Genetic aspects

Restrictive cardiomyopathy (RCM) is a heart muscle disease, characterized by diastolic dysfunction. The present dissertation is to understand the mechanisms underlyijng the initiation of diastolic dysfunction and the fast disease progression to early death in a RCM mouse model, the transgenic cTnI193His mouse... These data showed that myocardial ischemia occurred after diastolic dysfunction and before systolic dysfunction which proceeded congestive heart failure. The results demonstrate that myocardial ischemia causing cardiomycete death is a link between the initial diastolic dysfunction and late-stage systolic dysfunction, and accelerates the disease progression to fatal heart failure in the early age.

We attempted to understand the molecular regulators that impact inflammation using a rat model of human sensation-seeking/risk-taking trait for drug and stress vulnerability, based on their exploratory behavior displaying high rates (HRs) or low rates of locomotor reactivity (LRs) to environmental stress. We found that HRs have a pro-inflammatory phenotype as indicated by increased protein expression of the inflammatory cytokine TNF-(Sa(B. Furthermore, we found that HRs have a lower gene expression of the glucocorticoid receptor and histone deacetylase 2 which are known to play an immunosuppressive role. Autophagy (macroautophagy) is a homeostatic process needed for cell maintenance, growth and proliferation and known to assist in tumor survival. FYVE and coiled-coil domain containing 1 (FYCO1) is a novel protein implicated to assist in the plus-end directed trafficking and fusion of autophagosomes. In these studies, we show that FYCO1 gene expression among human breast cell lines of varying degrees of malignancy.

Cytogenetics is a study on the genetic considerations associated with structural and functional aspects of the cells with reference to chromosomal inclusions. Chromosomes are structures within the cells containing body's information in the form of strings of DNA. When atypical version or structural abnormality in one or more chromosomes prevails, it is defined as chromosomal aberrations (CA) depicting certain genetic pathogeny (known as genetic disorders). The present study assumes the presence of normal and abnormal chromosomal sets in varying proportions in the cytogenetic complex ; and, stochastical mixture theory is invoked to ascertain the information redundancy as a function of fractional abnormal chromosome population. This bioinformatic measure of redundancy is indicated as a track-parameter towards the progression of genetic disorder, for example, the growth of cancer. Lastly, using the results obtained, conclusions are enumerated, inferences are outlined and directions for future studies are considered.

Adult neurogenesis is affected in neurodegenerative diseases and also represents an important therapeutic target. The goal of this dissertation research was to test the hypothesis that regeneration of neurons and glia in the adult brain can be manipulated by neurotrophic drugs in the context of two mouse models of neurodegenerative disease : Parkinson's disease and Huntington's disease.... These findings have implications for the pathophysiology of Huntington's disease and neurodegeneration in general. Specific alterations to the SVZ neurogenic niche parallel some of the pre-motor symptoms of Parkinson's disease and Huntington's disease. This dissertation research contributes to the growing body of literature concerning the pharmacological modulation of SVZ-derived neurogenesis designed to attenuate the progressive loss of neurons in neurodegenerative diseases and perhaps delay the onset of symptoms.

Huntington's Disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the Huntingtin gene IT15. In this study we demonstrated that Bcl-2 interacting mediator of cell death Extra Long (BimEL) protein expression was significantly increased in cells expressing mutant Huntingtin (mHtt). Moreover, striatal BimEL expression remained high in an R6/2 HD mouse model throughout the disease progression. Utilizing novel BimEL phospho-mutants we demonstrated the phosphorylation of Ser65 to be important for the stabilization of BimEL. We provided evidence that impaired proteasome function, increased JNK activity and reduced striatal BDNF lead to changes in the phosphorylation of BimEL, thereby promoting its stabilization specifically within the striatum of R6/2 mice. Furthermore, knocking down BimEL expression prevented mHtt-induced cell death in a HD cell culture. Taken together, these findings suggest that BimEL may contribute to the selective neurodegeneration and pathogenesis of HD.

Supernumerary centrosomes can arise in a cell through a variety of methods. The presence of supernumerary centrosomes has been observed in nearly all types of cancer and promotes chromosomal instability, with rates of incident increasing as the cancer progresses. An oral squamous cell carcinoma line was treated with hydroxyurea to induce supernumerary centrosomes in the cells. NuMA was then knocked down using shRNA to promote centrosomal clustering and bipolar mitotic division in cells with supernumerary centrosomes. Immunofluorescence with an antibody against SAS 6 accuately stained the centrioles for observation. The cells exhibiting supernumerary centrosomes undergoing bipolar mitotic division were studied to look for a possible pattern in centrosomal clustering where the majority of centrosomes are at one pole with a single centrosome at the other pole. Initial results suggest the presence of such a mechanism, which would describe a previously unknown mechanism for cells to deamplify supernumerary centrosomes by centrosomal clustering.

Tumor cells are characterized by an increase in genomic instability, brought about by both chromosomal rearrangement and chromosomal instability. Both of these broad changes can be induced by exposure to carcinogens. During mitosis, cells can exhibit early and late lagging chromosomes, multipolar spindles or anaphase bridges, all of which contribute to genomic rearrantement. We have studied the link between exposure to carcinogen and prevalence of mitotic defect in both chromosomally stable and unstable cell lines as well as ecamined the restorative effects of antioxidants in preventing mitotic defects. We have exposed MES-SA uterine cancer cells to vinyl chloride followed by exposure to an antioxidant : ascorbic acid, B-carotene, or lycopene. Treated cells were then scored for the prevalence of mitotic defects within the population and compared to controls. We have also investigated whether pre-treatment with the antioxidants will weaken the effects of carcinogen exposure in these cell lines.

Aneuploid tumor cells have characteristically unstable genomes which can be caused by mitotic defects such as multipolar spindles. Multipolarity relies upon the presence of extra centrosomes to form. However, some cells, both cancerous and noncancerous are able to avoid the formation of multipolar spindles through centrosomal clustering. Previous research has shown that there are a large number of genes whose activity contributes to the clustering activity, making analysis of individual components of the process difficult. In order to better understand centrosomal clustering in cancer cells, we induced supernumerary centrosomes in a genomically normal cell line, RPE, to observe how the normal cells cope with extra centrosomes. Using colcemid to induce extra centrosomes in the RPE cell line, we observed an intact clustering mechanism in fixed cells. Further manipulation of the cells has allowed us to induce multipolarity in this cell line using various disrupters of cell-cycle checkpoint and dynein function.

Most tumors contain multiple karyotypes due to genomic instability gained through chromosomal segregational defects. The variability of genomic changes within a population makes it difficult to study specific processes without the existence of confounding mutations. My project is to create a model system for observation of mitotic defects, specifically multipolar spindles, in a normal cell line, where the genome is intact. Induction of centrosome amplification is required for formation of multipolar spindles. Treatments with colcemid showed a 10% increase in abnormal centrosome numbers over control. However, treatment with hydroxyurea and transfection of hMPSl showed little increase. Extra centrosomes are insufficient to drive multipolarity, therefore, I am using siRNA-mediated knockdown of Nek2 or HSET to decluster the extra centrosomes. Successful declustering will preferably show an increase in multipolar frequency, allowing us to study the formation and resolution of these structres to better understand how they contribute to aneuploidy and tumor progression.