Dissertations / Theses on the topic 'Citroe n'

Orientadores: Alessandra Alves de Souza, Marco Aurélio Takita
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Xylella fastidiosa é uma bactéria Gram-negativa que causa várias doenças em diferentes espécies de plantas, como a clorose variegada dos citros (CVC) em Citrus sinensis, cujos danos econômicos são da ordem de milhões de dólares anuais. O desenvolvimento dos sintomas tem sido associado ao bloqueio dos vasos do xilema causado pela formação de um biofilme pela bactéria. Recentemente foi verificado que o biofilme de X. fastidiosa é mais resistente a compostos antimicrobianos que a forma planctônica. Essa resistência tem se mostrado um fenômeno complexo que não pode ser explicado por um único mecanismo, e sim por multifatores. Nesse sentido, a proposta desse trabalho foi identificar, por microarranjos de DNA, genes possivelmente associados à adaptação do biofilme na presença de compostos antimicrobianos, em concentrações letais para células em crescimento planctônico, mas que não inibiram o crescimento do biofilme. Foram encontrados 223 (7,87%) CDS induzidas na presença de cobre e 150 (5,29%) reprimidas. Com tetraciclina foram 450 (15,89%) induzidas e 449 (15,85%) reprimidas. Muitas sequências codificadoras envolvidas com funções da síntese proteica, metabolismo energético, divisão celular e movimentação foram moduladas negativamente em ambas as situações, sugerindo que tais modificações contribuiriam para um provável estado de resistência. Com a adição da dose subinibitória de cobre também foi observada a indução de genes relacionados à adesão e produção de toxinas, que estão envolvidos com a virulência da bactéria em planta, o que sugere esta seria aumentada possivelmente induzindo maior severidade de sintomas. Já com tetraciclina o oposto foi observado, repressão de genes relacionados à formação do biofilme e produção de toxinas. No entanto, identificamos a indução de um possível mecanismo de resposta SOS, pela qual genes relacionados ao sistema toxina-antitoxina seriam superexpressos. Esse sistema provavelmente está envolvido com a morte celular programada e formação de células persistentes. Assim é possível concluir que doses subinibitórias de compostos antimicrobianos poderiam ao invés de matar, induzir a virulência da bactéria, como ocorrido com o cobre, ou a formação de células persistentes, como observado com a tetraciclina. Outra abordagem desse trabalho foi à realização de experimentos in vitro e in vivo com um análogo de cisteína, o N-acetyl-L-cysteina (NAC), que já vem sendo utilizado na medicina para desestruturação de biofilmes de bactériaspatógenas de humanos. Resultados da quantificação da massa celular, número de célulasviáveis e exopolissacarídeos totais revelaram que todas as doses de NAC (1, 2 e 6 mg/mL) testadas nos experimentos in vitro diminuiram a formação do biofilme e inibiram o crescimento de X. fastidiosa, o que indica um possível efeito tóxico dessa substância. Nos experimentos in vivo foi observada uma grande redução dos sintomas de CVC em plantas de C. sinensis infectadas e tratadas com diferentes doses de NAC. Esses estudos abrem uma real perspectiva ao uso dessa substância para o manejo da CVC.
Abstract: Xylella fastidiosa is a Gram-negative bacterium that causes several diseases in different plant species, including citrus variegated chlorosis (CVC) in sweet orange (Citrus sinensis L. Osbeck), whose economic damage is of millions of dollars annually. The symptoms development has been associated with the blockage of xylem vessels caused by bacterial biofilm formation. Recently it was found that the biofilm of X. fastidiosa is more resistant to antimicrobial compounds than the planktonic cells. This resistance has been considered as a complex phenomenon that cannot be explained by a single mechanism, but by multi-factors. Accordingly, the intent of this study was to identify through DNA microarray technology, genes possibly involved in adaptation of biofilm cells to the presence of antimicrobial compounds in concentrations that are lethal to cells in planktonic growth, but do not inhibit the cell growth in biofilm. We found 223 (7.87%) genes induced in presence of copper and 150 (5.29%) genes repressed. For tetracycline, there were 450 (15.89%) induced genes and 449 (15.85%) repressed ones. Many genes encoding proteins related to protein synthesis, energy metabolism and cell division were negatively modulated in both, copper and tetracycline treatments, suggesting that these changes could contribute to a state of resistance. When a subinhibitory dose of copper was applied we could also observe the induction of genes related to adhesion and thus biofilm formation and toxin production, suggesting that the bacterial virulence should be increased. The opposite was found for tetracycline. However, we observed the induction of a possible SOS response mechanism in which genes related to a toxin-antitoxin system was overexpressed. This system is probably involved with programmed cell death and formation of persistent cells. We then concluded that subinhibitory doses of antimicrobial compounds could induce bacterial virulence as occurred for copper, or the formation of persistent cells, as observed for tetracycline rather than kill the cells. Another approach of this work was to carry out experiments in vitro and in vivo with an analogue of cysteine, N-acetyl-L-cysteine (NAC), which has been already used in medicine as a drug for disruption of biofilms formed by human pathogenic bacteria. Results of cellular mass quantification, number of viable cells and total exopolysaccharide content revealed that all doses (1.0, 2.0 and 6.0 mg / mL) of NAC tested in in vitro experiments decreased the biofilm formation and inhibited growth of X. fastidiosa, which indicated that this substance could also be toxic for the bacteria. In vivo experiments showed a strong reduction in CVC symptoms in C. sinensis plants infected and treated with different doses of NAC. These studies open a real prospect for the use of this compound in CVC management.
Mestrado
Genetica de Microorganismos
Mestre em Genética e Biologia Molecular

The application of N-fertilizers mixed with a surface irrigation stream (surface N-fertigation) is widely practiced in the Yume Mesa. Guidelines for the efficient management of surface N-fertigation systems are needed. The purpose of the work reported herein is to evaluate the relative effectiveness of existing surface N-fertigation management practices in the Yuma Mesa. This has been accomplished through the following steps: (1) a complete set of performance indices that can be used to assess the relative merit of alternative management scenarios are identified and defined and Equations as well as solutions for quantifying the performance indices are proposed; (2) surface fertigation field experiments (using Br- as a tracer) were performed in two irrigation basins at the Yuma Mesa research farm of the University of Arizona during the fall season of 2000; (3) the spatial distribution as well as the application efficiency and adequacy of Br- applied with irrigation water was determined using the performance functions proposed herein; and (4) the results were analyzed to assess the merits and limitations of existing practices.

O objetivo deste trabalho foi avaliar os efeitos do biorregulador MBTA [cloridrato de N,N-dietil-2-(4-metilbenziloxi) etilamina] aplicado em diferentes épocas e concentrações sobre a produtividade e qualidade dos frutos das laranjeiras 'Hamlin', 'Valência' e 'Pêra' e em características fisiológicas da laranjeira 'Hamlin'. Em duas safras consecutivas, o MBTA foi aplicado em três diferentes concentrações (8; 16 e 32 mg L-1) e em duas diferentes fases fenológicas (25% e 100% de flores abertas) em árvores cítricas adultas, utilizando um volume de 7 litros de solução por planta acompanhada do adjuvante Silwett L-77 0,05%. Foram amostrados 20 frutos por planta em quatro diferentes épocas para cada cultivar estudado para determinar o teor de sólidos solúveis totais; acidez; quantidade de sólidos solúveis totais em caixa industrial de 40,8 kg; pH; rendimento de suco; cor do suco, massa média e diâmetro médio dos frutos. Os efeitos do MBTA variaram de acordo com o cultivar, concentração aplicada e com a fase fenológica de aplicação. Na laranjeira 'Hamlin', o MBTA na concentração de 16 mg L-1 aplicado com 25% de flores abertas incrementou o teor de sólidos solúveis totais, o rendimento de suco e a quantidade de sólidos solúveis totais por caixa de 40,8 kg. Este mesmo tratamento também reduziu a massa média dos frutos e não alterou a acidez, o "ratio", a cor do suco, o diâmetro médio dos frutos e a produtividade final da laranjeira 'Hamlin'. O incremento médio foi de 0,45 a 0,56% na concentração de sólidos solúveis totais e de 0,16 a 0,18 kg na quantidade de sólidos solúveis por caixa padrão de 40,8 kg. Na laranjeira 'Pêra', o MBTA na concentração de 8 mg L-1 aplicado com 25% de flores abertas incrementou o teor de sólidos solúveis totais, a acidez, a quantidade de sólidos solúveis totais por caixa de 40,8 kg e a produtividade. Este mesmo tratamento também reduziu a massa média dos frutos e não alterou o rendimento de suco, o ratio, a cor do suco e o diâmetro médio dos frutos. O incremento médio foi de 0,49 a 0,65% na concentração de sólidos solúveis totais, de 0,11 a 0,13 kg na quantidade de sólidos solúveis totais por caixa padrão de 40,8 kg e de 20,4 kg/planta na produtividade. Na laranjeira 'Valência', o MBTA na concentração de 16 mg L-1 aplicado com 25% ou 100% de flores abertas incrementou a quantidade de sólidos solúveis totais por caixa de 40,8 kg. Estes tratamentos não promoveram alterações consistentes sobre o teor de sólidos solúveis totais, o rendimento de suco, a acidez, o ratio, a massa média dos frutos, o diâmetro médio dos frutos, a cor dos frutos e a produtividade. O incremento médio foi de 0,11 a 0,14 kg na quantidade de sólidos solúveis totais por caixa industrial padrão de 40,8 kg. Aumentos na assimilação de CO2 e uma diminuição no conteúdo foliar de carboidratos foram observados nas folhas tratadas com MBTA na concentração de 16 mg L-1. Os efeitos do MBTA sobre a produtividade e a qualidade dos frutos estão possivelmente relacionados com a fotossíntese a com relações fonte-dreno.
The purpose of this research was to evaluate effects of MBTA [N,N-diethyl-2-(4- metylbenzyloxy) ethylamine hydrochloride] bioregulator applied on different times and concentrations on the yield and fruit quality of 'Hamlin', 'Pera' and 'Valencia' sweet orange and on physiological characteristics of 'Hamlin' sweet orange. In two consecutive harvest seasons, the MBTA was sprayed in three different concentrations (8; 16 and 32 mg L-1) and in two different phenological phases (25% and 100% open flowers) in citrus mature trees, using 7 L of spray per tree added with Silwett L-77 adjuvant at 0.05%. For each cultivar, twenty fruits per tree were sampled in four different times to determine the total soluble solids concentration, acidity, quantity of total soluble solids in 40.8 kg box, pH, juice concentration, juice color, fruit weight and diameter. The effects of MBTA varied according to the cultivar, concentration and phenological phase of application. In 'Hamlin' sweet orange, the MBTA at 16 mg L-1 concentration sprayed with 25% open flowers increased the total soluble solids concentration, juice concentration and the quantity of total soluble solids per 40,8 kg box. This same treatment decreased the fruit weight and didn´t affect the acidity, ratio, juice color, fruit diameter and yield. The average increase was from 0.45 to 0.56% on total soluble solids concentration and from 0.16 to 0.18 kg on the quantity of total soluble solids per 40.8 kg box. In 'Pera' sweet orange, the MBTA at 8 mg L-1 concentration sprayed with 25% open flowers increased the total soluble solids concentration, acidity, the quantity of total soluble solids per 40.8 kg box and the yield. This same treatment decreased the fruit weight and didn´t affect the juice concentration, ratio, juice color and fruit diameter. The average increase was from 0.49 to 0.65% on total soluble solids concentration, from 0.11 to 0.13 kg on the quantity of total soluble solids per 40.8 kg box and 20.4 kg/tree on yield. In 'Valencia' sweet orange, the MBTA at 16 mg L-1 concentration sprayed with 25% or 100% open flowers increased the quantity of total soluble solids per 40.8 kg box. These treatments didn't cause consistent changes in the total soluble solids concentration, acidity, fruit diameter, fruit weight, juice concentration, ratio, juice color and the yield. The average increase was from 0.11 to 0.14 kg on the quantity of total soluble solids per 40.8 kg box. An increase on the CO2 assimilation and a decrease on foliar carbohydrate content were observed in the leaves sprayed with MBTA at 16 mg L-1 concentration. The MBTA effects on the yield and fruit quality are possibly related to the photosynthesis and source-sink relations.

Much of the citrus produced in southwestern Arizona is grown on sandy soils. Because these soils have a low ion exchange capacity, are highly permeable to water, and are prone to nitrate leaching, achieving efficient N management presents a continuing challenge. A field study was conducted on a superstition sand to evaluate the response of lemons to combinations of soil and foliar applied N. Lemon yields significantly increased by soil applied N. Foliar N increased yields of lemons the first harvest at the lower soil N rates. However, there were no other significant responses to foliar N. Overall, there were few meaningful changes in fruit quality to N fertilization. The N content of the leaves increased linearly to soil N application

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Esta disserta??o se refere a um estudo interdisciplinar sobre as causas que levam pessoas ? confiss?o de atos il?citos penais n?o cometidos, a compreens?o das t?cnicas usadas em interrogat?rios e as infer?ncias psicossociais neles presentes que possam estimul?-las a tanto. O tema e tratado pelo confronto anal?tico e cr?tico de teorias de autores nacionais e estrangeiros e ? relacionado com os procedimentos do direito processual penal brasileiro. A confiss?o ? tomada como um comportamento autodestrutivo, e a compuls?o humana a ela, sustentada por Reik, ? estimulada pelo contexto religioso, que atingiu, subseq?entemente, as esferas inquisit?rias estatais. Neste tudo, constatou-se que indiv?duos sugestion?veis e com debilidades ps?quicas e/ou mnem?nicas s?o suscet?veis a confessar falsamente e que os conceitos de verdade material e formal s?o insuficientes ? reconstitui??o de um evento, pois essa verdade, de fato, e constru?da. Desse modo, o saber jur?dico, por si s?, n?o promove o entendimento do fen?meno, tornando imprescind?vel que se busque aux?lio em conhecimentos advindos de outras ?reas. Percebeu-se, outrossim, que inquiridores utilizam diversas estrat?gias para a obten??o da auto-acusa??o, o que leva ? presun??o de que toda confiss?o obtida judicialmente ou extrajudicialmente ? coagida, desde que n?o esteja plena e abundantemente corroborada por provas formalmente judicializadas, e ? de se destacar que, mesmo falsa, influencia julgadores no proferimento de senten?as condenat?rias.

Studies are being conducted which aim to improve the efficiency of irrigation and N fertilization for lemons produced on sandy soils in the low desert The first experiment evaluates the response of 'Lisbon' lemons to various flood irrigation intervals. Irrigation intervals are based on soil moisture depletion (SMD) as calculated from frequent neutron probe soil moisture measurements. Individual treatments were irrigated when total SMI) was 25 %, 40 %, 55 %, and 70 %, respectively. The second experiment compares the performance of young lemons produced under flood, trickle, and micro-spray irrigation systems. The third experiment evaluates the response of young lemons to water and N combinations (3 by 3 factorial) under micro - spray irrigation. The three irrigation rates were targeted for 30 cnbar, 20 cnbar, and 10 cnbar tension. The three N rates were 0.1, 0.2, and 0.4 kg N/tree. One flood irrigation treatment was added for comparative purposes. Overall, results obtained in experiment 1 during 1994, 1995, and 1996 indicate optimal fruit growth and yield is obtained at approximately 40% SMD. The results of experiment 2 show that after 3 years, only micro-jet irrigation produced less tree growth than flood irrigation. In 1995, first year fruit yields were significantly greater for pressurized irrigation compared to flood irrigation. However, by 1996 there were no differences in yield to irrigation treatment. Results from experiment 3 show a linear response in tree growth to irrigation. In 1994 and 1995, tree growth at the high micro - spray soil moisture regime was significantly greater than trees irrigated by flood. However, in 1996 where we failed to increase the micro-spay irrigation time to meet the increased water demand by the trees, the flood irrigation regime was superior. Yields were also increased to irrigation. There were no significant differences in tree growth to N fertilization rates in 1994 and 1995. However, there was increased tree growth in 1996 and a yield increase to N fertilizer rate at the highest soil moisture regime.

Much of the citrus produced in southwestern Arizona is grown on sandy soils. Because these soils have a low ion exchange capacity, are highly permeable to water, and are prone to nitrate leaching, achieving efficient N management presents a continuing challenge. Studies were conducted during 1999, 2000, and 2001 to evaluate the response of micro-sprinkler irrigated lemons to N rate (0, 1.8, and 3.6 kg N tree-1 yr-1) and N source (UN32, CAN-17, CN9, and mixed program) on Superstition Sand. Lemon yield increased by N rate during the first and second harvests in 1999, 2000, and 2001. In 1999, yields increased linearly to 3.6 kg N tree-1 yr-1 but in 2000 and 2001 yields were maximized at 1.8 kg N tree-1 yr-1. In 1999 where larger increments of N were applied over a smaller time period relative to the other seasons, UN32 seemed to decrease yields at the highest N rate. There were no significant effects to N source in 2000 and 2001.

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Introdu??o: O escarro induzido (EI) ? um m?todo n?o-invasivo ?til para avaliar a inflama??o de vias a?reas inferiores, caracter?stica principal observada na asma. Sabe-se que em adultos a inflama??o do tipo eosinof?lica ? a mais comumente encontrada, pois a maioria desses pacientes apresenta alergia como caracter?stica associada. Em crian?as, observa-se um n?mero significativo de asm?ticos com caracter?sticas n?o-al?rgicas e nestes casos ainda n?o est? bem definido qual o padr?o inflamat?rio predominante. H? poucos estudos comparando a inflama??o br?nquica entre estes diferentes fen?tipos de asma. Nosso estudo teve como objetivo avaliar caracter?sticas citol?gicas do EI em crian?as com asma at?pica e n?o-at?pica, comparando-as a um grupo controle sadio. M?todos: Dentre crian?as que participaram da fase II do estudo ISAAC no sul do Brasil, que tinham respondido ao question?rio de doen?as respirat?rias e haviam sido submetidas a testes cut?neos para al?rgenos ambientais comuns, uma sub-amostra aleat?ria foi selecionada. Assim definiram-se grupos representando dois fen?tipos cl?nicos de asma e um grupo controle de normais, a saber: asma at?pica (AA), asma n?oat?pica (ANA) e controles n?o-asm?ticos/n?o-at?picos (NANA). Todas estas crian?as tiveram escarro induzido atrav?s de solu??o salina a 4.5% e as caracter?sticas citol?gicas foram comparadas entre os tr?s grupos. Resultados: Noventa crian?as foram selecionadas a participar do estudo, sendo inclu?das setenta e seis crian?as (28 AA, 29 ANA e 19 NANA), com m?dia de idade de 13 (0.97) anos. O escarro foi induzido com sucesso em 55 (72.3%) crian?as (21 AA, 21 ANA e 13 NANA). Os dados demogr?ficos e a m?dia do VEF1 foram similares nos tr?s grupos. A mediana (intervalo interquartil) da propor??o de eosin?filos foi significativamente superior no escarro dos AA [9 (14)], comparado aos ANA [1 (2)] e aos NANA [0.5 (1)], p<0,001. A propor??o de crian?as com eosinofilia no escarro (eos ≥3%) foi tamb?m significativamente maior nos AA (81%) que nos ANA (23.9%), e n?o houve nenhum caso entre os NANA (p<0,001). A mediana (intervalo interquartil) da contagem de neutr?filos foi significativamente maior no escarro dos ANA [18 (5.6)], comparado aos AA [11 (2)] e aos NANA [(13 (7)], p<0.001. Conclus?es: Os resultados indicam que uma inflama??o eosinof?lica, assim como ocorre em pacientes adultos, pode ser detectada desde a pr?-adolesc?ncia em crian?as com asma at?pica. Por outro lado, asm?ticos n?o-at?picos n?o apresentam este perfil de resposta inflamat?ria, sendo neste grupo evidenciada uma maior propor??o de neutr?filos

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Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico
Carciniculture in Brazil occupies world-wide prominence due to shrimp culture, and the state of Rio Grande do Norte has presented the best results in the culture of the Litopenaeus vannamei in the last decade. This species has been shown to adapt easily to different environments and is between the five most cultivated penaeids of the world. The ponds are usually constructed in areas close to water courses and estuaries. Stock density and substrate ponds can pollute environment, causing losses in the growth and survival of the shrimps, being considered stress factors. Shrimps in inadequate densities and substrates can result reduced productivity of the farm; and favor diseases. So, it is important to verify how these variables influence the development of the animals in the culture farms. Our objective was to study the influence of the type of substrate and the stock density on the behavior and haemocyte count of the L. vannamei. Individually marked juvenile shrimps were kept in aquaria with 30 L of seawater and continuous aeration, in 12L-12D photoperiod. They were observed through Ad libitum and focal sampling instantaneous methods during thirty days, five times per week, six times per day (8:00 to 18:00) in windows of 15 minutes every two hours. The marking of carapace permitted quantifying molting and the feeding was supplied three times a day. Two experiments were carried out: the first one tested animals in the three different substrates (fine sand, smaller rocks-SPP and biggest rocks-SGR) with 33 shrimp/m2. In the second one, the animals were tested in three stock densities (26, 52 and 66 shrimp/m2) in fine sand substrate. At the end of experiment, biometry (first and second ones) and haemocyte count (second one) were made. The behavior of the L. vannamei seems to have been influenced by substrate and stocking density. In low granulometry of the substrate; the exploratory behavior became more frequent and inactivity of the shrimps was reduced. Burrowing was registered in sand substrate, specially in the initial period of the day. Cleaning was gradually higher along the day, presenting the biggest levels as the dark phase approached. The ingestion of feeding was more frequent in low density, and the animals were bigger and heavier at the end of the experiment. In the fine sand condition, the animals presented better growth, probably associated with the burrowing. The molting was equivalent in all types of substrate, but it was more frequent in high densities. Mortality of the shrimps was more frequent in high densities, and cannibalism and diseases were also registered in that condition. The clinical signals were similar to the ones of infectious mionecrosis (IMNV), generally associated with environment and physical stress. The haemocyte count was low for the hematologic standards of the penaeid, which we attributed for greater dilution of haemolymph in the postmolting phase. Smaller shrimps presented lower levels of haemocytes in relation to the bigger animals, count was also low in 26 shrimp/m2 density. The study demonstrates that stocking density and the granulometry of the substrate can affect the welfare, the health and the behavior of the L. vannamei. The sand substrate and low stocking density can be important tools in the management systems of shrimp production
A carcinicultura brasileira ocupa lugar de destaque mundial devido ? cria??o de camar?o, sendo o Rio Grande do Norte o maior produtor no cultivo de Litopenaeus vannamei. Essa esp?cie apresenta grande adapta??o ambiental e est? entre os cinco pene?deos mais cultivados do mundo. A cria??o ? realizada em viveiros nas ?reas pr?ximas a cursos d ?gua e estu?rios. Altas densidades populacionais e o substrato dos viveiros acarretam o aumento da polui??o e promovem perdas no crescimento e na sobreviv?ncia dos camar?es, sendo considerados fatores estressantes. Esses fatores podem comprometer a produtividade dos viveiros e favorecer doen?as, sendo importante verificar como essas vari?veis influenciam no desenvolvimento dos animais nas fazendas de cultivo. Nosso objetivo foi estudar a influ?ncia do tipo de substrato e da densidade populacional sobre o padr?o de atividades comportamentais e os n?veis de hem?citos (CHT) de L. vannamei. Em aqu?rios com 30 litros de ?gua salgada com aera??o cont?nua e em fotoper?odo 12C 12E (claro das 06:00 ?s 18:00), camar?es juvenis marcados individualmente foram observados pelos m?todos Ad libitum e focal instant?neo durante 30 dias, 5 vezes por semana, 6 observa??es di?rias (iniciando a primeira ?s 8:00 e a ?ltima ?s 18:00) em janelas de 15 minutos a cada duas horas, sendo registrado a cada minuto os seus comportamentos e localiza??o. Tamb?m foi feita a marca??o da carapa?a para quantificar as ecdises e a alimenta??o foi fornecida tr?s vezes ao dia. Dois experimentos foram realizados: tr?s substratos diferenciados (Areia, Seixos Pequenos e Seixos Grandes) com 33 animais/m2; e outro com tr?s densidades populacionais (26, 52 e 66 animais/m2) em substrato arenoso. Ao final dos experimentos, foram feitas a biometria dos camar?es e a contagem de hem?citos (apenas no 2? experimento). O padr?o geral de atividades comportamentais de L. vannamei n?o foi influenciado pelo substrato e densidade populacional. Contudo, ? medida que diminuiu a granulometria do substrato, o comportamento de explora??o tornou-se mais freq?ente e reduziu a inatividade dos camar?es. O enterramento foi registrado em substrato arenoso, sendo maior no per?odo inicial do dia. A limpeza distribuiu-se de forma crescente ? medida que a fase escura se aproximava, sendo maior ? noite. A ingest?o alimentar foi mais freq?ente em densidade baixa; acarretando maior crescimento nos camar?es. Houve maior ganho de peso nos animais em substrato arenoso, podendo estar associado com o enterramento e a taxa de crescimento dos animais. A taxa de ecdise foi igual em todos os substratos testados, mas a taxa de ecdise foi maior em maiores densidades. A mortalidade foi mais elevada em condi??es de maiores densidades, sendo registrados canibalismo e enfermidades nos animais. Os sinais cl?nicos foram semelhantes aos da mionecrose infecciosa (IMNV), geralmente associada ao estresse ambiental. Os n?veis de hem?citos foram baixos para os padr?es dos pene?deos, atribu?da pela maior dilui??o da hemolinfa na fase de p?s-ecdise. Camar?es menores apresentaram n?meros de hem?citos reduzidos em rela??o aos maiores, sendo tamb?m baixos em densidade de 26 camar?es/m2. O estudo demonstrou que os efeitos da densidade populacional elevada e a gramulometria do substrato podem afetar o bem estar, a sa?de e o comportamento de L. vannamei. O uso de substrato arenoso e baixa densidade populacional parecem ser medidas de manejo importantes em um sistema de produ??o de camar?o

Orientador: Marcia Ortiz Mayo Marques
Banca: Beatriz Helena Lameiro de Noronha Sales Maia
Banca: Filipe Pereira Giardini Bonfim
Resumo: Lippia alba (Mill.) N.E.Br, pertencente à família Verbenaceae é uma planta medicinal amplamente distribuída na América do Sul. A composição química do óleo essencial da espécie, a qual derivam suas propriedades terapêuticas mostra-se variável, com divisão em quimiotipos. Entre eles, o quimiotipo citral destaca-se pelas ações calmante e ansiolítica, sendo suas folhas empregadas na medicina popular na forma de chá. Dessa forma, visando o potencial uso econômico de Lippia alba, este estudo teve como objetivos avaliar clones de Lippia alba do quimiotipo citral quanto a produtividade de biomassa e óleo essencial, perfil sensorial do chá e composição química do óleo essencial pelas técnicas de cromatografia gasosa unidimensional (CG) e bidimensional abrangente (CG×CG). Entre os clones, dois mostraram-se promissores para inserção em sistemas de produção, com foco em maior produção de biomassa e óleo essencial. Os perfis químicos dos óleos essenciais dos clones são variáveis entre si. O emprego do sistema CG×CG resultou em maior capacidade de detecção dos constituintes dos óleos essenciais. Ao todo foram identificadas 47 substâncias por CG×CG, contra 26 no CG. A maior sensibilidade e resolução fazem do sistema CG×CG uma importante ferramenta metabolômica no estudo dos constituintes voláteis de Lippia alba. Os clones X6M?C e X6M7 se destacaram quanto as características sensoriais de aroma e sabor, com potencial de inclusão na indústria alimentícia na forma de chá.
Abstract: Lippia alba (Mill.) N.E.Br, from the Verbanaceae family is a medicinal plant widely distributed in the South America. The species's essential oil chemical composition, which derives its therapeutic properties, shows itself as variable, with division in chemotypes. Among them, the citral chemotype highlights itself for the soothing and anxiolytic actions, being its leaves used in the popular medicine in the form of tea. Thus, aiming the potential economic use of the Lippia alba, this work has as objectives evaluate the clones of the citral chemotype of Lippia alba in terms of productivity of biomass and essential oil, sensorial profile and chemical composition of the essential oil by using the one-dimensional gas Chromatography (CG) and comprehensive twodimensional gas chromatography(CGxCG). Among the clone, two stood out as promising to insertion in production systems, with focus on a bigger production of biomass and essential oil. The chemical profiles of the clones's essential oil are variable among themselves. The use of the CGxCG system have resulted in a bigger capacity of identification of the essential oils constituents. Altogether, 47 substances have been identified by CGxCG, against 26 in CG. The better sensibility and resolution are part of the CGxCG, an important metabolomics tool of the volatile constituents of Lippia alba. The X6M?C e X6M7 clones stood out in terms of sensorial features of flavor, with potential of inclusion in the food industry in the form of tea.
Mestre

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Introduction: Asthma is a disease with predominant presence of CD4 Th2 cells that affects children. The disease main characteristics is inflammation and airway hyperreactivity. Asthma has two main phenotypes depending on the presence of atopy. It was not yet fully described in asthmatic children of school age the presence of different profiles of Th1, Th2, Th17 and Treg cells in peripheral blood, which may be related to the pathogenesis of the disease. Aims: To analyze the phenotypic of CD4 T cells in asthmatics children in Porto Alegre/RS. Methods: This was a croos-sectional study when it was recruited children between 9 to 15 years old, asthmatics and controls. The PBMCs were isolated using Ficoll and the cells were cultured with anti-CD3/CD28antibodies, Derp1 or left unstimulated. After 24hs the cells were stained with antibodies specifics for transcription factors in order to analyze Th1, Th2, Treg and Th17 cells by flow cytometry and the supernatant was collected for cytokine analysis by CBA assay. Plasma was used to perform analysis of specific IgE. Results: It was analyzed 104 patients, 12 controls and 92 asthmatics. Atopic asthmatic patients presented higher frequency of Th2 cells compared with non-atopic and atopic controls. When we analyzed cells expressing more than one transcription factor it was observed that atopic asthmatics patients have a higher frequency of CD4+GATA3+FOXP3+, CD4+RORγT+GATA3+ and CD4+GATA3+Tbet+ compared with non-atopic patients and controls atopic. Analyzing the asthma severity it was seen a higher frequency of CD4+RORγT+GATA3+ in patients with moderate asthma compared with patients with mild asthma. Stimulating the cells from atopic asthma patients with anti-CD3 and anti-CD28 induced more Th1 profile while stimulating with DerP1 protein changed the profile of the cells mainly to Th17 and Treg cells. Conclusion: This is the first study in children that analyzes Th1, Th2, Treg and Th17 cells using the tag transcription factor by flow cytometry in asthmatic or atopic and non-atopic controls. We found a Th2 profile in atopic asthmatic children according to the literature. Interestingly non-atopic children showed no predominant profile. Children with moderate asthma have a profile that expresses both the transcription factor GATA3 and RORγT. Depending on the stimulus used it was induced different phenotypes in the cells of this group of patients with asthma.
Introdu??o: A asma ? uma doen?a com predomin?ncia da presen?a das c?lulas T CD4 do tipo Th2, que afeta das crian?as e tem como principais caracter?sticas a inflama??o e hiperreatividade das vias a?reas. A asma apresenta dois fen?tipos principais dependendo da presen?a de atopia. Ainda n?o foi totalmente descrito em crian?as asm?ticas com idade escolar a presen?a dos diferentes perfis de c?lulas Th1, Th2, Th17 e Treg no sangue perif?rico, que podem estar relacionados com a patog?nese da doen?a. Objetivo: Avaliar o perfil fenot?pico de c?lulas T CD4 em pacientes asm?ticos escolares do munic?pio de Porto Alegre/RS. M?todos: Este foi um estudo transversal realizado com o recrutamento de crian?as com 09 a 15 anos de idade, asm?ticas e controles. O sangue perif?rico foi coletado e as c?lulas mononucleares foram separadas utilizando Ficoll e colocadas em cultura com anticorpos anti-CD3/CD28. Ap?s 24hs as c?lulas foram marcadas com anticorpos espec?ficos para fatores de transcri??o para an?lise do perfil Th1, Th2, Th17 e Treg por citometria de fluxo e o sobrenadante foi recolhido para analise de citocinas por CBA. No plasma foi realizada a an?lise de IgE espec?ficas. Resultados: Foram analisados 104 pacientes, sendo 12 controles e 92 asm?ticos. Os pacientes asm?ticos at?picos apresentaram um perfil Th2 mais acentuado, comparado com os n?o-at?picos e controles at?picos. Quando analisamos as c?lulas que expressam mais de um fator de transcri??o foi observado que os pacientes asm?ticos at?picos apresentam maior frequ?ncia de c?lulas T CD4 positivas GATA3+FOXP3+, RORγT+GATA3+, GATA3+Tbet+ comparada com pacientes n?o-at?picos e controles at?picos. Analisando a severidade foi observado uma frequencia maior de c?lulas T CD4+ RORγT+ GATA3+ em pacientes com asma moderada comparado com pacientes com asma leve. O est?mulo com anti-CD3 e anti-CD28 nas c?lulas dos pacientes asm?ticos at?picos induziu mais um perfil Th1 enquanto que est?mulo com DerP1 mudou o perfil de algumas c?lulas principalmente de c?lulas Tregs e Th17. Conclus?o: Este ? o primeiro estudo em crian?as que analisa o perfil Th1, Th2, Th17 e Treg utilizando a marca??o de fatores de transcri??o por citometria de fluxo em crian?as asm?ticas at?picas ou n?o-at?picas e controles. Encontramos um perfil mais Th2 em crian?as asm?ticas at?picas de acordo com a literatura. Interessantemente as crian?as n?o-at?picas n?o apresentaram nenhum perfil predominante. As crian?as com asma moderadas apresentam um perfil que expressa ao mesmo tempo o fator de transcri??o RORgT e GATA3. Dependendo do estimulo s?o induzidas diferentes fen?tipos nas c?lulas deste grupo de pacientes com asma.

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior
Herpes simplex is a virus that can be transmitted sexually and is potentially associated with vertical transmission. This study evaluated the prevalence of genital infection by herpes simplex virus (HSV) types 1 and 2 in pregnant and nonpregnant care in the city of Natal / RN, including a total of 222 women, 92 pregnant and 130 nonpregnant. The participants answered a questionnaire to obtain data and socio-demographic characteristics, as well as potential risk factors for sexually transmitted diseases. After the interview, we collected two cervical specimens, one for the Pap test and the other for DNA extraction and analyzed by polymerase chain reaction (PCR) to detect both virus serotypes. Then the women underwent a clinical examination by colposcopy. For statistical analysis, we used the chi-square and logistic regression by SSPS 17.0 Statistic. Most women were up to 30 years of age, nonwhite ethnicity, married, elementary education, family income below the poverty level; initiated sexual activity with age up to 18 years; had more than one sexual partner lifelong and was not pregnant, but has had at least one child. The HSV-1 showed a prevalence of 26.1% among pregnant women and 30.0% in non-pregnant women. While HSV-2 prevalence was found with 10.9% and 19.2% in pregnant and nonpregnant women, respectively. The largest proportion of morphological changes of the uterine cervix was detected among nonpregnant women, both in cytology and in colposcopy. The women were nonwhite ethnicity, married, became pregnant aged less than or equal to 18 years and who had one to two pregnancies had a lower risk of acquiring genital HSV infection. There was a high prevalence of genital HSV infection, HSV-1 is more prevalent than HSV-2. No association was found between morphological changes of the uterine cervix and the presence of the virus in pregnant and nonpregnant women, nor between genital HSV infection and the classic risk factors for sexually transmitted diseases
O herpes simples ? um dos v?rus que pode ser transmitido sexualmente e est? potencialmente associado ? transmiss?o vertical. Este estudo avaliou a preval?ncia da infec??o genital pelo v?rus herpes simples (HSV) tipos 1 e 2 em mulheres gr?vidas e n?o gr?vidas atendidas no munic?pio de Natal/RN, incluindo um total de 222 mulheres, sendo 92 gr?vidas e 130 n?o gr?vidas. As participantes responderam um question?rio para a obten??o de dados e caracter?sticas s?cio-demogr?ficas, al?m de potenciais fatores de risco para doen?as sexualmente transmiss?veis. Ap?s a entrevista, foram coletados dois esp?cimes cervicais, um destinado ao exame citol?gico de Papanicolaou e o outro, para extra??o de DNA e analisado pela rea??o em cadeia da polimerase (PCR) para detectar os dois sorotipos do v?rus. Em seguida, as mulheres foram submetidas a um exame cl?nico por meio de colposcopia. Para an?lise estat?stica dos dados, utilizou-se o teste de qui-quadrado e de regress?o log?stica pelo programa SSPS Statistic 17.0. A maioria das mulheres tinha at? 30 anos de idade, etnia n?o branca, casada, grau de instru??o elementar, renda familiar de at? um sal?rio m?nimo; iniciou a atividade sexual com idade at? 18 anos; teve mais de um parceiro sexual ao longo da vida e n?o estava gr?vida, mas j? teve no m?nimo um filho. O HSV-1 apresentou preval?ncia de 26,1% nas mulheres gr?vidas e 30,0% nas n?o gr?vidas. Enquanto que o HSV-2 foi encontrado com preval?ncia de 10,9% e 19,2% nas mulheres gr?vidas e n?o gr?vidas, respectivamente. A maior propor??o de altera??es morfol?gicas da c?rvice uterina foi detectada entre as mulheres n?o gr?vidas, tanto no exame citol?gico quanto na colposcopia. As mulheres de etnia branca, casadas, que engravidaram com idade menor ou igual a 18 anos e que tiveram de uma a duas gesta??es apresentaram menor risco de adquirir infec??o genital pelo HSV. Constatou-se uma alta preval?ncia infec??o genital pelo HSV, sendo HSV-1 mais prevalente que o HSV-2. N?o se observou associa??o entre altera??es morfol?gicas da c?rvice uterina e a presen?a do v?rus nas mulheres gr?vidas e n?o gr?vidas, nem entre a infec??o genital pelo HSV e os fatores de risco cl?ssicos para doen?as sexualmente transmiss?veis

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This study compared the effectiveness of quick review 100%, 10% random review and revision of smears selected based on clinical criteria as a method of internal quality control of cervical smears negative on routine screening and found that the false-negative results identified by review rapid vary with the quality of the sample and the woman's age. Was based on the female population user of the Unified Health System in Goiânia-GO, which was subjected to cytological examination performed at the Laboratory of Clinical Analysis of the Romulus Rock School of Pharmacy, Federal University of Goiás The 5,530 smears classified as negative scrutiny underwent routine method for quick review 100%, then the smears were selected based on clinical criteria and 10% of negative smears and submitted to the respective revisães. Four cytologists alternated during revisions. After the revisions concordant results were considered final diagnosis, whereas the divergent were analyzed by a third cytologist, which consensus meeting defined the final diagnosis. All steps were blindly and the results classified according to the Bethesda System. For statistical analysis, the variables were analyzed descriptively and applied the chi-square test. A Quick Review of 100% smears identified 141 suspects, 84 of these (59.6%) tested positive for the final diagnosis, of which 36 (25.5%) were classified as atypical squamous cells of undetermined significance (ASC-US) five (3.5%) as atypical squamous cells can not exclude high-grade lesion (ASC-H), 34 (24.1%) as squamous intraepithelial lesions of low-grade (LSIL), six (4.3 %) as squamous intraepithelial lesion, high-grade (HSIL) and three (2.1%) atypical glandular cells (AGC). Smears of 84 suspected and confirmed the final diagnosis, 62 (73.8%) were classified as satisfactory and 22 (26.2%) satisfactory, but with some limitation, but no significant difference was with the woman's age. The method of rapid review identified 19 as unsatisfactory smears, 16 of these (84.2%) were confirmed in the final diagnosis. Of the 1,279 smears reviewed based on clinical criteria, 24 were considered abnormal. Of these 19 (79.2%) were confirmed as positive by the final diagnosis, nine (47.3%) classified as ASC-US, one (5.3%) ASC-H, eight (42.1%) and LSIL One (5.3%) HSIL. Of the 560 smears examined by reviewing 10%, 13 were considered abnormal, these six (46.2%) were confirmed in the final diagnosis, four (30.8%) classified as ASC-US, one (7.7%) and LSIL (7.7%) HSIL. Therefore, a quick review of 100% is a more efficient alternative for the detection of false-negative results of cervical screening as a method of internal quality control, than the random review of 10% and revision based on clinical criteria. It was observed also that the quick review showed better performance when the sample was classified as adequate for analysis, but does not vary with the age of the woman.
This study compared the effectiveness of quick review 100%, 10% random review and revision of smears selected based on clinical criteria as a method of internal quality control of cervical smears negative on routine screening and found that the false-negative results identified by review rapid vary with the quality of the sample and the woman's age. Was based on the female population user of the Unified Health System in Goiânia-GO, which was subjected to cytological examination performed at the Laboratory of Clinical Analysis of the Romulus Rock School of Pharmacy, Federal University of Goiás The 5,530 smears classified as negative scrutiny underwent routine method for quick review 100%, then the smears were selected based on clinical criteria and 10% of negative smears and submitted to the respective revisães. Four cytologists alternated during revisions. After the revisions concordant results were considered final diagnosis, whereas the divergent were analyzed by a third cytologist, which consensus meeting defined the final diagnosis. All steps were blindly and the results classified according to the Bethesda System. For statistical analysis, the variables were analyzed descriptively and applied the chi-square test. A Quick Review of 100% smears identified 141 suspects, 84 of these (59.6%) tested positive for the final diagnosis, of which 36 (25.5%) were classified as atypical squamous cells of undetermined significance (ASC-US) five (3.5%) as atypical squamous cells can not exclude high-grade lesion (ASC-H), 34 (24.1%) as squamous intraepithelial lesions of low-grade (LSIL), six (4.3 %) as squamous intraepithelial lesion, high-grade (HSIL) and three (2.1%) atypical glandular cells (AGC). Smears of 84 suspected and confirmed the final diagnosis, 62 (73.8%) were classified as satisfactory and 22 (26.2%) satisfactory, but with some limitation, but no significant difference was with the woman's age. The method of rapid review identified 19 as unsatisfactory smears, 16 of these (84.2%) were confirmed in the final diagnosis. Of the 1,279 smears reviewed based on clinical criteria, 24 were considered abnormal. Of these 19 (79.2%) were confirmed as positive by the final diagnosis, nine (47.3%) classified as ASC-US, one (5.3%) ASC-H, eight (42.1%) and LSIL One (5.3%) HSIL. Of the 560 smears examined by reviewing 10%, 13 were considered abnormal, these six (46.2%) were confirmed in the final diagnosis, four (30.8%) classified as ASC-US, one (7.7%) and LSIL (7.7%) HSIL. Therefore, a quick review of 100% is a more efficient alternative for the detection of false-negative results of cervical screening as a method of internal quality control, than the random review of 10% and revision based on clinical criteria. It was observed also that the quick review showed better performance when the sample was classified as adequate for analysis, but does not vary with the age of the woman.
Este estudo comparou a eficiência da revisão rápida de 100%, revisão aleatória de 10% e revisão de esfregaços selecionados com base em critérios clínicos como método de controle interno da qualidade dos esfregaços cervicais negativos no escrutínio de rotina e verificou se os resultados falso-negativos identificados pela revisão rápida variam com a adequabilidade da amostra e com a idade da mulher. Teve como base à população feminina usuária do Sistema Único de Saúde de Goiânia-GO, que se submeteu ao exame citopatológico realizado no Laboratório de Análises Clínicas Rômulo Rocha da Faculdade de Farmácia da Universidade Federal de Goiás. Os 5.530 esfregaços classificados como negativos no escrutínio de rotina foram submetidos ao método de revisão rápida de 100%, em seguida, foram selecionados os esfregaços com base em critérios clínicos e 10% do total de esfregaços negativos e submetidos às respectivas revisães. Quatro citologistas se alternaram durante as revisões. Após as revisões os resultados concordantes foram considerados como diagnóstico final, enquanto que, os divergentes foram analisados por um terceiro citologista, que em reunião de consenso definiu o diagnóstico final. Todas as etapas foram às cegas e os resultados classificados de acordo com o Sistema de Bethesda. Para análise estatística as variáveis foram estudadas de maneira descritiva e aplicado o Teste do Qui-quadrado. A revisão rápida de 100% identificou 141 esfregaços suspeitos, desses 84 (59,6%) foram considerados positivos pelo diagnóstico final, dos quais 36 (25,5%) foram classificados como células escamosas atípicas de significado indeterminado (ASC-US), cinco (3,5%) como células escamosas atípicas, não podendo excluir lesão de alto grau (ASC-H), 34 (24,1%) como lesão intra-epitelial escamosa de baixo grau (LSIL), seis (4,3%) como lesão intra-epitelial de alto grau (HSIL) e três (2,1%) células glandulares atípicas (AGC). Dos 84 esfregaços suspeitos e confirmados pelo diagnóstico final, 62 (73,8%) foram classificados como satisfatório e 22 (26,2%) satisfatórios, porém com alguma limitação, mas não observou diferença significativa com a idade da mulher. O método de revisão rápida identificou 19 esfregaços como insatisfatórios, desses 16 (84,2%) foram confirmados pelo diagnóstico final. Dos 1.279 esfregaços revisados com base em critérios clínicos, 24 foram considerados alterados. Desses 19 (79,2%) foram confirmados como positivos pelo diagnóstico final, sendo nove (47,3%) classificados como ASC-US, um (5,3%) ASC-H, oito (42,1%) LSIL e um (5,3%) HSIL. Dos 560 esfregaços analisados pela revisão de 10%, 13 foram considerados alterados, desses seis (46,2%) foram confirmados pelo diagnóstico final, sendo quatro (30,8%) classificados como ASC-US, um (7,7%) LSIL e um (7,7%) HSIL. Portanto, a revisão rápida de 100% é uma alternativa mais eficiente na detecção de resultados falso-negativos dos exames citopatológicos, como método de controle interno da qualidade, do que a revisão aleatória de 10% e revisão com base em critérios clínicos. Observou-se, também que a revisão rápida apresentou melhor desempenho quando a amostra foi classificada como satisfatória para análise, porém não varia com a idade da mulher.
Este estudo comparou a eficiência da revisão rápida de 100%, revisão aleatória de 10% e revisão de esfregaços selecionados com base em critérios clínicos como método de controle interno da qualidade dos esfregaços cervicais negativos no escrutínio de rotina e verificou se os resultados falso-negativos identificados pela revisão rápida variam com a adequabilidade da amostra e com a idade da mulher. Teve como base à população feminina usuária do Sistema Único de Saúde de Goiânia-GO, que se submeteu ao exame citopatológico realizado no Laboratório de Análises Clínicas Rômulo Rocha da Faculdade de Farmácia da Universidade Federal de Goiás. Os 5.530 esfregaços classificados como negativos no escrutínio de rotina foram submetidos ao método de revisão rápida de 100%, em seguida, foram selecionados os esfregaços com base em critérios clínicos e 10% do total de esfregaços negativos e submetidos às respectivas revisães. Quatro citologistas se alternaram durante as revisões. Após as revisões os resultados concordantes foram considerados como diagnóstico final, enquanto que, os divergentes foram analisados por um terceiro citologista, que em reunião de consenso definiu o diagnóstico final. Todas as etapas foram às cegas e os resultados classificados de acordo com o Sistema de Bethesda. Para análise estatística as variáveis foram estudadas de maneira descritiva e aplicado o Teste do Qui-quadrado. A revisão rápida de 100% identificou 141 esfregaços suspeitos, desses 84 (59,6%) foram considerados positivos pelo diagnóstico final, dos quais 36 (25,5%) foram classificados como células escamosas atípicas de significado indeterminado (ASC-US), cinco (3,5%) como células escamosas atípicas, não podendo excluir lesão de alto grau (ASC-H), 34 (24,1%) como lesão intra-epitelial escamosa de baixo grau (LSIL), seis (4,3%) como lesão intra-epitelial de alto grau (HSIL) e três (2,1%) células glandulares atípicas (AGC). Dos 84 esfregaços suspeitos e confirmados pelo diagnóstico final, 62 (73,8%) foram classificados como satisfatório e 22 (26,2%) satisfatórios, porém com alguma limitação, mas não observou diferença significativa com a idade da mulher. O método de revisão rápida identificou 19 esfregaços como insatisfatórios, desses 16 (84,2%) foram confirmados pelo diagnóstico final. Dos 1.279 esfregaços revisados com base em critérios clínicos, 24 foram considerados alterados. Desses 19 (79,2%) foram confirmados como positivos pelo diagnóstico final, sendo nove (47,3%) classificados como ASC-US, um (5,3%) ASC-H, oito (42,1%) LSIL e um (5,3%) HSIL. Dos 560 esfregaços analisados pela revisão de 10%, 13 foram considerados alterados, desses seis (46,2%) foram confirmados pelo diagnóstico final, sendo quatro (30,8%) classificados como ASC-US, um (7,7%) LSIL e um (7,7%) HSIL. Portanto, a revisão rápida de 100% é uma alternativa mais eficiente na detecção de resultados falso-negativos dos exames citopatológicos, como método de controle interno da qualidade, do que a revisão aleatória de 10% e revisão com base em critérios clínicos. Observou-se, também que a revisão rápida apresentou melhor desempenho quando a amostra foi classificada como satisfatória para análise, porém não varia com a idade da mulher.

碩士
國立成功大學
醫學檢驗生物技術學系
98
Citrin is a liver-type transporter for cytosolic glutamate and mitochondrial aspartate. The transported aspartate and cytosolic citrulline are converted into argininosuccinate through the catalysis of argininosuccinate synthetase (ASS). Patients with citrin deficiency showed increased oxidative stress, and decreased cytosolic nicotinamide adenine dinucleotide (NAD+) to NADH ratio, in accompany with reduced liver ASS activity. However, the mechanism of citrin deficiency-induced ASS downregulation is still unknown. Thus, the aim of this study is to establish liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for cytosolic NAD+/NADH and cell lysate GSH/GSSG measurement, and to investigate the effects of chemically or genetically-reduced ASS expression. And further to examine whether glycine or N-acetylcysteine (NAC) treatment would restore the changes. Huh-7 cells treated with menadione or citrin siRNA were analyzed for ASS protein expression and enzyme activity by western blotting and LC-MS/MS, respectively. Redox status was examined by cellular GSH/GSSG and NAD+/NADH ratio. We measured cells labeled by both Annexin V and propidium iodide, presenting apoptotic cells using flow cytometry. The LC-MS/MS methods showed the linear range from 5 to 5000 ng/mL for GSH and GSSG, 0.005 to 5 μmol/L for NAD+ and NADH, respectively. Imprecision (CV%) for these two methods were ＜6%. Menadione-treated cells exhibited a higher apoptosis rate and a decrease in cell viability. Moreover, menadione-induced oxidative stress resulted in depleted GSH (138.7±24.3 vs. 1.56±1.01, p＜0.01), decreased cytosolic NAD+ (2.67±0.42 vs. 0.67±0.32, p＜0.01), lower ASS expression (0.24±0.02 vs. 0.13±0.01, p＜0.01) and activity (0.49±0.05 vs. 0.10±0.02, p＜0.05). Similar to menadione-treated cells, citrin-knockdown cells showed lower ASS expression (0.12±0.02 vs. 0.05±0.001, p＜0.05), GSH (41.17±6.99 vs. 26.01±6.89) and cytosolic NAD+ level (6.94±0.68vs. 4.75±0.57, p＜0.05). Glycine or NAC pretreatment protected cells from oxidative stress-induced apoptosis and increased cell viability. However, only NAC administration prior to menadione-treatment or citrin-knockdown cells would restore the changes in GSH, NAD+ levels and ASS expression. Moreover, NAC supplement rescued these alterations caused by citrin-knockdown combined with menadione-treated cells, indicating the potency of NAC protection. Taken together, our data suggest that down-regulation of ASS expression in citrin-deficiency cells might be through the induction of oxidative stress. NAC, being able to restore the ASS expression, GSH/GSSG and NAD+/NADH ratio, might serve as a therapeutic strategy for patients with citrin deficiency.

碩士
東海大學
環境科學與工程學系
100
Chemical filters are commonly applied in conjunction with particulate filters to maintain the stringent air cleanliness demanded in the manufacturing processes of semiconductor and optoelectronic devices. Chemical filters, generally with activated carbon as the primary sorbent material, are responsible to remove gas-phase contaminants such as inorganic acid/base molecules and volatile/semi-volatile organic molecules. Conventional activated carbon filters, however, are less effective in removing molecular acids or bases as compared to the removal of organic molecules, therefore chemical impregnation promoting acid-alkali reactions have been studied to improve their capability to remove molecular acids or bases. This study targets the removal of N-methyl–2-pyrrolidone (NMP), an organic alkali compound commonly used as a photoresist stripper, by comparing the performance of chemical filters containing virgin activate carbon with those containing activate carbon impregnated with citric acid, an environmentally benign chemical. The quantity of citric acid impregnated on the activated carbon increased with the concentration of citric acid solution in which the activated carbon particles were immersed. The intensity of the carbonyl and hydroxyl functional group as measured by Fourier Transform Infrared analysis showed that the optimal immersion condition was 3M for three hours. In addition, the quantity of citric acid impregnated increased proportionally with the immersion concentration. The specific surface area of the impregnated carbon decreased linearly with the citric acid concentration between 0 and 3 M. For the NMP breakthrough studies, with a face velocity of 0.0092 m/s, the results showed that the amount of NMP adsorbed on activated carbon filter was 0.011 mg/g at an average influent concentration of 25±6 ppmv, 0.0074 mg/g at 56±11 ppmv, and 0.0089 mg/g at 60±18 ppmv. The non-Langmuir adsorption equilibrium showed that the physisorption was limited when the adsorbate was condensable. For the adsorption of NMP using citric acid-impregnated activated carbon, under similar loading concentration (face velocity = 0.0092 m/s; influent NMP concentrations ~70 ppmv), the adsorption quantities were 0.013 mg/g (when coated in 0.3M citric acid solution), 0.033 mg/g (1.0M), and 0.061 mg/g (3.0M). These results demonstrated that NMP was effectively adsorbed by the impregnated activated carbon, and that the amount of NMP adsorbed increased with the quantity of impregnated citric acid. Similar results were also obtained in the breakthrough tests using a face velocity of 0.0046 m/s. These results suggested that physisorption was not in effect as the NMP adsorption quantity was inversely related to the available specific surface area. In conclusion, this study successfully demonstrated the improved effectiveness of chemical filters containing citric acid-impregnated activated carbon for the removal of NMP from air.