MS, University of Cincinnati, 2012, Medicine: Genetic Counseling

Duchenne and Becker muscular dystrophy (DBMD) are progressive and debilitating neuromuscular disorders caused by X-linked recessive mutations in the dystrophin gene. DMD presents in childhood and limits life to the second or third decade. BMD, a relatively milder form, presents in childhood to adulthood. Although improvements in the care of patients with DBMD have enhanced patients' quality and duration of life, there has been no effect on long-term prognosis. Little is known of the psychological morbidity associated with DBMD on families and patients. Birth mothers of children with DBMD, who are themselves carriers of DBMD, may have increased psychological burden. The purpose of this analysis was to describe differences in adaptation and wellbeing between mothers who were carriers and those who were not carriers of DBMD. Data was collected from mothers with biological children with DBMD using a mixed-methods web-based survey. The primary outcome variable, adaptation score, was generated for each participant using the Psychological Adaptation to Illness Scale. Various other scales and investigator-developed questions were also used to measure secondary outcome variables. One hundred twenty-five participants completed the questionnaire and 116 responses were analyzed. Fifty-one (44%) were carriers of a DMD gene mutation, 47 (40%) were not carriers, and 18 (16%) did not know their carrier status. The mean adaptation score was 3.68 (SD=0.9) for carriers and 3.25 (SD=0.9) for non-carriers. Carriers showed better adaptation and higher perceived control than non-carriers (pooled t-test, p=0.02 and p=0.05, respectively). These results were limited by several factors including small sample size, recruitment bias, and increased risk of type 1 error resulting from multiple tests on the data. An open-ended question completed by the carriers revealed various positive and negative effects of being a carrier. In conclusion, carrier status may affect mothers' adaptation to DBMD an (open full item for complete abstract)

Committee: James Collins PhD (Committee Chair); Kathleen Kinnett MSN (Committee Member); Xue Zhang PhD (Committee Member); Martha Walker (Committee Member) Subjects: Genetics

MS, University of Cincinnati, 2013, Medicine: Genetic Counseling

Introduction: Duchenne / Becker muscular dystrophy (DBMD) is an x-linked condition with a wide variation of clinical presentation due to specific gene mutations and the gender of the affected individual. For families of the most severely affected male patients, care needs, natural history, and potential interventions are paramount. In contrast, reproductive risks may be important for less severely affected individuals as in the case of Becker phenotype or DBMD carrier females. The published literature has suggested the caregiver burden and poor prognosis of DBMD has an impact on the biological family as a whole. Additionally, published literature suggests a high disruption rate of adoptions that involve a child with special needs. However, the literature does not currently describe the role of genetic counselors in addressing the needs of families who have adopted a child with DBMD. Purpose: The purpose of this study was to determine the needs of adoptive families with sons diagnosed with DBMD, and how genetic counseling could be tailored to improve this population's experience. Methods: Participants were adoptive parents of males who were under age 18 and had a DBMD Diagnosis. They were recruited through Cincinnati Children's Hospital Medical Center or Duchenne Connect. Semi-structured qualitative interviews were conducted by telephone with use of an interview guide. The interview content was analyzed for recurrent themes using NVivo© software. These themes were organized into categories to summarize the findings. Results: Thirteen adoptive parents were interviewed. Their needs, relative to the diagnosis of DBMD, genetic counseling, and the genetics information, were not specific to adoptive families. In addition to the anticipated themes, 2 adoption specific points were described. Parents of adopted children with DBMD place importance on communicating the diagnostic implication of DBMD to the biological parents. Second, adoptive parents who questioned thei (open full item for complete abstract)

Committee: Robert Hopkin M.D. (Committee Chair); Martha Walker (Committee Member) Subjects: Surgery

Master of Science (MS), Wright State University, 2024, Biochemistry and Molecular Biology

Duchenne Muscular Dystrophy (DMD) is a devastating progressive muscular disorder caused by a mutation in the dystrophin gene affecting approximately 1 in 3,500 males. However, despite its prevalence, there is currently no cure for this disease. Mutations in dystrophin lead to enhanced inflammation, fibrosis, cell death, development, and decreased skeletal muscle function. Phosphatidylcholine, synthesized by choline, is a major phospholipid that functions in maintaining and synthesizing cell membranes and has previously been found to be decreased in DMD patients. This led us to hypothesize that the dystrophic phenotype could be improved through treatment with choline. In this study, we used concentrated choline to treat B10 wildtype and mdx mice for both short-term and long-term treatments. We found that compared to untreated groups, treatment with choline showed less inflammation, fewer macrophage markers, and reduced fibrosis development within the skeletal and cardiac tissue of mdx mice. We also found that within skeletal muscle, necroptotic protein markers were downregulated as a result of choline treatment. We further evaluated the effects of choline treatment on dystrophic skeletal and cardiac muscle function to explore the potential mechanism of action. This study determined whether choline could be a potential therapeutic agent for the treatment of DMD.

Committee: Hongmei Ren Ph.D. (Advisor); Michael Craig Ph.D. (Committee Member); Weiwen Long Ph.D. (Committee Member) Subjects: Biochemistry; Histology; Pathology; Scientific Imaging

Master of Science, The Ohio State University, 1969, Graduate School

Committee: Not Provided (Other) Subjects:

Master of Science, The Ohio State University, 1970, Graduate School

Committee: Not Provided (Other) Subjects:

Doctor of Philosophy, The Ohio State University, 2024, Biomedical Sciences

Plasma membrane repair is an evolutionary conserved cellular mechanism observed in multiple tissue types including striated muscle, skin, gastrointestinal tract, and peripheral nervous system. Plasma membrane repair is critical following damage to restore the barrier function of the membrane and avoid cell death, and many diseases have a defect in the membrane repair mechanism that contributes to disease progression. Some of these diseases have an intrinsic gene mutation which cause the defect in cell membrane repair, and others have an extrinsic factor causing the defect in cell membrane repair. Amyloid beta (A) is a hallmark protein implicated in Alzheimer's Disease (AD) and A has an intimate relationship with the neuronal plasma membrane by which it alters the integrity of the membrane and has been shown to damage and penetrate the membrane, which would require an effective repair response to compensate for the damage to remain viable. We hypothesized neuronal death in AD is due at least in part to a defect in neuronal plasma membrane repair in due to A deposition. We discovered a neuronal membrane repair defect in cell types treated with A and AD patient CSF, and in ex vivo APP/PS1 mice. We determined A was the cause of the induced repair defect in CSF treated cells because when it was depleted the repair defect was rescued. Furthermore, we identified a decrease in dysferlin protein expression, a membrane repair protein, which was due to overactive autophagy. Lastly, we tested a novel therapeutic, recombinant human MG53, as a viable therapeutic to increase membrane repair and reduces neurotoxicity in vitro. Limb Girdle Muscular Dystrophy (LGMD) is a genetic muscle disease caused by a mutation in dysferlin which leads to dysferlin deficiency in cells. LGMD is known to have an intrinsic membrane repair defect due to the loss of dysferlin protein expression. We hypothesized there was an extrinsic factor that exacerbated the membrane repair defect in LGMD mus (open full item for complete abstract)

Committee: Noah Weisleder (Advisor); Christoph Lepper (Advisor); Olga Kokiko-Cochran (Committee Member); Nuo Sun (Committee Member); Loren Wold (Committee Member) Subjects: Biomedical Research; Cellular Biology; Molecular Biology; Neurobiology

Bachelor of Science (BS), Ohio University, 2024, Neuroscience

Duchene muscular dystrophy (DMD) is an X-Linked recessive genetic disorder which occurs in approximately 1/5000 XY births and is caused by a mutation in the human dystrophin gene. DMD causes many physiological defects and drastically shortens the lifespan of those afflicted by it. Gene replacement therapies are in clinical trials, but traditional therapies are still needed whilst those are developed. The C. elegans dys-1 gene is highly conserved compared to human dystrophin, and mutations in C. elegans dys-1 produces a clinically relevant phenotype. By use of the C. elegans DMD model, the pathology of DMD can easily be studied in a laboratory setting, allowing various potential traditional treatments to be tested for effectiveness. Zoledronic Acid (ZA) is approved by the U.S. Food and Drug Administration (FDA) to treat osteoporosis in patients with DMD, and preliminary data suggests that ZA could have positive effects for muscular health in DMD patients without osteoporosis. In the present work, it was found that the drug ZA is effective in improving DMD health in the C. elegans DMD model. Additionally, it was found that ZA improves DMD health through lowering the increased Ca2+ levels classically found in dys-1 mutants, which along with further experimentation informs a potential mechanistic pathway through which ZA acts. With this data, it is hoped that a clinical trial for repurposing ZA for use in for DMD patients is conducted in order to lessen the suffering caused by this disorder whilst gene therapeutics are developed.

Committee: Nathaniel Szewczyk (Advisor); Corinne Nielsen (Advisor) Subjects: Neurosciences

Doctor of Philosophy, The Ohio State University, 2024, Molecular Genetics

Duchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutation of the Dystrophin gene resulting in complete absence of the dystrophin protein. Loss of dystrophin destabilizes the muscle membrane and increases muscle's susceptibility to injury. Over time, continuous rounds of muscle insult, muscle degeneration, and regeneration ultimately exhaust muscle's regenerative and reparative capacity and leads to loss of muscle function. There is currently no cure for DMD and there are many significant hurdles in the field for dystrophin-restoration based therapies. Thus, there is a critical need to develop new therapeutics. Genome-wide association studies (GWAS) have uncovered putative genetic modifiers of DMD. One identified class of modifiers are regulators of TGFb signaling. In Chapter 2, we use pharmacological and genetic means to investigate TGFb signaling as a modifier of DMD. Here I show that inhibition of TGFb using TGFb receptor inhibitors LY364947 and SB431542 leads to significant improvement in birefringence intensity, a read out of muscle organization, but causes developmental defects. Modifiers uncovered by GWAS include allelic variants that decrease expression of TGFb regulators latent TGFb binding protein 4 (LTBP4, ltbp4 in zebrafish) and thrombospondin-1 (THBS1, thbs1a and thbs1b in zebrafish) and correlate with decreased TGFb bioavailability and a less severe DMD phenotype. To model the decreased expression alleles of LTBP4 and THBS1 we used zebrafish mutants and CRISPR/Cas-9 F0 'crispants' of ltbp4, thbs1a, and thbs1b and show that mutation of thbs1b or ltbp4 improves survivorship in dmd mutant embryos and combinatorial mutation of thbs1a and ltbp4 leads to improved birefringence intensity, an indiciator of muscle organization. Together, these findings demonstrate that inhibition of TGFb signaling is protective in dmd mutant zebrafish. GWAS for DMD modifiers have also identified SNPS that lie in non-coding putative regulato (open full item for complete abstract)

Committee: Sharon Amacher (Advisor); Martin Haesemeyer (Committee Member); Harold Fisk (Committee Member); Susan Cole (Committee Member) Subjects: Biology; Genetics

MS, University of Cincinnati, 2024, Medicine: Genetic Counseling

Current literature has highlighted a diagnostic delay for Duchenne muscular dystrophy (DMD), clinical and familial needs for an early diagnosis, and increasing support for the inclusion of DMD on newborn screening (NBS) panels. Our aim was to investigate whether early diagnosis is associated with a delay in disease progression outcomes in male siblings with DMD. We conducted a retrospective chart review of 42 siblings sets and compared their disease courses. The primary predictor was age of diagnosis. Primary clinical disease outcomes included ages at loss of ambulation (LoA), first abnormal cardiac finding, first abnormal pulmonary finding, and North Star Ambulatory Assessment (NSAA) scores at age 8 years. The median age at last visit was 14.4 years and 10.5 years for the older and younger sibling cohorts, respectively. Median age of diagnosis in the younger sibling cohort was 2.04 years and was significantly earlier than the median age for the older sibling cohort, 4.96 years (p < 0.001). Corticosteroid treatment was initiated at a median age of 6.40 years for the older siblings and 4.45 years for the younger siblings (p <0.001). Age of diagnosis was not a predictor for the four primary outcomes. Although, advanced diagnostic age may be associated with increased time to perform the Gowers sign at age 8 years (p = 0.059). Though, we were unable to conclude that age of diagnosis is a predictor for disease progression outcomes in this cohort, further studies with longer follow-up times are warranted to provide a more accurate understanding of the impact of early diagnosis on disease course in this population.

Committee: Melanie Myers Ph.D. (Committee Chair); Chinmayee Bhimarao Nagaraj M.S. (Committee Member); Cuixia Tian M.D. (Committee Member); Valentina Pilipenko Ph.D. (Committee Member); Niki Armstrong M.S. (Committee Member) Subjects: Medicine

Doctor of Philosophy (PhD), Wright State University, 2023, Biomedical Sciences PhD

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder that affects 1 in 3500 male births worldwide. It is characterized by mutations in the dystrophin gene that results in the loss of functional dystrophin. Dystrophin deficiency leads to instability of the sarcolemma alongside increased inflammation, necrosis and fibrosis resulting in membrane rupture and eventual muscle fiber death. There are currently no effective treatments for DMD. Here we show that Lipin1 expression is significantly downregulated at the mRNA and protein levels in gastrocnemius muscle of mdx mice, the DMD mouse model. Lipin1 has a dual function as a phosphatidic acid phosphatase (PAP) regulating phospholipids and triacylglycerol biosynthesis, but also as a transcriptional cofactor. In this study, we evaluated the role of lipin1 in dystrophic muscle by characterizing two mouse models, Dystrophin/Lipin1-DKO mice and MDX:Lipin1Tg/0 transgenic mice. Further depletion of Lipin1 in our Dystrophin/Lipin1-DKO model showed worsened disease phenotype through increased inflammation, fibrosis, and necroptosis. In contrast, restoration of Lipin1 in our MDX:Lipin1Tg/0 model showed a significant improvement in skeletal muscle health through reduced inflammation, fibrosis, and necroptosis through improved membrane integrity. Altogether, our study showed that Lipin1 could be a potential therapeutic target for DMD.

Committee: Hongmei Ren Ph.D. (Advisor); Weiwen Long Ph.D. (Committee Member); Shulin Ju Ph.D. (Committee Member); Mark Rich Ph.D. (Committee Member); Michael Leffak Ph.D. (Committee Member) Subjects: Biochemistry; Biology; Cellular Biology; Molecular Biology

Master of Science (MS), Wright State University, 2023, Biochemistry and Molecular Biology

Duchenne muscular dystrophy (DMD) is a severe and progressive muscular dystrophy that develops in the skeletal muscles because of mutations in the dystrophin gene. Dystrophin stabilizes sarcolemma and assembles neuronal nitric oxide synthase (nNOS) into the dystrophin-associated protein complex on the sarcolemma. The absence of dystrophin triggers the delocalization of nNOS and contributes to the misregulation of muscle development, blood flow, muscle fatigue, and inflammation. Lipin1 was reduced in the skeletal muscles of patients with DMD and the mdx mouse model of DMD. In this study, we explored the role of lipin1 in the regulation of nNOS expression and function. We found that Lipin1 deficiency leads to the downregulation of nNOS protein and gene expression levels, while overexpression of lipin1 elevated nNOS protein and gene expression levels. We also found that Lipin1 upregulated nNOS gene expression by coactivating PPARα and binding to the promoter region of nNOS. Lipin1 deficiency leads to muscle fatigue in lipin1Myf5cKO mice, possibly due to the downregulation of nNOS. Most importantly, overexpressing lipin1 in dystrophic muscle improved muscle fatigue in our mdx:lipin1 transgenic mice which may be through the restoration of nNOS expression.

Committee: Hongmei Ren Ph.D. (Advisor); Michael Markey Ph.D. (Committee Member); Weiwen Long Ph.D. (Committee Member) Subjects: Biochemistry; Biology

Doctor of Philosophy (PhD), Wright State University, 2023, Engineering PhD

Blood flow dynamics plays a critical role in maintaining tissue health, as it delivers nutrients and oxygen while removing waste products. It is especially important when there is a disruption in cerebral autoregulation due to trauma, which can induce ischemia or hyperemia and can lead to secondary brain injury. Thus, there is a need for noninvasive techniques that can allow continuous monitoring of blood flow during intervention. Optical techniques have become increasingly practical for measuring blood flow due to their non-invasive, continuous, and relatively lower-cost nature. This research focused on developing a low-cost, scalable optical technique for measuring blood flow by implementing speckle contrast optical spectroscopy using a fiber-camera-based approach. This technique is particularly well-suited for measuring blood flow in deep tissues, such as the brain, which is challenging to access using traditional optical methods. A two-channel continuous wave speckle contrast optical spectroscopy device was developed, and the device was rigorously tested using phantoms. Then, it is applied to monitor blood flow changes in the brain following traumatic brain injury (TBI) in mice. The results indicate that trauma-induced significant blood flow decreases consistent with the recent literature. Overall, this approach provides noninvasive continuous measurements of blood flow in preclinical models such as traumatic brain injury.

Committee: Ulas Sunar Ph.D. (Advisor); Tarun Goswami Ph.D. (Committee Member); Keiichiro Susuki Ph.D. (Committee Member); Robert Lober M.D., Ph.D. (Committee Member) Subjects: Biomedical Engineering; Biomedical Research; Biophysics; Engineering; Optics

Master of Science (MS), Wright State University, 2022, Biochemistry and Molecular Biology

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder that is characterized by severe and progressive muscle wasting (Venugopal & Pavlakis, 2021). This disease is caused by a mutation in the largest known human gene which encodes the protein, dystrophin (Gao & McNally, 2015). Dystrophin connects the inner cytoskeleton to the extracellular matrix and is critical for maintaining the structural stability of muscle cells during contraction (Venugopal & Pavlakis, 2021). Mutations to the dystrophin gene result in myocyte membrane instability, contributing to the structural deterioration of the muscle tissue (Venugopal & Pavlakis, 2021). Progressive muscle degeneration and the replacement of muscle fibers with fibrotic tissue negatively impacts muscle contractility and is particularly detrimental to health when essential muscles such as the diaphragm are affected (Mann et al., 2011). Respiratory failure is a hallmark of DMD and is one of the leading causes of mortality associated with this disease (Venugopal & Pavlakis, 2021). Currently there is no cure for Duchenne Muscular Dystrophy, and gene therapy approaches are limited by the sheer size of the dystrophin gene which spans across 2,200 kb of DNA (Gao & McNally, 2015). Previous data generated from the laboratory has shown that the mdx mouse (used to model DMD) displays reduced expression of lipin1 (Unpublished data). Additionally, other works have shown that skeletal muscle specific lipin1 knockout mice present muscle membrane instability (Sattiraju et al., 2020). Collectively, these findings suggest the potential for lipin1 to serve as an alternative therapeutic target in the dystrophic diaphragm. Within the membrane of the endoplasmic reticulum, lipin1 functions as a phosphatidic acid phosphatase (PAP), which catalyzes the conversion of phosphatidic acid (PA) to diacylglycerol (DAG), a reaction important for membrane phospholipid and triacylglycerol synthesis (Chen et al., 2014). Current data sugg (open full item for complete abstract)

Committee: Hongmei Ren Ph.D. (Advisor); Weiwen Long Ph.D. (Committee Member); Michael Leffak Ph.D. (Committee Member) Subjects: Biochemistry; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2022, Biomedical Sciences

Muscle function consists of two critical components. The first is the level of force that is developed, and the underlying mechanisms, are well studied and well, albeit not completely, understood. The second component is kinetics, i.e., the speed at which force is developed and dissipates, which is less extensively studied, and less well understood. This work takes a multi-faceted approach in studying contractile kinetics in striated and smooth muscle in health and disease. In striated muscle, muscles function at sub-maximal levels in vivo, whereas maximal tetanic contractions are most commonly used to assess and report skeletal muscle function in muscular dystrophy studies. At submaximal activation, both the force and kinetics of contraction and relaxation are heavily impacted by the kinetics of the single twitch. To investigate the effect of muscle disease on twitch contraction kinetics, isolated diaphragm, and extensor digitorum longus (EDL) muscles of 10-, 20-week, "het" (dystrophin deficient and utrophin haplo-insufficient), and 52-week mdx (dystrophin deficient) mice were analyzed and compared to wild-type controls. We observed that twitch contractile kinetics are dependent on muscle type, age, and disease state. Differences in kinetics yielded greater statistical significance compared to previously published maximal tetanic force measurements, thus, using kinetics as an outcome parameter could potentially allow for use of smaller experimental groups in future study designs. We applied this knowledge to investigate if kinetics can be regulated via troponin-C (TnC) Ca2+ sensitivity. To investigate this key question, we measured the EDL and soleus force-frequency response (FFR) in a murine model with a TnC mutation (expressed after TnCL48QAAV or TnCD73NAAV injection) that causes an increased calcium sensitivity (L48Q) or a decreased calcium sensitivity (D73N) and compared the FFR to the wildtype counterparts. The kinetics of the twitch contraction are im (open full item for complete abstract)

Committee: Paul Janssen (Advisor); Philip Binkley (Committee Member); Mark Ziolo (Committee Member); Jill Rafael-Fortney (Committee Member); Douglas Kniss (Committee Member) Subjects: Physiology

Doctor of Philosophy, The Ohio State University, 2021, Biomedical Sciences

In muscle cells, action potentials originating from the neuromuscular junction cause release of calcium from intracellular stores that triggers muscle contraction, a process known as excitation contraction (EC) coupling. Formation of specialized membrane structures during skeletal muscle development collectively known as the triad junction is absolutely required for the regulation and maintenance of the calcium cycling that occurs during EC coupling. The triad junction is a well-organized anatomical structure consisting of the transverse-tubule invagination of the plasma membrane and the terminal cisternae of the sarcoplasmic reticulum that stores the intracellular calcium. Many skeletal muscle diseases such as limb girdle muscular dystrophy and miyoshi myopathy present muscle weakness and fatigue phenotypes. Skeletal muscle structure defects are observed in these diseases and are postulated to be an underlying pathophysiological cause. In this project we investigated whether over-expression of Mitsugumin 29 (MG29), a protein intricately involved the biogenesis of triad structures, can improve disease phenotypes in a mouse model of limb girdle muscular dystrophy (A/J mice). We have found that adeno-associated virus (AAV) delivery of FLAG tagged MG29 (FLAG-MG29) in skeletal muscle was able to significantly improve the transverse-tubule network and triad junction structure in the A/J mice. These structural developments correlated with improvements in skeletal muscle calcium signaling as well as increased skeletal muscle contractile capacity. In addition to the critical roles in skeletal muscle development, we also tested if MG29 contributes to skeletal muscle regeneration following injury. Using cardiotoxin (CTX) induced-skeletal muscle injury, we observed that MG29 deficient mice have impaired muscle regeneration capacity compared to wild type littermates. We present evidence that functional interaction between MG29 and Bin1, another resident triad protein that pro (open full item for complete abstract)

Committee: Jianjie Ma PhD (Committee Co-Chair); Hua Zhu PhD (Committee Co-Chair); Renzi Han PhD (Committee Member); Willa Hsueh MD (Committee Member); Bryan Whitson MD, PhD (Committee Member) Subjects: Biology; Biomedical Research; Physiology

Doctor of Philosophy, The Ohio State University, 2021, Neuroscience Graduate Studies Program

Congenital muscular dystrophies (CMDs) are debilitating and frequently lethal disorders, affecting an estimated 1 - 9 / 100,000 individuals, and often caused by disruption of the muscle cell membrane-extracellular matrix (ECM) interface. While recombinant adeno-associated virus (rAAV) gene therapy has recently made progress in treating diseases involving small genes at a single locus, it currently has limited use for CMDs which may involve large genes or multiple loci. The goal of this work was to use ECM-binding domains to create novel gene therapies for the CMDs. Two hypotheses were formed: (1, Chapters 2 and 4) rAAV-mediated expression of chimeric linker proteins comprised of ECM-binding domains and membrane-binding domains will be therapeutic in mouse models of CMDs, and (2, Chapter 3) adding ECM-binding domains to trophic factors will increase their overall therapeutic effect in a mouse model of CMD. To test each hypothesis, the heparin-binding (HB) domain of heparin-binding epidermal growth factor-like growth factor (HB-EGF) was used to bind the plentiful heparan sulfate proteoglycans in the skeletal muscle ECM. Chapter 1 of this document introduces concepts important for understanding the work in Chapters 2 - 5. Mutations in LAMA2 cause type 1A CMD (MDC1A). The product of LAMA2, laminin-α2 anchors myofibers to the ECM, in part by binding the sarcolemma with its five globular (G) domains, and its loss disrupts the myofiber-ECM connection. LAMA2 exceeds the rAAV packaging limit, and therefore cannot be delivered for direct gene replacement therapy. In Chapter 2 of this dissertation, I sought to restore the myofiber-ECM connection by creating micro-laminin genes, LAMA2(G1-5) and HB-LAMA2(G1-5). These were made by fusing segments of the HBEGF gene to segments of the LAMA2 gene. Micro-laminin genes are small enough to be delivered by rAAV, but were expected to retain some of the sarcolemma-ECM bridging activity of laminin-α2. I found that HB-LAMA2(G1-5) express (open full item for complete abstract)

Committee: Paul Martin (Advisor); Jill Rafael-Fortney (Committee Member); Nicolas Wein (Committee Member); Stephen Kolb (Committee Member) Subjects: Biology; Genetics; Molecular Biology

PhD, University of Cincinnati, 2020, Medicine: Molecular and Developmental Biology

Vertebrate skeletal muscle is formed by the fusion of mononucleated progenitor cells into multinucleated myofibers, which comprise the functional units of the tissue and perform both contractile and metabolic functions. Muscle cell fusion is driven by the skeletal muscle-specific membrane protein Myomaker, which is expressed during development and is reactivated following injury to drive regenerative fusion. Myonuclei within the resulting syncytial myofibers must coordinate activity in order to accomplish the distinct tasks necessary for cellular function, but understanding of myonuclear diversity has been lacking due to the technical difficulties of working with multinucleated cell types. We performed single-nucleus RNA-sequencing of skeletal muscle across the murine lifespan in order to achieve nuclear-level resolution of transcriptional dynamics during development, homeostasis, and aging. We uncovered the transient manifestation of distinct myonuclear transcriptional states in postnatal development, enriched for genes involved in myofibrillogenesis and sarcomere assembly, as well as their reemergence in aging muscle. In addition, our datasets comprise a nuclear atlas of skeletal muscle that serve as a platform for interrogation of rare myonuclear subpopulations such as the neuromuscular and myotendinous junctions, for which we identified numerous previously unknown enriched genes. Functional testing of novel postsynaptic genes using an siRNA knockdown screen in C2C12 myoblasts generated validated hits as well as proof-of-concept for our datasets as a resource for gene discovery and elucidation of cellular mechanisms. We also sought to investigate the consequences of ongoing cell fusion in chronic muscle pathology. Genetic muscle diseases such as Duchenne muscular dystrophy (DMD) are characterized by ongoing fusion of activated satellite cells (SCs). Using the mdx mouse model of DMD, we assessed the role of cell fusion by inducible knockout of Myomaker in eithe (open full item for complete abstract)

Committee: Douglas Millay Ph.D. (Committee Chair); Steven Crone Ph.D. (Committee Member); Jeffery Molkentin Ph.D. (Committee Member); Sakthivel Sadayappan Ph.D. (Committee Member); Kathryn Wikenheiser-Brokamp M.D. (Committee Member) Subjects: Molecular Biology

MS, University of Cincinnati, 2020, Medicine: Clinical and Translational Research

Background: Osteoporosis and vertebral fractures are common in patients with Duchenne Muscular Dystrophy (DMD) treated with glucocorticoids. Bisphosphonates (BP) have been used in pediatric patients for treatment of osteoporosis. However, the long-term effects of oral BP therapy on vertebral morphometry and fractures in patients with DMD and glucocorticoid-induced osteoporosis are not known. Methods: We retrospectively studied patients with DMD who had been treated with oral BP for glucocorticoid-induced osteoporosis at a tertiary-care pediatric center between 2010 and 2017. Demographic data, and glucocorticoid and oral BP treatment histories were obtained from electronic medical records. Treatment outcomes were changes in lumbar (L1-L4) vertebral morphometry and fractures, as assessed by the Genant semi-quantitative method. Patients were included if they had both baseline, within 6 months prior to start of oral BP treatment, and at least one follow-up spine radiograph available for review. The primary outcome was the prevalence of vertebral fractures and the secondary outcome was change in Genant grading during treatment. Results: Fifty-eight patients with DMD (median age 12.2 years, range 5.5-19.6 years) were treated with glucocorticoids for a median duration of 4.7 years (range 1.3-12.6 years) and 33% were non-ambulatory at BP start. Vertebral fractures, defined by Genant grading =1, were present in 6-19% of L1-L4 vertebrae at baseline. Among patients who had radiographs at baseline and 1 year-post BP start, the prevalence of L1-L4 vertebral fractures remained stable post treatment (n=43; 5-18% at baseline vs 8-18% at 1 year, p value 1.00). The prevalence of vertebral fractures at each year up to 5 years was also not statistically different from that of baseline (p-value 0.08-1.00). Among patients who had serial radiographs for comparison, longitudinal examination showed no change in Genant grading in the majority of vertebrae (64-80%) up (open full item for complete abstract)

Committee: Patrick Ryan Ph.D. (Committee Chair); Jane Khoury Ph.D. (Committee Member); Meilan Rutter (Committee Member) Subjects: Surgery

Doctor of Philosophy, The Ohio State University, 2020, Biochemistry Program, Ohio State

The plasma membrane repair response is a fundamental mechanism necessary for all living cells. Significantly, membrane repair is critical for individual cell survival, but it is also a contributing pathological factor in several disorders affecting multiple cell types and organs. Duchenne Muscular Dystrophy (DMD) and Limb-Girdle Muscular Dystrophy type 2B (LGMD2B) are two disorders that exhibit an exhausted or compromised membrane repair defect that exacerbates a patient's symptoms and outcome. This manuscript explores several projects that investigate new components of the membrane repair system generally, and specific applications to improve repair in cases of DMD and LGMD2B. A common strategy for membrane repair in mammalian cells, specifically in muscle derived fibers and cells, is the exocytosis of intracellular vesicles to the injury site. We find evidence that the GLUT4 containing vesicles and the glucose uptake regulators AS160, rab8A, and rab10 localize to injury sites and contribute to the membrane repair response in myoblasts and myofibers. We also find that by activating rab10, we can improve repair in skeletal muscle fibers of the DMD mouse model (mdx). Other major findings described in this manuscript are a panel of poloxamer compounds that can improve the membrane repair capacity of cells and myofibers. P188 is the most common poloxamer in this family used to alleviate symptoms in models of DMD and LGMD2B, but we find that other poloxamers share the same resealing properties as P188. In cases of LGMD2B and other dysferlinopathies where the membrane repair protein dysferlin is not expressed, gene therapy attempts to reintroduce the dysferlin gene have been unsuccessful, in part due to the large size of the dysferlin gene. We find evidence that truncated “nano-dysferlin” constructs can display membrane repair improvements similar to the wild type dysferlin protein. Additionally, the C2A domain of dysferlin, along with its transmembrane reg (open full item for complete abstract)

Committee: Noah Weisleder (Advisor); Jill Rafael-Fortney (Committee Member); Federica Accornero (Committee Member); Ross Dalbey (Committee Member) Subjects: Biochemistry; Biomedical Research; Cellular Biology; Physiology

Doctor of Philosophy, The Ohio State University, 2019, Molecular, Cellular and Developmental Biology

Duchenne muscular dystrophy is a severe childhood-onset striated muscle disease that has no cure. The current treatment for skeletal muscle weakness has substantial side-effects. We have previously shown that treatment with drugs that inactivate the mineralocorticoid receptor (MR) improves skeletal muscle function in muscular dystrophy mice. To determine if these MR antagonists work through direct mechanisms in the skeletal muscle, we conditionally knocked out the myofiber MR in muscular dystrophy mice. Genetic ablation of MR improved skeletal muscle force and reduced fibrosis in muscular dystrophy mice similar to that observed with MR antagonist drugs. Additionally, MR antagonists stabilize fragile dystrophic skeletal muscle membranes in a MR independent manner, suggesting that these drugs have many benefits for skeletal muscle in muscular dystrophy. We then evaluated previously identified candidate MR responsive genes for their role in muscular dystrophy. None of the evaluated genes appeared to be direct myofiber specific MR targets. To investigate the role of the myofiber MR in normal muscle biology, we acutely injured the skeletal muscle of myofiber MR conditional knockout mice on a wild-type background. We found for the first time that acutely injured skeletal muscle has MR hormonal regulation and genetic ablation of the MR temporarily stabilized damaged myofibers at four days after acute muscle injury. Pharmacological inhibition of the MR with MR antagonist treatment delayed normal muscle repair in acute injury, suggesting additional roles for MR in other cell types in skeletal muscle contribute to regeneration after acute injury. These results have implications for MR modulation after acute and chronic skeletal muscle injuries.

Committee: Jill Rafael-Fortney (Advisor); Lawrence Kirschner (Committee Member); Sharon Amacher (Committee Member); Paul Martin (Committee Member) Subjects: Cellular Biology; Molecular Biology; Physiology