Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--Ohio State University, 2007.

Mutations in the gene encoding the nuclear lamina protein lamin A/C (LMNA) and the associated integral inner nuclear membrane protein emerin (EMD) give rise to similar disease phenotypes and are both classified as Emery-Dreifuss muscular dystrophy (EDMD). However, the connection between the function of these nuclear envelope proteins and disease phenotype remains elusive. Given the consistent manifestation of dilated cardiomyopathy in EDMD, my investigation focused on deciphering the roles of these nuclear envelope proteins in the heart. To better understand their functions, I generated a set of isogenic human induced pluripotent stem cell (iPSC) lines with either LMNA mutation causing lamin A/C haploinsufficiency or EMD mutation causing emerin deficiency. I differentiated these iPSCs into cardiomyocytes (iPSC-CMs) and obtained their RNA transcript and protein expression profiles. I found that both mutant lines exhibited significant overlap in transcriptome and proteome changes. Analyzing alterations at both RNA and protein levels shed light on the potential functional roles of lamin A/C and emerin in cardiomyocytes and pathogenic mechanisms. To better understand the cardiac defects caused by loss of lamin A/C. I generated mice lines with tissue-specific and temporally regulated knockout of Lmna in the heart. The mutant mice experienced lethality due to heart failure, regardless of whether Lmna was knocked out at the embryonic or mature adult heart. This demonstrates that lamin A/C has a vital role in the normal function of cardiomyocytes.

La glycoprotéine M (gM) est une protéine virale transmembranaire qui est conservée dans la famille des Herpesviridae. Malgré son rôle non essentiel in vitro chez la plupart des virus de la sous-famille des Alphaherpesvirinae, dont l’herpès simplex de type 1 (VHS-1), gM est impliquée à plusieurs étapes de leur cycle viral et sa déplétion entraine une diminution de la production virale. Pour effectuer ses diverses fonctions, gM est ciblée dynamiquement à plusieurs compartiments cellulaires au cours de l’infection, dont le noyau, le réseau trans-Golgi et la membrane plasmique. Chez le VHS-1, gM est la première glycoprotéine détectée aux membranes nucléaires, et ce, dès 4 heures après le début de l’infection. Des expériences effectuées précédemment dans notre laboratoire ont démontré que la localisation de gM au noyau à 4hpi est un processus actif, viral-dépendant et spécifique qui succède sa traduction au réticulum endoplasmique. Or, sa fonction au noyau n’est toujours pas élucidée, ni le mécanisme lui permettant d’atteindre ce compartiment tôt durant l’infection. D’ailleurs, aucun des partenaires d’interaction connus de gM n’a été identifié comme participant à ce ciblage, soulevant des questions quant au mécanisme utilisé par la glycoprotéine virale pour atteindre le noyau. Notre hypothèse est que gM emprunte le transport nucléocytoplasmique de la cellule pour être activement ciblée à la membrane nucléaire interne par l’intermédiaire des importines. Afin d’étudier le rôle des importines dans la localisation de gM tôt dans l’infection, chaque importine a été déplétée par ARN interférent dans des cellules 143B. À la suite d’une infection de 4h, la localisation de gM a été déterminée par microscopie confocale suivie d’analyses qualitatives en 2D et en 3D. Les résultats obtenus suggèrent que les importines ne participent pas significativement au ciblage de gM aux membranes nucléaires à cette étape de l’infection. Ces observations ouvrent la porte à d’autres mécanismes de transport qui devront être étudiés afin de mieux comprendre le ciblage de gM à ce compartiment et, éventuellement, y déterminer son ou ses rôles dans le cycle viral de l’herpès. / Glycoprotein M (gM) is a viral transmembrane protein that is conserved in the Herpesviridae family. Despite its non-essential role in vitro in most viruses of the Alphaherpesvirinae subfamily, including herpes simplex virus type 1 (HSV-1), gM is involved at several stages of their viral cycle and its depletion leads to a decrease in viral production. To perform its various functions, gM is dynamically targeted to several cellular compartments during infection, including the nucleus, the trans-Golgi Network and the plasma membrane. In HSV-1, gM is the first glycoprotein detected at nuclear membranes as early as 4 hours after the onset of infection. Previous experiments conducted in our laboratory have shown that the localization of gM to the nucleus at 4hpi is an active, viral-dependent and specific process that follows its translation at the endoplasmic reticulum. However, its function at the nucleus is still not elucidated, nor is the mechanism by which it reaches this compartment early during infection. Moreover, none of gM's known interacting partners have been identified as participants in this targeting, raising questions about the mechanism used by the viral glycoprotein. Our hypothesis is that gM takes advantage of the nucleocytoplasmic transport of the cell to be actively targeted to the inner nuclear membrane via importins. In order to study the role of the importins in the localization of gM early in the infection, each importin was depleted by interfering RNA in 143B cells. After a 4-hour infection, gM localization was determined by confocal microscopy followed by 2D and 3D qualitative analysis. The results obtained from these experiments suggest that importins do not significantly participate in the targeting of gM to nuclear membranes at 4hpi. These observations open the door to other transport mechanisms that will need to be studied in order to better understand the targeting of gM to this compartment and to eventually determine its role(s) in the herpes viral cycle.

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