Les leucémies aiguës myéloblastiques (LAM) sont un groupe hétérogène de pathologies malignes représentant environ 70% des leucémies aiguës. Il existe une prolifération, dans le cadre des LAM de cellules immatures appartenant à la lignée myéloïde appelées myéloblastes ou communément blastes. Les traitements actuels reposent essentiellement sur la chimiothérapie antimitotique. L’homéostasie du fer est une cible dans le traitement des LAM en induisant la différentiation des blastes. Le mécanisme implique la modulation des ROS. Leur action est synergique avec celle de la Vitamine D (VD) au travers de l’activation de la voie des MAPK. Cette association a été utilisée chez plusieurs patients avec succès permettant un doublement de leur espérance de vie. Nous avons ensuite montré que l’expression du récepteur expression vitamine D (VD) est altérée dans les états indifférenciés / immatures sous-types de LAM et que la diminution de l'expression du VDR et de ces gènes cibles est corrélée à un mauvais pronostic chez les patients. Le mécanisme moléculaire entraînant le blocage de l'expression VDR implique la méthylation de son promoteur. Les souris invalidées pour le VDR ont une expansion du compartiment des cellules souches hématopoïétiques demeurant à un état quiescent ainsi qu’une diminution des niveaux du stress oxydatif en leur sein. En outre, la transformation maligne des cellules déficientes en VDR a abouti à une différenciation myéloïde limitée, à l'augmentation du nombre de progéniteurs hématopoïétiques précoces et ces cellules présentaient un potentiel d'auto-renouvellement accru et étaient résistantes aux inhibiteurs de la méthyltransférase et à la chimiothérapie. Enfin, l'induction de l'expression du VDR dans les modèles de LAM par un traitement combinant des agents de déméthylation et les agonistes de VDR a permis de diminuer la séminalité, de promouvoir la différenciation cellulaire, de bloquer la croissance tumorale et de restaurer la sensibilité à la chimiothérapie. Par conséquent, nous proposons que le VDR est un gène maître contrôlant la séminalité et la prolifération / différenciation cellulaire des cellules souches hématopoïétiques normales et leucémiques. Ainsi, la combinaison d'agents déméthylants et d’agonistes de VDR pourrait à l’avenir être proposée en thérapeutique pour traiter les LAM. / Acute myeloid leukemia (AML) is a heterogeneous group of malignancies representing approximately 70% of acute leukemias. There is a proliferation of immature cells belonging to the myeloid lineage commonly called myeloblasts or blasts. Current treatments are mainly based on antimitotic chemotherapy. Iron homeostasis is a target for the treatment of AML blasts inducing cell differentiation. The mechanism involves the modulation of ROS. Their action is synergistic with that of Vitamin D (VD) through the activation of MAPK. This association has been used successfully in several patients for a doubling of life expectancy. Then, we show that Vitamin D receptor (VDR) expression was impaired in undifferentiated/immature AML subtypes and that decreased expression of VDR and VDR-targeted genes was correlated with a negative prognosis of patients. Molecular mechanism resulting in the blockade of VDR expression involved VDR promoter methylation. VDR-deficient mice showed an expansion of the hematopoietic stem cell compartment which presented an improved quiescent status and decreased ROS levels that have been shown to be involved in both AML differentiation and stem cells longevity. Moreover, malignant transformation of VDR-deficient cells resulted in limited myeloid differentiation, increased numbers of early hematopoietic progenitors and those cells presented an enhanced self-renewal potential and were resistant to DNA methyltransferase inhibitors and to chemotherapy. Finally, induction of VDR expression in AML models by combined treatment of demethylating agents and VDR agonists decreased stemness, promoted cell differentiation, blocked tumor propagation and restored sensitivity to chemotherapy. Therefore, we propose that VDR is a master gene controlling stemness and proliferation/cell differentiation of normal hematopoietic stem cells and leukemic cells. Thus, combination of demethylation agents and VDR agonists may be used therapeutically to treat AML.

Recent developments in single-cell measurement technologies have yielded dramatic increases in throughput (measured cells per experiment) and dimensionality (measured features per cell). In particular, the introduction of mass cytometry has made possible the simultaneous quantification of dozens of protein species in millions of individual cells in a single experiment. The raw data produced by such high-dimensional single-cell measurements provide unprecedented potential to reveal the phenotypic heterogeneity of cellular systems. In order to realize this potential, novel computational techniques are required to extract knowledge from these complex data. Analysis of single-cell data is a new challenge for computational biology, as early development in the field was tailored to technologies that sacrifice single-cell resolution, such as DNA microarrays. The challenges for single-cell data are quite distinct and require multidimensional modeling of complex population structure. Particular challenges include nonlinear relationships between measured features and non-convex subpopulations. This thesis integrates methods from computational geometry and network analysis to develop a framework for identifying the population structure in high-dimensional single-cell data. At the center of this framework is PhenoGraph, and algorithmic approach to defining subpopulations, which when applied to healthy bone marrow data was shown to reconstruct known immune cell types automatically without prior information. PhenoGraph demonstrated superior accuracy, robustness, and efficiency, compared to other methods. The data-driven approach becomes truly powerful when applied to less characterized systems, such as malignancies, in which the tissue diverges from its healthy population composition. Applying PhenoGraph to bone marrow samples from a cohort of acute myeloid leukemia (AML) patients, the thesis presents several insights into the pathophysiology of AML, which were extracted by virtue of the computational isolation of leukemic subpopulations. For example, it is shown that leukemic subpopulations diverge from healthy bone marrow but not without bound: Leukemic cells are apparently free to explore only a restricted phenotypic space that mimics normal myeloid development. Further, the phenotypic composition of a sample is associated with its cytogenetics, demonstrating a genetic influence on the population structure of leukemic bone marrow. The thesis goes on to show that functional heterogeneity of leukemic samples can be computationally inferred from molecular perturbation data. Using a variety of methods that build on PhenoGraph's foundations, the thesis presents a characterization of leukemic subpopulations based on an inferred stem-like signaling pattern. Through this analysis, it is shown that surface phenotypes often fail to reflect the true underlying functional state of the subpopulation, and that this functional stem-like state is in fact a powerful predictor of survival in large, independent cohorts. Altogether, the thesis takes the existence and importance of cellular heterogeneity as its starting point and presents a mathematical framework and computational toolkit for analyzing samples from this perspective. It is shown that phenotypic and functional heterogeneity are robust characteristics of acute myeloid leukemia with clinically significant ramifications.

Acute lymphoblastic leukemia (ALL) is an aggressive hematologic malignancy that results from the unregulated growth of B-cell and T-cell lymphoid progenitors. Despite the implementation of risk-stratification and improved multi-agent therapeutic regimens, 20% of pediatric and 50% of adult patients fail to achieve remission and end up relapsing. NT5C2 (5’ cytosolic nucleotidase II) is the most frequently mutated gene specifically found in relapsed ALL. NT5C2 mutations are present in 20% of relapsed T-ALLs and 3-10% of relapsed B-ALLs and present as heterozygous gain of function alleles exhibiting increased nucleotidase activity. As NT5C2 can dephosphorylate and inactivate the cytotoxic metabolites generated by 6-mercaptopurine, a chemotherapy used in the treatment of ALL, these NT5C2 activating mutations can contribute to thiopurine chemotherapy resistance (Tzoneva, Perez-Garcia et al. 2013). Here we perform an extensive structure-function study to understand how relapse-associated NT5C2 mutations result in increased nucleotidase activity and contribute to chemotherapy resistance in ALL. Crystallization of 15 NT5C2 WT and mutant structures as well as enzymatic, structural modeling, and genetic screens identified three regulatory mechanisms of NT5C2, which are disrupted by these gain of function alleles. Class I NT5C2 mutations lock the protein in an active configuration through stabilization of the helixA region, which allows for substrate processing and catalysis. Class II NT5C2 mutations disrupt an intramolecular switch off domain involving the arm region and the intermonomeric positively charged pocket. And a single C-terminus truncating mutant creates a third class of mutations, which show increased nucleotidase activity due to the loss of the C-terminus blockade against allosteric activation. These studies provide new insight into the regulatory controls that mediate NT5C2 activity providing a framework for the development of targeted inhibitors for the treatment of relapsed ALL. In addition to looking at relapse associated NT5C2 mutations on a structural level, we also explored how NT5C2 mutations shape the clonal architecture and evolutionary dynamics during tumor initiation and disease progression in ALL. To formally address these questions, we developed a murine NOTCH1-driven T-ALL with conditional knock-in of the Nt5c2R367Q mutation, the most recurrent mutation found in relapsed ALL, from the endogenous locus. Using this model, we confirmed that Nt5c2+/R367Q lymphoblasts show increased resistance to 6-MP in vitro and in vivo. We also found that Nt5c2+/R367Q mutant lymphoblasts exhibit impaired cell fitness and decreased leukemia initiating cell capacity. Metabolomic profiling and guanosine rescue experiments show that this decrease in cell fitness is due to excess clearance of purine metabolites out of the cell as a result of deregulated Nt5c2 nucleotidase activity. However, in the context of 6-MP therapy, Nt5c2+/R367Q mutant cells are positively selected for in mixed population studies in vitro and in vivo. These results identify a clear selective advantage for NT5C2 mutant cells in the context of 6-MP chemotherapy. In addition, NT5C2 mutant chemoresistant cells show collateral sensitivity to inhibition of inosine monophosphate dehydrogenase (IMPDH) with mizoribine, which further disrupts guanosine production pointing to a potentially selective therapy against NT5C2 mutant cells. We also show here the initial development of a small molecule NT5C2 inhibitor for the treatment of relapsed ALL. Using a malachite green based NT5C2 nucleotidase assay, we performed a small molecule high throughput assay and identified HTP_2 as a lead compound with low micromolar inhibitory activity against NT5C2 R367Q mutant recombinant protein. HTP_2 can reverse 6-MP resistance in Nt5c2+/R367Q mouse lymphoblasts and NT5C2 R29Q mutant expressing human cell lines. Interestingly, HTP_2 treatment also results in increased sensitivity to 6-MP therapy in NT5C2 wild-type cells, suggesting a role for wild-type NT5C2 activity in the clearance of 6-MP and supporting a potential therapeutic use for NT5C2 inhibitors in potentiating the effects of 6-MP based chemotherapy in NT5C2 wild-type cells as well. NT5C2 knockdown cells and Nt5c2 knockout mice show no apparent toxicities suggesting that systemic inhibition of NT5C2 could be fairly well tolerated. In all, this work presents a framework for the development of a high affinity NT5C2 inhibitor for the reversal of 6-MP resistance in relapsed ALL patients. These studies presented here address the role of NT5C2 mutant proteins as drivers of resistance and as therapeutic targets in relapsed ALL. Improved understanding of the molecular mechanisms responsible for increased NT5C2 nucleotidase activity and on the process of clonal evolution during disease progression provide important insight into the mechanism driving ALL resistance and relapse. The identification of IMPDH inhibition as a collateral vulnerability in NT5C2 mutant ALL cells and the development of a first-in-class NT5C2 inhibitor serve as framework for the development of new combination therapies aimed at curtailing the emergence of these thiopurine-resistant relapse driving clones in ALL.

Chan Sin-Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 154-169). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iv / Abstract (Chinese Version) --- p.vi / Contents --- p.viii / Abbreviations --- p.xiii / List of Figures and Tables --- p.xvi / Chapter Chapter One --- General Introduction / Chapter 1.1 --- Hematopoiesis --- p.1 / Chapter 1.1.1 --- Ontogeny of hematopoiesis --- p.1 / Chapter 1.1.2 --- Hiercharay of hematopoiesis --- p.2 / Chapter 1.2 --- Regulation of hematopoiesis --- p.5 / Chapter 1.2.1 --- Bone marrow stromal cell --- p.5 / Chapter 1.2.2 --- Hematopoietic growth factor --- p.6 / Chapter 1.2.3 --- Hematopoietic growth factor receptors and signal transduction --- p.10 / Chapter 1.2.4 --- Transcriptional regulation of myeloid cell development --- p.11 / Chapter 1.3 --- Deregulated hematopoiesis - Leukemia --- p.20 / Chapter 1.3.1 --- Classification of leukemia --- p.20 / Chapter 1.3.2 --- Molecular basis of leukemia --- p.20 / Chapter 1.4 --- Prostaglandin E2 induced WEHI-3B JCS and JCS-14 cell differentiation --- p.22 / Chapter 1.4.1 --- Induced leukemia cell differentiation --- p.22 / Chapter 1.4.2 --- Inducer of cell differentiation - Prostaglandin E2 --- p.22 / Chapter 1.4.3 --- WEHI-3B JCS and subline JCS-14 cells --- p.24 / Chapter 1.5 --- The aims of study --- p.26 / Chapter Chapter Two --- Identification of differentially expressed genes during PGE2-induced WEHI-3B JCS-14 cell differentiation / Chapter 2.1 --- Introduction --- p.27 / Chapter 2.1.1 --- Strategy for studying PGE2-induced JCS-14 cell differentiation --- p.28 / Chapter 2.1.2 --- Method for studying differential gene expression: Microarry Technology --- p.29 / Chapter 2.2 --- Materials --- p.32 / Chapter 2.2.1 --- Cell line --- p.32 / Chapter 2.2.2 --- AtlasT M Mouse cDNA Expression Array --- p.32 / Chapter 2.2.3 --- Chemicals --- p.32 / Chapter 2.2.4 --- Solutions and buffers --- p.33 / Chapter 2.2.5 --- Reagents --- p.34 / Chapter 2.3 --- Methods --- p.35 / Chapter 2.3.1 --- Morphological study of PGE2-induced JCS-14 cell differentiation --- p.35 / Chapter 2.3.2 --- Preparation of total RNA from PGE2-induced JCS-14 cells --- p.35 / Chapter 2.3.2.1 --- Preparation of cell lysates --- p.35 / Chapter 2.3.2.2 --- Isolation of total RNA --- p.35 / Chapter 2.3.3 --- Preparation of cDNA probes --- p.36 / Chapter 2.3.3.1 --- Probe synthesis from total RNA --- p.36 / Chapter 2.3.3.2 --- Purification of the labeled cDNA probes --- p.37 / Chapter 2.3.4 --- Hybridization cDNA probes to the Atlas Array and stringency wash --- p.37 / Chapter 2.4 --- Results --- p.39 / Chapter 2.4.1 --- Morphological changes in PGE2-treated JCS-14 cells --- p.39 / Chapter 2.4.2 --- Analysis of total RNA from PGE2-induced JCS-14 cells --- p.43 / Chapter 2.4.3 --- Hybridization of cDNA probes to AtlasT M cDNA Expression Array --- p.45 / Chapter 2.5 --- Discussion --- p.73 / Chapter 2.5.1 --- Morphological study of JCS-14 cell differentiation --- p.73 / Chapter 2.5.2 --- Differentiation commitment of JCS-14 cell under PGE2 induction --- p.73 / Chapter 2.5.3 --- Gene expression profile by microarray --- p.74 / Chapter 2.5.4 --- Gene expression profile of 5 hours PGE2-induced JCS-14 cells --- p.74 / Chapter 2.5.5 --- Further analysis of regulatory genes in PGE2-induced JCS-14 cell differentiation --- p.77 / Chapter Chapter Three --- Expression profile of identified genes in WEHI-3B JCS-14 and JCS cell differentiation / Chapter 3.1 --- Introduction --- p.79 / Chapter 3.1.1 --- Quantitation of mRNA by Real time RT-PCR --- p.80 / Chapter 3.1.2 --- Relative quantitation of gene expression --- p.83 / Chapter 3.2 --- Materials --- p.85 / Chapter 3.2.1 --- Cell lines --- p.85 / Chapter 3.2.2 --- SYBR® Green PCR core kit --- p.85 / Chapter 3.2.3 --- Chemicals --- p.85 / Chapter 3.2.4 --- Solutions and buffers --- p.86 / Chapter 3.2.5 --- Enzymes and nucleic acids --- p.86 / Chapter 3.3 --- Methods --- p.88 / Chapter 3.3.1 --- Preparation of total RNA from PGE2-induced JCS-14 and JCS cells --- p.88 / Chapter 3.3.1.1 --- Preparation of cell lysates --- p.88 / Chapter 3.3.1.2 --- Isolation of total RNA --- p.88 / Chapter 3.3.2 --- Reverse transcription (RT) --- p.88 / Chapter 3.3.3 --- Design of real-time PCR primers --- p.88 / Chapter 3.3.4 --- Determination of relative efficiency of target and reference amplification by validation experiment --- p.89 / Chapter 3.3.5 --- Confirmation of expression profile of identified genes in JCS-14 and JCS cells by comparative CT method in real-time PCR --- p.90 / Chapter 3.4 --- Results --- p.91 / Chapter 3.4.1 --- Analysis of total RNA from PGE2-induced JCS-14 and JCS cells --- p.91 / Chapter 3.4.2 --- Validation experiment of real-time PCR primers --- p.93 / Chapter 3.4.3 --- Expression profile of specific genes in JCS-14 and JCS cells by comparative CT method --- p.101 / Chapter 3.5 --- Discussion --- p.114 / Chapter 3.5.1 --- Study of gene expression profiles in JCS-14 and JCS cell differentiation --- p.114 / Chapter 3.5.2 --- Transcription analysis by real-time PCR --- p.114 / Chapter 3.5.3 --- Gene expression profiles during PGE2-induced JCS-14 and JCS cell differentiation --- p.115 / Chapter Chapter Four --- Inhibition of specific gene expression in WEHI-3B JCS-14 and JCS cells using antisense blocking technique / Chapter 4.1 --- Introduction --- p.121 / Chapter 4.1.1 --- Antisense technique --- p.122 / Chapter 4.1.2 --- Design of antisense oligonucleotides --- p.125 / Chapter 4.1.3 --- Transfer of oligonucleotides to cells --- p.128 / Chapter 4.2 --- Materials --- p.129 / Chapter 4.2.1 --- Cell lines --- p.129 / Chapter 4.2.2 --- Chemicals --- p.129 / Chapter 4.2.3 --- Reagents --- p.129 / Chapter 4.2.4 --- Solutions --- p.129 / Chapter 4.3 --- Methods --- p.131 / Chapter 4.3.1 --- Design of antisense oligonucleotides --- p.131 / Chapter 4.3.2 --- Transfection of oligonucleotides into cells --- p.134 / Chapter 4.3.3 --- Morphological study of PGE2-induced JCS-14 and JCS cells --- p.134 / Chapter 4.4 --- Results --- p.135 / Chapter 4.4.1 --- Effect of antisense oligonucleotides on JCS-14 cell differentiation --- p.135 / Chapter 4.4.2 --- Effect of antisense oligonucleotides on JCS cell differentiation --- p.136 / Chapter 4.5 --- Discussion --- p.146 / Chapter 4.5.1 --- Effects of antisense B-myb on JCS-14 and JCS cell differentiation --- p.146 / Chapter 4.5.2 --- Effects of antisense thyroid hormone receptor (c-erbA) and transcription terminator factor (TTF-1) on JCS-14 and JCS cell differentiation --- p.147 / Chapter Chapter Five --- General Discussion / Chapter 5.1 --- Introduction --- p.148 / Chapter 5.2 --- Differentiation program triggered by Prostaglandin E2 --- p.148 / Chapter 5.2.1 --- Lineage preference during differentiation --- p.148 / Chapter 5.2.2 --- Differentially expressed genes during PGE2-induced JCS-14 cell differentiation --- p.149 / Chapter 5.2.3 --- Expression patterns of the three differentially expressed genes in PGE2-induced JCS-14 and JCS cells --- p.149 / Chapter 5.2.4 --- Antisense blocking during differentiation --- p.151 / Chapter 5.3 --- Further studies --- p.152 / References --- p.154

by Lau Kwok Wing, Wilson. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 166-188). / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.v / TABLE OF CONTENTS --- p.viii / Chapter CHAPTER 1 : --- GENERAL INTRODUCTION --- p.1 / Chapter (1.1) --- Hematopoiesis : An Overview --- p.2 / Chapter (1.1.1) --- Development of Blood Cells and Sites of Hematopoiesis --- p.2 / Chapter (1.1.2) --- Hematopoietic Cytokine Network --- p.4 / Chapter (1.1.3) --- Molecular Control of Hematopoietic Cell Development --- p.5 / Chapter (1.2) --- Leukemia : An Overview --- p.8 / Chapter (1.2.1) --- Leukemia : Abnormalities in Blood Cell Formation --- p.8 / Chapter (1.2.2) --- Pathophysiology and Etiology of Leukemia --- p.10 / Chapter (1.2.3) --- New Avenues for Therapy : Induction of Differentiation and Apoptosis --- p.12 / Chapter (1.3) --- Induction of Differentiation in Myeloid Leukemia Cells --- p.14 / Chapter (1.3.1) --- Inducers of Leukemic Cell Differentiation --- p.14 / Chapter (1.3.2) --- Cytokines as Inducers of Myeloid Leukemic Cell Differentiation --- p.17 / Chapter (1.3.3) --- Phenotypic Changes and Functional Characterizations --- p.20 / Chapter (1.3.4) --- Modulation of Gene Expression in Myeloid Leukemic Cell Differentiation --- p.22 / Chapter (1.4) --- Apoptosis and Leukemic Cell Death --- p.23 / Chapter (1.4.1) --- Apoptosis : An Overview --- p.23 / Chapter (1.4.2) --- Cytokines and Apoptosis in Myeloid Leukemia --- p.26 / Chapter (1.5) --- Objectives and Research Strategy --- p.27 / Chapter (1.5.1) --- The Murine Myeloid Leukemia Cell Line (Ml) as an Experimental Cell Model for Acute Myeloid Leukemia --- p.27 / Chapter (1.5.2) --- Leukemia Inhibitory Factor (LIF) as a Differentiation Inducer --- p.28 / Chapter (1.5.3) --- Aims and Scopes of This Investigation --- p.31 / Chapter CHAPTER 2 ： --- MATERIALS AND METHODS --- p.33 / Chapter (2.1) --- Materials --- p.34 / Chapter (2.1.1) --- Mice --- p.34 / Chapter (2.1.2) --- Cell Lines --- p.34 / Chapter (2.1.3) --- Recombinant Cytokines --- p.34 / Chapter (2.1.4) --- Monoclonal Antibodies --- p.36 / Chapter (2.1.5) --- Oligonucleotide Primers and Internal Probes --- p.37 / Chapter (2.1.6) --- "Buffers, Culture Medium and Other Reagents" --- p.39 / Chapter (2.1.7) --- Reagents and Solutions for Gene Expression Study --- p.41 / Chapter (2.2) --- Methods --- p.46 / Chapter (2.2.1) --- Culture of Myeloid Leukemia Cell Lines --- p.46 / Chapter (2.2.2) --- Induction of Leukemic Cell Differentiation --- p.46 / Chapter (2.2.3) --- Determination of Cell Growth and Proliferation --- p.46 / Chapter (2.2.4) --- Cell Morphological Study --- p.47 / Chapter (2.2.5) --- Assessment of Differentiation-Associated Characteristics --- p.48 / Chapter (2.2.5.1) --- Nitroblue Tetrazolium (NBT) Reduction Assay --- p.48 / Chapter (2.2.5.2) --- Phagocytosis Assay --- p.48 / Chapter (2.2.5.3) --- Assay of Plastic Adherence --- p.49 / Chapter (2.2.6) --- Flow Cytometric Analysis --- p.49 / Chapter (2.2.6.1) --- Surface Antigen Immunophenotyping --- p.49 / Chapter (2.2.6.2) --- Assay of Endocytic Activity --- p.50 / Chapter (2.2.6.3) --- Assay of Non-specific Esterase Activity --- p.50 / Chapter (2.2.6.4) --- Cell Cycle / DNA Content Evaluation --- p.51 / Chapter (2.2.7) --- Gene Expression Analysis --- p.52 / Chapter (2.2.7.1) --- Preparation of Cell Lysate --- p.52 / Chapter (2.2.7.2) --- RNA Isolation --- p.52 / Chapter (2.2.7.3) --- Reverse Transcription --- p.53 / Chapter (2.2.7.4) --- Polymerase Chain Reaction (PGR) --- p.54 / Chapter (2.2.7.5) --- Agarose Gel Electrophoresis --- p.55 / Chapter (2.2.7.6) --- 3' End Labelling of Oligonucleotide Probes --- p.56 / Chapter (2.2.7.7) --- Dot Blot Hybridization --- p.56 / Chapter (2.2.7.8) --- Digoxigenin (DIG) Chemiluminescent Detection --- p.57 / Chapter (2.2.8) --- DNA Fragmentation Analysis --- p.58 / Chapter (2.2.9) --- Statistical Analysis --- p.59 / Chapter CHAPTER 3 ： --- "EFFECTS OF LEUKEMIA INHIBITORY FACTOR ON THE PROLIFERATION, DIFFERENTIATION, AND APOPTOSIS OF MURINE MYELOID LEUKEMIA Ml CELLS" --- p.60 / Chapter (3.1) --- Introduction --- p.61 / Chapter (3.2) --- Results --- p.63 / Chapter (3.2.1) --- Induction of Growth Arrest in rmLIF-Treated Ml Cells --- p.63 / Chapter (3.2.2) --- Induction of Monocytic Differentiation of Ml cells by rmLIF --- p.66 / Chapter (3.2.2.1) --- Morphological Changes --- p.66 / Chapter (3.2.2.2) --- Induction of Plastic Adherence --- p.70 / Chapter (3.2.2.3) --- Surface Antigen Immunophenotyping --- p.70 / Chapter (3.2.2.4) --- NBT-Reducing Activity of rmLIF-Treated Ml Cells --- p.76 / Chapter (3.2.2.5) --- Non-specific Esterase Activity of rmLIF-Treated Ml Cells --- p.77 / Chapter (3.2.2.6) --- Endocytic Activity of rmLIF-Treated Ml Cells --- p.78 / Chapter (3.2.2.7) --- Phagocytic Activity of rmLIF-Treated Ml Cells --- p.79 / Chapter (3.2.3) --- Induction of Differentiation-Associated DNA Fragmentation --- p.80 / Chapter (3.2.4) --- Production of Differentiation-Inducing Factors --- p.84 / Chapter (3.3) --- Discussion --- p.88 / Chapter CHAPTER 4 : --- CYTOKINE INTERACTIONS IN REGULATING THE PROLIFERATION AND DIFFERENTIATION OF MURINE MYELOID LEUKEMIA Ml CELLS --- p.94 / Chapter (4.1) --- Introduction --- p.95 / Chapter (4.2) --- Results --- p.97 / Chapter (4.2.1) --- Synergistic Effect of LIF and IL-6 on the Proliferation and Differentiation of Ml Cells --- p.97 / Chapter (4.2.2) --- Regulation of Proliferation and Differentiation of Ml Cells by LIF and OSM --- p.101 / Chapter (4.2.3) --- Effects of LIF and TNF-α on the Proliferation and Differentiation of Ml Cells --- p.104 / Chapter (4.2.4) --- Synergistic Effect of LIF and IL-1 on the Proliferation and Differentiation of Ml Cells --- p.107 / Chapter (4.3) --- Discussion --- p.115 / Chapter CHAPTER 5 ： --- MODULATION OF CYTOKINE AND CYTOKINE RECEPTOR GENE EXPRESSION IN LIF- INDUCED DIFFERENTIATION OF MURINE MYELOID LEUKEMIA Ml CELLS --- p.120 / Chapter (5.1) --- Introduction --- p.121 / Chapter (5.2) --- Results --- p.123 / Chapter (5.3) --- Discussion --- p.152 / Chapter CHAPTER 6 ： --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.158 / REFERENCES --- p.166

by Betty Shuc Han Wills. / Year shown on spine: 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 117-128). / ACKNOWLEDGMENT --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iv / LIST OF APPENDICES --- p.viii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.6 / Parental Responses to the Diagnosis of Acute Lymphocytic Leukaemia (ALL) --- p.7 / Disclosure Of the Child's Diagnosis --- p.10 / Impact of Cancer Treatment on Parents --- p.14 / Sources of Support for Parents --- p.18 / Coping Strategies of Parents of Children With ALL --- p.20 / Coping With The Uncertainty of the Disease --- p.23 / Research Studies Involving Chinese Parents --- p.24 / Summary Of Issues From Literature Review --- p.27 / Chapter CHAPTER 3 --- METHODOLOGY / Research Design --- p.29 / Sampling --- p.31 / Data Collection Method --- p.32 / Data Collection Procedure --- p.34 / Ethical Considerations --- p.38 / Pilot Study --- p.40 / Data Analysis --- p.42 / Issues of Reliability and Validity --- p.45 / Validity --- p.45 / Reliability --- p.48 / Chapter CHAPTER 4 --- RESULTS & DISCUSSION / Introduction --- p.50 / Chapter (I). --- Parents' Profile --- p.51 / Demographic Characteristics Of The Parents / Chapter (II). --- Major categories Corresponding To Interviewing The Mothers --- p.54 / Initial Reactions of the Child's Confirmed Diagnosis --- p.55 / Unpreparedness for the child's Diagnosis / Suddenness of the Diagnosis --- p.56 / Physical and psychological reactions to the child's Diagnosis --- p.58 / Sources of Support for the Mothers --- p.62 / The mothers' main source of support / Other sources of support for the mothers --- p.64 / Disclosure Of Child's Diagnosis --- p.66 / Disclosure of the child's diagnosis to the child / Disclosure of the child's diagnosis to members of the immediate and extended families --- p.68 / Disclosure of child's diagnosis to non-family members --- p.70 / Uncertainty Brought On By The Illness --- p.71 / Waiting for confirmation of diagnosis / Uncertainty about the success of treatment --- p.73 / Uncertainty about the child's future --- p.74 / Changes In The Family Routine --- p.75 / Needed to normalise family life / Chapter (III). --- Major Categories Corresponding to Interviewing The Fathers Initial reactions to the child's confirmed diagnosis of ALL --- p.78 / Suddenness of diagnosis --- p.79 / Physical and psychological reactions to the diagnosis --- p.80 / Disclosure Of The Child's Diagnosis --- p.82 / Disclosure of the child's diagnosis to the child / Disclosure of the child's diagnosis to members of the immediate and extended family --- p.85 / Disclosure of the child's diagnosis to non-family members --- p.86 / Sources Of Support For The Fathers --- p.87 / Support from immediate and extended families / Support from medical professionals --- p.89 / Support from friends --- p.90 / Changes In The Family Routine --- p.91 / Coping Strategies Utilised By The Fathers --- p.92 / Open communication / Use of religious beliefs and rituals --- p.93 / Chapter (IV). --- Comparison Of Categories Found Between The Mothers And The Fathers --- p.95 / Initial reactions to the child's confirmed diagnosis of ALL / Disclosure of the child's diagnosis --- p.96 / Sources of support for the parents --- p.100 / Changes in the family routine --- p.101 / Summary of findings --- p.103 / Chapter (V). --- Differences between the initial and second interviews --- p.105 / Chapter CHAPTER FIVE --- CONCLUSION / Limitations Of The Study --- p.108 / Implications For Nursing Practice --- p.112 / Recommendations For Future Research --- p.114 / Conclusion --- p.115 / REFERENCES --- p.117 / APPENDIX I - PERSONAL DATA FORM --- p.129 / APPENDIX II - INTERVIEW SCHEDULE --- p.130 / APPENDIX III - CONSENT FORM --- p.134 / APPENDIX IV - SAMPLE SCRIPT OF INTERVIEWS WITH MOTHERS --- p.135 / APPENDIX V - MATRICES ON MOTHERS AND FATHERS --- p.140 / APPENDIX VI - SAMPLE OF FIELD NOTES FOR MOTHERS AND FATHERS --- p.143

Production of infectious retrovirus particles is a complex and poorly-understood process with multiple steps that are often linked to one another. Our aim in this study was to gain better understanding of the path the murine leukemia virus (MLV) structural protein Gag follows to assemble into immature capsid structures, the process of which is central to retroviral assembly and release. Extensive studies of human immunodeficiency virus type 1 (HIV-1) assembly have led to the development of a model proposing that the assembly of immature HIV-1 capsids proceeds sequentially through multiple intermediates, in association with an RNA granule containing some well-conserved cellular factors, such as ATP-binding cassette subfamily E member 1 (ABCE1) and DEAD-box helicase 6 (DDX6). In this work, we provided evidence suggesting that MLV Gag associates with endogenous ABCE1 in human cells expressing assembly-competent MLV, and can be found in at least three high-molecular weight complexes with sedimentation properties highly resembling the HIV-1 assembly intermediates. Furthermore, we assessed the Gag proteins of select assembly-defective MLV mutants in terms of their expression levels, ability to form viral particles, involvement in intracellular complexes, membrane association, and ABCE1 interaction. Our findings were not only consistent with a model of MLV assembly through host-mediated intermediates, but also provided novel information about the effects of various MLV Gag mutations that are associated with defects in particle production.

Acute lymphoblastic leukemia (ALL) predominates in children ages 0-14 years and has an excellent prognosis for cure with 5-year survival exceeding 90% in the United States. However, not all children experience such positive outcomes. The purpose of this quantitative, retrospective cohort study was to evaluate differences in survival of ALL among children who reside in the 32-county Texas-Mexico border region. While factors such as poverty and health insurance have been strongly associated with poorer cancer outcomes, additional factors such as geographic isolation and treatment disparities are not as well-documented in children. This study examined the association between use of Texas Children's Oncology Group (COG) pediatric research facilities and survival among children in Texas diagnosed with ALL. This study used cancer incidence data 1995-2009 from the Texas Cancer Registry. Differences in survival and use of COG facilities were investigated between children who reside within the 32-county Texas-Mexico border region and the combined remaining 222 Texas counties. Chi-square was used to analyze area of residence, gender, race/ethnicity, and poverty status between COG and non-COG reported cases. Logistic regression was used to examine ALL survival differences between COG and non-COG facilities controlling for multiple variables. COG affiliation alone was not a significant predictor of survival. An interaction between race/ethnicity, region, poverty status, and COG facility affiliation was observed as a significant predictor of poorer survival. The results of this study have the potential to promote positive social change by implementing interventions addressing access to equivalent pediatric cancer care in the 32-county Texas-Mexico border area.

The Rho family of GTPases plays a crucial role in the regulation of diverse cellular processes, including proliferation and actin cytoskeletal rearrangement to promote cell migration. However, dysregulation of RhoGTPases has been associated with disease, particularly cancers such as leukemia. Despite this, RhoGTPases are rarely mutated in cancer. Rather, dysregulation of their regulatory proteins through mutation or overexpression contributes to disease pathogenesis. RhoGTPases are activated through Rho guanine nucleotide exchange factors (GEFs). Although over eighty RhoGEFs have been identified that activate the 25 RhoGTPases, the pathological role of the majority of these proteins remains unclear. Further, whereas the majority of RhoGEFs are activated through tyrosine phosphorylation, a small subset can be activated through heterotrimeric G proteins, including through GΒ;Γ; subunits. However, the mechanism by which GΒ;Γ; induces RhoGEF activation remains unclear. PLEKHG2 is a Dbl family RhoGEF that was originally identified as a gene upregulated in a leukemia mouse model, and later shown to be activated by heterotrimeric G protein Β;Γ; subunits. However, its function and activation mechanisms remain elusive. Here we show that, as compared to primary human T cells, the expression of PLEKHG2 is upregulated in leukemia cell lines. Downregulation of PLEKHG2 by siRNAs specifically inhibited GΒ;Γ;-stimulated Rac and Cdc42, but not RhoA activation. Consequently, inhibition of PLEKHG2 blocked actin polymerization, protrusion formation, and leukemia cell migration in response to SDF1alpha;. Additional studies indicate that GΒ;Γ; likely activates PLEKHG2 by binding the N-terminus of PLEKHG2. This interaction results in the release of autoinhibition imposed by the C-terminus within a region encompassing the catalytic DH domain. As a result, overexpressing either the N-terminus of PLEKHG2 that binds GΒ;Γ; or the C-terminus that autoinhibits PLEKHG2 blocked GΒ;Γ;-stimulated Rac and Cdc42 activation and the ability of leukemia cell to form membrane protrusions and to migrate. Together, our results have demonstrated that PLEKHG2 functions as a novel GΒ;Γ; -stimulated RhoGEF that could contribute to chemokine-induced leukemia cell dissemination and leukemia pathogenesis.

Myeloid Leukemia Factor 1 (Mlf1) is the murine homolog of MLF1, which was identified as a fusion gene with Nucleophosmin (NPM) resulting from the (3;5)(q25.1;q34) translocation associated with acute myeloid leukemia and myelodysplastic syndrome (Yoneda-Kato et al., 1996). Mlf1 was independently isolated using cDNA representational difference to identify genes up-regulated when an erythroleukemic cell line underwent a lineage switch to display a monoblastoid phenotype (Williams et al., 1999). Mlf1 has been shown to enhance myeloid differentiation and suppress erythroid differentiation; however, its mechanism of action is unknown. A yeast two hybrid screen was employed to identify Mlf1-interacting proteins. This screen isolated a number of known protein, as well as several novel molecules, that bound Mlf1. One of these was 14-3-3ξ, a member of a family of molecules that bind phosphoserine motifs and regulate the subcellular localization of partner proteins. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and associated with 14-3-3ξ; via this phosphorylated motif (Lim et al., 2002). The aim of this thesis was to characterise a novel Mlf1-interacting protein that had some homology to protein kinases and was named Mlf1 Adaptor Molecule (Madm). Adaptor proteins are molecules that possess no enzymatic or transcriptional activity, but instead mediate protein-protein interactions. Madm is encoded by a gene consisting of 18 exons and promoter analysis suggested Madm expression might be widespread; indeed Northern blotting of adult tissues and in situ hybridization of embryos demonstrated ubiquitous Madm expression. Significantly, the Madm protein sequence is highly conserved across diverse species. / Madm formed dimers and although it contains a kinase-like domain, the protein lacks several critical residues required for catalytic activity, including an ATP-binding site. Purification of recombinant Madm revealed that the protein was not a kinase; however, studies in mammalian cells showed that Madm associated with a kinase and that Madm was phosphorylated on serine residues in vivo and in vitro. Madm also contains a nuclear localization sequence and nuclear export sequence and was shown to localise to both cytoplasm and nucleus by subcellular fractionation and confocal microscopy. The presence of two nuclear receptor binding motifs (consensus MILL) suggests that Madm may have a functional role in the nucleus. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, the Madm-associated kinase phosphorylated Mlf1 on serine residues, including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein NPM-MLF1 did not bind 14-3-3i; and localized exclusively in the nucleus. Although Madm co-immunoprecipitated with NPM-MLF1 the binding mechanism was altered. As Mlf1 is able to reprogram erythroleukemic cells to display a monoblastoid phenotype and potentiate myeloid maturation (Williams et al., 1999), the effects of Madm on myeloid differentiation was investigated. However, unlike Mlf1, ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation. / In summary, the data presented in this thesis reports on the cloning and characterization of a novel adaptor protein that is involved in the phosphorylation of the proto-oncoprotein MIM. Phosphorylation of Mlf1 is likely to affect its interaction with other proteins, such as 14-3-3~. Complex formation, therefore, may well alter the localization of Mlf1 and Madm, and influence hematopoietic differentiation.