Dissertationen zum Thema „Calcium Metabolism“

Cada cop més evidencies suggereixen que els astròcits participen en les altes funcions cerebrals, controlant des de la transmissió sinàptica fins a les ones cerebrals globals i els processos d’aprenentatge i memòria. Diferents mecanismes han sigut proposats com a responsables d’aquests processos mediats per astròcits, entre ells, l’alliberació de gliotransmissors a partir de les senyals de calci així com la de lactat semblen els principals efectors. L’existència d’aquest control de les funcions cerebrals per part dels astròcits suggereix que aquestes cèl·lules poden regular les funcions cerebrals en resposta a experiència tan com les neurones, constituint el fenomen de plasticitat astrocitària. En neurones s’ha demostrat que el conegut factor de transcripció CREB, coordina les plasticitats sinàptica i intrínseca. El fet que, en astròcits, l’activació de CREB també està regulada per activitat cerebral, situa aquest factor de transcripció com a la diana ideal per promoure canvis dependents d’activitat en astròcits. En aquesta tesi hem analitzat l’efecte de l’activació de la transcripció depenent de CREB en astròcits, centrant-nos en l’excitabilitat del calci i en el metabolisme d’aquestes cèl·lules. Hem demostrat que l’activació de la transcripció depenent de CREB redueix les senyals citosòliques de calci a través del mitocondri a la vegada que augmenta l’alliberació de lactat, dos canvis que poden tenir impacte en la transmissió sinàptica. Una altra contribució important d’aquest estudi es l’anàlisi molecular dels mitocondris dels astròcits, que ha revelat que aquestes cèl·lules poden utilitzar metabòlits que no són glucosa, com ara àcids grassos, per respondre a les necessitats metabòliques energètiques. Els nostres resultats estableixen el CREB en astròcits con un eix de la plasticitat astrocitària i revelen la interacció entre la plasticitat i el metabolisme energètic en astròcits. Aquests descobriments constitueixen un avenç mecanístic i conceptual en el coneixement de la biologia dels astròcits i com aquestes cèl·lules poden controlar l’aprenentatge i la memòria.
An increasing body of evidence suggests that astrocytes participate in higher-brain functions, controlling from synaptic transmission to global brain waves and learning and memory processes. Different mechanisms have been proposed to mediate these astrocyte-dependent processes, astrocytic lactate release and calcium-dependent gliotransmission being the main known effectors. The existence of control of brain functions by astrocytes suggests that astrocytes may shape brain functions in response to experience as much as neurons, thus constituting the phenomenon of astrocyte plasticity. In neurons, the transcription factor CREB is the best known coordinator of synaptic and intrinsic plasticity. The fact that, in astrocytes, CREB activation is also activity-dependent, positions CREB as an ideal target to promote plasticity-related changes in astrocytes, too. In this thesis, we have analyzed the effect of the activation of CREB-dependent transcription in astrocytes, specifically regarding calcium signals and metabolism. We have demonstrated that activation of CREB-dependent transcription reduces cytosolic calcium events via mitochondria and increases in lactate release, which may have impact on synaptic transmission. An important contribution of the study is the molecular analysis of astrocytic mitochondria, which has revealed that astrocytes may use fuels other than glucose such as fatty acids to meet basic energy metabolic demands. Taken together, our results establish astrocytic CREB as a hub in astrocyte-plasticity and shed light on the interplay between plasticity and energy metabolism in astrocytes; these findings constitute a conceptual and mechanistic advance in the knowledge of astrocytic biology and how these cells may control learning and memory.

The regulation of calcium (Ca²⁺) was examined during long-term changes of neuronal excitability in the mammalian CNS. The preparations under investigation included the kindling model of epilepsy, a genetic form of epilepsy and long-term potentiation (LTP) of neuronal activity. The study also includes a discussion of the possible roles of a neuron-specific calcium-binding protein (CaBP). The findings are summarized as follows: 1) The distribution of CaBP was determined in cortical areas of the rat using a specific radioimmunoassay. The protein was found to have an unequal distribution in various cortical areas with preponderence in ventral structures. 2) Extending previous studies on the role of CaBP in kindling-induced epilepsy, its decline was correlated to the number of evoked afterdischarges (AD's) during the process of kindling. 3) Marked changes in CaBP levels were also found in the brains of the epileptic strain of mice (El). The hippocampal formation and the dorsal occipital cortex contained significantly lower CaBP than the control (CF-1) strain. The induction of seizures further decreased the levels of CaBP in the El mice. These findings are indicative of a possible genetic impairment of neuronal Ca²⁺ homeostasis in the El strain. 4) The levels of total hippocampal Ca²⁺ and Zn²⁺ were measured by atomic absorption spectrophotometry in control and commissural-kindled animals. While no change was found in the total Ca²⁺ content of the region, hippocampal Zn²⁺ of kindled preparations was found to be significantly elevated. 5) To measure Ca²⁺ -homeostasis, the kinetic analysis of ⁴⁵Ca uptake curves was undertaken in the in vitro hippocampus. This technique was found to be a valid method for assessment of Ca²⁺-regulation in the CNS under both physiological and pathophysiological conditions. The effect of various extracellular Ca²⁺ concentrations, 2,3-dinitrophenol (DNP), calcitonin, nifedipine and 3-isobutyl-1-methylxanthine (IBMX) on ⁴⁵Ca uptake curves was examined in order to identify the two exchangeable Ca²⁺ pools derived through kinetic analysis. 6) The kinetic analysis of ⁴⁵Ca uptake curves revealed that Ca²⁺-regulation of the hippocampus is impaired following amygdala- and commissural kindling. The changes reflect an enhancement of a Ca²⁺ pool that includes free cytosolic Ca²⁺ and a concomitant decrease in the amount of buffered calcium probably as a result in the decrease of hippocampal CaBP levels. 7) A novel form of long-term potentiation (LTP) of neuronal activity in the CA1 region of the hippocampus is described. Perfusion of 100 uM of IBMX in the hippocampal slice preparation induced a long lasting increase in the amplitude of the stratum radiatum evoked population spike and EPSP responses with changes in synaptic efficacy as indicated by the altered input/output relationships. Intracellular correlates of IBMX-induced LTP included lowering of synaptic threshold and enhancement of the rate of rise of the EPSP with no alterations in the passive membrane characteristics of CA1 pyramidal neurons. The fact that IBMX was able to exert its effect even in the presence of the calcium-blocker cation Co²⁺, taken together with the drug's action on hippocampal exchangeable Ca²⁺, raises the possibility that the Ca²⁺ necessary for induction of LTP may be derived from an intraneuronal storage site. These studies indicate the significance of intracellular Ca²⁺ -regulatory mechanisms in long-term changes of neuronal excitability which occur in experimental models of epilepsy and long-term potentiation.
Medicine, Faculty of
Cellular and Physiological Sciences, Department of
Graduate

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A potência e a eficácia das terapias antirretrovirais (TARV) aumentaram a expectativa de vida dos pacientes infectados pelo vírus da imunodeficiência humana. Entretanto, resultados de vários estudos sugerem que estas terapias também podem produzir modificações metabólicas, tais como as doenças ósseas. O objetivo do presente estudo foi avaliar a qualidade óssea e marcadores de formação e reabsorção óssea em pacientes infectados pelo HIV em uso ou não da TARV. Foi realizado um estudo transversal, com 50 homens adultos, em tratamento ou não com antirretrovirais. Foram aplicados métodos de avaliação do consumo alimentar, medidas antropométricas e avaliação da composição corporal e óssea utilizando a dual energy x-ray absorptiometry (DXA). Para a avaliação bioquímica foram utilizados os marcadores: hormônio do folículo estimulante (FSH), hormônio luteinizante (LH), paratormônio (PTH), testosterona, cálcio total, fósforo, magnésio, albumina, cálcio 24h, creatinina, uréia, fator 1 de crescimento semelhante a insulina (IGF-I), 25 hidroxivitamina D, osteocalcina e deoxipiridinolina urinária. Os resultados obtidos foram escritos na forma de artigo científico, sendo que o primeiro abordou a influência do tempo de uso da TARV na densidade mineral óssea e mostrou uma redução na massa óssea na maioria dos participantes avaliados e o consumo de cálcio foi adequado apenas no Grupo B1 em tratamento há mais de dois anos com antirretrovirais. O segundo artigo abordou o impacto da TARV nos marcadores do metabolismo ósseo e mineral e mostrou valor aumentado no marcador de reabsorção óssea em todos os grupos do estudo. Concluímos que a maioria dos participantes apresentou uma desmineralização óssea, principalmente com maior tempo de uso da TARV da classe de inibidores de transcriptase reversa...
The potency and efficacy of antiretroviral therapies (ART) have increased the life expectancy of HIV-infected patients. However, the results of various studies suggest that these therapies may also produce metabolic modifications such as bone diseases. The objective of the present study was to evaluate the bone quality and the markers of bone formation and reabsorption in HIV-seropositive patients using or not ART. A cross-sectional study was conducted on 50 adult men treated or not with ART. Methods for the evaluation of food consumption were applied, as well as anthropometric measurements and the evaluation of body and bone composition using dual energy x-ray absorptiometry (DXA). The following markers were used for biochemical evaluation: follicle stimulating hormone (FSH), luteinizing hormone (LH), parathormone (PTH), testosterone, total calcium, phosphorus, magnesium, albumin, 24h calcium, creatinine, urea, insulin-like growth factor 1 (IGF-I), 25 hydroxyvitamin D, osteocalcin, and urinary deoxypyridinoline. The results obtained were presented in the form of scientific articles. The first concerned the influence of the time of ART use on bone mineral density and showed a reduction of bone mass in most of the participants evaluated, with calcium consumption being adequate only in Group B1 under treatment with ARVs for more than two years. The second concerned the impact of ART on the markers of bone and mineral metabolism and showed an increase in the bone resorption marker in all study groups. We conclude that most of the volunteers presented bone demineralization, especially after a longer time of use of ART of the nucleoside reverse transcriptase inhibitor class. The increased bone remodeling indicates a greater... (Complete abstract click electronic access below)

Although the therapeutic benefits of calcium supplementation have yet to be fully determined, popular literature has promoted this practice to combat osteoporosis. This study examined the potential bioavailability of calcium from various supplement and antacid forms. Four dosage levels were studied for each of five sources, including leading supplements Caltrate and Os-Cal, two "calcium rich" antacids, Tums and Rolaids, and a calcium carbonate control. These samples were subjected to four gastric treatments in an in vitro digestion procedure designed to observe the influence of acid secretion on calcium solubility. Depending on the degree of acidity of these solutions, solubility behavior of CaCO3 was highly variable. Normal and reduced acid treatments adequately solubilized between 85 and 88% of the total calcium, whereas negligible quantities were dissolved in deionized water or the achlorhydric solution. Relative availability of calcium from the various sources differed according to gastric treatment. Tums, Caltrate and Os-Cal had dissolution rates that were similar to reagent grade CaCO3. Rolaids provided a significantly lower amount of soluble calcium under all of the experimental conditions (P < 0.001). Amounts of available calcium increased with increasing dosages. The effectiveness of fortification techniques used to increase the calcium content of various food systems was also examined. Fortified orange juice and yogurt samples were found to contain a quantity of available calcium comparable to that found in milk. It was concluded that both of these approaches are reasonable for increasing calcium intakes in individuals with normal gastric secretion.
Master of Science

Bien que le cerveau n'occupe que 2 % de la masse corporelle, il consomme 20 % de l'énergie du corps. De la mouche au mammifère, la transmission synaptique neuronale consomme la plus grande quantité d'énergie (Attwell & Laughlin, 2001). La transmission synaptique est un processus très plastique, sujet à des changements dans l'intensité et la synchronisation des tirs neuronaux. Par conséquent, pour répondre à la demande synaptique changeante, la production d'énergie doit également être flexible et coordonnée avec l'activité synaptique. Ce processus de production d'énergie à la demande a lieu principalement dans les mitochondries et est piloté par l'entrée de calcium dans les mitochondries. Dans les presynapses en activité, une partie du calcium entrant par les canaux calciques voltage gated est absorbée par les mitochondries, où il se lie aux enzymes sensibles au calcium, augmentant ainsi la production d'ATP. Lorsque l'entrée du calcium dans les mitochondries est bloquée par le knockdown de l'uniporteur de calcium mitochondrial (MCU), les mitochondries sont incapables de reconstituer l'énergie consommée pendant l'activité neuronale. Ce déficit énergétique bloque l'endocytose des vésicules synaptiques et entraîne par la suite une rupture de la neurotransmission. La MCU dans les neurones contient des sous-unités spécifiques qui augmentent l'importation du calcium mitochondrial (mCa), ce qui entraîne des augmentations substantielles du mCa axonal pendant l'activité (Ashrafi et al., 2020). Cependant, il a été démontré que des quantités excessives de mCa perturbent la respiration mitochondriale et conduisent finalement à la mort cellulaire apoptotique (Nicholls & Budd, 2000). Par conséquent, après une stimulation, il devient essentiel pour les neurones de restaurer rapidement les niveaux de calcium de la matrice mitochondriale afin d'assurer leur bon fonctionnement et leur survie. Cependant, contrairement à l'importation de mCa, le processus d'exportation de calcium dans les mitochondries neuronales reste mal compris. Au cours de mon doctorat, nous avons étudié les mécanismes et l'importance de l'exportation de mCa axonales. En combinant des outils génétiques et optiques avec une stimulation électrophysiologique dans des neurones hippocampiques de rat en culture, nous avons montré que la protéine Letm1 (Leucine Zipper and EF-Hand Containing Transmembrane Protein 1), située sur la membrane mitochondriale interne, régule l'exportation de calcium. L'inactivation de Letm1 entraîne une augmentation du temps de séjour du mCa, ce qui se traduit par une augmentation du métabolisme neuronal, une production excessive d'ATP dépendante de l'activité et l'accumulation d'ATP présynaptique dans les synapses en activité. En ce qui concerne son effet sur la fonction neuronale globale, nous avons constaté que la régulation du métabolisme neuronal par l'abaissement de Letm1 dans les neurones du corps pédonculés de la drosophile adulte améliore la formation de la mémoire à long terme. Au niveau synaptique, l'inactivation de Letm1 dans les neurones hippocampiques de rat en culture n'a pas modifié la fusion ou d'endocytose des vésicules, mais a étonnamment conduit à une libération excessive du neurotransmetteur glutamate. Des recherches plus approfondies sur ce mécanisme ont révélé que le calcium mitochondrial ne dirige pas seulement la production d'énergie dans les neurones, mais coordonne également la synthèse du neurotransmetteur glutamate en fonction de l'activité. Dans l'ensemble, nos résultats démontrent le rôle central de la gestion du calcium mitochondrial pour assurer le bon fonctionnement des neurones dans le cerveau. Ces résultats peuvent aider à comprendre comment une gestion anormale du calcium mitochondrial, signalée dans diverses maladies neurologiques telles que l'épilepsie, la maladie d'Alzheimer et la maladie de Parkinson, contribue à leur physiopathologie (Folbergrová & Kunz, 2012 ; Jung et al., 2020)
Even though the brain occupies only 2 percent of the body mass, it disproportionately uses a large amount of the body’s energy (up to 20 percent). From flies to mammals, neuronal synaptic transmission is thought to consume the most amount of energy (Attwell & Laughlin, 2001). Synaptic transmission is a highly plastic process, subject to changes in neuronal firing intensity and timing. Therefore, to meet the changing demand at synapses, energy production must also be flexible and coordinated with synaptic activity. In the pre-synapse, this process of on-demand energy production takes place mainly in the mitochondria and is driven by calcium entry into the mitochondria. In firing synapses, part of the calcium entering through the voltage-gated calcium channels is taken up by the mitochondria, where it binds to calcium-sensitive enzymes in the TCA cycle, upregulating ATP production. When calcium entry to the mitochondria is blocked through the knockdown of the Mitochondrial Calcium Uniporter (MCU), mitochondria are unable to replenish the energy consumed during neuronal activity. This energy deficit blocks the endocytosis of synaptic vesicles and subsequently leads to a failure in neurotransmission. Interestingly, MCU in neurons contains neuron-specific subunits that enhance mitochondrial calcium import, which results in substantially large increases in axonal mitochondrial calcium during activity (Ashrafi et al., 2020). Excessive amounts of mitochondrial calcium, however, have been shown to disrupt mitochondrial respiration and ultimately lead to apoptotic cell death (Nicholls & Budd, 2000). Therefore, after stimulation, it becomes critical for neurons to rapidly restore matrix calcium levels to ensure proper functioning and survival. However, in contrast to mitochondrial calcium import, the process of calcium export in neuronal mitochondria remains poorly understood. During my Ph.D., we investigated the mechanisms and importance of calcium export in axonal mitochondria. Combining genetic and optical tools with electrophysiological stimulation in cultured rat hippocampal neurons, we show that the protein Letm1 (Leucine Zipper and EF-Hand Containing Transmembrane Protein 1), located on the inner mitochondrial membrane, regulates calcium export. Knockdown of Letm1 leads to increased mitochondrial calcium dwell time, resulting in the upregulation of neuronal metabolism, excessive activity-dependent ATP production and the accumulation of presynaptic ATP in firing synapses. With respect to its effect on overall neuronal function, we found that upregulating neuronal metabolism through Letm1 knockdown in mushroom body neurons of adult Drosophila improves the formation of long-term memory. At the synaptic level, the knockdown of Letm1 in rat hippocampal neurons did not change the vesicle fusion or endocytosis rates but surprisingly lead to an excessive release of the neurotransmitter glutamate. Further investigations into this mechanism, revealed that mitochondrial calcium not only drives energy production in neurons but also coordinates activity-dependent synthesis of the neurotransmitter glutamate. Overall, our findings demonstrate the central role of mitochondrial calcium handling in ensuring proper neuronal function in the brain. These findings can help understand how abnormal mitochondrial calcium handling reported in various neurological diseases such as epilepsy, Alzheimer’s disease, and Parkinson’s disease contributes to their pathophysiology (Folbergrová & Kunz, 2012; Jung et al., 2020)

The purpose of this study was to evaluate the problem solving method applied to a self-instructional material in nutrition education. A comparative evaluation design was employed to determine the value of a problem solving model reflected in a commercially developed material, referred to as the 'Calcium Calculator'. Data were collected using a quasi-experimental randomized group pretest, posttest research design. A panel of judges then evaluated the impact of the problem solving method in nutrition education. Three research questions were generated for the purpose of this study. The first involved comparing impacts produced by the two forms of the 'Calcium Calculator'. Measures of impact, selected based on learner objectives of the 'Calcium Calculator', were learners': attitudes toward dietary calcium and osteoporosis; perception of problem solving ability and self-reported dietary calcium intake. The second research question was posed to investigate the nature of relationships between learners' levels of self-esteem and measures of instructional impact. Influences of selected biodemographic variables on change in the measures of impact were explored in the third research question. Eighteen groups of women (n=241) from community centres were randomly assigned to one of three intervention groups: Groups A and B were exposed to active problem solving methods while group C viewed a film, a passive information-oriented instructional technique. The latter group was included in.the study since active learning was hypothesized to result in greater impact than passive learning. Pretest data were collected using a self-administered questionnaire and food intake form. Posttest data were collected an average of 4.7 weeks later using a modification of the pretest questionnaire which included a self-esteem scale, along with the food intake form. Forty-four percent of women (n=l06) who completed pretest questionnaires returned for the posttest session. Participants in all three intervention groups experienced increases in attitude scores from pre- to posttest, and these changes were significant within groups B and C. Perceptions of problem solving ability were maintained within intervention groups B and C, yet decreased significantly within intervention group A. Significant increases in self-reported dietary calcium intakes occurred in all three intervention groups among non-pregnant women whose pretest dietary calcium intakes were below their Recommended Nutrient Intake. Impacts produced by form A and B of the 'Calcium Calculator' were significantly different on only one dependent variable: perception of problem solving ability (p≤0.05). Changes in the dependent variables produced by problem solving versus non-problem solving interventions were not significantly different. Changes in dietary calcium intake and attitude toward dietary calcium and osteoporosis were not significantly correlated with self-esteem levels. However, positive significant correlations were identified between learners' levels of self-esteem and change in learners' perceptions of their problem solving ability (p≤0.0l). Measures of impact were infrequently influenced by the biodemographic variables. Of the associations that were identified, most involved dietary characteristics of participants. Yet change in perception of problem solving ability was also affected by a combination of three demographic variables: age, employment status and education. A panel of users (n=9) of educational materials was asked to make judgements on selected study results. Although judges did not distinguish between impacts produced by the two problem solving materials, they acknowledged that: (1) an important relationship exists between self-esteem and learners' perceptions of their problem solving ability and (2) the problem solving method is valuable when directed to specific kinds of learners. The quasi-experimental research design used in this study appeared appropriate for the evaluation of innovative instructional methods. Two main advantages of the design were its comparative nature and its use of a panel of experts to judge the relative effectiveness of both forms of the 'Calcium Calculator' as well as the value of the problem solving method and self-esteem in material design.
Education, Faculty of
Curriculum and Pedagogy (EDCP), Department of
Graduate

Calcium status is a major factor in the regulation of reproductive activity in the hen. Restriction of dietary calcium (Ca) or vitamin D (D) is assumed to cause cessation of ovulation through decreased plasma calcium concentrations. Several studies suggest that there may be a threshold level of ionized calcium (Cai) below which ovulation will not proceed. The objectives of this thesis were to determine how Cai concentration is involved in the process of ovulation by comparing Ca and D-deficient hens, that had ceased laying, with control birds that were laying normally. A secondary objective was to determine the effects of multiple blood sampling (MBS) on the hen's ovulatory cycle. SCWL hens were divided into three groups-control, Ca-deficient and D-deficient groups and fed respective diets. Control birds were serially sampled every two hrs for 24-26 hrs immediately after oviposition until the next oviposition. Deficient birds, that had ceased laying for 10 to 14 days, were sampled at the same time. MBS was achieved with an indwelling vascular access port. Six birds/experimental group were used. Control birds were bled two weeks later from late afternoon until the following day at the same time. Whole blood was analyzed for Cai. Separated plasma was analyzed for total calcium (Cat), inorganic phosphorus (Pi), estrogen (E₂), progesterone (P₄) and l,25(OH)₂D₃ concentrations. Tibiae were ashed for mineral content. In expt. 1, the effect of MBS on the ovulatory pattern of hormones and ions was observed by sampling control birds twice, using two different time courses. Patterns and concentrations of the hormones and ions, regardless of time course, were similar to previous studies. Overall treatment effects were only significant between treatments with regards to total calcium and estradiol concentrations. The large loss of plasma proteins during the bleeding regime resulted in a steady decline in Cat over the 26 hrs, however, it was still within the physiological range of laying birds. . E₂ concentrations were also affected due to interruption of the laying sequence. However, this can be avoided since some birds continued to lay. In expt. 2, the control group had significantly higher mean plasma Cat and Pi concentrations and bone ash than both the deficient groups. Control and D-deficient groups had similar mean Cai concentrations, however, the ovulatory profile of the control group had a significant cyclic pattern over the 24-26 hrs, whereas, both deficient groups did not vary significantly over the 24 hrs. Plasma Pi concentration in the control group, not previously described, was cyclic in nature, related to the egg laying cycle. Plasma l,25(OH)₂D₃ concentrations were significantly higher in the Ca-deficient group than the control group. D-deficient birds had detectable levels of plasma 1,25(OH)₂D₃, but it was significantly lower than the control group. Plasma E₂ and P₄ concentrations were significantly higher in the control group In conclusion, it would appear that an inter-relationship exists among Cai, Pi and 1,25(0H)₂D₃ and the reproductive hormones. A threshold concentration of Cai may be the trigger for ovulation, perceived at the level of the pituitary, hypothalamus or ovary. A threshold of Pi and a window of l,25(OH)₂D₃ concentration may also have permissive roles in ovulation. In addition, MBS, regardless of time course, can be used as a method for determining ovulatory profiles in individual birds without seriously affecting ionic and hormonal concentrations and patterns.
Land and Food Systems, Faculty of
Graduate

Kyoto University (京都大学)
0048
新制・論文博士
博士(農学)
乙第12384号
論農博第2719号
新制||農||975(附属図書館)
学位論文||H21||N4493(農学部図書室)
27319
UT51-2009-K693
(主査)教授 松井 徹, 教授 久米 新一, 教授 廣岡 博之
学位規則第4条第2項該当

AMP-activated protein kinase (AMPK) is a major regulator of skeletal muscle metabolism with relevance to agriculture and human health. During the conversion of muscle to meat, the rate and extent of postmortem metabolism and pH decline largely determine pork quality development. Pigs with the AMPKγ3 R200Q mutation generate pork with low ultimate pH (pHu); this is attributed to high glycogen content, and greater â potentialâ to produce lactate and H+. We hypothesized that decreasing muscle phosphocreatine and creatine would decrease ATP buffering capacity, resulting in earlier termination of glycolysis and pH decline. Dietary supplementation with the creatine analogue, β-GPA, decreased muscle total creatine but negatively affected performance. Another experiment was conducted using control or β-GPA diet and wild type and AMPKγ3R200Q pigs in a 2à 2 factorial design. The loss of muscle total creatine was important in maintenance of ATP levels in AMPKγ3R200Q muscle early postmortem. Moreover, elevated glycogen did not affect pHu, supporting that energetic modifications induced by feed restriction and β-GPA supplementation influence extent of pH decline. Next, we utilized a line of pigs selected for differences in pHu. Another AMPKγ3 mutation (V199I), which is associated with higher pHu and lower glycolytic potential, was prevalent. The 199II genotype increased pHu in castrated males only. The wild type VV genotype increased glycolytic potential, but neither glycolytic potential nor lactate predicted pHu. In humans, AMPK activation is at least partly responsible for the beneficial effects of exercise on glucose transport and increased oxidative capacity in skeletal muscle. An inverse relationship exists between skeletal muscle fiber cross-sectional area and oxidative capacity, which suggests muscle fibers hypertrophy at the expense of oxidative capacity. Therefore, we utilized pigs possessing mutations associated with increased oxidative capacity (AMP-activated protein kinase, AMPKγ3R200Q) or fiber hypertrophy (ryanodine receptor 1, RyR1R615C) to determine if these events occur in parallel. RyR1R615C increased muscle fiber size; AMPKγ3R200Q increased oxidative capacity, evidenced by enhanced enzyme activity, mitochondrial function, and expression of mitochondrial proteins. Thus, pigs with both AMPKγ3R200Q and RyR1R615C possess increased fiber size and oxidative capacity, suggesting hypertrophy and oxidative capacity can occur simultaneously in skeletal muscle.
Ph. D.

The purpose of this investigation was to determine the relationship between calcium and glucose uptake following muscle contraction with the use of the calcium channel blocker dantrolene. In previous studies an exercise model has been used to investigate the role of calcium during post-exercise glucose uptake. This study utilized electrical stimulation. It has been shown that exercise-induced glucose uptake is calciummediated, but to date no one has shown that glucose transport induced by electrical stimulation is calcium-mediated. Twenty four male Sprague Dawley rats weighing 140 g were sacrificed and their epitrochlearis muscles were removed. Four treatment groups were established: control, muscle incubated in glucose (4mM); insulin, muscles incubated in glucose (4mM) and insulin (1000uU/ml); electrical stimulation, at 50 Hz for two five minute intervals separated by one minute rest periods; insulin (1000uU/ml) and electrical stimulation at 50 Hz for two five minute intervals separated by one minute intervals. Each group consisted of contain 8-10 muscle preparations. Glucose uptake was measured through the use of a double label of radioactive mannitol and 3-O-methylglucose and analyzed using liquid scintillation. This project followed a randomized group design. Treatments were measured with a one way ANOVA.
School of Physical Education

At fertilisation in mammals a series of Ca2+ oscillations are initiated that activate development. These Ca2+ oscillations cause the reduction of mitochondrial NAD+ and flavoproteins, suggesting that they might also stimulate changes in cytosolic ATP levels. Many events at fertilisation are triggered that require ATP; however, the changes in ATP during fertilisation are poorly defined. In this thesis intracellular C a2+ and ATP levels in individual m ouse eggs were m easured by monitoring the fluorescence of a C a 2+ dye (Oregon green bapta dextran) and lum inescence of firefly luciferase. During fertilisation of m ouse eggs it w as found that there are two phases of increase in ATP in both the cytosol and the mitochondria, during the series of sperm-induced Ca2+ oscillations. The increase in ATP is Ca2+ dependent since it did not occur when Ca2+ oscillations were prevented by BAPTA injection and, were abrogated by extracellular Ca2+ chelation. Additionally, it w as not seen when eggs were activated by cycloheximide, which does not cause a Ca2+ increase. The ATP increase is likely to be caused by oxidative phosphorylation by the mitochondria since the ATP levels in substrate free media are recovered by the addition of pyruvate. This recovery is blocked by the pyruvate uptake inhibitor ar-Cyano-4-hydroxycinnamic acid. T hese data suggest that mammalian fertilisation is associated with a sudden but transient increase in cytosolic ATP via oxidative phosphorylation, and that Ca2+ oscillations are both necessary and sufficient to cause this increase in ATP. Work in this thesis has also investigated the functionality of the sperm factor PLC?. Using luciferase tagged PLC constructs, the Ca2+ oscillation inducing ability of a series of PLC? truncated constructs, PLC5 and PLCy have been established. Results show that PLC? activation of m ouse eggs cannot be reproduced by other PLCs and that the C2, EF1 and catalytic site on the X domain are all essential for causing C a2+ oscillations.

Fatigue during exercise is associated with reduced muscle glycogen. However, evidence linking glycogen content to fatigue is lacking. In this study we examined whether reduced muscle glycogen content limited SR function or muscle performance. Two groups of female Sprague-Dawley rats were fasted for 24 hr and exercised for 90 min to reduce muscle glycogen; rats fasted after exercise formed the low glycogen (LG) group. Rats in the high glycogen (HG) group were allowed free access to food and a 5% sucrose solution. The LG group had 42% less muscle glycogen and 90% less glycogen associated with the sarcoplasmic reticulum (SR) than the HG group. Notably, time to exhaustion during a subsequent treadmill run (21 m/min at 10% grade) was markedly lower in the LG group (35 vs. 166.75 min). Despite less glycogen, the LG group had a higher SR Ca2+ uptake rate (45%) and Ca2+-stimulated ATPase activity (51%) possibly due to a 33% greater SERCA content. Surprisingly, in situ gastrocnemius initial twitch and tetanic forces were not different between groups although the rates of relaxation were higher in the LG group. The force responses to fatigue-inducing stimulus trains (20 Hz for 333 ms every 1 sec for 30 min) also were similar for both groups as were twitch and tetanic forces in the fatigued state. These results suggest that despite reduction in exercise performance, reduced muscle glycogen does not limit muscle performance or SR function.
Ph. D.

Orientador: Anderson Marliere Navarro
Banca: Alcyone Artioli Machado
Banca: Flavia Queiroga de Almeida
Resumo: A potência e a eficácia das terapias antirretrovirais (TARV) aumentaram a expectativa de vida dos pacientes infectados pelo vírus da imunodeficiência humana. Entretanto, resultados de vários estudos sugerem que estas terapias também podem produzir modificações metabólicas, tais como as doenças ósseas. O objetivo do presente estudo foi avaliar a qualidade óssea e marcadores de formação e reabsorção óssea em pacientes infectados pelo HIV em uso ou não da TARV. Foi realizado um estudo transversal, com 50 homens adultos, em tratamento ou não com antirretrovirais. Foram aplicados métodos de avaliação do consumo alimentar, medidas antropométricas e avaliação da composição corporal e óssea utilizando a dual energy x-ray absorptiometry (DXA). Para a avaliação bioquímica foram utilizados os marcadores: hormônio do folículo estimulante (FSH), hormônio luteinizante (LH), paratormônio (PTH), testosterona, cálcio total, fósforo, magnésio, albumina, cálcio 24h, creatinina, uréia, fator 1 de crescimento semelhante a insulina (IGF-I), 25 hidroxivitamina D, osteocalcina e deoxipiridinolina urinária. Os resultados obtidos foram escritos na forma de artigo científico, sendo que o primeiro abordou a influência do tempo de uso da TARV na densidade mineral óssea e mostrou uma redução na massa óssea na maioria dos participantes avaliados e o consumo de cálcio foi adequado apenas no Grupo B1 em tratamento há mais de dois anos com antirretrovirais. O segundo artigo abordou o impacto da TARV nos marcadores do metabolismo ósseo e mineral e mostrou valor aumentado no marcador de reabsorção óssea em todos os grupos do estudo. Concluímos que a maioria dos participantes apresentou uma desmineralização óssea, principalmente com maior tempo de uso da TARV da classe de inibidores de transcriptase reversa... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The potency and efficacy of antiretroviral therapies (ART) have increased the life expectancy of HIV-infected patients. However, the results of various studies suggest that these therapies may also produce metabolic modifications such as bone diseases. The objective of the present study was to evaluate the bone quality and the markers of bone formation and reabsorption in HIV-seropositive patients using or not ART. A cross-sectional study was conducted on 50 adult men treated or not with ART. Methods for the evaluation of food consumption were applied, as well as anthropometric measurements and the evaluation of body and bone composition using dual energy x-ray absorptiometry (DXA). The following markers were used for biochemical evaluation: follicle stimulating hormone (FSH), luteinizing hormone (LH), parathormone (PTH), testosterone, total calcium, phosphorus, magnesium, albumin, 24h calcium, creatinine, urea, insulin-like growth factor 1 (IGF-I), 25 hydroxyvitamin D, osteocalcin, and urinary deoxypyridinoline. The results obtained were presented in the form of scientific articles. The first concerned the influence of the time of ART use on bone mineral density and showed a reduction of bone mass in most of the participants evaluated, with calcium consumption being adequate only in Group B1 under treatment with ARVs for more than two years. The second concerned the impact of ART on the markers of bone and mineral metabolism and showed an increase in the bone resorption marker in all study groups. We conclude that most of the volunteers presented bone demineralization, especially after a longer time of use of ART of the nucleoside reverse transcriptase inhibitor class. The increased bone remodeling indicates a greater... (Complete abstract click electronic access below)
Mestre

The objective of this dissertation was to assess the dynamics of calcium (Ca) and phosphorus (P) metabolism in dairy cattle. Hypocalcemia, or a drop in blood Ca, is a common condition near parturition. All cows experience some degree of hypocalcemia. Maintenance of blood Ca within the acceptable range of 8 to 10 mg/dl is a balancing act between the demand for Ca for milk production and the cow's homeostatic mechanisms to maintain blood Ca. These homeostatic mechanisms include bone resorption that is driven by Ca demand however both Ca and P are released when bone is resorbed. These times of bone resorption and bone mineral replenishment have not been accounted for in current mineral recommendations. For the first study, it was postulated that dairy producers could administer 25-hydroxyvitamin D₃ (25-OH) in the prepartum period to prevent hypocalcemia. Twenty-seven multiparous Jersey cows were randomly assigned to receive an oral bolus containing corn starch (control, CON) or corn starch plus 15 mg of 25-hydroxyvitamin D₃ (25-OH) or 15 mg of vitamin D₃ (D₃) at 6 d prior to expected parturition. Jugular blood samples were collected at -14, -13, -5, -4, -3, -2, -1 d prior to expected calving, on the day of calving, and 1, 3, 5, 7, 9, 11, 13, 28, 56, and 84 d with respect to calving. Samples were analyzed for 25-OH, Ca, P, magnesium, osteocalcin (OC), and parathyroid hormone (PTH). Blood Ca, P, and Mg decreased near the time of calving and then increased over time. Serum 25-hydroxyvitamin D₃ was higher for cows dosed with 25-OH (119.0 pg/ml) compared with those dosed with D₃ (77.5 pg/ml) or CON (69.3 pg/ml). Cows dosed with 25-OH tended to have lower serum PTH concentration, but treatments did not affect serum Ca, P, or Mg. Serum OC was higher in second lactation cows compared with cows entering their third or fourth lactation but OC was unaffected by treatment. Although results indicated a 60% increase in serum 25-OH due to a single oral dose of 25-OH prior to calving, the amount administered in this study apparently was not sufficient for initiation of any improvement in Ca homeostasis at parturition. Due to the intimate relationship of Ca and P in bone, it was postulated for the second study that dietary Ca would affect bone mobilization and Ca and P balance in the lactating dairy cow. Eighteen Holstein cows were blocked by parity and calving date and randomly assigned to one of three dietary treatments: high (1.03%, HI), medium (0.78%, MED), or low (0.52%, LOW) dietary Ca. Dietary P was 0.34% in all diets. Total collection of milk, urine, and feces was conducted 2 wk prior to calving and in wk 2, 5, 8, 11, and 20 of lactation. Blood samples were collected at -14 and -10 d prior to calving and 0, 1, 3, 5, 10, 14, 21, 28, 35, 56, 70, 84, 98, and 140 d after calving. Blood samples were analyzed for Ca, P, PTH, OC, and deoxypyridinoline (DPD). Rib bone biopsies were conducted within 10 d of calving and during wk 11 and 20 of lactation. Dietary Ca concentration affected Ca balance, with cows consuming the HI Ca diet in positive Ca balance for all weeks with the exception of wk 11. Interestingly, all cows across all treatments had a negative Ca balance at wk 11, possibly the result of timed estrous synchronization that occurred during wk 11. At wk 20, Ca balances were 61.2, 29.9, and 8.1 g/d for the HI, MED, and LOW diets, respectively. Phosphorus balances across all treatments and weeks were negative. Dietary Ca concentration did not affect P balance in the weeks examined for this study but there was a clear effect of parity on balance, markers of bone metabolism, and bone P. Regardless of dietary treatment, serum OC concentration peaked around d 35 of lactation. Simultaneously, DPD concentration began to decrease, which may indicate a switch from net bone resorption to net bone formation after day 35. This was not reflected in balance measures however, this information may help refine dietary mineral recommendations for lactating dairy cows and ultimately reduce P excretion into the environment. Ultimately from the first study it is clear that oral dosing with 25-OH at 6 d prior to expected calving is not justified. However, we learned that parity has an effect on bone formation with younger animals resorbing and forming more bone and that net formation appears to occur after 30 days in milk. Both of these points were corroborated in the second study. Additionally, the second study demonstrated that dietary Ca content has no effect on P balance from 2 to 20 wk of lactation. Finally, the rib bone does not appear to be a sensitive indicator of bone metabolism or at least not at the time points we measured.
Ph. D.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive form of cancer with poor prognosis and limited treatment options. Since many patients present with metastatic disease and are thus ineligible for surgical resection, PDAC is almost ubiquitously fatal; new treatment options are therefore needed to combat this disease. A key hallmark of many cancers, including PDAC, is metabolic reprogramming and a shift towards a high glycolytic rate, known as the Warburg effect. This allows cancer cells to generate ATP in the face of hypoxia and to meet the increased metabolic requirements associated with rapid proliferation. We hypothesised that this shift towards glycolytic metabolism has important implications for the regulation of cytosolic Ca2+ ([Ca2+]i) in PDAC, since the plasma membrane Ca2+ ATPase (PMCA), which is critical for maintaining low [Ca2+]i and thus cell survival, is dependent on ATP to extrude cytosolic Ca2+. The relative contributions of mitochondrial vs glycolytic ATP in fuelling the PMCA in human PDAC cell lines (PANC-1 and MIA PaCa-2) were therefore assessed. Moreover, the effects of numerous mechanistically distinct metabolic inhibitors on key readouts of cell death, [Ca2+]i and ATP were investigated. Treatment with glycolytic inhibitors induced significant ATP depletion, PMCA inhibition, [Ca2+]i overload and cell death in both PANC-1 and MIA PaCa-2 cells, while mitochondrial inhibitors had no effect. Subsequently, these experiments were repeated on PDAC cells cultured in media formulated to "switch" their highly glycolytic phenotype back to one more reliant on mitochondrial metabolism. Culture in nominal glucose-free media supplemented with either galactose (10 mM) or alpha-ketoisocaproate (KIC, 2 mM) resulted in a switch in metabolism in MIA PaCa-2 cells, where proliferation rate and glycolysis were significantly decreased, and in the case of cells cultured in KIC, oxidative phosphorylation rate was preserved (assessed using Seahorse XF technology). Following culture of MIA PaCa-2 cells in either galactose or KIC, glycolytic inhibition failed to recapitulate the profound ATP depletion, PMCA inhibition and [Ca2+]i overload observed in glucose-cultured MIA PaCa-2 cells. These data demonstrate that in PDAC cells exhibiting a high rate of glycolysis, glycolytically-derived ATP is important for fuelling [Ca2+]i homeostasis and thus is critical for survival. Finally, using a cell surface biotinylation assay, the keyglycolytic enzymes LDHA, PFKP, GAPDH, PFKFB3 and PKM2 were all found to associate with the plasma membrane in MIA PaCa-2 cells, possibly in a tyrosine phosphorylation-dependent manner. To investigate whether the dynamic membrane-association of glycolytic enzymes provides a privileged supply of ATP to the PMCA in PDAC, the effects of tyrosine kinase inhibitors was assessed on PMCA activity. However, while these inhibited PMCA activity, this occurred without accompanying global ATP depletion. These data indicate that glycolytic ATP is critical for the regulation of [Ca2+]i by the PMCA in PDAC, and that the glycolytic regulation of the PMCA may be an important therapeutic locus. However, further research is required to determine whether membrane-bound glycolytic enzymes regulate its activity.

Calcium (Ca) intakes may affect cardiovascular disease risk by altering body weight/fat and serum lipid profile, but results have been inconsistent and the underlying mechanisms are not well understood. Thus, the effects of dietary Ca on body composition and lipid metabolism were examined in male Sprague-Dawley rats. Rats were fed high-fat, high-energy diets containing (g/kg) low (0.75Ca, 0.86 ± 0.05; 2Ca, 2.26 ± 0.02), normal (5Ca, 5.55 ± 0.08) or high (10Ca,11.03 ± 0.17; 20Ca, 21.79 ± 0.15) Ca for 10 weeks. At the end of the study the 0.75Ca group had lower (p < 0.05) body weight and fat mass compared to other groups. Rats fed the high Ca diets had lower serum total and LDL cholesterol compared to rats fed normal or low Ca. Liver total cholesterol was lower in rats fed high compared to low Ca. In general, liver mRNA expression of the LDLR and genes involved in cholesterol synthesis (HMGCR and HMGCS1), fatty acid oxidation (CPT2) and cholesterol esterification (ACAT2) were higher in rats fed higher Ca. Apparent digestibility of total trans, saturated, monounsaturated and polyunsaturated fatty acids was lower in rats fed the high compared to the low Ca diets, but the differences were greatest for trans and saturated fatty acids. Fecal excretion of cholesterol and total bile acids was highest in rats fed the 20Ca diet. The results suggest little effect of dietary Ca on body composition unless Ca intakes are very low. Decreased bile acid reabsorption and reduced absorption of neutral sterols and trans and saturated fatty acids may contribute to the improved serum lipid profile in rats fed higher Ca.

Experiments were conducted to investigate the effect of reducing dietary anion-cation balance (AC-balance), defined as meq $ sp circ$(Sodium + Potassium)-(Chloride + Sulfur) S, on calcium (Ca) metabolism. Reducing the dietary AC-balance from +314 to $-22$ (meq kg$ sp{-1}$ dry matter (DM)) did not affect plasma Ca level but reduced Ca retention mainly due to a 10-fold increase in urinary Ca excretion with similar rate of apparent absorption. It also decreased urine pH and titratable acidity. The response to induced hypocalcemia created by the infusion of 4.6% EDTA solution revealed that reducing dietary AC-balance from +354 to +37 (meq kg$ sp{-1}$DM) did not affect the volume of the compartment, within which there was rapid equilibration of free Ca but tended to increase the rate of Ca mobilization from it during the infusion. The Ca kinetic study with a four-compartment model indicated that reducing dietary AC-balance from +338 to $-127$ (meq kg$ sp{-1}$DM) during the eucalcemic period and from +429 to $-147$ (meq kg$ sp{-1}$DM) during an EGTA-infusion period (simulated lactational Ca loss) caused hypercalciuria and increased ionized form of plasma Ca. Increased true intestinal Ca absorption and reduced bone accretion were observed in the lowest AC-balance diet only during the EGTA-infusion period. There were no differences in the size of total exchangeable Ca pool but the amount of Ca movement between them tended to increase in reduced AC-balance diets during both periods. Results indicated that feeding reduced AC-balanced diet may have a beneficial role in preventing parturient paresis (milk fever) by increasing Ca flux through the exchangeable Ca pool and Ca mobilization capability.

The association of hyperparathyroidism with Paget’s disease, the changes in bone mineral density after parathyroidectomy, the surgical experience with a large number of patients with hyperparathyroidism, the findings of stable bone mineral density and renal function in hyperparathyroid patients during long term follow up, and, most importantly, in a large study continued over several decades, the reduced life expectancy of patients with primary hyrperparathyroidism are described. The effective use of salmon calcitonin in a large number of patients with Paget’s disease, the use of pentagastrin to stimulate calcitonin release in persons with suspected occult medullary thyroid carcinoma, the evaluation of the impact of quantified exercise on circulating calcitonin, the evaluation of the interaction of circulating calcitonin with osteoblastic and osteoclastic function in bone in patients with chronic renal failure are described. The usefulness of the measurement of bone mineral density in the diagnosis and follow up of patients with osteoporosis, the value of the use of intravenous pamidronate in the treatment of osteoporosis, the finding that bone mineral density is a predictor of mortality in patients with chronic renal failure, the finding that long term treatment of postmenopausal women with formononetin reduces serum LDL cholesterol but does not preserve bone mineral density, the finding of low bone mineral density in persons with chronic fatigue syndrome, the failure of supplemental vitamin D to preserve bone mineral density in post-menopausal women, the effect of inhaled steroids on the bone mineral density of children, the finding that the heterozygous C282Y HFE mutation in postmenopausal women is associated with low bone mineral density and that venesection in a premenopausal woman with haemochromatosis improved bone mineral density are described. The findings of a high serum vasopressin in persons with hypercalcaemia and a high serum 1,25-OH vitamin D in a person with T-cell lymphoma are described. An extensive study of the role of FGF-23 in the clinical manifestations of oncogenic osteomalacia, and a unique study of cell function in a patient with fibrogenesis imperfecta ossium are described. The first description of diabetes insipidus in a patient with hypoparathyroidism is presented.

The plasma membrane calcium-transport ATPase plays a major role in maintaining the low cytosolic calcium concentrations required for normal cellular function. Calcium, magnesium, calmodulin and lanthanum have been shown to alter the activity of the calcium-stimulated, magnesium-dependent ATPase activity in human erythrocytes. In an attempt to examine the reaction sequence of the (Ca²⁺ + Mg²⁺)-ATPase, the effects of these agents on the kinetics of calcium dependent phosphoprotein formation, the first step in the partial reaction sequence, were examined. Calmo-dulin-depleted erythrocyte membranes were prepared by hypotonic lysis in the presence of EDTA, according to the method of Carafoli et al (1980). Calcium-dependent formation of the phosphorylated intermediate was biphasic; the high calcium-affinity component was associated with low levels of E.Ca.P and a shallow response to changing calcium concentrations, whereas in the region of the low calcium-affinity component, E.Ca.P rose sharply in response to increasing calcium concentrations. The low affinity component of E.Ca.P lies in the range of calcium concentrations which inhibit (Ca²⁺ + Mg²⁺)-ATPase activity. When analyzed on LiDS acid PAGE, both components of calcium-dependent phosphoprotein formation were due to hydroxylamine-sensitive phosphorylation of a 135,000-145,000 dalton protein. Hence, the low calcium-affinity component of phosphoprotein formation and calcium-dependent inhibition of (Ca²⁺ + Mg²⁺)-ATPase activity were likely due to calcium-inhibition of dephosphorylation. Kinetic studies of calcium-dependent phosphoprotein formation, at two different calcium concentrations (1.0 μM, 0.4 mM), indicated that a steady-state was reached much sooner at higher calcium concentrations. Lanthanum, which is known to block dephosphorylation of the intermediate complex, increased both the apparent rate of formation and the steady-state level of the phosphorylated intermediate. Calmodulin, which has previously been shown to increase both the maximum velocity and the calcium affinity of the (Ca²⁺ + Mg²⁺)-ATPase, did not affect either calcium-dependent inhibition of (Ca²⁺ + Mg²⁺ )-ATPase activity or the biphasic nature of calcium-dependent phosphoprotein formation. At low calcium concentrations, calmodulin increased the apparent rate of phosphoprotein formation, whereas at higher calcium concentrations (0.4 mM) calmodulin reduced the steady-state level of the phosphoprotein; the apparent rate of formation was unaffected. In the presence of lanthanum, calmodulin increased both the apparent rate of formation and steady-state level of the phosphoprotein, suggesting that the true rate of formation was increased by calmodulin at higher calcium concentrations, but this was normally hidden by a simultaneous increase in the rate of dephosphorylation. Removal of endogenous magnesium, using trans-1,2-diamino-cyclohexane tetraacetic acid (CDTA) did not alter the calcium sensitivity or rate of formation of the phosphorylated intermediate, however turnover of the intermediate was markedly reduced. In the absence of free magnesium, both the velocity and calcium sensitivity of the (Ca²⁺ + Mg²⁺)-ATPase were also found to be lower. The low calcium-affinity component of calcium-dependent phosphoprotein formation, which Schatzmann (1982) has attributed to an action of calcium at a "magnesium-specific" site, was not affected by magnesium concentrations as high as 1 mM. Furthermore, this phosphoprotein could be dephosphorylated along either the forward or reverse pathways. These results indicate that the transformation from E₁.Ca.P to E₂.Ca.P may not be the site of the calcium-dependent inhibition of dephosphorylation. Calmodulin-depleted membrane fragments were prepared from the erythrocytes of cystic fibrosis patients as well as age- and sex-matched controls. Under conditions in which dephosphoryla-tion is inhibited, phosphoprotein formation and (Ca²⁺ + Mg²⁺)-ATPase activities were determined. Both (Ca²⁺ + Mg²⁺)-ATPase activity and phoshoprotein formation were found to be significantly reduced in the preparations derived from patients with cystic fibrosis. Turnover of the phosphorylated intermediate did not differ significantly between the two groups. A reduction in (Ca²⁺ + Mg²⁺)-ATPase activity and phosphoprotein formation suggests that there may be fewer active calcium-pumping sites in the erythrocyte membranes of cystic fibrosis patients compared to normal subjects.
Pharmaceutical Sciences, Faculty of
Graduate

A metabolic study was conducted with 23 men to determine the effects on plasma lipids and lipoproteins of a high calcium intake from two sources compared to a normal level of calcium intake over an eight week controlled feeding period. Three diet treatments were examined: 1) high dietary calcium mainly from dairy sources (1600-1800 mg/day), 2) high dietary calcium supplied by a CaCO₃ supplement (1600-1800 mg/day), and 3) normal dietary calcium intake from mixed sources (600-800 mg/day). Fat, carbohydrate and protein were provided in the ratio of percent kcal as 40:49:11. Nutrient, cholesterol levels (500 mg) and polyunsaturated to saturated (P/S) fatty acid ratio (0.446) were held constant for all diet treatments. There were no significant differences between treatments or across time in plasma TC, LDL, HDL or VLDL-cholesterol. Levels remained similar throughout the controlled diet treatment and the pre- and post-treatment periods. At the level of fat content and P/S ratio tested, there was no hypocholesterolemic effect of elevated calcium intake on plasma lipids.
Master of Science

Identified differences of calcium metabolism indicate signs of calcium malabsorption in DM patients and the processes of bone resorption intensification. This allows to conclude that calcium metabolism status (in particular, the severity of bone resorption) has significance at the development of diabetic hard tissue foot lesions as well as diabetic neuropathies and angiopathies. Insulin has a significant anabolic effect. It is known that a insulin production decreasing leads to the bone mineralization decreasing, reducing of the calcium blood serum level, increasing of the calcium urine secretion. The bone tissue trophics is broken – this is a result of the carbohydrate metabolism changes at cells and vascular lesions during insulin deficiency. Calcium metabolism and bone resorption changes defined at the study point to the necessity of the calcium drugs including to the complex therapy of DM.

Glucose is a primary and essential energy source for humans. It is broken down from complex carbohydrates in the diet and absorbed across the gut epithelium into the blood stream. Glucose homeostasis is important as hyperglycermia causes damage of pancreatic and peripheral cells. In response to a meal glucose is principally taken up by fat and muscle tissues and this response is activated by insulin release from pancreatic beta cells. Insulin stimulates the translocation of GLUT4 from the intracellular storage vesicles to the plasma membrane in fat and muscle cells. Although many proteins have been implicated in this process, the key insulin-regulated substrate has not been determined yet. In the present study, the phosphoserine/threonine binding protein 14-3-3 was used as a tool to affinity-purify insulin-stimulated phosphoproteins from 3T3-L1 adipocytes. By using mass spectrometry 38 proteins were identified, reflecting the important role of 14-3-3 in mediating many insulin-regulated processes. Among the potential phosphoproteins was Myosin 1C (Myo1c), an actin-associated molecular motor, which has previously been implicated in insulin-stimulated GLUT4 trafficking in adipocytes. I showed that insulin stimulates the activation of CaMKII which phosphorylates Myo1c at S701 in a Ca2+/PI3K-dependent manner. Myo1c phosphorylation induced its interaction with 14-3-3-proteins, reduced calmodulin-binding and stimulated its in vitro ATPase activity. Insulin-dependent stimulation of Myo1c phosphorylation and its ATPase activity were both required for GLUT4 translocation. By using yeast two-hybrid techniques, I identified a candidate ligand of the Myo1c tail, Armcx5, and demonstrated the in vivo interaction in 3T3-L1 adipocytes. The siRNA-mediated knockdown of Armcx5 inhibited insulin-stimulated glucose uptake and GLUT4 translocation. These results suggest that the regulation of Myo1c and its ligand Armcx5 are essential in insulin-regulated GLUT4 trafficking, possibly playing a key role in vesicle fusion.

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.

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A soja é uma leguminosa rica em proteínas, fonte de cálcio e isoflavonas. Um dos possíveis efeitos das isoflavonas é de reduzir a perda óssea quando há uma deficiência de estrogênio. A proposta desse estudo foi analisar a influência da dieta suplementada com extrato hidrossolúvel de soja (EHS) na massa óssea de ratos adultos jovens de ambos os sexos. Foram utilizados 40 ratos Novergicus Albinus linhagem Wistar adultos jovens e saudáveis, aleatoriamente separados em quatro grupos: macho controle (MC), macho suplementado (MS), fêmea controle (FC) e fêmea suplementada (FS) que permaneceram por 11 semanas em gaiola comum recebendo 500 mL de água e 300g de ração diariamente, os grupos suplementados (MS e FS), ao qual foi oferecido além de água e ração, 500 ml de EHS, durante o mesmo período experimental do controle. O consumo de ração e a ingestão de líquidos foram mensurados, bem como a massa dos animais. Ao final do período os animais foram eutanasiados. Os ossos foram submetidos à densitometria óssea– DXA (osso inteiro e região cortical) e ensaio mecânico, para avaliação da densidade mineral óssea - DMO (g/cm2), Força Máxima (N) e Rigidez (kN/m). Na análise de grau de mineralização foi utilizado as cinzas para quantificar de concentração de cálcio e fósforo. O consumo de EHS provocou diminuição da DMO no grupo MS, porém não houve alteração nas propriedades biomecânicas, força máxima e rigidez. A concentração de cálcio e fósforo nas cinzas dos fêmures do grupo MS foi significantemente menor que o grupo controle. Conclui-se assim, que o extrato hidrossolúvel de soja influenciou negativamente o conteúdo mineral ósseo de ratos machos jovens adultos diminuindo a mineralização, sem alterar as características mecânicas.
Soybeans are a rich legume protein, a source of calcium and isoflavones. One of the possible effects of isoflavones is to reduce bone loss when it was estrogen deficiency. The purpose of this study was to analyze the influence of diet supplemented with soybean’s hydro soluble extract (SHE) in bone mass in young adults rats of both genders. Were used 40 rats Novergicus Albinus Wistar young and healthy, randomly separated into four groups: male control (MC) and female control(FC), which remained for 11 weeks in common cage receiving 500 ml of water and 300 g of feed daily, and the others supplemented groups; male supplemented (MS) and female supplemented, that was offered in addition to water and feed 500ml of SHE. During the experimental period, feed intake and intake of liquids were measured as well as the mass of animals. At the end, the animals were euthanized. The bones underwent bone densitometry – DXA(whole bone and cortical area) and mechanical testing for bone mineral density - BMD (g / cm 2) Maximum Strength (N) and stiffness (kN / m), respectively. To analyze the mineralization degree was used bone ashes to quantify calcium and phosphorus concentration. The consumption of SHE caused decreased of BMD in MS group, but there were no changes in the bone biomechanical properties, maximum strength and rigidity. Concluding that the water extract of soy beans negatively affected the bone mineral content of young adult males rats decreasing the mineralization degree without changing the mechanical characteristics.

Orientador: Mario Jefferson Quirino Louzada
Banca: Paulo César Ciarlini
Banca: Cáris Maroni Nunes
Resumo: A soja é uma leguminosa rica em proteínas, fonte de cálcio e isoflavonas. Um dos possíveis efeitos das isoflavonas é de reduzir a perda óssea quando há uma deficiência de estrogênio. A proposta desse estudo foi analisar a influência da dieta suplementada com extrato hidrossolúvel de soja (EHS) na massa óssea de ratos adultos jovens de ambos os sexos. Foram utilizados 40 ratos Novergicus Albinus linhagem Wistar adultos jovens e saudáveis, aleatoriamente separados em quatro grupos: macho controle (MC), macho suplementado (MS), fêmea controle (FC) e fêmea suplementada (FS) que permaneceram por 11 semanas em gaiola comum recebendo 500 mL de água e 300g de ração diariamente, os grupos suplementados (MS e FS), ao qual foi oferecido além de água e ração, 500 ml de EHS, durante o mesmo período experimental do controle. O consumo de ração e a ingestão de líquidos foram mensurados, bem como a massa dos animais. Ao final do período os animais foram eutanasiados. Os ossos foram submetidos à densitometria óssea- DXA (osso inteiro e região cortical) e ensaio mecânico, para avaliação da densidade mineral óssea - DMO (g/cm2), Força Máxima (N) e Rigidez (kN/m). Na análise de grau de mineralização foi utilizado as cinzas para quantificar de concentração de cálcio e fósforo. O consumo de EHS provocou diminuição da DMO no grupo MS, porém não houve alteração nas propriedades biomecânicas, força máxima e rigidez. A concentração de cálcio e fósforo nas cinzas dos fêmures do grupo MS foi signific... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Soybeans are a rich legume protein, a source of calcium and isoflavones. One of the possible effects of isoflavones is to reduce bone loss when it was estrogen deficiency. The purpose of this study was to analyze the influence of diet supplemented with soybean's hydro soluble extract (SHE) in bone mass in young adults rats of both genders. Were used 40 rats Novergicus Albinus Wistar young and healthy, randomly separated into four groups: male control (MC) and female control(FC), which remained for 11 weeks in common cage receiving 500 ml of water and 300 g of feed daily, and the others supplemented groups; male supplemented (MS) and female supplemented, that was offered in addition to water and feed 500ml of SHE. During the experimental period, feed intake and intake of liquids were measured as well as the mass of animals. At the end, the animals were euthanized. The bones underwent bone densitometry - DXA(whole bone and cortical area) and mechanical testing for bone mineral density - BMD (g / cm 2) Maximum Strength (N) and stiffness (kN / m), respectively. To analyze the mineralization degree was used bone ashes to quantify calcium and phosphorus concentration. The consumption of SHE caused decreased of BMD in MS group, but there were no changes in the bone biomechanical properties, maximum strength and rigidity. Concluding that the water extract of soy beans negatively affected the bone mineral content of young adult males rats decreasing the mineralization degree without c... (Complete abstract click electronic access below)
Mestre

Le Cuivre (Cu) appartient aux métaux majeurs contaminant les écosystèmes dulcicoles européens. Les bivalves sont des filtreurs qui bioaccumulent fortement les métaux lourds comme le Cu, lequel, en excès cause de graves troubles métaboliques, et physiologiques. Le but de cette thèse est d'étudier les mécanismes de toxicité et de détoxication du cuivre chez Anodonta cygnea et Anodonta anatina, bivalves largement répandus dans les eaux continentales. L'effet du Cu a d'abord été étudié sur le transport cellulaire du calcium (Ca). Le Ca intervient dans de nombreux processus biologiques et le métabolisme calcique est connu pour être perturbé par le Cu chez les mollusques. L'inhibition par le Cu de l'activité de la Ca2+-ATPase de la membrane plasmique (PMCA) a été observée après 4 jours d'exposition. La récupération totale de l'activité enzymatique a été notée dans les reins et les branchies, après 7 jours d'exposition à concentration environnementale, mais pas à plus forte concentration. L'inhibition des PMCA pourrait entraîner des perturbations de l'osmorégulation et de la réabsorption rénale du calcium chez les moules et conduire à une carence. La récupération de l'activité enzymatique suggère l'induction de mécanismes de détoxications. Les composés riches en thiol, impliqués dans l'homéostasie et la détoxication des métaux ont donc été étudiés dans une seconde étape. Nos résultats montrent la présence d'un peptide riche en cystéine, différent du glutathion, et détecté dans la glande digestive et les branchies des bivalves exposés ou non au Cu. Ce peptide, augmente significativement dans les branchies, après exposition au Cu suggérant son implication dans sa détoxication
Copper (Cu) belongs to the major metals causing environmental contamination in European fresh water ecosystems. Filter feeding bivalves have a high bioaccumulation potential for heavy metals as Cu, which at high concentration can cause serious metabolic, physiological impairments. The objectives of this thesis are to acquire knowledge about toxic effects and detoxification mechanisms of Cu in Anodonta cygnea and Anodonta anatina, two mussels species widely distributed in continental waters. Because calcium (Ca) plays a fundamental role in numerous biological processes, Cu effect was studied first on cellular calcium transport which is known to be disturbed by Cu in molluscs. In a second step, investigations have been carried out on Cu detoxification mechanisms by thiol compounds known to be involved in homeostasis of essential trace metals and in metals cell detoxification. Cu inhibited plasma membrane Ca2+-ATPase (PMCA) activities upon 4 days of exposure was obtained. Total recovery was observed at day 7 in kidneys and gills under exposure to environmental Cu concentration but not at higher level of exposure. PMCA inhibition may entail disturbance of osmo-regulations and calcium renal re-uptake in mussels and lead to continuous under-supply of Ca. Recovery of the enzyme function suggests that metal-detoxification is induced. Our results showed the presence of a cysteine-rich peptide different from glutathione, detected in the digestive gland and the gills from controls and Cu exposed bivalves. This peptide, observed in absence of metal exposure in both organs, increased significantly in gills after Cu exposure suggesting its implication in Cu detoxification mechanisms

Includes bibliographical references (leaves 226-273.) Investigates the regulation of the expression of CYP27B1, CYP24 and vitamin D receptor (VDR) mRNA, both in the bone and in the kidney, with the aim to determine whether the regulation of the vitamin D metabolism in the bone is independent from that in the kidney. The effects of age, dietary calcium and vitamin D status on the expression of these genes in both the kidney and the bone, as well as on a number of biochemical factors known to regulate the renal metabolism of 1,25D, such as PTH, calcium and 1,25D itself, were examined. CYP27B1 mRNA expression was also studied in histological sections of rat femoral bone.

Calcium (Ca2+) plays an important role as a second messenger, transmitting the message of arrival of stimuli such as hormones and neurotransmitters to the intracellular system that carries out the cellular response to the stimulus. The universality of Ca2+ as an intracellular messenger depends on its enormous versatility. This versatility is exploited to control processes as diverse as fertilization, proliferation, development, learning and memory, contraction and secretion, and must be accomplished within the context of Ca2+ being highly toxic. Ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs) are Ca2+ -release channels located on intracellular membranes of the endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) that perform essential functions as key targets of hormone/neurotransmitter action to initiate intracellular Ca2+ signals. The purpose of this project was to study the role of RyR2 in Ca2+ signaling in the NG115-401L neuronal cell line. siRNA transfection methods were employed to knockdown RyR2 expression levels in NG115-401L cells. We used reverse transcription and real-time PCR to evaluate RyR2 gene expression in transfected/untransfected cells. We also evaluated cytosolic Ca2+ changes induced by RyR activators or regulators, using fura-2 AM as the Ca2+ indicator. Successful RyR2 gene knockdown allowed us to carry out some initial experiments to characterize the specific roles played by the RyR2 receptor isoform. We examined cell responses to FK-506 under the condition of RyR2 knockdown, finding that RyR2 appears to confer selectivity to this response. Finally, the effects of siRNA transfection and FK-506 treatment on NG115-401L cell growth were evaluated. These experimental results may contribute to future studies of RyR2, and help develop novel treatments for RyR2-base d dysfunctional diseases.