The obesity epidemic is associated with an increase in the prevalence of a number of chronic diseases including type 2 diabetes, cardiovascular disease, hypertension and some cancers and has been described by the World Health Organisation as one of the greatest public health challenges of the 21st century. Obesity is characterised by excessive expansion of adipose tissue mass underpinned by adipocyte hyperplasia. Central to this is the process of adipogenesis, which encompasses the proliferation and terminal differentiation of fibroblastic preadipocytes, contained within adipose tissue, to mature adipocytes. Despite the pivotal role of this process in obesity our understanding of the regulatory mechanisms governing adipocyte development, either in physiological or pathophysiological settings, is limited. Studies aimed at understanding this complex process are integral to development of more effective strategies for the prevention and/or treatment of obesity and obesity related diseases. Our laboratory recently identified a putative role for inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, in the dynamic regulation of lipid accumulation. Upon treatment of a variety of cell types with insulin or oleic acid IMPDH translocates to lipid droplets and inhibition of this translocation is correlated with reduced lipid accumulation. As lipid droplet formation and lipid accretion are defining features of adipogenesis, it was hypothesised that IMPDH may facilitate efficient lipid accumulation during adipose conversion of preadipocytes. In vitro systems have been used extensively to dissect the molecular and cellular events involved in adipogenesis. Therefore the aim of this project was to extend these investigations to examine the requirement for IMPDH activity during adipogenesis, using the well characterised murine 3T3-L1 cell line and primary human preadipocytes (phPAs). IMPDH expression and activity were transiently increased during differentiation of the 3T3-L1 cells although IMPDH did not associate with lipid droplets under these conditions. Pharmacological inhibition of IMPDH, using mycophenolic acid (MPA; 1 µM), reduced intracellular GTP by 60%, and blocked mitotic clonal expansion (MCE) and adipogenesis. Supplementation with guanosine (60 µM), a substrate in the nucleotide salvage pathway, restored both GTP levels and adipogenesis. These observations indicated that IMPDH activity is required for efficient differentiation of 3T3-L1 preadipocytes. Preliminary studies, involving differentiation of phPAs in standard serum-free medium (SFM) suggested that phPAs were resistant to MPA. To afford better comparison between the phPAs and the 3T3-L1 cells, which are differentiated in serum-containing medium (SCM), a modified 3T3-L1 like protocol facilitating efficient differentiation of the phPAs in SCM was established. Under these conditions phPAs displayed considerable variation in sensitivity to MPA which gave a trend towards decreased differentiation (reduced by 26%; p=0.07). Supplementation with guanosine significantly reduced adipogenesis (by 37%; p<0.05) in the phPAs independent of MPA. Furthermore, cells that were MPA resistant were also refractory to guanosine suggesting greater plasticity of guanine metabolism in phPAs from those subjects. A major difference between the cell types was that phPAs differentiated with high efficiency in the absence of MCE. Collectively, these data indicate that MCE is required for efficient differentiation of 3T3-L1 cells but not phPAs, even when differentiated under similar conditions, and suggest that the involvement of MCE underpins the differences in sensitivity to MPA between cell types. The differential effects of guanosine suggest there are additional differences with respect to the effects of manipulation of guanine nucleotides between cell types. In summary, the work presented in this thesis demonstrated inhibition of IMPDH blocked adipogenesis of murine 3T3-L1 cells and reduced differentiation of phPAs in some subjects. These observations provided novel insights into differences between differentiation of 3T3-L1 cells and phPAs, including their relative sensitivities to alterations in guanine nucleotides, and have implications for adipose tissue biology especially those factors involved in guanine metabolism. Ultimately this knowledge may form the basis for development of novel therapeutic strategies aimed at reduction of obesity and associated complications such as insulin resistance and type 2 diabetes.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 4 uppsatser.

Thesis (M.S.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on April 21, 2009) Includes bibliographical references.

Thesis (Ph.D.) -- University of Rhode Island, 2008. / Typescript. Includes bibliographical references (leaves 107-114).

Most eukaryotic mRNAs have a sequence of polyadenylic acid [poly(A)] at their 3'-termini. Although it has been almost two decades since the discovery of these poly(A) tracts, their function(s) have yet to be clarified. Earlier results from our laboratory led us to propose that poly(A) has a role in translation. More specifically, we proposed that an interaction of the cytoplasmic poly(A)-binding protein (PABP) with a critical minimum length of poly(A) facilitates the initiation of translation of poly(A)+, but not poly(A)-, mRNAs. The results of several different experimental approaches have provided evidence which indirectly supports this hypothesis. These results include: 1) the correlation of specific changes in mRNA poly(A) tail length with translational efficiency in vivo and in vitro; 2) correlations between the abundance and stability of PABPs and the rate of translational initiation in vivo and in vitro; and 3) the demonstration that exogenous poly(A) is a potent and specific inhibitor of the in vitro translation of poly(A)+, but not poly(A)-mRNAs. To evaluate the hypothesis that the 3'-poly(A) tract of mRNA plays a role in translational initiation, we have constructed derivatives of pSP65 which direct the in vitro synthesis of mRNAs with different poly(A) tail lengths and compared, in reticulocyte extracts, the relative efficiencies with which such mRNAs are translated, degraded, recruited into polysomes, and assembled into mRNPs or intermediates in the translational initiation pathway. Relative to mRNAs which are polyadenylated, we find that poly(A)- mRNAs have a reduced translational capacity which is not due to an increase in their decay rates, but is attributable to a reduction in their efficiency of recruitment into polysomes. The defect in poly(A)- mRNAs affects a late step in translational initiation, is distinct from the phenotype associated with cap-deficient mRNAs, and results in a reduced ability to form 80S initiation complexes. Moreover, poly(A) added in trans inhibits translation from capped poly(A)+ mRNAs, but stimulates translation from capped poly(A)- mRNAs. We suggest that poly(A) is the formal equivalent of a transcriptional enhancer, i.e., that poly(A)-binding protein (PABP) bound at the 3'-end of mRNA may facilitate the binding of an initiation factor or ribosomal subunit at the mRNA 5'-end.

Made available in DSpace on 2015-05-14T16:53:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-11Bitstream added on 2015-05-14T16:59:17Z : No. of bitstreams: 1 000817853.pdf: 598063 bytes, checksum: acf178d90222a37997b7fb5f7129bec3 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os objetivos desta pesquisa foram avaliar os efeitos da utilização de nucleotídeos (NU) e ácido glutâmico (AG) na dieta de leitões sobre o desempenho e perfil de lipídeos plasmáticos (colesterol total - CT, triglicérides - TRI, HDL colesterol e LDL+VLDL colesterol) (experimento I) e turnover do 13C na mucosa do duodeno e jejuno (meia-vida do 13C) (experimento II). Em ambos os experimentos foi utilizado delineamento experimental de blocos ao acaso, com esquema fatorial 2x2 dos tratamentos, sendo dois níveis de NU (0 e 0,1%) e dois níveis de AG (0 e 1%), com sete repetições e três animais por parcela. O primeiro experimento teve duração de 35 dias e foram utilizados 84 leitões desmamados aos 21 dias de idade e com peso médio inicial de 6,54±0,70kg. No segundo experimento, nos dias 3, 6, 9, 14, 21, 34 e 49 após o desmame, foram abatidos três leitões por tratamento e no dia do desmame (dia zero) foram abatidos três leitões, totalizando 87 animais com peso inicial de 6,28±0,13kg. Não houve interação dos fatores nas variáveis analisadas e nem efeitos de NU e do AG no consumo diário de ração, ganho diário de peso, CT, HDL colesterol, TRI, LDL + VLDL colesterol e no turnover do 13C na mucosa intestinal. O uso de NU na dieta piorou (P≤0,05) a conversão alimentar no período de 0 a 27 dias, mas esta variável não foi influenciada no período total de experimento (0 a 35 dias). Ocorreu redução (P<0,0001) na concentração plasmática de todas as variáveis estudadas aos 56 dias em relação aos 21 dias de idade e na mucosa do jejuno a meia-vida do 13C foi maior nos leitões mais leves (P≤0,05). Conclui-se, portanto, que a adição de NU e AG nas dietas não interferem no desempenho, no perfil lipídico plasmático e no turnover do 13C na mucosa do duodeno e jejuno de leitões recém-desmamados no período total de creche / The objectives of this research were to evaluate the effects of nucleotides (NU) and glutamic acid (AG) in the diet of pigs on performance and plasma lipid profile (total cholesterol - CT, triglycerides - TRI, HDL cholesterol and LDL + VLDL cholesterol ) (experiment I) and turnover of 13C in duodenal and jejunum mucosa (13C half-life) (experiment II). In both experiments we used experimental design of randomized blocks, with 2x2 factorial treatments, with two levels of NU (0 and 0.1%) and two levels of AG (0 and 1%), each treatament had seven replicated and three pigs per pen. The first experiment lasted 35 days and were used 84 piglets weaned at 21 days of age and average initial weight of 6.54 ± 0,70kg. In the second experiment, on days 3, 6, 9, 14, 21, 34 and 49 after weaning, were slaughtered three pigs per treatment and the day of weaning (day zero) were slaughtered three piglets, totaling 87 animals with initial weight 6.28 ± 0,13kg. There was no interaction of factors in the analyzed variables and not effects of NU and AG in daily feed intake, daily weight gain, CT, HDL cholesterol, TRI, LDL + VLDL cholesterol and 13C turnover in the intestinal mucosa. The use of NU in the diet has worsened (p≤0.05) feed conversion in the period 0-27 days, but this variable was not affected in the total period (0-35 days). We observed a decrease (P<0.0001) in plasma concentration of all variables after 56 days compared to 21 days of age and in the jejunal mucosa the 13C half-life was higher in lighter piglets (p≤0.05). It can be concluded therefore that the addition of AG and NU in the diets did not affect performance, the plasma lipid profile and 13C turnover in the duodenum and jejunum mucosa of weaned piglets in total nursery period

Made available in DSpace on 2014-06-11T19:27:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-06-02Bitstream added on 2014-06-13T19:35:41Z : No. of bitstreams: 1 zavarize_kc_me_botfmvz.pdf: 548332 bytes, checksum: 367c8c738a0d44b4cd3cc7d0af8fbc10 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / Glutamina e nucleotídeos são importantes para estimular o crescimento e diferenciação das células intestinais e imunes, controlando a instalação de agentes antimicrobianos e prevenindo quedas no desempenho. A pesquisa teve por objetivo verificar o efeito do nível de inclusão de glutamina e nucleotídeos na dieta sobre o desempenho, rendimento de carcaça e partes, peso de órgãos e o desenvolvimento da mucosa intestinal de frangos de corte criados no sistema alternativo. Foram utilizados 600 pintos de corte machos distribuídos em um delineamento em blocos casualizados, no esquema fatorial 3x2 (três níveis de glutamina na dieta: 0,0; 0,5 e 1,0% e dois níveis de nucleotídeos purificados na dieta: 0,0 e 0,04%), totalizando seis tratamentos, com quatro repetições de 25 aves cada. Foram obtidos os dados de desempenho (peso corporal, ganho de peso, consumo de ração, conversão alimentar e mortalidade). Ao final do experimento foram abatidas 20 aves/tratamento para determinação do rendimento de carcaça e partes e 4 aves/tratamento para avaliação do peso de órgãos e morfologia intestinal. A suplementação da dieta com glutamina e nucleotídeos não afetou as características de desempenho, rendimento de carcaça e partes, peso de órgãos e o desenvolvimento da mucosa intestinal de frangos de corte criados no sistema alternativo. Porém, na fase pré-inicial, a suplementação da dieta com 1,0% glutamina favoreceu o desempenho destas aves, melhorando o ganho de peso. Termos para indexação: criação alternativa, desempenho... / Glutamine and nucleotides play roles in stimulating growth and differentiating intestinal and immune cells, thereby controlling antimicrobial resistance and preventing growth impairment. The experiment was conducted to evaluate the effect of supplementing glutamine and nucleotides on growth performance, carcass yield, parts, abdominal fat, organs weight and intestinal morphological in broiler chicks. Six-hundred 1-day old chicks were randomly assigned to one of six treatments. The diets were created on a 3x2 factorial, for a total of 6 treatments, consisting of a uniform basal diet supplemented with: 0.0, 0.5 or 1.0% glutamine, and 0.0 or 0.04% nucleotides. The collected data were submitted to analysis of variance and comparison between pairs of means through the Tukey test. The effect of these treatments, when significant, was displayed. It was demonstrated that feeding 1.0% glutamine improved growth performance of broiler birds at seven days of age. However, glutamine and nucleotides supplementation did not affect growth performance in broiler chicks in the period one to 21 and one to 42 days old... (Complete abstract click electronic access below)

O objetivo deste trabalho foi avaliar a utilização da levedura hidrolisada como fonte de nucleotídeos para frangos de corte. As aves foram suplementadas com diferentes níveis de inclusão nas dietas iniciais de 1 a 14 dias de idade. Foram utilizados um total de 576 pintos da linhagem Ross 708, os quais foram distribuídos em 6 tratamentos com 8 repetições (12 aves por box). Os tratamentos consistiram da inclusão de 0, 2, 4, 6, 8 e 10 kg de levedura hidrolisada/tonelada de ração. A levedura hidrolisada foi adicionada na dieta no lugar do material inerte da ração. A dieta inicial foi fornecida na forma triturada enquanto as dietas de crescimento, final e de retirada foram fornecidas na forma de pellets. Durante todo o experimento o acesso à água e ração foi ad libitum. Todas as dietas foram feitas à base de milho, farelo de soja e gordura de frango e foram formuladas para atender as exigências nutricionais recomendadas pelo manual de recomendações nutricionais Ross 708. Aos 42 dias, as aves alimentadas com 1% de levedura hidrolisada tiveram maior peso corporal e ganho de peso quando comparadas aos demais tratamentos (P<0,05). Não houveram diferenças estatísticas entre os tratamentos para as variáveis mortalidade e densidade de vilos. Contudo aves que não foram suplementadas com levedura hidrolisada (tratamento controle) apresentaram menor profundidade de cripta e a suplementação de 1% de levedura resultou em maior altura de vilos. Aves as quais receberam dietas com 0,2% de inclusão de levedura hidrolisada apresentaram menor rendimento de peito que as aves que receberam os demais níveis de levedura, mas foram iguais as aves do tratamento controle. Contudo, o rendimento de carcaça, sassami e gordura abdominal não foram influenciados pelos tratamentos experimentais. Estes resultados demostraram a eficácia da utilização de levedura hidrolisada na dieta de frangos de corte no período de 1 a 14 dias sobre as características de produção. / The objective of this work was to evaluate hydrolyzed yeast utilization as nucleotides source to broilers. Birds were supplemented with different inclusion levels on starter diets from 1 to 14 days of age. A total of five hundred seventy six Ross 708 chicks were allotted to 6 experimental treatments with 8 replications (12 broilers per pen). Birds were randomly distributed in following treatments: 0, 2, 4, 6, 8 and 10 kg hydrolyzed yeast/ton of feed. Hydrolyzed yeast was added to the test diet in place of filler. Starter diets were supplied in crumbled form while grower, finisher, and withdrawal were supplied in pellet form. Throughout experiment water and feed were supplied ad libitum. All diets were based on corn, soybean meal and poultry fat, and were formulated to achieve nutritional requirements from recommendations guide for Ross x Ross 708 broilers. At 42 d chicks fed 1% hydrolyzed yeast demonstrated higher body weight and body weight gain over birds fed other treatments (P<0.05). Mortality and villous density did not differ among treatments. However birds fed control treatment showed lower crypt depth and 1% hydrolyzed yeast supplementation promoted higher villous high. Birds fed 0.2% hydrolyzed yeast showed lower breast meat yield than birds received other yeast levels, but were equals to control treatment control. However, carcass and tender yield, and abdominal fat were not influenced by treatments. These results indicated efficacy of hydrolyzed yeast utilization on broiler diets from 1 to 14 on production characteristics.