The overall objective of this research was to define the cellular basis underlying heritable subfertility in roosters homozygous for the rose comb allele (R/R). Fertilization in the hen is preceded by the ascension of motile sperm through the vagina and sperm sequestration within sperm storage tubules (SST). The objective of the first set of experiments was to determine if reduced sperm sequestration could account for subfertility. Sperm sequestration differed between genotypes following intravaginal insemination (p<0.0001). However, sperm sequestration did not differ between genotypes when sperm were incubated with SST in vitro (p>0.05). Therefore, subfertility was attributed to reduced sperm transport within the vagina. To test this hypothesis, an assay was developed to evaluate fowl sperm motility in vitro. Based upon this assay, ejaculates from subfertile males contained smaller subpopulations of highly motile sperm than the ejaculates from controls (p<0.001). The objective of the next set of experiments was to characterize the motility of individual sperm and to identify a mechanism that could account for the genotypic difference in sperm cell motility. Computer-assisted sperm motion analysis evaluation revealed that ejaculates from controls contained 91% motile sperm whereas ejaculates from subfertile males contained 62% motile sperm (p<0.001). The ATP concentration in sperm from subfertile males was 63% less than that of sperm from controls (p<0.001). A link between sperm ATP concentration and immotility was investigated. First, sperm metabolism was evaluated using motility as an endpoint. The genotypic difference in sperm motility persisted when ATP synthesis was limited to glycolysis (p<0.001). Consequently, mitochondrial respiration could not account for the genotypic difference in sperm motility. In contrast, sperm uptake of [1,2-��H] 2-deoxy-D-glucose did differ between genotypes (p<0.001). The activity of key glycolytic enzymes, creatine kinase, and dynein ATPase did not differ between genotypes (p>0.05). Therefore reduced sperm motility did not appear to be due to ATP synthesis, allocation of high energy phosphate bonds along the axoneme, or ATP consumption (p>0.05). In conclusion, subfertility of roosters homozygous for the rose comb allele was attributed to decreased spermatozoal glucose transport. / Graduation date: 1997

We have developed a system to hunt and reuse special gene integration sites that allow for high and stable gene expression. A vector, named pRGFP8, was constructed. The plasmid pRGFP8 contains a reporter gene, gfp2 and two extraneous DNA fragments. The gene gfp2 makes it possible to screen the high expression regions on the chromosome. The extraneous DNA fragments can help to create the unique loci on the chromosome and increase the gene targeting frequency by increasing the homology. After transfection into Chinese hamster ovary cells (CHO) cells, the linearized pRGFP8 can integrate into the chromosome of the host cells and form the unique sites. With FACS, 90 millions transfected cells were sorted and the cells with strongest GFP expression were isolated, and then 8 stable high expression GFP CHO cell lines were selected as candidates for the new host cell. Taking the unique site created by pRGFP8 on the chromosome in the new host cells as a targeting locus, the gfp2 gene was replaced with the gene of interest, human ifngamma, by transfecting the targeting plasmid pRIH-IFN. Then using FACS, the cells with the dimmest GFP fluorescence were selected. These cells showed they had strong abilities to produce the protein of interest, IFN-gamma. During the gene targeting experiment, we found there is positive correlation between the fluorescence density of the GFP CHO host cells and the specific production rate of IFN-gamma. This result shows that the strategy in our expression system is correct: the production of the interesting protein increases with the increase fluorescence of the GFP host cells. This system, the new host cell lines and the targeting vector, can be utilized for highly expressing the gene of interest. More importantly, by using FACS, we can fully screen all the transfected cells, which can reduce the chances of losing the best cells. / Singapore-MIT Alliance (SMA)

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.

Thesis (Ph.D.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (February 27, 2007) Vita. Includes bibliographical references.

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.

<p>Over the past decades, transitions from repressive rule to democracy have increased all over the world, aiming at establishing disclosure and accountability for the crimes perpetrated. One way of assessing the &ldquo / solidity&rdquo / of these new democracies is to look at their provisions on freedom of expression, one of the most precious and fragile rights of man. The right to freedom of expression was recognised by classical traditional liberal theory as from the eighteenth century. It considered it as a useful tool to enhance true statements within the &ldquo / marketplace of ideas&rdquo / . Liberals also believed that such right was a prerequisite for individual autonomy and selffulfillment. They claimed that it strengthened democracy, by allowing individuals to receive all information on issues of public concern which they needed to vote intelligently. Lastly, they argued that it promoted the ideal of tolerance. Since then, the right to freedom of expression has been considered a cornerstone of democracy and protected as such by international instruments among which the International Covenant on Civil and Political Rights of 1966, the African Charter for Human and Peoples&rsquo / Rights of 1981 and the European Convention for the Protection of Human Rights and Fundamental Freedoms of 1950.</p>

The innovation of DNA analysis techniques has enabled arevolution in the field of molecular biology. In the 70’s,first technologies for sequence determination of DNA wereinvented and these techniques enormously increased thepossibilities of genetic research. A large proportion ofmethods for DNA sequencing is based on enzymatic DNA synthesiswith chain termination followed by electrophoretic separationand detection. However, alternative approaches have beendeveloped and one example of this is the pyrosequencingtechnology, which a four-enzyme DNA sequencing method based onreal-time monitoring of DNA synthesis. Currently, the method is limited to analysis of short DNAsequences and therefore it has primarily been used for mutationdetection and single-nucleotide polymorphism analysis. In orderto expand the use of the pyrosequencing technology, the readlength obtained in the methods needs to be improved. However,it was previously shown that the data quality in pyrosequencingtechnology could be significantly increased by addition ofEscherichia coli single-stranded DNAbinding protein, SSB, tothe sequencing reaction. Since little was known about themechanism of this enhancement, we performed a systematic effortto analyse the effect of SSB on 103 clones randomly selectedfrom a cDNA library. We investigated the effect of SSB on theobtained read length in pyrosequencing and identified thecauses of low quality sequences. Moreover, the effciency ofprimer annealing and SSB binding for individual cDNA clones wasinvestigated by use of real-time biosensor analysis. Resultsfrom these experiments show that templates with highperformance in pyrosequencing without SSB possess effcientprimer annealing and low SSB affnity. To minimise the cost of the pyrosequencing system, effcientand scaleable procedures for production and isolation of theprotein components are required. Therefore, protocol foreffcient expression in E.coliand rapid isolation of native SSB was developed.Moreover, by use of a gene fusion strategy, Klenow polymerasewas produced in fusion with the Zbasic domain at high levels inE. coli. This highly charged protein handle enables selectiveand effcient ion exchange purification at physiological pH.Furthermore, active Apyrase was expressed in Methyltropic yeastPichia pastoris and purified by two chromatographic steps. Since pyrosequencing analysis mainly is performed in a96-sample plate format, an increase in sample capacity would bevery beneficial. One approach to achieve this would be to usemicromachined filter chamber arrays where nano-liter samplescan be monitored in real-time. However, to enable accuratepyrosequencing analysis of parallel samples, the produced lightshould preferable be docked to the correct DNA template.Therefore, two different gene fusion strategies were utilisedbased on directed immobilisation of the light-harvesting enzymeLuciferase on the DNA molecules. The thermostable variant ofthe enzyme was genetically fused to a DNA binding protein(either SSB or Klenow) and the Zbasic purification handle, which could beselectively removed by protease cleavage. A protocol wasdeveloped for effcient expression in E.coliand purification by Ion Exchange Chromatography.The proteins were analysed by complete extension of DNAtemplates immobilised on magnetic beadspyrosequencing monitoredby pyrosequencing chemistry. Results from these experimentsshow that the proteins bound selectively to the immobilised DNAand that their enzymatic domains were active. In summary, the work presented in this thesis pinpointsfeatures in the pyrosequencing technology that needs to befurther developed. Moreover, various protein-based strategiesare presented in order to overcome these limitations. <b>Keywords:</b>pyrosequencing, SSB, Zbasic, Klenow, Apyrase, expression, purification,Biacore, DNA template length, Luciferase, affnity, gene fusion,immobilisation.