Thesis advisor: Thomas N. Seyfried / Thesis advisor: Charles Hoffman / Bis(monoacylglycero)phosphate, BMP, is a negatively charged glycerol-phospholipid with an unusual sn-1;sn-1’ structural configuration. BMP is primarily enriched in endosomal/lysosomal membranes. BMP is thought to play a role in glycosphingolipid degradation and cholesterol transport. It constitutes only about 1-2% of the total phospholipids in most mammalian cells, but is abundant in lung alveolar macrophages where it can comprise up to 16% of the total phospholipids. BMP also accumulates in tissues of humans and animals with lysosomal storage disorders. However, little information is available on BMP levels in gangliosidosis brain tissue. In this work, I found that total BMP content was significantly greater in cells of macrophage/microglial origin than in cells of macroglial origin (astrocyte, oligodendrocyte progenitor), whether normal or tumorigenic. I also observed that BMP in brain was significantly greater in humans and in animals (mice, cats, American black bears) with either GM1 or GM2 ganglioside storage diseases, than in brains of normal subjects. Since BMP is associated with macrophages, I also analyzed the BMP levels in relation to disease-associated inflammation in gangliosidoses. I found that BMP levels were increased due to accumulation of primary storage material gangliosides, rather than an outcome of disease-associated inflammation. In addition, in this thesis I also explored the effect of new ketogenic diet formula from Solace Nutrition (KetoGen) on the growth and metastatic spread of the VM-M3 tumor. Most current drug therapies for cancer are toxic and only marginally effective in providing long-term management. Respiratory insufficiency with compensatory aerobic fermentation (Warburg effect) is the hallmark biochemical phenotype of nearly all neoplastic cells within tumors. Calorie restriction, which lowers blood glucose and elevates ketone bodies, is known to reduce tumor growth to a certain extent, however it does not reduce systemic metastasis. Tumor bearing VM mice were fed either a standard lab chow diet in unrestricted amounts (SD-UR), a standard lab chow restricted to obtain an 18% reduction in body weight (SD-R), or the KetoGen diet restricted (KG-R) to match the body weights of the SD-R group. Tumor size was significantly smaller and organ metastasis was significantly less in the KG-R group than in the SD-UR or SD-R groups. Even though blood glucose was reduced similarly in both the SD-R and KG-R groups, blood ketones were 3-fold higher in the KG-R group than in the SD-R group. These results show that VM-M3 tumor growth and systemic metastasis were managed better with the restricted KetoGen KD than with calorie restriction of a high carbohydrate standard diet. As all human and mouse tumors cells suffer from respiratory insufficiency, my findings suggest that the restricted KetoGen diet should be an effective non-toxic therapy against tumor growth and systemic metastatic cancer. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

<p>  </p> <p>Niemann-Pick Type C (NPC) disease is a rare lysosomal storage disorder that occurs in about 1/89,000 to 1/120,000 live births and is characterized by an aberrant accumulation of cholesterol within the late endosome/lysosome of cells. Symptoms of this disease include splenomegaly, neurological deterioration, and often death before adulthood. Mutations in the membrane bound NPC1 or luminal NPC2 proteins lead to a decrease in cholesterol efflux within the lysosomes by which excess cholesterol crystallizes within membranes resulting in cell death. It has been demonstrated that increasing the amount of the lysosomal specific phospholipid Bis(monoacylglycerol)phosphate (BMP), also known as Lysobisphosphatidic acid (LBPA), in cells increases the rate of cholesterol transport in <em>npc1</em>-/- cells, but not in <em>npc</em>2-/- cells, indicating a strong synergistic relationship between the NPC2 protein and the lysosomal membranes. Increasing the amount of phosphatidyl glycerol (PG), a hypothesized precursor to BMP, has also shown an increase in cholesterol egress. While it is hypothesized that the increase in cholesterol clearance in the latter is due to the biosynthesis of LBPA from PG, there is no study to directly confirm this phenomenon. Therefore, we set out to synthesize diastereochemically pure PG containing isotopically labeled oleyl acyl chains to examine LBPA levels using lipidomic analysis of <em>npc1-/-</em> cells post treatment with PG. </p> <p>Initially, efforts centered around the use of phosphoramidite methodology commonly encountered in DNA oligonucleotide synthesis. While this route proved to be successful in making PG in modest yield (52%), reproducibility of this route with consistent yields was hindered due to the use of tetrabutylammonium fluoride (TBAF) in the final global deprotection step. Thus, we set out to discover a phosphorylated intermediate that did not require TBAF in the final step or contain easily hydrolysable protecting groups. It was discovered that H-phosphonate methodology using diphenyl phosphite for phosphorylation of the glycerol headgroup and backbone proved to be robust enough for PG synthesis. In this strategy, PG can be isolated in two steps from the final protected intermediate by first oxidizing the H-phosphonate from PIII to PV followed by deprotection of the glycerol head group under acidic conditions. Additionally, the H-phosphonate strategy also allowed us to omit headgroup modification prior to phosphorylation which reduced the number of synthetic steps from 11 steps to 7 steps. As a result, we were able to synthesize diastereochemically pure PG more consistently than the previous route in 75% yield. The route was further modified further to incorporate asymmetric acyl chains allowing the selective installation of a labeled acyl chain on the <em>sn</em>-1 or <em>sn</em>-2 positions of the phosphoglycerol backbone. The results from the lipidomic experiments indicate that increased LBPA concentrations in cells rise upon incubation with labeled PG. Additionally, increases in lyso-PG and acyl-PG are also observed leading to several hypotheses on how LBPA might be synthesized from PG. Future directions on this effort include identification of phospholipid species made from PG containing asymmetrically labeled acyl chains.  Synthesis of photoaffinity labeled PG is also underway to determine the protein partners involved in PG metabolism.</p>