response, protect the membrane by replacing the water around the lipid headgroups while also interacting with other sugars to form a glass atop the bilayer. One of the most successful anhydrobiotic sugars has been trehalose, although other sugars have been evaluated and are capable of protecting lipid bilayers minimally. The experimental section of this thesis involves the creation of SLBs that are examined with and without the presence of sugar molecules. Essentially, the SLB was created, exposed to sugar solutions, dried, and subsequently rehydrated. Successful experiments occurred when rehydrated bilayers exhibited little damage and were mobile and functional. In addition to trehalose, several other mono- and disaccharides were used as were glycolipids, lipids with sugar headgroups. Upon the completion of all experiments it was clear that trehalose afforded the most protection of all species tested and that glycolipids do not sufficiently protect the membrane during rehydration. Therefore, the addition of a sugar such as trehalose to an SLB could allow for the creation of an air-stable biosensor that would be both practical and require little maintenance.

This dissertation focuses on the applications of solid supported phospholipid membranes as mimics of the cellular membrane using lab-on-a-chip devices in order to study biochemical events such as ligand-receptor binding and the chemical mechanism for the preservation of the biomembrane. Supported lipid bilayers (SLBs) mimic the native membrane by presenting the important property of two-dimensional lateral fluidity of the individual lipid molecules within the membrane. This is the same property that allows for the reorganization of native membrane components and facilitates multivalent ligand-receptor interactions akin to immune response, cell signaling, pathogen attack and other biochemical processes. The study is divided into two main facets. The first deals with developing a novel lipopolymer supported membrane biochip consisting of Poly(ethylene glycol) (PEG)-lipopolymer incorporated membranes. The formation and characterization of the lipopolymer membranes was investigated in terms of the polymer size, concentration and molecular conformation. The lateral diffusion of the PEG-bilayers was similar to the control bilayers. The air-stability conferred to SLBs was determined to be more effective when the PEG polymer was at, or above, the onset of the mushroom-to-brush transition. The system is able to function even after dehydration for 24 hours. Ligandreceptor binding was analyzed as a function of PEG density. The PEG-lipopolymer acts as a size exclusion barrier for protein analytes in which the binding of streptavidin was unaffected whereas the binding of the much larger IgG and IgM were either partially or completely inhibited in the presence of PEG. The second area of this study presents a molecular mechanism for in vivo biopreservation by employing solid supported membranes as a model system. The molecular mechanism of how a variety of organisms are preserved during stresses such as anhydrobiosis or cryogenic conditions was investigated. We investigated the interaction of two disaccharides, trehalose and maltose with the SLBs. Trehalose was found to be the most effective in preserving the membrane, whereas maltose exhibited limited protection. Trehalose lowers the lipid phase transition temperature and spectroscopic evidence shows the intercalation of trehalose within the membrane provides the chemical and morphological stability under a stress environment.

Thesis (Masters)--California Institute of Technology, 1974. / Advisor name found in the Acknowledgements pages of the thesis. Title from home page. Viewed 12/02/2009. Includes bibliographical references.

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The doubly labelled water technique (DLW) allows the estimation of the carbon dioxide production (r<SUB>co2</SUB>) of a free-living animal from which, with knowledge of the respiratory quotient (RQ), energy expenditure may be predicted. The aim of this project was to assess the performance of this technique in ruminant animals using sheep as a model. In a series of preliminary experiments physiological processes which may affect this technique were examined. It was found the administered isotopes equilibrated throughout the body water after 6 hours following either an oral or intravenous dosing. <SUP>2</SUP>H lost in the form of methane was estimated to lead to errors in r<SUB>co2</SUB> of between -3.27 and -6.54% and may be corrected for by use of a methane to urine enrichment ratio of 0.6536. Losses of <SUP>2</SUP>H in exchangeable sites in faeces were estimated to lead to errors in r<SUB>co2</SUB> of between -5.35 to -6.54%, again corrections factors were proposed. Sequestration of <SUP>2</SUP>H into carcase fat was also quantified, a maximum error of 0.67% was estimated on r<SUB>co2</SUB>, although more <SUP>2</SUP>H was detected in the fat free carcase this was assumed to be exchangeable <SUP>2</SUP>H. A maximum error of -2.39% on water turnover (r<SUB>H20</SUB>) was estimated to arise from the recycling of <SUP>2</SUP>H due to the catabolism of labelled body solids, although this was probably an overestimate. No convincing evidence was found to suggest the fractionation factors applied to human subjects are not equally applicable to ruminant animals. The baseline body water enrichment was altered by a change in diet from synthetic concentrates to fresh chopped grass. Isotopic estimates of r<SUB>co2</SUB> were compared with respiration chamber measurements of r<SUB>co2</SUB>. A number of curve fitting procedures were considered together with three multi-point, three ratio plot and three two-point estimations of r<SUB>co2</SUB>. Using a multi-point calculation and after making corrections for fractional water loss, loss of <SUP>2</SUP>H in methane and faecal solids, r<SUB>H20</SUB> was underestimated by 16.24%. The isotopic r<SUB>co2</SUB> estimates were on average 1.52% (SD 15.31) lower than the chamber measurements, although one animal was markedly different from the others, omitting this animal led to an average error of + 5.95% (SD 4.34). In applying this technique to animals in the field the influence of a free-living existence on the performance of the method was assessed. A number of r<SUB>co2</SUB> calculation procedures were again considered. Preliminary chamber measurements were used to estimate methane production and faecal dry matter output in the field. Estimates of r<SUB>H2O</SUB> in the field (mean 7.218.1. day<SUP>-1</SUP>) were lower than those measured in the chambers (mean 9.812 1.day<SUP>-1</SUP>). From predictions of dry matter intake in the field RQ was estimated (mean 1.067), using the isotopic r<SUB>co2</SUB> estimates (mean 590 1.day<SUP>-1</SUP>) energy expenditure was then predicted (mean 11586 kJ.day<SUP>-1</SUP>). In the light of this work it was concluded the DLW technique may be usefully applied to ruminant animals and a number of practical suggestions were made.

Major interest has focused on the identification of regulatory factors involved in lipid biosynthesis. This study examined the B.napus β-Keto-ACP reductase 5' sequence for potential regulatory cis-acting elements. The 5' sequence of the most highly expressed Brassica napus β-Keto-ACP reductase isoform was fused to the reporter gene β-glucuronidase (GUS) and its expression pattern examined within transgenic Arabidopsis. The construct was shown to act as a functional promoter and direct transcription within embryos, cotyledons and roots. There was no apparent staining within the true leaves, but staining was visible within the cotyledons. Overlapping fragments of the promoter were analysed in gel mobility shift assays and all six showed the formation of protein-DNA complexes. Competition analysis suggested that the same trans-acting factor binds to a number of regions along the promoter. The protein-DNA complex appeared to be competed away by the Arabidopsis enoyl-ACP reductase (EnR) promoter sequence, but not the lipid transfer protein (LTP) promoter. A common 9bp cis-element (CGCANTAAA) was identified in four of the six promoter fragments. Deletion analysis of the β-Keto-ACP reductase promoter intransient expression experiments into B.napus tissue, suggested the promoter could still direct transcription upon deletion to 132bp within embryos. The GUS expression appeared to show more than one decrease in expression upon subsequent deletions of the promoter within embryos, suggesting that more than one cis-element may be involved in the control of transcription. At least one of these suspected decreases con elated with the deletion of one of the 9bp boxes identified. Differences were observed for expression of the constructs within leaves and embryos suggesting that different elements may be involved in transcriptional control within these tissues. The identification of a potential cis-acting element within this study could be used to isolate a potential regulatory trans-acting factor that binds to the B.napus β-Keto-ACP reductase promoter.