Made available in DSpace on 2014-06-11T19:27:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-25Bitstream added on 2014-06-13T20:36:07Z : No. of bitstreams: 1 domingues_chf_me_jabo.pdf: 336764 bytes, checksum: 96a84ad26e208c18fbcdc65dde25a4fe (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O presente estudo teve por objetivo avaliar o efeito do uso de diferentes níveis de lisina e de metionina + cistina digestíveis durante o período pós - muda, sobre a recuperação corporal, desempenho, qualidade de ovos e morfometria do aparelho reprodutor, fígado e pâncreas de poedeiras comerciais no segundo ciclo de produção. Foram utilizadas 432 poedeiras comerciais da linhagem Isa Brown, com 72 semanas de idade, distribuídas em 54 parcelas, em um delineamento inteiramente casualizado com seis tratamentos e nove repetições de oito aves cada. Durante o descanso foram utilizados seis rações cujos níveis de lisina e metionina + cistina digestíveis variaram: 0,48% de lisina digestível e 0,43% de metionina+cistina digestíveis; 0,48% de lisina digestível e 0,47% de metionina+cistina digestíveis; 0,48% de lisina digestível e 0,52% de metionina+cistina digestíveis; 0,56% de lisina digestível e 0,50% de metionina+cistina digestíveis; 0,56% de lisina digestível e 0,56% de metionina+cistina digestíveis; 0,56% de lisina digestível e 0,62% de metionina+cistina digestíveis.Os dados obtidos foram submetidos à análise de variância e em caso de efeito significativo, a comparação de médias foi realizada a 5% de probabilidade através do teste de Tukey. Os diferentes níveis de lisina e de metionina+cistina digestíveis das dietas de descanso, determinaram efeitos significativos sobre os parâmetros de desempenho das aves. Observou-se que, o nível de 0,56% de lisina e 0,56% de metionina + cistina digestíveis, proporcionou maior peso dos ovos durante o segundo ciclo de produção / This study was conducted to evaluate the effect of using different levels of lysine and methionine + cystine, about the body recovery, performance and egg quality of laying hens in the post molt. It was used four hundred and thirty two hens of Isa Brown strain, with 72 weeks of age, distributed in 54 cages in a completely randomized design with six treatments and nine replicates of eight birds each. During the rest period, were used six diets with different levels of digestible lysine and methionine + cystine. The values ranged from: 0.48% digestible lysine and 0,43% methionine + cystine; 0.48% digestible lysine and 0.47% methionine + cystine; 0.48% digestible lysine and 0.52% methionine + cystine; 0.56% digestible lysine and 0.50% methionine + cystine; 0.56% digestible lysine and 0, 56% methionine + cystine; 0.56% digestible lysine and 0.62% methionine + cystine. The data were subjected to analysis of variance and in case of significant effect, the comparison of means was performed at 5% probability by Tukey test. The different levels of lysine and methionine + cystine diets of rest have determined significant effects on the performance parameters of laying hens. It was observed that the level of 0.56% lysine and 0.56% methionine + cystine, resulted in greater weight of eggs during the second production cycle

Leukemia is the most common cancer of children, moreover it is also not uncommon of elderly patients. Research has focused on the development of specific antileukemic drugs in recent years. Abnormalities in tumor cell metabolism that can be targeted during treatment appear to be the key. Natural 1,4-naphthoquinones, including quambalarin B produced as a secondary metabolite by the basidiomycetes of Quambalaria cyanescens, are known for their therapeutic effects. Not surprisingly, Quambalarine B has also been shown to inhibit cell proliferation in some leukemic cell lines and subsequently caused cell death. In the present thesis, I tried to observe changes in amino acid metabolism by monitoring amino acid levels in the intracellular and extracellular environment of leukemic cells after treatment with Quambalarine B using amino acid analysis with fluorescence detection. The observation was performed in Jurkat, Ramos and THP-1 cell lines, each of these lines represents another type of leukemic disease. [IN CZECH] Key words Amino acid analysis, amino acid metabolism, Quambalarine B, leukemia

System L (SL), a basolateral amino acid transporter, transports large neutral amino acids (LNAAs) in a Na+-independent manner. Previously, we identified two isoforms of transporters: L-type amino acid transporter 1 (LAT1) and 2 (LAT2) and revealed their distinct substrate selectivity and transport properties. In this study, to establish more stable human LAT1 (hLAT1) and LAT2 (hLAT2) in vitro assay systems, we established mouse cell lines stably expressing hLAT1 (S2-LAT1) and hLAT2 (S2-LAT2). Real-time quantitative RT-PCR analysis revealed that S2-LAT1 and S2-LAT2 cells express hLAT1 and hLAT2 mRNAs at 20 - 1000-fold higher levels than those of endogenous mouse Lat1 and Lat2. S2-LAT1 and S2-LAT2 mediated [14C]L-leucine transport properties were measured and corresponded to results observed via Xenopus oocytes. Using these cells, the data demonstrate that hLAT1 and hLAT2 exhibit different characters in the acceptance of α-methyl amino acids and amino acid-related compounds with bulky side chains such as thyroid hormones and melphalan. S2-LAT1 and S2-LAT2 cells are expected to facilitate hLAT1 and hLAT2 substrate recognition research and contribute to drug development by providing an efficient assay system to screen for chemical compounds that interact with hLAT1 and hLAT2.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18331号 / 農博第2056号 / 新制||農||1022(附属図書館) / 学位論文||H26||N4838(農学部図書室) / 31189 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 三上 文三, 教授 小川 順 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Amino acids are necessary for protein synthesis and specialized metabolism in plants. Yet very little is known about how plants sense and regulate when and where to allocate amino acids to meet the demand for nitrogen in growing tissues. In particular, while characterized in yeast and mammals, no amino acid sensor has been identified in plants. Amino Acid Permease 1 (AAP1) has been previously characterized and was shown to mediate amino acid uptake from the soil. aap1 knockout plants and several EMS mutants affected in AAP1 sequence display enhanced tolerance to toxic concentrations of amino acids. Yet, two of the corresponding variant proteins appear to be functional transporters, effectively dissociating amino acid transport and phenotype. To understand this apparent discrepancy, I precisely studied AAP1 localization of expression at the plant and cellular level, and in specific tissue types of the root where AAP1 function is required for the tolerance phenotype and the amino acid uptake activity. I showed that AAP1 protein is present in the endoplasmic reticulum of the cortex in wild type plants Yet, its ectopic expression in root tip and phloem increased amino acid uptake, while expression in cortex could not. This and other of my results do not support the current model of AAP1 functioning in amino acid uptake by the root. I propose that the main effect of mutations in AAP1 is a disturbance in amino acid metabolism, possibly triggered by altered amino acid sensing. In this new model, AAP1 would be necessary for sensing amino acid status of cortex cells, possibly in the endoplasmic reticulum, and adjust amino acid metabolic activity and uptake to current availability. In effect, disruption of the sensing function, either by complete loss of AAP1 function (knockout) or by uncoupling the transport and sensing function (EMS mutants), would lead to the various characteristics of the phenotype of the aap1 mutants I observed. My main hypothesis is that AAP1 is a transporter endowed with sensing function, i.e., an amino acid transceptor. / Doctor of Philosophy / Changing environments create challenges for plants to grow under harsher, nutrient limiting conditions. Nitrogen is an essential nutrient for plant growth, used for the synthesis of amino acids and other nitrogen-containing metabolites. Amino acids are necessary for protein synthesis and other specialized metabolism – being targets for manipulation for improving agronomic traits. Protein content is a complex trait that involves many genes, possibly including amino acid transporters. In addition, the amount of nitrogen needed by and available to the plant increases or decreases depending on the environment conditions. How plants control nitrogen need and use at the molecular level is not well understood. The data presented here challenge a current model and I report how a protein (AAP1) involved in the acquisition of amino acids from the soil provides regulatory control over these processes. . This valuable information is useful for better understanding how plants use nitrogen and more precise breeding methods can be used to improve traits, such as protein content in agronomically important crops.

No / The minerals involved in the formation of metazoan skeletons principally comprise glassy silica, calcium phosphate or carbonate. Because of their ancient heritage, glass sponges (Hexactinellida) may shed light on fundamental questions such as molecular evolution, the unique chemistry and formation of the first skeletal silica-based structures, and the origin of multicellular animals. We have studied anchoring spicules from the metre-long stalk of the glass rope sponge (Hyalonema sieboldi; Porifera, Class Hexactinellida), which are remarkable for their size, durability, flexibility and optical properties. Using slow-alkali etching of biosilica, we isolated the organic fraction, which was revealed to be dominated by a hydroxylated fibrillar collagen that contains an unusual [Gly-3Hyp-4Hyp] motif. We speculate that this motif is predisposed for silica precipitation, and provides a novel template for biosilicification in nature.

DISC1, a strong genetic candidate for psychiatric illness, is a molecular scaffold residing in multiple subcellular compartments, where it regulates the function of interacting proteins with key roles in neurodevelopment and plasticity. Both common and rare DISC1 missense variants are associated with risk of mental illness and/or brain abnormalities in healthy carriers, but the underlying mechanisms are unclear. In this thesis, I initially examine the effect of a panel of common and rare single amino acid substitutions on DISC1 subcellular targeting, establishing that the rare mutation R37W and the common variant L607F disrupt DISC1 nuclear targeting in a dominant-negative fashion. This finding predicts that DISC1 nuclear expression is severely impaired in 37W and 607F carriers. In addition, I show that the L607F substitution results in aberrant cytoplasmic and cytoskeletal distribution of DISC1. In the nucleus, DISC1 interacts with the transcription factor ATF4, which is involved in the regulation of cellular stress responses and memory consolidation. Here I show that at basal cAMP levels, wild-type DISC1 strongly inhibits the transcriptional activity of ATF4, and this effect is ablated by 37W and 607F, most likely as a consequence of their defective nuclear targeting. 607F additionally reduces DISC1/ATF4 interaction, which likely contributes to its weakened inhibitory effect. I also demonstrate that DISC1 modulates transcriptional responses to endoplasmic reticulum stress, and that this modulatory effect is also ablated by 37W and 607F. By providing evidence that single amino acid substitutions of DISC1 associated with psychiatric illness impair its regulatory function on ATF4-dependent transcription, I highlight a potential mechanism by which these protein variants may impact on molecular pathways underlying cognition and stress responses, two processes of direct relevance to psychiatric disease.

The Solute Carrier (SLC) superfamily is the largest group of membrane-bound transporters, currently with 456 transporters in 52 families. Much remains unknown about the tissue distribution and function of many of these transporters. The aim of this thesis was to characterize select SLCs with emphasis on tissue distribution, cellular localization, and function.       In paper I, we studied the leucine transporter B0AT2 (Slc6a15). Localization of B0AT2 and Slc6a15 in mouse brain was determined using in situ hybridization (ISH) and immunohistochemistry (IHC), localizing it to neurons, epithelial cells, and astrocytes. Furthermore, we observed a lower reduction of food intake in Slc6a15 knockout mice (KO) upon intraperitoneal injections with leucine, suggesting B0AT2 is involved in mediating the anorexigenic effects of leucine.     In paper II, we studied the postnatal, forebrain-specific deletion of Slcz1, belonging to the SLC18 family, in conditional KO mice (cKO). We observed a decreased response to diazepam and a higher neuronal activity in cortex and hippocampus of cKO mice, as well as an impairment in short-term recognition memory. Intracellular expression was found in neurons but not astrocytes with IHC, indicating SLCZ1 is implicated in neuronal regulation of locomotion and memory.    In paper III, we performed the first detailed histological analysis of PAT4, a transporter belonging to the SLC36 family, involved in the activation of mTOR complex 1 on lysosomes. We found abundant Slc36a4 mRNA and PAT4 expression in mouse brain, using ISH and IHC. We used IHC to localize PAT4 to both inhibitory and excitatory neurons and epithelial cells. We also found both intracellular- and plasmalemmal expression and partial colocalization of PAT4 with lysosomal markers.    Lastly, in paper IV, we provided the first tissue mapping of orphan transporter MCT14 (SLC16A14). Using qPCR, we detected moderate to high Slc16a14 mRNA in the central nervous system and kidney. We found widespread Slc16a14 and MCT14 in mouse brain using ISH and IHC. We also found MCT14 to have intracellular and plasmalemmal expression in mainly excitatory but also inhibitory neurons, as well as epithelial cells. We found MCT14 to be most closely related to MCT8, MCT2 and MCT9, suggesting a similar role for this transporter.

The amino acid requirements of growing horses are currently unknown, and studies suggest that threonine is a limiting amino acid in common horse diets. Thus, the objective of this study was to determine the threonine requirement of growing horses fed two different forage to concentrate ratios using the indicator amino acid oxidation method. The study consisted of a high concentrate phase (HC; 60% concentrate and 40% forage) and a high forage phase (HF; 25% concentrate and 75% forage). Within each phase, 6 female yearling Thoroughbred horses were randomly assigned each of 6 dietary treatments in a 6 x 6 Latin square design. All 6 treatments were identical, apart from varying equimolar ratios of threonine to glutamate. After 6 days of adaptation, blood samples were collected before and after the morning meal for plasma urea nitrogen (PUN) and amino acid analysis. On day 7, horses underwent the IAAO protocol, during which regular breath and blood samples were collected. Phenylalanine flux, oxidation, non-oxidative disposal, and release from body protein, as well as total carbon dioxide production were calculated using plateau enrichment of samples. There was a significant linear effect of threonine intake on plasma threonine concentrations, and PUN had a significant linear response during the HC phase. There was no significant effect of treatment on phenylalanine oxidation during either phase (P ≥ 0.05). It is unlikely that threonine was limiting in the experimental diets.

Microvitellogenin is a female specific yolk protein from the tobacco hornworm moth Manduca sexta. A cDNA library was constructed from poly (A)⁺ RNA isolated from adult female fat body. cDNA clones of mRNA for microvitellogenin were isolated by screening the cDNA library with antiserum against microvitellogenin. The results of Northern blot analysis and hybrid selection indicated that the cDNA clone was specific for microvitellogenin. The complete nucleotide sequence of the 834 base pair cDNA insert has been determined by the dideoxy chain termination method. The deduced amino acid sequence was compared with the N-terminal sequence determined by Edman degradation, an amino terminal extension of 17 amino acids appeared to be a signal peptide. The cDNA sequence predicts that the mature microvitellogenin is a protein of 232 amino acids with a calculated molecular weight of 26,201. A comparison of the translated amino acid sequence with the sequences in National Biomedical Research Foundation protein library did not establish any sequence similarity with known proteins. The microvitellogenin gene begins to be expressed in the fat body on the first day of the wandering (prepupal) females as determined by using the cDNA insert as a probe to hybridize with the mRNA for microvitellogenin. The cDNA probe was also used to screen a genomic library of M. sexta, yielding three genomic clones for microvitellogenin. One of them was characterized and it contained the complete microvitellogenin gene. The gene sequence was determined. Comparison to the cDNA sequence showed that the microvitellogenin gene contains an intron near the 5'-end of the non-coding region. The 5'-flanking sequence of the gene has been compared to the same regions of yp genes of Drosophila and vitellogenin genes of locust, some similar sequences have been observed and discussed.