The work presented in this thesis has developed an existing in vitro culture system used for expansion of cord blood progenitors to one that can be applied to expand significant numbers of erythroblasts from peripheral blood mononuclear cells isolated from healthy donor blood samples or patients with haemolytic anaemia. These erythroblasts can be further differentiated to enucleated reticulocytes synchronously producing homogeneous populations of cells at each of the morphologically defined stages of erythroid differentiation in quantities amenable to biochemical experimentation. Expression profiles of proteins that constitute the band 3 based multiprotein complex were monitored throughout-erythropoiesis by a variety of methods and these studies were extended to investigate the spatial and temporal establishment of critical interactions required for multiprotein complex assembly during differentiation of erythroblasts to reticulocytes. Protein 4.2, 'a key component of this complex was found to be expressed early during terminal erythroid differentiation in basophilic erythroblasts where it interacts with band 3 in an intracellular compartment prior to delivery to the plasma membrane. Rh and its associated glycoprotehRhAG which form the core of the band 3 associated Rh complex were also found to interact at this stage at the plasma membrane simultaneous to the onset of Rh dependency on RhAG. Expansion and differentiation of erythroblasts derived from a protein 4.2 null hereditary spherocytosis patient was conducted for the first time. This demonstrated that the hallmarks of protein 4.2 absence observed in erythrocytes begin to manifest within 48 hours of differentiation concomitant with the onset of initial complex assembly in basophilic erythroblasts and within a window of major membrane protein synthesis and delivery. Initial culture of erythroblasts from CDAII patients suggests that the onset of a glvcosvlation defect also occurs from the onset of terminal differentiation whilst additional characteristic phenotypes of this disease develop later.

When CD47 is ligated with a specific ligand, phosphatidylserine exposure is observed on the surface of the erythrocyte in a process known as eryptosis, which is similar to apoptosis. The aim of this study was to identify new proteins within the CD47 receptor mediated phosphatidylserine pathway and to elucidate the functions of any newly identified proteins. Previous research had suggested a link between protein 4.1R and CD47. Therefore, diseased protein 4.1R deficient erythrocytes were used as a molecular tool to help explore the role of protein 4.1R in the CD47 pathway. To achieve this aim, flow cytometry to measure phosphatidylserine exposure, 1D and 2D protein immunoblotting, 1D and 2D electrophoresis, RS100 ProteinChips coupled to SELDI-TOF, Q-TOF and MALDI-TOF, calcium influx, 2D serine/threonine phosphoblots and protein kinase A and protein kinase C inhibition were employed. Results showed increased phosphatidylserine exposure in 4.1R deficient erythrocytes and protein immunoblots highlighted a novel interaction between CD44 and protein 4.1R. RS100 ProteinChip experiments also confirmed an interaction between CD44 and protein 4.1R. Phosphatidylserine exposure in normal erythrocytes was increased when protein kinase A was inhibited. Conversely phosphatidylserine exposure was decreased when protein kinase C was inhibited. In 4.1R deficient erythrocytes, protein kinase A inhibition further increased phosphatidylserine exposure and protein kinase C inhibition further decreased phosphatidylserine exposure. Phosphorylation of protein 4.1R and calcium influx was also observed in response to CD47 ligation. The study concludes that protein 4.1R, protein kinase A and protein kinase C have a direct effect on phosphatidylserine exposure and can therefore be assumed to be a part of the CD47 pathway. Calcium influx and phosphorylation of protein 4.1R also play a critical role in the CD47 pathway. The novel interaction between CD44 and protein 4.1R and the role that CD44 might play in the CD47 pathway is worthy of further research.

Erratum is pasted onto back endpaper. / Bibliography: leaves 189-219. / xxiii, 221 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Current understanding of metastatic processes and the clinical significance of these processes is discussed. The study of circulating solid tumour cells in peripheral blood is reviewed from an historical perspective. Newer molecular and cell biological techniques which may facilitate more reliable tumour evaluation are reviewed with special reference given to polymerase chair reaction (PCR) / Thesis (M.D.)--University of Adelaide, Dept. of Surgery, 1997?

Erratum is pasted onto back endpaper. / Bibliography: leaves 189-219. / xxiii, 221 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Current understanding of metastatic processes and the clinical significance of these processes is discussed. The study of circulating solid tumour cells in peripheral blood is reviewed from an historical perspective. Newer molecular and cell biological techniques which may facilitate more reliable tumour evaluation are reviewed with special reference given to polymerase chair reaction (PCR) / Thesis (M.D.)--University of Adelaide, Dept. of Surgery, 1997?

An arterialblood gas analysis which is conducted in critical care areas contributes to the assessment of a patient's ventilatory status and acid -base balance. The purpose of this research was to determine the relationship of time delays and temperature on the result of a blood gas analysis. The objective was to either accept or refute the null hypothesis, that there is no relationship between temperature and time delays and an arterial blood gas result Fifteen subjects were randomly selected. The researcher drew three samples of arterial blood from each subject. Ethical principles were observed. An inferential non-parametric statistic was used. The chi-squared test was used to test the hypothesis and the Friedman and the Wilcoxon signed ranks test were used to test the differences between the means. The results revealed that there was a relationship between time delays, temperature and the arterial blood gas result. The null hypothesis was rejected. / Health Stusies / M.A. (Health Studies)

L’acide valproïque (VPA) est un inhibiteur des histones désacétylases (HDACi), qui présente des propriétés anti-tumorales nsur différents types de cancers. Son utilisation depuis plusieurs décennies comme médicament antiépileptique a révélé des effets secondaires, notamment sur le système hématopoïétique. Dans la présente étude, nous nous sommes intéressés à l’effet du VPA sur les réseaux microARN (miR)/Facteurs de transcription (FT) spécifiquement impliqués dans la régulation des voies de différenciation érythro-mégacaryocytaires. Nous montrons que le VPA est capable d’inhiber la différenciation érythroïde dans les cellules érythroleucémiques humaines TF1 et K562 et dans les cellules souches hématopoïétiques (CSH) CD34+, stimulées par l’érythropoïétine recombinante (Epo) ou par l’aclacinomycine A. Cette inhibition se traduit par une diminution de l’expression de la glycophorine A, de la γ-globine, des miR-144/451 et du FT GATA-1. L’inhibition du pré-miR-144 suggère que le VPA est capable de réguler l’expression du gène miR-144/451 au niveau transcriptionnel, via GATA-1. Dans les cellules Epo/CD34+, le VPA induit l’augmentation du FT PU.1 en accord avec l’inhibition du miR-155 et favorise son interaction avec GATA-1 pour inhiber son activité. L’utilisation d’un analogue du VPA, sans activité HDACi (Valpromide) et d’un inhibiteur d’HDAC de classe I, le MS-275, a montré que l’activité HDACi du VPA n’est pas requise pour l’inhibition de la différenciation érythroïde. Le VPA affecte également la voie mégacaryocytaire issue d’un progéniteur commun aux cellules érythroïdes. Dans la lignée mégacaryoblastique Meg-01, le VPA induit des modifications morphologiques du type mégacaryocytaire, une augmentation du marqueur CD61, du FT GATA-2 et du miR-27a. En revanche, l’expression du FT GATA-1 et des miR-144/451 diminuent. L’augmentation du miR-27a coïncide avec la diminution de l’expression de l’ARNm du FT RUNX1, en accord avec l’induction de la voie mégacaryocytaire. En conclusion, le VPA est capable de moduler le programme de différenciation érythro-mégacaryocytaire, à travers un micro réseau de régulation miR/FT / Valproic acid (VPA), a histone deacetylase inhibitor (HDACi), exhibits anti-cancer properties against several tumor types. Its use as an anti-epileptic drug for several decades reveled side effects at the hematological level. In this study, we analyzed the effect of VPA on an erythro-megakaryocyte-specific miR/transcription factors network. VPA inhibited erythroid differentiation in the erythroleukemia cell lines TF1 and K562 as well as in CD34+/hematopoietic stem cells (HSCs), induced by the recombinant erythropoietin (Epo) or aclacinomycin. This inhibition was characterized by glycophorin-A, γ-globin and GATA-1/miR-144/451 down-regulation. Inhibition of pre-miR-144 expression suggested that VPA regulates transcription of the miR-144/451 gene through GATA-1. In Epo-stimulated HSCs, VPA induced PU.1 expression in correlation with miR-155 inhibition and promoted GATA-1/PU.1 interaction. The use of valpromide, a VPA analogue without HDACi activity and the class-I HDACi MS-275, showed that HDAC inhibition by VPA was not required for its inhibitory activity on erythropoiesis. VPA also induced megakaryocyte features in Meg-01 cells, at both cellular and molecular levels. Notably, CD61, GATA-2 and miR-27a were over-expressed. RUNX1 mRNA expression and GATA-1/miR-144/451 axis decreased in accordance with megakaryocyte differentiation. In conclusion, VPA is able to modulate erythro-megakaryocytic differentiation program, through a regulatory micro-network involving miRs and TFs

An arterialblood gas analysis which is conducted in critical care areas contributes to the assessment of a patient's ventilatory status and acid -base balance. The purpose of this research was to determine the relationship of time delays and temperature on the result of a blood gas analysis. The objective was to either accept or refute the null hypothesis, that there is no relationship between temperature and time delays and an arterial blood gas result Fifteen subjects were randomly selected. The researcher drew three samples of arterial blood from each subject. Ethical principles were observed. An inferential non-parametric statistic was used. The chi-squared test was used to test the hypothesis and the Friedman and the Wilcoxon signed ranks test were used to test the differences between the means. The results revealed that there was a relationship between time delays, temperature and the arterial blood gas result. The null hypothesis was rejected. / Health Stusies / M.A. (Health Studies)

Le facteur 4 plaquettaire (PF4/CXCL4) est un tétramère constitué de quatre sous-unités identiques de 7,8 kDa qui est libéré en grande quantité par les plaquettes lors de l’hémostase primaire (ensemble des phénomènes permettant un colmatage initial d’une lésion vasculaire). L’étude de la formation d’un caillot de fibrine en présence de PF4 montre une augmentation de la turbidité finale du caillot : le PF4 modifie le réseau formé. Etant donné que la plupart des acteurs de la fibrinolyse se lie au caillot de fibrine et que le PF4 modifie sa structure, nous avons pensé qu’il serait intéressant de rechercher si le PF4 influençait aussi la fibrinolyse. La lyse d'un caillot est effectuée par la plasmine issue de l'activation du plasminogène par son activateur d’origine tissulaire (t-PA) en présence d’un cofacteur qui n'est autre que la fibrine. Nous avons étudié la lyse de caillots de plasma, obtenus par activation de la cascade de la coagulation, en condition statique et à l'aide d'un modèle de thrombose artérielle (système Chandler loop). Dans les deux cas, une diminution du temps de demi-lyse a été observée en présence de PF4. Cependant, la lyse de caillots préparés par simple ajout de thrombine sur du fibrinogène ne permet pas de retrouver cet effet du PF4. Ceci suggère que l’influence du PF4 sur la structure du caillot n’est pas à l’origine de l’effet sur sa lyse et que le PF4 n’influence pas (ou très peu) l'activation du plasminogène, ainsi que l'activité de la plasmine résultante. Cette hypothèse a été confirmée par l’étude de l’activité amydolytique du t-PA et de la plasmine (quelle soit ajoutée ou générée). En système purifié, les inhibiteurs plasmatiques de la fibrinolyse sont absents. Les deux principaux sont l'inhibiteur de l'activateur du plasminogène de type 1 (PAI-1) et l’α2-antiplasmine (α2-AP). La lyse de caillots préparés à partir de plasma déficient en α2-AP montre une diminution du temps de demi-lyse en présence de PF4 (comme pour le plasma normal), alors qu’avec le plasma dépourvu de PAI-1 le temps de demi-lyse n'est plus influencé. De plus, l’ajout de PAI-1 dans le système purifié entraine une diminution du temps de demi-lyse en présence de PF4. Ceci suggère que le PF4 prévient directement ou indirectement l'inhibition du t-PA par PAI-1. L’étude de la cinétique d'inhibition de l’activité amidolytique du t-PA par le PAI-1, la détermination de la stœchiométrie de cette inhibition, et l’analyse de ces cinétiques par immuno-empreinte montrent que le PF4 est un modulateur de la fibrinolyse qui agit en retardant la formation d'un complexe initial entre le t-PA et le PAI-1. Cette nouvelle fonction du PF4 est cohérente, et vient en complément de celle décrite récemment d’inhibiteur de l'activation du TAFI. / Platelet factor 4 (PF4/CXCL4) is a tetramer constituted of four identical 7,8 kDa subunits released in large quantities by platelets during primary heamostasis (allowing initial clogging of a vascular injury). Study of fibrin clot formation in the presence of PF4 shows an increase of the final clot turbidity: PF4 modifies the formed network. Given that most fibrinolysis actors are bound to the fibrin clot and that PF4 modifies its structure we thought it would be interesting to investigate if PF4 also influences fibrinolysis. Clot lysis is performed by plasmin originating from activation of its precursor by tissue plasminogen activator (t-PA) with fibrin itself as cofactor of the reaction. We have studied lysis of plasma clots formed by activation of the coagulation cascade in static condition and in a Chandler loop model mimicking arterial thrombosis. Half-times of lysis decreased in the presence of PF4 in both systems. However, PF4 had no longer detectable influence on the half-time of lysis with clots formed by direct addition of thrombin on purified fibrinogen. Observation suggested that the observed decrease of the half-time of lysis induced by PF4 did not originate from its influence on fibrin clot formation and that PF4 had little effect if any on plasminogen activation or plasmin activity. We confirmed this hypothesis by comparing amydolytic activities of t-PA and plasmin (added or generated through plasminogen activation). In purified system, fibrinolysis inhibitors are absent. The two main inhibitors are plasminogen activator inhibitor-1 (PAI-1) and α2-antiplasmin (α2-AP). Lysis of clots obtained from α2-AP deficient plasma showed a decrease of the half-time of lysis in the presence of PF4 (as in normal plasma), whereas in PAI-1 deficient plasma half-time of lysis was unchanged. Moreover if PAI-1 was added to the purified system, half-time of lysis decreased in the presence of PF4. Observations therefore suggested that PF4 prevented directly or indirectly t-PA inhibition by PAI-1. Kinetics of the amidolytic activity of t-PA inhibition by PAI-1 in the presence or not of PF4, determination of its stoichiometry and Western blot analysis of these inhibition kinetics revealed that PF4 is a fibrinolysis modulator which delays formation of the initial (Michaelis) complex between t-PA and PAI-1. This new feature of PF4 is consistent and complementary with its recently described role as a modulator of TAFI activation.

L’existance de cellules souches leucémiques (CSL) dans la Leucémie Myéloïde Chronique (LMC) prédit que que seule la destruction des CSL conduirait à une guérison. Une proportion importante de patients atteints de LMC développe une résistance aux drogues, environ ~ 30% des cas, Les mécanismes de résistance aux inhibiteurs de tyrosine kinase (TKI) dans la leucémie myéloïde chronique (LMC) restent souvent obscures. Les cellules souches leucémiques de la LMC pourraient rester viables et en repos, malgré la présence de facteurs de croissance ou de médicaments qui semblent les protéger de l'apoptose. Nous avons montré dans la première parti de cette étude que certains transporteurs telles ABCG2, hOCT-1 pourraient jouer un rôle avec le micro environnement dans la résistance des lignées LMC en adhésion au stoma médullaire. De plus, dans deuxième parti nous avons montré que le gène TWIST-1 (est un acteur clé de l'embryogenèse) est déréglementé dans les cellules de LMC innée des résistants à l'imatinib, et que la sur-expression de l’oncogène TWIST-1 pourrait représenter un nouveau facteur clé de pronostiques potentiellement utiles pour améliorer la guérison de LMC aux TKI. De plus, nous avons pu également montrer que le gène TP73 est impliqué dans la résistance des CSL de LMC. Ce gène pourrait être un facteur prédictif pour identifier une résistance potentielle des patients de LMC au moment du diagnostic. Nous avons montré également que ce gène est régulé par le micro-environnement. Nous avons montrés une sur-expression des isoformes tronquées dans les lignées LMC avec l’adhésion au stroma. Les résultats suggèrent que les molécules intrinsèques comme TWIST-1, les transporteurs et les isoformes de p73 sont dérégulées dans les CSL par des mécanismes extrinsèques qui interviennent avec le micro environnement leucémique par le mécanisme d’adhésion dans le phénomène de résistance aux traitements. Ce mécanisme spécial de résistance due à la conservation de ces CSL dans l’état immature en adhésion avec son micro environnement. / The existence of Leukemia stem cells (CSL) in chronic myelogenous leukemia (CML) predicts that only the destruction of CSL lead to a cure. A significant proportion of CML patients develop resistance to drugs, ~ 30% cases, mechanisms of resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML) often remain obscure. Leukemic stem cells of CML could remain viable and quiet, despite the presence of growth factors or drugs that seem to protect them from apoptosis. We have shown in the first part of this study that some carriers such as ABCG2, hOCT could to be play a role with the microenvironment in the resistance among CML adhesion of stoma Bone marrow. Furthermore, in the second part we showed that the gene TWIST-1 (is a key player of the embryogenesis) is deregulated in cells of CML innately resistant to imatinib, and that overexpression of the oncogene TWIST-1 could represent a new prognostic factor key potentially useful for improving the querison CML to TKI. In addition, we also could show that the TP73 gene is involved in the resistance of CSL CML. This gene could be a predictor to identify potential resistance of CML patients at diagnosis. We have also shown that this gene is regulated by the microenvironment. We have shown an overexpression of truncated isoforms in CML cell lines with the accession to the stroma. The results suggest that intrinsic molecules such as TWIST-1 carriers and p73 isoforms are deregulated in CSL by extrinsic mechanisms involved with the leukemia microenvironment by the mechanism for participation in the phenomenon of drug resistance. This mechanism with its microenvironment.