Genome-wide association studies (GWAS) have identified up to 30 loci that associate with increased risk of coronary artery disease or myocardial infarction. Here, I tested the function of one locus that changed the amino acid sequence of a mitochondrial matrix protease called paraplegin (SPG7) that performs critical quality assurance functions. Loss-of-function mutations in this protease are associated with hereditary spastic paraplegia. Here, I show that this variant that changes an arginine to a glutamine at position 688 within the protease domain is a gain-of-function. Cells bearing this variant have increased mitochondrial fusion and number, produce higher levels of reactive oxygen species and have increased cellular proliferation. Importantly, when expressed in yeast, the Q688 variant of SPG7 rescues the growth arrest caused by a protease-deficient mutation in AFG3L2. My study identifies a novel functional variant of SPG7 and highlights the need to go beyond the GWAS paradigm.

Thesis (Ph.D.) in Biochemistry and Molecular Biology--University of Maine, 2006. / Includes vita. Advisory Committee: Lucy Liaw, Scientist, Maine Medical Center Research Institute, Advisor; Carla Mouta-Bellum, Scientist, Maine Medical Center Research Institute; Jeong Yoon, Scientist, Maine Medical Center Research Institute; Dorothy E. Croall, Professor of Biochemistry; Thomas Gridley, Senior Scientist, The Jackson Laboratory. Includes bibliographical references (leaves 97-112 ).

No description available.

Despite advances breast cancer management, it remains a leading cause of death in women globally. Breast cancer is molecularly heterogeneous with some subtypes that are challenging to therapeutically target. This necessitates identification and validation of novel targets for breast cancer therapy. This study hypothesised that Popeye domain-containing (POPDC) proteins are dysregulated to promote breast malignancy. The study aimed to determine the potential of POPDC proteins as novel targets for inhibiting breast cancer cell migration and proliferation. Western blot and immunofluorescence assays demonstrated that POPDC1 and POPDC2 were significantly suppressed in malignant breast cells relative to non-malignant breast cells. In ductal carcinoma tissues, POPDC1 was significantly suppressed without correlation to clinical progression. In contrast, POPDC2 and POPDC3 were overexpressed in ductal carcinoma tissues and significantly correlated to HER2+ status and high tumour grade. Secondly, cell membrane expression of POPDC1 and POPDC2 were significantly reduced in malignant cells instead shifted to endosomal trafficking vesicles. Thirdly, suppression and gain of function studies showed that POPDC suppression significantly promoted cell migration and proliferation, while gain of POPDC function significantly inhibited cell migration and proliferation. The study also demonstrated that cAMP interacted with POPDC1, regulates POPDC1 expression levels and potentially controls POPDC1-mediated inhibition of cell migration and proliferation in breast cancer. Finally, this study showed for the first time that EGFR negatively regulates POPDC1 expression in breast cancer cells and the overexpression of POPDC1 can reduce EGFR-mediated cell migration and proliferation in breast cancer cells. In conclusion, POPDC protein expression, localisation and functions change in breast cancer. POPDC1 was also identified as a novel therapeutic target for inhibiting breast cancer cell migration and proliferation that could potentially be targeted to inhibit EGFR-driven breast malignancy. The study also demonstrated POPDC2 and POPDC3 are dysregulated in breast cancer, but in a less consistent and more complex manner.

Genome-wide association studies (GWAS) have identified up to 30 loci that associate with increased risk of coronary artery disease or myocardial infarction. Here, I tested the function of one locus that changed the amino acid sequence of a mitochondrial matrix protease called paraplegin (SPG7) that performs critical quality assurance functions. Loss-of-function mutations in this protease are associated with hereditary spastic paraplegia. Here, I show that this variant that changes an arginine to a glutamine at position 688 within the protease domain is a gain-of-function. Cells bearing this variant have increased mitochondrial fusion and number, produce higher levels of reactive oxygen species and have increased cellular proliferation. Importantly, when expressed in yeast, the Q688 variant of SPG7 rescues the growth arrest caused by a protease-deficient mutation in AFG3L2. My study identifies a novel functional variant of SPG7 and highlights the need to go beyond the GWAS paradigm.

No description available.

Cellular transformation is accomplished in vitro through the concerted action of growth factors and oncogenes. This association has demonstrated that malignant growth results from aberrations in pathways that normally operate to control proliferation. Activation of genes that code for growth factors, their receptors, and/or molecules essential in the transduction of signals from the cell surface to the nucleus are all potential mechanisms by which tumor cells could establish a selective growth advantage over normal cells. This dissertation addresses the question of what oncogenic mechanisms are important in the development and progression of human melanoma. These studies show that melanoma growth is regulated by endogenous substances produced by the melanoma cells themselves (autocrine stimulation), as well as by exogenous substances supplied by neighboring cells and platelets (paracrine stimulation). These factors work to drive the expression of the transformed phenotype for melanoma as evidenced by induction of serum-free soft agar growth. Human platelets were found to the the richest source of paracrine growth promoters. The factor from human platelets was characterized and partially purified. Melanoma cells respond to this 60,000 molecular weight, disulfide-bond-containing protein in colony formation assays. In addition, the protein has endothelial cell growth factor activity. Purified fractions which promoted optimal colony formation for human melanoma cells also maximally stimulated monolayer growth of bovine aortic endothelial cells, while melanocytes were nonresponsive. This implies that melanoma cells are expressing receptors for a protein which plays no known or apparent role in the normal growth of melanocytes. Melanoma cells are sensitive to growth regulatory molecules of autocrine and paracrine nature. This dissertation provides clues to the genetic lesions which have occurred in these melanoma cells to influence their proliferation. The aberrations appear to reside in those genes important in growth factor pathways at the level of endogenous production and misguided response to exogenous factors through receptor expression. We can not hope to fully inhibit the proliferation of tumor cells until we identify and understand those forces which drive their growth. These studies have increased our knowledge of those signals which stimulate melanoma cellular proliferation, and thus provide insight into important therapeutic targets.

published_or_final_version / abstract / Surgery / Master / Master of Philosophy

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

No description available.