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In this thesis optical microscopy techniques are used to consider aspects of viral and bacterial infections. In part 1, the physical effects of cytomegalovirus on lipid droplet dynamics in live cells are studied; in part 2, the effects of an antimicrobial peptide on the fluidity of model bacterial membranes are studied. The optical microscopy techniques used to study the effects of murine-cytomegalovirus (mCMV) on lipid droplets in live NIH/3T3 fibroblast cells in real-time are coherent anti- Stokes Raman scattering (CARS), two-photon fluorescence (TPF) and differential interference contrast (DIC) microscopies. Using a multimodal CARS and TPF imaging system, the infection process was monitored by imaging the TPF signal caused by a green fluorescent protein (GFP)-expressing strain of mCMV, where the amount of TPF detected allowed distinct stages of infection to be identified. Meanwhile, changes to lipid droplet configuration were observed using CARS microscopy. Quantitative analysis of lipid droplet numbers and size distributions were obtained from live cells, which showed significant perturbations as the infection progressed. The CARS and TPF images were acquired simultaneously and the experimental design allowed incorporation of an environmental control chamber to maintain cell viability. Photodamage to the live cell population was also assessed, which indicated that alternative imaging methods must be adopted to study a single cell over longer periods of time. To this end, DIC microscopy was used to study the lipid droplet dynamics, allowing lipid droplet motion to be tracked during infection. In this way, the effects of viral infection on the mobility and arrangement of the lipid droplets were analysed and quantified. It was found that the diffusion coefficient of the lipid droplets undergoing diffusive motion increased, and the droplets undergoing directed motion tended to move at greater speeds as the infection progressed. In addition, the droplets were found to accumulate and cluster in infected cells. The second part of this thesis presents a study on the effects of an antimicrobial peptide on model bacterial membranes. Giant unilamellar vesicles (GUVs) were produced as a simple model of E. Coli membrane using a 3:1 mixture of DPPC and POPG lipids. Incorporating Laurdan fluorescent dye into the lipid membrane of the GUVs allowed the membrane fluidity to be probed and visualised using TPF microscopy, whereby the fluidity was quantified by determining the general polarization (GP) values. Studying GUVs comprising single lipid and mixed lipid compositions over a temperature range from 25 C to 55 C enabled the lipid phase bands to be identified on the basis of GP value as gel phase and liquid crystalline phase. As such, the changes in lipid phase as a result of interaction with AMP were quantified, and phase domains were identified. It was found that the amount of liquid crystalline phase domains increased significantly as a result of AMP interaction.

Obesity is the most prevalent nutritional disorder encountered in small animal medicine. Problems related with obesity are the higher incidence of morbidity and mortality. Nutritional and physical activity interventions have been common strategies employed; however, they have shown low compliance rates. Because of it more attention has been given to the nutrient composition of diets. Using the canine model, three experiments were conducted to examine the effect of fish oil or barley on protein and lipid metabolism, as well as postprandial glycemia, and nutrient digestibility in mature and in young adult dogs. In Exp. 1, seven female dogs were randomly assigned to one of two isonitrogenous and isocaloric diets, control (CO) or fish oil (FO), in a crossover design. Animals fed the FO diet tended to be more sensitive to glucose, showing a lower glucose half life. Cholesterol and HDL decreased (p<0.05) on the FO treatment. Overall, the supplementation of fish oil may improve glucose clearance rate and is effective in decreasing cholesterol in mature overweight dogs. In Exp. 2, eight female Beagles were randomly assigned to one of two isonitrogenous and isocaloric diets, control (CO) or fish oil (FO), in a crossover design. Overall, feeding a FO containing diet showed a protective effect against the rise of plasma CHOL and it increased plasma ghrelin levels. However, it did not appear to improve protein metabolism or postprandial glycemia in adult lean dogs. In Exp. 3, sixteen female dogs were randomly assigned to four experimental diets; control (40% corn) or three levels of barley (10, 20, 40%). The data suggest that inclusion of barley up to 40% in diets for adult dogs is well tolerated and does not negatively impact nutrient digestibility of the diets. However, inclusion of barley did not improve aspects related to fecal odor, postprandial glycemia, or plasma cholesterol. Overall, the research presented herein suggests that different nutritional strategies - dietary lipid or carbohydrate manipulation - may be beneficial in ameliorating health issues (e.g., hyperlipidemia) or in improving the health status of dogs (e.g., gut health by increased SCFA production).

<p>Obesity is today a problem that has reached epidemic proportions. One of the causes of obesity is the over-consumption of energy. Fat is the most energy-dense nutrient, where the quality seems to be more important for the development of the metabolic diseases than the quantity. The fatty acid composition in serum lipid fractions can be used to mirror the dietary fat quality.</p><p>Stearoyl-CoA-desaturase (SCD) is an enzyme that converts saturated to monounsaturated fatty acids. A surrogate measure of SCD activity can be estimated as a fatty acid ratio; 16:1/16:0 (palmitoleic acid/palmitic acid) and 18:1/18:0 (oleic acid/stearic acid). The aim of this project was to investigate the intra-day variation in the SCD-ratio in humans eating a standardized diet. The results showed that triacylglycerol and nonesterified fatty acid fractions in serum lipids had a significant variance in the 16:1/16:0 ratio during the day, whereas 18:1/18:0 ratio in the same fractions did not exhibit the same pattern. In this study 16:1/16:0 ratio also seems to be a better marker than 18:1/18:0 ratio for estimating SCD activity. For further evaluation of the intra-day variation there need to be a more long-term study of the SCD-activity for a larger group of subjects.</p>

Anesthesia, a state of being absent of sensation and consciousness, has been recognized since antiquity. Even today anesthesia is still best characterized by the lack of consciousness and sensations. Since anesthetic potency is correlated with lipid solubility, the site of action of general anesthetics has been thought to be hydrophobic in nature and to involve excitable membranes critical for interneuronal communications. Thus, general anesthetics may interact directly with functionally-relevant membrane proteins (via hydrophobic pockets) or indirectly, with the lipids surrounding these proteins. To better understand the details of general anesthetic action, I examined how halothane interacts with a functional synaptic protein, the acetylcholine receptor channel embedded in the membranes of cultured Xenopus myocytes. Next, I examined how changing the lipid composition, of these membranes, affected this interaction. Using the extracellular patch-clamp technique, I found that halothane, at clinically-relevant concentrations, shortened the burst duration of single receptor channels without affecting their conductance. Moreover, the halothane-induced reduction of burst durations was significantly attenuated after pretreatment with cholesterol-rich lipsomes which increased significantly the cholesterol content of these cells. These findings provide the first direct support for the role of membrane lipids in the mechanism of GA action. In particular, I demonstrated that increases in membrane cholesterol antagonize the anesthetic action of halothane. Although direct action of cholesterol on synaptic proteins cannot be ruled out, my data strongly suggest that membrane lipids are involved at a critical, but as yet undefined, site with which GAs interact. The exact manner by which increases in membrane cholesterol antagonize GA action remains to be eludicated.

In vitro studies have shown that the major membrane phospholipid phosphatidylcholine (PC) can positively influence the incorporation of cholesterol in lipid membranes. The influence of PC on the cellular trafficking of LDL-derived free cholesterol was investigated. Sterol regulatory-defective (SRD)-4 cells are Chinese hamster ovary (CHO)-derived fibroblasts that display vastly elevated rates for the synthesis and catabolism of PC. SRD-4 cells harbor two known gene mutations: a mutation in the functional allele for SCAP, resulting in defective feedback suppression of cholesterol biosynthesis; and a loss-of-function mutation in the functional allele for acyl-CoA:cholesterol acyl transferase (ACAT), an endoplasmic reticulum (ER)-localized enzyme that esterifies free cholesterol. Incubation of SRD-4 cells with 50 µg/ml low density lipoprotein (LDL) for 18 h resulted in lysosomal accumulation of free cholesterol as revealed by filipin staining. This accumulation was not evident following LDL treatment of parental CHO7 cells, and was blunted in SRD-2 cells that express a constitutively-active form of SREBP-2 and overproduce cholesterol but have functional ACAT activity. Treatment of SRD-2 cells with LDL in the presence of an ACAT inhibitor 58-035 resulted in robust lysosomal cholesterol accumulation that was reversible upon drug washout, supporting that cholesterol trafficking in cholesterol-loaded cells is dependent on ACAT activity and, more specifically, ER free cholesterol levels. Lysosomal accumulation of LDL-derived cholesterol was prevented in SRD-4 cells supplemented with lyso-PC (50 µM), a substrate for PC synthesis through the reacylation pathway, and also in cells treated with bromoenol lactone (BEL), an inhibitor of phospholipase A2 implicated in bulk PC turnover. In a counter study, lysosomal LDL-derived cholesterol accumulation was induced in parental CHO-7 cells using R-propranolol, which inhibits the conversion of phosphatidic acid to diacylglycerol (DAG), a substrate in the CDP-choline pathway. This blockage was also relieved through co-treatment with lyso-PC. These studies support that PC to free cholesterol ratios in downstream organellar membranes can influence cholesterol trafficking out of lysosomal compartments in cholesterol-loaded cells.

In this thesis we study the interactions of ions and cationic peptides with a negatively charged lipid bilayer in an ionic solution where the electrostatic interactions are screened. <br /><br /> We first examine the problem of charge renormalization and inversion of a highly charged bilayer with low dielectric constant. To be specific, we consider an asymmetrically charged lipid bilayer, in which only one layer is negatively charged. In particular, we study how dielectric discontinuities and charge correlations among lipid charges and condensed counterions influence the effective charge of the surface. When counterions are monovalent, e. g. , Na⁺, our mean-field approach implies that dielectric discontinuities can enhance counterion condensation. A simple scaling picture shows how the effects of dielectric discontinuities and surface-charge distributions are intertwined: Dielectric discontinuities diminish condensation if the backbone charge is uniformly smeared out while counterions are localized in space; they can, however, enhance condensation when the backbone charge is discrete. In the presence of asymmetric salts such as CaCl₂, we find that the correlation effect, treated at the Gaussian level, is more pronounced when the surface has a lower dielectric constant, inverting the sign of the charge at a smaller value of Ca²⁺ concentration. <br /><br /> In the last chapter we study binding of cationic peptides onto a lipid-bilayer membrane. The peptide not only interacts electrostatically with anionic lipids, rearranging their spatial distributions, but it can also insert hydrophobically into the membrane, expanding the area of its binding layer (i. e. , the outer layer). We examine how peptide charges and peptide insertion (thus area expansion) are intertwined. Our results show that, depending on the bilayer's surface charge density and peptide hydrophobicity, there is an optimal peptide charge yielding the maximum peptide penetration. Our results shed light on the physics behind the activity and selective toxicity of antimicrobial peptides, i. e. , they selectively rupture bacterial membranes while leaving host cells intact.

The lipid composition of cotton (Gossypium hirsutum, L) fibers was determined. Fatty acid profiles revealed that linolenate and palmitate were the most abundant fatty acids present in fiber cells. Phosphatidylcholine was the predominant lipid class in fiber cells, while phosphatidylethanolamine, phosphatidylinositol and digalactosyldiacylglycerol were also prevalent. An unusually high amount of phosphatidic acid was observed in frozen cotton fibers. Phospholipase D activity assays revealed that this enzyme readily hydrolyzed radioactive phosphatidylcholine into phosphatidic acid. A profile of expressed sequence tags (ESTs) for genes involved in lipid metabolism in cotton fibers was also obtained. This EST profile along with our lipid metabolite data was used to predict lipid metabolic pathways in cotton fiber cells.

Receptor tyrosine kinases (RTKs) are a major class of cell surface receptors, important in cell signalling events associated with a variety of functions. High-throughput (HTP), coarse-grained molecular dynamics (CG-MD) simulations have been used to investigate the dimerization of the transmembrane (TM) domain of selected RTKs, including epidermal growth factor receptor (EGFR) and muscle-specific kinase (MuSK). EGFR activation requires not only a specific TM dimer interface, but also a proper orientation of its juxtamembrane (JM) domain. Phosphatidylinositol 4,5-bisphosphate (PIP₂) is known to abolish EGFR phosphorylation through interaction with basic residues within the JM domain. Here, a multiscale approach was used to investigate anionic lipid clustering around the TM-JM junction and how such clustering is modulated by the mutation of basic residues. The simulations demonstrated that PIP₂ may help stabilize the JM-A antiparallel dimer, which may in turn help stabilize TM domain helix packing of the N-terminal dimerization motif. A proximal TM domain residue has been implicated in the inhibition of ganglioside GM3 in phase-separated membranes. Here, CG simulations were used to explore the dynamic behaviour of the EGFR TM domain dimer in GM3-containing and GM3-depleted bilayers designed to resemble lipid-disordered (Ld) and phase-separated (Ld/Lo) membranes. The simulations suggest that the presence of GM3 in Ld/Lo bilayers can disrupt and destabilize the TM dimer, which helps to explain why GM3 may favour monomeric EGFR in vivo. To gain insights into the dynamic nature of the intact EGFR, a nearly complete EGFR dimer was modelled using available structural data and embedded in an asymmetric compositional complex bilayer, which resembles the mammalian plasma membrane. The results demonstrated the dynamic nature of the EGFR ectodomain and its predicted interactions with lipids in the local bilayer. Strong protein-lipid interactions, as well as lipid-lipid interactions, affect the local clustering of lipids and the diffusion of lipids in the vicinity of the protein on both leaflets.

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