Introdução Os feijões comuns, da espécie Phaseolus vulgaris, são amplamente produzidos e consumidos no Brasil. As variedades, carioca e preto ganham destaque na região Sudeste do país. Encontra-se descrita na literatura a ação hipocolesterolemizante de algumas leguminosas, tais como, soja, tremoço e feijão caupi, que podem estar associados à redução do risco de doenças cardiovasculares. Objetivo Avaliar o potencial efeito da adição de farinhas de feijões carioca e preto (Phaseolus vulgaris) no metabolismo lipídico de hamsters alimentados com dieta contendo gordura saturada e colesterol. Métodos A produção das farinhas dos feijões envolveu as etapas de autoclavagem, congelamento, liofilização e moagem. As propriedades hipocolesterolemizantes destas farinhas foram avaliadas por meio de dois ensaios biológicos. Foram utilizados hamsters Golden Syrian, machos com 21 dias, pesando 60 ± 4g, que receberam as dietas experimentais ad libitum. No Ensaio A, os animais foram separados em 3 grupos, diferenciados pela dieta. Todas as dietas eram hipercolesterolemizantes [13.5 por cento de gordura de coco e 0.1 por cento colesterol] e tinham as mesmas quantidades de proteínas, carboidratos, fibras, vitaminas e minerais. O Grupo Controle (C) tinha como fonte protéica a caseína; no Grupo Feijão Carioca (FC) a farinha de feijão carioca representou 15 por cento do peso total da dieta e no Grupo Feijão Preto a farinha de feijão preto representou 15 por cento do peso total da dieta. No Ensaio B, os animais foram separados em três grupos novamente. Desta vez, a única diferença entre os grupos foi quanto a fonte protéica, para o grupo controle (C) somente caseína, para o grupo feijão carioca (FC), 67 por cento de feijão e 7,5 por cento de caseína e para o grupo feijão preto (FP), 62 por cento de feijão e 7,5 por cento de caseína. Nos dois ensaios, após 21 dias de experimento, foi realizada coleta de materiais biológicos (plasma, fígado e fezes). Resultados O processo de produção das farinhas de feijões cozidas liofilizadas não alterou a composição centesimal das matérias-primas. A análise de fibras alimentares revelou que não há diferenças entre os cultivares Pérola e Uirapuru. No Ensaio A, as concentrações de colesterol não HDL e HDL colesterol foram maiores nos grupos que receberam feijão de maneira significativa. Quanto aos demais parâmetros plasmáticos não foram observadas diferenças entre os grupos. No Ensaio B as concentrações plasmáticas de triglicerídeos foram maiores no grupo FP. As concentrações de HDL colesterol foram maiores nos grupos FP e FC, sendo estatisticamente significativa para o feijão carioca em relação ao grupo controle. As excreções fecais de ácidos biliares foram maiores no grupo FC e a de colesterol no grupo C. A determinação de lipídeos totais no fígado não revelou diferenças entre os grupos, dados que corroboraram com a análise do grau de esteatose nos fígados, a qual demonstrou desenvolvimento de acúmulo de lipídeos nos hepatócitos dos animais dos três grupos. O teste qui quadrado mostrou que as variáveis grau de esteatose e tipo de dieta, assim como tipo de dieta e grau de inflamação portal hepática são independentes. Já o grau de inflamação parenquimatosa hepática está associado ao tipo de dieta e o feijão carioca mostrou-se capaz de reduzir em 30 por cento o risco de desenvolver esteatoepatite severa. Conclusões Os feijões não foram capazes de proteger contra o aumento do colesterol total, triglicérides e colesterol não HDL no plasma, mas mesmo na presença de gordura saturada e colesterol na dieta, o feijão carioca foi capaz de aumentar a HDL, mostrando que o mecanismo de remoção do colesterol plasmático foi preservado. O feijão carioca mostrou-se eficaz na proteção contra a inflamação parenquimatosa hepática severa. / Carioca and black beans are the varieties of Phaseolus vulgaris most consumed on Brazil Southwest. It is well described that some legumes, as soy and cowpea beans, have hypocholesterolaemic effects. To test cholesterol-lowering properties of carioca and black beans, two biological assays were conducted. Golden Syrian hamsters, 21 days old, were housed individually under 12 h light-dark cycle and temperature-controlled environment, with free access to food and water. There was a adaptation period of 6 days, before the start of experimental period. In Assay A, the animals (n=19) were randomly assigned to three distinct groups. All groups received a hypercholesterolaemic diet (13.5 per cent coconut oil and 0.1 per cent cholesterol) and similar amounts of proteins, carbohydrates, fiber, vitamins and minerals to suit the animal requirements. Control group received casein as the only protein source; Carioca bean group received 15 per cent of carioca bean flour and casein to complement protein requirement and Black bean group received 15 per cent of black bean flour and casein to complement protein requirement. After 21 days, the experimental period was over and liver, blood and feces were collected. In Assay B, all groups also received a hypercholesterolaemic diet (13.5 per cent coconut oil and 0.1 per cent cholesterol). In this assay the only difference between groups (n=27) was protein source: casein for control group, and the others received carioca (67 per cent ) or black bean whole seed flour (62 per cent ) plus 7,5 per cent of casein. The beans flours obtained showed no differences in chemical composition. In Assay A, plasma HDL cholesterol and non-HDL cholesterol were higher in Carioca bean group and Black bean group. The other plasma parameters had no differences. In assay B, plasma triglyceride was higher in Black bean group. The HDL cholesterol was increased in both beans groups, and was significant in Carioca group. Fecal excretion of bile acids was higher in animals of Carioca bean group. Fecal excretion of cholesterol was higher in Control group. There were no differences between groups in total liver lipid concentration, data supporting the steatosis analysis in livers. The chi-square test showed that the type of experimental diet and steatosis grade were independents, also the portal hepatic inflammation was not associated with the experimental diets. The parenchymal inflammation of the liver was associated with Carioca bean group, which showed that the chance of developing severe inflammation was 30 per cent lower in carioca bean group compared with Control group. Beans had no cholesterol-lowering effect, but the HDL increases in plasma and lower inflammation in Carioca bean group deserves further investigation.

Cytosolic LPA acyltransferase in yeast An isooctane tolerant strain of S. cerevisiae KK-12 was reported to have increased saturated fatty acid content (Miura et. al., 2000). Amongst the various genes upregulated on isooctane treatment, ICT1 (Increased Copper Tolerance 1) was found to have maximal expression (Miura et. al., 2000; Matsui et. al., 2006). This gene in S. cerevisiae is encoded by YLR099C annotated as Ict1p. However, the physiological significance of Ict1p was not understood. Here we showed that an increase in the synthesis of phosphatidic acid (PA) is responsible for enhanced phospholipid synthesis, which confers organic solvent tolerance to S. cerevisiae. This increase in the PA formation is due to the upregulation of Ict1p, a soluble oleoyl-CoA dependent lysophosphatidic acid (LPA) specific acyltransferase. Analysis of Δict1 strain by in vivo [32P]orthophosphate labeling showed a drastic reduction in PA, suggesting the role of Ict1p in phospholipid biosynthesis. Overexpression of Ict1p in S. cerevisiae showed an increase in PA and the overall phospholipid content on organic solvent exposure. The purified recombinant enzyme was found to specifically acylate LPA. Specific activity of Ict1p was found to be higher for oleoyl-CoA as compared to palmitoyl-CoA and stearoyl-CoA. The study therefore, provides a mechanistic basis of solvent tolerance in S. cerevisiae.It is well known that phosphatidic acid (PA) is formed by the acylation of LPA by LPA acyltransferase. However, all the LPA acyltransferases characterized till date have distinct transmembrane domains and form a member of membrane bound biosynthetic machinery of phospholipid biosynthesis. They have a conserved signature motif, H(X)4D. Phosphatidic acid is an important precursor for the synthesis of glycerophospholipids and triacylglycerols. PA enters the biosynthetic pathway of phospholipids through a CTP-dependent activation catalyzed by CDPdiacylglycerol synthase. This enzyme forms CDP-diacylglycerol, which serves as a direct precursor for phosphatidylinositol, phosphatidylglycerol and cardiolipin. PA can also be dephosphorylated by phosphatidic acid phosphatase yielding diacylglycerol, which serves as a precursor for the formation of PE and PC through the CDP-ethanolamine and CDP-choline pathway or for the triacylglycerol synthesis through a dephosphorylation step followed by an acylation establishing it as a supreme molecule for the acylglycerol biosynthesis. Since, PA is an important intermediate and that there are mechanisms to synthesize PA, other than the conventional membrane bound pathways, we wanted to understand whether such a mechanism of PA biosynthesis is conserved across the plant and animal kingdom. Therefore, we resorted to analyze Ict1p like proteins in Arabidopsis and human whose complete genome sequence is available. Cytosolic LPA acyltransferase in Arabidopsis Homology search with ICT1 in Arabidopsis thaliana genome, led to the identification of At4g24160 as a close relative. In order to gain an insight into the significance of such proteins in plants we performed a genome wide survey of At4g24160 like proteins in Arabidopsis. We identified that A. thaliana genome encodes twenty four At4g24160 like proteins, most of which belong to the α/β- hydrolase family of proteins and possess a distinct lipase motif (GXS/NXG). Interestingly, amongst these twenty four, only At4g24160 has a conserved HX4D motif. Domain analysis of these proteins suggests a wide functional diversification during evolution. Gene expression studies revealed their importance during various abiotic stress. Bacterial expression of At4g24160 followed by its purification using Ni2+-NTA column chromatography and characterization revealed it to be a LPA acyltransferase. Expression analysis showed that it is highly expressed in the pollen grains followed by the root cap. In addition, the gene was found to be upregulated under salt stress conditions. Direct correlation between salt stress and phospholipid biosynthesis is well known in the literature. We envisage that At4g24160 might be one of the gene products involved in membrane repair when exposed to such a stressCytosolic LPA acyltransferase in human Homology search with Ict1p revealed another interesting candidate protein in Homo sapiens known as Comparative Gene Identification–58 (cgi-58). Mutations in CGI- 58 are known to be the causative reason for a rare autosomal recessive genetic disorder known as Chanarin-Dorfman syndrome characterized by the excessive TG accumulation and defective membrane phospholipid regulation in several tissues. It is known to be a coactivator of adipose triglyceride lipase (ATGL), promoting lipolysis of TG (Lass et. al., 2006). However, the exact biochemical role remains unknown. To understand the biochemical function of cgi-58, the gene was overexpressed in E. coli and the purified, recombinant protein was found to specifically acylate lysophosphatidic acid in an acyl-CoA dependent manner. Overexpression of CGI-58 in Δict1 rescued the metabolic defect of the strain. Heterologous overexpression of CGI-58 in S. cerevisiae followed by metabolic labeling with [32P]orthophosphate showed an increased biosynthesis of membrane phospholipids. Analysis of neutral lipid biosynthesis by [14C]acetate labeling showed an increase in DG and free fatty acids. However, marked decrease in the TG biosynthesis was seen. Decrease in TG was confirmed by ESI-MS. In addition, physiological significance of cgi-58 in the mice white adipose tissue is reported in this thesis. We found soluble lysophosphatidic acid acyltransferase activity in the mice white adipose tissue. Immunoblot with anti-Ict1p antibodies followed by MALDI-TOF analysis of the cross reacting protein in lipid droplets revealed its identity as cgi-58. These observations suggest the existence of an alternate cytosolic phosphatidic acid biosynthetic pathway in the white adipose tissue. Collectively, our observations suggest a possible involvement of cgi-58 in the phospholipid biosynthesis of adipocytes and its probable role in maintaining the TG homeostasis. In conclusion, the study reveals the significance of cytosolic lipid metabolic enzymes having conserved biochemical function, in maintaining homeostasis in living organisms across phylogeny.

Intestinal diseases affect millions of people worldwide. Recently, a number of proteins have been shown to be upregulated in the intestinal cells of patients that contribute to disease progression. Therefore, these diseases could be amenable to RNA interference technology (RNAi). Utilizing RNAi to deliver short interfering ribonucleic acid (siRNA) to intestinal cells shows promise for the treatment of diseases by specifically suppressing the expression of disease relevant proteins. A class of lipid nanoparticles termed lipidoid nanoparticles (LNPs) have been shown previously to potently deliver siRNA to several cell types in vitro and in vivo. Here, we seek to establish the utility of lipidoid nanoparticles (LNPs) in the context of oral siRNA delivery to intestinal cells for the treatment of intestinal diseases. Initial in vitro studies demonstrated that the siRNA-loaded LNPs mediated potent, dose dependent, and durable gene silencing in Caco-2 intestinal cells without inducing significant cytotoxicity or altering intestinal barrier function. LNP stability studies revealed that LNPs in an aqueous buffer remained stable for long periods of time when stored in the refrigerator (2 °C) compared to the freezer (-20 °C) or at room temperature. In addition, LNPs remained stable upon lyophilization with the addition of trehalose or sucrose to the LNP solution before freeze-drying. To determine potential for oral LNP delivery, we studied LNP stability under gastrointestinal (GI) tract conditions. LNPs remained potent and stable following exposure to solutions of varied pH, including pH values as low as 1.2. However, efficacy decreased following exposure to increasing concentrations of pepsin and bile salts. Mouse oral biodistribution studies indicated that siRNA-loaded lipid nanoparticles were retained in the GI tract for at least 8 hours. Confocal microscopy confirmed that nanoparticles entered the epithelial cells of the mouse small intestine and colon. Oral LNP therapeutic efficacy was measured in an inflammatory bowel disease (IBD) mouse model by targeting the upregulated genes myosin light chain kinase (MLCK) and Interleukin 18 receptor (IL18R) and were found to prevent some IBD disease progression. Lastly, a formulation for the co-delivery of siRNA and messenger RNA (mRNA) was developed and it was discovered that a negatively charged polymer can be used to improve LNP efficacy. Together, these studies have advanced our knowledge of lipid nanoparticle stability, and potential as an orally delivered intestinal therapeutic.

Lipid peroxidation is well established to contribute to the etiology of many deteriorative conditions including neurodegeneration, cardiovascular disease, cancer, aging, and recently in ferroptosis—a regulated, necrotic modality of cell death that results from the accumulation of lipid hydroperoxides. Recent high-throughput screening efforts have uncovered ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1) as two premiere inhibitors of ferroptosis. We propose that these compounds function as radical trapping antioxidants. We employ a systematic methodology of evaluating inherent radical trapping antioxidant (RTA) activity of Lip-1, Fer-1, and various aryl amine and aryl nitroxide analogues to put forward a biologically relevant mechanism of action based on our previous work in the field. Joining these observations with the efficacy of tetrahydronaphthyridinols (THNs), the results support a clear role of autoxidation in the execution of ferroptosis. Next, we expand the THN repertoire by targeting the payload towards the engine of our cells, the mitochondria. Decades of research have implicated mitochondrial dysfunction brought about by the peroxidation of mitochondrial membranes and the leaking of downstream oxidants, in the death of their symbiotic host cells. Isolated successes in the field have been demonstrated academically, though viable drugs remain to be developed, partially due to the lack of effective diagnostic tools. We endeavor to address some of these issues by investigating mitochondrially-targeted THNs (MitoTHNs) as a targeted chain-breaking antioxidant of unparalleled potency. Furthermore, we advance development of the THNs towards therapeutic applications by demonstrating their biodistribution in mice.

Abstract A data base search using the amino acid sequence of Saccharomyces cerevisiae Etr1p, the last enzyme of mitochondrial fatty acid synthesis type II (FAS II), revealed a highly similar human protein, NRBF-1. Expression of NRBF-1 in a yeast etr1Δ strain rescued its respiratory deficiency. NRBF-1 resides in mitochondria in cultured HeLa cells. The recombinant NRBF-1 is enzymatically active, reducing 2E-enoyl-CoAs to acyl-CoAs in an NADPH-dependent manner. Altogether, our data showed that NRBF-1 is a mitochondrial 2-enoyl-CoA reductase/2-enoyl thioester reductase (MECR/ETR1), the human functional counterpart of yeast Etr1p. In addition, MECR was also isolated from bovine heart. It turns out that mammals contain a mitochondrial FAS II pathway, in addition to cytoplasmic FAS I. To investigate the functional mechanism of MECR/ETR1 at the molecular level, the protein was crystallized and the crystal structure determined. The apo-structure of MECR/ETR1 contains two sulfates in the nucleotide binding site and the domain arrangement resembles the NADPH-containing holo-structure of yeast Etr1p. The predicted mode of NADPH-binding and kinetic data suggest that Tyr94 and Trp311 play critical roles in catalysis. A pocket was found in the structure extending away from the catalytic site that can accommodate fatty acyl chains up to 16 carbons. An acyl carrier protein (ACP) binding site was also suggested. To study the physiological function of mouse Mecr, two lines of transgenic mice overexpressing Mecr were generated. The Mecr transgenic mice developed cardiac and mitochondrial abnormalities. The phenotyping was carried out using echocardiography, heart perfusion, histology, and endurance testing. Our results suggest Mecr plays a role in mitochondrial and heart function. Therefore, inappropriate expression of the genes of FAS II may result in the development of cardiomyopathy.

The membrane-spanning phospholipids of the domain Archaea are postulated to provide membrane stability; this thesis reports the design and synthesis of a synthetic membrane-spanning macrocyclic lipid to test this hypothesis. Protected glutaric anhydride reacted with 10-undecyn-1-ol to produce a glutarate monoester. Copper-mediated azide-alkyne coupling (CuAAC) using 1,5-diazido-3-oxapentane(bis-azide) afforded a dicarboxylicacid with a hydrophobic chain of sufficient length to span a 35 Å bilayer membrane. The carboxylic acids were each esterified with an equivalent of 10-undecyn-1-ol. After optimization an 87% yield was obtained in the closure of the 72-membered macrocycle with the bis-azide using CuAAC. Deprotection and coupling with p-nitrophenyl phosphorodichloridate completed the synthesis. Two other phospholipids, a linear bolaamphiphile derived from the precursor to the second click reaction, and a linear amphiphile created from a glutarate bis-dodecyl ester, were also synthesized to provide controls for probing the orientation of the macrocyclic phospholipid in the bilayer membrane of vesicles. The amphiphile, linear bolaamphiphile, and macrocyclic bolaamphiphile were synthesized in 4, 5, and 6 steps with yields of 19, 4, and 5% respectively. The hydrophilic head group of the macrocyclic phospholipid was designed to release p-nitrophenolate in basic conditions from the p-nitrophenylphosphate head group to produce an absorbance at 400 nm. This assay was expected to elucidate the membrane-spanning orientation of the phospholipid in the bilayer membrane of vesicles. The final target compound failed to release p-nitrophenolate under basic conditions and underwent phosphate elimination to produce an α, β-unsaturated ester instead. Although the macrocyclic lipid produced associates with membranes and may be membrane-spanning, this lipid design was unable to reveal its membrane orientation. / Graduate

Magister Scientiae - MSc / Several abnormal conditions, including some cancers, have been associated with changes in the membrane lipid and FA composition. Dietary fat serves as a major source of lipids and FA, particularly the polyunsaturated fatty acids (PUFA), n-6 and n-3. High intakes of n-6 PUFA have been linked to the development of colon cancer in association with low n-3 PUFA intake. Therefore understanding the differences in the lipid and FA profiles between cancer and normal cells in the colon, and the role diet plays in these factors may be invaluable in understanding their role in carcinogenesis. This study compares the lipid profile of azoxymethane (AOM) induced colon polyps to that of the surrounding mucosa tissue in rats fed a diet high in n-6 PUFA. Male Fischer rats were fed the AIN-76A diet containing sunflower oil that has high n-6 PUFA content for a period of nine months. Results indicate that the lipid and FA content of the colon polyps differs significantly from the surrounding mucosa. Colon polyps had an increase in membrane phopholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Changes in membrane fluidity were indicated by the decrease (0.05) in the PC/PE and cholesterol/phospholipids (chol/PL) ratios, and increase (0.05) in the polyunsaturated FA/saturated FA (P/S) ratio. Metabolism of FA was significantly altered in the polyps favouring n-6 FA metabolism and the production of prostaglandin E2. No clear indication of impaired & Delta;6-desauturase enzyme activity was noticed. Increases in the n-6 PUFA content could be a reflection of the dietary FA intake that increases FA incorporation in the polyps. Changes in the FA parameters of the polyps, particularly an increase in C20:4n-6 and the n6/n3 ratio have been shown to contribute to the rapid growth of cancer tissue. These lipid changes associated with the development of colon polyps could provide unique targets for developing strategies in chemoprevention by dietary manipulation. / South Africa

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Conjugated linoleic acids (CLA) are geometrical and positional isomers of n-6 octadecadenoic acid (linoleic acid, LA, 18:2n-6), which form part of a family of essential polyunsaturated fatty acids (PUFA). There are 28 identified CLA isomers that mostly found in the meat and milk from ruminant animals. CLA has shown to possess a number of health benefits including; reduction in body fat and increased lean body mass, prevention of atherosclerosis, hypertension, increased immune function and in particular the prevention of cancer. The effects of CLA on cancer cell lines will be evaluated to discover the mechanisms that are employed to achieve this great phenomenon on cell growth. The aim of this study was to determine the effect of CLA on various parameters that are essential in the development of cancer cell phenotype. The objectives were to evaluate the effect of CLA on iron-induced lipid peroxidation of microsomes isolated from rat liver cells and in vitro cytotoxicity, cell proliferation and apoptosis in HepG2 hepatocarcinoma cells. The Fatty acid incorporation in HepG2 cells was also assessed.

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