Indiana University-Purdue University Indianapolis (IUPUI) / Bone is a dynamic organ that responds to its external environment. Cell signaling cascades are initiated within bone cells when changes in mechanical loading occur. To describe these molecular signaling networks that sense a mechanical signal and convert it into a transcriptional response, we proposed the mechanosome model. “GO” and “STOP” mechansomes contain an adhesion-associated protein and a nucleocytoplasmic shuttling transcription factor. “GO” mechanosomes functions to promote the anabolic response of bone to mechanical loading, while “STOP” mechanosomes function to suppress the anabolic response of bone to mechanical loading. While much work has been done to describe the molecular mechanisms that enhance the anabolic response of bone to loading, less is known about the signaling mechanisms that suppress bone’s response to loading. We studied two adhesion-associated proteins, Src and Pyk2, which may function as “STOP” mechanosomes. Src kinase is involved in a number of signaling pathways that respond to changes in external loads on bone. An inhibition of Src causes an increase in the expression of the anabolic bone gene osteocalcin. Additionally, mechanical stimulation of osteoblasts and osteocytes by fluid shear stress further enhanced expression of osteocalcin when Src activity was inhibited. Importantly, fluid shear stress stimulated an increase in nuclear Src activation and activity. The mechanism by which Src participates in attenuating anabolic gene transcription remains unknown. The studies described here suggest Src and Pyk2 increase their association in response to fluid shear stress. Pyk2, a protein-tyrosine kinase, exhibits nucleocytoplasmic shuttling, increased association with methyl-CpG-binding protein 2 (MBD2), and suppression of osteopontin expression in response to fluid shear stress. MBD2, known to be involved in DNA methylation and interpretation of DNA methylation patterns, may aid in fluid shear stress-induced suppression of anabolic bone genes. We conclude that both Src and Pyk2 play a role in regulating bone mass, possibly through a complex with MBD2, and function to limit the anabolic response of bone cells to fluid shear stress through the suppression of anabolic bone gene expression. Taken together, these data support the hypothesis that “STOP” mechanosomes exist and their activity is simulated in response to fluid shear stress.

Indiana University-Purdue University Indianapolis (IUPUI) / Seven human alcohol dehydrogenase genes (which encode the primary enzymes involved in alcohol metabolism) are grouped into classes based on function and sequence identity. While the Class I ADH isoenzymes contribute significantly to ethanol metabolism in the liver, Class IV ADH isoenzymes are involved in the first-pass metabolism of ethanol. It has been suggested that the ability to efficiently oxidize ethanol occurred late in primate evolution. Kinetic data obtained from the Class I ADH isoenzymes of marmoset and brown lemur, in addition to data from resurrected ancestral human Class IV ADH isoenzymes, supports this proposal--suggesting that two major events which occurred during primate evolution resulted in major adaptations toward ethanol metabolism. First, while human Class IV ADH first appeared 520 million years ago, a major adaptation to ethanol occurred very recently (approximately 15 million years ago); which was caused by a single amino acid change (A294V). This change increases the catalytic efficiency of the human Class IV enzymes toward ethanol by over 79-fold. Secondly, the Class I ADH form developed 80 million years ago--when angiosperms first began to produce fleshy fruits whose sugars are fermented to ethanol by yeasts. This was followed by the duplication and divergence of distinct Class I ADH isoforms--which occurred during mammalian radiation. This duplication event was followed by a second duplication/divergence event which occurred around or just before the emergence of prosimians (some 40 million years ago). We examined the multiple Class I isoforms from species with distinct dietary preferences (lemur and marmoset) in an effort to correlate diets rich in fermentable fruits with increased catalytic capacity toward ethanol oxidation. Our kinetic data support this hypothesis in that the species with a high content of fermentable fruit in its diet possess greater catalytic capacity toward ethanol.

<p dir="ltr">Skeletal muscle accounts for 40% of total body weight and the homeostasis of muscle tissue is critical in maintaining proper body function. Skeletal muscle develops during the embryonic stages from the muscle progenitor cells derived from the dermomyotome structure. The myogenic progenitor cells contribute to the primary myogenesis by forming the primary myotubes which are the founding structures that the secondary myogenesis continues to build on. A portion of the myogenic progenitor cells makes up the adult muscle stem cells residing in homeostatic muscle tissue. The adult muscle stem cells contribute substantially for the adult muscle regeneration. Due to the significance of the muscle tissue and the importance of muscle stem cells, dysregulation of the muscle homeostasis or the muscle stem cell homeostasis will result in severe pathological conditions such as myopathy.</p><p dir="ltr">Mitochondria are cellular organelles that are responsible for generating energy needed for cellular processes, especially for muscle tissue where muscle contraction requires the presence of ATP. On the other hand, mitochondria also serve as signaling molecules and provide macromolecules for the biosynthesis. FAM210A (Family With Sequence Similarity 210 Member A) protein was shown to impact the lean mass of human subjects yet a detailed study on the effect of FAM210A in skeletal muscle was not performed, nor has the molecular mechanisms through which FAM210A function been elucidated. Therefore, I take on the task to unveil the function of FAM210A in muscle development, muscle homeostasis and muscle stem cell behavior by using a combination of mouse models with different myogenic promoters to target <i>Fam210a</i> at different developmental stages.</p><p dir="ltr">In the first part of the thesis, I investigated the role of FAM210A in post differentiation myofibers. Using the <i>Myl1</i>^<em>Cre</em> driven deletion of <i>Fam210a</i>, I found that <i>Fam210a</i>^<em>MKO</em> had normal development before 3 weeks of age, but the growth was stagnant from 4 weeks on, and the mice did not survive past 8 weeks of age. I found that the assembly of the ribosomes in the <i>Fam210a</i>^<em>MKO</em> was defective, leading to impaired translation which attenuated the muscle atrophy phenotype. I identified through proteomics that the mitochondrial autophagy and proteostatic control pathways were significantly induced yet mitochondrial organization and energetic proteins were downregulated. Metabolomics analysis showed that the signaling metabolite acetyl-CoA was increased in the <i>Fam210a</i>^<em>MKO</em> which led to increased protein acetylation, specifically, we showed that the ribosomal proteins were hyperacetylated, and that the acetylation increase was elicited by the <i>Fam210a</i>-null mitochondria.</p><p dir="ltr">In the second part of the thesis, I investigated the function of FAM210A in muscle progenitor cells. In the <i>FamMKO</i> mice, I found that deletion of <i>Fam210a</i> from embryonic myogenic progenitor cells led to developmental arrest and postnatal death at day 6. In the <i>FamPKO</i> mice, I found that <i>Fam210a</i> is needed for adult muscle stem cell to contribute to regeneration. Loss of <i>Fam210a</i> leads to the regenerative defects when the muscle was exposed to injury cues. We further showed that <i>Fam210a</i> deletion in muscle stem cells resulted in disruption of the proteostatic control over muscle stem cell activation, thereby forbidding the translational increase necessary to facilitate activation and proliferation. Furthermore, I showed that <i>Fam210a</i> deletion leads to excessive OPA1 cleavage, which contributes to the regenerative failure of muscle stem cells as fusion is required for the mitochondrial network remodeling during regeneration. Therefore, <i>Fam210a</i> safeguards the mitochondrial network and proteostasis during regeneration.</p><p dir="ltr">In summary, my studies characterized the functional contribution of FAM210A during embryonic muscle development, muscle mass maintenance and adult muscle stem cell homeostasis. The regulation of FAM210A in these three processes impinge on the translational regulation. My studies further demonstrated the importance of mitochondrial regulated protein translation in skeletal muscle and muscle stem cells.</p>

<p dir="ltr">Newborn animals undergo a lot of early-life stress that heavily impact on their long-term growth, performance, and welfare. Typically, the stress would indirectly interfere with the capacity of these neonates to utilize dietary nutrients and consequently impact tissue growth and development. In piglets, weaning is a stressful situation characterized by disruption of intestinal epithelial cell development which causes poor digestion of solid feed and a negative impact on absorption of nutrients especially in the post-gastric region. In addition, weaning in piglets could cause an increase in cellular assault by reactive oxygen species thereby potentially causing gut leakiness and paracellular loss of nutrients along the intestinal tract. In broiler chickens, access to feed may take up to 72 h following hatching which may affect their gut development as well as their gut microbiota. After the first feed ingestion, there is a sharp increase in the gut microbiota which triggers an increase in the development of the immune system as well as the gut. There is continuous attention on the strategies and nutritional interventions to mitigate or ameliorate the adverse effects of early life stressors in these food animals, especially in broiler chickens and piglets. In the studies described in this dissertation, myo-inositol (purely supplemented or phytase-induced) and luteolin were tested as nutritional strategies to mitigate the effects of early-life stressors on growth and the potential mechanisms by which myo-inositol and luteolin regulate growth were investigated.</p><p dir="ltr">In study I, the effect of myo-inositol on growth in 128 postweaning piglets fed protein-deficient corn-soy diets was tested. There were 4 dietary treatments in a randomized complete block design with body weight as the blocking factor. The treatments consisted of 1 positive control (PC) diet formulated to meet all the nutrient requirements of the piglets with a 20% crude protein (CP); the remaining 3 diets were the negative control (NC) diets with a 3% reduction in CP, a 2 g/kg myo-inositol supplemented negative control diet (NC+INO), and phytase (3,000 FTU/kg) supplemented negative control (NC+PHY) diet. The results showed that phytase enhanced the apparent total tract digestibility (ATTD) of P in the weanling pigs. Myo-inositol supplementation in a protein-deficient diet improved (P < 0.05) porcine plasma myo-inositol concentration while an in vitro myo-inositol incubation with intestinal epithelial cells increased the expression of genes that encode for Claudin-1, Claudin-3, Claudin-4, ZO-1, NaPiIIb, GLUT2, and SLC7A2. The in vitro analysis of tight junction integrity in the IPEC-J2 cells indicated by the transepithelial electrical resistance and FITC-Dextran permeability showed an enhancement in response to myo-inositol treatment. Although the in vivo study found that myo-inositol did not improve growth performance or ATTD, the in vitro myo-inositol enhanced markers of gut health and function.</p><p dir="ltr">In study II, the effect of myo-inositol on the growth of broiler chickens was tested. In this study, there were 6 experimental treatments based on two dietary protein levels (PC and NC) and three supplement types (BASAL, INO, and PHY) resulting in a 2 x 3 factorial arrangement in a completely randomized design. A total of 384 broiler chickens comprising 6 treatments with eight replicates per treatment and 8 birds per replicate were used. The birds were fed a common starter diet for the initial 7 days after they arrived at the poultry unit followed by a 14-day trial. The protein-deficient diet decreased the feed efficiency of the birds. Phytase addition increased (P < 0.05) the apparent ileal digestibility (AID) and ATTD of P and Ca in both PC and NC groups. The jejunal gut morphology was enhanced by supplemental phytase as indicated by an increase in villus height and the ratio of the villus height-to-crypt depth, coupled with an increase in serum myo-inositol concentration caused by both myo-inositol and phytase. In conclusion, myo-inositol showed a differential influence on growth performance, nutrient digestibility, and gut morphology.</p><p dir="ltr">In study III, the effects of luteolin on weanling pigs and IPEC-J2 cells were examined. A total of 48 piglets were randomly allotted to two dietary treatments consisting of a control group and a luteolin (LUT)-supplemented dietary group for a 4-week trial. A weekly assessment of the growth performance and expression of specific proteins in the jejunal mucosa was performed. In each dietary group, 8 piglets were slaughtered at weeks 1, 2, and 4 postweaning to collect blood, jejunal and ileal mucosa, and tissues. Luteolin supplementation numerically improved the ADG and G:F of the pigs. Luteolin feeding altered the jejunal and ileal gut morphology with increased villi height (P < 0.05) and villus height-to-crypt depth ratio (VCR, P < 0.05) in the jejunum and decreased crypt depth in the ileum. The effect of luteolin on IPEC-J2 global proteome and phosphor-proteome showed that luteolin could potentially improve intestinal barrier integrity by enhancing the abundance of proteins important in cell growth and survival. </p><p dir="ltr">In summary, dietary supplementation with myo-inositol and luteolin could regulate growth and nutrient digestibility in broiler chickens and weanling pigs by enhancing the integrity of intestinal cells and facilitating the expression of nutrient transporters that are significant in the uptake of nutrients across the lining of the gastrointestinal tract. Phytase supplementation improves the P release from phytate in the diets thereby alleviating its loss.</p>

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