I proposed the study of two distinct aspects of Ten-Eleven Translocation 2 (TET2) protein for understanding specific functions in different body systems. In Part I, I characterized the molecular mechanisms of Tet2 in the hematological system. As the second member of Ten-Eleven Translocation protein family, TET2 is frequently mutated in leukemic patients. Previous studies have shown that the TET2 mutations frequently occur in 20% myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN), 10% T-cell lymphoma leukemia and 2% B-cell lymphoma leukemia. Genetic mouse models also display distinct phenotypes of various types of hematological malignancies. I performed 5-hydroxymethylcytosine (5hmC) chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) of hematopoietic stem/progenitor cells to determine whether the deletion of Tet2 can affect the abundance of 5hmC at myeloid, T-cell and B-cell specific gene transcription start sites, which ultimately result in various hematological malignancies. Subsequent Exome sequencing (Exome-Seq) showed that disease-specific genes are mutated in different types of tumors, which suggests that TET2 may protect the genome from being mutated. The direct interaction between TET2 and Mutator S Homolog 6 (MSH6) protein suggests TET2 is involved in DNA mismatch repair. Finally, in vivo mismatch repair studies show that the loss of Tet2 causes a mutator phenotype. Taken together, my data indicate that TET2 binds to MSH6 to protect genome integrity. In Part II, I intended to better understand the role of Tet2 in the nervous system. 5-hydroxymethylcytosine regulates epigenetic modification during neurodevelopment and aging. Thus, Tet2 may play a critical role in regulating adult neurogenesis. To examine the physiological significance of Tet2 in the nervous system, I first showed that the deletion of Tet2 reduces the 5hmC levels in neural stem cells. Mice lacking Tet2 show abnormal hippocampal neurogenesis along with 5hmC alternations at different gene promoters and corresponding gene expression downregulation. Through the luciferase reporter assay, two neural factors Neurogenic differentiation 1 (NeuroD1) and Glial fibrillary acidic protein (Gfap) were down-regulated in Tet2 knockout cells. My results suggest that Tet2 regulates neural stem/progenitor cell proliferation and differentiation in adult brain.

The immune systems of cnidaria are important to study for two reasons: to gain a better understanding of the evolution of immune responses, and to provide a basis to partially redress the precipitous world-wide die-offs of reef corals, some of which have been attributed to diseases and stress. Many immune responses share ancient evolutionary origins and are common across many taxa. Using Swiftia exserta, an azooxanthellate ahermatypic local octocoral, as a proxy model organism to study aspects of innate immunity in corals and cnidaria allows us to address both of the reasons listed above while not using endangered species. Utilizing a coral that does not contain symbiotic dinoflagellates (zooxanthellae) simplifies the system by restricting the source of proteins to a single genome. The lack of zooxanthellae in Swiftia exserta also allows the animal’s simple adaptation to lab settings. This study of the innate immune system of an octocoral demonstrates: 1) a novel understanding of the microanatomy of octocoral tissues; 2) that Swiftia exserta has at least two cell types that function as constitutive immunocytes; and 3) the presence of two potent antibacterial peptides, one with a mass between 4694 and 4696 Daltons. My report on the microanatomy of the coenenchyme, the tissue between polyps, advances the understanding of octocoral anatomy by systematically comparing histology sections with electron micrographs. Applying various techniques of enzyme histochemistry, coupled with cryo-preservation, to the coenenchyme I have identified at least two populations of constitutive immunocytes in Swiftia exserta. Two antibacterial proteins are identified by protein purification and antimicrobial testing techniques. The more active protein is partially characterized with modern hyphenated mass-spectrometry techniques, and can be the focus of future study.

Previously, our lab demonstrated that heterologous immunity could result in either gain or loss of protective immunity and alteration in immune pathology following infection by a second un-related pathogen. One of the prototypical models to study T cell-mediated heterologous immunity involves two distantly related arenaviruses, namely lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PV). Each virus encodes a cross-reactive CD8 epitope that has six out of eight in amino acid (aa) similarity with respect to its counterpart at the position 205-212 of the nucleoprotein (NP205). Heterologous challenge between LCMV and PV results in 1) expansion of the cross-reactive NP205-specific CD8 T cell responses and alteration of the immunodominance hierarchy and 2) partial protective immunity (heterologous immunity). Our lab showed that cross-reactive NP205-specific CD8 T cell receptor (TCR) repertoires become extremely narrowed following a heterologous challenge between LCMV and PV. Therefore, I questioned if LCMV NP205 epitope escape variants could be isolated during a dominant but narrowed crossVI reactive NP205-specific CTL response. In the first part of my thesis, I describe the isolation of a LCMV NP-V207A CTL escape variant in vivo using PV-immune animals challenged with LCMV clone 13. The LCMV NP-V207A variant contains a point mutation, which results in the switching of valine to alanine at the third non-anchoring residue of the LCMV NP205 CD8 epitope. Immunization of mice with the LCMV NP-V207A variant results in a significantly diminished cross-reactive NP205-specific CD8 T cell response. This suggests that the point mutation is responsible for the loss in the immunogenicity of the LCMV NP205 CD8 epitope. In addition, an in vitrorescued(r) recombinant LCMV variant (r/V207A) that encodes the original mutation also induces a highly diminished cross-reactive NP205-specific CD8 T cell response in mice. In agreement with the result obtained from the intracellular cytokine assays (ICS), MHC-Ig dimers loaded with the LCMV NP205 (V-A) peptide could only detect a minute population of cross-reactive NP205-specific CD8 T cells in mice infected with r/V207A variant virus. All the data indicate that the point mutation results in a significant loss in immunogenicity of the LCMV NP205 CD8 epitope. So far, no direct link between the cross-reactive NP205-specific CD8 T cells and heterologous immunity had been established in this system. Therefore, we immunized mice with either LCMV WT or the LCMV NP-V207A variant virus and showed that a significant loss of heterologous immunity is associated with the group immunized with LCMV NP-V207A variant virus. Again, r/V207Aimmune animals also displayed a significant loss in heterologous immunity following PV challenge. This suggests that the cross-reactive NP205-specific CD8 T cells mediate the majority of heterologous immunity between LCMV and PV in vivo. In comparison to the PV-immune control group, PV clearance kinetics mediated by the cross-reactive NP205-specific CD8 T cells were significantly delayed. Finally, these data also suggest that bystander activation plays very little role in heterologous immunity between LCMV and PV. Many studies in murine systems and humans suggest that cross-reactive T cells are often associated with immune pathology. We showed that in mice that were sequentially immunized with PV and LCMV (PV+LCMV WT double immune mice), there was a development of a high incidence and high level of immune pathology known as acute fatty necrosis (AFN) following a final PV challenge. The data suggest that these cross-reactive NP205-specific CD8 T cells might play an important role in immune pathogenesis. Therefore, we asked if the cross-reactive NP205-specific CD8 T cells play a role in immune pathogenesis by comparing the incidence of AFN between the (PV+LCMV WT) and the (PV+LCMV NP-V207A) double immune mice following a final PV challenge. In agreement with our hypothesis, the result showed the (PV+LCMV NP-V207A) double immune mice developed a significantly lower incidence of AFN compared to the (PV+LCMV WT) double immune mice. However, linear correlation studies comparing the frequency of different antigen-specific CD8 T cell populations within the (PV+LCMV WT) double immune mice before challenge and the severity of AFN following the PV challenge suggest that two opposing antigen-specific CD8 T cell populations are involved in determining the final outcome of the immune pathology. The PV NP38-45-specific CD8 T cell response (PV NP38) appears to be more protective than the cross-reactive NP205-specific CD8 T cell response. In addition, a positive linear correlation between the ratio of cross-reactive NP205 to PV NP38 and the severity of AFN seem to suggest that these cross-reactive populations are important contributors to immune pathogenesis. Peptide titration studies examining the functional avidities to different antigenic specificities suggest that both populations consist of high avidity TCR and peptide MHC (TCR:pMHC) interactions. However, skewing within the cross-reactive NP205 specific CD8 T cell response towards the LCMV NP205 epitope response in one of the (PV+LCMV WT) double immune mice suggests that cross-reactive NP205 specific CD8 T cells could constitute a sub-optimal response to a PV challenge. In summary, I questioned what might be some of the immunological consequences of heterologous immunity in this model. First of all, we have established a direct link between the cross-reactive NP205-specific CD8 T cell response and heterologous immunity in LCMV and PV. Second of all, I demonstrated that a LCMV NP205 epitope escape variant could be selected in vivo under the conditions of heterologous immunity. In addition, I showed that PV clearance kinetic was significantly delayed in cross-reactive NP205-mediated heterologous immunity as compared to homologous challenge. Finally, we demonstrated that cross-reactive NP205-specific CD8 T cells could play an important role in immune pathogenesis in this model. However, correlation data indicate that two opposing antigen-specific CD8 T cell populations could ultimately decide the outcome and magnitude of immune pathology in each individual mouse. All the data presented above strongly suggest that the cross-reactive NP205 CD8 T cells play a crucial role in immune pathology in this model system by 1) interfering with the regular establishment of immunodominance hierarchy orders, or 2) exhibiting a sub-optimal protective immunity due to the nature of the cross-reactive epitope.

The Tec family tyrosine kinase, Itk, plays an important role in signal transduction following T cell receptor engagement. Several prior studies have established the importance of Itk in immune system processes, such as T cell development and T cell activation. Additional biochemical studies have found that Itk specifically functions within a multi-molecular signalosome complex, which ultimately functions to provide a platform by which Itk can phosphorylate and activate PLC-γ1, a crucial step in T cell activation. To further study how Itk regulates distinct immune outcomes via T cell effector processes within the peripheral immune system, and to further understand how Itk functions in T cells in response to a physiological ligand-receptor interaction, I crossed Itk-deficient mice to mice transgenic for a TCR specific for a moth cytochrome C peptide. My studies have established a unique role for Itk in several important aspects of T cell function. Following T cell activation, I identified an imperative role for Itk in activation-induced cell death via FasL, a mechanism of immune homeostasis. Furthermore, I found Itk plays a unique role in the process of T cell differentiation, where Itk positively regulates the induction of cytokine genes, such as IL-4, while negatively regulating the induction of T-bet, a transcription factor important for Th1 differentiation. Lastly, following T cell differentiation, I found that Itk mRNA and protein are up-regulated during Th2 differentiation, while Rlk, a related Tec kinase, disappears rapidly from Th2 cells, indicating a critical role for Itk in Th2 cell function. Collectively, my thesis work has more clearly defined an important function for Itk not only in TCR signaling, but also in immune processes such as T cell differentiation and activation-induced cell death that are required for proper immune function.

Itk and Rlk are members of the Tec kinase family of non-receptor protein tyrosine kinases that are preferentially expressed in T cells. Numerous previous studies have demonstrated that these proteins play an important a role in the regulation of signalling processes downstream of TCR activation in CD4+ T cells, particularly in the phosphorylation of PLCγl. In addition, Itk and Rlk have both been shown to be important for CD4+ T cell development, differentiation, function and homeostasis following TCR activation. In the absence of Itk and Rlk, CD8+ SP thymocytes and T cells develop a memory/previously activated phenotypic profile, however, very little is known about the influence of Itk and Rlk on CD8+ T cell development and function. This study illustrates a previously unappreciated role for Itk and Rlk in the regulation of cytokine signals during CD8+ SP thymocyte maturation, and in the development of the memory CD44hi profile of Itk -/- and Itk -/- Rlk -/- CD8+ SP thymocytes and CD8+ T cells. This study also provides the first detailed study of the role of loss of Itk and particularly both Itk and Rlk in CD8+ signalling and function and shows that these Tec kinase family members play an important role in the maintenance of CD8+ T cell fitness and function, particularly in the ability of CD8+ T cells to accumulate in response to infection. Collectively, my studies demonstrate a critical role for Itk and Rlk in the generation of optimal CD8+ T cell responses. They also raise the novel observation that these proteins may be involved on the regulation of cytokine signals in T cells.

Heavy chain isotype switch recombination is preceded by the appearance of RNA initiating 5' of the specific switch region which will undergo recombination. In an effort to understand the potential function of germline transcripts in switch recombination and the degree to which the regulation of germline transcripts correlates with the regulation of switching, we studied this process in the murine B-lymphoma cell line I.29μ, which in the presence of bacterial lipopolysaccharide (LPS) switches primarily to IgA and less frequently to IgE. Levels of α-germline transcripts initiating upstream of α switch (Sα) sequences are elevated in clones of this line which switch well as compared to clones which switch less frequently. TGFβ1 has been shown to increase α-germline transcripts and switching to IgA expression in LPS-stimulated murine splenic B-cells. We now demonstrate in I.29μ cells that TGFβ also increases switching to IgA and increases the level of α-germline transcripts 5 to 9 fold. Nuclear run-on analysis shows that this increase is at the level of transcription. Thus, TGFβ appears to direct switching to IgA by inducing transcription from the unrearranged Sα- CαDNA segment. Germline α RNA is quite stable in I.29μ cells, having a half life of about 3 to 5 hours, and we find only slight stabilization in the presence of TGFβ. Levels of ε-germline transcripts are not increased by TGFβ . IL-4, which modestly increases switching to IgA in I.29μ cells, slightly increases trancription of α-germline RNA. However, we present evidence suggesting that endogenously produced IL-4 may also act at additional levels to increase switching to IgA. IFNγ, which reduces IgA expression in these cells, also reduces the level of α-germline transcripts. IFNγ also reduces the level of ε-germline transcripts induced by IL-4. Our results support the hypothesis that the regulation of transcription of particular switch sequences by cytokines in turn regulates the specificity of recombination. In studies aimed at identifying other signalling pathways that promote class switching, we discovered that inhibitors of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) increase lipopolysaccharide (LPS)-induced switching to IgA in the B cell lymphoma I.29μ and to IgG1 in LPS + IL-4-treated splenic B cells. PARP, which binds to and is activated by DNA strand breaks, catalyzes the removal of ADP-ribose from NAD+ and poly(ADP-ribosylation) of chromatin-associated acceptor proteins. This enzyme is believed to function in cellular processes involving DNA strand breaks as well as in modulating chromatin structure. In I.29μ cells, PARP inhibitors increase IgA switching by day 2 and cause a 5-fold average increase in switching on day 3 as assayed by immunofluorescence microscopy. The PARP inhibitor, nicotinamide, also causes a reduced intensity of hybridization of Cμ and Cα specific probes to genomic DNA fragments containing the expressed VDJ-Cμ and the unrearranged Sα - Cα segments, respectively, indicating that PARP inhibition increases rearrangment of these fragments. Induction of switching by PARP inhibitors is not mimicked by treatment with cAMP analogs or reduced by inhibitors of protein kinase A (PKA). Induction of switching by PARP inhibitors does not appear to involve increased levels of transcription of the unrearranged Cα gene, although TGFβ is required for optimal induction by PARP inhibitors, consistent with a requirement for transcription of the unrearranged CH gene. PARP inhibitors do not overcome the requirement for endogenously produced IL-4.

T lymphocytes are essential components of the immune system and as such are continually regulated by a variety of factors. Every step of their development, survival and function is tightly monitored to ensure their ability to recognize most foreign agents and mount adaptive immune responses during pathogenic infections, while remaining tolerant to self-antigens. Among the many factors that participate in the regulation of T cell development and function are the cytokines. Cytokines that signal through the common gamma (γс) chain and the Janus kinase 3 (Jak3) include IL-2, -4, -7, -9, -15, and -21 and have been implicated in the regulation of every stage in the life of a T cell. Therefore, it is not surprising that mutations in the γс chain or Jak3 lead to a SCID condition in humans and mice. Specifically, Jak3-deficient mice are characterized by a reduction in thymic cellularity and dysregulated T cell homeostasis. They have an expansion of memory-like CD4+ mature T cells and an almost complete absence of mature CD8+ T cells. By investigating the TCR repertoire of CD4+ T cells in the thymus and spleen of Jak3-/- mice, I deduced that the CD4+ T cell activation and expansion is TCR-specific and takes place in the periphery of the mice. After crossing Jak3-deficient mice to Bcl-2 transgenic mice I showed that the developmental block observed in Jak3-/- mice could not be rescued by the anti-apoptotic factor, despite the fact that its expression did increase, slightly, the total numbers of developing thymocytes. The enforced expression of Bcl-2 was also not sufficient to revert the dysregulation of T cell homeostasis in Jak3-/- mice. Finally, in order to further understand the role played by γс cytokines during T cell function, I investigated the ability of mature Jak3-/- CD8+ T cells to become activated and differentiate into effector cells in response to a viral infection. My results indicate that CD8+ T cells are activated and proliferate in response to a viral infection, but their survival, as well as their ability to proliferate and differentiate into effector cells are greatly impaired, resulting in the inability of Jak3-deficient mice to mount a protective response.

The immune response to viral infection involves a complexity of both innate and adaptive pathways at the cellular and the molecular level. There are many approaches to begin to define the pathways at work to control viral pathogenesis. The approach favored in this thesis was to conduct a broad screen of the innate immune response at the gene expression level of infected cells. The innate immune response is critical to the control of viral infections. Type I interferons (IFN), IFNα and IFNβ, are antiviral proteins that are an integral part of the innate immune response. Furthermore, by virtue of their effects on maturation and activation of antigen-presenting cells, IFNs are a pivotal link between the innate and adaptive immune systems. Most cell types produce type-I IFN when exposed to viruses. However, viruses have evolved multiple strategies to suppress IFN production or signaling. It is imperative to understand the virus-host interaction at the molecular level in order to identify as yet unknown mechanisms of the host antiviral response; these additional pathways may be useful in counteracting the viral suppression of IFN. Type-I IFNs regulate expression of at least five hundred genes, suggesting a complex network of signaling pathways. Depending on the cell type different proteins regulate the induction of IFN or the expression of IFN-inducible genes. Identification of proteins that induce selected IFN-inducible genes may provide synergistic activity with or may have an advantage over type-I IFN for anti-viral therapy in the future. Many diseases are untreatable if identified late in their progression. In resource-limited countries, many diseases are diagnosed clinically, which can lead to incorrect or delayed diagnosis and treatment. The identification of biomarkers of disease has the potential to guide the correct therapy in a timely fashion. The objective of this thesis was to identify novel anti-viral therapies and disease biomarkers for dengue virus (DENV) infection. DENV is a mosquito-borne positive-sense single-stranded RNA virus, which causes an estimated 50 million infections annually. Most DENV infections result in a febrile illness called Dengue fever (DF). Less frequently, infections cause Dengue hemorrhagic fever (DHF), a potentially fatal vascular leakage syndrome associated with the production of pro-inflammatory cytokines. At present patients infected with DENV can only be treated by intravenous fluid support to prevent hypovolemia and hypotensive shock. This treatment is less effective in severe cases if the diagnosis is delayed. Identification of therapeutics with both antiviral and immune-modulatory activity may lower patient mortality and reduce the burden of DENV on society. DENV infection is cleared in most individuals after a short period of viremia {Libraty, 2002 #2225}. Based on in vitro and mouse models, type-I and type-II IFN signaling pathways are thought to be critical in the regulation of DENV infection. Higher serum levels of type I and type II IFNs during acute DENV infection in patients lend support to the above hypothesis {Kurane, 1993 #2152; Libraty, 2002 #2225}. To understand the DENV-human host cell interaction at the molecular level, we performed global gene expression analysis on DENV-infected primary human cells using Affymetrix GeneChips (HG-U133A). We studied dendritic cells (DC), monocytes, B cells and human umbilical vein endothelial cells (HUVECs), all of which are known to be permissive to DENV infection. We first identified genes commonly regulated in multiple cell types in response to DENV infection; we hypothesized that understanding this common gene expression profile would identify signaling pathways involved in regulation of viral spread, activation of immune cells or induction of inflammation. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), one of the 23 common response genes, was identified as a key link between type I and type II interferon response genes. Pretreatment of cells with recombinant TRAIL (rTRAIL) inhibited DENV replication in monocytes, B cells, HUVECs and DCs. Using the DC infection model, we showed that this inhibition of viral replication was apoptosis-independent. Type-I IFN receptor (IFNR) blocking experiments showed that signaling through the type-I IFN receptor played an important role in the antiviral activity of exogenous rTRAIL. Furthermore, TRAIL also significantly reduced the expression of mRNA and protein of pro-inflammatory cytokines (TNFα, MIP-1β and IFNα) and chemokines (MCP-2, IP-10 and IL-6) in response to DENV infection. The data that TRAIL inhibits both viral replication and pro-inflammatory cytokine production suggest that TRAIL has therapeutic value in dengue. The endothelial cell is the site of pathology in DENV infection in vivo (vascular permeability and plasma leakage). To understand the direct effect of DENV infection on endothelial cells and its role in the induction of genes regulating vascular permeability, we compared gene expression in DENV-infected HUVECs to that of uninfected cells and cells infected with other RNA and DNA viruses, including flaviviruses (West Nile, yellow fever, and Japanese encephalitis viruses), bunyaviruses (Sin Nombre and Hantaan viruses), Epstein-Barr virus and vaccinia virus. Among the genes confirmed for their differential expression, ST2 (Interkeukin-1 receptor-like-1 protein-IL1RL1) and indoleamine 2,3-dioxygenase (IDO) were identified to be upregulated specifically in response to DENV infection. Higher serum soluble ST2 (sST2) levels were detected in DENV-infected patients than in patients with other febrile illnesses (OFI) at the end of the febrile stage and at defervescence (p=0.0088 and p=0.0004, respectively). In addition, patients with secondary DENV infections had higher serum sST2 levels compared with patients with primary DENV infections (p=0.047 at the last day of fever and p=0.030 at defervescence). Higher levels of IDO activity (pIn conclusion, global gene expression analysis identified novel proteins with promising characteristics for the treatment and/or diagnosis of DENV infection. Although further studies will be needed to validate the clinical utility of TRAIL, sST2, and IDO, these studies demonstrate the utility of this unbiased genomics approach to identify therapies to currently incurable diseases.

Purpose: The purpose of this study was to determine the usefulness of SBAR-cued web-based communication skills training and address study participants' perceptions of the training. Specific Aims: Evaluate the usefulness and accuracy of participants to answer prompts of SBAR-cued communication responses. Describe individuals' perspectives of the acceptability of using SBAR patient-HCP communication simulation to better prepare for ED visits during a SCC. Framework: This study was guided by The Theory of Self-Care Management for Sickle Cell Disease (SCMSCD). Design: A secondary analysis was conducted using a qualitative descriptive approach. Inter-rater reliability (IRR) of qualitative data was used to evaluate the usefulness and accuracy of participants to answer prompts of SBAR-cued communication responses. Content analysis was also utilized to describe individuals' perspectives of the acceptability of using SBAR patient-HCP communication simulation to better prepare for ED visits during a SCC. Results: IRR between raters ranged from 64%-94% with predominant themes of (1) Patient-Provider Communication and Interaction, (2) Patients want to be Heard and Believed, (3) Accuracy of the ED Experience and Incorporating the Uniqueness of each Patient and (4) Overall Usefulness of the Video Trainer emerging. Conclusions: This secondary analysis supported how SBAR can be effectively used to assist patients in a SCC to communicate with their HCP. Participants' responses indicated the training module facilitated communication between patients and HCPs.

Chronic myeloid leukemia (CML) is a disease characterized by the expansion of granulocytic cells. The BCR-ABL tyrosine kinase inhibitor imatinib, the frontline treatment for Ph+ leukemias, can induce complete hematologic and cytogenetic response in most chronic phase CML patients. Despite the remarkable initial clinic effects, it is now recognized that imatinib will unlikely cure patients because a small cell population containing leukemic stem cells (LSCs) with self-renewal capacity is insensitive to tyrosine kinase inhibitors. In Chapter I, I briefly review the BCR-ABL kinase and its related signaling pathways. BCR-ABL kinase activates several signaling pathways including MAPK, STAT, and JNK/SAPK. BCR-ABL also mediates kinase-independent pathways through SRC family kinases. I will also discuss pathways involving β-catenin, hedgehog, FoxO and Alox5 are critical to the regulation of self-renewal and differentiation in LSC of CML. As detailed in Chapter II, I describe our work evaluating the effects of omacetaxine, a novel CML drug inducing cell apoptosis by inhibition of protein synthesis, on self-renewal and differentiation of LSCs and BCR-ABL-induced CML and acute lymphoblastic leukemia (B-ALL) in mice. We found that treatment with omacetaxine decreased the number of LSCs and prolonged the survival of mice with CML or B-ALL. In chapter III, I describe that Alox5 is an essential gene in the function of LSCs and CML development. We show evidence that Alox5 affects differentiation, cell division, and survival of long-term LSCs. Treatment of CML mice with a 5-LO inhibitor also impaired the function of LSCs similarly and prolonged survival. In chapter IV, I present evidence of our work showing a further dissection the Alox5 pathway by comparing the gene expression profiles of wild type and Alox5-/- LSCs. We show that Msr1 deletion causes acceleration of CML development. We also show that Msr1 affects CML development by regulating the PI3K-AKT pathway and β-catenin. Taken together, these results demonstrate that some pathways including Alox5 and Msr1 play an important role in regulating the self-renewal and differentiation of LSC. More efforts should be put into developing the novel strategies that may effectively target LSCs and thus cure CML.