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To investigate the effects of proliferation on metabolism and cell product yields, proliferating and growth-limited EPOBF7 hybridoma cells have been compared as to their growth rates, energy demand, relative energy distributions, and monoclonal antibody (MAb) yield. Medium deprivation of leucine or serum was used to prevent growth. Energy consumption rates were determined in cell suspensions from rates of glucose consumption, lactate production, and oxygen utilization. In addition, the energy consumption of pathways critical to cell growth and survival were estimated from the relative decreases occurring in oxygen and glucose consumption upon pathway inhibition. The overall rate of energy consumption was significantly lower among growth-limited cultures. In addition, the distributions of oxidative and glycolytic energy among cellular synthetic pathways differed significantly between the culture conditions. Non-growing cultures also produced significantly lower antibody yields. Cell growth was also investigated using ³¹p nuclear magnetic resonance (NMR) spectroscopy of cells grown and maintained in bioreactor culture. Saturation transfer methods detected measurable transfer between inorganic phosphate (P(i)) and the gamma resonance of ATP. This transfer rate could be correlated with cellular growth rates within the reactor. Transfer of magnetization from the gamma resonance of ATP to P(i) was also detected, although the rate of this transfer did not appear to be related to the growth rate. The ratio of these transfer rates was consistently near 4. This information is believed to suggest the importance of other reactions through which ATP may donate its terminal phosphate. Cells in bioreactor culture were found to grow more slowly and produce lower levels of monoclonal antibody when compared to cells proliferating in suspension. such phenomena may be a function of diffusion limitations within the reactor such that the cells cannot obtain the nutrient supply required for optimal cell growth or product formation.

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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.

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Levels of retinoic acid (RA), the active metabolite of vitamin A, are tightly regulated throughout vertebrate CNS development by RA synthesising and catabolising enzymes. However, increasing evidence suggests that similar regulatory mechanisms exist in the adult brain to maintain RA at the optimum level. The hippocampus is one of very few regions where neurons continue to be born. Furthermore, the hippocampus is one of the regions in which RA regulates function. RA is synthesised in the region of the hippocampus by the enzyme, retinaldehyde dehydrogenase 2 (RALDH2), expressed in the adjacent meninges. CYP26B1 has been previously shown to be present by in situ hybridisation in the CA4/3 region between the two blades of the dentate gyrus. We hypothesised that a gradient was set up between the source and sink of RA in the adult hippocampus. To test this, we disrupted the balance using exogenous RA and using inhibitors to its catabolising enzymes. A reporter mouse was used to detect RA signalling and significantly more lacZ expression was detected in the infrapyramidal blade (closest to the meninges) compared to the suprapyramidal blade. Furthermore, administration of RA equalised lacZ expression between the two blades. RA is a potent differentiation agent; however, its effects on cell proliferation are less clear. In order to examine the direct effects RA on cell proliferation, an organotypic hippocampal slice culture technique was optimised and it was found that RA inhibits cell proliferation specifically in the dentate gyrus in a dose dependent manner. Taken together, this thesis provides insight for the first time into a parallel regulatory mechanism in the adult hippocampus to the embryo where RA is tightly regulated by its synthesising and catabolising enzymes and this mechanism is involved in the regulation of cell proliferation in the adult dentate gyrus.