The nuclear matrix (NM) is proposed to be a permanent network of core filaments underlying thicker fibres, present regardless of transcriptional activity. It is found to be both RNA and protein rich; indeed, numerous important nuclear proteins are components of the structure. In addition to mediating the organisation of entire chromosomes, the NM has also been demonstrated to tether telomeres via their TTAGGG repeats. In order to examine telomeric interactions with the NM, a technique known as the DNA halo preparation has been employed. Regions of DNA that are tightly attached to the structure are found within a so-called residual nucleus, while those sequences forming lesser associations produce a halo of DNA. Coupled with various FISH methodologies, this technique allowed the anchorage of genomic regions by the NM, to be analysed. In normal fibroblasts, the majority of chromosomes and telomeres were extensively anchored to the NM. Such interactions did not vary significantly in proliferating and senescent nuclei. However, a decrease in NM-associated telomeres was detected in quiescence. Since lamin A is an integral component of the NM, it seemed pertinent to examine chromosome and telomere NM-anchorage in Hutchinson-Gilford Progeria Syndrome (HGPS) fibroblasts, which contain mutant forms of lamin A. Indeed, genome tethering by the NM was perturbed in HGPS. In immortalised HGPS fibroblasts, this disrupted anchorage appeared to be rescued; the implications of this finding will be discussed. This study also suggested that telomere-NM interactions are aberrant in X-linked Emery-Dreifuss Muscular Dystrophy (X-EDMD), which is caused by mutant forms of emerin, another NM-associated protein. The positioning of selected genes in control and X-EDMD cell lines was examined in un-extracted nuclei using 2D and 3D FISH. Subtle shifts in the organisation of these genes were detected in diseased cells; however, their expression levels remained unaltered. Furthermore, in order to examine the architectural integrity of the nuclear lamina in lamin A and emerin mutant cell lines, scanning electron microscopy (SEM) was employed. This work revealed that such structures were indeed compromised in disease. The findings presented in this thesis highlight the importance of lamin A and emerin in mediating the organisation of the genome and taken together, promote the hypothesis that dysfunctional NM dynamics may well contribute to disease pathology.

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.