In simple terms, copy number variations or CNVs are replications or deletions in the DNA which, in humans, changes it from the normal number of two gene copies. These CNVs are caused by inherited or de novo structural changes such as duplications, insertions or deletions of repeated portions of genetic material (Fig. 1). These duplications can vary from one to ten or more copies and range in size from 50 DNA base pairs to several million . Since their discovery in 1987 by Nakamura et al. , when they were initially named variable number tandem repeats, many studies have investigated their association with rare and common human diseases. Throughout evolution, some of these changes in copy number were beneficial such as the globin gene number duplication, while others such as the CNVs that cause Huntington's disease were not. In 2004, two landmark studies by Iafrate et al.  and Sebat et al.  found that large-scale copy-number variations, ranging in size from 100 kb to 2 Mb are common throughout the human genome, and that a high proportion of them are in known genes. These findings roused several association studies between CNVs and disease
Zn2+ is a broadly utilised ion in biology that has important catalytic, structural and regulatory roles within the cell. Zn2+ distribution in cells is maintained by zinc transporters, Zips and ZnTs, and disruptions in levels of Zn2+ have been associated with insulin resistance and muscle atrophy disorders. Zn2+ and reactive oxygen species (ROS) interact through inhibition of protein tyrosine phosphatases and ROS-mediated oxidation of the metal-binding metallothioneins (Mts) causing release of bound Zn2+, however the precise mechanisms are unclear. In the first study of this thesis addition of inhibitors of ROS-generating enzymes, superoxide dismutase 1 (SOD1) and NADPH oxidase 1 (NOX1) showed that only SOD1 inhibition increased short-term insulin-mediated Zn2+ release and increased the expression of Mt1 and 2. These results may suggest that ROS, in particular O2- accumulation through inhibition of SOD1, plays a role in insulin-mediated Zn2+ release. Inhibiting SOD1 prevents the conversion of O2- to H2O2 causing an accumulation of O2- in the cell which oxidises Mts to release Zn2+, thereby increasing Zn2+ levels within the cell. Manipulation of the expression of the zinc transporter Zip-7 has previously been shown to modulate cell signalling and glucose metabolism in C2C12 skeletal muscle cells, warranting further investigation into the role of Zn2+ within insulin signalling. Reducing Zip-7 expression when NOX1 was inhibited caused a decrease in Mt2 expression in response to insulin suggesting an interaction between insulin, Zip-7 and NOX1 activity but this requires further investigation. Skeletal muscle atrophy is a clinical symptom of insulin resistance and diabetes. Muscle atrophy is associated with increases in circulating glucocorticoid levels and accumulation of Zn2+ in muscle. This study investigates if Zn2+ homeostasis is disrupted in glucocorticoid-induced atrophy using C2C12 skeletal muscle cells treated with Dexamethasone (DEX) and iv insulin. Results demonstrate DEX-induced atrophy significantly increased the gene expression of the Mt1&2 and decreased glycogen accumulation when treated with insulin. Both confocal microscopy and flow cytometry showed significant increases in free cellular Zn2+ after DEX treatment. Notably, free Zn2+ levels observed with confocal microscopy increased after insulin treatment in control cells but decreased in DEX treated cells. Total cellular Zn2+ was increased by DEX treatment. This demonstrates that DEX causes Zn2+ accumulation in muscle cells and disrupts both Zn2+ homeostasis through blocking insulin-induced Zn2+ release, and insulin-induced glycogen synthesis. This raised the question of whether the same effects of atrophy on Zn2+ homeostasis apply to other cell systems. To investigate this, we examined adipose cells given that these too are involved in insulin resistance and muscle atrophy disorders. In this study we found similar increases in mRNA abundance of Mt1 & 2. Confocal microscopy revealed that DEX treatment caused changes in the distribution of free Zn2+ within peri-nuclear and cytosolic regions of the cell upon stimulation with insulin. Furthermore, investigation into morphometric changes using Oil Red O staining and particle analysis through Coherent Anti-Stokes Ramen Spectrophotometry (CARS) microscopy showed changes in cell and lipid droplet size consistent with reduced lipid turnover in DEX treated cells. These results highlight a potential mechanistic role for Zn2+ in the development of atrophy in 3T3-L1 adipocytes where increased free Zn2+ and its redistribution in cells may inhibit lipid metabolism downstream of insulin signalling. These findings show that insulin-induced Zn2+ release is disrupted by glucocorticoids and this is associated with insulin resistance. Restoring control of Zn2+ homeostasis, possibly through controlling oxidation or manipulating Zn2+ levels directly, may prove beneficial in metabolic disease states such as diabetes.