Browsing by Subject "Lipid"
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Item Assembly of colloidal nanocrystals into phospholipid structures and photothermal materials(2012-08) Rasch, Michael; Korgel, Brian Allan, 1969-There has been growing interest in developing colloidal metal and semiconductor nanocrystals as biomedical imaging contrast agents and therapeutics, since light excitation can cause the nanocrystals to fluoresce or heat up. Recent advances in synthetic chemistry produced fluorescent 2-4 nm diameter silicon and 1-2 nm diaemeter CuInSSe nanocrystals, as well as 16 nm diameter copper selenide (Cu₂₋[subscript x]Se) nanocrystals exhibiting strong absorbance of near infrared light suitable for biomedical applications. However, the syntheses yield nanocrystals that are stabilized by an adsorbed layer of hydrocarbons, making the nanocrystals hydrophobic and non-dispersible in aqueous solution. Encapsulating these nanocrystals in amphiphilic polymer micelles enables the nanocrystals to disperse in water. Subsequently, the Si nanocrystals were injected into tissue to demonstrate fluorescence imaging, the photothermal transduction efficiency of copper selenide nanocrystals was characterized in water, and the copper selenide nanocrystals were used enhance the photothermal destruction of cancer cells in vitro. The polymer-encapsulated copper selenide nanocrystals were found to have higher photothermal transduction efficiency than 140 nm diameter Au nanoshells, which have been widely investigated for photothermal therapy. Combining the optical properties of metal and semiconductor nanocrystals with the drug-carrying capability of lipid vesicles has received attention lately since it may create a nanomaterial capable of performing simultaneous drug delivery, optical contrast enhancement, and photo-induced therapy. Hydrophobic, dodecanethiol-coated Au nanocrystals were dispersed in water with phosphatidylcholine lipids and characterized using cryo transmission electron microscopy. 1.8 nm diameter Au nanocrystals completely load the bilayer of unsaturated lipid vesicles when the vesicles contain residual chloroform, and without chloroform the nanocrystals do not incorporate into the vesicle bilayer. 1.8 nm Au nanocrystals dispersed in water with saturated lipids to form lipid-coated nanocrystal agglomerates, which sometimes adhered to vesicles, and the shape of the agglomerates varied from linear nanocrystal chains, to flat sheets, to spherical clusters as the lipid fatty acid length was increased from 12 to 18 carbons. Including squalene formed lipid-stabilized emulsion droplets which were fully loaded with the Au nanocrystals. Results with 4.1 nm Au and 2-3 nm diameter Si nanocrystals were similar, but these nanocrystals could not completely load the bilayers of unsaturated lipids.Item Characterization of atherosclerotic plaques using ultrasound guided intravascular photoacoustic imaging(2011-05) Wang, Bo, 1981-; Emelianov, Stanislav Y.; Sokolov, Konstantin; Smalling, Richard; Litovsky, Silvio; Dunn, Andrew; Aglyamov, SalavatRupture of atherosclerotic plaque is closely related to plaque composition. Currently, plaque composition cannot be clinically characterized by any imaging modality. The objective of this dissertation is to use a recently developed imaging modality – ultrasound-guided intravascular photoacoustic (IVPA) imaging – to detect the distribution of two critical components in atherosclerotic plaques: lipid and phagocytically active macrophages. Under the guidance of intravascular ultrasound imaging, spectroscopic IVPA imaging is capable of detecting the spatially resolving optical absorption property inside a vessel wall. In this study, contrast in spectroscopic IVPA imaging was provided by either the endogenous optical property of lipid or optically absorbing contrast agent such as gold nanoparticles (Au NPs). Using a rabbit model of atherosclerosis, this dissertation demonstrated that ultrasound guided spectroscopic IVPA imaging could simultaneously image lipid deposits as well as macrophages labeled in vivo with Au NPs. Information of macrophage activity around lipid rich plaques may help to identify rupture-prone or vulnerable plaques. The results show that ultrasound guided IVPA imaging is promising for detecting plaque composition in vivo. Clinical use of ultrasound guided IVPA imaging may significantly improve the accuracy of diagnosis and lead to more effective treatments of atherosclerosis.Item Development of an Algal Oil Separation Process(2012-10-19) Samarasinghe, NalinMicroalgae surpass the lipid productivity of terrestrial plants by several folds. However, due to the high moisture content and rigidity of algal cell walls, extraction of lipids from algae is still a significant technological challenge. In this research, an attempt was made to develop an algal lipid separation process which is energy efficient and effective. Algal related research requires a unique set of knowledge in areas of algae culturing, measuring cell concentration, harvesting, cell rupturing and lipid quantification. The first section of this thesis focuses on the state of the art as well as knowledge gained during preliminary studies. The second section of this thesis focuses on selecting a suitable measurement technique for quantification of algal cell disruption induced by homogenization. The selected method, hemocytometry was used to measure the degree of algal cell disruption induced by homogenization. In the third section, various homogenization treatments were evaluated for determining the fraction of cells disrupted during the homogenization. Finally, lipid extraction efficiency of homogenized algae was evaluated using different extraction solvents under different homogenization conditions. Preliminary research concluded that using cell counting is the most suitable technique to measure the effect of high pressure homogenization on concentrated microalgae. It was observed that higher pressure and increased number of passes increase the degree of cell disruption. Concentrated, non stressed samples show best response to homogenization. Out of the three solvents used for solvent extraction, chloroform gave a higher extraction yield at low intensity homogenizations. However at harsher homogenization levels the advantage of chloroform was not significant. Lipid extraction efficiency increases with increased levels of homogenization. However, a significant increase in lipid yields was not detected beyond 20 000 psi and 2 passes of homogenization treatment.Item Experimental and computation study of protein interactions with lipid nanodomains(2013-05) Qiu, Liming; Cheng, Kelvin K.; Vaughn, Mark W.; Sanati, Mahdi; Khare, Rajesh; Quitevis, Edward L.Protein lipid interactions are significantly relevant to understanding of a wide variety of biological phenomena in general. In particular, human beta-amyloid protein is closely related to the pathogenesis of Alzheimer's disease. Due to its high propensity to self-aggregate, beta-amyloid protein is difficult to study with experiments. Molecular dynamics simulations is capable of providing atomistic details of the protein lipid interactions; therefore, is an important theoretical tool to investigate these subtle interactions and offer insights to the pathogenesis of Alzheimer's disease. In this dissertation, I studies the protein lipid interactions with several systems with different lipid composition and protein conformations. I developed computational tools to quantitatively analyze lipid perturbations due to protein interactions, since it is commonly believed that the neurotoxicity of beta-amyloid protein is through perturbation of the lipid membrane. I discovered that for the case of a beta-amyloid dimer on the surface of lipid bilayers, the perturbation effect of protein is correlated to the degree of disorder of the protein in term of its secondary structure. Meanwhile, for a system where a beta-amyloid protein was partially inserted into the bilayer, the protein insertion rate was regulated by both the secondary structure of the protein and the lipid environment. Especially, a scaling relation between the insertion rate and degree of disorder was found. Even though molecular dynamics simulations is a powerful tool in studying atomistic protein lipid interactions, it is not efficient in sampling the free energy landscape of the system; hence results are biased by the initial structure of the system. I developed a multiscale molecular simulation scheme to increase the efficiency in free energy landscape sampling by switching the system between different spatial resolutions, i.e., atomistic and coarse-grain representations of the system. Using this method, I discovered a novel protein lipid orientation, which has implications in understanding the biochemical pathway of the protein as well as developing therapeutic interventions. Finally, I also developed a Monte Carlo method to estimate molecule volumes accurate to atomistic scale. This method is directly applicable to lipid membrane system with heterogeneous components including proteins; it is a useful tool for not only investigating protein lipid interactions but also calibration of force field parameters for classical molecular dynamics simulations.Item Growth Rate of Marine Microalgal Species using Sodium Bicarbonate for Biofuels(2013-08-05) Gore, MatthewWith additional research on species characteristics and continued work towards cost effective production methods, algae are viewed as a possible alternative biofuel crop to current feedstocks such as corn. Current open pond production methods involve bubbling carbon dioxide (CO_(2)) gas into the media to provide a carbon source for photosynthesis, but this can be very inefficient releasing most CO_(2) back into the atmosphere. This research began by investigating the effect of sodium bicarbonate (NaHCO_(3)) in the growth media as an alternative carbon source to bubbling CO_(2) into the cultures. The second part examined if NaHCO_(3) could act as a lipid trigger in higher (10.0 g/L) concentrations. The microalgae species Dunaliella tertiolecta (Chlorophyta), Mayamaea spp. (Baciallariophyta) and Synechoccocus sp. (Cyanophyta) were grown with 0.0 g/L, 0.5g/L, 1.0 g/L, 2.0 g/L and 5.0 g/L dissolved NaHCO_(3) in modified seawater (f/2) media. To investigate effects of NaHCO_(3) on lipid accumulation, growth media cultures were divided into two ?lipid phase? medias containing either 0.0g/L (non-boosted) or 10.0 g/L (boosted) NaHCO_(3) treatments. Culture densities were determined using spectrophotometry, which showed both all three species are able to successfully grow in media ameliorated with these high NaHCO_(3) concentrations. Highest growth phase culture densities occurred in NaHCO_(3) concentrations of 2.0 g/L for D. tertiolecta and Mayamaea spp., and the 5.0 g/L treatment for Synechoccocus sp. Highest growth rates occurred in the 5.0 g/L NaHCO_(3) concentration treatments for D. tertiolecta, Mayamaea spp., and Synechoccocus sp. (0.205 d-1 ?0.010, 0.119 d-1 ?0.004, and 0.372 d-1 ?0.003 respectively). As a lipid accumulation trigger two of the three species (D. tertiolecta and Mayamaea spp) had their highest end day oil indices in a 10.0 g/L treatment. Highest oil indices occurred in boosted 5.0 g/L Dunaliella tertiolecta and 2.0 g/L Mayamaea spp. (13136 ? 895 and 62844 ? 8080 respectively (relative units)). The results obtained indicate NaHCO3 could be used as a photosynthetic carbon source for growth in all three species and a lipid trigger for D. tertiolecta and Mayamaea spp.Item Harnessing Yarrowia lipolytica’s potential as a lipid and alkane production platform(2013-08) Blazeck, John James; Alper, Hal S.; Contreras, Lydia; Ellington, Andrew; Georgiou, George; Maynard, JenniferEngineering cellular phenotype can enable the in vivo synthesis of renewable fuels, industrial precursors, and pharmaceuticals. Achieving economic viability requires the use of a cellular platform that generates high titers independent of fermentation condition, through either native or imported biosynthetic metabolism. While lacking fully developed genetic tools, the oleaginous yeast Yarrowia lipolytica has the native capacity to produce large titers of lipids and citric acid cycle intermediates. However, unlocking this biosynthetic capacity requires complete rewiring of native metabolism. To this end, this work focuses on the development and engineering of the yeast Y. lipolytica to rewire native metabolism and enable the production of lipids, alkanes, and itaconic acid. Precise control of gene expression is a requisite to enable metabolic and pathway engineering applications for any host organism. However, Y. lipolytica lacks promoter elements strong enough to manipulate intracellular metabolism. Thus, we utilized a hybrid promoter engineering approach to produce libraries of high-expressing, tunable promoters, seven-fold stronger than promoters previously characterized in Y. lipolytica 1,2. We successfully applied this approach to Saccharomyces cerevisiae, expanding transcriptional capacity of the strongest constitutive to highlight our hybrid approach as a generalizable method to increase expression capacity in eukaryotic organisms 3. We utilized our novel Y. lipolytica hybrid promoters to drive intracellular metabolism towards lipid production and to overexpress heterologous enzymes that enable alkane and itaconic acid production. Specifically, we implemented a global rewiring of Y. lipolytica’s native metabolism to increase lipogenesis more than sixty fold to 25.3g/L (the highest lipid production ever reported) and generated cells nearly 90% lipid content. We further expressed a lipoxygenase enzyme to catalyze the novel microbial production of the short-chain n-alkane, pentane. Finally, we exploited Y. lipolytica’s capacity to accumulate citric acid cycle intermediates by expressing a heterologous cis-aconitic acid decarboxylase enzyme to produce itaconic acid. Increasing substrate availability through media optimization and genomic engineering increased pentane and itaconic acid production threefold and eightfold, respectively 4. Collectively, these studies have facilitated the utilization of Y. lipolytica as an industrially relevant microbial platform, and represent a generic approach towards enabling biosynthetic control in microbial hosts will ill-defined gene expression technology.