Tesis sobre el tema "Adenosine triphosphatase"

The Na,K-ATPase, or sodium pump, is a membrane-associated protein complex comprising two subunits, alpha and beta, both of which can exist as one of several isoforms. It generates and maintains the electrochemical Na + and K+ gradients across the plasma membrane of animal cells. These gradients are the driving force for a variety of ubiquitous and specialized cell functions, such as transport of solutes, as well as maintenance of membrane potential and cell volume. Agents that modulate the kinetic behaviour of the pump enable cells to adapt to changing needs. Distinct substrate activation profiles in various tissues presumably underlie the specialized functions of the sodium pump in these tissues. Although the tissue-specific distribution of various isoforms accounts for some of the differences in kinetic behaviour of the enzyme, other factors are also important determinants of such behaviour. This study describes the characterization of two mechanisms of sodium pump modulation. The first involves alterations in the susceptibility of the enzyme to competitive inhibition by K+ at Na+ binding sites. Studies on the alpha1 and alpha3 isoform of various tissues and cells have revealed that there exist tissue-specific components that determine the extent to which the two cations compete with each other for cytoplasmic binding sites. Specifically, pumps that are highly susceptible to K +/Na+ antagonism also bind and occlude K+ much more readily on the cytoplasmic site, and this behaviour is abrogated upon fusion of pumps into another membrane environment, that of the red blood cell. The second mechanism of regulation of pump behaviour described in this thesis involves modulation of the apparent affinity of the enzyme for ATP by the gamma subunit. This membrane protein had been previously cloned and sequenced, but very little was known about its function. This study shows that the gamma subunit is expressed uniquely in kidney tubules, and has a C-terminus-in, N-terminus-

Thesis (Ph. D.)--University of Oregon, 2005.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 85-95). Also available for download via the World Wide Web; free to University of Oregon users.

ATP synthase (F_1 F_O-ATPase) is an essential enzyme for life. Powered by an electrochemical proton gradient, it catalyzes ADP and phosphate into ATP. The F_1-subunit of ATP synthase is called F_1-ATPase as it also independently catalyzes the reverse reaction in absence of F_O-part. The nearly 100% energy conversion efficiency of the molecular motor has attracted the attention of many physicists and biologists to explore the underlying thermodynamics. Recently, a new nonequilibrium equality derived by Harada and Sasa (Harada & Sasa, 2005) was applied to the experimental time series data on F_1-ATPase to extract heat flow to the environment. A phenomenological model for rotary motion was proposed and shown to reproduce key experimental features. Interested in the high efficiency of F_1-ATPase and the good performance of the corresponding model, we carried out a detailed computational study of the model to understand its behavior in a broader range of parameter values. We solved the model using a modified Gillespie algorithm for stochastic simulation and by integrating the Fokker-Planck equation. Various physical properties of the model, such as the relation between rotational velocity and parameters characterizing angular dependence (q) and ATP switching rates (W), the relation between two kinds of dissipation and rotational velocity, the negative heat flow from environment to system through ATP binding etc. are analyzed in detail. Importantly, we modified the driving potential to investigate the factors affecting the efficiency. Additionally, we found some inconsistences between properties of this model and previous studies and we could unify them by some adjustments, which may be useful for constructing more precise models in the future.

The tonoplast H$ sp+$-ATPase of Beta vulgaris L. was partially purified by Triton X-100 solubilization and Sepharose 4B chromatography resulting in the enrichment of two polypeptides (57 and 67 kDa). Kinetic analysis of ($ alpha$-$ sp{32}$P) BzATP labeling identified the 57 kDa polypeptide as a nucleotide-binding subunit with a possible regulatory function. In addition, ($ sp{14}$C) DCCD-labeling identified a 16 kDa polypeptide as a putative transmembrane proton channel. It is concluded that the tonoplast H$ sp+$-ATPase is a multimer composed of at least three polypeptides.
Anti-57 and anti-67 kDa sera reacted with polypeptides of the corresponding size in bovine chromaffin granules, bovine clathrin-coated vesicles, and yeast vacuolar membranes, suggesting common structural features and common ancestry for endomembrane H$ sp+$-ATPases of different organelles and different phyla. Anti-57 serum was used to isolate a cDNA encoding the corresponding subunit from Arabidopsis. Protein sequence analysis revealed homologies between endomembrane, F$ sb0$F$ sb1$ and archaebacterial ATPases, suggesting that these different classes of ATPases have evolved from a common ancestor.

VCP/p97 works as a segregase to extract the ubiquitylated proteins from protein complexes, lipid membranes and chromosomes, thereby promoting their degradation or recycling. VCP/p97 plays essential roles in ubiquitin-dependent proteasome degradation, ERAD, autophagy, endocytosis, reassembly of ER, Golgi and nuclear envelop, and cell cycle regulation. In ubiquitin dependent proteasome degradation pathway, VCP/p97, as a special ubiquitin binding-shuttle factor, is required for the successful cell cycle progression in regulating IκB, CDT-1, Aurora B, and CDC25A. Here, we studied the role of VCP/p97 in G1 to S phase transition in MCF-7 human breast cancer cells. We found that VCP/p97 knockdown or inhibition by DBeQ, a potent VCP/p97 inhibitor, decreased cell proliferating rates and reduced S phase cell percentages in asynchronized MCF-7 cells.VCP/p97 inhibition by DBeQ also arrested cells at G1 phase in synchronized MCF-7 cells. These data suggest that VCP/p97 is required for G0/G1 to S phase transition in MCF-7 cells. In addition, in either asynchronized or synchronized MCF-7 cells, VCP/p97 knockdown or DBeQ treatment resulted in the accumulation of p21 and p27, two CDK inhibitors. Moreover, p27, not p21, knockdown in MCF-7 cells rescued the defects of S phase entry caused by VCP/p97 knockdown or DBeQ treatment. Taken together, our results suggest that VCP/p97 regulates the timely degradation of p27 to promote G1 to S phase transition in MCF-7 cells.
published_or_final_version
Physiology
Master
Master of Philosophy

Cytochemical localization of nuclear-envelope nucleos-side triphosphatase was exhibited in the nuclear membrane and nuclear pores of the Necturus, maculosus oocyte. This enzyme is thought to promote nucleocytoplasmic transport of mRNA through the nuclear pores. Various tissue preparations were performed to assure optimum results of reaction product formation and preservation of tissue ultrastructure.Incubation of frozen oocyte sections in a modified Wachstein-Meisel medium resulted in positive staining of the nuclear membrane and the nucleoli as evidenced in light and electron micrographs. Whole oocytes were incubated in the Wachstein-Meisel medium and then embedded in Epon for electron microscopy. The whole oocytes contained reaction product associated with microvilli and plasma membranes. Exposure of manually isolated nuclei to the same experimental medium resulted in lead deposits in the nuclear envelope, the nuclear pores, and randomly dispersed among chromatin granules. Thus, these nuclear structures may play a role in transport of mRNA to the cytoplasm.
Department of Physiology and Health Science

Thesis (Ph. D.)--University of Oregon, 2006.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 93-99). Also available for download via the World Wide Web; free to University of Oregon users.

The Na,K-ATPase, or Na+ pump is an integral membrane protein found in the cells of virtually all higher eukaryotes and is one of the most important systems in cellular energy transduction. Na,K-ATPase catalyzes the electrogenic exchange of three intracellular Na+ for two extracellular K+ ions coupled to the hydrolysis of one molecule of ATP. The research described in this thesis concerns the regulation of the Na,K-ATPase by FXYD 2, a member of the FXYD family of small single transmembrane proteins. FXYD 2, commonly known as the gamma modulator, is located primarily in the kidney and has a role in modulating the enzyme's apparent affinities for ligands. This study has addressed several aspects of gamma structure and function, namely its function in intact cells, the function of the gamma transmembrane domain, and delineation of regions of the enzyme's catalytic alpha subunit with which gamma interacts. Transport assays using intact transfected HeLa cells showed that the two gamma variants, gammaa and gammab, cause (i) an increase in K+/Na+ antagonism, seen as an increase in K'Na at high K+ concentration, and (ii) an increase in apparent ATP affinity seen as an increase in ouabain-sensitive K+ influx as a function of ATP concentration. These results are consistent with those obtained earlier with unsided membrane preparations. The present study also showed a gamma-mediated increase in steady-state intracellular Na+ concentration and, in contrast to assays using permeabilized membranes, a gamma-mediated increase in apparent affinity for extracellular K+. Experiments with synthetic gamma transmembrane (gamma-TM) peptides provided insight into the role of the TM region such that incubation of these peptides with membranes containing alphabeta pumps modulated K'Na similarly to transfected full-length gamma, indicating that the TM domain alone can cause an increase in K'Na at high K+ concentration. Results with gamma-TM bearing the Gly41→Arg missense mutation associated with familial renal hypomagnesemia provided direct evidence that this mutation prevents gamma association with alphabeta pumps. In a study aimed to identify regions of alpha critical for the functional effects of gamma, interactions of alpha1/alpha2 (and the reverse alpha2/alpha1) chimeras with gamma showed the importance of the carboxy terminus, particularly TM 9. The chimera data also indicate that interactions of transmembrane regions of the catalytic alpha subunit with FXYD proteins are not necessarily the sole determinants of the kinetic effects of gamma on Na+ affinity since the extramembranous L7/8 loop of a appears to modulate intramembranous alpha-gamma interactions that mediated the increase in K+/Na+ antagonism.

V-ATPases are enzymes found in all eukaryotic cells. They are organized into a peripheral membrane complex (V1) and an integral membrane complex (V0). VI is responsible for ATP hydrolysis and generates the energy used by Vo to pump protons from the cytosol into the vacuole. Subunit d is a component of Vo possibly located at the interface between V 1 and V. in the V-ATPase complex. We hypothesize that subunit d could be involved in the structural and functional coupling of VI and Vo. This was tested by generating point mutations along the open reading frame of subunit d from yeast. The mutations F94A, H128A, D173A, D217A, D261A, E317A, W325A, E328A and C329A, all in conserved regions of the protein sequence, were characterized by examining their growth phenotype and by assessing their ATPase specific activity, proton transport and V1Vo assembly in purified vacuolar membranes. The mutations E317A, W325A, E328A and C329A had reduced ATPase and proton transport activities. In addition, V1Vo assembly was compromised by the mutation W325A. Our results suggest that residues at the carboxyl-end of subunit d are important for ATPase activity, proton pumping and V1Vo assembly at the membrane.
Department of Chemistry

Thesis (Ph. D.)--West Virginia University, 2007.
Title from document title page. Document formatted into pages; contains xi, 82 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

The sarcoplasmic reticulum Ca²⁺ -pumping ATPase is the primary system responsible for the removal of calcium from the sarcoplasm during relaxation of skeletal and cardiac muscles. Since the rat heart SR is used frequently in our laboratory to study the Ca²⁺ -transport defects in disease states, the Ca²⁺ - ATPase activity of this system was characterized. Calmodulin (CaM) and cAMP-dependent protein kinase (cAMP-PK) are known to regulate the dog cardiac SR Ca²⁺ -pump. The effects of these regulators on the rat heart SR Ca²⁺ -pump were studied. Studies were also carried out to investigate the effects of Triton X-100 on SR Ca²⁺ -ATPase activity and the regulation of this activity by CaM. The rat heart SR Ca²⁺-ATPase was stimulated in a concentration-dependent manner by both Ca²⁺ and Mg²⁺ in the complete absence of the other cation. Magnesium produced a concentration-dependent increase in the basal ATPase activity without affecting the maximal ATPase activity. This appeared to result in a gradual disappearance of the Ca²⁺ dependency of the ATPase activity. Addition of 100µM CDTA (trans-1,2-diaminocyclo- hexane-N,N,N',N'-tetraacetic acid), in the absence of added magnesium, produced no effect on Ca²⁺ stimulation of ATPase activity. The results appear to indicate the presence of a low affinity non-specific divalent cation-stimulated ATPase. At a constant Mg: ATP ratio, ATP simulated the SR Ca²⁺-ATPase activity in a concentration-dependent manner. Double-reciprocal plots of the data suggest that the true substrate for rat heart SR Ca²⁺-ATPase may be ATP and not Mg.ATP. In the crude SR, CaM did not stimulate total or Ca²⁺-stimulated ATPase activity over a range of Ca²⁺ and Mg²⁺ concentrations. CaM also failed to stimulate membrane phosphorylation over a range of Mg²⁺ concentrations. Furthermore, CaM did not produce a significant effect on calcium transport into SR vesicles. The catalytic subunit of cAMP-dependent protein kinase was also ineffective in stimulating membrane phosphorylation and Ca²⁺ -ATPase activity. Two CaM antagonists, trifluperazine and compound 48/80, did not affect the rat heart SR ATPase activity. The ATPase activity in Triton-washed SR membranes appeared to be increased at low Triton concentrations. This effect was probably due to the removal of non-intrinsic proteins, leaky vesicles or altered membrane fluidity. At higher Triton X-100 concentrations, the ATPase activity was lost, probably due to loss of the phospholipid environment. When SR membranes phosphorylated under conditions similar to those used for the ATPase assay were analysed by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) followed by autoradiography, a single phosphorylated protein of 7,500-9,000 dalton was observed. This protein may represent the monomeric form of phospholamban. CaM, however, appeared to have no effect on the phosphorylation of this 7,500-9,000 dalton protein in either untreated or Tritan-washed SR membranes. It is speculated that the rat heart SR contains tightly bound CaM which cannot be removed by treatment with Triton X-100.
Pharmaceutical Sciences, Faculty of
Graduate

The adult nervous system is a network of neurons connected to each other by thin processes, called axons. During development, axons are guided to their targets by patterned chemical cues. The leading tip of an axon, called the growth cone, pulls the axon to its target with cell surface receptors that sense these cues and trigger biochemical cascades which extend, retract or turn the axon. This thesis examines mechanisms involved in the guidance of axons to the cue netrin-1. I test the hypothesis that netrin-1 may function as an adhesive ligand. As well, I examine the role of two signaling pathways, the cyclic adenosine monophosphate (cAMP) and the rho guanine triphosphatase (Rho GTPase), in the guidance of axons to netrin-1. I report that neurons efficiently attach to netrin-1 and that their growth cones display morphological changes consistent with its function as an adhesive ligand. Inhibition of the RhoA subfamily of Rho GTPases or elevation of cAMP levels increases the plasma membrane presentation of netrin-1's receptor DCC and promotes axon outgrowth and turning to netrin-1. This observation is consistent with the possibility that these two biochemical pathways are linked. However, an important difference exists in how these pathways are regulated by netrin-1; while netrin-1 inhibits RhoA activation, it does not affect cAMP levels. Studies by others have reported that inhibiting RhoA activity or elevating cAMP concentration promotes axon regeneration following injury. These studies did not, however, examine the axon's guidance decisions past the injury site; while overcoming an inhibitory injury site likely involves ignoring or switching the response to inhibitory cues, guidance to their appropriate targets requires axons to sense and respond appropriately to the cues in their environment. Results presented here indicate that inhibiting RhoA or augmenting cAMP levels promote axonal attraction to the netrin-1 guidance cue.
Le système nerveux adulte comprend un réseau de neurones connectés les uns aux autres par des prolongements minces que l'on nomme des axones. Pendant le développement, les axones sont guidés vers leurs cibles par des signaux moléculaires. L'extrémité d'un axone, appelé cône de croissance, tire celui-ci vers sa cible grâce à des récepteurs de surface qui détectent les signaux moléculaires environnementaux et déclenchent des cascades biochimiques qui font s'allonger, se rétracter ou tourner l'axone. Cette thèse examine les mécanismes impliqués dans le guidage des axones vers le signal moléculaire nétrine-1. J'ai investigué l'hypothèse voulant que nétrine-1 puisse fonctionner comme ligand adhésif. De plus, j'ai examiné le rôle de deux cascades biochimiques : l'adénosine monophosphate cyclique (cAMP) et la rho guanine triphosphatase (Rho GTPase), dans le guidage de l'axone vers nétrine-1. Le présent travail démontre que les neurones s'attachent efficacement à nétrine-1 et que leurs cônes de croissance axonaux affichent des changements morphologiques conformes à une fonction de ligand adhésif pour nétrine-1. L'inhibition de RhoA, une sous-famille de Rho GTPases, ou l'élévation des niveaux de cAMP augmente la présentation du récepteur de nétrine, DCC, à la surface de la cellule, promeut la croissance axonale et fait tourner l'axone vers nétrine-1. Ces observations suggèrent la possibilité que ces deux voies biochimiques soient inter-reliées. Cependant, une différence importante existe dans la façon dont ces voies biochimiques sont régulées par nétrine-1 : nétrine-1 diminue l'activation de RhoA, mais n'affecte pas les niveaux de cAMP. Des études précédentes avaient rapporté que le fait de diminuer l'activité de RhoA ou d'élever la concentration de cAMP promeut la régénération des axones après une lésion. Cependant, ces études n'ont pas examiné les décisions de l'axone, une fois passé le site de lésion

Thesis (PH.D.)--Michigan State University. Comparative Medicine and Integrative Biology, 2008.
Title from PDF t.p. (viewed on Aug. 11, 2009) Includes bibliographical references. Also issued in print.

The plasma membrane calcium-transport ATPase plays a major role in maintaining the low cytosolic calcium concentrations required for normal cellular function. Calcium, magnesium, calmodulin and lanthanum have been shown to alter the activity of the calcium-stimulated, magnesium-dependent ATPase activity in human erythrocytes. In an attempt to examine the reaction sequence of the (Ca²⁺ + Mg²⁺)-ATPase, the effects of these agents on the kinetics of calcium dependent phosphoprotein formation, the first step in the partial reaction sequence, were examined. Calmo-dulin-depleted erythrocyte membranes were prepared by hypotonic lysis in the presence of EDTA, according to the method of Carafoli et al (1980). Calcium-dependent formation of the phosphorylated intermediate was biphasic; the high calcium-affinity component was associated with low levels of E.Ca.P and a shallow response to changing calcium concentrations, whereas in the region of the low calcium-affinity component, E.Ca.P rose sharply in response to increasing calcium concentrations. The low affinity component of E.Ca.P lies in the range of calcium concentrations which inhibit (Ca²⁺ + Mg²⁺)-ATPase activity. When analyzed on LiDS acid PAGE, both components of calcium-dependent phosphoprotein formation were due to hydroxylamine-sensitive phosphorylation of a 135,000-145,000 dalton protein. Hence, the low calcium-affinity component of phosphoprotein formation and calcium-dependent inhibition of (Ca²⁺ + Mg²⁺)-ATPase activity were likely due to calcium-inhibition of dephosphorylation. Kinetic studies of calcium-dependent phosphoprotein formation, at two different calcium concentrations (1.0 μM, 0.4 mM), indicated that a steady-state was reached much sooner at higher calcium concentrations. Lanthanum, which is known to block dephosphorylation of the intermediate complex, increased both the apparent rate of formation and the steady-state level of the phosphorylated intermediate. Calmodulin, which has previously been shown to increase both the maximum velocity and the calcium affinity of the (Ca²⁺ + Mg²⁺)-ATPase, did not affect either calcium-dependent inhibition of (Ca²⁺ + Mg²⁺ )-ATPase activity or the biphasic nature of calcium-dependent phosphoprotein formation. At low calcium concentrations, calmodulin increased the apparent rate of phosphoprotein formation, whereas at higher calcium concentrations (0.4 mM) calmodulin reduced the steady-state level of the phosphoprotein; the apparent rate of formation was unaffected. In the presence of lanthanum, calmodulin increased both the apparent rate of formation and steady-state level of the phosphoprotein, suggesting that the true rate of formation was increased by calmodulin at higher calcium concentrations, but this was normally hidden by a simultaneous increase in the rate of dephosphorylation. Removal of endogenous magnesium, using trans-1,2-diamino-cyclohexane tetraacetic acid (CDTA) did not alter the calcium sensitivity or rate of formation of the phosphorylated intermediate, however turnover of the intermediate was markedly reduced. In the absence of free magnesium, both the velocity and calcium sensitivity of the (Ca²⁺ + Mg²⁺)-ATPase were also found to be lower. The low calcium-affinity component of calcium-dependent phosphoprotein formation, which Schatzmann (1982) has attributed to an action of calcium at a "magnesium-specific" site, was not affected by magnesium concentrations as high as 1 mM. Furthermore, this phosphoprotein could be dephosphorylated along either the forward or reverse pathways. These results indicate that the transformation from E₁.Ca.P to E₂.Ca.P may not be the site of the calcium-dependent inhibition of dephosphorylation. Calmodulin-depleted membrane fragments were prepared from the erythrocytes of cystic fibrosis patients as well as age- and sex-matched controls. Under conditions in which dephosphoryla-tion is inhibited, phosphoprotein formation and (Ca²⁺ + Mg²⁺)-ATPase activities were determined. Both (Ca²⁺ + Mg²⁺)-ATPase activity and phoshoprotein formation were found to be significantly reduced in the preparations derived from patients with cystic fibrosis. Turnover of the phosphorylated intermediate did not differ significantly between the two groups. A reduction in (Ca²⁺ + Mg²⁺)-ATPase activity and phosphoprotein formation suggests that there may be fewer active calcium-pumping sites in the erythrocyte membranes of cystic fibrosis patients compared to normal subjects.
Pharmaceutical Sciences, Faculty of
Graduate

Thesis (Ph.D.)--University of Missouri-Columbia, 2007.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on March 19, 2009) Vita. Includes bibliographical references.

Thesis (M.S.)--University of Missouri-Columbia, 2008.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on August 28, 2008) Vita. Includes bibliographical references.

P1B-type ATPases transport a number of monovalent and divalent heavy metals (Cu+, Cu2+, Ag+, Zn2+, Cd2+, Pb2+ and Co+2) across biological membranes. These ATPases are found in archea, bacteria and eukaryotes and are one of the key elements required for maintaining metal homeostasis. Plants have an unusually high number of P1B-type ATPases with distinct metal selectivity compared to other eukaryotes that usually have one or two Cu+-ATPases. Higher plants are the only eukaryotes where Zn2+-ATPases have been identified. Towards understanding the physiological roles of plant Zn2+-ATPases, we characterized Arabidopsis thaliana HMA2. We expressed HMA2 in yeast and measured the metal dependent ATPase activity in membranes. We showed that HMA2 is a Zn2+-ATPase that is also activated by Cd2+. Zn2+ transport determinations showed that this enzyme drives the efflux of metal from the cytoplasm. Analysis of HMA2 mRNA levels showed that the enzyme is present in all plant organs. We analyzed the effect of removal of HMA2 full-length transcript in whole plants by gene knock out. Although hma2 mutants did not show a different visible phenotype from the wild type plants, we observed increased levels of Zn2+ or Cd2+. The observed phenotype of hma2 mutants and plasma membrane location of HMA2, mainly in vasculature (Hussain et al., 2004), indicates that this ATPase might have a central role in Zn2+ uploading into the phloem. P1B-type ATPases have cytoplasmic regulatory metal binding domains (MBDs) in addition to transmembrane metal binding sites (TMBDs). Plant Zn2+-ATPases have distinct sequences in both their N- and C-termini that might contribute to novel metal binding sites. These ATPases contain long C-terminal sequences rich in CC dipeptides and His repeats. Removal of the C-terminus (C-MBD) of HMA2 leads to a 50% reduction in the enzyme turnover suggesting a regulatory role for this domain. Atomic Absorption Spectroscopy (AAS) analysis showed that Zn2+ binds to C-MBD with a stoichiometry of three (3 Zn/C-MBD). Chemical modification studies and Zn K-edge X-ray Absorption Spectroscopy (XAS) of Zn-C-MBD showed that Zn2+ is likely coordinated by His in two sites and the third site slightly differs from the others involving a Cys together with three His. All plant Zn2+-ATPases lack the typical CXXC signature sequences observed in Cu+-ATPases and some bacterial Zn2+-ATPases N-terminus metal binding domains (N-MBDs). Instead, these have conserved CCXXE sequences. Truncation of HMA2 N-MBD results in a 50% decrease in enzyme Vmax suggesting that N-MBD is also a regulatory domain. The results indicate that the N-MBD binds Zn2+ with a stoichiometry of one (1 Zn/N-MBD). Metal binding analysis of individual N-MBD mutants Cys17Ala, Cys18Ala and Glu21Ala/Cys prevented Zn+2 binding to HMA2 N-MBD suggesting the involvement of all these residues in metal coordination. ATPase activity measurements with HMA2 carrying the mutations Cys17Ala, Cys18Ala and Glu21Ala/Cys showed a reduction in the enzyme activity similar to that observed the truncated protein indicating that the enzyme activity reduction observed in the N-terminus truncated forms of the enzyme is related to the removal of the metal binding capability. Summaryzing, these studies show the central role of HMA2 in plant Zn2+ homeostasis. They also describe the mechanism and direction of Zn2+ transport. Finally, they establish the presence of novel metal binding domains in the cytoplasmic portion of the enzyme. Metal binding to these domains is required for full enzymatic activity.

The synthesis of nanostructured materials, especially metallic nanoparticles, has accrued utmost interest over the past decade owing to their unique properties that make them applicable in different fields of science and technology. The limitation to the use of these nanoparticles is the paucity of an effective method of synthesis that will produce homogeneous size and shape nanoparticles as well as particles with limited or no toxicity to the human health and the environment. The biological method of nanoparticle synthesis is a relatively simple, cheap and environmentally friendly method than the conventional chemical method of synthesis and thus gains an upper hand. The biomineralization of nanoparticles in protein cages is one of such biological approaches used in the generation of nanoparticles. This method of synthesis apart from being a safer method in the production of nanoparticles is also able to control particle morphology. In this study, a comparative biological synthesis, characterization and biomedical effects of metallic nanoparticles of platinum, gold and silver were investigated. Metallic nanoparticles were biologically synthesized using cage-like (apoferritin), barrel-like (GroEL) and non-caged (ribonuclease) proteins. Nanoparticles generated were characterized using common techniques such as UV-visible spectroscopy, scanning and transmission electron microscopy, inductively coupled optical emission spectroscopy, Fourier transform infra-red spectroscopy and energy dispersion analysis of X-rays (EDAX). Nanoparticles synthesised biologically using apoferritin, GroEL and RNase with exhibited similar chemical and physical properties as thoses nanoparticles generated chemically. In addition, the metallic nanoparticles fabricated within the cage-like and barrel-like cavities of apoferritin and GroEL respectively, resulted in nanoparticles with relatively uniform morphology as opposed to those obtained with the non-caged ribonuclease. The enzymatic (ferroxidase) activity of apoferritin was found to be greatly enhanced with platinum (9-fold), gold (7-fold) and silver (54-fold) nanoparticles. The ATPase activity of GroEL was inhibited by silver nanoparticles (64%), was moderately activated by gold nanoparticles (47%) and considerably enhanced by platinum nanoparticles (85%). The hydrolytic activity of RNase was however, lowered by these metallic nanoparticles (90% in Ag nanoparticles) and to a higher degree with platinum (95%) and gold nanoparticles (~100%). The effect of synthesized nanoparticles on the respective enzyme activities of these proteins was also investigated and the potential neurotoxic property of these particles was also determined by an in vitro interaction with acetylcholinesterase. Protein encapsulated nanoparticles with apoferrtin and GroEL showed a decreased inhibition of acetylcholinesterase (<50%) compared with nanoparticles attached to ribonuclease (>50%). Thus, it can be concluded that the cavities of apoferitin and GroEL acted as nanobiofactories for the synthesis and confinement of the size and shape of nanoparticles. Furthermore, the interior of these proteins provided a shielding effect for these nanoparticles and thus reduced/prevented their possible neurotoxic effect and confirmed safety in their method of production and application. The findings from this study would prove beneficial in the application of these nanoparticles as a potential drug/drug delivery vehicle for the prevention, treatment/management of diseases associated with these enzymes/proteins.

The aim of the present investigation was to determine whether a plasma membrane high affinity Ca²+-ATPase plays an integral role in the maintenance of cytoplasmic free Ca²+ in pancreatic acinar cells. To achieve this, the Ca²+-transport and Ca²+-ATPase activities were characterized and their properties compared. Plasma membranes from guinea-pig pancreatic acini were shown to contain an ATP-dependent high affinity Ca²+-pump and a high affinity Ca²+-dependent ATPase activity. In addition, a low affinity ATPase activity was also observed. The high affinity Ca²+-ATPase activity as well as the Ca²+-transport were found to be dependent on Mg²+, whereas the low affinity ATPase activity appeared to be inhibited by Mg²+. The high affinity ATPase activity was 7-fold greater in magnitude than the Ca²+-transport. Whereas the Ca²+-transport was very specific for ATP as a substrate, the high affinity Ca²+-ATPase showed little specificity for various nucleotide triphosphates. These data would suggest that the Ca²+-transport and the high affinity Ca²+-dependent ATPase in guinea-pig pancreatic acinar plasma membranes may be two distinct activities To further investigate whether the two activities were related, we investigated how the Ca²+-transport and Ca²+-ATPase activities were regulated by intracellular mediators. Regulation of the two activities by calmodulin, cyclic AMP-dependent protein kinase, Protein kinase C and inositol phosphates was investigated. Calmodulin failed to stimulate either activity. In addition, calmodulin antagonists, trifluoperazine and compound 48/80 produced a concentration-dependent inhibition of Ca²+-transport. These data suggested the presence of endogenous calmodulin. Both antagonists failed to influence the Ca²+-dependent ATPase activity. Experiments using boiled extracts from guinea-pig pancreatic acinar plasma membranes and erythrocyte plasma membranes Ca²+-ATPase confirmed the presence of endogenous calmodulin. The catalytic subunit of cyclic AMP-dependent protein kinase stimulated Ca²+ transport, suggesting that cyclic AMP may have a role in the regulation of Ca²+-pump-mediated Ca²+ efflux from pancreatic acini. Ca²+-dependent ATPase activity, on the other hand, was not affected by the catalytic subunit. HA 1004, a specific inhibitor of cAMP-dependent protein kinase, failed to inhibit the Ca²+-transport and Ca²+-dependent ATPase activities. Since, this inhibitor was also ineffective at inhibiting the catalytic-subunit-stimulated Ca²+ transport, it may be concluded that HA 1004 is ineffective in blocking the actions of cAMP-dependent protein kinase in pancreatic acinar plasma membranes. In our studies, purified protein kinase C, the phorbol ester TPA and the diacylglycerol derivative, SA-DG, failed to stimulate the Ca²+-uptake activity. However, these agents produced stimulation of the Ca²+-dependent ATPase activity in the presence of phosphatidylserine. CGP 41 251, a potent and selective inhibitor of protein kinase C, did not inhibit the Ca²+-transport or Ca²+-dependent ATPase activities. These observations suggest that protein kinase C may not be involved in the regulation of the plasma membrane Ca²+-pump in guinea-pig pancreatic acinar cells. These results also point to another difference between Ca²+-transport and the Ca²+-ATPase activities in guinea-pig pancreatic acinar plasma membranes. Neither inositol trisphosphate nor inositol tetrakisphosphate produced a statistically significant effect on Ca²+-uptake, suggesting that IP₃- and/or IP₄-mediated Ca²+ releasing pathways may not operate in the isolated guinea-pig pancreatic acinar plasma membrane vesicles. In summary, the results presented here provide evidence to suggest that the high affinity Ca²+-ATPase is not the biochemical expression of plasma membrane Ca²+-transport in panreatic acini. Our results imply a role for calmodulin and cAMP-dependent protein kinase, but not protein kinase C, in the regulation of Ca²+ efflux from pancreatic acinar cells.
Medicine, Faculty of
Anesthesiology, Pharmacology and Therapeutics, Department of
Graduate

[Truncated abstract] Osteoclasts are multinucleated giant cells responsible for the resorption of the mineralized bone matrix during the process of bone remodelling. During activation towards bone resorption, polarization of the osteoclast results in the formation of a unique plasma membrane, the ruffled border, the actual resorptive organelle of the osteoclast. Through this domain protons are actively pumped into the resorption lacuna creating an acidic microenvironment that favours the dissolution of the mineralized bone matrix. The polarised secretion of protons is carried out by the action of the vacuolar-type (H+)-ATPase (V-ATPase), composed of functionally and structurally distinct subunits of the V1 and V0 domains. The general structure of the V-ATPase complex is highly conserved from yeast to mammals, however, multiple isoforms for specific V-ATPase subunits do exist exhibiting differential subcellular, cellular and tissue-specific localizations. This study focuses on the molecular identification and characterization of V-ATPase accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit in osteoclasts. Using the techniques of cDNA Subtractive Hybridization and DNA Micro-Array analyses respectively, the accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit were identified in RAW264.7-cells derived OcLs. ... Using web-based computational predictions, two possible transmembrane domains, an N-terminus 'signal anchor' sequence and a C-terminus dilysine- like endoplasmic reticulum (ER) retention signal were identified. By confocal microscopy, EYFP-tagged e was found to localize to the perinuclear region of transfected COS-7 cells in compartments representing the ER and Golgi apparatus with some localization in late endosomal/lysosomal-like vesicles. ER truncation of e did not alter its subcellular localization but exhibited significantly weaker association with Ac45 compared to the wild-type as depicted by BRET analyses. Association with the other V0 subunits remain unaffected. This may hint at a possibility that Ac45 may play a role in the masking of the ER signal of e following it's incorporation into the V0 domain. Although no solid evidence for a role in the assembly of the mammalian VATPase have been established, subunit e still represents a potential candidate whose role in the V-ATPase complex requires further investigation. Collectively, the data presented in this thesis has provided further insight into the composition of the osteoclast V-ATPase proton pump by: 1) identifying an accessory subunit, Ac45 which shows promise as a potential candidate for the regulation and/or targeting of the V-ATPase complex in osteoclasts and truncation of its targeting signal impairs osteoclastic bone resorption; 2) identification and preliminary characterization of the d2 isoform of the V0 domain d subunit whose exact role in the V-ATPase complex and in osteoclasts remains to be determined, although its has been implicated to be essential for osteoclastic function; and 3) Preliminary characterization of subunit-e, a potential assembly factor candidate for the mammalian V-ATPase V0 domain.

Two enzymatic systems from the lactic acid bacterium Oenococcus oeni, isolated from wine, have been studied. The first one is the H+-ATPase for which the activity was characterized under various conditions of growth. The activity gradually increased by l.6 to 1.9-fold upon inoculation at pH 3.5. The H+-ATPase activity did not vary significantly in function of the growth rate or with and without malic acid. However, acidification of the medium in the absence of malic acid induced the activity by 1.5 to 2.2-fold depending on the initial pH. The partially cloned H+-ATPase genes shared high homologies with those from other bacterial F0F1-ATPases. A mRNA of about 7 kb was detected by Northern blot and its size suggests that the genetic organization of O. oeni atp operon is similar to most F0F 1-ATPases. Furthermore, the amount of atp mRNA was shown to increase in acidic conditions. O. oeni H +-ATPase activity was pH-inducible and regulation of the expression seems to occur at the level of mRNA synthesis. Thus, the results confirmed the proposed role of the H+-ATPase in acid tolerance in O. oeni.
The second system studied was a chromosome-encoded P-type ATPase (CopB) and its putative transcriptional regulator (CopR). The copB gene encodes a protein showing great similarities with other Cu2+-ATPases of the CPx-type family of heavy-metal ATPases like Enterococcus hirae copB. Another gene (copR) was found 250 bp upstream of copB and displays great similarities with proteins of the MecI/BlaI family of transcriptional regulators, including En. hirae CopY repressor. O. oeni was shown to be highly resistant to copper and growth occurred in up to 30 mM CuSO4. Northern blot analyses indicated that the amount of copB mRNA increased upon a 0.2 to 4.0 mM copper stress suggesting that expression of the enzyme might be regulated at the level of mRNA synthesis. Whether CopR is involved in this regulation remains to be determined, but the results suggest that copRB genes might be involved in copper resistance in O. oeni.

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Abamectina é uma lactona macrocíclica pertencente à família das avermectinas, utilizada mundialmente como agente antiparasitário em animais de criação e estimação, além do emprego agrícola como princípio ativo dos inseticidas e nematicidas. Mitocôndrias são responsáveis pela conversão da energia liberada pelo transporte de elétrons e armazenamento como energia de ligação na molécula de ATP, um componente metabólico essencial. Interferências em sua síntese ou utilização caracterizam mecanismos pelos quais os xenobióticos podem expressar toxicidade aguda ou crônica. Neste trabalho, os efeitos da abamectina na bioenergética de mitocôndrias isoladas de fígado de rato foram avaliados. Nas concentrações utilizadas (5 a 25 µM), abamectina causou inibição da cadeia respiratória, sem afetar a atividade das enzimas NADH desidrogenase, succinato desidrogenase e o potencial de membrana, comportando-se de maneira semelhante à oligomicina e ao atractilosídeo. A principal atuação da abamectina foi reduzir o potencial mitocondrial de fosforilação oxidativa, diminuindo os níveis de ATP provavelmente como resultado de sua ação direta sobre a FoF1-ATPase, uma vez que inibiu a atividade desta enzima, e/ou sobre o translocador de ADP/ATP. A inibição mais acentuada da atividade fosfohidrolase em mitocôndrias intactas desacopladas do que em mitocôndrias rompidas juntamente com a inibição da despolarização do potencial de membrana induzida pelo ADP sugerem que a abamectina atuou inibindo mais especificamente o translocador de ADP/ATP do que a FoF1-ATPase
Abamectin is a macrocyclic lactone belonging to the avermectin family, used worldwide as antiparasitic agent in farm animals and pets, and agricultural employment as the active ingredient of insecticides and nematicides. Mitochondria are responsible for converting the energy released by electron transport and storage as the binding energy molecule ATP, an essential metabolic component. Interference in its synthesis or utilization characterize mechanisms by which xenobiotics can express acute or chronic toxicity. In this study, the effects of abamectin in the bioenergetics of mitochondria isolated from rat liver were evaluated. At the concentrations used (5-25 mM), abamectin caused inhibition of the respiratory chain without affecting the activity of enzymes NADH dehydrogenase, succinate dehydrogenase and the membrane potential, behaving similarly to oligomycin and Atractyloside. The main activity of abamectin was to reduce the potential of mitochondrial oxidative phosphorylation, decreasing ATP levels probably as a result of its direct action on the Fo-F1 ATPase, since it inhibited the activity of this enzyme, and/or the ADP/ATP translocator. The more pronounced inhibition of the fosfohydrolase activity in intact uncoupled mitochondria than in disrupted mitochondria, in addition to the inhibition of the ADP-stimulated depolarization of mitochondrial membrane potential suggest that abamectin acted more specifically by inhibiting the ADP/ATP translocator than the FoF1-ATPase

Orientador: Fabio Erminio Mingatto
Banca: Flavia Thomaz Verechia Pereira
Banca: Tiago Rodrigues
Resumo: Abamectina é uma lactona macrocíclica pertencente à família das avermectinas, utilizada mundialmente como agente antiparasitário em animais de criação e estimação, além do emprego agrícola como princípio ativo dos inseticidas e nematicidas. Mitocôndrias são responsáveis pela conversão da energia liberada pelo transporte de elétrons e armazenamento como energia de ligação na molécula de ATP, um componente metabólico essencial. Interferências em sua síntese ou utilização caracterizam mecanismos pelos quais os xenobióticos podem expressar toxicidade aguda ou crônica. Neste trabalho, os efeitos da abamectina na bioenergética de mitocôndrias isoladas de fígado de rato foram avaliados. Nas concentrações utilizadas (5 a 25 µM), abamectina causou inibição da cadeia respiratória, sem afetar a atividade das enzimas NADH desidrogenase, succinato desidrogenase e o potencial de membrana, comportando-se de maneira semelhante à oligomicina e ao atractilosídeo. A principal atuação da abamectina foi reduzir o potencial mitocondrial de fosforilação oxidativa, diminuindo os níveis de ATP provavelmente como resultado de sua ação direta sobre a FoF1-ATPase, uma vez que inibiu a atividade desta enzima, e/ou sobre o translocador de ADP/ATP. A inibição mais acentuada da atividade fosfohidrolase em mitocôndrias intactas desacopladas do que em mitocôndrias rompidas juntamente com a inibição da despolarização do potencial de membrana induzida pelo ADP sugerem que a abamectina atuou inibindo mais especificamente o translocador de ADP/ATP do que a FoF1-ATPase
Abstract: Abamectin is a macrocyclic lactone belonging to the avermectin family, used worldwide as antiparasitic agent in farm animals and pets, and agricultural employment as the active ingredient of insecticides and nematicides. Mitochondria are responsible for converting the energy released by electron transport and storage as the binding energy molecule ATP, an essential metabolic component. Interference in its synthesis or utilization characterize mechanisms by which xenobiotics can express acute or chronic toxicity. In this study, the effects of abamectin in the bioenergetics of mitochondria isolated from rat liver were evaluated. At the concentrations used (5-25 mM), abamectin caused inhibition of the respiratory chain without affecting the activity of enzymes NADH dehydrogenase, succinate dehydrogenase and the membrane potential, behaving similarly to oligomycin and Atractyloside. The main activity of abamectin was to reduce the potential of mitochondrial oxidative phosphorylation, decreasing ATP levels probably as a result of its direct action on the Fo-F1 ATPase, since it inhibited the activity of this enzyme, and/or the ADP/ATP translocator. The more pronounced inhibition of the fosfohydrolase activity in intact uncoupled mitochondria than in disrupted mitochondria, in addition to the inhibition of the ADP-stimulated depolarization of mitochondrial membrane potential suggest that abamectin acted more specifically by inhibiting the ADP/ATP translocator than the FoF1-ATPase
Mestre

Thesis (Ph. D.)--University of Oregon, 2006.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 63-68). Also available for download via the World Wide Web; free to University of Oregon users.

Despite over a century of inquiry, the mechanisms that achieve the close matching of oxygen supply to demand during exercise remain elusive. It has been proposed that in addition to its role as the primary oxygen carrier, the red blood cell (RBC) functions as a roving oxygen sensor, linking the oxygen demand at the muscle with oxygen delivery via the circulation (Ellsworth et al. 1995). It is hypothesised that the RBC would release adenosine triphosphate (ATP) in proportion to the number of unoccupied binding sites on the haemoglobin molecule as it traverses regions of high oxygen demand such as the microcirculation of active skeletal muscle. ATP would then stimulate the release of vasodilatory substances from the endothelium which would diffuse to neighbouring vascular smooth muscle resulting in vasodilation and an increase in blood flow in accordance with the oxygen demand set by the muscle. The first step in establishing a role for this mechanism during exercise in humans is to determine whether ATP increases in the venous blood draining an active muscle bed. Based on the handful of published studies, there is an increase in ATP concentration in the femoral vein during knee extensor exercise. However the response has not been studied in other vascular beds in humans. As such, the main aim of this thesis was to measure the ATP response to dynamic handgrip exercise. Secondary aims were to determine whether the response was modified by hypoxia, and to provide information about the timing of the changes in ATP concentration during a bout of handgrip exercise. These questions were addressed in Studies 3 and 4. Because blood flow is central to this hypothesis, a substantial portion of this thesis was also associated with the measurement of forearm blood flow (FBF) using venous occlusion strain gauge plethysmography (VOSGP), and this was conducted in Studies 1 and 2. VOSGP is based on the assumption that with venous outflow prevented, any increase in limb volume is proportional to the rate of arterial inflow. The rate of arterial inflow is determined as the slope of the change in limb volume over time. The slope must be calculated over the initial linear portion of this relationship, when arterial inflow is unaffected by the inevitable rise in venous pressure associated with venous occlusion. VOSGP was initially used to measure blood flow at rest and in response to pharmacological interventions which produced only modest increases in arterial inflow (Joyner et al. 2001). However, measurement of the high rates of arterial inflow that occur with exercise may challenge the limits of this technique. Tschakovsky et al. (1995) reported a marked reduction in arterial inflow over the first four cardiac cycles during venous occlusion following static handgrip exercise that elevated blood flow to 22-24 mL/min/100mL. Only during the first cardiac cycle was arterial inflow unaffected by cuff inflation. As such, the window for measuring high rates of arterial inflow may be very brief. Therefore Study 1 aimed to determine whether blood flow could be measured using VOSGP across the range of arterial inflows that occur with dynamic handgrip exercise. Participants (n = 7) completed four, five-minute bouts of dynamic handgrip exercise at 15, 30, 45, and 60% of maximum voluntary contraction (MVC). FBF was measured using VOSGP at rest, and following five minutes of dynamic handgrip exercise. The slope of the change in limb volume was measured over the first one, two, three, and four consecutive cardiac cycles following the onset of occlusion. FBF was 2.5 ± 0.5 at rest, and 16.5 ± 4.9, 24.9 ± 9.4, 44.1 ± 22.0, and 57.8 ± 14.9 mL/min/100mL following five minutes of exercise at 15, 30, 45, and 60% MVC, respectively. At rest, arterial inflow decreased across the four cardiac cycles (P = 0.017 for the main effect), however post-hoc pairwise comparisons revealed no significant differences between any of the cardiac cycles. In contrast, the inclusion of two, three, or four cardiac cycles at 30 and 60% MVC, and three or four cardiac cycles at 15 and 45% MVC resulted in reductions in calculated arterial inflow compared with using the first cardiac cycle alone (P > 0.05). The inclusion of just two cardiac cycles resulted in a 9-26% reduction in calculated arterial inflow depending on the workload. This reduction was even more pronounced when three (19-40%) or four (26-50%) cardiac cycles were included. In conclusion, resting FBF can be calculated over at least four cardiac cycles during venous occlusion at rest. However, exercising FBF should be calculated from the first cardiac cycle only following dynamic handgrip exercise across the range of intensities used in this study. This extends the findings of Tschakovsky et al. (1995) who demonstrated this effect following handgrip exercise at a single intensity. Study 2 was designed to establish the FBF response to dynamic handgrip exercise, whether the workloads produced different blood flow responses, and to establish the within- and between-day reproducibility of FBF measured using VOSGP. In Part A (within-day reproducibility), participants (n = 7) completed three trials of dynamic handgrip exercise at four intensities (15, 30, 45, and 60% MVC), with each exercise trial separated by 10 minutes of rest. In Part B (between-day reproducibility) participants (n = 7) completed three trials of dynamic handgrip exercise at 15, 30, and 45% MVC on three separate days within a two week period. FBF was measured at rest, and each minute of exercise during brief (5-7 second) pauses in contractions. FBF response. FBF increased from rest at all workloads (P > 0.05), and then plateaued between Minutes 1 to 5 at the 15 and 30% MVC workloads and between Minutes 2 and 5 at the 45% workload (P > 0.05 for each minute compared to Minute 5). Too few participants completed the 60% workload to permit any statistical analysis. FBF reached values of 13.0 ± 2.0, 26.8 ± 8.4, 44.8 ± 14.9, and 52.9 ± 5.1 mL/min/100mL in the final minute of exercise at the 15, 30, 45, and 60% MVC workloads. FBF was different between the 15, 30, and 45% workloads by Minute 3 (P > 0.05). Reproducibility. The within-day test-retest reliability of exercising FBF was poor to moderate (ICC = 0.375-0.624) with individual coefficients of variation (CVs) ranging from 6-25%, 9-23%, and 9-31% for the 15, 30, and 45% MVC workloads, respectively. The between-day test-retest reliability for resting FBF was moderate (ICC = 0.644, P > 0.05; individual CVs between 1 and 31%). Between-day test-retest reliability for exercising FBF was poor to moderate (ICC = 0.381-0.614), with individual CVs ranging from 14-24%, 8-23%, and 6-18% for the 15, 30, and 45% workloads, respectively. It was concluded from this study that VOSGP provides adequately reproducible measurements to detect changes in FBF of the magnitude seen between workloads in this study. However, the variability in the measurement precludes its use when smaller differences are of interest. Based on the previous findings reporting an increase in ATP concentration during dynamic knee extensor exercise in the leg (Gonzalez-Alonso et al. 2002; Yegutkin et al. 2007), Study 3 was designed to determine whether ATP concentration increased in the venous effluent during dynamic handgrip exercise in the forearm. Since the deoxygenation of haemoglobin is a primary stimulus for ATP release from red blood cells, a further aim was to determine whether this response was augmented by systemic hypoxia. Participants (n = 6) completed four, five-minute bouts of dynamic handgrip exercise at 30, 45, 65, and 85% MVC under normoxia (inspired oxygen fraction = 0.21) and hypoxia (inspired oxygen fraction = 0.12). Blood samples for the determination of ATP concentration were drawn at rest and 180 seconds after the onset of exercise at each workload from a catheter inserted into a forearm vein. Venous plasma ATP concentration at rest was 0.28 ± 0.11 μM/L and remained unchanged during exercise at workloads up to 85% MVC (P > 0.05). Systemic hypoxia, sufficient to reduce arterial oxygen saturation to 83 ± 2%, also failed to alter the plasma ATP concentration (P = 0.148). The lack of a change in ATP concentration was unexpected but there are several possible explanations. It is possible, although unlikely, that ATP was not released in the forearm microcirculation. The previous demonstration that ATP increased in response to static handgrip exercise (Forrester and Lind 1969) would suggest that this was probably not the case. When considered in the context of the findings from Study 4, the most plausible explanation is that a less than optimal blood sampling site may have hindered the measurement of a change in ATP. The blood flow response at the onset of dynamic exercise in the forearm is at least biphasic; Phase 1 describes the immediate, large increase in blood flow within 2 seconds of the onset of exercise and is believed to be governed by mechanical factors whereas Phase 2 has a latency of ~20 seconds and describes a further, slower increase until blood flow reaches steady state (Saunders et al. 2005b). The temporal characteristics of Phase 2, along with the fact that blood flow during this phase is closely related to the metabolic rate of the muscle, suggest regulation by metabolic factors. Currently there is scant evidence detailing the time course of vasodilator release, although it is important to demonstrate that the release of a vasodilatory substance precedes the blood flow response it is proposed to influence (Delp 1999). ATP is released from red blood cells in proportion to the offloading of oxygen and a reduction in the oxygen content of venous blood draining a muscle bed occurs within 10 seconds of the onset of exercise. Thus the release of ATP should follow soon thereafter. As such, Study 4 was designed to determine whether ATP increased in the venous effluent of the forearm following 30 and 180 seconds of dynamic handgrip exercise at 45% MVC; and whether this increase corresponded with a decrease in venous oxygen content. Participants (n = 10) completed two bouts of dynamic handgrip exercise at 45% MVC; the first was one minute in duration, and the second was four minutes in duration. Venous blood samples for the determination of ATP and venous oxygen content were drawn at rest and during exercise from a catheter inserted in a retrograde manner into the median cubital vein. Arterialised samples for the estimation of arterial blood gases and ATP concentration were obtained from the non-exercising hand. ATP concentration in arterialised blood from the non-exercising arm was 0.79 ± 0.30 μM/L at rest and remained unchanged at both time points during exercise (P > 0.05). ATP concentration in the venous blood of the exercising arm increased from 0.60 ± 0.17 μM/L at rest to 1.04 ± 0.33 μM/L 30 seconds after the onset of exercise (P > 0.05), and remained at this higher level after 180 seconds (0.92 ± 0.26 μM/L, P > 0.05 versus rest). This corresponded with a decrease in venous oxygen content from 103 ± 23 mL/L at rest to 68 ± 16 mL/L 30 seconds after the onset of exercise (P > 0.05) and 76 ± 15 mL/L (P > 0.05 versus rest) 180 seconds into exercise. Furthermore, at 180 seconds of exercise, ATP concentration was moderately and inversely related to venous oxygen content (r = -0.651, p > 0.05). In conclusion, this study provides the first evidence that ATP concentration is increased in the blood draining the exercising forearm muscles in response to dynamic handgrip exercise. The finding that ATP concentration was increased just 30 seconds after the onset of exercise is also novel, and particularly interesting in the context of the recently reported dynamic response characteristics of the forearm blood flow response. In conclusion, the work contained within this thesis provides several important findings. The first study has provided evidence that measuring high rates of arterial inflow using VOSGP is possible, but that the window for making these measurements is small, probably as brief as a single cardiac cycle. The second study demonstrated that while the reproducibility of forearm blood flow measurements using VOSGP is poor, it is adequate to detect the large changes that occurred between workloads. However, VOSGP cannot be used to detect more modest differences. Common to both Study 3 and 4 was the measurement of ATP at rest, and 180 seconds after the onset of dynamic handgrip exercise at 45% MVC. The primary difference was the position of the catheter which was inserted in an antegrade manner in Study 3, and in a retrograde manner in Study 4. Since ATP was unchanged in Study 3 but increased under similar conditions in Study 4, it is likely that ATP was also released during exercise in Study 3, but that a less than optimal blood sampling site precluded its measurement. This illustrates the necessity to sample blood from as close as possible to the probable site of ATP release, the muscle microcirculation. The most important and novel findings from this body of work come from Study 4. This is the first study to demonstrate an increase in ATP concentration in the forearm in response to dynamic handgrip exercise. However, the most novel finding was that ATP concentration was elevated just 30 seconds after the onset of exercise. Such an early increase has not previously been reported during dynamic exercise in any vascular bed. This is an important finding since establishing the time course for the release of vasodilatory substances is critical to our understanding of the mechanisms that regulate blood flow during exercise.

Despite over a century of inquiry, the mechanisms that achieve the close matching of oxygen supply to demand during exercise remain elusive. It has been proposed that in addition to its role as the primary oxygen carrier, the red blood cell (RBC) functions as a roving oxygen sensor, linking the oxygen demand at the muscle with oxygen delivery via the circulation (Ellsworth et al. 1995). It is hypothesised that the RBC would release adenosine triphosphate (ATP) in proportion to the number of unoccupied binding sites on the haemoglobin molecule as it traverses regions of high oxygen demand such as the microcirculation of active skeletal muscle. ATP would then stimulate the release of vasodilatory substances from the endothelium which would diffuse to neighbouring vascular smooth muscle resulting in vasodilation and an increase in blood flow in accordance with the oxygen demand set by the muscle. The first step in establishing a role for this mechanism during exercise in humans is to determine whether ATP increases in the venous blood draining an active muscle bed. Based on the handful of published studies, there is an increase in ATP concentration in the femoral vein during knee extensor exercise. However the response has not been studied in other vascular beds in humans. As such, the main aim of this thesis was to measure the ATP response to dynamic handgrip exercise. Secondary aims were to determine whether the response was modified by hypoxia, and to provide information about the timing of the changes in ATP concentration during a bout of handgrip exercise. These questions were addressed in Studies 3 and 4. Because blood flow is central to this hypothesis, a substantial portion of this thesis was also associated with the measurement of forearm blood flow (FBF) using venous occlusion strain gauge plethysmography (VOSGP), and this was conducted in Studies 1 and 2. VOSGP is based on the assumption that with venous outflow prevented, any increase in limb volume is proportional to the rate of arterial inflow. The rate of arterial inflow is determined as the slope of the change in limb volume over time. The slope must be calculated over the initial linear portion of this relationship, when arterial inflow is unaffected by the inevitable rise in venous pressure associated with venous occlusion. VOSGP was initially used to measure blood flow at rest and in response to pharmacological interventions which produced only modest increases in arterial inflow (Joyner et al. 2001). However, measurement of the high rates of arterial inflow that occur with exercise may challenge the limits of this technique. Tschakovsky et al. (1995) reported a marked reduction in arterial inflow over the first four cardiac cycles during venous occlusion following static handgrip exercise that elevated blood flow to 22-24 mL/min/100mL. Only during the first cardiac cycle was arterial inflow unaffected by cuff inflation. As such, the window for measuring high rates of arterial inflow may be very brief. Therefore Study 1 aimed to determine whether blood flow could be measured using VOSGP across the range of arterial inflows that occur with dynamic handgrip exercise. Participants (n = 7) completed four, five-minute bouts of dynamic handgrip exercise at 15, 30, 45, and 60% of maximum voluntary contraction (MVC). FBF was measured using VOSGP at rest, and following five minutes of dynamic handgrip exercise. The slope of the change in limb volume was measured over the first one, two, three, and four consecutive cardiac cycles following the onset of occlusion. FBF was 2.5 ± 0.5 at rest, and 16.5 ± 4.9, 24.9 ± 9.4, 44.1 ± 22.0, and 57.8 ± 14.9 mL/min/100mL following five minutes of exercise at 15, 30, 45, and 60% MVC, respectively. At rest, arterial inflow decreased across the four cardiac cycles (P = 0.017 for the main effect), however post-hoc pairwise comparisons revealed no significant differences between any of the cardiac cycles. In contrast, the inclusion of two, three, or four cardiac cycles at 30 and 60% MVC, and three or four cardiac cycles at 15 and 45% MVC resulted in reductions in calculated arterial inflow compared with using the first cardiac cycle alone (P > 0.05). The inclusion of just two cardiac cycles resulted in a 9-26% reduction in calculated arterial inflow depending on the workload. This reduction was even more pronounced when three (19-40%) or four (26-50%) cardiac cycles were included. In conclusion, resting FBF can be calculated over at least four cardiac cycles during venous occlusion at rest. However, exercising FBF should be calculated from the first cardiac cycle only following dynamic handgrip exercise across the range of intensities used in this study. This extends the findings of Tschakovsky et al. (1995) who demonstrated this effect following handgrip exercise at a single intensity. Study 2 was designed to establish the FBF response to dynamic handgrip exercise, whether the workloads produced different blood flow responses, and to establish the within- and between-day reproducibility of FBF measured using VOSGP. In Part A (within-day reproducibility), participants (n = 7) completed three trials of dynamic handgrip exercise at four intensities (15, 30, 45, and 60% MVC), with each exercise trial separated by 10 minutes of rest. In Part B (between-day reproducibility) participants (n = 7) completed three trials of dynamic handgrip exercise at 15, 30, and 45% MVC on three separate days within a two week period. FBF was measured at rest, and each minute of exercise during brief (5-7 second) pauses in contractions. FBF response. FBF increased from rest at all workloads (P > 0.05), and then plateaued between Minutes 1 to 5 at the 15 and 30% MVC workloads and between Minutes 2 and 5 at the 45% workload (P > 0.05 for each minute compared to Minute 5). Too few participants completed the 60% workload to permit any statistical analysis. FBF reached values of 13.0 ± 2.0, 26.8 ± 8.4, 44.8 ± 14.9, and 52.9 ± 5.1 mL/min/100mL in the final minute of exercise at the 15, 30, 45, and 60% MVC workloads. FBF was different between the 15, 30, and 45% workloads by Minute 3 (P > 0.05). Reproducibility. The within-day test-retest reliability of exercising FBF was poor to moderate (ICC = 0.375-0.624) with individual coefficients of variation (CVs) ranging from 6-25%, 9-23%, and 9-31% for the 15, 30, and 45% MVC workloads, respectively. The between-day test-retest reliability for resting FBF was moderate (ICC = 0.644, P > 0.05; individual CVs between 1 and 31%). Between-day test-retest reliability for exercising FBF was poor to moderate (ICC = 0.381-0.614), with individual CVs ranging from 14-24%, 8-23%, and 6-18% for the 15, 30, and 45% workloads, respectively. It was concluded from this study that VOSGP provides adequately reproducible measurements to detect changes in FBF of the magnitude seen between workloads in this study. However, the variability in the measurement precludes its use when smaller differences are of interest. Based on the previous findings reporting an increase in ATP concentration during dynamic knee extensor exercise in the leg (Gonzalez-Alonso et al. 2002; Yegutkin et al. 2007), Study 3 was designed to determine whether ATP concentration increased in the venous effluent during dynamic handgrip exercise in the forearm. Since the deoxygenation of haemoglobin is a primary stimulus for ATP release from red blood cells, a further aim was to determine whether this response was augmented by systemic hypoxia. Participants (n = 6) completed four, five-minute bouts of dynamic handgrip exercise at 30, 45, 65, and 85% MVC under normoxia (inspired oxygen fraction = 0.21) and hypoxia (inspired oxygen fraction = 0.12). Blood samples for the determination of ATP concentration were drawn at rest and 180 seconds after the onset of exercise at each workload from a catheter inserted into a forearm vein. Venous plasma ATP concentration at rest was 0.28 ± 0.11 μM/L and remained unchanged during exercise at workloads up to 85% MVC (P > 0.05). Systemic hypoxia, sufficient to reduce arterial oxygen saturation to 83 ± 2%, also failed to alter the plasma ATP concentration (P = 0.148). The lack of a change in ATP concentration was unexpected but there are several possible explanations. It is possible, although unlikely, that ATP was not released in the forearm microcirculation. The previous demonstration that ATP increased in response to static handgrip exercise (Forrester and Lind 1969) would suggest that this was probably not the case. When considered in the context of the findings from Study 4, the most plausible explanation is that a less than optimal blood sampling site may have hindered the measurement of a change in ATP. The blood flow response at the onset of dynamic exercise in the forearm is at least biphasic; Phase 1 describes the immediate, large increase in blood flow within 2 seconds of the onset of exercise and is believed to be governed by mechanical factors whereas Phase 2 has a latency of ~20 seconds and describes a further, slower increase until blood flow reaches steady state (Saunders et al. 2005b). The temporal characteristics of Phase 2, along with the fact that blood flow during this phase is closely related to the metabolic rate of the muscle, suggest regulation by metabolic factors. Currently there is scant evidence detailing the time course of vasodilator release, although it is important to demonstrate that the release of a vasodilatory substance precedes the blood flow response it is proposed to influence (Delp 1999). ATP is released from red blood cells in proportion to the offloading of oxygen and a reduction in the oxygen content of venous blood draining a muscle bed occurs within 10 seconds of the onset of exercise. Thus the release of ATP should follow soon thereafter. As such, Study 4 was designed to determine whether ATP increased in the venous effluent of the forearm following 30 and 180 seconds of dynamic handgrip exercise at 45% MVC; and whether this increase corresponded with a decrease in venous oxygen content. Participants (n = 10) completed two bouts of dynamic handgrip exercise at 45% MVC; the first was one minute in duration, and the second was four minutes in duration. Venous blood samples for the determination of ATP and venous oxygen content were drawn at rest and during exercise from a catheter inserted in a retrograde manner into the median cubital vein. Arterialised samples for the estimation of arterial blood gases and ATP concentration were obtained from the non-exercising hand. ATP concentration in arterialised blood from the non-exercising arm was 0.79 ± 0.30 μM/L at rest and remained unchanged at both time points during exercise (P > 0.05). ATP concentration in the venous blood of the exercising arm increased from 0.60 ± 0.17 μM/L at rest to 1.04 ± 0.33 μM/L 30 seconds after the onset of exercise (P > 0.05), and remained at this higher level after 180 seconds (0.92 ± 0.26 μM/L, P > 0.05 versus rest). This corresponded with a decrease in venous oxygen content from 103 ± 23 mL/L at rest to 68 ± 16 mL/L 30 seconds after the onset of exercise (P > 0.05) and 76 ± 15 mL/L (P > 0.05 versus rest) 180 seconds into exercise. Furthermore, at 180 seconds of exercise, ATP concentration was moderately and inversely related to venous oxygen content (r = -0.651, p > 0.05). In conclusion, this study provides the first evidence that ATP concentration is increased in the blood draining the exercising forearm muscles in response to dynamic handgrip exercise. The finding that ATP concentration was increased just 30 seconds after the onset of exercise is also novel, and particularly interesting in the context of the recently reported dynamic response characteristics of the forearm blood flow response. In conclusion, the work contained within this thesis provides several important findings. The first study has provided evidence that measuring high rates of arterial inflow using VOSGP is possible, but that the window for making these measurements is small, probably as brief as a single cardiac cycle. The second study demonstrated that while the reproducibility of forearm blood flow measurements using VOSGP is poor, it is adequate to detect the large changes that occurred between workloads. However, VOSGP cannot be used to detect more modest differences. Common to both Study 3 and 4 was the measurement of ATP at rest, and 180 seconds after the onset of dynamic handgrip exercise at 45% MVC. The primary difference was the position of the catheter which was inserted in an antegrade manner in Study 3, and in a retrograde manner in Study 4. Since ATP was unchanged in Study 3 but increased under similar conditions in Study 4, it is likely that ATP was also released during exercise in Study 3, but that a less than optimal blood sampling site precluded its measurement. This illustrates the necessity to sample blood from as close as possible to the probable site of ATP release, the muscle microcirculation. The most important and novel findings from this body of work come from Study 4. This is the first study to demonstrate an increase in ATP concentration in the forearm in response to dynamic handgrip exercise. However, the most novel finding was that ATP concentration was elevated just 30 seconds after the onset of exercise. Such an early increase has not previously been reported during dynamic exercise in any vascular bed. This is an important finding since establishing the time course for the release of vasodilatory substances is critical to our understanding of the mechanisms that regulate blood flow during exercise.

This study has been concerned with the design and synthesis of A TP analogues with the potential to act as inhibitors of glutamine synthetase - a novel target for therapeutic intervention in the treatment of tuberculosis. Using a structural -analogy approach, various 3-indolylalkanoic acid, benzimidazole and pyrazolo[3,4-dJpyrimidine derivatives have been prepared and characterized. Alkylation of the heterocyclic bases using 4-(bromomethyl)-2,2-dimethyl-1 ,3-d ioxolane, 2-(bromomethoxy)ethyl acetate and 2-(chloroethoxy)ethanol in the presence of either NaH or BulOK afforded the corresponding N-alkylated derivatives of benzimidazole and 4-aminopyrazolo[3,4-dJpyrimidine (4-APP). Similar reactions with 3-indo lylalkanoic esters resulted in O-alkyl cleavage with the formation of new esters. Alkylation of benzimidazole with allyl bromide, 4-bromobutene and 2-methylbut-2-ene has also been shown to afford the corresponding l-alkenylbenzimidazoles in moderate to excellent yield (43-96%). Subsequent oxidation of these products using CTAP, gave the dihydroxy derivatives in poor to good yields (26-77%). Phosphorylation of various hydroxy derivatives of benzimidazole and 4-APP has been achieved using diethyl chlorophosphate to afford the corresponding monophosphate and 1,2-diphosphate esters. Glycosylation of each of the heterocyclic bases has been successfully achieved using 1,2,3,4,6-penta-O-acetyl-D-glucopyranose and SnCl4 in acetonitri le, while methanolysis of the resulting tetraacetates, using methanolic NaOMe, afforded the hydroxy derivatives in good yield (50-70%). Various 1- and 2-dimensional NMR spectroscopic methods (e.g., IH, 13C, lip, COSY, HSQC and HMBC) have been used to confirm the structures of the synthesized A IP analogues. The application of NMR prediction programmes has been explored, permitting assessment of their agreement with the experimental data and confirmation of assigned structures. High-resolution electron impact (EI) mass spectrometric data have been used to explore the mass fragmentation pathways exhibited by selected derivatives, and certain common fragmentations have been identified. Molecular modelling of selected products as potential glutamine synthetase ligands has been performed on the Accelrys Cerius2 platform, and interactive receptor-ligand docking studies have been conducted using the Ligand-Fit module. These studies have revealed possible hydrogen-boding interactions between the selected analogues and various amino acid residues in the glutamine synthetase active site.

Orientador: Alberto Vazquez Saa
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Matemática, Estatística e Computação Científica
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Resumo: O ATP (adenosina tri-fosfato) é uma molécula chave para a fisiologia, atuando como fonte de energia para diversos processos celulares. ...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital
Abstract: ATP (adenosine triphosphate acts as an "energy currency" providing energy to several physiological processes. ...Note: The complete abstract is available with the full electronic document
Mestrado
Matematica Aplicada
Mestre em Matemática Aplicada