The interaction between the glycolytic metabolism and fatty acid and glycerolipid biosynthesis in pea root (Pisum sativum L.) plastids was assessed in this study. When various glycolytic intermediates were used to substitute for the APT requirement for fatty acid synthesis from acetate, phosphoenolpyruvate, 2-phosphoglycerate, fructose-6-phosphate and glucose-6-phosphate each gave 48, 17, 23 and 17%, respectively, of the ATP-control activity. Similarly, in the absence of exogenously supplied ATP, the optimized triose-phosphate shuttle, which consists of 2 mM dihydroxyacetone phosphate, 2 mM oxaloacetic acid and 4 mM inorganic phosphate, gave up to 44% the ATP-control activity in promoting fatty acid synthesis from acetate. These results suggest that 3-phosphoglycerate kinase and pyruvate kinase in these plastids can function in intraplastidic ATP production through substrate level phosphorylation. However, in all cases, exogenously supplied ATP gave the greatest rates of fatty acid and glycerolipid synthesis. Radiolabeled pyruvate, glucose, glucose-6-phosphate, and malate in comparison to acetate were all variously utilized for fatty acid and glycerolipid biosynthesis by the root plastid. At the highest concentrations tested (3-5 mM), the rates of incorporation of pyruvate, glucose-6-phosphate and acetate into fatty acids were 183, 154, 125 nd 99 nmol $ rm cdot h sp{-1} cdot mg sp{-1}$, respectively. Malate was the least effective precursor, giving less than 55 nmol $ rm cdot h sp{-1} cdot mg sp{-1}$. Acetate incorporation was approximately 55% dependent on exogenously supplied reduced nuclotides (NADPH and NADH), whereas the utilization of the remaining precursors was only approximately 10-20% dependent on NAD(P)H. These results indicate that the entire pathway of carbon flow from glycolysis, including pyruvate dehydrogenase (PDHase), to fatty acids is operating in pea root plastids. Further, the intraplastidic glycolytic pathway plays an important role in provi

Fatty acid biosynthesis from (1-$ sp{14}$C) acetate was optimized in plastids isolated from primary root tips of 7-day-old germinating pea seeds. Fatty acid synthesis was maximum at 82.3 nmol/hr/mg protein in the presence of 200$ mu$M acetate, 0.5mM each of NADH, NADPH and CoA, 6mM each of ATP and MgCl$ sb2$, 1mM each of MnCl$ sb2$ and glycerol-3-phosphate (G3P), 15mM KHCO$ sb3$, and 0.1M Bis tris propane, pH 8.0 incubated at 35C. At the standard incubation temperature of 25C, fatty acid synthesis was linear for up to 6 hours with 80 to 120 $ mu$g/ml plastid protein. ATP and CoA were absolute requirements, whereas divalent cations, potassium bicarbonate and reduced nucelotides all improved activity by 2 to 10 fold. Mg$ sp{2+}$ and NADH were the preferred cation and nucleotide, respectively. G3P and dihydroxyacetone phosphate had little effect, and dithiothreitol and detergents generally inhibited incorporation of $ sp{14}$C-acetate into fatty acid. / Glycerolipid synthesis was obtained from $ sp{14}$C-acetate, (U-$ sp{14}$C) G3P and (U-$ sp{14}$C) glycerol at relative rates of 3.7:1.0:0.1, respectively. (Abstract shortened by UMI.)

Lipid metabolism in Trypanosoma brucei, the causative agent of African sleeping sickness, differs from its human host, allowing a plethora of novel drug targets to be discovered and validated. Cytidine diphosphate diacylglycerol (CDP-DAG) is a central lipid intermediate produced by the enzyme CDP-DAG synthase (CDS), but nothing was known about CDS in T. brucei. Only one gene encodes CDS in Trypanosoma brucei (Tb927.7.220) and this was shown to encode a functional CDS by overexpression in E. coli and complementation of a yeast CDS null, which was created during this study. Expression and activity of TbCDS was confirmed in T. brucei, and was shown to be essential in both life cycle stages. Disruption of TbCDS altered the lipid profile of T. brucei, confirming a central role for CDP-DAG in phospholipid synthesis. Biochemical and morphological characterisation of mutants in TbCDS expression elucidated at least two separately localised and regulated pools of CDP-DAG and phosphatidylinositol in T. brucei. In bloodstream form these pools are localised to the Golgi and the ER, however in procyclics it is possible that both of these pools are localised to the Golgi, since no phosphatidylinositol synthase protein was detected in the ER of procyclics. Reduction in TbCDS was shown to affect cell cycle regulation and Golgi segregation possibly due to a depletion of phosphorylated phosphatidylinositols (PIPs). These studies also indicate that phosphatidylglycerol may be synthesised by the phosphatidylglycerol-phosphate synthase which may be capable of using phosphatidylserine as a substrate in a headgroup swapping reaction. TbCDS has now been genetically validated as a drug target, and has highlighted novel aspects of lipid biosynthesis in T. brucei. Collectively, these findings highlight the central role played by TbCDS and the new knowledge gained here may lead to the discovery and validation of other novel drug targets against African sleeping sickness.

No description available.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Many cancers, including breast cancer, often develop resistance to chemotherapeutic drugs over a course of treatment. Many factors, including ABC transporter-mediated drug efflux, have been shown to play a role in acquired drug resistance. Fatty acid synthase (FASN), the key enzyme of lipid synthesis pathway, was found to be over-produced in an Adiamycin resistant breast cancer cell line MCF7/AdrVp3000, compared to its parental drug sensitive MCF7 cell line. Inhibition of FASN expression increased the drug sensitivity in breast cancer cells (MCF7/AdrVp3000 and MDA-MB-468), but not in the normal breast epithelia cell line MCF10A1. Enforced overexpression of FASN in MCF7 breast cancer cells decreased its drug sensitivity. These results indicated that FASN overexpression can induce drug resistance in breast cancers. Ectopic overexpression of FASN in MCF7 cells did not affect cell membrane permeability, transporter activity, nor did it affect cell proliferation rate. However, FASN overexpression protects cancer cells from drug-induced apoptosis by decreasing caspase-8 activation. In FASN over-expressing MCF7 cells, I discovered the positive feedback relationship between FASN and activation of Akt as previously reported. However, activation of Akt did not mediate FASN-induced drug resistance. Together with the findings that FASN expression associates with poor prognosis in several types of cancers, my investigations suggest that FASN overexpression is a novel mechanism of drug resistance in breast cancer chemotherapy. Inhibitors of FASN can be used as sensitizing agents in breast cancer chemotherapy.

The thesis entitled “Novel Cationic Gemini Lipids, Click Chemistry Based Adducts and Amphiphile-Capped Silver Nanostructures: Synthesis, Aggregation and Biological Properties” elucidates the design, synthesis, aggregation and gene transfection properties of novel gemini cationic lipids based on cholesterol and pseudoglyceryl backbone, and click chemistry based adducts. This thesis also elucidates the synthesis and aggregation properties of silver nanoparticles loaded cationic liposomes and silver nanorods stabilized by micellar solutions of gemini surfactants. The work has been divided into six chapters. Chapter 1: Introduction: Membrane Formation from Cholesterol-based Cationic Lipids and their use as Non-Viral Gene Delivery Agents This chapter describes the importance of cholesterol in biological membranes, the aggregation properties of cholesterol-based cationic lipids and their interactions with phospholipid membranes. This chapter also gives a comprehensive account of the research towards the development of novel cationic cholesterol-based monomeric and gemini lipids. It also reviews the utilization of cholesterol-based cationic monomeric and gemini lipids in gene transfection properties. Chapter 2A: Effect of Hydroxyl group on the Cationic Headgroups of Cholesterol-based Gemini Lipids on their Aggregation, DNA Binding Properties and Interaction with Phospholipid Membranes This chapter describes the syntheses and aggregation properties of two series of cholesterol-based monomeric and gemini cationic lipids with and without hydroxyl functionality (Figure 1). The gemini lipids of a given series differ in their spacer polymethylene -(CH2)n- chain lengths between the cationic headgroups. Figure 1. Molecular structures of non-hydroxylated and hydroxylated cationic cholesterol-based gemini lipids and their monomeric counterparts. All monomeric and gemini lipids were found to generate stable suspensions in aqueous media. Electron microscopic studies showed that all the lipids form vesicular aggregates in aqueous media. The structures seen under TEM for the non-hydroxylated series of monomeric (C-M) and gemini lipids are of variable sizes, they appeared like separated vesicular aggregates. For the hydroxylated series of lipids, however, both the monomeric lipid aggregates (CH-M) and aggregates of their gemini counterparts were found to be ‘connected’ with each other to form elongated chain of aggregates of different length scales. XRD studies with the cast films of lipids revealed that the monomeric lipids of either series have higher bilayer width than the corresponding gemini lipids. Incorporation of the -(CH2)n- spacer units at the head group level joining the two monomeric units lowered the bilayer thicknesses of both series of the lipid aggregates. Thus the monomeric lipids (C-M and CH-M) appear to form nearly regular bilayer type arrangements whereas gemini lipids form interdigited and tilted bilayer arrangements in their aggregates. Calorimetry studies of the coaggregates showed that ~10 mol-% of most of the cholesterol gemini lipids is enough to abolish the phase transition of DPPC membranes whereas more than 10 mol-% is required in case of their monomeric counterparts. Further these thermotropic properties depend upon the length of the spacer of the gemini lipid included in the mixture. We have observed greater quenching of the thermal phase transition of DPPC membranes with 10 mol-% of C-M as compared to CH-M doped liposomes. At 10 mol-% of all the cationic lipid doped DPPC covesicles, only CG-3 doped liposomes showed an observable transition temperature. Maximum broadening of the DPPC transition peak was observed in the case of the gemini lipids, CHG-6 and CHG-12. DNA binding and release studies show that the interactions between gemini lipids and DNA depend upon the nature of the head group as well as the length of the spacer between the cationic head groups. For the non-hydroxylated cholesterol-based cationic lipid series, the monomeric liposomes of C-M facilitates the dissociation of EB from the EB-DNA complex to an extent of 93% at a maximum lipid:DNA ratio of 3.0 whereas the liposomes of CG-4 and CG-12 showed the lowest extent of maximum EB exclusion (~74%) from the EB-DNA complex at lipid:DNA ratio of 3.0. For hydroxylated cholesterol-based cationic lipid series, the monomeric liposomes of CH-M facilitate the dissociation of EB from the intercalated EB-DNA complex to an extent of 81 % whereas the liposomes of CHG-3 showed the minimum binding to DNA. Thus the two monomeric liposomes C-M and CH-M were the more efficient formulations that allow dissociation of DNA from the corresponding lipoplexes. These findings have important being in their gene transfection activity compared their respective gemini lipid counterparts. Chapter 2B: Novel Cholesterol-based Cationic Gemini Lipids possessing Hydroxyethyl group on the Headgroup: Transfection Efficacy and Cell Toxicity Properties This chapter describes the transfection efficacy and cell toxicity properties of five cholesterol based gemini cationic lipids possessing hydroxyethyl functionality on each head group, which differ in the length of the polymethylene spacer [-(CH2)n-] chain (Figure 2). These gemini lipids are important to gene delivery processes as they possess pre-optimized molecular features, e.g., cholesterol backbone, ether linkage and a variable spacer chain between both the headgroups of the gemini lipids. Cationic liposomes were prepared from each of these lipids individually and as a mixture of individual cationic gemini lipid and 1,2-dioleoylphosphatidylethanolamine (DOPE). The gemini lipid with a hydroxyethylated headgroup and a -(CH2)5- spacer, CHG-5 showed the highest transfection activity at N/P (lipid/DNA) ratio of 0.5 and lipid:DOPE molar ratio of 2. Upon comparison of the relevant parameters, e.g., % transfected cells, the amount of DNA transfected to each cell and % cell viability all together against LipofectAMINE 2000, one of the most potent commercially available transfecting agents, the optimized lipid formulation based on CHG-5/DOPE was found to be comparable. In terms of its ability to induce gene-transfer in presence of serum and shelf-life CHG-5/DOPE liposome was found to be better than its commercial counterpart. Recording of confocal images confirmed that in presence of 10% serum using 1.2 µg DNA per well and lipid:DOPE ratio of 1:4 and N/P charge ratio of 0.75, CHG-5 is better than LipofectAMINE 2000. These properties render them to be reagents of practical value for various gene delivery applications. Figure 2. Molecular structures of cholesterol-based cationic monomeric lipid and gemini lipids possessing hydroxyethyl group on the headgroup synthesized. Chapter 3: Bilayer Membrane and Stable Monolayer Forming Properties of Cationic Pseudoglyceryl Gemini Lipids having Polymethylene Spacers and Oxyethylene Linkages This chapter describes the synthesis of five new cationic pseudoglyceryl gemini lipid versions of their monomeric counterpart (Figure 3). Each cationic lipid aggregate in aqueous media was found to form vesicular structures as evidenced from the negatively stained TEM experiments and DLS measurements. XRD experiments with their cast films of aqueous dispersions revealed that introduction of the polymethylene -(CH2)n-spacer chain joining the two monomers decreased the bilayer widths of the gemini lipid aggregates. The inter-lipidic packing and the hydration of the lipid vesicles were examined using fluorescence anisotropy and generalized polarization measurements using membrane-soluble probes, DPH and Paldan respectively. Fluorescence anisotropy measurements showed that the aggregates of lipid 2c with -(CH2)5- spacer chain were highly packed and ordered in the vesicular aggregates than that of the other cationic lipid aggregates in the series. Paldan hydration studies showed that incorporation of the polymethylene -(CH2)n- spacer chains joining two monomeric units lowered the hydration of the gemini lipid aggregates in the solid gel state. Each of these cationic lipid aggregates showed sharp transition temperatures (Tm) as observed from differential scanning calorimetric studies. DSC studies further revealed that the incorporation of oxyethylene group at the linker region of cationic pseudoglyceryl gemini lipid 2a with (CH2)3- spacer chain length lowered the thermotropic phase transition temperature (Tm) of the aggregates in aqueous media when compared with the corresponding gemini analogue without oxyethylene linkages. Langmuir film balance studies showed that each cationic gemini lipid and their monomeric counterpart were able to form stable monolayers at the air-water interphase. We observed that the mean molecular area (collapse area) of each of the cationic lipid obtained from the Langmuir monolayer studies increased with increase in the spacer chain lengths. Figure 3. Molecular structures of the cationic pseudoglyceryl gemini lipids and their monomeric counterpart. Chapter 4: Vesicle and Stable Monolayer Formation from Simple ‘Click’ Chemistry Adducts in Water This chapter describes successful use of Cu(I) catalyzed “Click Chemistry” for the syntheses of a series of hitherto unknown amphiphilic adducts (M1, M2, D1 and T1) which on dispersal in water afforded vesicular aggregates as evidenced from dye entrapment, TEM, SEM, AFM and DLS studies (Figure 4). Figure 4. Molecular structures of triazole based adducts. XRD experiments with their cast films of aqueous suspensions indicate the formation of a tilted bilayer arrangement for the aggregates of M1 whereas regular bilayer structures are predominant for the aggregates derived from M2, D1 and T1. Measurement of pKa values using UV-Vis spectroscopy showed that the aggregates of monomeric click adducts (M1 and M2) possess less pKa value than that of the aggregates of dimeric (D1) and tetrameric (T1) analogues and the values lie within the range of 2.8-3.2. The hydrodynamic diameter of the aggregates of each click adduct increased with decrease in the pH of the media. Thus, the protonation of the triazole groups in the aggregates of each click adduct increased the hydrodynamic diameter. Dye entrapment studies showed that each click chemistry based adduct formed closed vesicular aggregates with inner aqueous compartment in aqueous media. The temperature induced order-to-disorder transitions of the aggregates and the accompanying changes in hydration were examined using high sensitive DSC, fluorescence anisotropy and generalized polarization measurements using a membranesoluble probe, DPH and Paldan respectively. In the solid state, M1 remains as the most hydrated species whereas in the fluidized phase, D1 maintains as the most hydrated aggregate. Clearly simple variation in the adduct molecular architecture bring about significant changes in their packing in aggregates and also the hydration of the resulting vesicles. Langmuir monolayer studies confirmed that these click adducts do form stable monolayers as well on water subphase at the air-water interface. We also calculated the mean molecular areas from the Langmuir monolayer studies and as perhaps expected the adduct T1 has the highest head group area. Thus click chemistry based simple triazole adducts, which can be very easily prepared, are good candidates for further investigations involving syntheses of novel self-assembling structures. Chapter 5: Lipid Mediated Synthesis of Silver Nanoparticles, their Physical Characterizations and DNA Binding Abilities In this chapter, work on the Ag-NP (silver nanoparticle) loaded liposomes preparation using four cationic lipids (1-4) in which the Ag-NPs were entrapped within lipid bilayer has been described. A novel method was developed to synthesize the Ag-NPs where the lipid itself capped and stabilized the Ag-NPs. Consequently there was no need of inclusion of any other capping agents like citrate. Confocal microscopy confirmed that these Ag-nanoparticles are fluorescent in character. It was also demonstrated that silver nanoparticles are indeed entrapped in lipid bilayer with transmission electron microscopy (TEM). DLS experiments provided information about the hydrodynamic diameter of the lipid vesicles which increased with the increase in Ag concentrations. This could be due to the ‘loosening’ of the lipid packing in vesicles. Zeta potential measurements showed that the zeta potential value decreased with the increase in the concentration of Ag-NPs in the cationic lipid vesicles. XRD studies with the cast films of the lipid or Ag-NP loaded lipid suspensions revealed that when the Ag-NPs get entrapped into the bilayer of the multilamellar vesicles of the lipid in the aqueous media, the unit bilayer thickness of the aggregates increased. Paldan experiments showed that with the incorporation of Ag-NPs in the lipid vesicles, the hydration of the lipid vesicles increased to a significant extent but the phase transition temperatures remained practically unaltered for all the lipids. Fluorescence anisotropy experiments revealed that the hydrocarbon chain packing of the lipid vesicles ‘loosens’ with the incorporation of Ag-NPs. Ag-NP loaded liposomes showed enhanced DNA binding ability and also the presence of Ag-NPs in cationic liposomes induced the release of DNA from silver nanoparticle-loaded lipoplexes more effectively. Figure 5. Molecular structures of the cationic lipids mentioned in the present chapter. Chapter 6: Dependence of Spacer Chain Lengths in the Synthesis of Ag-Nanorods in Gemini Cationic Surfactant Micelles Figure 6. Chemical structures of cationic gemini surfactants. This chapter describes the synthesis of Ag-nanospecies by seed-mediated wet synthesis method using four gemini surfactants (16-2-16, 16-4-16, 16-5-16 and 16-1216) as the capping agents (Figure 6). For this, we first synthesized Ag-nanoseeds of diameter ~7 nm stabilized by trisodium citrate (as capping agent). Then the solution containing Ag-nanoseeds was used to synthesize Ag-nanorods of different aspect ratios. It was that with decreasing Ag-nanoseed concentration, the aspect ratios of Agnanorods stabilized by gemini surfactants (16-2-16 and 16-4-16) increased gradually as evidenced from TEM images. These Ag-nanoseeds and Ag-nanorods were further characterized using UV-Vis spectroscopy (to know the surface plasmon bands), transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDAX) and X-ray diffraction (XRD). It was observed that when gemini surfactant 164-16 was used to stabilize Ag-nanorods, the λmax of the longitudinal band shifted more towards the red region (red-shift) as observed by UV-Vis spectroscopy when compared to that of gemini surfactant with shortest spacer, 16-2-16. Thus the gemini surfactants with shorter -(CH2)2- and -(CH2)4- spacer chains promoted the growth of Ag-nanorods in their micellar solutions whereas -(CH2)5- and -(CH2)12- spacer chains of gemini surfactants did not. So, the growth of Ag-nanorods in micellar solutions is found to be highly spacer-chain length specific. TEM micrographs revealed that the aspect ratios of Ag-nanorods stabilized by gemini surfactants 16-4-16 are larger than those compared to the Ag-nanorods stabilized by gemini surfactants 16-2-16 at a particular amount of Agnanoseed solution. TEM images of the samples containing micellar solutions of gemini surfactants 16-5-16 and 16-12-16 showed that the formation of only Ag-nanoparticles of larger sizes (compared to Ag-nanoseeds stabilized by trisodium citrate) and Agnanoprisms irrespective of the amount of Ag-nanoseed solution added. No Ag-nanorod formation in the micellar solutions of gemini surfactants 16-5-16 and 16-12-16 was observed. Gemini surfactants (16-2-16 and 16-4-16) formed bilayer arrangements to facilitate the growth and stabilization of Ag-nanorods in aqueous media where the inner layer is attached to the Ag-nanorod surface through the gemini surfactant ammonium headgroups. X-ray diffraction (XRD) studies showed that these Ag-nanorods stabilized by gemini surfactants 16-2-16 and 16-4-16 crystallized in the aqueous media via (111), (220) and (222) lattice faces. Thus this study demonstrated the way one can control structures and shapes of the silver nanoobjects using gemini surfactant micelles. (For structural formula pl refer the thesis)