25 resultados para CA2 HOMEOSTASIS
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FCM: UC Bioqumica I - PhD Thesis
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Dissertation presented to obtain the Doctorate degree (Ph.D.) in Biology at Instituto de Tecnologia Qumica e Biolgica da Universidade Nova de Lisboa
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Dissertation presented to obtain the Ph.D degree in Biology
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Dissertation for a degree in Doctor in Sustainable Chemistry
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Febs Journal (2009)276:1776-1786
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The S100 proteins are 10-12 kDa EF-hand proteins that act as central regulators in a multitude of cellular processes including cell survival, proliferation, differentiation and motility. Consequently, many S100 proteins are implicated and display marked changes in their expression levels in many types of cancer, neurodegenerative disorders, inflammatory and autoimmune diseases. The structure and function of S100 proteins are modulated by metal ions via Ca2+ binding through EF-hand motifs and binding of Zn2+ and Cu2+ at additional sites, usually at the homodimer interfaces. Ca2+ binding modulates S100 conformational opening and thus promotes and affects the interaction with p53, the receptor for advanced glycation endproducts and Toll-like receptor 4, among many others. Structural plasticity also occurs at the quaternary level, where several S100 proteins self-assemble into multiple oligomeric states, many being functionally relevant. Recently, we have found that the S100A8/A9 proteins are involved in amyloidogenic processes in corpora amylacea of prostate cancer patients, and undergo metal-mediated amyloid oligomerization and fibrillation in vitro. Here we review the unique chemical and structural properties of S100 proteins that underlie the conformational changes resulting in their oligomerization upon metal ion binding and ultimately in functional control. The possibility that S100 proteins have intrinsic amyloid-forming capacity is also addressed, as well as the hypothesis that amyloid self-assemblies may, under particular physiological conditions, affect the S100 functions within the cellular milieu.
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RESUMO: As concentraes circulantes de clcio so notavelmente constantes a despeito das variaes dirias na absoro intestinal e na eliminao renal deste elemento. A regulao da calcmia um sistema complexo que compreende vrios factores controladores (a calcmia, a fosformia, as concentraes circulantes de paratormona (PTH) e calcitriol alm de muitos outros factores como hormonas esterides em geral, outros ies como o magnsio e outros factores hormonais) e vrios rgos alvo (glndulas paratiroideias, osso, rim e intestino). As respostas dos rgos alvo tambm so muito variadas. No caso mais simples, a cristalizao de sais de clcio corresponde a uma mudana de fase em que participam molculas orgnicas que a iniciam, aceleram ou inibem. Em geral a combinao de um factor controlador com o respectivo receptor de membrana (para polipeptdeos ou ies) ou intracelular (hormonas esterides) apenas o primeiro passo de uma cadeia bioqumica que introduz uma enorme amplificao na resposta. A esta variedade de mecanismos de resposta correspondem grandes diferenas nos tempos de resposta que podem ser de minutos a semanas. hoje possvel observar (medir) com aprecivel rigor nos lquidos biolgicos (sangue, urina, fezes, etc.) os factores mais importantes do sistema de regulao da calcmia (clcio, fsforo, paratormona e calcitriol) assim como administrar estes factores em experincias agudas. Esta possibilidade reflecte se na literatura neste campo que tem vindo a crescer. O advento das tcnicas da biologia molecular tem permitido a caracterizao molecular de algumas das disfunes da homeostase do clcio e de esperar um diagnstico fisiopatolgico cada vez mais rigoroso dessas disfunes. Com o avano dos conhecimentos nesta rea que no cessa de aumentar temos cada vez maiores capacidades para fazer diagnsticos e cada vez mais difcil interpretar com rigor os correspondentes quadros metablicos. A anlise ou sntese de sistemas complexos a actividade mais nobre dos engenheiros que lhes permite desenhar pontes, diques, barcos, avies ou automveis. Com o aparecimento de computadores de mdio ou grande porte foi lhes possvel utilizar descries matemticas no s para desenhar sistemas como ainda para interpretar eventuais falhas na sua operao. Essas descries matemticas consistem numa sequncia de operaes realizadas num computador segundo um programa informtico que receberam a designao genrica de modelos, por analogia com as famosas leis (equaes) da fsica que foram deduzidas a partir de um certo nmero de postulados e que permitem representar matematicamente processos fsicos. As famosas leis de Newton so talvez os exemplos mais famosos de modelos de sistemas fsicos. A introduo de modelos matemticos em biologia e particularmente em medicina s se deu recentemente.MTODOS No trabalho que aqui se apresenta construiu - se um modelo simplificado da homeostase do clcio destinado ao clculo de variveis observveis (concentraes de clcio, fsforo, PTH e calcitriol) de modo a poderem comparar-se valores calculados com valores observados. A escolha dos componentes do modelo foi determinada pela nossa experincia clnica e pela informao fisiopatolgica e clnica publicada. Houve a preocupao de construir o modelo de forma modular de modo a ser possvel a sua expanso sem grandes transformaes na descrio matemtica (e informtica) j existente. Na sua fase actual o modelo no pode ser usado como instrumento de diagnstico. antes uma ferramenta destinada a esclarecer em princpio mecanismos fisiopatolgicos. Usou se o modelo para simular um certo nmero de observaes publicadas e para exemplificar a sua eventual aplicao clnica na simulao de situaes hipotticas e na anlise de possveis mecanismos fisiopatolgicos responsveis por situaes de hipo ou hipercalcmias. Simultaneamente fez se uma anlise dos dados acumulados relativos a doentes vistos no Servio de Endocrinologia do Instituto Portugus de Oncologia de Francisco Gentil Centro Regional Oncolgico de Lisboa, S.A. CONCLUSES Numa populao de 894 doentes com patologias variadas do Instituto Portugus de Oncologia de Lisboa os valores da calcmia tiveram uma distribuio normal unimodal com uma mdia de 9.56 mg/dl, e um erro padro de 0.41 mg/dl. Estas observaes sugerem que a calcmia est sujeita a regulao. A partir dos resultados publicados em que o metabolismo do clcio foi perturbado por infuses de clcio, calcitriol ou PTH, de estudos bioqumicos e fisiolgicos sobre os mecanismos de aco de factores controladores da calcmia e do estudo do comportamento de rgos alvo (paratiroideias, intestino, osso e rim) foi possvel construir um modelo matemtico de parmetros concentrados do sistema de regulao da calcmia. As expresses analticas usadas foram baseadas na cintica enzimtica de modo a que os seus parmetros tivessem um significado fsico ou fisiolgico simples. O modelo revelou aprecivel robustez e flexibilidade. estvel quando no perturbado e transita entre estados estacionrios quando perturbado. Na sua forma actual gera simulaes que reproduzem satisfatoriamente um nmero aprecivel de dados experimentais colhidos em doentes. Isto no significa que possa ser usado como instrumento de diagnstico aplicvel a doentes individuais. O desenho do modelo comporta a adio posterior de novas relaes quando surgirem situaes para as quais se revele insuficiente. A utilizao exaustiva do modelo permitiu explicitar aspectos do metabolismo do clcio que ou no esto contidas na sua formulao actual o aparecimento de hipertrofia ou de adenomas das paratiroideias e as alteraes na estrutura ssea , a participao de outros factores controladores magnsio, ou esto insuficientemente descritas alteraes do metabolismo do fsforo nos hipoparatiroidismos. A anlise dos dados relativos aos doentes do Servio de Endocrinologia do IPO permitiu o incio da caracterizao dos tipos de patologia que representam e de possveis mecanismos fisiopatolgicos subjacentes. Estas observaes so o ponto de partida para anlises futuras. So exemplos das relaes encontradas: a distribuio dos doentes por dois grandes grupos conforme a calcmia determinada pelas concentraes circulantes de PTH ou estas so determinadas pela calcmia; a distribuio sazonal das concentraes de Vit. D25. no sangue; a correlao negativa entre estas e as concentraes de PTH no sangue. Tambm foi possvel extrair a cintica do controlo da PTH sobre a sntese de calcitriol. O estudo dos nveis circulantes de PTH no ps-operatrio imediato de doentes paratiroidectomizados permitiu determinar as suas taxas de degradao metablica. O modelo permitiu simular as relaes Ca/PTH no sangue, Ca/Fraco excretada da carga tubular, Ca/P no sangue para valores normais ou altos de Ca. Foram feitas simulaes de situaes fisiopatolgicas (em doentes virtuais): infuses crnicas de clcio, PTH e calcitriol; alteraes no comportamento de receptores. Estas simulaes correspondem a experincias que no podem ser realizadas em humanos. So exemplos da utilizao do modelo na explorao de possveis mecanismos fisiopatolgicos atravs da observao de resultados quantitativos inacessveis intuio. O modelo foi til em duas fases do trabalho: Primeiro, durante a sua sntese implicou uma escolha criticamente selectiva de informao, sua anlise quantitativa e processamento, uma explicitao rigorosa (analtica) das relaes funcionais entre os controladores e as variveis e da sua integrao numa estrutura global; Segundo, a simulao de situaes experimentais ou clnicas (dados do Servio de Endocrinologia do IPO) em doentes obrigou a explicitar raciocnios fisiopatolgicos habitualmente formulados em bases puramente intuitivas. Esta prtica revelou comportamentos bvios aps as simulaes aco reduzida das infuses PTH (simulao de hiperparatiroidismos primrios) enquanto no h inibio total da respectiva secreo, necessidade de aumento da massa secretora da paratiroideia nas insuficincias renais avanadas, etc. A sntese e utilizao do modelo no implicaram uma preparao matemtica avanada e foram possveis merc da disponibilidade de software interactivo especificamente desenhado para a simulao de sistemas dinmicos em que os programas se escrevem em ingls usando a simbologia simples da lgebra elementar. A funo nobre de modelos desta natureza semelhante dos modelos usados pelos fsicos desde o sculo XVII: permitir explicaes de carcter geral funcionando como uma ferramenta intelectual para manipulao de conceitos e para a realizao de experincias pensadas (thought experiments) respeitando certos princpios fsicos (princpios de conservao) que estabelecem as fronteiras da realidade. -------ABSTRACT: Calcium blood levels are remarkably constant despite great variations in calcium daily intake, intestinal absorption and renal excretion. The regulation of the calcium concentration in the blood is achieved by a complex system that includes several controller factors (mainly the serum levels of calcium, phosphorus, parathyroid hormone (PTH) and calcitriol but also of steroid hormones, ions such as magnesium and other hormonal factors) and several target organs (parathyroid glands, bone, kidney and intestine). The functional response to the controlling factors obeys a variety of kinetics. The precipitation of calcium salts is a simple phase transition in which organic molecules may provide nucleation centres or inhibit the process. The combination of a controller factor with its receptor located in the cell membrane (for peptides or ions) or in the nucleus (for steroid hormones) is only the first step of a biochemical chain that introduces a huge amplification in the response. To this great variability of response we have to add the times of response that vary from minutes to weeks. It is possible to observe (measure) with great accuracy in biological fluids (blood, urine, faeces, etc.) the most important factors intervening in the calcium regulation (calcium, phosphorus, PTH and calcitriol). The response of the system to acute infusions of the controlling factors has also been studied. Using molecular biology techniques it has been possible to characterize some calcium homeostasis dysfunctions and better physiopathological diagnosis are expected. With the increasingly new knowledge in this area we have better capacity to diagnose but it is harder to explain correctly the underlying metabolic mechanisms. The analysis or synthesis of complex systems is the noble activity of engineers that enables them to draw bridges, dams, boats, airplanes or cars. With the availability of medium-large frame computers it was possible to use mathematical descriptions not only to draw systems but also to explain flaws in its operations. These mathematical descriptions are generally known as models by analogy with the laws (equations) of physics that allow the mathematical description of physical processes. In practice it is not possible to find general solutions for the mathematical descriptions of complex systems but (numeric) computations for specific situations can be obtained with digital computers. The introduction of mathematical models in biology and particularly in medicine is a recent event. METHODS In this thesis a simplified model of calcium homeostasis was built that enables the computation of observable variables (concentrations of calcium, phosphorus, PTH and calcitriol) and allows the comparison between the simulation values and observed values. The choice of the models components was made according to our clinical experience and to the published clinical and physiopathological data. The model has a modular design that allows future expansions with minor alterations in its structure. In its present form the model cannot be used for diagnosis. It is a tool designed to enlighten physiopathological processes. To exemplify its possible clinical application in the simulation of hypothetical situations and in the analysis of possible mechanisms responsible for hypo or hypercalcemias the model was used to simulate a certain number of published observations. An analysis of clinical and laboratory data from the Endocrinology Department of the Portuguese Cancer Institute (I.P.O.F.G.-C.R.O.L.,S.A.) is also presented. CONCLUSIONS In a population of 188 patients without an identifiable disease of the calcium metabolism at the Portuguese Cancer Institute the calcemia levels had a unimodal distribution with an average of 9.56 mg/dL and a S.E.M of 0.41 mg/dL. This observation confirms that serum calcium is regulated. Using published data; in which calcium metabolism was disrupted by calcium, PTH or calcitriol infusions; from biochemical and physiological studies of the action of controller factors on the calcemia; in which the response of target organs (parathyroid glands, intestine, bone, kidney) was studied it was possible to build a mathematical model of concentrated parameters of the calcium homeostasis. Analytical expressions used were based on enzymatic kinetics. The model is flexible and robust. It is stable when not disturbed and changes between steady states when disturbed. In its present form it provides simulations that reproduce closely a number of experimental clinical data. This does not mean that it can be used as a diagnostic tool for individual patients. The exhaustive utilisation of the model revealed the need of future expansions to include aspects of the calcium metabolism not included in its present form hypertrophy or adenomas of the parathyroid glands, bone structure changes, participation of other controller factors such as magnesium or insufficiently described phosphate metabolism in hypoparathyroidism. The analysis of the data collected from the I.P.O.s Endocrinology Department allowed the initial characterization of the different pathologies represented and of their possible physiopathological mechanisms. These observations are a starting point for future analysis. As examples of the relations found were: the distribution of patients in two groups according to the dependency of calcium by PTH levels or PTH levels by calcium concentration; the seasonal distribution of the serum concentrations of D25; its negative correlation with PTH concentration. It was also possible to extract the kinetics of the control of the synthesis of calcitriol by PTH. The analysis of immediate post-surgical levels of PTH in parathyroidectomized patients allowed the determination of its metabolic clearance. The model also allowed the simulation of the relations between Ca/PTH in blood, serum Ca/Fraction of tubular load excreted and Ca/P in blood for normal and high values of calcium. Simulations were made of pathological situations (in virtual patients): chronic infusions of calcium, PTH and calcitriol; changes in the characteristics of receptors. These simulations are not possible in real persons. They are an example of the use of this model in exploring possible mechanisms of disease through the observation of quantitative results not accessible to simple intuition. This model was useful in two phases: Firstly, its construction required a careful choice of data, its quantitative analysis and processing, an analytical description of the relations between controller factors and variables and their integration in a global structure. Secondly, the simulation of experimental or clinical (I.P.O.s Endocrinology Department) data implied testing physiopathological explanations that previously were based on intuition. The construction and utilisation of the model didnt demand an advanced mathematical preparation since user-friendly interactive software was used. This software was specifically designed for the simulation of dynamic systems. The programs are written in English using elementary algebra symbols. The essential function of this type of models is identical to that of those used by physicists since the XVII century which describe quantitatively natural processes and are an intellectual tool for the manipulation of concepts and the performance of thought experiments based in certain physical principles (conservation principles) that are the frontiers of reality.------------------RESUME: Les concentrations circulantes de calcium sont constantes mme pendant des variations de labsorption intestinale et de llimination rnale de cet lment. La rgulation de la calcmie est un systme complexe qui comprend plusieurs lments contrleurs (la calcmie, la phosphormie, les concentrations circulantes de lhormone parathyrodienne (PTH) e du calcitriol et dautres comme les hormones strodes ou des ions comme le magnsium) et plusieurs organes (glandes parathyrodiennes, los, le rein et lintestin). Les rponses de ces organes sont varies. Dans le cas plus simple, la cristallisation des sels de calcium correspond un changement de phase dans lequel y participent des molcules organiques que la dbutent, lacclrent ou linhibent. Gnralement la combinaison dun lment contrleur avec leur rcepteur de membrane (pour les peptides ou les ions) ou intracellulaire (pour les hormones strodes) nest que le premier pas dune chane biochimique quintroduit une grande amplification de la rponse. A cette varit de rponses correspondent des grandes diffrences des temps de rponses quy vont des minuits a semaines. Il est possible observer (mesurer) dans les fluides biologiques (sang, urine, fces, etc.) les lments plus importants du systme de rgulation de la calcmie (calcium, phosphate, PTH et le calcitriol) et les administrer en exprimentes aigus. Cette possibilit est visible dans la littrature publie dans ce domaine qui est en croissance permanente. Lavenir des techniques de biologie molculaire a permis caractriser des nombreuses dysfonctions de la rgulation de la calcmie et on attend un diagnostique physiopathologique de ces dysfonctions chaque fois plus rigoureuses. Les connaissances dans ce domaine sagrandissent et on a de plus de capacits pour faire des diagnostiques et il est chaque fois plus difficile les interprter. Lanalyse ou synthse de systmes complexes est lactivit plus noble des ingnieurs qui les permit dessiner des ponts, bateaux, avions ou automobiles. Avec des ordinateurs de mdium ou grand port il les est possible utiliser descriptions mathmatiques pour dessiner les systmes et interprter des ventuelles fautes dopration. Ces descriptions mathmatiques sont une squence doprations ralises dans un ordinateur selon un programme informatique qui ont reu la dsignation gnrique de modles, pour analogie avec les quations de la physique qui ont t dduits dun nombre de postules et quont permit reprsenter des processus physiques en quations mathmatiques. Les fameuses quations de Newton sont peut-tre les exemples plus connus des systmes physiques. Lintroduction des modles mathmatiques en biologie et en particulier en mdecine est un vnement rcent. Dans ce travaille, on a construit un modle simplifi de lhomostasie du calcium pour calculer les variables observables (concentrations de calcium, phosphate, PTH et calcitriol) pour les comparer. Les choix des components a t dtermins par notre exprience clinique et par linformation physiopathologique et clinique publie. Le modle a t construit de faon modulaire ce que permit leur postrieur expansion sans des grandes altrations dans la description mathmatique et informatique dj existante. Dans cette forme le modle ne peut tre utilis comme un instrument de diagnostique. Il est un outil pour clairer la physiopathologie. Le modle a t utilis pour simuler un certain nombre dobservations publies et pour exemplifier leur possible utilisation clinique dans la simulation des hypothses et de la physiopathologie des situations dhypo ou hypercalcmie. On a fait une analyse des lments des procs cliniques des malades observes dans le Service dEndocrinologie de lIPOFG-CROL, SA. Dans une population de 894 malades avec des diffrentes pathologies les valeurs de calcmie on une distribution uni modale avec une Mdie de 9.56 mg/dL et une erreur standard de 0.41 mg/dL. Ces observations suggrent que la calcmie soit sujette de rgulation. En utilisant des rsultats de travaux publis dans lesquels le mtabolisme du calcium a t chang par des infusions de calcium, calcitriol ou PTH, des tudes biochimiques et physiologiques sur des mcanismes daction des lments contrleurs de la calcmie et de ltude du comportement des organes cible (parathyrodes, intestin, rein, os), il a t possible de construire un modle mathmatique de paramtres concentrs du systme de rgulation de la calcmie. Les expressions analytiques utilises ont t bases sur la cintique enzymatique de faon que les paramtres aient eu une signification physique ou biologique. Le modle est stable quand il nest pas perturb et transit entre tats stationnaires quand il est sujet a des perturbations. A ce moment il fait des simulations qui reproduisent de faon satisfaisant un nombre dobservations exprimentales. La construction du modle permit laddiction de nouvelles relations dans les cas ou il est insuffisant. Lutilisation exhaustive du modle a permit expliciter des aspects du mtabolisme du calcium qui y ne sont pas compris lhyperplasie ou la formation des adnomes des parathyrodes, les altrations de la structure des os, la participation doutres lments rgulateurs (magnsium), ou sont insuffisamment dcrites les altrations du mtabolisme des phosphates dans lhypoparathyroidism. Lanalyse de linformation des malades du Service dEndocrinologie a permit caractriser les pathologies reprsentes et leurs possibles mcanismes physiopathologiques. Ces observations sont le point de dpart pour les analyses futures. Sont des exemples des relations trouves: la distribution des malades par deux groupes: ceux dans lequel la calcmie est dtermine par la PTH ou ceux dans lesquels la PTH est dtermine par la calcmie; la distribution sazonale de la concentration de la vitamine D; la corrlation ngative entre la vitamine D et la PTH. On a eu la possibilit de dduire la cintique de control de la PTH sur la synthse du calcitriol. Ltude des niveaux circulants de PTH sur des sujets parathyroidectomises a permit dduire leur taux de dgradation mtabolique. Le modle a permit simuler les relations Ca/PTH dans le sang, Ca/fraction limine par le rein, Ca/P dans le sang pour des valeurs normales ou hautes de calcium. On a fait des simulations de situations physiopathologiques (dans malades virtuelles): Infusions chroniques de calcium, PTH ou calcitriol; altrations des rcepteurs. Ces simulations ne peuvent pas tre ralises dans les humains. Sont des exemples dutilisation du modle dans lexploration des possibles mcanismes de la physiopathologie en observant des rsultats quantitatifs inaccessibles lintuition. Le modle a t utile pendant deux tapes des travaux: La premire, dans sa construction on a choisi linformation disponible, son analyse quantitative, lexplicitation rigoureuse (analytique) des relations fonctionnelles entre les contrleurs et les variables et sa intgration dans une structure globale. La deuxime, la simulation de situations exprimentales ou cliniques (du Service dEndocrinologie) a oblig dexpliciter des raisonnements physiopathologiques gnralement formuls utilisant lintuition. Cette pratique a montr des comportements action rduite des infusions de PTH (jusqu linhibition totale de leur respective scrtion), ncessit daugmenter la masse scrteuse de la parathyrode dans les insuffisants rnales, etc. La synthse et utilisation du modle nont pas besoin dune formation avance en mathmatique et sont possibles grce un programme interactif qui a t conu pour la simulation des systmes dynamiques dans lesquels le programme se construit en anglais en utilisant la symbolique lmentaire de lalgbre. La fonction noble de ces modles est semblable celles des physiques du XVII sicle: Permettre tablir explications gnrales en fonctionnant comme un outil intellectuel pour manipuler des concepts et pour la ralisation dexprimentes penses en respectant certains principes de la physique (principe de la conservation) qutablissent les frontires de la ralit.
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Dissertao para obteno do Grau de Mestre em Biotecnologia
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Dissertao para obteno do Grau de Mestre em Gentica Molecular e Biomedicina
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RESUMO: Sessenta e trs derivados de hidantona foram utilizados para avaliar possveis efeitos de modulao na actividade das bombas de efluxo (BE) na Salmonella NCTC 13349 utilizando um mtodo fluorimtrico semi-automtico. Nenhum dos compostos apresentaram actividade anti-bacteriana at concentraes de 240 mg/L. Entre todos os compostos, SZ-7 demonstrou possuir propriedades de modulao de effluxo na presena de glucose. Para testar esta actividade, estirpes de Salmonella resistentes ciprofloxacina, induzidas a elevados nveis de resistncia com sobre-expresso de BE, foram expostas ao SZ-7. Este derivado afectou a susceptibilidade das estirpes ciprofloxacina. Uma vez que os 63 compostos estudados apresentaram pouco efeito inibitrio /cumulativo, apesar de serem conhecidos pelos seus efeitos moduladores de BE-dependentes de ies em eucariotas, foi questionado o papel dos ies na regulao de BE bacterianas, que podero influenciar a eficcia de novos compostos. Para este estudo, utilizamos a Escherichia coli AG100 como modelo, devido ao extenso conhecimento no que respeita a estrutura e actividade das BE. Devido importncia de ies de clcio (Ca2+) nos canais de transporte membranar e na actividade de ATPases, a sua actividade na modulao do efluxo foi investigada. De resultados anteriormente obtidos concluiu-se que a pH 5 o efluxo independente de energia metablica; contudo, a pH 8 absolutamente dependente, sendo que o Ca2+ indispensvel para manter a actividade das ATPases bacterianas. A acumulao/effluxo de EtBr pela E. coli AG100 foi determinada na presena/ausncia de Ca2+, clorpromazina (inibidor de ligao de Ca2+ a protenas), e cido etilenodiamino tetra-actico (quelante de Ca2+). Acumulao/effluxo aumentou a pH 8, contudo o Ca2+ reverte estes efeitos evidenciando a sua importncia no funcionamento das BE bacterianas. Em resumo este trabalho colocou em evidncia que muitos aspectos bioqumicos e bioenergticos devem ser tomados em considerao no estudo da resistncia bacteriana mediada por BE.------- ABSTRACT: Sixty-three hydantoin derivatives were evaluated for their modulating effects on efflux pump (EP) activity of Salmonella NCTC 13349 utilizing a semi-automatic fluorometric method. None of the compounds presented antibacterial activities at concentrations as high as 240 mg/L. Among all compounds, SZ-7 showed possible efflux modulating activity in the presence of glucose, indicative of a potential EP inhibitor. To verify its potential effects, ciprofloxacin-resistant Salmonella strains, induced to high level resistance with over-expressing EPs, were exposed to SZ-7. This derivative affected the susceptibility of the ciprofloxacin-resistant strains. Since the 63 compounds studied had very low inhibitory/accumulative effects, even though their known for being efficient in modulating ion-driven eukaryotic EPs, we questioned whether ions had a leading role in regulating bacterial EPs, influencing the effectiveness of new compounds. For this study we used Escherichia coli AG100 as a model, due to the extensive knowledge on its EPs structure and activity. Owing the importance of calcium ions (Ca2+) for membrane transport channels and activity of ATPases, the role of Ca2+ was investigated. From previous results we concluded that at pH 5 efflux is independent of metabolic energy; however, at pH 8 it is entirely dependent of metabolic energy and the Ca2+ ions are essential to maintain the activity of bacterial ATPases. Accumulation and efflux of ethidium bromide (EtBr) by E. coli AG100 was determined in the presence and absence of Ca2+, chlorpromazine (inhibitor of Ca2+-binding to proteins), and ethylenediaminetetraacetic acid (Ca2+ chelator). Accumulation of EtBr increased at pH 8; however Ca2+ reversed these effects providing information as to the importance of this ion in the regulation of bacterial EP systems. Overall this work puts in evidence that many biochemical and bioenergetic aspects related to the strains physiology need to be taken into consideration in bacterial drug resistance mediated by EPs.
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Resumo: RodZ um componente do sistema morfogentico das clulas bacterianas. uma protena transmembranar que localiza em bandas ao longo do eixo longitudinal da clula. Em Bacillus subtilis, RodZ consiste numa poro citoplasmtica, RodZn, e em uma parte extra-citoplasmtica, RodZc. RodZn contm um domnio em helixturn- helix (HTH), enquanto que RodZc pode ser dividido num domnio coiled-coil e num domnio terminal C, de funo desconhecida. Um segmento transmembranar (TM) nico separa RodZn de RodZc. A eliminao de rodZ causa alongamento do nucleide e leva produo de clulas polares nucleadas. Aqui, mostramos que RodZn estruturado, estvel e em hlice . Descobrimos que as substituies Y32A e L33A na suposta hlice de reconhecimento (3) do motivo HTH, bem como as substituies Y49A e F53A, fora do motivo HTH (4), causam diviso assimtrica, mas apenas as ltimas levam deslocalizao sub-celular de RodZ. Sugerimos que as hlices 3 e 4 so utilizadas para uma interaco protena-protena ou protena- DNA essencial para diviso celular enquanto que 4 deve contactar um componente do citosqueleto, possivelmente MreB, uma vez que a correcta localizao sub-celular de RodZ depende desta protena. Em todos os mutantes as clulas polares so anucleadas, pelo que conclumos que o alongamento do nucleide no um prrequisito para diviso assimtrica. RodZc largamente no estruturado mas com contedo de folha , sendo estabilizado pelo domnio coiled-coil. Mostramos uma relao homloga entre RodZc e a bomba de transporte Na+/Ca2+ NCX1 e identificmos dois resduos no domnio C, G265 e N275, essenciais para a manuteno da forma celular. Estes resduos fazem parte de um motivo em gancho que pode actuar como um local de interaco com um ligando desconhecido. RodZn e RodZc so monomricos em soluo. Contudo, na membrana, RodZ interage consigo prpria num sistema de dois hbridos (Split-Ubiquitin) em levedura, sugerindo que possa formar multmeros in vivo.-----------ABSTRACT: RodZ is a transmembrane component of the bacterial core morphogenic apparatus. RodZ localizes in bands long the longitudinal axis of the cell, and it is though to functionally link the cell wall to the actin cytoskeleton. In Bacillus subtilis, RodZ consists of a cytoplasmic moiety, RodZn, and an extracytoplasmic moiety, RodZc. RodZn contains a predicted helix-turn-helix domain, whereas RodZc is thought to contain a coiled-coil region and a terminal C domain of unknown function. A single transmembrane domain separates RodZn from RodZc. Deletion of rodZ causes elongation of the nucleoid and leads to the production of polar minicells containing DNA. Here, we have studied the structure and function of RodZn and RodZc. We show that RodZn is a stable, folded, -helical domain. We discovered that the Y32A and L33A substitutions within the presumptive recognition helix (3) of the HTH motif, as well as the Y49A and F53A substitutions outside of the HTH motif (in 4) cause asymmetric cell division. However, only the substitutions in 4 cause sub-celular delocalization of RodZ. We suggest that 3 and 4 are used for a protein-protein or protein-DNA interaction important for cell division, whereas 4 is likely to contact a cytoskeletal component, presumably MreB. The polar cells formed by all the mutants are anucleate. We conclude that nucleoid elongation is not a prerequisite for asymmetric division. RodZc appears to be a largely unstructured domain, with some -sheet content, and is stabilized by the coiled-coil region. We show a homology relationship between RodZc and the NCX1 Na+/Ca2+ transporter and we found two residues within the C domain, G265 and N275, that are important for cell shape determination. These residues are predicted to be essential determinants of a claw-like motif, which may act as a binding site for an unknown ligand. Both the isolated RodZn and RodZc proteins are monomeric in solution. However, because full-length RodZ interacts with itself in a split-ubiquitin yeast two-hybrid assay, we suggest that it may dimerize or form higher order multimers in vivo.
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Dissertation presented to obtain the Ph.D degree in Biology
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Inorg Chem. 2008 Jul 7;47(13):5677-84. doi: 10.1021/ic702405d
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Dissertao para obteno do Grau de Mestre em Engenharia Qumica e Bioqumica
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Dissertao apresentada para obteno do Grau de Doutor em Biologia, na especialidade de Gentica Molecular, pela Universidade Nova de Lisboa, Faculdade de Cincias e Tecnologia
