Orientador: Maria Cristina Cintra Gomes Marcondes / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T04:03:57Z (GMT). No. of bitstreams: 1 Inocencio_AlineTatianeToneto_M.pdf: 4201268 bytes, checksum: d04253942aceb6648e341e3f059a1864 (MD5) Previous issue date: 2012 / Resumo: O câncer-caquexia é caracterizado pelo desenvolvimento de anorexia, astenia, perda de peso, saciedade prematura, anemia e principalmente alteração no metabolismo de carboidratos, gorduras e proteínas dos pacientes com neoplasia. O aminoácido leucina atua promovendo a sinalização celular, inibindo o processo de catabolismo protéico e estimulando o processo de síntese protéica no músculo esquelético, principalmente em animais experimentais. O exercício físico proporciona alterações metabólicas voltadas, principalmente, à síntese protéica. Desse modo, estudamos os efeitos do treinamento físico associado à dieta rica em leucina sobre o processo de adaptação metabólica e hormonal durante a evolução do tumor de Walker em ratos. Ratos Wistar foram submetidos ao treinamento físico (natação por 45 minutos diários, 5 dias por semana) durante 6 semanas e após esse período receberam implante de células do carcinossarcoma de Walker 256, no subcutâneo. Após 21 dias de crescimento tumoral, os ratos foram sacrificados para obtenção de soro para determinação de glicocorticoides e catecolaminas e ressecção de tecidos e órgãos (adrenais, fígado, coração e músculo). Avaliação da cultura primária da glândula adrenal desses animais pôde-se observar a síntese de catecolaminas e liberação de citocinas, no meio de cultura, em função do crescimento tumoral modulados pelos efeitos da dieta rica em leucina associada ao exercício físico. O crescimento tumoral promoveu alterações metabólicas, bioquímicas e hormonais no hospedeiro do câncer como perda de peso e aumento de hormônios catabólicos como ACTH e glucagon. O exercício promoveu diminuição de IL-6 e INF-? no meio de cultura e aumento sérico de IL-4 e IL-10. O exercício e a suplementação com leucina proporcionaram a manutenção das concentrações plasmáticas de proteínas totais séricas e albumina, provavelmente em função da leucina, que é aminoácido conhecido como sinalizador celular e estimulador do metabolismo protéico. Nos animais tumor leucina exercitado (WLE), observamos manutenção de proteinas totais e albumina, manutenção dos níveis de ACTH e glucagon, além de diminuição nos níveis séricos de catecolaminas, em relação ao grupo LE. As análises bioquímicas, hormonais e de cultura de células mostraram que o exercício e a suplementação de leucina proporcionaram à melhora do estado caquético desses animais / Abstract: The cancer-cachexia is characterized by the development of anorexia, asthenia, weight loss, early satiety, anaemia and especially changes in the metabolism of carbohydrate, fat and protein in these cancer patients. The amino acid leucine acts promoting cell signalling by inhibiting the protein catabolism and stimulating the protein synthesis in skeletal muscle, especially in experimental animals. The exercise provides metabolic changes mainly in protein turnover. Thus studies that concern cancer-cachexia, physical exercise and nutritional supplementation can come up with best ways to support cancer treatment. In this work we evaluated the effects of physical training associated with leucine-rich diet on the metabolic processes and hormonal changes during Walker tumour growth in rats. Wistar rats (45 days-old) were submitted to physical training (swim section for 45 minutes daily, five days a week for 6 weeks). After 6 weeks of training, the rats received subcutaneous implant of Walker carcinoma cells. After 21 days of tumour growth, animals were sacrificed and collected blood, tissues and organs (adrenal, liver, heart and muscle). Cell primary culture of adrenal gland analysed the catecholamine synthesis and cytokines release in the culture medium, which was altered under tumour growth effects and modulated by the effects of leucine-rich diet associated with exercise physical. Tumour growth promoted metabolic, biochemical and hormonal changes in host such as weight loss and increased catabolic hormones release (ACTH and glucagon). The exercise decreased IL-6 and IFN-? content in culture medium and increased IL-4 and IL-10 serum content. Exercise and leucine supplementation provided maintenance of total serum protein and albumin, probably due to leucine which is known as cell signalling and stimulates protein metabolism. In addition, tumour-bearing-exercised animals (WLE) maintained serum protein and albumin, and also the ACTH and glucagon levels, and reduced serum catecholamine levels, in relation to LE group. The biochemical analyses, hormonal and cell culture have shown that exercise and leucine supplementation could improve the cachectic state in experimental animals / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Urofacial syndrome (UFS; MIM# 236730) is a rare autosomal recessive condition characterised by urinary bladder and bowel voiding dysfunction with a pathognomonic abnormality of facial movement with expression. UFS can be caused by biallelic putative loss-of-function mutations in HPSE2, which encodes heparanase 2. Failure to discover HPSE2 mutations in all cases of UFS suggests genetic heterogeneity. The urinary tract features of UFS overlap those seen in the spectrum of non-syndromic non-neurogenic voiding dysfunction and vesicoureteric reflux (VUR). This overlap suggests there may be some aspects of pathogenesis in common. The project aimed to define the genotypic and phenotypic spectrum associated with mutations in HPSE2 by Sanger sequencing and multiplex ligation-dependent probe amplification (MPLA) in newly referred cases of UFS and making comparison to a review of mutations and phenotypes seen in the literature. This work discovered five further families with HPSE2 associated UFS increasing known mutations whilst, reinforced that this is an under-recognised condition and emphasised the previously under-reported feature of facial weakness. The failure to discover HPSE2 mutations in all cases referred provided further evidence of genetic heterogeneity. The project also aimed to discover further genes associated with UFS. Autozygosity mapping and whole exome sequencing was carried out in cases of UFS without mutations in HPSE2. This led to the recognition that UFS is also caused by biallelic putative loss-of-function mutations in LRIG2 encoding the leucine-rich repeats and immunoglobulin-like domains 2 (LRIG2) protein in three families. Failure to identify LRIG2 mutations in all HPSE2 negative families suggests further genetic heterogeneity. To address the question of whether the pathogenesis of UFS overlaps more common conditions with a similar spectrum of urinary tract abnormalities I aimed to examine whether pathogenic variants in HPSE2 and LRIG2 were seen in these phenotypes. Unexpectedly this led to the discovering of further families affected by UFS but failed to show an association of variants in UFS genes with non-syndromic urinary tract abnormalities. However, variants of potential interest were discovered. As part of work toward understanding the pathogenesis of UFS and designing a model to test the pathogenesis of sequence variants expression studies in a Xenopus tropicalis hpse2 knock-down model of UFS were carried out. The knock-down model provided valuable insight in to the likely pathogenesis of UFS with evidence pointing towards a congenital peripheral neuropathy with failure of correct nerve path finding. Understanding the pathogenesis of UFS has the potential to direct further research in to therapeutic intervention.

Orientador: Maria Cristina Cintra Gomes Marcondes / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T06:12:33Z (GMT). No. of bitstreams: 1 Miyaguti_NataliaAngelodaSilva_M.pdf: 7149513 bytes, checksum: e738b40c2027a8507e7b5ae2e65321e8 (MD5) Previous issue date: 2015 / Resumo: O Resumo poderá ser visualizado no texto completo da tese digital / Abstract: The Abstract is available with the full electronic digital document / Mestrado / Fisiologia / Mestra em Biologia Funcional e Molecular

Estudos demonstram que a indução do estresse oxidativo pelo peptídeo beta amiloide (A?) exerce um importante papel no desencadeamento da excitotoxicidade neuronal o que pode resultar no desenvolvimento de doenças neurodegenerativas. A formação do peptídeo A? se deve a alterações na proteína precursora de amiloide (APP) que é clivada para a formação do peptídeo A?. Por sua vez, os mecanismos de ação do A? no S.N.C. ocorrem através da sinalização do receptor NMDA (N-metil D-aspartato) receptor este que quando ativado pelo glutamato exerce importante papel fisiológico no S.N.C., visto que apresenta atividade ionotrópica que permite o influxo de Na+ e Ca2+ para as células neuronais, auxiliando nos processos de formação da memória e aprendizagem. Entretanto, apesar do seu papel fisiológico, a ativação excessiva do receptor NMDA é fortemente correlacionada com lesões no S.N.C. decorrente da excessiva permeabilidade do íon Ca2+ para o citosol das células neuronais. Com isso as concentrações de glutamato na fenda sináptica são estritamente controladas para que não haja ativação excessiva dos receptores com atividade glutamatérgica, como o receptor NMDA. Estudos indicam que o transporte de glutamina/glutamato através da barreira hematoencefálica é menor do que de outros aminoácidos, sendo que cerca de 25% a 30% do transporte de aminoácidos dos vasos sanguíneos para o cérebro através da barreira hematoencefálica é ocupado pelo aminoácido leucina, sendo este um grande responsável pela síntese de glutamato/glutamina no S.N.C. Com isso, estudos tem demonstrado que dietas enriquecidas com aminoácidos de cadeia ramificada, dentre eles a leucina, é responsável por alterar o metabolismo do glutamato e aumentar a susceptibilidade à excitotoxicidade de células neurais. A fim de testar esta hipótese utilizamos um modelo de cultura de células de neuroblastoma humano e realizamos o tratamento com diferentes concentrações de aminoácido leucina associado com o tratamento de peptídeo beta-amilóide. Realizamos as analises de citotoxicidade (LDH), viabilidade celular (MTT) e apoptose celular por citometria de fluxo (marcação com PE Anexina V e 7-AAD). Nossos resultados indicam que houve diferenças apenas entre o controle em relação aos demais grupos de tratamento / Studies demonstrate that induction of oxidative stress by beta amyloid peptide (A?) plays an important role in triggering neuronal excitotoxicity which can result in the development of neurodegenerative diseases. The formation of A? peptide are due to changes in the amyloid precursor protein (APP) which is cleaved to form the peptide A?. On the other hand, the mechanisms of action of A? in the C.N.S. occur through signaling of the NMDA (N-methyl-D-aspartate) receptor that when activated by glutamate plays an important physiological role in the C.N.S., as has inotropic activity that allows the influx of Na+ and Ca2+ into the neuronal cells, assisting in procedures of memory formation and learning. However, despite its physiological role, the excessive activation of the NMDA receptor is strongly correlated with C.N.S. lesions due to excess permeability of Ca2+ ions into the cytosol of neuronal cells. Thus the concentrations of glutamate in the synaptic cleft are strictly controlled so that there is excessive activation of receptors with glutamatergic activity, as the NMDA receptor. Studies indicate that the transport of glutamine/glutamate across the blood brain barrier is lower than that of other amino acids, of which about 25% to 30% of the amino acid transport blood vessels to the brain through the blood brain barrier is occupied by leucine this being one largely responsible for the synthesis of glutamate/glutamine in the C.N.S. Thus, studies have shown that diets enriched in branched chain amino acids, including leucine, are responsible for altering the metabolism of glutamate and excitotoxic increase susceptibility to neural cells. To test this hypothesis we used a cell culture model of human neuroblastoma and carry out the treatment with different concentrations of leucine associated with the processing of amyloid-beta peptide. We performed analysis of cytotoxicity (LDH), cell viability (MTT assay) and apoptosis using flow cytometry (Annexin V staining with PE and 7-AAD). Our results indicate that there were differences only between the control compared to the other treatment groups

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22636号 / 医博第4619号 / 新制||医||1044(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 古川 壽亮, 教授 村井 俊哉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Unlike most other host-specific mycoplasmas, Mycoplasma gallinarum exists as a commensal with a host range including most poultry as well as some mammals. This property of M. gallinarum may reflect unique mechanisms for its colonization and persistence in hosts. Whereas M. gallinarum shows leucine and arginine aminopeptidase activity, the genes encoding the enzymes had not been cloned and characterized. We identified an aminopeptidase gene (APN) by oligonucleotide hybridization to a genomic library of M. gallinarum in lambda ZAPII bacteriophage. Nucleotide sequence analysis of overlapping phage clones identified a 1,362 bp open reading frame (ORF) encoding a putative leucine aminopeptidase gene. Database searches indicate that this ORF has 68% nucleotide identity and 51% amino acid identity with the M. salivarium leucine aminopeptidase gene. The active sites of the leucine aminopeptidases in other eukaryotes and prokaryotes were conserved in the cloned aminopeptidase gene. Northern-blot hybridization analysis showed that this ORF is expressed as a 1.5 kb transcript. Southern-blot hybridization analysis demonstrated this gene was present as a single copy in M. gallinarum. Characterization of the leucine aminopeptidase demonstrated that it is a metallo-aminopeptidase (EC 3.4.11.1) and is located in the cytoplasm with a weak interaction with the cell membrane. The subcellular location was further confirmed by immunoblotting with polyclonal anti-recombinant APN serum and M. gallinarum Triton-114 partitions. Immunoblotting results with sera from three chickens experimentally infected with M. gallinarum showed that there were very few proteins in M. gallinarum exposed to the host immune responses and that leucine aminopeptidase was not able to stimulate production of specific humoral antibody. Our results suggest that this leucine aminopeptidase play a role in nutrition supply for the host adaptation of M. gallinarum and that the enzyme was not strongly immunogenic.

No description available.

Soybean is a dominant oilseed cultivated worldwide for its use in multiple sectors such as food and feed industries, animal husbandry, cosmetics and pharmaceutical sectors, and more recently, in production of biodiesel. Increasing demand of soybean, changing environmental conditions, and evolution of pathogens pose challenges to soybean production in limited acreage. Genetic research is the key to ensure the continued growth in soybean production, with enhanced yield and quality, while reducing the losses due to diseases and pests. This research is focused on the understanding of transcriptional regulation of two economically important agronomic traits of soybean: low seed phytic acid and resistance to Soybean mosaic virus (SMV), using the 'transcriptomics' and 'genomics' approaches. The low phytic acid (lpa) soybean is more desirable than conventional soybean, as phytic acid is an anti-nutritional component of seed and is associated with phosphorus pollution. Despite the eco-friendly nature of the lpa soybean, it shows poor emergence, which reduces soybean yield. This research is mainly focused on addressing the impact of lpa-causing mutations on seed development, which is suspected to cause low emergence in lpa soybeans. The differences in transcriptome profiles of developing seeds in lpa and normal phytic acid soybean are revealed and the biological pathways that may potentially be involved in regulation of seed development are suggested. The second research project is focused on Rsv3-type resistance, which is effective against most virulent strains of Soybean mosaic virus. The Rsv3 locus, which maps on to soybean chromosome 14, contains 10 genes including a cluster of coiled coil-nucleotide binding-leucine rich repeat (CC-NB-LRR) protein-encoding genes. This dissertation employed a comparative sequencing approach to narrow down the list of Rsv3 gene candidates to the most promising CC-NB-LRR gene. The evidence provided in this study clearly indicates a single CC-NB-LRR gene as the most promising candidate to deliver Rsv3-type resistance. / Ph. D.

Soybean mosaic virus (SMV) is the most common member of the viral genus Potyvirus to infect soybeans (Glycine max [L.] Merr.) worldwide. SMV has been traditionally controlled by the deployment of single dominant, strain specific resistance genes, referred to as Rsv genes. Rsv1 is the most widely used form of SMV resistance with nine different alleles conferring resistance only to the lower numbered less virulent strains, G1 to G3. Rsv3 gives resistance to higher numbered more virulent strains G5 to G7. Soybean lines containing Rsv4, are resistant to all seven currently recognized North American SMV strains. In this study, the recently released soybean whole genome sequence was used to design molecular markers for fine mapping Rsv3 to a ~150 kb genomic region containing four coiled-coil nucleotide-binding leucine-rich repeat proteins. In a related study a large population segregating at the Rsv3 locus was screened for resistance to facilitate future characterization of this region. The markers identified in this study will allow for more accurate marker-assisted selection of Rsv3. / Master of Science