Dissertations / Theses on the topic 'Cellular control mechanisms'

Im ersten Teil meiner Arbeit habe ich den molekularen Mechanismus beschrieben, mit dem endogene neuronale Vorläuferzellen antitumorigen gegen Gliomstammzellen wirken. Unsere Forschungsgruppe hat in bereits veröffentlichten Arbeiten gezeigt, dass neuronale Vorläuferzellen zu experimentellen Gehirntumoren migrieren und Tumorzelltod induzieren können. In der nun vorliegenden Arbeit zeige ich, dass die neuronalen Vorläuferzellen nicht nur benefiziell gegen die Hauptpopulation der Tumorzellen wirken, sondern darüber hinaus auch die kleinere Population der sehr aggressiven Tumorstammzellen – mittels Sekretion von BMP7 – supprimieren. Insgesamt zeigt meine Arbeit, dass neuronale Vorläuferzellen die Pathogenität der Gliomstammzellen unterdrücken. Im zweiten Teil meiner Arbeit habe ich einen zellautonomen Mechanismus untersucht, der Gliomzellen in vitro und in vivo vermehrt expandieren lässt. Meine Ergebnisse zeigen, dass die Familie der ets-Transkriptionsfaktoren Gliomzellen zur Proliferation anregen, indem sie die Expresion eines Eisentransporters (dem Transferrin-Rezeptor-1) induzieren und damit die intrazelluläre Akkumulation von Eisenionen begünstigen. Die Veränderung des Redox-Gleichgewichts in den Gliomzellen regt die Tumore zu verstärkter Sekretion von Glutamat an. Dadurch werden die Gliome sehr zytotoxisch und induzieren Zelltod in den Zellen des tumorumgebenden Parenchyms. Das untergegangene Nervengewebe schafft damit den Platz, den der Tumor zur Expansion braucht. Insgesamt zeigt meine Arbeit, dass die ets1-induzierte CD71 Expression nicht nur das Tumorwachstum befördert, sondern auch den Platz zum Tumorwachstum schafft.
In my first part,Gliomas cells with stem-like properties (GSCs) control tumor growth and recurrence. Here, I showed that endogenous neural precursor cells (NPCs) perform an anti-tumor response by specifically targeting GSCs: In vitro, NPCs predominantly expressed BMP7; BMP7 was constitutively released from neurospheres and induced canonical BMP-signaling in GSCs. Exposure of human and murine GSCs to neurosphere-derived BMP7 increased GSC differentiation, attenuated GSC-marker expression, GSC self-renewal and the ability for tumor initiation.This anti-tumor response of NPCs protect the brain from gliomas by releasing BMP7, which acts as a paracrine tumor suppressor that represses proliferation, self-renewal and tumor-initiation of GSCs. In the 2nd part, Transferrin receptors (TfR) are overexpressed in brain tumors, but the pathological relevance has not been fully explored. Here, I showed that TfR is an important downstream effector of ets transcription factors that promotes glioma proliferation and increases glioma-evoked neuronal death. TfR mediates iron accumulation and reactive oxygen formation and thereby enhanced proliferation in clonal human glioma lines. TfR-induced oxidant accumulation modified cellular signaling by inactivating a protein tyrosine phosphatase (low-molecular-weight protein tyrosine phosphatase), activating mitogen-activated protein kinase and Akt and by inactivating p21/cdkn1a and pRB. Inactivation of these cell cycle regulators facilitated S-phase entry. Besides its effect on proliferation, TfR also boosted glutamate release, which caused NMDA mediated reduction of neuron cell mass. Overall my results indicate that TfR promotes glioma progression by two mechanisms, an increase in proliferation rate and glutamate production, the latter mechanism providing space for the progressing tumor mass.

Upon cell stimulation with hormones and other mitogens, a variety of biochemical and physiological responses occur within the first few minutes, including turnover of inositol phospholipids, activation of a number of kinases, and changes in intracellular pH (pHⁱⁿ) and calcium ([Ca²⁺]ⁱⁿ). Changes in both pHⁱⁿ and [Ca²⁺](in) are prominent and play a major role in the signal transduction mechanism leading to the physiological response, i.e. secretion, neurotransmission, proliferation and differentiation. The intracellular pH changes that follow mitogenic activation are complex and may reflect several different H⁺ transporting mechanisms. There are at least three main systems involved in the regulation of pHⁱⁿ in eukaryotic cells: (a) the mitogen stimulated Na⁺/H⁺ exchange, which electroneutrally raises pHⁱⁿ and can be inhibited by amiloride and its derivatives; (b) a variety of HCO₃⁻-based mechanisms which can alkalinize or acidify the cytosol, and can be inhibited by stilbene disulfonate derivatives; (c) and a plasma membrane H⁺-ATPase, which represents the least understood mechanism of pHⁱⁿ regulation. Under non-pathological conditions, pHⁱⁿ regulation is generally achieved by Na⁺/H⁺ exchange and HCO₃⁻-based mechanisms. Missexpression of a H⁺-ATPase in the plasma membrane can lead to a chronically high pHⁱⁿ in some tumor cells and might contribute to carcinogenesis. Chapter I explains the dissertation format and the relationship of the manuscripts included in three appendices. This chapter also indicates my contribution to each of these manuscripts. Chapter II is a summary of the most important findings in these manuscripts. Appendix I deals with the role of Na⁺/H⁺ exchange and Cl⁻/HCO₃⁻ exchange in the regulation of pHⁱⁿ. Appendix II deals with the role of H⁺-ATPase in the maintenance of a chronically high pHⁱⁿ and its possible involvement in tumorigenesis. Appendix III describes a technique to simultaneously measure pHⁱⁿ and [Ca²⁺]ⁱⁿ by fIuorescence spectroscopy. This appendix also describes the application to study the role of pHⁱⁿ and Ca²⁺ in the regulation of cell growth and progesterone secretion.

Due to the diverse functions of Fc eRII, such as its roles in cellular adhesion, growth and differentiation of B and T lymphocytes, rescue of B cells from apoptosis and release of cytotoxic mediators, it is clear why it is believed to be a central molecule in allergic response. Because of its important role in the regulation of IgE production, FceRII may be the primary cause of certain allergic conditions. This study attempted to express and purify a recombinant human soluble FceRII to test its effect on a B-lymphoblastoid (RPMI 8866) and a monocytic (U937) cell line. The protein was expressed in Escherichia coli inclusion bodies, before being refolded and purified in a single gel chromatography step. This pure protein was then tested for biological activity by testing its IgE binding func tion. Once proven functional, it was used to test its effect on the cell lines at three concentrations for its apoptotic rescue properties and its cytokine effects. The recombinant protein did not seem to have any significant effect on the apoptotic rescue of either cell line. While the recombinant sFceRII appeared to have a slight effect on the stimulation of IL-1ß and TNFa in the RPMI 8866 cells, there was no apparent effect on the production of NF?B. In U937 cells, the protein did not seem to have any effect on the stimulation of IL-1ß, TNFa or NF?B. However, the cytokine effects of the recombinant protein were tested on isolated PBMCs from a healthy individual and a hyper-IgE syndrome patient. The recombinant protein was able to stimulate the production of cytokines in both individuals’ PBMCs, proving that it has the same effect as the natural protein. The upregulation of these cytokines indicates that the recombinant protein is able to stimulate the immune system. Therefore, this recombinant soluble FceRII protein could possibly be used for immune therapy.

Two important membrane transport processes involved in the regulation of intracellular pH (pH) and Na* activity (ave) are the plasmalemmal Na*-H* exchanger and Na*-K* pump. In several non-cardiac tissues exposure to a hyperosmolar or hypoosmolar (anisosmolar) extracellular milieu modulates the activity of the Na*-H* exchanger. The response of the Na*-K* pump to alterations of extracellular osmolarity is, however, largely unknown. In cardiac muscle the effects of changes in extracellular osmolarity and hence cell volume on the activity of the Na*-H* exchanger and Na*-K* pump has not been studied. Such a study is desirable since pH, and am are important determinants of contractility in cardiac muscle and because the myocardium may be exposed to an anisosmolar milieu in several clinical settings. The studies described in this thesis examine the effect of shrinkage and swelling of cardiac tissue during exposure to anisosmolar solutions on the regulation of pH. and a'. by the sarcolemmal Na*-H* exchanger and Na*-K* pump. Ion-selective microelectrodes were used to determine pH; and a'v. in guinea-pig ventricular muscle. Exposure of tissue to HEPES-buffered Tyrode's solution made hyperosmolar by the addition of 100 mM sucrose (418 mosM) produced an intracellular alkalinization of O.10 units and hyperpolarization of the membrane potential. When the Na*-H* exchanger was inhibited by 5-(N,N-dimethyl) amiloride (DMA) or by reducing extracellular Na* to 15 mM (choline substituted) exposure to hyperosmolar solution caused a small decrease in pH. The Na+-dependence and DMA-sensitivity of the intracellular alkalinization in hyperosmolar solution together with the demonstration of a DMA-sensitive rise in ax, during such exposure suggested that the sarcolemmal Na*-H* exchanger was activated by cell shrinkage in hyperosmolar solution. Further evidence for osmotic activation of the Na*-H* exchanger was obtained from experiments showing that the recovery of pH, from an intracellular acidosis, induced by brief exposure to NIL, CI, was faster in hyperosmolar than in isosmolar solution.

Thesis (Ph. D.)--University of Oregon, 2000.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 106-112). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003.
Includes bibliographical references (leaves 139-153). Also available in electronic version. Access restricted to campus users.

The cellular regulation of acidification and intracellular ph (pHi) was studied in the integument of Rana pipiens, a model renal epithelium. Acidification was enhanced by : (1) chronic metabolic acidosis, (2) high salinity adaptation, and (3) ibuprofen treatment.

The aims of this thesis were to: (i) determine the regulatory properties of human follicular fluid (FF) on the activities and expression of 11(3HSD, (ii) investigate the effects of progesterone on llpHSD activity and expression (iii) investigate the effects of prostaglandins on basal and endocrine-stimulated llpHSD activity and expression (iv) to predict the topology and tertiary structure of 11 pHSDl. Human FF contains hydrophilic and hydrophobic compounds that stimulate and inhibit, respectively, the NADP(H)-dependent activities of 11 pHSDl. The predominant lipid inhibitors of 11(3HSD1 in FF are unlikely to be steroids or prostaglandins, and the effects of the FF components on enzyme activities occur without changes in 11 PHSDl protein expression. Inhibition of progesterone production by aminoglutethimide increased 1 lpHSD activities, without affecting 1 lpHSDl protein expression in human granulosa-lutein cells. Pharmacological inhibitors of prostaglandin-H synthase (PGHS)-2 (meclofenamic acid (MA) and NS-398) decreased cortisol-cortisone inter-conversion by up to 50%, suggesting that prostaglandins may stimulate ovarian llpHSD activities in a paracrine/autocrine manner. Furthermore, PGHS-2 inhibitors prevented human chorionic gonadotrophin (hCG) and IL-1(3 from stimulating 11(3HSD activities in human granulosa-lutein cells, implicating eicosanoids in the stimulation of ovarian llpHSD by gonadotrophins and cytokines. Since MA and NS-398 had no effect on 1 IpHSDl protein expression, the stimulation of 11 pHSD activities by eicosanoids occurs at the post-translational level. Finally, the structure of 1 lpHSDl protein was modelled to develop molecular mechanisms by which prostaglandins, gonadotrophins and cytokines might regulate enzyme activities. Although the primary sequence of 1 lpHSDl, which is highly conserved across 10 mammalian species, contains several consensus phosphorylation sites for serine/threonine and tyrosine kinases, models of 11 pHSDl topology predict that most phosphorylation sites would lie in the lumen of the endoplasmic reticulum. 1 lpHSDl includes hydrophobic a-helices neighbouring the active site, which may provide regions for the allosteric control of glucocorticoid metabolism by progesterone and eicosanoids in ovarian cells.

This thesis presents mathematical modeling and control techniques that can be used to predict and specify performance of biologically inspired actuation systems called cellular actuators. Cellular actuators are modular units designed to be connected in bundles in manner similar to human muscle fibers. They are characterized by inherent compliance and large numbers of on-off discrete control inputs. In this thesis, mathematical tools are developed that connect the performance to the physical manifestation of the device. A camera positioner inspired by the human eye is designed to demonstrate how these tools can be used to create an actuator with a useful force-displacement characteristic. Finally, control architectures are presented that use discrete switching inputs to produce smooth motion of these systems despite an innate tendency toward oscillation. These are demonstrated in simulation and experiment.

Slits and their Roundabout (Robo) receptors were identified based on their role in regulating axon guidance, but are known to play multiple roles in development, including regulating heart development and myoblast migration. There are 3 vertebrate Slits (Slit1 – 3) and 4 Robos (Robo1 – 4), and previous work has demonstrated expression of Slit and Robo family members in and around developing joints where their function is unclear. Mutations in human Robo3 have been linked to degenerative joint disorders, such as scoliosis and rheumatoid arthritis. Misregulation of other members of the Slit/Robo signalling pathway is also reported in cells from arthritic joints. This suggests that Slit/Robo signalling is required for normal joint development and/or maintenance, though our understanding of their roles in these processes is rudimentary. The central question of my thesis is to determine the role/s of Slit/Robo signalling in limb and joint development. In situ hybridisation confirmed strong expression of Slits and Robos throughout mouse limb and joint development, though no expression of Slit1 or Robo3 was detected. Analysis of Slit1/2, Slit3 and Robo1 mutant (loss-of-function) mice revealed normal limb development, however misexpression of dominant-negative Robo2 during chicken limb development caused shortening of cartilage elements. To begin to identify molecular changes that may compensate for the loss of Slit/Robo signalling I demonstrated members of the Sema3/PlexinA/Nrp axon guidance family are expressed in patterns comparable to those of Robo1, Robo2 and Slit3. I discovered that PlexinA1 is downregulated in Slit3 mutant mouse limbs. My results suggest the role for Silt/Robo signalling may be more complex than previously thought and do not define a clear role for signalling during limb development. My results suggest the role for Silt/Robo signalling may be more complex than previously thought and do not define a clear role for signalling during limb development. Previous work has linked Slit/Robo signalling to development of degenerative joint disorders, and I propose some hypotheses as to how Slit/Robo signalling could cause bone and joint defects.

En la actualidad, la Internet de las Cosas (Internet of Things, IoT) es una tecnología esencial para la próxima generación de sistemas inalámbricos. La conectividad es la base de IoT, y el tipo de acceso requerido dependerá de la naturaleza de la aplicación. Uno de los principales facilitadores del entorno IoT es la comunicación machine-to-machine (M2M) y, en particular, su enorme potencial para ofrecer conectividad ubicua entre dispositivos inteligentes. Las redes celulares son la elección natural para las aplicaciones emergentes de IoT y M2M. Un desafío importante en las redes celulares es conseguir que la red sea capaz de manejar escenarios de acceso masivo en los que numerosos dispositivos utilizan comunicaciones M2M. Por otro lado, los sistemas celulares han experimentado un tremendo desarrollo en las últimas décadas: incorporan tecnología sofisticada y nuevos algoritmos para ofrecer una amplia gama de servicios. El modelado y análisis del rendimiento de estas redes multiservicio es también una tarea desafiante que podría requerir un gran esfuerzo computacional. Para abordar los desafíos anteriores, nos centramos en primer lugar en el diseño y la evaluación de las prestaciones de nuevos mecanismos de control de acceso para hacer frente a las comunicaciones masivas M2M en redes celulares. Posteriormente nos ocupamos de la evaluación de prestaciones de redes multiservicio y proponemos una nueva técnica analítica que ofrece precisión y eficiencia computacional. Nuestro principal objetivo es proporcionar soluciones para aliviar la congestión en la red de acceso radio cuando un gran número de dispositivos M2M intentan conectarse a la red. Consideramos los siguientes tipos de escenarios: (i) los dispositivos M2M se conectan directamente a las estaciones base celulares, y (ii) forman grupos y los datos se envían a concentradores de tráfico (gateways) que les proporcionan acceso a la infraestructura. En el primer escenario, dado que el número de dispositivos añadidos a la red aumenta continuamente, esta debería ser capaz de manejar el considerable incremento en las solicitudes de acceso. El 3rd Generation Partnership Project (3GPP) ha propuesto el access class barring (ACB) como una solución práctica para el control de congestión en la red de acceso radio y la red troncal. El ajuste correcto de los parámetros de ACB de acuerdo con la intensidad del tráfico es crítico, pero cómo hacerlo de forma dinámica y autónoma es un problema complejo cuya solución no está recogida en las especificaciones del 3GPP. Esta tesis doctoral contribuye al análisis del rendimiento y al diseño de nuevos algoritmos que implementen efectivamente este mecanismo, y así superar los desafíos introducidos por las comunicaciones masivas M2M. En el segundo escenario, dado que la heterogeneidad de los dispositivos IoT y las arquitecturas celulares basadas en hardware imponen desafíos aún mayores para permitir una comunicación flexible y eficiente en los sistemas inalámbricos 5G, esta tesis doctoral también contribuye al diseño de software-defined gateways (SD-GWs) en una nueva arquitectura propuesta para redes inalámbricas definidas por software que se denomina SoftAir. Esto permite manejar tanto un gran número de dispositivos como el volumen de datos que estarán vertiendo en la red. Otra contribución de esta tesis doctoral es la propuesta de una técnica novedosa para el análisis de prestaciones de redes multiservicio de alta capacidad que se basa en un nuevo enfoque del modelizado analítico de sistemas que operan a diferentes escalas temporales. Este enfoque utiliza el análisis del transitorio de una serie de subcadenas absorbentes y lo denominamos absorbing Markov chain approximation (AMCA). Nuestros resultados muestran que para un coste computacional dado, AMCA calcula los parámetros de prestaciones habituales de un sistema con mayor precisión, en comparación con los resultados obtenidos por otr
Nowadays, Internet of Things (IoT) is an essential technology for the upcoming generation of wireless systems. Connectivity is the foundation for IoT, and the type of access required will depend on the nature of the application. One of the leading facilitators of the IoT environment is machine-to-machine (M2M) communication, and particularly, its tremendous potential to offer ubiquitous connectivity among intelligent devices. Cellular networks are the natural choice for emerging IoT and M2M applications. A major challenge in cellular networks is to make the network capable of handling massive access scenarios in which myriad devices deploy M2M communications. On the other hand, cellular systems have seen a tremendous development in recent decades; they incorporate sophisticated technology and algorithms to offer a broad range of services. The modeling and performance analysis of these large multi-service networks is also a challenging task that might require high computational effort. To address the above challenges, we first concentrate on the design and performance evaluation of novel access control schemes to deal with massive M2M communications. Then, we focus on the performance evaluation of large multi-service networks and propose a novel analytical technique that features accuracy and computational efficiency. Our main objective is to provide solutions to ease the congestion in the radio access or core network when massive M2M devices try to connect to the network. We consider the following two types of scenarios: (i) massive M2M devices connect directly to cellular base stations, and (ii) they form clusters and the data is forwarded to gateways that provide them with access to the infrastructure. In the first scenario, as the number of devices added to the network is constantly increasing, the network should handle the considerable increment in access requests. Access class barring (ACB) is proposed by the 3rd Generation Partnership Project (3GPP) as a practical congestion control solution in the radio access and core network. The proper tuning of the ACB parameters according to the traffic intensity is critical, but how to do so dynamically and autonomously is a challenging task that has not been specified. Thus, this dissertation contributes to the performance analysis and optimal design of novel algorithms to implement effectively this barring scheme and overcome the challenges introduced by massive M2M communications. In the second scenario, since the heterogeneity of IoT devices and the hardware-based cellular architectures impose even greater challenges to enable flexible and efficient communication in 5G wireless systems, this dissertation also contributes to the design of software-defined gateways (SD-GWs) in a new architecture proposed for wireless software-defined networks called SoftAir. The deployment of these SD-GWs represents an alternative solution aiming at handling both a vast number of devices and the volume of data they will be pouring into the network. Another contribution of this dissertation is to propose a novel technique for the performance analysis of large multi-service networks. The underlying complexity of the network, particularly concerning its size and the ample range of configuration options, makes the solution of the analytical models computationally costly. However, a typical characteristic of these networks is that they support multiple types of traffic flows operating at different time-scales. This time-scale separation can be exploited to reduce considerably the computational cost associated to determine the key performance indicators. Thus, we propose a novel analytical modeling approach based on the transient regime analysis, that we name absorbing Markov chain approximation (AMCA). For a given computational cost, AMCA finds common performance indicators with greater accuracy, when compared to the results obtained by other approximate methods proposed in the literature.
En l'actualitat, la Internet de les Coses (Internet of Things, IoT) és una tecnologia essencial per a la propera generació de sistemes sense fil. La connectivitat és la base d'IoT, i el tipus d'accés requerit dependrà de la naturalesa de l'aplicació. Un dels principals facilitadors de l'entorn IoT és la comunicació machine-to-machine (M2M) i, en particular, el seu enorme potencial per oferir connectivitat ubiqua entre dispositius intel · ligents. Les xarxes mòbils són l'elecció natural per a les aplicacions emergents de IoT i M2M. Un desafiament important en les xarxes mòbils que actualment está rebent molta atenció és aconseguir que la xarxa siga capaç de gestionar escenaris d'accés massiu en què una gran quantitat de dispositius utilitzen comunicacions M2M. D'altra banda, els sistemes mòbils han experimentat un gran desenvolupament en les últimes dècades: incorporen tecnologia sofisticada i nous algoritmes per oferir una àmplia gamma de serveis. El modelatge i análisi del rendiment d'aquestes xarxes multiservei és també un desafiament important que podria requerir un gran esforç computacional. Per abordar els desafiaments anteriors, en aquesta tesi doctoral ens centrem en primer lloc en el disseny i l'avaluació de les prestacions de nous mecanismes de control d'accés per fer front a les comunicacions massives M2M en xarxes cel · lulars. Posteriorment ens ocupem de l'avaluació de prestacions de xarxes multiservei i proposem una nova tècnica analítica que ofereix precisió i eficiència computacional. El nostre principal objectiu és proporcionar solucions per a alleujar la congestió a la xarxa d'accés ràdio quan un gran nombre de dispositius M2M intenten connectar-se a la xarxa. Considerem els dos tipus d'escenaris següents: (i) els dispositius M2M es connecten directament a les estacions base cel · lulars, i (ii) formen grups i les dades s'envien a concentradors de trànsit (gateways) que els proporcionen accés a la infraestructura. En el primer escenari, atès que el nombre de dispositius afegits a la xarxa augmenta contínuament, aquesta hauria de ser capaç de gestionar el considerable increment en les sol · licituds d'accés. El 3rd Generation Partnership Project (3GPP) ha proposat l'access class barring (ACB) com una solució pràctica per al control de congestió a la xarxa d'accès ràdio i la xarxa troncal. L'ajust correcte dels paràmetres d'ACB d'acord amb la intensitat del trànsit és crític, però com fer-ho de forma dinàmica i autònoma és un problema complex, la solució del qual no està recollida en les especificacions del 3GPP. Aquesta tesi doctoral contribueix a l'anàlisi del rendiment i al disseny de nous algoritmes que implementen efectivament aquest mecanisme, i així superar els desafiaments introduïts per les comunicacions massives M2M en les xarxes mòbils actuals i futures. En el segon escenari, atès que l'heterogeneïtat dels dispositius IoT i les arquitectures cel · lulars basades en hardware imposen desafiaments encara més grans per permetre una comunicació flexible i eficient en els sistemes sense fil 5G, aquesta tesi doctoral també contribueix al disseny de software-defined gateways (SD-GWS) en una nova arquitectura proposada per a xarxes sense fils definides per programari que s'anomena SoftAir. Això permet gestionar tant un gran nombre de dispositius com el volum de dades que estaran abocant a la xarxa. Una altra contribució d'aquesta tesi doctoral és la proposta d'una tècnica innovadora per a l'anàlisi de prestacions de xarxes multiservei d'alta capacitat que es basa en un nou enfocament del modelitzat analític de sistemes que operen a diferents escales temporals. Aquest enfocament utilitza l'anàlisi del transitori d'una sèrie de subcadenes absorbents i l'anomenem absorbing Markov chain Approximation (AMCA). Els nostres resultats mostren que per a un cost computacional donat, AMCA calcula els paràmetres de prestacions habituals d
Tello Oquendo, LP. (2018). Design and Performance Analysis of Access Control Mechanisms for Massive Machine-to-Machine Communications in Wireless Cellular Networks [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/107946
TESIS

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A transcrição realizada pela RNA polimerase (RNAP) é um processo cuidadosamente controlado no desenvolvimento e na manutenção das funções vitais dos organismos. O desenvolvimento de novas técnicas e equipamentos para seu estudo, como as técnicas de pinça ótica ou magnética, a microscopia de força atômica e a fiuorescência de molécula única, complementaram os resultados dos estudos bioquímicos tradicionais e nos levaram a um maior entendimento do processo. A ocorrência de pausas em sítios específicos durante o alongamento, já observadas na década de 80, passou a ser estudada com maior interesse devido a sua importância biológica: acredita-se que essas pausas assegurem que a transcrição e a tradução ocorram simultaneamente em bactérias, permitam o dobramento correto das estruturas secundárias e terciárias do R A, facilitem a ligação de reguladores de alongamento e precedam a etapa de terminação transcricional. Modelos teóricos baseados na estabilidade termodinâmica do complexo de alongamento transcricional foram bem sucedidos na previsão da cinética do alongamento. Seus resultados indicaram que a RNAP pode ser vista como um motor molecular e sua motilidade possui características do modelo de catraca browniana. Entretanto, esses modelos consideram a presença de apenas uma polimerase realizando a transcrição. Experimentos recentes mostraram que a ocorrência de colisões entre essas enzimas durante a transcrição múltipla de um mesmo gene altera seu comportamento. Baseados nesses resultados, propomos a generalização de um dos modelos estocásticos que consideram a sequência molde para o estudo desse fenômeno. Em nossa aproximação, colisões entre as moléculas RNAP modificam a taxa de ocorrência da transcrição. A implementação do modelo foi realizada em...
The transcription of the information encoded within the DNA to the RNA molecule is exquisitely controlled during the development of the organisms and to its vital functions and has as the protagonist the RNA polymerase enzyme (RNAP). The development of single-molecule techniques, such as the magnetic and optical tweezers, atomic-force microscopy and single-molecule uorescence, increased our understanding of the process, complementing traditional biochemical studies. The non-homogeneity of the RNAP movement due to the occurrence of \pauses at speci c sites during elongation was revealed using electrophoresis gels. It is believed that these pauses ensure concurrency between transcription and translation in bacteria, allow the correct folding of RNA secondary and tertiary structures, facilitate the binding of regulating factors during elongation and preceding the transcriptional termination step. Theoretical models have been proposed to explain and predict the RNAP kinetics during the polymerization. Models based on the thermodynamic stability of the transcription elongation complex recover much of the kinetics and indicate that its movement has a Brownian ratchet mechanism. However, experiments showed that if more than one RNAP molecule initiate from the same promoter, their behavior changes and new phenomenona are observed. We proposed and implemented a theoretical model that considers collisions between RNAP molecules and predicts their cooperative behavior during multi-round transcription. The model generalizes a stochastic sequence-dependent model. In our approach, collisions between elongating enzymes modify their transcription rate values. We performed the simulations in Mathematica and compared the results of the single and the multiple-molecule transcription with experimental... (Complete abstract click electronic access below)

Orientador: Ney Lemke
Banca: Scheila de Ávila e Silva
Banca: José Luiz Rybarczyk Filho
Resumo: A transcrição realizada pela RNA polimerase (RNAP) é um processo cuidadosamente controlado no desenvolvimento e na manutenção das funções vitais dos organismos. O desenvolvimento de novas técnicas e equipamentos para seu estudo, como as técnicas de pinça ótica ou magnética, a microscopia de força atômica e a fiuorescência de molécula única, complementaram os resultados dos estudos bioquímicos tradicionais e nos levaram a um maior entendimento do processo. A ocorrência de "pausas" em sítios específicos durante o alongamento, já observadas na década de 80, passou a ser estudada com maior interesse devido a sua importância biológica: acredita-se que essas pausas assegurem que a transcrição e a tradução ocorram simultaneamente em bactérias, permitam o dobramento correto das estruturas secundárias e terciárias do R A, facilitem a ligação de reguladores de alongamento e precedam a etapa de terminação transcricional. Modelos teóricos baseados na estabilidade termodinâmica do complexo de alongamento transcricional foram bem sucedidos na previsão da cinética do alongamento. Seus resultados indicaram que a RNAP pode ser vista como um motor molecular e sua motilidade possui características do modelo de catraca browniana. Entretanto, esses modelos consideram a presença de apenas uma polimerase realizando a transcrição. Experimentos recentes mostraram que a ocorrência de colisões entre essas enzimas durante a transcrição múltipla de um mesmo gene altera seu comportamento. Baseados nesses resultados, propomos a generalização de um dos modelos estocásticos que consideram a sequência molde para o estudo desse fenômeno. Em nossa aproximação, colisões entre as moléculas RNAP modificam a taxa de ocorrência da transcrição. A implementação do modelo foi realizada em ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The transcription of the information encoded within the DNA to the RNA molecule is exquisitely controlled during the development of the organisms and to its vital functions and has as the protagonist the RNA polymerase enzyme (RNAP). The development of single-molecule techniques, such as the magnetic and optical tweezers, atomic-force microscopy and single-molecule uorescence, increased our understanding of the process, complementing traditional biochemical studies. The non-homogeneity of the RNAP movement due to the occurrence of \pauses" at speci c sites during elongation was revealed using electrophoresis gels. It is believed that these pauses ensure concurrency between transcription and translation in bacteria, allow the correct folding of RNA secondary and tertiary structures, facilitate the binding of regulating factors during elongation and preceding the transcriptional termination step. Theoretical models have been proposed to explain and predict the RNAP kinetics during the polymerization. Models based on the thermodynamic stability of the transcription elongation complex recover much of the kinetics and indicate that its movement has a Brownian ratchet mechanism. However, experiments showed that if more than one RNAP molecule initiate from the same promoter, their behavior changes and new phenomenona are observed. We proposed and implemented a theoretical model that considers collisions between RNAP molecules and predicts their cooperative behavior during multi-round transcription. The model generalizes a stochastic sequence-dependent model. In our approach, collisions between elongating enzymes modify their transcription rate values. We performed the simulations in Mathematica and compared the results of the single and the multiple-molecule transcription with experimental... (Complete abstract click electronic access below)
Mestre

ABF -1 is a human class II bHLH transcription factor that is expressed predominantly in activated B cells and EBV immortalized cell lines. A portion of this study sought to characterize the homolog of ABF- l in Caenorhabditis e/egans. The nematode gene product, ceABF -1, is capable of forming heterodimers with E2A gene products and binding E box binding sites. HeLa cells transfected with ceABF-1 reveal that it is capable of blocking E2A mediated gene transcription. In order to maintain full repression capabilities, two conserved amino acid residues within helix I ofthe HLH domain are required. These results show a conserved mechanism of gene repression between invertebrates and vertebrates. This study also sought to analyze ABF-1 mediated regulation of both ld3 and cellular growth. Using a human ABF-1 stably transfected cell line, ID3 protein levels and transcript levels were shown to increase in response to overexpression of ABF-1 via western and northern blot, respectively. Flow cytometry analysis and Real-time PCR revealed that ABF-1 programs a slow down in the cell cycle, however this growth arrest is not mediated by ID3.

Receptor-activated phosphatidylinositol (PtdIns) hydrolysis was examined in AR42J rat pancreatic acini. Cholecystokinin-octapeptide (CCK₈) and bombesin induced a dose-dependent accumulation of [³H] inositol monophosphate ([³H]InsP₁). Manganese (Mn²⁺), a known calcium channel blocker, did not alter basal PtdIns hydrolysis. In contrast, when added 5 minutes prior to the addition of agonists for 60 minutes, Mn²⁺ markedly inhibited secretagogue-mediated [³H]InsP1 formation. Mn²⁺ also attenuated the CCK₈-mediated increase in biologically active inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. These inhibitory effects of Mn²⁺ were mimicked by lanthanum and by EGTA. Addition of calcium to EGTA-treated cells abolished the inhibitory effects of extracellular calcium depletion. Mn²⁺, La³⁺ and EGTA exerted similar inhibitory effects on PtdIns hydrolysis in pancreatic acini. These findings suggest that receptor-activated calcium influx is required for full activation of the CCK₈-mediated signal transduction pathway that is coupled to PtdIns hydrolysis.

The cellular stress response (CSR) is one of the most highly conserved mechanisms among all organisms. Cellular stress can be defined as damage or the threat of damage to proteins, macromolecules and/or DNA. The response to damage can involve cell cycle regulation, protein chaperoning, DNA repair or, if macromolecular damage is too severe, apoptotic mechanisms can be initiated. This thesis details experiments that were designed to examine the cellular response to non-lethal environmental stressors at the protein level, using two fish species as study models. Two proteins that can cause cell cycle arrest and apoptosis mechanisms were examined. Expression of the CCAAT enhancer binding protein-delta (C/EBP-[delta]) was examined in the zebrafish, Danio rerio, exposed to acute, non-lethal hypoxic conditions. While C/EBP-[delta] was expressed constitutively in control individuals during all time points, exposure to hypoxic conditions did not have a consistent significant effect on C/EBP-[delta] expression (two-way ANOVA, P>0.05) in zebrafish white muscle tissue. In a second study, the expression of the growth arrest and DNA damage 45-alpha protein (gadd45-[alpha], a mediator of cell cycle arrest and perhaps apoptosis was examined in heat-stressed liver tissue of an extremely cold-adapted Antarctic fish, Trematomus bernacchii. Gadd45-[alpha] levels were higher in fish exposure to 2°C across all time points (one-way ANOVA; P

Regulatory T cells (Tregs) are a subpopulation of T cells with immuno-suppressive properties. Tregs play a key role in immune response regulation and tolerance to antigens, thereby preventing autoimmunity, and may be partly responsible for the lack of an appropriate immune response against tumor cells. However, a human microRNA (miR) Treg signature has not been described yet. We investigated human natural Tregs and identified a signature composed of five microRNAs (-21, -31, -125a, -181c and -374). Among those, two were considerably under-expressed (miR-31 and miR-125a). We identified a functional target sequence for miR-31 in the 3’ untranslated region (3’ UTR) of FOXP3 mRNA. Using lentiviral transduction of fresh cord blood T cells, we demonstrated that miR-31 and miR-21 had opposite effects on FOXP3 expression. We showed that miR-31 negatively regulates FOXP3 expression by binding directly to its potential target site in the 3’ UTR of FOXP3 mRNA. We next demonstrated that miR-21 acted as a positive, though indirect, regulator of FOXP3 expression.

Recent reports have shown that histone deacetylase inhibitors increased FOXP3 expression in T cells. We therefore decided to investigate in non-Treg CD4-positive cells, the mechanisms by which an aspecific opening of the chromatin could lead to an increased FOXP3 expression. We focused on the binding of potentially activating transcription factors to the promoter region of FOXP3 and on modifications in the five miRs constituting the Treg signature. Valproate treatment induced binding of Ets-1 and Ets-2 transcription factors to the FOXP3 promoter and acted positively on its expression, by increasing the acetylation of histone H4 lysines. Valproate treatment also induced the acquisition of the miRs of Treg signature. To elucidate whether the changes in the miRs expression could be due to the increased FOXP3

expression, we transduced these non-Tregs with a FOXP3 lentiviral expression vector, and found no changes in miRs expression. Therefore, the modification in their miR expression profile is not due to an increased expression of FOXP3 but directly results from histone deacetylase inhibition. Rather, the increased FOXP3 expression results from the additive effects of Ets factors binding and the change in the expression level of miR-21 and miR-31. These data, by allowing a better understanding of the molecular phenomena underlying the number and function of Tregs, could open the door to novel therapeutic approaches in cancer immunotherapy and treatment of autoimmune disorders.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Sphingolipids are a complex family of molecules that participate in many aspects of cell structure and function, including an essential cellular process known as autophagy. Autophagy is a degradation and recycling pathway whereby intracellular components are sequestered into double-membrane vesicles, known as autophagosomes, for subsequent fusion with lysosomes and degradation. Autophagy takes part in cell survival, host immune defense against pathogens, and other biological processes, but is also sometimes lethal. Ceramide, sphingosine 1-phosphate, and more recently dihydroceramide have been shown to induce autophagy, which opens an interesting new field of cell regulation by sphingolipids. This dissertation describes two new cases in which sphingolipids participate in the induction of autophagy: a) RAW264.7 cells treated with Kdo2-Lipid A, a lipopolysaccharide sub-structure with endotoxin activity equal to LPS; and b) MCF7 cells treated with fenretinde, a chemotherapeutic agent which has shown success in clinical trials. It also analyzes the structural properties of fenretinide that contribute to its ability to modulate sphingolipid metabolism through inhibition of dihydroceramide desaturase, thereby elevating dihydroceramide and induction of autophagy. Autophagy was monitored by following the redistribution of GFP-LC3 into discrete punctate vesicles in response to the agents and by Western blotting; in parallel, the sphingolipid composition of the cells was monitored by liquid chromatography, electrospray ionization tandem mass spectrometry. These analyses revealed that Kdo2-Lipid A and fenretinide induce profound changes in sphingolipid metabolism in RAW264.7 and MCF7 cells, respectively, and that one of the purposes for increased de novo biosynthesis is to enable the production of autophagosomes, as the autophagic response was inhibited by myriocin. These studies have uncovered a direct link between sphingolipid metabolism and autophagy, which could pave the way for new therapeutic interventions for the treatment of pathogenic infection and be clinically useful in enhancing the efficacy of current cancer treatment strategies.

The 1980's heralded the discovery and identification of extra-pituitary sources of the neurohypophysial hormone oxytocin in non-neural tissues of several animal species. The presence, location and biosynthesis of significant amounts of oxytocin in the ovarian corpus luteum was followed by the immunocytochemical demonstration of an oxytocin-like peptide in the testicular interstitial cells. Leydig cells, which comprise up to 80% of the testicular intertubular cell population, are known to synthesize testosterone in situ. Indirect evidence indicated that an oxytocin-like peptide was also present in Leydig cells. The question arose whether this peptide was synthesized de novo by Leydig cells or was taken up and stored by the cells following biosynthesis at some other intra- and/or extra-gonadal source(s). Since luteinizing hormone (LH) and ascorbate are known to augment the production of oxytocin in ovarian granulosa cells, varying concentrations of these two stimulants were used to monitor the biosynthesis of oxytocin from isolated Leydig cells in culture.
Ph. D.

Anti-oestrogens act via the oestrogen receptor whether they compete with the hormone for binding to the receptor and therefore interfere with DNA binding or inhibit transcriptional activity. These receptors exist as a large 85 complex and/or a small 45 form on sucrose density gradients. High performance ion-exchange chromatography has confirmed that the oestrogen and progestin complex is present in various isoforms. Progestin receptor heterogeneity could be influenced by the presence of oestrogens and anti-oestrogens in the culture media of hormone-dependent neoplastic cells. Cell culture methods offer the opportunity to test effects of specified components in repeated experiments on a homogeneous population of cells. MCF-7 and T47-D human breast cancer cell lines were conditioned to grow in a serum-free environment. There was no difference in cell proliferation rates, nor in their oestrogen or progestin receptor levels when compared to the same cells grown in conventional media. Receptors were present mainly in the large molecular 85 form. Both the MCF-7 and T47-D breast cancer cells showed an increase in proliferation rate with the addition of oestrogen or diethylstilbestrol. There was a corresponding loss of progestin receptor levels and an alteration in the high performance ion-exchange isoforms. Flow cytometry confirmed differences in the S-phase components of the cells following exposure to oestrogens. The proliferation rates of the cell lines as well as their progestin receptor levels decreased when treated with tamoxifen or the hydroxylated tamoxifen. There were marked changes on high performance ion-exchange chromatography profiles. DNA ploidy and S-phase showed signs of toxicity and there was an increase in cellular debris. The MCF-7 and T47-D human breast cancer cell line retained response to antioestrogen saturation.