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 (Ph. D.)--University of Hawaii at Manoa, 1996. / Includes bibliographical references (leaves 185-196). / Microfiche. / xii, 196 leaves, bound ill. (some col.) 29 cm

Thesis--Helsinki. / Includes five papers on which the present dissertation is based (p. 61-131). Includes bibliographical references.

Thesis (Ph. D.)--Ohio state University, 1923. / Autobiography.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1969. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliography.

Techn. Hochsch., Diss., 2001--Aachen.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 132-151).

Thesis (Master of Engineering in Chemical Engineering)--Cape Peninsula University of Technology, 2018. / Electrochemical and non-enzymatic fructose detection has evoked keen interest in the scientific literature. Several authors have reported on different methods of electrode preparation for fructose sensors. However, little systematic study has been conducted to design a cheap, efficient method of depositing metal oxides to detect fructose. To address the challenge, a Co3O4 thin film was fabricated using a simple solution step deposition on Fluorine doped Tin oxide (FTO) glass electrode. In this study, a report on the selective oxidation of fructose on Co3O4 thin film electrode surface is presented. Electrode characterization was done using X-ray diffraction (XRD), High Resolution Transmission Electron Microscopy (HR-TEM), Scanning Electron Microscope (SEM), Atomic Fluorescence Microscopy (AFM), and Electrochemical Impedance Spectroscopy (EIS). All cyclic voltammetry (CVs) and chronoamperometry tests were carried out by the use of an AUTOLAB POTENTIOSTAT 302 N, controlled by Nova 2.0 software instrumentation using a customized 50 cm3 electrochemical cell. The cell consisted of a graphite rod as the counter electrode (CE), 3 M Ag/AgCl reference electrode (RE) and the fabricated Co3O4/FTO as the working electrode (WE). All experiments were carried out at 25±2 ⁰C. From the results, the constructed sensor exhibited two distinctive linear ranges in the ranges of 0.021 – 1.74 mM and from 1.74 - ~15 mM, covering a wide linear range of up to ~15 mM at an applied potential of +0.6V vs. Ag/AgCl in 0.1M NaOH solution. The sensor demonstrated a high, reproducible and repeatable sensitivity of 495 (lower concentration range) & 53 (higher concentration range) μA cm-2 mM-1 for a low R.S.D of 5 %. The Co3O4 thin film produced a low detection limit of ~1.7 μM for a signal to noise ratio of 3 (S/N = 3); a fast response time of 6s and long term stability. The repeatability and stability of the electrode resulted from the chemical stability of Co3O4 thin film. The study showed that the sensor was highly selective towards fructose compared to the presence of other key interferences i.e. AA, AC, and UA. Because of such a favourable electrocatalysis of the Co3O4 sensor towards fructose, the ease of the electrode fabrication and reproducibility makes it a future candidate for commercial applications in the food and beverages sector.

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 2014-06-11T19:28:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2001Bitstream added on 2014-06-13T19:57:13Z : No. of bitstreams: 1 barreiros_rc_me_botfm.pdf: 2089159 bytes, checksum: 5458c167603553aa52e8877b42426d13 (MD5) / O metabolismo da frutose, bem como o seu nível sangüíneo em recém-nascidos não está bem esclarecido. A frutose é uma hexose encontrada normalmente no organismo humano e tem seu metabolismo associado à glicose e ao sorbitol. As principais fontes de frutose são os vegetais e o mel. O leite materno não contém frutose. O metabolismo desse açúcar é independente da insulina o que o torna uma boa opção para utilização em pacientes com deficiência desse hormônio. Além da independência da insulina, o metabolismo da frutose é caracterizado por uma rápida fosforilação e rápida conversão em glicogênio e glicose ou conversão em glicerol, para posterior utilização no metabolismo lipídico. A frutose pode ser produzida endogenamente, a partir da glicose, através da via do sorbitol. Apesar de ser utilizado há tempos clinicamente como uma alternativa à glicose, existem poucos trabalhos na literatura determinando os níveis normais em humanos. Isso ocorreu em grande parte devidas dificuldades na dosagem desse carboidrato: em virtude de ser difícil a sua diferenciação da glicose, outra hexose, além da frutose ser encontrada em pouca quantidade em fluídos orgânicos... / The metabolism of fructose as well the blood-levels of fructose in newborn infants are not yet well established. Fructose is an hexose found naturally in fruits, vegetables and honey and its metabolism is associated with glucose and sorbitol. The human milk does not contain fructose. The metabolism of fructose is insulin independent, which makes it an alternative to glucose. Besides its independence of insulin, fructose is rapidly metabolized primarily in the liver, where occurs phosphorylation and conversion to glycogen and glucose or to glycerol, to further utilization in lipid metabolism. Fructose, also, can be produced by the human organism, originating from glucose by the sorbitol pathway. Although fructose is utilized since 1893 for medical purposes, there are few studies in the literature about normal levels of fructose in humans. This lack of studies is due in part to difficulties to determine this sugar in human fluids: glucose interferes in the final results and levels of fructose in biological fluids are very low. Our main goal was to establish the normal levels of fructose in newborn infants at 48 hours of life, with adequate weight for gestational age, breast-fed exclusively and to correlate the level of fructose with the levels of glucose and sorbitol. For this purpose we used the High performance liquid chromatography was used. Our study group was selected among breast-fed term newborn... (Complete abstract click electronic access below)