In vivo analyses of the Von Willebrand Factor (VWF) promoter previously demonstrated that a fragment spanning sequences -487 to +247 targets promoter activation to brain vascular endothelial cells. This fragment is active in all embryonic vessels of transgenic mice but in adult mice its activity is restricted to brain vascular endothelial cells, while endogenous VWF gene is expressed in vasculature of all major organs. In this study we demonstrate that a DNase I hypersensitive (HSS) sequences in intron 51 of the VWF gene contain cis-acting elements that are necessary for the VWF gene transcription in a subset of lung endothelial cells in vivo. Our results demonstrated that Nuclear Factor 1 (NF1) and Nuclear transcription Factor Y (NFY) repressors contribute to VWF organ-specific regulation. Mutation of the NF1 binding site resulted in promoter activation in lung and heart, while mutation of the repressor corresponding to a novel binding site for NFY resulted in promoter activation in kidney vasculature. / Experimental Medicine

Hypoxia in the marine environment is a growing environmental concern, and can have profound impacts on organisms. This dissertation seeks to understand the physiologically induced changes in gene expression, the relationship between gene expression and metabolism, and how these parameters vary among populations, in response to hypoxic stress. By comparing evolved intraspecific variation in gene expression and physiological parameters among populations from multiple regions in the Gulf of Mexico we seek to determine the physiologically induced changes that are essential to hypoxic survival. First, whole body metabolism, measured as oxygen uptake, was profiled across seven decreasing oxygen concentrations. Metabolism and the critical oxygen tension (PO2crit) were compared between populations from across the Gulf of Mexico. This study demonstrated a significant interaction of body mass with the hypoxic response. Additionally, populations only differed in their metabolism at the lowest oxygen concentration, 1.8 kPa. PO2crit did not differ between populations, but was body mass dependant. Next, the effects of hypoxia on gene expression were examined. These studies examined the effects of hypoxia on gene expression over time and at different hypoxic doses, utilizing a 384 gene microarray. In the first studies individuals were subjected to 0, 4, 8, 12, 24, 48, or 96 hours of hypoxia. Different genes had different times for peak gene expression, with most changes occurring after 96 hours of exposure. However, only 14 genes had significant changes in gene expression. To determine the effect of differing hypoxic dose, individuals were exposed to normoxia, 7.8 kPa O2 (moderate hypoxia), or 1.8 kPa (severe hypoxia) for 4 or 48 hours. Sixty-nine genes had significant changes in gene expression for either dose or time. To elucidate the relationship between effect of time and dose, genes were examined for dose response within each time. The maximum number of changes occurred at 1.8 kPa after 48 hours of exposure. Interestingly different sets of genes had changes in gene expression at either 7.8 or 1.8 kPa. Finally, to ascertain the difference among populations, for thousands of genes, individuals from six populations of Fundulus grandis were exposed to hypoxia (1.8 kPa) for 4 or 96 hours. Hypoxia had a significant effect on the expression of 609 genes, while population affected the expression of 355 genes. Genes with significant differences in expression among populations reflect geographic separation. For the 59 genes with significant differences in expression for both hypoxia response and population, shared hypoxic histories appears to be more important than simply the neutral patterns expected with geographic distance. The majority of significant changes for the 609 hypoxia responsive genes take place after 96 hours of hypoxia exposure. This research demonstrates that F. grandis cope with hypoxia through changes in metabolism and gene expression. Overall, the response to hypoxia is dependent on an individual's size (body mass), the ambient oxygen concentration, and the duration of hypoxia exposure. Additionally, there appear to be some differences between populations with differing exposure history to hypoxia in the Gulf of Mexico.

Undergraduate honors paper--Mount Holyoke College, 2006. Program in Biochemistry. / Includes bibliographical references (leaves 69-74).

Thesis (Ph.D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves. Also available in print.

Steroid hormones, such as estrogen, are produced in one tissue and carried through the blood stream to target tissues in which they bind to highly specific nuclear receptors and trigger changes in gene expression and metabolism. Industrial chemicals, such as bisphenol A and many agricultural chemicals, including permethrin and fervalerate, are known to have estrogenic potential and therefore are estrogen mimics. Widely used agricultural chemicals, Enable (fungicide) and Diazinon (insecticide), were evaluated to examine their toxicity and estrogenicity. MCF-7 cells, an estrogen-dependent human breast cancer line, were utilized for this purpose. MCF-7 cells were treated with 0.033-3.3 ppb (ng/ml) of Enable and 0.3-67 ppm of Diazinon and gene expression was compared to that in untreated cells. Microarray analysis showed down-regulation of eight genes and up-regulation of thirty four genes in cells treated with 3.3 ppb of Enable, compared to untreated cells. Similarly, in cells treated with 67 ppm of Diazinon, there were three genes down-regulated and twenty seven genes up-regulated. For both chemicals, specific genes were selected for special consideration. RT-PCR confirmed results obtained from analysis of the microarray. These studies were designed to provide base-line data on gene expression-altering capacity of specific chemicals and will allow assessment of the deleterious effects caused by exposure to the aforementioned chemicals.

Gene expression analysis has evolved as an extensive toolfor elucidation of various biological and molecular eventsoccurring in different organisms. A variety of techniques andsoftware tools have been developed to enable easier and morerapid means of exploring the genetic information. A moreeffective approach than exploring the whole content of genesexpressed under certain conditions is to study fingerprintassays or to use subtracted cDNA libraries to identify onlydifferentially expressed genes. The objective for the work in this thesis has been toexplore differentially expressed genes in atherosclerosis. Thiswas done by applying and modifying a protocol for thesubtractive approach RDA (Representational Difference Analysis)in different model systems. Initially, the molecular effects of an anti-atheroscleroticdrug candidate were elucidated. In addition, two alternativeapproaches to identify differentially expressed genes obtainedafter iterative rounds of RDA subtraction cycles wereevaluated. This revealed that in most cases, the shotgunapproach in which the obtained gene fragments are clonedwithout any prior selection has clear advantages compared tothe more commonly used selection strategy, whereby distinctbands are excised after gel electrophoresis. A key process in the atherosclerotic plaque initiation isthe phenotypic change of macrophages into foam cells, which canbe triggered in a model system by using macrophages exposed tooxidised LDL. To investigate the genes expressed in thisprocess, the RDA technique was combined with microarrayanalysis, which allows for selectivity and sensitivity throughRDA, as well as rapid high-throughput analysis usingmicroarrays. The combination of these techniques enablessignificant differences in gene expression to be detected, evenfor weakly expressed genes and the results to be reliablyvalidated in a high throughput manner. Finally, investigation of the focal nature ofatherosclerotic lesions and gene expression profiling werestudied using in vivo aortic tissues from ApoE-/- and LDLR -/-mice. The study was based on a comparison between localisationsthat are likely, and others that are unlikely, to developatherosclerotic plaques, and the RDA technique was employed toexplore differential gene expression. <b>Keywords:</b>Representational Difference Analysis,atherosclerosis, gene expression profiling

The E2F family of transcription factors regulate cellular growth, death and differentiation, but their role in cardiac biology remains to be fully explored. We hypothesized that the balance of the E2F pathway would determine cardiac development and function. We provide evidence for this via modulation of the E2F6 repressor, in a transgenic (Tg) mouse model. Targeted expression of E2F6 in the heart led to dilated cardiomyopathy (DCM) and death. Microarray analysis revealed that E2F responsive pathways were activated in Tg mice. Furthermore, we found that E2F6 and YY1 (E2F-co-factor) were translocated to the nucleus in Tg mice, providing a potential mechanism for the observed transcriptional activation. We also observed a marked decrease of Connexin43 protein in the myocardium, and reduced atrial conductivity in Tg mice which may lead to reduced cardiac function. The data demonstrates a novel role for E2F pathway outside of cell cycle control in the heart.

The dynamic changes in chromatin play a significant role in lineage commitment and differentiation. These epigenetic modifications control gene expression through recruitment of transcription factors. While the active mark H3K4me3 is present around the transcription start site on the gene, the function of the H3K36me3 mark is unknown. A number of H3K36-specific histone methyltransferases (HMTs) have been identified, however the focus of this study is the HMT Hypb. To elucidate the role of H3K36me3 in mediating expression of developmentally-regulated loci, native chromatin immunoprecipitation (N-ChIP) was performed at a subset of genes. Upon differentiation, we observe that H3K36me3 becomes enriched at the 3’ end of several muscle-specific genes. To further investigate the role of H3K36me3 in myogenesis, a lentiviral-mediated knockdown of the H3K36 HMT Hypb was performed in muscle myoblasts using shRNA. Upon Hypb knockdown, we were surprised to observe enhanced myogenesis. N-ChIP was also performed on differentiated Hypb knockdown cell lines in order to look at H3K36me3 enrichment on genes involved in muscle differentiation. N-ChIP data show a drop in H3K36me3 enrichment levels on myogenin and Ckm genes. The possible occupancy of Hypb on the coding regions of muscle-specific genes was experimentally observed by cross-linked chromatin immunoprecipitation (X-ChIP) on differentiated C2C12 cells and subsequently confirmed by X-ChIP on knockdown lines where the occupancy was lost. A model is proposed that links the observed phenotype with H3K36me3.

The unspliced 9kb HIV-1 RNA (encoding Gag and GagPol) can undergo multiple splicing events to produce members of the 4kb (encoding Env, Vif, Vpr, and Vpu) or 2kb (encoding Tat, Rev and Nef), respectively. The incompletely spliced 9 and 4kb viral RNAs are exported by HIV-1 Rev which interacts with the RRE (Rev responsive element) in these RNAs as well as the nuclear export receptor Crm1. Several proteins can modulate Rev function and/or HIV-1 gene expression, including the nuclear phosphorprotein Sam68. We have found that overexpression of Sam68 stimulates HIV-1 structural gene expression and increases the proportion of unspliced, 3’ end processed viral RNA. This activity requires the RNA binding activity of Sam68. Surprisingly, Sam68 overexpression does not increase the proportion of unspliced, cleaved RNA found in the cytoplasm, suggesting that Sam68 alters the viral RNP to increase its translation. The Sam68 related proteins Slm1 and Slm2 also stimulate 3’ end cleavage and expression of unspliced HIV-1 RNAs. Sam68 and Slm2 were expressed in Hela cells, whereas Slm1 was not. Therefore, we reduced Sam68 expression alone or in combination with Slm2 to determine if these proteins were required for HIV-1 RNA processing or expression. Knockdown of Sam68 and/or Slm2 had little to no effect on viral RNA cleavage or structural gene expression from transiently transfected reporters. Furthermore, depletion of Sam68 only slightly reduced Gag expression from a stably expressed proviral reporter. These results suggest that additional redundant proteins may be present that functionally replace Sam68 and Slm2. We defined a region encompassing the N-terminal GSG (GRP33, Sam68, Gld1) and KH RNA binding motif as the minimal region of Sam68 required to stimulate HIV-1 gene expression in 293 and 293T cells. The minimal mutant enhanced unspliced RNA cleavage in 293T, but not in 293 cells suggesting that Sam68 may act at other stage of the viral lifecycle to increase gene expression.

A method for Agrobacterium-mediated transformation of hybrid poplar (Populus alba x P. grandidentata) suspension cultures and regeneration of transformed plants is described. The best protocol was one in which suspension cultures were inoculated with Agrobacterium tumefaciens to a density of 10⁷ cfu's/ml, cocultivated for 48 hours, plated to cellulose acetate filters at a density of 14 colonies/mm², and cultured on medium containing 1 mg/1 2,4-D. Although cefotaxime inhibited callus growth, it was used in the plating medium to suppress proliferation of Agrobacterium. Selection appeared to be more reliable using hygromycin as compared to kanamycin or geneticin (G418). Transgenic plants were regenerated by culturing the calli on media containing thidiazuron, but no shoots could be regenerated using BA. / Graduation date: 1992