The increasing prevalence of diabetes and the severity of long-term complications have emphasized the need for continuous glucose monitoring. Optically-based methods are advantageous as they have potential for noninvasive or minimally invasive detection. Fluorescence-based affinity assays, in particular, can be fast, reagentless, and highly specific. Poly(ethylene glycol) (PEG) microspheres have been used to encapsulate such fluorescently labeled molecules in a hydrogel matrix for implantation into the body. The matrix is designed to retain the sensing molecules while simultaneously allowing sufficient analyte diffusion. Sensing assays which depend upon a spatial displacement of molecules, however, experience limited motility and diminished sensor response in a dense matrix. In order to overcome this, a process of hydrogel microporation has been developed to create cavities within the PEG that contain the assay components in solution, providing improved motility for large sensing elements, while limiting leaching and increasing sensor lifetime. For an implanted sensor to be successful in vivo, it should exhibit long-term stability and functionality. Even biocompatible materials that have no toxic effect on surrounding tissues elicit a host response. Over time, a fibrous capsule forms around the implant, slowing diffusion of the target analyte to the sensor and limiting optical signal propagation. To prevent this biofouling, a thermoresponsive nanocomposite hydrogel based on poly(N-isopropylacrylamide) was developed to create a self-cleaning sensor membrane. These hydrogels exist in a swollen state at temperatures below the volume phase transition temperature (VPTT) and become increasingly hydrophobic as the temperature is raised. Upon thermal cycling around the VPTT, these hydrogels exhibit significant cell release in vitro. However, the VPTT of the original formula was around 33-34 degrees C, resulting in a gel that is in a collapsed state, ultimately limiting glucose diffusion at body temperature. The hydrogel was modified by introducing a hydrophilic comonomer, N-vinylpyrrolidone (NVP), to raise the VPTT above body temperature. The new formulation was optimized with regard to diffusion, mechanical strength, and cell releasing capabilities under physiological conditions. Overall, this system is a promising method to translate a glucose-sensitive assay from the cuvette to the clinic for minimally invasive continuous glucose sensing.

This study determined the effects of gestational obesity on the long-term metabolic status of the mother and if obesity management during or after pregnancy could attenuate these effects. At 120 days prior to estrus, 8 ewes received 100 percent of NRC nutrient requirements (control group) and 24 ewes had free access to feed (obesity induction). Beginning on day 42 of gestation, 8 obese ewes were restricted to 65 percent of NRC nutrient requirements. Following parturition, controls and all but one group of obese ewes were fed 100 percent of NRC nutrient requirements. At postpartum days (PPD) 1 and 150, glucose tolerance tests were administered to ewes. At both PPD1 and PPD150, obesity resulted in insulin resistance, impairment of whole-body glucose utilization, increased levels of circulating leptin, and altered profiles of amino acids in plasma; however, these effects were diminished in ewes receiving obesity management during or after gestation. Additionally at PPD150, obesity increased the circulating levels of ammonia and urea in ewes, which was prevented by realimentation to 100 percent NRC requirements. These results indicate that weight reduction in obese dams during pregnancy or after parturition can beneficially ameliorate the adverse effects of gestational obesity on the mother.

The equilibria of adsorption on calcium and hydrogen forms of zeolite Y by equimolar solutions of 12.5 %, 20%, 25%, 30% and 35% w/v of mixtures of glucose, G and fructose, F / also the non-equimolar mixtures of 20% w/v glucose - 30% w/v fructose, 30% w/v glucose - 20% w/v fructose, 25% w/v glucose &ndash / 35% w/v fructose, and 35% w/v glucose-25% w/v fructose solutions, which were prepared 24 hours in advance at the experimental temperature, have been studied batch wise at 50&ordm / C. Glucose adsorption, in solutions that had adsorption differences, was fast on both zeolites, on the contrary of slow adsorption of fructose with the stable dynamics. Both adsorptions had small amounts of adsorption changes after minute 30. The treatments made under the same conditions with the same mixtures showed Ca-Y zeolite had better separation capacity compared to H-Y zeolite. Some trials were repeated with CaCl2 added to the solutions. The slowed down affection of fructose adsorption in spite of the small change of glucose adsorption led to better separation. Samples were analyzed by classical methods, not HPLC. All the data were considered with various models and their convergence numbers were tested for their closeness to reality. The models were analyzed by response surface methodology and some of those models had correlation factors as high as 88% at the equilibrium points at 30th minutes. Besides, time dependent models have been considering the lag times with a time dependent variable included all the data of all treated solutions with correlation as high as 79.5%.

The aim of this study is to develop a metabolically engineered P. pastoris strain for production of an active extracellular thermostable glucose isomerase (GI) enzyme by using genetic engineering techniques. For this purpose, research program was performed in two sub-programs. In the first sub-program, xylA gene from Thermus thermophilus was amplified and inserted into pPICZ&alpha / -A expression vector. Thereafter, this pPICZ&alpha / -A::xylA vector was cloned into AOX1 locus in P. pastoris genome and expressed under alcohol oxidase promoter which is induced by methanol. After constructing the recombinant P. pastoris strains, the best producing strain was selected according to the specific enzyme activity assay and SDS-PAGE analyses in batch shaker-bioreactor experiments. The selected recombinant P. pastoris clone carrying xylA gene in its genome was named as eP20. Using recombinant P. pastoris eP20 clone, effects of salt and sorbitol concentration on the cell growth and recombinant GI activity were investigated. The data obtained from the experiments showed that the maximum cell and GI activity values were obtained in production medium that contained 30 g L-1 sorbitol, 4.35 g L-1 ammonium sulphate, 0.1 M potassium phosphate buffer (pH 6.0), 14.9 g L-1 MgSO4&bull / 7H2O, 1.17 g L-1 CaSO4 &bull / 2H2O, 1 ml L-1 chloramphenicol and 4.35 ml L-1 PTM1 / where, the maximum biomass and recombinant GI activity were calculated , respectively, as 6.3 g L-1 and 3.21 U L-1. Moreover, the research program related with the effect of initial sorbitol concentration shows that optimum initial sorbitol concentration, CS0 is 50 g L-1 that resulted a cell concentration and recombinant GI activity which are 7.32 g L-1 and 3.6 U L-1, respectively. In the second part of the M.Sc. of the study, a pilot scale bioreactor experiment in a working volume of 1 L was performed in controlled bioreactor system. The variations in the cell growth, recombinant GI activity, AOX activity, total protease activity and organic acid concentrations throughout the fermentation were analyzed whereas the specific growth rates, yield coefficients and specific consumption rates were also calculated. The results showed that a pH and oxygen controlled operation enabled an important increase in recombinant GI activity. In this context, recombinant GI activity was increased as 56.1-fold and resulted in 202.8 U L-1 at t=12 whereas the maximum biomass concentration was obtained as 85.2 g L-1 at t=36. In this study, an active thermostable recombinant GI enzyme was produced extracellularly by a yeast cell, i.e. recombinant P. pastoris, for the first time.

"Uniaxial Cyclic Stretch-Stimulated Glucose Transport Is Mediated by a Ca2+-Dependent Mechanism in Cultured Skeletal Muscle Cells" Pathobiology, v.74, n.3, pp.159-168を、博士論文として提出したもの。 / 名古屋大学博士学位論文 学位の種類:博士（リハビリテーション療法学）（課程）学位授与年月日:平成19年3月23日

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Heart-type Fatty Acid-Binding Protein (H-FABP) is required for high rates of skeletal muscle long chain fatty acid (LCFA) oxidation and esterification. Here we assessed whether H-FABP affects soleus muscle glucose uptake when measured in vitro in the absence of LCFA. Wild type and H-FABP null mice were fed a standard chow or high fat diet before muscle isolation. With the chow, the mutation increased insulin-dependent deoxyglucose uptake by 141% (P<0.01) at 0.02 mU/ml of insulin, but did not cause a significant effect at 2 mU/ml insulin; skeletal muscle triglyceride and long chain acyl-CoA (LCACoA) levels remained normal. With the fat diet, the mutation increased insulin-dependent deoxyglucose uptake by 190% (P<0.01) at 2 mU/ml insulin, thus partially preventing insulin resistance, and completely prevented the threefold (P<0.001) diet-induced increase of muscle triglyceride levels; however, muscle LCACoA levels showed little or no reduction. With both diets, the mutation reduced the basal (insulinindependent) soleus muscle deoxyglucose uptake by 28% (P<0.05). These results establish a close relationship of FABP-dependent lipid pools with insulin sensitivity, and indicate the existence of a non-acute, antagonistic, and H-FABP-dependent fatty acid regulation of basal and insulin-dependent muscle glucose uptake. Liver fatty acid binding protein (L-FABP) has been proposed to limit the availability of chain LCFA for oxidation and for peroxisome proliferator-activated receptor (PPAR-alpha), a fatty acid binding transcription factor that determines the capacity of hepatic fatty acid oxidation. Here, we used L-FABP null mice to test this hypothesis. Under fasting conditions, this mutation reduced &#946;-hydroxybutyrate (BHB) plasma levels as well as BHB release and palmitic acid oxidation by isolated hepatocytes. However, the capacity for ketogenesis was not reduced: BHB plasma levels were restored by octanoate injection; BHB production and palmitic acid oxidation were normal in liver homogenates; and hepatic expression of key PPAR-alpha target (MCAD, mitochondrial HMG CoA synthase, ACO, CYP4A3) and other (CPT1, LCAD) genes of mitochondrial and extramitochondrial LCFA oxidation and ketogenesis remained at wild-type levels. These results suggest that under fasting conditions, hepatic L-FABP contributes to hepatic LCFA oxidation and ketogenesis by a nontranscriptional mechanism.

Le paludisme, maladie provoquée par le parasite Plasmodium, tue chaque année de 1,5 à 2,7 millions de personnes dans le monde. La découverte d'une protéine spécifique (PfHT) responsable du transport du sucre nécessaire au parasite pour se développer au sein des globules rouges qu'il infecte, laisse envisager que cette protéine puisse devenir une nouvelle cible thérapeutique.<br />Le présent travail a concerné la préparation d'une trentaine de dérivés du glucose diversement substitués en -3, afin de mieux connaître l'influence de la nature, ainsi que de l'hydrophilie ou de l'hydrophobie de ces substituants, sur l'inhibition de la protéine de transport des sucres chez Plasmodium. Par ailleurs, un même substituant (le motif undec-10-enyle) a été introduit de façon systématique sur les autres positions du glucose, tant sur les oxygènes que sur les carbones.

Thesis (Ph. D.)--Case Western Reserve University, 2004. / [School of Medicine] Department of Pathology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph. D.)--Institute of Paper Chemistry, 1964. / Includes bibliographical references (p. 79-82).