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favorable since differentiation would utilize metabolic energy, which bears mustconserve during hibernation. In fact, although osteoblast activity remains balanced withosteoclast activity, activities of both cell types dramatically decrease during hibernation,as is seen in histomorphometric data [18, 20] and serum levels of BSALP (Table 5.3).Finally, regression analysis was used to determine what hormones may beresponsible for reduced osteoblast differentiation and caspase-3/7 activation duringhibernation. Serum levels of the neuropeptide NPY increase in the serum of hibernatingbears, and are inversely correlated with caspase-3/7 response to apoptotic threat inosteoblasts (Table 5.4). Although there are no known studies demonstrating arelationship between NPY and osteoblast caspase-3 and -7 response, NPY is known tosuppress osteoblast differentiation [246]. Furthermore, NPY is negatively correlated toserum BSALP, suggesting a possible relationship between NPY and bone turnover inthe bear. Another potential effector of differentiation is adiponectin, which decreases inthe serum of hibernating bears and is positively correlated to caspase-3/7 response toapoptotic threat in osteoblasts (Table 5.4). Again, although there are no known studiesdemonstrating a relationship between adiponectin and caspase-3 and -7 response,adiponectin has been shown to stimulate osteoblast proliferation, differentiation, andexpression of BSALP in cultured osteoblasts [202]. Adiponectin levels are positivelycorrelated to serum BSALP in the bears, suggesting that it, too, could be regulatingturnover during hibernation.Bears have a unique ability to maintain bone despite prolonged mechanicalunloading (i.e. hibernation). The aim of this study was to demonstrate that a factor in theserum of hibernating bears would protect cultured osteoblasts from apoptosis. Such aprevention of osteoblast and osteocyte apoptosis in the bones of hibernating bears mayhelp maintain bone during this period of disuse. Compared to cells cultured in activebear serum, osteoblasts cultured in serum from hibernating bears had reduced caspase-3 and -7 activity and a higher percentage of living cells after 6 hours of serum starvation.These findings suggest that the osteoblast response to apoptotic stimulus may bereduced during hibernation. This effect did not appear to be PKA dependent or stronglysensitive to pertussis toxin. Based upon regression analysis, two possible hormones areNPY and adiponectin. Both hormones have an impact on osteoblast differentiation,82
which is dependent upon caspase-3 activation. It is likely that more than one serumfactor is involved in the maintenance of bone mass during hibernation in bears; however,determining a few possible effector hormones could pinpoint new signaling pathways aspotential drug targets. This study has taken a step towards that long-term goal.5.5 Tables and figuresTable 5.1—Summary of assaysThe number of bears and assays that were performed in each hibernation season. Thenumber of bears that gave birth to cubs is in parentheses. Unknown values are indicated as“UK.”SeasonBearsAge(years)Assays2006‐2007 2 (1) UK Caspase‐3, 6day qPCR (OCN, Runx2)2007‐2008 4 (3) 5‐14 Caspase‐32008‐2009 5 (3) 2‐21SB206553 Caspase‐3, Caspase‐3,Pertussis Toxin Caspase‐3, H‐89Caspase‐3, 10kd and 30kd FilterCaspase‐3Data LocationFigure 5.2Tables 5.2, 5.3Figure 5.2Tables 5.2, 5.3Figures 5.3‐5.7Table 5.2, 5.3Table 5.2—Cell responses to bear serumData are represented as LSM+SE. The total number of samples included in the ANOVA isindicated below the means. Letters (A, B, C) represent values that are significantly differentfrom each other at α=0.05. The p-value reported is for the partial F-test for the serum factor.CellResponseFall(Active)Winter(Hibernation)Spring(Active)pCaspase-3/7OCNRunx2LSM+SE 5.66+0.23 B 3.82+0.20 C 7.89+0.41 AN samples 40 53 14
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EXPLORATION OF THE ROLE OF SERUM FA
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Table of contentsIndex of Figures .
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Chapter Four - Attempts to find an
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Index of figures1.1. Simplified sch
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6.17. Longitudinal analysis of OPG
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6.49. Longitudinal analysis of ColI
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A.2. Complete list of BSALP correla
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List of abbreviationsα-MEM. Alpha-
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PCR. Polymerase chain reactionPer1
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G2/M checkpoint. The point in the c
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Species names13-lined ground squirr
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Chapter One - Background and signif
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1.3 Bone turnover is unbalanced dur
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pro-apoptosis regulator BAX. An imp
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low-dose administration of parathyr
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decrease in bone trabecular thickne
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experience periodic arousals in whi
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ears must have evolved a mechanism
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1.7.6 Hypothesis 2: OCN interacts w
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ain, heart, and femoral muscle of h
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Hypothesis 3a: Serum concentrations
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were released. Behavior indicating
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2.3 ResultsSerum activity of the bo
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(p
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post-hibernation sampling in the be
Page 53 and 54: also unclear whether ucOCN may affe
Page 55 and 56: atios of intact to fragmented OCN m
Page 57 and 58: Table 2.3—Bone marker “seasonal
Page 59 and 60: Table 2.7—Chemistry panel “seas
Page 61 and 62: ABNormalized BSALPBSALP (U/L)CO N D
Page 63 and 64: Total Calcium (mg/dL)A1.21.151.11.0
Page 65 and 66: ABTotal OCN (ng/mL)150100500O N D J
Page 67 and 68: Adiponectin (ng/mL)AB40003500300025
Page 69 and 70: ABNormalized InsulinInsulin (μU/mL
Page 71 and 72: surrounding and encompassing hibern
Page 73 and 74: decreased from 788+30 ng/mL in preh
Page 75 and 76: 120-122]. Most small hibernators ar
Page 77 and 78: ears. Similarly, serum NPY increase
Page 79 and 80: Table 3.2—Serum factor longitudin
Page 81 and 82: Leptin (ng/mL)ABO N D J F M A M O N
Page 83 and 84: Norepinephrine (ng/mL)A807060504030
Page 85 and 86: IGF‐1 (ng/mL)AN D J F M A MB10009
Page 87 and 88: 2008. Samples were collected as des
Page 89 and 90: sampling points. It is possible to
Page 91 and 92: C‐Terminal PTH (Relative)AB504030
Page 93 and 94: Chapter Five — Serum from hiberna
Page 95 and 96: Synergy HT Multi-Detection Micropla
Page 97 and 98: gene specific primers and 12.5 μL
Page 99 and 100: involved in the reduced caspase-3/7
Page 101 and 102: hibernation cycles without problem
Page 103: filtered seasonal bear serum and fo
Page 107 and 108: Caspase‐3/7 ActivityO N D J F M A
Page 109 and 110: 6Caspase‐3/7 Activity54321**0Vehi
Page 111 and 112: ABCaspase‐3/7 Activity9876543210*
Page 113 and 114: eyond the G1/S checkpoint, thus ind
Page 115 and 116: ears, it is possible that tissue se
Page 117 and 118: PTH1R, RANKL, Runx2, Smurf1, TLR4,
Page 119 and 120: factors Runx2 and OCN also increase
Page 121 and 122: Table 6.3—Gene expression 3 hour
Page 123 and 124: Table 6.5—Gene expression 6 day d
Page 125 and 126: Cyclin D1 Gene ExpressionO N D J F
Page 127 and 128: Smurf1 Gene ExpressionO N D J F M A
Page 129 and 130: BAK Gene ExpressionO N D J F M A MM
Page 131 and 132: M‐CSF Gene ExpressionO N D J F M
Page 133 and 134: Per1 Gene ExpressionO N D J F M A M
Page 135 and 136: Akt Gene ExpressionO N D J F M A MM
Page 137 and 138: Bcl‐2 Gene ExpressionO N D J F M
Page 139 and 140: Cyclin D1 Gene ExpressionO N D J F
Page 141 and 142: OPN Gene ExpressionO N D J F M A MM
Page 143 and 144: 2PTH1R Gene Expression1.510.50O N D
Page 145 and 146: TLR4 Gene ExpressionO N D J F M A M
Page 147 and 148: Bcl‐2 Gene ExpressionO N D J F M
Page 149 and 150: OCN Gene ExpressionO N D J F M A MM
Page 151 and 152: PTH1R Gene ExpressionO N D J F M A
Page 153 and 154: TLR4 Gene ExpressionO N D J F M A M
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condition in renal patients in whic
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hibernation. This report brings to
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14. Kaneps, A.J., S.M. Stover, and
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41. Chowdhury, I., B. Tharakan, and
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68. Ashe, M.C., et al., Bone geomet
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95. Perrien, D.S., et al., Aging al
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122. Lesser, R.W., et al., Renal re
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151. Confavreux, C.B., R.L. Levine,
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178. Toribara, T.Y., A.R. Terepka,
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206. Luo, X.H., et al., Adiponectin
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233. Florant, G.L., et al., Fat-cel
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259. Bhasin, S., et al., Older men
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284. Miura, M., et al., A crucial r
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Appendix A—Complete tables of cor
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Table A.5—Complete list of ionize
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Table A.7—Complete list of adipon
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Table A.12—Complete list of norep
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