This thesis examines the hypothesis that human erythrocyte net sugar transport is the sum of two serial processes: sugar translocation followed by interaction of newly imported sugar with an intracellular binding complex from which sugar dissociates into the bulk cytosol. This hypothesis suggests that steady-state transport measurements in the human erythrocyte do not accurately reflect the intrinsic catalytic features of the glucose transporter and unless correctly interpreted, may lead to apparent inconsistencies in the operational behavior of the human erythrocyte sugar transport system. Our results support this proposal by demonstrating that although sugar transport measurements in human red blood cells suggest that transport is catalytically asymmetric, ligand binding measurements indicate that transport must be symmetric. In order to examine the serial compartments hypothesis, we set out to determine the following: 1) identify the component(s) of the proposed sugar binding complex, 2) determine whether cytosolic ATP levels and transporter quaternary structure affect sugar binding to the sugar binding complex, and 3) determine whether the sugar binding site(s) are located within or outside the cell. We present findings which support the hypothesis that the sugar binding complex is in fact the sugar transport protein, GLUT1. The number of sugar binding sites and the release of sugar from the GLUT1 complex are regulated by ATP and by GLUT1 quaternary structure. The sugar binding sites are located on a cytoplasmic domain of the GLUT1 complex. We show how these observations can account for the apparent complexity of erythrocyte sugar transport and its regulation by ATP.

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Deuteration is the replacement of a hydrogen atom by deuterium atom in a molecule. The replacement begins at the most acidic hydrogen in the molecule. In ATP, the deshielded hydrogen is C8-H which is the first replaced during deuteration. During ATP deuteration some of the ATP is hydrolysed to ADP concurrently. Using kinetic analysis, it was confirmed that the ATP hydrolysis that occurs is 1st order in ATP concentration, while the hydrogen replacement is 2nd order. The ATP and its C8 deuterated analogue were tested against three enzymes shikimate kinase (SK), acetate kinase (AK) and glutamine synthetase (GS) to determine if a kinetic isotope effect (KIE) exists in these systems. With AK and GS, the KIED increased as the KIEH decreased, while with SK the KIED decreased as the KIEH increased as the concentration of the ATP or deuterated analogue increased. Deuteration of imidazole and purine compounds reduced the specific activity of AK or SK at low concentrations in an enzyme-catalysed reaction. From a library of imidazole-containing compounds that inhibited SK, three compounds were selected and their IC50 values were determined on the SK-catalysed reaction. These compounds show a differential potency and efficiency between their protonated and deuterated analogues when compared in a 1:1 mixture. Synthesized purines incorporating three different substituents at N-9 were tested against AK or SK for their ability to lower the specific activity of the enzymes used / Physics / M. Sc. (Physics)

Deuteration is the replacement of a hydrogen atom by deuterium atom in a molecule. The replacement begins at the most acidic hydrogen in the molecule. In ATP, the deshielded hydrogen is C8-H which is the first replaced during deuteration. During ATP deuteration some of the ATP is hydrolysed to ADP concurrently. Using kinetic analysis, it was confirmed that the ATP hydrolysis that occurs is 1st order in ATP concentration, while the hydrogen replacement is 2nd order. The ATP and its C8 deuterated analogue were tested against three enzymes shikimate kinase (SK), acetate kinase (AK) and glutamine synthetase (GS) to determine if a kinetic isotope effect (KIE) exists in these systems. With AK and GS, the KIED increased as the KIEH decreased, while with SK the KIED decreased as the KIEH increased as the concentration of the ATP or deuterated analogue increased. Deuteration of imidazole and purine compounds reduced the specific activity of AK or SK at low concentrations in an enzyme-catalysed reaction. From a library of imidazole-containing compounds that inhibited SK, three compounds were selected and their IC50 values were determined on the SK-catalysed reaction. These compounds show a differential potency and efficiency between their protonated and deuterated analogues when compared in a 1:1 mixture. Synthesized purines incorporating three different substituents at N-9 were tested against AK or SK for their ability to lower the specific activity of the enzymes used / Physics / M. Sc. (Physics)

Combined atomic force microscopy-scanning electrochemical microscopy (AFM-SECM) is capable of providing simultaneous topographical and electrochemical imaging at sample surfaces. Integration of amperometric biosensors at tip-integrated electrodes recessed from the apex of the AFM tip further enhances the versatility of such bifunctional probes. Of particular interest to this work was the detection of adenosine triphosphate (ATP) at a cellular level, since ATP is involved in many biologically relevant processes. There are challenges concerning the integration of biosensors into bifunctional AFM-SECM probes. This thesis focuses on addressing and advancing several of these limitations. Thin insulation layers are important for AFM-SECM based applications to enhance AFM and SECM performance. Plasma-polymerized fluorocarbon membranes are introduced as novel thin film insulation materials for AFM-SECM probes. Insulation layers with a thickness of < 300 nm were found to exhibit excellent insulating properties and satisfying temporal stability for successful application in AFM-SECM experiments. Furthermore new approaches for increasing the electrode area in conventionally focused ion beam (FIB) fabricated AFM-SECM probes were implemented, since enhancement of the current response in conjunction with biosensing experiments is required. Ion beam induced deposition (IBID) was used to generate platinum carbon (PtC) deposits at AFM-SECM probes, thereby successfully increasing the tip-integrated electrode area. PtC composites were thoroughly characterized in terms of their physical and electrochemical properties. Since a high carbon fraction in the PtC composite was inhibiting the charge transfer kinetics at the electrode surface for certain analytes, several pre-treatment strategies were investigated including annealing, UV/ozone treatment, and FIB milling. FIB milling proved to be the most promising procedure improving charge transfer properties at the electrode along with fabrication compatibility at AFM-SECM probes. The last part of this thesis aimed at providing fundamental studies on AFM-SECM application at live epithelial cell monolayers. AFM was used in different imaging modes to characterize the topography of epithelial cells. ATP detection at epithelial cells was achieved with amperometric biosensors combined with non-invasive SECM. Biosensors were further miniaturized at batch-fabricated AFM-SECM probes enabling laterally-resolved detection of ATP at epithelial cells. Additionally, PtC composite materials were evaluated for applicability as transducer platforms for enzymatic biosensors.

Thèse (M.Ress.Renouv.)-- Université du Québec à Chicoutimi, 2002. / Document électronique également accessible en format PDF. CaQCU

In diverse eukaryotic organisms, double-stranded RNA (dsRNA) induces robust silencing of cellular RNA cognate to either strand of the input dsRNA; a phenomenon now known as RNA interference (RNAi). Within the RNAi pathway, small, 21 nucleotide (nt) duplexed RNA, dubbed small interfering RNAs (siRNAs), derived from the longer input dsRNA, guide the RNA induced silencing complex (RISC) to destroy its target RNA. Due to its ability to silence virtually any gene, whether endogenous or exogenous, in a variety of model organisms and systems, RNAi has become a valuable laboratory tool, and is even being heralded as a potential therapy for an array of human diseases. In order to understand this complex and unique pathway, we have undertaken the biochemical characterization of RNAi in the model insect, Drosophila melanogaster. To begin, we investigated the role of ATP in the RNAi pathway. Our data reveal several ATP-dependent steps and suggest that the RNAi reaction comprises as least five sequential stages: ATP-dependent processing of double-stranded RNA into siRNAs, ATP-independent incorporation of siRNAs into an inactive ~360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, ATP-dependent activation of RISC following siRNA unwinding, and ATP-independent recognition and cleavage of the RNA target. In addition, ATP is used to maintain 5´ phosphates on siRNAs, and only siRNAs with these characteristic 5´ phosphates gain entry into the RNAi pathway. Next, we determined that RISC programmed exogenously with an siRNA, like that programmed endogenously with microRNAs (miRNAs), is an enzyme. However, while RISC behaves like a classical Michaelis-Menten enzyme in the presence of ATP, without ATP, multiple rounds of catalysis are limited by release of RISC-produced cleavage products. Kinetic analysis of RISC suggests that different regions of the siRNA play distinct roles in the cycle of target recognition, cleavage and product release. Bases near the siRNA 5´ end disproportionately contribute to target RNA-binding energy, whereas base pairs formed by the central and 3´ region of the siRNA provide helical geometry required for catalysis. Lastly, the position of the scissile phosphate is determined during RISC assembly, before the siRNA encounters its RNA target. In the course of performing the kinetic assessment of RISC, we observed that when siRNAs are designed with regard to 'functional asymmetry' (by unpairing the 5´ terminal nucleotide of the siRNA's guide strand, i.e. the strand anti-sense to the target RNA), not all of the RISC formed was active for target cleavage. We observed, somewhat paradoxically, that increased siRNA unwinding and subsequent accumulation of single-stranded RNA into RISC led to reduced levels of active RISC formation. This inactive RISC did not act as a competitor for the active fraction. In order to characterize this non-cleaving complex, we performed a series of protein-siRNA photo-crosslinking assays. From these assays we found that thermodynamic stability and termini structure plays a role in determining which proteins an siRNA will associate with, and how association occurs. Furthermore, we have found, by means of the photo-crosslinking assays, that siRNAs commingle with components of the miRNA pathway, particularly Ago1, suggesting overlapping functions or crosstalk for factors thought to be involved in separate, distinct pathways.

Orientador: Patricia Maria de Paula / Banca: Luciane Helena Gargaglioni Batalhão / Banca: Lisandra Brandino de Oliveira / Resumo: Estudos recentes demonstram que os receptores purinérgicos estão presentes no núcleo parabraquial lateral (NPBL), uma estrutura pontina envolvida no controle da ingestão de sódio. No presente estudo, investigamos os efeitos das injeções do, -methyleneadenosine 5 -triphosphate (, -metileno ATP, agonista dos receptores P2X) sozinho ou combinado com o ácido piridoxalfosfato-6-azofenil-2',4'-disulfônico (PPADS, antagonista dos receptores P2X) ou suramin (antagonista não seletivo dos receptores P2) no NPBL sobre a ingestão de NaCl 1,8% induzida por depleção de sódio. Também investigamos os efeitos da injeção de, -metileno ATP sozinho ou combinado com o PPADS no NPBL sobre a pressão arterial média (PAM) e freqüência cardíaca (FC) em ratos saciados e depletados de sódio. Foram utilizados ratos Holtzman com implante de cânulas implantadas bilateralmente em direção ao NPBL. A depleção de sódio foi induzida pelo tratamento com o diurético furosemida (20 mg/kg do peso corporal) acompanhado de uma dieta deficiente em sódio por 24 horas. As injeções bilaterais de, -metileno ATP (2,0 e 4,0 nmol/0,2 μL) no NPBL aumentaram a ingestão de NaCl 1,8% induzida por depleção de sódio (25,3 ± 0,8 e 26,5 ± 0,9 mL/2 h, respectivamente, vs. salina: 15,2 ± 1,3 mL/2 h). O pré-tratamento com o suramin (2,0 nmol/0,2 μL) ou com o PPADS (4,0 nmol/0,2 μL) no NPBL aboliu os efeitos do, -metileno-ATP na ingestão de NaCl 1,8% (15,2 ± 1,2 e 16,9 ± 0,9 mL/2 h, respectivamente). As injeções de PPADS sozinho no NPBL não alteraram a ingestão de NaCl 1,8% (14,6 ± 0,8 mL/2 h vs. salina: 18,3 ± 1,8 mL/2 h). No entanto, as injeções de suramin sozinho no NPBL quase aboliram a ingestão de NaCl 1,8% (5,7 ± 1,9 mL/120 min, vs. salina: 15,5 ± 1,1 mL/120 min) e aumentaram a ingestão de sacarose 2% somente no tempo de 90 minutos (7,1 ± 1,3 vs. salina: 5,3 ± 0,8 mL/90 min) sem alterar...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Recent studies have shown that purinergic receptors are present in the lateral parabrachial nucleus (LPBN), a pontine structure involved in the control of sodium intake. In the present study, we investigated the effects of, -methyleneadenosine 5 -triphosphate (, - methylene ATP, selective P2X purinergic agonist) alone or combined with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, P2X purinergic antagonist) or suramin (non-selective P2 purinergic antagonist) injected into the LPBN on sodium depletion-induced by 1.8% NaCl intake. We also investigated the effects of, -methylene ATP alone or combined with PPADS injected into the LPBN on mean arterial pressure (MAP) and heart rate (HR) on replete and sodium depleted rats. Male Holtzman rats with stainless steel cannulas implanted into the LPBN were used. Sodium depletion was induced by treating rats with the diuretic furosemide (20 mg/kg of body weight) followed by 24 h of sodium-deficient diet. Bilateral injections of, -methylene ATP (2.0 and 4.0 nmol/0.2 μL) into the LPBN increased sodium depletion-induced 1.8% NaCl intake (25.3 ± 0.8 and 26.5 ± 0.9 mL/2 h, respectively, vs. saline: 15.2 ± 1.3 mL/2 h). Pre-treatment with suramin (2.0 nmol/0.2 μL) or PPADS (4 nmol/0.2 μL) into the LPBN abolished the effects of, - methylene-ATP on 1.8% NaCl intake (15.2 ± 1.2 and 16.9 ± 0.9 mL/2 h, respectively). Injections of PPADS alone into the LPBN did not change 1.8% NaCl intake (14.6 ± 0.8 ml/2 h vs. saline: 18.3 ± 1.8 mL/2 h). However, injections of suramin alone into the LPBN strongly reduced 1.8% NaCl intake (5.7 ± 1.9 mL/120 min, vs. saline: 15.5 ± 1.1 mL/2 h) and increased the 2% sucrose intake only at 90 min (7.1 ± 1.3 vs. saline: 5.3 ± 0.8 mL/90 min), without changing 24h water deprivation-induced water intake (16.7 ± 1.8 mL/2 h vs. saline: 15.0 ± 2.1 mL/2 h)... (Complete abstract click electronic access below) / Mestre

Orientador: Antônio Fernando Ribeiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T16:58:35Z (GMT). No. of bitstreams: 1 Kmit_Arthur_M.pdf: 1478515 bytes, checksum: 34c7bf6fcce0ab6ab1b63b0927edcf98 (MD5) Previous issue date: 2013 / Resumo: O receptor purinérgico P2X7 é um canal iônico permeável a cátions pertencente da família P2X (P2X1-P2X7). Ele é ativado por altas concentrações (100?M - 1000?M) de ATP (Adenosine 5?-triphosphate), apresentando duas distintas respostas: 1) uma rápida ativação do canal, 2) uma segunda ativação, lenta e continua, que gera largos poros na membrana celular, permeáveis a grande moléculas (900 Da). O receptor P2X7 está envolvido em processos como morte celular, formação de células gigantes e secreção de citocinas como IL-1? e está predominantemente presente em células imunes. Neste estudo foi examinado como as altas correntes do P2X7 são geradas e qual o mecanismo necessário para serem ativadas em oócitos de Xenopus laevis. Os oócitos foram cirurgicamente retirados de uma rã adulta de Xenopus laevis e injetamos o cRNA do P2X7 para expressa-los na membrana celular. Medimos a condutância através da técnica de Voltage Clamp (TEVC). A incubação dos Oócitos superexpressos com P2X7 em BABTA-AM demonstrou que o Ca2+ extracelular, e não intracelular, é necessário para gerar altas correntes não seletivas através do P2X7, e a reposição de íons extracelular (Cl- e Na+) demonstrou regula-las. A mutação de truncamento da cauda C-terminal na proteína P2X7 gerou uma corrente menor após a estimulação com 1mM de ATP. E ainda três bloqueadores de canais o Ácido Tânico, o AO1 e o NPPB inibiram significativamente as correntes geradas pelo P2X7. Nós concluímos que (i) Os oócitos de Xenopus que expressam P2X7 produzem altas correntes não seletivas após estimulação com ATP, (ii) A ativação do P2X7 requer tanto o influxo de Ca2+ e a cauda C-terminal, e (iii) as correntes do P2X7 são regulados por cátions e ânions extracelulares / Abstract: The purinergic P2X7 receptor is an ion channel permeable to cations which belong to the P2X family (P2X1-P2X7). It is activated by high concentrations (100?M - 1000?M) of ATP (adenosine 5'-triphosphate), presenting two distinct responses: 1) a rapid activation of the channel, 2) a second activation, slow and continuous, which generates a large pore in the cell membrane permeable to large molecules (900 Dalton). The P2X7 receptor is involved in processes such as cell death, giant cell formation and secretion of cytokines such as IL-1? and is present predominantly on immune cells. In this study we examined how the P2X7 high currents are generated and what is the mechanism required to be activated in Xenopus laevis oocytes. Oocytes were surgically removed from an adult frog Xenopus laevis and injected with cRNA to express the P2X7 in the cell membrane. We measure the conductance through the Voltage Clamp technique (TEVC). Incubation of oocytes overexpressed with P2X7 receptors in BABTA-AM demonstrated that extracellular Ca2+, and do not intracellular, it is necessary to generate nonselective high currents through P2X7, and replacing extracellular ions (Cl- and Na+) showed regulate them. The truncation mutation in C-terminal tail of the P2X7 protein generated a smaller current after stimulation with 1 mM ATP. And three channel blockers Tannic Acid, AO1 and NPPB significantly inhibited the generated currents by P2X7. We conclude that (i) Xenopus oocytes expressing P2X7 produce a nonselective high currents after stimulation with ATP (ii) Activation of the P2X7 requires both the influx of Ca2+ and C-terminal tail, and (iii) the currents of the P2X7 are regulated by extracellular cations and anions / Mestrado / Saude da Criança e do Adolescente / Mestre em Ciências