The present research examined the effects of compound bows and crossbows on the remains of Sus scrofa and Odocoileus virginianus. Isolated pig heads and white-tailed deer necks were impacted by three forms of arrow heads: the broad-head tip, conical field-tip, and bullet field-tip from both the compound bow and the crossbow. The structural design of the arrowheads was examined to understand their level of impact, as well as, the velocities of the compound bow and crossbow were calculated and compared. The total number of impact marks for the experiment was 55. It was hypothesized that the compound bow would have a greater extent of trauma to bone than the crossbow due to the higher velocity created from a longer power stroke. It was also hypothesized that the broad-head arrow tip will create larger fracture patterns on bone due to the three-blade-prong design compared to the oval shape of both the conical field-tip and bullet field-tip. Through the use of one-way ANOVA and Pearson’s Chi-Square, the results show no direct correlation between the difference in the type of weapon used or the arrow tip used. The results show the vast majority of impacts are penetration with shapes that roughly resemble the cross-section of the type of tip used. The results, however, did not support both hypotheses due to the limited number of impact marks and sample size of the experiment. Further experiments are required to assess the extent to which it is possible to distinguish between arrow related osseous trauma.

Mitochondrial dysfunction is a common feature in a wide range of disorders and diseases from obesity, diabetes, cancer to cardiovascular diseases. The overall goal of my doctoral research has been to investigate mitochondrial metabolic dysfunction in skeletal and cardiac muscles in the context of chronic disease development. Perinatal nutrition is well known to affect risk for insulin resistance, obesity, and cardiovascular disease during adulthood. The underlying mechanisms however, are poorly understood. Previous research from our lab showed that the in utero maternal undernutrition mouse model is one in which skeletal and cardiac muscle physiology and metabolism is impaired. Here we used this model to study the impact of in utero undernutrition on offspring skeletal primary muscle cells and to determine if there is a cell autonomous phenotype. Metabolic analyses using extracellular flux technologies revealed a shift from oxidative to glycolytic metabolism in primary myotubes. Gene expression profiling identified significant changes in mRNA expression, including an upregulation of cell stress and OXPHOS genes and a downregulation of cell division genes. However, there were no changes in levels of marker proteins for mitochondrial oxidative phosphorylation (OXPHOS). Findings are consistent with the conclusion that susceptibility to metabolic disease in adulthood can be caused at least in part by muscle defects that are programmed in utero and mediated by impaired mitochondrial function. In my second project, the effects of the absence of glutaredoxin-2 (Grx2) on redox homeostasis and on mitochondrial dynamics and energetics in cardiac muscle from mice were investigated. Previous work in our lab established that Grx2-deficient mice exhibit fibrotic cardiac hypertrophy, and hypertension, and that complex I of OXPHOS is defective in isolated mitochondria. Here we studied the role of Grx2 in the control of mitochondrial structure and function in intact cells and tissue, as well as the role of GRX2 in human heart disease. We demonstrated that the absence of Grx2 impacts mitochondrial fusion, ultrastructure and energetics in mouse primary cardiomyocytes and cardiac tissue and that provision of the glutathione precursor, N-acetylcysteine (NAC) did not restore glutathione redox or prevent impairments. Furthermore we used data from the human Genotype-Tissue Expression consortium to show that low GRX2 expression is associated with increased fibrosis, hypertrophy, and infarct in the left ventricle. Altogether, our results indicate that GRX2 plays a major role in cardiac mitochondrial structure and function, and protects against left ventricle pathologies in humans. In my third project, we collaborated with cardiac surgeon, Dr. Calum Redpath, of the Ottawa Heart Institute to study atrial mitochondrial metabolism in atrial fibrillation patients with and without type 2 diabetes (T2DM). T2DM is a major risk factor for atrial fibrillation, but the causes are poorly understood. Atrial appendages from coronary artery bypass graft surgery were collected and analyzed. We showed an impaired complex I respiration in diabetic patients with atrial fibrillation compared to diabetic patients without atrial fibrillation. In addition, and for the first time in atrial fibrillation patients, mitochondrial supercomplexes were studied; results showed no differences in the assembly of the “traditional” complexes but a decrease in the formation of “high oligomeric” complexes. A strong trend for increased protein oxidation was also observed. There were no changes in markers for OXPHOS protein levels. Overall findings reveal novel aspects of mitochondrial dysfunction in atrial fibrillation and diabetes in humans. Overall, our results reveal that in utero undernutrition affects the programming of skeletal muscle primary cells, thereby increasing susceptibility to metabolic diseases. In addition, we show that GRX2 impacts cardiac mitochondrial dynamics and energetics in both mice and humans. Finally, we show impaired mitochondrial function and supercomplex assembly in humans with atrial fibrillation and T2DM. Ultimately, understanding the mechanisms causing mitochondrial dysfunction in muscle tissues during chronic disease development will increase our capacity to identify effective prevention and treatment strategies.

Bilateral mandibular ostectomies were performed between premolars 3 and 4 in 10 adult canine specimens. A type I external fixator incorporating a full interdental pin was placed stabilizing a 0.5 cm fracture gap. Four different pin configurations were tested in dorsoventral bending five separate times on each of the ten mandibles: 1) intact mandibular bodies with fixator; 2) ostectomized mandibular bodies and complete fixator; 3) ostectomized mandibular bodies with the caudal pins of the rostral fragment cut; 4) ostectomized mandibular bodies with all pins of the rostral fragment cut. The full interdental pin remained intact in all configurations. Total stiffness and gap stiffness were then determined for each fixation geometry on a materials testing machine. The mean total stiffness(Nm/rads) for the four configurations was 1) 1543.6, 2) 301.6, 3) 290.5, 4) 267.0. The mean gap stiffness(Nm/rads) for the right hemimandible was: 2) 2041.1, 3) 1763.5, 4) 1679.9. The mean gap stiffness of the left hemimandible was: 2) 2110.8, 3)1880.1, 4)1861.1. There was no gap stiffness for the first configuration since a fracture gap was not present. Two-way ANOVA was performed on the gap stiffness and the total stiffness. There was a significant decrease in total stiffness between intact mandibles and ostectomized mandibles regardless of external fixator configuration. However, there was not a significant difference in total stiffness or gap stiffness among the different external fixator configurations applied to ostectomized mandible. External fixator configurations with only the full interdental pin engaging the rostral fragment were as stiff as configurations which had two or four additional pins in the rostral fragment for the applied loads. External fixators for rostral mandibular fractures may be rigidly secured with rostral fragment implants applied extracortically avoiding iatrogenic trauma to teeth and tooth roots. / Master of Science

The emergence of new technologies and improved computing power helped to introduce a renewed vitality in morphological research in recent decades. This is especially apparent in the new advances made in understanding the evolutionary morphology of the skeletal system in extinct and extant squamate reptiles. The new data generated as a result of the recent increase in attention are relevant not only for systematic analyses but also are valuable in their own right for contributing to holistic perspectives on organismal evolution, mosaic evolution in the rates of change in different anatomical systems, and broader patterns of macroevolution. A global community of morphological researchers now can share data through online digital collections, but opportunities for continued advance are hindered because we lack even basic data on patterns of variation of the skeletal system for virtually all squamate lineages. Most work on skeletal morphology of squamates is based on a sample size of n=1; this is an especially noticeable phenomenon for studies relying on X-ray computed tomography technology. We need new collections of skeletal specimens, both material and digital, and new approaches to the study of skeletal morphology. Promising areas for continued research include the recent focus on skeletal elements not traditionally included in morphological studies (especially systematic analyses based upon morphological data) and efforts to elucidate patterns of variation and phylogenetically informative features of disarticulated skeletal elements. Anat Rec, 297:344-348, 2014.

The nerve-derived trophic factor neuregulin (NRG) is a prime candidate molecule for modulating muscle fiber growth. NRG regulates signal transduction in skeletal muscle through activation of ErbB receptors present at the neuromuscular junction. In this study, we hypothesize that NRG increases protein synthesis in maturing muscle via a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. NRG signal transduction and its ability to stimulate protein synthesis (measured by incorporation of [3H]phenylalanine into the protein pool) were investigated in differentiated C2C 12 myotubes and rat diaphragm muscle (DIAm). In C2C 12 myotubes, NRG dose dependently increased phosphorylation of ErbB3 and recruitment of the p85 subunit of PI3K. NRG also increased phosphorylation of Akt, a downstream effector of PI3K. NRG treatment increased total protein synthesis by 35% compared with untreated control myotubes. This NRG-induced increase in Akt phosphorylation and protein synthesis was completely blocked by wortmannin, an inhibitor of PI3K but was unaffected by PD-98059, an inhibitor of MEK. In DIAm obtained from 3-day-old rat pups, Akt phosphorylation increased ∼30-fold with NRG treatment (vs. untreated DIAm). NRG treatment also significantly increased protein synthesis in the DIAm by 29% after 3 h of incubation with [3H]phenylalanine (vs. untreated DIAm). Pretreatment with wortmannin abolished the NRG-induced increase in protein synthesis, suggesting a critical role for PI3K in this response. The results of the present study support the hypothesis that nerve-derived NRG contributes to the regulation of skeletal muscle mass by increasing protein synthesis via activation of PI3K.

Background. Bisphenol A (BPA) and BPS are environmental pollutants that are associated with the development of insulin resistance and type 2 diabetes (T2D). Although skeletal muscle and adipose tissue dysfunction are involved the development of insulin resistance, there are few studies that have investigated the effects of bisphenols on their metabolism. In this study, we investigated the effects of BPA and BPS exposure on skeletal muscle and adipose tissue metabolism to determine how they contribute to the development of T2D. Methods. L6 muscle cells were treated with BPA during the last 24 hours of differentiation, and mitochondrial function and glucose metabolism was measured. Human subcutaneous adipose tissue was incubated for 24 or 72 hours with BPA or BPS, and adipokine gene expression and glucose metabolism was measured in adipose tissue. Results. L6 muscle cells treated with high concentrations of BPA (10⁵ nM) had mitochondrial dysfunction and a compensatory increase in glucose metabolism; however, there were no effects at environmentally-relevant concentrations. Adipose tissue treated with BPA for 24 hours had reduced expression of proinflammatory cytokines and adipokines, and reduced insulin-stimulated glucose uptake. Conclusions. BPA exposure for 24 hours did not alter L6 muscle cell mitochondrial function and glucose metabolism at environmentally-relevant concentrations; however, adipose tissue had altered proinflammatory expression and glucose metabolism at low concentrations. This has important implications in regulatory guidelines in the use of BPA in the manufacturing of consumer products.

As dense connective tissues, tendons play a vital role in the transmission of contractile forces from muscle to bone. This link between muscles and bones provides the means in transferring tensile forces produced by muscles on to the connected bone. During movement, tendons slide over surrounding bony and articular surfaces and are thus commonly subjected to shear and compression forces in addition to tensile force. Fibrillar collagen, proteoglycan and various glycoproteins make up the composition of tendinous tissue and contribute to its ability to withstand these forces. Tendons contain a distinct population of cells, called tenocytes. Tenocytes undertake a flattened morphology within the tendon matrix and contain cytoplasmic projections which extend longitudinally and laterally towards other tenocytes. An intercellular network of cells thus maintains the extracellular environment of the tendon and allows a coordinated response to external mechanical stimuli. Defects to load-bearing connective tissue elements such as tendons whether due to trauma, overuse, age-related diseases or degenerative diseases, are often limited in their healing potential and thus contributes often to persistent, chronic clinical symptoms. Chronic disease, overuse or acute injuries damages the tendon. This damage compromises the transmission of tensile forces and because of the hypovascularity of some tendinous tissues and many other reasons, a healing response often is severely insufficient in regenerating tissue back to its original constitution. Even the best treatment options for such tendinopathies, supplemented with the body’s own healing response fail to produce quality outcomes. An understanding of the molecular, cellular and mechanical characteristics of tenocytes, tendon matrix and the tendon system as a whole will be vital for the development of effective therapies for all tendinopathies. It is the goal of this current work to outline the current molecular, cellular, mechanical and clinical understanding of tendons. A broad address to tendon biology should help illustrate the key dimensional aspects that must be considered when attempting the effective translation of research into useful clinical therapies.

Indiana University-Purdue University Indianapolis (IUPUI) / Background: Pulmonary Hypertension (PH) is a devastating disease characterized by pulmonary arterial remodeling, right ventricular dysfunction and ultimately right heart failure. Increased emphasis has been given to skeletal muscle dysfunction in PH, and to its implication in the severe exercise intolerance that is a hallmark of the condition. In this dissertation, skeletal muscle blood flow was measured via the microsphere technique at rest and during exercise (Aim 1), with an acute dose of dietary nitrate via beetroot juice (BRJ) gavage used to determine if supplementation could improve muscle blood flow and alter energetics (Aim 2). VO2max, voluntary running and grip strength tests were used to determine the effect of disease on performance, and to test for an ergogenic effect of BRJ vs. placebo (PL) in healthy and PH rats (Aim 3). Methods: A prospective, randomized, counterbalanced, placebo-controlled trial was used to examine the aforementioned aims across four groups; PH rats (induced with monocrotaline, MCT, 60mg/kg, s.q., 4 weeks) supplemented with BRJ (MCT BRJ, n=9); PH rats supplemented with placebo (MCT PL, n=9); healthy control rats (vehicle, s.q.) supplemented with BRJ (CON BRJ, n=8); healthy control rats supplemented with placebo (CON PL, n=9). Results: Monocrotaline induced a severe PH phenotype evidenced by increased RV wall thickness, RV hypertrophy, RVSP and reduced cardiac output and stroke volume compared to controls (p=<0.001). MCT rats demonstrated lower muscle blood flow at rest, and more prominently during exercise compared to controls (p=0.007-0.047), regardless of supplementation. MCT rats displayed a greater reliance on anaerobic metabolism, demonstrated by increased blood lactate accumulation (p=<0.001), and this was significantly related to reduced blood flow during exercise (r=-0.5879, p=0.001). BRJ supplementation resulted in increased plasma nitrate and nitrite compared to PL (p=<0.001), but at the skeletal muscle level, only nitrate was increased after BRJ. BRJ did not have a significant effect on blood flow, with no improvement during exercise shown vs. PL. Similarly, BRJ did not significantly improve exercise function in MCT or CON rats. Conclusion: MCT rats demonstrated a reduction in muscle blood flow, with BRJ supplementation not resulting in improved flow or exercise performance.

Indiana University-Purdue University Indianapolis (IUPUI) / Type 2 diabetes mellitus (T2DM) is a growing problem globally and is associated with increased fracture risk and delayed bone healing. Novel approaches are needed in the treatment of T2DM and the resulting diabetic osteopathy. Recent studies highlight the role of bone as an endocrine organ producing factors that communicate with distant tissues to modulate systemic glucose metabolism. Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) is a potent regulator of whole-body energy metabolism, inflammation, bone remodeling and fracture healing. Genetic ablation of CaMKK2 protects from diet-induced obesity, insulin resistance and inflammation, while enhancing pancreatic β cell survival and insulin secretion. Deletion or inhibition of CaMKK2 promotes bone accrual by stimulating osteoblast-mediated bone formation and suppressing osteoclast-mediated bone resorption; however, its specific role in osteocytes, the master regulator of bone remodeling remains unknown. Here we demonstrate that conditional deletion of CaMKK2 from osteocytes enhances bone mass in 3-month-old female, but not male mice, due to suppression of osteoclasts. Conditioned media experiments and proteomics analysis revealed that female osteocytes lacking CaMKK2 suppressed osteoclast formation and function through enhanced secretion of calpastatin, a potent inhibitor of calpains, which are calciumdependent cysteine proteases that support osteoclasts. Further, to determine if CaMKK2- deficient osteocytes regulate whole-body glucose homeostasis, we placed these mice on a high-fat diet (HFD) for a period of 16 weeks. Although the diet did not significantly impact bone mass or strength, we found that conditional deletion of CaMKK2 in osteocytes enhanced bone microarchitecture in 6-month-old male and female mice. We also observed that conditional deletion of CaMKK2 from osteocytes protected male and female mice from HFD-induced obesity and insulin insensitivity. Taken together, these findings highlight CaMKK2 as a potent regulator of osteocyte-mediated modulation of bone remodeling and whole-body energy metabolism. / 2024-08-02

Among vertebrates, oviparous animals lay eggs with a calcified eggshell and eggs are laid in an external environment, while viviparous animals retain eggs in oviduct until they give birth to live young. Viviparity has evolved in the lineage of snakes and lizards (squamates) over 100 times, more than all other vertebrate groups. Embryos of oviparous squamates obtain calcium from both yolk and eggshell while their viviparous counterparts lack a calcified eggshell and must obtain their calcium solely from yolk, or from yolk plus placental transfer. During embryonic development, squamates rely on calcium to build the skeleton before hatching. The extent of skeletal ossification at hatching or birth varies considerably among vertebrates. This study aims to determine if skeletal development/ossification varies in association with reduced eggshell calcium in embryos of viviparous squamate species. We studied the amount of ossification and overall size of embryos and hatchlings from an oviparous and a viviparous population of the reproductively bimodal lizard Zootoca vivipara. Previous studies suggested that limb development is delayed, and that overall size is reduced in viviparous squamates. We tested the hypothesis that viviparous embryos and hatchlings are more skeletally immature and smaller in size than oviparous embryos and hatchlings in squamates. To achieve this, specimens from both populations, spanning multiple stages of embryonic development including hatchlings, were cleared & stained to reveal skeletal cartilage and bone. Lengths of total body, humerus, femur, skull and Meckel’s cartilage were measured from photographs of cleared & stained specimens taken with a Cannon EOS 70D camera on a Motic, Leica MZ9 Compound Microscope, with a measurement reference in each picture. Photos were calibrated to the measurement reference and total length measurements were obtained using iSolutionLite® software. In addition to total measurements, the lengths of ossified portions on the humerus and femur were also measured. Preliminary results revealed that total length of the skull and body are reduced in embryos and hatchlings of viviparous specimens. Total length of the limbs and of Meckel’s cartilage are not significantly different between populations. However, it appears that the amount of ossification in the limbs is reduced in oviparous specimens. These preliminary findings do not support our hypothesis and indicate that reduction in eggshell calcium in embryos of viviparous populations does not negatively impact ossification during development but does influence overall size.