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El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / Chylothorax is defined as the accumulation of chyle within the pleural space. Originally described in 1917 by Pisek, it is the most common cause of pleural effusion in the neonatal period. The leading cause of chylothorax is laceration of the thoracic duct during surgery, which occurs in 0.85% to 6.6% of children undergoing cardiothoracic surgery. Few authors of reports in the literature have looked at etilefrine, a relatively unknown sympathomimetic, as an option for the medical treatment of chylothorax. In this case report, we review the clinical course of 2 infants with type III esophageal atresia who developed chylothorax after thoracic surgery and were successfully treated with intravenous etilefrine after failing initial dietary and pharmacological management. / Revisión por pares

Plasma triacylglycerols (TG) are elevated in diabetes, metabolic syndrome, obesity, and dyslipidemia. Very-low density lipoprotein (VLDL) is the main plasma carrier of TG and the direct metabolic precursor of low-density lipoprotein (LDL), the main carrier of plasma cholesterol and the major causative risk factor for atherosclerosis. Binding of LDL to heparan sulfate on the arterial wall initiates retention and modifications of LDL in the arterial intima, triggering atherosclerosis. Studies presented in this dissertation show that variations in TG levels and lipoprotein binding to heparin, a model for heparan sulfate, alter the structural and biochemical stability of VLDL and LDL, and increase their atherogenic potential. The molecular consequences of variations in the lipoprotein TG content and LDL-heparin binding were determined by combining heparin affinity chromatography with biochemical, spectroscopic and electron microscopic techniques. Remodeling of human VLDL and LDL by thermal denaturation was used to mimic key aspects of lipoprotein remodeling in vivo. Our studies revealed that increasing the TG content in VLDL promotes changes in the lipoprotein size and release of the exchangeable apolipoproteins. Similarly, increased TG content in LDL promotes lipoprotein remodeling and fusion. Additionally, an increase in TG content increases lipoprotein susceptibility to oxidation and lipolysis, thereby promoting the generation of free fatty acids that augment fusion. Consequently, TG-induced destabilization may be a general property of plasma lipoproteins. Our studies showed that binding to heparin initiates irreversible pro-atherogenic remodeling of human LDL. As a result of heparin binding, LDL showed decreased structural stability and increased susceptibility to hydrolysis and fusion. Further, phospholipid hydrolysis and/or glycation of LDL (as occurs in diabetes) increased the proteolytic susceptibility of apolipoprotein (apo)B (the major apolipoprotein of VLDL and LDL) and its heparin binding affinity. LDL derived from hyperglycemic patients with type-2 diabetes, became particularly destabilized following heparin binding causing apoB fragmentation and LDL fusion. In summary, binding to heparin alters apoB conformation and triggers pro-atherogenic LDL modifications including proteolysis, lipolysis and structural destabilization. Furthermore, phospholipid lipolysis and glycation of LDL in vitro strengthen its binding to heparin. Together, these findings help establish a mechanistic link between diabetes and atherosclerosis.

This work identifies transcription factors (TF) controlling triacylglycerol (TAG) synthesis and accumulation in plant tissues. TAG plays vital role in plants and are used by humans. Most plants accumulate oil in the seed, but some species accumulate oil in other tissues. The Wrinkled1 (WRI1) TF has been shown to regulate oil accumulation in multiple species and tissues. Here, four WRI homologues in avocado were identified, their phylogeny was examined and three of them were cloned into expression vectors for further characterization. However, WRI1 likely does not act alone in regulation of TAG accumulation in plants. Additional candidate TFs were identified by using transcriptome data from a variety of species, and cloned into expression vectors. Future studies will be able to use this information to better understand regulation of TAG accumulation, which will allow increased oil accumulation in plants for various human uses.

Triacylglycerols are the predominant storage form of energy in eukaryotes. As obesity has become a worldwide problem and excessive accumulation of triacylglycerols in adipose tissue causes obesity, enzymes catalyzing the synthesis of triacylglycerols are of great interest. Acyl CoA:diacylglycerol acyltransferase (DGAT), including the isoforms DGAT1 and DGAT2, catalyze the final and committed step in triacylglycerol synthesis. Proteins that physically interact with DGAT1 may provide information regarding the metabolic role of DGAT1. We chose HEK-293T cell line to express DGAT1 and used mass spectrometry to identify proteins that co-immunoprecipitated with DGAT1. We confirmed that DGAT2 and ACAT1 did interact with DGAT1. The interaction of DGAT1 with DGAT2 appeared to interrupt the synthesis of triacylglycerol since the co-expression of DGAT1 and DGAT2 was expected to increase triacylglycerol synthesis. This implied that DGAT1 and DGAT2 might serve different functional roles. On the other hand, DGAT1 overexpression may increase the synthesis of cholesterol esters that was the product of ACAT1. Additionally, ACAT1 overexpression did increase triacylglycerol synthesis and ACAT1 disruption by siRNA did decrease triacylglycerol synthesis. Our findings indicated that DGAT1 and ACAT1 might be involved in the same lipid-synthesizing protein complex.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Timothy Durrett / In plants including the members of Brassicaceae family, such as Arabidopsis thaliana and Brassica juncea, seed storage reserves, which include lipids and proteins, accumulate in seeds during development. Triacylglycerols (TAG) are the major storage lipids found in the developing seeds, petals, pollen grains, and fruits of plants. In Arabidopsis seeds, acyl-CoA: diacylglycerol acyltransferase 1 (DGAT1) is the major enzyme contributing to TAG biosynthesis. In Arabidopsis, dgat1-1 mutants retain 60-80% seed TAG content due to the involvement of phospholipid: diacylglycerol acyltransferase (PDAT1) in acyl-CoA independent TAG biosynthesis. My study focuses on the elucidation and functional characterization of novel genes involved in the regulation of the TAG biosynthesis pathway. In developing seeds of the dgat1-1 mutant, altered fatty acid composition was observed with reduced TAG content and increased polar lipid content as compared to wild type. RNA-Seq of developing Arabidopsis seeds was employed to detect differentially expressed genes in dgat1-1. An empirical analysis for differential gene expression revealed a significant number of differentially expressed genes among all developmental stages in dgat1-1. Significant changes in gene expression profile were detected in lipid-related genes such as lipases and desaturases. RT-PCR was used to confirm the differential expression of major lipid-related genes including DGAT1, PDAT, and FAD2. Lipid profiling of T-DNA insertion mutants for differentially expressed genes revealed significant changes in lipid content and composition. Mutations in a member of the α, β-hydrolase family, encoded by gene named PLIP1, resulted in smaller seed and an altered seed oil phenotype. Also, combining the dgat1-1 and plip1-2 mutations resulted in a lethal phenotype, demonstrating the important role of this enzyme in embryo development and TAG biosynthesis. To identify key components in the regulation of storage lipid biosynthesis, correlation analysis using differential transcript abundance and lipid profile during different stages of seed development from dgat1-1 and wild type lines of Arabidopsis was performed. Using clustering analysis with Pearson correlation coefficient and single linkage identified one cluster of genes which included PLIP1, FAD2, FAD3, and PDCT . Similar analysis using combined data from the neutral and polar fractions resulted in clustering of lipids containing polyunsaturated fatty acids. To investigate the reduced seed germination phenotype for mature seeds of dgat1-1 and non-germinating green seed phenotype of dgat1-1 plip1-2 lines, differential expression (DE) analysis for genes involved in hormone metabolism was performed. Upregulation of expression was observed for genes involved in promoting abscisic acid (ABA) response, which led us to specuate the role of altered hormone metabolism in delayed germination of dgat1-1 seeds. Development of allopolyploid Brassica species from its diploid progenitors involves duplication, loss, and reshuffling of genes leading to massive genetic redundancy. It leads to selective expression or newly acquired role for duplicated homeologs. Differential expression (DE) analysis for homoeologous genes from A and B subgenomes of allopolyploid B. juncea implicated in FA synthesis, acyl editing, and TAG biosynthesis and metabolism was performed. Differential expression (DE) analysis identified the transcriptional dominance of A subgenome homoeologs. Identification of these homoeologs will enable their use in breeding programs directed towards improvement of lipid content and composition in seeds.

Triacylglycerols (TAG) play vital roles in plant development and are also utilized by humans for consumption, chemical and industrial feed stocks, and production of biofuels. Their primary function in plants is as an energy reserve stored in seeds to allow for the growth of the developing plant after germination. While many plants accumulate significant quantities of TAGs in the developing embryo or other parts of the seed, significant quantities of TAG accumulation also occurs in nonseed tissues such as the mesocarp of avocado and oil palm, the roots of nutsedge, and the fruits of bay berry. In Arabidopsis the transcription factor Wrinkled 1 (WRI1) was shown to be responsible for regulating the accumulation of TAG in seeds, and homologues of WRI1 have been shown to have similar functions in other plants and plant tissues. However, WRI1 likely does not act alone to control the synthesis and accumulation of TAG in plants. This project aims to identify additional transcription factors that are responsible for controlling the synthesis and accumulation of TAG in nonseed tissues by utilizing transcriptome data from a variety of plant species. The transcriptome data also revealed three homologues of WRI1 that are highly expressed in Avocado mesocarp. Their function in Avocado will be determined by complementing Arabidopsis wri1 mutants and examining TAG accumulation in the resulting transgenic plants. This study is aimed at identifying genes that play a role in the accumulation of high levels of TAG in various plant tissues and confirming the function of the WRI homologues in Avocado. Future studies will be able to use this information to better understand regulation of TAG accumulation in plants which will allow increased oil accumulation and yield in plants for various commercial and industrial applications.

Modern society’s demand for oil has resulted in depletion of resources and caused higher oil prices. Therefore, natural oil resources of plants are gaining the spotlight and are expected to increase twice that of current use by 2030. Plants are able to accumulate up to 90% oil by dry weight in the form of triacylglycerol (TAG) and it is derived from fleshy part of the fruits, such as mesocarp of oil palm, avocado, and olive. In seed tissues, an acyl CoA-dependent enzyme, diacylglycerol acyltransferase (DGAT) participates in conversion of diacylglycerol (DAG) to TAG. However, TAG can also be formed by an acyl CoA- independent enzyme, phospholipid:diacylglycerol acyltransferase (PDAT). Avocados (Persea americana) store up to ~70% oil in mesocarp and I am interested in identifying the acyltransferase involved in oil biosynthesis in mesocarp tissues. Based on the transcriptome data available, I hypothesize that unlike in seeds, both DGAT and PDAT are associated with TAG biosynthesis in developing mesocarp of avocado. To test this hypothesis, I will determine 1) TAG content and composition and 2) expression levels for DGAT and PDAT genes in mesocarp and seed tissues of five stages of developing fruits of avocado (n=5). Total lipids will be extracted by isopropanol-chloroform method and analyzed for composition as methyl esters on GC-FID. Total RNA, for expression analysis, will be extracted by Trizol method and analyzed with gene-specific primers by real-time PCR. Statistical significance in change in oil content in association with gene expression during fruit development between mesocarp and seed tissues will be analyzed by ANOVA repeated measures. Comparison of temporal gene expression pattern of oil accumulation mesocarp, to that of seed, will allow us to differentiate the acyltransferase(s) specifically associated with TAG biosynthesis. The proposed research work will take the field of plant lipid biochemistry a step forward in understanding TAG synthesis in fruit tissue. Specifically, I will be able to clearly demonstrate the association of a particular acyltransferase to increasing lipid content in a non-seed (mesocarp) tissue. Understanding differences in oil regulation of a basal angiosperm (avocado) in relation to a monocot (oil palm) and a dicot (olive) also will provide additional insights into fundamental changes in TAG biosynthesis during the evolution of flowering plants. My research is part of a global project that includes a group in California, Florida and Israel. Data generated from my proposed work will be used to develop a joint Binational Agricultural and Research proposal. Therefore, my research work will subsequently contribute to development of ways to manipulation of extent and timing of oil accumulationa direct benefit to avocado growers. Furthermore, this study will be a pivotal step to understanding TAG synthesis that will lead to bioenergy crop.