Evolutionarily conserved Pumilio (Pum) RNA-binding proteins act as translational repressors during embryo development and cell fate specification. Previous work in the lab has shown that over-expression of Pum2 (Pum2-EML) supports maintenance and suppresses mutilineage differentiation of murine multipotent HSC/MPP-like cell line EML. The subsequent analysis of HSC markers and functional analysis has revealed that wt EML cells share the LKS CD34 positive phenotype, whereas the majority Pum2-EML cells are similar to LKS CD34 negative. The CD34 positive wt EML cells can be divided into CD34low, CD34med and CD34high subpopulations, whereas Pum2-EML CD34 positive cells correspond to CD34low subpopulation. Colony forming assays have revealed that the overall multilineage differentiation of wt EML and Pum2-EML cells strongly correlates with the CD34 expression levels. Multiple experiments have revealed that purified CD34 negative and CD34 positive wt EML cells can generate each other and among CD34 positive wt EML cells the CD34low cells have the highest capacity to give rise to CD34 negative EML cells. We have proposed a model in which CD34 negative EML cells are more primitive cells in an "inactive" (differentiation inhibited) state, that give rise to CD3low "active" (differentiation ready) EML cells. The CD34low EML cells can revert back to the CD34 negative state or give rise to CD34med/high cells that can readily differentiate into multiple lineages. Based on that model, the over-expression of Pum2 leads to increased maintenance of cells in inactive CD34 negative state, and blocks development of CD34 positive cells past the CD34low stage. Cumulatively, these results support the notions that Pum2 could be involved in maintaining the balance between inactive and active state of multipotent hematopoietic cells. The c-kit receptor plays a vital role in self-renewal and differentiation of hematopoietic stem cells (HSC) and multipotent progenitors (MPPs). We have discovered that besides c-kit, the murine multipotent HSC/MPP-like cell line EML expresses the transcript and protein for a truncated form of c-kit, called tr-kit. Notably, the tr-kit transcript and protein levels were down-regulated during cytokine induced differentiation of HSC/MPP-like cell line EML into myelo-erythroid lineages. RT-PCR results show tr-kit is transcribed solely in cell populations enriched for LTR-HSC, STR-HSC and MPPs. The observation that tr-kit is co-expressed with c-kit only in more primitive, HSC and MPP-enriched cell populations raises an exciting possibility that tr-kit functions either as a new component of SCF/c-kit pathway, or is involved in a novel signaling pathway, present exclusively in HSC and MPPs. These findings necessitate functional characterization of tr-kit, and analysis of its potential role in the self-renewal, proliferation and/or differentiation of HSC and multipotent progenitors.

Antibodies specific for DNA, with varying degrees of sequence specificity, are common in many autoimmune diseases including systemic lupus erythematosus. The presence of anti-DNA antibodies is a useful determinant in arriving at a prognosis in these conditions. Given the prevalence of these diseases in both the developing and developed world and the difficulty that often accompanies diagnosis of autoimmune diseases, it is desirable to have sensitive, rapid, and inexpensive diagnostic tools for these diseases. Because of the great sensitivity of electrochemical techniques and their potential utility in characterizing interactions between macromolecules, electrochemistry has great potential as a diagnostic tool for any disease involving antibodies. Anti-DNA antibodies are present in many autoimmune diseases, notably systemic lupus erythematosus. Since DNA is a stable and well-characterized antigen, an electrochemical-based assay is particularly useful for diagnosis of these diseases. <p>The impedance of a gold surface was measured in the presence and absence of single- and double-stranded DNA monolayers. The DNA monolayer was diluted with butanethiol in order to provide a surface with more accessible binding sites than an undiluted monolayer. The change in impedance of the DNA monolayer following exposure to various small molecules and macromolecules was assessed. The molecules used included polyamines that induce conformational changes in DNA, proteins which bind DNA specifically, proteins which bind DNA non-specifically, and proteins which do not bind DNA. The presence of a DNA monolayer, whether single- or double-stranded, increased the impedance of the gold surface and dilution of the monolayers by butanethiol decreased the impedance, as expected. When exposed to polyamines, the impedance of the DNA monolayer decreased further. This could be due to lowered charge repulsion, to DNA condensation, or to a combination of both. When methylated bovine serum albumin was exposed to the monolayer, there was an increase in impedance. Conversely, when bovine serum albumin was exposed to the monolayer, the impedance was only increased at very high concentrations of protein. The increase following exposure to high concentrations of bovine serum albumin was likely due to deposition of protein on to the monolayer. The specificity of these interactions was illustrated by experiments with the antibody Hed 10, which binds single-stranded but not double-stranded DNA. Exposure to Hed 10 only caused a significant change in impedance when exposed to monolayers of single-stranded DNA.<p>The decreased impedance of the DNA monolayer caused by the presence of polyamines is consistent with the known structural perturbations induced by these molecules as measured with other methods. Similarly, the increase in impedance caused by the presence of proteins which bind DNA is consistent with increased steric interference by the protein-DNA complex. The failure of proteins which do not bind DNA to affect the impedance of the monolayer indicated that the effects in the experiments with DNA-binding proteins were due to protein binding and not other factors. The specificity of the assay as demonstrated by the results of the experiments with Hed 10 suggest that impedance-based measurements may provide the basis for a reliable, sensitive, and inexpensive assay for detecting the presence of anti-DNA antibodies in the serum of autoimmune disease patients.

Antibodies specific for DNA, with varying degrees of sequence specificity, are common in many autoimmune diseases including systemic lupus erythematosus. The presence of anti-DNA antibodies is a useful determinant in arriving at a prognosis in these conditions. Given the prevalence of these diseases in both the developing and developed world and the difficulty that often accompanies diagnosis of autoimmune diseases, it is desirable to have sensitive, rapid, and inexpensive diagnostic tools for these diseases. Because of the great sensitivity of electrochemical techniques and their potential utility in characterizing interactions between macromolecules, electrochemistry has great potential as a diagnostic tool for any disease involving antibodies. Anti-DNA antibodies are present in many autoimmune diseases, notably systemic lupus erythematosus. Since DNA is a stable and well-characterized antigen, an electrochemical-based assay is particularly useful for diagnosis of these diseases. <p>The impedance of a gold surface was measured in the presence and absence of single- and double-stranded DNA monolayers. The DNA monolayer was diluted with butanethiol in order to provide a surface with more accessible binding sites than an undiluted monolayer. The change in impedance of the DNA monolayer following exposure to various small molecules and macromolecules was assessed. The molecules used included polyamines that induce conformational changes in DNA, proteins which bind DNA specifically, proteins which bind DNA non-specifically, and proteins which do not bind DNA. The presence of a DNA monolayer, whether single- or double-stranded, increased the impedance of the gold surface and dilution of the monolayers by butanethiol decreased the impedance, as expected. When exposed to polyamines, the impedance of the DNA monolayer decreased further. This could be due to lowered charge repulsion, to DNA condensation, or to a combination of both. When methylated bovine serum albumin was exposed to the monolayer, there was an increase in impedance. Conversely, when bovine serum albumin was exposed to the monolayer, the impedance was only increased at very high concentrations of protein. The increase following exposure to high concentrations of bovine serum albumin was likely due to deposition of protein on to the monolayer. The specificity of these interactions was illustrated by experiments with the antibody Hed 10, which binds single-stranded but not double-stranded DNA. Exposure to Hed 10 only caused a significant change in impedance when exposed to monolayers of single-stranded DNA.<p>The decreased impedance of the DNA monolayer caused by the presence of polyamines is consistent with the known structural perturbations induced by these molecules as measured with other methods. Similarly, the increase in impedance caused by the presence of proteins which bind DNA is consistent with increased steric interference by the protein-DNA complex. The failure of proteins which do not bind DNA to affect the impedance of the monolayer indicated that the effects in the experiments with DNA-binding proteins were due to protein binding and not other factors. The specificity of the assay as demonstrated by the results of the experiments with Hed 10 suggest that impedance-based measurements may provide the basis for a reliable, sensitive, and inexpensive assay for detecting the presence of anti-DNA antibodies in the serum of autoimmune disease patients.

Thesis (M. Sc)--York University, 2000. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 87-100). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ59208.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 186-201). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "December 2001. Typescript. Vita. Includes bibliographical references (leaves 326-347). Also available on the Internet.

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Nutritional Sciences." Includes bibliographical references (p. 149-161).

Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Pharmacology and Toxicology. Title from title-page of electronic thesis. Bibliography: leaves 130-143.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (p. 103-113).

Thesis (Ph. D.)--University of Virginia, 2001. / Includes bibliographical references (leaves 123-140). Also available online through Digital Dissertations.