Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily

The pathogenesis-related (PR) protein superfamily is widely distributed in the animal, plant, and fungal kingdoms and is implicated in human brain tumor growth and plant pathogenesis. The precise biological activity of PR proteins, however, has remained elusive. Here we report the characterization, cloning and structural homology modeling of Tex31 from the venom duct of Conus textile. Tex31 was isolated to >95% purity by activity-guided fractionation using a para-nitroanilide substrate based on the putative cleavage site residues found in the propeptide precursor of conotoxin TxVIA. Tex31 requires four residues including a leucine N-terminal of the cleavage site for efficient substrate processing. The sequence of Tex31 was determined using two degenerate PCR primers designed from N-terminal and tryptic digest Edman sequences. A BLAST search revealed that Tex31 was a member of the PR protein superfamily and most closely related to the CRISP family of mammalian proteins that have a cysteine-rich C-terminal tail. A homology model constructed from two PR proteins revealed that the likely catalytic residues in Tex31 fall within a structurally conserved domain found in PR proteins. Thus, it is possible that other PR proteins may also be substrate-specific proteases.

Caracterização do papel da adrenalina na maturação dos adrenorreceptores B2

As respostas pós-juncionais mediadas por adrenorreceptores β2 (ARβ2), responsáveis pelo relaxamento do músculo liso, na veia safena do cão, estão ausentes à nascença. Pelo contrário, no rato recém-nascido já se verifica a estimulação da adenilil ciclase pela activação dos ARβ2. Não existem ainda estudos no coelho recém-nascido. O principal objectivo deste trabalho é avaliar as respostas pós-juncionais mediadas pelos ARβ2 em coelhos recém-nascidos e jovens e relacionar essas respostas com a adrenalina produzida nas glândulas supra-renais. Traçaram-se curvas de dose-resposta à isoprenalina (agonista β) utilizando-se anéis de aorta montados em banho de órgãos isolados ligado a um transdutor de força isométrica. As catecolaminas das supra-renais foram quantificadas por RP-HPLC-ED. Em aortas pré-contraídas com fenilefrina (agonista α1), a isoprenalina causou relaxamento total apenas em coelhos recém-nascidos (n=10). O relaxamento máximo nos coelhos jovens foi de 21±4% (n=23). A potência da isoprenalina foi maior nos recém-nascidos (EC50=1.15×10-8±7.2×10-10 M, n=10) do que nos coelhos jovens (EC50=1.29×10-7 ±4.7×10-9 M, n=23). O relaxamento máximo com isoprenalina, em aortas pré-contraídas com prostaglandina F2α (PGF2α), no grupo de coelhos recém-nascidos foi de 95±3.6% (n=16). O relaxamento máximo nos coelhos jovens foi de 43.7±8.6% (n=9). Na pré-contracção com PGF2α a potência da isoprenalina registou-se maior nos recémnascidos (EC50=9.59×10-9±4.0×10-10 M, n=16) do que nos coelhos jovens (EC50=2.13×10- 8±3.8×10-9 M, n=9), estando concordante com os resultados da pré-contracção com fenilefrina. Nas supra-renais dos recém-nascidos, o conteúdo de noradrenalina foi de 586±128 nmol/mg e da adrenalina foi de 1915±356 nmol/mg (n=4) e nos coelhos jovens foi de 112±12 nmol/mg e de 3644±403 nmol/mg (n=6), respectivamente. As respostas mediadas por ARβ2 no coelho desenvolvem-se mais cedo do que no cão, pois já estão presentes no nascimento. Tal como no rato, no coelho a adrenalina é já a catecolamina em maior quantidade à nascença, enquanto no cão é vestigial. Há uma relação temporal entre a síntese da adrenalina, a única catecolamina biogénica com alta afinidade para os ARβ2 e a maturação das respostas pós-juncionais mediadas por esses receptores. Um protocolo para experiências futuras destinadas a testar esta hipótese, com base no knockdown da Feniletanolamina-N-metiltransferase por RNAi foi elaborado e incluído neste documento.

Development of TNBS-induced colitis: animal model to test new pharmacological approaches

IBD is a gastro-intestinal disorder marked with chronic inflammation of intestinal epithelium, damaging mucosal tissue and manifests into several intestinal and extra-intestinal symptoms. Currently used medical therapy is able to induce and maintain the patient in remission, however no modifies or reverses the underlying pathogenic mechanism. The research of other medical approaches is crucial to the treatment of IBD and, for this, it´s important to use animal models to mimic the characteristics of disease in real life. The aim of the study is to develop an animal model of TNBS-induced colitis to test new pharmacological approaches. TNBS was instilled intracolonic single dose as described by Morris et al. It was administered 2,5% TNBS in 50% ethanol through a catheter carefully inserted into the colon. Mice were kept in a Tredelenburg position to avoid reflux. On day 4 and 7, the animals were sacrificed by cervical dislocation. The induction was confirmed based on clinical symptoms/signs, ALP determination and histopathological analysis. At day 4, TNBS group presented a decreased body weight and an alteration of intestinal motility characterized by diarrhea, severe edema of the anus and moderate morbidity, while in the two control groups weren’t identified any alteration on the clinical symptoms/signs with an increase of the body weight. TNBS group presented the highest concentrations of ALP comparing with control groups. The histopathology analysis revealed severe necrosis of the mucosa with widespread necrosis of the intestinal glands. Severe hemorrhagic and purulent exsudates were observed in the submucosa, muscular and serosa. TNBS group presented clinical symptoms/signs and histopathological features compatible with a correct induction of UC. The peak of manifestations became maximal at day 4 after induction. This study allows concluding that it’s possible to develop a TNBS induced colitis 4 days after instillation.

Coral snake venoms: mode of action and pathophysiology of experimental envenomation

Coral snakes, the New World Elapidae, are included in the genera Micniroides and Micrurus. The genus Mlcrurus comprises nearly all coral snake species and those which are responsible for human snake-bite accidents. The following generalizations concerning the effects induced by their venoms, and their venom-properties can be made. Coral snake venoms are neurotoxic, producing loss of muscle strenght and death by respiratory paralysis. Local edema and necrosis are not induced nor blood coagulation or hemorrhages. Proteolysis activity is absent or of very low grade. They display phospholipase A2 activity. Nephrotoxic effects are not evoked. The main toxins from elapid venoms are postsynaptic and presynaptic neurotoxins and cardiotoxins. Phospholipases A2 endowed with myonecrotic or cardiotoxin-like properties are important toxic components from some elapid venoms. The mode of action of Micrurus frontalis, M. lemniscatus, M. corallinus and M. fulvius venoms has been investigated in isolated muscle preparations and is here discussed. It is shown that while M. frontalis and M. lemniscatus venoms must contain only neurotoxins that act at the cholinergic end-plate receptor (postsynaptic neurotoxins), M. corallinus venom also inhibits evoked acetylcholine release by the motor nerve endings (presynaptic neurotoxin-like effect) and M. fulvius induces muscle fiber membrane depolarization (cardiotoxin-like effect). The effects produced by M. corallinus and M. fulvius venoms in vivo in dogs and M. frontalis venom in dogs and monkeys are also reported.

Methemoglobinemia associated with loxoscelism

In twenty five patients who presented the cutaneous form of loxoscelism, serum haptoglobin and lactic dehydrogenase, erythrocyte glucose-6-phosphate dehydrogenase, glutathione reductase, glutathione peroxidase, methemoglobin, bilirubin and reticulocytes were investigated after bite. No hemolysis was detected but an increase in methemoglobin was found in 54% of the cases; in 7% it was between 1.1% and 2%, in 27% it ranged from 2.1% to 4%, and in 20% from 4.1% to 8%. Blood samples of a normal, blood group 0 individual and of a patient who exhibited methemoglobinemia after Loxosceles bite were incubated separately with antisera against Loxosceles gaucho, Crotalus terrificus, Bothrops jararaca, with Loxosceles gaucho venom and 0.3% phenol. No methemoglobin was found after 1, 4,8 and 15 days in both sets of samples. At the 25th day all the samples, including the controls, exhibited similar methemoglobin reductase decrease. The data suggest that the methemoglobinemia which occurs in 50% of the patients probably arises from in vivo venom metabolism, inasmuch as the crude venom does not induce methemoglobinemia.

Hepatobiliary alterations in massive biliary ascariasis: histopathological aspects of an autopsy case

Hepatobiliary alterations found in an autopsy case of massive Biliary Ascariasis, are reported on histological grounds. Severe cholangitis was the main finding, but other changes were also detected, such as pyloric and intestinal metaplasia, hyperplasia of the epithelial lining, with intraductal papillomas and adenomatous proliferation. Remnants of the worm were observed tightly adhered to the epithelium, forming microscopic intrahepatic calculi. Mucopolysaccharides, especially acid, showed to be strongly positive on the luminal border, and in proliferated glands around the ducts. The authors discuss the similarity between such findings and Oriental Cholangiohepatitis, and suggest that inflammation and the presence of the parasitic remnants are responsible for the hyperplastic and metaplastic changes, similarly with what occurs in chlonorchiasis, fascioliasis and schistosomiasis.

Accumulation, responses and genotoxicity of trace elements in octopus vulgaris

Dissertação apresentada para obtenção do Grau de Doutor em Bioquímica pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Lance-headed viper (Bothrops moojeni) bite wounding the eye

A 5-year-old girl was bitten in her left eye by a lance-headed viper identified as Bothrops moojeni, measuring 115 cm of length. There was severe facial swelling and left exophthalmus, and enucleation of the eye was necessary. The patient apparently had mild systemic envenoming, but local inflammatory signs and histological evidence of necrosis suggest that both the mechanical trauma and the local action of the venom had a role in the genesis of the eye lesion. It is arguable if the loss of the eye could be prevented even if the antivenom was administered earlier.

Severe scorpion envenomation in Brazil: clinical, laboratory and anatomopathological aspects

Scorpion stings in Brazil are important not only because of their incidence but also for their potential ability to induce severe, and often fatal, clinical situations, especially among children. In this report we present the clinical and laboratory data of 4 patients victims of scorpion stings by T. serrulatus, who developed heart failure and pulmonary edema, with 3 of them dying within 24 hours of the sting. Anatomopathologic study of these patients revealed diffuse areas of myocardiocytolysis in addition to pulmonary edema. The surviving child presented enzymatic, electrocardiographic and echocardiographic changes compatible with severe cardiac involvement, which were reversed within 5 days. These findings reinforce the need for continuous monitoring of patients with severe scorpion envenoming during the hours immediately following the sting.

Philodryas patagoniensis bite and local envenoming

A 5-year-old boy bitten by a specimen of Philodryas patagoniensis, a colubrid snake currently classified as nonvenomous, developed signs of local envenoming characterized by swelling and warmth on the bitten limb. This is the first time that local envenoming following Philodryas patagoniensis bite is recognized. Based on the clinical findings and misidentification of the snake, the patient was treated as a victim of Bothrops bite, having received unnecessarily the specific antivenom. Educational efforts to make doctors and health workers capable to identify correctly venomous snakes are necessary, to avoid inappropriate indication of antivenom and decrease the risk of its potentially harmful untoward effects. Examination of the bite site can be useful to the differential diagnosis between pit viper and colubrid bites.

Liver dysfunction in patients bitten by Crotalus durissus terrificus (Laurenti, 1768) snakes in Botucatu (State of São Paulo, Brazil)

Thirty-two patients bitten by venomous snakes sixteen by Bothrops spp. and sixteen by Crotalus durissus terrificus were studied. The group comprised thirty males and two females, aged eight to sixty-three years (mean 33±15). Bromsulphalein tests were increased in the majority of patients bitten by Crotalus durissus terrificus. The correlation coefficient of Spearman was positive between bromsulphalein tests and alanine aminotransferase levels, and between alanine aminotransferase and aspartate aminotransferase levels only in the Crotalus group. The only patient who died was bitten by Crotalus durissus terrificus and showed hydropic degeneration and mitochondrial injury in the liver. It was concluded that the hepatic damage might have been caused by at least two possible mechanisms: venom effect on liver mitochondria and cytokine effects on hepatocyte, specially interleukin-6.

Development of Schistosoma mansoni in Biomphalaria tenagophila, Biomphalaria straminea and Biomphalaria glabrata

A comparative study of the development of Schistosoma mansoni during the intra-molluscan phase was made by means of histological sections of Biomphalaria tenagophila, B. straminea and B. glabrata from Brazil. Two hundred snails of each species were individually exposed to 50 miracidia of the S. mansoni, AL line. No larvae were observed in the snails fixed 72 h after exposure. In specimens shedding cercariae, 31 days after exposure tissue reactions encapsulating the larvae were seen in B. tenagophila and B. straminea, in the head-foot, mantle collar and renal ducts. No tissue reactions occurred in the digestive glands of these two species. In B. glabrata the presence of numerous sporocysts and cercariae without tissue reactions was observed in the digestive gland, and other organs. The levels of infection of the snails and the average numbers of cercariae shed per day were 32.6% and 79±90 respectively for B. tenagophila, 11.3% and 112±100 for B. straminea and 75.3% and 432±436 for B. glabrata. The lower levels of infection and average numbers of cercariae shed by B. tenagophila and B. straminea are thus related to their more potent internal defense systems.

Schistosoma mansoni Sambon, 1907: comparative morphologica studies of some Brazilian strains

The morphology of Schistosoma mansoni adult male worms from three strains which have been maintained in albino mice for several generations, was compared to a strain that has been isolated from the natural host Nectomys squamipes (Rodentia: Muridae) captured in Sumidouro (Rio de Janeiro State) and have been maintained in the same sylvatic rodent under laboratory conditions. Total length of specimens, distance between suckers, the number of testes and extention of testes grouping were the taxonomic characters analysed. The worms recovered from N. squamipes showed expressive differences (p< 0.01) compared to the other strains regarding the considered morphological characters. The strains that were maintained in mice presented statistical differences (p< 0.01) in several characters. Some adult worms besides the normal position of the testes also showed an atypical arrangement of these glands. It can be concluded that the morphology of adult worms may be used to distinguish S. mansoni strains and that morphological changes in adult worms are not induced by successive inoculations of a strain in mice.

Neostigmine in the treatment of snake accidents caused by Micrurus frontalis: report of two cases

Antivenom in order to be effective in the treatment of coral snake accidents must be injected very soon after the bite owing to the rapid rate of absorption of the venom neurotoxins. As this is not always possible, other forms of treatment besides serotherapy must be employed to avoid asphyxia and death. Neostigmine and artificial respiration are used for this purpose. Neostigmine restores neuromuscular transmission if the venom-induced blockade results from a reversible interaction of its neurotoxins with the end-plate receptors. This is the mechanism of the neuromuscular blockade produced by the venom of M. frontalis snakes from centereastern and southern Brazil, and Argentine. Neostigmine is able, therefore, to antagonize the blockade, and has been shown to be very effective in the treatment of the experimental envenomation of dogs and monkeys. In the present communication, two cases of M. frontalis accidents treated with antivenom and neostigmine are reported. In both, neostigmine was successful in producing regression of the paralysis, confirming the effectiveness shown in the treatment of the poisoning induced in animals by M. frontalis venom.

A regulação endócrina dos balanços de cálcio

RESUMO: As concentrações circulantes de cálcio são notavelmente constantes a despeito das variações diárias na absorção intestinal e na eliminação renal deste elemento. A regulação da calcémia é um sistema complexo que compreende vários factores controladores (a calcémia, a fosforémia, as concentrações circulantes de paratormona (PTH) e calcitriol além de muitos outros factores como hormonas esteróides em geral, outros iões como o magnésio e outros factores hormonais) e vários órgãos alvo (glândulas paratiroideias, osso, rim e intestino). As respostas dos órgãos alvo também são muito variadas. No caso mais simples, a cristalização de sais de cálcio corresponde a uma mudança de fase em que participam moléculas orgânicas que a iniciam, aceleram ou inibem. Em geral a combinação de um factor controlador com o respectivo receptor de membrana (para polipeptídeos ou iões) ou intracelular (hormonas esteróides) é apenas o primeiro passo de uma cadeia bioquímica que introduz uma enorme amplificação na resposta. A esta variedade de mecanismos de resposta correspondem grandes diferenças nos tempos de resposta que podem ser de minutos a semanas. É hoje possível “observar” (medir) com apreciável rigor nos líquidos biológicos (sangue, urina, fezes, etc.) os factores mais importantes do sistema de regulação da calcémia (cálcio, fósforo, paratormona e calcitriol) assim como administrar estes factores em experiências agudas. Esta possibilidade reflecte – se na literatura neste campo que tem vindo a crescer. O advento das técnicas da biologia molecular tem permitido a caracterização molecular de algumas das disfunções da homeostase do cálcio e é de esperar um diagnóstico fisiopatológico cada vez mais rigoroso dessas disfunções. Com o avanço dos conhecimentos nesta área que não cessa de aumentar temos cada vez maiores capacidades para fazer diagnósticos e é cada vez mais difícil interpretar com rigor os correspondentes quadros metabólicos. A análise ou síntese de sistemas complexos é a actividade mais nobre dos engenheiros que lhes permite desenhar pontes, diques, barcos, aviões ou automóveis. Com o aparecimento de computadores de médio ou grande porte foi – lhes possível utilizar descrições matemáticas não só para desenhar sistemas como ainda para interpretar eventuais falhas na sua operação. Essas descrições matemáticas consistem numa sequência de operações realizadas num computador segundo um “programa informático” que receberam a designação genérica de modelos, por analogia com as famosas leis (equações) da física que foram deduzidas a partir de um certo número de postulados e que permitem representar matematicamente processos físicos. As famosas leis de Newton são talvez os exemplos mais famosos de “modelos” de sistemas físicos. A introdução de modelos matemáticos em biologia e particularmente em medicina só se deu recentemente.MÉTODOS No trabalho que aqui se apresenta construiu - se um modelo simplificado da homeostase do cálcio destinado ao cálculo de variáveis observáveis (concentrações de cálcio, fósforo, PTH e calcitriol) de modo a poderem comparar-se valores calculados com valores observados. A escolha dos componentes do modelo foi determinada pela nossa experiência clínica e pela informação fisiopatológica e clínica publicada. Houve a preocupação de construir o modelo de forma modular de modo a ser possível a sua expansão sem grandes transformações na descrição matemática (e informática) já existente. Na sua fase actual o modelo não pode ser usado como instrumento de diagnóstico. É antes uma ferramenta destinada a esclarecer “em princípio” mecanismos fisiopatológicos. Usou – se o modelo para simular um certo número de observações publicadas e para exemplificar a sua eventual aplicação clínica na simulação de situações hipotéticas e na análise de possíveis mecanismos fisiopatológicos responsáveis por situações de hipo ou hipercalcémias. Simultaneamente fez – se uma análise dos dados acumulados relativos a doentes vistos no Serviço de Endocrinologia do Instituto Português de Oncologia de Francisco Gentil – Centro Regional Oncológico de Lisboa, S.A. CONCLUSÕES Numa população de 894 doentes com patologias variadas do Instituto Português de Oncologia de Lisboa os valores da calcémia tiveram uma distribuição normal unimodal com uma média de 9.56 mg/dl, e um erro padrão de 0.41 mg/dl. Estas observações sugerem que a calcémia está sujeita a regulação. A partir dos resultados publicados em que o metabolismo do cálcio foi perturbado por infusões de cálcio, calcitriol ou PTH, de estudos bioquímicos e fisiológicos sobre os mecanismos de acção de factores controladores da calcémia e do estudo do comportamento de órgãos alvo (paratiroideias, intestino, osso e rim) foi possível construir um modelo matemático de parâmetros concentrados do sistema de regulação da calcémia. As expressões analíticas usadas foram baseadas na cinética enzimática de modo a que os seus parâmetros tivessem um significado físico ou fisiológico simples. O modelo revelou apreciável robustez e flexibilidade. É estável quando não perturbado e transita entre estados estacionários quando perturbado. Na sua forma actual gera simulações que reproduzem satisfatoriamente um número apreciável de dados experimentais colhidos em doentes. Isto não significa que possa ser usado como instrumento de diagnóstico aplicável a doentes individuais. O desenho do modelo comporta a adição posterior de novas relações quando surgirem situações para as quais se revele insuficiente. A utilização exaustiva do modelo permitiu explicitar aspectos do metabolismo do cálcio que ou não estão contidas na sua formulação actual – o aparecimento de hipertrofia ou de adenomas das paratiroideias e as alterações na estrutura óssea , a participação de outros factores controladores – magnésio, ou estão insuficientemente descritas – alterações do metabolismo do fósforo nos hipoparatiroidismos. A análise dos dados relativos aos doentes do Serviço de Endocrinologia do IPO permitiu o início da caracterização dos tipos de patologia que representam e de possíveis mecanismos fisiopatológicos subjacentes. Estas observações são o ponto de partida para análises futuras. São exemplos das relações encontradas: a distribuição dos doentes por dois grandes grupos conforme a calcémia é determinada pelas concentrações circulantes de PTH ou estas são determinadas pela calcémia; a distribuição sazonal das concentrações de Vit. D25. no sangue; a correlação negativa entre estas e as concentrações de PTH no sangue. Também foi possível extrair a cinética do controlo da PTH sobre a síntese de calcitriol. O estudo dos níveis circulantes de PTH no pós-operatório imediato de doentes paratiroidectomizados permitiu determinar as suas taxas de degradação metabólica. O modelo permitiu simular as relações Ca/PTH no sangue, Ca/Fracção excretada da carga tubular, Ca/P no sangue para valores normais ou altos de Ca. Foram feitas simulações de situações fisiopatológicas (em “doentes virtuais”): infusões crónicas de cálcio, PTH e calcitriol; alterações no comportamento de receptores. Estas simulações correspondem a experiências que não podem ser realizadas em humanos. São exemplos da utilização do modelo na exploração de possíveis mecanismos fisiopatológicos através da observação de resultados quantitativos inacessíveis à intuição. O modelo foi útil em duas fases do trabalho: Primeiro, durante a sua síntese implicou uma escolha criticamente selectiva de informação, sua análise quantitativa e processamento, uma explicitação rigorosa (analítica) das relações funcionais entre os controladores e as variáveis e da sua integração numa estrutura global; Segundo, a simulação de situações experimentais ou clínicas (dados do Serviço de Endocrinologia do IPO) em doentes obrigou a explicitar raciocínios fisiopatológicos habitualmente formulados em bases puramente intuitivas. Esta prática revelou comportamentos óbvios após as simulações – acção reduzida das infusões PTH (simulação de hiperparatiroidismos primários) enquanto não há inibição total da respectiva secreção, necessidade de aumento da massa secretora da paratiroideia nas insuficiências renais avançadas, etc. A síntese e utilização do modelo não implicaram uma preparação matemática avançada e foram possíveis mercê da disponibilidade de “software” interactivo especificamente desenhado para a simulação de sistemas dinâmicos em que os programas se escrevem em inglês usando a simbologia simples da álgebra elementar. A função nobre de modelos desta natureza é semelhante à dos modelos usados pelos físicos desde o século XVII: permitir explicações de carácter geral funcionando como uma ferramenta intelectual para manipulação de conceitos e para a realização de “experiências pensadas” (“thought experiments”) respeitando certos princípios físicos (princípios de conservação) 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.------------------RESUMÉE: Les concentrations circulantes de calcium sont constantes même pendant des variations de l’absorption intestinale et de l’élimination rénale de cet élément. La régulation de la calcémie est un système complexe qui comprend plusieurs éléments contrôleurs (la calcémie, la phosphorémie, les concentrations circulantes de l’hormone parathyroïdienne (PTH) e du calcitriol et d’autres comme les hormones stéroïdes ou des ions comme le magnésium) et plusieurs organes (glandes parathyroïdiennes, l’os, le rein et l’intestin). Les réponses de ces organes sont variées. Dans le cas plus simple, la cristallisation des sels de calcium correspond à un changement de phase dans lequel y participent des molécules organiques que la débutent, l’accélèrent ou l’inhibent. Généralement la combinaison d’un élément contrôleur avec leur récepteur de membrane (pour les peptides ou les ions) ou intracellulaire (pour les hormones stéroïdes) n’est que le premier pas d’une chaîne biochimique qu’introduit une grande amplification de la réponse. A cette variété de réponses correspondent des grandes différences des temps de réponses qu’y vont des minuits a semaines. Il est possible « observer » (mesurer) dans les fluides biologiques (sang, urine, fèces, etc.) les éléments plus importants du système de régulation de la calcémie (calcium, phosphate, PTH et le calcitriol) et les administrer en expérimentes aigus. Cette possibilité est visible dans la littérature publiée dans ce domaine qui est en croissance permanente. L’avenir des techniques de biologie moléculaire a permis caractériser des nombreuses dysfonctions de la régulation de la calcémie 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 capacités pour faire des diagnostiques et il est chaque fois plus difficile les interpréter. L’analyse ou synthèse de systèmes complexes est l’activité plus noble des ingénieurs qui les permit dessiner des ponts, bateaux, avions ou automobiles. Avec des ordinateurs de médium ou grand port il les est possible utiliser descriptions mathématiques pour dessiner les systèmes et interpréter des éventuelles fautes d’opération. Ces descriptions mathématiques sont une séquence d’opérations réalisées dans un ordinateur selon « un programme informatique » qui ont reçu la désignation générique de modèles, pour analogie avec les équations de la physique qui ont été déduits d’un nombre de postulées et qu’ont permit représenter des processus physiques en équations mathématiques. Les fameuses équations de Newton sont peut-être les exemples plus connus des systèmes physiques. L’introduction des modèles mathématiques en biologie et en particulier en médecine est un évènement récent. Dans ce travaille, on a construit un modèle simplifié de l’homéostasie du calcium pour calculer les variables observables (concentrations de calcium, phosphate, PTH et calcitriol) pour les comparer. Les choix des components a été déterminés par notre expérience clinique et par l’information physiopathologique et clinique publiée. Le modèle a été construit de façon modulaire ce que permit leur postérieur expansion sans des grandes altérations dans la description mathématique et informatique déjà existante. Dans cette forme le modèle ne peut être utilisé comme un instrument de diagnostique. Il est un outil pour éclairer la physiopathologie. Le modèle a été utilisé pour simuler un certain nombre d’observations publiées et pour exemplifier leur possible utilisation clinique dans la simulation des hypothèses et de la physiopathologie des situations d’hypo ou hypercalcémie. On a fait une analyse des éléments des procès cliniques des malades observées dans le Service d’Endocrinologie de l’IPOFG-CROL, SA. Dans une population de 894 malades avec des différentes pathologies les valeurs de calcémie on une distribution uni modale avec une Médie de 9.56 mg/dL et une erreur standard de 0.41 mg/dL. Ces observations suggèrent que la calcémie soit sujette de régulation. En utilisant des résultats de travaux publiés dans lesquels le métabolisme du calcium a été changé par des infusions de calcium, calcitriol ou PTH, des études biochimiques et physiologiques sur des mécanismes d’action des éléments contrôleurs de la calcémie et de l’étude du comportement des organes cible (parathyroïdes, intestin, rein, os), il a été possible de construire un modèle mathématique de paramètres concentrés du système de régulation de la calcémie. Les expressions analytiques utilisées ont été basées sur la cinétique enzymatique de façon à que les paramètres aient eu une signification physique ou biologique. Le modèle est stable quand il n’est pas perturbé et transit entre états stationnaires quand il est sujet a des perturbations. A ce moment il fait des simulations qui reproduisent de façon satisfaisant un nombre d’observations expérimentales. La construction du modèle permit l’addiction de nouvelles relations dans les cas ou il est insuffisant. L’utilisation exhaustive du modèle a permit expliciter des aspects du métabolisme du calcium qui y ne sont pas compris – l’hyperplasie ou la formation des adénomes des parathyroïdes, les altérations de la structure des os, la participation d’outres éléments régulateurs (magnésium), ou sont insuffisamment décrites – les altérations du métabolisme des phosphates dans l’hypoparathyroidism. L’analyse de l’information des malades du Service d’Endocrinologie a permit caractériser les pathologies représentées et leurs possibles mécanismes physiopathologiques. Ces observations sont le point de départ pour les analyses futures. Sont des exemples des relations trouvées: la distribution des malades par deux groupes: ceux dans lequel la calcémie est déterminée par la PTH ou ceux dans lesquels la PTH est déterminée par la calcémie; la distribution sazonale de la concentration de la vitamine D; la corrélation négative entre la vitamine D et la PTH. On a eu la possibilité de déduire la cinétique de control de la PTH sur la synthèse du calcitriol. L’étude des niveaux circulants de PTH sur des sujets parathyroidectomisées a permit déduire leur taux de dégradation métabolique. Le modèle a permit simuler les relations Ca/PTH dans le sang, Ca/fraction éliminée 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; altérations des récepteurs. Ces simulations ne peuvent pas être réalisées dans les humains. Sont des exemples d’utilisation du modèle dans l’exploration des possibles mécanismes de la physiopathologie en observant des résultats quantitatifs inaccessibles à l’intuition. Le modèle a été utile pendant deux étapes des travaux: La première, dans sa construction on a choisi l’information disponible, son analyse quantitative, l’explicitation rigoureuse (analytique) des relations fonctionnelles entre les contrôleurs et les variables et sa intégration dans une structure globale. La deuxième, la simulation de situations expérimentales ou cliniques (du Service d’Endocrinologie) a obligé d’expliciter des raisonnements physiopathologiques généralement formulés utilisant l’intuition. Cette pratique a montré des comportements – action réduite des infusions de PTH (jusqu’à l’inhibition totale de leur respective sécrétion), nécessité d’augmenter la masse sécréteuse de la parathyroïde dans les insuffisants rénales, etc. La synthèse et utilisation du modèle n’ont pas besoin d’une formation avancée en mathématique et sont possibles grâce à un programme interactif qui a été conçu pour la simulation des systèmes dynamiques dans lesquels le programme se construit en anglais en utilisant la symbolique élémentaire de l’algèbre. La fonction noble de ces modèles est semblable à celles des physiques du XVII siècle: Permettre établir explications générales en fonctionnant comme un outil intellectuel pour manipuler des concepts et pour la réalisation d’expérimentes pensées en respectant certains principes de la physique (principe de la conservation) qu’établissent les frontières de la réalité.