Orientador: Fernando Broetto / Banca: Roberto Lyra Villas Bôas / Banca: Magali Ribeiro da Silva / Banca: Marcia Ortiz Mayo Marques / Banca: Otto Jesu Crocomo / Resumo: Um dos mais importantes fatores ambientais e agronômicos que afetam a produção de metabólitos secundários em plantas é a água. Quantidade limitada de água tem efeito negativo sobre o crescimento e desenvolvimento das plantas. Entretanto, a deficiência hídrica (DH), muitas vezes tem se mostrado positiva no acúmulo de constituintes ativos em espécies medicinais, aromáticas e condimentares. Verificou-se a influência da DH na participação de assimilados, acúmulo e na composição do óleo essencial em manjericão (Ocimum basilicum L.). Paralelamente, foram avaliados parâmetros bioquímicos relacionados com a resposta das plantas a DH. Os tratamentos foram constituídos por dois blocos, T1 (controle) que foi mantido sempre irrigado e T2 (DH) submetido a três níveis de DH. Nas plantas dos tratamentos do bloco T2 (DH), foi suspenso o fornecimento de água quando 50% das plantas iniciaram seu florescimento. Observou-se que as plantas cultivadas sob DH, responderam com aumento da concentração de óleo essencial na inflorescência e nas folhas. O maior rendimento de óleo por planta, foi encontrado nas plantas do controle, principalmente no tecido da inflorescência, inversamente do observado para o tecido foliar. Em termos qualitativos, a análise dos componentes do óleo revelou que os diferentes níveis de DH não alteraram o padrão conhecido para esta espécie. Constatou-se que todos os parâmetros biométricos foram influenciados negativamente pela DH. A análise da atividade enzimática indicou que o estresse causado por DH ativou todas as enzimas relacionadas ao sistema de resposta antioxidativo, além de induzir ao acúmulo de L-prolina. / Abstract: The water is one of the most important agronomic and environmental factors that affect the production of secondary metabolites in plants. Limited amount of water has negative effect on growth and development of plants. However, the hydric deficiency (HD), a lot of times have been shown positive in the active constituents accumulation in medicinal species, aromatic and spices. The influence of HD was verified in participation of assimilate, accumulation and in the essential oil composition of basil (Ocimum basilicum L.). At the same time, biochemical parameters related with HD in plants were studied. The treatments were constituited by two blocks, T1 (control) that was maintained well irrigated and T2 (HD) submitted to three levels of HD. In T2 (HD) treatments, the water supply was suspended when 50% of the plants began flowering. In plants cultivated under HD was observed that the essential oil concentration was increased in the inflorescences and leaves. The largest oil income was found in the plants of control, mainly in inflorescences material, inversely of observed in leaves material. In qualitative terms, the oil components analysis revealed that the different levels of HD didn't alter the known standarts for this species. It was verified that all of the biometrical parameters were negatively influenced by HD. The enzymatic activity analysis indicated that the stress caused by HD had activated all of the enzymes related to the antioxitative response system, besides inducing the accumulation of L-proline. / Doutor

A banana é considerada um bom modelo para o estudo da transformação amido-sacarose, já que acumula um alto teor de amido durante o desenvolvimento que é rapidamente degradado durante o amadurecimento. Várias enzimas e provavelmente mais de uma via metabólica estão envolvidas neste processo. Com isso, o objetivo deste trabalho foi estudar as características estruturais dos grânulos, bem como, a atuação das enzimas envolvidas em sua degradação. Os grânulos de amidos foram isolados de bananas controle (não tratadas) e submetidas a diferentes tratamentos: etileno, 1-MCP, frutos mantidos a 13°C e frutos tratados com etileno e mantidos a 13°C. Os resultados obtidos mostraram alta atividade de enzimas α e β-amilases ligadas ao grânulo tanto por ensaios in vitro como por géis de eletroforese contendo amilopectina como substrato. Os resultados obtidos para Western blot utilizando anticorpos produzidos contra essas enzimas, indicaram que a α-amilase atua no início da degradação enquanto a β-amilase foi encontrada no momento em que o amido estava em pleno processo. de degradação. Nos estudos de imunolocalização observou-se que as proteínas associadas aos grânulos de amido e em cortes do fruto demonstraram que estas enzimas estão localizadas na superfície do grânulo. As técnicas utilizadas para observação dos grânulos de amido foram a de Microscopia Óptica, Microscopia Eletrônica de Varredura e Microscopia de Varredura Confocal a Laser. Os grânulos apresentaram um padrão de degradação diferenciado para cada tratamento realizado nos frutos. O teor de amilose encontrado para os amidos foi ao redor de 15%, não variando durante a degradação. O padrão de difração de Raios-X foi do tipo B em amidos de banana recém-colhidas e tipos A e B foram encontrados em amidos degradados. O grau de cristalinidade aumentou de 15% para 17% nos amidos em degradação. / Banana fruit is considered a good example for studying the starch-sucrose transformation, accumulating high starch content during the development being rapidly degraded during the ripening. Several enzymes and, probably more then one metabolic way are involved in this processo Then, the aim of this work was to study the structural characteristics of starch granules and the action of the enzymes involved in its degradation. Starch granules were isolated from bananas control (fruits without treatment), and exposed to different treatments, such as: ethylene, 1-MCP, stored fruits to 13°C and stored fruits to 13°C + ethylene. Results obtained showed high activities of enzymes α and β-amylases associated to starch granules, measured by in vitro assay and native PAGE containing amylopectin like substrate. Results obtained by Western blot using antibodies against these enzymes, indicated that α-amylase is responsible for the initial attack on the starch, and β-amylase was localized at moment that starch was in degradation processo Results of the immunolocalization of proteins associated on starch granule and proteins on banana tissue confirmed that these enzymes are localized on granule surface. When techniques of microscopy were used to starch, such as Optical Microscopy, Scanning Electron Microscopy and Confocal Laser Scanning Microscopy, was observed that granules showed a different degradation pattern, for each treatment made on fruits. Amylose content obtained for starch was around 15%, not changing during degradation. B-type diffraction pattern was found for green banana starch, and A and. B-type patterns for degraded starch. Degree of crystallinity increased from 15% to 17% for starches during degradation.

A banana é considerada um bom modelo para o estudo da transformação amido-sacarose, já que acumula um alto teor de amido durante o desenvolvimento que é rapidamente degradado durante o amadurecimento. Várias enzimas e provavelmente mais de uma via metabólica estão envolvidas neste processo. Com isso, o objetivo deste trabalho foi estudar as características estruturais dos grânulos, bem como, a atuação das enzimas envolvidas em sua degradação. Os grânulos de amidos foram isolados de bananas controle (não tratadas) e submetidas a diferentes tratamentos: etileno, 1-MCP, frutos mantidos a 13°C e frutos tratados com etileno e mantidos a 13°C. Os resultados obtidos mostraram alta atividade de enzimas α e β-amilases ligadas ao grânulo tanto por ensaios in vitro como por géis de eletroforese contendo amilopectina como substrato. Os resultados obtidos para Western blot utilizando anticorpos produzidos contra essas enzimas, indicaram que a α-amilase atua no início da degradação enquanto a β-amilase foi encontrada no momento em que o amido estava em pleno processo. de degradação. Nos estudos de imunolocalização observou-se que as proteínas associadas aos grânulos de amido e em cortes do fruto demonstraram que estas enzimas estão localizadas na superfície do grânulo. As técnicas utilizadas para observação dos grânulos de amido foram a de Microscopia Óptica, Microscopia Eletrônica de Varredura e Microscopia de Varredura Confocal a Laser. Os grânulos apresentaram um padrão de degradação diferenciado para cada tratamento realizado nos frutos. O teor de amilose encontrado para os amidos foi ao redor de 15%, não variando durante a degradação. O padrão de difração de Raios-X foi do tipo B em amidos de banana recém-colhidas e tipos A e B foram encontrados em amidos degradados. O grau de cristalinidade aumentou de 15% para 17% nos amidos em degradação. / Banana fruit is considered a good example for studying the starch-sucrose transformation, accumulating high starch content during the development being rapidly degraded during the ripening. Several enzymes and, probably more then one metabolic way are involved in this processo Then, the aim of this work was to study the structural characteristics of starch granules and the action of the enzymes involved in its degradation. Starch granules were isolated from bananas control (fruits without treatment), and exposed to different treatments, such as: ethylene, 1-MCP, stored fruits to 13°C and stored fruits to 13°C + ethylene. Results obtained showed high activities of enzymes α and β-amylases associated to starch granules, measured by in vitro assay and native PAGE containing amylopectin like substrate. Results obtained by Western blot using antibodies against these enzymes, indicated that α-amylase is responsible for the initial attack on the starch, and β-amylase was localized at moment that starch was in degradation processo Results of the immunolocalization of proteins associated on starch granule and proteins on banana tissue confirmed that these enzymes are localized on granule surface. When techniques of microscopy were used to starch, such as Optical Microscopy, Scanning Electron Microscopy and Confocal Laser Scanning Microscopy, was observed that granules showed a different degradation pattern, for each treatment made on fruits. Amylose content obtained for starch was around 15%, not changing during degradation. B-type diffraction pattern was found for green banana starch, and A and. B-type patterns for degraded starch. Degree of crystallinity increased from 15% to 17% for starches during degradation.

Indiana University-Purdue University Indianapolis (IUPUI) / Fatty acid elongation is the extension of de novo synthesized fatty acids through a series of four reactions analogous to those of fatty acid synthase. ELOs catalyze the first reaction in the elongation pathway through the condensation of an acyl group with a two carbon unit derived from malonyl-CoA. This study uses the condensing enzyme, EloA, from the cellular slime mold, Dictyostelium discoideum as a model for the family of ELOs. EloA has substrate specificity for monounsaturated and saturated C16 fatty acids and catalyzes the elongation of 16:1Δ9 to 18:1Δ11. Site-directed mutagenesis was used to change residues highly conserved among the ELO family to examine their potential role in the condensation reaction. Mutant EloAs were expressed in yeast and fatty acid methyl esters prepared from total cellular lipids were analyzed by gas chromatography/mass spectrometry. Sixteen out of twenty mutants had a decrease in 18:1Δ11 production when compared to the wild-type EloA with little to no activity observed in ten mutants, four mutants had within 20% of wild-type activity, and six mutants had 10-60% of wild-type activity. Immunoblot studies using anti-EloA serum were used to determine if the differences in elongation activity were related to changes in protein expression for each mutant. Analysis of immunoblots indicated that those mutants with little to no activity, with the exception of T130A and Q203A, had x comparable protein expression to the wild-type. Further research included the solubilization of the His6-ELoA fusion protein and preliminary work toward the isolation of the tagged protein and the use of a radiolabeled condensation assay to determine the activity of the eluted protein. Preliminary results indicated that the protein was solubilized but the eluted protein showed no activity when examined by a condensation assay. The work presented here contributes to a better understanding of the role of certain amino acid residues in the activity of EloA and serves as a stepping-stone for future EloA isolation work.

Indiana University-Purdue University Indianapolis (IUPUI) / The PRL family of enzymes constitutes a unique class of protein tyrosine phosphatase, consisting of three highly homologous members (PRL-1, PRL-2, and PRL-3). Family member PRL-3 is highly expressed in a number of tumor types and has recently gained much interest as a potential prognostic indicator of increased disease aggressiveness and poor clinical outcome for multiple human cancers. PRL-1 and PRL-2 are also known to promote a malignant phenotype in vitro, however, prior to the present study, little was known about their expression in human normal or tumor tissues. In addition, the biological function of all three PRL enzymes remains elusive and the underlying mechanisms by which they exert their effects are poorly understood. The current project was undertaken to expand our knowledge surrounding the normal cellular function of the PRL enzymes, the signaling pathways in which they operate, and the roles they play in the progression of human disease. We first characterized the tissue distribution and cell-type specific localization of PRL-1 and PRL-2 transcripts in a variety of normal and diseased human tissues using in situ hybridization. In normal, adult human tissues we found that PRL-1 and PRL-2 messages were almost ubiquitously expressed. Only highly specialized cell types, such as fibrocartilage cells, the taste buds of the tongue, and select neural cells displayed little to no expression of either transcript. In almost every other tissue and cell type examined, PRL-2 was expressed strongly while PRL-1 expression levels were variable. Each transcript was widely expressed in both proliferating and quiescent cells indicating that different tissues or cell types may display a unique physiological response to these genes. In support of this idea, we found alterations of PRL-1 and PRL-2 transcript levels in tumor samples to be highly tissue-type specific. PRL-1 expression was significantly increased in 100% of hepatocellular and gastric carcinomas, but significantly decreased in 100% of ovarian, 80% of breast, and 75% of lung tumors as compared to matched normal tissues from the same subjects. Likewise, PRL-2 expression was significantly higher in 100% of hepatocellular carcinomas, yet significantly lower in 54% of kidney carcinomas compared to matched normal specimens. PRL-1 expression was found to be associated with tumor grade in the prostate, ovary, and uterus, with patient gender in the bladder, and with patient age in the brain and skeletal muscle. These results suggest an important, but pleiotropic role for PRL-1 and PRL-2 in both normal tissue function and in the neoplastic process. These molecules may have a tumor promoting effect in some tissue types, but inhibit tumor formation or growth in others. To further elucidate the signaling pathways in which the PRLs operate, we focused on PRL-1 and used microarray and microRNA gene expression profiling to examine the global molecular changes that occur in response to stable PRL-1 overexpression in HEK293 cells. This analysis led to identification of several molecules not previously associated with PRL signaling, but whose expression was significantly altered by exogenous PRL-1 expression. In particular, Filamin A, RhoGDIalpha, and SPARC are attractive targets for novel mediators of PRL-1 function. We also found that PRL-1 has the capacity to indirectly influence the expression of target genes through regulation of microRNA levels and we provide evidence supporting previous observations suggesting that PRL-1 promotes cell proliferation, survival, migration, invasion, and metastasis by influencing multi-functional molecules, such as the Rho GTPases, that have essential roles in regulation of the cell cycle, cytoskeletal reorganization, and transcription factor function. The combined results of these studies have expanded our current understanding of the expression and function of the PRL family of enzymes as well as of the role these important signaling molecules play in the progression of human disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Epigenetic alterations have been shown to control cell type specification and differentiation leading to the changes in chromatin structure and organization of many genes. HDACs have been well documented to play an important role in both neurogenesis and gliogenesis in ganglionic eminence and cortex-derived cultures. However, the role of HDACs in glial cell type specification and differentiation in the optic nerve has not been well described. As a first step towards understanding their role in glial cell type specification, we have examined histone acetylation and methylation levels as well as the expression levels and patterns of the classical HDACs in both murine and chick optic nerve. Analysis of mRNA and protein levels in the developing optic nerve indicated that all 11 members of the classical HDAC family were expressed, with a majority declining in expression as development proceeded. Based on the localization pattern in both chick and murine optic nerve glial cells, we were able to group the classical HDACs: predominantly nuclear, nuclear and cytoplasmic, predominantly cytoplasmic. Nuclear expression of HDACs during different stages of development studied in this project in both murine and chick optic nerve glial cells suggests that HDACs play a role in stage-dependent changes in gene expression that accompany differentiation of astrocytes and oligodendrocytes. Examination of localization pattern of the HDACs is the first step towards identifying the specific HDACs involved directly in specification and differentiation of glia in optic nerve.

Indiana University-Purdue University Indianapolis (IUPUI) / In mammalian cells DNA double strand breaks (DSBs) are highly variable with respect to sequence and structure all of which are recognized by the DNA- dependent protein kinase (DNA-PK), a critical component for the resolution of these breaks. Previously studies have shown that DNA-PK does not respond the same way to all DSBs but how DNA-PK senses differences in DNA substrate sequence and structure is unknown. Here we explore the enzymatic mechanism by which DNA-PK is activated by various DNA substrates. We provide evidence that recognition of DNA structural variations occur through distinct protein-protein interactions between the carboxy terminal (C-terminal) region of Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Discrimination of terminal DNA sequences, on the other hand, occurs independently of Ku 80 C-terminal interactions and results exclusively from DNA-PKcs interactions with the DNA. We also show that sequence differences in DNA termini can drastically influence DNA repair through altered DNA-PK activation. Our results indicate that even subtle differences in DNA substrates influence DNA-PK activation and ultimately Non-homologous End Joining (NHEJ) efficiency.