Thesis (M. Sc.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 103-111). Also available via the World Wide Web.

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Genome wide association studies have shown that common variants in the human fat mass and obesity-associated (FTO) gene predispose to obesity and increased fat mass. Mice globally lacking Fto are lean, while mice globally overexpressing Fto have increased body weight due to increased fat mass. FTO protein was shown to localise to the nucleus and demethylate ssDNA and ssRNA. However, the mechanisms by which FTO mediates its effects on body phenotype remain unknown. In this thesis, I found that native FTO can be detected in both the nucleus and the cytoplasm during interphase, and that nuclear FTO was exported through the nuclear membrane during early prophase of the mitotic cell division. I developed co-immunoprecipitation (Co-IP) protocol to pull-down native FTO and identified a large number of new candidate binding partners (CBPs). Computational analysis predicted a role for FTO, and many CBPs, in RNA post-transcriptional modification and processing. I confirmed that the E3 ubiquitin-protein ligase TRIM21 interacts with FTO in multiple mouse tissues and binds FTO through its SPRY domain. Importantly, TRIM21 ubiquitinated FTO which did not lead to its degradation. FTO partially co-localised with TRIM21 and the decapping enzyme DCP2 in mRNA processing bodies (p-bodies). Overexpression of TRIM21 led to the accumulation of FTO outside the nucleus, but was reversed when both proteins were overexpressed. Additionally, I created a muscle specific Fto knock-out mouse model and found that lack of FTO in muscle did not result in the body composition phenotype reported in global Fto knock-out mice. Taken together, FTO can function in both the cytoplasm and the nucleus, where it interacts with TRIM21 which ubiquitinates FTO and potentiates its cytoplasmic localisation. Moreover, function of FTO in muscle does not mediate the obesity phenotype in mice.

by Brian C. Freeman. / Typescript. / Thesis (M.S.)--Florida State University, 1991. / Includes bibliographical references.

Isocitrate Dehydrogenase-1 has been found to be mutated in over 70% of lower grade gliomas and has become an important diagnostic tool for tumor prognosis, however its role in glioma development, and its impact on response to therapy, is still not fully understood. Unmutated IDH1 functions to convert isocitrate to alpha-ketoglutarate (a-KG) in the tricarboxylic acid (TCA) cycle. Mutated IDH1R132H changes the enzymatic equilibrium and converts a-KG to 2-hydroxyglutarate (2-HG), an oncometabolite. IDH1R132H mutated tumors show an elevated production of 2-HG and epigenetic alterations in DNA and histone methylation. This mutation is predominantly seen in the proneural glioma subtype in which oligodendrocytes progenitors (OPCs) are considered the cell of origin due to phenotypic similarities. The effect of IDH1R132H mutation in cellular transformation has not been fully established. Epigenetic modifications connect genotype to phenotype by genetic expression alterations and epigenetic modifications are necessary for OPC differentiation. Tri-methylation of lysine residue K27 on histone H3 (H3K27me3) is a repressive mark associated with cell pluripotency. H3K27me3 is trimethylated by Enhancer of Zeste 2 (EZH2) and demethylated by the a-KG-dependent demethylases UTX/KMD6A and JMJD3/KDM6B. 2-HG is a competitive inhibitor of a-KG-dependent demethylases, providing a mechanistic link between IDH mutations and increases H3K27me3 by inhibiting demethylation. In this thesis, we evaluated the epigenetic changes in mouse models of IDH mutant and wildtype glioma and genetically-transformed OPCs and tested the effects of drugs that target specific epigenetic marks. We developed a mouse model of glioma to compare IDH1R132H cells to wildtype glioma cells and found that although there was no difference in survival, IDH1R132H gliomas have increased levels of 2-HG by MALDI-IMS and metabolomic analysis. Interestingly, based on RNA-sequencing analysis our IDH1R132H model has a more OPC-restricted expression profile compared the wildtype glioma model which have higher enrichment of genes from other cell lineages, including neurons, astrocytes, myelinating oligodendrocytes and microglia. We used the EZH2 inhibitor (Tazemetostat, EPZ-6438) and found that this treatment was not cytotoxic or cytostatic to our cells although H3K27me3 was reduced. Interestingly, Tazemetostat treatment increased the expression of non-OPC genes (genes normally expressed by other lineages as assessed using the Barres transcriptomic database). To better understand how IDH1R132H influences OPC transformation, we transformed OPCs in vitro. OPCs were isolated from floxed p53 postnatal day 5 mice from and retrovirally infected with viruses to delete p53 alone or to also express IDH1R132H. OPCs that express IDH1R132H had increased levels of 2-HG by metabolomics and showed alteration in H2K27 methylation and acetylation that resembled those seen in glioma cells. Standard methods of western blot analysis consist of analyzing whole cell lysate, cytoplasmic and nuclear fractionation, or histone acid extraction. To analyze both the cytoplasmic fraction as well as histone modification, I developed a cellular extraction method in which cells were fractionated and the nuclear fraction was acid extracted. This method allows for the analysis of both cytoplasmic proteins as well as histone modifications by western blot. Using this method, we found that treating glioma cells or OPCs with synthetic cell permeable octyl-2HG, or expressing IDH1R132H, caused cells to have increased H3K27me3, while treatment with Tazemetostat caused a decrease in H3K27me3. Based on the RNA-sequencing data we found that increased H3K27me3 (ID1R132H mutation) express more OPC-like phenotype while reduced H3K27me3 (Tazemetostat treatment) induced an upregulation of genes associated with other lineages making them less restricted to the OPC transcriptional phenotype. We found that in both the glioma cells and OPCs, Tazemetostat treatment decreased H3K27me3 and increased H3K27ac. Based on the increase of H3K27ac after Tazemetostat treatment, we hypothesized that a Histone deacetylase inhibitor (HDACi) would be synergistic. We found that although the HDACi Panobinostat was less cytotoxic to IDH1R132H mutated glioma cells and OPCs, co-treatment with Tazemetostat is synergistic in mutant and wildtype models. We also saw that in IDH1R132H ex vivo slices, the co-treatment reduced tumor marker composition. These findings point to a novel therapeutic strategy for IDH1-mutated proneural gliomas that targets the specific epigenetic alteration in these tumors.

The emergence of life depended on the ability of the first biopolymer populations to thrive and approach larger population sizes and longer sequences. The evolution of these populations likely occurred under circumstances under which Muller's Ratchet in synergism with random drift could have caused large genetic deterioration of the biopolymers. This deterioration can drive the populations to extinction unless there is a mechanism to counteract it. The effect of the mutation rate and the effective population size on the time to extinction was tested on clonal populations of B16-19 ligase ribozymes, evolved with the continuous evolution in vitro system. Populations of 100, 300, 600 and/or 3000 molecules were evolved with and without the addition of Mn(II). The times to extinction for populations evolved without Mn(II) were found to be directly related to the effective size of the population. The small populations approached extinction at an average of 24.3 cycles; while the large populations did so at an average of 44.5 cycles. Genotypic characterization of the populations showed the presence of deleterious mutations in the small populations, which are the likely cause of their genetic deterioration and extinction via mutational meltdown. These deleterious mutations were not observed in the large populations; in contrast an advantageous mutant was present. Populations of 100 and 3000 molecules were evolved with Mn(II). None of the populations showed signs of genetic deterioration nor did they become extinct. Genotypic characterization of the 100-molecule population indicated the presence of a cloud of mutants closely genetically-related, forming a "quasispecies" structure.. The close connectedness of the mutants facilitates the recovery of one from another in the event of being removed from the population by random genetic drift. Thus, quasispecies shift the target of selection from individual to group. The total fitness of the molecules was measured by identifying the fitness component of the system that effect the ligase replication cycles: ligation, reverse transcription and transcription. It was found that the strength of the three components of fitness varied and that each one has a differential effect in the total absolute fitness of the ligases.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. Johannesburg, 17 May 1994. / Two black South African subjects presenting with hereditary elliptocytosis were investigated and the defect defined as Spol/74, a previously described spectrin variant leading to defective heterodimer self-association and instability of the erythrocyte membrane. [Abbreviated Abstract. Open document to view full version]

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