Skeletal muscle can intrinsically repair itself in response to injury. This repair process has been shown to be mediated through signaling of the innate immune system. The immune response caused during repair helps to clear away debris in damage and promotes the activation and proliferation of muscle stem cells (MuSCs) that will repair the damage muscle. Dysregulation of this inflammation leads to fibrosis and decreased efficacy of the repair process. Despite the requirement of inflammatory signaling during muscle repair, muscle’s contribution during inflammation as only recently started to be explored. The objective of this dissertation is to assess the contribution of muscle in the early inflammatory response during repair as well attempting to modulate this inflammation during disease to ameliorate disease pathology in a model of Duchenne’s muscular dystrophy. I tested the hypotheses that 1) muscle is an active participant in the early inflammatory response, 2) the transcription factor Mohawk (Mkx) is a regulator of the early inflammatory response and, 3) If this inflammation can be modulated with a virally derived serine protease inhibitor in a model of muscle disrepair and chronic inflammation. I found that muscle is actively participating in the establishment early inflammation in repair through the production of chemokines used to promote infiltration of immune cells. As well as the identification of a new muscle subtype that produces more chemokines compared to the average MuSC and upregulated genes in the Interferon signaling pathway. I also discovered that presence of this muscle subtype is linked to the expression of Mkx. In Mkx null mice this population is not present, and these cells are deficient in chemokine expression compared to WT mice. I subsequently found that, using the myxomavirus derived serine protease inhibitor, Serp-1 I was able to modulate the chronic inflammation that is common in those affected with Duchenne’s muscular dystrophy (DMD) utilizing a high-fidelity mouse model of the disease. The result of this dissertation provides an expanded role for muscle in inflammation and gives a potential new class of therapeutics to be used in disease associated with chronic inflammation.

Skeletal muscle injury, whether acute or chronic, is characterized by influxes of pro- and anti-inflammatory cells that coordinate with muscle to precisely control the reparative process. This intricate coordination is facilitated by a signaling feedback loop between satellite cells and extravasated immune cells. Regulation of the cytokines and chemokines that mediate healthy repair is critical for the overall success of fiber regeneration and thus provides a prospective direction for the development of therapeutics aimed at fine-tuning the local inflammatory response. This work describes (1) the contribution of non-myogenic cells in skeletal muscle regeneration, (2) the role of the transcription factor Mohawk (Mkx) in regulating inflammation following acute muscle injury and the identification of an overarching requirement for Mkx in the establishment of a pro-inflammatory response, and (3) characterization of eosinophils in acute and chronic muscle damage. Mice deficient for Mkx exhibited delayed muscle regeneration, accompanied by impaired clearance of necrotic fibers and smaller regenerated fibers. This diminished regenerative capacity was associated with a reduction in the recruitment of pro-inflammatory macrophages to the site of damage. In culture, Mkx-/- bone marrow-derived macrophages displayed reduced proliferative capacity but retained the ability to polarize in response to a pro-inflammatory stimulus. The necessity of Mkx in mounting a robust immune response was further confirmed by an immunological challenge in which Mkx-/- mice exhibited increased susceptibility to Salmonella enterica serovar Typhimurium infection. Significant downregulation of key cytokine and chemokine expression was identified throughout the course of muscle repair in Mkx-/- mice and represents one mechanism in which Mkx regulates the establishment of an inflammatory response. Previous research discovered that Mkx is highly expressed in eosinophils, a type of innate immune cell that participates in disease-fighting and inflammation, however the role of eosinophils in muscle repair is not well described. This work outlines the contribution of eosinophils in muscle repair following acute and chronic injury. In healthy mice, eosinophils were found to inhibit efficient muscle repair following acute injury. Utilizing the mdx-/-utrn-/- muscular dystrophy mouse model, eosinophil depletion via administration of anti-IL-5 antibody significantly improved diaphragm fiber diameter and increased the survival rate during the course of treatment.

The Numb gene encodes an adaptor protein that has been shown to play a role in muscle repair, cell proliferation, and the determination of cell fate in satellite cells. Ablation of this gene in satellite cells results in an up-regulation of myostatin and p21, which inhibit the proliferation of myoblasts. These results indicate that the regulation of numb and myostatin could be used to amplify muscle regeneration. This would function as a therapeutic approach to degenerative muscle diseases, such as muscular dystrophy. There are four mammalian NUMB proteins produced through alternative splicing of the Numb mRNA transcript. Only two isoforms are present in adult mammalian muscle, indicating some form of muscle-specific post-transcriptional control of the gene. Additionally, the presence of two polyadenylation sites, and multiple miRNA seed sequences within the 3’ untranslated region (UTR) of mouse Numb indicate the possibility of regulation by a muscle specific miRNA.

Multicellular organisms use precise gene regulation, executed throughout development, to build and sustain various cell and tissue types. Post-transcriptional gene regulation is essential for metazoan development and acts on mRNA to determine its localization, stability, and translation. MicroRNAs (miRNAs) and RNA binding proteins (RBPs) are the principal effectors of post-transcriptional gene regulation and act by targeting the 3'untranslated regions (3'UTRs) of mRNA. MiRNAs are small non-coding RNAs that have the potential to regulate hundreds to thousands of genes and are dysregulated in many prevalent human diseases such as diabetes, Alzheimer's disease, Duchenne muscular dystrophy, and cancer. However, the precise contribution of miRNAs to the pathology of these diseases is not known.

MiRNA-based gene regulation occurs in a tissue-specific manner and is implemented by an interplay of poorly understood and complex mechanisms, which control both the presence of the miRNAs and their targets. As a consequence, the precise contributions of miRNAs to gene regulation are not well known. The research presented in this thesis systematically explores the targets and effects of miRNA-based gene regulation in cell lines and tissues.

I hypothesize that miRNAs have distinct tissue-specific roles that contribute to the gene expression differences seen across tissues. To address this hypothesis and expand our understanding of miRNA-based gene regulation, 1) I developed the human 3'UTRome v1, a resource for studying post-transcriptional gene regulation. Using this resource, I explored the targets of two cancer-associated miRNAs miR-221 and let-7c. I identified novel targets of both these miRNAs, which present potential mechanisms by which they contribute to cancer. 2) Identified in vivo, tissue-specific targets in the intestine and body muscle of the model organism Caenorhabditis elegans. The results from this study revealed that miRNAs regulate tissue homeostasis, and that alternative polyadenylation and miRNA expression patterns modulate miRNA targeting at the tissue-specific level. 3) Explored the functional relevance of miRNA targeting to tissue-specific gene expression, where I found that miRNAs contribute to the biogenesis of mRNAs, through alternative splicing, by regulating tissue-specific expression of splicing factors. These results expand our understanding of the mechanisms that guide miRNA targeting and its effects on tissue-specific gene expression.

Amongst the most studied of the social insects, the honey bee has a prominent place due to its economic importance and influence on human societies. Honey bee colonies can have over 50,000 individuals, whose activities are coordinated by chemical signals called pheromones. Because these pheromones are secreted from various exocrine glands, the proper development and function of these glands are vital to colony dynamics. In this thesis, I present a study of the developmental ontogeny of the exocrine glands found in the head of the honey bee. In Chapter 2, I elucidate how the larval salivary gland transitions to an adult salivary gland through apoptosis and cell growth, differentiation and migration. I also explain the development of the hypopharyngeal and the mandibular gland using apoptotic markers and cytoskeletal markers like tubulin and actin. I explain the fundamental developmental plan for the formation of the glands and show that apoptosis plays an important role in the transformation toward an adult gland.

Duchenne Muscular Dystrophy (DMD) is a muscular degenerative disease characterized by striated membrane instability that stimulates continuous cycles of muscle repair. Chronic activation of the innate immune response necessary for muscle repair leads to a pathological accumulation of fibrotic materials that disrupt muscle function. During healthy tissue repair, a balance between pro-inflammatory macrophage (M1) and anti-inflammatory macrophage (M2) promotes clearance of necrotic fibers (myolysis) followed by tissue repair. This is regulated by an intricate feedback loop between muscle, neutrophils and macrophages mediated by Th1 and Th2 cytokines and chemokines. During chronic inflammation, there is an imbalance in an M2 species that produces high levels of extracellular matrix that leads to fibrosis. Finding treatments that ameliorate fibrosis are essential to limiting the muscle pathology that reduces ambulation of DMD patients. Previous studies have shown that Mohawk, (Mkx) a homeobox transcription factor, is essential for the initiation of the inflammation response during acute muscle injury. This study aims to examine whether Mkx regulates inflammation during chronic damage associated with muscular dystrophy. The mdx mouse is a well-studied mouse model that recapitulates muscle necrosis, chronic inflammatory response and fibrosis associated with muscular dystrophy. Utilizing quantitative RT-PCR and histological analysis, the diaphragms and Quadriceps of adult Mkx-/-/mdx and Mkx+/+/mdx mice were analyzed at three critical time points (4 weeks, 3 months and 7 months). In contrast to what was anticipated, there was evidence of increased muscle damage in the absence of Mkx. There was a consistent reduction in the diameter of muscle fibers found in both types of tissue in Mkx-/-/mdx versus Mkx+/+/mdx mice without a difference in the number of fibers with centralized nuclei at 4 weeks and 1 year between the two genotypes, suggesting that the Mkx mutation influences the maturation of fibers forming in response to muscle damage. Fibrosis was higher in the diaphragm of the Mkx-/-/mdx mice at 4 weeks and 3 months, while at1 year there did not appear to be a difference. Overall, the results predict that the absence of Mkx exacerbates the instability of muscle fibers in the mdx mouse. Future studies will be needed to understand the relationship between Mkx and the dystrophin gene.

Numb is a gene that encodes an adaptor protein which has been characterized for its role cell migration, cell adhesion, endocytosis, and cell fate determination through asymmetrical division in various embryonic and adult tissues. In vertebrates, several Numb isoforms are produced via alternative splicing. In the Mus musculus genome, one Numb gene on chromosome 12 is alternatively spliced to produce four distinct protein isoforms, characterized by an 11 amino acid insert in the phosphotyrosine binding domain and a 49 amino acid insert in the proline rich region. Two poly adenylation sites in the currently published Numb 3' UTR exist, thus, the possibility that various 3' UTRs containing different miRNA seed sites is a possible posttranscriptional mechanism by which differential expression is observed. In an attempt to elucidate this hypothesis, PCR was performed to amplify the 3' UTR of murine neural tube cells, the products of which were subsequently cloned and sequenced. Multiple fragment sizes were consistently observed in the PCR data, however, sequencing demonstrated that these bands did not reveal an association with Numb.

Sarcopenia, a disease defined by age-related muscle loss and function, impacts each and every one of us as we age. Medical research over the past 40 years has identified dozens of factors that contribute to Sarcopenia, including, hormonal changes, deficiencies in nutrition, denervation, changes in physical activity and diseases. Developing effective therapeutic treatments for Sarcopenia is dependent on identifying the mechanisms by which these factors affect muscle loss and understanding the interrelationship of these mechanisms. I conducted my research by compiling and analyzing several previous studies on many different mechanisms that contribute to Sarcopenia. Of these mechanisms, I determined the most significant mechanisms and mapped them out on a visual presentation. In addition to the contributing factors listed above, I found that dysregulated cell signaling, mitochondrial abnormalities, impaired autophagy/protein regulation, altered nitric oxide production, and systemic inflammation all contribute to Sarcopenia. Their impact on skeletal muscle is manifested by reduced satellite function, reduced regenerative capacity, loss of muscle mass, accumulation of damaged products, and fibrosis. My research clearly demonstrated that there was not a one-to-one correlation between factors and specific pathological characteristics of Sarcopenia. Instead, factors funneled into a discrete number of cellular processes, including cell proliferation, protein synthesis, and autophagy and apoptosis. Based on my findings, the overall cause of Sarcopenia appears to be a loss of balance between these pathways. The results of my thesis indicate that Sarcopenia is a multifactorial disorder, and therefore, effective therapy should consist of those that prevent necrosis associated with autophagy and apoptosis.

The development of skeletal muscle during embryogenesis and repair in adults is dependent on the intricate balance between the proliferation of myogenic progenitor cells and the differentiation of those cells into functional muscle fibers. Recent studies demonstrate that the Drosophila melanogaster transcription factor CG9650 is expressed in muscle progenitor cells, where it maintains myoblast numbers. We are interested in the Mus musculus orthologs Bcl11a and Bcl11b (C2H2 zinc finger transcription factors), and understanding their role as molecular switches that control proliferation/differentiation decisions in muscle progenitor cells. Expression analysis revealed that Bcl11b, but not Bcl11a, is expressed in the region of the mouse embryo populated with myogenic progenitor cells; gene expression studies in muscle cell culture confirmed Bcl11b is also selectively transcribed in muscle. Furthermore, Bcl11b is down-regulated with differentiation, which is consistent with the belief that the gene plays a role in cell proliferation.