Production, purification and characterization of laccase from pleurottus ostreatus grown on tomato pomace

A strain of Pleurotus ostreatus was grown in tomato pomace as sole carbon source for production of laccase. The culture of P. ostreatus revealed a peak of laccase activity (147 U/L of fermentation broth) on the 4th day of culture with a specific activity of 2.8 U/mg protein. Differential chromatographic behaviour of laccase was investigated on affinity chromatographic matrices containing either urea, acetamide, ethanolamine or IDA as affinity ligands. Laccase exhibited retention on such affinity matrices and it was purified on a Sepharose 6B-BDGE-urea column with final enzyme recoveries of about 60%, specific activity of 6.0 and 18.0 U/mg protein and purification factors in the range of 14-46. It was also possible to demonstrate that metal-free laccase did not adsorb to Sepharose 6B-BDGE-urea column which suggests that adsorption of native laccase on this affinity matrix was apparently due to the specific interaction of carbonyl groups available on the matrix with the active site Cu (II) ions of laccase. The kinetic parameters (V (max), K (m) , K (cat), and K (cat)/K (m) ) of the purified enzyme for several substrates were determined as well as laccase stability and optimum pH and temperature of enzyme activity. This is the first report describing the production of laccase from P. ostreatus grown on tomato pomace and purification of this enzyme based on affinity matrix containing urea as affinity ligand.

Synthesis and structural characterization of new Piano-stool Ruthenium(II) complexes bearing 1-Butylimidazole heteroaromatic ligand

New cationic ruthenium(II) complexes with the formula [Ru(eta(5)-C5H5)(LL)(1-BuIm)] [Z], with (LL) = 2PPh(3) or DPPE, and Z = CF3SO3-, PF6-, BPh4-, have been synthesized and fully characterized. Spectroscopic and electrochemical studies revealed that the electronic properties of the coordinated 1-butylimidazole were clearly influenced by the nature of the phosphane coligands (LL) and also by the different counter ions. The solid state structures of the six complexes determined by X-ray crystallographic studies, confirmed the expected distorted three-legged piano stool structure. However the geometry of the 1-butylimidazole ligand was found considerably different in all six compounds, being governed by the stereochemistry of the mono and bidentate coligands (PPh3 or DPPE).

Neutral C60 effusive source for atomic collisions with fullerene

Review of scientific instruments, Vol.72, N��9

Electrical ageing studies of polymeric insulation for power cables (estudo do envelhecimento el��ctrico do isolante polim��rico de cabos el��ctricos

Disserta����o apresentada para obten����o do Grau de Doutor em Engenharia F��sica - F��sica Aplicada pela Universidade Nova de Lisboa, Faculdade de Ci��ncias e Tecnologia

Y Trinuclear Cu-II structural isomers coordination, magnetism, electrochemistry and catalytic activity towards the oxidation of alkanes

The reaction of the Schiff base (3,5-di-tert-butyl-2-hydroxybenzylidene)-2-hydroxybenzohydrazide (H3L) with copper(II) nitrate, acetate or metaborate has led to the isomeric complexes [Cu-3(L)(2)(MeOH)(4)] (1), [Cu-3(L)(2)(MeOH)(2)]2MeOH (2) and [Cu-3(L)(2)(MeOH)(4)] (3), respectively, in which the ligand L exhibits dianionic (HL2-, in 1) or trianionic (L3-, in 2 and 3) pentadentate 1O,O,N:2N,O chelation modes. Complexes 1-3 were characterized by elemental analysis, IR spectroscopy, single-crystal X-ray crystallography, electrochemical methods and variable-temperature magnetic susceptibility measurements, which indicated that the intratrimer antiferromagnetic coupling is strong in the three complexes and that there exists very weak ferromagnetic intermolecular interactions in 1 but weak antiferromagnetic intermolecular interactions in both 2 and 3. Electrochemical experiments showed that in complexes 1-3 the Cu-II ions can be reduced, in distinct steps, to Cu-I and Cu-0. All the complexes act as efficient catalyst precursors under mild conditions for the peroxidative oxidation of cyclohexane to cyclohexyl hydroperoxide, cyclohexanol and cyclohexanone, leading to overall yields (based on the alkane) of up to 31% (TON = 1.55x10(3)) after 6 h in the presence of pyrazinecarboxylic acid.

Alternative chelating agents: Evaluation of the ready biodegradability and complexation properties

The ready biodegradability of four chelating agents, N,N -(S,S)-bis[1-carboxy-2-(imidazol-4-yl)ethyl]ethylenediamine (BCIEE), N - ethylenedi-L-cysteine (EC), N,N -bis (4-imidazolymethyl)ethylenediamine (EMI) and 2,6-pyridine dicarboxylic acid (PDA), was tested according to the OECD guideline for testing of chemicals. PDA proved to be a readily biodegradable substance. However, none of the other three compounds were degraded during the 28 days of the test. Chemical simulations were performed for the four compounds in order to understand their ability to complex with some metal ions (Ca, Cd, Co, Cu, Fe, Mg, Mn, Ni, Pb, Zn) and discuss possible applications of these chelating agents. Two different conditions were simulated: (i) in the presence of the chelating agent and one metal ion, and (ii) in the simultaneous presence of the chelating agent and all metal ions with an excess of Ca. For those compounds that were revealed not to be readily biodegradable (BCIEE, EC and EMI), applications were evaluated where this property was not fundamental or even not required. Chemical simulations pointed out that possible applications for these chelating agents are: food fortification, food process, fertilizers, biocides, soil remediation and treatment of metal poisoning. Additionally, chemical simulations also predicted that PDA is an efficient chelating agent for Ca incrustations removal, detergents and for pulp metal ions removal process.

Desidrata����o/oxida����o seletiva de metanol recorrendo a catalisadores heterog��neos para a obten����o de biocombust��veis (DME e DMM)

Trabalho Final de Mestrado para obten����o do grau de Mestre em Engenharia Qu��mica e Biol��gica

Desenvolvimento de m��todos de an��lise f��sico-qu��mica em ��guas usando um analisador multiparam��trico AQUAKEM

Trabalho Final de mestrado para obten����o do grau de Mestre em Engenharia Qu��mica e Biol��gica

Novel coordination polymers with (Pyrazolato)-based tectons: catalytic activity in the peroxidative oxidation of alcohols and cyclohexane

Coupling five rigid or flexible bis(pyrazolato)based tectons with late transition metal ions allowed us to isolate 18 coordination polymers (CPs). As assessed by thermal analysis, all of them possess a remarkable thermal stability, their decomposition temperatures lying in the range of 340-500 degrees C. As demonstrated by N-2 adsorption measurements at 77 K, their Langmuir specific surface areas span the rather vast range of 135-1758 m(2)/g, in agreement with the porous or dense polymeric architectures retrieved by powder X-ray diffraction structure solution methods. Two representative families of CPs, built up with either rigid or flexible spacers, were tested as catalysts in (0 the microwave-assisted solvent-free peroxidative oxidation of alcohols by t-BuOOH, and (ii) the peroxidative oxidation of cydohexane to cydohexanol and cydohexanone by H2O2 in acetonitrile. Those CPs bearing the rigid spacer, concurrently possessing higher specific surface areas, are more active than the corresponding ones with the flexible spacer. Moreover, the two copper(I)-containing CPs investigated exhibit the highest efficiency in both reactions, leading selectively to a maximum product yield of 92% (and TON up to 1.5 x 10(3)) in the oxidation of 1-phenylethanol and of 11% in the oxidation of cydohexane, the latter value being higher than that granted by the current industrial process.

Bioatividade de L��quidos I��nicos derivados do ��cido Valpr��ico em bact��rias Gram-negativa

Os L��quidos I��nicos (LIs) s��o sais org��nicos constitu��dos exclusivamente por i��es e possuem pontos de fus��o inferiores a 100��C. As suas propriedades ��nicas e o facto de ser poss��vel ajustar as suas propriedades f��sicas, qu��micas e biol��gicas, de acordo com o objetivo pretendido, tornam esta classe de compostos, um grande objeto de estudo de in��meros investigadores. Desde os in��cios da sua aplica����o at�� �� atualidade, a investiga����o nesta ��rea expandiu o seu raio de a����o, estando j�� descrito o seu potencial como agentes antimicrobianos e, mais recentemente, como compostos farmac��uticos ativos. Atualmente muitas das suas aplica����es s��o baseadas nas suas propriedades biol��gicas. Esta Tese teve como objetivo avaliar a influ��ncia que os LIs podem exercer a n��vel do crescimento bacteriano e estudar alternativas de combater a resist��ncia bacteriana. Todos os LIs utilizados neste trabalho tinham como ani��o o ��cido valpr��ico, sendo utilizados cati��es org��nicos de am��nio e de imidaz��lio. Foram utilizadas 4 bact��rias e avaliou-se a atividade biol��gica e a respetiva taxa de crescimento. O estudo da sua atividade biol��gica foi feito atrav��s da determina����o da Concentra����o M��nima Inibit��ria (CMI) e a an��lise das suas curvas de crescimentos na presen��a e aus��ncia de composto. Com este trabalho foi poss��vel verificar que dentro dos compostos em estudo, LIs derivados do valproato, o Valproato com o cetilperid��nio [valp] [cetylpir] foi o que influenciou o crescimento de todas as bact��rias estudadas. Este estudo demonstrou o potencial antibacteriano de alguns compostos, podendo desta forma vir a ser utilizados para fins farmac��uticos

Electron driven reactions in complexes embedded in superfluid helium droplets

A thesis submitted to the University of Innsbruck for the doctor degree in Natural Sciences, Physics and New University of Lisbon for the doctor degree in Physics, Atomic and Molecular Physics

Natural and amyloid self-assembly of S100 proteins: structural basis of functional diversity

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.

A regula����o end��crina dos balan��os de c��lcio

RESUMO: As concentra����es circulantes de c��lcio s��o notavelmente constantes a despeito das varia����es di��rias na absor����o intestinal e na elimina����o renal deste elemento. A regula����o da calc��mia �� um sistema complexo que compreende v��rios factores controladores (a calc��mia, a fosfor��mia, as concentra����es circulantes de paratormona (PTH) e calcitriol al��m de muitos outros factores como hormonas ester��ides em geral, outros i��es como o magn��sio e outros factores hormonais) e v��rios ��rg��os alvo (gl��ndulas paratiroideias, osso, rim e intestino). As respostas dos ��rg��os alvo tamb��m s��o muito variadas. No caso mais simples, a cristaliza����o de sais de c��lcio corresponde a uma mudan��a de fase em que participam mol��culas org��nicas que a iniciam, aceleram ou inibem. Em geral a combina����o de um factor controlador com o respectivo receptor de membrana (para polipept��deos ou i��es) ou intracelular (hormonas ester��ides) �� apenas o primeiro passo de uma cadeia bioqu��mica que introduz uma enorme amplifica����o na resposta. A esta variedade de mecanismos de resposta correspondem grandes diferen��as nos tempos de resposta que podem ser de minutos a semanas. �� hoje poss��vel ���observar��� (medir) com apreci��vel rigor nos l��quidos biol��gicos (sangue, urina, fezes, etc.) os factores mais importantes do sistema de regula����o da calc��mia (c��lcio, f��sforo, paratormona e calcitriol) assim como administrar estes factores em experi��ncias agudas. Esta possibilidade reflecte ��� se na literatura neste campo que tem vindo a crescer. O advento das t��cnicas da biologia molecular tem permitido a caracteriza����o molecular de algumas das disfun����es da homeostase do c��lcio e �� de esperar um diagn��stico fisiopatol��gico cada vez mais rigoroso dessas disfun����es. Com o avan��o dos conhecimentos nesta ��rea que n��o cessa de aumentar temos cada vez maiores capacidades para fazer diagn��sticos e �� cada vez mais dif��cil interpretar com rigor os correspondentes quadros metab��licos. A an��lise ou s��ntese de sistemas complexos �� a actividade mais nobre dos engenheiros que lhes permite desenhar pontes, diques, barcos, avi��es ou autom��veis. Com o aparecimento de computadores de m��dio ou grande porte foi ��� lhes poss��vel utilizar descri����es matem��ticas n��o s�� para desenhar sistemas como ainda para interpretar eventuais falhas na sua opera����o. Essas descri����es matem��ticas consistem numa sequ��ncia de opera����es realizadas num computador segundo um ���programa inform��tico��� que receberam a designa����o gen��rica de modelos, por analogia com as famosas leis (equa����es) da f��sica que foram deduzidas a partir de um certo n��mero de postulados e que permitem representar matematicamente processos f��sicos. As famosas leis de Newton s��o talvez os exemplos mais famosos de ���modelos��� de sistemas f��sicos. A introdu����o de modelos matem��ticos em biologia e particularmente em medicina s�� se deu recentemente.M��TODOS No trabalho que aqui se apresenta construiu - se um modelo simplificado da homeostase do c��lcio destinado ao c��lculo de vari��veis observ��veis (concentra����es de c��lcio, f��sforo, PTH e calcitriol) de modo a poderem comparar-se valores calculados com valores observados. A escolha dos componentes do modelo foi determinada pela nossa experi��ncia cl��nica e pela informa����o fisiopatol��gica e cl��nica publicada. Houve a preocupa����o de construir o modelo de forma modular de modo a ser poss��vel a sua expans��o sem grandes transforma����es na descri����o matem��tica (e inform��tica) j�� existente. Na sua fase actual o modelo n��o pode ser usado como instrumento de diagn��stico. �� antes uma ferramenta destinada a esclarecer ���em princ��pio��� mecanismos fisiopatol��gicos. Usou ��� se o modelo para simular um certo n��mero de observa����es publicadas e para exemplificar a sua eventual aplica����o cl��nica na simula����o de situa����es hipot��ticas e na an��lise de poss��veis mecanismos fisiopatol��gicos respons��veis por situa����es de hipo ou hipercalc��mias. Simultaneamente fez ��� se uma an��lise dos dados acumulados relativos a doentes vistos no Servi��o de Endocrinologia do Instituto Portugu��s de Oncologia de Francisco Gentil ��� Centro Regional Oncol��gico de Lisboa, S.A. CONCLUS��ES Numa popula����o de 894 doentes com patologias variadas do Instituto Portugu��s de Oncologia de Lisboa os valores da calc��mia tiveram uma distribui����o normal unimodal com uma m��dia de 9.56 mg/dl, e um erro padr��o de 0.41 mg/dl. Estas observa����es sugerem que a calc��mia est�� sujeita a regula����o. A partir dos resultados publicados em que o metabolismo do c��lcio foi perturbado por infus��es de c��lcio, calcitriol ou PTH, de estudos bioqu��micos e fisiol��gicos sobre os mecanismos de ac����o de factores controladores da calc��mia e do estudo do comportamento de ��rg��os alvo (paratiroideias, intestino, osso e rim) foi poss��vel construir um modelo matem��tico de par��metros concentrados do sistema de regula����o da calc��mia. As express��es anal��ticas usadas foram baseadas na cin��tica enzim��tica de modo a que os seus par��metros tivessem um significado f��sico ou fisiol��gico simples. O modelo revelou apreci��vel robustez e flexibilidade. �� est��vel quando n��o perturbado e transita entre estados estacion��rios quando perturbado. Na sua forma actual gera simula����es que reproduzem satisfatoriamente um n��mero apreci��vel de dados experimentais colhidos em doentes. Isto n��o significa que possa ser usado como instrumento de diagn��stico aplic��vel a doentes individuais. O desenho do modelo comporta a adi����o posterior de novas rela����es quando surgirem situa����es para as quais se revele insuficiente. A utiliza����o exaustiva do modelo permitiu explicitar aspectos do metabolismo do c��lcio que ou n��o est��o contidas na sua formula����o actual ��� o aparecimento de hipertrofia ou de adenomas das paratiroideias e as altera����es na estrutura ��ssea , a participa����o de outros factores controladores ��� magn��sio, ou est��o insuficientemente descritas ��� altera����es do metabolismo do f��sforo nos hipoparatiroidismos. A an��lise dos dados relativos aos doentes do Servi��o de Endocrinologia do IPO permitiu o in��cio da caracteriza����o dos tipos de patologia que representam e de poss��veis mecanismos fisiopatol��gicos subjacentes. Estas observa����es s��o o ponto de partida para an��lises futuras. S��o exemplos das rela����es encontradas: a distribui����o dos doentes por dois grandes grupos conforme a calc��mia �� determinada pelas concentra����es circulantes de PTH ou estas s��o determinadas pela calc��mia; a distribui����o sazonal das concentra����es de Vit. D25. no sangue; a correla����o negativa entre estas e as concentra����es de PTH no sangue. Tamb��m foi poss��vel extrair a cin��tica do controlo da PTH sobre a s��ntese de calcitriol. O estudo dos n��veis circulantes de PTH no p��s-operat��rio imediato de doentes paratiroidectomizados permitiu determinar as suas taxas de degrada����o metab��lica. O modelo permitiu simular as rela����es Ca/PTH no sangue, Ca/Frac����o excretada da carga tubular, Ca/P no sangue para valores normais ou altos de Ca. Foram feitas simula����es de situa����es fisiopatol��gicas (em ���doentes virtuais���): infus��es cr��nicas de c��lcio, PTH e calcitriol; altera����es no comportamento de receptores. Estas simula����es correspondem a experi��ncias que n��o podem ser realizadas em humanos. S��o exemplos da utiliza����o do modelo na explora����o de poss��veis mecanismos fisiopatol��gicos atrav��s da observa����o de resultados quantitativos inacess��veis �� intui����o. O modelo foi ��til em duas fases do trabalho: Primeiro, durante a sua s��ntese implicou uma escolha criticamente selectiva de informa����o, sua an��lise quantitativa e processamento, uma explicita����o rigorosa (anal��tica) das rela����es funcionais entre os controladores e as vari��veis e da sua integra����o numa estrutura global; Segundo, a simula����o de situa����es experimentais ou cl��nicas (dados do Servi��o de Endocrinologia do IPO) em doentes obrigou a explicitar racioc��nios fisiopatol��gicos habitualmente formulados em bases puramente intuitivas. Esta pr��tica revelou comportamentos ��bvios ap��s as simula����es ��� ac����o reduzida das infus��es PTH (simula����o de hiperparatiroidismos prim��rios) enquanto n��o h�� inibi����o total da respectiva secre����o, necessidade de aumento da massa secretora da paratiroideia nas insufici��ncias renais avan��adas, etc. A s��ntese e utiliza����o do modelo n��o implicaram uma prepara����o matem��tica avan��ada e foram poss��veis merc�� da disponibilidade de ���software��� interactivo especificamente desenhado para a simula����o de sistemas din��micos em que os programas se escrevem em ingl��s usando a simbologia simples da ��lgebra elementar. A fun����o nobre de modelos desta natureza �� semelhante �� dos modelos usados pelos f��sicos desde o s��culo XVII: permitir explica����es de car��cter geral funcionando como uma ferramenta intelectual para manipula����o de conceitos e para a realiza����o de ���experi��ncias pensadas��� (���thought experiments���) respeitando certos princ��pios f��sicos (princ��pios de conserva����o) 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 model���s 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 didn���t 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.------------------RESUM��E: Les concentrations circulantes de calcium sont constantes m��me pendant des variations de l���absorption intestinale et de l�����limination r��nale de cet ��l��ment. La r��gulation de la calc��mie est un syst��me complexe qui comprend plusieurs ��l��ments contr��leurs (la calc��mie, la phosphor��mie, les concentrations circulantes de l���hormone parathyro��dienne (PTH) e du calcitriol et d���autres comme les hormones st��ro��des ou des ions comme le magn��sium) et plusieurs organes (glandes parathyro��diennes, l���os, le rein et l���intestin). Les r��ponses de ces organes sont vari��es. Dans le cas plus simple, la cristallisation des sels de calcium correspond �� un changement de phase dans lequel y participent des mol��cules organiques que la d��butent, l���acc��l��rent ou l���inhibent. G��n��ralement la combinaison d���un ��l��ment contr��leur avec leur r��cepteur de membrane (pour les peptides ou les ions) ou intracellulaire (pour les hormones st��ro��des) n���est que le premier pas d���une cha��ne biochimique qu���introduit une grande amplification de la r��ponse. A cette vari��t�� de r��ponses correspondent des grandes diff��rences des temps de r��ponses qu���y vont des minuits a semaines. Il est possible �� observer �� (mesurer) dans les fluides biologiques (sang, urine, f��ces, etc.) les ��l��ments plus importants du syst��me de r��gulation de la calc��mie (calcium, phosphate, PTH et le calcitriol) et les administrer en exp��rimentes aigus. Cette possibilit�� est visible dans la litt��rature publi��e dans ce domaine qui est en croissance permanente. L���avenir des techniques de biologie mol��culaire a permis caract��riser des nombreuses dysfonctions de la r��gulation de la calc��mie et on attend un diagnostique physiopathologique de ces dysfonctions chaque fois plus rigoureuses. Les connaissances dans ce domaine s���agrandissent et on a de plus de capacit��s pour faire des diagnostiques et il est chaque fois plus difficile les interpr��ter. L���analyse ou synth��se de syst��mes complexes est l���activit�� plus noble des ing��nieurs qui les permit dessiner des ponts, bateaux, avions ou automobiles. Avec des ordinateurs de m��dium ou grand port il les est possible utiliser descriptions math��matiques pour dessiner les syst��mes et interpr��ter des ��ventuelles fautes d���op��ration. Ces descriptions math��matiques sont une s��quence d���op��rations r��alis��es dans un ordinateur selon �� un programme informatique �� qui ont re��u la d��signation g��n��rique de mod��les, pour analogie avec les ��quations de la physique qui ont ��t�� d��duits d���un nombre de postul��es et qu���ont permit repr��senter des processus physiques en ��quations math��matiques. Les fameuses ��quations de Newton sont peut-��tre les exemples plus connus des syst��mes physiques. L���introduction des mod��les math��matiques en biologie et en particulier en m��decine est un ��v��nement r��cent. Dans ce travaille, on a construit un mod��le simplifi�� de l���hom��ostasie du calcium pour calculer les variables observables (concentrations de calcium, phosphate, PTH et calcitriol) pour les comparer. Les choix des components a ��t�� d��termin��s par notre exp��rience clinique et par l���information physiopathologique et clinique publi��e. Le mod��le a ��t�� construit de fa��on modulaire ce que permit leur post��rieur expansion sans des grandes alt��rations dans la description math��matique et informatique d��j�� existante. Dans cette forme le mod��le ne peut ��tre utilis�� comme un instrument de diagnostique. Il est un outil pour ��clairer la physiopathologie. Le mod��le a ��t�� utilis�� pour simuler un certain nombre d���observations publi��es et pour exemplifier leur possible utilisation clinique dans la simulation des hypoth��ses et de la physiopathologie des situations d���hypo ou hypercalc��mie. On a fait une analyse des ��l��ments des proc��s cliniques des malades observ��es dans le Service d���Endocrinologie de l���IPOFG-CROL, SA. Dans une population de 894 malades avec des diff��rentes pathologies les valeurs de calc��mie on une distribution uni modale avec une M��die de 9.56 mg/dL et une erreur standard de 0.41 mg/dL. Ces observations sugg��rent que la calc��mie soit sujette de r��gulation. En utilisant des r��sultats de travaux publi��s dans lesquels le m��tabolisme du calcium a ��t�� chang�� par des infusions de calcium, calcitriol ou PTH, des ��tudes biochimiques et physiologiques sur des m��canismes d���action des ��l��ments contr��leurs de la calc��mie et de l�����tude du comportement des organes cible (parathyro��des, intestin, rein, os), il a ��t�� possible de construire un mod��le math��matique de param��tres concentr��s du syst��me de r��gulation de la calc��mie. Les expressions analytiques utilis��es ont ��t�� bas��es sur la cin��tique enzymatique de fa��on �� que les param��tres aient eu une signification physique ou biologique. Le mod��le est stable quand il n���est pas perturb�� et transit entre ��tats stationnaires quand il est sujet a des perturbations. A ce moment il fait des simulations qui reproduisent de fa��on satisfaisant un nombre d���observations exp��rimentales. La construction du mod��le permit l���addiction de nouvelles relations dans les cas ou il est insuffisant. L���utilisation exhaustive du mod��le a permit expliciter des aspects du m��tabolisme du calcium qui y ne sont pas compris ��� l���hyperplasie ou la formation des ad��nomes des parathyro��des, les alt��rations de la structure des os, la participation d���outres ��l��ments r��gulateurs (magn��sium), ou sont insuffisamment d��crites ��� les alt��rations du m��tabolisme des phosphates dans l���hypoparathyroidism. L���analyse de l���information des malades du Service d���Endocrinologie a permit caract��riser les pathologies repr��sent��es et leurs possibles m��canismes physiopathologiques. Ces observations sont le point de d��part pour les analyses futures. Sont des exemples des relations trouv��es: la distribution des malades par deux groupes: ceux dans lequel la calc��mie est d��termin��e par la PTH ou ceux dans lesquels la PTH est d��termin��e par la calc��mie; la distribution sazonale de la concentration de la vitamine D; la corr��lation n��gative entre la vitamine D et la PTH. On a eu la possibilit�� de d��duire la cin��tique de control de la PTH sur la synth��se du calcitriol. L�����tude des niveaux circulants de PTH sur des sujets parathyroidectomis��es a permit d��duire leur taux de d��gradation m��tabolique. Le mod��le a permit simuler les relations Ca/PTH dans le sang, Ca/fraction ��limin��e 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; alt��rations des r��cepteurs. Ces simulations ne peuvent pas ��tre r��alis��es dans les humains. Sont des exemples d���utilisation du mod��le dans l���exploration des possibles m��canismes de la physiopathologie en observant des r��sultats quantitatifs inaccessibles �� l���intuition. Le mod��le a ��t�� utile pendant deux ��tapes des travaux: La premi��re, dans sa construction on a choisi l���information disponible, son analyse quantitative, l���explicitation rigoureuse (analytique) des relations fonctionnelles entre les contr��leurs et les variables et sa int��gration dans une structure globale. La deuxi��me, la simulation de situations exp��rimentales ou cliniques (du Service d���Endocrinologie) a oblig�� d���expliciter des raisonnements physiopathologiques g��n��ralement formul��s utilisant l���intuition. Cette pratique a montr�� des comportements ��� action r��duite des infusions de PTH (jusqu����� l���inhibition totale de leur respective s��cr��tion), n��cessit�� d���augmenter la masse s��cr��teuse de la parathyro��de dans les insuffisants r��nales, etc. La synth��se et utilisation du mod��le n���ont pas besoin d���une formation avanc��e en math��matique et sont possibles gr��ce �� un programme interactif qui a ��t�� con��u pour la simulation des syst��mes dynamiques dans lesquels le programme se construit en anglais en utilisant la symbolique ��l��mentaire de l���alg��bre. La fonction noble de ces mod��les est semblable �� celles des physiques du XVII si��cle: Permettre ��tablir explications g��n��rales en fonctionnant comme un outil intellectuel pour manipuler des concepts et pour la r��alisation d���exp��rimentes pens��es en respectant certains principes de la physique (principe de la conservation) qu�����tablissent les fronti��res de la r��alit��.

Heavy-metal resistance in Marinobacter aquaeolei 617 insights into copper resistance

Disserta����o apresentada na Faculdade de Ci��ncias e Tecnologia da Universidade Nova de Lisboa para obten����o do grau de Mestre em Biotecnologia

Recycling old screen-printed electrodes with newly designed plastic antibodies on the wall of carbon nanotubes as sensory element for in situ detection of bacterial toxins in water

Using low cost portable devices that enable a single analytical step for screening environmental contaminants is today a demanding issue. This concept is here tried out by recycling screen-printed electrodes that were to be disposed of and by choosing as sensory element a low cost material offering specific response for an environmental contaminant. Microcystins (MCs) were used as target analyte, for being dangerous toxins produced by cyanobacteria released into water bodies. The sensory element was a plastic antibody designed by surface imprinting with carefully selected monomers to ensure a specific response. These were designed on the wall of carbon nanotubes, taking advantage of their exceptional electrical properties. The stereochemical ability of the sensory material to detect MCs was checked by preparing blank materials where the imprinting stage was made without the template molecule. The novel sensory material for MCs was introduced in a polymeric matrix and evaluated against potentiometric measurements. Nernstian response was observed from 7.24 �� 10���10 to 1.28 �� 10���9 M in buffer solution (10 mM HEPES, 150 mM NaCl, pH 6.6), with average slopes of ���62 mVdecade���1 and detection capabilities below 1 nM. The blank materials were unable to provide a linear response against log(concentration), showing only a slight potential change towards more positive potentials with increasing concentrations (while that ofthe plastic antibodies moved to more negative values), with a maximum rate of +33 mVdecade���1. The sensors presented good selectivity towards sulphate, iron and ammonium ions, and also chloroform and tetrachloroethylene (TCE) and fast response (<20 s). This concept was successfully tested on the analysis of spiked environmental water samples. The sensors were further applied onto recycled chips, comprehending one site for the reference electrode and two sites for different selective membranes, in a biparametric approach for ���in situ��� analysis.