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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
11

Efeito da superexpress?o do fator de transcri??o OsDof25 sobre a efici?ncia de absor??o de nitrog?nio em Orysa sativa L. / Effect of superexpression of the transcription factor OsDof25 on the efficiency of nitrogen uptake in Orysa sativa L.

SILVA, Renata Aparecida Costa 15 February 2012 (has links)
Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-05-26T20:35:35Z No. of bitstreams: 1 2012 - Renata Aparecida Costa Silva.pdf: 1687897 bytes, checksum: 4d1af8e117daa1842ae1766319ddf8d6 (MD5) / Made available in DSpace on 2017-05-26T20:35:35Z (GMT). No. of bitstreams: 1 2012 - Renata Aparecida Costa Silva.pdf: 1687897 bytes, checksum: 4d1af8e117daa1842ae1766319ddf8d6 (MD5) Previous issue date: 2012-02-15 / CAPES / Nitrogen is one of the nutrient elements most limiting for plant growth. Thus, increasing plant nitrogen usage efficiency (NUE) is an essential factor for sustainable agriculture, leading to an increased food production with less fertilizer input and less environment impact. The aim of this study was to evaluate the effect of OsDof25 overexpression on N-NO3- and N-NH4+ uptake. In transgenic rice plants, OsDof25 was expressed under control of maize ubiquitin promoter (UBIL:OsDof25:3xHA). Two experiments were conducted: one to evaluate the kinetic parameters Vm?x and KM, and another to analyze the expression level of nitrate (NRT2.1~2.2 and NAR) and ammonium transporters (AMT1.1~1.3), both under high and low NO3- and NH4+ supply. The untransformed plants showed higher growth that transformed lineages. The L1 and L2 showed a lower value of the KM in the resupply treatment of 0.2 mM N-NO3-. In the resupply with 0.2 mM N-NH4 + the L4 showed higher Vmax and L1 lower KM. There were no large variations in uptake kinetics between the transformed and untransformed plants. At the root the NRT2 showed low expression in lineages L1 and L4, when under constant supply of N-NO3-, in contrast, in the treatment under resupply with 0.2 mM N-NO3-was increased expression of OsNTR2.1 ~ 2.2, and NAR in both transformed lineages, but in the root the concentration of NO3- was opposed to the expression of NRT2 and NAR, in both conditions. In the leaves, the line L4 showed high expression of the transporter OsNRT2.1 with the resupply of 0.2 and 2.0 mM N-NO3-. In plants grown under constant supply of N-NH4+, L1 showed lower expression of AMT1 in the root compared to L4 and untransformed plants. When subjected to nitrogen deficiency, there was an increased expression of the OsAMT1.2. However, there was no correlation between N transporter expression levels and NO3- and NH4+ content in the transformed plants, indicating a possible change in enzyme activity and reduction or assimilation of N in these plants. The transformed plants when subjected to resupply with low levels of nitrate and ammonium showed better response parameters Vmax and KM compared to the untransformed. In the plants transformed the resupply with nitrate at low concentration resulted in increasing the gene expression of the transporters (OsNTR2.1 ~ 2.2 and protein OsNAR2.1), and the treatment with constant supply provided greatest nitrate accumulation in these plants. The results of both kinetic parameters and accumulation of fresh matter suggest that plants transformed for the expression of the OsDof25 presented highest tolerance to nutritional stress. / O nitrog?nio ? um dos elementos minerais que mais limita o desenvolvimento das plantas. Assim, aumentar a efici?ncia de uso de nitrog?nio (EUN) ? um fator ? essencial para uma agricultura sustent?vel, levando a um aumento da produ??o de alimentos com menor uso de insumos e menos impactos ao ambiente. Este trabalho teve por objetivo avaliar o efeito da superexpress?o do fator de transcri??o OsDof25 sobre a absor??o de nitrog?nio em duas linhagens transformadas de arroz (L1 e L4) da variedade Nipponbare comparando-as com plantas n?o transformadas (WT). Nas plantas transformadas, o OsDof25 foi expresso sob o controle do promotor da ubiquitina 1 de milho (UBIL:OsDof25:3xHA). For feitos dois experimentos: um para avaliar os par?metros cin?ticos Vm?x e KM, sob condi??es de alto e baixo suprimento de N-NO3- e N-NH4+, e outro para analisar a express?o dos transportadores de NO3- (NRT2.1~2.2 e NAR) e NH4+ (AMT1.1~1.3) tamb?m sob alto e baixo suprimento desses ?ons. As plantas n?o transformadas apresentaram maior crescimento do que as linhagens transformadas. As L1 e a L2 mostraram menor valor de KM no tratamento com ressuprimento de 0,2 mM de N-NO3-. No ressuprimento com 0,2 mM de N-NH4+ a L4 apresentou maior Vm?x e L1 menor KM, mas, n?o houve grandes varia??es nos par?metros cin?ticos de absor??o entre as plantas transformadas e n?o transformadas. Na raiz os NRT2 mostraram baixa express?o nas linhagens L1 e L4 quando submetidas ao suprimento constante de N-NO3-, em contrapartida, no tratamento sob ressuprimento com 0,2 mM de N-NO3- ocorreu aumento de express?o dos OsNTR2.1~2.2 e NAR nas duas linhagens transformadas, por?m na raiz a concentra??o de N-NO3- foi oposta a express?o dos NRT2 e NAR, em ambas as situa??es. Nas folhas, a linhagem L4 apresentou alta express?o do transportador OsNRT2.1 com o ressuprimento de 0,2 e 2,0 mM de N-NO3-. Nas plantas submetidas ao suprimento constante de N-NH4+, a L1 apresentou menor express?o dos AMT1 na raiz quando comparada a L4 e a planta n?o transformada. Quando submetida a defici?ncia de N-NH4+, a express?o do OsAMT1.2 aumentou nas ra?zes de todas as plantas. Entretanto, n?o houve correla??o positiva entre a express?o dos transportadores de N e os teores de NO3- e NH4+ nas linhagens transformadas, indicando uma poss?vel altera??o na atividade das enzimas de redu??o e ou assimila??o de N. As plantas transformadas quando submetidas ao ressuprimento com baixos teores de nitrato e am?nio apresentaram melhor resposta dos par?metros Vm?x e KM em rela??o a n?o transformadas. Nas plantas transformadas o ressuprimento com nitrato em baixa concentra??o resultou em maior express?o dos genes dos transportadores OsNTR2.1~2.2 e da prote?na OsNAR2.1 e o tratamento com suprimento constante proporcionou maior ac?mulo de nitrato nestas plantas. Os resultados tanto dos par?metros cin?ticos quanto do ac?mulo de mat?ria fresca sugerem que as plantas transformadas para express?o do OsDof25 apresentaram maior toler?ncia ao estresse nutricional.
12

Ação da insulina na captação de beta-alanina pelo músculo esquelético: efeito sobre o conteúdo de beta-alanina muscular e mecanismos envolvidos / Insulin action on beta-alanine uptake by skeletal muscle: effect on muscle beta-alanine content and mechanisms involved

Gonçalves, Lívia de Souza 23 May 2019 (has links)
A disponibilidade de beta-alanina é o fator limitante para a síntese intramuscular de carnosina. Dessa maneira, aumentar a disponibilidade de beta-alanina para o músculo esquelético é a estratégia mais eficaz para aumentar o conteúdo de carnosina muscular. Postula-se que o transportador de beta-alanina (TauT) possa ser estimulado pela insulina. Para testar essa hipótese, examinamos se a captação de beta-alanina pelo músculo esquelético de humanos é influenciada pela hiperinsulinemia, controlando as concentrações de insulina e beta-alanina no plasma através de infusão intravenosa aguda de beta-alanina. Realizamos um estudo crossover e contrabalanceado em 12 homens jovens e saudáveis (27,5±5,1 anos). Os participantes compareceram ao laboratório em duas ocasiões separadas por 10 semanas de whashout. A beta-alanina foi infundida por via intravenosa em ambos os ensaios por 150 min a uma taxa de 0,11 g.kg.min-1. Em um ensaio, a técnica de clamp euglicêmico hiperinsulinêmico foi usada para obtermos concentrações elevadas de insulina (AI), enquanto que no outro ensaio, foram mantidas concentrações de insulina em jejum (BI). Antes e 30 minutos após a infusão de beta-alanina, amostras de músculo (biópsias percutâneas) foram coletadas para determinar o conteúdo de beta-alanina e carnosina. Coletas sanguíneas foram realizadas antes (0), 10, 30, 60, 90, 120, 150 e 30 min (180) após a infusão para análise de insulina e beta-alanina plasmáticas. Urina 24 h foi coletada após o período de infusão para análise de beta-alanina. Não houve diferenças significantes entre os ensaios na concentração de beta-alanina plasmática (p=0,20), de beta-alanina muscular (p=0,72), de carnosina muscular (p=0,82) e de beta-alanina urinária (p= 0,92). A hiperinsulinemia não aumentou a captação de beta-alanina para o músculo esquelético, nem aumentou a retenção de beta-alanina corporal, pelo menos quando as concentrações de beta-alanina excederam a Vmax do TauT. Nossas descobertas sugerem que as estratégias de suplementação de beta-alanina que manipulam as concentrações de insulina provavelmente apresentam relevância clínica limitada / Beta-alanine availability is limiting for the intramuscular synthesis of carnosine. Thus, increasing beta-alanine availability to skeletal muscle is the most effective strategy to increase muscle carnosine content. It has been postulated that the transmembrane transporter of beta-alanine (TauT) could be stimulated by insulin. To test this hypothesis, we examined whether the beta-alanine uptake by human muscle is influenced by hyperinsulinemia by controlling both insulin and beta-alanine concentrations in plasma via intravenous infusion of beta-alanine. We conducted a counterbalanced crossover study in 12 young men (27.5 ± 5.1 yr). Participants attended to the laboratory on two separated occasions, 10 weeks apart. beta-alanine was intravenously infused on both trials for 150 min at a rate of 0.11 g.kg.min-1. In one trial, a hyperinsulinemic-euglycemic clamp was used to main high insulin concentrations (HI) whereas fasting insulin concentrations (LI) was maintained in the other trial. Before and 30 min after infusion, muscle samples (percutaneous biopsies) were taken to determine beta-alanine and carnosine content. Blood samples were taken before (0), 10, 30, 60, 90, 120, 150 e 30 min (180) after the infusion for plasma insulin and beta-alanine analysis. 24 h urine was colleted after infusion for beta-alanine analysis. No significant differences in plasma beta-alanine (p=0.20), muscle beta-alanine (p=0.72), muscle carnosine (p=0.82) and urinary beta-alanine (p=0.92) were shown between conditions. Hyperinsulinemia did not increase beta-alanine uptake to muscle tissue and bodily tissues, nor did it increase whole-body beta-alanine retention, at least when beta-alanine concentrations exceed the Vmax of TauT. Our findings suggest that beta-alanine supplementation strategies that maniupulate insulin concentrations are probably of limited clinical relevance
13

O transportador ABC de Trypanosoma cruzi TcABCG1 potencialmente envolvido na resistência a benznidazol: características e filogenia. / The ABC transporter of Trypanosoma cruzi TcABCG1 potentially involved in benznidazole resistance: characteristics and phylogeny.

Carvalho Junior, Jaques Franco de 29 April 2014 (has links)
Benznidazol (BZ), fármaco utilizado para o tratamento da doença de Chagas, apresenta eficácia limitada na fase crônica da doença. Falhas terapêuticas foram atribuídas majoritariamente a diferenças na suscetibilidade a BZ entre as cepas do T. cruzi. Resultados prévios de nosso grupo indicam que o gene de um transportador ABC da subfamília G, TcABCG1, encontra-se super-expresso em cepas resistentes a BZ. Transportadores ABCG foram associados a resistência a drogas em vários organismos. O objetivo central do presente estudo foi caracterizar o gene TcABCG1 em cepas de diferentes linhagens e cuja suscetibilidade a BZ foi definida. A sequência do gene TcABCG1 (1.998 pb) de 14 cepas foi determinada. Observamos algumas variações de aminoácidos na proteína ABC entre as cepas. Análises genealógicas de TcABCG1 definiram quatro clados (TcI, TcII, TcIII e Tcbat). Os dois haplótipos das cepas híbridas TcV e TcVI agruparam com os clados TcII e TcIII. Dados de imunofluorescência indireta em epimastigotas indicam que TcABCG1 está localizado em vesículas intracelulares. / Benznidazole (BZ), drug employed for Chagas disease treatment, has limited efficacy in the chronic phase of the disease. Treatment failures have been attributed mostly to differences in BZ susceptibility among T. cruzi strains. Previous data from our group indicate that one ABC transporter gene of the G subfamily, named TcABCG1, is overexpressed in BZ-resistant strains. ABCG transporters have been associated to drug resistance in several organisms. The central goal of the present study was to characterize TcABCG1 gene in strains belonging to different lineages and of defined BZ susceptibility. TcABCG1 gene sequence (1,998 bp) of 14 strains was determined. Few amino acid substitutions were detected in the ABC transporter protein among the strains. Genealogic analyses of TcABCG1 showed four distinct clades (TcI, TcII, TcIII and Tcbat). The two haplotypes of TcV and TcVI hybrid strains clustered with TcII and TcIII clades. Indirect immunofluorescence analysis in epimastigote forms indicated that TcABCG1 is localized to intracellular vesicles.
14

Estudo de transportador de poliaminas, PotD, e seus híbridos como antígenos vacinais contra Streptococcus pneumoniae. / The study of the polyamine transporter, PotD, and it hybrids as vaccine antigens against Streptococcus pneumoniae.

Converso, Thiago Rojas 10 February 2017 (has links)
A Proteína Transportadora de Poliaminas (PotD) é um antígeno importante para a virulência de Streptococcus pneumoniae in vivo, capaz de proteger camundongos imunizados contra infecção sistêmica, além de reduzir a colonização da nasofaringe dos animais. Porém, visando ampliar a cobertura vacinal, a combinação com outros antígenos da bactéria se faz necessária. Este trabalho teve como objetivo aprofundar o estudo sobre a resposta imune gerada contra a proteína PotD, sozinha ou em fusão com duas outras proteínas pneumocócicas: o derivado de Pneumolisina, PdT, e a proteína de superfície de pneumococo A (PspA). Para tanto, os genes potD, pdT e pspA foram clonados e expressos, sozinhos ou fusionados, gerando as proteínas híbridas rPotD-PdT e rPspA-PotD. As proteínas recombinantes e os híbridos foram utilizados na imunização subcutânea de camundongos BALB/c, gerando elevados níveis de anticorpos. O soro dos animais imunizados foi capaz de reconhecer e se ligar à superfície de diferentes isolados de pneumococos, e de ampliar a fagocitose da bactéria por células peritoneais murinas in vitro. Em todos os ensaios, os híbridos se mostraram mais eficazes do que as proteínas isoladas, induzindo anticorpos capazes de potencializar a fagocitose dos pneumococos. A resposta imune celular foi caracterizada pela produção de INF-γ, IL-2 e IL-17 pelos esplenócitos, e um aumento na produção de NO pelos fagócitos peritoneais dos animais imunizados. Apesar dos resultados promissores in vitro, a proteína rPotD-PdT não foi capaz de induzir proteção em nenhum dos modelos avaliados; em contraste, a fusão rPspAPotD foi capaz de proteger os camundongos contra sepse por dois isolados virulentos de pneumococo, além de reduzir a colonização na nasofaringe. Por fim, demonstramos que a adição das poliaminas transportadas por PotD, espermidina e putrescina, à cultura de pneumococos interfere na formação de biofilme in vitro. Cnsiderando o importante papel da formação de biofilmes na colonização, este resultado sugere um possível mecanismo de ação da PotD durante a colonização por pneumococo. Em conjunto, os resultados deste estudo sugerem que a utilização de uma formulação híbrida, rPspA-PotD, compreende uma estratégia vacinal promissora, capaz de proteger contra colonização e sepse pneumocócica, pela produção de anticorpos opsonizantes e ativação de citocinas protetoras, como IL-17. / Polyamine Transporter D (PotD) is an important antigen for Streptococcus pneumoniae virulence in vivo, protecting immunized mice against systemic infection and reducing the bacterial load in the nasopharynx of immunized animals. However, in order to extend vaccine coverage, the combination of PotD with other antigens of the bacterium is required. The present study aimed at expanding the investigation of the immune response generated against PotD alone or fused with two other pneumococcal proteins: the Pneumolysin derivative, PdT and Pneumococcal Surface Protein A (PspA). Therefore, the potD, pdt and pspA genes were cloned and expressed, either alone or in fusion, generating the hybrid proteins rPotD-PdT and rPspA-PotD. The recombinant proteins and hybrids were used for subcutaneous immunization of BALB/c mice, generating high levels of antibodies. Sera from immunized animals were able to recognize and bind onto the surface of different pneumococcal strains, and to enhance phagocytosis of the bacterium in vitro. In all tests, the hybrids were more effective than the isolated proteins. The cellular immune response was characterized by the production of INF-γ, IL-2 and IL-17 by splenocytes and increased production of NO by peritoneal cells of the immunized animals. Despite promising results in vitro, rPotD-PdT protein was not able to induce protection in any of the tested challenge models. In contrast, rPspA-PotD fusion was able to protect mice against sepsis with two virulent isolates of pneumococcus and led to reduction in bacterial loads in the nasopharynx of challenged animals. Finally, we demonstrate that the addition of exogenous polyamines, spermidine, and putrescine, in the pneumococcal culture interfered with biofilm formation in vitro. Considering the important role of biofilm formation for successful colonization, this result suggests a possible mechanism of action of PotD during colonization by pneumococcus. Taken together, the results suggest that the use of the hybrid rPspA-PotD comprises a promising vaccine strategy, able to protect against colonization and pneumococcal sepsis, through the production of opsonizing antibodies and activation of protective cytokines, such as IL-17.
15

Síntese, caracterização e estudos em solução de terpolímeros antififílicos termo-sensíveis ao pH: uma perspectiva para obtenção de sistemas transportadores de fármacos

Blaz Vieira, Neide Aparecida [UNESP] 17 February 2006 (has links) (PDF)
Made available in DSpace on 2014-06-11T19:30:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-02-17Bitstream added on 2014-06-13T21:01:34Z : No. of bitstreams: 1 blazvieira_na_dr_sjrp.pdf: 2164319 bytes, checksum: f4c71268208cbd270c732d15862f8233 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A presente tese envolveu a síntese e caracterização de duas séries de polímeros anfifílicos inteligentes. A utilização de quatro monômeros foi feita com o objetivo de se conferir aos polímeros sensibilidade à temperatura e ao pH . Na primeira série, ou série 1, foram sintetizados terpolímeros anfifílicos termo-sensíveis a partir dos monômeros Nisopropilacrilamida (NIPAm), monometacrilato de poli(etilenoglicol)metil éter (PEG) e ndodecilmetacrilato (DOMA), cuja termo-sensibilidade foi controlada variando-se a proporção molar dos monômeros. Os polímeros foram caracterizados utilizando-se as técnicas de ressonância magnética nuclear e cromatografia de permeação em gel (GPC). As propriedades das soluções foram estudadas empregando-se espectrofotometria, fluorescência e espalhamento de luz dinâmico. Os polímeros exibiram temperatura de turvação em solução aquosa entre 17 e 52ºC, dependendo da composição. A proporção do monômero hidrofílico (PEG) foi variada com o objetivo de se obter termo-sensibilidade próxima à temperatura do corpo humano (37° C). O aumento da força iônica das soluções pela adição de KCl reduziu a temperatura de turvação pelo efeito salting out. Os polímeros formaram, em solução aquosa, microdomínios hidrofóbicos, cuja CAC variou de 1,2x10-3 a 1x10-2 g dm-3 , com o aumento da proporção molar de PEG de 5 a 35%. Os agregados hidrofóbicos tipo micelar apresentaram um tamanho que variou de 50 a 140 nm, dependendo da proporção dos monômeros, e mostraram capacidade para incorporar moléculas hidrofóbicas, como pireno e doxorubicina. As quantidades máximas incorporadas foram de, respectivamente, 6,1 mg/g polímero e 13,5 mg/g polímero para pireno e doxorubicina. Agregados mais hidrofóbicos incorporaram uma quantidade maior do hidrófobo. / In the present thesis the synthesis and characterization of two series of intelligent polymers were performed aiming to construct potential drug carriers. Four monomers were employed to provide thermo and pH sensitivity to the systems. In the first series terpolymers of N-isopropylacrylamide, dodecyl methacrylate (DOMA) and poly(ethylene glycol)(PEG) methacrylate, were synthesized by random copolymerization, and the composition was controlled to achieve systems having different thermosensitivities. 1HNMR spectra and gel permeation chromatography (GPC) were employed to characterize the different samples obtained. The solution properties were studied by employing spectrophotometry, fluorescence, and dynamic light scattering techniques. The chemical compositions in the final terpolymers are close to those in the feed and the composition was controlled aiming to adjust the thermosensitivity close to that of the human body temperature (370C). The polymers exhibited cloud point temperatures (Tcs) varying from 17 to 52 0C. Micropolarity studies using I1/I3 ratio of the vibronic bands of pyrene show the formation of amphiphilic aggregates capable of incorporating hydrophobic drugs as the polymer concentration is increased. The critical aggregation concentration (CAC) increases from 1.2x10-3 to 1x10-2 g.L-1 with the PEG content varying from 5 to 35 mol%. Anisotropy measurements confirm the results obtained by pyrene fluorescence and show that the aggregates resulting from intermolecular interactions present different organizations. The hydrodynamic diameters (Dh) of the aggregates determined by dynamic light scattering (DLS) vary from 40 to 150 nm depending on the terpolymer composition. The Tcs and Dh values decreased with the ionic strength, and this behavior was attributed to the dehydration of the polymeric micelles. The capacity of solubilization of the aggregates was evaluated by employing pyrene and adryamicin and the obtained results confirm th
16

Estudo da imunoexpressão dos transportadores de glicose 1 e 3 e do índice angiogênico em tumores odontológicos ceratocísticos isolados e associados à Síndrome de Gorlin

Leite, Rafaella Bastos 30 July 2014 (has links)
Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2016-03-15T13:08:48Z No. of bitstreams: 1 PDF - Rafaella Bastos Leite.pdf: 1326501 bytes, checksum: 57e6fffb3bbe74433cba4f469a5b6545 (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2016-07-21T21:05:02Z (GMT) No. of bitstreams: 1 PDF - Rafaella Bastos Leite.pdf: 1326501 bytes, checksum: 57e6fffb3bbe74433cba4f469a5b6545 (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2016-07-21T21:05:12Z (GMT) No. of bitstreams: 1 PDF - Rafaella Bastos Leite.pdf: 1326501 bytes, checksum: 57e6fffb3bbe74433cba4f469a5b6545 (MD5) / Made available in DSpace on 2016-07-21T21:05:12Z (GMT). No. of bitstreams: 1 PDF - Rafaella Bastos Leite.pdf: 1326501 bytes, checksum: 57e6fffb3bbe74433cba4f469a5b6545 (MD5) Previous issue date: 2014-07-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The keratocystic odontogenic tumor (KOT) stands out among the other odontogenic lesions in view of the potentially aggressive biological behavior and its association in some cases, with the Gorlin syndrome. Some studies have suggested a more aggressive biological behavior for KOTs associated with Gorlin syndrome, compared to isolated KOTs, characterized by greater growth capacity and bone infiltration and higher tendency to recur. The present study aimed to evaluate, descriptively and comparatively, by means of immunohistochemistry, the expression of glucose transporter-1 (GLUT-1) and -3 (GLUT-3) and the angiogenic index (CD34) in isolated primary and recurrent KOTs and in KOTs associated with Gorlin syndrome. The sample was composed by 21 isolated KOTs (14 primary and 7 recurrent) and 14 KOTs associated with Gorlin syndrome. The expression of GLUTs was evaluated in the epithelial component of the lesions, establishing the percentage of immunopositive cells, according to the scores: score 0 (negative), score 1 (≤ 25% of positive cells), score 2 (26% - 50% of positive cells), score 3 (51% - 75% of positive cells), and score 4 (≥ 76% positive cells). For the angiogenic index, the microvessel count (MVC) technique was applied, quantifying the microvessels immunoreactive to anti-CD34 antibody. Regarding the median scores for immunopositivity for GLUT-1 and the angiogenic index, comparisons between groups were performed using the nonparametric Kruskal-Wallis test. For GLUT-3, the data obtained from the evaluation of epithelial expression of this protein were submitted to descriptive statistical analysis. Possible correlations between the scores of immunopositivity for GLUT-1 and angiogenic index in the lesions were evaluated using the Spearman correlation test. The level of significance was set at 5% (p <0.05). The analysis of epithelial GLUT-1 immunoreactivity revealed predominance of score 4 in isolated primary KOTs (n = 9, 64.3%) and in KOTs associated with Gorlin syndrome (n = 8; 57.1%). In isolated recurrent KOTs, it was identified a slightly higher frequency of cases with scores 4 (n = 3; 42.9%) and 2 (n = 2; 28.6%). The nonparametric Kruskal-Wallis test showed no statistically significant difference between groups (p = 0.406). Regarding the GLUT-3, all groups showed higher frequency of negative cases. The few KOTs positive for GLUT-3 were classified as score 1 (≤ 25% of positive cells), showing a low expression of this protein in the epithelial component. The mean number of microvessels was 63.80 in isolated primary KOTs, 61.11 in KOTs associated with the Gorlin syndrome, and 65.88 in isolated recurrent KOTs, without significant differences between groups (p = 0.965). The results of this study suggest that the differences in biological behavior of isolated KOTs and KOTs associated with Gorlin syndrome may not be related to the expression of GLUTs-1 and -3, or to the angiogenic index in the lesions. The high expression of GLUT-1 in KOTs suggests an important role for this protein in glucose uptake by the epithelial cells of these tumors. / O tumor odontogênico ceratocístico (TOC) se destaca entre as demais lesões odontogênicas em virtude do comportamento biológico potencialmente agressivo e por sua associação, em alguns casos, à síndrome de Gorlin. Pesquisas tem sugerido um comportamento biológico mais agressivo para os TOCs associados à síndrome de Gorlin, em comparação aos TOCs isolados, caracterizado por maior capacidade de crescimento e infiltração óssea e maior tendência a recorrência. O presente estudo se propôs a avaliar, descritiva e comparativamente, a imunoexpressão dos transportadores de glicose-1 (GLUT-1) e -3 (GLUT-3) e o índice angiogênico (CD34) em TOCs isolados primários e recorrentes e TOCs associados à síndrome de Gorlin. A amostra foi composta por 21 TOCs isolados (14 primários e 7 recorrentes) e 14 TOCs associados à síndrome de Gorlin. A expressão dos GLUTs foi avaliada no componente epitelial das lesões, estabelecendo-se o percentual de células imunopositivas, de acordo com os escores: escore 0 (negativo), escore 1 (≤ 25% das células positivas), escore 2 (26% - 50% das células positivas), escore 3 (51% - 75% das células positivas) e escore 4 (≥76% das células positivas). Para o índice angiogênico, foi empregada a técnica da contagem microvascular (MVC), quantificando-se os microvasos imunomarcados pelo anticorpo anti-CD34. Em relação às medianas para os escores de imunopositividade para GLUT-1 e para o índice angiogênico, as comparações entre os grupos foram realizadas por meio do teste não paramétrico de Kruskal-Wallis. Para o GLUT-3, os dados obtidos com a avaliação da expressão epitelial desta proteína foram submetidos apenas à análise estatística descritiva. Possíveis correlações entre os escores de imunopositividade para GLUT-1 e o índice angiogênico nas lesões foram avaliadas por meio do teste de correlação de Spearman. O nível de significância foi estabelecido em 5% (p < 0,05). A análise da imunoexpressão epitelial de GLUT-1 revelou predomínio de casos com escore 4 nos TOCs isolados primários (n = 9; 64,3%) e nos TOCs associados à síndrome de Gorlin (n = 8; 57,1%). Nos TOCs isolados recorrentes, foi identificada frequência discretamente maior para os casos com escores 4 (n = 3; 42,9%) e 2 (n = 2; 28,6%). O teste não paramétrico de Kruskal-Wallis revelou ausência de diferença estatisticamente significativa entre os grupos (p = 0,406). Em relação ao GLUT-3, todos os grupos estudados revelaram maior frequência de casos negativos. Os poucos TOCs positivos para GLUT-3 foram classificados como escore 1 (≤ 25% das células positivas), revelando uma baixa expressão desta proteína no componente epitelial. O número médio de microvasos foi de 63,80 nos TOCs isolados primários, 61,11 nos TOCs associados a síndrome de Gorlin e 65,88 nos TOCs isolados recorrentes, sem diferenças significativas entre os grupos (p = 0,965). Os resultados do presente estudo sugerem que as diferenças no comportamento biológico de TOCs isolados e TOCs associados à síndrome de Gorlin não foram relacionadas com a expressão de GLUTs-1 e -3 ou com o índice angiogênico (CD34) nas lesões. A alta expressão de GLUT-1 em TOCs sugere um importante papel para esta proteína na captação de glicose pelas células epiteliais destes tumores.
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Estudo da homeostase de zinco no fungo patogênico Histoplasma capsulatum var. capsulatum: Abordagens proteômica e funcional / Study of zinc homeostasis in the pathogenic fungus Histoplasma capsulatum var. capsulatum: Proteomic and functional approaches

Siqueira, Janaina Gomes de 30 November 2016 (has links)
Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-01-09T12:02:26Z No. of bitstreams: 2 Dissertação - Janaina Gomes de Siqueira - 2016.pdf: 2965200 bytes, checksum: 015ab0c28746d09363a65c8817dd4068 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-01-09T12:03:04Z (GMT) No. of bitstreams: 2 Dissertação - Janaina Gomes de Siqueira - 2016.pdf: 2965200 bytes, checksum: 015ab0c28746d09363a65c8817dd4068 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-01-09T12:03:04Z (GMT). No. of bitstreams: 2 Dissertação - Janaina Gomes de Siqueira - 2016.pdf: 2965200 bytes, checksum: 015ab0c28746d09363a65c8817dd4068 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-11-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Histoplasma capsulatum var. capsulatum is a pathogenic fungus that causes the systemic disease known as histoplasmosis. This mycosis presents endemicity in the regions of the Mississippi and Ohio rivers in the United States, also found in several countries of South America. In Brazil, the number of people infected by this fungus has been growing in recent years, especially in immunocompromised people. H. capsulatum is a thermodymorphic microorganism, found in two morphotypes: mycelium at 25ºC and yeast at 37ºC. During the infection process the pathogenic microorganism needs to obtain nutrients so that it grows and develops within the tissue of the host. Among the essential nutrients, zinc is an essential metal ion, used in the maintenance of numerous metabolic pathways. Zinc is used as catalytic and structural cofactor of numerous metalloproteins. Zinc uptake is characterized as a virulence factor in H. capsulatum. In order to study the molecular mechanisms of zinc homeostasis in H. capsulatum, the in silico analysis of zinc homeostasis related proteins. The ZafA transcription factor, which acts on the regulation of genes in response to the zinc level in the medium and the transcription factor PacC that regulates genes in response to pH, were also found. ZrfA and ZrfC membrane transporters were also found ZrfF, ZrcA and ZrcC vacuolar transporters. The transcriptional profile analysis of these genes was measured by RT-PCR in infected macrophages and in vitro. During the infection the ZafA and ZrfA genes were induced, suggesting that these genes were expressed in order to supply the fungal cell with zinc, due to the metal deficiency caused by the infection. In vitro, ZafA, ZrfC, ZrfF, ZrcA and ZrcC were induced at zinc deprivation condition, compared with control condition. According with proteomic data of fungus under conditions of zinc deprivation, it was suggested the induction of proteins related to rescue, defense and virulence, related mainly to oxidative stress, besides other metabolic pathways such as gluconeogenesis. These data suggest that the fungus undergoes a metabolic remodeling in response to the oxidative stress caused by zinc deprivation. / Histoplasma capsulatum var. capsulatum é um fungo patogênico causador da doença sistêmica conhecida como histoplasmose. Essa micose apresenta endemicidade nas regiões do rio Mississipi e Ohio nos Estados Unidos, encontrado também em vários países da América do Sul. No Brasil, o número de pessoas infectadas por este fungo vem crescendo nos últimos anos, principalmente em pessoas imunocomprometidas. H. capsulatum é um micro-organismo termodimórfico, encontrado em dois morfotipos: micélio a 25ºC e levedura a 37ºC. Durante o processo de infecção o micro-organismo patogênico necessita obter nutrientes para que cresça e se desenvolva dentro do tecido do hospedeiro. Dentre os nutrientes essenciais, o zinco é um íon metal essencial, utilizado na manutenção de inúmeras vias metabólicas. O zinco é utilizado como cofator catalítico e ou estrutural de inúmeras metaloproteínas. A captação de zinco é caracterizada como um fator de virulência em H. capsulatum. Com o objetivo de estudar os mecanismos moleculares da homeostase de zinco em H. capsulatum, foi realizada primeiramente a análise in silico das proteínas relacionadas a homeostase de zinco. Foram encontrados o fator de transcrição ZafA que atua na regulação de genes em resposta ao nível de zinco no meio e o fator de transcrição PacC que atua na regulação de genes em resposta ao pH, adicionalmente foram encontrados transportadores de membrana ZrfA e ZrfC, além dos transportadores vacuolares ZrfF, ZrcA e ZrcC. A análise do perfil transcricional destes genes foi mensurada por RT-PCR em macrófagos infectados e in vitro. Durante a infecção os genes ZafA e ZrfA foram induzidos, sugerindo que estes genes foram expressos a fim de suprir a célula fúngica com zinco, devido a deficiência do metal causada pela infecção. In vitro, ZafA, ZrfC, ZrfF, ZrcA e ZrcC foram induzidos na condição de privação de zinco, comparado com a condição controle. De acordo com os dados proteômicos do fungo em condições de privação zinco, foi sugerido a indução de proteínas relacionadas ao resgate, defesa e virulência, relacionadas principalmente ao estresse oxidativo, além de outras vias metabólicas como gliconeogênese. Estes dados sugerem que o fungo sofre um remodelamento metabólico em resposta ao estresse oxidativo causado pela privação de zinco.
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Caracterización génica y bioquímica de una ciclodextrin glucanotransferasa, enzima implicada en el metabolismo del almidón en Haloferax mediterranei

Bautista, Vanesa 07 June 2010 (has links)
No description available.
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The first 3D structural model of an eukaryotic heteromeric aminoacid transporter / Primer model estructural en 3D d’ un transportador heteromèric d’aminoàcids eucariota

Costa i Torres, Meritxell 04 May 2012 (has links)
Introduction Heteromeric amino acid transporters (HATs) are composed of a heavy subunit (rBAT or 4F2hc) and a light subunit (b0 + AT, ASC1, LAT1, LAT2, y + LAT1, y + LAT2 and xCT), joined by a disulfide bridge (Chillaron et al. 2001). rBAT and 4F2hc are type II membrane glycoproteins (N-terminal cytoplasmic). Both have a single transmembrane segment, an N-terminal intracellular tail and an extracellular domain (ectodomain). As far as we know, the role of the heavy subunit is facilitating the transit of the light subunit to the plasma membrane. The light subunits are polytopic proteins unglycosylated, with 12 transmembrane segments, and the N-and C-terminal intracellular. The light subunit is the catalytically active subunit which confers specificity to the heterodimer on the transport system (Reig et al. 2002): LAT1 and LAT2 for system L , y+LAT1 and y+LAT2 for system y + L, asc for system ASC1; xCT for system Xc -, and b0+at for system b0, + (Chillaron et al. 2001). Results Overexpression of these human proteins was carried out with the methylotrophic yeast Pichia pastoris (strain KM71H) as expression system (based on Long et al. 2005). The main objective was to generate enough protein in a high level of purity to study the structure and check their function by transport assays. The different subunits, light and heavy, were cloned into the expression vector pPICZ (Invitrogen). To facilitate the purification of the different proteins, a cluster of 10 histidines was introduced by PCR at the N-terminus of the heavy subunits and a StrepTagII (IBA) at the N-terminus of the light subunits. 4F2hc is a glycoprotein with 4 possible targets for glycosylation. The glycosylations confer heterogeneity to protein, thus glycosylation targets were eliminated by directed mutagenesis. From all these human heavy and light subunits and heterodimers, only 4F2hc for the heavy subunits, LAT2 for the light subunits, and the heterodimer 4F2hc/LAT2 were overexpressed and extracted from the yeast membrane in enough amounts to continue with the purification step. The light subunit LAT2 was successfully purified but when the stability was analysed by size exclusion chromatography showed a clear profile of aggregation, concluding further studies. In contrast, the heavy subunit 4F2hc was stable after the exclusion chromatography for two days. The heterodimer 4F2hc/LAT2 proved to be stable after gel filtration analysis during one day. Thus, the heterodimer was significantly more stable than the light subunit alone, which allowed us to make an important statement. The catalytic subunit LAT2 needed their heavy subunit (i.e. 4F2hc) to increase the stability. This statement contrasted with the results for the heterodimer rBAT/b0+AT, in which was the heavy subunit rBAT the one who needed its light subunit b0+AT to a correct folding during its biogenesis (Bartoccioni et al. 2008; Rius et al. 2011). Functional studies with human heterodimer 4F2hc/LAT2 were set up to check the role of 4F2hc in the transport. Firstly the functionality of the heterodimer 4F2hc/LAT2 and the light subunit LAT2 in the living cell was checked successfully, meaning a correct folding at expression level. The apparent KM in both cases was the same, remaining unanswered the role of the heavy subunit 4F2hc in the transport function. Next, reconstitution in liposomes was carried out successfully for 4F2hc/LAT2 but not for LAT2, due to the high aggregation tendency. 4F2hc/LAT2 showed the typical overshoot for an amino acid transporter. To carry out the structural studies and due to the difficulty to maintain a stable soluble heterodimer, it was decided to carry out the technique of Single particle -negative staining (SP-NS) in the laboratory of Prof. Fotiadis in the University of Bern (Switzerland). The 3D model technique based on transmission electron microscopy (TEM) is relatively new and has been imposed for mammalian membrane proteins, allowing structural analysis with relatively small concentration of protein. The pure heterodimer was stained in a grid with uranyl formate at 0.75% (two drops optimized for 1 second, washing with water twice). This sample was analysed by transmission electron microscopy (TEM). Different images of projections in different orientations for 4F2hc/LAT2 were kept in a library of 11,000 picks. The refinement of the whole library allowed the 3D reconstruction of this protein by Mr. Meury. The model showed two asymmetric particles, one smaller, in which the crystal of the human ectodomain 4F2hc (Fort et al. 2007) fitted pretty well, and other bigger, which showed a black hole. Thus, the smaller particle was recognized as the heavy subunit, located on top of the light subunit. The resolution was 19 amstrongs, which was in the normal range for this method (from 16 amstrongs to 25 amstrongs). Discussion It was observed that the heavy subunit was located on top of the light subunit LAT2, and not in contact with the cell membrane as was firstly though. The size for the heavy subunit coincided with the existing 3D crystals of the human ectodomain which can fit quite accurately, always assuming the presence of the transmembrane segment in the 3D model. By contrast, the light subunit did not fit with the crystal structure of the prokaryotic homolog AdiC in the APA family (APC superfamily) (Gao et al. 2009) (Kowalczyk et al. 2011) due to the large amount of detergent surrounding this highly hydrophobic subunit in SP-NS method. In spite of that, when the size was compared with AdiC and Stet (a prokaryotic homolog in the LAT family with 30% of homology) studied in the same SP-NS method (Casagrande et al. 2009) the light subunit LAT2 coincided in size with its homologs, demonstrating that the increased volume was due to the detergent effect. Supporting the 4F2hc/LAT2 model, interaction studies with integrins (Feral et al 2005; Feral et al. 2007) and other membrane proteins involved in cell growth (ICAMI; Liu et al. 2003) and / or overexpressed in tumours (CD147/MCT1; Xu et al. 2005) suggest an effect in the transport function through the heavy subunit 4F2hc, which may be explained with an orientation on top of the light subunit and interaction by the external loops. New Evidences: Recently, the 4F2hc/LAT2 heterodimer model in which the heavy subunit is located on top of the light subunit has been corroborated by cross-linking experiments by Miss Helena Alvarez in our laboratory. This fact, allow us to imagine how interactions between both subunits will carry out also when the disulphide bridge is missing. Analyzing the external loops in AdiC atomic structure (the closest paradigm with LATs at present) is found that the external loop 3 and the external loop 4 are the longest (around 25 residues). These loops are even longer in LAT2, which make possible the interaction between both subunits being the separation of 16 amstrongs in the 3D model. Both loops have important roles in the transport cycle based in LeuT fold. The external loop 3 has an important movement in the transition from outward-open conformation to occluded-outward conformation due to the tilt of 40o of the transmembrane 6. The external loop 4, moves down to lid the substrate pathway during the transition from occluded-outward conformation to the occluded-inward conformation. Our new 3D model of a human heteromeric aminoacid transporter offers the opportunity to study new aspects about the role of the heavy subunit in the holotransporter. If the external loops join 4F2hc and LAT2 modulating the transport function in presence of other transmembrane proteins, or if 4F2hc only acts as a bollard of a multiproteic complex, rest to be studied in the future. / Els transportadors heteromèrics d'aminoàcids (HATS) de metazous estan formats per una subunitat pesada (4F2hc o rBAT) (N-glicoproteïna amb 1 segment transmembrana i un gran ectodomini en el seu extrem C-terminal), i una subunitat lleugera (d'entre 10) unides covalentment per un pont disulfur, fent aquests transportadors únics entre els metazous. En humans, 6 subunitats lleugeres es troben formant heterodímers amb 4F2hc (LAT1, LAT2, y+ LAT1, y + LAT2, XCT i asc1) i una (b0, + AT) amb rBAT. Els HATs tenen incidència en la salut, ja que mutacions en qualsevol de les subunitats ocasionen aminoacidúries (cistinúria, lisinúria amb intolerància a proteïnes), són receptors virals o estan sobre expressats en cèl • lules tumorals. El nostre grup va determinar l'estructura de l'ectodomini de 4F2hc a 2.1 Å (Fort J et al. 2007), i recentment ha resolt l'estructura d'un homòleg procariota (AdiC d' E. coli, amb ~17% d´homologia) de les subunitats lleugeres a 3.0 Å de resolució (Kowalczyk et al. 2011). Per contra no hi ha informació estructural sobre els holo-transportadors HAT. El present treball ens mostra el primer model estructural a 19 Å d'un transportador HAT humà, el transportador 4F2hc/LAT2. La importància de 4F2hc, a part de tenir un paper important en immunologia, es troba en la seva sobreexpressió en cèl•lules tumorals, el que la converteix en una important diana per a tractaments i desenvolupament de vacunes contra el càncer. El model ens mostra com en aquest transportador, l´ectodomini de 4F2hc està situat sobre LAT2, suggerint interacció amb els bucles extracel•lulars del transportador i nos sols interacció a través del pont disulfur del segment transmembrana com es pensava anteriorment. Aquesta nova topologia explica la necessitat i la importància de que l'ectodomini de 4F2hc formi part de l´heterodímer 4F2hc/LAT2 i presenta un escenari estructural per al paper "chaperone-like" de 4F2hc sobre les subunitats lleugeres.
20

Diagnóstico clínico, bioquímico y molecular en pacientes con defecto del transportador de creatina. Opciones terapéuticas

Fons Estupiñá, M. Carmen 19 February 2010 (has links)
Los defectos de creatina cerebral (DCC) constituyen un grupo de enfermedades neurometabólicas hereditarias, descritas recientemente y hasta ahora infradiagnosticadas, tanto por la falta de procedimientos adecuados para su diagnóstico, como por el amplio e inespecífico espectro de síntomas con que se manifiestan.Se ha reportado que el defecto de transportador de creatina (CRTR) constituye una de las causas más frecuentes de retraso mental ligado al cromosoma X. Por ello, uno de nuestros objetivos ha sido determinar la prevalencia en nuestro medio del defecto de CRTR, mediante el cribaje de metabolitos de la Cr en orina en una población de pacientes afectos de retraso mental y/o autismo. Además se realizaron estudios bioquímicos para evaluar qué factores dietéticos pueden influir en la alteración de los resultados bioquímicos en orina y así dificultar el diagnóstico bioquímico de esta entidad.Posteriormente con el objetivo de incrementar el conocimiento del fenotipo clínico en pacientes con defecto de CRTR, evaluamos específicamente las características de la epilepsia, síntoma frecuente y que asocia complicaciones graves. Hemos estudiado los mecanismos fisiopatogénicos de la epilepsia y la eficacia de los respectivos tratamientos antiepilépticos además de evaluar la correlación fenotipo-genotipo respecto a la gravedad de la epilepsia.Dado que el defecto de CRTR, es una enfermedad que no dispone de tratamiento efectivo en la actualidad, gran parte de nuestro trabajo se ha basado en ensayar diferentes estrategias terapéuticas en pacientes con defecto de CRTR, y evaluar la respuesta a estos tratamientos.

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