Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Jaquelin P. Dudley. Vita. Includes bibliographical references. Also available from UMI.

Thesis supervisor: Kevin G. Rice. Includes bibliographical references (p. 170-181).

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 134-158). Also available in print.

In multicellular organisms, cell growth and differentiation is controlled in part by apoptosis. One major apoptotic pathway is triggered by Fas receptor (Fas)-Fas ligand (FasL) interaction. Resistance to Fas-mediated apoptosis is regulated through the production of a soluble Fas isoform (sFas), created by exclusion of transmembrane spanning sequences encoded within exon 6 (FasΔ6) that can bind FasL and block apoptosis. Long noncoding RNAs (lncRNAs) are >200-nucleotide sequences that are important regulators of cellular programs. However, their role in erythropoiesis is just beginning to be appreciated with studies limited to murine systems. I studied the potential role of lncRNAs during human red blood cell development using RNA from cultured CD34+ purified from fetal liver (FL), cord blood (CB), or adult bone marrow (BM) to screen 82 documented lncRNAs. This screen revealed that the Fas Anti-sense (Saf) was consistently increased during maturation and levels high for BM compared to FL or CB. Next, I characterized the regulatory sequence of Saf and by In silico analysis identified canonical binding sites for the erythroid-specific transcription factors KLF1 and GATA1. Chromatin immunoprecipitation (ChIP) assays confirmed binding of both factors to their target sequences and luciferase reporter constructs revealed synergistic activity evidenced by increase in luciferase expression relative to controls. Genome wide expression analysis using cells with overexpression of Saf showed no effect on global gene transcription, suggesting Saf function at post transcriptional levels. Saf was shown to participate in alternative splicing of Fas pre-mRNA through unknown mechanisms. Using a combination of biochemical assays, overexpression and knockdown studies, I showed how this occurs. Cell fractionation and RT-PCR demonstrated that Saf is localized in the nucleus. I found that Saf directly interacted with Fas pre-mRNA to form RNaseA-resistant double-stranded RNA intermediates at regions that flank Exon6. Post-transcriptional function of Saf was confirmed by qRT-PCR demonstrating significantly increased levels of sFas for Saf overexpressing cells. Enrichment for sFas RNA coincided with reduced Fas on the cell surface and increased sFas protein levels when conditioned supernatants were assayed by ELISA. Conversely, siRNA-mediated knockdown of Saf significantly reduced sFas production compared to non-targeting siRNA controls. Saf-interacting proteins were identified by mixing in vitro transcribed and biotin-labeled Saf RNA with nuclear lysates followed by mass spectrometry analysis. This screen identified human splicing factor 45 (SPF45) which has a known role in Fas pre-mRNA alternative splicing. Specific SPF45/Saf interaction was confirmed by RNA pulldown and western blot with SPF45-specific antibodies and the ability to detect sequences for Saf, Fas and sFas by RT-PCR of RNA that immunoprecipitated with SPF45. SPF45 knockdown decreased sFas transcripts and this reduction corresponded to limited production of sFas and increased sensitivity to Fas-mediated apoptosis when cells were exposed to the Fas-activating antibody CH11. Importantly, overexpression of Saf in SPF45 knockdown cells failed to rescue production of sFas supporting the hypothesis that Saf and SPF45 co-participate in modulating Fas pre-mRNA splicing. Protein phosphorylation modulates the interaction of the splicing factors with RNA. The effect of phosphorylation on the Saf-SPF45 interaction was evaluated using stable cell lines expressing a myc-tagged SPF45 protein or versions modified to introduce alanine in place of threonine 71 (T-71-A) or serine 222 (S-222-A) to prevent phosphorylation. Mutation in S-222-A reduced the interaction of SPF45 with Saf. I conclude that Saf interacts with Fas pre-mRNA at sequences that flank exon 6 and recruits phosphorylated SPF45 as mechanism to recognize exon 6 and allow for alternative splicing of Fas Pre-mRNA. Collectively, these studies reveal a novel mechanism to regulate apoptosis that may be responsible for cell proliferation and drug resistance.

The obligate methylotroph, Methylophilus methylotrophus, used by ICI for its Single Cell Protein production, may represent a valuable alternative host organism to E. coli for the commercial production of heterologous gene products. The organism has the advantages of being safe, coupled with an ability to grow well, in large quantities, on a cheap carbon source. This thesis describes the construction, characterisation and analysis of a series of plasmid cloning vectors designed for use in M.methylotrophus. The vectors are based on the IncQ plasmid R300B and maintain a broad host range with an increased capacity for easy-to-use cloning sites mainly derived from the E.coli plasmids pBR322 and pBR328. All the vectors thus carry antibiotic- resistance genes containing restriction sites which could lead to insertional inactivation as a means of detecting recombinants. One plasmid in particular, pGSS33, has four antibiotic-resistance genes all of which contain at least one such restriction site. The first demonstration of expression of a eukaryotic coding sequence, murine dihydrofolate reductase (DHFR), in M.methylotrophus has been described. This has been followed by expression of E.coli B-galactosidase and synthetic human ?-1 interferon, all making use of pGSS vectors. The pGSS15-DHFR plasmid, pDHFR2.43, may turn out to be a valuable test plasmid for studying the stability of cloned eukaryotic coding sequences in both E.coli and M.methylotrophus; it is readily detected (trimethoprim-resistant) and can be grown with or without selection. A start has been made towards providing increased expression from vectors carrying strong E.coli promoters (lacUV5 and synthetic trp) which have been demonstrated to work well in M.methylotrophus. Broad host range cosmid vectors have been constructed which have the potential to be used for the production of gene-banks in Gram-negative organisms other than E.coli. Copy numbers of the vector plasmids have been determined in E.coli strains and several different methods of measurement reviewed in an attempt to find one suitable for use with M.methylotrophus. An encouraging lead has been identified in the search for a high copy number plasmid, which coupled with a strong promoter, could provide the basis for a very efficient batch production process. Thus with the availability of easy-to-use cloning vectors, convenient delivery systems and the accumulated evidence of strong E.coli promoters working efficiently in M.methylotrophus, this organism can seriously be considered as a safe alternative host to E.coli.

The purpose of this paper is to make a detailed study of vector spaces and a certain vector-like system.

archives@tulane.edu / Brazil was at the epicenter of the 2015-2016 Zika virus (ZIKV) epidemic, with approximately 60% of globally reported cases within its borders. This dissertation explored how contextual factors were relevant to women’s ability and desire to adhere to the ZIKV recommendations made by the Brazilian Ministry of Health during the epidemic. More specifically this dissertation focused on women’s response to the recommendation to avoid pregnancy during this epidemic. Methods utilized in this dissertation included a rapid assessment and a series of case studies of pregnancy narratives from women in Fortaleza, Brazil. By more deeply exploring women’s fertility decision making during the ZIKV epidemic, a more comprehensive understanding of the underlying contextual factors influencing women’s fertility desires, decisions, and behaviors was developed. Among the findings, we document that the health recommendations promoted during the epidemic were made without the use of best practices in health communication and without acknowledgement of the context in which the epidemic was taking place. For example, these recommendations assumed women had the power to adhere to health authorities’ directives to prevent ZIKV. Furthermore, we document how these generic recommendations ignored the unique conditions of vulnerable populations constrained by income, class, and gender. Analysis showed that participants perceived the recommendations as unrealistic, recycled, unhelpful, or not directed toward them. Case studies and pregnancy narratives illuminated major gaps in the healthcare and support provided to pregnant women during the outbreak, as well as to the mothers of children born with Congenital Zika Syndrome (CZS). ZIKV is the latest of an inevitable influx of emerging infectious diseases that are being brought on by deforestation, urbanization, and global warming. With these diseases on the horizon, it is important to acknowledge and research the underlying contextual factors that permeate these epidemics and continue to devastate the most marginalized within societies. / 1 / Jeni Stolow

It is a universal goal of mankind to seek or to create sacred spaces. What is a sacred space, and what are the qualities which describe that space? For thousands of years, the grove has possessed qualities which have made its space sacred. What defines a grove and the space it makes and how does this form relate to us today? In September 2002 Congress passed legislation for the establishment of Living Memorials to honor and commemorate the losses of September 11th, 2001. Through a program developed by the United States Forest Service and American Forests, these Living Memorials are taking the form of memorial groves. Washington, D.C. chose Kingman Island, located in the Anacostia River, as the location for their 9/11 Memorial Grove. Using the Kingman Island site, this thesis investigates the qualities of sacred space, and the qualities of the grove, to discover how a contemporary grove can create a sacred space that reflects the traditions of the Living Memorial, and honors the losses of September 11th. / Master of Landscape Architecture

No description available.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [154]-164).