This thesis aims to address issues relating to genome architecture and base composition. The first part of this thesis addresses questions relating to codon usage. Initially I will investigate thousands of bacterial species using a detailed analysis of strengths of selection acting upon codons usage while also investigating patterns of optimal codon changes with respect to genomic base composition and tRNA abundance. I report that selection on codon usage increases throughout the length of highly expressed genes, in particular, the first quarter of genes have significantly lower selection. Further, it is clear that factors affecting genomic base composition can eventually lead to changes in optimal codons if the change in base composition is strong enough, however these patterns differ substantially between amino acids. The debate over translational efficiency vs. accuracy was addressed by comparing sites of differing conservation. Differing conservation were defined using a phylogenetic method, allowing sites to change in their extent of conservation throughout the tree. The results show that translational accuracy acts strongly on the top 10% of conserved sites, however is relatively weak when compared to the efficiency for other sites. Also detected is a reduction in apparent selection on codon usage on the bottom 10% of conserved sites which is likely to be caused by conflicting positive selection on amino acids. Finally, although differences in patterns are observed between amino acids, the general relationship to conservation is similar. As much of the variation in codon usage is determined by variation in base composition, this aspect of base composition is investigated in the second part of the thesis. The observed variation in intragenomic base composition in bacteria was found to be far higher than expected for GC-rich bacteria. The non-core part of the genome contributes to this variation to a greater extent than the core part, suggesting that processes such as AT-rich horizontal gene transfer may be involved. Secondly, base composition is modelled under Brownian motion and as an extension, the Ornstein- Uhlenbeck process, which allows for multiple optima throughout the tree. The model including optima fits the data better than standard Brownian motion or Brownian motion with multiple diffusion coefficients. Finally, I investigate a case where a previous codon usage analysis has been seriously confounded by an unusual genome architecture of abnormal regional base composition in two species of eukaryotic parasites in the genus Theileria. In both species, the background G+C content is 37% at most, out of the four syntenic chromosomes. In many orthologous regions however, T.annulata has a decreased G+C content of 28% while T.parva has an increased G+C content of 41%. Various factors coincide with this remarkable divergence: increased divergence at all types of site, recombination hot spots in T.parva, an increased frequency of tandem repeats and DNA sequence motifs in both species. The evolutionary origins of these unusual patterns will be discussed.

In this thesis, two potential applications of transdermal iontophoresis in the field of sports medicine were studied: (1) the local delivery of dexamethasone phosphate (Dex-Phos), a corticosteroid used to treat musculoskeletal inflammation, and (2) the extraction of systemic amino acids (AAs), potential biological markers of fatigue in athletes. The iontophoretic delivery of Dex-Phos was studied, in vitro, in order to evaluate the effects of competing ions and electroosmosis, and identify the optimal conditions for its delivery. The iontophoretic extraction of AAs from the skin was first studied in vitro, before evaluating the method in a group of human volunteers. Dex-Phos was best delivered by iontophoresis from the cathode in absence of background electrolyte in the drug solution. In this situation, the delivery of Dex-Phos is limited principally by the competition with counter-ions (mainly Na+) present subdermally and the small mobility of the drug inside the membrane. The accumulation of Cl-, released by the Ag/AgCl cathode in the drug solution during current passage, can also reduce Dex-Phos delivery. The extraction of zwitterionic AAs from the skin during iontophoresis was highly influenced by their presence in the outermost layer of the skin, the stratum corneum (SC). In the pig skin model, the amount of the AAs extracted during a short extraction period (1 hour) correlated with their abundance in the SC. Once this ‘reservoir’ was emptied (after ~3 hours of iontophoresis), the subdermal compartment could be sampled, suggesting that the method could be used to monitor systemic levels of AAs. The experiments in human volunteers revealed, however, that a 4-hour iontophoretic extraction period was insufficient to deplete the AAs SC ‘reservoir’. It follows that the method can be used to evaluate the abundance of AAs in the SC, but is unpractical for the clinical monitoring of their systemic levels.

No desvio portossistêmico, o sangue proveniente do trato gastrintestinal não é direcionado ao fígado para ser metabolizado e sim desviado para a circulação sistêmica. O acúmulo dessas substâncias tóxicas associado às alterações funcionais do órgão pode resultar em encefalopatia hepática. O manejo nutricional é o ponto chave para o sucesso da terapia. Dessa forma, o presente estudo objetivou avaliar os efeitos do emprego de um alimento hipoproteico nas concentrações séricas aminoacídicas de cães com desvio portossistêmico. Foram incluídos nove cães com diagnóstico de desvio portossistêmico (Shunt) e nove cães saudáveis (Controle). Os animais foram avaliados no dia um (T0) e após 60 dias (T60) de ingestão de uma dieta hipoproteica comercial. Nos dois momentos foram coletadas amostras de sangue para realização de hemograma, leucograma, bioquímica sérica, determinação dos aminoácidos séricos e amônia. A análise estatística dos resultados referentes aos hemogramas e exames bioquímicos foi baseada em um modelo misto que considerou efeito fixos de grupo, tempo e interação e, para os aminoácidos somente efeitos fixos de grupo (p<0,05). Os animais do grupo controle apresentaram maiores valores de proteína total (p<0,0001), ureia (p<0,0001), creatinina (p=0,0163), albumina (p<0,0001) e colesterol (p=0,0012) quando comparado ao grupo Shunt que apresentou maiores valores de hematócrito (p=0,0213), ALT e FA (p=0,0253 e p=0,0004, respectivamente), amônia em jejum (p=0,0083) e amônia pós-prandial (p=0,0036). Em relação aos aminoácidos séricos, o grupo Shunt apresentou valores maiores quando comparados ao grupo Controle nas concentrações dos aminoácidos fenilalanina (p=0,0054), glutamato (p=0,0066), serina (p=0,0054) e tirosina (p=0,0106), já o aminoácido sérico alanina (p=0,0280), a razão de Fischer (p=0,0093) e a razão da concentração sérica de aminoácidos de cadeia ramificada e tirosina (p=0,0243) foram maiores no grupo Controle. Concluiu-se que o manejo dietético foi eficiente em prevenir a progressão da doença e controlar os sinais clínicos, sem promover aumento nas concentrações séricas dos aminoácidos de cadeia ramificada. / In the portosystemic shunt, the blood coming from the gastrointestinal tract is not directed to the liver to be metabolized but rather diverted to the systemic circulation. The accumulation of these toxic substances associated with functional alterations of the organ can result in hepatic encephalopathy. Nutritional management is the key to successful therapy. Thus, the present study aimed to evaluate the effects of the use of a hypoproteic food on the protein and amino acid profile of dogs with portosystemic shunt. For this, nine dogs with a diagnosis of portosystemic shunt (Shunt) and nine healthy dogs (Control) were included. The dogs were evaluated on day one (T0) and after 60 days (T60) feeding a commercial hypoprotein diet. Blood samples were collected for hemogram, leukogram, serum biochemistry, determination of serum amino acids and ammonia. For the analysis of hemograms and biochemistry, a mixed model was used considering group fixed effects, time and interaction, and for amino acids only group fixed effects (p<0.05). The results showed that the animals in the Control group presented higher values of total protein (p<0.0001), urea (p<0.0001), creatinine (p=0.0163), albumin (p<0.0001) and cholesterol (p=0.0012) when compared to the Shunt group that presented higher values of hematocrit (p=0.0213), ALT and FA (p=0.0253 and p=0.0004, respectively), fasting ammonia (p=0.0083) and postprandial ammonia (p=0.0036). Serum amino acids (p=0.0054), glutamate (p=0.0066), serine (p=0.0054) and tyrosine (p=0.0054) were higher in the Shunt group than in the Control group (0.0106). The Fischer ratio (p=0.0093) and the ratio of serum concentration of branched-chain amino acids and tyrosine (p=0.0243) were higher for the Control group. In conclusion, that dietary management was efficient in preventing disease progression and controlling clinical signs without increasing branched-chain amino acids.

A produção de tomate (Solanum lycopersicum L.) no estado de São Paulo é de grande importância para a produção brasileira, pois representa 21,5 % da produção total e é o principal estado produtor de tomate de mesa. O tomate de mesa é cultivado por tutoramento das plantas e o cultivo em casas de vegetação é uma prática crescente no Brasil, trazendo muitas vantagens a produção, como proteção às chuvas e pragas. Porém em muitos casos, dependendo da estrutura utilizada, pode ocorrer aumento da temperatura interna das casas de vegetação. O tomateiro é uma planta sensível as altas temperaturas, e alterações dos níveis ótimos podem causar danos no metabolismo e consequentemente na produtividade da cultura. Altas temperaturas no cultivo de tomate são prejudiciais ao seu desenvolvimento, pois alteram a homeostase celular e desregulam funções vitais das plantas, como fotossíntese, gerando em excesso espécies reativas de oxigênio (EROs) como os radicais superóxidos, peróxido de hidrogênio e radicais hidroxilas, que são tóxicos às plantas e causam estresse oxidativo, levando até a sua morte. Plantas estressadas acumulam solutos osmoprotetores, entre eles a L-prolina, que auxiliam na proteção das estruturas celulares, na eliminação das EROs e também ativam o metabolismo antioxidante das plantas. Além da acumulação endógena de L-prolina, a aplicação exógena desse aminoácido pode trazer vantagens ao alívio do estresse térmico. Esse estudo teve por objetivo avaliar a eficiência da L-prolina nas doses de 0, 50, 100, 200, 400, 800 e 1600 mg/L na nutrição foliar, parâmetros fotossintéticos, enzimas do sistema antioxidante, malonaldeído e peróxido de hidrogênio, produtividade e características pós colheita dos frutos. Os dados climáticos durante o cultivo do tomateiro evidenciaram a ocorrência de temperaturas elevadas durante o ciclo de produção. Assim, foi possível observar que a aplicação exógena de L-prolina nas doses de 50 e 100 mg/L aumentaram a eficiência do uso da água, diminuíram o conteúdo de malonaldeído e aumentaram a produtividade comercial e total de frutos por planta. O uso de 50 mg/L desse aminoácido também proporcionou aumento do número de frutos comerciais por planta. A aplicação de L-prolina, independente da dose, proporcionou diminuição no conteúdo de peróxido de hidrogênio e elevou a atividade das enzimas do sistema antioxidante, além de melhorar a qualidade do fruto, diminuindo a acidez e aumentando a relação SST/ATT. Dessa maneira, constatou-se o efeito positivo do uso exógeno da L-prolina no alívio dos danos causados por altas temperaturas, principalmente em plantas tratadas com 50 e 100 mg/L. / The production of tomato (Solanum lycopersicum L.) in the state of São Paulo is of great importance for the Brazilian production, it represents 21.5% of total production and is the main state table tomato producer. The fresh tomato is grown by staking of plants and cultivation in greenhouses is a growing practice in Brazil, bringing many benefits to production, as protection of rain and pests. However, in many cases, depending on the structure used, there may be an increase in the internal temperature of the greenhouse. The tomato is a plant sensitive to high temperatures, and changes in optimal levels can cause damage to metabolism and consequently crop productivity. High temperatures in tomato cultivation are detrimental to their development, as they alter cellular homeostasis and deregulate vital plant functions, such as photosynthesis, generating in excess reactive oxygen species (ROS) such as superoxide radicals, hydrogen peroxide and hydroxyl radicals, which are toxic to plants and cause oxidative stress, leading to their death. Stressed plants accumulate osmoprotective solutes, including proline, which help protect cell structures, eliminate ROS and also activate plant antioxidant metabolism. In addition to the accumulation of endogenous proline, exogenous application of this amino acid can bring advantages to the relief of heat stress. The objective of this study was to evaluate the efficiency of L-proline at doses of 0, 50, 100, 200, 400, 800 and 1600 mg / L by foliar spray, in photosynthetic parameters, antioxidant system enzymes, malonaldehyde and hydrogen peroxide, productivity and post-harvest characteristics of fruits. The climatic data during tomato cultivation evidenced the occurrence of high temperatures during the production cycle. Thus, it was possible to observe that the exogenous application of L-proline at doses of 50 and 100 mg / L increased the efficiency of water use, decreased malonaldehyde content and increased commercial and total fruit yield per plant. The use of 50 mg / L of this amino acid also provided an increase in the number of commercial fruits per plant. The application of L-proline, regardless of the dose, resulted in a decrease in the hydrogen peroxide content and increased the activity of the antioxidant system enzymes, besides improving fruit quality, decreasing the acidity and increasing the SST/ATT ratio. Thus, there was the positive effect of the exogenous use of L-proline in alleviating the damage caused by high temperatures, especially in plants treated with 50 and 100 mg / L.

Objetivou-se avaliar o efeito da suplementação de um complexo enzimático, composto principalmente por endo 1,4 xilanase e endo 1,3(4) β-glucanases (CE), sobre o desempenho produtivo, a digestibilidade de aminoácidos (AA) e a utilização da energia em frangos de corte. Foram alojados 2.016 frangos de corte machos Cobb x Cobb 500 de um dia de idade em 72 boxes experimentais. As aves foram distribuídas em delineamento inteiramente casualizado com 9 tratamentos, 8 repetições e 28 aves por unidade experimental. Os tratamentos consistiram de uma dieta controle, formulada com níveis usuais de energia metabolizável aparente (EMA) e aminoácidos digestíveis (AA dig.) sem a inclusão do CE; e 8 dietas com reduções de EMA (-80 e -120 kcal/kg) e de AA dig. (-3% e 6%), suplementadas ou não com 50 mg/kg do CE. Foi utilizado um programa alimentar de 3 fases: inicial (1 a 21 d), crescimento (22 a 35 d) e final (36 a 42 d). Todas as dietas foram formuladas a base de milho e farelo de soja e com 1% de Celite, utilizado como indicador indigestível. Ganho de peso, consumo de ração e conversão alimentar corrigida para mortalidade (CA) foram avaliados aos 21, 35 e 42 d. Nos dias 21 e 42, 4 aves por unidade experimental foram sacrificadas para coleta de conteúdo ileal e posterior determinação da energia digestivel ileal (EDI) e da digestibilidade de AA. Os dados foram submetidos à análise de variância e, quando significativas, as médias foram comparadas pelo teste de Student-Newman-Keuls a 5% de significância. Contrastes ortogonais foram realizados entre os tratamentos com reduções nutricionais com e sem adição do CE. Frangos alimentados com dietas com reduções de energia e de AA dig. sem a adição do CE apresentaram maior CA e menor EDI quando comparados aos frangos que receberam a suplementação do CE. A suplementação do CE nas dietas aumentou a digestibilidade total de AA aos 21 d, porém não houve efeito aos 42 dias. A maior liberação de energia proporcionada pelo CE foi observada aos 21 d em frangos de corte alimentados com dietas com -120 kcal/kg AME e -6% AA dig., sendo 5,8% maior quando comparados aos alimentados com a dieta análoga sem enzima. Conclui-se que a adição do CE melhorou o desempenho produtivo e resultou em aumento da digestibilidade de AA e do aproveitamento da energia em frangos de corte aos 21 e aos 42 d. / The objective of this study was to evaluate the effect of supplementation of an enzyme complex, composed of endo 1,4 xylanase and endo 1,3 (4) β-glucanases (EC), on the productive performance, amino acid digestibility (AA) and the use of energy in broilers. A total of 2,016 one-day-old Cobb x Cobb 500 male broilers were allocated in 72 floor pens. Chicks were distributed into 9 treatments with 8 replicates of 28 birds each in a completely randomized design. Treatments consisted of a control diet formulated with usual apparent metabolizable energy (AME) and nutrient levels without enzyme supplementation as well as 8 diets having reductions in formulated AME (-80 and -120 kcal/kg) and digestible amino acids (dig. AA; -3 and -6%) supplemented or not with 50 mg/kg of the supplemental enzyme complex. A 3 phases feeding program was used: Starter (1 to 21 d), Grower (22 to 35 d) and Finisher (36 to 42 d). All diets were corn-soybean meal based with 1% of Celite, used as marker. Body weight gain, feed intake and feed conversion corrected for the weight of dead birds (FCR) were evaluated at 21, 35 and 42 d. On days 21 and 42, four birds per experimental unit were sacrificed for ileal content collection and subsequent determination of ileal digestible energy (IDE) and AA digestibility. Data were submitted to analysis of variance and, when significant, the means were compared by the Student-Newman-Keuls test at 5% significance. Orthogonal contrasts were performed between treatments with nutritional reductions with and without EC addition. Diets with AME and dig. AA reductions without enzyme had higher FCR and lower IDE (P < 0.05). The supplemental enzyme increased total AA digestibility at 21 d; however, no effects were observed at 42 d. The highest energy improvement provided by the supplemental enzyme was observed at 21 d on broilers fed diets with -120 kcal/kg AME and -6% dig. AA, being 5.8% higher when compared to those fed the analogous diet without enzyme. Summarizing, the addition of EC led to improvements on growth performance, digestibility of AA and energy utilization on broilers at 21 and 42 d.

Orientador: Luciano Hauschild / Coorientador: Ines Andretta / Banca: Edney Pereira da Silva / Banca: Charles Kiefer / Resumo: Numerosos estudos estão sendo publicados para avaliar os efeitos do consumo de lisina sobre o desempenho de frangos de corte, no entanto, tais estudos diferem em seus resultados, principalmente devido à variabilidade existente nas condições experimentais. Uma meta-análise foi realizada para conduzir a informação disponível neste campo de pesquisa e estudar a resposta de lisina em dietas para frangos de corte. Os dados de diferentes níveis de lisina 104 experimentos dose-resposta oriundos de 66.726 frangos de corte foram utilizados neste banco de dados. Codificações foram usadas com critérios de agrupamento qualitativo e como moderador nas variáveis analisadas. O banco de dados foi dividido em quatro grupos: machos em fase inicial (de 1 a 21 dias de idade); machos em fase de crescimento (22-42 dias de idade); fêmeas em fase inicial (de 1 a 21 dias de idade); e fêmeas em fase de crescimento (22-42 dias de idade). Análises gráficas foram usadas para explorar a base de dados, ao passo que equações foram utilizadas para estudar o estudo das respostas das variáveis dependentes em cada grupo anteriormente descrito. Essas equações foram geradas através de análises sequenciais usando modelos de efeitos fixos e de efeitos aleatórios. Menores variâncias foram observados em modelos equipados usando dados de frangos de corte machos, comparando com os dados de fêmeas. A utilização de componentes aleatórios permitiu que os modelos explicassem a variabilidade na base de dados e reduzir a variação de 67% em média para machos e fêmeas nos períodos: inicial e crescimento, em comparação com o modelo de efeito fixo. O ganho de peso médio diário foi maximizado num... / Abstract: Numerous publications are being published evaluating the effects of lysine intake on broiler performance, however, showing differing results mainly due to the variability in the experimental conditions. A meta-analysis was conducted to address the available information in this research field and study the response of lysine in diets for broilers. Data of 104 dose-response experiments testing different dietary lysine levels for 66.726 broilers were used in this database. Codifications were used with qualitative grouping criteria and as moderating variables in the analysis. The database was subdivided into four groups, which were: males in initial phase (from 1 to 21 days of old); males in growth phase (from 22 to 42 days of old); females in initial phase (from 1 to 21 days of old); and females in growth phase (from 22 to 42 days of old). Graphical analyses were used to explore the database, whereas equations were used to study study the response of dependent variables in each group previously described. These equations were generated through sequential analysis using fixed-effects and random-effects models. Lower variance responses were observed in models fitted using data of male broilers comparing to the female treatments. Using random components allowed the models to account for the variability in the database and to reduce the variance by 67% in average for males and females in initial and growing comparing to the fixed-effect model. Average Daily Gain was maximized at dietary digestible lysine intake of 0,526 and 1,364 grams day-1 for males in the initial and growth phases ... / Mestre

No desvio portossistêmico, o sangue proveniente do trato gastrintestinal não é direcionado ao fígado para ser metabolizado e sim desviado para a circulação sistêmica. O acúmulo dessas substâncias tóxicas associado às alterações funcionais do órgão pode resultar em encefalopatia hepática. O manejo nutricional é o ponto chave para o sucesso da terapia. Dessa forma, o presente estudo objetivou avaliar os efeitos do emprego de um alimento hipoproteico nas concentrações séricas aminoacídicas de cães com desvio portossistêmico. Foram incluídos nove cães com diagnóstico de desvio portossistêmico (Shunt) e nove cães saudáveis (Controle). Os animais foram avaliados no dia um (T0) e após 60 dias (T60) de ingestão de uma dieta hipoproteica comercial. Nos dois momentos foram coletadas amostras de sangue para realização de hemograma, leucograma, bioquímica sérica, determinação dos aminoácidos séricos e amônia. A análise estatística dos resultados referentes aos hemogramas e exames bioquímicos foi baseada em um modelo misto que considerou efeito fixos de grupo, tempo e interação e, para os aminoácidos somente efeitos fixos de grupo (p&lt;0,05). Os animais do grupo controle apresentaram maiores valores de proteína total (p&lt;0,0001), ureia (p&lt;0,0001), creatinina (p=0,0163), albumina (p&lt;0,0001) e colesterol (p=0,0012) quando comparado ao grupo Shunt que apresentou maiores valores de hematócrito (p=0,0213), ALT e FA (p=0,0253 e p=0,0004, respectivamente), amônia em jejum (p=0,0083) e amônia pós-prandial (p=0,0036). Em relação aos aminoácidos séricos, o grupo Shunt apresentou valores maiores quando comparados ao grupo Controle nas concentrações dos aminoácidos fenilalanina (p=0,0054), glutamato (p=0,0066), serina (p=0,0054) e tirosina (p=0,0106), já o aminoácido sérico alanina (p=0,0280), a razão de Fischer (p=0,0093) e a razão da concentração sérica de aminoácidos de cadeia ramificada e tirosina (p=0,0243) foram maiores no grupo Controle. Concluiu-se que o manejo dietético foi eficiente em prevenir a progressão da doença e controlar os sinais clínicos, sem promover aumento nas concentrações séricas dos aminoácidos de cadeia ramificada. / In the portosystemic shunt, the blood coming from the gastrointestinal tract is not directed to the liver to be metabolized but rather diverted to the systemic circulation. The accumulation of these toxic substances associated with functional alterations of the organ can result in hepatic encephalopathy. Nutritional management is the key to successful therapy. Thus, the present study aimed to evaluate the effects of the use of a hypoproteic food on the protein and amino acid profile of dogs with portosystemic shunt. For this, nine dogs with a diagnosis of portosystemic shunt (Shunt) and nine healthy dogs (Control) were included. The dogs were evaluated on day one (T0) and after 60 days (T60) feeding a commercial hypoprotein diet. Blood samples were collected for hemogram, leukogram, serum biochemistry, determination of serum amino acids and ammonia. For the analysis of hemograms and biochemistry, a mixed model was used considering group fixed effects, time and interaction, and for amino acids only group fixed effects (p&lt;0.05). The results showed that the animals in the Control group presented higher values of total protein (p&lt;0.0001), urea (p&lt;0.0001), creatinine (p=0.0163), albumin (p&lt;0.0001) and cholesterol (p=0.0012) when compared to the Shunt group that presented higher values of hematocrit (p=0.0213), ALT and FA (p=0.0253 and p=0.0004, respectively), fasting ammonia (p=0.0083) and postprandial ammonia (p=0.0036). Serum amino acids (p=0.0054), glutamate (p=0.0066), serine (p=0.0054) and tyrosine (p=0.0054) were higher in the Shunt group than in the Control group (0.0106). The Fischer ratio (p=0.0093) and the ratio of serum concentration of branched-chain amino acids and tyrosine (p=0.0243) were higher for the Control group. In conclusion, that dietary management was efficient in preventing disease progression and controlling clinical signs without increasing branched-chain amino acids.

No description available.

Piu Wong. / Thesis submitted in: July 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 126-146). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.viii / General abbreviations --- p.xi / Abbreviations of chemicals --- p.xiii / List of figures --- p.xv / List of Tables --- p.xvii / Table of contents --- p.xviii / Chapter 1 --- Literature review / Chapter 1.1 --- Overviews --- p.1 / Chapter 1.1.1 --- Nutritional and economical significance of aspartate family amino acidsin human and animal nutrition --- p.1 / Chapter 1.1.2 --- Synthesis of aspartate family amino acids in plants --- p.2 / Chapter 1.2 --- Regulation of aspartate family amino acids between sink and source organs --- p.6 / Chapter 1.2.1 --- Co-ordination of genes/enzymes involved in amide amino acid metabolism to channel aspartate for aspartate family amino acid synthesis --- p.6 / Chapter 1.2.2 --- Sink-source regulation as a general mechanism in plants --- p.9 / Chapter 1.3 --- Source regulation at free amino acid level --- p.11 / Chapter 1.3.1 --- Regulation of free methionine synthesis --- p.11 / Chapter 1.3.1.1 --- Competition for OPHS between TS and CGS --- p.11 / Chapter 1.3.1.2 --- Turnover of CGS mRNA --- p.12 / Chapter 1.3.1.3 --- Post-translational regulation of CGS enzyme --- p.13 / Chapter 1.3.2 --- Regulation of lysine synthesis and catabolism --- p.15 / Chapter 1.3.2.1 --- Feedback regulation loop --- p.15 / Chapter 1.3.2.2 --- Possible intracellular compartmentalization of enzymes and metabolitesin regulating lysine level --- p.21 / Chapter 1.3.2.3 --- Co-ordination of gene/enzyme in aspartate kinase pathway in regulating flux to Lys --- p.21 / Chapter 1.3.3 --- Significance of lysine catabolism in mammals and plants --- p.24 / Chapter 1.3.3.1 --- Complex developmental regulation and stress response of LKR/SDH gene expression --- p.28 / Chapter 1.3.3.2 --- Regulation through a novel composite locus LKR-SDH --- p.28 / Chapter 1.3.3.3 --- Post-translational control of LKR-SDH activity --- p.31 / Chapter 1.3.3.4 --- Implication of two metabolic flux in Lys catabolism --- p.34 / Chapter 1.4 --- Source (free lysine) enhancement in transgenic plants --- p.36 / Chapter 1.4.1 --- Expression of feedback insensitive enzyme in transgenic plants to enhance free lysine supply in transgenic plant --- p.36 / Chapter 1.4.2 --- Reducing or eliminating lysine catabolism to enhance free lysine poolin transgenic plants --- p.40 / Chapter 1.5 --- Sink regulation --- p.41 / Chapter 1.5.1 --- Engineering transgenic plants through expression of seed storage protein (sink) --- p.41 / Chapter 1.5.2 --- "Dynamic relationship between sink protein, nitrogen metabolism and sulphur metabolism" --- p.45 / Chapter 1.6 --- Transgenic plants with improved source or enhanced sinks related to aspartate family amino acids available for our research --- p.47 / Chapter 1.6.1 --- Enhanced source: ASN1 over-expressers --- p.47 / Chapter 1.6.2 --- Enhanced source: metL transgenic plants --- p.47 / Chapter 1.6.3 --- Altered source: RNAi line --- p.47 / Chapter 1.6.4 --- Effective sink: LRP transgenic plants --- p.48 / Chapter 1.7 --- Overall concept of this study --- p.48 / Chapter 2 --- Materials and methods --- p.50 / Chapter 2.1 --- Materials and growth conditions --- p.50 / Chapter 2.1.1 --- "Plants, bacterial strains and vectors" --- p.50 / Chapter 2.1.2 --- Chemicals and reagents used --- p.53 / Chapter 2.1.3 --- Solutions used --- p.53 / Chapter 2.1.4 --- Commercial kits used --- p.53 / Chapter 2.1.5 --- Equipment and facilities used --- p.53 / Chapter 2.1.6 --- Growth condition --- p.53 / Chapter 2.1.7 --- Tagging of A. thaliana siliques of different developmental stage --- p.54 / Chapter 2.2 --- Methods --- p.55 / Chapter 2.2.1 --- Expression pattern analysis --- p.55 / Chapter 2.2.1.1 --- RNA extraction --- p.55 / Chapter 2.2.1.2 --- Generation of single-stranded DIG-labelled ASN1 DNA probes --- p.55 / Chapter 2.2.1.3 --- Testing the concentration of DIG-labelled probes --- p.56 / Chapter 2.2.1.4 --- Northern blot --- p.57 / Chapter 2.2.1.5 --- Hybridization --- p.58 / Chapter 2.2.1.6 --- Stringency washes --- p.58 / Chapter 2.2.1.7 --- Chemiluminescent detection --- p.58 / Chapter 2.2.2 --- Amino acid analysis and nitrogen determination --- p.60 / Chapter 2.2.2.1 --- Free amino acids in A. thaliana --- p.60 / Chapter 2.2.2.2 --- Phloem exudates collection from A. thaliana --- p.60 / Chapter 2.2.2.3 --- Soluble Protein quantitation --- p.61 / Chapter 2.2.2.4 --- Extraction of salt and water soluble protein from A. thaliana seeds --- p.61 / Chapter 2.2.2.5 --- Purification and amino acid analysis of protein extracts from A. thaliana seeds --- p.62 / Chapter 2.2.2.6 --- Total amino acid determination in mature dry seeds --- p.63 / Chapter 2.2.3 --- Generation of crossing progenies --- p.64 / Chapter 2.2.3.1 --- Artificial crossing of A. thaliana --- p.64 / Chapter 2.2.3.2 --- CTAB extraction of genomic DNA --- p.64 / Chapter 2.2.3.3 --- PCR screening for successful crossing --- p.65 / Chapter 2.2.4 --- Generation of transgenic plants --- p.67 / Chapter 2.2.4.1 --- Cloning of E.coli dapA gene --- p.67 / Chapter 2.2.4.2 --- Preparation of recombinant plasmid --- p.68 / Chapter 2.2.4.3 --- Gene sequencing --- p.68 / Chapter 2.2.4.4 --- Homology search of differentially expressed genes --- p.69 / Chapter 2.2.4.5 --- Construction of chimeric dapA genes (TP-Phas-dapA) --- p.69 / Chapter 2.2.4.6 --- Transformation of electro-competent Agrobacterium cell --- p.73 / Chapter 2.2.4.7 --- Transformation of A. thaliana through vacuum infiltration --- p.73 / Chapter 2.2.4.8 --- Selection of hemizygous and homozygous transgenic plants --- p.74 / Chapter 2.2.4.9 --- Expression analysis of homozygous LRP/dapA transgenic plants --- p.75 / Chapter 3 --- Results --- p.77 / Chapter 3.1 --- Characterization of ASN1 over-expressers --- p.77 / Chapter 3.1.1 --- Overexpression of the ASN1 gene enhances the sink-source relationship of asparagine transport under regular daylight cycle --- p.88 / Chapter 3.1.2 --- Spatial distribution of total free amino acids under normal daylight cycle --- p.88 / Chapter 3.1.3 --- Over-expression of the ASN1 gene affects free amino acid level quantitatively under normal daylight cycle --- p.89 / Chapter 3.1.4 --- Over-expression of the ASN1 gene affects composition of total amino acid under normal daylight cycle --- p.89 / Chapter 3.2 --- Construction of dapA transgenic Arabidopsis --- p.91 / Chapter 3.2.1 --- Construction of chimeric gene for expression of the dapA gene --- p.91 / Chapter 3.2.2 --- Transformation of p1300/Phas-dapA into Arabidopsis and selection of homozygous progenies --- p.91 / Chapter 3.3 --- Generation of transgenic plants with altered sink-source relationship through crossing and in-planta transformation --- p.96 / Chapter 3.3.1 --- Rationale in methods for generating transgenic plants with different combination of sources and sinks --- p.96 / Chapter 3.3.2 --- Screening for double homozygous progenies through crossing --- p.98 / Chapter 3.3.3 --- Screening for F1 progenies of successful crossing --- p.100 / Chapter 3.3.4 --- Selection of homozygous crossing progenies --- p.102 / Chapter 3.3.5 --- Screening for homozygous dapA/LRP transgenic plants --- p.104 / Chapter 3.4 --- Amino acid composition analysis --- p.109 / Chapter 3.4.1 --- The change of aspartate family amino acids in mature seeds of transgenic plants with altered sources --- p.113 / Chapter 3.4.2 --- The change of aspartate family amino acids in mature seeds of transgenic plants with improved sink --- p.114 / Chapter 3.4.3 --- The change of aspartate family amino acids in mature seeds of transgenic plants with improved sink --- p.115 / Chapter 4. --- Discussion / Chapter 4.1 --- Characterization of ASN1 over-expressers --- p.116 / Chapter 4.1.1 --- Possible regulation of ASN1 mRNA stability through level of asparagine --- p.117 / Chapter 4.1.2 --- Over-expression of ASN1 gene may improve nitrogen remobilisation from source to sink tissues --- p.118 / Chapter 4.1.3 --- Over-expression of ASN1 gene has modified the composition of amino acidsin sink organs --- p.119 / Chapter 4.2 --- ASN1 RNAi transgenic plants increases the relative contents of lysine in the seeds --- p.122 / Chapter 4.2.1 --- Role of ASN1 in supplying or competing aspartate in developing seeds --- p.122 / Chapter 4.2.2 --- Possible role of glutamate receptor --- p.123 / Chapter 4.3 --- Lysine catabolism may strictly control the level of lysine --- p.123 / Chapter 4.3.1 --- Possible role of lysine-tRNA in protein synthesis --- p.124 / Chapter 5. --- Conclusion and prospective --- p.125 / References --- p.126 / Appendix --- p.147

Made available in DSpace on 2014-06-11T19:22:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2006Bitstream added on 2014-06-13T20:48:33Z : No. of bitstreams: 1 garcia_vcl_me_botfm_prot.pdf: 957486 bytes, checksum: f860c8e0d2f9c1906e798f8bcd79542f (MD5) / o paciente com câncer de esôfago tem a desnutrição protéico-energética como principal fator de risco. A taurina é um composto sulfurado que participa de funções fisiológicas importantes, como a manutenção do sistema de defesa do organismo. O objetivo deste trabalho foi estudar o metabolismo da taurina e seus precursores e a associação destes, com os indicadores nuíricionais de pacientes com câncer de esôfago. Para tanto, realizou-se estudo prospectivo com corte vertical e grupo controle, em 18 pacientes (43-73 anos) portadores de neoplasia maligna de esôfago, e 20 voluntários (27-65 anos) controles sadios. Em todos foram realizadas dosagens plasmáticas de taurina, cisteína e homocisteína e avaliação do estado nutricional antropométrico e bioquímico. Paralelamente, foram coletados dados referentes à história e ao diagnóstico clínico e período de sobrevivência dos pacientes. Os dados foram analisados por meio do teste t de Sfudenf e correlação de Pearson. O câncer de esôfago foi mais predominante no sexo masculino e na raça branca. Houve maior freqüência do carcinoma espino celular e localização no terço superior. A maioria dos pacientes. no momento do diagnóstico, apresentou estágio avançado da doença (estadio IV). A perda de peso nos pacientes foi de 14,9%, entretanto, variáveis CMB e %GC não apresentaram diferença estatística com o controle. Adicionalmente, no grupo de estudo, foram observadas hipoalbuminemia e elevação da PCR (55,5% e 50% dos pacientes, respectivamente). Os níveis de Hb, Ht, Colesterol total, HOL e cisteína foram menores, e os de TGO, TGP, taurina e homocisteína maiores significativamente do que o grupo controle (p<0,05). A taurina se correlacionou positivamente com CTL (r=0,49 e p=O,03) e a sobrevida... / The main risk factor of the esophagus' cancer patient is pratein-energetic malnourishment. Taurine is a sulphur-containing amino acid which takes part in important physiological functions such as organíc defense system maintenance. The objective of this work was to study the metabolism of taurine and its precursors and their association with nutritional indicators in patients with esophagus cancer whose main risk factor is protein-energy undemutrition. This was a prospective study with a vertical cut and control group with 18 malignant esophageal neoplasia (4373yrs) and 20 healthy volunteers (27-65yrs). Ali individuais were scrutinized with respect to their plasma levels of taurine, cysteine, and homocysteine and underwent nutritional, anthropometrical, and biomedical state evaluation. Also data were collected on patient history, clínical diagnosis, and survival time. Data were analyzed by Student t and Pearson Correlatíon tests. Esophagus cancer was more predominant in white males. Squamous cel! carcinoma and superior third location were frequent Most patients were in the advanced stage when diagnosed (Stage IV). Patient weight loss was 14.9%, however, AMC and %BF were not statistically different to contrais. Additionally hypoalbuminemia and elevaíed PCR (in 55.5% & 50% of patients, respectively). Hb, Ht, total cholesterol, HDL, and cysteine were significantly lower, and GOT, GPT, taurine, and homocysteine significantly higher than controls (p<0.05). Taurine positively correlated with CTL... (Complete abstract click eletronic address below)