Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 154 pages. Includes vita. Includes bibliographical references.

Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references (leaves 123-154). Also available online.

Les maladies rénales chroniques (MRC) et l'insuffisance rénale aiguë (IRA) sont des problèmes essentiels de santé publique en raison de l'augmentation continue de leur fréquence et du manque de solutions thérapeutiques contre ces maladies. L'acide lysophosphatidique (LPA) est un lysophospholipide bioactif qui induit un large éventail de réponses cellulaires par le biais de récepteurs membranaires spécifiques (LPA1 à LPA6) couplés aux protéines G. Dans ce travail, nous nous sommes intéressés aux effets biologiques et au métabolisme du LPA dans les MRC et l'IRA. Des travaux antérieurs de l'équipe avaient montré que le LPA contribuait, via le récepteur LPA1, au développement de la fibrose tubulointerstitio (TIF) dans un modèle de MRC chez la souris : l'obstruction urétérale. Dans la première partie de la thèse nous avons étudié l'implication du LPA dans un modèle plus avancé de MRC: la néphrectomie subtotale (SNX) chez la souris. Nos travaux ont montré que 5 mois après chirurgie les souris (SNX) développaient une albuminurie massive associée à une TIF sévère et à une hypertrophie glomérulaire. Chez ces souris la concentration en LPA mesurée par chromatographie liquide en spectrométrie de masse en tandem était augmentée dans l'urine et étroitement corrélée à l'albuminurie et à la TIF. En parallèle, nous avons observé une diminution de l'expression rénale des Lipid-Phosphate Phosphatases (LPP 1, 2 et 3) responsables de l'inactivation du LPA. Nous avons également observé que l'expression rénale des récepteurs LPA1, 2, 3 et 4 était diminuée chez les souris Snx. Nous avons conclu que les effets délétères éventuels du LPA dans le développement de la MRC chez les souris SNX était vraisemblablement lié à une augmentation de sa production rénale plutôt qu'à une sensibilité accrue du rein au LPA. Des travaux antérieurs avaient montré que l'injection de LPA protégeait contre l'apparition des lésions rénales induites par ischémie/reperfusion chez la souris. Une autre étude avait montré que le LPA permettait d'atténuer l'inflammation systémique et les dommages aux organes induits par un choc septique. Dans la deuxième partie de la thèse, nous avons étudié l'influence du LPA sur l'IRA induite par une endotoxémie au LPS (lipopolysaccharide) chez la souris. Nous avons observé que l'injection de LPA permettait d'atténuer l'élévation d'urée et de créatinine plasmatiques, ainsi que l'augmentation d'expression rénale de cytokines inflammatoires (IL-6, TNFa, MCP-1) induites par le LPS. Le LPA a également empêché la baisse d'expression rénale du facteur PGC1a ainsi que les altérations ultra-structurales des mitochondries rénales induites par le LPS. In vitro, le LPA atténue l'augmentation d'expression des cytokines pro-inflammatoires (TNFa et MCP-1) induite par le LPS dans les macrophages RAW264. Enfin, nos travaux ont montré que l'endotoxémie au LPS chez la souris entrainait une réduction de la concentration urinaire de LPA associée à une réduction des enzymes anaboliques LPA (autotaxine et acylglycérol kinase) et une élévation de l'expression de de LPP2, dans le cortex rénal. Nous en avons conclu que l'IRA associée à l'endotoxémie pourrait être liée, au moins en partie, à une réduction de la production rénale de LPA et, par voix de conséquence, de ses effets anti-inflammatoires protecteurs de la fonction rénale. En conclusion, notre travail montre que les variations de production rénale de LPA pourraient jouer un rôle important dans le développement des maladies rénales. L'augmentation du LPA dans les MRC favoriserait ses effets délétères (fibrose, inflammation). Sa réduction dans l'IRA réduirait ses effets anti-inflammatoires. Cibler le catabolisme LPA pourrait donc être une approche intéressante dans le traitement des maladies rénales. / Both chronic kidney diseases (CKD) with consecutive development of end stage renal disease (ESRD) and acute kidney injury (AKI) represent worrying problems for healthcare system due to its increased frequency and the lack of efficient treatments. Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that elicits a wide range of cell responses (proliferation, migration, transformation, contraction etc.) through the activation of specific G protein-coupled receptors (LPA1 to LPA6). In this work we were interested in involvement of the LPA and changes in its metabolism in CKD and AKI. Previous works showed that LPA exerts pro-fibrotic activity and contributes to development of tubulointerstitioal fibrosis (TIF) after ureteral obstruction through activation of LPA1 receptors. In the first part of the thesis we were interested whether LPA signalization is involved in more advanced model of the disease. We found that 5 months after subtotal nephrectomy (SNX) mice develop massive albuminuria, TIF and glomerular hypertrophy compared to control animals. LPA concentration measured by liquid chromatography tandem mass spectrometry was increased in urine but not in plasma of animals. That increase in LPA significantly correlated with albuminuria and TIF. In addition we found a decreased renal expression of lipid phosphate phosphatases (LPP1, 2 and 3) that are responsible for the degradation of LPA by dephosphorylation. Moreover, the expression of LPA1-LPA4 receptors is down-regulated, whereas LPA5 and LPA6 are unchanged. We concluded here that the possible deleterious effect of LPA in the development of CKD in SNX mice was likely related to its increased production rather than an increased sensitivity of the kidney to LPA. Since LPA was reported previously to protect kidney damage in the course of ischemia/reperfusion injury, and that it was able to mitigate the systemic inflammation and organ damage in sepsis, we were interested in second part of the thesis to determine whether exogenous and/or endogenous LPA might protect against sepsis-associated AKI. C57BL/6 mice were treated with exogenous LPA 18:1 1 hour before being injected with the lipopolysaccharide (LPS) and AKI was analyzed after 24h. LPA pre-treatment significantly mitigated the LPS-induced elevation of plasma urea and creatinine, lessened the up-regulation of inflammatory cytokines (IL-6, TNFa, MCP-1) and completely prevented the down-regulation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1a) in kidney. LPA also prevented LPS-mediated alterations of renal mitochondria ultrastructure. In vitro, pre-treatment with LPA 18:1 (10 µM) significantly attenuated LPS-induced up-regulation of the pro-inflammatory cytokines (TNFa and MCP-1) in RAW264 macrophages. In addition we found that LPS led to the reduction of urinary LPA concentration that was associated with a reduction in LPA anabolic enzymes (autotaxin and acylglycerol kinase), and an elevation in LPA catabolic enzyme (lipid phosphate phosphatase 2) expression in kidney cortex. We concluded hereby that exogenous LPA exerts protection against endotoxemia-induced kidney injury. Moreover, the observation that LPS reduces the renal production of LPA suggests that sepsis-associated AKI could be mediated, at least in part, by alleviation of the protective action of endogenous LPA. In general our work shows that LPA local metabolism is altered in both forms of kidney diseases. In course of sepsis-induced AKI LPS leads to increased local catabolism of LPA leading to low availability of the phospholipid and alleviating its protective effect whereas in advanced CKD the local catabolism of the phospholipid is decreased with subsequent increase of urine LPA that favors development of the disease. Targeting LPA catabolism can be an interesting approach in treatment of kidney diseases.

Soybean (Glycine max (L.) Merr) is the largest oil and protein crop in the world and it is grown for both oil and protein. To address the needs of both the edible oil market and industrial applications of soybean oil, fatty acid modification has been a focus of soybean breeding programs. Natural variation, mutagenesis and genetic engineering have been used to alter the fatty acid profile. Several genes, mostly desaturases, have been associated with altered fatty acid profiles but enzymes in the Kennedy Pathway have yet to be studied as another source of genetic variation for altering the fatty acid profiles. The Kennedy Pathway is also known as the oil producing pathway and consists of four enzymes: glycerol-3-phosphate acyltransferase (G3PAT); lysophosphatidic acid acyltransferase (LPAAT); phosphatidic acid phosphatase (PAP); and diacylglycerol acyltransferase 1 (DGAT1). The starting material for this pathway is glycerol-3-phosphate, which is produced from glycerol by glycerol kinase (GK), and the product of this pathway is triacylglycerol (TAG). The overall objective of this study was to elucidate the role that the Kennedy Pathway plays in determining the fatty acid profile in two ways: (1) sequencing the transcribed region of the genomic genes encoding the enzymes of GK, G3PAT, LPAAT, and DGAT1 in soybean genotypes with altered fatty acid profiles; and (2) studying their expression over seed development, across three growing temperatures. The genetic material for the study consisted of four soybean genotypes with altered fatty acid profile: RG2, RG7, RG10, and SV64-53. Results from sequencing showed that the mutations identified in G3PAT, LPAAT, and DGAT1 in the four soybean genotypes did not explain the differences in the fatty acid profiles. The expression of G3PAT, LPAAT, and DGAT1 over seed development showed that G3PAT had the lowest levels, followed by LPAAT, then DGAT1, across the growing temperatures. The differences in expression among genotypes corresponded to differences in fatty acid accumulation, suggesting that expression rather than genetic mutations in the transcribed region of the genes influenced the fatty acid profile of the genotypes in this study. In conclusion, the enzymes of the Kennedy Pathway appear to contribute to the altered fatty acid profiles observed in the soybean mutant genotypes. / Ontario Ministry of Economic Development and Innovation (formerly Ontario Ministry of Research and Innovation), BioCar Initiative, Grain Farmers of Ontario, SeCan

京都大学 / 新制・課程博士 / 博士(薬学) / 甲第23847号 / 薬博第854号 / 新制||薬||242(附属図書館) / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 金子 周司, 教授 土居 雅夫, 教授 竹島 浩 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

The imidazoline I1 receptor (I1-R) is a novel receptor found primarily in the brain and nervous tissue where it modulates neurotransmission. It is named for its high affinity for compounds with an imidazoline structure such as the anti-hypertensive drugs, clonidine and moxonidine. The imidazoline receptor antisera-selected protein (IRAS) is the putative clone of the I1-R. IRAS has a unique structure, which does not resemble any other receptor protein. IRAS is present throughout the body with highest levels in the brain. There is a growing body of research examining the physiological roles of IRAS as an I1-R, in cell survival, migration and protein trafficking. However, there is little research into its neuronal functions. IRAS interacts with other membrane receptors: the mouse homologue of IRAS reorganises the actin cytoskeleton through interaction with the α5β1 fibronectin receptor. IRAS also binds insulin receptor substrate 4 and enhances insulin-induced extracellular signal-regulated kinase1/2 (ERK1/2) activation. Actin reorganisation and ERK1/2 activation are important for the development of neurites during neuronal differentiation. Therefore, the work described in this thesis aimed to investigate the effects of IRAS on neuronal differentiation. Studies reported in this thesis also aimed to investigate whether IRAS affected ERK1/2 signalling of other receptors involved in neuronal differentiation such as the NGF receptor, TrkA, and lysophospholipid receptors. The above aims were carried out in neuronal model PC12 cells transfected with either IRAS or a vector plasmid. Fluorescence microscopy and Western blotting techniques were used to examine the effect of IRAS on cell morphology and ERK1/2 signalling. The work described in this thesis found that IRAS reorganises the actin cytoskeleton and enhances growth cone development in PC12 cells. This study also shows that IRAS differentially enhances or inhibits NGF-induced PC12 cell differentiation depending on the presence or absence of serum in the media. In full-serum conditions, IRAS enhanced neurite outgrowth and this was accompanied by an increase in ERK1/2 activation. In serum-starved cells, IRAS inhibited neurite outgrowth with similar levels of ERK1/2 activation observed in vector- and IRAS-transfected cells. Finally, studies presented in this thesis found that IRAS enhances lysophosphatidic acid-induced ERK1/2 activation and that IRAS interacting with lysophospholipid receptor agonists present in serum is a potential mechanism by which it enhances NGF-induced ERK1/2 activation in full-serum conditions. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1345359 / Thesis (Ph.D.) - University of Adelaide, School of Medical Sciences, 2008

L'acide lysophosphatique est un biolipide naturel actif capable de réguler diverses fonctions biologiques et d'agir en tant que facteur de croissance, via l'activation de six différents récepteurs de surfaces couplées aux protéines G (LPA1-6). Notre laboratoire a montré que le ciblage thérapeutique du récepteur LPA1 bloque de façon remarquable la dissémination métastatique des cellules de cancer du sein. Les mécanismes moléculaires et génétiques impliqués dans ce processus sont cependant encore inconnus. De plus, la plupart des cellules de mammifères co-expriment plusieurs formes de récepteurs du LPA. La réponse cellulaire est la résultante de l'activation de multiples voies de signalisation, parfois synergiques ou opposées, compromettant la validation chez le patient de l'efficacité des thérapies ciblant ces récepteurs. Au cours de cette thèse, nous avons dans un premier temps montré que HB-EGF est un marqueur spécifique de l'activité de LPA1. Le blocage pharmacologique de ce récepteur via des antagonistes des récepteurs LPA1-3 (Ki16425/Debio0719) ou l'invalidation de son expression par une technique d'ARN interférence entraine une inhibition de la surexpression en HB-EGF. Le ciblage thérapeutique de LPA1 via l'antagoniste Ki16425, dans notre modèle animal préclinique de xénogreffe de cancer de la prostate PC3, conduit également à une diminution de l'expression en ARNm de HB-EGF au niveau de la tumeur primaire et à une diminution des concentrations en HB-EGF humain circulants dans le sérum. Dans un deuxième temps, nous nous sommes intéressé au rôle des miRNAs, qui sont impliqués dans la régulation de l'expression de gènes. Grâce à l'analyse de 1488 patients atteins de cancers du sein référencés sur des bases de données publiques, nous avons pu établir une corrélation entre le gène LPA1 et le gène ZEB1. Nous avons également trouvé que le coefficient de corrélation entre ZEB1 et LPA1 était supérieur au niveau des tumeurs mammaires basales / Lysophosphatidic acid (LPA) is a natural bioactive lipid with growth factor-like functions due to activation of a series of six G protein-coupled receptors (LPA1-6). It has been demonstrated that blocking LPA1 activity in vivo inhibits breast cancer cell metastasis, however, activated genes involved in LPA-induced metastasis have not been defined yet. In addition most mammalian cells co-express multiple LPA receptors, resulting in the co-activation of multiple intracellular signaling pathways with potential redundant or opposite effects impairing the validation of target inhibition in patients because of missing LPA receptor-specific biomarkers. In the first part of this thesis I found that HB-EGF is a specific biomarker of LPA1 activity. HB-EGF upregulation was inhibited by LPA1-3 antagonists (Ki16425, Debio0719) and by stably silencing LPA1. Using a human xenograft prostate tumors mouse model with PC3 cells, we found that a five-day treatment with Ki16425 significantly decreased both HB-EGF mRNA expression at the primary tumor site and circulating human HB-EGF concentrations in serum. In the second part of experimental work, we focused our attention on miRNAs that are master gene regulators. We carried out correlation studies in 1488 human primary breast tumors from publically available databases and found ZEB1 as the most correlated gene with LPAR1. The coefficient of correlation between ZEB1 and LPAR1 was higher in human basal tumors than in non basal tumors. In three different basal cell lines LPA up-regulated ZEB1 through an LPA1/Phosphatidylinositol-3-Kinase (Pi3K)/AKT-dependent pathway. Based on microarray and real-time PCR analyses we found that LPA up-regulated the oncomiR miR-21 through an LPA1/Pi3K/AKT/ZEB1-dependent mechanism. MirVana miR-21 inhibitor, silencing LPA1 or silencing ZEB1 totally blocked in vitro LPA-induced cell migration and invasion, and in vivo tumor cell bone colonization. In all cases, basal breast cancer cell functions were rescued with mirVana miR-21 mimic. All together our results identify HB-EGF as a new and relevant biomarker with potentially high value in quantifying LPA1 activation state in patients receiving anti-LPA1 therapies

Indiana University-Purdue University Indianapolis (IUPUI) / Polycystic kidney diseases (PKD) are genetic disorders characterized by fluid filled cysts in the kidney tubules and liver bile ducts. There are two forms of PKD, autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD). The focus of the studies in this thesis has been on ADPKD. The disease progresses slowly and the fluid-filled cysts grow in size due to increased rates of cell proliferation and fluid secretion into the cyst lumen. The expanding cysts compromise the normal kidney function and result in a decrease of renal function to the point of end-stage renal failure in midlife. Cyst enlargement is due, at least in part, to chloride secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Currently therapy is limited to renal cyst aspiration, dialysis, and eventually renal transplantation after organ failure, thus it has critical to determine possible drug therapies for the treatment of PKD. Previous studies showed that cyst fluid caused a secretory response in cells lining the cysts. We hypothesized that once the cyst have expanded and become so large that they burst or leak, which could also occur due to renal injury or aging, the cyst fluid may stimulate additional cyst growth. Lysophosphatidic Acid (LPA) was determined to be the active component of human cyst fluid, and we investigated the LPA stimulated signaling pathway. Our data suggest that the LPA stimulates chloride and fluid secretion by a combination of CFTR and Calcium-Activated chloride channels (CaCC) and that the two channels may functionally be linked to each other. The secretion is not occurring through a cAMP stimulated pathway, and it is possible that TMEM16A, a CaCC, plays a larger role than previously expected. Previous studies demonstrated that PPARγ agonists, insulin sensitizing drugs used to treat diabetes, inhibit chloride secretion by the collecting duct principal cells by decreasing CFTR synthesis. It was logical therefore to considered PPARγ agonists as long-term treatment for PKD. The first preclinical studied showed that high (20 mg/kg BW) dose pioglitazone, a PPARγ agonist, inhibited cyst growth in the PCK rat model, a slow progressing model, of PKD. To continue to look at the effects of the PPARγ agonists another preclinical study was completed, which tested if there was a class action of PPARγ agonists and if a lower dose was effective in treating the cystic burden. Using the PCK rat model, and another PPARγ agonist, rosiglitazone, a 24 week study was completed using 3 doses (4, 0.4, and 0.04 mg/kg BW). 4 mg/kg BW rosiglitazone is analogous to 20 mg/kg BW pioglitazone. The data indicated that the rosiglitazone is effective in lowering the cystic burden, and importantly the low dose proved to be effective. An additional rat model, the W-WPK rapidly progressing model was used to determine efficacy across multiple models, and to determine if there was a way to track the progress of the disease in a manner analogous to that used in human patients. The animals were treated with pioglitazone using 2 doses (2 and 20 mg/kg BW), and were imaged using CT scans to track the progress of the disease. The data suggest that pioglitazone was not as effective in the W-WPK rat model as it was the PCK rat model. There was a trend however, that low dose PPARγ agonist was as effective ad high dose. Even more important, the CT scans proved to be an effective way to track the progress of the disease in animal models.

Le léiomyome utérin est la pathologie utérine la plus fréquente chez les femmes en âges de procréer. Des résultats précédent obtenus avec les cellules ELT3, une lignée de cellules de léiomyomes de rat, ont montré que l’acide lysophosphatidique (LPA) activait les MAP kinases ERK1/2 via le récepteur LPA1 couplé à la protéine Gi et l’activation de Raf, Ras et de MEK. Durant ce travail, nous avons caractérisé l’activité phosphatase responsable de la dégradation du LPA dans cette lignée de cellules ELT3. Nous avons montré que le LPA était dégradé exclusivement par la lipide-phosphate phosphatase 1 (LPP1), seule isoforme exprimé dans les cellules ELT3. Dans un deuxième temps nous nous somme intéressés aux effets du diacylglycerol pyrophosphate (DGPP). Le DGPP est un médiateur lipidique qui, sous sa forme dioctanoyl (DGPP8:0), est décrit comme un antagoniste des récepteurs LPA1 et LPA3 chez les mammifères. Dans cette étude, nous montrons que le DGPP8:0 n’a pas d’effet antagoniste sur l’activation du module MAP kinase ERK1/2 par le LPA mais qu’il induit une activation de ERK1/2 dans plusieurs lignées de cellules de mammifères. En effet le DGPP active ERK1/2 à travers l’activation des PKC, Raf et MEK. De plus, nous montrons que l’activation induite par le DGPP repose sur sa déphosphorylation catalysée par une LPP. Nous montrons également que l’inhibition de LPP1 par le VPC32183 ou l’utilisation de siRNA dirigé contre la lipide phosphate-phosphatase 1 (LPP1) réduit l’activation ERK1/2 induite par le DGPP. Ceci montre que le DGPP active ERK1/2 via sa déphosphorylation en acide phosphatidique (PA8:0), catalysée par la LPP1. Enfin dans une dernière partie nous montrons que le myomètre sain, contrairement aux cellules ELT3, exprime à la fois la LPP1 et la LPP3. En étudiant l’effet de la surexpression de la LPP3 dans les cellules ELT3, nous avons observé que la LPP3 interagissait avec la LPP1 et qu’elle pourrait la séquestrer dans des compartiments membranaires internes. Cette séquestration entraine une diminution de l’actvité ecto-LPP au profit de l’activité intracellulaire qui pourrait réguler négativement la production de seconds messagers phospholipidiques. Ces résultats montrent l’importance des LPP dans la régulation des effets des phospholipides bioactifs et suggère un lien entre le caractère tumorale des cellules de léiomyomes et l’absence de la LPP3. / Leiomyoma is the most common uterine disease in women in age of procreation. Previous result have shown that in ELT3 cells, which is a rat leiomyoma-derived cell line, lysophosphatidic acid (LPA) was able to activate ERK1/2 MAP kinases through the activation of the LPA1 receptor via the classical MAP kinase pathway involving Raf, Ras and MEK. We have observed that LPA was dephosphorylated in ELT3 cells by Lipid-phosphate phosphatase 1 (LPP1) which is the only LPP isoform expressed in these cells. In a second part, we investigated the effect of diacylglycerol pyrophosphate (DGPP). DGPP, in its octanoyl form (DGPP8:0), is described as an LPA1, 3-antognist. Here, we show that DGPP had no antagonistic effect on LPA-ERK activation, but was able to induce ERK activation by itself. This activation occurred through the activation of PKC, Raf and MEK. On the other hand, we show that DGPP-ERK activation required its dephosphorylation by LPP. Next we observed that the DGPP-ERK activation was inhibited by VPC32183, which we showed to inhibit LPP activity, and by siRNA treatment targeting LPP1. This results show that DGPP needs to be dephosphorylated into PA to induce ERK phosphorylation, and this dephosphorylation is catalyzed by LPP1. Finally, in the third part of this study, we were interested in the differential LPP expression between ELT3 cells and myometrium cells. Indeed, we have previously observed that ELT3 cell express only LPP1 while myometrial cells express LPP1 and LPP3. We observed that when LPP3 is expressed in ELT3 cells, it can interact with LPP1 and may restrain its plasma membrane localization reducing ectoLPP activity in favor of intracellular LPP activity. This may result in a negative regulation of intracellular phospholipidic second messengers. All these results show the significance of LPP in the regulation of bioactive phospholipid effects and suggest a relationship between the tumoral phenotype of leiomyoma cells and the absence of LPP3.

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.