Radial chromosome positioning in interphase nuclei is non-random and can alter according to developmental, differentiation, proliferation or disease status. The aim of this thesis is to understand how chromosome re-positioning is elicited and to identify the nuclear structures that assist this re-localisation event. By positioning all human chromosomes in primary fibroblasts that have left the proliferative cell cycle, the study within this thesis has demonstrated that in cells made quiescent by reversible growth arrest, chromosome positioning is altered considerably. Upon removal of serum from the culture medium, chromosome re-positioning took less than 15 minutes, required energy and was inhibited by drugs affecting the polymerization of myosin and actin. The nuclear distribution of nuclear myosin 1β was dramatically different in quiescent cells as compared to proliferating cells. If the expression of nuclear myosin 1β was suppressed using interference RNA procedures the movement of chromosomes after 15 minutes in low serum was inhibited. When high serum was restored to the serum starved cultures chromosome repositioning was only evident after 24-36 hours that coincided with a return to a proliferating distribution of nuclear myosin 1β.

The eukaryotic interphase nucleus is well organised and the genome positioned non-randomly. Nuclear structure is an important regulator of genome behaviour and function. Genome organisation and nuclear structure are compromised in diseases such as cancer and laminopathies. This study was to find out and to determine if there is any functional relationship between nuclear structure and genome mis-organisation in cancer cells. I have assessed the presence and distribution of specific nuclear structural proteins (A-type, B-type lamins and its receptor LBR, many of their binding proteins such as MAN1, LAP2α, LAP2, and Emerin and other nuclear proteins (PML, Nucleolin, and Ki67) using indirect immunofluorescence. From this study, it is found that the nuclear structure of breast cancer cells is often altered. The most severely affected proteins are the nuclear lamins B1 and B2 and they found as large foci within the nucleoplasm with little LBR expression to localise the lamin B. I also assessed the chromosome positioning (HSA 7, 10, 11, 14 and 17) and gene positioning (AKT1, CCND1, HSP90AA1, EGFR, ERRBB2/HER2 and PTEN) in breast cancer cell lines (T-47D, GI-101, Sk-Br-3 and BT-474) and in normal breast cell lines (MCF-10A) using 2D-FISH technique. I also assessed the position of the genes in nuclei and correlated with gene expression using qRT-PCR. Breast cell lines have treated with a drug named lovastatin and it was found that the cells have restored LBR expression and localisation of lamin B, leading to altered gene positioning and changed expression of breast cancer genes. Since the drug (lovastatin, 12 μM/48 hours) affects the prenylation as a post-translation modification process and lamins B biosythensis, it is found that B-type lamins and its receptor expression and distribution were improved and increased in expression by 2-fold in expression levels in the most affected cells (T-47D, and BT-474) compared to the normal cells (MCF-10A) and these cells also showed abnormal nuclei and dead cells. When analysing the nuclear positioning of the genes (AKT1, HSP90AA1 and ERRBB2/HER2), it is found that AKT1 was positioned periphery in BT-474 and T-47D cells and interiorly in the normal cells (MCF-10A) before treatment whereas the same gene was positioned periphery in T-47D and MCF-10A cells and interiorly in BT-474 after treatment with lovastatin. It is also found that HSP90AA1 was positioned periphery in MCF-10A and T-47D cells and interiorly in BT-474 cells before and after treatment (no change). Moreover, ERRBB2/HER2 gene was positioned periphery in T-47D and BT-474 cells and interiorly in MCF-10A cells before treatment whereas the same gene was positioned periphery in MCF-10A and T-47D cells and interiorly in BT-474 after treatment with the same drug. Regarding LMNB1, LMNB2, and LBR genes, the study focussed only on their expression levels and no work has done on their chromosome positioning as well as gene position before and after treatment. These three genes were over expressed when assessed by measuring the relative and fold changes in expression. Therefore, it is suggestive that 2D-FISH experiment to assess their localisation and their specific chromosome territories is required. The results shown in this thesis demonstrate the importance and roles of nuclear architecture specifically nuclear lamins and the integral nuclear membrane proteins (B-type lamins and LBR) in mediating correct genome organisation and function. The breast normal (immortalised cells) and cancerous cell lines showed different nuclear structures as lamin B affect the position of specific target chromosomes and genes. These results will strength the finding that the nuclear lamina is a significant nuclear structure which associates, organises, and regulates numerous vital nuclear processes and the stability of the genome.

Mutations in the gene encoding nuclear lamin A (LA) cause the premature aging disease Hutchinson-Gilford Progeria Syndrome. The most common of these mutations results in the expression of a mutant LA, with a 50-aa deletion within its C terminus. In this study, we demonstrate that this deletion leads to a stable farnesylation and carboxymethylation of the mutant LA (LAΔ50/progerin). These modifications cause an abnormal association of LAΔ507 progerin with membranes during mitosis, which delays the onset and progression of cytokinesis. Furthermore, we demonstrate that the targeting of nuclear envelope/lamina components into daughter cell nuclei in early G 1 is impaired in cells expressing LAΔ50/ progerin. The mutant LA also appears to be responsible for defects in the retinoblastoma protein-mediated transition into S-phase, most likely by inhibiting the hyperphosphorylation of retinoblastoma protein by cyclin D1/cdk4. These results provide insights into the mechanisms responsible for premature aging and also shed light on the role of lamins in the normal process of human aging.