Thesis (Ph. D.)--University of Texas at Austin, 2003. / Vita. Includes bibliographical references.

Ethanol is a widely used drug, yet an understanding of its sites and mechanisms of action remains incomplete. Among the protein targets of ethanol are glycine receptors (GlyRs). In addition to ethanol, zinc also modulates GlyR function. Although the individual effects of zinc and alcohols on GlyR function have been well studied, the combined effects of these agents have not been thoroughly examined. This project investigated the effects of zinc on alcohol action at the glycine receptor (GlyR). In Aim 1, the effects of zinc on ethanol modulation of GlyR function were tested and characterized in three GlyR [alpha] subunits ([alpha]1-3). Aim 2 explored a site of action for the augmenting effects of zinc on ethanol action at the GlyR. Mutant D80A GlyRs, which lack a zinc binding site (D80), were constructed and allowed us determine if this zinc binding site is important for the zinc/ethanol interactions that were observed in Aim 1. The effects of ethanol were reduced in mutant D80A GlyRs compared to wild type (WT). In addition, manipulating zinc levels in our buffers either by adding or chelating zinc did not change the magnitude of ethanol enhancement of mutant D80A GlyRs as it did in WT GlyRs suggesting that the D80 position is important for zinc modulation of ethanol action. Finally, Aim 3 extended the findings from Aims 1 and 2 by evaluating the effects of a GlyR point-mutation on alcohol consumption and other behavioral tests in mice. Glra1(D80A) knock-in mice provided an animal model for behavioral studies of zinc/ethanol interactions at the GlyR and showed decreased alcohol consumption and preference compared to their WT littermates. In addition, D80A KI mice had increased startle responses compared to their WT littermates. Other behavioral tests were also conducted including tests of ethanol motor incoordination and strychnine induced convulsions; there were no differences detected between KI and WT mice in these assays. Overall, our findings demonstrate that zinc is critical in determining the effects of ethanol at GlyRs and suggest that zinc signaling at the D80 position may be important for mediating the behavioral effects of ethanol action at GlyRs. / text

There is a great need for new analgesics. The current problem in treatment of severe pain is that side effects limit the effectiveness of therapy. Glycine receptors are important in modulation of nociception, suggesting a novel class of analgesics. Previous studies in rats show that intrathecal administration of glycine agonists and amino acids structurally similar to glycine have antinociceptive effects. The effects of isovaline, a unique, non-proteogenic glycine-like aminoacid, have not been studied. Isovaline is absorbed from the gut and transported across the blood-brain-barrier. We examined the hypothesis that isovaline produces antinociception in mice. Administration of strychnine, an antagonist at glycine receptors, into the cisterna magna or lumbar intrathecal space resulted in allodynia, localized to the somatotopic distribution of the trigeminal and lumbar nerves. These findings provided a basis for models of lumbar and trigeminal neuralgia. Racemic isovaline blocked strychnine induced allodynia in both models without apparent side effects. We next investigated the antinociceptive effects of glycine-like amino acids in formalin foot assay, a conventional rodent model of acute and chronic pain. Antinociceptive effects were demonstrated on intrathecal administration of glycine, beta-alanine, and isovaline. Intravenous isovaline produced significant antinociceptive effects in the formalin foot model. The toxicity of isovaline and related amino acids were determined. Exploratory behavior, gait, and responses to stimuli were used to assess sedation. The rotarod test was used to examine central nervous system (CNS) and neuromuscular toxicities of intravenous isovaline. Lumbar administration of glycine and beta-alanine caused scratching and/or lower body weakness. Isovaline at 7-times intrathecal ED50 produced lower body weakness in some animals. None of the amino acids produced sedation comparable to morphine. At 6-times ED50, beta-alanine produced weakness. Both glycine (ED50) and beta-alanine (3x ED50) but not isovaline produced local nerve irritation. Intracisternal injection of glycine did not reverse allodynia and resulted in death. Neither R nor S enantiomers of isovaline impaired performance on the rotarod test. Isovaline has significant antinociceptive properties. Given the absence of apparent CNS or motor toxicity, isovaline has potential as a clinical analgesic.

No description available.

Thesis (Ph. D.)--University of Adelaide, 1984. / Includes bibliographical references (leaves [i]-xvi).

Thesis (Ph.D.) -- University of Adelaide, Department of Entomology, 1978.

Visando aumentar a resistência a moléstias fúngicas, o presente trabalho teve como objetivo introduzir um gene (chit1) que codifica uma quitinase do fungo Metarhizium anisopliae em cultivares de soja [Glycine max (L.) Merrill]. A co-transformação foi a estratégia escolhida, visando a obtenção de plantas livres de transgenes marcadores na progênie das plantas transformadas. A co-transformação foi realizada via biolística, tendo como tecido-alvo conjuntos de embriões somáticos globulares das cultivares MG/BR46 Conquista e IAS-5. O plasmídeo pGusHyg, que contém o gene repórter gusA e o gene marcador hpt, foi bombardeado concomitantemente com o plasmídeo pMOG463chit1, que porta o gene chit1. Os conjuntos de embriões bombardeados foram transferidos para meio seletivo contendo higromicina, visando a obtenção de material estavelmente transformado. Os conjuntos embriogênicos higromicina-resistentes foram transferidos seqüencialmente para meios de proliferação D-20 (sem higromicina), maturação e regeneração. No total, foram obtidos 387 e 380 embriões histodiferenciados das cultivares MG/BR46 Conquista e IAS-5, respectivamente. Plantas transgênicas adultas e férteis foram regeneradas. Para avaliar a eficiência da estratégia de cotransformação, foram realizadas análises moleculares de embriões histodiferenciados e de plantas regeneradas. Os resultados obtidos neste trabalho permitiram o cálculo da taxa de co-transformação de 44% para os embriões histodiferenciados da cultivar MG/BR46 Conquista e de 50% para plantas de IAS-5. Não existem, até o momento, relatos de trabalhos em soja utilizando embriões somáticos globulares em proliferação como alvo para estudos de co-transformação. / Aiming the increase of plant resistance to fungal disease, the present work was carried out with the objective of introducing a gene (chit1) coding a chitinase from Metarhizium anisopliae in soybean cultivars [Glycine max (L.) Merril]. Co-transformation was the strategy elected, in order to obtain marker-free transgenic plants in the progeny of transformed plants. Co-transformation was performed via biolistic using somatic globular embryo clusters of MG/BR46 Conquista and IAS-5 cultivars as target tissues. Plasmid pGusHyg, containg the reporter gusA gene and the selectable marker hpt gene, was co-delivered with the pMOG463chit1 plasmid containg the chit1 gene. Bombarded embryo clusters were transferred to selective medium containg hygromycin, aiming to obtain stable transformed material. Hygromycin-resistant embryogenic clusters were sequentially transferred to proliferation D20 (without hygromycin), maturation and regeneration media. A total of 387 and 380 histodifferentiated embryos were respectively obtained from MG/BR46 Conquista and IAS-5 cultivars. Transgenic adult fertile plants were regenerated. To evaluate the co-transformation eficiency, DNA from histodifferentiated embryos and from regenerated fertile plants was submitted to molecular analysis. Data obtained allowed us to calculate co-transformation frequencies of 44% and 50% for MG/BR46 Conquista and IAS-5 cultivars, respectively. As far as we are concerned there is no studies utilizing soybean somatic embryos for cotransformation via biolistic.

A soja é uma das culturas mais importantes a nível mundial, devido à produção de óleo e a seu alto teor proteico. A fase reprodutiva é a mais importante para a produtividade da soja, visto que seu cultivo se destina principalmente à produção de grãos. Os microRNAs (miRNAs) desempenham funções essenciais em diversos aspectos do desenvolvimento reprodutivo, incluindo o florescimento, a fertilidade e o desenvolvimento da semente. A função destes pequenos RNAs (sRNAs) endógenos não codificantes é regular a expressão gênica, principalmente através de clivagem e inibição da tradução de mRNAs alvos. A identificação de miRNAs ainda não está saturada e, em soja, não há trabalhos relacionando-os aos diferentes órgãos florais, que são fundamentais na produtividade desta cultura. Neste estudo, amostras de flores, carpelos, estames e pétalas de soja foram usadas na construção de quatro bibliotecas de sRNAs sequenciadas utilizando a plataforma Solexa, gerando um total de 13.557.795 sequências. Através da análise do mapeamento das sequências de sRNAs das bibliotecas em candidatos a precursores de miRNAs de soja identificados, 276 foram considerados precursores autênticos, incluindo 143 precursores novos. Foram identificados 235 miRNAs maduros, dos quais 51 são miRNAs inéditos, pertencentes a 40 novas famílias. Os demais miRNAs identificados pertencem a 64 famílias conhecidas de miRNAs de plantas, das quais três ainda não tinham sido reportadas em soja. Todas as famílias de miRNAs que estão envolvidas na regulação do florescimento foram identificadas entre as mais frequentes nos tecidos florais de soja. Na análise de expressão pela frequência de sequências nas bibliotecas de sRNAs de carpelos, estames e pétalas, 67.2% (158) dos miRNAs identificados foram diferencialmente expressos. Análises de expressão por PCR quantitativa (RT-qPCR) comprovaram a expressão diferencial de 19 miRNAs. O miRNA inédito denominado NF13 apresentou a maior diferença entre os tecidos, sendo fortemente induzido nos carpelos. Para os miRNAs com expressão diferencial comprovada por RT-qPCR foi feita a predição computacional dos genes alvos, para muitos dos quais já foram descritas funções relacionadas ao processo reprodutivo em plantas. O estudo da regulação destes genes pelos miRNAs em diferentes tecidos e estádios de desenvolvimento floral contribuirá para o entendimento dos mecanismos moleculares envolvidos na reprodução da soja. / Soybean is one of the most important crops worldwide, due to the production of oil and its high protein content. The reproductive phase is considered the most important for the yield of soybean, which is mainly intended to produce the grains. MicroRNAs (miRNAs) play essential roles in various aspects of reproductive development, including flowering, fertility and seed development. The function of these endogenous small non-coding RNAs (sRNAs) is to regulate gene expression, mainly through cleavage and translation inhibition of target mRNAs. The identification of miRNAs is not yet saturated in soybeans, and there are no studies linking them to the different floral organs, which are fundamental in the productivity of this crop. In this study, samples of flowers, carpels, petals and stamens of soybeans were used in the construction of four sRNA libraries sequenced using the platform Solexa, generating a total of 13,557,795 sequences. The sRNAs sequences from four libraries were mapped in precursors candidates. Among them, 276 were considered authentic precursors, including 143 new precursors. 235 mature miRNAs were identified, of which 51 are novel miRNAs belonging to 40 new families. The other identified miRNAs belongs to 64 known plant miRNA families, of which three had not yet been reported in soybean. All miRNAs families which are involved in regulating flowering were identified among the most frequent floral tissue of soybean. Expression analysis based on the frequency of sequences in the libraries of sRNAs of carpels, stamens and petals demonstrated that 67.2% (158) corresponded to differentially expressed miRNAs. Most of 22 and 24 nt miRNAs that were differentially expressed was induced in carpels, suggesting that these miRNAs sizes are important in regulating the processes occurring specifically in these organs. Analysis of expression by quantitative PCR (RT-qPCR) confirmed the differential expression of 19 miRNAs. The novel miRNA named NF13 showed the greatest difference between the tissues and is strongly induced in the carpels. A computational prediction of targets for miRNAs with differential expression confirmed by RT-qPCR was performed. Many of the predicted targets have described functions related to the reproductive process in plants. The study of regulation of these genes by miRNAs in different tissues and stages of flower development will contribute to understanding the molecular mechanisms involved in reproduction of soybean.

Ureases (ureia amido-hidrolases; EC 3.5.1.5) são metaloenzimas, dependentes de níquel, produzidas por plantas, fungos, bactérias e invertebrados, que catalisam a hidrólise da ureia em amônia e dióxido de carbono. Em plantas e fungos, as ureases são hexâmetros formados por seis subunidades idênticas, e, em bactérias, são formadas por duas a três subunidades distintas. A presença de dois íons de níquel no sítio ativo das ureases é essencial para sua atividade catalítica. Em bactérias, ureases atuam como fatores de virulência em infecções do trato urinário e gastrointestinal. Em plantas, ureases são encontradas principalmente nas sementes, mas estão distribuídas em todos os tecidos. A soja produz duas isoenzimas: a urease embrião-específica, sintetizada no embrião, e a urease ubíqua, presente em todos os tecidos da planta, em menor quantidade que a embrião-especifica. Em estudos prévios realizados pelo grupo foram identificadas proteínas de plastídios que co-imunoprecipitaram associadas à urease, o que despertou o interesse em estudar a presença de ureases na organela. Nesse trabalho, estabeleceu-se o protocolo para purificação e enriquecimento de cloroplastos de folhas de soja para investigar a provável presença de ureases na organela. Ensaios de atividade enzimática, ELISA e cromatografia de troca iônica foram realizados com o extrato obtido de cloroplastos após o processo de extração e purificação e indicaram a presença de ureases nestes plastídeos. Na tentativa de identificar-se as isoformas de ureases presentes no cloroplasto, realizou-se ensaios de espectrometria de massas, porém não se obteve sucesso devido a quantidade da enzima presente no extrato. O aprimoramento do processo de enriquecimento de cloroplastos possibilitará prosseguir nos estudos para identificar as isoformas presentes na organela, bem como detectar sua localização nestas organelas. / Ureases (urea amido-hydrolases; EC 3.5.1.5) are nickel dependent metalloenzymes, produced by plants, fungi, bacteria and invertebrates, that catalyze the hydrolysis of urea into ammonia and carbon dioxide. In plants and fungi, ureases are hexamers formed by two or three identical subunits, while in bacterias are formed by two to three different subunits. The catalytic activity of urease is due to the presence of two nickel ions in its active site. Bacterial ureases are known virulence factors in urinary and gastrointestinal tracts. In plants urease are mainly found in the seeds, but are widely distributed in all tissues. Soybean produces two isoenzymes: embryo-specific urease is synthesized in the embryo during development and ubiquitous urease is present in all plant tissues, in a smaller amount compared to embryo-specific. Previous studies have shown that many plastid proteins co-immunoprecipitate with urease, which sparked interest in studying the presence of ureases in the organelle. In this study, we established the protocol for purification and enrichment of soybean leaves chloroplasts in order to investigate the presence of urease in the organelle. Enzymatic activity, ELISA and ion exchange chromatography assays were conducted with the organelle and indicated the presence of ureases. In an effort to identify which ureases isoforms were present in the chloroplast, we performed mass spectrometry analysis. This effort was unsuccessful due to small amount of enzyme obtained from the extract. Improving the enrichment process will enable further studies regarding the identification of the urease isoforms as their location in the plastid.

Aquaporinas, também conhecidas como Major Intrinsic Proteins (MIPs), são proteínas de membrana distribuídas em todos os domínios da vida, responsáveis pelo transporte de água e pequenos solutos. Em plantas superiores, essa família é particularmente diversa e pode ser classificada em cinco subfamílias: proteínas intrínsecas de membrana plasmática, proteínas intrínsecas de tonoplasto, proteínas intrínsecas do tipo nodulina-26, proteínas intrínsecas pequenas e proteínas intrínsecas X. Esses canais apresentam uma estrutura bastante conservada, exibindo, no centro do poro, duas regiões importantes para a seletividade: os motivos NPA e o filtro seletivo. No presente trabalho, sessenta e seis genes de aquaporinas foram identificados no genoma da soja e classificados por homologia de sequência nas cinco subfamílias estabelecidas. As estruturas tridimensionais das MIPs de soja foram determinadas por modelagem comparativa e o raio dos poros e os resíduos importantes para a especificidade foram identificados. Ensaios funcionais utilizando expressão heteróloga em Saccharomyces cerevisiae foram realizados para quatro aquaporinas, GmTIP1;9, GmTIP2;5, GmTIP3;2 e GmNIP2;2, demonstrando que esses genes codificam canais funcionais capazes de transportar água, peróxido de hidrogênio e ureia. A ureia possui um papel muito importante na agricultura por ser o fertilizante nitrogenado mais utilizado mundialmente. A identificação de transportadores de ureia em plantas ainda é recente e, além de um transportador específico, somente as aquaporinas estão relacionadas ao transporte de ureia em plantas. Apesar disso, a relevância fisiológica desse transporte pelas MIPs é ainda motivo de debate. Empregando uma abordagem baseada em PCR quantitativo, foi demonstrado que os níveis de expressão de GmTIP1;9, GmTIP3;2 e GmNIP2;2, mas não de GmTIP2;5, são alterados em raízes de soja cultivada em solução nutritiva contendo ureia como a única fonte de nitrogênio. Além disso, os níveis dos transcritos de GmNIP2;2 são aumentados na parte aérea das plantas, indicando o possível envolvimento dessa aquaporina na redistribuição de ureia nos tecidos. Outros genes de aquaporinas de soja também apresentaram maior expressão nessas condições. Esses resultados sugerem o envolvimento de aquaporinas no metabolismo de ureia em plantas e apontam essas proteínas como potenciais alvos para manipulação genética visando o aumento da eficiência do uso de nitrogênio em sistemas adubados com ureia e a melhoria da produção agrícola. / Aquaporins, also known as Major Intrinsic Proteins (MIPs), are membrane proteins distributed in all kingdoms of life, responsible for water and small solutes transport. In higher plants, this family is particularly diverse and can be classified into five subfamilies: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin-26-like intrinsic proteins (NIPs), small basic intrinsic proteins (SIPs) and X intrinsic proteins (XIPs). These channels present a highly conserved structure, displaying, in the center of the pore, two regions important for selectivity: the NPA motifs and the selective filter. In the present work, sixty-six aquaporin genes were identified in soybean genome and classified by sequence homology into the five established subfamilies. The three-dimensional structure of soybean MIPs was determined by homology modeling and the pore radius and residues influencing substrate specificity were identified. Functional assays using heterologous expression in Saccharomyces cerevisiae were performed for four aquaporins, GmTIP1;9, GmTIP2;5, GmTIP3;2 and GmNIP2;2, indicating that these genes codify functional channels capable of transporting water, hydrogen peroxide and urea. Urea plays an important role in agriculture because it is the most used nitrogen fertilizer worldwide. Plant urea transporters are just beginning to be identified and, to date, besides a specific transporter, only aquaporins are linked to urea transport in plants. Nevertheless, the physiological relevance of this transport is still in debate. Using a quantitative PCR approach, we have shown that the expression levels of three urea transporting MIPs, GmTIP1;9, GmTIP3;2 and GmNIP2;2, but not GmTIP2;5, are altered in soybean roots when urea is the sole nitrogen source. Moreover, transcript levels of GmNIP2;2 were also up-regulated in shoots, indicating the possible involvement of this aquaporin in urea redistribution to other plant tissues. Several other soybean aquaporin genes were up-regulated in this condition as well. Taken together, these results suggest the involvement of aquaporins in urea metabolism in plants and place these proteins as potential targets for plant manipulation to improve urea based nitrogen use efficiency and crop production.