An experiment was designed to study in the same animal any preferential absorption of a free fatty acid in the presence of a triglyceride of the same fatty acid. Rats were administered a mixture of free fatty acid and its triglyceride labeled with carbon-13 and carbon-14 respectively. Each isotope in the fed lipid and in the lipid recovered from the gastrointestinal tract was measured. The isotope effect, if any, was studied by administering a mixture of palmitic acid-1-C13 and palmitic acid-1-C14.

SPIONs have unique material properties: ultra-small size and high magnetic susceptibility. These properties give SPIONs advantages to be used in a wide range of applications such as NMR/MRI contrast agents, cancer hyperthermia therapy, and cell- targeted and magnetically-triggered intracellular uptake. However, research has found that it is challenging to develop a SPION system that has controllable colloidal stability. This thesis endeavored to develop a SPION surface coating that allowed creation of a SPION system with known surface properties, hence enabling the study of its colloidal stability with a combination of computational and experimental methods. With the knowledge of colloidal behavior of nanoparticles, this thesis further explored the magnetic properties of SPIONs by designing lipid nanoparticles encapsulating (LNPs) that target and isolate a rare type of immune cell.

Liposomes are promising drug delivery vesicles due to their biodegradibility, large volume and biocompatibility towards both hydrophilic and hydrophobic drugs. They suffer, however, from poor stability which limits their use in controlled delivery applications. Herein, a novel method was devised for modification of liposomes with small molecules, polymers or nanoparticles to afford stimuli responsive systems that release on demand and stay relatively stable in the absence of the trigger.. This dissertation discusses thermosensitive, pH sensitive, light sensitive and magnetically triggered liposomes that have been prepared for controlled drug delivery application. RAFT polymerization was utilized for the preparation of thermosensitive liposomes (Cholesterol-PNIPAm) and acid-labile liposomes (DOPE-PAA). With low Mw Cholesterol-PNIPAm, the thermosensitive liposomes proved to be effective for controlled release and decreased the cytotoxicity of PNIPAm by eliciting the polymer doses. By crosslinking the DOPE-PAA on liposome surface with acid-labile diamine linkers, DOPE-PAA liposomes were verified to be sensitive at low pH. The effects of polymer structures (linear or hyperbranched) have also been studied for the stability and release properties of liposomes. Finally, a dual-responsive Au@SPIO embedded liposome hybrid (ALHs) was prepared with light-induced “on-and-off” function by photo-thermal process (visible light) and instant release properties triggered by alternating magnetic field, respectively. The ALH system would be further applied into the cellular imaging field as MRI contrast agent.

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The presented thesis is a continuation of the bachelor work, in which the effects of monovalent ions on neutral model lipid membranes were characterized. Herein physical properties of physiologically relevant anionic membranes in the presence of monovalent cations and oxidized lipids were studied. Hydration and mobility of the lipid bilayer at glycerol level were investigated using fluorescent solvent relaxation technique. In the first part of this work the interactions of cations (Na+ , K+ , Cs+ ) with negatively charged POPC/POPS lipid mixture, which is a good model of inner leaflet of cellular membrane, were studied. The presence of cations resulted in dehydration and substantial hinderence of mobility of hydrated lipids at the glycerol level probed by Laurdan. Clear specificity of those effects, which correlated with Hofmeister series have been observed. In the second part of the work truncated oxidized phospholipids, oxPLs (PazePC, PoxnoPC, PGPC, POVPC), which are known to be important in pathophysiology of numerous diseases, were investigated. 10 mol% of each oxPL was incorporated into neutral and anionic lipid bilayers, the hydration and mobility of which were measured in water or in KCl solution. The results reveal complex interactions between oxPLs, negatively charged lipids, and K+ . In...

Lipids are the main constituents of the cell membrane and maintain its fluidity. Plants undergo various changes in lipids under environmental stresses and alter the membrane fluidity and permeability. Membrane lipids mostly contain a polar or neutral head group and fatty acid tails that vary in length and degree of unsaturation. The composition of membrane affects its physicochemical properties and ability to tolerate stress. The moss Physcomitrium patens is an early land plant with unique ability to tolerate stressors like cold and dehydration. During its life cycle, for the most part, mosses remain as gametophytes, multiplying asexually. The period from germination of spores into filamentous protonema, which give rise to gametophyte is transient. They enter reproductive sporophytes stage only under cold temperatures. Because of the diverse roles of these developmental stages and the time span they are exposed to the environment, we hypothesized that these stages share distinct lipid content and composition. To this extent, using LC-MS/MS methods we carried out lipidomic analyses of five developmental stages of the moss. We identified and quantified the major and minor lipid classes (types) and their acyl composition of protonema, early, mid and late gametophyte and sporophyte tissues. Galactolipids, which typically occur in the plastid were predominant in green tissues and thus most abundant in the vegetative tissues but not in sporophytes. Throughout the life cycle, among the phospholipids, phosphatidylcholine was the abundant lipid, a feature that is typical of plant membranes. Sporophyte tissues, however, were distinct from gametophyte and protonema and also other vascular plants with high amounts of phosphatidic acid (PA). In plants, PA typically accumulates in response to stress; it is likely that the low temperature cue necessary for sporophyte formation is associated with spike in PA and needs further investigation. In comparing the acyl composition of the various lipid classes, we identified that in addition to 34C and 36C lipids, moss lipids also contain 38C and 40C, which are not represented in vascular plants. We predict that the occurrence of long-chain, highly unsaturated lipids might contribute to the dynamic nature of the membrane and stability under stress. This study serves as a primary resource to further investigate the role of specific lipids and acyl groups in maintaining membrane properties. Overall, it aids to our understanding of the evolution of stress tolerance in early land plants that coped through harsh environmental conditions during their transition from water to land.

Indiana University-Purdue University Indianapolis (IUPUI) / Coxiella burnetii is an intracellular bacterium that causes the human disease Q fever. C. burnetii is transmitted from infected animals to humans through inhalation of infectious droplets. Acute Q fever is a flu-like illness lasting 10-14 days. Patients often have respiratory symptoms and present with pneumonia. Patients with suppressed immune systems or valvular heart disease can develop chronic Q fever, which causes endocarditis and vasculitis long after initial infection. Chronic Q fever is difficult to treat, and if untreated, is typically fatal. Currently, the United States lacks any vaccine for Q fever. In order to better prevent and treat this disease, it is important to understand how C. burnetii interacts with mammalian cells. Within the host cell, C. burnetii forms a large, acidic Coxiella-containing vacuole (CCV) and uses a Type 4B secretion system (T4SS) to secrete effector proteins into the host cell cytoplasm. While the CCV membrane is rich in sterols, cholesterol accumulation in the CCV is bacteriolytic, suggesting that C. burnetii regulation of lipid transport is critical for infection. The mammalian lipid transport protein ORP1L localizes to the CCV membrane and mediates CCV-ER membrane contact sites. ORP1L functions in lipid transport, including cholesterol efflux from late endosomes/lysosomes. Its sister isoform ORP1S binds cholesterol but localizes to the cytoplasm and nucleus. In ORP1- null cells, we found that CCVs were smaller than in wildtype cells, highlighting the importance of ORP1 in CCV development. CCVs in ORP1-null cells had higher cholesterol content than CCVs in wildtype cells, suggesting ORP1 functions in cholesterol efflux from the CCV. ORP1-null MH-S cells do not accumulate lipid droplets upon C. burnetii infection, supporting our hypothesis that ORP1 promotes cholesterol transfer from the CCV to the ER, as lipid droplets form from neutral lipids in the ER. While the absence of ORP1 led to a C. burnetii growth defect in MH-S cells, there was no growth defect in HeLa cells. Together, our data demonstrate that C. burnetii uses the host sterol transport protein ORP1 to promote CCV development, potentially by using ORP1 to facilitate cholesterol efflux from the CCV to diminish the bacteriolytic effects of cholesterol.

Modification of the lipid A domain of lipopolysaccharide (LPS) has been reported to contribute to the virulence and pathogenesis of various Gram-negative bacteria. The Kdo (3-deoxy-D-manno-octulosonic acid)-lipid A domain of Helicobacter pylori LPS shows several differences to that of Escherichia coli. It has fewer acyl chains, a reduced number of phosphate groups, much lower immunobiological activity, and only a single Kdo sugar is attached to the disaccharide backbone. However, H. pylori synthesizes a minor lipid A species resembling that of E. coli, which is both bis-phosphorylated and hexa-acylated suggesting that the major species results from the action of specific modifying enzymes. This work describes two enzymes, a lipid A phosphatase and a phosphoethanolamine transferase, involved in the periplasmic modification of the 1-position of H. pylori lipid A. Furthermore, we report a novel Kdo trimming enzyme that requires prior removal of the 1-phosphate group for enzymatic activity. Discovery of the enzymatic machinery involved in the remodeling of H. pylori LPS will help unravel the importance of these modifications in H. pylori pathogenesis.d.

Lipopolysaccharide (LPS) is the major surface molecule of Gram-negative bacteria and consists of three distinct structural domains: O-antigen, core, and lipid A. The lipid A (endotoxin) domain of LPS is a unique, glucosamine-based phospholipid that serves as the hydrophobic anchor of LPS and is the bioactive component of the molecule that is associated with Gram-negative septic shock. The structural genes encoding the enzymes required for the biosynthesis of Escherchia coli lipid A have been identified and characterized. Lipid A is often viewed as a constitutively synthesized structural molecule. However, determination of the exact chemical structures of lipid A from diverse Gram-negative bacteria shows that the molecule can be further modified in response to environmental stimuli. These modifications have been implicated in virulence of pathogenic Gram-negative bacteria and represent one of the molecular mechanisms of microbial surface remodeling used by bacteria to help evade the innate immune response. The intent of this review is to discuss the enzymatic machinery involved in the biosynthesis of lipid A, transport of the molecule, and finally, those enzymes involved in the modification of its structure in response to environmental stimuli.

Niveoscincus ocellatus is an important species in historical analyses of the evolution of viviparity because it is the species upon which the type II chorioallantoic placenta was based. Here we describe the net nutrient uptake across the placenta of N. ocellatus for comparison with other species of skinks with complex placentae. N. ocellatus is highly placentotrophic, with neonates being 1.68-times larger in dry matter than the fresh eggs. There is an increase of nitrogen from 6.3 ± 0.2 mg to 9.2 ± 0.6 mg, and ash from 3.8 ± 0.3 mg to 6.7 ± 0.6 mg. The increase in ash is made up by a more than two-fold increase in the amounts of calcium, potassium and sodium. There is no significant difference in lipids in the neonates compared to fresh eggs, so considerable lipid must have crossed the placenta to provide energy for embryonic development. N. ocellatus is significantly more placentotrophic than Niveoscincus metallicus, which also has a complex chorioallantoic placenta. Discovery of substantial placentotrophy in this genus confirms that two lineages of Australian lygosomine skinks (represented by the genera Pseudemoia and Niveoscincus) have evolved this pattern of embryonic nutrition and supports the hypothesis that the evolution of reptilian placentotrophy involves specialisations in addition to structural modifications of the chorioallantoic placenta.