Orientadores: Everardo Magalhães Carneiro, Camila Aparecida Machado de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T15:32:11Z (GMT). No. of bitstreams: 1 CostaJunior_JoseMaria_M.pdf: 1708636 bytes, checksum: 8f21b938177c9819646df6957696b6c4 (MD5) Previous issue date: 2012 / Resumo: Neste trabalho investigamos os efeitos do treinamento físico aeróbio de longa duração, associado ou não com a suplementação de leucina, sobre o metabolismo protéico e glicídico em músculo soleus de camundongos Swiss. Para isso, parte dos camundongos (T) realizou protocolo de 12 semanas de exercício de natação, com 1h de duração, 5 dias/semana, sem sobrecarga, e a outra parte permaneceu sedentária (C). Metade dos animais dos grupos C e T foram suplementados com leucina (1,5%) na água para beber (grupos CL e TL, respectivamente) ao longo do experimento. Os dados foram analisados pela ANOVA Two-Way (variáveis exercício e suplementação com leucina) e o teste post hoc de Newman-Keus foi empregado nos casos de interação das variáveis. Foi adotado um valor de p<0,05 como estatisticamente significativo. Resultados: A área abaixo da curva glicêmica durante o teste de tolerância à glicose foi maior nos grupos suplementados com leucina (CL e TL). Já a sensibilidade à insulina, estimada pelo kITT, foi maior por efeito do exercício. A fosforilação da AS160, etapa distal da cascata de sinalização que leva a captação de glicose no músculo, foi maior no grupo T em relação aos demais tanto na condição basal quanto estimulada com insulina, provavelmente via AMPK, cuja fosforilação foi maior por efeito do exercício em ambas as condições, mas diminuída por efeito da suplementação com leucina após estimulação com insulina. A fosforilação da Akt não foi afetada pelo exercício, mas foi menor no grupo CL em relação aos demais. Apesar do maior peso do músculo soleus por efeito do exercício, a síntese protéica não diferiu entre os grupos, mesmo com a maior fosforilação da mTOR na condição basal no grupo CL, e a redução por efeito do exercício após estimulação com insulina. A degradação protéica no referido músculo, contudo, foi reduzida por efeito do exercício. A expressão gênica de isoformas específicas de E2 e E3 ligases, integrantes da via proteolítica ubiquitina-proteossoma, também foi menor por efeito do exercício. Todos os indicadores de resposta ao treinamento aeróbio foram aumentados por efeito do exercício: tempo até a exaustão em teste de esforço, oconsumo máximo de oxigênio e atividade da enzima citrato sintase. Alguns destes indicadores também sofreram interferência da suplementação. Concluímos que a suplementação com leucina pode prejudicar a homeostase glicêmica e reduzir os efeitos positivos do exercício sobre a sinalização insulínica. O exercício aumentou o peso do músculo soleus ao diminuir a degradação protéica por inibição da via proteolítica ubiquitina-proteossomo, enquanto a síntese protéica não foi afetada por nenhum tratamento (exercício ou suplementação com leucina) / Abstract: We investigated the effects of chronic physical training, associated or not with leucine supplementation, on protein and glucose metabolism in soleus muscle of Swiss mice. Half of the mice (T) performed a 12 weeks protocol of swimming exercise, 1h/day, 5 days/week, bearing no overload, and the other half remained sedentary (C). Additionally, half of the C and T mice were supplemented with leucine (1,5%) into the drinking water (groups CL and TL, respectively) throughout the experiment. Data were analysed by Two-Way ANOVA (variables exercise and leucine supplementation) and the Newman-Keus post hoc test was used in the cases of interaction between the variables. A p<0,05 was considered statistically significant. Results: The area under glucose curve during glucose tolerance test was increased in the leucine supplemented groups (CL and TL). Insulin sensitivity, estimated by kITT, was higher as an effect of exercise. AS160 phosphorylation, a distal step in the signaling pathway which leads to muscle glucose uptake, was increased in the T group compared to the others in both basal and insulin-stimulated condition, probably via AMPK, whose phosphorylation was increased by exercise in both conditions, but it was reduced by an effect of leucine supplementation following insulin stimulation. Akt phosphorylation was not affected by exercise, but it was reduced in the CL group compared to the other groups. Despite the increased weight of soleus muscle caused by an effect of exercise, protein synthesis was similar among the groups, even with the increased mTOR phosphorylation at the basal condition in the CL group, and the reduction caused by exercise following insulin stimulation. Protein degradation in the soleus muscle was reduced by an effect of exercise. Gene expression of specific isoforms of E2 and E3 ligases, members of the ubiquitin-proteosome proteolytic pathway, was also reduced by an effect of exercise. All the indicators of response to aerobic training were increased by exercise: time to exhaustion in the effort test, maximal oxygen uptake and citrate synthase enzyme activity. Some of these indicators were also affected by leucine supplementation. We conclude that leucine supplementation may impair glucose homeostasis and reduce the positive effects of exercise on insulin signaling. Exercise increased soleus muscle weight by reducing protein degradation via an inhibition of the ubiquitin-proteasome proteolytic pathway, whereas protein synthesis was not affected by either of the treatments (exercise or leucine supplementation) / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

African horse sickness virus (AHSV) affects equine populations around the world. It is the cause of a high rate of morbidity and associated large economic losses in affected regions. The virus is a segmented double stranded RNA virus and a member of Orbivirus genus in the Reoviridae family. The prototype member of the orbiviruses is bluetongue virus (STY) and other members include Chuzan virus and St. Croix River virus. These viruses are all characterized by a genome of ten dsRNA segments that encode at least ten different proteins. Three of the minor core proteins are found within the core of BTV. These are all associated with the RNA transcription complex and the enzymatic activities with which they are associated include an RNA polymerase (VP1), an RNA capping enzyme (VP4) and an RNA helicase (VP6). Genes homologous to the BTV genes that encode these proteins are found in all members of the Orbivirus genus. The aim of this thesis is to characterize VP4 of AHSV, the capping enzyme candidate, and to compare it to other orbivirus capping enzymes. Possible functional motifs and regions of importance within the orbivirus capping enzymes will be identified. The gene will also be expressed and used to perform assays to characterize the different enzymatic activities of VP4. The VP4 cDNA of AHSV serotype 3 was cloned and sequenced. From the full-length verified nucleotide sequence an open reading frame was identified and used to predict the amino acid sequence. These were compared to other orbivirus species including STY, Chuzan virus and St. Croix River virus. These alignments identified a number of highly conserved regions, consisting of four or more amino acids conserved between all the sequences analyzed. A fibronectin type 3-like motif, containing 12 conserved amino acids, was identified which could be responsible for protein binding. This motif contains 12 conserved amino acids making it a good candidate for a functional motif. Conservation does not, however, always predict regions of importance. In BTV a lysine-containing motif was identified to be responsible for GMP binding. This region is not conserved between the different viruses. AHSV has a motif containing a lysine residue similar to the motif identified in rotavirus and reovirus. Two other motifs described in BTV were also not conserved in the other viruses. One of them, a leucine zipper, was shown to dimerize BTV VP4. Phylogenetically, AHSV and Chuzan virus are the most closely related while BTV is more distant and St. Croix River virus forms a distinct out-group when the different VP4 sequences are compared. AHSV-3 VP4 was expressed as a histidine-tagged protein in the baculovirus expression system. Not unexpectedly, the protein was found to be insoluble, similar to BTV VP4 produced by means of the same system. However, whereas BTV VP4 could be solubilized by the addition of salt the AHSV VP4 remained insoluble at high salt concentrations. Several adjustments were made. Cells were lysed in a high salt buffer, the pH of the buffers was adjusted and sucrose cushions were used but none of the methods was found to improve the yield of soluble VP4 significantly. However, the pellet containing VP4 was relatively empty of contaminating protein and, therefore, a number of enzymatic assays were performed with the pellet. Assays for inorganic phosphatase and nucleotide phosphatase were performed. Strikingly, both assays indicated the presence of active phosphatases in the WT and VP4 pellets. Also, an assay was performed for guanylyltransferase activity but no activity was observed for this assay. The sequence data therefore points to VP4 as the probable capping enzyme although it may have a different structural complex. The failure to produce a reliable source of soluble purified AHSV VP4 made it impossible to provide evidence to confirm the associated enzymatic activities. / Dissertation (MSc(Genetics))--University of Pretoria, 2005. / Genetics / unrestricted

Orientador: Laurecir Gomes, Maria Cristina Cintra Gomes Marcondes / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T20:59:10Z (GMT). No. of bitstreams: 1 Barbosa_AlexandreWesleyCarvalho_M.pdf: 9272160 bytes, checksum: d577f5e5dbcbe2f820746ed64790cbfa (MD5) Previous issue date: 2006 / Resumo: o tendão apresenta-se como um dos integrantes essenciais do complexo de movimentação angular nas articulações e tem sido estudado em sua estrutura e composição bioquímica quando sob tensão, compressão, após exercício e durante o envelhecimento. Em condições de estresse, a constituição bioquímica da matriz extracelular do tendão é alterada no tipo e/ou na quantidade do colágeno, proteoglicanos e glicosaminoglicanos. A desnutrição severa afeta de forma significativa à síntese protéica e a capacidade de resposta corporal à atividade física. A utilização de dieta rica em leucina tem demonstrado influência no implemento da síntese protéica e na recuperação corpórea após desnutrição severa. O presente estudo se propôs a submeter ratos às condições: (1) desnutrição protéica severa; (2) recuperação nutricional com dieta normal (C); (3) recuperação nutricional com dieta rica em leucina (L); e (4) exercício físico na água, e analisar os conteúdo de colágeno e as propriedades biomecânicas do tendão flexor profundo dos dedos de ratos. Após período de desnutrição, grupos recuperados com leucina apresentaram concentrações elevadas de hidroxiprolina em relação aos demais grupos. Quanto à carga, grupos exercitados obtiveram valores idênticos ao controle. O grupo desnutrido apresentou menor valor entre os demais. Na tensão, o controle, o desnutrido e o recuperado com leucina não diferiram entre si, diferindo apenas do controle exercitado.O grupo renutrido com dieta C e exercitado sofreu a menor tensão. Os maiores valores de deformação foram observados no controle, recuperado com dieta L e recuperados com dieta C (exercitado e sedentário). Os grupos desnutrido, recuperado com leucina e exercitado e o controle exercitado, que obtiveram os menores valores, diferiram dos grupos recuperado com leucina e renutrido com dieta C e exercitado, estes semelhantes ao controle. Os resultados sugerem que, após desnutrição protéica, a dieta L promove implementação na síntese de colágeno do tendão, no entanto, para níveis nonnais de colágeno, a aplicação da dieta C é suficiente. O exercício físico na água favorece a síntese de colágeno, principalmente quando associado à dieta L, mas as propriedades biomecânicas são afetadas por microtraumas cumulativos resultantes do protocolo empregado. Uma menor intensidade no programa de exercícios pode favorecer a resposta à tração / Abstract: Tendon is an essential component ofthe angular motion complex and it has been studied on its structure and biochemical composition under tension, compression, after exercise and during ageing. Under stress, the biochemical constitution of the extracellular matrix is altered in kind and the amount of collagen, proteoglycans and glycosaminoglycans. In this way, severe protein-caloric malnourishe alters the protein synthesis and the body response to exercise. Leucine-rich diet increases the proteins synthesis and improves the body recovery and response to severe under nutrition. Leucine-rich diet groups showed greater hydroxyproline amount. Analyzing the load (biomechanical properties), control exercised group (CT), undemourished (D), recovered and exercised (DRT), recovered with leucine- rich diet and exercised (DRLT) groups achieved identical statistic values to control group (C). The D group showed the minor leveI among the others, statistically decreased when compared to C, recovered group (DRC) e recovered with leucine-rich diet (DRL). On the stress results, C, D and DRL had the same contents, differing only to CT. DRT group suffered the minor stress. DRC, DRLT and CT were different to C group. The highest strain levels were observed on C, DRL, DRT and DRC groups. D, DRLT and CT groups, achieving the lowest values, differed statistically from DRL and DRT groups, and these identical to C group. The resuIts suggest that after the under nutrition period, the leucine-rich diet improves the tendon collagen synthesis, but the control diet is sufficient to improves collagen normal levels. The exercise protocol raises the collagen synthesis response when associated to leucine-rich diet, but biomechanical properties are affected by tendon overuse. These mechanical properties levels could improve when associated to lower exercises program / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

This study was designed to explore the hormonal regulation of CNS opioid peptide levels in female Sprague Dawley rats. Forty-eight animals were divided into 2 equal groups for acute and chronic studies. Each group was further divided into 4 subgroups, each containing 6 animals. Each rat in the control group received an inert pill (in 0.25 ml corn oil daily by gavage); the second group, 15 μg norethindrone (NE, a potent progestin present in the oral contraceptive Micronor®); the third group, 15 μg NE and 1 μg ethinyl estradiol, EE2 (present in the oral contraceptive Modicon®) and the fourth group, 10 times the dose of the third group. Rats were treated either acutely for 5 days or chronically for 7 weeks. Opioid peptides were estimated by radioimmunoassay. Acute administration of 150 μg NE + 10 μg EE2 decreased the levels of methionine-enkephalin (ME), leucine-enkephalin (LE), dynorphin (DYN) and β-endorphin like immunoreactivity (β-EI) by about 50% in the pituitary. The same dose on chronic administration also decreased DYN, but increased the levels of ME and LE in the pituitary by 331 and 69%, respectively. In the hypothalamus, chronic administration of NE + EE2 increased the level of ME (155%) and LE (87%) as well as of DYN (97%). In the striatum, the levels of LE (33%) and DYN (115%) were elevated during chronic administration. It is concluded that the acute administration of NE + EE2, in general, reduces the levels of ME, LE, DYN and β-EI. The extent of this decrease is about the same in the pituitary, hypothalamus and striatum. Chronic administration of these hormones, however, results in a reversal of this decrease (except for β-EI) and actually can increase the levels of ME, LE and DYN in all three tissues.

Indiana University-Purdue University Indianapolis (IUPUI) / Mutations in the gene encoding for leucine rich repeats kinase 2 (LRRK2) are commonly found in Parkinson’s disease. Recently, we found that the disease-associated point mutations at residue R1441 in the G domain (ROC) of LRRK2 resulted in perturbation of its GTPase activity. In this study, we compare the biochemical and biophysical properties of the ROC domain of LRRK2 carrying the PD-associated mutations at residue R1441 with those of the wild-type. We found that the disease-associated mutations (R1441C/G/H) showed marked quaternary structure compared to wild-type, in that the latter existed in solution in both monomeric and dimeric conformations dynamically regulated by GDP/GTP binding state, while we detected only monomeric conformation for three disease-associated mutants. To understand the structural basis for this plasticity and the activity reduction in the mutants, we solved a 1.6 Å crystal structure of the wild type ROC that shows a stable dimeric conformation in which the switch motifs and inter-switch regions mediate extensive interactions at the dimer interface. Residue R1441, where PD-associated mutations occur, forms exquisite interactions at the interface, thus suggesting a critical role of this residue in maintaining a dynamic dimer-monomer interconversion and conformational flexibility of the switch motifs. Consistently, substituting R1441 for other arbitrary mutations (R1441K/S/T) lead to similar perturbation of GTPase activity and dimerization defects as observed in the disease-associated mutants. Locking the ROC domain in either dimeric or monomeric conformations by engineered disulfide bond alters the binding affinity to GTP (but not GDP) and significantly reduce GTPase activity, thus suggesting that the dynamic dimer-monomer interconversion and conformational plasticity are essential for ROC function as a molecular switch modulating the kinase activity of LRRK2.

This study has investigated the characteristics of a leucine aminoacyl transfer RNA synthetase enzyme from Tritrichomonas augusta. Differential centrifugation and DEAE-cellulose column chromatography were used for partial enzyme purification. The column purification increased the synthetase activity 125-fold over the unfractionated cell extract. The conditions for maximum [3H] leucine charging were 37°C for 20 min, with protein at 180 μg ml-1 using yeast leucine tRNA as an acceptor. The optimal reaction conditions were 14 mM-Mg acetate, 3 mM-ATP, 3 mM-spermidine and 5.5 mM-putrescine. Acceptor activity with T. augusta transfer RNA was 8-fold higher than with yeast transfer RNA and 25-fold higher than with Escherichia coli transfer RNA. The partially purified enzyme fraction had comparable changing activities for both leucine and valine.

Novel terpolymers of leucine (L), aspartic acid (D), and valine (V) were developed as targeting drug delivery carriers that specifically interact with the α 4 β 1 integrin, which is over-expressed in human malignant melanoma cells. As such, copolymerization and terpolymerization of leucine-N-carboxyanhydride (NCA), β-benzyl-aspartate-NCA, and valine-NCA, in dioxane, initiated with triethylamine, were investigated to determine the random nature of the terpolymer composition, for potential application as a targeting drug delivery carrier. The reactivity ratio of each monomer was determined by using Fineman-Ross, Kelen-Tüdös, and nonlinear least-squares curve fitting methods. The product of the estimated reactivity ratios from the monomers in the binary copolymerizations indicated that random polymers were predominantly formed. Based on the reactivity ratios determined from the binary copolymers, Alfrey-Goldfinger equations were used to estimate the composition of the terpolymers. There was no statistical difference between the actual monomer compositions and the calculated compositions of the terpolymers, which validate the randomness of the terpolymers. Therefore, the poly (Leucine-Aspartate-Valine) synthesized in this study is primarily a random terpolymer. The probability of the appearance of LDV sequences occurring within the random polymers were assessed by analyzing the influence of tacticity on the 13 C NMR signals of LDV terpolymers, a statistical method based on the terminal terpolymerization model, and the Poisson distribution, with the highest probability (∼13%) of LDV occurring in a random terpolymer of LDV to be approximately 8 to 9 triad units. Poly (LDV) polymers were shown to exhibit strong binding affinity for A-375 human malignant melanoma cells. The effectiveness of poly (LDV) to target malignant melanoma was evaluated and it showed that 21.3 ± 2.10% of melanoma cells adhered to poly (LDV) films compared to 39.0 ± 3.90% with the positive control fibronectin, whereas binding to HTB-129 human breast carcinoma cells and NHEK normal human keratinocytes were not significant. Poly (LDV)-doxorubicin conjugates displayed excellent selectivity and cytotoxicity in the delivery of doxorubicin. It was shown that the poly (LDV)doxorubicin conjugates exhibited cytotoxicity toward human malignant melanoma cells, but were less toxic than free doxorubicin. In addition, poly (LDV)-doxorubicin conjugates displayed a substantial reduction in toxicity per mole of doxorubicin against normal human keratinocyte cells when compared to free doxorubicin. Fluorescence microscopy showed poly (LDV)-FITC conjugates bound to the A-375 cells and were internalized within 30 minutes. Scatchard plots of poly (LDV)-doxorubicin conjugates were generated, which determined the association constants and established that there is one class of binding sites. It was shown that poly (LDV) could be internalized in a target specific manner by human malignant melanoma cells, which is dependent on the number of LDV targeting moieties in the polymer. These results established that poly (LDV) could be used as a drug delivery carrier that specifically targets the α 4 β 1 integrin.

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