Дисертації з теми "Glucose"

Since the pioneering work of Altman in the late 60's, much has been learned about the generation of neurons in the adult brains of several species, including mice, rats, and humans. An underlying assumption is that these newborn neurons acquire their energy, in the form of glucose, in a similar manner to mature neurons: via glucose transporters. Using BRDU and double immunohistochemistry, we investigated the relationship between hippocampal neurogenesis and glucose transporters, as well as monocarboxylate transporters. Unexpectedly, the results suggest that newborn neurons do not acquire their energy via the major glucose transporters (1, 3, 4, and 8), nor via either monocarboxylate transporter tested (1 and 2). Future studies will have to resolve whether lesser known glucose transporters carry this function or if other mechanisms are used to provide metabolic energy to newborn neurons.

The electrooxidation of glucose has attracted a lot of interest due to its applications in blood glucose sensors and biological fuel cells. Glucose sensors optimization is highly necessary to improve the treatment of Diabetes Mellitus, a chronic disease affecting millions of people around the world, while biological fuel cells have been studied in order to explore new, renewable energy sources alternative to fossil fuels. There are three main ways to perform glucose electrooxidation, depending on the active oxidant agent or mediator employed: enzymatic electrooxidation utilizes enzymes such as glucose oxidase and glucose dehydrogenase in their isolated forms; abiotical electrooxidation makes use of non-biological catalysts e.g., noble metals and microbial electrooxidation employs the whole enzymatic system of an electroactive microorganism. Many researchers in the past focused on the utilization of enzymes to facilitate the process of glucose oxidation, however the limited enzyme stability and difficult immobilization procedures impede long term applications. During my PhD training I worked on both abiotical and microbial electrocatalysis; the former (at gold electrodes) applied to glucose sensing and glucose-gluconate fuel cells, the latter for the development of microbial fuel cells (MFCs). The complex oxidation of glucose at the surface of gold electrodes was studied in detail in different conditions of pH, buffer and halide concentration. As observed in previous studies, an oxidative current peak occurs during the cathodic sweep showing a highly linear dependence on glucose concentration, when other electrolyte conditions are unchanged. The effect of the different conditions on the intensity of this peak has stressed the limitations of the previously proposed mechanisms. A mechanism able to explain the presence of this oxidative peak was proposed in which the key step is the competitive adsorption at the active sites of the ionic species present in the solution (phosphate buffer, chlorides and OH-) and the substrate (glucose). Simulations of the proposed mechanism have supported the plausibility of the mechanism. The application of the above mentioned peak in blood glucose sensing has been hindered by the presence of inhibitors: chlorides amino acids and human albumin. Among them chlorides are the most problematic because of their high concentration in the blood (about 0.1 M) and the difficulty inherent in trying to separate them from glucose. In order to overcome this problem we developed a four-step, three electrode (silver gauze, gold pin and platinum counter electrode) technique. In the first step a silver gauze working electrode is oxidized to silver chloride, while water is reduced at the platinum counter electrode. In the overall reaction, for every chloride removed, a hydroxide ion is generated shifting the solution pH from 7.4 to 11.5. In the second step the gold pin electrode surface is oxidized to gold hydroxide and subsequently reduced in the third step: once the gold surface is regenerated, glucose can be re-adsorbed and oxidized giving rise to the sensing peak. In the last (fourth) step, the silver gauze (partially covered with silver chloride from step 1) is reduced and regenerated, ready for the next sensing. For the first time, an electrochemical glucose sensor able to work in the presence of chlorides and with higher accuracies and sensitivities than an enzymatic device was proposed. All the materials used in the prototype (silver, platinum and gold) are fully bio-compatible thus prefiguring an application in implantable glucose sensors, the future glucose meters technology. The direct oxidation of glucose to produce electrical energy has been widely investigated because of renewability, abundance, high energy density and easy handling of the carbohydrate. Most of the previous studies were conducted in extreme conditions in order to achieve complete glucose oxidation to CO2, neglecting the carbohydrate chemical instability that generally leads to useless by-products mixtures. Instead the partial oxidation to gluconate, originally studied for implantable fuel cells, has the advantage of generating a commercially valuable chemical. For this reason, we started optimizing fuel composition and operating conditions in order to selectively oxidize glucose to gluconate, maximizing the power density output of a standard commercial platinum based anode material. A deep electrochemical characterization concerning reversible potential, cyclic voltammetry and overpotential measurements have been carried out at 25°C in the D-(+)-glucose concentration range 0.01 to 1.0M. NMR and EIS investigation clarified the role of the buffer (Na2HPO4/NaH2PO4) in enhancing the electrochemical performance: it changes the reaction rates and steps; it increases the amount of β form of glucose; it increases the conductivity of the solution; it may adsorb at the platinum surface of platinum and subtract active sites for the electrooxidation of glucose as already highlighted for gold electrodes. Moreover the presence of the buffer not only stabilizes the potential, but also improves the electrochemical performances of the anode in term of exchange current density. Such behavior is not ascribable to the chemical interaction with glucose, as demonstrated by NMR measurements, but to the interaction with the anode material as indicated by the decrease of all the resistive components in the EIS measurement. In order to improve the anode performance of the previously discussed glucose-gluconate FCs, the electrocatalytic properties of nanostructured gold electrodes were investigated, by cyclic voltammetry, and compared with commercially available polycrystalline gold electrodes. These nanostructured electrodes were prepared by depositing gold nanoparticles from a colloidal dispersion (sol) onto different carbonaceous conductive supports: glassy carbon, carbon cloth and graphite paper. The gold sol was prepared reducing an aqueous solution of tetrachloroauric acid with sodium borohydride. The gold particles (average size 100 nm) exhibit better electrocatalytic properties with respect to commercially available polycrystalline electrode for glucose oxidation. A surface treatment of the carbonaceous conductive supports with warm concentrated nitric acid resulted in an improved adhesion of the gold nanoparticles. Gold on treated carbon cloth turned out to be a very promising anode for glucose electrooxidation. On the basis of the information acquired in the above mentioned studies, we came out with a new anode for glucose-gluconate direct oxidation fuel cells prepared by electrodeposition of gold nanoparticles on what we called a “conductive energy textile” realized by conformally coating polyester textile substrates with single walled carbon nanotubes (SWNT). The electrodeposition conditions have been optimized in order to obtain uniform distribution of gold nanoparticles in the 3D porous structure of the conductive textile. The electrochemical characterization, carried out by means of cyclic voltammetry, showed higher current densities with respect to the previously reported materials. As previously mentioned I also worked on microbial glucose electrooxidation applied to microbial fuel cells. As a new member in the fuel cell family, microbial fuel cells (MFCs) are devices that convert chemical energy into electrical energy by the catalytic activity of microorganisms. The most promising application of this technology is to harvest energy from undesirable fuel sources, such as the organic matter in domestic wastewater, marine sediment, or human excrement in space. A novel carbon nanotube-cotton (CNT-cotton) composite material with high conductivity and high porosity was proposed to be used as anode for achieving high-performance MFCs. Scanning electron microscope (SEM) images of microorganisms growing on the CNT layer provided the direct evidence to support the biocompatibility of the CNTs and their feasibility to be used as the anode in MFCs. The randomly intertwined CNT-cotton fibers create a 3D active space for biofilm growth, and meanwhile, the incompact macroporous structure allows efficient mass transfer for microbial metabolism inside the anode. Furthermore, the coated CNTs significantly improve the mechanical binding as well as the electrical conductivity between the exoelectrogenic microorganisms and the CNT-cotton anode. Compared to commercial carbon cloth anode, the CNT-cotton anode achieves 64% higher power density and 75% higher energy recovery efficiency in MFCs. Air is considered to be the most suitable oxidant for field scale MFCs, because it is free and inexhaustible. However, the oxygen reduction efficiency is highly constrained by the specific operating conditions of MFCs, such as ambient temperature and mostly neutral pH. Thus, cathode performance often limits the power output of MFCs. Moreover, cathode usually accounts for the greatest part of the total capital cost of a MFC, mainly because of the use of precious metal catalyst like Pt. Therefore, improving the cathode performance decreasing the catalyst loading represents a critical issue for researchers working on MFCs. A new CNT-textile-Pt cathode specially designed for aqueous-cathode MFCs was obtained by electrochemically depositing Pt nanoparticles on a macroporous CNT-textile substrate. This CNT-textile-Pt cathode shows two orders higher of surface area utilization efficiency than the commercial carbon cloth (CC)-Pt cathode. Assisted by the additional catalytic activity of CNTs, the MFCs equipped with CNT-textile-Pt cathodes achieve higher power density (2.14-fold) with lower Pt loading (19.3%). Moreover, the synthesis process of CNT-textile-Pt is simple and scalable. Thus, CNT-textile-Pt is promising to function as cathodes for large scale high performance aqueous-cathode MFCs. In parallel to glucose electrooxidation a new stretchable, porous conductive energy textile has been developed. Recently there is strong interest in lightweight, flexible and wearable electronics to meet the technological demands of modern society. Integrated energy storage devices of this type are a key area that is still significantly underdeveloped. We developed wearable power devices using everyday textiles as the platform. With an extremely simple “dipping and drying” process using single-walled carbon nanotube (SWNT) ink, we produced highly conductive textiles with conductivity of 125 S cm-1 and sheet resistance less than 1 Ω/sq. Such conductive textiles show outstanding flexibility and stretchability, and demonstrate strong adhesion between the SWNTs and the textiles of interest. Supercapacitors (SC) made from these conductive textiles show high areal capacitance, up to 0.48 F cm-2, and high specific energy. We demonstrated that the loading of pseudocapacitor materials into these conductive textiles leads to a twenty four-fold increase of the areal capacitance of the device. Moreover, supercapacitors have been fabricated using the conductive energy textile as both active material and current collector (resistance lower than 1 Ω/sq). The device has excellent cycling performance (good capacity retention after 35,000 cycles) and high specific capacitance (70–80 F g-1 at 0.1 mA cm–2). The as prepared device is fully wearable since both the textile (cotton) and the lithium sulfate electrolyte are compatible with the human body. It can also be integrated into wearable devices. By means of impedance spectroscopy and differential curves, we have highlighted an additional reversible capacitance due to a slow ionsorption process.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2011.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 157-164).
A holy grail of bioelectronics is to engineer biologically implantable systems that can be embedded without disturbing their local environments, while harvesting from their surroundings all of the power they require. As implantable electronic devices become increasingly prevalent in scientific research and in the diagnosis, management, and treatment of human disease, there is correspondingly increasing demand for devices with unlimited functional lifetimes that integrate seamlessly with their hosts in these two ways. This thesis presents significant progress toward establishing the feasibility of one such system: A brain-machine interface powered by a bioimplantable fuel cell that harvests energy from extracellular glucose in the cerebrospinal fluid surrounding the brain. The first part of this thesis describes a set of biomimetic algorithms and low-power circuit architectures for decoding electrical signals from ensembles of neurons in the brain. The decoders are intended for use in the context of neural rehabilitation, to provide paralyzed or otherwise disabled patients with instantaneous, natural, thought-based control of robotic prosthetic limbs and other external devices. This thesis presents a detailed discussion of the decoding algorithms, descriptions of the low-power analog and digital circuit architectures used to implement the decoders, and results validating their performance when applied to decode real neural data. A major constraint on brain-implanted electronic devices is the requirement that they consume and dissipate very little power, so as not to damage surrounding brain tissue. The systems described here address that constraint, computing in the style of biological neural networks, and using arithmetic-free, purely logical primitives to establish universal computing architectures for neural decoding. The second part of this thesis describes the development of an implantable fuel cell powered by extracellular glucose at concentrations such as those found in the cerebrospinal fluid surrounding the brain. The theoretical foundations, details of design and fabrication, mechanical and electrochemical characterization, as well as in vitro performance data for the fuel cell are presented.
by Benjamin Isaac Rapoport.
Ph.D.

Critically ill patients are known to experience stress-induced hyperglycemia. Inhibiting the physiological response to increased glycaemic levels in these patients are factors such as increased insulin resistance, increased dextrose input, absolute or relative insulin deficiency, and drug therapy. Although hyperglycemia can be a marker for severity of illness, it can also worsen outcomes, leading to an increased risk of further complications. Recent studies have shown that tight control can reduce mortality up to 43%. Metabolic modelling has been used to study physiological behaviour and/or to control glycaemia for a long time and many successful approximate system models have been developed. Due to the malfunction of medical equipments, clinical measurements obtained usually come with noise. In addition, the few such systems currently available can have errors in excess of 20-30%. Therefore, to fully simulate the clinical data, the system model also needs to couple with a successful noise model. This research has developed a new noise model that better fits the current available statistical description of the noise profile and therefore can be applied to achieve better simulation results. The research also designed a filter algorithm that is capable of reducing the sensor measurement error down to an acceptable value. Achieving such a goal is a significant step towards fully automated adaptive control of hyperglycaemia in critically ill patients and would therefore reduce mortality.

Over the past decade, there has been unprecedented increase in the number of genetic loci associating with type 2 diabetes (T2D) risk and related glycemic traits, thanks to advances in sequencing technologies and access to large sample sizes. Identification of associated genetic variants across the frequency spectrum can provide valuable insight into disease pathophysiology. However, the translation into biological insights has been slow often due to uncertainties over the underlying effector transcripts. G6PC2/ABCB11 is one locus characterised by common non-coding variants that are strongly associated with fasting plasma glucose (FG) levels in healthy adults. The work presented in this thesis aims to understand how protein-coding variants in glycemic trait loci such as G6PC2 contribute to the variability of glycemic traits and in addition gain further insight into the physiological role of G6PC2. To evaluate the role of coding variants in glycemic trait variation, an exome array genotyping study of non-diabetic European individuals (n=33,407) reported multiple coding variants in G6PC2 that were independently associated with FG. I designed and conducted in vitro assays to functionally assess these variants and showed that they result in loss of function (LOF) due to reduced protein stability. This established G6PC2 as the effector transcript influencing FG and highlighted a critical role for G6PC2 (encoding the islet-specific glucose-6-phosphatase catalytic subunit) in glucose homeostasis. To investigate the role of low frequency (MAF=1-5%) and rare (MAF<1%) coding variants in influencing glycemic traits, recent large-scale exome array meta-analyses and whole exome sequencing were carried out as part of MAGIC (n=144,060) and the T2D-GENES/GoT2D consortia (n=12,940) respectively. G6PC1, a gene homolog of G6PC2 that primarily acts through the liver, was uncovered as a novel glycemic locus. My functional follow-up studies demonstrated that rare coding variants in G6PC1 exhibited LOF to influence both FG and FI levels. As rare variation in G6PC2 not previously identified could also affect G6PC2 function and modulate glycemic traits, I also functionally characterised a suite of rare non-synonymous G6PC2 variants. Most of the variants tested exhibited markedly reduced protein levels and/or loss of glycosylation. Several variants were also found to impact on enzymatic activity through inactivating or activating mechanisms to influence FG levels. Finally, to gain better understanding of the function of G6PC2 I performed gene knockdown studies in the EndoC-βH1 human beta cell model followed by insulin secretion analyses. G6PC2 knockdown resulted in increased insulin secretion at sub-threshold glucose stimulation levels, consistent with studies in knockout mouse models. In addition, expression of LOF G6PC2 variants were found to upregulate ER stress responses. These results warrant further studies of the precise roles that G6PC2, an ER-resident protein, plays in regulating insulin secretory function and ER homeostasis in the beta cell. Overall, my work described multiple rare coding variants in both G6PC1 and G6PC2 that alter protein function to regulate glucose metabolism through diverse mechanisms in different tissues. Improved understanding of these effector transcripts will open up opportunities for the exploration of new therapeutic targets for glucose regulation and T2D.

The prevalence of diabetes mellitus (DM) continues to be a global concern among health care practitioners. Without collaboration and interventions, this chronic disease, which poses a significant financial burden for health care institutions, will continue to be problematic. Promoting the use of glycemic control measures among diabetic patients is an intervention, which has the potential to reduce diabetic complications and improve outcomes. The purpose of this doctoral project was to explore available evidence through a systematic review of the best practices for glucose management. The chronic care model served as the theoretical framework. The evidence based practice question was, What is the current evidence supporting the utilization of a computer-based glucose management system (CBGMS) for inpatient diabetic adults in acute and critical care settings? A systematic review was conducted, yielding 532 studies in which 3 of the studies related to CBGMSs published from 2008 to 2017 were critically appraised. The John Hopkins Nursing Evidence Appraisal Tool with specific inclusion and exclusion criteria was utilized. Participants were adult patients (aged 18 and over) with DM in inpatient care settings who were English speaking. Interventions included the traditional paper-based sliding scale regimen versus the utilization of a CBGMS. Outcome measures included decreased length of stay, reduced cost, and glucose optimization. A conclusion was the implementation of a CBGMS has the potential to improve patient outcomes with additional research that exhibits overall benefits and implement into practice. Thus, implementation of a CBGMS can lead to positive social change by aiding in a change in practice that will ultimately ameliorate patient health outcomes.

This thesis investigates the ability of rainbow trout to modulate hepatic glucose production (Ra) and disposal (Rd). My goals were to determine: (1) if resting trout can modulate fluxes to cope with exogenous glucose; (2) how fluxes change during graded swimming; (3) how exogenous glucose affects swimming kinetics; and (4) if exogenous glucose affects cost of transport or performance. Results show that resting trout suppress Ra completely and stimulate Rd from 10.6 to 27.6 μmol kg-1 min-1. During swimming, fluxes increase from 15.6 to 21.9 μmol kg-1 min-1, but only at speeds >2.4 BL s-1. When given glucose, trout suppress Ra from 16.4 to 4.1 μmol kg-1 min-1 and stimulate Rd from 16.4 to 40.1 μmol kg-1 min-1. Glucose lowers metabolic rate but does not affect critical swimming speed. Therefore, this research shows that rainbow trout have a much better capacity for glucoregulation than generally suggested by current literature.

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.

The relationship between human erythrocyte glucose transporter (GLUT1) oligomeric structure and function was studied. GLUT1 was purified from human erythrocytes in the absence (GLUT1-DTT) or the presence (GLUT1+DTT) of dithiothreitol. Chemical cross-linking studies of lipid bilayer-resident purified GLUT1 and hydrodynamic studies of cholate-solubilized GLUT1 support the view that GLUT1-DTT is a homotetramer and GLUT1+DTT is a homodimer. Parallel studies on human erythrocyte, and studies employing conformation-specific antibodies (anti-GLUT1-DTT antibodies, ∂-IgGs), indicate that erythrocyte-resident GLUT1 resembles GLUT1-DTT (a homotetramer). While the D-glucose binding capacities of GLUT1-DTT and GLUT1+DTT are indistinguishable, GLUT1-DTT presents at least two population of binding sites to D-glucose whereas GLUT1+DTT presents only one population of sugar binding sites. The cytochalasin B (CCB) binding capacity of GLUT1-DTT (0.4 sites/monomer) is one half of that of GLUT1+DTT. GLUT1-DTT and GLUT1+DTT contain 2 and 6 free sulfhydryls per monomer respectively. The subunits (monomers) of tetrameric and dimeric GLUT1 are not linked by disulfide bridges. Erythrocyte resident GLUT1 presents at least two binding sites to D-glucose and binds CCB with a molar stoichiometry of 0.55 sites per GLUT1 monomer. Following treatment with high pH plus dithiothreitol, the sugar binding capacity of erythrocyte membrane resident transporter is unaltered but the transporter now presents only one population of binding sites to D-glucose, binds CCB with molar stoichiometry of 1.3 sites per GLUT1 monomer and displays significantly reduced affinity for ∂-IgGs. These findings demonstrate that erythrocyte resident glucose transporter is GLUT1-DTT (a GLUT1 tetramer) and that GLUT1 oligomeric structure determines GLUT1 functional properties. A model which rationalizes these findings is proposed.

Thesis (Ph. D.)--University of California, San Diego, 2007.
Title from first page of PDF file (viewed June 21, 2007). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 269-276).

Glucose absorbs electromagnetic energy in the near infrared (NIR) spectrum at characteristic wavelengths. An NIR beam is transmitted and as it passes through a solution this absorbance causes detectable spectral changes. The amount of spectral absorbance can be correlated to the concentration of glucose in the solution. In this study we used NIR Spectroscopy and micro sensors and detectors in order to detect glucose in small volume samples with economic instrumentation. The goal was to accurately measure the concentration of glucose in a solution held in a glass capillary vessel.

Approximately 450 000 people have diabetes in Sweden today, and the number of diabetics only rises. Monitoring blood sugar several times a day is a fundamental part of managing the disease, and reducing the risks of complications. Today’s glucose monitoring devices are invasive and require small needle sticks for a measurement. Providing a painless method of monitoring the blood sugar level would relieve the lives of diabetics world-wide.   The objective of this project was to investigate the absorption spectra of aqueous glucose concentrations (100 to 5000 mg/dl) in the mid infrared region with Fourier Transform spectroscopy (FTIR), and finally implementing a hand-held monochromatic spectrometer to demonstrate a non-invasive concept. The method chosen for implementing the hand-held demo is due to the commercial availability of diodes and detectors at those wavelengths.   The results from the FTIR showed a trend among concentrations in all wavelengths, in between 1180 to 980 cm-1, specifically at 1035 cm-1, but also in the region 2920 to 2850 cm-1. The hand-held spectrometer did not register any transmittance of the glucose samples. For future implementations, 1035 cm-1 should be investigated more in-depth for a hand-held device.

GLUT1-mediated, facilitated sugar transport is proposed to be an example of transport by a carrier that alternately presents exofacial (e2) and endofacial (e1) substrate binding sites, commonly referred to as the alternating access carrier model. This hypothesis is incompatible with observations of co-existent exo- and endofacial ligand binding sites, transport allostery, and e1 ligand (e.g. cytochalasin B) induced GLUT1 sugar occlusion. The fixed-site carrier model proposes co-existent, interacting e2 and e1 ligand binding sites but involves sugar translocation by geminate exchange through internal cavities. Demonstrations of membrane-resident dimeric and tetrameric GLUT1 and of e2, e1 and occluded GLUT conformations in GLUT crystals of monodisperse, detergent-solubilized proteins suggest a third model. Here, GLUT1 is an alternating access carrier but the transporter complex is a dimer of GLUT1 dimers, in which subunit interactions produce two e2 and two e1 conformers at any instant. The crystallographic structures in different conformations can be utilized to further understand the transport cycle, ligand binding behavior and complex kinetics observed in GLUT1. Specifically, the GLUT1 crystal structure and homology models based upon related major facilitator superfamily proteins were used in this study, to understand inhibitor binding, ligand binding induced GLUT1 transport allostery and the existence of helix packing/oligomerization motifs and glycine induced flexibility. These studies suggest that GLUT1 functions as an oligomeric allosteric carrier where cis-allostery is an intramolecular behavior and trans-allostery is an intermolecular behavior. Additionally, mutations of a dynamic glycine affect the turnover of the transporter while mutations to helix packing motifs affect affinity.

The primary role of the incretin hormone, glucose-dependent insulinotropic polypeptide (GIP) is to potentiate the secretion of insulin in a glucose-dependent manner. In humans and rat models with type 2 diabetes, however, this action is impaired as a result of attenuated GIP receptor (GIPR) expression in the pancreatic β-cell. It has been previously reported that glucose strongly downregulates GIPR mRNA expression in rat insulinoma FNS-1 (832/13) cells and rat islets in a concentration-dependent manner. Since chronic hyperglycemia was proposed to be a primary cause for GIP resistance one objective of my studies was to attempt reversal of this process by pharmacologically lowering blood glucose. However, treatment studies on the effects of the dipeptidyl peptidase (DPIV) inhibitor P32/98 and a DPIV-resistant analogue D-Ala²-GIP on VDF and ZDF rats respectively, showed only moderate improvements in glucose homeostasis, and therefore were not appropriate models for determining the potential reversal effects of lowering blood glucose on islet GIPR expression. The mechanism for the glucose-induced GIPR downregulation is not clear; however it has been previously reported that peroxisome proliferated activated receptor a (PPARa), an important transcription factor involved in fatty acid metabolism, plays an important role. The second objective of this study therefore, was to investigate the effects of a PPARa overexpression system on GIPR expression in INS-1 (832/13) cells. Unexpectedly PPARa did not significantly upregulate our gene of interest; however these studies and others suggest that stimulation of PPARa alone is not sufficient for regulation, and thus also requires an increase in availability of PPARa's co-activator retinoid X receptor (RXR). The third objective of this thesis was to examine the regulation of GIPR in adipose tissue and to determine whether GIPR is differentially expressed in fat and pancreatic islet. Glucose concentration-dependent studies on INS-1 (832/13) β-cell lines and 3T3L1-adipocytes as well as an in vivo characterization study of lean (Fa/?) versus fatty (fa/fa) VDF rat models, collectively demonstrated a tissue-specific pattern of GIPR expression. In all, the findings of this thesis therefore prompt new questions and provide a basis for future experimentation.
Medicine, Faculty of
Cellular and Physiological Sciences, Department of
Graduate

Le métabolisme glucidique de l’oiseau est important pour maitriser la croissance de l’animal et la qualité de la viande. Nous avons étudié l’utilisation périphérique du glucose chez le poulet au niveau musculaire en identifiant et en caractérisant les principaux acteurs impliqués. Nous avons identifié et caractérisé un nouveau transporteur de glucose chez les oiseaux, GLUT12. Il est exprimé dans les muscles, son expression est régulée in vivo par l’insuline et il peut être enrichi dans les membranes plasmiques des cellules suite à une stimulation insulinique. In vitro, une augmentation du transport de glucose est mesurée sur le même pas de temps que la translocation de GLUT12. Comme pour GLUT4 chez les mammifères, la PI3K est impliquée dans la translocation de GLUT12. L’expression des GLUTs musculaires varie avec l’âge des animaux mais aussi avec leur état physiologique, le type métabolique et/ou la fonction du muscle. L’ensemble de nos résultats explique en partie le métabolisme glucidique atypique des oiseaux et laisse entrevoir le développement de nouvelles stratégies d’élevage pour répondre à une demande croissante de produits avicoles de qualité
Glucose metabolism in birds is important to control animal growth and meat quality. We studied peripheral glucose utilization in chicken muscle by identifying and characterizing a new glucose transporter in birds, GLUT12. This transporter is expressed in muscles, its expression is regulated in vivo by insulin and it can be enriched in cells plasma membranes following insulin stimulation. In vitro an increase of glucose transport is measured in the same time than GLUT12 translocation. As for GLUT4 in mammals, PI3K pathway is involved in GLUT12 translocation. Expression of muscular GLUTs varies depending on animals’ age but also depending on their physiological state and on the metabolic type and/or function on the muscle. All of our results partly explain the atypical glucose metabolism in birds and let us foresee development of new farming strategies in order to answer to increased demand of avian quality products

Hepatic glucose-6-phosphatase (G6Pase) catalyses the final step in blood glucose production by the liver. It is a multicomponent system: the catalytic subunit is on the luminal surface of the endoplasmic reticulum membrane; there are transport proteins for glucose-6-phosphate, inorganic phosphate and glucose across the ER membrane; and there is a calcium-binding stabilising protein associated to the catalytic subunit. In fasted and diabetic humans (and rat models) kinetic analysis has shown that the capacity of the glucose-6-phosphate transport protein is the rate-limiting step in G6Pase activity, making this protein vital in controlling hepatic glucose output. However, deficiency of any part of this system will lead to hypoglycaemia and other possible metabolic upsets (type 1 glycogen storage diseases). The structure and known regulation of the G6Pase system are reviewed in the introduction to this thesis. The aims of the work presented here are to investigate the human glucose-6-phosphatase system by studying adult patients newly diagnosed with abnormalities of the G6Pase system, and tissues not previously proven to contain G6Pase in healthy adults thereby improving understanding of the enzyme, its regulation and physiological role and to look for a tissue more accesible than liver in which to study human G6Pase activity. A unique series of eight adult patients each with an abnormality of hepatic G6Pase (two with previously unrecorded defects) is presented and the features of these cases are discussed with reference to the existing literature on type 1 glycogen storage diseases. The cases demonstrate how difficult it can be to prove hypoglycaemia in adults; the diversity of presenting symptoms and signs; the use of a screening test (blood glucose response to a 1mg intramuscular dose of glucagon) for such patients; and the benefits of developing reliable assays for the protein components of the G6Pase system. This series of patients also give further clues to the physiological role of glucose-6-phosphatase in extra-hepatic tissues and the regulation of the hepatic G6Pase system. The diagnosis and subsequent follow-up of the above patients would have been eased by being able to study a more accessible tissue than liver. Intestinal mucosa and neutrophils have been described as abnormal in G6Pase deficiencies. Therefore G6Pase activity was sought in these tissues from normal adult humans.

Biomass transformation into high-value chemicals has attracted attention according to the “green chemistry” principles. Low price and high availability make biomass one of the most interesting renewable resources as it provides the means to create sustainable alternatives to the oil-derived building blocks of the chemical industry In recent year, the need for alternative environmentally friendly routes to drive chemical reactions has in photocatalytic processes an interesting way to obtain valuable chemicals from various sources using the solar light as energy source. The purpose of this work was to use supported noble metal nanoparticles in the selective photo-oxidation of glucose through using visible light. Glucose was chosen as model molecule because it is the cheapest and the most common monosaccharide. Few studies about glucose photo oxidation have been conducted so far, and reaction mechanism is still not totally explained. The aim of this work was to systematically analyze and assess the impact of several parameters (eg. catalyst/substrate ratio, reaction time, effect of the solvent and light source) on the reaction pathway and to monitor the product distribution in order to draw a general reaction scheme for the photo oxidation of glucose under visible light. This study regards the reaction mechanism and the influence of several parameters, such as solvent, light power and substrate concentration. Furthermore, the work focuses on the influence of gold and silver nanoparticles and on the influence of metal loading. The glucose oxidation was monitored through the mass balance and the products selectivity. Reactions were evaluated in terms of glucose conversion, mass balance and selectivities towards arabinose and gluconic acid. In conclusion, this study is able to demonstrate that the photo oxidation of glucose under visible light is feasible; the full identification of the main products allows, for the first time, a comprehensive reaction mechanism scheme.

Glucose, as the most plentiful sugar in nature, is a renewable resource and possesses excellent record in health safety. Levulinic acid is a platform chemical which plays an important role  in  biomass transformation and reactive intermediates. Both glucose and levulinic acid can be produced by biomass conversion with green processing techno logies. Due to the rising needs for bio-based, eco-friendly and non-toxic plasticizers, glucose levulinates as bio­ plasticizers were synthesized from glucose and levulinic acid, by utilizing microwave radiation or conventional condensation reaction (direct-heating method ). Acid number for the reaction liquor was measured by acid-base titration to follow the decrease of acid groups due to the reaction and the trend in  the acid number within reaction time displayed the process of esterification and possible sensitivity of the reaction rate to reaction scale. It showed that microwave radiation had superior ability in  enhancing reaction speed but it was also more sensitive to reaction scale and generated more diverse prod ucts  than the direct-heating method. Besides, the process of reaction and formation  of ester  bonds was  followed  and confirmed by FT IR. The achieved levulinate products were extracted by 2-pro panol and ethyl acetate. The practices showed several serio us problems in 2-propanol extraction, including high dosage required  for  NaCl and solvent and difficulties in purification. The ethyl acetate proved to be a suitable solvent for this study and the  extrac ted  product s  from  the Con-24hrs  and Micro-3/4/5/6/7hrs  were  characterized  by  1H  NMR,  13C N :tvlR. and LDI-MS. The results from spectrum suggested the presence of GL,. and G J .'l. type of levulinates. That means the glucose levulinates were  successfully  synthesized  although  the  dehydration side reaction of glucose was inevitable leading to the generation of glucosidic bonds. In addition, BG (mixture of glucose and glycosidic levulinates) was evaluated by so lution casting of starch and PVC. In order to minimize the microbial contaminations in solution casting of  starch, a  modified  method  was raised and applied. The results showed that 40% BG had goo d miscibility with starch and the conclusion was further proved by DSC measurements, while the BG performed poor miscibility with  PVC.

Diabetes is one of the leading causes of death and disability in the world. Even though insulin was discovered in 1920, an intense research on diabetes has been conducted during the last five decades and this is because of the market size. The huge demand is creating the need for the development of new approaches. This project involved the research aimed at better understanding and improvements in performance of glucose biosensors. In general, high surface area electrodes are desired as the high surface area provides more active sites for electrochemical reactions, and hence higher kinetic rate capability. Therefore, the determination of the active electrochemical surface area of the electrode is very important. A study has been conducted to determine the real electrochemical surface area of the Pelikan screen printed electrodes (SPEs) and a method has been optimised and established by Pelikan for the evaluation of their SPEs. Another very important issue that most of the current blood glucose monitoring tests are facing is the haematocrit effect, since the haematocrit differences observed in the blood samples can significantly affect glucose measurements. Therefore a study has been conducted in order to observe the absorption of the blood samples into the working electrode paste according to the haematocrit level. The second part of the study included the characterisation of the novel conjugated polymer made of N-(N, N’ diethyldicarbamoyl ethyl amido ethyl) aniline (NDDEAEA), the optimization of the conditions for the electrochemical polymerization, their application in grafting and finally the development of NDDEAEA based glucose biosensor. The new conducting polymer, acted as a matrix for the biosensor fabrication in this study, possesses macroiniferter properties and is capable of initiation free radical initiated addition polymerisation after formation of the polyaniline (PANI) material while preserving or even enhancing some of the PANI’s electrochemical properties. This material can potentially be used in the construction of novel Pelikan electrodes with enhanced integration functionalities, e.g. grafting non adhesive polymer coatings to assure that the poor performance in sensors as a result of impact of blood components can be mitigated. The final study included the development and optimisation of the reaction conditions for grafting a hyperbranched polymer onto the surface of the multi walled carbon nanotubes (MWCNT), using the A3 and B2 approach (described below). The aim of this work was achieving further increase in the sensitivity of Pelikan sensors.

This Thesis covers the investigation into the feasibility of monitoring blood glucose non-invasively. The work carried out involved the development of an in-vitro instrument through a series of four stages, each stage of development being an improvement on the previous one. Using these instruments it was shown that by using an appropriate wavelength, glucose could be detected down to 156 mg/dL repeatedly in distilled water, saline and a non-opaque blood analogue. It was also demonstrated that this wavelength could be used to detect the difference between blood samples with different glucose levels. The instruments were also used to demonstrate that a appropriate wavelength could be used as a reference wavelength. In addition to the in-vitro instrument, a basic in-vivo instrument was developed so that physiological data could be taken from either a person's ear or little finger non-invasively. It was clearly demonstrated that the instrument could detect a physiological change in a person whilst the person carried out a 75 g oral glucose to tolerance test.

Miniaturized glucose-oxygen biofuel cells are useful to power implantable medical devices such as biosensors. They are small, more biocompatible and run continuously on glucose and oxygen, providing cleaner energy at neutral environment. A typical glucose-oxygen biofuel cell consists of an anode with glucose oxidase (GOx) and a cathode with various oxygen reducing catalysts. This thesis describes experimental investigations of the major issues of such systems, viz.: complex electrode fabrication, enzyme instability and inefficient oxygen reduction. Electrodes were built using the simple and scaleable bulk modification method, where all the material was simply mixed and bound together into composites with epoxy resin. For the anodes, the composite made of 10% GOx with 7:7 TTF-TCNQ was found optimal. The GOx electrodes were modified with various enzyme stabilisers (PEI, DTT, PEG, GLC, FAD and mixture of PEI:DTT and PEI:FAD) and 2% of PEI-DTT (1:1 w/w) was most effective in the presence of O2. Its maximum output current density was 1.8 x 10-2 ± 9.9 x 10-3 A.m-2. It also showed the resistant against O2 electron deprivation and enzyme inhibition. Its KM.was 5 mM. For the cathodes, various oxygen reducing catalysts (metalised carbon, anthroquinone modified carbon, laccase and bilirubin oxidase) were incorporated into graphite composite and the electrodes were pretreated in different media in order to enhance their catalytic activity. None showed four-electron O2 reduction. NaOH-pretreated cobalt (II) salophen composite electrodes showed two-electron O2 reduction and were most catalytic. Its standard catalytic rate constant was 1.2 x 10-5 ± 1.2 x 10-6 m.s-1. Of the catalysts examined, metal complex composites gave the best results for oxygen-reducing cathodes and their pretreatment led to the synergetic effect because it increased the concentration of catalytic surface oxygen groups and enhanced oxygen reduction.