A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg / Throughout the world, many traditional plant treatments for diabetes exist and therein lies a hidden wealth of potentially useful natural products for diabetes contrl. Despite this, few traditional antidiabetic plants have received scientific or medical scrutiny, and the World Health Organisation ( 1980 ) recommended accordingly that this area warrants further evaluation. / IT2018

Thesis advisor: Charles Hoffman / The fission yeast Schizosaccharomyces pombe senses environmental glucose through a cAMP-signaling pathway. Elevated cAMP levels activate protein kinase A (PKA) to inhibit transcription of genes involved in sexual development and gluconeogenesis, including the fbp1⁺ gene, which encodes fructose-1,6-bisphosphatase. Glucose-mediated activation of PKA requires the function of nine git genes (git=glucose insensitive transcription), encoding adenylate cyclase, the PKA catalytic subunit and seven “upstream” proteins required for glucose-triggered adenylate cyclase activation. This thesis describes the cloning and characterization of the git10⁺ gene, which is identical to swo1⁺ and encodes the S. pombe Hsp90 chaperone protein. This discovery is consistent with the previous identification of the Git7 protein as a member of the Sgt1 Hsp90 co-chaperone family. Glucose repression of fbp1⁺ transcription is impaired by both hsp90⁻ and git7⁻ mutant alleles, as well as by chemical inhibition of Hsp90 activity and temperature stress. Unlike the swo1⁻ and git7⁻ ts mutant alleles, the git10-201 allele and git7-93 allele support cell growth at 37º and show no cytokinesis defect, while severely reducing glucose repression of an fbp1-lacZ reporter, suggesting a separation-of-function defect. A physical interaction between Git7 and Hsp90 in S. pombe was also detected and findings in this thesis suggest their involvement in the initial assembly of the cAMP complex. / Thesis (PhD) — Boston College, 2008. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Thomas C. Chiles / B-lymphocytes respond to environmental cues for their survival, growth, and differentiation through receptor-mediated signaling pathways. Naïve Blymphocytes must acquire and metabolize external glucose in order to support the bioenergetics associated with maintaining cell volume, ion gradients, and basal macromolecular synthesis. The up-regulation of glycolytic enzyme expression and activity via engaged B-cell receptor mediated-events was glucose-dependent. This suggests an essential role for glucose energy metabolism in the promotion of B cell growth, survival, and proliferation in response to extracellular stimuli. In addition, the activity of ATP-citrate lyase (ACL) was determined to be crucial for ex vivo splenic B cell differentiation to antibody-producing cells wherein B cells undergo endomembrane synthesis and expansion. This investigation employed knockout murine models as well as chemical inhibitors to determine the signaling components and enzymes responsible for glucose utilization and incorporation into membrane lipids. These results point to a critical role for phosphatidylinositol 3- kinase (PI3K) in orchestrating cellular glucose energy metabolism and glucosedependent de novo lipogenesis for B lymphocyte responses. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Welkin Johnson / Thesis advisor: Thomas Chiles / B cell activation is an energetically demanding process during which B lymphocytes undergo reprogramming and shift from a resting state to a highly proliferative, metabolically active state. Little is known about the metabolic reprogramming process or the role extracellular nutrients play in the activation response. Here we demonstrate that there are distinct requirements for the nutrients L-glutamine and glucose during activation. We show that cells activated in glucose-depleted conditions are still able to undergo growth and signaling events. In contrast, we show that extracellular L-glutamine is essential for all but the earliest activation events, and cells cultured in L-glutamine-deprived conditions are unable to enter the cell cycle. Consistently, we show that extracellular supplementation of the cell-permeable derivative of α-ketoglutarate (α-KG), a glutaminolytic product, is able to rescue cell activation in the absence of glutamine. We also show the induction of the high affinity amino acid transporter ASCT2 is required for glutamine uptake following B cell receptor (BCR) crosslinking. Specifically, we found that halting glutamine uptake or processing by inhibiting ASCT2 or the glutaminolytic enzyme glutaminase causes activation defects that parallel those observed in glutamine deprived conditions, indicating a requirement for glutaminolysis during the very early stages of activation. We found that -KG does not contribute to epigenetic remodeling, but is necessary for mammalian target of rapamycin complex 1 (mTORC1) activation. In turn, mTORC1 activity is required for upregulation of the glucose transporter Glut1 during the initial 24 hours of activation, as well as increased glucose uptake. These findings indicate a distinct metabolic profile that begins with glutamine uptake, and acts through mTORC1 signaling to later promote glucose uptake. Finally, we show that nutrients contribute to functional differentiation events during B cell activation. Glucose is required to support biogenesis of the endoplasmic reticulum as well as differentiation into plasma-like cells, while glutamine is required to support differentiation into IL-10 secreting regulatory B cell subsets. The requirement for glutamine for in vitro B10 cell differentiation is the first reported link between nutrient signaling and regulatory B cell development, and is a novel finding in the field. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Leung Siu Wai. / Bibliography: leaves 135-150 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1984

The calorie restriction (CR) and insulin/IGF-I-like signalling (IIS) are two pathways regulating the lifespan of C. elegans. Recent studies showed that glucose restriction extends the lifespan of C. elegans while excessive glucose shortens the lifespan of the worms. The first step of the glucose metabolism is the transport of glucose across the plasma membrane by the glucose transporters. The work described in this thesis aims to identify glucose transporters in C. elegans and to provide a primary investigation of the in vitro and in vivo function of the identified glucose transporter. Nine putative transporters have been cloned and expressed. Out of the nice cloned putative transporters in the C. elegans genome, H17B01.1 (H17) only is identified as a fully functional glucose transporter using an oocyte expression system in which glucose transport activity is directly measured. The two transcripts of H17 are both capable of transporting glucose with high affinity, as well as transporting trehalose. Heterologous expression of H17 in mammalian CHO-T cells suggests that the protein is localised both on the plasma membrane and in the cytosol. In vitro studies of H17 show that the protein does not respond to insulin stimulation when expressed in mammalian CHO-T cell and rat primary adipocyte systems. In vivo functional studies using H17 RNAi indicate that the worm’s lifespan is not affected by the H17 knockdown. However, glucose metabolism of C. elegans (as measured by glucose oxidation to CO2 and incorporation into fat reserves) is influenced by the decreased expression of H17, especially in the daf-2 mutant strain, e1370. However, the increase of glucose metabolism caused by H17 knockdown observed in daf-2 mutant is inhibited in the age-1 and akt-1 mutant strains. The findings reported in this thesis suggest that the H17 glucose transporter may play an important role glucose metabolism in C. elegans and that this transport and metabolism is influenced by insulin receptor activity and serine kinase cascades.

Stress is defined as a challenge to homeostatic equilibrium by physical or psychological events, generating a coping response consisting of central and peripheral changes, with the aim of exerting control over the threatening events. Corticotropin-releasing factor (CRF) is well known as a hypothalamic factor which controls the hypothalamic-pituitary-adrenocortical (HPA) axis during basal activity and stress. CRF also serves a neurotransmitter function in the brain, where it is implicated in a range of stress-related behaviours. The measurement of local cerebral glucose utilisation (LCGU) using radiolabelled 2-deoxy-D-glucose (2DG) provides an estimate of cellular activity in the brain. 2DG competes with glucose in its metabolic pathway, but is not fully metabolised, instead accumulating within astrocytes where it can be quantified. After consideration of available modifications to the LCGU technique, the effect of manipulating the CRF system on LCGU was studied, in order to test the hypothesis that CRF and other endogenously expressed CRF-related peptides would induce different patterns of LCGU, and to examine the involvement of CRF receptors in any response. The CRF1 receptor has been implicated in the mediation of stress- and anxiety-related behaviour, while recent evidence has suggested a role for CRF2 in mediating the delayed effects of stress, although it has previously been postulated that CRF2 may be involved in the attenuation of stress-related behaviour. CRF and the endogenous CRF-related peptide Urocortin 1 both induced increases in LCGU in a number of brain regions associated with the CRF system, with concomitant activation of the HPA axis. CRF induced increases in LCGU in the dissected hypothalamus, thalamus, cerebellum and hippocampus, while Urocortin 1 induced a significant increase in LCGU in a dissected hindbrain region, with trend-like effects in frontal cortex and hippocampus. These regions contain components of the CRF system, or receive projections from regions involved in the CRF system, and have been implicated in stress-related function. The effects of CRF on LCGU appear to be mediated by the CRF2 receptor, as they were abolished by the selective CRF2 antagonist antisauvagine-30, but persisted in mice lacking CRF1 and were unaffected by a selective CRF1 antagonist. However, neither of the endogenous CRF-related peptides selective for CRF2, Urocortin 2 and Stresscopin, affected LCGU, which may indicate ligand-specific effects within the CRF system. In contrast to the effects of CRF, restraint stress reduced LCGU, while activating the HPA axis, and this response was unaffected by a selective CRF1 antagonist. This data suggests that the role of CRF receptors in restraint-induced LCGU changes may be overshadowed by effects on other neurotransmitter systems. These studies support the hypothesis that CRF and other endogenously expressed CRF-related peptides would induce different patterns of LCGU, and highlight the involvement of particular brain regions in the response to CRF receptor stimulation. Furthermore, these studies provide evidence suggesting ligand-specific effects within the CRF system.

The main technology currently used in diabetic care, monitors blood glucose and involves an invasive “fingerstick” step. However, low patient compliance and non-continuous glucose monitoring imply poor management of diabetes through this technology, which could lead to adverse and potentially life threatening conditions. In this context, non-invasive glucose sensing appears as an alternative that can bring a change in the prevention and management of the diabetic condition, promising to eliminate patient resistance towards more frequent monitoring and, hence, considerably improving diabetic’s control over glycaemia. However, no non-invasive technology has yet succeeded on the market over the long term. The research field is therefore open to innovative and performant non-invasive technologies. This thesis presents the development of a non-invasive biosensor which as a core principle accesses individual, privileged glucose pathways in the skin (such as hair follicles), allowing the extraction of glucose directly from the interstitial fluid, via reverse iontophoresis (RI). The transdermally extracted glucose is then electrochemically detected in a small size sensor with very high sensitivity. A fully developed technology based on this principle will not require fingerpricking and would thus eliminate users’ main barrier to glucose monitoring. The developed sensor is enzymatic (using glucose oxidase), which electrochemically detects the produced H2O2; while the electrode material is graphene produced by Chemical Vapour Deposition, a promising carbon nanomaterial platform for biofunctionalisation and biosensing. The sensor is a miniature one (typically of 9 mm2 area, containing 24 μL of gel encasing the enzyme), with demonstrated performance parameters that are highly competitive (sensitivity of 2.89 μA.mM-1.cm-2 and limit of detection down to 1 μM), with high specificity towards glucose. The combination of this sensor with glucose extraction by reverse iontophoresis was then validated (with proportionality between subdermal and extracted glucose concentrations demonstrated); as well as enhanced extraction through targeting of hair follicles with the miniature device. The electrochemical determination of glucose concentration was further confirmed by 1H quantitative-NMR detection of glucose. Finally, several such sensors were integrated in a multiplex configuration, and independent sensing, with no cross-talk was demonstrated. The steps demonstrated and implemented so far are proof-of-concept of a highly promising non-invasive, transdermal, future technology for diabetic care.

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A pesquisa na área de biossensores de glicose tem crescido muito nos últimos anos, devido a sua grande importância no monitoramento contínuo da glicemia em pessoas com diabetes. O estudo da utilização de novos materiais nestes dispositivos, como os polímeros condutores e nanopartículas de ouro, tem sido alvo de extensas pesquisas. Neste trabalho, a polianilina (PAni), um dos polímeros condutores mais estudados, foi sintetizada quimicamente na presença de poli(óxido de etileno) (PEO) e também na presença de PEO e de ácido cloroáurico (HAuCl4), para a formação de nanopartículas de ouro (NPAu). Estes nanocompósitos foram utilizados na fabricação de um biossensor eficiente para glicose, servindo de suporte para a imobilização da enzima glicose oxidase (GOx) e de facilitadores do transporte de elétrons. Os polímeros foram caracterizados por infravermelho com transformada de Fourier (FT-IR), microscopia eletrônica de transmissão (MET), microscopia eletrônica de varredura (MEV), espectroscopia UV-visível, voltametria cíclica e pelo método padrão de 4 pontas. Para a produção dos biossensores, parâmetros como a quantidade de polímero a ser aplicada sobre os eletrodos, a concentração da GOx, o pH do eletrólito de realização dos ensaios eletroquímicos e a quantidade de mediador no eletrólito, foram avaliadas previamente por voltametria cíclica, a fim de encontrar a máxima resposta eletroquímica do biossensor. Além dos ensaios de voltametria cíclica, os biossensores foram caracterizados por espectroscopia de impedância eletroquímica e por cronoamperometria. Através dos ensaios de cronoamperometria foi verificado que o biossensor de PAni-PEO detecta glicose em uma faixa de concentrações de 1 a 10 mM, com sensibilidade de 16,04 μA mM-1 cm-2, e o de PAni-PEO-NPAu, na faixa de 0,1 a 5,5 mM, com sensibilidade de 5,5 e 0,76 μA mM-1 cm-2, nas faixas de concentração de 0,1 a 0,5 e de 1,5 a 5,5 mM, respectivamente. Além disso, ambos os biossensores apresentaram seletividade a interferentes como ácido ascórbico e ácido úrico, confirmando que o sinal gerado nos ensaios eletroquímicos refere-se efetivamente à detecção da glicose. / Research in the area of glucose biosensors has grown tremendously in recent years due to their great importance in continuous glucose monitoring in patients with diabetes. The study of the use of new materials in these devices, such as conductive polymers and gold nanoparticles, has been the subject of extensive research. In this work, polyaniline (PAni), one of the most studied conductive polymers, was chemically synthesized in the presence of polyethylene oxide (PEO) and also in the presence of PEO and chloroauric acid (HAuCl4) for the formation of gold nanoparticles (AuNP). These nanocomposites were used in the manufacture of an efficient glucose biosensor, serving as support for the immobilization of the enzyme glucose oxidase (GOx) and as electron transport facilitators. The polymers were characterized by Fourier transform infrared (FT-IR), transmission electron microscopy (TEM), scanning electron microscopy (SEM), UV-visible spectroscopy, cyclic voltammetry and standard 4-point pobe method. For the production of the biosensors, parameters such as the amount of polymer to be applied on the electrodes, the concentration of GOx, the electrolyte’s pH of the electrochemical tests and the amount of mediator in the electrolyte were previously evaluated by cyclic voltammetry to find the maximum electrochemical response of the biosensor. In addition to the cyclic voltammetry tests, the biosensors were characterized by electrochemical impedance spectroscopy and chronoamperometry. Through the chronoamperometry assays, it was verified that PAni-PEO biosensor detected glucose in a range of 1 to 10 mM, with a sensitivity of 16,04 μA mM-1 cm-2 and PAni-PEO-NPAu biosensor, in the range of 0,1 to 5,5 mM, with sensitivity of 5,5 and 0,76 μA mM-1 cm-2 in the 0,1 to 0,5 and 1,5 to 5,5 mM ranges, respectively. In addition, both biosensors presented selectivity to interferents such as ascorbic acid and uric acid, confirming that the signal generated in the electrochemical tests effectively refers to the detection of glucose.