Orientador: Telma Teixeira Franco / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química. / Made available in DSpace on 2018-08-20T23:25:42Z (GMT). No. of bitstreams: 1 Vidotti_AnnamariaDoriaSouza_M.pdf: 2006384 bytes, checksum: cae5b036525c63c6b285732d6f067476 (MD5) Previous issue date: 2012 / Resumo: Rotas heterotróficas a partir de microalgas apresentam ganhos significativos de produtividade em biomassa quando comparadas aos sistemas fotossintéticos convencionais, estando a glicose e o acetato entre as fontes de carbono mais comuns desse tipo de cultivo. Neste contexto, o objetivo do presente estudo foi desenvolver cultivos heterotróficos axênicos para a microalga Chlorella vulgaris, utilizando a glicose e o acetato de sódio como fontes de carbono exógeno, avaliando o efeito de inibição por substrato, bem como a modelagem dos perfis de crescimento de biomassa e consumo de substrato. A influência da concentração inicial de substrato na produção de biomassa pela C. vulgaris foi investigada, sendo obtidas cinéticas de crescimento do microrganismo submetido a concentrações iniciais de glicose entre 2 e 100 g.L-1, e de acetato de sódio, entre 2 e 20 g.L-1. Foi verificado que a concentração inicial de substrato influenciou significativamente o rendimento celular final, e que apesar dos dois substratos avaliados terem se mostrado fontes de carbono adequadas, a utilização de maiores concentrações iniciais, 100 g.L-1 e 20 g.L-1 de glicose e acetato de sódio, respectivamente, acarretaram na inibição do crescimento da C. vulgaris. Os modelos matemáticos testados representaram adequadamente a cinética de inibição, sendo que os resultados indicaram que a concentração ótima de glicose para o cultivo heterotrófico da C. vulgaris foi 5,8 ± 0,3 g.L-1 e de acetato de sódio foi 3,5 ± 0,2 g.L-1. Os resultados evidenciaram ainda que os procedimentos adotados no controle da contaminação foram efetivos para a manutenção da axenia dos cultivos. Pela comparação do desempenho cinético, foi constatada uma superioridade (maior que 50%) da glicose como substrato em comparação com o acetato de sódio. E no estudo de aumento de produtividade de biomassa para cultivos com acetato, foi obtido um aumento de 70% neste parâmetro com a batelada alimentada, e uma concentração final de biomassa 2,5 vezes maior do que a melhor concentração celular alcançada em shaker / Abstract: Heterotrophic microalgal routes show significant productivity gains in biomass when compared with the conventional photosynthetic systems, being glucose and acetate among the most common carbon sources such in this kind of cultivation. In this context the objective of this study was to develop axenic cultures for heterotrophic microalgae Chlorella vulgaris using glucose and sodium acetate as exogenous carbon sources, evaluating the effect of substrate inhibition, as well as the modeling of the biomass growth profiles and substrate consumption. The influence of the initial substrate concentration in the biomass production by C. vulgaris was investigated, being obtained kinetics growth of the microorganism subjected to initial glucose concentrations between 2 and 100 g/L-1, and sodium acetate between 2 and 20 g/L-1. It was found that the initial substrate concentration significantly affected the final cell yield and that although the two have been shown to be tested substrates carbon sources suitable, the use of larger initial concentrations of 100 and 20 g.L-1 of glucose and sodium acetate, respectively, resulted in inhibiting the growth of C. vulgaris. The mathematical models tested represented adequately the kinetics of inhibition, and the results indicated that the optimum concentration of glucose to heterotrophic cultivation of C. vulgaris is 5,8 ± 0,3 g.L-1 and for the sodium acetate this value is 3,5 ± 0,2 g.L-1. The results showed also that the procedures used in contamination control have been effective for the maintenance of the axenic of crops. By comparing the performance, kinetic superiority was observed (more than 50%) for the systems using glucose as substrate in comparison with the systems using sodium acetate. Finally, in the study of increasing of productivity of biomass for crops with acetate, was obtained a 70% increase in this parameter with the fed batch operation, and a final biomass concentration 2.5 times greater than the best cell concentration achieved in shake flasks / Mestrado / Desenvolvimento de Processos Químicos / Mestra em Engenharia Química

Nutrient metabolism consists of a series of chemical processes concerned with supplying energy to the body. This enables the body to perform various physiological processes and to maintain homeostasis (Guyton and Hall, 2000). In healthy, non-diabetic subjects, plasma glucose concentrations are held within a narrow range throughout the day, despite wide fluctuations in nutritional intake and physical exercise, as well as other physiological, psychological and iatrogenic influences (Owens, 2002). The purpose of this research study was to determine the effect of the homoeopathic preparations, Saccharum officinale 9cH and 200cH, on glucose metabolism in healthy, non-diabetic humans. This research study was a double blind, placebo-controlled clinical trial. A group of thirty participants were required to undergo an oral glucose tolerance test for three hours. Timing started as soon as the participants started drinking the glucose solution. In addition, ten drops of the homoeopathically prepared medicine or a placebo were administered at each of the following times: -15 minutes, 27 minutes, 87 minutes and 147 minutes. Group one received the placebo (20% alcohol), Group two received Saccharum officinale 9cH and Group three received Saccharum officinale 200cH. Blood glucose concentrations were measured, using capillary blood samples and a glucose meter, at the following times: -30 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, and 180 minutes. Vital signs were measured at: 10 minutes, 50 minutes, 110 minutes and 170 minutes in order to ascertain any detrimental changes in health. Data was expressed as mean ± standard error. Differences between the groups were determined using the one-way repeated measures analysis of variance method. The hypoglycaemic effect of Saccharum officinale 9cH and 200cH was not proven to be effective in reducing the rate of glucose disposal in the body. Even though a slight difference between the experimental groups and the control group was observed, these changes could not be attributed to the therapeutic effect of the remedy and was regarded as statistically insignificant. / Dr. Natasha Wolf Mr. Neil de Villiers

2-deoxy-D-glucose (2DG), a non-metabolizable glucose analog, is currently being used in clinical trials to determine its efficacy in augmenting radiotherapy and chemotherapy of various cancers. It is thought to kill cancer cells by inducing glucose deprivation state. However, 2DG only inhibits glycolysis by 15-40%, not sufficient to simulate glucose deprivation. Further, 2-flour-deoxy-D-glucose (2FDG), which is a more potent inhibitor of glycolysis than 2DG, is less effective than 2DG in killing cancer cells. These observations suggest that glucose deprivation is not the mechanism by which 2DG kills cancer cells. On the other hand, it has been shown that treatment of cancer cells with 2DG leads to increased reactive oxygen species (ROS) production, indicating that cytotoxic effect of 2DG is due to increase ROS. Our lab previously found that inhibition of aldose reductase (AR) activity attenuated 2DG-induced ROS in cardiomyocytes (Tang, et al., 2010). We therefore propose that 2DG-induced ROS increase in cancer cells is a consequence of the depletion of GSH in the process of reduction of 2DG by AR and/or by a related aldose reductase-like enzyme (ARL). These two enzymes are often overexpressed in cancer cells. This proposed project is to test our hypothesis that the cytotoxicity of 2DG is due to the depletion of GSH as a consequence of the reduction of 2DG by AR or ARL. We found that HepG2, SKOV3, HCT116 and CaCo2 cells were sensitive to 2DG, and these cells over-express AR and/or ARL proteins. However, HT29 cells and SW480 cells, which were not sensitive to 2DG, had low level of AR and ARL proteins, indicating that there is a close relationship between sensitivity to 2DG toxicity and the level of AR/ARL in these cells. Further, when AR/ARL activity were inhibited in HepG2, SKOV3, HCT116 and CaCo2 cells by AR/ARL inhibitors fidarestat or tolrestat, the cells were protected against 2DG cytotoxicity. Tolrestat or fidarestat significantly restored the drop of GSH levels in 2DG sensitive cancer cells induced by 2DG. On the other hand, MG-132 and bortezomib, which increased the expression of AR/ARL in HT29 and SW480 cells, made HT29 and SW480 cells more sensitive to 2DG. These experiments confirmed our hypothesis that 2DG toxicity in cancer cells was due to oxidative stress induced by AR/ARL. 2DG is not an efficient substrate for AR/ARL enzymes and it is not very efficient in killing cancer cells. Based on our hypothesis, better AR/ARL substrates should be more toxic to cancer cells that overexpress AR/ARL than 2DG. The cytotoxic effects of glyceraldehyde and diacetyl, which were better substrates for AR/ARL than 2DG, were tested. Both glyceraldehyde and diacetyl were more efficient in killing cancer cells that over-express AR and/or ARL (HepG2, SKOV3, HCT116 and CaCo2) than cancer cells with low levels of AR and ARL proteins (HT29 and SW480). Glyceraldehyde and diacetyl were more efficient in lowering the GSH level in cancer cells that over-express AR and/or ARL. In order to further develop glyceraldehyde and diacetyl as anti-cancer drugs, animal studies were carried out to determine their anti-cancer effects. Both glyceraldehyde and diacetyl significantly inhibited the tumor growth in nude mice tumor xenograft model. In conclusion, this thesis proposed and proved that 2DG kills cancer cells by lowering intracellular GSH levels as a consequence of its reduction by AR/ARL activities, rather than by inhibition of glycolysis. This novel mechanism predicts that better substrates for AR/ARL than 2DG would be more effective in killing cancer cells than 2DG. This was confirmed by using glyceraldehyde and diacetyl. We believe that this would lead to the development of more efficient anti-cancer drugs.

The long terra effects of glucose on the rate of (pro) insulin biosynthesis and the amount of preproinsulin mRNA in rat islets maintained in tissue culture were investigated. The rate of (pro)insulin synthesis was 35 times greater in islets cultured for 6 days in 8 mM glucose than it was in islets cultured in 4 mM glucose. The preproinsulin mRNA content at this time was 2 fold greater in islets incubated with 8 mM glucose compared to 4 mM glucose. The rate of (pro)insulin synthesis and the preproinsulin mRNA content of islets cultured at 8 mM glucose were maximal since no further significant increases were observed in islets cultured at 16 mM glucose for 6 days. These results indicate that the long term effects of glucose on the rate of (pro)insulin synthesis in rat islets of Langerhans is mediated both by transcriptional and translational events and that translational events exert the major controlling influence.

Class of 2013 Abstract / Specific Aims: The objective of this study is to evaluate the effect of early blood glucose correction in the Emergency Department (ED) on hospital length of stay. Methods: This study has received institutional review board approval. This is a retrospective cohort study conducted in an academic medical institution. Diabetic patients with hyperglycemia in the ED between June 1st, 2011 and June 30th, 2012 were included. Patients were excluded if they were less than 18 or greater than 89 years of age, not admitted, had diabetic ketoacidosis or hyperglycemic hyperosmolar state, treated with insulin for hyperkalemia, trauma patients, or had an initial blood glucose value of 200 mg/dL or less. Patients were categorized into two groups based on blood glucose control achieved within the first 24 hours from triage. The primary outcome of this study was to compare hospital length of stay between the groups. Main Results: A total of 161 patients were included in this study. Baseline demographics between groups were statistically similar with the exception of gender (p=0.635), ethnicity (p = 0.149), and co-morbidities calculated by the Charlson Co-Morbidity Score (p = 0.112). Blood glucose values in the ED did not statistically correlate to hospital length of stay (p = 0.299), however, co-morbidities were predictive of hospital length of stay (p = 0.025). Conclusion: Early correction of blood glucose values in the ED are not associated with hospital length of stay.

No description available.

A novel glucose oxidase from Penicillium canescens (Tt42) was isolated, purified and characterised. The P. canescens Tt42 was cultivated using an optimised growth medium from literature, and maximum glucose oxidase activities of 11.5 U/ml and 6.9 U/ml for the intra- and extracellular fractions were obtained. Maximum glucose oxidase production was achieved after 72 hours at 28°C which coincided with glucose depletion. A total of 1104 U (from 60ml) of glucose oxidase was produced with a biomass specific glucose oxidase activity of 1.08 Umg[superscript -1] Four methods of cell disruption were evaluated for release of intracellular glucose oxidase from P. canescens Tt42 cells. These methods were; sonication, French press, Freeze-Thaw and a high pressure cell disrupter (Z-Plus Series) from Constant systems. All the methods were successful in releasing the intracellular glucose oxidase from P. canescens Tt42. The use of the Constant Systems high pressure cell disrupter was preferred, since it was the simplest and most rapid method. Ammonium sulphate precipitation was shown to be effective as an initial purification step for extracellular glucose oxidase from P. canescens Tt42. Comparison of the intra- and extracellular glucose oxidase fractions using isoelectric focusing showed 2 isoenzymes in both fractions. The pI values of the isoenzymes were determined to be 4.30 and 4.67, with the former being dominant. Since both the intra- and extracellular fractions contained the same isoenzymes of glucose oxidase, further purification studies were performed using the extracellular fraction. The glucose oxidase from P. canescens Tt42 was purified using 3 main techniques: ammonium sulphate precipitation (60% - 70% cut), anion exchange chromatography (Super Q 650M) and size exclusion chromatography (Sephadex S200HR). The glucose oxidase was determined to be ±80% pure by size exclusion chromatography. The final purified glucose oxidase was lyophilised, and an overall purification yield of 10.3% was achieved with an 8.6-fold purification. The purified glucose oxidase was confirmed to be catalase free. Glucose oxidase from P. canescens Tt42 was determined to be a dimeric protein (M[subscript r] ±148kDa) likely consisting of 2 equal subunits (M[subscript r] ± 70kDa). The temperature optimum range was shown to be 25-30°C. The optimum pH for the oxidation of β-D-glucose was pH 7. The enzyme was shown to be stable at 25°C for 10 hours, with a half life of approximately 30 minutes at 37°C. The lyophilised enzyme was stable at -20°C for 6 months. The properties of glucose oxidase from Tt42 were comparable to alternative glucose oxidase enzymes from Aspergillus and other Penicillium species. Glucose oxidase from P. canescens Tt42 was shown to have distinct kinetic characteristics. The V[subscript max] and K[subscript m] were shown to be 651 Umg[superscript -1] and 18.4 mM towards β-D-glucose. The catalytic kcat and specificity k[subscript cat]/K[subscript m] constants for glucose oxidase from P. canescens Tt42 were shown to be 791 s[superscript -1] and 40 s[superscript -1]mM[superscript -1] each respectively. The specificity constant (k[subscript cat]/K[subscript m]) of glucose oxidase from P. canescens Tt42 was determined to be 1.3-fold higher than that that of A. niger (Sigma Type VII) and 8.7-fold lower than that of P. amagasakiense (ATCC 28686) from literature.

Triprolidine hydrochloride (C₁₉H₂₂N₂.HCl.H₂O) (TPH) is a well-known antihistamine drug. It melts between 118°C and 122°C and the amount of water present is 4.5 mass percent. TPH is reported as being photosensitive and must be stored in sealed, light-tight containers. The thermal stabilities of TPH and of 1:1 molar and 1:1 mass ratio physical mixtures of TPH with beta-cyclodextrin (BCD) and with glucose have been examined using DSC, TG and TG-FTIR, complemented by X-ray powder diffraction (XRD) and infrared spectroscopic (IR) studies. Thermal studies of the solid TPH/BCD mixtures indicated that interaction between the components occurs and it is possible that the TPH molecule may be least partially accommodated in the cavity of the BCD host molecule. XRD results support this indication of inclusion. The results for mixtures of TPH/glucose also suggest that there is interaction between the two components. The results of molecular modelling suggest that TPH is most likely to be accommodated in the BCD cavity as a neutral triprolidine molecule with the toluene portion of the molecule entering first. There is also an indication that the Z-isomer should be accommodated slightly more readily than the E-isomer. Photostability studies were done by irradiating thin layers of solid samples of TPH and its mixtures for various times at 40°C using an Atlas Sun test CPS lamp operating at 550 W h m⁻². An analytical method using HPLC was developed and validated to determine the amounts of any photodegradants. DSC, TG, FTIR, XRD and IR were also used examine the irradiated samples. XRD results showed that changes in the TPH crystal structure occurred during irradiation and that these changes increased with the time of irradiation. Irradiation for 20 hours with UV or exposure to sunlight showed the presence of degradants. The results obtained illustrate the general stability of TPH, especially in the solid state. Although the potential for isomerization to the pharmaceutically inactive Z-isomer exists, this transformation would require extreme light conditions. The study has also shown TPH to be compatible with both glucose and BCD, which are potential excipients both in solid and liquid dosage forms. The presents of these excipients in dosage forms will thus not adversely affect the stability and the therapeutic efficacy of TPH. . An analytical method using HPLC was developed and validated to determine the amounts of any photodegradants. DSC, TG, FTIR, XRD and IR were also used examine the irradiated samples. XRD results showed that changes in the TPH crystal structure occurred during irradiation and that these changes increased with the time of irradiation. Irradiation for 20 hours with UV or exposure to sunlight showed the presence of degradants.

Diabetes Mellitus is a common chronic disease that is an ever-increasing public health issue. Continuous glucose monitoring has been shown to help diabetes mellitus patients stabilize their glucose levels, leading to improved patient health. Hence, a glucose sensor, capable of continuous real-time monitoring, has been a topic of research for three decades. Current methods of glucose monitoring, however, require taking blood samples several times a day, hence patient compliance is an issue. Optical methods are one of the painless and promising methods that can be used for blood glucose predictions. However, having accuracies lower than what is acceptable clinically has been a major concern. To improve on the accuracy of the predictions, the signal-to-noise ratio in the spectrum can be increased, for which the use of thermally tunable vertical cavity surface emitting lasers (VCSELs) as the light source to obtain blood absorption spectra, along with a multivariate technique (Partial Least Square (PLS) techniques) for analysis, is proposed. VCSELs are semiconductor lasers with small dimensions and low power consumption, which makes them suitable for implants. VCSELs provide higher signal-to-noise ratio as they have high power spectral density and operate within a small spectrum. In the current research, experiments were run for the preliminary investigations to demonstrate the feasibility of the proposed technique for glucose monitoring. This research involves preliminary investigations for developing a novel optical system for accurate measurement of glucose concentration. Experiments in aqueous glucose solutions were designed to demonstrate the feasibility of the proposed technique for glucose monitoring. In addition, multivariate techniques, such as PLS, were customized for various specific purposes of this project and its preliminary investigation. This research will lead to the development of a small, low power, implantable optical sensor for diabetes patients, which will be a major breakthrough in the area of treating diabetes patients, upon successful completion of this research and development of the device. / Applied Science, Faculty of / Graduate

Diabetes associated complications make management during exercise complex (Brugnara, Vinaixa, Murillo, Samino, Rodriguez, Beltran, Lerin, Davison, Correig & Novials, 2012). Research on the prevention of such challenges is of paramount importance. The aim of this study was to determine the effects of three different carbohydrate supplementation protocols on blood glucose levels after every 10 minutes of a 60 minute exercise bout at 65 to 75 % HRR on the treadmill as well as every half hour during a two hour post exercise recovery period. The three protocols implemented after a standardized pre-exercise meal were: control protocol (no carbohydrate supplementation), protocol 1 (one carbohydrate supplementation of 15 grams given at 30 minutes) and protocol 2 (two carbohydrate supplementation of 15 grams given at 30 minutes and 45 minutes). A total of 32 participants took part in the study (Mean age: 32.84 ±12.12). All participants were submitted to all three protocols. Statistical and practical significant differences were found between blood glucose levels of protocol 0 and protocol 1 (MDIF = 2.62 ± 3.99 mmol.L--‐1) at 20 minutes of the exercise duration (p=.024;d=0.42). Statistical and practical significant differences in blood glucose levels with protocol 0 rendering the higher glucose values were also found between protocols 0 and 2 at 10 minutes (MDIF = 3.44 ± 5.54 mmol.L--‐1; p=.001;d=0.62), 20 minutes (MDIF = 3.32 ± 5.23 mmol.L--‐1; p=.001;d=0.63) and 30 minutes of exercise (MDIF = 2.81 ± 5.40 mmol.L--‐1; p=.006;d=0.52) as well as between the mean minimum (M0 = 9.49 ± 4.51 mmol.L--‐1 and M2 = 7.28 ± 4.07 mmol.L--‐1; p=.013;d=0.46), mean maximum (M0 = 12.73 ± 5.51 mmol.L--‐1 and M2 = 10.07 ± 4.63 mmol.L--‐1; p=.015;d=0.46) and overall mean (M0 = 9.07 ± 4.88 mmol.L--‐1 and M2 = 8.53 ± 4.25 mmol.L--‐1; p=.011;d=0.48) with protocol 0 rendering the higher glucose values in all these comparisons. It was concluded that carbohydrate supplementation during exercise affects blood glucose levels positively particularly considering the significant difference found between protocol 0 and 2. Whilst protocol 2 also resulted in less fluctuations in the blood glucose levels during exercise and minimum, overall mean and maximum blood glucose values were closer to “normal/safe” range, there was no conclusive evidence that protocol 2 was better than protocol 1.