Existe atualmente uma restrição muito forte ao uso de antibióticos como promotores de crescimento na alimentação avícola, uma vez que tais produtos passaram a ser vistos como fatores de risco para a saúde humana. Estudos com produtos alternativos tornaram-se necessários e, neste sentido, as leveduras vem sendo exploradas, principalmente, seu conteúdo celular. Dessa forma, o presente estudo teve como objetivo avaliar o uso de levedura hidrolisada, fonte de nucleotídeos, sobre o desempenho e imunidade de frangos de corte, suplementados no período de 1 a 14 dias de idade. Foram utilizados 576 pintos de corte, machos, linhagem Ross 308, criados até 42 dias de idade sobre cama reutilizada. O delineamento foi inteiramente casualizado com 6 tratamentos: controle negativo (sem suplementação); controle positivo (com antibiótico) e diferentes níveis de adição de levedura hidrolisada (0,25%; 0,50%; 0,75% e 1,00%), sendo 8 repetições/tratamento. Considerando-se o período total de criação, foram observados efeitos significativos para os parâmetros de ganho de peso (GP) e conversão alimentar (CA). Por sua vez, o parâmetro de consumo de ração (CR) não apresentou efeitos significativos entre os tratamentos. Com relação à imunidade, a suplementação com levedura hidrolisada apresentou efeito sobre a resposta a vacinação contra a doença de Newcastle, produção de água oxigenada, óxido nítrico e influenciou também no peso relativo dos órgãos linfóides, com destaque para o nível 0,75% de suplementação. Diante dos resultados obtidos no presente estudo, pode-se concluir que a levedura hidrolisada, suplementada durante o período de 1 a 14 dias de idade, promove efeitos significativos no desempenho durante todo o período de criação e também influencia na imunidade das aves. / There is currently a strong restriction on the usage of antibiotics as growth promoters in broiler feed, since such products were seen as risk factors for human health. Research with alternative products has became necessary, and the yeast has been exploited, particularly its cellular content. Thus, this experiment was aimed at evaluating the use of hydrolyzed yeast, source of nucleotides on performance and immunity of broiler chickens supplemented during the period of 1 to 14 days old. A total of 576 male chicks, Ross 308 were raised up to 42 days old, on reused litter. The design was completely randomized with six treatments: negative control (no supplementation), positive control (with antibiotic) and different levels of hydrolyzed yeast (0.25%; 0.50%; 0.75% e 1.00%), with 8 replicates per treatment. Considering the whole breeding period, significant effects were observed for parameters of weight gain (WG) and feed conversion (FC). However, the parameter of feed consumption (FC) had no significant effect among the treatments. Regarding to immunity, the hydrolyzed yeast supplementation had an effect on the response to vaccination against Newcastle disease, production of hydrogen peroxide, nitric oxide and also influenced the relative weight of lymphoid organs, especially the 0.75% level of supplementation. With the results obtained in this experiment it is possible to conclude that the hydrolyzed yeast supplementation, during the period of 1 to 14 days old, promotes a significant effect on performance throughout the whole rearing period and also influences the immunity of the broiler chicks.

O Experimento foi realizado com o objetivo de avaliar a substituição parcial e total de plasma bovino por levedura hidrolisada na dieta de leitões desmamados no período de 21 a 47 dias de idade. Foram utilizados 1600 leitões da linhagem PIC, distribuídos em blocos ao acaso, com quatro tratamentos. As dietas foram divididas em quatro fases (pré-inicial I 22 a 28 dias; pré-inicial II 29 a 35 dias; inicial I - 36 a 47 dias e inicial II 48 a 63 dias). A inclusão percentual, Plasma: Levedura, nas dietas foi: T1 (6:0; 4:0; 2:0 e 0:0); T2 (3: 4; 2: 3; 1: 2 e 0: 0); T3 (1,5: 6; 1: 4,5; 0,5: 3 e 0: 0) e T4 (0: 8; 0: 6; 0: 4 e 0: 0). Cada tratamento teve 10 repetições (cinco de machos e cinco de fêmeas) totalizando 40 unidades experimentais com 40 animais cada. As variáveis zootécnicas avaliadas durante o período experimental foram: consumo de ração, ganho de peso, e conversão alimentar. Foram tomadas amostras de sangue de 5 animais por tratamento no dia de início do experimento, e de 10 animais por tratamento 25 dias depois. Foram quantificadas a IgA e a IgG. Os dados obtidos foram submetidos a análise de variância ANOVA, utilizando-se o teste Tukey para comparação das médias ao nível de significância de 5% utilizando o pacote estatístico SAS. Não houve diferença estatística nas quantidades de Ig A e Ig G circulante, nem na variação destas imunoglobulinas no tempo, entre os tratamentos. Considerando a análise dos dados conclui-se que nas condições estudadas, a utilização da relação percentual de (1,5: 6; 1: 4,5; 0,5: 3 e 0: 0) de plasma: levedura hidrolisada resultou um maior consumo de ração e ganho de peso dos animais, em comparação às outras proporções, e consequentemente podendo trazer um maior lucro para o produtor / The experiment was performed with the objective of evaluating the partial and complete substitution of bovine plasma for hydrolyzed yeast in the diet of weaned piglets from 21 to 47 days old. 1600 piglets of PIC lineage were used and randomly distributed in blocks where they received four treatments. Their diets were divided into four phases (pre-initial I: 22 to 28 days; pre-initial II- 29 to 35 days; initial I- 36 to 47 days and initial II-48 to 63 days). The percentage inclusion Plasma:Yeast in the diets were: T1 (6:0; 4:0; 2:0 and 0:0); T2 (3: 4; 2: 3; 1: 2 and 0: 0); T3 (1.5: 6; 1: 4.5; 0.5: 3 and 0: 0) and T4 (0: 8; 0: 6; 0: 4 and 0: 0). Each treatment had 10 repetitions (five times with the males and five times with the females), totaling 40 experimental units with 40 animals each. Husbandry variables evaluated during the trial period were: feed intake, body weight gain and feed conversion. Blood samples of 5 animals per treatment were taken at the beginning of the experiment day, and 10 animals per treatment 25 days later. IgA and IgG were quantified. The data obtained were analyzed by ANOVA analysis of variance, using the Tukey test to compare means at the 5% significance level, using the statistical package SAS. There was no statistical difference in the quantities of Ig A and Ig G circulating, or the variation of these immunoglobulins in the time, between treatments. Considering the analysis of the data it was concluded that the conditions studied, the percentage utilization ratio of (1.5: 6; 1: 4.5: 0.5: 3 and 0: 0) plasma: hydrolyzed yeast resulted in a higher feed intake and weight gain of animals compared to other proportions, and consequently can bring higher profits to the producer

O avanço tecnológico dos laboratórios de biologia molecular tem proporcionado um grande aumento no volume de seqüências de nucleotídeos armazenadas em bancos de dados biológicos, introduzindo o desafio de pesquisar eficientemente estes dados. Neste contexto, a árvore de sufixo é um método de acesso utilizado por muitas aplicações que envolvem pesquisa em dados biológicos. Entretanto, o custo de construção das árvores de sufixo é alto devido ao tamanho da estrutura de indexação gerado e à necessidade da árvore de sufixo caber em memória principal para ser construída com complexidade linear em relação ao tempo. Esta dissertação propõe o Perseus, uma nova técnica para tratar árvores de sufixo persistentes. A técnica Perseus apresenta os seguintes diferenciais. Ela introduz uma abordagem que realiza a construção de árvores de sufixo persistentes cujos tamanhos podem exceder a capacidade da memória principal. Além disso, ela provê um algoritmo que constrói árvores de sufixo por meio do particionamento destas árvores somente quando necessário. Esta construção também permite que o usuário escolha quais subseqüências de uma seqüência devem ser indexadas, de acordo com os requisitos particulares de suas aplicações. Por fim, a técnica proposta também introduz um algoritmo de casamento exato que permite a busca por uma seqüência de consulta em árvores de sufixo que podem estar particionadas. A validação do Perseus foi realizada por meio de testes de desempenho considerando genomas de vários organismos, os quais possuem diferentes ordens de magnitude de tamanho. Os resultados obtidos foram comparados com a técnica Trellis+, a qual representa o estado da arte nesta linha de pesquisa. Os testes indicaram que o Perseus construiu árvores de sufixo mais rapidamente do que o Trellis+, reduzindo o tempo total gasto na construção em até 24%. Perseus também criou árvores de sufixo mais compactas, atingindo uma redução média de 27% no espaço de memória secundária utilizado. Já com relação ao tempo total gasto no processamento de consultas, Perseus sempre produziu os melhores resultados, respondendo consultas em média 49% mais rápido do que o seu principal concorrente. Com relação à indexação de subseqüências escolhidas pelo usuário, comparando os resultados obtidos com o Trellis+, os testes mostraram que Perseus proveu uma redução no tempo de construção de árvores de sufixo de 97% na média e uma redução no tempo gasto no processamento de consultas de genes de 93% na média / Due to the technological advances in molecular biology laboratories, biological databases are extremely voluminous and tend to become more voluminous as data on new genome organisms are available. This introduces the challenge of searching nucleotide sequences efficiently. The suffix tree is an access method used for several applications that search for these data. However, the cost of building suffix trees is high, since they are extremely large data structures and they should fit in the main memory to be constructed in linear time. In this masters thesis, we propose the Perseus, a novel technique that handles persistent suffix trees. The Perseus introduces the following distinctive good properties. It is based on an approach that constructs persistent suffix trees whose sizes may exceed the main memory capacity. Furthermore, it provides an algorithm that allows for users to indicate which substrings of the input string should be indexed, according to the requirements of their applications. Moreover, it proposes an extended exact matching algorithm that searches for a query string into suffix trees that may be partitioned. The Perseus was validated through performance tests using genomes of several organisms of different sizes. The results were compared with the Trellis+ technique, which represents the state-of-the-art in this field. The tests showed that the Perseus reduced the time spent on constructing suffix trees by 24%. The Perseus also constructed compacter suffix trees, providing an average reduction in the secondary memory storage of 27%. Furthermore, the Perseus reduced the time spent on query processing of nucleotide sequences by up to 49%. As for the functionality of indexing substrings according to the users requirements, the Perseus greatly improved the query performance in comparison to the Trellis+. The results showed that the Perseus reduced the time spent on constructing suffix trees by 97% on average and the time spent on query processing of genes by 93% on average

The strain promoted azide alkyne cycloaddition (SPAAC) of azido nucleobase modified nucleosides and nucleotides with cyclooctynes to give fluorescent triazoles has been relatively unexplored. Thus, SPAAC between azido-nucleobases and various cyclooctynes in aqueous solution at ambient temperature resulted in the efficient formation (3 min - 2 h) of triazole products with inherent fluorescent properties. The 2- and 8-azidoadenine nucleosides reacted with fused cyclopropyl cyclooctyne, dibenzylcyclooctyne or monofluorocyclooctyne to produce click products functionalized with hydroxyl, amino, N-hydroxysuccinimide, or biotin moieties. The previously unexplored 5-azidouridine and labile 5-azido-2'-deoxyuridine were similarly converted to the analogous triazole products in quantitative yields in less than 5 minutes. The 8-azido-ATP quantitatively afforded the triazole product with fused cyclopropyl cyclooctyne (3 h). Addition of a triazole ring at the 2 or 8 position of adenine or 5-position of uracil induces fluorescent properties which were used for direct imaging with fluorescent microscopy in MCF-7 cancer cells without the need for traditional fluorogenic reporters. Fluorescent lifetime imaging microscopy of the click adducts in live cells were used to determine the lifetime of each fluorophore in the cellular nuclei demonstrating the potential utility of the synthesized triazole adducts for dynamic measuring and tracking of events inside single living cancer cells. The SPAAC methodology developed has also been applied to study the cellular targets in protozoal parasite, Trichomonas vaginalis and bacteria, Pseudomonas aeruginosa. The 9-(2-deoxy-2-fluoro-β,D-arabino-furanosyl)adenine (arabino-F-Ado) was modified with an azido moiety at the C8 position for use in click chemistry. Tagging and subcellular localization studies using azido modified arabino-F-Ado could provide insight into the mechanism of action of arabino-F-Ado. An activated analogue of S-adenosyl-L-methionine (SAM) with an EnYn group on the sulfur instead of a methyl group was prepared to study the transfer of the methyl group from SAM. I found that the EnYn group was transferred from SAM to a guanosine on tRNA by methytransferase Trm1. Thus, AdoEnYn is a competitive inhibitor of SAM and can be incorporated into tRNA in place of SAM.

The cAMP-second messenger hypothesis for β-adrenoceptor-induced relaxation of uterine smooth muscle was tested in high-K+ depolarized rat uterus. At 10⁻⁸ M concentration, Isoproterenol, a β -adrenergic agonist, could cause relaxation of the depolarized uterus without Increasing tissue cAMP levels. Further, although increases in cAMP levels were associated, in some cases, with -isoproterenol (10⁻⁸ M or 10⁻⁴ M)-Induced relaxation, there was no quantitative correlation between the Increases in cAMP and relaxation. Pretreatment of the tissue with a phosphodiesterase Inhibitor, RO 20-1724 (10⁻⁴ M), did not potentiate the relaxation response to Isoproterenol. These results suggested that there Is no simple cause and effect relationship between β -adrenoceptor-Induced Increases in cAMP levels and relaxation in uterine smooth muscle. The dissociation between cAMP and relaxation found ln the present study was also extended to cGMP, since no changes in cGMP levels were observed with isoproterenol-induced relaxation. It is generally accepted that the ionic environment of the cell affects the cellular responses of the tissue. It was demonstrated that hlgh-K*" depolarization of uterine smooth muscle caused an impairment of the ability of isoproterenol to induce cAMP accumulation. This was found to be related to Increased Ca++-Influx known to occur during depolarization. This Is because pretreatment of the tissue with 10⁻⁵M D-600, an Inhibitor of Ca++-lnflux, restored the stimulation of cAMP by Isoproterenol ln the depolarized muscle to a level similar to that observed ln non-depolarized muscle. Furthermore, there was an Inverse relationship between [ca++] ex in the depolarizing medium (range 0.9 to 7.2 mM) and increases in cAMP produced by isoproterenol (10⁻⁴ M). It was also found that exposure of the rat uterus to a Ca++-deficient solution (Ca++-free with 0.2 mM EGTA) accentuated the Increase of tissue cAMP content produced by isoproterenol (10⁻⁸ M). The studies on ionic interactions demonstrated that the presence of Na+(80 mM) or high Mg++(2.5 mM) in the depolarizing medium could overcome the blockade of lsoproterenol-induced increases in cAMP levels by high-K+ depolarization. The studies on the mechanism of this effect of Na+ on the cAMP response revealed that Na+ exerted this effect probably by reducing the Increase In Ca++-influx occurring during depolarization. A similar type of interaction between Mg++ and Ca++ was also observed. These studies have pointed out a possible regulatory role of Ca++ in isoproterenol-lnduced Increases in cAMP levels in uterine smooth muscle. Since it was also demonstrated that cAMP Is not an obligatory requirement In order for Isoproterenol to produce relaxation, these data have raised the question as to whether the Increases ln cAMP produced by β-adrenoceptor stimulation Is an event secondary to the changes in Ca++ movements produced by the agonist. The electrophysiological studies showed that isoproterenol (10 M) could inhibit spontaneous contractility of the rat uterus without causing hyperpolarlzation. In hlgh-K+ depolarized muscle, Isoproterenol (10⁻⁶M) produced relaxation without any change in membrane potential. These data suggested that hyperpolarlzation of cell membranes is not a prerequisite for β-adrenoceptor-med-lated relaxation of uterine smooth muscle. / Pharmaceutical Sciences, Faculty of / Graduate

Pyridine nucleotide transhydrogenase catalyzes the reversible transfer of hydride ion equivalents between NADP(H) and NAD(H). In this study, the activity of the enzyme was measured by following the rate of reduction of an analogue of NAD⁺ , 3-acetylpyridxne-NAD⁺ (APNAD⁺ ) by NADPH. The enzyme was solubilized by detergents such as lysolecithin, sodium cholate (in the presence of ammonium sulphate) or Triton X-100. The molecular size of the solubilized enzyme was examined using sucrose density gradient centrifugation in the presence of Brij 58. These detergents gave soluble fragments of different sizes. That solubilized by Triton X-100 or sodium cholate (in the presence of ammonium sulphate) existed as large aggregates with sedimentation coefficients of 24.5 to 25.4S, whereas that obtained with lysolecithin consisted mainly of a species with a sedimentation coefficient of 7.3 to 16.5S. The fragment resulting from the solubilization with Triton X-100 could be cleaved into a smaller species (8.4S) by lysolecithin. Analysis by chromatography on Sepharose 6B of the enzyme preparation solubilized by sodium cholate (in the presence of ammonium sulphate), revealed the presence of other constituents of the membrane, such as succinate dehydrogenase, ATPase and cytochrome b₁. The molecular weight of the aggregate was estimated to be between 0.25 x 10⁶ and 4 x 10⁶. The enzyme in this preparation could not be further disaggregated by Tween 80, Brij 3 5 or Triton X-100. Chromatography of this preparation on DEAE-Sepharose CL-6B yielded a maximum purification of 37 to 68-fold over that of the membrane particle suspension. The specific activity of the enzyme was 8.8 to 15.7 umol per min per mg protein. Analysis of the partially purified enzyme on poly-acrylamide gels in the presence of sodium dodecyl sulphate revealed enrichment of several major polypeptide bands of molecular weights 90 000, 57 000, 50 000 and 40 000, coinciding with the transhydrogenase activity. The partially purified enzyme could be activated by detergents of the Tween or Brij series and by lysolecithin, palmitic acid and phospholipid extracts from E. coli. Measurements of the steady-state kinetics of the membrane-bound enzyme gave values of 45.6 and 106.7 uM for the substrates APNAD+ and NADPH, and dissociation constants of 3.6 and 16.2 uM, respectively. Lineweaver-Burk plots for each substrate at different fixed concentrations of the other substrate revealed a unique pattern of lines that is characteristic of rapid equilibrium random bireactant mechanisms with two dead-end products. In this type of mechanism each substrate is able to interact at the binding site of the other substrate to cause inhibition of enzyme activity. This mechanism was confirmed by kinetic studies using the alternate substrates deamino-NADPH and NAD⁺ , as well as by product inhibitxon studies. The adenine nucleotides 5’-AMP and ADP were competitive inhibitors of the APNAD+-binding site, while 2'-AMP was a competitive inhibitor of the NADPH-binding site on the enzyme. Studies on the active site using 2,3-butanedione or phenyl glyoxal revealed the presence of one modifiable arginyl residue per active site on the enzyme. Protection against modification by 2,3-butanedione was afforded by 2'-AMP, 5'-AMP, NAD+ and NADP+. Inhibition by 2,3-butanedione was enhanced in the presence of low concentrations of NADH or NADPH suggesting that binding of the reduced pyridine nucleotides, possibly at an allosteric site, causes a conformational change in the enzyme. Enhancement of in-activation of the enzyme by TPCK-trypsin was also observed in the presence of reduced pyridine nucleotides. NAD(P)H was oxidized by 2,3-butanedione in the presence of light. The rate of photooxidation was greatest at pH 7 and when the wavelength of incident light was 410 nm. This indicates that absorption of light by the diketone was necessary for the occurrence of the photooxidation reaction. The stochiometry of the reaction between NADH and 2,3-butanedione was 1:1. The possible nature of the reaction product is discussed in the thesis. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Burkholderia cepacia is a common soil bacterium of significance in agriculture and bioremediation. B. cepacia is also an opportunistic pathogen of humans causing highly communicable pulmonary infections in cystic fibrosis and immunocompromized patients. The pyrB gene encoding ATCase was cloned and ATCase was purified by the glutathione S-transferase gene fusion system. The ATCase in B. cepacia has been previously classified as a class A enzyme by Bethell and Jones. ATCase activity gels showed that B. cepacia contained a holoenzyme pyrBC complex of 550 kDa comprised of 47 kDa pyrB and 45 kDa pyrC subunits. In the course of purifying the enzyme, trimeric subunits of 140 kDa and 120 kDa were observed as well as a unique proteolysis of the enzyme. The 47 kDa ATCase subunits were cleaved to 40 kDa proteins, which still demonstrated high activity as trimers. The proteolysis site is between Ser74 and Val75 residues. To confirm this, we converted the Ser74 residue to an Ala and to an Arg by site-directed mutagenesis. After this primary sequence changed, the proteolysis of ATCase was not observed. To further investigate the characteristics of B. cepacia pyrB gene, a pyrB knock-out (pyrB-) was constructed by in vitro mutagenesis. In the assay, the 550 kDa holoenzyme and 140 kDa and 120 kDa trimers disappeared and were replaced with a previously unseen 480 kDa holoenzyme pyrB- strain. The results suggest that B. cepacia has two genes that encode ATCase. ATC1 is constitutive and ATC2 is expressed only in the absence of ATC1 activity. To check for the virulence of these two strains, a eukaryotic model virulence test was performed using Caenorhabditis elegans (C. elegans). The pyrB1+pyrB2+ (wild type) B cepacia killed the nematode but pyrB1-pyrB2+ B. cepacia had lost its virulence against C. elegans. This suggests that ATC1 (pyrB1) is involved in virulence in B.cepacia and ATC2 (pyrB2) is not.

The pyrimidine biosynthetic pathway is an essential pathway for most organisms. Previous research on the pyrimidine pathway in Pseudomonas aeruginosa (PAO1) has shown that a block in the third step of the pathway resulted in both a requirement for exogenous pyrimidines and decreased ability to produce virulence factors. In this work an organism with a mutation in the second step of the pathway, aspartate transcarbamoylase (ATCase), was created. Assays for pyrimidine intermediates, and virulence factors were performed. Results showed that the production of pigments, haemolysin, and rhamnolipids were significantly decreased from PAO1. Elastase and casein protease production were also moderately decreased. In the Caenorhabditis elegans infection model the nematodes fed the ATCase mutant had increased mortality, as compared to nematodes fed wild type bacteria. These findings lend support to the hypothesis that changes in the pyrimidine biosynthetic pathway contribute to the organism's ability to effect pathogenicity.

This study focused on the enzyme, aspartate transcarbamoylase (ATCase) from A. hydrophila, a Gram-negative bacterium found in fresh water. The molecular mass of the ATCase holoenzyme from A. hydrophila is 310 kDa. The enzyme is likely composed of 6 catalytic polypeptides of 34 kDa each and 6 regulatory polypeptides of 17 kDa each. The velocity-substrate curve for A. hydrophila ATCase is sigmoidal for both aspartate and carbamoylphosphate. The Km for aspartate was the highest to date for an enteric bacterium at 97.18 mM. The Km for carbamoylphosphate was 1.18 mM. When heated to 60 ºC, the specific activity of the enzyme dropped by more than 50 %. When heated to 100 ºC, the enzyme showed no activity. The enzyme's activity was inhibited by ATP, CTP or UTP.

AMPA receptors mediate excitatory synaptic transmission throughout the central nervous system via activation by their natural agonist glutamate. Several other molecules have been recognized as receptor agonist or antagonist, and recently allosteric modulators have been developed that potentiate the currents generated by these receptors. The goal of this thesis has been to address specific and as yet unresolved questions regarding the binding interactions between the AMPA receptors and these classes of molecules. For instance AMPA receptors are seemingly converted to have lower affinity for agonist as they move towards synapses and we evaluate two hypotheses put forward to explain the molecular mechanisms responsible for this. Additionally, guanine nucleotides competitively inhibit AMPA receptors and a second goal has been to further characterize guanine nucleotide binding, and to create mutations that selectively diminish this so that the function of the inhibition can be evaluated. A third goal has been to characterize the molecular factors that influence the effects of the allosteric modulators in order to explain why their efficacy differs greatly between brain regions. Experiments pertaining to these three goals were carried out sequentially and are described below as Projects 1 (guanine nucleotide inhibition), Project 2 (agonist affinity), and Project 3 (allosteric modulators). Project 1. Guanine nucleotides competitively inhibit AMPA-Rs (AMPA receptors) and because this inhibition is ubiquitous among virtually all types of glutamate receptors from fish to mammals, it likely serves a physiological function. Evaluation of this would be greatly facilitated if nucleotide binding could be eliminated through mutations without altering other aspects of receptor function, or if compounds were discovered that selectively prevent nucleotide binding. It was previously reported that a lysine in the chick kainate binding protein (cKBP) is specifically involved in guanine nucleotide binding. Therefore we mutated the homologous lysine (K445) in AMPA-R subunit GluR1 plus 12 additional residues around the glutamate binding pocket with the expectation that this would reduce nucleotide binding even further. Nucleotide affinity was determined by measuring the displacement of [3H]fluorowillardiine. As expected, the guanine nucleotide affinity was decreased about five-fold in R1-K445A mutants and the agonist affinity was seemingly unchanged. However, when tested by electrophysiology, characteristics of the mutant such as desensitization and the EC50 for glutamate were found to be altered. None of the other mutations were more successful at decreasing nucleotide affinity selectively. Nonetheless, these studies have given new insight into the docking mode of guanine nucleotides. The loss of binding in R1-K445A was much larger for GTP and GDP than for GMP, and guanosine binding, which is much lower, was unaffected by the mutation. These data suggest that the first phosphate of GMP determines the higher affinity of the phosphorylated nucleotides, and that K445 stabilizes the binding of the second and third phosphates of GDP and GTP. This along with various other observations suggest that the guanine base docks deep within the agonist binding pocket and that bulky additions, such as the phosphates, are accommodated by projecting out of the cleft in the vicinity of lysine 445. However, the exact docking mode of guanine nucleotides would have to be determined by crystallography. Project 2. Agonist binding to AMPA-R in brain consists of a high and low affinity components with KDs of 9-28 nM and 190-700 nM. Previous studies have suggested that newly synthesized receptors have high affinity and are converted to lower affinity by a secondary process. Two particular processes have been implicated, namely the conversion of receptor glycosylation from immature to complex, and modulation by receptor associated proteins. Both hypotheses were evaluated in this project using homomeric receptors GluR1-4 expressed in HEK 293 cells. The role of glycosylation was tested mostly with GluR4 receptors because they are expressed in distinct populations that exhibit either immature or complex glycans and their binding consists of high and low affinity components similar to those previously seen in brain receptors. Cells were treated with castanospermine or deoxymannojirimycin to decrease the proportion of receptors with complex glycosylation, or with cycloheximide plus chloroquine to increase the number of receptors with complex glycosylation. Although 70% of receptors from cells treated with cyloheximide/chloroquine exhibited complex glycans compared to <5% with other treatments, the affinity decreased at most 2-fold. Also, the low affinity component was nearly 80% of the total binding in receptors that exhibited virtually no complex glycans. Taken together these data indicate that complex glycosylation is not the key factor that confers low affinity. To test the second hypothesis GluR1i or GluR2i were co-expressed with stargazin which associates to receptors in neurons and affects their kinetics and trafficking. Considering the affinities of the two components seen in brain, we expected stargazin to cause a 20-fold or greater decrease in binding affinity. This was not the case, however our results did suggest that stargazin caused the appearance of a low affinity component but this was small and remained largely masked by the more abundant high affinity component. Recently, experiments with brain membranes have revealed preliminary evidence that an associated protein of ~85kDa may cause receptors to have low affinity. This hypothesis is currently under investigation. Project 3. Ampakines are cognitive enhancers that potentiate AMPA receptor currents at excitatory synapses. The efficacy of these drugs varies substantially among neurons in different brain regions, being for example about three times larger in the hippocampus than in the thalamus. Binding assays have shown that these compounds also increase the affinity of receptors for agonists. Importantly, the efficacy of these drugs to increase synaptic responses and agonist binding exhibit a positive correlation. Indeed, we have found that the increase in agonist binding (Emax) induced by the prototypical ampakine CX546 is highly variable across eight brain regions and that there is a 3-fold difference between the hippocampus and the thalamus which is similar to the difference reported for physiological efficacy. Therefore, binding assays or receptor autoradiography can potentially be used to predict the physiological efficacy of these drugs in a particular brain region. An important goal of this project has been to identify factors that may be responsible for the regionally different efficacies. Ampakines show some preference for receptor subunits but various considerations suggest that other factors must be involved. In this project we evaluated the role of a novel class of proteins called TARPs (transmembrane AMPA receptor regulatory proteins) that have recently been discovered to be tightly associated with AMPA receptors and to regulate their kinetics. Four of these proteins, named lambda;2(stargazin),λ3,λ4,and λ8 are abundant in the brain, but they exhibit highly selective regional distribution. We determined the maximum increase in agonist binding (Emax ) caused by saturating CX546 in three different AMPA receptor subunits, GluR1i, GluR2i, and GluR4i without and with co-expression of the four TARPs. Without TARPs, both Glu2i and GluR4i showed an Emax value of 100% over baseline binding. Co-expression of TARPs increased the Emax in GluR2i and this was largest for λ3 and λ8 (~130%). However, TARPs decreased the Emax of CX546 in GluR4i and this was most notable with λ2 and λ4 (~72%). Agonist binding in GluR1i was increased by only 15% and it was not significantly changed by TARPs. The expression patterns of TARPs and AMPA-R subunits in the brain have been partially characterized in the literature. Thus, it was previously reported that GluR4i transcripts are abundant in the thalamus but minor in the hippocampus. Using western blots we confirmed that this is also true for protein content; in the thalamus expression of GluR1, GluR2, GluR3, and GluR4 was 4%, 33%, 40%, and 147% respectively, of that in the hippocampus. When considering the known expression patterns of TARP variants, the hippocampus can be described as being enriched in GluR2, λ3 and λ8 while GluR4, λ2 and λ4 are prevalent in the thalamus. In comparison between these specific subunit/TARP combinations, the Emax values for those representative of the hippocampus (GluR2i/λ3 or λ8) were ~2-times larger than the Emax values of thalamic combinations (R4i/λ2 or λ4). Thus we can conclude that the differences in the expression of both TARP variants and AMPA-R subunits are critical factors for determining the variable efficacy of ampakines across brain regions.