<p><p>Hematopoiesis is the process in which billions of blood cells are produced on a daily basis, and is vital for sustaining life. This process is tightly regulated by a dynamic balance between hematopoietic stem cell (HSC) self-renewal and differentiation, and maintenance of this balance is of critical importance as dysregulation of HSCs can lead to hematopoietic deficiencies or malignancies such as leukemogenesis. While the signaling mechanisms that regulate HSC homeostasis and function are not well understood, our previous studies have identified a calcium/calmodulin (CaM)-dependent protein kinase, CaMKIV, that is intrinsically required for regulating normal proliferation and survival in HSCs. These findings suggest not only the importance of calcium-initiated pathways including CaMKIV-dependent signaling in hematopoietic cells, but also the potential for other calcium/CaM-dependent effector proteins such as other CaM-kinases to be involved in regulating HSCs and hematopoiesis.</p> </p><p><p>The first major section of this dissertation work presented herein was based on the usage of RNA interference (RNAi) technology to specifically deplete HSCs of growth factor erv1-like (Gfer), a gene whose expression appeared to be absent in CaMKIV null HSCs based on comparative microarray analysis with wild-type HSCs, and seemed a potential target of CaMKIV. We showed that depletion of Gfer in HSCs compromised their <i>in vivo</i> engraftment potential and triggered a hyper-proliferative response that led to their exhaustion. We further assessed Gfer-depleted HSCs by using microscopy techniques and found that these cells possessed significantly reduced levels of the cyclin-dependent kinase inhibitor (CDKI) p27^kip1. In contrast, ectopic over-expression of Gfer in HSCs resulted in significantly elevated total and nuclear p27^kip1. We next performed immunoprecipitation-immunoblot analyses to determine whether alteration of Gfer levels would affect p27^kip1's binding with its inhibitor, the COP9 signalosome subunit jun activation-domain binding protein 1 (Jab1), that would subsequently lead to its ubiquitination, and determined that depletion of Gfer resulted in enhanced binding of p27^kip1 to Jab1. Conversely, over-expression of Gfer resulted in its enhanced binding to Jab1 and inhibition of the Jab1-p27^kip1 interaction. Furthermore, normalization of p27^kip1 in Gfer-KD HSCs rescued their <i>in vitro</i> proliferation deficits. These results provide evidence for a novel Gfer-Jab1-p27^kip1 pathway present in HSCs that functions to restrict abnormal proliferation.</p> </p><p><p>The second major section of this dissertation work describes our studies of a CaMKIV kinase, CaMKK2, and its role in HSCs and hematopoietic development. These studies were largely based on the usage of mice genetically ablated for the <i>Camkk2</i> gene in the germline. Herein, we identified a role for CaMKK2 in the restriction of granulocytic fate commitment and differentiation of myeloid progenitor cells. We performed bone marrow transplantation studies and discovered that engraftment by <i>Camkk2^-/-</i> donor cells resulted in the increased production of mature granulocytes in the bone marrow and peripheral blood. Similarly, we used fluorescence activated cell sorting (FACS) to determine that <i>Camkk2^-/-</i> mice possessed elevated numbers of common myeloid progenitor cells, and exhibited an accelerated granulopoietic phenotype in the bone marrow. Expression of ectopic CaMKK2 in <i>Camkk2^-/-</i> common myeloid progenitors was sufficient to rescue aberrant granulocyte differentiation, and when over-expressed in 32Dcl3 cells was also sufficient to impede granulocyte differentiation in a kinase activity-dependent manner. Collectively, our results reveal a novel role for CaMKK2 as an inhibitor of granulocytic fate commitment and differentiation in early myeloid progenitors.</p></p><p><p>While our original intent was to identify and link a downstream target and upstream kinase to CaMKIV in HSCs, our results ultimately did not suggest that Gfer or CaMKK2 function in the same pathway in HSCs as discussed in the following chapters. Nonetheless, our findings represent a considerable advance in identifying and characterizing the functions of two novel regulators, Gfer and CaMKK2, that are important for HSC proliferation and the commitment and early differentiation steps of granulopoiesis, respectively.</p></p> / Dissertation

Membrane proteins function as important communication channels of the cell and its environment that aid in regulating the overall homeostasis of organisms. Understanding the pathways by which these proteins adopt their three-dimensional structures can provide us with key insights into their functions. Failure of a membrane protein to fold into its native structure can lead to disruption of their functions and cause diseases. Through an understanding of the folding mechanisms of membrane proteins it may be possible to identify avenues for the treatment of such diseases. Towards these goals, this thesis describes the biophysical characterization of denatured states of rhodopsin, a model system selected to study helical membrane protein folding. The first contribution of this thesis was to establish approaches that can be used to identify suitable conditions for studying membrane protein folding in vitro. This required screening different denaturing conditions to obtain maximum unfolding without causing aggregation of rhodopsin. 30% SDS and 3% SDS + 8 M urea were found to be the most suitable denaturing conditions. Next, structural features of largely unfolded states of rhodopsin under optimized denaturing conditions were systematically characterized focussing on three levels of structural resolution: global, local and site-specific. Global tertiary structure changes upon SDS denaturation were observed to correlate with SDS micellar structure changes and also hinted at formation of compact intermediate states. Local structural dynamics, probed by NMR spectroscopy, showed that the cytoplasmic domain is more flexible than extracellular and transmembrane domains taken together in spite of an overall increase in flexibility with denaturation. Mobility studies probing site-specific changes by EPR spectroscopy, showed that specific extracellular residues retain more rigidity than cytoplasmic residues in denatured states. These results indicate that the former domain is involved in more stable interactions forming a possible folding core like structure, the location of which correlates with that described by the long-range interaction model of folding. Finally, the importance of dynamics in understanding folding mechanisms of rhodopsin led us to contribute to the development of two novel methodologies: terahertz spectroscopy to detect global motions and 19F NMR using new monofluoro labels to quantify residue specific motions.

A chemical screen of approximately 2000 small molecules in zebrafish embryos identified a compound that generated pericardial edema, suggesting aberrant renal development. Treatment with this compound, 4-(phenylthio)butanoic acid (PTBA), increased the size of the pronephric kidney in zebrafish. Earlier in development, PTBA expanded the expression of renal progenitor cell markers, including lhx1a, pax2a, and pax8. Blocking DNA synthesis with hydroxyurea and aphidicolin before PTBA treatment decreased its efficacy, suggesting that PTBA-mediated renal progenitor expansion is proliferation dependent. Structure-activity analysis revealed that PTBA was an analog of the known histone deacetylase inhibitors (HDACis) 4-phenylbutanoic acid (PBA) and trichostatin A (TSA). Like PTBA, PBA and TSA both demonstrated the ability to expand lhx1a expression in treated embryos. PTBA was subsequently confirmed to function as an HDACi both in vitro and in vivo. HDACis are hypothesized to stimulate retinoic acid (RA) signaling by decreasing the concentration of RA necessary to activate RA receptors (RARs) on target genes. Indeed, treatment with PTBA affected the expression of the RA-responsive genes, cyp26a1 and cmlc2, in a manner consistent with increased RA signaling. Furthermore, blocking the RA pathway with a dominant-negative RAR alpha construct decreased PTBA efficiency. Therefore, PTBA appears to stimulate renal progenitor cell proliferation by activating the RA-signaling pathway. HDACis have been shown to improve renal recovery following acute kidney injury. Since PTBA increases renal progenitor cell proliferation, it may exert similar effects on the multipotent cells involved in regeneration. In an effort to improve PTBA efficacy for pharmacological applications, analogs were generated by modifying the key structural elements of the general HDACi pharmacophore. These were tested along with a panel of known HDACis for their ability to increase lhx1a expression in treated embryos. Several compounds were characterized that function at nanomolar concentrations and do not cause toxicity in kidney cell culture. These second generation PTBA analogs are excellent candidates for development as potential renal therapeutics.

Tuberculosis kills nearly 2 million people each year, and more than one-third of the world�s population is infected with the causative agent, Mycobacterium tuberculosis. Mycobacteriophages, or bacteriophages that infect Mycobacterium species including M. tuberculosis, are already being used as tools to study mycobacteria and diagnose tuberculosis. More than 60 mycobacteriophage genomes have been sequenced, revealing a vast genetic reservoir containing elements useful to the study and manipulation of mycobacteria. Mycobacteriophages also encode proteins capable of fast and efficient killing of the host cell. In most bacteriophages, lysis of the host cell to release progeny phage requires at minimum two proteins: a holin that mediates the timing of lysis and permeabilizes the cell membrane, and an endolysin (lysin) that degrades peptidoglycan. Accessory lysis proteins have also been discovered, often with functions specific to that phage�s host. Many lysins of phages infecting Gram-positive bacteria are proving to be potent antibacterials. Further, lysis proteins can provide insight into the properties and composition of the host cell wall. Given the complexity of the mycobacterial cell wall and its medical relevance in tuberculosis as an immunogenic barrier that complicates treatment, as well as the urgent need for new therapeutic options, the mycobacteriophage lysins clearly warrant further scientific investigation. This work focuses on the mycobacteriophage lysin LysA and the accessory lysis protein LysB. Bioinformatic characterizations show that LysA proteins posess a variety of domains arranged in modular organizations, reflecting extensive recombination within the mycobacteriophage population. In addition to known peptidoglycan-hydrolytic activities, novel cell wall-binding domains are identified, as well as several domains of unknown function found only in mycobacteriophages. LysB proteins are unique to mycobacteriophages and perform a singular role as mycolylarabinogalactan esterases that sever the connection between the mycobacterial outer membrane and the peptidoglycan cell wall complex to ensure efficient lysis and progeny phage release. There is also preliminary evidence of peptidoglycan hydrolytic ability, inducible cell lysis, and growth inhibition of Mycobacterium smegmatis by LysA and LysB proteins. These studies suggest that mycobacteriophage lysis proteins can be exploited as useful tools, both in the laboratory and clinical setting.

Merkel cell polyomavirus (MCV) has been recently described as the cause for most human Merkel cell carcinomas. MCV is similar to simian virus 40 (SV40) and encodes a nuclear large T (LT) oncoprotein that is usually mutated to eliminate viral replication among tumor-derived MCV. In search of novel cellular interactors for MCV LT, we identified the hVam6p cytoplasmic protein involved in lysosomal processing as a binding partner with MCV LT but not SV40 LT. We have shown that hVam6p binds through its clathrin heavy chain homology domain to a unique region of MCV LT adjacent to the retinoblastoma protein (pRB) binding motif. hVam6p and pRB have discrete binding sites on LT. Intriguingly, MCV LT translocates hVam6p to the nucleus, sequestering it from involvement in lysosomal trafficking. A naturally occurring, tumor-derived mutant LT (MCV350) lacking a nuclear localization signal binds hVam6p but fails to inhibit hVam6p-induced lysosomal clustering, suggesting MCV has evolved a novel mechanism to target hVam6p that may contribute to viral uncoating or egress through lysosomal processing during virus replication. In addition, we have investigated the effect of LT-hVam6p interaction on MCV virion production and viral replication. Mutation of the MCV LT-hVam6p binding site enhances encapsidated virion production, which is confirmed by both elevated subgenomic DNA synthesis and viral particle production. Remarkably, overexpression of hVam6p reduces MCV virion production by >90%, suggesting a previously unrecognized role for this protein in regulating virus replication. Collectively, identification of novel binding partners for MCV LT has provided new insights into the mechanisms underlying the MCV lifecycle.

Thesis (Ph. D.)--Cornell University, January, 2007. / Vita. Includes bibliographical references (leaves 163-181).

Thesis (Ph.D.)--Bowling Green State University, 2009. / Document formatted into pages; contains xx, 271 p. : ill. Includes bibliographical references.

Thesis (M.Sc.)--York University, 2008. Graduate Programme in Biology. / Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR38742

Thesis (Ph.D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3342. Advisers: Steven M. Finkbeiner; Graeme Davis.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3351. Adviser: Philip M. Best. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.