A selection system for yeast cells expressing mutated invertase (EC 3.2.1.26, SUC2) with altered fructotransferase activity was developed based on the survival of a fructose-intolerant strain in the presence of suitable acceptor substrates and sucrose. Cells of such a strain expressing a wild-type hydrolase activity will not grow due to the release of free fructose from sucrose. Cells expressing an inactive invertase mutant will not grow since they cannot cleave the sucrose, the sole carbon source. Only cells expressing sucrose fructosyl-transferase activity will thrive, growing on the released glucose, the fructosyl moiety not being released. A strain of Saccharomyces cerevisiae was engineered to be intolerant of the presence of fructose in its growth media. This was achieved by inducing a condition in yeast similar to liver cells of humans suffering from hereditary fructose intolerance (MIM 22960). This disorder results from a deficiency of aldolase B (EC 4.1.2.13), and phosphorylation of fructose by ketohexokinase (EC 2.7.1.3) results in an accumulation of fructose 1-phosphate, with a consequent depletion of cytoplasmic phosphate and ATP. Thus, cells in which ketohexokinase phosphorylates fructose, but which lack aldolase B, are intolerant of fructose. Yeast possess neither of these enzymes, and so expression of ketohexokinase in yeast would result in fructose-intolerance. A strain of yeast, for ketohexokinase expression, was initially bred to be unable to metabolize sucrose or fructose, yet remain capable of utilizing glucose, as well as lacking non-specific phosphatases, to prevent remobilization of sequestered fructose 1-phosphate. Rat liver ketohexokinase was purified to heterogeneity, and the partial amino acid sequence subsequently generated exploited to amplify a region of the ketohexokinase cDNA by PCR. This was used to probe a cDNA library, yielding clones encoding the entire ketohexokinase coding region. This was cloned into pMA91, and subsequent expression in yeast resulted in a strain intolerant of fructose in its growth medium, although still capable of growing on glucose. In order to produce a stable fructose-intolerant selection strain, a vector (pIADl) was constructed that allowed multiple integration of an expression cassette containing ketohexokinase cDNA into the rDNA locus of yeast chromosome XII. Expression of wild type invertase from the episomal plasmid pIAD3 in this strain resulted in sucrose-intolerance. A preliminary programme of mutagenesis of the SUC2 gene yielded eight libraries of about one hundred clones each. None of these contained any mutants showing solely sucrose fructosyl-transferase activity, although this system would clearly provide an ideal selection for such mutants from a much larger library.

Thesis (MSc)--University of Stellenbosch, 2005. / Bibliography. Also available via the Internet.

Universiẗat, Diss., 2003--Stuttgart.

Increased sugar consumption is considered to be a contributor to the worldwide epidemics of obesity and diabetes and the consequent cardio metabolic risks. These include a significant increase for Type II diabetes and associated multiple comorbidities such as non-alcoholic fatty liver disease (NAFLD). The accumulation of excess triglycerides characterizes NAFL with a prevalence of up to 53% in morbidly obese populations. While in itself benign, fatty liver can progress to non-alcoholic steatohepatitis (NASH), which is characterized by apoptosis, inflammation and fibrosis in 10-20% of individuals. Progression to NASH increases the risk of further deterioration to cirrhosis and hepatocellular carcinoma (HCC). However, progression is unpredictable in any given individual and no risk factors predisposing to progression have been identified. Variation in a limited number of genes, such as patatin-like phospholipase (PNPLA3) and transmembrane 6 superfamily member 2 (TM6SF2), have been linked to an increased susceptibility to NAFLD. Recently, fibroblast growth factor 21 (FGF21) was reported to be a potential predictor for NAFLD as it significantly increases in patients with obesity and NAFL. Multiple lines of evidence indicate that FGF21 plays an important role in liver metabolism in mice and humans, playing a key role in carbohydrate and lipid metabolism. FGF21 was originally identified as an endocrine member of the fibroblast growth factor family as it can be released into the circulation. FGF21 was initially assigned a purely metabolic role as infusions led to weight loss and increased glucose clearance through induced expression of the GLUT1 transporter. However, FGF21 biology is now understood to be extremely complex, as it is expressed in many metabolically active tissues including, liver, white (WAT) and brown adipose tissue (BAT), muscle and pancreas. Functions of FGF21 are distinct in all these tissues. In the previous studies from our lab, we have seen fructose consumption, but not glucose, leads to an increase in serum FGF21 levels both in humans and mice. In general, sugar is typically consumed by humans in the form of sucrose or high fructose corn syrup (HFCS), both of which consist of nearly equal amounts of the simple sugars, glucose and fructose. Although attention has been focused on sucrose and fructose in many studies, no direct comparison was found to study fructose, glucose and sucrose. The current study aims to expand on the role of FGF21 in mediating the effects of chronic consumption of these refined sugars in mice. Wildtype (WT) and FGF21 knockout (KO) mice were fed with one of these diets for 20 weeks and in general, mice eating diets with high refined sugars gained less weight than mice eating chow, although calorie consumption was the same. In terms of body composition, sucrose fed FGF21 KO mice had less fat mass compared to chow fed animals. Dextrose fed and fructose fed mice had comparable fat mass reduction in WT and KO mice. Interestingly, glucose tolerance tests (GTT) showed increased glucose sensitivity in dextrose fed WT and KO mice after four weeks, however glucose tolerance decayed after 12 weeks on the diet. At 16 weeks fructose fed KO mice had significant increased glucose sensitivity compared to controls. Insulin tolerance tests showed similar results between all cohorts and a larger sample size would be needed to elicit any differences. Pyruvate tolerance tests (PTT) showed significantly increased hepatic gluconeogenesis in fructose fed KO mice compared to controls but not in dextrose or sucrose fed mice. Energy expenditure was measured by indirect calorimetry. No significance changes were observed in dextrose fed mice compared to chow controls in terms of VO2 or heat production. Both WT and KO dextrose fed mice had a higher RER, consistent with utilization of carbohydrates over fat for baseline energy expenditure. Sucrose fed mice showed marked increases in VO2 over an averaged 24-hour period and similarly fructose fed mice FGF21 KO mice had increased energy expenditure. Significant increases in RER were observed in both WT and KO sucrose fed mice controls and a similar trend was observed in WT and KO fructose fed mice. Overall, we see differential metabolic effects of all the high carbohydrate diets on the mice. Chronic consumption of dextrose only affected glucose sensitivity. Whereas chronic consumption of sucrose influences glucose and insulin sensitivity and energy expenditure suggesting internal metabolic changes while fructose consumption additionally showed increased hepatic gluconeogenesis without the marked increase in insulin sensitivity. However, detailed tissue analysis is required to determine specific physiological and molecular changes between refined sugar cohorts and the role of FGF21 in this context.

Made available in DSpace on 2014-06-11T19:29:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-25Bitstream added on 2014-06-13T18:38:46Z : No. of bitstreams: 1 santos_lb_me_sjrp.pdf: 1031549 bytes, checksum: b1556827453125236e6acc8a6094143f (MD5) / A sacarose é um dos produtos químicos puro mais abundante produzido no mundo. A determinação do ponto de fusão trata-se de um parâmetro importante para elucidar a pureza do cristal de açúcar, sendo sugerido, recentemente, como um parâmetro para assegurar a qualidade do produto. No entanto, uma ampla gama de pontos de fusão para a sacarose cristalina foi relatada, intervalo de 160 a 191 ºC, e referente ao mecanismo de decomposição de açúcares de alta pureza. Neste sentido, no presente trabalho pretendeu-se abordar os aspectos gerais da presença da água e produtos de degradação em sacarose de cana-de-açúcar, estudando materiais de referência (material de referência de consenso), comercial e pré-formulado (purificado), para o estabelecimento de relação entre impurezas e a variabilidade do ponto de fusão, que conduzem ao aparecimento de coloração amarela no produto e a formação de pedras, o que imprime grandes perdas quando se trata de comércio, principalmente do comércio exterior. Para tanto, utilizou-se técnicas de análise térmica amplamente difundidas: Termogravimetria (TG), a Análise Térmica Diferencial (DTA) e a Calorimetria Exploratória Diferencial (DSC). A análise térmica dos componentes estudados foi realizada em um aparelho SDT 2960-simultâneo TG-DTA, da TA Instruments , nas seguintes condições: ∆T 30-700 ºC; β = 20, 10 e 0,5 ºC min-1; cadinho de α-alumina; massa de amostra da ordem de 7 mg; atmosfera de ar sintético e nitrogênio . Para a obtenção das curvas de DSC, foi usado o DSC modelo DSC1 Stare Sistem, da Mettler Toledo, nas seguintes condições: ∆T 25-220 ºC; β = 20, 10, 5, 2, 1 e 0,5°C min – 1; cadinho de alumínio vazio (perfurado Φ = 1mm); massa de amostra da ordem de 3 mg; atmosfera de nitrogênio. Os resultados obtidos em termogravimetria (TG/DTG) e análise térmica diferencial (DTA) se mostraram análogas para todas... / Sucrose is one of the most abundant pure chemicals produced worldwide. Determining the melting point of this is an important parameter to elucidate the purity of crystal sugar, being suggested recently as a parameter to ensure product quality. However, a wide range of melting points for crystalline sucrose has been reported, range from 160 to 191 ° C, and for the mechanism of decomposition of sugars in high purity. In this sense, the present study was intended to address the general aspects of the presence of water and degradation products of sucrose in cane sugar, study reference materials (reference material of consensus), commercial and pre-formulated (purified) for the establishment of a relationship between impurities and variability of the melting point, leading to the appearance of yellow in the product and the formation of stones, which prints great loss when it comes to trade, especially foreign trade. To this end, we used thermal analysis techniques widespread: Thermogravimetry (TG), Differential Thermal Analysis (DTA) and Differential Scanning Calorimetry (DSC). Thermal analysis of the components studied was performed on a unit-simultaneous SDT 2960 TG-DTA, TA Instruments, under the following conditions: ∆T 30-700 ° C, β = 20, 10 and 0.5 ° C min-1; crucible α -alumina, sample mass around 7 mg; atmosphere of synthetic air and nitrogen. For the curves of DSC, DSC model was used DSC1 Stare Sistem, Mettler Toledo, the following conditions: ∆T 25-220 ° C Dt, β = 20, 10, 5, 2, 1 and 0.5 ° C min - 1; empty aluminum crucible (drilled Φ = 1 mm), sample mass of about 3 mg, nitrogen atmosphere. The results of thermogravimetry (TG / DTG) and differential thermal analysis (DTA) were similar for all samples of sucrose, where the first mass loss corresponds to the fusion followed by loss of water or volatilization... (Complete abstract click electronic access below)

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abstract: The unpleasant bitter taste found in many nutritious vegetables may deter their consumption. While bitterness suppression by prototypical tastants is well-studied in the chemical and pharmacological fields, mechanisms to reduce the bitterness of foods such as vegetables remain to be elucidated. Here tastants representing the taste primaries of salty and sweet were investigated as potential bitterness suppressors of three types of Brassicaceae vegetables. The secondary aim of these studies was to determine whether the bitter masking agents were differentially effective for bitter-sensitive and bitter-insensitive individuals. In all experiments, participants rated vegetables plain and with the addition of tastants. In Experiments 1-3, sucrose and NNS suppressed the bitterness of broccoli, Brussels sprouts, and cauliflower, whereas NaCl did not. Varying concentrations of NaCl and sucrose were introduced in Experiment 4 to assess the dose-dependency of the effects. While sucrose was a robust bitterness suppressor, NaCl suppressed bitterness only for participants who perceived the plain Brussels sprouts as highly bitter. Experiment 5, through the implementation of a rigorous control condition, determined that some but not all of this effect can be accounted for by regression to the mean. Individual variability in taste perception as determined by sampling of aqueous bitter, salty, and sweet solutions did not influence the degree of suppression by NaCl or sucrose. Consumption of vegetables is deterred by their bitter taste. Utilizing tastants to mask bitterness, a technique that preserves endogenous nutrients, can circumvent this issue. Sucrose is a robust bitter suppressor whereas the efficacy of NaCl is dependent upon bitterness perception of the plain vegetables. / Dissertation/Thesis / Doctoral Dissertation Psychology 2014

Orientador: Marcelo Menossi Teixeira / Texto em português e inglês / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T15:47:59Z (GMT). No. of bitstreams: 1 Farani_TiagoLuz_D.pdf: 5590005 bytes, checksum: 5a826906d504777afc8a4763b6863d0c (MD5) Previous issue date: 2013 / Resumo: A cana-de-açúcar é uma das mais importantes culturas vegetais do mundo, sendo o Brasil o principal produtor. A relevância mundial desta planta tem aumentado e muito tem sido investido na obtenção de variedades de cana-de-açúcar com maiores teores de sacarose e mais resistentes a estresses bióticos e abióticos. O presente trabalho teve como objetivo caracterizar funcionalmente o gene ScCIPK8, relacionado ao acúmulo de sacarose em cana-de-açúcar. Com a finalidade de elucidar as redes de interação da proteína ScCIPK8, ensaios de duplo-híbrido em leveduras foram realizados. Foi identificado que a proteína ScCIPK8 interage com as proteínas ScCBL1, ScCBL3 e ScCBL6, mas não interage com as proteínas ScCBL2, ScCBL9 e ScCBL10. As interações identificadas em leveduras foram confirmadas in planta por ensaios de BiFC. No intuito de identificar alvos de interação diferentes das proteínas CBLs, foram realizados screenings de duas bibliotecas de cDNA transformadas em leveduras. Foram identificadas a interação da proteína ScCIPK8 com as porções C-terminal de uma provável proteína do tipo PP2C e de uma provável proteína MYC2. Avaliamos adicionalmente a resposta à seca do gene ScCIPK8 e dos seus alvos de interação através de ensaios de PCR em tempo real. O gene ScCIPK8 é induzido em uma variedade de cana-de-açúcar tolerante à seca após dois dias de estresse hídrico e é induzido após quatro dias de estresse hídrico, tanto na variedade sensível quanto na tolerante à seca. Por outro lado, o gene ScPP2C-1 é induzido apenas na variedade tolerante à seca após dois dias de estresse hídrico. As expressões dos genes ScCBL1 e ScCBL6 não foram alteradas de forma significativa nas condições testadas. Por outro lado, a expressão do gene ScCBL3 foi reprimida nas plantas da variedade tolerante sob quatro dias de seca e manteve-se inalterado em todas as outras condições testadas. Por fim, plantas transgênicas de Arabidopsis thaliana transformadas com o gene ScCIPK8 foram obtidas e avaliadas quanto à resistência a estresses hídrico e salino. Não foi verificada diferença significativa na tolerância das plantas transformadas com o gene ScCIPK8 em relação aos controles utilizados / Abstract: Sugarcane is one of the most important crop plants in the world and Brazil is the leading manufacturer. The world importance of sugarcane is increasing and much has been invested on obtaining better plants with increasing sucrose content and resistance to biotic and abiotic stresses. The present work aims to characterize the sugarcane ScCIPK8 gene involved in sucrose accumulation. To gain insights on the ScCIPK8 protein-protein interaction network, yeast two-hybrid assays were conducted. It was shown that ScCIPK8 protein interacted with ScCBL1, ScCBL3 and ScCBL6, but not with ScCBL2, ScCBL9 and ScCBL10. BiFC assays were conducted to confirm in planta the interactions observed in yeast cells. Yeast two-hybrid screenings using two different cDNA libraries were conducted to identify unknown targets of ScCIPK8 protein. It was shown that ScCIPK8 interacted with the C-terminal portions of a putative PP2C and a putative MYC2 protein. We also performed qPCR assays in order to evaluate the drought response of ScCIPK8 gene and its target genes. ScCIPK8 gene expression was induced in a tolerant sugarcane variety after two days of drought stress and was induced in both the sensitive and tolerant sugarcane varieties after four days of drought stress. On the other hand, ScPP2C-1 gene expression was induced in the tolerant variety after two days of drought stress. The expression of ScCBL1 and ScCBL6 was not significantly changed under the tested conditions, while ScCBL3 expression was repressed in the tolerant variety after four days of drought stress. Finally, Arabidopsis thaliana transgenic plants overexpressing ScCIPK8 were obtained and evaluated for their resistance to drought and salinity stresses. We could not observe any significant difference regarding drought tolerance between control and transgenic plants transformed with ScCIPK8 / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

Binge Eating Disorder (BED) is a deadly, psychiatric condition which affects about 10 million people in the USA. It is characterized by discrete and recurrent binge eating episodes consisting of rapid consumption of excessive amounts of highly palatable, energy-dense food (e.g. rich in sugars and fats) within discrete periods of time. Our laboratory has been focusing on the understanding of the behavioral, metabolic, and neurobiological factors underlying BED, through the development of an animal model of binge-like eating. This model is based on a limited access schedule in which rats are exposed 1-hour/day to a high-sucrose diet (HSD) in operant conditioning self-administration boxes. However, the consummatory and metabolic outcomes of exposing rats to a high-fat diet (HFD) in the same procedure are unknown. The aim of this thesis was to test the consummatory and metabolic effects of 1-hour limited access to either a HSD or a HFD in an operant rat model of binge-like eating. For this purpose, female rats were subjects of the binge-like eating procedure by limiting access to a HSD, a HFD, or a standard Chow diet. Our results show that limiting access to either a HSD or a HFD promoted binge-like eating as compared to control Chow diet. HSD binge-like eating was based on a true increase in the amount of food consumed, that is, an increased eating rate. Such suggests increase in palatability and a decrease in the home-cage standard chow intake, likely due to a negative contrast effect. Conversely, binge-like eating of the high-fat diet resulted from passive energy consumption due to the high energy-density of the food. Also, HFD binge-like eating was accompanied by neither increased eating rate nor rejection of the home-cage chow. Moreover, while HSD rats consumed less energy than HFD rats, the former were more energy efficient and gained more body weight than the latter. These results provide information on how the quality of food can deeply influence the behavioral and metabolic outcomes of binge-like eating. / 2022-06-07T00:00:00Z

Iron deficiency is the most prevalent nutrient deficiency in the world, leading to long-term developmental and health consequences in populations at risk. Also known as prebiotics, non-digestible oligosaccharides such as fructooligosaccharide (FOS), inulin, galactooligosaccharide (GOS) and lactulose resist digestion by gastric acid and pancreatic enzymes in vivo, but are preferentially fermented by beneficial intestinal bacteria once they reach the colon. Prebiotics have been shown to increase the absorption of minerals such as iron from diets, but results from studies reported in the literature at times are contradictory, and mechanisms involved are still unclear. A better understanding of the role of FOS and other prebiotics in iron absorption may lead to new dietary modification strategies to increase intake of iron absorption enhancers in plant-based diets. The objectives of this study were therefore to determine the effects of prolonged FOS, as well as Synergy 1 (a combination of long- and short-chain FOS), inulin, GOS and lactulose supplementation on iron status of anemic rats; and to assess the enhancing effects of FOS on iron absorption and elucidate the regulatory mechanism involved using the Caco-2 cell culture model. In our animal studies, male Sprague-Dawley rats were first fed a low-iron diet for 14 days prior to prebiotics supplementation to achieve an iron-deficient status. Rats receiving the low-iron diet (12 ppm Fe) showed significantly lower non-heme iron concentrations in liver, spleen and kidney, as well as lower hemoglobin level than rats receiving a normal diet (45 ppm Fe), confirming iron-deficiency anemia. At the onset of the feeding trials, anemic rats were further divided into groups with or without supplementation of prebiotics. Prebiotics were provided to the rats by dissolving in water at 5% (w/v). Rats were kept on their respective test diets for 28 or 35 days, and all had free access to food and water during the feeding trials. The results showed significantly higher hemoglobin and non-heme iron levels in anemic rats with FOS or GOS supplementation, suggesting that both FOS and GOS could have positive effects on the iron status of anemic subjects with a low-iron intake. Rat colon contents also showed significant changes in short-chain fatty acid (SCFA) concentrations, presumably due to fermentation of prebiotics by intestinal microflora. Changes in the expression of Duodenal cytochrome b (Dcytb) and Divalent metal transporter 1 (DMT-1) in Caco-2 cells were measured by Western Blot and Real Time PCR. Our results confirmed that Caco-2 cells 14 days post confluence provided a stable research model for gene expression studies related to iron absorption. At low iron level, especially with FOS or SCFA supplementation, Dcytb and DMT-1 expression levels were increased in Caco-2 cells. While at high iron level, expression of Dcytb or DMT-1 was mostly down-regulated. Effects of SCFA were much more pronounced than FOS at different iron concentrations, suggesting that any effects of dietary FOS on improving iron status would require fermentation by the intestinal microflora. Further studies on other prebiotics (e.g., GOS and lactulose) and different combinations of SCFA are warranted.