Cationic lipids are efficient tools to introduce nucleic acids and proteins into cells. Elucidation of the mechanism and cellular pathways associated to such a transport has been relatively slow, even though significant progress has been made in the characterization of the intracellular trafficking of cationic lipid/DNA complexes. Surprisingly, little is known about the effects of these delivery vectors on cell functioning. In the present thesis, we show that cationic lipids and cationic lipid/DNA complexes strongly increase the intracellular Ca2+ concentration. The end point of the Ca2+ increase was ~400 nM from a basal level of ~100 nM. The [Ca2+]i increase was studied using K562 and Jurkat cells cultured in vitro. This effect is weakened following addition of DNA to cationic liposomes, although remaining very large at cationic lipid/DNA ratios commonly used for cell transfection experiments. Removal of extracellular Ca2+ did not abolish this effect significantly and preincubating K562 cells with the Ca2+-ATPase inhibitor thapsigargin strongly abolished intracellular Ca2+ concentration increase, indicating that Ca2+ was released mainly from internal Ca2+ stores sensitive to thapsigargin. Pretreatment of the cells with the phospholipase C inhibitor U73122 blocked the intracellular Ca2+ concentration rise, suggesting an inositol pathway-dependent mechanism. LDH release assay indicates that in the conditions used for fluorescence measurement and in those used to transfer DNA into cells, cationic liposomes diC14-amidine and DOTAP had no massive cytotoxic effects. Cationic liposomes showed more toxicity than their corresponding complexes; this toxicity decreases in the presence of serum. The effect of cationic lipids on phosphatidylinositol-specific phospholipase C (PI-PLC) was quantitatively assessed using phosphatidylinositol (PI) and radiolabeled phosphatidylinositol ([3H]-PI). Incorporation of diC14-amidine into PC/PI vesicle activated PI-PLC and was shown to activate the hydrolysis of PI and [3H]-PI. Our data may suggest that mobilization of intracellular Ca2+ by complex could have an effect on the transfection process itself. These results indicate for the first time that cationic lipids and cationic lipid/DNA complexes are not inert and can affect the functioning of the cells by increasing their intracellular Ca2+.

The oxidation of lipids and antioxidants has been extensively studied in human plasma but little attention has been given to how plasma proteins are oxidised. Proteins make up the majority of biomolecules in cells and plasma and therefore are the most likely reactants with oxidants and free radicals. Previous studies in the laboratory had shown that peroxyl radicals generated by the thermolytic decay of 2-azobis (2-amdinopropane) dihydrochloride (AAPH) generated significant amounts of protein hydroperoxides, but only after a six hour lag period. In this study the existence of the six hour lag period was confirmed and shown by dialysis of the plasma to be due to the presence of low molecular weight antioxidants. The addition of both uric acid and ascorbic acid to the dialysed plasma restored the lag phase suggesting that in vivo these antioxidants act to prevent protein hydroperoxide formation. Lipid oxidation was also observed in the plasma but only after a two hour lag phase. This was the first time lipid oxidation has been observed in the absence of protein oxidation. The lipid lag phase was also abolished by dialysis of the plasma and restored by the addition of ascorbic acid and uric acid. The kinetics of tocopherol loss suggests that the tocopherol radicals act to inhibit lipid oxidation by transferring the electrons to the water-soluble ascorbate. The loss of ascorbate appears to cause the formation of a tocopherol radical mediate the lipid peroxidation process. Overall the data shows ascorbic acid scavenging the peroxyl radicals while uric acid acts to reduce the overall AAPH generated radical flux. In a separate investigation, the production of protein-bound DOPA (PB-DOPA) on albumin during X-ray radiolysis and copper mediate Fenton oxidation was investigated using a fluorescence based derivatisation method (ED-DOPA), which was compared with the more specific acid hydrolysis and HPLC analysis method. The ED-DOPA method consistently gave a much higher reading that the HPLC based methods, suggesting that the ED-DOPA method was measuring DOPA plus DOPA oxidation products. This was confirmed by oxidising X-ray radiolysis generated PB-DOPA with Cu++ to cause DOPA oxidation. The Cu++ treatment drastically increased the level of signal given by the ED-DOPA assay while HPLC analysis showed all the DOPA had been oxidised.

It would be beneficial to incorporate transmembrane proteins (TMPs) into biosensors, because TMPs are important for cell function in healthy and diseased states. These devices would employ an artificial cell membrane to maintain TMP function since cell membranes, which are mostly lipids, are necessary for the TMPs to function. These artificial lipid membranes must be robust for sensor applications. The ruggedness of these artificial membranes can be increased by using polymerizable lipids. Some polymerized lipid membranes exhibit increased stability, while successfully incorporating TMPs.Some polymerized membranes do not support the activity of certain TMPs, while maintaining the function of others. It is believed the physical properties of the membranes are important for TMP function. Some important physical properties of polymerizable lipid membranes have not yet been measured. Here, fluidity and mechanical properties of polymerizable dienoylPC lipid membranes were investigated.Fluorescence Recovery After Photobleaching was used to measure the fluidity of polymerizable dienoylPC membranes. Unpolymerized, UV-polymerized, and redox-polymerized membranes were investigated. Three types of membranes were found: fluid, partially fluid, and immobile. Unpolymerized and some polymerized membranes were fluid, while only polymerized membranes were partially fluid or immobile. Polymer size is believed to cause the differences in fluidity. This study highlights how polymerization parameters can influence membrane fluidity.Micropipette Aspiration was used to measure the mechanical properties of Giant Unilamellar Vesicles (GUVs) composed of dienolyPC lipids. Unpolymerized and UV-polymerized GUVs were investigated. Strength measurements showed that denoylPC GUVs were stronger than sorbylPC GUVs. Area expansion moduli of denoylPCs and mono-SorbPC GUVs were slightly lower than SOPC GUVs, while bis-SorbPC GUVs were substantially easier to stretch. The bending moduli of all GUVs was similar. UV-polymerization had no significant effect on the parameters. The difference in strength between denoylPCs and sorbylPCs is hypothesized to be due to the porous nature of sorbylPCs. It is thought UV-polymerization of these GUVs created polymers too small to significantly alter mechanical properties.It was demonstrated that some stable membranes are also fluid, which is important for the function of certain TMPs. A correlation cannot be made between the bending and stretching moduli of polymerizable membranes and function of TMPs.

Microalgae has shown to be an ideal choice for biofuel industry. Algae has high oil productivity, a short growth cycle and survives in a wide variety of water sources including high salinity and waste water. For this project, four different species of marine microalgae were screened based on oil content. They were Dunaliella tertiolecta (CCMP364), Nannochloropsis gaditana (CCMP527), Tetraselmis sp (CCMP 908) and Nannochloropsis salina (CCMP1776). Experimental results showed that CCMP 527 and 1776 strains had higher lipid content and better fatty acids profile than the other two. Further investigations were carried on CCMP 527 in order to maximize biomass productivity and lipid content. Nutrients, salinity, pH, temperature, light intensity and aging of the culture can all affect both lipid content and fatty acid profile and were investigated. Nutrient stress is the easiest way to manipulate lipid composition and increase lipid content. Hence, various carbon and nitrogen sources were investigated to determine the range and amount of substrates that may be feasible for cultivation. For supplying lipid for biodiesel production, the optimum culture conditions for strain Nannochloropsis gaditana are using CO₂ enriched air bubbling, f/2-Si medium, pH control, and nitrate as the nitrogen source. Use of other fertilizers is feasible as well, however, the nitrogen source greatly affects lipid productivity, but trace amounts of organics in ground water do not.A model which predicts cell growth, nitrogen concentration, and lipid yield in batch systems is developed that is applicable for low nitrogen conditions. Plus, a sensitivity analysis of three major parameters was done to validate how variations in these key parameters affect simulation results. The fatty acid profile as a function of time was shown not to vary from mid-exponential to stationary phase. The model describes reactor behavior well, therefore it can be applied to the genus of Nannochloropsis to predict biomass yield and lipid accumulation, and be a useful tool to optimize and compare bioreactor systems for the biofuel industry. In addition, effects of nitrate and urea under repletion condition on microalgae growth, lipid yield and fatty acids profile for microalgae Nannochloropsis gaditana were investigated. Replacing nitrate by urea didn't show positive influence on lipid content and yield compared to normal medium. The major fatty acids for these two mediums were palmitic acid (C16:0) and palmitioleic acid (C16:1). Nannochloropsis gaditana still shows to be ideal candidate for biodiesel production using urea or nitrate enriched agriculture wastewater.

Invadolysin (inv) is a member of the M8 class of zinc-metalloproteases and is conserved throughout metazoans. It is essential for development and invadolysin homozygous Drosophila mutants are third instar larval lethal. These larvae exhibit a reduced larval brain size and an absence of imaginal discs. Detailed analysis showed that inv mutants exhibit pleiotropic effects, including defects with chromosome architecture, cell cycle progression, spindle assembly, nuclear envelope dynamics, protein turnover and problems with germ cell migration. These findings indicated that Invadolysin must have a critical role in Drosophila. In order to better understand these roles, I set out to identify genetic interactors of invadolysin. I performed a genetic screen scoring for enhancer/suppressor modification of a ‘rough eye’ phenotype induced by invadolysin overexpression. Screening against the Drosdel ‘deficiency kit’ identified numerous genetic interactors including genes linked to energy regulation, glucose and fatty acid pathways. Immunofluorescence experiments in cultured cells showed that H. sapiens Invadolysin localises to the surface of lipid droplets (LD), and subcellular fractionation confirmed its enrichment to these structures. Lipid droplets are highly dynamic organelles involved not only in energy storage but also in protein sequestration, protein and membrane trafficking, and cell signaling. Drosophila fat bodies are enriched in LDs and therefore important energy stores. In addition, they are nutritional sensors and regulators, which are proposed to be the ortholog of vertebrate liver and adipose tissue. Mutant inv fat bodies appeared smaller and thinner than wild type fat body, and accumulated lower levels of triacylgylcerides. This indicated that the loss of invadolysin might be affecting lipid metabolism and storage, confirming the genetic data. However, it was not clear whether these effects were due to the direct action of Invadolysin. Hence, transgenic fly lines expressing either HA, RFP or FLAG tagged forms of Invadolysin were generated to identify physical interactors of Invadolysin. Subsequent mass spectrometry analysis detected ATP synthase-α, -β and -d as interactors. This result suggested that Invadolysin might play a role in regulating mitochondrial function, which might then be manifest in the fat body as the defects previously observed. Energy levels are known to affect the cell cycle, cell growth, lipid metabolism and inevitably development. Further in vivo and in vitro experiments confirmed this hypothesis. Genetic crosses confirmed the interaction of invadolysin with ATP-synthase subunit-α, whilst staining of mitochondria in mutant third instar larval fat bodies suggested decreased mitochondrial activity. Mutants also showed lower ATP levels and an accumulation of reactive oxygen species, hence indicating the possibility of a dysfunctional electron transport chain. Lipid droplets are known to interact with mitochondria, whilst ATP synthase has been found on lipid droplets by proteomic studies in Drosophila. Therefore, based on these data, we propose that Invadolysin is found, with ATP synthase, on lipid droplets, where Invadolysin (likely acting as a protease) could be aiding the normal processing or assembly of ATP synthase. This interaction is vital for the proper functioning of ATP synthase, and hence mitochondria. In this scenario, cellular ATP needs are not met, energy levels drop which results in an inhibition of fatty acid synthesis, cell and organismal growth defects.

Transgenic tobacco plants were developed containing a partial PLD clone in antisense orientation. The PLD isoform targeted by the insertion was identified. A PLD clone was isolated from a cDNA library using the partial PLD as a probe: Nt10B1 shares 92% identity with PLDβ1 from tomato but lacks the C2 domain. PCR analysis confirmed insertion of the antisense fragment into the plants: three introns distinguished the endogenous gene from the transgene. PLD activity was assayed in leaf homogenates in PLDβ/g conditions. When phosphatidylcholine was utilized as a substrate, no significant difference in transphosphatidylation activity was observed. However, there was a reduction in NAPE hydrolysis in extracts of two transgenic plants. In one of these, a reduction in elicitor- induced PAL expression was also observed.

Cardiovascular magnetic resonance imaging (MRI) is a well established technique in clinical cardiology. Different MRI sequences are routinely used to assess cardiac anatomy, function, viability and other parameters that aid diagnosing cardiac disease. Conversely, cardiac magnetic resonance spectroscopy (MRS), the only available method for a non-invasive study of human cardiac metabolism, has not evolved into a clinical tool yet. The combination of both techniques holds great potential to gain insight into the causality of cardiomyopathy diseases or other medical conditions with high cardiovascular risk profile, like diabetes or obesity and improve the clinical management of cardiac diseases. Nowadays, high field clinical MR systems have the great potential of improving the low spatial and temporal resolution and reproducibility of MRS. The aim of this thesis was to develop and implement a cardiac 1H-MRS method at 3 T that can be applied in clinical routine for the assessment of creatine and lipid levels in the human myocardium. The methodological developments to advance cardiac MRS are presented first. A robust 1H-MRS method comprising an optimized single-voxel technique, phased-array coil combination routine, optimized water suppression, breath-hold averaging and post-processing methods were developed. First, reproducibility and feasibility of the method were validated in vivo by acquiring 1H-MRS of the liver in almost one hundred healthy subjects. Subsequently, myocardial lipids levels were obtained in healthy volunteers by single breath-hold 1H-MRS triggered to mid-diastole, showing good reproducibility in an acquisition time less than 12 s. The good spectral resolution achieved using this method was demonstrated by the ability to differentiate for the first time two pools of myocardial lipids in spectra from the septum of patients with suspected myocardial lipid excess. Finally, creatine levels for healthy volunteers were investigated using multiple breath-hold acquisitions. Thus, this study shows the practicality and feasibility to incorporate this rapid cardiac 1H-MRS method into clinical studies of the human myocardium.

Bedrock features represent various economic, social, and symbolic aspects of past societies, but have historically received little study, particularly in North America. Fortunately, new techniques for analyzing spatial configurations, use-wear, and organic residues are beginning to unlock more of the interpretive potential of these features. Though preliminary in nature, the present study contributes to this trend by documenting an application of lipid analysis to bedrock features in a dry rockshelter. Results of this initial application indicate that bedrock features in dry rockshelters may provide especially favorable conditions for the preservation and interpretation of ancient organic residues. Abundant lipids, comparable to concentrations present in some pottery sherds, were extracted from a bedrock grinding surface at Gila Cliff Dwellings National Monument and analyzed using gas chromatography-mass spectrometry. Though the lipids were highly oxidized, degradation products indicative of former unsaturated fatty acids were retained. Comparisons to experimentally aged residues, and absence of a known biomarker for maize, indicate that the bulk of the lipids preserved in the milling surface probably derive from processing an oily nut or seed resource, and not from processing maize. Substantially lower amounts of lipids were recovered from a small, blackened cupule. It is hypothesized that some portion of the lipids in the blackened cupule was deposited from condensed smoke of cooking and heating fires in the caves. Potential for the preservation of organic residues in similar sheltered bedrock contexts is discussed, and a practical method for sampling bedrock features in the field is described.

Electrochemical Impedance Spectroscopy (EIS) was used to develop a novel methodology for studying ionic interaction with lipids arranged in a lipid cubic phase (LCP). Studying different types of ions, both cations and anions, validated the method. A free-standing LCP membrane was formed between two cell compartments and impedance experiments were carried out in a 2-electrode setup to estimate dielectric properties of the membrane, exposed to the following electrolyte solutions at different concentrations: KCl, CsBr, CaCl2, MgCl2, CsCl, NaCl, NaOAc and NaTryptophan. Two different LCP were used in this setup, i.e: Monoloein/water and the ternary system of monoolein/dioleoylphosphatidylcholine/water (MO/DOPC/H2O). SAXRD measurements were performed to determine the space group of the cubic phase and confirm the stability of the LCP during measurements. Membrane resistances and capacitances were found from equivalent circuit fitting to the impedance data. The membrane resistance was shown to be related to ionic interaction with the lipid head group in the water channels of the LCP. Membrane capacitance were correlating to condensing and swelling effect of LCP due to the exposure of ions. The results correlated well with the SAXRD results and earlier published data. The results also indicate that these membranes become less permeable to ions as they increase in size as well as in charge or polarity.  Cyclic voltammetry was used to study the applications of a LCP for modification of the bioanode in a biofuel cell. The monoolein cubic phase was used to host Glucose oxidase (GOx) and a freely diffusing ferrocene carboxylate was used as mediator. The supported cubic phase had an intrinsic resistance in the same order of magnitude as the freestanding MO-LCP membrane as measured with EIS. / Elektrokemisk impedans spektroskopi har använts för att utveckla en ny metod för att studera joners växelverkan med lipider som bildat en kubisk fas. Olika typer av joner, både positiva och negativa, användes för att validera metoden. Ett fristående membran uppbyggt av en kubisk fas separerade två avdelningar i en elektrokemisk cell. Cellen fylldes med elektrolyt-lösningar och impedansmätningar kunde utföras mellan två platina elektroder placerade i vardera avdelning. Membranet exponerades för följande elektrolytlösningar av olika koncentration: KCl, CsBr, CaCl2, MgCl2, CsCl, NaCl, NaOAc and NaTryptofan. Två olika kubiska faser användes i denna uppställning, dvs: Monoloein/vatten och det ternära systemet monoolein/dioleoylfosfatidylkolin/vatten(MO/DOPC/H2O). Med hjälp av SAXRD kunde den kubiska fasens kristallstruktur bestämmas och dess stabilitet under mätningarna bekräftas. De dielektriska egenskaperna hos membranet bestämdes genom att anpassa impedansspektrat till en ekvivalent krets bestående av resistanser, kapacitanser och konstant-faselement. Membranresistansen visade sig vara relaterad till jonernas växelverkan med lipidhuvudgruppen i vattenkanalerna i kubiska fasen. Ju starkare växelverkan desto högre var resistansen. Membrankapacitansen kunde korreleras med kondenserande och uppsvällande effekter på kubiska fasen förorsakade av exponeringen till joner. Resultaten bekräftades av SAXRD mätningar och även tidigare publicerade data. Resultaten indikerar också tydligt att permeabiliteten hos membranet minskar med ökad jonstorlek, jonladdningoch polaritet hos jonen. Cyklisk voltammetri användes för att studera en tillämpning av kubiska fasen i en tänkt applikation som bioanod i en biobränslecell. Elektroden modifierades med en kubisk fas innehållande GOx och tillsammans med en fritt diffunderande ferrocen karboxylat som mediator, där oxidation av glukos studeras. Det visade sig att den kubiska fasen hade en resistans av samma storleksordning som det fristående membranet uppmätt med impedansspektroskopi.

The present study was undertaken to investigate the effects of the T-type calcium channel blocker, Mibefradil, on white adipocytes. Unexpected for a T-type channel blocker, Mibefradil was found to increase intracellular calcium levels, cause lipid droplet fusion, and result in cell death. Calcium imaging of white adipocytes showed an increase of calcium concentration by Mibefradil at concentrations ranging from10-50 µM. The elevation in calcium by Mibefradil was significantly reduced by pretreatment of cells with Thapsigargin, an endoplasmic reticulum (ER) specific Ca ATPase inhibitor. Additionally, lipid droplet fusion and cell death were also attenuated by Thapsigargin pretreatment in white adipocytes. We conclude that Mibefradil elevated intracellular calcium levels, induced lipid droplet fusion and cell death in white adipocytes via mobilizing intracellular calcium stores from the ER. These results describe novel effects of Mibefradil on white adipocytes and may provide new insight into how this drug might be repurposed in obesity research.