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Most studies of insect Malpighian tubules (MTs) have examined transport of monovalent ions (K^+, Na^+, Cl^-). Isolated Drosophila melanogaster MTs also transport Ca^2+ from bath to lumen and transport is stimulated by cAMP. The lower segment of the MTs transports Ca^2+ at a higher rate per unit length than does the main segment known to produce the primary urine. This study examines both whole animal calcium regulation in larvae, pupae and adults and the mechanisms of Ca^2+ transport by isolated MTs. Drosophila melanogaster appears to regulate its calcium content and haemolymph calcium level. Calcium content of the whole fly only increased 10% with a 6.2-fold increase in dietary calcium. Anterior MTs can contain as much as 50% of the whole animal calcium content. The difference in MTs accumulation is due primarily to the enlarged initial segment of the anterior MTs. This segment, absent from the posterior MT, contains calcium-containing concretions. Whole fly calcium content does not increase continuously with the age implying that calcium is eventually being excreted. Haemolymph calcium concentrations do not change in response to changes in dietary calcium, suggesting that calcium concentration is regulated either by the rate of absorption or by the rate of excretion. The midgut and the enlarged initial segment of the anterior MTs may play important roles in haemolymph calcium regulation. Isolated MTs show sensitivity to both Ca^2+ channel blockers and Ca^2+ -ATPAse inhibitors on the basolateral and apical membranes respectively. Voltage-gated calcium channels appear to mediate calcium movement from bath to cell. A ruthenium red sensitive Ca^2+ -ATPAse may be used to transport calcium against the electrochemical gradient from cell to lumen. Lastly, the dissolution of luminal concretions plays a large role in net calcium secretion. / Thesis / Master of Science (MSc)

In the absence of X-ray structure of L-type Ca²⁺ channel (LCC), we have built a homology model of LCC based on the crystal structure of KcsA channel and have performed a series 0° docking simulations. The search for lowest-energy conformations was performed by the Monte Carlo energy-minimization method. To obtain the conformation with the lowest energy where dihydropyridine (DHP) ligand forms optimal contacts with the DHP-sensing residues of the channel, we have tested different sequence alignments between KcsA and LCC, and have docked the ligand inside the pore of the channel as well as into the interface between repeats IIIS5-IIIS6-IVS6. The LCC ligand tetrandrine was used during the studies of the selectivity filter of the channel. Conformational studies of the drug and its interaction with Ca²⁺ ions in a non-polar solution were performed by NMR spectroscopy. These experiments have demonstrated the binding of Ca²⁺ ions to the ligand. In the model based on the alignment proposed by Lipkind and Fozzard (2000), the DHP ligand nifedipine fits inside the pore and forms favorable contacts with several hydrophobic DHP-sensing residues, and forms hydrogen bonds with conserved tyrosines in repeats HI and IV. These interactions stabilize the portside-down docking mode of nifedipine, in which this blocker exposes its hydrophobic methoxy group to the bracelet of hydrophobic residues forming the gate of the channel near the crossing of the bundle of helices. The stabilizes the closed state of the channel. In contrast, the agonist has the hydrophilic group at its portside. The favorable interaction of this group with hydrated Ca²⁺ ion facilitates its permeation through hydrophobic gate. We have simulated the passage of the hydrated ion along the pore with the agonist bound inside and determined several residues crucial for this passage. The role of these residues can be tested experimentally. / Thesis / Master of Science (MSc)

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.

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.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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4 pp. / Healthy bone growth and maintenance requires adequate calcium intake. You can meet your calcium needs from foods, beverages, and if necessary, supplements. This publication contains the calorie and calcium content of some foods from each group of the Food Guide Pyramid which includes bread, cereal, rice, & pasta group; vegetable group; fruit group; milk, yogurt, & cheese group; meat, poultry, fish, dry beans, eggs, & nuts group; and fats, oils & sweets.