Thesis (Ph.D.)--McMaster University, 2003. / Advisor: David Andrews. Includes bibliographical references (leaves 206-242). Also available via World Wide Web.

A thimet oligopeptidase (EP24.15) foi originariamente descrita como uma enzima metabolizadora de neuropeptídeos que não possui um peptídeo sinal para entrada na via secretória clássica, mas é secretada pelas células através de um mecanismo não-convencional. Nesse trabalho, identificamos uma nova interação cálcio-dependente entre EP24.15 e calmodulina I (CaM), que é importante para a secreção estimulada, mas não constitutiva, da EP24.15. A superexpressão da CaM em células HEK293 aumenta a secreção estimulada da EP24.15, podendo ser inibida pelo inibidor da CaM. O inibidor específico da PKA reduz a secreção estimulada de EP24.15. Nossos dados sugerem que a interação entre EP24.15 e calmodulina é regulada e relevante para a secreção estimulada da EP24.15 em células HEK293. Surpreendentemente, experimentos com slices (fatias) de cérebros de ratos sugerem que, fisiologicamente, a EP24.15 é secretada predominantemente de forma constitutiva, embora o tratamento com A23187 e forskolin sejam capazes de aumentar modestamente a secreção dessa enzima nessas preparações. / Thimet oligopeptidase (EC3.4.24.15; EP24.15) was originally described as a neuropeptide-metabolising enzyme that lacks a typical signal-peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. Here, we identify a novel calcium-dependent interaction between EP24.15 and calmodulin I (CaM) that is important for the stimulated, but not constitutive, secretion of EP24.15. Overexpression of CaM in HEK293 cells increase the stimulated secretion of EP24.15, which can be inhibited by the CaM inhibitor. The specific inhibition of PKA with reduced the A23187-stimulated secretion of EP24.15. Our data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells. Surprising, the rats brain slices experiments showed that, physiological, EP24.15 has a constitutive secretion, although the A23187 and forskolin treatment are able to increase a little this enzyme secretion in these preparations.

Orientadores: Antonio Marcos Orsi, Sergio Luis Felisbino / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T10:45:56Z (GMT). No. of bitstreams: 1 Domeniconi_RaquelFantin_D.pdf: 10868266 bytes, checksum: 8bdfe8a912508f6afd1b6131f9f8934e (MD5) Previous issue date: 2007 / Resumo: Estudos recentes têm identificado família de proteínas denominadas aquaporinas (AQP), relacionadas à alta permeabilidade de água em várias membranas biológicas. As AQP1, AQP2, AQP7, AQP8 e AQP9 são as principais AQPs identificadas no sistema genital masculino, sendo a sua localização espécie-específica e região-específica. Em vista da importância do fluido luminal na via espermática para a integridade morfofuncional dos espermatozóides, bem como dos componentes que os constituem, tais como a água e proteínas, é importante estudar a distribuição das AQPs ao longo da via espermática. Assim, este trabalho teve como objetivos principais estudar no cão as AQP1, AQP2, AQP7, AQP8 e AQP9, visando identificá-las e localizá-las, através de imuno-histoquímica e ¿Western blotting¿ na via espermática. No cão, a AQP1 foi notada na rede testicular, ductos eferentes e em vasos, sugerindo sua importância na rápida absorção de fluido testicular. Pela primeira vez a AQP2 foi detectada na rede testicular, ductos eferentes e epidídimo, e a AQP7 no epitélio epididimário e ducto deferente em mamíferos. Porém, o papel funcional dessas AQPs no sistema genital masculino do cão permanece desconhecido. A AQP8 não foi detectada ao longo dos ductos extratesticulares do cão. A AQP9 foi abundantemente expressada ao longo da via espermática do cão, que representa um importante caminho apical para o fluxo transmembrana de água e solutos. Portanto, os resultados confirmam o padrão de expressão espécie-específica e região-específica das AQPs, sugerindo variações de atividades de absorção de fluidos e solutos ao longo da via espermática. O conhecimento destas variações torna-se relevante para estudos clínicos de infertilidade, bem como para tecnologias de reprodução assistida / Abstract: Recent studies have identified proteins called aquaporins (AQP) related to the fast water permeability in some biological membranes. AQPs are small, intrinsic membrane proteins that are present in many cell types involved in fluid transport. AQP1, AQP2, AQP7, AQP8 and AQP9 had been the main AQPs identified in the male reproductive tract, being their localization species-specific and region-specific. In view of the importance of the luminal fluid to sperm maturation and integrity of the spermatozoa, it is important to study the distribution of the AQPs throughout the spermatic way. Thus, the aim of this study was to examine the expression of AQP1, AQP2, AQP7, AQP8 e AQP9 in epithelial cells in the adult dog efferent ducts, epididymis and vas deferens, using immunohistochemistry and estern blotting methods to characterize the aquaporins in male reproductive tract. In dog, AQP1 was noted in rete testis, efferent ducts and in vessels in intertubular space, suggesting that AQP1 is important for rapid absorption of testicular fluid. For the first time the AQP2 was detected in rete testis, efferent ducts and epididymis and the AQP7 was expressed in the epithelium epididymidis and in vas deferens in mammals. But its functional role in the male dog reproductive tract, remain unknown. No specific staining for AQP8 was detected in epithelial cells of excurrent ducts in dog testis. AQP9 was abundantly expressed in dog male reproductive tract, in which it is an important apical pathway for transmembrane flow of water and neutral solutes. Thus the results confirm that the AQPs are species-specific and region-specific, suggesting activity variations related with the fluid and solute absorption throughout male excurrent ducts. Investigations of AQP biology could be relevant to clinical studies of the male reproductive tract, as well as to technologies for assisted procreation / Doutorado / Anatomia / Doutor em Biologia Celular e Estrutural

Abstract Beta-lactoglobulin (β-lg) is the main whey protein in bovine milk. It belongs to the lipocalin protein family, and it is one of the main milk allergens. Resistance to hydrolysis is a particular feature of β-lg making it possible that β-lg reaches the small intestine in its native form. Heat treatments during milk processing may change the native structure of bovine β-lg and change its intestinal transport properties. Heat induced conformational alterations may also expose new antigenic sites. However, there have been no previous studies on the effects of heat treatment on the transport of β-lg or on its sensitizing properties. Cow's milk allergy is one of the most important food allergies affecting about 2.4% of infants. Milk proteins, including β-lg, in breast milk substitute formulas are often the earliest foreign antigens in the diet of newborns. According to the hygiene hypothesis, natural infections and vaccinations may modify the immunological balance and decrease the risk of allergy. Isoelectric precipitations followed by anion exchange and gel filtration were used to purify bovine milk β-lg in its native form. Transport of native and heat-denatured β-lg was compared in two in vitro cell models, Caco-2 and M-cells. Sensitization properties of native and heat-denatured β-lg were studied with an animal model using Hooded-Lister rats. Effects of BCG vaccination in combination with the native β-lg were also studied. Effects of different sensitizations were assessed by antibody levels in serum and inflammation locally in the gastrointestinal tract. Heat denaturation of β-lg made its transport slower in both enterocytes and M-cells. M-cells were more effective transporters of both native and heat-denatured β-lg than caco-2 cells. Animals generated higher levels of IgE when sensitized with native β-lg, but heat-denatured β-lg induced a more intense inflammatory cell reaction in the gastrointestinal tract. Vaccination with BCG decreased serum IgE concentration and modified the predominant site of the inflammatory cell response in intestine. The results indicate that, heat denaturation of β-lg and BCG vaccination, change both the systemic and the mucosal response to bovine milk β-lg. The reasons for this remain speculative. The effect of BCG vaccination is consistent with the hygiene hypothesis. The observed alteration of transport properties could be one mechanism by which heat denaturation modifies the allergenic properties of this protein, but additional studies are necessary to assess whether other mechanisms, such as exposure of new antigenic determinants are also relevant.

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

Membrane traffic in polarised cells was investigated by growing Madin-Darby canine kidney (MOCK) cells on ·permeable polycarbonate filter supports which allowed access to both sides of the cell monolayer. Membrane glycoconjugates on the apical and basolateral cell surfaces were labelled enzymatically with ³H- and ¹⁴C-galactose, respectively, to provide covalent membrane markers. Experiments were done to quantitate membrane traffic during endocytosis at the respective plasma membrane domains and that due to transcytosis. Internalized label was quantitatively distinguished from label on the respective cell surface by its resistance to removal by glycosidases.

No description available.

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.

We investigate the relaxation dynamics of two distinct non-equilibrium processes: relaxation of three-dimensional antiferromagnetic lattice spin models with Heisenberg interaction following a critical quench, and a one-dimensional exclusion process inspired by the gear-like motion of molecular motors. In a system of three-dimensional Heisenberg antiferromagnets the non-conserved staggered magnetization components couple non-trivially to the conserved magnetization densities inducing fully reversible terms that enter the Langevin dynamic equation. We simulate the exact microscopic dynamics of such a system of antiferromagnets by employing a hybrid simulation algorithm that combines the reversible spin precession implemented by the fourth-order Runge-Kutta integration method with the standard relaxational dynamics at finite temperatures using Monte Carlo updates. We characterize the dynamic universality class of this system by probing the early temporal window where the system exhibits aging scaling properties. We also verify an earlier renormalization group prediction that the temporal decay exponent in the two-time spin autocorrelation function exhibits non-universality, specifically it depends on the width of the initial spin orientation distribution. We employ a similar numerical technique to study the critical dynamics of an anisotropic Heisenberg antiferromagnet in the presence of an external field. The phase diagram of this system exhibits two critical lines that meet at a bicritical point. We study the aging scaling dynamics for the model C critical line, probe the model F critical line by investigating the system size dependence of the characteristic spin-wave frequencies near criticality, and measure the dynamic critical exponents for the order parameter including its aging scaling at the bicritical point. We introduce a one-dimensional non-equilibrium lattice gas model representing the processive motion of dynein molecular motors over the microtubule. We study both dynamical and stationary state properties for the model consisting of hardcore particles hopping on the lattice with variable step sizes. We find that the stationary state gap-distribution exhibits striking peaks around gap sizes that are multiples of the maximum step size, for both open and periodic boundary conditions, and verify this using a mean-field calculation. For open boundary conditions, we observe intriguing damped oscillator-like distribution of particles over the lattice with a periodicity equal to the maximum step size. To characterize transient dynamics, we measure the mean square displacement that shows weak superdiffusive growth with exponent γ≈ 1.34 for periodic boundary and ballistic growth ( γ≈ 2) for open boundary conditions at early times. We also study the effect of Langmuir dynamics on the density profile. / Doctor of Philosophy / Most systems found in nature are out of equilibrium. In this dissertation we investigate the relaxation dynamics of two such non-equilibrium systems: 1. We investigate a three-dimensional antiferromagnetic system relaxing towards equilibrium from an initial state that is driven far away from equilibrium at the point in the parameter space where the system undergoes a second-order phase transition. We devise a novel simulation method that captures emerging dynamic universal features and scaling features at these points of continuous phase transition in the early times of relaxation when the system is still far away from equilibrium. 2. Cytoplasmic dyneins are one of three kinds of motor proteins that move on tubular structures called microtubules carrying and transporting cellular cargo inside the cells. Unlike the other molecular motors that move forward with fixed step sizes, the dyneins have been experimentally observed to vary their step size depending on the amount of cargo they are carrying. We model an exclusion process in a one-dimensional lattice inspired by the motion of the dynein molecular motors where the motors can hop from one to four steps depending on their internal states. We study the effect of this variable step size on the dynamics of a collection of dyneins. We observe intriguing oscillating density profiles and discrete peaks in the distribution of empty sites. Our results suggest self-organization among the motors and the empty sites.

No description available.