Dissertationen zum Thema „Glycicne“

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Fitopatologia, 2008.
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A soja (Glycine max), percentualmente, foi a cultura que mais cresceu nos últimos anos. A produção (26%) brasileira é a segunda maior do mundo, ficando atrás somente dos EUA (37%). O potencial produtivo da soja poderia ser maior se não fosse os danos causados pelas doenças nesta cultura. A mancha parda ou septoriose (Septoria glycines), uma das principais “doenças de final de ciclo”, tem provocado danos em lavouras comerciais de diversas regiões brasileiras, podendo reduzir o rendimento em mais de 30%. A busca para encontrar cultivares de soja resistentes a S. glycines vem de três a quatro décadas atrás, porém até os dias atuais ainda não foram encontradas cultivares com resistência satisfatória à doença. Assim sendo, o controle desta doença é baseado na aplicação de fungicidas. Diante da importância da cultura e da doença os objetivos deste trabalho foram: (a) avaliar em campo a reação de genótipos convencionais e transgênicos à doença e o efeito da época e do local de plantio; (b) avaliar o efeito de fungicidas no controle da mancha parda. Os experimentos foram conduzidos no campo (Cristalina, GO) durante as safras 2004/2005 e 2005/2006 e em casa de vegetação (Brasília, DF) e laboratório nos anos de 2006 a 2008. Em uma primeira etapa foram testados 16 genótipos, três épocas de semeadura e quatro tratamentos com fungicidas. Os resultados indicaram que dos genótipos avaliados, a menor quantidade da doença e maior produtividade foram apresentados pelos genótipos Msoy8411 e GT01-308, e conforme foi se atrasando o plantio, de outubro para dezembro, a intendidade da mancha parda foi aumentado. Na segunda etapa do trabalho avaliou-se a reação de 92 genótipos de soja à mancha parda, entre estes genótipos existiam cultivares de ciclo precoce, médio e tardio. Entre os genótipos avaliados observou-se existir uma variação nos níveis de susceptibilidade, 43,4% apresentaram menores valores de severidade quando comparados ao genótipo padrão (Msoy8001). Na análise da severidade da doença, AACPD e produtividade em relação aos ciclos das culturas, observou-se que as cultivares precoces apresentaram maiores valores de severidade e AACPD e menores valores de produtividade. Em uma terceira etapa do trabalho avaliou-se a reação à mancha parda de genótipos de soja transgênica e convencional. Não foram observadas diferenças de severidade de doença entre soja transgênica e convencional. Houve variação na susceptibilidade à doença entre os genótipos, porém não houve nenhum com resistência. Na quarta etapa do estudo analisou-se a resposta de genótipos de soja à mancha parda em diferentes localidades. Neste experimento foram semeados 58 genótipos em quatro localidades (Cristalina, Orizona, Morrinhos e Piracanjuba/ GO). Dos 58 genótipos avaliados, 31 apresentaram severidade semelhante ao padrão Msoy8001, considerado moderadamente suscetível. Os genótipos restantes mostraram maiores valores de severidade. Em todos os locais avaliados observaram-se diferenças entre produtividade. Durante a terceira etapa do trabalho, subdividiu-se o estudo em dois: (a) o primeiro trata da avaliação de fungicidas e da época de aplicação sobre a mancha parda da soja, e o segundo; (b) estudou-se a resposta do uso de fungicidas (tetraconazol) e fitorreguladores (ácido índolbutírico 0,005%, cinetina 0,009% e ácido giberélíco, como GA3 0,005%) na intensidade da mancha parda. Na primeira sub-etapa (a), conclui-se que dos dez produtos testados, Chlorotalonil (500g i.a./L) + Tetraconazole (20g i.a./L) na dose de 1,75 L/ha foi eficiente na redução da mancha parda. Na sub-etapa (b) não houve redução na doença, nem incremento na produtividade da soja devido aos tratamentos. Finalmente, um estudo foi realizado com a finalidade de padronizar o meio de cultura e temperatura para a obtenção de inoculo de S. glycines. Testaram-se cinco meios de cultura e três temperaturas de incubação. O meio mais adequado para a produção de conídios (1,53 x 107conídios / ml) foi o de extrato de folha de soja ágar (Folha de soja 200g; sacarose 10g; ágar 15g; 1l água destilada) a 20°C de incubação. Em seguida procurou-se definir um protocolo para o uso do “método da folha destacada” para avaliar sintomas da mancha parda. Após a análise dos resultados dos experimentos, verificou-se que a técnica de pincelamento de conídios foi mais prática do que a da pulverização. Concentrações acima de 1 x 105 conídios/ml foram eficientes na produção de sintomas. O estádio foliar mais adequado para inoculação foi o V2. Todas as temperaturas (20 a 30°C) incitaram a manifestação dos sintomas e não foram observadas diferenças de severidade da doença entre inoculações feitas na face adaxial ou abaxial das folhas. _______________________________________________________________________________ ABSTRACT
In recent years, soybean (Glycine max) had one of the greatest production increase among the cultivated crops. Brazil is the second largest soybean world producer (26%), staying only behind of the United States of America (37%). The incidence of diseases reduces the potential production of soybean. The brown spot (Septoria glycines) is one of the diseases that cause reduction on soybean yield. The Brazil’s soybean yield reduction due to brown spot might reach losses higher than 30%. From the last three to four decades, the search for brown spot resistant soybean cultivars was not satisfactorily successful. Therefore, the major brown spot control method is the application of fungicides. Based on above information the objectives of this study were: (a) assess the reaction to brown spot of conventional and transgenic soybean on different time of and different planting locations, and; (b) evaluate the effect of fungicide application brown spot. The experiments were performed in the field (Cristalina, GO, Brazil) during the growing seasons of 2004/2005 and 2005/2006, and in greenhouse (Brasília, DF, Brazil) and laboratory in 2006 to 2008. In the first part of the study were tested 16 soybean genotypes, three planting times, and four different treatments with fungicides. The results indicated that Msoy8411 and GT01-308 genotypes presented lowest amount of brown spot and the highest yield. In addition, the December planting time favored the increase of disease (severity and incidence) if compared to October and November planting time. In the second part of the study a brown spot reaction evaluation of 92 soybean genotypes with contrasting growing response was conducted. Among these genotypes, 43.4% of then presented lower disease ratings than the traditional standard genotype (Msoy8001). The cultivars with smaller growing cycles presented higher disease severity, lower yield than the ones with longer cycles. In the third part of the study is the reaction to brown spot of transgenic and conventional soybean genotypes was evaluated. There were no significant disease differences between transgenic and conventional soybeans. There were degrees of susceptibility to brown spot among the genotypes, but no resistant genotypes. In the fourth stage of the study, 58 genotypes were planted in four localities (Cristalina, Orizona, Morrinhos, and Piracanjuba, GO, Brazil). From these genotypes, 31 showed similar disease severity to the standard genotype (Msoy8001) considered moderately susceptible. The remaining genotypes showed higher values of severity. In all four evaluated places there were differences on genotype yield. In addition, during the third part stage this study, two evaluations were made: (a) evaluation of different fungicides and timing of application on brown spot, and; (b) evaluation of fungicide (tetraconazole) and growth regulators (IBA 0005% 0009% kinetin and gibberellic acid, GA3 as 0005%) on brown spot intensity. From these tests, Chlorotalonil (500g ai / L) + tetraconazole (20g ai / L) at a dose of 1.75 L / ha was effective to reduce brown spot. In the second sub-step (b) no differences on soybean disease and yield due to treatments. Finally, a study was carried out to standardize a culture media and temperature to satisfactorally produce S. glycines conidia. Five culture media and three incubation temperatures were tested.The media considered more suitable for the production of conidia (1.53 x 107 conidia / ml) was the extract of soybean leaves agar (Soybean Leaf 200g, sucrose 10g, agar 15g, 1l distilled water) at 20° C of incubation. Also, a protocol to induce brown spot symptoms, using the "method of detached leaf”, was studie After seven experiments were performed the analysis of the results showed: (a) the technique of brushing conidia suspension on leaves was more practical than spraying. Concentrations above 1 x 105 conidia / ml were effective in producing symptoms. The best leaf phonological stage for inoculation was V2. All tested (20 to 30°C) temperatures that were used to incubate the inoculated leaves showed symptoms, and no disease differences were observed between inoculations made on above and under side of leaves.

Soybean (Glycine max) yield is significantly affected by soybean cyst nematode (SCN), Heterodera glycines, and causes an annual loss of billions of US dollars. In this study, Glycine max xyloglucan endotransglycosylase/hydrolase gene (Gm-XTH52) was transformed into a nematode susceptible G. max [Williams 82/PI 518671] variety of soybean to test whether the protein expression has a role in resistance to H. glycines, and possible chemical changes the expression may cause in the plant composition. Expression level of the Gm-XTH52 gene was three times higher than in controls. Significant reduction in the number of SCN cysts suggested suppression of H. glycines parasitism upon transformation. While total sugar amounts did not significantly differ between the transformed and control plants, xyloglucan amounts of loosely bound sugars of genetically mosaic plants were significantly lower in comparison to controls. Control plants showed lower molecular weight sugars than the transformed plants not subjected to H. glycines infection.

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Soybean is the most important oilseed crop grown in the world and Brazil is the second largest producer. Many pest problems can affect soybean, including the soybean cyst nematode, Heterodera glycines. The search for efficient management alternatives of this nematode and understanding of their biology have been largely studied, but still lacking information. Experiments were conducted under greenhouse conditions and naturally infested field aiming to better understand the behavior of H. glycines and to propose management alternatives. The first study had the purpose to evaluate the penetration and life cycle of H. glycines, race 3 (HG Type 0 -) in soybean cultivars resistant (BRSGO 8860RR) and susceptible (BRS Valiosa RR) under greenhouse conditions. Evaluations in stained roots were made at 2, 4, 6, 9, 12, 15, 18, 21, 24 and 30 days after inoculation (DAI). The penetration of H glycines occurred throughout the period of evaluation in both cultivars. The H. glycines life cycle was completed in 15 days, both in the susceptible and resistant cultivars. Resistant cultivar had the peak period of formation of J3, J4 and females delayed in comparison with the susceptible cultivar. The second study aimed to evaluate effectiveness of the resistance to H. glycines under high inoculum concentration, and the effect of increasing inoculum rates on the penetration of juveniles and H. glycines survival. Two experiments were conducted using three soybean cultivars (BRS Valiosa RR, susceptible to H. glycines; BRSGO Chapadoes and BRSGO 8860RR, both resistant to H. glycines) under four inoculum concentrations (1.000, 2.500, 5.000 and 10.000 eggs and J2 per plant). The increase in the H. glycines inoculum concentration increased the final nematode population in the susceptible cultivar. The cultivar resistance was not affected by high inoculum concentration. The root penetration of J2 increased as the inoculum concentration increased regardless the cultivar. The survival rate was higher in the susceptible cultivar decreasing with increasing of inoculum concentration. The third study evaluated the effect of seed treatment two soybean cultivars (resistant BRSGO 8860RR and susceptible BRS Valiosa RR). Two experiments were conducted, one in a naturally infested field and other under greenhouse conditions. The seed treatments did not affect the stand and the plant height of the resistant soybean cultivar. There was no effect of seed treatments on the nematode population in the field experiment. Under greenhouse, seeds treated with abamectin (50 and 75 mL a.i. 100 kg seed-1) + thiamethoxam (70 mL a.i.), imidacloprid + thiodicarb (105+315 mL a.i.) and thiodicarb + imidacloprid with carbendazim + thiram (75+225 e 30+70 mL a.i.) reduced the number of females per gram of roots on the susceptible cultivar. Seed treatment with imidacloprid + thiodicarb (75+225 mL a.i.) also reduced the number of eggs per female on the resistant cultivar BRSGO 8860RR.
A soja é a mais importante oleaginosa cultivada no mundo sendo o Brasil o segundo maior produtor. Diversos problemas fitossanitários afetam a cultura da soja, dentre eles o nematoide de cisto da soja, Heterodera glycines. A busca por alternativas de manejo eficiente desse nematoide e a compreensão de sua biologia têm sido temas muito difundidos, mas ainda carente de estudos. Dessa forma, foram conduzidos experimentos em condições de casa de vegetação e campo naturalmente infestado visando conhecer melhor o comportamento de H. glycines e propor alternativas para seu manejo. O primeiro estudo teve como objetivo avaliar a penetração e a duração do ciclo de H. glycines, raça 3 (HG tipo 0-), em cultivares de soja resistente (BRSGO 8860RR) e suscetível (BRS Valiosa RR) em condições controladas de casa de vegetação. Foram realizadas avaliações de coloração de raiz aos 2, 4, 6, 9, 12, 15, 18, 21, 24 e 30 dias após a inoculação (DAI). A penetração do H. glycines ocorreu durante todo o período de avaliação nas duas cultivares. O ciclo do H. glycines se completou em 15 dias, tanto na cultivar suscetível como na cultivar resistente. Na cultivar resistente o período de pico de formação de J3, J4 e fêmeas foi atrasado em relação a cultivar suscetível. O segundo estudo teve como objetivo avaliar se a resistência ao H. glycines é eficiente na redução do desenvolvimento do nematoide, mesmo sob elevada concentração de inóculo, bem como se esse aumento na concentração de inóculo e a resistência afetam a penetração de juvenis e a taxa de sobrevivência do H. glycines. Foram conduzidos dois ensaios utilizando-se três cultivares de soja, uma suscetível (BRS Valiosa RR) e duas resistentes (BRSGO Chapadões e BRSGO 8860RR), submetidas a quatro concentrações de inóculo (1.000, 2.500, 5.000 e 10.000 ovos e J2 por vaso). O aumento na concentração inicial de inóculo de H. glycines resultou no aumento da população final do nematoide na cultivar suscetível. A reação de resistência das cultivares não foi afetada por altas concentrações de inóculo e a penetração de J2 nas raízes aumentou com o aumento da concentração de inóculo independentemente da cultivar. A taxa de sobrevivência do nematoide foi maior na cultivar suscetível diminuindo com o aumento da concentração do inóculo, não sendo influenciada pela concentração de inóculo para cultivares resistentes. O terceiro estudo teve como objetivo avaliar o efeito do tratamento de sementes de duas cultivares de soja resistente (BRSGO 8860RR) e suscetível (BRS Valiosa RR), com produtos a base de abamectina e tiodicarbe no manejo de H. glycines. Foram conduzidos dois experimentos, sendo um em campo naturalmente infestado e outro em condições de casa de vegetação. Os tratamentos de sementes não afetaram o estande e a altura das plantas de soja. Não foi observado efeito dos tratamentos de sementes sobre o nematoide no experimento conduzido em campo. Em condições de casa de vegetação os tratamentos de sementes com produtos a base de abamectina, nas dosagens de 50 e 75 mL i.a. 100 kg sementes-1 acrescido de tiametoxam (70 mL i.a.) e os tratamentos imidacloprido + tiodicarbe (105+315 mL i.a.) e imidacloprido + tiodicarbe e carbendazim + tiram (75+225 e 30+70 mL i.a.) reduziram o número de fêmeas por grama de raiz na cultivar suscetível. O tratamento de sementes com imidacloprido + tiodicarbe (75+225 mL i.a.) também reduziu o número de ovos por fêmea na cultivar resistente, BRSGO 8860RR.

Glycine receptors (GlyR) are anion-permeable channels that belong to the pentameric ligand-gated ion channel family. Different GlyR subtypes are known. The main synaptic form is thought to be α1β heteropentamers which mediate fast synaptic inhibition in the adult spinal cord and brainstem. Data on recombinant receptors suggest two possible stoichiometries for this subtype, 2α1:3β and 3α1:2β. Evidence for the first comes from experiments on oocytes, whereas a study in mammalian cells favours the latter, raising the possibility that stoichiometry depends on the expression system. Here, we assess the stoichiometry of α1β GlyRs in Xenopus oocytes using two different electrophysiological approaches. The first involves the use of a reporter mutation at the conserved 9΄ position of the pore-lining domain. In other receptors, this mutation shifts agonist sensitivity in proportion to the number of mutated subunits. Recordings from mutant receptors failed to point towards one or the other stoichiometry. The second approach involved single-channel recordings from conductance mutants. This approach was also inconclusive for stoichiometry. However, we provide evidence that oocytes are not a suitable expression system for the study of heteromeric glycine receptors as they are highly prone to contamination by homomers. α2 homomeric GlyRs are predominant early in development and are replaced by α1 subunits in the first postnatal days. We investigated the activation mechanism of these channels in HEK293 cells by maximum likelihood fitting of single-channel data, at a wide range of glycine concentrations. The mechanism we propose suggests that α2 channels can open only when all binding sites are occupied by glycine, and only after the channel undergoes a conformational change ('flip') that links binding to gating. Macroscopic data favour a two binding site model. The scheme can describe adequately macroscopic currents from fast concentration jumps experiments when desensitization is included in the model.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
Fusarium solani f. sp. glycines, agente causal da síndrome da morte súbita em soja, é um importante patógeno no Brasil e em outras partes do mundo. Foram obtidos 15 isolados de F. solani f. sp. glycines a partir de 57 fragmentos de raíz de soja coletados em diferentes localidades no Brasil. A diversidade genética destes 15 isolados e de outros 4 isolados cedidos pelo Departamento de Fitotecnia da Universidade Federal de Viçosa, foi avaliada por RAPD. As amplificações com 15 oligonucleotídeos resultaram em 175 fragmentos polimórficos e 5 monomórficos. As distâncias genéticas entre os isolados variaram de 12,2 a 85%, revelando alta variabilidade genética. Não houve agrupamento dos isolados de acordo com seu local de coleta. Esta alta variabilidade genética provavelmente se deve à presença de transposons e de ciclo parassexual do patógeno. Protoplastos de Fusarium solani f. sp. glycines foram obtidos por digestão enzimática do micélio, na presença de MgSO 4 1,2 M como estabilizador osmótico. Foram liberados 2,4 X 10 7 protoplastos/mL após 4 horas de digestão do micélio a 28°C e 80 rpm. A concentração ideal de enzima lítica para a protoplastização foi de 15 mg/mL. A maior taxa de regeneração dos protoplastos foi de 5,5% em meio BDA estabilizado com sacarose 1,0 M.
Fusarium solani f. sp. glycines, the causative agent of sudden death syndrome in soybean, is an important pathogen in Brazil and in other parts of the world. Fifteen F. solani f. sp. glycines isolates were obtained out of 57 root fragments collected in different growing regions in Brazil. The genetic diversity of these isolates and that of four isolates obtained from the Department of Plant Sciences of the Federal University of Viçosa were analyzed by RAPD. Amplification with 15 primers resulted in 175 polymorphic and 5 monomorphic DNA fragments. The genetic distances among the isolates ranged from 12.2 to 85%. No grouping was obtained based on geographic localization, certainly because there were not enough differentiation. This high genetic variability is probably due to the activity of transposons and the presence of the parassexual reproduction. Fusarium solani f. sp. glycines protoplasts were obtained by enzymatic digestion of micelium, using 1,2 M MgSO 4 as osmotic stabilizer. Aproximately 2.4 X 10 7 protoplasts/mL were obtained after 4 hours of micelium digestion at 28°C e 80 rpm. The optimum enzyme concentration was 15 mg/mL. The highest protoplast regeneration rate was 5.5% in BDA stabilized with 1,0 M sucrose.
Dissertação importada do Alexandria

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This study evaluated the effect of herbicides on the population density of H. glycines in soybean cultivars, (one resistant and three susceptible) in natural infestation conditions. Experiments were done in Campo Alegre de Goiás and Gameleira de Goiás, agricultural year 2006/07, using randomized blocks, with three repetitions. The treatments were arranged in a factorial design 4 x 4 + 1, with four cultivars (BRSGO Ipameri, BRSGO Luziânia, BRSGO Jataí BRS Silvânia RR), four forms of weed control (hand weeding, chlorimuron-ethyl + lactofen, chlorimuron-ethyl and haloxyfop-methyl), and additional treatment represented by the combination of transgenic cultivar BRS Silvânia RR and herbicide glyphosate. In agricultural year 2009/2010, another test was conducted in Gameleira de Goiás, with four repetitions, in a factorial design 2 x 4 + 2, involving two cultivars (BRSGO Chapadões and BRSGO 8360), four forms of weed control (hand weeding, lactofen, chlorimuron-ethyl; haloxifop-r), and two additional treatments represented by the combination of transgenic cultivar BRS Valiosa RR with control manual weed and chemical control via herbicide glyphosate. In the agricultural year 2006/07, in Campo Alegre de Goiás, eighty days after planting, there was less formation of viable cysts in cultivar BRS Silvânia RR associated with the application of clorimuron + lactofen, compared to manual control. In Gameleira de Goias, it was found, forty days after planting, increase in the number of viable cysts using herbicides, compared with manual control, in cultivar susceptible BRSGO Luziânia, BRSGO Ipameri, resistant to H. glycines, the number of viable cysts was lower when applied herbicides clorimuron+lactofen or haloxyfop, compared the application of clorimuron. The herbicides had no effect on the number of females in susceptible and resistant cultivars in the year 2009/10. In cultivar susceptible BRSGO 8360, the herbicide haloxifop led to a smaller number of females, in comparison with clorimuron, 45 days after sowing. The herbicide lactofen, in the cultivar BRSGO Chapadões (resistant) was associated with increase in the number of viable cysts 45 days after sowing. This herbicide affects negatively the biomass of leaves, fresh green beans and dry bean in the cultivars BRSGO Chapadões and BRSGO 8360.
O trabalho objetivou avaliar o efeito de herbicidas sobre a densidade populacional de H. glycines em cultivares de soja (uma resistente e três suscetíveis), em condições naturais de infestação. Experimentos foram conduzidos em Campo Alegre de Goiás e Gameleira de Goiás, safra 2006/07, utilizando delineamento de blocos casualizados, com três repetições. Os tratamentos foram arranjados em esquema fatorial 4x4+1, sendo: quatro cultivares (BRSGO Ipameri, BRSGO Luziânia, BRSGO Jataí, BRS Silvânia RR); quatro formas de controle de plantas daninhas (arranquio manual, chlorimuron-etil+lactofen, chlorimuron-etil e haloxyfop-metil); e o tratamento adicional representado pela combinação da cultivar transgênica BRS Silvânia RR e o herbicida glifosato. Na safra 2009/10, outro ensaio foi conduzido em Gameleira de Goiás, com quatro repetições, em esquema fatorial 2x4+2, envolvendo duas cultivares (BRSGO Chapadões e BRSGO 8360) quatro formas de controle de plantas daninhas (arranquio manual, lactofen, chlorimuron-etil e haloxifop-r), e os dois tratamentos adicionais que consistiram das combinações entre a cultivar BRS Valiosa RR com o controle manual das plantas daninhas e com o controle químico via herbicida glifosato. Na safra 2006/07, em Campo Alegre de Goiás, aos oitenta dias após o plantio, observou-se menor formação de cistos viáveis na cultivar BRS Silvânia RR, associado à aplicação de clorimuron + lactofen, em comparação ao controle manual. Em Gameleira de Goiás, verificou-se, aos quarenta dias após o plantio, aumento do número de cistos viáveis com o uso de herbicidas, em comparação com o controle manual, na cultivar suscetível BRSGO Luziânia, já na cultivar BRSGO Ipameri, resistente a H. glycines, o número de cistos viáveis foi menor quando se aplicou os herbicidas clorimuron+lactofen ou haloxifop, em comparação à aplicação de clorimuron. Na safra 2009/10, os herbicidas não influenciaram o comportamento das cultivares (resistente ou suscetíveis) quanto ao número de fêmeas. Na cultivar suscetível BRSGO Luziânia, o herbicida haloxifop possibilitou a formação de menor número de fêmeas, em comparação com clorimuron, aos 45 dias após o plantio. O herbicida lactofen, em BRSGO Chapadões, resistente, esteve associado com o aumento na formação de cistos viáveis, aos 45 dias após o plantio. Este herbicida afetou negativamente a produção de biomassa de planta seca, vagem fresca e vagem seca nas cultivares BRSGO Chapadões e BRSGO 8360.

Reduced inhibitory glycinergic neurotransmission is implicated in a number of neurological conditions including chronic pain and hyperekplexia and restoring glycinergic signalling may be an effective method of treating these pathologies. Glycine transporters (GlyTs) control synaptic and extra-synaptic glycine concentrations. Slowing the reuptake of glycine using non-competitive GlyT inhibitors will increase extracellular glycine concentrations and increase glycine receptor (GlyR) activation. The general approach to drug development, of modulating a single target with high affinity and efficacy, has not produced useful novel treatments and therapeutic outcomes for sufferers of chronic pain. Modulation of multiple related targets to induce robust, systemic modulation is suggested as a potential way to improve therapeutic outcomes. I propose that a multi-target drug approach could be taken to modulate two key proteins in a restricted system, GlyT2 and GlyRα1, to improve glycinergic inhibitory signalling and provide good leads for the development of novel analgesics. Furthermore, hyperekplexia is a disease caused by mutations in glycinergic proteins and I proposed that multi-target drugs could also be investigated for use in treating this condition. To study the dual modulation of the glycinergic synapse, in Chapter 3 I characterise a system where GlyTs are co-expressed with GlyRs in Xenopus laevis oocytes and show that GlyTs can reduce the concentration of glycine sensed by GlyRs and alter the efficacy of receptor activation. I use two-electrode voltage-clamp electrophysiology and confocal microscopy to measure the impact of GlyTs on GlyRs and show that increases in GlyT density near GlyRs increasingly diminish receptor currents. Reductions in GlyR mediated currents are not observed when non-transportable GlyR agonists are applied or when Na+ is not available, validating changes in GlyR activation as related to glycine uptake by GlyTs. GlyTs create diffusion-limited, concentration gradients across different agonist concentration ranges. Due to these diffusion barriers, the actual glycine concentrations sensed at the membrane by GlyRs can be greatly reduced and these are estimated. Full receptor currents can be restored when GlyTs are blocked with selective inhibitors. Modulation of glycinergic neurotransmission is a novel way of increasing inhibitory signalling in the CNS, however, attempts to pharmacologically target this system have thus far failed in the clinical treatment of chronic pain. Glycine activates inhibitory GlyRs, however it is also a co-agonist at excitatory NMDA receptors. Full inhibition of GlyTs has also been shown to cause motor defects and lead to death. Targeted and potent inhibition of GlyTs to increase synaptic levels of glycine may therefore have unintended and opposite effects. Bioactive lipid compounds developed in our lab have actions as both GlyT inhibitors and GlyR positive allosteric modulators (PAMs). In Chapter 4, I assess the usefulness of these compounds in the co-expression system characterised in Chapter 3, exploring the feasibility of a multi-target approach to drug design at the glycinergic synapse and show that features of glycinergic modulators which are not apparent when studied at single targets can be elucidated. Hyperekplexia is a rare hereditary disease caused by mutations at various glycinergic proteins, however the majority of hyperekplexia cases are caused by mutations in the GlyRα1 gene. GlyRs are modulated by alcohols and volatile anaesthetics, and a serine at position 267 has been identified as an important residue that mediates their actions. The S267 residue has also been implicated in a hyperekplexic family carrying the dominant missense hyperekplexic mutation GlyRα1S267N, and heterologous expression of this mutant GlyR displays decreased sensitivity to glycine and to potentiation by ethanol. In Chapter 5, I co-express the GlyRα1S267N mutant with GlyT2 and show that GlyT2 does not modulate this mutant receptor. I then show that although dual modulation is an efficacious approach at WT GlyRα1/GlyT2, robust positive potentiation of GlyRα1S267N/GlyT2 currents is only achieved by a high efficacy GlyR PAM. These results highlight the need to understand how disease mechanisms change the interaction between target proteins of interest when designing multi-target drugs. Finally, in Chapter 6 I undertake a joint investigation into the modulation of GlyRs by glutamate. In contradiction to a previous report by Liu and colleagues in 2010, we show that glutamate does not allosterically modulate GlyRs. Reciprocal glutamatergic modulation of GlyRs in the central nervous system (CNS) would imply crosstalk between these two signalling systems, which would have fundamental implications for our understanding of nociceptive circuits and approaches to modulating them. However, GlyRs have multiple sites of allosteric modulation and are known to be modulated by large number of compounds, and we suggest the presence of a contaminating substance as the basis for this discrepancy. This study highlights the importance of independent assessment of novel neurobiological concepts. In this thesis, I have described a novel way of assessing dual action pharmacological modulation of the glycinergic synapse which can be adapted to model diseases states. This work will support the development of good leads into novel glycinergic modulators with better tolerability, efficacy and therapeutic outcomes.

Nos últimos anos tem se intensificado o uso de produtos com a finalidade de aumentar a produtividade na cultura da soja. Dentre estes estão os bioestimulantes que podem possuir em sua constituição extratos de algas, aminoácidos e hormônios. No entanto, pouco se sabe sobre o efeito isolado de cada um destes constituintes. Frente a isto, esta pesquisa teve por objetivos avaliar o efeito da aplicação de aminoácidos isolados em plantas de soja. Para isto, o trabalho foi dividido em três experimentos. No primeiro, foi realizada a aplicação via sementes de glutamato, cisteína, fenilalanina e glicina em doses variáveis. Essa etapa foi conduzida em sistema de canteiros e foi avaliada a emergência, índice de velocidade de emergência, acúmulo de massa de matéria seca, metabolismo antioxidante (enzimas superóxido dismutase - SOD, catalase - CAT, peroxidase - POD, teor de peróxido de hidrogênio - H2O2, prolina e peroxidação lipídica - PL) e produtividade. A partir da seleção das melhores doses obtidas na primeira etapa, foi realizado o segundo experimento, conduzido em casa de vegetação. As aplicações desse experimento foram realizadas no tratamento de semente, via foliar ou em ambas as épocas, além disso, foi realizada a aplicação de todos os aminoácidos em associação. Nesse experimento foram avaliados metabolismo antioxidante, enzimas de resistência (polifenoloxidase - PFO e fenilalanina amônia-liase - PAL), metabolismo do nitrogênio (enzimas nitrato redutase e urease, teor de NO3-, NH4+, N-Aa, ureídeos e N-Total), variáveis de crescimento de raíz, acúmulo de massa de matéria seca e produtividade. Já o experimento III foi realizado em campo, utilizando os mesmos tratamentos do experimento II. Foram avaliados o metabolismo antioxidante, enzimas de resistência, metabolismo do nitrogênio, massa de matéria seca e produtividade. Todos os experimentos foram conduzidos em delineamento em blocos casualizados com quatro repetições para cada tratamento. Todos os aminoácidos proporcionaram efeito positivo em diversas variáveis fisiológicas analisadas. O uso de glutamato, fenilalanina, cisteína e glicina de forma isolada repercutiram em melhores efeitos quando a aplicação é realizada somente no tratamento de sementes. A partir da aplicação desses aminoácidos ocorreu incremento da assimilação de nitrogênio e no acúmulo de massa de matéria seca, o que levou a maior produtividade dessas plantas. O maior efeito na produtividade foi observado por meio da aplicação de fenilalanina em todos os experimentos, quando comparados com os demais aminoácidos. Com relação ao metabolismo antioxidante o uso de cisteína no tratamento de sementes proporcionou aumento da atividade das enzimas SOD e PAL e redução da PL. O uso de fenilalanina no tratamento de sementes induz ao incremento da CAT e SOD e o glutamato induz o aumento de PAL e SOD. A utilização de todos os aminoácidos em associação somente foi eficiente na aplicação foliar, o que proporcionou maior desenvolvimento de raíz, maior assimilação de nitrogênio, acúmulo de massa de matéria seca e produtividade. Portanto, foi possível perceber que o glutamato, cisteína, fenilalanina e glicina apresentam importante papel de sinalização em plantas, pois pequenas doses já são suficientes para induzir ao incremento de parâmetros fisiológicos e, consequentemente aumentar a produtividade.
In recent years, the use of products to increase productivity in soybean has been intensified. Bio-stimulants can have in their constitution algae extracts, amino acids and hormones. However, little is known about the isolated effect of each of these constituents. Facing this problem, this research aimed to evaluate the effect of the application of single amino acids to soybeans. For this, the work was divided into three experiments. In the first, the application of amino acids was performed via glutamate, cysteine, phenylalanine and glycine to seeds. This stage was carried out on planting beds and the following variables were evaluated: emergency, emergency speed index, dry matter accumulation, antioxidant metabolism (superoxide dismutase - SOD, catalase - CAT, peroxidase - POD, hydrogen peroxide - H2O2 - content, proline and lipid peroxidation - PL) and productivity. From the selection of the best rates obtained in the first stage, the second experiment was carried out in a greenhouse. The applications of this experiment were performed as seed treatments, foliar application and both procedures; furthermore, the application of all amino acids in combination was also performed. In this experiment, the following variables were evaluated: antioxidant metabolism, resistance enzymes (polyphenol oxidase - PFO and phenylalanine ammonia lyase - PAL), nitrogen metabolism (nitrate reductase and urease, NO3- content, NH4+, N-Aa, ureide and N-Total), root growth, dry matter accumulation and productivity. The third experiment was carried out in the field using the same treatments of the second experiment. The following variables were evaluated: antioxidant metabolism, resistance enzymes, nitrogen metabolism, dry matter and productivity. All experiments were carried out in a randomized block design with four replications for each treatment. All amino acids provided positive effect on several physiological variables. The use of glutamate, phenylalanine, cysteine, and glycine alone lead to the best effect when the application was done only as seed treatment. From the application of these amino acids, the nitrogen assimilation was increased and the dry matter accumulation, which led to higher productivity of the plants. The greatest effect on productivity was observed by application of phenylalanine in all experiments, when compared with other amino acids. Regarding the antioxidant metabolism, cysteine use in seed treatment increased SOD and PAL activity and PL reduction. The phenylalanine use in seed treatment increased CAT and SOD activities and glutamate induced an increase of PAL and SOD activities. The use of all amino acids in association was only effective in foliar application, which provided further development of root, greater assimilation of nitrogen, dry matter accumulation and productivity. So, it was possible to conclude that glutamate, cysteine, phenylalanine and glycine have an important signaling role in plants, because small rates are enough to induce the increase of physiological parameters and consequently increase productivity.

Glycine is the third most abundant of the amino acids released by muscle. Perfused rat hind-limb and sheep diaphragm preparations were employed to study the origin of glycine produced by non-ruminant and ruminant muscle. Neither the degradation of muscle and erythrocyte glutathione nor the 'leaching out' of the intracellular glycine pool contributed to the glycine released by either muscle. When the perfusions were carried out with the medium free of amino acids, the proteolysis accounted for 57% of the total glycine release by the rat hind-limb and 38% by the sheep diaphragm. Minimum de novo synthesis of glycine was 12.3 umol/3 h/30 g in the rat muscle and 10.3 umol/3 h/30 g in the sheep muscle. Addition of serine to the perfusion medium stimulated significantly both the rate of glycine efflux and total glycine production in the rat hind-limb. Similar results were obtained with the sheep diaphragm; however, the increases were not statistically significant. Addition of 5-formyl tetrahydrofolate, a specific inhibitor of serine hydroxymethyltransferase, SHMT (EC 2.1.2.1) significantly decreased the rate of glycine efflux from both the muscles. The observations using cold serine were confirmed with the experiments employing radioisotopes. Up to 40% of total glycine produced by the rat hind-limb was derived from serine, whereas in the sheep diaphragm it was only 4%. In both the muscles synthesis of glycine from serine was by SHMT and not glycine synthase (EC 2.1.2.10). Synthesis of glycine from threonine was negligible in both the muscles. SHMT activity increased in liver, diaphragm and hind-limb muscle of female rats treated with trenbolone acetate or testosterone, anabolic agents. Both the muscles incorporated 14C from (U-14C) serine and (3-14C) serine to methionine, cystine, alanine, aspartate and glutamate + glutamine. The label from (U-14C) glucose was recovered in serine and glycine in the rat hind-limb but not in the sheep diaphragm. A 'serine-glycine' cycle involving kidney and muscle is proposed. The possible significance of glycine released by muscle is discussed. Development of a system for the perfusion of sheep diaphragm with erythrocyte-free medium, and a method for the determination of radioactivity in C-2 of glycine also form a part of the thesis.

Doutoramento em Nanociências e Nanotecnologia
Bioorganic ferroelectrics and piezoelectrics are becoming increasingly important in view of their intrinsic compatibility with biological environment and biofunctionality combined with strong piezoelectric effect and switchable polarization at room temperature. Here we study piezoelectricity and ferroelectricity in the smallest amino acid glycine, representing a broad class of non-centrosymmetric amino acids. Glycine is one of the basic and important elements in biology, as it serves as a building block for proteins. Three polymorphic forms with different physical properties are possible in glycine (α, β and γ), Of special interest for various applications are non-centrosymmetric polymorphs: β-glycine and γ-glycine. The most useful β-polymorph being ferroelectric took much less attention than the other due to its instability under ambient conditions. In this work, we could grow stable microcrystals of β-glycine by the evaporation of aqueous solution on a (111)Pt/Ti/SiO2/Si substrate as a template. The effects of the solution concentration and Pt-assisted nucleation on the crystal growth and phase evolution were characterized by X-ray diffraction analysis and Raman spectroscopy. In addition, spin-coating technique was used for the fabrication of highly aligned nano-islands of β-glycine with regular orientation of the crystallographic axes relative the underlying substrate (Pt). Further we study both as-grown and tip-induced domain structures and polarization switching in the β-glycine molecular systems by Piezoresponse Force Microscopy (PFM) and compare the results with molecular modeling and computer simulations. We show that β-glycine is indeed a room-temperature ferroelectric and polarization can be switched by applying a bias to non-polar cuts via a conducting tip of atomic force microscope (AFM). Dynamics of these in-plane domains is studied as a function of applied voltage and pulse duration. The domain shape is dictated by both internal and external polarization screening mediated by defects and topographic features. Thermodynamic theory is applied to explain the domain propagation induced by the AFM tip. Our findings suggest that β-glycine is a uniaxial ferroelectric with the properties controlled by the charged domain walls which in turn can be manipulated by external bias. Besides, nonlinear optical properties of β-glycine were investigated by a second harmonic generation (SHG) method. SHG method confirmed that the 2-fold symmetry is preserved in as-grown crystals, thus reflecting the expected P21 symmetry of the β-phase. Spontaneous polarization direction is found to be parallel to the monoclinic [010] axis and directed along the crystal length. These data are confirmed by computational molecular modeling. Optical measurements revealed also relatively high values of the nonlinear optical susceptibility (50% greater than in the z-cut quartz). The potential of using stable β-glycine crystals in various applications are discussed in this work.
Piezo e ferroeléctricos biorgânicos são materiais que estão a atrair para si uma importância crescente por força da sua compatibilidade intrínseca com ambientes biológicos e uma biofuncionalidade aliada a um forte efeito piezoeléctrico e polarização controlada, a temperature ambiente. Aqui estudamos a piezo e ferroelectricidade no mais pequeno aminoácido, a glicina, representando uma ampla classe de aminoácidos nao-centrosimétricos. A glicina é um elemento básico e extremamente importante em biologia, uma vez que serve de unidade base de construção para proteínas. Três formas polifórmicas com diferentes propriedades são possíveis na glicina (α, β e γ). De especial interesse para várias aplicações são as estruturas não-centrosimétricas: β-glycina e γ-glycina. A mais interessante β-polimorfa está a ser alvo de uma atenção reduzida, comparativamente às outras, por motivos de uma maior instabilidade a temperatura ambiente. Neste trabalho, Podemos crescer microcristais estáveis de glicina-β pela evaporação da solução aquosa num substrato (111)Pt/Ti/SiO2/Si que funciona como "template". Os efeitos da concentração da solução e da nucleação Pt-assistida no crescimento do cristal e evolução da fase foram estudados com recurso à difracção Raio-X e espectroscopia Raman. Adicionalmente, a técnica de "spin-coating" foi utilizada para a fabricação de nano-ilhas de glicina-β altamente alinhadas, com a orientação dos eixos cristalográficos normalizada pelo substrato de Pt. Estudamos a indução de domínios estruturais por meio da ponta do AFM e a variação da polarização nos sistemas moleculares da β-glicina através da técnica PFM (Microscopia de Piezo Força), comparando os resultados obtidos com modelação molecular e simulações computacionais. Mostramos que a β-glycina é de facto um piezoeléctrico à temperatura ambiente e a polarização pode ser controlada por aplicação de uma tensão a cortes não polares. A dinâmica destes domínios complanares é estudada como função da tensão aplicada e duração do pulso. A forma do domínio é ditada pela polarização interna e externa, cujo rastreio é mediado por defeitos e características topográficas. A teoria termodinâmica é aplicada para explicar a propagação dos domínios induzidos pela ponta do AFM. As nossas descobertas sugerem que a β-glycina é um ferroeléctrico uniaxial com propriedades controladas pelas fronteiras dos domínios (electronicamente carregadas), que em seu turno podem ser manipuladas por tensão externa. Adicionalmente, propriedades ópticas não-lineares da β-glycina foram investigadas por um método de segunda geração harmonica (SHG). Este método confirmou que a simetria axial é preservada em cristais crescidos sem pós-tratamento, reflectindo a esperada simetria P21 da fase β. A direcção da polarização espontânea mostrou ser paralela ao eixo monoclínico [010] e direccionada no comprimento do cristal. Estes dados foram confirmados por modelação computacional molecular. Medições ópticas revelaram também um valor relativamente elevado para a susceptabilidade óptica não-linear (50% maior que no quartzo com corte em z). O pontencial uso de cristais de β-glycina estáveis em diversas aplicações são também discutidos.

Alcohol is one of the most widely used drugs, yet its targets in the brain have not been reliably established, and effective treatments for alcohol addiction are lacking. Various ligand-gated ion channels (LGICs) are established targets of ethanol in the central nervous system, including NMDA receptors and the Cys-loop γ-aminobutyric acid type A (GABAA), 5-HT3, nicotinic acetylcholine (nACh), and glycine (Gly) receptors (Harris et al 2008, Spanagel 2009). Historically, the role of GABAA receptors in alcohol’s sedative, anxiolytic, and depressant effects has been supposed. More recently, extrasynaptic δ subunit-containing GABAA receptors have come to light as targets of behaviourally relevant concentrations of alcohol in the brain (Olsen et al 2007). Glycine receptors (GlyRs) have also been implicated in ethanol’s (EtOH) effects on motor coordination, reward pathways, sensory processing and perception. Furthermore, glycine receptors may be involved in regulating alcohol consumption (Perkins et al 2010, Soderpalm et al 2017). Ethanol may interact with the receptor via residues in transmembrane M2 and M3 regions, in addition to residues in Loop 2 of the extracellular domain (Burgos et al 2015, Crawford et al 2008, Horani et al 2015). Loop 2 residues of the extracellular domain may mediate signal transduction in the channel of both GABAA and glycine receptors (Cederholm et al 2010), and may be important in ethanol’s actions at these receptors (Crawford et al 2007). Furthermore, Loop 2 of the GABAA receptor δ subunit may be necessary for its high sensitivity to ethanol, and an ethanol ultra-sensitive mutant GlyR may be created through insertion of this sequence into the α subunit (Perkins et al 2009). Recent behavioural and electrophysiological experiments have demonstrated that the neuropeptide oxytocin (OT) attenuates ethanol-induced motor impairment in rats, and prevents ethanol-induced potentiation of GABA-gated currents via selective and specific interaction at δ-GABAA receptors (Bowen et al 2015). Although oxytocin’s binding site remains unclear, its interactions at these receptors presents a useful tool for investigation of the receptor conformations necessary for both alcohol and oxytocin’s actions. The actions of oxytocin at glycine receptors have not yet been characterised, although the functional and structural similarities between GABAA and Gly receptors - particularly as inhibitory LGIC targets of ethanol - suggested that a similar oxytocin-ethanol interaction could be possible at glycine receptors. Overall, this project aimed to investigate the interactive effects of ethanol and oxytocin at recombinant human homomeric α1 and α2 GlyRs. The role of Loop 2 in oxytocin’s effects and interactions with ethanol at ethanol-sensitive mutant α1δL2 GlyRs was also a focus of this investigation. To achieve this, human wild type α1, α2, and mutant α1δL2 homomers were expressed recombinantly in Xenopus laevis oocytes. Two-electrode voltage clamp electrophysiology was then used to evaluate the effects of EtOH and OT at these receptors. Presently, ethanol was found to potentiate α1 and α2 GlyRs at pharmacologically relevant concentrations, with the threshold concentration for potentiation greatly reduced (from 30 mM EtOH to as low as 1 mM) in the α1δL2 mutant receptor. At all of these receptors, oxytocin significantly attenuated ethanol-induced potentiation of glycine-gated current when co-applied at low micromolar concentrations. Importantly, neither ethanol nor oxytocin activated the receptors independently, and oxytocin did not modulate glycine currents in the absence of ethanol. The structurally similar neuropeptide arginine vasopressin (AVP) did not modulate ethanol potentiation when tested in place of OT at α2 receptors, suggesting structural moieties unique to the oxytocin molecule may allow modulation of ethanol’s actions at these receptors. In addition to their significantly increased sensitivity to ethanol, Loop 2-mutated GlyRs were more sensitive to glycine, although the general function of the receptor appeared unchanged. Importantly, the modulatory effects of oxytocin upon ethanol was preserved across wild type α1 and mutant α1δL2 receptors, indicating that Loop 2 is unlikely to be involved in mediating oxytocin’s interactions with the glycine receptor. However, the binding site for oxytocin at glycine and GABAA receptors has not been identified, necessitating further investigation into this novel action of oxytocin in the brain.

Many soybean [Glycine max (L.) Merrill] cultivars have narrow leaflet shape but it is not known if all of these lines derive this trait from the ln gene or another locus. This project was conducted to determine the inheritance of the narrow leaflet trait in several soybean genotypes and wild [Glycine soja Sieb. et Zucc.] accessions, and also to determine the allelism of the genes for this trait in the selected lines. The parents, F1, F2 and F2:3 generations were grown at Kentland Research Farm near Blacksburg, VA or in the greenhouse. The F2 and F2:3 generations (where available) were observed for segregation in leaflet shape. The populations were scored as having either broad or narrow leaflets using visual classification and leaf measurements when necessary. 'Camp' was crossed with broad leaflet parent 'Essex' to study the inheritance of the narrow leaflet trait in Camp. Observation of the F2 and F2:3 generations lead to the conclusion that a single recessive gene controls leaflet shape in Camp. Narrow leaf parents 'SRF 400' and Camp were crossed with lines having the ln gene (T41, S56, and D64-4731). None of the crosses among Camp, T41, SRF 400, S56 and D64-4731 segregated for leaflet shape in the F2 generation leading to the conclusion that they all have the ln allele at the same locus controlling lanceolate leaflet shape. T313, a line containing a gene for narrow rugose leaflets (lnr), was crossed with Camp to study allelism between the lnr and ln genes. Segregation for leaflet shape was observed in the F2 and F2:3 generations allowing the conclusion that the lnr gene controlling the narrow rugose leaflet trait in T313 is at a locus independent from the ln gene. A deficiency of narrow rugose plants was observed in all of the populations with T313 as a parent, and was theorized as being caused by selection against lnr gametes. After adjustment for the lnr deficiency, the F2 data appeared to fit a 9 broad : 3 narrow : 4 narrow rugose ratio. Three G. soja lines were crossed to broad and narrow leaflet parents and the F2 generations were examined to determine the inheritance of the very narrow leaf phenotype. The results indicate that there are one or two recessive genes controlling narrow leaflet shape in the G. soja accessions, which are not allelic to the ln gene. Since these populations were not advanced to the F3 generation, definite conclusions cannot be drawn about the genetics of the very narrow leaf phenotype.
Master of Science

Thesis (Ph.D.)--University of Missouri-Columbia, 2006.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (February 27, 2007) Vita. Includes bibliographical references.

Glutathione transferases, also known as Glutathione S-transferases (GSTs), are a diverse group of enzymes that catalyse the conjugation of the tri-peptide glutathione to a wide range of electrophilic substrates. Their biological function in endogenous metabolism in plants is not well characterised, although their role in herbicide metabolism and herbicide selectivity is well documented. Many herbicides used in soybean. Glycine max (L.) Merr., are selective against weeds due to their rapid detoxification in the crop through conjugation with homoglutathione (γ-glu-cys-β-ala), the predominant free thiol in many legumes. However, an in depth characterisation of the GSTs which can potentially catalyse these reactions in soybean has never been performed. This work describes the biochemical and molecular characterisation of GSTs in soybean with emphasis on the identification of specific isoenzymes involved in herbicide metabolism. GST activity toward the chloroacetanilide herbicides acetochlor and metolachlor, the diphenyl ethers acifluorfen and fomesafen and the sulphonyl urea chlorimuron-ethyl were all detected in crude protein extracts from five-day-old suspension cultured soybean cells. GST activity was also determined in five-day-old soybean seedlings, though this activity was significantly lower than that observed with the cell suspension cultures. Treatment of soybean plants with herbicides and herbicide safeners resulted in increased GST activity toward the model substrate l-chloro-2,4-dinitrobenzene (CDNB), but no change in activity toward herbicide substrates. In both plant and cell cultures GST-catalysed conjugation of the diphenyl ethers acifluorfen and fomesafen was over five-fold greater in the presence of homoglutathione as compared with glutathione. The preferential detoxification of these herbicides in the presence of homoglutathione appeared to be an important determinant of their rapid detoxification in soybean and an important factor in herbicide selectivity. GSTs were purified from five-day-old soybean cell cultures using S-hexylglutathione affinity chromatography and anion-exchange chromatography. A combination of reversed-phase HPLC, SDS-PAGE and MALDI-TOF mass spectrometry of the purified fractions indicated the presence of nine putative GST subunits, each with a molecular mass between 25 and 29 kDa. Soybean GST cDNA clones were obtained using a combination of RT-PCR, utilising degenerate oligonucleotides designed to conserved regions within plant GSTs, and screening of cDNA libraries prepared from soybean plants and cell cultures. This process failed to identify any theta-type GSTs, the class associated with herbicide detoxification in maize. In contrast, seven distinct tau-type GSTs were isolated together with a number of clones showing minor variations in individual sequences. Expression of these cDNAs in Escherichia coli showed the purified recombinant GSTs were active toward a diverse range of substrates, and possessed additional glutathione peroxidase activity. GST activities for each recombinant enzyme varied with substrate and thiol type, with a marked preference for homoglutathione with selected substrates. From the work reported in this study it would appear that the tau-type GSTs of soybean are at least as complex as those previously reported in cereals and have an important role in determining herbicide metabolism and selectivity in this major crop.

The glycine cleavage system (GCS) is a multi-enzyme complex localised in the mitochondria and serves as the main catabolic pathway for glycine. It contributes to supply of one-carbon units into folate one-carbon metabolism (FOCM) which utilises them for vital processes such as purines and thymidylate biosynthesis and methylation reactions. This thesis focuses on the role of glycine decarboxylase (Gldc), a member of the GCS, in embryonic development of the brain. It utilises two loss-of-function mouse models for Gldc which were found to exhibit two distinct disease phenotypes: non-ketotic hyperglycinemia (NKH) and neural tube defects (NTDs). The aims of this project are to investigate what effects GCS deficiency has on FOCM, the developmental mechanisms underlying NTDs caused by loss of Gldc expression, and suitability of the Gldc mice models as animal models for classical NKH. NKH is a rare metabolic disease caused by mutations of GCS genes (mainly GLDC) and characterised by accumulation of glycine in body fluids, resulting in severe neurological dysfunction and poor survival. Gldc-deficient mice exhibited features of NKH including elevated glycine, early post-natal lethality, and hydrocephalus. Enlargement of the brain ventricles was found to already be present at late-foetal stage, while glycine levels in whole embryos were already elevated shortly after neurulation. Gldc-deficient embryos also displayed NTDs, a common birth defect of the central nervous system that result from failure of the neural tube to close. Gldc-deficient embryos displayed abnormal folate metabolism, growth retardation and reduced cell proliferation. Supplementation with one-carbon units through dietary means was able to normalise folate profiles, completely rescue the NTDs, and normalise proliferation and growth in Gldc-deficient embryos. Diet-induced folate deficiency and interactions with the Mthfr mutation (which results in a methylation defect) did not exacerbate the NTDs caused by the Gldc mutation. This study provides the first mouse model for classical NKH and suggests that the pathology of NKH begins earlier in development than suspected. It also suggests that Gldc deficiency causes NTDs by reducing the supply of glycine-derived, mitochondrial one-carbon units for FOCM reactions.

The ionotropic receptors including nicotinic acetylcholine (nAChR), y-aminobutyric acid type A and C (GABAa/c), serotonin (5HT-3) and glycine receptors (GlyR) all comprise the cys-loop receptor family. A pentameric configuration is adopted by these receptors featuring a large extra cellular domain, four transmembrane (M) spanning domains and an intracellular compartment primarily contributed from the M3-4 loop. The recent advent of an X-ray crystal structure of the related ACh binding protein from snail, Lymnaea stagnalis, and an electron micrograph nearing atomic resolution of the transmembrane domains for the Torpedo nAChR provide the opportunity to homology model the structures of all cys-loop ligand-gated receptors and investigate critical elements of receptor function with unprecedented accuracy. By using homology modelling to guide site-directed mutagenesis in combination with whole-cell patch clamp electrophysiology, molecular elements that mediate the biphasic modulation of glycine receptors by Zn2+ were investigated. The structure of a previously identified low sensitivity inhibitory Zn2+ site was clarified revealing critical determinants of GlyR subtype selectivity for Zn2+ mediated inhibition. Intriguingly these experiments also revealed a novel functional asymmetry at this site suggesting that one specific side of the site directs downstream transduction of the inhibitory Zn2 effect. This enabled the elucidation of a hydrophobic pathway leading directly through the core of the GlyR from the inhibitory Zn2+ site to the agonist binding site suggesting a mechanism for Zn2+ mediated inhibition. This study also investigated the structural basis for Zn2* enhancement of GlyR function. A comparative study of GlyR al and a2 followed by a directed mutagenesis regional scan led to the identification of a complete and novel site for Zn2+ potentiation. Further studies also elucidated a potential mechanism of action for this site by identifying an important gating component of the GlyR, which exhibited a strong interaction with this novel potentiation site for Zn2+. These studies demonstrate the power of a homology modelling strategy to resolve key elements of cys-loop ligand-gated receptor function. Identifying and understanding receptor modulation by allosteric effectors is a key requisite to understanding receptor function. This work describes the key structural determinants involved in the biphasic Zn2+ modulation of GlyRs both in terms of actual binding sites and downstream effector mechanisms.

Glycine receptor antibodies have been identified in a few patients with progressive encephalomyelitis with rigidity and myoclonus (PERM), a highly disabling disorder characterised by rigidity, spasm and brainstem symptomatology. The clinical characteristics of patients with glycine receptor antibodies have not yet been fully described and it is not clear whether GlyR-Abs are pathogenic or just an epiphenomenon. This study examined the clinical features and immunotherapy responses of 45 patients; characterised the GlyR-Ab pathogenicity, subunit specificity and binding to different brain region in vitro, and examined mice injected with GlyR-Abs to model the disease in vivo. Most of the patients were classified as PERM but some patients had symptomatology beyond the classical motor manifestations and there were four patients with tumours (thymomas and lymphomas). GlyR-Ab titres were varied in serum and CSF, but there was intrathecal synthesis in the six patients with suitable samples. Most patients were very disabled but almost all showed excellent responses to immunotherapies. The antibodies were mainly IgG1 and IgG3 subclasses, activated complement on glycine receptor-transfected HEK cells at room temperature, and caused internalisation and lysosomal degradation of the glycine receptors at 37°C. GlyR-Abs bound to rodent spinal cord and brainstem co-localising with monoclonal antibodies to GlyRα1 on the surface of neurons. GlyR-IgG injected intra-peritoneally led to impairment in forced walking ability, sensorimotor function and coordination. Analysis of the brain showed that animals injected with patients' IgG, but not control IgG, had antibodies bound to the brainstem, spinal cord, cerebellum and caudate, co-localising with GlyRα1 monoclonal antibody. Intra-cerebroventricular injection of GlyR-IgG caused an anxiety-like behaviour in mice but no evident motor disturbances. These results provide the first evidence of in vitro and in vivo pathogenicity of the GlyR-Abs, supporting the use of long term immunosuppression in these patients to provide them with a good prognosis.

Diversos fatores foram investigados a fim de se determinar uma metodologia para regeneração de plantas de soja (Glycine max (L.) Merrill) via embriogênese somática, tendo como base o germoplasma brasileiro de soja. Para isso, cotilédones imaturos foram cultivados in vitro sob diversas condições (regime hormonal, tipo e. concentração de açúcar, etc.). Foram avaliados os parâmetros referentes à embriogênese e a calogênese, assim como observações organográficas e anatômicas. Os resultados obtidos mostraram que altas concentrações (10-25 mg/l) de 2,4-D ou 2,4,5-T (11,5-17,5 mg/l), interrompem a ontogênese do embrióide no estádio globular de desenvolvimento, provavelmente por assegurar o estado meristemático das células. Baixas concentrações destas auxinas promoveram um desenvolvimento precoce dos embrióides. Nessas condições os embrióides eram em sua grande maioria morfologicamente anormais e não puderam ser convertidos em plântulas. A vacuolização precoce das células foi um dos eventos celulares associados a esse processo. Os embrióides tiveram origem multicelular a partir de células da epiderme e sub-epiderme do explante. A sacarose, em concentrações variando de 1,0 a 2,0% foi o açúcar que proporcionou maior eficiência de embriogênese. O processo de indução de embriogênese foi fortemente inibido pela irradiação do explante com raios ϒ. A adição de L-glutamina, L-prolina, caseína hidrolisada, extrato de malte ou extrato de levedura não estimulou a indução de embriogênese. A suplementação com ABA ou BA inibiu a indução de embriogênese e não favoreceu a maturação dos embrióides. As maiores eficiências de embriogênese foram obtidas cultivando-se os explantes na posição abaxial. Tanto a indução de embriogênese quanto a maturação dos embrióides foram fortemente afetadas pelo genótipo da planta doadora do explante. Não foi evidenciada a existência de interação entre genótipo e o meio de cultura (tipo e concentração de auxina). A luz favoreceu a indução de embriogênese, sob altas concentrações de auxina, mas inibiu completamente a maturação dos embrióides. Os embrióides induzidos pelo 2,4,5-T apresentaram uma maior frequência de maturação. Com base nesses resultados foi proposto um protocolo reproduzível para regeneração de plântulas assim como um modelo de origem dos embrióides. A aplicação dessa metodologia na obtenção de somaclones e de plantas de soja transgênicas foi discutida.
Several factors were studied in order to stabilish a methodology for soybean Glycine max (L.) Merrill) plant regeneration through somatic embryogenesis, im Brazilian soybean germplasm. Immature cotyledons were cultured in vitro, under several conditions (type and concentrations of plant regulators, sugars, etc.). Parameters of embryogenesis, callus formation, morphological and anatomical observations were assessed qualitative and quantitatively. The results threin analysed showed that high concentrations (10-25 mg/l) of 2,4-D or 2,4,5-T (11,5-17,5 mg/l) 2,4,5-T suspended embryoid ontogenesis at the globular stage, probably by maintaining the meristematic state of the cell. Low auxin concentrations promoted premature embryo development. In this condition, mostly embryoids were morphological abnormal and could not be converted into plantlets. Early vacuolation of the cells was an event associated with this process. Somatic embryos had multicellular origin from epidermal and su-epidermal explant cells. Concentrations of sucrose ranging from 1.0 to 2.0% presented the highest embryogenesis efficiency. The process of embryogenesis induction was strongly inhibited by explant gamma rays irradiation. Addition of L-glutamine, L-proline, casein hydrolysate, malt extract, or yeast extract to culture medium, did not enhance embryonic induction. Medium supplementation with ABA or BA inhibited embryogenesis induction and did not favour embryoid maturation. The highest embryogenesis efficiency was obtained culturing the explants with the abaxial surface in contact with the medium Either embryogenesis induction and embryoid maturation was strongly influenced by the genotype of the explant donor plant. It was not found any interaction between genotype and culture medium. The presence of light favoured embryogenesis induction, under high auxin concentration, but completely supressed embryoid maturation. Embryoids formed by the addition of 2,4,5-T showed a high maturation frequency. Regarding these results, a reproducible procedure for plantlet regeneration was proposed, as well as a model for embryoid origin. The potential application of this metodolog in the obtention of somaclones and transgenic soybean plants is discussed.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2002.
On t.p. "A̳" is subsript. Includes bibliographical references (leaves 160-177). Also available in electronic version. Access restricted to campus users.

Inhibitory glycine receptors (GlyRs) undergo developmental regulation with α2 subunits most abundant at prenatal and early postnatal stages, and α1 subunits predominating in adults. In comparing the relative amounts of mRNA for two known splice variants of the α2 subunit, α2A and α2b, in the rat spinal cord at different stages of development, I found evidence for the existence of an additional, novel variant. This variant, missing exon 3, I have termed "α2N." Examination of the RNA from spinal cords of different-aged rats indicated that α2N undergoes dramatic down-regulation during prenatal development. Its mRNA forms a significant portion of the α2 subunit pool at E14, but is nearly undetectable at the time of birth. I also examined the developmental changes in two factors that can regulate GlyR α2 pre-mRNA splicing, the splicing factor neurooncological ventral antigen-1 (Nova-1) and the brain isoform of the polypyrimidine tract binding protein (brPTB). Treatment of neurons in culture with antisense oligonucleotides to "knock down" one of the Nova-1 variants altered the expression of GlyR α2N. These results suggest that the relative levels of the variants of Nova-1 and brPTB may play a role in the developmental regulation of GlyR α2N. Based on these results I propose a model for the developmental regulation of GlyR α2N. These results provide evidence for a novel splice variant of the GlyR α2 subunit that undergoes dramatic developmental regulation, and reveal the developmental profiles of two possible regulators of its expression during development.

The slow leach rate of copper from chalcopyrite in acid solutions has often been attributed to the formation of a poorly soluble “passive” layer on the surface. For this study, it has been shown that no such passive layer exists in alkaline glycine solutions. Surface analyses have shown these species exist, but are not passivating. The leach rate is instead determined by the electronic structure of chalcopyrite.

A demanda por óleos vegetais tem sido crescente, principalmente para utilização como fonte de energia renovável na forma de biodiesel. Somando-se a isso, a podridão vermelha das raízes da soja (PVR), ou síndrome da morte súbita, causada pelo fungo Fusarium solani f.sp. glycines, tornou-se uma doença preocupante para os sojicultores, técnicos e pesquisadores, sendo uma estratégia recomendada a adoção de um sistema de controle integrado em que a utilização de cultivares tolerantes é um componente indispensável. Assim, este trabalho teve por objetivo verificar a possibilidade de se reunir em uma mesma planta de soja genes para alta produtividade de óleo e para tolerância a PVR. O sistema genético compreendeu um dialelo parcial 7x7, envolvendo sete genitores com alta produtividade de óleo e sete genitores tolerantes a PVR. A maioria dos genitores compreendeu linhagens experimentais desenvolvidas no Setor de Genética Aplicada às Espécies Autógamas (ESALQ/USP). Os experimentos envolveram as plantas F2, na safra 2008/2009 e progênies F2:3, na safra 2009/2010, para avaliação de caracteres agronômicos, do teor de óleo e produtividade de óleo em campo experimental; já a avaliação da reação a PVR foi realizada em progênies F2:4, em casa de vegetação com inoculação artificial do patógeno, por meio de uma escala de notas para a severidade dos sintomas radiculares. Além disso, por meio de marcadores microssatélites fez-se um estudo de genética de associação entre os marcadores e os caracteres reação a PVR e teor de óleo em três populações. Os resultados evidenciaram a existência de variabilidade nos genitores e nas progênies para todos os caracteres avaliados. O genitor com a maior capacidade geral de combinação e média de teor de óleo foi a cultivar A 7002; por outro lado, os menores valores foram verificados em PI 520733 e IAC 100. Os cruzamentos mais produtivos em óleo foram aqueles que envolveram o genitor A 7002, exceto quando este foi cruzado com IAC 100. Dez cruzamentos (USP 70004 com USP 14-10-38, USP 14-01-20, USP 14-13-16 e M-Soy 8001; USP 14-10-38 com USP 70057 e USP 70080; M-Soy 8001 com USP 70006, USP 70080 e USP 70123; e USP 14-01-20 x USP 70006) destacaram-se por originar a maioria de suas progênies com as melhores características em todos os caracteres, ou seja, apresentaram ciclo precoce ou semi-precoce, altura média, plantas eretas e com valor agronômico bom ou médio, alta produtividade de grãos e de óleo, alto teor de óleo e tolerância a PVR. Nenhum cruzamento originou a maioria de suas progênies com as piores características em todos os caracteres. Teor de óleo apresentou baixa correlação genética com altura da planta na maturidade, acamamento, valor agronômico, produtividade de grãos e reação a PVR. PVR apresentou baixa correlação genética com acamamento, produtividade de grãos, teor de óleo, produtividade de óleo; entretanto, foi possível obter progênies com alta produtividade de grãos e de óleo e tolerantes a PVR. Os marcadores microssatélites mostraram-se polimórficos e, foram associados dois locos SSR ao caráter teor de óleo e outros dois locos ao caráter reação a PVR.
The demand for vegetable oils has increased, mainly because of its use as a renewable energy source like biodiesel. Moreover, the sudden death syndrome (SDS) in soybean, caused by the fungus Fusarium solani f.sp. glycines, has become a worrying disease for soybean producers, technicians and researchers, and the adoption of an integrated control system using tolerant cultivars as an essential component has been a recommended strategy. This work aimed to verify the possibility of gathering genes for high oil yield and tolerance to SDS in the same soybean plant. The genetic system comprised a partial diallel 7x7 involving seven parents with high oil yield and seven parents tolerant to SDS. Most of the parents were experimental lines developed at the Sector of Genetics Applied to Self-Pollinated Species (ESALQ/USP). The 2008/2009 trials involved the F2 plants, and the 2009/2010 trials involved the evaluation of F2:3 progenies traits, such as agronomic characters, oil content and oil yield in the experimental field; whereas the analysis of the SDS reaction was tested in F2:4 progenies, in a greenhouse with artificial inoculation of the pathogen through a rating scale for severity of radicular symptoms. Moreover, a study of genetic association among microsatellite markers and the characters reaction to SDS and oil content in three populations was performed. The results showed the existence of variability in parents and progenies for all characters. The parent who had the highest general combining ability and average oil content was the cultivar A 7002; on the other hand, the lowest values were found in PI 520733 and IAC 100. The crosses that originated the best oil content progenies have involved the parent A 7002, except when it was crossed with IAC 100. Ten crosses (USP 70004 with USP 14-10-38, USP 14-01-20, USP 14-13-16 and M-Soy 8001; USP 14-10-38 with USP 70057 and USP 70080; M-Soy 8001 with USP 70006, USP 70080 and USP 70123; USP 14-01-20 x USP 70006) distinguished from the others by originating the majority of their progenies with the best features in all traits, in other words, showed early and semi-early maturity, medium height, upright plants, good or average agronomic value, high grain and oil yield, high oil content and tolerance to SDS. None of the crosses originated the majority of their progeny with the worst characteristics in all traits. Oil content showed low genetic correlation with plant height at maturity, lodging, agronomic value, yield and reaction to SDS. SDS showed a low genetic correlation with lodging, grain yield, oil content, oil yield. However, it was possible to obtain progenies with high grain and oil yield and tolerance to SDS. The microsatellite markers were polymorphic and, moreover, two SSR loci were associated with the character oil content and other two loci to the character SDS reaction.

USA soybean germplasm has a narrow genetic base that could be augmented by alleles from the wild species Glycine soja which positively influence agronomic traits. The objective of this study was to identify such alleles for yield and yield component QTL (quantitative trait loci). Two populations of 150 BC2F4 lines were generated from a mating between recurrent parent Glycine max 7499 and donor parent Glycine soja PI 245331 with one line in each population tracing back to the same BC2 plant. Population A was used for the QTL identification analysis and population B was used for the QTL verification test. The population A lines were genotyped at 120 SSR marker loci and one phenotype marker, covering a total map length of 1506 cM in 20 linkage groups with an average interval size of 12.5 cM. There were nine putative QTL significantly (Pandlt;0.0001, LODandgt;3.0) associated with yield and yield component traits across 3 environments. One QTL for seed yield was identified using the combined data; the G. soja allele at satt511 on LG-A1 was associated with increased seed yield (LOD=4.3) with an additive yield effect of 190 235 kg ha-1 depending on the QTL analysis method. The phenotypic variance accounted for by the QTL at satt511 was 12%. This QTL also provided a significant yield increase across environments in the validation population; lines that were homozygous for the G. soja allele at satt511 demonstrated a 6.3% (P=0.037) yield increase over lines that were homozygous for the G. max allele. One seed filling period QTL was identified at satt335 (LOD=4.0) on LG-F with an additive effect of +1 day. This QTL also provided a +1 day additive effect (LOD=3.3) on maturity. These results demonstrate the potential of using exotic germplasm to improve soybean yield.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico
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Soyatoxina 2 (SYTX-2) à uma proteÃna tÃxica isolada de sementes de soja [Glycine max (L.) Merril], genÃtipo BR-10, sendo letal para camundongos quando administrada por via intraperitoneal (DL50 4,5  0,1 mg/Kg de peso corpÃreo). Os sintomas observados apÃs sua administraÃÃo foram piloereÃÃo, taquicardia, estiramento de patas e da cauda, diurese e convulsÃes tÃnico-clÃnicas, que precederam a morte do animal. A SYTX-2 foi purificada do extrato bruto de soja por fracionamento com sulfato de amÃnio, cromatografias de troca iÃnica (DEAE-celulose), de afinidade (Sepharose-4B-tripsina) e de filtraÃÃo em gel (Superdex 200HR 10/30). A inclusÃo de ditiotreitol 0,005 M no tampÃo de extraÃÃo e em outros tampÃes utilizados durante o processo de purificaÃÃo foi crucial para obtenÃÃo da SYTX-2 num estado altamente homogÃneo e preservaÃÃo da toxicidade. A SYTX-2 nÃo à uma glicoproteÃna e apresenta massa molecular aparente de 28,0 e 25,4 kDa, quando avaliada por PAGE-SDS e filtraÃÃo em gel em coluna de Superdex 200HR 10/30, respectivamente, e coeficiente de extinÃÃo molar (ε1%1cm) de 16,9. Sua seqÃÃncia NH2-terminal exibiu uma identidade de 75%, quando relacionada com aquela de uma endoquitinase acÃdica da classe III de sementes de soja. Ao lado de sua atividade tÃxica, a SYTX-2 apresentou atividade quitinÃsica de 1,34 nKat/mgP. Essa toxina, quando em contato com o nervo ciÃtico de ratos foi capaz de aumentar a amplitude pico-a-pico, comprovando sua aÃÃo neurotÃxica. A SYTX-2 tambÃm mostrou atividade prÃ-inflamatÃria, induzindo migraÃÃo de neutrÃfilos para a cavidade peritoneal de ratos, cujo pico se deu 8 h apÃs sua administraÃÃo. AvaliaÃÃo de seu papel fisiolÃgico, mostrou que a SYTX-2 exerceu efeitos negativos sobre o desenvolvimento do inseto-peste de feijÃo-de-corda Callosobruchus maculatus, particularmente na emergÃncia do adulto e no tempo mÃdio de desenvolvimento larval. Adicionalmente, SYTX-2 foi tÃxica para ninfas de Dysdercus peruvianus, causando reduÃÃo no ganho de peso corpÃreo e na taxa de sobrevivÃncia. A SYTX-2 tambÃm atuou negativamente sobre o nematÃide Meloidogyne incognita, impedindo sua mobilidade. Por outro lado, a SYTX-2 nÃo foi capaz de inibir a germinaÃÃo de esporos e nem o desenvolvimento de hifas dos fungos Colletotrichum lindemuthianum, C. gloesporioides, Rhizoctonia solani, Cercospora kikuchii, Fusarium solani, F. oxysporum e Aspergillus niger, nas condiÃÃes de ensaio realizadas no presente trabalho. Em resumo, este trabalho apresenta, pela primeira vez, dados de uma proteÃna neurotÃxica contendo uma atividade quitinÃsica nÃo usual, cujo papel parece estar relacionado aos mecanismos de defesa da planta. Nesse contexto, SYTX-2 possui potencial para aplicaÃÃo biotecnolÃgica como um novo agente de defesa vegetal contra insetos-peste e nematÃide-parasitas
Soyatoxin 2 (SYTX-2) is a toxic protein isolated from soybean seeds [Glycine max (L.) Merril], genotype BR-10, lethal to mice upon intraperitoneal injection (LD50 4.5 Â 0.1 mg/Kg body weight). The symptoms observed upon administration were pilloerection, tachycardia, paw and tail extending, diuresis and tonic-clonic convulsions prior to death. The SYTX-2 was purified from soybean crude extract by ammonium sulfate fractionation, ion-exchange (DEAE-cellulose), affinity (Sepharose-4B-trypsin) and gel filtration (Superdex 200HR 10/30) chromatography. Inclusion of 0.005 M dithiothreitol in the extracting and other buffers used during the purification process was crucial to obtain highly homogenous SYTX-2 and to preserve the toxicity. SYTX-2 is not a glycoprotein and has apparent molecular masses of 28.0 and 25.4 kDa as measured by SDS-PAGE and gel filtration in Superdex 200HR 10/30 column, respectively, and a molar extinction coefficient (ε1% 1cm) of 16.9. Its NH2-terminal sequence shows 75% identity with a class III acidic endochitinase from soybean seeds. Therefore, besides to its toxic properties, SYTX-2 presented chitinase activity of 1.34 nKat/mgP. This toxin, when in contact with the sciatic nerve of rats was able to increase the peak-to-peak amplitude, confirming its neurotoxic action. Moreover, SYTX-2 presented pro-inflammatory activity, inducing neutrophil migration into the peritoneal cavity of rats which peaked at 8 h after administration. Evaluation of its physiological role showed that SYTX-2 exerted negative effect on the development of the cowpea pest insect Callosobruchus maculates, particularly on adult emergence and in the mean larval development time. Additionally, SYTX-2 was toxic to Dysdercus peruvianus nymphs, as it caused reduction of both the body weight gain and survival rate. SYTX-2 also acted negatively on the root-knot nematode Meloidogyne incognita by hindering its mobility. On the other hand, SYTX-2 did not inhibit neither the spore germination nor the hyphal development of the phytopathogenic fungi Colletotrichum lindemuthianum, C. gloesporioides, Rhizoctonia solani, Cercospora kikuchii, Fusarium solani, F. oxysporum and Aspergillus niger, under the assay conditions carried out in this present work. In summary this study presents, for the first time, data on a neurotoxin protein with additional unusual chitinase activity, whose physiological role is likely related to the plant defense mechanisms. Accordingly, SYTX-2 possesses potential biotechnological application as a novel plant defensive agent against insect pests and parasitic nematodes.

As the global population rises, the demand for food increases which underscores a need for improvement in food security. Disease pressures are a major concern surrounding sustainable agriculture. Static crop populations, containing little to no genetic diversity, are vulnerable to diverse pathogen populations. Wild relatives of crop plants are a reservoir for new disease resistance traits that can be introgressed into cultivated crops. The identification of novel disease resistance is of paramount importance because pathogen co-evolution is not only defeating current resistance genes (R genes) but chemical controls as well. Phytophthora sojae (P. sojae), the causal agent of Phytophthora root and stem rot disease, reduces soybean harvests worldwide. We developed an approach to screen for new R genes that recognize core effectors from P. sojae. We expect R genes identified by these screens to be durable because P. sojae requires core effectors for virulence. We utilized effector-based screening to probe Glycine soja germplasm with core RXLR effectors from P. sojae to search for novel R genes. We developed segregating populations from crosses of P. sojae resistant G. soja germplasm with susceptible G. max cultivar Williams to determine inheritance of potential R genes in germplasm that responded to core effectors. We are using marker assisted breeding to map disease resistance traits in recombinant inbred (RI) lines. To better understand pathosystems, we examined host resistance and susceptibility using bioinformatics. We analyzed the interaction between Arabidopsis thaliana ecotype Col-0 and Hyaloperonospora arabidopsidis isolate Emwa1 using a publicly available RNA time-course experiment. We describe a new algorithm to sort genes into time-point specific clusters using activation and repression parameters. Gene ontology annotations were used to identify defense genes with unique expression profiles, and A. thaliana null mutants for these genes were significantly more susceptible to Emwa1 than wild-type. We plan to use these tools to rapidly identify and guide introgression of durable disease resistance into crop species.
Ph. D.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Objetivou-se estudar diferentes metodologias de inoculação de Fusarium tucumaniae em folhas destacadas e em plântulas de soja, visando à caracterização da reação de resistência de cultivares de soja à F. tucumaniae. O trabalho foi desenvolvido em diferentes etapas relacionadas a seguir: obtenção e manutenção do inóculo; avaliação do desenvolvimento do patógeno em diferentes meios de cultura e diferentes luminosidades; avaliação de diferentes métodos de inoculação em folhas destacadas acondicionadas em papel de filtro e em solo, incubadas em duas temperaturas; avaliação de métodos de inoculação em plântulas cultivadas em casa de vegetação (primavera/verão e outono/inverno), reação de cultivares de soja a F. tucumaniae avaliadas em folhas destacadas, reação de cultivares de soja a F. tucumaniae avaliadas em plântulas (outono/inverno). As análises dos resultados permitiram concluir maior esporulação do fungo em meio de grãos de sorgo. As metodologias de inoculação de plântulas foram bem sucedidas na época de outono/inverno 2005, observando-se infecção das plântulas, principalmente de variedades suscetíveis, e os sintomas típicos da doença ocorreram em todos os métodos utilizados. FT-Cristalina apresentou maior nível de infecção da planta, para o método de grãos de sorgo (GS). O método de inoculação, em folhas destacadas, com grãos de aveia, promoveu maior infecção nas folhas e, o ambiente mais propício ao desenvolvimento dos sintomas da doença foi o solo a 23ºC. A cultivar FT-Estrela apresentou 90,2% de plântulas mortas, e CAC-1 46,9% (método GS). No método da folha destacada não foi possível caracterizar a reação das cultivares estudadas.
Detached leaves and seedlings of some cultivars were inoculated with F. tucumaniae using different methodologies in order to evaluated their resistance reaction. The conduction of the research work consisted of the following phases: obtainment and maintenance the isolate, evaluation of the pathogen growth and sporulation in different cultura media and luminosity; evaluation of different inoculation methods in detached leaves conditioned either in filter paper and soil being, incubated under two temperatures; evaluation of inoculation methods in soybean seedlings developed in greenhouse (Spring/Summer and Autumn/Winter); reaction of soybean cultivars to F. tucumaniae evaluated in detached leaves; reaction of soybean cultivars to F. tucumaniae evaluated in seedling (Autumn/Winter). The obtained results showed that the fungus sporulation was more intense when grown in a sorghum grain medium. Inoculation methodologies in seedlings showed best results when carried out during the 2005 Autumn/Winter period, because seedlings infection was observed, mainly in the susceptible cultivars, and all the employed methods allowed observation of the disease typical symptoms. FT-Cristalina was the cultivar in which the highest level of plant infection was observed, when the sorghum grain method (GS) was employed. The oat grain inoculation method in detached leaves, led to the highest leaf infection index, and the environmental condition which allowed the highest degree of leaf infection was in the soil under 23oC. FT-Estrela cultivar had 90.2% of their seedlings dead whereas for the CAC-1 cultivar that value was of 46.9% (method GS). Cultivars reaction could not be evaluated by means of the detached leaf method.

Soybean [Glycine max (L.) Merr.] seeds contain isoflavones that have positive impacts on human health. Field and greenhouse experiments were conducted in Quebec Canada to determine the effects of management and environmental factors [seeding date (late May and mid June), row spacing (20-, 40- and 60-cm), weeds (presence or absence), irrigation levels (low, moderate, and high) and genotypes (Proteina, Orford, and Golden)] and of foliar applications of elicitor compounds (i.e., LCOs, chitosan, and actinomycetes spores), on the isoflavone concentrations of mature soybean seeds, and other important seed characteristics. Our results indicated that environmental and agronomical factors have a great impact on soybean seed isoflavone concentrations of early maturity soybean cultivars. Year, seeding date, and weeds affected total and individual isoflavone concentrations, row spacing had no effect. Total isoflavone concentration was greater in 2003 than 2004. Seeding in mid June increased isoflavone concentration by 38%, compared to seeding in May. The presence of weeds increased total isoflavone concentrations by 9%. Isoflavone concentrations were significantly affected by cultivars and irrigation levels. In both of two growing seasons, Proteina had significantly greater isoflavone concentrations compared to Orford. Irrigation effects on isoflavone concentrations differed between years and cultivars. However, most responses were observed with the lower of the two irrigation levels, which increased isoflavone concentrations by as much as 60% compared to a non-irrigated control. Our results suggest that under greenhouse conditions most biotic elicitors tested increased the concentration of individual and total isoflavones in soybean seeds when compared to untreated control plants. LCOs proved to be the most effective in studies contrasting various elicitors. Response of field-grown plants was more variable than that of greenhouse-grown plants.

Rapidly proliferating cancer cells consume significantly more glycine than slow growing cancer cells and rapidly proliferative normal cells. Extracellular glycine supports high growth rates of tumour cells, being utilised in purine and glutathione synthesis. Glutathione, the major cellular antioxidant, is involved in chemotherapy resistance and cancer progression. It was observed that glycine transport via a specific glycine transporter GLYT1 is utilised to maintain adequate glutathione levels under cellular stress. Additionally, GLYT1 is regulated by the transcription factor ATF4, itself upregulated under cellular stress and also linked to cancer progression. Thus, extracellular glycine may play an essential role in tumour cell survival and proliferation and inhibiting its uptake could be a possible target in association with current cancer treatments. To address this hypothesis the work described in this thesis evaluated the role of a specific glycine transporter, GLYT1, in supporting tumour cell proliferation utilising molecular and pharmacological approaches. A549, a non-small cell lung cancer (NSCLC) cell line, and HT29, a colorectal cancer cell line, were utilised as models of rapidly proliferating tumour cells. A498, a renal cancer cell line, and HUVEC, a human umbilical vein endothelial cell, were utilised as models of slow growing tumour cell and non-transformed fast growing cell line, respectively. Following GLYT1 knockdown (kd), tumour cells presented a significant reduction in glycine uptake and cell proliferation. When compared to control cells, rapidly proliferating GLYT1kd tumour cells showed a reduction of ~30% in cell number over time and a decrease of ~50% in DNA replication rate, whereas it had only a minor or no effect on the other cell lines. GLYT1kd downregulated expression of mRNA of other amino acid transporters able to transport glycine, PAT1 and SNAT2. GLYT1kd also reduced glutathione levels of all tumour cells. A reduction in mRNA levels of cystine transporter subunit, xCT, essential to maintain intracellular levels of cysteine, another component of glutathione, was also observed following GLYT1 downregulation. When cells were treated with a specific GLYT1 inhibitor, ALX-5407, in a pharmacological approach, a reduction in cell proliferation was observed after 96h treatment with a prolonged doubling time for the fast growing tumour cells, while A498 and HUVEC cells proliferation were not affected by the inhibitor, when compared to untreated cells. ATF4 effect on cell proliferation and glycine uptake was also evaluated. ATF4kd decreased significantly GLYT1 mRNA levels, but had only a mild influence on glycine uptake of all cell types analysed. There was a significant reduction in proliferation of ATF4kd tumour cells, ~40% for A549 and HT29 cells and 26% for A498 cell; ATF4kd in HUVEC cells increased cell proliferation by ~11%, when compared to control cells. ATF4 was also important to maintain intracellular levels of glutathione. ATF4kd dowregulated the transcription factors involved in stress response (ATF3, ATF5 and ATF6) suggesting a connection between them. Data from this work show that glycine uptake by GLYT1 is required to maintain proliferation rates of highly proliferative cancer cells, but not slowly proliferating and nontumour cells. This suggests that targeting GLYT1 may be a possible new approach to cancer treatment.

Crystallisation under confinement conditions can produce nanocrystals with high surface-to-volume ratio, altered intrinsic properties (e.g. melting point depression), preferred orientation, selective polymorphic form and well-controlled morphology. The confinement conditions are normally achieved by nanopores prepared from polymers, which provide physical constraints on the size of the crystal. In this thesis, microemulsions, which have been extensively used for nanoparticle synthesis, are employed to achieve the 3D-nanoconfinement, owing to their spherical morphology to provide volume control and excellent droplet size control (< 50 nm) via the addition of dispersed phase. Thermodynamic control has been achieved during the crystallisation of glycine under the 3D-nanoconfinement generated by microemulsions. These microemulsions were prepared from heptane, Span 80/Brij 30, glycine solution, and methanol antisolvent. Polymorph selection has been obtained by varying the microemulsion compositions. A majority of y-glycine was observed when the microemulsion droplet size was in the range of 3.3-4.2 nm. This is when the microemulsions just had sufficient glycine for crystallization. Below this, no crystallization was observed. The microemulsions comprised of 0.25 g 4% glycine solution and 0.8-1.5 g methanol per sample, with crystallisation yielding visible crystals in 2-3 weeks. Higher concentration of glycine solution (> 4.5%) yielded mostly the α form and excess amount of methanol (> 1.5 g) produced the β form, due to the loss of confinement within the microemulsion and the participation of solvent templating effects introduced by methanol. The polymorph outcome was not affected by the surfactant-continuous phase ratio. Following on from the success of glycine and other organic/pharmaceutical compounds studied within the research group, dipicolinic acid (DPA) was chosen to study the thermodynamic control of hydrates within 3D-nanoconfinement. The hydration or solvation control was demonstrated in the microemulsions. In particular, hydration was only observed when the droplet sizes were greater than 4.3 nm with 15 mg/ml of DPA solution in the AOT system. However unexpected formation of metal salts occurred, i.e. Na+ and K+ ions from impurities participated in the crystal structures, regardless of the surfactants or solvents employed. Upon treatment with 2M HCl solution, metal salt participation in DPA crystallisation could be prevented. From the acidified microemulsions employing Trion X-100/1-hexanol as surfactants, no macroscopic sized crystals were seen. Instead, square plate-shaped nanoaggregates (30–100 nm in dimension) of 2–10 nm nanoparticles were obtained for samples with a droplet size of 4.01 nm, prepared from 18 mg/ml DPA in 2M HCl solution. Despite the strong electron density contrast across the nanocrystal surface, single crystal-like diffraction pattern (DP) was seen from these crystals, which was considered to be consistent with these nanocrystals being iso-oriented crystals/mesocrystals. The size and morphology of these nanoparticles can be controlled by varying the microemulsion composition. Lower DPA concentrations of 10-12 mg/ml and smaller droplet sizes of 2.40-3.15 nm produced much less organised nanoaggregates, with 'arcing' and 'rings' appearing in the DP, indicating the polycrystalline nature, whereas the highest DPA concentration of 18 mg/ml and the largest droplet of 4.01 nm could produce organized nanoaggregates that were barely distinguishable from single crystal structure.

La glycine est le neuromediateur inhibiteur majeur dans la moelle epiniere et le tronc cerebral. Elle exerce son action en se liant au recepteur de la glycine (glyr), un canal ionique permeable aux ions chlorures. Nous avons etudie les proprietes fonctionnelles des glyrs exprimes dans des ovocytes de xenope ou dans des cellules transfectees. Nous avons montre que les sous-unites de poisson zebre z1 et z2 forment des glyrs homomeriques fonctionnels avec des profils de desensibilisation differents. Glyrz1 a par ailleurs une forte sensibilite pour la taurine et est active par le gaba deux resultats inatendus compte tenu des donnees anterieures obtenues sur des glyrs humains par schmieden et al. (1992, 1993). Nous avons donc reexamine les proprietes des glyrs humains glyrh1 et glyrh2 et avons montre qu'il sont actives de maniere similaire par la taurine et le gaba. Les reponses de ces agonistes sont cependant variables puisque (i) leurs ec 5 0s varient d'un facteur 10 selon la cellule tout en restant proportionnels a l'ec 5 0gly et (ii) les rapports i m a xtau/i m a xgly et i m a xgaba/i m a xgly augmentent lorsque diminue l'ec 5 0gly. Ces donnees impliquent que le role presume des residus f-159/y-161 et de i-111 pour l'activation des glyrs par le gaba et la taurine a ete surevalue. Elles nous ont aussi conduit a rechercher les mecanismes capables de moduler l'ec 5 0 du glyr. Nous avons observe que l'entree massive de calcium dans des cellules exprimant des glyrs produit une potentialisation transitoire des courants glycinergiques. Cet effet s'etablit rapidement (en e-moins de 100 ms) grace a une baisse de l'ec 5 0 des glyrs qui se traduit par une augmentation de la duree d'ouverture du canal. Nos experiences indiquent que le ca 2 + n'interagit pas directement avec le glyr mais plutot via une proteine cytoplasmique ca 2 + -dependante. Ces resultats suggerent que le ca 2 + intracellulaire pourrait etre un important modulateur postsynaptique de la neurotransmission glycinergique.

La multiplication vegetative in vitro du soja et en particulier les methodes de regeneration doivent etre parfaitement maitrisees afin de mettre en uvre ulterieurement des techniques de transformation genetique, dans le cadre de l'amelioration des cultivars americains ou canadiens introduits en france. Dans ce but, des segments de cotyledons immatures sont cultives in vitro. Sur des milieux riches en auxines, ils peuvent produire directement des embryons somatiques. L'age de l'explant, le cultivar ainsi que l'auxine presente dans le milieu de culture jouent un role preponderant au cours de l'etape d'initiation de l'embryogenese somatique. Par ailleurs, les auxines employees au cours de cette phase influencent les autres etapes du procede de regeneration: la proliferation des embryons somatiques par embryogenese secondaire n'est obtenue qu'avec du 2,4d; la conversion des embryons somatiques est plus aisee quand ces derniers ont ete inities en presence d'ana. L'application temporaire de basses temperatures (10 a 14c) permet d'ameliorer les taux de conversion. Ce resultat est nouveau dans le cadre des travaux concernant l'embryogenese somatique chez le soja. Les plantes regenerees ainsi que leurs descendances paraissent conformes dans l'ensemble. La discussion traite des aspects quantitatifs et qualitatifs des differentes etapes du procede de regeneration: initiation des embryons somatiques; proliferation par embryogenese secondaire, croissance et developpement jusqu'au stade cotyledonnaire; conversion. Ceux-ci sont envisages en relation avec la nature et la concentration des auxines employees dans les milieux d'initiation de l'embryogenese

As pequenas proteínas de choque térmico (HSP20) são frequentemente associadas com a resposta das plantas ao estresse causado por fatores abióticos e, mais recentemente, têm sido também associadas a resposta aos estresses bióticos. Nas plantas, os genes Hsp20 representam a classe mais abundante dentre as proteínas de choque térmico, mas ainda pouco se conhece sobre essa família de genes em soja. Devido à suas aparentes multifuncionalidades, essas proteínas são alvos promissores para o desenvolvimento de variedades agrícolas melhor adaptadas a condições de estresses abióticos e bióticos, até mesmo quando combinados. Dessa forma, o presente trabalho realizou a caracterização molecular in silico das regiões codificadoras e reguladoras da família de genes codificadores para HSP20 de soja, com foco em sua distribuição no genoma, localização subcelular, divisão em subfamílias, estrutura secundária e regulação frente a estresses bióticos e abióticos, além da identificação de padrões de cis elementos, potencialmente envolvidos na resposta da soja a nematóides. Após a prospecção de genes anotados em bancos de dados do genoma da soja como Hsp20, foram obtidos 76 modelos gênicos. Dentre estes, apenas 52 modelos gênicos fizeram parte dos potenciais candidatos a GmHsp20 (Glycine max-Hsp20), devido as suas características estruturais, de cis elementos e de expressão. Em seguida, foram identificados, a partir das análises in vivo, 45 genes como Hsp20 de soja, distribuídos em 11 subfamílias. Para cada uma dessas, foi possível observar padrões de estrutura secundária específicos. Dentre os 45 genes GmHsp20 responsivos ao estresse de calor, 5 foram também responsivos ao estresse de frio e outros 5 ao estresse por infecção pelo nematóide M. javanica. Além disso, foram observados mais dois genes responsivos ao estresse biótico, mas não ao choque térmico. Obtiveram-se modelos operacionais de promotores para os genes responsivos a cada tipo de estresse analisado. Entre os cis elementos identificados nos Hsp20, que respondem a infecção por M. javanica, estão os Wbox, CAAT box, ABRE e MYB, além do elemento HSE/Heat. Os promotores responsivos ao estresse biótico seguiram padrões de composição e distribuição de cis elementos, descritos na literatura como relacionados a esse tipo de estresse e outros. Tais resultados irão auxiliar na geração de tecnologias de expressão dirigida, ainda mais avançadas, e novos genótipos de soja cada vez mais adaptados a condições combinadas de estresse.
The small heat shock proteins (HSP20) are often associated in plant stress response caused by abiotic factors and, more recently, have also been associated with response to biotic stresses. The Hsp20 genes represent, in plants, the most abundant class among the heat shock proteins, but little is known about this gene family in soybean. Due their apparent multifunctionality, these proteins are promising targets to the crop varieties development for better conditions adapted to biotic and abiotic stresses, even when they are combined. Thus, the present study conducted an in silico molecular characterization of regulatory and coding regions of HSP20 genes from soybean, focus in its genome distribution, subcellular localization, division into subfamilies, secondary structure and regulation under biotic and abiotic stresses, besides the identification patterns to cis elements potentially involved in the response to nematodes. After the exploration of Hsp20 genes annotation in soybean genome databases, 76 gene models were obtained. After in silico analysis, just 52 gene models were part of the GmHsp20 potencial candidates due to their structural characteristics of cis elements and expression profile. In addition, based on in vivo analysis, 45 soybean Hsp20 genes were identified, distributed in 11 subfamilies, for which is possible to observe a specific secondary structure for each one. Among the 45 GmHsp20 genes heat stress responsives, 5 genes were cold stress responsive and other five were nematode infection by M. javanica responsive. Moreover, two genes were observed being responsive to biotic stress, but they weren't responsive to thermal shock. Operational Models of Hsp20 promoters were obtained to responsive genes to each stress condition examined in this study. Among the identified cis elements in Hsp20 soybean genes that were responsive to M. javanica infection were W box, CAAT box, ABRE and MYB, besides the HSE / Heat element. Promoters responsive to biotic stress in soybean follows composition and distribution standards of cis elements, as described in the literature to be related to this type of stress. These results, such as responsive genes and promoters to many different stresses, can assist in generation of expression directed technologies even more advanced and new soybean genotypes more adapted to under combined stress conditions.

In recent years, much research has focused on Algal High Rate Oxidation Ponds as both an economic means for wastewater treatment and as a system for the mass production of algae. With the advent of these systems for the treatment of saline organic effluents, the extreme halophile, Dunaliella salina was considered. In this study, the growth and productivity of a number of Dunaliella species (and strains thereof) was evaluated in hide soak liquor tannery effluent. Hide soak liquor, diluted to 20% with water, proved to be highly suitable as a growth medium for the majority of the Dunaliella species under study and in some instances, resulted in enhanced growth rates and higher biomass yields compared to those obtained in defined inorganic medium. A few Dunaliella species failed to grow in this effluent. A correlation was observed between the lack of growth displayed by these species in this organio-rich medium and their failure to utilise organic compounds. Glycine, a major component of this effluent, possibly stimulates the growth of Dunaliella. Studies on the mechanism of growth stimulation by glycine revealed that an algal-bacterial relationship existed whereby the bacteria mineralised the amino acid, releasing ammonia which was then utilised by the alga. Results of this work revealed significant variations in the intracellular glycerol content amongst the Dunaliella species under study. Large differences were also observed between the glycerol contents of effluent-grown and control Dunaliella cells, where the effluent-grown cells were characterised by greatly reduced intracellular glycerol content. These reduced glycerol levels are assumed to have arisen from the glycine-induced stimulation of glycerol release which was observed in this study, where the high glycine content of the hide soak liquor is proposed to have induced glycerol release. This enhanced glycerol release in tatmery effluent could play a central role in the fimction of Dunaliella-based High Rate Oxidation Ponding systems, by stimulating bacterial activity. Observed glycerol productivities were therefore proposed to be a fimction of the type and concentration of the organic constituents of the medium. A similar medium-induced phenomenon was observed in the starch content of Dunaliella cells.

The Gate Control Theory of Pain highlights an interconnected network of myelinated and unmyelinated fibres within the dorsal horn (Melzack & Wall, 1965). Currently there are a number of proposed theories to explain the mechanism by which pain is developed, one of which is the theory of disinhibition whereby a reduction of inhibitory neurotransmission leads to over-excitation within the central nervous system (Guo & Hu, 2014; Zeilhofer, 2005). Evidence within my supervisor’s laboratory has identified glycinergic inhibitory neurotransmission to be a key component in the development of neuropathic pain. This study aimed to identify the main reasons for these reductions in inhibitory neurotransmission shown within lamina II of the dorsal horn. It is hypothesised that inhibitory neurotransmission is lost following injury leading to over-excitation within the dorsal horn. The experiments performed within this study were optimised through following vigorous testing procedures often requiring multiple trial and error attempts prior to the development of both valid and reliable results. PKCγ immunoreactive neurons are found within lamina II of the dorsal horn are classified into distinct morphological categories, and are involved in the filtering of nociceptive and mechanical input (Martin, Liu, Wang, Malmberg & Basbaum, 1999). This study showed distinct changes of PKCγ immunoreactive neurons following a neuropathic pain injury suggestive of the possibility of changes to the neural circuitry within the dorsal horn. Furthermore GAD67 neurons in lamina II of the dorsal horn was analysed to identify whether this can be used as a method of identifying changes in presynaptic neurons following neuropathic injury but failed to yield significant results. In conclusion immunohistological studies are found to be limited in providing physiological information on neurons within the dorsal horn. As a result future experimentations seeking alternative techniques must be sought to confirm the significance the results in the study.