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archives@tulane.edu / Off-the-shelf implantable biomaterials for tissue engineering and regenerative medicine (TERM) applications can treat numerous damaged organs/tissues. Such biomaterials have been investigated for their natural and synthetic biomimetic material properties to induce tissue regeneration in situ. Engineering biomaterials based off their mechanical properties like that of tensile strengths, porosity, nanofiber widths, or moduli can aid in tissue repair. However, tuning biomechanical properties alone is not always sufficient for biomaterials to induce effective tissue regeneration of large tissue/organ defects. The inclusion of biological properties to certain biomaterials such as stem/progenitor cells, growth factors, and/or cytokines can attenuate inflammation and stimulate proper tissue regeneration. In order to develop better off-the-shelf biomaterial implants for tissue repair, both the biomechanical and biological properties of the engineered biomaterial implant need to be assessed. In this dissertation, a decellularized polycaprolactone/chitosan hollow tube grown with MSC2s (De-PCL/CS) is investigated as a novel off-the-shelf tissue engineered graft (OTS-TEG) to repair hollow tubular organs like that of the esophagus. It is demonstrated that De-PCL/CS can retain extracellular matrix (ECM) factors from anti-inflammatory MSC2s post freeze-thaw decellularization. De-PCL/CS exhibits similar esophageal compressive biomechanics and is shown in an in vivo murine omentum model to be anti- inflammatory while robustly recruiting gastrointestinal (GI) cells. This gives great insight into De-PCL/CS as an immune modulating biomaterial to attenuate inflammation while promoting tissue repair for GI applications. Severe esophageal malignancies like esophageal cancer (EC), advanced Barrett’s Esophagus (BE), and pediatric atresia require GI reconstructive surgery (i.e. gastric pull up). De-PCL/CS has the translational capability as an implantable OTS-TEG to regenerate damaged esophageal tissue avoiding a gastric pull up. This not only increases the quality of life of the patient receiving this treatment but has an immediate potential impact of over $2.2 billion saved annually on the U.S. healthcare system. Nevertheless, the unique bioengineering patent-pending methods in developing De-PCL/CS allow it to be a platform OTS-TEG for treating a plethora of damaged organs/tissues. Translationally, De-PCL/CS can have a great therapeutic impact in the TERM global market that is expected to accrue over $100 billion by the mid 2020s. / 1 / Derek Cyrus Dashti

Zebrafish, danio rerio, are bony fish in the Teleost class. Zebrafish can regenerate most organs, including their fins. The caudal fin of zebrafish adults has bundles of rigid, uncalcified fibrils, termed actinotrichia, at the distal end of each lepidotrichia. During embryonic and early larval stages of fin development, actinotrichia are synthesized between the fin fold epidermis. Actinotrichia remain present in the fins of adult fish and during fin regeneration. They provide structural support and facilitate mesenchymal cell migration during fin development and regeneration. The importance of the role of actinotrichia for fin formation and regeneration can be studied through loss of function analysis of structural components of the actinotrichia. Our lab identified two genes, actinodin1 and actinodin2 (and1, and2), that are coding for structural proteins of the actinotrichia. CRISPR/Cas9 mediated knockout of and1 and and2 lead to loss of actinotrichia in the embryonic fins. Immunohistochemistry with anti-And1 and anti-ColII antibodies, in addition to controlled tissue degradation to observe actinotrichia fibers revealed the absence of actinotrichia in the caudal fins of adult and1/2 double homozygous mutants. We hypothesized that loss of actinotrichia will lead to the disruption of mesenchymal cell migration during development and regeneration, resulting in fin growth and morphology defects. The intact adult caudal fin of and1/2 double mutants have fewer rays (lepidotrichia) and these lepidotrichia are shorter and wavy when compared to WT. Along the lepidotrichia of actinodin mutants, there is an uneven distribution of joints revealing that there are defects in ray patterning. We carried out a regeneration time course analysis to compare caudal fin regeneration in WT and and1/2 double homozygous mutants. The and1/2 double homozygous mutants have a delay in bone re-formation and defects in joint patterning are observed in regenerating rays. From these results, and1 and and2 are shown to be required for actinotrichia formation. In addition, morphological analyses identified that actinotrichia is required for proper caudal fin growth and patterning during development and regeneration.

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Numerous studies highlighted sharp declines in abundance of red oak species (Quercus spp., Section Erythrobalanus) in the southeastern United States. Red oaks are major components of bottomland forests, provide important ecological services, and are a critical source of hard mast for wildlife and high-value timber (Oliver et al. 2005). Bottomland hardwoods are usually managed with natural regeneration, and maintaining a component of red oak can challenge forest managers, given sporadic acorn production (masting behavior), and lack of advance regeneration establishment prior to disturbance. This study investigated the development of hardwood advance regeneration in relation to understory light availability and stand structure in mature closed canopy stands following silvicultural treatments. Improved understanding of red oak natural regeneration can better clarify any relationship between seedling abundance, available understory light, and residual basal area. Results will aid in selection of appropriate management techniques to sustain dominance of red oaks within bottomland hardwood forests.

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The robust regenerative capacity of muscle stem cells (MuSCs) and their progenitors depends on their ability to undergo rapid and vast changes to their transcriptome during cell state changes. While transcription factors and epigenetic remodelling proteins are critical to render genes permissive for transcription, often these genes are found to have paused promoter proximal RNA Polymerase II (Pol II) which remains in a rate-limiting poised state. Indeed, while prior studies have shown poised Pol II is often regulated by the Negative Elongation Factor (NELF) to induce rapid changes in gene expression, the specific need for NELF in somatic stem cell populations has not been previously examined. In this thesis, we identify a specific requirement for NELF-dependent promoter proximal Pol II pausing in proliferating myogenic progenitors. Here, NELF stabilizes nascent transcripts associated with the paused RNA Pol II at genes required to maintain muscle progenitors in cell cycle. This promotes expansion of the pool of myogenic progenitors required to adequately repair damaged skeletal muscle. Our molecular analysis suggests that in proliferating progenitors, NELF-bound Pol II ensures the stabilization of transcripts, and continued expression of genes that prevent p53-mediated cell cycle withdrawal and terminal differentiation. Unexpectedly, this work revealed a previously unappreciated contribution of proliferating myogenic progenitors to replenish the stem cell niche in support of MuSC self-renewal during skeletal muscle regeneration. Based on our results, new therapeutic avenues which could treat muscle wasting disease are also discussed.