Thesis (Ph. D.)--University of Montana, 2007. / Title from title screen. Description based on contents viewed Oct. 11, 2007. Includes bibliographical references (p. 109-128).

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (p. 154-168).

Judicious addition of photolabile caging groups (PCGs) to protect biologically im- portant molecules, has enabled the development of many powerful chemical tools for the study of biological processes. These tools have the potential to be activated in a cellular setting by irradiation with light of appropriate wavelengths, restoring functionality, with excellent spatial and temporal control. This D. Phil. dissertation highlights two biologically relevant examples where PCGs can be applied: (i) 4,5-dimethoxynitrobenzyl (DMNB) caged derivatives of GSH and ESG, were synthesised and uncaging of the DMNB group at 350 nm to reveal the free α-carboxylic acid of the glycine residue was demonstrated. These molecules have the potential to probe the binding mode within the KefC KTN binding domain, a ligand-gated K+ efflux system, critical for bacterial response to electrophilic assault. The molecules are currently with collaborators awaiting further evaluation. (ii) The wavelength-dependent application of PCGs toward the study of protein post- translational modifications (PTMs) was developed. PTMs modulate protein function and have a ubiquitous role in a diverse range of cellular functions. A wavelength selective sequential pair of PCGs was developed and demonstrated in a tripeptide, using the diethylamino coumarin (DEACM), which was cleaved at 420 nm, and the DMNB caging group, which was subsequently cleaved at 350 nm. A chromatically orthogonal pair of PCGs, with possible applications in both organic synthesis and biological systems, incorporating the BODIPY-based and DEACM caging groups, which could be cleaved at 530 nm and 420 nm, respectively, was also developed. Photolysis was conducted on a tripeptide and hexapeptide and an in vitro application was demonstrated where when caged, the peptide was not susceptible to peptidolysis when incubated with enzymes. Following irradiation at 420 nm, the DEACM group was uncaged and the peptide underwent peptidolysis in the presence of Endoproteinase AspN, whereas following irradiation at 530 nm to uncage the BODIPY group, the peptide underwent peptidolysis in the presence of Endoproteinase LysC. These results not only provide conclusive evidence of the ability of this pair of caging groups to act in a wavelength orthogonal sense but also demonstrates the potential applicability of the pair to biochemical studies. This pair of PCGs represents the first example of an orthogonal pair of caging groups where both PCGs could be cleaved, at wavelengths longer than 400 nm, in a sequence independent manner.

Foram avaliados os efeitos do fornecimento de dietas com três densidades de aminoácidos (AA) no desempenho e rendimento de carcaça de frangos de corte. Um total de 1.200 pintos de um dia Cobb × Cobb 500 foram distribuídos em 6 tratamentos com um arranjo fatorial 2 × 3 (sexo × densidade AA). Cada tratamento teve 8 repetições com 25 aves cada. As dietas foram formuladas tendo como base milho e farelo de soja, satisfazendo as fases de alimentação de 1 a 7, 8 a 21, 22 a 35 e 36 a 40 dias de idade, mantendo as mínimas relações entre AA essenciais e lisina (Lis) em base digestível (dig.) verdadeira: Lis, 100%; AA sulfurados totais, 75%; treonina, 65%, valina, 75% (de 1 a 21 dias) e 78% (de 22 a 42 dias). Os tratamentos dietéticos foram com baixa, moderada e alta densidades de AA, com a densidade média tendo Lis dig. de 1,25%, 1,19 %, 1,09 % e 1,05 % da dieta pré-inicial até a retirada respectivamente. As dietas com baixa e alta densidade tiveram redução e aumento de 12% no nível de Lis dig. em relação à dieta moderada. Não houve efeito dos tratamentos sobre a mortalidade (P>0,05) e os machos tiveram melhor desempenho zootécnico que as fêmeas (P<0,05). Não foram observadas interações entre sexo × densidade de AA, com exceção para a deposição de gordura abdominal, que foi mais reduzida nos machos com o aumento da densidade de AA do que nas fêmeas (P<0,01). O ganho de peso foi reduzido para as dietas com densidade baixa (P<0,05) quando comparadas com as densidades alta e moderada, porém entre estas últimas não houve diferença (P>0,05). A conversão alimentar (CA) melhorou (P<0,05) com o aumento da densidade de AA. As porcentagens de rendimento de carcaça, peito e deposição de gordura abdominal foi maior para as fêmeas (P<0,05), entretanto não foram encontradas respostas para a densidade de AA da dieta, com exceção da gordura abdominal, que reduziu com o aumento da densidade de AA da dieta (P<0,05). As melhorias obtidas na CA com o aumento das densidades de AA na dieta não foram estendidas para os ganhos econômicos nas condições econômicas utilizadas nesse estudo. / An evaluation of the effects of feeding programs with three amino acid (AA) densities given to an early growth broiler breed was conducted. Growth performance, carcass yield and an economic evaluation was done using current figures at the time of the study. A total of 1,200 Cobb X Cobb 500 were distributed across 6 treatments in a 2 X 3 factorial arrangement (Sex X AA Density). Each treatment had 8 replications of 25 birds per pen. Maize-soybean meal diets, provided from 1 to 7, 8 to 21, 22 to 35 and 36 to 42 d of age, were formulated maintaining minimum ratios between essential AA and Lysine (Lys) in a true digestible (dig) basis as follow: Lys, 100%; total sulfur AA, 75%; threonine), 65%, valine, 75% (from 1 to 21 d) and 78% (from 22 – 42 d). Experimental dietary treatments were Low, Moderate and High AA density diets with Moderate having dig Lys of 1.25%, 1.19 %, 1.09 % and 1.05 % in from starter to withdrawal, respectively. Low and High diets had reductions and increases of 12% on dig Lys in relation to the Moderate diet. There were no effects of treatments on mortality (P > 0.05) and males had an overall better live performance than females (P < 0.01). No interactions were observed between Sex x AA density, with the exception of abdominal fat which was further reduced in males as AA density increased than with females (P < 0.01). Body weight gain was reduced for Low diets (P < 0.05) when compared to High and Moderate, which were not different (P > 0.05). Feed conversion ratio (FCR) was improved (P < 0.05) as AA density increased. Percent yield of carcass, breast meat and abdominal fat were higher for females (P < 0.01); however, there were no responses for AA density on processing measures. One exception was abdominal fat, which was reduced with the High diet (P<0.05). Improvements obtained in feed FCR with the increase in AA density were not extended to economic gains under the economic conditions used in this study. Thus, the decision making process to change AA density in the diet towards a higher density AA diet is expected to depend on the ratio between feed ingredients and meat market prices.

MANSO, Helena Emília Cavalcanti da Costa Cordeiro. Avaliação da glutamina sintetase e da concentração da glutamina no terço final da gestação e na lactação de camundongos fêmeas e matrizes suínas primíparas. 2006. 106 f. Tese (doutorado em zootecnia)- Universidade Federal do Ceará, Fortaleza-CE, 2006. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-04-07T20:30:52Z No. of bitstreams: 1 2006_tese_hecccmanso.pdf: 2703402 bytes, checksum: 12ef7f4302a4af1a6a8de3b3cfed3d9e (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-05-25T19:50:44Z (GMT) No. of bitstreams: 1 2006_tese_hecccmanso.pdf: 2703402 bytes, checksum: 12ef7f4302a4af1a6a8de3b3cfed3d9e (MD5) / Made available in DSpace on 2016-05-25T19:50:44Z (GMT). No. of bitstreams: 1 2006_tese_hecccmanso.pdf: 2703402 bytes, checksum: 12ef7f4302a4af1a6a8de3b3cfed3d9e (MD5) Previous issue date: 2006 / The glutamine (Gln), a non-essential amino acid, normally it isn’t included in the animal feed. The metabolic properties of glutamine made it an essential conditionally amino acid during stress and catabolism, when the body has no ability to attend the nutritional requirements. The present research was divided in two parts; the first one had the objective to characterize the glutamine (Gln) and the glutamine synthetase (GS) during lactation of primiparous mice and the second one, the aim was to evaluate the effects of glutamine supplementation, given in two forms, pure (L-glutamine) and in association with glutamic acid (AminoGut® by Ajinomoto) for primiparous gilts. In the first experiment was characterized the importance of GS and Gln, and the important role on the animal metabolism during a catabolism. In the second experiment was demonstrated the importance of Gln to maintain the gilt health. More studies should have to establish the adequate level of Gln supplementation and to evaluate the reproductive performance of gilts as well as the piglets development after weaning. / A glutamina (Gln), aminoácido por ser não-essencial, não é tradicionalmente incluído nas rações animais. As propriedades metabólicas da Gln fazem dela um aminoácido condicionalmente essencial em períodos de estresse e catabolismo, quando o organismo não tem a habilidade de suprir em tempo hábil sua demanda nutricional. A presente pesquisa foi dividida em 2 partes, a primeira delas teve por objetivo de avaliar o metabolismo da glutamina (Gln) e da glutamina sintetase (GS) no período lactacional de camundongos fêmeas primíparas, a segunda parte teve o objetivo de avaliar os efeitos da suplementação da Gln na dieta, fornecida em 2 formas, uma delas, pura (L-glutamina) e uma outra associada ao ácido glutâmico (AminoGut® Ajinomoto) para matrizes suínas primíparas. No primeiro experimento caracterizou-se a importância da GS e da Gln e evidenciou-se o seu importante papel no metabolismo, quando o animal se encontrava em catabolismo. No segundo experimento, a glutamina mais uma vez demonstrou sua importância na manutenção da higidez da matriz. Mais estudos devem ser feitos no intuito de estabelecer níveis adequados de suplementação e de avaliar a subsequente performance reprodutiva de matrizes suínas assim como o desempenho dos leitões após o desmame.

A resolução temporal é uma questão-chave em Paleontologia, uma vez que a sua magnitude define a precisão dos estudos não somente paleoecológicos como também evolutivos. A resolução temporal é estimada pela magnitude de time-averaging (mistura de gerações em uma camada, uma amostra). Tais estimativas têm sido amplamente conduzidas em ambientes marinhos recentes. A plataforma continental do sul do Brasil (PSB; 22°S – 34°S) tem sido um laboratório natural para estudos desta natureza desde o início do século XXI. Consequentemente, possui um amplo acervo de dados disponíveis para comparação. Neste contexto, esta tese visou responder (i) qual a magnitude do time-averaging em acumulações de bivalves da PSB? (ii) como este processo varia ao longo de gradientes espaciais? e (iii) como o time-averaging reflete na informação biológica preservada no registro fóssil? Para isto, mais de 140 espécimes de bivalves foram datados integrando racemização de aminoácidos e 14C AMS. Além disto, análises tafonômicas foram realizadas em todas as amostras datadas, incluindo mais sete amostras em sedimentos lamosos. A resolução temporal (time-averaging) e a variabilidade total de idades (mistura temporal) basearam-se em uma nova abordagem numérica, a estatística bayesiana, que integra os erros e as incertezas derivadas da distribuição posterior dos resíduos associados com os modelos resultantes das calibrações das idades. As tendências onshore-offshore — aumento da mediana e da uniformidade das curvas de frequência de distribuição de idades, redução da variabilidade tafonômica, ainda que a escala do time-averaging seja invariante — provavelmente refletem a interação entre as mudanças do nível relativo do mar e da bioprodutividade mais elevada em águas menos profundas. / The temporal resolution of the fossil record plays a key role in paleontology because it determines the scale and the precision of paleoecological and evolutionary studies. The temporal resolution of the fossil record is estimated by the magnitude of time-averaging (non-contemporaneous generations preserved in a single layer, a bulk-sample). Quantitative estimates of time-averaging have been conducted primarily on mollusk shells from modern shallow-water marine settings. Most of them have been addressed in the Southern Brazilian continental shelf (SBS; 22°S up to 34°S), which is considered a natural laboratory for several similar studies since the earlier of current century (XXI). Consequently, the SBS has several available datasets that allow comparisons of the new results displayed here with those previous data. Thus, this thesis aimed answer (i) what is the magnitude of time-averaging on SBS mollusk death assemblages? (ii) how does time-averaging vary across spatial gradients? and (iii) how does time-averaging can reflect on the preservation of the fossil record? Here, >140 specimens were individually dated using amino acid racemization calibrated using radiocarbon ages (14C). In addition, taphonomic analyses were conducted in all samples, including more seven muddy sites. The time-averaging and the total age variability was based on a Bayesian approach that integrates the estimation errors and uncertainties derived from the posterior distribution associated with the 14C–AAR calibration average model. The onshore-offshore trends — increased median age, decreased skewness of age distributions, decreased taphonomic variation, yet the invariant scale of time-averaging — likely reflect the interplay between sea-level changes and elevated bioproductivity in shallower water settings.

A resolução temporal é uma questão-chave em Paleontologia, uma vez que a sua magnitude define a precisão dos estudos não somente paleoecológicos como também evolutivos. A resolução temporal é estimada pela magnitude de time-averaging (mistura de gerações em uma camada, uma amostra). Tais estimativas têm sido amplamente conduzidas em ambientes marinhos recentes. A plataforma continental do sul do Brasil (PSB; 22°S – 34°S) tem sido um laboratório natural para estudos desta natureza desde o início do século XXI. Consequentemente, possui um amplo acervo de dados disponíveis para comparação. Neste contexto, esta tese visou responder (i) qual a magnitude do time-averaging em acumulações de bivalves da PSB? (ii) como este processo varia ao longo de gradientes espaciais? e (iii) como o time-averaging reflete na informação biológica preservada no registro fóssil? Para isto, mais de 140 espécimes de bivalves foram datados integrando racemização de aminoácidos e 14C AMS. Além disto, análises tafonômicas foram realizadas em todas as amostras datadas, incluindo mais sete amostras em sedimentos lamosos. A resolução temporal (time-averaging) e a variabilidade total de idades (mistura temporal) basearam-se em uma nova abordagem numérica, a estatística bayesiana, que integra os erros e as incertezas derivadas da distribuição posterior dos resíduos associados com os modelos resultantes das calibrações das idades. As tendências onshore-offshore — aumento da mediana e da uniformidade das curvas de frequência de distribuição de idades, redução da variabilidade tafonômica, ainda que a escala do time-averaging seja invariante — provavelmente refletem a interação entre as mudanças do nível relativo do mar e da bioprodutividade mais elevada em águas menos profundas. / The temporal resolution of the fossil record plays a key role in paleontology because it determines the scale and the precision of paleoecological and evolutionary studies. The temporal resolution of the fossil record is estimated by the magnitude of time-averaging (non-contemporaneous generations preserved in a single layer, a bulk-sample). Quantitative estimates of time-averaging have been conducted primarily on mollusk shells from modern shallow-water marine settings. Most of them have been addressed in the Southern Brazilian continental shelf (SBS; 22°S up to 34°S), which is considered a natural laboratory for several similar studies since the earlier of current century (XXI). Consequently, the SBS has several available datasets that allow comparisons of the new results displayed here with those previous data. Thus, this thesis aimed answer (i) what is the magnitude of time-averaging on SBS mollusk death assemblages? (ii) how does time-averaging vary across spatial gradients? and (iii) how does time-averaging can reflect on the preservation of the fossil record? Here, >140 specimens were individually dated using amino acid racemization calibrated using radiocarbon ages (14C). In addition, taphonomic analyses were conducted in all samples, including more seven muddy sites. The time-averaging and the total age variability was based on a Bayesian approach that integrates the estimation errors and uncertainties derived from the posterior distribution associated with the 14C–AAR calibration average model. The onshore-offshore trends — increased median age, decreased skewness of age distributions, decreased taphonomic variation, yet the invariant scale of time-averaging — likely reflect the interplay between sea-level changes and elevated bioproductivity in shallower water settings.

Foram avaliados os efeitos do fornecimento de dietas com três densidades de aminoácidos (AA) no desempenho e rendimento de carcaça de frangos de corte. Um total de 1.200 pintos de um dia Cobb × Cobb 500 foram distribuídos em 6 tratamentos com um arranjo fatorial 2 × 3 (sexo × densidade AA). Cada tratamento teve 8 repetições com 25 aves cada. As dietas foram formuladas tendo como base milho e farelo de soja, satisfazendo as fases de alimentação de 1 a 7, 8 a 21, 22 a 35 e 36 a 40 dias de idade, mantendo as mínimas relações entre AA essenciais e lisina (Lis) em base digestível (dig.) verdadeira: Lis, 100%; AA sulfurados totais, 75%; treonina, 65%, valina, 75% (de 1 a 21 dias) e 78% (de 22 a 42 dias). Os tratamentos dietéticos foram com baixa, moderada e alta densidades de AA, com a densidade média tendo Lis dig. de 1,25%, 1,19 %, 1,09 % e 1,05 % da dieta pré-inicial até a retirada respectivamente. As dietas com baixa e alta densidade tiveram redução e aumento de 12% no nível de Lis dig. em relação à dieta moderada. Não houve efeito dos tratamentos sobre a mortalidade (P>0,05) e os machos tiveram melhor desempenho zootécnico que as fêmeas (P<0,05). Não foram observadas interações entre sexo × densidade de AA, com exceção para a deposição de gordura abdominal, que foi mais reduzida nos machos com o aumento da densidade de AA do que nas fêmeas (P<0,01). O ganho de peso foi reduzido para as dietas com densidade baixa (P<0,05) quando comparadas com as densidades alta e moderada, porém entre estas últimas não houve diferença (P>0,05). A conversão alimentar (CA) melhorou (P<0,05) com o aumento da densidade de AA. As porcentagens de rendimento de carcaça, peito e deposição de gordura abdominal foi maior para as fêmeas (P<0,05), entretanto não foram encontradas respostas para a densidade de AA da dieta, com exceção da gordura abdominal, que reduziu com o aumento da densidade de AA da dieta (P<0,05). As melhorias obtidas na CA com o aumento das densidades de AA na dieta não foram estendidas para os ganhos econômicos nas condições econômicas utilizadas nesse estudo. / An evaluation of the effects of feeding programs with three amino acid (AA) densities given to an early growth broiler breed was conducted. Growth performance, carcass yield and an economic evaluation was done using current figures at the time of the study. A total of 1,200 Cobb X Cobb 500 were distributed across 6 treatments in a 2 X 3 factorial arrangement (Sex X AA Density). Each treatment had 8 replications of 25 birds per pen. Maize-soybean meal diets, provided from 1 to 7, 8 to 21, 22 to 35 and 36 to 42 d of age, were formulated maintaining minimum ratios between essential AA and Lysine (Lys) in a true digestible (dig) basis as follow: Lys, 100%; total sulfur AA, 75%; threonine), 65%, valine, 75% (from 1 to 21 d) and 78% (from 22 – 42 d). Experimental dietary treatments were Low, Moderate and High AA density diets with Moderate having dig Lys of 1.25%, 1.19 %, 1.09 % and 1.05 % in from starter to withdrawal, respectively. Low and High diets had reductions and increases of 12% on dig Lys in relation to the Moderate diet. There were no effects of treatments on mortality (P > 0.05) and males had an overall better live performance than females (P < 0.01). No interactions were observed between Sex x AA density, with the exception of abdominal fat which was further reduced in males as AA density increased than with females (P < 0.01). Body weight gain was reduced for Low diets (P < 0.05) when compared to High and Moderate, which were not different (P > 0.05). Feed conversion ratio (FCR) was improved (P < 0.05) as AA density increased. Percent yield of carcass, breast meat and abdominal fat were higher for females (P < 0.01); however, there were no responses for AA density on processing measures. One exception was abdominal fat, which was reduced with the High diet (P<0.05). Improvements obtained in feed FCR with the increase in AA density were not extended to economic gains under the economic conditions used in this study. Thus, the decision making process to change AA density in the diet towards a higher density AA diet is expected to depend on the ratio between feed ingredients and meat market prices.

abstract: The biological and chemical diversity of protein structure and function can be greatly expanded by position-specific incorporation of non-natural amino acids bearing a variety of functional groups. Non-cognate amino acids can be incorporated into proteins at specific sites by using orthogonal aminoacyl-tRNA synthetase/tRNA pairs in conjunction with nonsense, rare, or 4-bp codons. There has been considerable progress in developing new types of amino acids, in identifying novel methods of tRNA aminoacylation, and in expanding the genetic code to direct their position. Chemical aminoacylation of tRNAs is accomplished by acylation and ligation of a dinucleotide (pdCpA) to the 3'-terminus of truncated tRNA. This strategy allows the incorporation of a wide range of natural and unnatural amino acids into pre-determined sites, thereby facilitating the study of structure-function relationships in proteins and allowing the investigation of their biological, biochemical and biophysical properties. Described in Chapter 1 is the current methodology for synthesizing aminoacylated suppressor tRNAs. Aminoacylated suppressor tRNACUAs are typically prepared by linking pre-aminoacylated dinucleotides (aminoacyl-pdCpAs) to 74 nucleotide (nt) truncated tRNAs (tRNA-COH) via a T4 RNA ligase mediated reaction. Alternatively, there is another route outlined in Chapter 1 that utilizes a different pre-aminoacylated dinucleotide, AppA. This dinucleotide has been shown to be a suitable substrate for T4 RNA ligase mediated coupling with abbreviated tRNA-COHs for production of 76 nt aminoacyl-tRNACUAs. The synthesized suppressor tRNAs have been shown to participate in protein synthesis in vitro, in an S30 (E. coli) coupled transcription-translation system in which there is a UAG codon in the mRNA at the position corresponding to Val10. Chapter 2 describes the synthesis of two non-proteinogenic amino acids, L-thiothreonine and L-allo-thiothreonine, and their incorporation into predetermined positions of a catalytically competent dihydrofolate reductase (DHFR) analogue lacking cysteine. Here, the elaborated proteins were site-specifically derivitized with a fluorophore at the thiothreonine residue. The synthesis and incorporation of phosphorotyrosine derivatives into DHFR is illustrated in Chapter 3. Three different phosphorylated tyrosine derivatives were prepared: bis-nitrobenzylphosphoro-L-tyrosine, nitrobenzylphosphoro-L-tyrosine, and phosphoro-L-tyrosine. Their ability to participate in a protein synthesis system was also evaluated. / Dissertation/Thesis / Ph.D. Chemistry 2013

Neste trabalho são apresentados estudos de Cu(&#945-AIB)2 utilizando-se a técnica de RPE à temperatura ambiente nas freqüências de 9,7 GHz e 34 GHz. Os espectros de R. P. E. mostram uma única ressonância tanto em banda X (9,7 GHz) quanto em banda Q (34 GHz), devido ao efeito de estreitamento por troca. Os valores das componentes do tensor g e da largura de linha foram determinados a partir dos espectros obtidos variando o ângulo entre H e os eixos do cristal a´, b e c em três planos a´b, a´c, e bc. O tensor g reflete as propriedades moleculares do complexo, com o íon Cu(II) em uma simetria axial e também a orientação destas moléculas dentro da cela unitária do cristal. A variação angular da largura de linha é analisada em termos da simetria do íon Cu(II) na rede cristalina e das contribuições das interações dipolar e Zeeman Residual. O parâmetro da interação de troca |J\'|, é obtido através da contribuição da interação Zeeman residual na largura de linha, |J\'| ~ 0,34K. É também observada uma característica magnética bidimensional no complexo Cu(&#945-AIB)2 concordando com os resultados cristalográficos. / In this work is presented a study of the complex Cu(&#945-AIB) 2 using EPR spectroscopy at room temperature, in two frequency bands (9.7 and 34 GHz). The EPR spectra, in both bands and any direction of the extremal magnetic field consist of a single resonance line. This fact can be understood considering the exchange narrowing between non-equivalent Cu(II) íons. The elements of the g tensor and line width were determined from the angular dependence of the EPR spectrum, in three ortogonal crystal planes a´b, a´c and ab (a´=b x c). The angular dependence of the g tensor reflects the molecular properties of the complex Cu(&#945-AIB)2 the axial symmetry of the molecule and the orientation on the crystal unit cell. The most important contributions to the line width were found to be: 2D dipolar interactions, the residual Zeeman effect and defects compatible to the symmetry of the crystal. The Exchange parameter, |J\'| ~ 0.34K, was obtained from the residual Zeeman contribution to the line width (Q band). The low dimension found for dipolar interations agrees with crystallographic results.