C - Michigan Technological University

PTH1R, RANKL, Runx2, Smurf1, TLR4, and ubiquitin. Cyclophilin was used as thereference gene.All statistical analyses were performed using JMP 7 software. Data werepresented as the LSM + SE. The units were relative since all samples were normalizedto reference genes and to a reference (FBS) sample. All available samples from allbears were grouped by season (i.e. prehibernation, hibernation, and post-hibernation)and analyzed with a single-factor ANOVA, blocking by bear, with a Tukey’s post-hoc.Graphs are represented with a point for each serum sample, and a smoothed averagecurve generated with JMP software by a cubic spline.6.3 ResultsIn the first condition (undifferentiated pre-osteoblasts stimulated for 3 hours inseasonal bear serum), there were no seasonal trends (Table 6.2). In the second study(differentiated osteoblasts stimulated for 3 hours in seasonal bear serum), ubiquitinexpression increased from 0.998+0.058 in osteoblasts cultured in prehibernation serumto 1.32+0.10 in osteoblasts cultured in post-hibernation serum (p=0.0341). Expression ofubiquitin in osteoblasts cultured in hibernation serum was not different from either activeseason (Table 6.3). In the third study (undifferentiated pre-osteoblasts stimulated for 24hours in seasonal bear serum) (Table 6.4), Akt expression increased from 0.796+0.102in cells cultured in hibernating bear serum to 1.55+0.182 in cells cultured in posthibernationserum; there was no significant difference in Akt gene expression in cellscultured in prehibernation bear serum compared to hibernation serum (p=0.0046).Cyclin D1 gene expression increased from 0.950+0.032 in cells cultured in hibernationserum to 1.12+0.05 in cells cultured in post-hibernation serum; Cyclin D1 expressionwas not different in cells cultured in prehibernation serum compared to those inhibernation nor post-hibernation serum (p=0.0177). In the fourth condition (differentiatedin bear serum for 6 days) (Table 6.5), expression of the markers for osteoblastdifferentiation Runx2 and OCN were upregulated in post-hibernation compared toprehibernation and hibernation seasons. Runx2 increased from 0.741+0.069 in cellscultured in hibernation serum to 1.13+0.12 in cells cultured in post-hibernation serum;Runx2 gene expression did not change in cells cultured in prehibernation serum95