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JMJD3 acts as a tumor suppressor by disrupting cytoskeleton in pancreatic ductal adenocarcinoma cells. / CUHK electronic theses & dissertations collectionJanuary 2013 (has links)
Xiao, Zhangang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 118-131). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.
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Hidrogéis contendo tretinoína associada a nanocápsulas de núcleo lipídico : influência da secagem das suspensões nas propriedades físico-químicas e biofarmacêuticasZuglianello, Carine January 2015 (has links)
Este estudo tem como objetivo central avaliar a influência da secagem por aspersão de nanocápsulas de núcleo lipídico contendo tretinoína nos perfis in vitro de liberação e de penetração cutânea deste fármaco a partir de hidrogéis. Esses experimentos foram conduzidos empregando-se células de difusão de Franz, pele de abdome de porcos (fêmeas), regime de aplicação de doses infinitas e meio receptor composto por tampão fosfato pH 7,4 e etanol (70:30). A secagem por aspersão das suspensões de nanocápsulas, utilizando PVP e lactose (1:1, m/m) a 10% como adjuvantes, forneceu produtos com bons perfis de dispersão em água, bons rendimentos (próximos a 70%), baixos teores de substâncias voláteis, e teores do fármaco acima de 92%. O tipo de produto intermediário, suspensão aquosa ou respectivo pó, utilizado na produção de hidrogéis (G-LNC-TTN e G-LNC-TTN-SD, respectivamente) não influenciou no perfil de liberação in vitro da tretinoína, que se ajustou ao modelo de Higuchi. No estrato córneo houve diferenças nas quantidades de tretinoína penetradas a partir das duas formulações. O G-LNC-TTN levou a uma retenção exponencial do fármaco nessa camada, enquanto para o G-LNC-TTN-SD isso não ocorreu. Essa diferença foi associada à forma de organização das nanocápsulas na matriz do gel. Na epiderme e na derme, ambas as formulações permitiram a chegada de pequenas e constantes quantidades de tretinoína. No compartimento receptor da célula de Franz o fármaco não foi detectado. A pequena permeação da tretinoína para as camadas mais profundas da pele e para o meio receptor são indicativos de baixa absorção sistêmica, e também podem contribuir para a diminuição dos efeitos adversos associados à terapia tópica com essa substância. A secagem das suspensões de nanocápsulas de núcleo lipídico, nas condições utilizadas, forneceu um intermediário em potencial para a produção de formas farmacêuticas semissólidas contendo tretinoína. / This study’s central goal is to assess the influence of spray-drying lipid core nanocapsules on tretinoin in vitro release profiles as well as skin penetration/permeation from hydrogels. These experiments were conducted employing Franz diffusion cells, pig abdominal skin (female), infinite doses regimen and receptor medium composed of phosphate buffer pH 7.4 and ethanol (70:30). Spray-drying of the nanocapsules suspensions, using PVP and lactose (1:1, m/m) at 10% (m/v) as drying adjuvant provided powders with good water dispersion profiles, good yields (around 70%), low volatile substances contents, in addition to drug contents above 92%. Interchanging intermediate products, aqueous suspension or respective powder, used in hydrogel formulation (G-LNC-TTN and G-LNC-TTN-SD, respectively) caused no influence on tretinoin in vitro release profile which was adjusted by Higuchi model. In corneum stratum there were differences in tretinoin quantities which penetrated from those formulations. The G-LNC-TTN provided an exponential retention of the drug on this skin’s layer, although G-LNC-TTN-SD did not. This difference was associated with the nanocapsules organization form in hydrogel matrix. In epidermis and dermis both formulations allowed permeation of constant and low tretinoin quantities. Moreover, at receptor fluid the drug was not detected. The low tretinoin permeation for deeper skin layers and for receptor fluid is low systemic absorption indicative, furthermore, may contribute in reducing adverse effects associated with tretinoin topical therapy. In given conditions, spray-drying of lipid core nanocapsules provided a potential intermediate for production of semi solids pharmaceutical forms containing tretinoin.
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Adolescence scarifiée : traces et mouvements symboliques d'un groupe à médiation écriture / Scarified adolescence : traces and symbolic movements in a writing-mediated groupBoinet, Pauline 30 January 2018 (has links)
La spécificité de l’adolescence est la venue au premier plan de la génitalité. Cela entraîne à la fois une métamorphose du corps et un remaniement psychique. À l’adolescence, le corps est aussi un lieu de contradiction que tantôt il attaque, tantôt il embellit dans un érotisme effréné. En effet, la contradiction peut se traduire sous forme symptomatique et manifester l’encombrement que l’adolescent ressent devant son corps qui lui échappe. Il ne sait pas repérer ce qu’il ressent, il ne sait pas le nommer et même il ne sait pas qu’il ne sait pas. Parfois le rapport qu’il entretient avec son corps est suffisamment paradoxal pour qu’il ne puisse pas se l’approprier. C’est cette étrangeté qui va provoquer un clivage chez l’adolescent. Les deux perspectives qui émanent de ces positions sont assez différentes quant à l’avenir du sujet. Ce travail s’inscrit dans la continuité de divers travaux de recherche sur la problématique adolescente. Il vient rendre compte de la mise en place dans un service de Pédiatrie générale d’un groupe thérapeutique à médiation écriture avec des adolescentes qui se scarifient. C’est en considérant ces scarifications comme un langage du corps au coeur de la problématique adolescente et pubertaire, et au regard des difficultés que ces adolescentes ont à mettre des mots sur l’indicible de leur douleur, que le groupe a été créé. La mise en place de celui-ci correspond plus globalement à une réflexion sur le processus d’adolescence, de subjectivation, et le rapport au corps, notamment à travers le travail de symbolisation à l’adolescence. Nous interrogeons également à l’endroit de notre réflexion, la médiation écriture en tant que dépôt d’une trace sur un support, qui au même titre que la rencontre de la lame sur la peau, viendrait comme une butée (Le Breton, 2002) ; la rencontre avec la feuille par analogie au corps viendrait recréer et offrir un contenant à la souffrance psychique. Le groupe quant à lui pourrait être vécu comme espace transitionnel au sens où l'entend Winnicott, au fondement de l’expérience créatrice et rassurante pour l’adolescente. Le corps aurait alors une fonction semblable dans ce qu'il incarnerait une frontière entre un dedans et un dehors. Une des fonctions de la pratique scarificatoire serait alors de restaurer les limites du Soi dans une lutte contre un possible effondrement. / The specificity of adolescence is the coming to the fore of genitality. This involves both a metamorphosis of the body and a psychic reworking. The body is also a place of contradiction that sometimes attacks, sometimes it embellishes in a frenzied eroticism. Indeed, the contradiction can be translated in symptomatic form and manifest the clutter that the adolescent feels in front of his body that escapes him. He does not know how to identify what he feels, he does not know how to name it and he does not even know he does not know. Sometimes the relationship he has with his body is sufficiently paradoxical that he can't appropriate it. It is this strangeness that will cause a cleavage in the adolescent. The two perspectives that emanate from these positions are quite different as to the future of the subject. This work is a continuation of various research works on the adolescent problem. He reports on the establishment of a therapeutic group in writing in a Pediatric General Service, writing with teenagers who are scarifying themselves. It is by considering these scarifications as a body language at the heart of the adolescent and pubertal problem, and in view of the difficulties that these teenagers have to put words on the indescribable of their pain, that the group was created. The setting up of this one corresponds more generally to a reflection on the process of adolescence, of subjectivation, and the relation with the body, in particular through the work of symbolization in adolescence. We also question the place of our reflection, the mediation writing as a deposit of a trace on a support, which as well as the meeting of the blade on the skin, would come as a stop (Le Breton, 2002); the encounter with the sheet by analogy with the body would come to recreate and offer a container for psychic suffering. The group could be seen as a transitional space in Winnicott's sense, at the root of the creative and reassuring experience for the teenager. The body would then have a similar function in what it would incarnate a border between an inside and an outside. One of the functions of the scarificatory practice would then be to restore the limits of the Self in a fight against a possible collapse.
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Microphysiometry in the evaluation of cytotoxic drugs with special emphasis on the novel cyanoguanidine CHS 828Ekelund, Sara January 2001 (has links)
<p>This thesis describes the use of a new technology, the Cytosensor<sup>®</sup> microphysiometer, in the in vitro evaluation of cytotoxic drugs, using the lymphoma cell line U-937 GTB and primary cultures of tumour cells from patients as model systems. The method was specifically applied to study the metabolic effects of the novel cyanoguanidine N-(6-(4-chlorophenoxy)hexyl)-N’-cyano-N’’-4-pyridylguanidine, CHS 828, currently in phase I/II clinical trials. </p><p>The Cytosensor<sup>®</sup> measures metabolic effects as changes in the rate of extracellular acidification of cells exposed to a drug by perfusion. A number of standard cytotoxic drugs were found to produce typical and reproducible acidification response patterns during observation times up to 20 h. There seemed to be a relationship between a decrease in acidification and cytotoxicity, measured in the fluorometric microculture cytotoxicity assay (FMCA), after 20-24 h of continuous drug exposure.</p><p>In U-937 cells, CHS 828 induced a cytotoxic effect characterised by a steep concentration-response relationship followed by a plateau. After 24 h of incubation the DNA and protein synthesis were turned off. CHS 828 was found to produce a rapid and prolonged increase in extracellular acidification and lactate production similar to that of the structurally related mitochondrial inhibitor m-iodobenzylguanidine (MIBG). The CHS 828 induced acidification was observed in cell lines as well as in cells from various tumour types from patients and probably originates from increased glycolytic flux. The effects may be secondary to block of oxidative phosphorylation in the mitochondria, but the relevance of the early acidification is not clear. CHS 828 seemed to induce a late, at approximately 15 h, inhibition of the glycolysis followed by loss of ATP and subsequent cell death. After exposure to MIBG the loss of ATP and cell death occurred earlier and in parallel. The effects of CHS 828 were not found to resemble those of the structurally related polyamine biosynthesis inhibitor methylglyoxal-bis(guanyl-hydrazone) (MGBG). Thus, CHS 828 may represent a new and, thus, interesting mode of cytotoxic action worthwhile for further development.</p><p>In combinatory studies, a synergistic interaction was demonstrated between CHS 828 and the non-toxic drug amiloride. Additive-to-synergistic effects were also seen between CHS 828 and the bioreductive cytotoxic drug mitomycin C. In U-937 cells as well as in tumour cells from patients, CHS 828 demonstrated synergistic interactions in combination with melphalan and etoposide. </p><p>It is concluded that measurement in the Cytosensor<sup>®</sup> microphysiometer of early cellular metabolic changes is a feasible and potentially valuable complement to more conventional methods used in the evaluation of anticancer agents. </p>
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Microphysiometry in the evaluation of cytotoxic drugs with special emphasis on the novel cyanoguanidine CHS 828Ekelund, Sara January 2001 (has links)
This thesis describes the use of a new technology, the Cytosensor® microphysiometer, in the in vitro evaluation of cytotoxic drugs, using the lymphoma cell line U-937 GTB and primary cultures of tumour cells from patients as model systems. The method was specifically applied to study the metabolic effects of the novel cyanoguanidine N-(6-(4-chlorophenoxy)hexyl)-N’-cyano-N’’-4-pyridylguanidine, CHS 828, currently in phase I/II clinical trials. The Cytosensor® measures metabolic effects as changes in the rate of extracellular acidification of cells exposed to a drug by perfusion. A number of standard cytotoxic drugs were found to produce typical and reproducible acidification response patterns during observation times up to 20 h. There seemed to be a relationship between a decrease in acidification and cytotoxicity, measured in the fluorometric microculture cytotoxicity assay (FMCA), after 20-24 h of continuous drug exposure. In U-937 cells, CHS 828 induced a cytotoxic effect characterised by a steep concentration-response relationship followed by a plateau. After 24 h of incubation the DNA and protein synthesis were turned off. CHS 828 was found to produce a rapid and prolonged increase in extracellular acidification and lactate production similar to that of the structurally related mitochondrial inhibitor m-iodobenzylguanidine (MIBG). The CHS 828 induced acidification was observed in cell lines as well as in cells from various tumour types from patients and probably originates from increased glycolytic flux. The effects may be secondary to block of oxidative phosphorylation in the mitochondria, but the relevance of the early acidification is not clear. CHS 828 seemed to induce a late, at approximately 15 h, inhibition of the glycolysis followed by loss of ATP and subsequent cell death. After exposure to MIBG the loss of ATP and cell death occurred earlier and in parallel. The effects of CHS 828 were not found to resemble those of the structurally related polyamine biosynthesis inhibitor methylglyoxal-bis(guanyl-hydrazone) (MGBG). Thus, CHS 828 may represent a new and, thus, interesting mode of cytotoxic action worthwhile for further development. In combinatory studies, a synergistic interaction was demonstrated between CHS 828 and the non-toxic drug amiloride. Additive-to-synergistic effects were also seen between CHS 828 and the bioreductive cytotoxic drug mitomycin C. In U-937 cells as well as in tumour cells from patients, CHS 828 demonstrated synergistic interactions in combination with melphalan and etoposide. It is concluded that measurement in the Cytosensor® microphysiometer of early cellular metabolic changes is a feasible and potentially valuable complement to more conventional methods used in the evaluation of anticancer agents.
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Determination of monophosphate nucleotides, sulfur-containing amino acids, arsenic species and various oxidation states of iron, vanadium and chromium by capillary electrophoresis inductively coupled plasma mass spectrometryYeh, Ching-fen 15 July 2005 (has links)
Capillary electrophoresis (CE) is in comparison with other chromatographic techniques, CE has several advantages such as high resolving power, small sample volume requirement, minimal buffer consumption and high sample throughtput. As a detection technique, inductively coupled plasma mass spectrometry (ICPMS) provides the advantages of low detection limit, multielement detection, and element- and isotope-specific detection capabilities. Therefore, the use of CE as a high resolution separation technique with ICP-MS as a sensitive element specific detector is of growing interest for analytical research. Four studies in our research are described below, respectively.
A preliminary study of a modified microconcentric nebulizer (CEI-100, CETAC) as the sample introduction device of capillary electrophoresis inductively coupled plasma mass spectrometry (CE-ICP-MS) for the determination of monophosphate nucleotides is described. The monophosphate nucleotides studied include adenosine 5¡¦-monophosphate (AMP), guanosine 5¡¦-monophosphate (GMP), uridine 5¡¦-monophosphate (UMP) and inosine 5¡¦-monophosphate (IMP). The species studied were well separated using a 70 cm length ¡Ñ 75 £gm id fused silica capillary while the applied voltage was set at -22 kV and a 20 mmol/L ammonium citrate/citric acid buffer (pH 4.0) containing 0.1% m/v cationic polymer (hexadimethrine bromide, Polybrene) was used as the electrophoretic buffer. The electroosmotic flow was reversed by flushing the fused silica capillary with 0.2% m/v Polybrene to accelerate separation. The detection limit of various species studied was in the range of 0.036~0.054 £gg P/mL, which corresponded to the absolute detection limit of 1.1~1.6 pg P based on the injection volume of 30 nl. We determined the concentrations of nucleotides in two IG-enriched monosodium glutamates purchased from the local market. The recovery was in the range of 100~112% for various species, and the concentrations of IMP and GMP in these samples were in the range of 0.15¡V0.18% m/m.
Capillary electrophoresis dynamic reaction cellTM inductively coupled plasma mass spectrometry (CE-DRC-ICP-MS) for the determination of sulfur-containing amino acids is described. The sulfur-containing amino acids studied include L-cysteine, L-cystine, DL-homocystine and L-methionine. The species studied were well separated using a 70 cm length ¡Ñ 75 £gm i.d. fused silica capillary while the applied voltage was set at +22 kV and a 10 mmol/L disodium tetraborate buffer (pH 9.8) containing 0.1 mmol/L EDTA and 0.5 mmol/L Triton X-100 was used as the electrophoretic buffer. The sulfur-selective electropherogram was determined at m/z 48 as 32S16O+ by using its reaction with O2 in the reaction cell. The method avoided the effect of polyatomic isobaric interferences at m/z 32 caused by 16O16O+ and 14N18O+ on 32S+ by detecting 32S+ as the oxide ion 32S16O+ at m/z 48, which is less interfered. The detection limit of various species studied was in the range of 0.047~0.058 £gg S/mL, which corresponded to the absolute detection limit of 1.3~1.6 pg S based on the injection volume of 27 nl. We determined the concentrations of selected sulfur-containing amino acids in urine and nutritive complement samples. The recovery was in the range of 92~128% for various species.
Capillary electrophoresis-dynamic reaction cell inductively coupled plasma mass spectrometry (CE-DRC-ICP-MS) for the speciation of iron (III/II), vanadium (V/IV) and chromium (VI/III) is described. Two different CE migration modes were employed for separating the six metal ions using pre-capillary complexation. One is counter-electroosmotic mode in which iron (III/II) and vanadium (V/IV) ions were well separated using a 60 cm ¡Ñ 75 £gm i.d. fused silica capillary. The voltage was set at +22 kV and a 15 mmol/L tris(hydroxymethyl)aminomethane (Tris) buffer (pH 8.75) containing 0.5 mmol/L ethylenediaminetetraacetic acid (EDTA) and 0.5 mmol/L ortho-phenanthroline (phen) was used as the electrophoretic buffer. The other is co-electroosmotic mode in which chromium (VI/III) ions were well separated while the applied voltage was set at −22 kV and a 10 mmol/L ammonium citrate buffer (pH 7.7) containing 0.5 mmol/L diethylenetriaminepentaacetic acid (DTPA) and 0.01% polybrene was used as the electrophoretic buffer. The mass spectra were measured at m/z 51, 52 and 56 for V, Cr and Fe, respectively. The interfering polyatomic ions of 35Cl16O+, 40Ar12C+ and 40Ar16O+ on 51V+, 52Cr+ and 56Fe+ determination were reduced in intensity significantly by using NH3 as the reaction cell gas in the DRC. The detection limits were in the range of 0.1~0.5, 0.4~1.3 and 1.2~1.7 £gg/L for V, Cr and Fe, respectively. Applications of the method for the speciation of V, Cr and Fe in wastewater were demonstrated. The recoveries were in the range of 92~120% for various species.
A capillary electrophoresis-inductively coupled plasma-mass spectrometric (CE-ICPMS) method for the speciation of six arsenic compounds, namely arsenite [As(III)], arsenate [As(V)], monomethyl arsonic acid, dimethylarsinic acid, arsenobetaine and arsenocholine is described. The separation has been achieved on a 70 cm length ¡Ñ 75 £gm ID fused-silica capillary. The electrophoretic buffer used was 15 mmol/L Tris (pH 9.0) containing 15 mmol/L sodium dodecyl sulfate (SDS), while the applied voltage was set at +22 kV. The arsenic species in biological tissues were extracted into 80% v/v methanol-water mixture, put in a closed centrifuge tube and kept in a water bath, using microwaves at 80¢J for 3 min. The extraction efficiencies of individual arsenic species added to the sample at 0.5 mg As/g level were between 96% and 107%, except for As(III), for which it was 89% and 77% for oyster and fish samples, respectively. The detection limits of the species studied were in the range 0.3~0.5 £gg As/L. The procedure has been applied for the speciation analysis of two reference materials, namely dogfish muscle tissue (NRCC DORM-2) and oyster tissue (NIST SRM 1566a), and two real-world samples.
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Simultaneous Determination of Sulfhydryl and Disulfide Containing Amino Acids by Capillary Electrophoresis with Electrochemical Detection at Au/Hg MicroelectrodeHsu, Kai-Chih 31 August 2005 (has links)
None.
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Effect Of Medicinal Plants Epilobium Hirsutum L. And Viscum Album L. On Rat Liver Flavin-containing Monooxygenase Activity And ExpressionCelebioglu, Hasan Ufuk 01 July 2012 (has links) (PDF)
Epilobium hirsutum L. (Onagraceae), a medicinal plant known as hairy willow herb, has been used by people all around the world for treatment or prevention of inflammation, adenoma, rectal bleeding, menstrual disorders, constipates, and prostate. It contains polyphenolics including steroids, tannins such as gallic, ellagic, and p-coumaric acids and flavonoids such as myricetin, isomyricetin, and quercetin. Polyphenols have been known for their multiple biological health benefits, including antioxidant activities.
Viscum album L. (Loranthaceae), a species of mistletoe, contains lectins, polypeptides, mucilage, sugar alcohols, flavonoids, lignans, triterpenes, and phenylallyl alcohols. The leaves and twigs of Viscum album L., taken as tea, have been traditionally used for hypertension, stomachache, diarrhea, diabetes, dysuria and also as analgesic and cardiotonic agent in Anatolia, Turkey. In addition, in Europe, sterile extracts of Viscum album L. are among the most common herbal extracts applied in cancer treatment and have been used as prescription drugs, while in US, considered as dietary supplement.
Flavin-containing monooxygenases are FAD-containing phase I enzymes responsible for the oxidation of wide-range of nucleophilic nitrogen, sulfur, phosphorus, and selenium heteroatom-containing drugs such as tamoxifen,
v
methimazole and imipramine, pesticides, neurotoxins, and other chemicals using NADPH as cofactor.
The aim of this study was to determine the in vivo effects of Epilobium hirsutum L. and Viscum album L. (subspecies growing on pine trees-subsp. austriacum (Wiesb.) Vollmann) on FMO activity, mRNA and protein expressions in rat liver. The water extracts of Epilobium hirsutum L. (37.5 mg/kg body weight) and Viscum album L. (10 mg/kg body weight) were injected intraperitonally (i.p) into Wistar albino rats for 9 consecutive days. Following the decapitation, the livers were removed and microsomal fractions were prepared by differential centrifugation. Rat liver microsomal FMO activity using methimazole as substrate, mRNA expression by quantitative Real-Time PCR, and protein expression by Western Blot were determined.
The results showed that water extract of Epilobium hirsutum L. has no significant effect on FMO activity / however, it decreased significantly (p< / 0.05) FMO3 protein and mRNA expression 27.71% and 1.41 fold, respectively, compared as controls. Water extract of Viscum album L. decreased mRNA (2.56 fold), and protein expressions (27.66%) as well as enzyme activity (19%) of FMO with respect to controls. In conclusion, our current data suggest that the metabolism of xenobiotics including drug molecules by FMO-catalyzed reactions may be altered due to the changes in FMO expression and activity by medicinal plants Epilobium hirsutum L. and Viscum album L.
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Influence of changing patterns of sucrose consumption on industrial users : response by manufacturers of soft drinks, biscuits, cereals, cakes confectionery, ice-cream, jams, canned products and other sugar-containing foods to the U.K. dietary guidelines that relate to sucrose consumptionHeasman, Michael Kenneth January 1988 (has links)
Sugar is intrinsically linked with the modern food system. Large sections of the U. K. food industry are dependent on its use and functional qualities. Supplies of sucrose entering the food chain have declined 25% between the 1950's and 1980's and currently stand around 37 kg/person/year. Furthermore, U. K. dietary guidelines over the past 14 years have consistently suggested caution over how much sugar is eaten, especially in manufactured foods. Dietary guidelines such as the NACNE report (1983) recommend average sugar consumption should be no more than 20 kg/person/year. Currently, two-thirds of sugar supplies are bought for use in food and drink manufacture. Continued pressures on sugar consumption and negative consumer attitudes to sugar may be reflected in lost sales of sugar-containing foods. The available information on U. K. sugar consumption is critically assessed. Although the main sources of sugar supply are identified, individual sugar consumption is shown to vary by considerable amounts. The place of sucrose is examined in relation to other sweeteners and why and where sugars and sweeteners are used in food systems. The promotion of "no added sugar" and "sugar free" products is examined since the publication of the NACNE report to the end of 1987. To further test the impact of changing patterns of sugar consumption on food and drink manufacturers a national survey of manufacturers who use sugar was carried out in early 1988. This was an attitudinal postal questionnaire and responses to the issue of sugar, diet and health were analysed. Respondents bought an estimated 650,000 tonnes of sugar in 1986, around 45% of the total industrial market. While the survey aggregate were fully supportive of sucrose, respondents reported that the majority of consumers were worried about sugar being bad for health and were actively cutting down on individual intakes. There were significant differences to the issue of sugar, diet and health dependent on company size, whether a company manufactured for a retailer's own label and if products had already been marketed at a "healthy eating" segment. However, in general, while manufacturers considered consumer attitudes to sugar to be important they had to be put in the context of other factors. So far the impact of changing patterns of sugar consumption is not reflected in the total average industrial purchases of sugar, although substantial "sugar-free" and "sugar-reduced" product niches have been established.
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Evaluation and optimization of four real-time PCRs, using TaqMan-probes, for detection of and discrimination between barley, oat, rye and wheatBjörklund, Kristofer January 2008 (has links)
Coeliac disease is a chronic inflammatory disease treated with a gluten-free diet, excluding barley, rye and wheat. Hence, there is a demand for methods able to detect gluten in foods in order to ensure correct labeling of products. According to the Codex Alimentarius Commission, 20ppm gluten is the maximum amount allowed in food labeled gluten-free. PCR can detect DNA from cereals in food. Four real-time PCR-systems, using TaqMan®-probes for detection of barley, oat, rye and wheat were optimized and evaluated. Evaluations were carried out using seeds. Primers were targeted to genes coding for prolamines, seed storage proteins. PCR-systems targeted to barley, oat and wheat were shown to be specific for the cereals corresponding to each system. The system targeted to rye showed cross-reactions with durum wheat and spelt wheat. Detection limits were 50pg, corresponding to <10 haploid genome copies for each cereal. All systems were able to detect 250ppm amounts of DNA, most likely even smaller amounts are detectable. All systems showed an amplification efficiency of ≥95%. Systems for detection of barley, oat and wheat are ready for further evaluation, using food products as samples. The rye system however, needs to be re-designed before further evaluation can take place.
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