Cytotoxic chemotherapy is extremely important in adjuvant treatment of breast cancer. Yet, tumours frequently acquire chemoresistance that correlates with increased aggressiveness and poor prognosis. Three theories exist describing how the resistance develops: genetic, epigenetic and karyotypic theory. The epigenetic theory is the least explored. Here we analyzed the role of the epigenetic phenomena in the acquisition of drug resistance. To do so, we employed genome wide screens of microRNA and gene expression, DNA methylation and complete genome hybridization. We identified three novel microRNA interactions involved in the chemoresistant phenotype. These three microRNAs displayed depressed expression in the resistant cell lines and we were able to re-establish some level of drug sensitivity through ectopic expression of these under expressed microRNAs. In addition, we described the role of DNA methylation in impacting expression of a wide range of genes, thus, contributing to the phenotype of chemoresistance. Furthermore, we revealed a distorted global DNA methylation pattern that coincides with massive instability of the resistant genome. Finally, our results present a striking similarity between gene expression, epigenetic profiles and chromosomal aberrations in two different drug resistant cell lines. Taken together, this project suggests that the acquisition of chemoresistant phenotype is epigenetic in nature and may arise with a predictable pattern. Elucidating the specifics of this pattern may in the future prove useful in developing treatment and prognostic chemoresistance biomarkers. / xiii, 116 leaves : ill. (some col.) ; 29 cm

Previous findings showed that mild chronic restraint stress causes motor impairments in rats. These behavioural impairments might be related to molecular changes in brain areas that regulate motor functions, such as the cerebellum. Little is known about the role of the cerebellum in stress-induced behavioural alteration. We hypothesized that alteration in animal behaviour after chronic restraint stress is due to brain-specific changes in miRNA and proteins encoding gene expression. Our results revealed that expression of three miRNAs and 39 mRNAs was changed significantly after two weeks of stress. Furthermore, we verified one putative target for one of the changed miRNAs and expression of four randomly selected genes. Changes in gene expression disappeared after two weeks of recovery from stress. These findings provide a novel insight into stress-related mechanisms of gene expression underlying altered behavioural performance. The observations bear implications for the prevention and treatment of stress-related disorders and disease. / xii, 109 leaves. : ill. ; 29 cm

Indiana University-Purdue University Indianapolis (IUPUI) / The epigenetic state of any cell is, in part, regulated by the interaction of DNA with nuclear histones. Histone tails can be modified in a number of ways that impact on the availability of DNA to interact with transcriptional complexes, including methylation, acetylation, phosphorylation, ubiquituination, and sumoylation. Histones are acetylated by a large family of enzymes, histone acetyl transferases (HATs), and deacetylated by the histone deacetylases (HDACs). Acetylated histones are generally considered markers of genomic regions that are actively being transcribed, whereas deacetylated and methylated histones are generally markers of regions that are inactive. The goal of the present study was to 1) study the epigenetic state with regard to the presence of euchromatin and heterochromatin in the developing chick and murine retina, 2) study and compare the localization patterns of the classical HDACs in the developing chick and murine retina with respect retinal progenitors and early differentiated cell types 3) to test the hypothesis that overall HDAC activity is required for dividing retinal progenitors to leave the cell cycle and differentiate. Our results showed that the classical HDACs were ubiquitously expressed in the developing chick and murine retinas. Species specific differences as well as stage dependent variations were observed in the localization of the HDACs in the cell types that were studied in the chick and murine retina. Our preliminary results also showed that HDAC inhibition may lead to the inability of the cell types to leave the cell cycle and a subsequent increase in the number of progenitor cells present in the developing chick retina.

Indiana University-Purdue University Indianapolis (IUPUI) / Epigenetic alterations have been shown to control cell type specification and differentiation leading to the changes in chromatin structure and organization of many genes. HDACs have been well documented to play an important role in both neurogenesis and gliogenesis in ganglionic eminence and cortex-derived cultures. However, the role of HDACs in glial cell type specification and differentiation in the optic nerve has not been well described. As a first step towards understanding their role in glial cell type specification, we have examined histone acetylation and methylation levels as well as the expression levels and patterns of the classical HDACs in both murine and chick optic nerve. Analysis of mRNA and protein levels in the developing optic nerve indicated that all 11 members of the classical HDAC family were expressed, with a majority declining in expression as development proceeded. Based on the localization pattern in both chick and murine optic nerve glial cells, we were able to group the classical HDACs: predominantly nuclear, nuclear and cytoplasmic, predominantly cytoplasmic. Nuclear expression of HDACs during different stages of development studied in this project in both murine and chick optic nerve glial cells suggests that HDACs play a role in stage-dependent changes in gene expression that accompany differentiation of astrocytes and oligodendrocytes. Examination of localization pattern of the HDACs is the first step towards identifying the specific HDACs involved directly in specification and differentiation of glia in optic nerve.

Indiana University-Purdue University Indianapolis (IUPUI) / Fetal alcohol spectrum disorders (FASD) is the leading neurodevelopment deficit in children born to women who drink alcohol during pregnancy. The hippocampus and cortex are among brain regions vulnerable to alcohol-induced neurotoxicity, and are key regions underlying the cognitive impairment, learning and memory deficits shown in FASD individuals. Hippocampal and cortical neuronal differentiation and maturation are highly influenced by both intrinsic transcriptional signaling and extracellular cues. Epigenetic mechanisms, primarily DNA methylation and histone modifications, are hypothesized to be involved in regulating key neural development events, and are subject to alcohol exposure. Alcohol is shown to modify DNA methylation and histone modifications through altering methyl donor metabolisms. Recent studies in our laboratory have shown that alcohol disrupted genome-wide DNA methylation and delayed early embryonic development. However, how alcohol affects DNA methylation in fetal hippocampal and cortical development remains elusive, therefore, will be the theme of this study. We reported that, in a dietary alcohol-intake model of FASD, prenatal alcohol exposure retarded the development of fetal hippocampus and cortex, accompanied by a delayed cellular DNA methylation program. We identified a programed 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC) cellular and chromatic re-organization that was associated with neuronal differentiation and maturation spatiotemporally, and this process was hindered by prenatal alcohol exposure. Furthermore, we showed that alcohol disrupted locus-specific DNA methylation on neural specification genes and reduced neurogenic properties of neural stem cells, which might contribute to the aberration in neurogenesis of FASD individuals. The work of this dissertation suggested an important role of DNA methylation in neural development and elucidated a potential epigenetic mechanism in the alcohol teratogenesis.

Indiana University-Purdue University Indianapolis (IUPUI) / Multiple sclerosis (MS) is a disabling disease that afflicts an estimated two million people worldwide. The disease is characterized by degradation of the myelin sheath that insulates neurons of the central nervous system manifesting as a heterogeneous collection of symptoms. Two enzymes, protein arginine deaminases type 2 and 4 (PAD2 and PAD4) have been implicated to play an etiologic role in demyelination and neurodegeneration by catalyzing a post-translational modification of arginine peptide residues to citrulline. The pathogenesis of MS is poorly understood, though vitamin D deficiency is a well-associated risk factor for developing the disorder. Using the experimental autoimmune encephalomyelitis (EAE) model of MS we demonstrate vitamin D treatment to attenuate over-expression of PAD 2 and 4 in the brain and spine during EAE. In addition, we identify two molecules produced by peripheral immune cells, IFNɣ and IL-6, as candidate signaling molecules that induce PAD expression in the brain. We demonstrate vitamin D treatment to inhibit IFNɣ mediated up regulation of PAD2 and PAD4 both directly within the brain and by modulating PAD-inducing cytokine production by infiltrating immune cells. These results provide neuroprotective rational for the supplementation of vitamin D in MS patients. More importantly, these results imply an epigenetic link between vitamin D deficiency and the pathogenesis of MS that merits further investigation.