Thesis advisor: Eric S. Folker / Thesis advisor: David R. Burgess / Skeletal muscle is a multinucleated cell type in which the many nuclei are precisely positioned to maximize the distance between adjacent nuclei. In order to reach this final positioning, nuclei undergo an elaborate set of movements during muscle development. The disruption of this process is evident throughout muscular dystrophies and myopathies. However, the contribution of aberrant nuclear positioning toward disease progression is unclear and the mechanisms regulating nuclear movement and positioning are poorly defined. The goal of this thesis is to determine the contribution of disease-linked genes to the regulation of nuclear movement and positioning and how these mechanisms are coordinated in skeletal muscle. In this thesis, we utilize Drosophila melanogaster skeletal muscle as an in vivo model system to investigate nuclear positioning throughout muscle development and correlate aberrant nuclear positioning with a decrease in muscle function. We provide the first evidence of distinct mechanisms that are independently regulated by genes that are associated with two different muscle diseases, Emery-Dreifuss muscular dystrophy and Centronuclear myopathy (Chapter 2). We also provide evidence that Emerin-dependent regulation of the LINC complex is a critical determinant of nuclear positioning and for the first time demonstrate a division of Emerin functions among the two Drosophila emerin homologs, bocksbeutel and otefin (Chapter 3). Finally, we conduct a proof-of-concept screen to identify novel regulators of muscle development and function (Chapter 4). Together, the work presented in this thesis provides a framework to further our understanding of the mechanisms regulating nuclear movement and positioning as well as muscle development as a whole. Using the tools and techniques developed throughout this thesis, we provide novel insight into the mechanisms regulating nuclear movement and positioning and strengthen Drosophila as an in vivo model for investigating muscle development and function. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Myotonic dystrophy type 1 (DM1) is the most common form of adult muscular dystrophy (1:8000) and is caused by an abnormal expansion of CTG repeats in the 3’ untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The expanded repeats of the DMPK mRNA forms hairpin structures which sequester RNA-binding proteins (RBP) in intranuclear foci, such as the splicing regulator muscleblind-like 1 (MBNL1), which results in aberrant splicing of several mRNAs and underlie, at least in part, DM1 pathogenesis. It has been previously shown that disaggregating these RNA foci repletes free and thus functional MBNL1, rescuing DM1 spliceopathy and alleviating associated signs and symptoms such as myotonia. Importantly, the direct upregulation of MBNL1 has comparable beneficial outcomes. The focus of this thesis was to develop novel and practical therapeutic avenues for DM1 by employing high-throughput screening technology to identify key pathways and small molecule candidates which reduce CUG foci in patient cells, and ultimately correct DM1 spliceopathy and associated signs in vivo. First, a high-throughput kinome screen using an siRNA library targeting 692 kinase subunits identified PACT, HIPK4, and PKA2β as candidates for reducing CUG foci in patient fibroblasts. Knockdown of each gene resulted in a partial reduction in CUG foci, but ultimately did not correct aberrant splicing of insulin receptor (IR) or sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA1), two genes which are typically misspliced in DM1. A second set of screens focused on testing small molecules, several of which are FDA-approved for clinical use, in an effort to expedite drug discovery. One approach was to data-mine from a previously completed chemical screen, which used system-wide RNA sequencing to establish drug-gene interactions in mouse neuronal cultures treated with blood brain barrier-penetrant drugs, and specifically look for compounds which downregulate DMPK mRNA or upregulate MBNL mRNA (MBNL1 and MBNL2). No compounds were found to downregulate DMPK mRNA. However, several compounds upregulated MBNL mRNAs; the activity of one of these, nilotinib, was validated in human DM1 fibroblasts and converted myoblasts, mediating a small correction in SERCA1 spliceopathy. Administration of nilotinib to unaffected mice did not result in in vivo MBNL gene upregulation in mouse skeletal muscle, as was seen in vitro. Further testing of nilotinib in DM1 in vivo models is required. A final set of chemical screens in patient myoblasts using an FDA-approved drug library and a chemogenomic drug library identified several HDAC inhibitors which reduced foci and rescued SERCA1 spliceopathy in vitro in DM1 differentiated myoblasts. Of these, vorinostat (SAHA) was further tested in a mouse model of DM1 (HSALR), proving safe and effective in correcting aberrant muscle pathology as well as splicing defects of RYR1, SERCA1, and CLCN1. Functional validation, such as myotonia, remains to be completed, but given the strong evidence for CUG foci reduction and splicing correction, vorinostat has emerged as a promising novel candidate for DM1 therapy.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18870号 / 医博第3981号 / 新制||医||1008(附属図書館) / 31821 / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 瀬原 淳子, 教授 中畑 龍俊 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(社会健康医学) / 甲第20057号 / 社医博第75号 / 新制||社医||9(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 山田 亮, 教授 小泉 昭夫, 教授 松田 文彦 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20788号 / 医博第4288号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 良輔, 教授 高橋 淳, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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