by Yip Mei Chu Pandora. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 207-233). / Abstract also in Chinese. / STATEMENT --- p.i / ACKNOWLEDGEMENTS --- p.ii / ABSTRACT --- p.iii / ABSTRACT (CHINESE VERSION) --- p.v / TABLE OF CONTENTS --- p.vii / ABBREVIATIONS --- p.xiii / LIST OF FIGURES AND TABLES --- p.xvii / Chapter CHAPTER ONE ... --- GENERAL INTRODUCTION / Chapter 1.1 --- the blood cells formation - hematopoiesis --- p.1 / Chapter 1.1.1 --- Hierarchy of hematopoiesis --- p.2 / Chapter 1.1.2 --- Malfunction in the process of hematopoiesis - hematologic neoplasia - Leukemia --- p.6 / Chapter 1.1.2.1 --- Classification of leukemia --- p.7 / Chapter 1.1.2.2 --- Differentiation therapy ´ؤ a new hope in the treatment of leukemia --- p.9 / Chapter 1.2 --- Understanding the pathogenesis of leukemia --- p.12 / Chapter 1.2.1 --- General regulation of hematopoiesis --- p.12 / Chapter 1.2.2 --- Regulation of the differentiation of myeloid lineage --- p.15 / Chapter 1.2.2.1 --- Regulation of myeloid cell differentiation by hematopoietic regulatory protein --- p.16 / Chapter 1.2.2.2 --- Signal transduction pathways in myeloid cell differentiation --- p.20 / Chapter 1.2.2.3 --- Gene regulation of myeloid cell differentiation --- p.22 / Chapter 1.2.2.3.1 --- Transcription factors --- p.23 / Chapter 1.2.2.3.2 --- Myeloid specific genes --- p.31 / Chapter 1.2.2.3.3 --- Protooncogenes and tumor suppressor genes --- p.37 / Chapter 1.2.2.3.4 --- Homeobox genes --- p.42 / Chapter 1.2.2.3.5 --- Cell cycle control in myeloid growth and differentiation --- p.47 / Chapter 1.3 --- Induction of differentiation in myeloid leukemia cell --- p.48 / Chapter 1.3.1 --- Induced myeloid leukemia cell differentiation --- p.48 / Chapter 1.3.2 --- Inducers of myeloid cell differentiation --- p.52 / Chapter 1.3.3 --- Chemical inducers ´ؤ Flavonoids --- p.57 / Chapter 1.3.4 --- Murine myeloid leukemia cell ´ؤ WEHI-3B JCS --- p.60 / Chapter 1.4 --- Aim of study --- p.53 / Chapter CHAPTER TWO ... --- ISOLATION OF GENES THAT ARE DIFFERENTIALLY EXPRESSED DURING BIOCHANIN A INDUCED WEHI-3B (JCS) MYELOID LEUKEMIA CELL DIFFERENTIATION / Chapter 2.1 --- Introduction --- p.65 / Chapter 2.1.1 --- Strategy for searching differentially expressed genes - RNA fingerprinting by arbitrarily primed polymerase chain reaction (RAP- PCR) --- p.65 / Chapter 2.1.2 --- Reamplification of PCR products by Touchdown PCR --- p.67 / Chapter 2.1.3 --- Methods for eliminating false positives : Dot blot hybridization screening --- p.68 / Chapter 2.2 --- Materials --- p.70 / Chapter 2.2.1 --- "Cell line, Bacterial strain and Vector" --- p.70 / Chapter 2.2.2 --- Chemicals --- p.70 / Chapter 2.2.3 --- Reagents and nucleic acids --- p.71 / Chapter 2.2.4 --- Kits --- p.72 / Chapter 2.2.5 --- Solutions --- p.72 / Chapter 2.2.6 --- Equipments --- p.73 / Chapter 2.3 --- Methods --- p.74 / Chapter 2.3.1 --- Induction of murine myeloid leukemia cell line -WEHI-3B (JCS) cells by biochanin-A --- p.74 / Chapter 2.3.2 --- Isolation of total RNA by guanidium thiocyanate cesium chloride ultracentrifugation --- p.74 / Chapter 2.3.3 --- RNA fingerprinting by arbitrarily primed PCR --- p.75 / Chapter 2.3.3.1 --- Synthesis of first strand cDNA --- p.75 / Chapter 2.3.3.2 --- Normalization of RNA samples --- p.75 / Chapter 2.3.3.3 --- RAP-PCR --- p.76 / Chapter 2.3.3.4 --- Reamplification of differentially amplified fragment --- p.77 / Chapter 2.3.4 --- First round dot blot hybridization screening --- p.78 / Chapter 2.3.4.1 --- Dot blot --- p.78 / Chapter 2.3.4.2 --- Preparation of cDNA probe --- p.79 / Chapter 2.3.4.3 --- 32P-labelling of cDNA probe --- p.79 / Chapter 2.3.4.4 --- Removal of unincorporated probe by NICK´ёØ column --- p.80 / Chapter 2.3.4.5 --- Estimation of 32P labelling efficiency by scintillation counting --- p.80 / Chapter 2.3.4.6 --- Prehybridization and hybridization --- p.81 / Chapter 2.3.4.7 --- Quantitation of hybridization signal by scanning densitometry --- p.81 / Chapter 2.3.5 --- Second round dot blot hybridization screening --- p.81 / Chapter 2.3.5.1 --- Subcloning of differentially amplified fragments --- p.82 / Chapter 2.3.5.1.1 --- Preparation of vector DNA --- p.82 / Chapter 2.3.5.1.2 --- Synthesis of blunt end PCR product --- p.84 / Chapter 2.3.5.1.3 --- Blunt end ligation --- p.34 / Chapter 2.3.5.1.4 --- Transformation --- p.85 / Chapter 2.3.5.1.5 --- Selection and confirmation by polymerase chain reaction --- p.85 / Chapter 2.3.5.2 --- Dot blot hybridization screening --- p.85 / Chapter 2.4 --- Results --- p.87 / Chapter 2.4.1 --- Spectrophotometric analysis of total RNA --- p.87 / Chapter 2.4.2 --- Normalization of RNA samples --- p.88 / Chapter 2.4.3 --- RNA fingerprinting by arbitrarily primed PCR --- p.39 / Chapter 2.4.4 --- Reamplification of isolated RAP-PCR products --- p.91 / Chapter 2.4.5 --- First round of dot blot hybridization screening --- p.92 / Chapter 2.4.6 --- Subcloning of differentially amplified fragments --- p.100 / Chapter 2.4.7 --- Second round of dot blot hybridization screening --- p.102 / Chapter 2.4.8 --- Comparison of the first and second round of dot blot hybridization screening --- p.106 / Chapter 2.5 --- Discussion --- p.108 / Chapter 2.5.1 --- RNA fingerprinting by arbitrarily primed PCR --- p.108 / Chapter 2.5.2 --- Limitation of RAP-PCR --- p.110 / Chapter 2.5.3 --- Two rounds of dot blot hybridization screening --- p.111 / Chapter CHAPTER THREE... --- CHARACTERIZATION OF THE ISOLATED GENE FRAGMENTS / Chapter 3.1 --- Introduction --- p.113 / Chapter 3.1.1 --- Automated DNA sequencing and analysis --- p.113 / Chapter 3.1.2 --- GenBank and the BLAST homology search --- p.115 / Chapter 3.2 --- Materials --- p.118 / Chapter 3.2.1 --- Selected recombinant plasmids --- p.118 / Chapter 3.2.2 --- Chemicals --- p.118 / Chapter 3.2.3 --- Reagents --- p.118 / Chapter 3.2.4 --- Kits --- p.119 / Chapter 3.2.5 --- Solutions --- p.119 / Chapter 3.2.6 --- Equipment --- p.119 / Chapter 3.3 --- Methods --- p.120 / Chapter 3.3.1 --- Preparation of selected recombinant plasmid DNA --- p.120 / Chapter 3.3.2 --- Restriction digestion of recombinant plasmid DNA --- p.120 / Chapter 3.3.3 --- Automated DNA sequencing --- p.120 / Chapter 3.3.3.1 --- Primer annealing to template --- p.120 / Chapter 3.3.3.2 --- Sequencing reactions --- p.121 / Chapter 3.3.3.3 --- Polyacrylamide gel electrophoresis --- p.121 / Chapter 3.3.3.4 --- Data analysis by ALF manager and DNAsis --- p.122 / Chapter 3.3.4 --- Sequence homology search with databases --- p.122 / Chapter 3.4 --- Results --- p.123 / Chapter 3.4.1 --- Spectrophotometric analysis of selected recombinant plasmid DNAs subcloned with differentially amplified fragments --- p.123 / Chapter 3.4.2 --- Restriction digestion of selected recombinant plasmid DNA --- p.124 / Chapter 3.4.3 --- Sequences of the subcloned differentially amplified fragments --- p.126 / Chapter 3.4.4 --- Sequence analysis of the subcloned differentially amplified fragments --- p.144 / Chapter 3.5 --- Discussion --- p.157 / Chapter 3.5.1 --- Sequence analysis of the isolated gene fragment --- p.157 / Chapter CHAPTER FOUR … --- "EXPRESSION PROFILE OF ISOLATED GENES FRAGMENTS IN MYELOID LEUKEMIA CELL, MOUSE EMBRYO, AND TISSUES" / Chapter 4.1 --- Introduction --- p.162 / Chapter 4.1.1 --- Quantitation of mRNA by Reverse transcription-polymerase chain reaction --- p.162 / Chapter 4.1.2 --- Internal primer design by OLIGO´ёØ ver 34 --- p.167 / Chapter 4.2 --- Materials --- p.168 / Chapter 4.2.1 --- Mice --- p.168 / Chapter 4.2.2 --- Cell lysate --- p.168 / Chapter 4.2.3 --- Total RNAs --- p.168 / Chapter 4.3 --- Methods --- p.169 / Chapter 4.3.1 --- Internal primer design by OLIGO´ёØ ver 34 --- p.169 / Chapter 4.3.2 --- "Isolation of total RNA from biochanin A induced JCS cells, mouse embryos and tissue" --- p.169 / Chapter 4.3.2.1 --- Preparation of cell lysate from mouse embryo and postnatal mouse brain --- p.169 / Chapter 4.3.2.2 --- Isolation of RNA by guanidium thiocyanate cesium chloride method --- p.170 / Chapter 4.3.3 --- Preparation of saggital section of mouse embryo --- p.170 / Chapter 4.3.4 --- Confirmation of differential expression of isolated genes fragments during biochanin A and midazolam induced WEHI 3B (JCS) differentiation and the expression profile in mouse tissues and during mouse embryo development by reverse transcription-polymerase chain reaction --- p.171 / Chapter 4.4 --- Results --- p.173 / Chapter 4.4.1 --- Internal primer design of the sequenced fragments --- p.173 / Chapter 4.4.2 --- Spectrophotometric analysis of total RNA --- p.175 / Chapter 4.4.3 --- Saggital section of mouse embryo --- p.176 / Chapter 4.4.4 --- Normalization of RNA samples --- p.180 / Chapter 4.4.5 --- Analysis of mRNA expression of differentially amplified fragmentsin biochanin A or midazolam induced JCS cells and mouse embryos by RT- PCR --- p.182 / Chapter 4.4.5.1 --- "Genes downregulated at 1 hour, 5 hours and 48 hours after biochanin A induction of JCS cells" --- p.183 / Chapter 4.4.5.2 --- Genes up-regulated at 48 hours after biochanin A induction --- p.183 / Chapter 4.4.5.3 --- Genes constitutively expressed during the course of biochanin A treatment --- p.184 / Chapter 4.4.5.4 --- Genes showing undetectable level of expression in biochanin A induced JCS cells --- p.184 / Chapter 4.4.6 --- Tissue expression of the biochanin A induced-differentially expressed fragments by RT-PCR --- p.188 / Chapter 4.5 --- Discussion --- p.191 / Chapter 4.5.1 --- Expression profiles of isolated differentially amplified fragments --- p.191 / Chapter 4.5.2 --- Comparison of the expression profiles of the isolated gene fragments analyzed by dot blot hybridization screening and RT-PCR --- p.197 / Chapter CHAPTER FIVE ... --- GENERAL DISCUSSION --- p.200 / REFERENCES --- p.207 / APPENDIX --- p.234

The tumour microenvironment plays a key role in tumour progression. In this thesis acute myeloid leukaemia (AML) was used as a model system to investigate the interplay between stromal and cancer cells. AML is a heterogeneous clonal disorder of haematopoietic undifferentiated progenitor cells or 'blast cells', which accumulate in the bone marrow and lead to the reduced output of crucial haematopoietic elements. Due to its heterogeneity (at least in part), treatment of the disease has not witnessed great innovation in the past 30 years. The bone marrow microenvironment (BMM) has a key role in the haematological malignancies contributing to the survival of leukaemic blasts. Relapse in AML occurs because of residual disease and evidence suggests that this resistance is facilitated through leukaemic cells ability to reside in BMM niches. To understand the precise role of the BMM in AML progression and therefore target any supportive mechanisms requires knowledge of how AML cells communicate with their microenvironment. In the work presented in this thesis I undertook a multi-proteomic approach that utilised liquid chromatography tandem mass spectrometry (LC-MS/MS) to assess the interplay between AML and BMM cell signalling. This thesis shows the results of a secretomic analysis of stromal cell lines, which identified a previously uncharacterized panel of six stromal secreted proteins (BMP-1, CSF-1, CTGF, HGF, S100-A4 and S100-A11) that support primary AML cell survival and proliferation in culture. Comparison of AML cell signalling (using global phosphoproteomic methods) following treatment with the newly identified growth factors revealed that these signalling proteins elicit multi-nodular activation of signalling networks with known anti-apoptotic activity. Consistent with the cell signalling proteomics data, cell viability studies as a function of pharmacological kinase inhibitor treatment determined that the sensitivity of AML to targeted kinase inhibitors was modulated by the supportive stromal conditioned media. To investigate heterotypic signalling between cell populations, AML/stromal cell co-cultures were designed, tested and optimised. These studies identified additional activated pathways in AML cells that were only present when AML cells had physical interaction with stroma. Complementary analysis of the stromal cells which had been first cultured with AML cells revealed that despite heterogeneity there is an emerging stromal phospho-proteomic signature that is different in BMM independent AML cells vs BMM interactive AML cells. Collectively these findings evidence the influence that the BMM can have on AML signalling. Although evidence for the influence of BMM in modulating AML resistance to standard chemotherapy exists, this study highlights specific BMM components that contribute to the ability of AML cells to circumvent current treatments based on kinase targeted drugs. These observations have implications for designing future therapies for AML.

The mammalian target of rapamycin (mTOR) signalling pathway takes part in both extracellular and intracellular signals. It is a major regulator of cell metabolism, growth, proliferation and survival. mTOR also regulates critical processes such as cytoskeletal organization, ribosomal biogenesis, transcription and protein synthesis. The mTOR pathway has been implicated in many diseases such as cancer, neurodegeneration and diabetes, which impact homeostasis and cellular functions. Moreover, mTOR has also been shown to play a critical role in immune cell regulation of T and B cells together with neutrophils and antigen presenting cells, as it integrates signals between them extending to the entire immune microenvironment. The aim of my study was to investigate the role of a component of the mTOR complex 1, Raptor, in myeloid cells. My findings show that the absence of Raptor knock out (KO) does not affect bone marrow derived macrophage (BMDM) differentiation and maturation. However, the absence of Raptor influences BMDM polarisation towards an inflammatory phenotype, at least at the level of transcription as observed by increases in mRNA expression of inflammatory cytokines such as TNFα, IL-12β, and IL-6. This finding was consolidated by an increase in NFκΒ pathway signalling in Raptor KO BMDMs. Downstream intracellular signalling in myeloid cells was affected by deletion of Raptor as I found reduced S6K phosphorylation in Raptor KO BMDMs compared to wild type (WT) BMDMs. As a consequence of Raptor absence in BMDMs, STAT3 phosphorylation was also reduced. Raptor deletion did not impact the PI3K/Akt signalling pathway, but decreased phosphorylation of ERK. BMDMs lacking Raptor had reduced phagocytic activity as they were also observed to migrate less towards a pancreatic cancer cell line. However preliminary experiments in pancreatic cancer models did not indicate a major role for Raptor in the activity of tumour associated myeloid cells. My results demonstrate that Raptor and by implication mTORC1, is involved in macrophage polarisation and function.

L’hématopoïèse regroupe l'ensemble des mécanismes qui assurent le renouvellement continu et régulé des cellules sanguines, à partir des cellules souches hématopoïétiques. La différenciation des cellules souches en cellules matures est un phénomène finement orchestré par divers signaux (facteurs de croissance, hormones, cytokines et microenvironnement médullaire) capables de stimuler la prolifération de cellules quiescentes ainsi que leur engagement dans diverses voies de la maturation hématopoïétique. Ces processus sont généralement régulés via l’activation de facteurs de transcription, mais également par des mécanismes épigénétiques. Le gène RINF/CXXC5 a initialement été décrit comme essentiel pour les processus de différenciation granulocytaire normale. Toutefois, son implication dans d’autres voies de l’hématopoïèse n’avait pas été étudiée. Afin de définir son rôle dans la différenciation et l’engagement vers des lignages hématopoïétiques autres que la voie granulocytaire, notamment érythroïde et monocytaire, sa contribution fonctionnelle dans des lignées hématopoïétiques et dans des cellules primaires CD34+ de moelle osseuse (donneurs sains) a été étudiée par une approche de perte ou gain de fonction. Ces expériences, dans des modèles de la voie granulocytaire (lignée NB4 et HL60 traitées par les rétinoïdes) et de la voie érythroïde (lignées K562 et UT7/GM traitées par l’hémine ou l’EPO) démontrent l’implication de RINF dans la maturation terminale de ces deux lignages. Ces données ont ensuite été validées dans des progéniteurs hématopoïétiques (tests clonogéniques) où l’expression de RINF favorise la différenciation granuleuse et interfère négativement avec la différenciation érythroïde, sans impacter la voie monocytaire. En effet, l’extinction de son expression diminue le nombre des colonies granuleuses et augmente le nombre de colonies érythroïdes. Des dérégulations au niveau de l’expression des facteurs ou co-facteurs de trancription qui régulent l’hématopoïèse peuvent aboutir à des hémopathies, telles que les Leucémies Aiguës Myéloïdes. Ces pathologies sont la résultante de l’association d’une augmentation de la prolifération cellulaire et d’un blocage des processus de différenciation. Au vu de son rôle important au cours de la granulopoïèse et de l’érythropoïèse, l’hypothèse que des dérégulations de RINF pourraient intervenir dans les processus de leucémogenèse a été testée par l’étude de son niveau d’expression dans les différentes Leucémies Aiguës Myéloïdes. Ainsi, il a été démontré que parmi les patients dont le niveau d’expression de RINF est le plus élevé, le pronostic vital est mauvais, associé à une résistance aux traitements chimiothérapeutiques. L’ensemble de ces données a permis d’aboutir à la conclusion que des dérégulations de l’expression de RINF pourraient contribuer au processus de leucémogenèse, et qu’ainsi RINF pourrait être une potentielle cible thérapeutique dans le cadre des LAM. D’un point de vue moléculaire, le mode d’action de la protéine RINF reste à ce jour du domaine de l’hypothèse, mais la présence d’un doigt de zinc de type CXXC lui permettrait d’intervenir dans des mécanismes des régulations épigénétiques, tels que la méthylation de l’ADN. Une meilleure compréhension des mécanismes régulés par la protéine pourrait permettre à terme une meilleure compréhension des régulations de l’hématopoïèse, voire des processus de leucémogenèse. / During hematopoiesis, hematopoietic stem cells (HSC) differentiation is orchestred by different signals, able to stimulate cell proliferation of quiescent cells, and their commitment in the different hematopoietic lineages. These process are regulated by transcription factors activation, as well by epigenetic mecanisms. By a microarray approach, we have identified a novel retinoid-responsive gene (CXXC5) encoding a Retinoid-Inducible Nuclear Factor (RINF) that plays an essential role during in vitro human hematopoiesis. Indeed, expression studies and gene silencing experiments both demonstrate RINF requirement during in vitro terminal differentiation of myeloid leukemia cells (NB4, HL60), but also during normal myelopoiesis of bone marrow progenitors (CD34+ HSPC cells in presence of cytokines). In the present study, we demonstrate that in cell lines, RINF overexpression provokes an earlier myeloid differentiation under retinoids treatement and slow-downs erythroid maturation induced by hemin whereas its down-regulation accelerates erythroid terminal differentiation. In normal CD34+ HSCP, we demonstrated that RINF down regulation (1) promotes differentiation in erythroid lineage at the expense of granulocyte lineage, and (2) accelerates terminal erythroid differentiation. Overexpression, contribute to promote myeloid pathway even though cells are in erythroid conditions. Because of its role during hematopoiesis regulation and its gene localization in 5q31.2, we investigated CXXC5/RINF expression in primary human acute myeloid leukemia (AML) cells derived from 594 patients. A wide variation in CXXC5/RINF mRNA levels was observed in the immature leukemic myeloblasts. Furthermore, patients with low-risk cytogenetic abnormalities showed significantly lower levels compared to patients with high-risk abnormalities, and high RINF/CXXC5/ mRNA levels were associated with decreased overall survival for patients receiving intensive chemotherapy for newly diagnosed AML. CXXC5/RINF knockdown in AML cell lines caused increased susceptibility to chemotherapy-induced apoptosis, and regulation of apoptosis also seemed to differ between primary human AML cells with high and low RINF expression. The association with adverse prognosis together with the antiapoptotic effect of CXXC5/RINF suggests that targeting of CXXC5/RINF should be considered as a possible therapeutic strategy, especially in high-risk patients who show increased expression in AML cells compared with normal hematopoietic cells.

Les Leucémies Aigues Myéloïdes (LAM) sont des hémopathies au pronostic sombre. A l’exception des Leucémies Aigues Promyélocytaires (LAP), un sous type minoritaire de LAM, la plupart des patients sont traités par une chimiothérapie d’induction composée de la cytarabine et d’une anthracycline. Cependant, une fraction importante (20-30%) des patients ne répond pas à la chimiothérapie d’induction et les taux de rechutes sont très élevés.Il n’existe actuellement pas d’outils disponibles au diagnostic permettant de prédire la réponse des patients aux chimiothérapies. De tels tests permettraient d’adapter les doses utilisées pour augmenter le taux de réponse et limiter la toxicité des chimiothérapies, très importante chez les personnes âgées.Les protéines de la famille de l’Ubiquitine (UbL), dont l’ubiquitine, SUMO (-1,-2,-3) et Nedd8 sont les membres les plus étudiés, sont des modificateurs post-traductionnels peptidiques conjugués de façon covalente sur des milliers de protéines. Elles sont impliquées dans la plupart des fonctions cellulaires et dérégulées dans de nombreuses pathologies. De nombreux travaux suggèrent que leur dérégulation est associée à la chimiorésistance des LAM.La première partie de nos travaux a consisté à identifier un ensemble de protéines dont le niveau de modification par l’ubiquitine, SUMO et NEDD8 pourrait constituer des biomarqueurs de la réponse des LAM aux drogues chimiothérapeutiques. Nous avons de plus développé un test permettant d’évaluer leur niveau de modification par des extraits de cellules de patients de manière simple et rapide, utilisable en clinique au diagnostic.Le ciblage des voies Ubiquitine, SUMO et Nedd8 est en train d’apparaître comme une stratégie thérapeutique prometteuse dans les cancers et plusieurs molécules ciblant ces voies ont été récemment décrites. Le développement clinique de ces molécules nécessite la mise au point de tests compagnons pour suivre leur efficacité sur leurs cibles moléculaires. Nous avons ainsi développé un test d’activité permettant de mesurer de façon simple et quantitative l’efficacité des molécules ciblant les modificateurs de la famille de l’ubiquitine,Enfin, compte tenu du potentiel thérapeutique du ciblage de la SUMOylation, nous avons débuté le développement d’un inhibiteur de la SUMOylation basé sur l’inhibition de l’interaction entre les enzymes E1 et E2 par des peptides agrafés. / Acute Myeloid Leukemias (AML) are severe haematological malignancies with a poor prognosis. Except Acute Promyelocytic Leukemia (APL), a minor subtype of AML, most patients receive an induction chemotherapy consisting of cytarabine and an anthracycline. However, a large number (20-30%) of patients do not respond to this induction chemotherapy, and relapses are frequent.There are currently no tools available at diagnosis to predict patient response to chemotherapy. Such tests would make it possible to adapt the doses used to increase the response rate and limit the toxicity of chemotherapies, which is significant in older patients.Ubiquitin-like modifiers (UbL), among which ubiquitin, SUMO (-1, -2, -3) and Nedd8 are the most studied members, are post-translational modifiers covalently conjugated to thousands of proteins. They are involved in most cellular pathways, and deregulated in several pathologies. Many studies suggest that their deregulation is associated with LAM chemoresistance.The first part of our work consisted in identifying a set of proteins whose level of modification by ubiquitin, SUMO and NEDD8 could constitute biomarkers of AMLs response to chemotherapeutic drugs. We have also developed a test to evaluate their level of modification by extracts of patient cells in a simple and rapid manner, usable in clinical diagnosis.Targeting of the Ubiquitin, SUMO and Nedd8 pathways is emerging as a promising therapeutic strategy in cancers and several molecules targeting these pathways have recently been described. The clinical development of these molecules requires the development of companion tests to monitor their efficacy on their molecular targets. We have developed an activity test to measure in a simple and quantitative way the efficacy of molecules targeting ubiquitin-like modifiers.Finally, given the therapeutic potential of targeting SUMOylation, we have started the development of a SUMOylation inhibitor based on the inhibition of the interaction between E1 and E2 enzymes by stapled peptides.

A Leucemia Mielóide Crônica (LMC) é uma doença rara. No entanto, os avanços nas pesquisas básica e clínica nos últimos anos, colocaram a LMC em evidência sendo hoje uma neoplasia maligna potencialmente curável. O diagnóstico e tratamento desta doença são, no entanto, extremamente caros. Não havendo dados sistemáticos nem registros de incidência da LMC no Rio Grande do Sul ou no Brasil, o levantamento de dados baseado em registros dos centros de referência se justifica também para planejar ações em saúde. Entre 1996 e 2000, 276 casos foram diagnosticados. A estimativa de casos novos anuais foi de aproximadamente 0,6:100.000 habitantes, e a idade média no momento do diagnóstico foi 42 anos e 4 meses (±16 anos e 2 meses). Quanto ao tratamento e evolução destes pacientes, dos 257 avaliados, 56 (21,8%) foram submetidos ao transplante alogênico de medula óssea, com taxa de sobrevida em 5 anos de 75% e 27% para as fases crônica e acelerada/blástica, respectivamente. O tempo médio de sobrevida para os 257 pacientes foi de 47,7 meses (IC 43,3 - 52,1). Comparando ao relatado na literatura, encontramos um menor número anual de novos casos e também uma média de idade no diagnóstico mais baixa. Isto poderia ser explicado pela menor referência de idosos a serviços terciários de saúde. Para os pacientes transplantados, os resultados foram semelhantes aos relatados na literatura. / Although rare, the advances made in basic and clinical research throughout the last years have thrown a spotlight on CML. Diagnosis and treatment of CML is of high cost. Since there is no systematic data or information about the incidence of CML in Rio Grande do Sul or Brazil, the data obtained from reference centers serve to estimate the number of CML cases in our state to better plan health actions. Between 1996 and 2000, 276 cases were diagnosed. The annual estimate of new cases was approximately of 0,6:100,000 inhabitants, and the median age at diagnosis was 42 years and 4 months (±16 years and 2 months). The mean overall survival time for the 257 patients was 47,7 months (CI 43,3-52,1). That could be explained by the lack of referral for older patients. Regarding treatment and evolution, of the 257 valuable patients, 56 (21,8%) were submitted to allogeneic BMT with a five-year survival of 75% and 27% for chronic and accelerated/blastic phases, respectively. In conclusion, we found a lower estimated incidence and a lower median age at diagnosis. For the transplanted patients the results were similar to those reported in the literature.

by Kong Lai Ping, Ada. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 171-189). / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.v / TABLE OF CONTENTS --- p.ix / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- An Overview on Hematopoiesis --- p.1 / Chapter 1.1.1 --- Development of Hematopoietic Stem Cells and Sites of Hematopoiesis --- p.1 / Chapter 1.1.2 --- Role of Cytokines in the Control of Hematopoiesis --- p.3 / Chapter 1.2 --- Leukemia and Cell Differentiation --- p.5 / Chapter 1.2.1 --- Leukemia as Abnormalities in Hematopoietic Cell Development --- p.5 / Chapter 1.2.2 --- Classification and Etiology of Leukemia --- p.6 / Chapter 1.2.3 --- Current Modalities for the Treatment of Leukemia --- p.9 / Chapter 1.2.4 --- Leukemia Cell Lines as In Vitro Models for the Study of Myeloid Leukemia --- p.10 / Chapter 1.2.5 --- Cytokines as Inducers of Myeloid Leukemia Cell Differentiation --- p.12 / Chapter 1.2.6 --- The Murine Myeloid Leukemia Cell Line (WEHI- 3B JCS) as an Experimental Cell Model --- p.13 / Chapter 1.3 --- Flavonoids: Properties and Biological Activities --- p.15 / Chapter 1.3.1 --- Chemical Structure and Classification of Flavonoids --- p.15 / Chapter 1.3.2 --- Occurrence and Distribution of Flavonoids --- p.16 / Chapter 1.3.3 --- Biological Properties and Action Mechanisms of Flavonoids --- p.17 / Chapter 1.3.4 --- Effects of Flavonoids on Leukemia --- p.20 / Chapter 1.4 --- Aims and Scopes of This Investigation --- p.23 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.26 / Chapter 2.1.1 --- Cell Lines --- p.26 / Chapter 2.1.2. --- Mice --- p.28 / Chapter 2.1.3 --- Flavonoids --- p.28 / Chapter 2.1.4 --- Recombinant Cytokines --- p.30 / Chapter 2.1.5. --- Physiological Differentiation Inducers ´ؤ Vitamin Analogs --- p.31 / Chapter 2.1.6 --- Monoclonal Antibodies --- p.31 / Chapter 2.1.7 --- "Buffers, Culture Medium and Other Reagents" --- p.33 / Chapter 2.1.8 --- Oligonucleotide Primers and Internal Probes --- p.36 / Chapter 2.1.9 --- Reagents for Cytokine Gene Expression Study --- p.38 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- Culture of Tumor Cell Lines --- p.44 / Chapter 2.2.2 --- Determination of Cell Growth and Proliferation --- p.45 / Chapter 2.2.3 --- Colony Assay --- p.46 / Chapter 2.2.4 --- In vivo Tumorigenicity Assay --- p.46 / Chapter 2.2.5 --- Induction of Leukemic Cell Differentiation --- p.47 / Chapter 2.2.6 --- Cell Morphological Study --- p.47 / Chapter 2.2.7 --- Assessment of Differentiation Associated Characteristics --- p.48 / Chapter 2.2.7.1 --- Nitroblue Tetrazolium (NBT) Reduction Assay --- p.48 / Chapter 2.2.7.2 --- Assay of Plastic Adherence --- p.48 / Chapter 2.2.8 --- Flow Cytometric Analysis --- p.49 / Chapter 2.2.8.1 --- Surface Antigen Immunophenotyping --- p.49 / Chapter 2.2.8.2 --- Assay of Non-specific Esterase Activity --- p.50 / Chapter 2.2.8.3 --- Assay of Phagocytic Activity --- p.50 / Chapter 2.2.8.4 --- Assay of Endocytic Activity --- p.51 / Chapter 2.2.8.5 --- Cell Cycle/DNA Content Evaluation --- p.52 / Chapter 2.2.9 --- Gene Expression Analysis --- p.53 / Chapter 2.2.9.1 --- Cell Lysate Preparation --- p.53 / Chapter 2.2.9.2 --- Total RNA Isolation by cesium chloride isopycnic gradient --- p.53 / Chapter 2.2.9.3 --- Reverse Transcription --- p.54 / Chapter 2.2.9.4 --- Polymerase Chain Reaction (PCR) --- p.55 / Chapter 2.2.9.5 --- Agarose Gel Electrophoresis --- p.56 / Chapter 2.2.9.6 --- DIG 3，End Labeling of Oligonucleotide Probes --- p.57 / Chapter 2.2.9.7 --- Dot Blot Hybridization --- p.57 / Chapter 2.2.9.8 --- DIG Chemiluminescent Detection --- p.58 / Chapter 2.2.10 --- DNA Fragmentation Analysis --- p.59 / Chapter 2.2.11 --- Statistical Analysis --- p.60 / Chapter CHAPTER 3: --- EFFECTS OF FLAVONOIDS ON THE PROLIFERATION AND APOPTOSIS OF MYELOID LEUKEMIA CELLS / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Results --- p.63 / Chapter 3.2.1 --- Growth-Inhibitory Effects of Flavone on Murine Myeloid Leukemia JCS Cells --- p.63 / Chapter 3.2.2 --- Cytotoxic Effects of Flavone on Murine Lymphocytes and Myeloid Leukemia JCS Cells --- p.67 / Chapter 3.2.3 --- Effects of Different Flavonoids on the Proliferation of Leukemia JCS Cells --- p.70 / Chapter 3.2.4 --- Anti-proliferative Effect of Flavonoids on Different Tumor Cell Lines --- p.74 / Chapter 3.2.5 --- Effects of Flavone and Flavonol on the Cell Cycle Kinetics of JCS Cells --- p.86 / Chapter 3.2.6 --- Induction of DNA Fragmentation of JCS cells by Flavone --- p.89 / Chapter 3.2.7 --- Effect of Flavone on the Clonogenicity of JCS Cells In Vitro and Tumorigenicity In Vivo --- p.92 / Chapter 3.3 --- Discussion --- p.94 / Chapter CHAPTER 4: --- EFFECTS OF FLAVONOIDS ON THE DIFFERENTIATION OF MURINE MYELOID LEUKEMIA JCS CELLS / Chapter 4.1 --- Introduction --- p.98 / Chapter 4.2 --- Results --- p.100 / Chapter 4.2.1 --- Morphological Changes in Flavonoid-Treated JCS Cells --- p.100 / Chapter 4.2.2 --- Induction of Plastic Adherence in Flavonoid- Treated JCS Cells --- p.106 / Chapter 4.2.3 --- Surface Antigen Immunophenotyping of Differentiating JCS Cells --- p.106 / Chapter 4.2.4 --- NBT-Reducing Activity of Flavonoid-Treated JCS Cells --- p.114 / Chapter 4.2.5 --- Non-specific Esterase Activity of Flavonoid- Treated JCS Cells --- p.115 / Chapter 4.2.6 --- Endocytic Activity of Flavonoid-Treated JCS Cells --- p.116 / Chapter 4.2.7 --- Phagocytic Activity of Flavonoid-Treated JCS Cells --- p.117 / Chapter 4.3 --- Discussion --- p.118 / Chapter CHAPTER 5: --- MECHANISTIC STUDIES ON THE ANTI- PROLIFERATIVE AND DIFFERENTIAION-INDUCING ACTIVITIES OF FLAVONE ON MURINE MYELOID LEUKEMIA JCS CELLS / Chapter 5.1 --- Introduction --- p.122 / Chapter 5.2 --- Results --- p.125 / Chapter 5.2.1 --- Combinations of Flavone with Physiological Differentiation Inducers on the Proliferation and Differentiation of JCS Cells --- p.125 / Chapter 5.2.1.1 --- Modulatory Effects of Flavone and All-Trans Retinoic Acid (ATRA) on the Proliferation and Differentiation of JCS Cells --- p.125 / Chapter 5.2.1.2 --- "Modulatory Effects of Flavone and 1,25- dihydroxyvitamin D3 on the Proliferation and Differentiation of JCS Cells" --- p.130 / Chapter 5.2.2 --- Combinations of Flavone and Cytokines on the Proliferation and Differentiation of JCS Cells --- p.134 / Chapter 5.2.2.1 --- Modulatory Effects of Flavone and rmlFN-γ on the Proliferation and Differentiation of JCS Cells --- p.134 / Chapter 5.2.2.2 --- Synergistic Effects of Flavone and rmIL-1 on the Proliferation and Differentiation of JCS Cells --- p.137 / Chapter 5.2.3 --- Modulation of Cytokine Gene Expressionin Flavone-Treated JCS Cells --- p.144 / Chapter 5.3 --- Discussion --- p.159 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.165 / REFERENCES --- p.171

by Szeto Yuk Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 257-283). / Statement --- p.iii / Acknowledgments --- p.iv / Abbreviations --- p.vi / Abstract --- p.ix / Contents --- p.xi / Chapter Chapter One --- General Introduction / Chapter 1.1 --- Hematopoies --- p.is / Chapter 1.1.1 --- Ontogeny of the hematopoietic system --- p.1 / Chapter 1.1.2 --- Hierarchy of hematopoietic cells --- p.3 / Chapter 1.1.3 --- Characteristics of a functional blood system and the need for regulation --- p.11 / Chapter 1.1.4 --- Interrupted hematopoiesis -- Leukemia --- p.13 / Chapter 1.2 --- Regulation of myeloid cell differentiation / Chapter 1.2.1 --- Regulation of hematopoiesis --- p.16 / Chapter 1.2.2 --- Models of hematopoiesis --- p.18 / Chapter 1.2.3 --- Genes regulation of myeloid cell differentiation and its study --- p.21 / Chapter 1.2.4 --- Genes differentially expressed and involved in myeloid cell differentiation --- p.24 / Chapter 1.3 --- Induced myeloid cell differentiation / Chapter 1.3.1 --- Induced myeloid cell differentiation --- p.46 / Chapter 1.3.2 --- WEHI-3B JCS cells --- p.48 / Chapter 1.3.3 --- Chemical inducers -- Flavonoids and benzodiazepines --- p.51 / Chapter 1.4 --- The aim of study --- p.59 / Chapter Chapter Two --- Cytokine Expression in Biochanin A- and Midazolam-treated JCS cells / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- Cytokine and myeloid differentiation --- p.62 / Chapter 2.1.2 --- Phenotypic studies biochanin A- and midazolam-treated JCS cells --- p.65 / Chapter 2.1.3 --- Cytokine regulation at transcriptional level --- p.68 / Chapter 2.1.4 --- Cytokine mRNA phenotyping by a semi-quantitative approach --- p.69 / Chapter 2.2 --- Materials / Chapter 2.2.1 --- Cell line --- p.72 / Chapter 2.2.2 --- Chemicals and buffers --- p.72 / Chapter 2.2.3 --- DIG system --- p.73 / Chapter 2.2.4 --- Enzymes and nucleic acids --- p.73 / Chapter 2.2.5 --- Solutions --- p.74 / Chapter 2.3 --- Methods / Chapter 2.3.1 --- Isolation of total RNA by guanidinium thiocyanate/cesium chloride isopycnic gradient --- p.75 / Chapter 2.3.2 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.76 / Chapter 2.3.3 --- Southern blotting --- p.79 / Chapter 2.3.4 --- Cycle titration and dot blotting --- p.79 / Chapter 2.3.5 --- DIG 3' end labeling of probes --- p.81 / Chapter 2.3.6 --- Hybridization and stringency wash --- p.81 / Chapter 2.3.7 --- Chemiluminescent detection --- p.82 / Chapter 2.3.8 --- Quantitation by densitometry --- p.82 / Chapter 2.4 --- Results / Chapter 2.4.1 --- Analysis of total RNA --- p.83 / Chapter 2.4.2 --- mRNA phenotyping --- p.85 / Chapter 2.4.3 --- Summary of mRNA phenotyping results --- p.98 / Chapter 2.5 --- Discussion / Chapter 2.5.1 --- mRNA phenotyping --- p.100 / Chapter 2.5.2 --- Cytokine gene regulation --- p.106 / Chapter 2.5.3 --- mRNA quantitation using the current method --- p.108 / Chapter Chapter Three --- Identification and Isolation of Genes that are Differentially Expressed during Midazolam-induced JCS Cell Differentiation / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- Methods for studying differentially expressed genes --- p.110 / Chapter 3.1.2 --- RNA fingerprinting by arbitrarily-primed PCR (RAP-PCR) and differential display (DDRT-PCR) --- p.113 / Chapter 3.1.3 --- Re-amplification of PCR products by touchdown PCR --- p.118 / Chapter 3.1.4 --- Strategies to avoid false positives --- p.119 / Chapter 3.2 --- Materials / Chapter 3.2.1 --- Cell line and bacterial culture --- p.121 / Chapter 3.2.2 --- Chemicals --- p.121 / Chapter 3.2.3 --- Enzymes and nucleic acids --- p.122 / Chapter 3.2.4 --- Kits --- p.122 / Chapter 3.2.5 --- Solutions --- p.122 / Chapter 3.3 --- Methods / Chapter 3.3.1 --- Isolation of total RNA --- p.124 / Chapter 3.3.2 --- First strand cDNA synthesis --- p.124 / Chapter 3.3.3 --- RNA fingerprinting by arbitrarily-primed PCR --- p.124 / Chapter 3.3.4 --- First round cDNA probe screening --- p.126 / Chapter 3.3.5 --- Subcloning of differentially amplified fragments --- p.129 / Chapter 3.3.6 --- Second round cDNA probe screening --- p.133 / Chapter 3.4 --- Results / Chapter 3.4.1 --- Spectrophotometric analysis of total RNA --- p.134 / Chapter 3.4.2 --- Normalization of samples --- p.135 / Chapter 3.4.3 --- RNA fingerprinting of arbitrarily-primed PCR --- p.136 / Chapter 3.4.4 --- Re-amplification of PCR products --- p.138 / Chapter 3.4.5 --- First round cDNA probe screening --- p.139 / Chapter 3.4.6 --- Subcloning of the differentially amplified fragments --- p.143 / Chapter 3.4.7 --- Second round cDNA probe screening --- p.145 / Chapter 3.4.8 --- A comparison of the first and second screening --- p.149 / Chapter 3.5 --- Discussion / Chapter 3.5.1 --- Towards the steps to isolate differentially expressed genes --- p.151 / Chapter 3.5.2 --- Expression profiles predicted at different stage of the procedures --- p.156 / Chapter 3.5.3 --- Representation of the total mRNA in the cell --- p.158 / Chapter 3.3.4 --- Comparison of the original and modified protocol of RAP-PCR --- p.159 / Chapter 3.3.5 --- Advantages of the modified protocol and further refinements --- p.163 / Chapter Chapter Four --- Characterization of the Putative Differentially Expressed Genesin Midazolam-induced JCS cells / Chapter 4.1 --- Introduction / Chapter 4.1.1 --- DNA sequencing --- p.165 / Chapter 4.1.2 --- Automated DNA sequencing and analysis --- p.168 / Chapter 4.1.3 --- Genbank and BLAST homology search --- p.171 / Chapter 4.1.4 --- Internal primer design for RT-PCR --- p.174 / Chapter 4.1.5 --- Genes involved in both myeloid cell differentiation and embryonic development --- p.177 / Chapter 4.2 --- Materials / Chapter 4.2.1 --- Selected recombinant plasmids --- p.180 / Chapter 4.4.2 --- Total RNAs --- p.180 / Chapter 4.2.3 --- Chemicals --- p.180 / Chapter 4.2.4 --- Enzymes and nucleic acids --- p.181 / Chapter 4.2.5 --- Kits --- p.181 / Chapter 4.2.6 --- Solutions --- p.181 / Chapter 4.3 --- Methods / Chapter 4.3.1 --- Preparation of selected recombinant plasmid DNA --- p.182 / Chapter 4.3.2 --- Sequencing --- p.182 / Chapter 4.3.3 --- Data analysis and assessment by ALF manager and DNAsis --- p.184 / Chapter 4.3.4 --- Sequence search by BLASTN program --- p.185 / Chapter 4.3.5 --- Primer design by Oligo´ёØ ver. 34 --- p.186 / Chapter 4.3.6 --- Differential expression confirmed by RT-PCR --- p.186 / Chapter 4.4 --- Results / Chapter 4.4.1 --- Analysis of selected recombinant plasmid DNA --- p.187 / Chapter 4.4.2 --- Sequencing results --- p.191 / Chapter 4.4.3 --- BLASTN search results --- p.212 / Chapter 4.4.4 --- Primer design of the sequenced fragments --- p.222 / Chapter 4.4.5 --- "Expression profile of the isolated genes in midazolam-, biochanin A- induced JCS cells and mouse embryos" --- p.223 / Chapter 4.5 --- Discussion / Chapter 4.5.1 --- Sequence analysis of the isolated gene fragments --- p.233 / Chapter 4.5.2 --- Expression profiles of the isolated genes --- p.236 / Chapter Chapter Five --- General Discussion / Chapter 5.1 --- Studies on leukemic cell differentiation / Chapter 5.1.1 --- Differentiation pathways revealed by different inducers --- p.241 / Chapter 5.1.2 --- Lineage preference during differentiation --- p.243 / Chapter 5.2 --- Differentiation program triggered by midazolam / Chapter 5.2.1 --- Signaling pathways initiated by biochanin A and midazolam --- p.245 / Chapter 5.2.2 --- Differentially expressed genes during midazolam-induced differentiation --- p.247 / Chapter 5.2.3 --- Expression patterns of the isolated differentially expressed genesin midazolam and biochanin A-induced JCS cells --- p.248 / Chapter 5.2.4 --- Myeloid genes in embryonic development --- p.250 / Chapter 5.3 --- Future studies of the isolated fragments --- p.252 / Chapter 5.4 --- Conclusion --- p.256 / Reference --- p.257 / Append --- p.ix / Chapter A1. --- Ambiguity codes for sequencing --- p.i / Chapter A2. --- Myeloid cell lines --- p.ii / Chapter A3. --- Details of manufacturer's products --- p.iii / Chapter A4. --- List of machine and equipment --- p.v

Chiu Lai-Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 137-152). / Abstracts in English and Chinese. / Acknowledgement --- p.II / Abstract --- p.IV / Contents --- p.VIII / Abbreviations --- p.XIV / Chapter Chapter One --- General introduction / Chapter 1.1 --- Haemopoiesis --- p.1 / Chapter 1.1.1 --- Background --- p.1 / Chapter 1.1.2 --- Regulation --- p.2 / Chapter 1.1.2.1 --- Stromal cells --- p.2 / Chapter 1.1.2.2 --- Haemopoietic regulator --- p.3 / Chapter 1.1.2.3 --- Haemopoietic regulator receptors and signal transduction --- p.5 / Chapter 1.2 --- Disorder of haemopoiesis --- p.9 / Chapter 1.2.1 --- Causes --- p.9 / Chapter 1.2.2 --- Types of leukemia --- p.9 / Chapter 1.2.3 --- Treatment of leukemia --- p.10 / Chapter 1.3 --- Prostaglandins --- p.13 / Chapter 1.3.1 --- Introduction --- p.13 / Chapter 1.3.2 --- Types and biosynthesis --- p.14 / Chapter 1.3.3 --- Prostaglandin receptors --- p.15 / Chapter 1.3.4 --- Prostaglandins and cell differentiation --- p.17 / Chapter 1.3.4.1 --- PGD2 and cell differentiation --- p.19 / Chapter 1.3.4.2 --- PGE2 and cell differentiation --- p.20 / Chapter 1.3.4.3 --- PGJ2 and cell differentiation --- p.22 / Chapter 1.4 --- WEHI-3B JCS cells --- p.25 / Chapter 1.5 --- Aims of study --- p.27 / Chapter Chapter Two --- Roles of Prostaglandin D2，E2 and J2 in WEHI-3B JCS myeloid leukemia cell differentiation / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.1.1 --- Morphological studies of JCS cells --- p.28 / Chapter 2.1.2 --- Methods in determining cell proliferation --- p.29 / Chapter 2.1.3 --- Methods in determining differentiated cells --- p.31 / Chapter 2.2 --- Materials --- p.33 / Chapter 2.2.1 --- Cell line --- p.33 / Chapter 2.2.2 --- Chemicals --- p.33 / Chapter 2.2.3 --- Solutions and buffers --- p.34 / Chapter 2.3 --- Methods --- p.36 / Chapter 2.3.1 --- Microscopic studies of the JCS cells --- p.36 / Chapter 2.3.1.1 --- Histochemical staining of JCS --- p.36 / Chapter 2.3.1.2 --- Transmission electronic microscopic --- p.36 / Chapter 2.3.2 --- [3H]-thymidine incorporation assay --- p.37 / Chapter 2.3.3 --- MTT assay --- p.37 / Chapter 2.4 --- Results --- p.38 / Chapter 2.4.1 --- Histochemical staining of JCS cells --- p.38 / Chapter 2.4.2 --- Electron microscopy --- p.40 / Chapter 2.4.3 --- "Effect of PGD2, E2 and J2 on JCS cells proliferation" --- p.44 / Chapter 2.4.4 --- "Effect of PGD2, E2 and J2 on JCS cells differentiation" --- p.48 / Chapter 2.5 --- Discussion --- p.53 / Chapter 2.5.1 --- Morphological differentiation of JCS cells --- p.53 / Chapter 2.5.2 --- The ultra-structures of JCS cells --- p.53 / Chapter 2.5.3 --- "Effect of PGD2, E2 and J2 on JCS cells proliferation" --- p.54 / Chapter 2.5.4 --- "Effect of PGD2, E2 and J2 on JCS cells differentiation" --- p.55 / Chapter Chapter Three --- Roles of Prostaglandin E2 in normal haemopoiesis and the detection of PGE2 receptors expression in JCS and bone marrow cells / Chapter 3.1 --- Introduction --- p.57 / Chapter 3.1.1 --- Colony assay --- p.57 / Chapter 3.1.2 --- The use of RT-PCR --- p.58 / Chapter 3.1.3 --- Prostaglandin E receptors --- p.59 / Chapter 3.2 --- Materials --- p.62 / Chapter 3.2.1 --- Bone marrow cells --- p.62 / Chapter 3.2.2 --- Cell line --- p.62 / Chapter 3.2.3 --- Chemicals --- p.62 / Chapter 3.2.4 --- Primers --- p.63 / Chapter 3.2.5 --- Solutions and buffers --- p.64 / Chapter 3.2.6 --- Enzymes and reagents --- p.65 / Chapter 3.3 --- Methods --- p.66 / Chapter 3.3.1 --- Titration of mouse IL-3 --- p.66 / Chapter 3.3.2 --- Determination of suitable IL-3 concentration for growth of bone marrow cells in colony assay --- p.66 / Chapter 3.3.2.1 --- Preparation of bone marrow cells --- p.66 / Chapter 3.3.2.2 --- Preparation of culture medium for colony assay --- p.67 / Chapter 3.3.3 --- Investigation of the effect of PGE2 on normal haemopoiesis by colony assay --- p.68 / Chapter 3.3.4 --- Detection of PGE2 receptors expression on JCS cells and bone marrow cells --- p.68 / Chapter 3.3.4.1 --- Preparation of cell lysates --- p.68 / Chapter 3.3.4.2 --- Preparation of total RNA of JCS cells and bone marrow cells --- p.68 / Chapter 3.3.4.3 --- RT-PCR --- p.69 / Chapter 3.4 --- Results --- p.71 / Chapter 3.4.1 --- Titration of mouse IL-3 --- p.71 / Chapter 3.4.2 --- Effect of mouse IL-3 on normal haemopoiesis --- p.73 / Chapter 3.4.3 --- Effect of PGE2 on mouse IL-3 driven normal bone marrow cell differentiation --- p.76 / Chapter 3.4.4 --- Analysis of total RNA prepared from uninduced JCS cells and bone marrow cells --- p.79 / Chapter 3.4.5 --- "Expression of gapdh in heart, liver, spleen, JCS and bone marrow cells" --- p.81 / Chapter 3.4.6 --- "Expression of PGE2 receptors in heart, liver, spleen, JCS and bone marrow cells" --- p.82 / Chapter 3.5 --- Discussion --- p.84 / Chapter 3.5.1 --- Effect of PGE2 on IL-3 driven normal bone marrow cells differentiation --- p.84 / Chapter 3.5.2 --- "Expression of PGE2 receptors in heart, liver, spleen, JCS and bone marrow cells" --- p.85 / Chapter Chapter Four --- Gene expression profile of JCS cells under 5 hours of PGE2 induction / Chapter 4.1 --- Introduction --- p.88 / Chapter 4.1.1 --- Review of methods studying differential gene expression --- p.88 / Chapter 4.1.2 --- The choice of method studying differential gene expression --- p.92 / Chapter 4.1.3 --- The microarray --- p.93 / Chapter 4.2 --- Materials --- p.95 / Chapter 4.2.1 --- Cell line --- p.95 / Chapter 4.2.2 --- Kits --- p.95 / Chapter 4.2.3 --- Chemicals --- p.95 / Chapter 4.2.4 --- Solutions and buffers --- p.96 / Chapter 4.2.5 --- Reagents --- p.97 / Chapter 4.3 --- Methods --- p.98 / Chapter 4.3.1 --- Preparation of total RNA from PGE2 induced JCS cells --- p.98 / Chapter 4.3.2 --- Preparation of cDNA probes --- p.98 / Chapter 4.3.2.1 --- Probe synthesis from total RNA --- p.98 / Chapter 4.3.2.2 --- Column chromatography --- p.99 / Chapter 4.3.3 --- Hybridizing cDNA probes to the Atlas Array --- p.99 / Chapter 4.4 --- Results --- p.101 / Chapter 4.4.1 --- Spectrophotometric analysis of total RNA after ethanol precipitation --- p.101 / Chapter 4.4.2 --- Hybridization of cDNA probes to Atlas Array --- p.102 / Chapter 4.5 --- Discussion --- p.121 / Chapter 4.5.1 --- Genes with increased expression --- p.121 / Chapter 4.5.2 --- Genes with decrease expression --- p.127 / Chapter 4.5.3 --- Study of gene expression profile by microarray --- p.128 / Chapter Chapter Five --- General discussion / Chapter 5.1 --- Introduction --- p.131 / Chapter 5.2 --- Roles of PGE2 in JCS cells differentiation --- p.131 / Chapter 5.3 --- Roles of PGE2 in normal haemopoiesis --- p.134 / Chapter 5.4 --- Further studies --- p.135 / References --- p.137

Les leucémies aiguë myéloïde (LAM) sont un groupe d’hémopathies malignes, dont le traitement est généralement composé de deux génotoxiques : la cytarabine (Ara-C) et la daunorubicine (DNR). Nous avons montré que l’Ara-C et la DNR induisent la déconjugaison rapide de SUMO (Small Ubiquitin-related Modifier) de ses protéines cibles. Cette deSUMOylation est dûe à l'inactivation des enzymes E1 et E2 de SUMOylation par les espèces réactives de l'oxygène (ROS) produites par l’Ara-C et la DNR et est impliquée dans l'activation de l'apoptose. En outre, cet axe ROS/SUMO est anergisé dans les LAM chimiorésistantes. Cependant, il peut être réactivé par des pro-oxydants ou par inhibition de la voie SUMO par l'acide anacardique. Pour identifier les protéines contrôlées par l’axe ROS/SUMO nous avons effectué une approche de spectrométrie de masse quantitative (SILAC). Parmi les 1000 protéines SUMOylées identifiées, la plupart des 114 protéines qui perdent leur SUMOylation lors du traitement sont impliquées dans la régulation de l'expression des gènes. De plus, un ChIP-Seq avec des anticorps anti SUMO-2 a permis de montrer que les génotoxiques, en particulier la DNR, induisent une diminution massive de la présence de protéines SUMOylées sur la chromatine. La recherche de motifs au sein des séquences fixant SUMO a permis d’identifier le motif de liaison de CTCF à l’ADN. De plus, CTCF a été trouvé dans la SILAC comme l’une des protéines déSUMOylées par les traitements. En utilisant des données publiques de Chip-Seq pour CTCF, nous avons identifié 55 gènes qui fixent à la fois CTCF et SUMO et dont l’expression est régulée par les traitements. Dans la dernière partie de ce travail, nous avons étudié le groupe de 19 protéines dont la SUMOylation augmente suite aux traitements génotoxiques. Parmi ces protéines, nous avons trouvé diverses protéines centromériques, y compris CENP-B et CENP-C. En utilisant le PLA (Proximity Ligation Assay) nous avons pu montrer que CENP-B et CENP-C colocalisent avec SUMO et yH2AX après traitement. Cela suggère que la SUMOylation des protéines centromériques se produit sur les sites de cassure et pourrait jouer un rôle dans la réparation des dommages de l'ADN. / Acute Myeloid Leukemias (AML) are a group a severe hematological malignancies, which treatment is generally composed of two genotoxics: Cytarabine (Ara-C) and Daunorubicin (DNR). We have shown that these drugs induce the rapid deconjugation of the Small Ubiquitin-related Modifier (SUMO) from its target protein. This is due to the inactivation of SUMO E1 and E2 enzymes by Reactive oxygen species (ROS). This deSUMOylation participated in the activation of specific genes and is involved the induction of apoptosis. In addition, this ROS/SUMO axis is anergized in chemoresistant AMLs. However, it can be reactivated by pro-oxidants or inhibition of the SUMO pathway with anacardic acid, an inhibitor of the SUMO E1. To identify which proteins are regulated by this ROS/SUMO axis, we performed a quantitative mass spectrometry approach. Among the 1000 identified SUMO targets, most of the 114 proteins, which SUMOylation decrease upon treatment, are involved in the regulation of gene expression. In addition, we showed by ChIP-Seq with SUMO-2 antibodies that genotoxics, in particular DNR, induce a massive decrease of the presence of SUMOylated proteins on the chromatin. Motif search analysis of the SUMO binding sequences in these genes identified CTCF binding motif. Interestingly, CTCF was found in the SILAC as deSUMOylated by the drugs. Using publicly available ChIP-Seq data for CTCF, we found 55 genes which are occupied by both SUMO-2 and CTCF and which expression is regulated by the drugs. In the last part of this work, we got interested in the 19 proteins that get up-SUMOylated upon treatment. Among them, we found centromeric proteins, including CENP-B and CENP-C. Using Proximity Ligation Assay, we could show that CENP-B and CENP-C colocalize with both SUMO and yH2AX upon DNR treatment. Altogether, this suggests that centromeric protein up-SUMOylation occurs at sites of DNA damage and might play a role in DNA damage repair.