THE INTERACTION OF ANDROGENIC HORMONE AND CRANIOFACIAL VARIATION: RELATIONSHIP BETWEEN EPIGENETICS AND THE ENVIRONMENT ON THE GENOME WITH AN EYE TOWARD NON-SYNDROMIC

Recent research suggests that diversity in craniofacial morphology is produced by a complex interaction of environmental variables including 1) muscle function, 2) genetic factors related to skull growth, 3) timing and heritability of suture fusion (cessation of growth of the joints connecting the bones of the skull), 4) growth and morphology of the brain, and 5) other non-genetic factors including hormones of the endocrine system. How these factors interact in cranial growth and development is not well understood. This dissertation investigated the influence of androgenic hormone on suture bone biology. Methodology used including in vitro cellular challenges, protein analyses, and in vivo therapies. The work described here utilized a large sample size to establish the role of testosterone as a modulator of bone morphogenetic protein and subsequent effects on osteoblast differentiation. Testosterone increased the effect of BMP on osteoblasts, increasing differentiation. The increased differentiation effect was successfully blocked using flutamide, an androgen receptor blocker. Bone cells harvested from non suture calvaria in craniosynostotic rabbits were most susceptible to flutamide administration. The presence of androgen receptors in cells harvested from the suture and non suture bone of craniosynostotic or wild type rabbits could not be confirmed due to a lack of an effective antibody. In vivo administration of flutamide to the coronal suture of craniosynostotic rabbits resulted in greater growth across the coronal suture. However, no correction of craniofacial growth was observed. These results suggest 1) an alternative pathway for dihydrotestosterone's and testosterone's effect on the suture, similar to the adrenal androgens, via the MAP kinase pathway, 2) lack of an effective delivery system of the flutamide treatment, or 3) that an androgen receptor blocker-based therapy is not effective for delaying the eventual fusion of the coronal suture in this model

Molecular Analysis of Twist1 and FGF Receptors in a Rabbit Model of Craniosynostosis: Likely Exclusion as the Loci of Origin

Craniosynostosis is the premature fusion of the cranial vault sutures. We have previously described a colony of rabbits with a heritable pattern of nonsyndromic, coronal suture synostosis; however, the underlying genetic defect remains unknown. We now report a molecular analysis to determine if four genes implicated in human craniosynostosis, TWIST1 and fibroblast growth factor receptors 1-3 (FGFR1-3), could be the loci of the causative mutation in this unique rabbit model. Single nucleotide polymorphisms (SNPs) were identified within the Twist1, FGFR1, and FGFR2 genes, and the allelic patterns of these silent mutations were examined in 22 craniosynostotic rabbits. SNP analysis of the Twist1, FGFR1, and FGFR2 genes indicated that none were the locus of origin of the craniosynostotic phenotype. In addition, no structural mutations were identified by direct sequence analysis of Twist1 and FGFR3 cDNAs. These data indicate that the causative locus for heritable craniosynostosis in this rabbit model is not within the Twist1, FGFR1, and FGFR2 genes. Although a locus in intronic or flanking sequences of FGFR3 remains possible, no direct structural mutation was identified for FGFR3

Effects of Thyroxine Exposure on Osteogenesis in Mouse Calvarial Pre-Osteoblasts

The incidence of craniosynostosis is one in every 1,800-2500 births. The gene-environment model proposes that if a genetic predisposition is coupled with environmental exposures, the effects can be multiplicative resulting in severely abnormal phenotypes. At present, very little is known about the role of gene-environment interactions in modulating craniosynostosis phenotypes, but prior evidence suggests a role for endocrine factors. Here we provide a report of the effects of thyroid hormone exposure on murine calvaria cells. Murine derived calvaria cells were exposed to critical doses of pharmaceutical thyroxine and analyzed after 3 and 7 days of treatment. Endpoint assays were designed to determine the effects of the hormone exposure on markers of osteogenesis and included, proliferation assay, quantitative ALP activity assay, targeted qPCR for mRNA expression of Runx2, Alp, Ocn, and Twist1, genechip array for 28,853 targets, and targeted osteogenic microarray with qPCR confirmations. Exposure to thyroxine stimulated the cells to express ALP in a dose dependent manner. There were no patterns of difference observed for proliferation. Targeted RNA expression data confirmed expression increases for Alp and Ocn at 7 days in culture. The genechip array suggests substantive expression differences for 46 gene targets and the targeted osteogenesis microarray indicated 23 targets with substantive differences. 11 gene targets were chosen for qPCR confirmation because of their known association with bone or craniosynostosis (Col2a1, Dmp1, Fgf1, 2, Igf1, Mmp9, Phex, Tnf, Htra1, Por, and Dcn). We confirmed substantive increases in mRNA for Phex, FGF1, 2, Tnf, Dmp1, Htra1, Por, Igf1 and Mmp9, and substantive decreases for Dcn. It appears thyroid hormone may exert its effects through increasing osteogenesis. Targets isolated suggest a possible interaction for those gene products associated with calvarial suture growth and homeostasis as well as craniosynostosis. © 2013 Cray et al

Lawson criterion for ignition exceeded in an inertial fusion experiment

For more than half a century, researchers around the world have been engaged in attempts to achieve fusion ignition as a proof of principle of various fusion concepts. Following the Lawson criterion, an ignited plasma is one where the fusion heating power is high enough to overcome all the physical processes that cool the fusion plasma, creating a positive thermodynamic feedback loop with rapidly increasing temperature. In inertially confined fusion, ignition is a state where the fusion plasma can begin "burn propagation" into surrounding cold fuel, enabling the possibility of high energy gain. While "scientific breakeven" (i.e., unity target gain) has not yet been achieved (here target gain is 0.72, 1.37 MJ of fusion for 1.92 MJ of laser energy), this Letter reports the first controlled fusion experiment, using laser indirect drive, on the National Ignition Facility to produce capsule gain (here 5.8) and reach ignition by nine different formulations of the Lawson criterion

REFERENCE INTERVALS FOR ERYTHROCYTE SEDIMENTATION RATE, LACTATE, FIBRINOGEN, HEMATOLOGY, AND PLASMA PROTEIN ELECTROPHORESIS IN CLINICALLY HEALTHY CAPTIVE GOPHER TORTOISES ( GOPHERUS POLYPHEMUS)

Currently available tests for the diagnosis of inflammatory disease in reptiles are limited and poorly sensitive. However, a number of hematological and plasma biochemical analytes are validated in the diagnosis of inflammation in mammals. The objective of this study was to establish reference intervals for erythrocyte sedimentation rate, lactate, heat-precipitated fibrinogen, hematology, and plasma protein electrophoresis based on total protein by biuret method in 23 clinically healthy, captive gopher tortoises ( Gopherus polyphemus) after successful rehabilitation and to determine differences by age, sex, and season. In order to investigate biological differences, samples were collected in April, July, and November. There were no sex differences in any measured analyte; however, there were significant differences by age and season. Immature animals (<2 kg) had significantly higher total protein, albumin : globulin ratio, pre-albumin, albumin, and α-1 globulin than adults. Tortoises sampled in the spring season had significantly higher total solids (refractometer) and lower eosinophils compared with animals sampled in the summer. Further investigation is required to determine the clinical utility of these analytes in the diagnosis of inflammation in this species

DIAGNOSTIC PERFORMANCE OF INFLAMMATORY MARKERS IN GOPHER TORTOISES ( GOPHERUS POLYPHEMUS)

The lack of sensitive and specific markers of inflammation poses a diagnostic challenge in sick or injured reptile patients. The objective of this study was to investigate the diagnostic performance of blood analytes associated with inflammation in healthy ( n = 24) and sick ( n = 38) gopher tortoises ( Gopherus polyphemus). Receiver operating characteristic analysis identified the following as the best-performing diagnostic tests: erythrocyte sedimentation rate (area under the curve [AUC] = 0.812; 95% confidence interval [CI] = 0.693, 0.900), absolute mature heterophils (AUC = 0.771; 95% CI = 0.646, 0.869), total leukocytes (AUC = 0.767; 95% CI = 0.642, 0.866), lactate (AUC = 0.766; 95% CI = 0.641, 0.864), and absolute immature heterophils (AUC = 0.755; 95% CI = 0.628, 0.856). These results support the clinical application of additional tools for the diagnosis and monitoring of inflammatory disease in gopher tortoises. Clinicians may consider adding erythrocyte sedimentation rate and lactate to the minimum database for this species

Targeted Osteogenic MicroArray, Genes Dysregulated after Thyroxine Treatment.

<p>Targeted Osteogenic MicroArray, Genes Dysregulated after Thyroxine Treatment.</p

Gene-Chip MicroArray, Genes Dysregulated after 7 days of Thyroxine Treatment.

<p>Gene-Chip MicroArray, Genes Dysregulated after 7 days of Thyroxine Treatment.</p

<i>Proliferation</i>: Cell proliferation assay after thyroxine treatment.

<p>Reference line indicates control (untreated) response and experimental group plotted as percent response compared to reference (error bars = standard error of the mean). Note the increases in proliferation after 3 and 7 days of treatment with the exception of the highest dose after 7 days.</p

Quantitative Polymerase Chain Reaction Primer Data.

<p>Quantitative Polymerase Chain Reaction Primer Data.</p