Neurodegeneration in Alzheimer's disease is characterised by multiple pathologies including disrupted calcium homeostasis and elevated Zn2+ levels. Calbindin D28k (CB-D28k), which buffers Ca2+ and can bind Zn2+, was suspected to be involved in these abnormalities. The PDB structure of this EF-hand protein shows that not all hands are well formed. Docking and molecular dynamics calculations were employed to achieve the two goals in this project. The first goal was to get a better structure of CB-D28k to improve our understanding of its behavior. Calculations improved the structure protein: helix-loop-helix sequences were formed in all hands and most canonical interactions were formed in the four functional hands. The second goal was to test the Ca2+ binding capacity of Zn2+-bound CB-D28k. Analysis of calculated structures showed that the Ca2+ binding capability of Zn2+ bound protein was significantly compromised, permitting only half of the correct canonical interactions with the loop residues.

Neurodegeneration in Alzheimer's disease is characterised by multiple pathologies including disrupted calcium homeostasis and elevated Zn2+ levels. Calbindin D28k (CB-D28k), which buffers Ca2+ and can bind Zn2+, was suspected to be involved in these abnormalities. The PDB structure of this EF-hand protein shows that not all hands are well formed. Docking and molecular dynamics calculations were employed to achieve the two goals in this project. The first goal was to get a better structure of CB-D28k to improve our understanding of its behavior. Calculations improved the structure protein: helix-loop-helix sequences were formed in all hands and most canonical interactions were formed in the four functional hands. The second goal was to test the Ca2+ binding capacity of Zn2+-bound CB-D28k. Analysis of calculated structures showed that the Ca2+ binding capability of Zn2+ bound protein was significantly compromised, permitting only half of the correct canonical interactions with the loop residues.

Since the development of knockout and transgenic mouse models, mice have become the most widely used mammalian animal model to study bone. Despite the advances in knowledge of bone biology and function that have occurred from use of mouse models, many studies use primarily qualitative techniques, which may result in overlooking important subtle pathophysiologic changes. The hypothesis of this dissertation is that quantitative techniques to measure bone structure and function could identify the physiologic role of carbonic anhydrase II and calbindin-D28k in mouse bone, despite earlier qualitative studies indicating mice without these proteins have normal bone structure and function. Furthermore, a method to quantify bone function in vivo will be tested in a mouse model.Although carbonic anhydrase II deficient mice are less severely affected than patients, the mice demonstrate features of osteopetrosis including metaphyseal widening and a 50% increase in trabecular bone volume. The mice partially compensate for inhibited osteoclast function by increasing osteoclast number.Calbindin-D28k knockout mice demonstrated an increase in bone volume that results from additional bone at the endosteal surfaces. The higher bone volume results in increased stiffness and failure loads, highlighting the potential use of drugs that inhibit calbindin-D28k to treat diseases such as osteoporosis.Finally, calcium phosphate ceramic and hydroxyapatite particles used as strain gauge coatings demonstrated bone bonding to mouse femora after two months in vivo. The use of hydroxyapatite particles to coat strain gauges is the first time this method has been used with all commercially available materials, and will allow other research groups to use this technique. The major limitation to in vivo bone strain measurement in mice is the relatively large size of the sensors, which resulted in increased second moments of inertia in the implanted bones.Overall, this dissertation demonstrates that the use of quantitative techniques, including histology, histomorphometry, µCT imaging, and mechanical testing can measure subtle changes in bone properties that have been previously overlooked. Development of additional quantitative methods to study bone biomechanics in mouse models may encourage other research groups to quantify bone properties if no changes are noted using primarily qualitative methods.

Calbindin-D28K is a cytosolic calcium binding protein found in a variety of cells that transport calcium. The chorioallantoic membrane and yolk sac of oviparous squamate reptiles (lizards and snakes) transport calcium from the eggshell and yolk to the developing embryo. I used immunohistochemistry to localize calbindin-D28K expression in the chorioallantoic membrane and yolk sac of two species of oviparous scincid lizards, Plestiodon fasciatus and Saproscincus mustelinus. Calbindin-D28K was detected in the chorioallantoic membrane and yolk sac of both lizard species by a polyclonal anti-snake calbindin antibody and a monoclonal anti-cow calbindin antibody. Calbindin-D28K was localized in the chorionic epithelium and allantoic epithelium of the chorioallantoic membrane and in endodermal cells scattered throughout the yolk mass of both species. This is the first demonstration of calbindin-D28K in allantoic epithelium and in endodermal cells of the yolk sac of lizards.

One hypothesis explaining the numerous independent evolutionary transitions from oviparity to viviparity among squamates (snakes and lizards) proposed that squamate embryonic development is independent of eggshell calcium. Recent research showed at least 25% of the calcium in hatchling oviparous squamates is extracted from the shell. Though not a direct test, these results are inconsistent with the hypothesis. To directly test the hypothesis, we removed eggshell calcium (through peeling) early in development of Pantherophis guttatus (corn snake) eggs. Survivorship to hatching did not differ between peeled and intact eggs. Yet hatchlings from peeled eggs were shorter (273.6 ± 3.4 vs. 261.0 ± 3.7 mm, p=0.0028, n=16), lighter (6.36 ±0.22 vs. 5.75 ± 0.23 g, p=0.0158, n=16), and had reduced calcium (40.8 ± 1.7 vs. 30.5 ± 1.8 mg, p