Thesis (M.A.)--Monterey Institute of International Studies, 2002.

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Thesis (M.S.)--Central Connecticut State University, 1999. / Thesis advisor: Kathy Martin-Troy. " ... in partial fulfillment of the requirements for the degree of Master of Science in Biology." Includes bibliographical references (leaves 56-62).

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Indiana University-Purdue University Indianapolis (IUPUI) / Fanconi anemia (FA) is a genetic disorder characterized by bone marrow (BM) failure. Previous studies suggest that FA patients exhibit alterations in immunologic function. However, it is unclear whether the immune defects are immune cell autonomous or secondary to leucopenia from evolving BM failure. The aim of the current study was to determine whether FA type C deficient (Fancc-/-) macrophages exhibit impaired function and contribute to an altered inflammatory response. In this study, primary peritoneal macrophage function and the inflammatory response of Fancc-/- immune cells after in vivo intraperitoneal (IP) administration of lipopolysaccharide (LPS) were assessed. Fancc-/- peritoneum exhibit normal macrophage distribution at baseline. However, Fancc-/- macrophages exhibit reduced adhesion both on fibronectin and endothelial cells, impaired migration toward monocyte chemotactic protein-1 (MCP-1) and macrophages-colony stimulating factor (M-CSF), and altered phagocytosis of E.coli and ImmunoglobulinG (IgG)-labeled latex beads compared to WT. An altered F-actin reorganization and impaired activation of RhoA were observed in Fancc-/- macrophages. After single LPS injection IP, Fancc-/- mice exhibited decreased macrophage recruitment, reduced peripheral inflammatory monocytes and impaired myeloid colony formation in presence of M-CSF. Upon M-CSF stimulation, Fancc-/- BM derived macrophages (BMDM) showed a decreased phosphorylation of AKT and ERK compared to WT, leading to reduced proliferation. Collectively, these data suggest that Fancc-/- macrophages and subsequent defects in adhesion, migration, phagocytosis, and recruitment in vivo. These data also support a Fancc-/- macrophage cells autonomous defect predisposing to an altered inflammatory response.

Homologous Recombination (HR) can promote genome stability through its capacity to faithfully repair DNA gouble 2trand !;!reak2 (DSBs) and preventing the demise of stalled replication forks in part by catalysing template switching to enable DNA polymerase to bypass lesions. Despite these beneficial roles, inappropriate or untimely HR events can have deleterious consequences. HR can cause genome instability by recombining "inappropriate" homologous sequences, especially if the recombination intermediates are resolved to form crossovers. Over the past few years, study of the rare inherited chromosome instability disorder, Eanconi Anaemia (FA), has uncovered a novel DNA damage response pathway. Although the FA pathway is required primarily for interstrand DNA cross link repair, its precise role in DNA repair reactions is still unclear. FA.Qomplementation group M (FANCM) is the sole component within the FA core complex which possesses a DNA helicase/ATPase domain and an endonuclease domain (albeit non-functional), suggesting that FANCM could translocate along DNA and target the FA core complex to blocked replication forks. To further elucidate the role of FANCM in HR, I have purified Fm11, the FANCM orthologue in the fission yeast Schizosaccharomyces pombe and tested its activity on a range of synthetic replication and recombination intermediates in vitro. Fml1 binds both replication forks and Holliday Junctions (HJs) which are key intermediates of HR.

Indiana University-Purdue University Indianapolis (IUPUI) / The dawn of the genetic era has allowed for investigation of gene transfer therapy as a treatment for certain diseases. Fanconi anemia (FA) is a rare genetic disorder in which the majority of patients develops progressive bone marrow failure (BMF) and require bone marrow transplantation. A possible alternative treatment is autologous gene therapy; however, original clinical trials involving gene transfer for FA were unsuccessful. This has led to re-evaluation of the gene transfer protocols, the vectors and also a deeper investigation of the FA pathway itself. My work has focused on illuminating these areas to further advance gene transfer therapy for FA. Many gene transfer protocols require the hematopoietic stem and progenitor cells (HSC/HPC) to be collected and then transduced ex vivo. The most common collection method is mobilization of the HSC/HPC to the peripheral blood (PB) using granulocyte colony-stimulating factor (G-CSF) and collection via apheresis. In FA patients G-CSF fails to mobilize a sufficient number of HSC/HPC. This has led to research into agents such as AMD3100, a CXCR4 antagonist, which may replace or augment G-CSF mobilization. These data show in two FA murine models that AMD3100 synergizes with G-CSF resulting in a significant increase in mobilization as compared to G-CSF alone. Previous work in our lab has shown that prototype foamy virus (FV) is an efficient gene transfer vector. Here a modified FV vector is used to transduce mobilized FA cells. The data indicate that long-term repopulating cells mobilized with both G-CSF and AMD3100 can be efficiently transduced by our FV vector. Clinically, FA is characterized mainly by BMF, but also by myelodysplasia (MDS) and acute myeloid leukemia (AML). However, current FA murine models do not display these disease phenotypes. These data show that double-mutant Fancc-/-;Fancg-/- mice spontaneously develop BMF, MDS and complex random chromosomal abnormalities that the single-mutant mice do not. Importantly, this model closely recapitulates the phenotypes found in FA patients and may be useful as a preclinical platform to evaluate the molecular pathogenesis of spontaneous BMF and MDS in FA and novel gene transfer protocols for FA.