L’objectif de notre travail était de déterminer le rôle joué par l’érythropoïétine et la thrombopoïétine, respectivement, dans l’anémie et la thrombocytopénie observées chez des enfants souffrant d’une hémopathie maligne. Par le dosage simultané de la forme soluble du récepteur de la transferrine et de l’érythropoïétine dans le sérum nous avons montré que l’anémie observée chez ces patients est bien la conséquence d’une réduction du nombre de progéniteurs érythropoïétiques (atteinte médullaire centrale) mais que celle-ci n’est pas la conséquence d’une production insuffisante d’érythropoïétine. Nous avons fait la même observation chez des enfants souffrant d’une tumeur solide non hématologique et chez des patients en cours de traitement par chimiothérapie. Chez ces derniers patients, en appliquant un modèle de culture de moelle à long terme, nous avons pu démontrer l’existence d’une altération du microenvironnement médullaire, probablement induite par la chimiothérapie, se traduisant par une réduction de son aptitude à supporter le développement de la lignée érythroïde. Ceci expliquant au moins partiellement l’inadéquation de la réponse érythropoïétique observée chez ces patients en réponse à l’anémie. Dans la dernière partie du travail, nous avons montré que la thrombocytopénie très fréquemment observée chez les patients leucémiques s’accompagne dans la majorité des cas d’une élévation exponentielle de la concentration de thrombopoïétine, excepté dans les cas de leucémies de la lignée myéloïde. Chez ces derniers la concentration de thrombopoïétine est proche des valeurs observées chez des sujets normaux alors qu’elle devrait être 10 à 100 fois plus élevée compte tenu du nombre de plaquettes extrêmement bas. Nous avons pu montrer que ces taux très bas sont la conséquence de la liaison de la thrombopoïétine à un récepteur spécifique et fonctionnel présent à la surface des cellules leucémiques myéloïdes qui, en l’utilisant comme facteur de croissance, (stimulant leur prolifération et retardant leur mort cellulaire) « consomment » la thrombopoïétine présente dans le sérum.

I have studied the calpain system in acute myeloid leukemia using the 32D and 32Dkit cell lines. Specifically, I characterized the calpain system in the cell lines, and performed calpastatin overexpression and knockdown studies. I found that calpain activity is elevated in the 32D and 32Dkit cells, and calpain inhibition causes apoptosis. Both μ- and m-calpain contribute to the calpain activity in these cell lines. The 32Dkit cells have higher calpain activity than the 32D cells, which I have shown is partially attributed to basal ckit activation. Calpastatin was present in both cell lines, but exists mainly in a degraded form. Calpastatin overexpression lowered calpain activity and provided a growth disadvantage to the 32Dkit cells, but had no effect on 32D cells. Calpastatin knockdown caused a significant increase in calpain activity in the 32D cells, which changed the cell cycle distribution but had no other major effects.

Thesis (Ph. D.)--University of Florida, 2006. / Title from title page of source document. Document formatted into pages; contains 227 pages. Includes vita. Includes bibliographical references.

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Medicine / Master / Master of Philosophy

The SOX (Sry-related HMG box) genes belong to a family of transcription factors containing a High-Mobility-Group box domain. In an initial screen, SOX7 was uniquely down-regulated in myeloid malignancies compared with most cases of precursor B-cell acute lymphoblastic leukemia (ALL) and normal bone marrow cell, leading us to examine the expression and function of SOX7 during normal hematopoietic differentiation and in acute lymphoblastic leukemia. By studying human umbilical cord blood (UCB), SOX7 expression in different hematopoietic lineages was evaluated by RT-PCR. SOX7 was preferentially expressed in CD34+CD38- compared with CD34+CD38+ population and in CD34-CD19+ compared with CD34-CD33+ cells. SOX7 expression was down-regulated in colonies in CFU assay and in engrafting myeloid cells in NOD/SCID mouse transplantation. Transfecting SOX7 siRNA into CD34+ cells reduced cell growth and the CD34+CD33+ population in 3-day culture; induced cell-cycle arrest at G1 phase; reduced clonogenic activities but had no effect on apoptosis. Overall engraftment into NOD/SCID mice were not affected but the engrafting myeloid populations were reduced. In acute lymphoblastic leukemia, SOX7 was robustly expressed, compared with that in normal UCB and acute myeloid leukemia (AML). In 5 ALL patients in whom the coding sequence of SOX7 was examined, 3 of them showed mutations (amino acid change) in the SOX C-terminal transactivation domain. No mutation was observed in the β-catenin binding site. Knockdown of SOX7 with specific siRNA significantly increased appoptosis and decreased cell proliferation. SOX7 knockdown by shRNA in a precursor B-cell ALL cell line Nalm20 significantly reduced its engraftment into NOD/SCID mice. In summary, SOX7 is preferentially expressed in early hematopoietic stem and progenitor cells and is important for the maintenance of myeloid progenitor. It is also expressed in the primitive population of ALL and is important for leukemia initiation in ALL. The present study has generated important information about the regulation of normal hematopoiesis and acute lymphoblastic leukemia / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

No description available.

I have studied the calpain system in acute myeloid leukemia using the 32D and 32Dkit cell lines. Specifically, I characterized the calpain system in the cell lines, and performed calpastatin overexpression and knockdown studies. I found that calpain activity is elevated in the 32D and 32Dkit cells, and calpain inhibition causes apoptosis. Both μ- and m-calpain contribute to the calpain activity in these cell lines. The 32Dkit cells have higher calpain activity than the 32D cells, which I have shown is partially attributed to basal ckit activation. Calpastatin was present in both cell lines, but exists mainly in a degraded form. Calpastatin overexpression lowered calpain activity and provided a growth disadvantage to the 32Dkit cells, but had no effect on 32D cells. Calpastatin knockdown caused a significant increase in calpain activity in the 32D cells, which changed the cell cycle distribution but had no other major effects.

Acute Lymphoblastic Leukemia (ALL) is the most common cancer in children under the age of 15. At present, diagnosis, prognosis and treatment decisions are made based upon blood and bone marrow laboratory testing. With advances in microarray technology it is becoming more feasible to perform genetic assessment of individual patients as well. We used Singular Value Decomposition (SVD) on Illumina SNP, Affymetrix and cDNA gene-expression data and performed aggressive attribute se- lection using random forests to reduce the number of attributes to a manageable size. We then explored clustering and prediction of patient-specific properties such as disease sub-classification, and especially clinical outcome. We determined that integrating multiple types of data can provide more meaningful information than individual datasets, if combined properly. This method is able to capture the cor- relation between the attributes. The most striking result is an apparent connection between genetic background and patient mortality under existing treatment regimes. We find that we can cluster well using the mortality label of the patients. Also, using a Support Vector Machine (SVM) we can predict clinical outcome with high accu-racy. This thesis will discuss the data-mining methods used and their application to biomedical research, as well as our results and how this will affect the diagnosis and treatment of ALL in the future. / Thesis (Master, Computing) -- Queen's University, 2010-01-12 18:40:44.2

Haematopoiesis requires strict regulation in order to maintain a balanced production of the various blood cell components. Escape from this regulation contributes to the development of cancers such as leukemia. SOCS2 is a member of the Suppressor of cytokine signalling (SOCS) family, and normally functions as a negative regulator of the JAK/STAT pathway. I examined gene expression and promoter methylation in acute myeloid leukemia (AML) cell lines and patient samples. SOCS2 expression was quite variable in AML patients, and very low in acute promyelocytic leukemia (APL) patients. Promoter hyper-methylation was found in these patients, particularly those with high white blood cell count and a FLT3-ITD. I speculate that SOCS2 interacts with an aspect of the signalling complex to inhibit cell growth in these patients, and silencing SOCS2 is necessary for leukemia progression. Treating these patients with a de-methylating agent, such as decitabine, may show promise in the clinic.