Plant development is complex and highly regulated. Tens of thousands of genes have been sequenced for the model plant <em>Arabidopsis thaliana</em>, yet few have been functionally annotated and characterized. This thesis describes the expression analysis and characterization of four genes in <em>Arabidopsis</em>. Three of these belong to the eukaryotic translation initiation factor 5A (eIF5A) gene family, and the fourth encodes diacylglycerol acyltransferase 1 (DGAT1). Putative roles for these genes in the development of <em>Arabidopsis thaliana</em> are described. <br /><br /> eIF5A is the only known protein to contain the amino acid hypusine. It has been demonstrated previously that eIF5A acts as a shuttle protein, moving specific mRNAs from the nucleus to the cytoplasm for translation. In <em>Arabidopsis thaliana</em> (At), there are three isoforms of eIF5A, and it is clear from the present study that they each have a unique temporal and spatial expression pattern. AteIF5A-1 and -2 are up-regulated during natural senescence and wounding/pathogenesis, respectively, and it is proposed that they regulate the onset of programmed cell death during these events. AteIF5A-3 is up-regulated in elongating meristem of the root, and it is proposed that this isoform is involved in cell growth. <br /><br /> Over-expression of the individual <em>AteIF5A</em> isoforms <em>in planta</em> resulted in pleiotropic phenotypes. When <em>AteIF5A-1</em> or <em>AteIF5A-2</em> was over-expressed, the phenotypes observed were indicative of their putative roles in the translation of proteins required for programmed cell death. When <em>AteIF5A-3</em> was over-expressed, the phenotypes were indicative of a role for this protein in the regulation of cell and tissue elongation. <br /><br /> Lipid analysis of rosette leaves from <em>Arabidopsis thaliana</em> revealed an accumulation of triacylglycerol with advancing leaf senescence coincident with an increase in the abundance and size of plastoglobuli. The terminal step in the biosynthesis of triacylglycerol in <em>Arabidopsis</em> is catalyzed by DGAT1. When gel blots of RNA isolated from rosette leaves at various stages of development were probed with the <em>Arabidopsis</em> EST clone, E6B2T7, which has been annotated as DGAT1, a steep increase in DGAT1 transcript levels was evident in the senescing leaves coincident with the accumulation of triacylglycerol. The increase in DGAT1 transcript correlated temporally with enhanced levels of DGAT1 protein detected immunologically. Two lines of evidence indicated that the triacylglycerol of senescing leaves is synthesized in chloroplasts and sequesters fatty acids released from the catabolism of thylakoid galactolipids. First, triacylglycerol isolated from senescing leaves proved to be enriched in hexadecatrienoic acid (16:3) and linolenic acid (18:3), which are normally present in thylakoid galactolipids. Second, DGAT1 protein in senescing leaves was found to be associated with chloroplast membranes. These findings collectively indicate that DGAT1 plays a role in senescence by sequestering fatty acids de-esterified from galactolipids into triacylglycerol.

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Made available in DSpace on 2014-06-11T19:22:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-06-13Bitstream added on 2014-06-13T20:49:39Z : No. of bitstreams: 1 galvao_fc_me_arafcf.pdf: 2113855 bytes, checksum: e2ee31898ce2604cafc3f1e7c7c0c0bd (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / PROPG / O fator de início de tradução 5A (eIF5A) é altamente conservado de arqueas a mamíferos e é essencial para a viabilidade celular. Este fator é a única proteína conhecida que sofre uma modificação pós-traducional única e necessária para a função de eIF5A, em que uma lisina específica é convertida em um resíduo de hipusina pela ação das enzimas desoxi-hipusina sintase (Dys1) e desoxi-hipusina hidroxilase (Lia1). Inicialmente, eIF5A foi relacionada à etapa do início da tradução, porém, dados recentes sugerem a sua atuação na etapa de elongação ao invés de início. No entanto, além do fato de a função específica de eIF5A na célula não ser conhecida, o papel da hipusinação para o funcionamento de eIF5A também não é conhecido. Diante disso, o objetivo deste trabalho é caracterizar mutantes condicionais para o gene da desoxi- hipusina sintase e, dessa forma, contribuir para o entendimento não só da função da hipusinação sobre eIF5A, mas também para o entendimento da função específica de eIF5A na célula. Para isso, foram iniciadas análises de caracterização fenotípica com os alelos dys1Δ1-28 e dys1W75R/T118A/A147T (dys1-1). Inicialmente, foi realizada a subclonagem do alelo dys1Δ1-28 , uma vez que, por ter sido identificado em um rastreamento de duplo-híbrido, este alelo estava em fusão com a região codificadora do domínio de ativação de Gal4. Porém, após realização da subclonagem, ou seja, quando na ausência do domínio de ativação, este alelo não apresentou o fenótipo condicional de crescimento inicialmente observado. Portanto, o mutante se tornou impróprio para a realização dos ensaios subsequentes e foi descartado. Em seguida, foram iniciadas as análises com o alelo dys1-1, nas quais foi observada diminuição nos níveis totais de Dys1 mutada, e consequentemente, diminuição nos níveis de hipusinação. Devido a isso... / The translation initiation factor 5A (eIF5A) is highly conserved from archaea to mammals and is essential for cell viability. This factor is the only known protein that undergoes an unique and essential post-translational modification, in which a specific lysine residue is converted into hypusine by the action of the enzymes deoxyhypusine synthase (Dys1) and deoxyhypusine hydroxylase (Lia1). Initially, eIF5A was related to the initiation step of translation, however, recent data suggest a role in the elongation step of translation. However, besides the fact that the specific function of eIF5A in the cell is still obscure, the role of hypusination in eIF5A function is unknown. Thus, the goal of this project is to characterize conditional mutants of the deoxyhypusine synthase gene and thereby contribute to the understanding not only the function of hypusination in eIF5A, but also of the specific role of eIF5A in the cell. We started a phenotypic characterization of two different alleles: dys1Δ1-28 and dys1W75R/T118A/A147T (dys1-1). Initially, we performed a subcloning of the allele dys1Δ1-28 , once the allele was fused with the coding region of GAL4 activation domain, due to the fact that this allele is devived of a two-hybrid screening. However, after performing the subcloning, that is, in the absence of the activation domain, this allele showed no conditional growth phenotype as originally observed. Therefore, this mutant has become improper to carry out the subsequent analysis and was discarted. Then, the analyses with dys1-1 allele were initiated, in which it was observed a decrease in total levels of Dys1 and, consequently, a decrease in the hypusination levels. Because of that, this allele shows a decrease in cell growth rate and growth arrests after 24 hours in medium lacking the osmotic regulator. However, this growth arrest is not followed by cell lysis. Furthermore, the mutant ... (Complete abstract click electronic access below)

Orientador: Cleslei Fernando Zanelli / Resumo: eIF5A é um fator de tradução envolvido com vários processos celulares, como o controle da proliferação celular e inflamação. Esse fator sofre uma modificação pós- traducional totalmente conservada e específica para sua ativação. Essa modificação é chamada de hipusinação e consiste na modificação de uma lisina específica em duas etapas: primeiro, a porção aminobutil da poliamina espermidina é transferida para o grupamento amino da cadeia lateral da lisina, pela ação da enzima desoxi-hipusina sintase; e, em seguida, o radical transferido sofre uma hidroxilação na posição beta, pela ação da desoxi-hipusina hidroxilase. Assim como eIF5A, desoxi-hipusina sintase é altamente conservada em arquea e eucariotos e é essencial em Saccharomyces cerevisiae. A desoxi-hipusina sintase é inibida por N1-Guanyl-1,7-diaminoheptane (GC7), um análogo estrutural da espermidina. No entanto, estudos mais recentes demonstraram que o composto GC7 apresenta efeitos celulares independentes de eIF5A, o que demonstra que este possui outros alvos. Além disso, não é conhecido como se dá o processo de transporte do GC7 para o meio intracelular ou se existem mecanismos de compartimentalização celular ou externalização do GC7. Por fim, também não são conhecidos mecanismos de metabolização celular do GC7. Desta forma, o presente projeto teve como objetivo a busca de novos alvos celulares para o composto GC7 utilizando rastreamento quimio-genético utilizando a c... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

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Indiana University-Purdue University Indianapolis (IUPUI) / Traditionally, the exocrine and endocrine cellular compartments of the pancreas have been considered distinct functional systems. However, recent studies suggest a more intricate relationship between the exocrine and endocrine, which may impact pancreatic growth and health. Additionally, translational control mechanisms have been linked to organ development. Our lab has shown that the mRNA translation factor eukaryotic initiation factor 5A (eIF5A), when in its post-translationally modified “hypusinated” form, plays a role in pancreas development. The hypusination of eIF5A requires the rate-limiting enzyme deoxyhypusine synthase (Dhps) to post- translationally modify a critical lysine residue which in turn produces the active form of eIF5A that functions in mRNA translation. When we generated animals with a deletion of Dhps in the pancreatic progenitor cells, there was no alteration in islet mass but significant exocrine insufficiency at embryonic (E) day 18.5 concomitant with downregulation of proteins required for exocrine pancreas development and function. Resultantly these animals died by 6 weeks-of-age. These observations prompted the question, is the phenotype caused by the absence of hypusinated eIF5A or the increase of unhypusinated eIF5A? To address this, we generated a mouse model wherein Eif5a is deleted in the pancreas (eIF5A∆PANC) and these mutant animals also display exocrine insufficiency. Interestingly, beta cell mass is increased at E18.5, and the mutant animals maintain euglycemia and survive up to 2 years. Ongoing analyses are interrogating the differences between these animal models with the goal to determine if mRNA translation facilitates cellular communication between the exocrine and endocrine pancreas.

<p>  </p> <p>Traditionally, the exocrine and endocrine cellular compartments of the pancreas have been considered distinct functional systems. However, recent studies suggest a more intricate relationship between the exocrine and endocrine, which may impact pancreatic growth and health. Additionally, translational control mechanisms have been linked to organ development. Our lab has shown that the mRNA translation factor eukaryotic initiation factor 5A (eIF5A), when in its post-translationally modified “hypusinated” form, plays a role in pancreas development. The hypusination of eIF5A requires the rate-limiting enzyme deoxyhypusine synthase (<em>Dhps</em>) to post-translationally modify a critical lysine residue which in turn produces the active form of eIF5A that functions in mRNA translation. When we generated animals with a deletion of <em>Dhps</em> in the pancreatic progenitor cells, there was no alteration in islet mass but significant exocrine insufficiency at embryonic (E) day 18.5 concomitant with downregulation of proteins required for exocrine pancreas development and function. Resultantly these animals died by 6 weeks-of-age. These observations prompted the question, is the phenotype caused by the absence of hypusinated eIF5A or the increase of unhypusinated eIF5A? To address this, we generated a mouse model wherein <em>Eif5a</em> is deleted in the pancreas (eIF5A∆PANC) and these mutant animals also display exocrine insufficiency. Interestingly, beta cell mass is increased at E18.5, and the mutant animals maintain euglycemia and survive up to 2 years. Ongoing analyses are interrogating the differences between these animal models with the goal to determine if mRNA translation facilitates cellular communication between the exocrine and endocrine pancreas.</p>

A proteína eIF5A apresenta um resíduo exclusivo de aminoácido chamado hipusina formado por modificação pós-traducional envolvendo espermidina como substrato. Neste estudo, observamos que a expressão de eIF5A é intensificada ao longo da diferenciação de células-tronco progenitoras de fibras musculares (células satélites) e que a inibição da hipusinação com GC7 bloqueia a diferenciação. Associado a esse bloqueio encontramos aumento do consumo de glicose e produção de lactato, diminuição da descarboxilação de glicose e palmitato, redução da proliferação celular e alteração do perfil traducional, efeitos que podem estar envolvidos na inibição do programa de diferenciação. Em seguida, o músculo tibial anterior de ratos foram criolesados e após severa supressão da expressão de eIF5A (período agudo de lesão) a mesma foi retomada ao longo da regeneração, chegando a quantidades superiores ao encontrado em músculos não lesados. Verificamos que a L-arginina, um supressor parcial do fenótipo distrófico e precursor de espermidina, reverte parcialmente o efeito de GC7. / eIF5A protein contains an exclusive amino acid residue named hypusine produced by a post-translational modification involving spermidine as substrate. In this study, we observed that eIF5A expression is raised during muscle fiber stem cells (satellite cells) differentiation and the hypusination inhibition by GC7 abolished the differentiation process. In association with this blockage, an increase in glucose consumption and lactate production, a decrease in glucose and palmitate decarboxylation, a reduction in cell proliferation and an alteration in translational profile were observed. These changes may be involved in the inhibition of the differentiation induced by GC7. The rat tibialis anterior muscle was injured and a marked reduction of eIF5A expression (acute injury period) was found. The expression of eIF5A was reestablished during regeneration, reaching higher levels than that observed in non injured muscle. We also verified that L-arginine, a partial suppressor of muscle dystrophic phenotype condition and precursor of spermidine, partially abolished the GC7 effects.

Deoxyhypusine synthase (DHS) catalyzes the first step in the hypusination of eukaryotic translation initiation factor 5A (eIF5A). In human cells, two eIF5A isoforms are present, eIF5A-1 and eIF5A-2, and DHS catalyzes the hypusination of both. Since both eIF5As are substrates for DHS, the biological functions of DHS are likely to be exerted through the various post-translational forms of these two eIF5As. The lysine form of eIF5A-1 has been associated with apoptosis, while the hypusinated form of eIF5A-1 has been associated with cell viability and proliferation. eIF5A-2 has been found to be over-expressed in certain cancers and has been proposed to function as an oncogene. Dhs is also over-expressed in certain human cancers and is a metastatic signature gene. The purpose of the present study was to investigate the role of DHS in cancer cell invasiveness, cell proliferation, and apoptosis using RNA interference. The main finding of the study is that DHS siRNA treatment decreases invasiveness of PC3 cells in vitro. Both DHS 0 siRNA treatment and DHS 1/b siRNA treatment significantly reduced cell invasiveness of PC3 cells as measured by the Matrigel invasion assay. Potential confounding variables, such as differences in cell proliferation or differences in apoptosis in response to DHS siRNA treatment, were assessed using the XTT cell proliferation assay and the Annexin V/Pi apoptosis assay, and they were found not to have an effect. In the absence of serum, DHS siRNA treatment did not result in significant decrease in cell proliferation compared to the control siRNA treatment. Furthermore, DHS siRNA treatment did not induce apoptosis in PC3 cells under the present experimental conditions. In conclusion, depletion of DHS with RNAi reduces invasiveness, but does not induce apoptosis in PC3 cells. The significance of the research is that the anti-invasiveness effect of DHS depletion in metastatic cancer cells is shown for the first time in the present study. Thus, DHS depletion may be useful to combat cancer in conjunction with L-eIF5A-1 over-expression.

Deoxyhypusine synthase (DHS) catalyzes the first step in the hypusination of eukaryotic translation initiation factor 5A (eIF5A). In human cells, two eIF5A isoforms are present, eIF5A-1 and eIF5A-2, and DHS catalyzes the hypusination of both. Since both eIF5As are substrates for DHS, the biological functions of DHS are likely to be exerted through the various post-translational forms of these two eIF5As. The lysine form of eIF5A-1 has been associated with apoptosis, while the hypusinated form of eIF5A-1 has been associated with cell viability and proliferation. eIF5A-2 has been found to be over-expressed in certain cancers and has been proposed to function as an oncogene. Dhs is also over-expressed in certain human cancers and is a metastatic signature gene. The purpose of the present study was to investigate the role of DHS in cancer cell invasiveness, cell proliferation, and apoptosis using RNA interference. The main finding of the study is that DHS siRNA treatment decreases invasiveness of PC3 cells in vitro. Both DHS 0 siRNA treatment and DHS 1/b siRNA treatment significantly reduced cell invasiveness of PC3 cells as measured by the Matrigel invasion assay. Potential confounding variables, such as differences in cell proliferation or differences in apoptosis in response to DHS siRNA treatment, were assessed using the XTT cell proliferation assay and the Annexin V/Pi apoptosis assay, and they were found not to have an effect. In the absence of serum, DHS siRNA treatment did not result in significant decrease in cell proliferation compared to the control siRNA treatment. Furthermore, DHS siRNA treatment did not induce apoptosis in PC3 cells under the present experimental conditions. In conclusion, depletion of DHS with RNAi reduces invasiveness, but does not induce apoptosis in PC3 cells. The significance of the research is that the anti-invasiveness effect of DHS depletion in metastatic cancer cells is shown for the first time in the present study. Thus, DHS depletion may be useful to combat cancer in conjunction with L-eIF5A-1 over-expression.