Chez la souris, le Leukemia Inhibitory Factor (LIF) joue un rôle clé dans le maintien des cellules souches embryonnaires (ES) à l’état pluripotent. Le LIF agit en activant le facteur de transcription STAT3 via les kinases Jak. Cette activation est nécessaire et suffisante au maintien des cellules ES en autorenouvellement en présence de sérum. Une étude du transcriptome de STAT3 réalisée au laboratoire a permis d’identifier plusieurs gènes cibles de ce facteur, parmi lesquels plusieurs gènes inconnus. L’un d’eux, le gène 1456160_at, est fortement exprimé dans les cellules ES de souris et son expression diminue après induction de la différenciation. Ce gène a été appelé Asgard pour Another Self-renewal GuARDian. La caractérisation et le séquençage de ce gène ont permis de mettre en évidence qu'Asgard code pour un microARN. De nombreux microARNs jouent un rôle clé dans le maintien de l'autorenouvellement des cellules ES et dans le contrôle de la différenciation. Des expériences d’inhibition et de surexpression ont permis de montrer que Asgard est impliqué dans la régulation de la différenciation endoderme versus mésoderme. Des analyses préliminaires ont permis d’identifier Pbx3, FoxA2 et Sox17 comme cibles potentielles. Bien que les mécanismes d’action du microARN Asgard restent à confirmer, ce travail a permis d’identifier un nouveau gène clé de l'autorenouvellement des cellules ES de souris / The Leukemia Inhibitory Factor (LIF) activates the transcription factor STAT3, which results in the maintenance of mouse embryonic stem cells in the undifferentiated state by inhibiting mesodermal and endodermal differentiation. We identified several target genes of STAT3 by transcriptomic analysis. Among them, we focused on an unknown gene referred as 1456160_at on Affymetrix array. This gene is highly expressed in embryonic stem cells and its expression level decreases during differentiation. We named this gene Asgard for Another Self-renewal GuARDian. Its characterization and sequencing revealed that Asgard encodes for a microRNA sequence. Several microRNAs have been shown to play key role in the maintenance of self-renewal of mouse ES cells and in the control of differentiation. Inhibition and overexpression assays showed that Asgard inhibits endodermal differentiation in order to maintain self-renewal. Through preliminary analysis, we identified Pbx3, FoxA2 and Sox17 as potential targets of the microRNA Asgard. Our work enables us to identify a new key gene of self-renewal of mouse ES cells

Indiana University-Purdue University Indianapolis (IUPUI) / The development and function of hematopoietic stem cells (HSCs) is regulated by numerous signaling pathways including Akt-mechanistic target of rapamycin complex1 (mTORC1) pathway. Dysregulation of this pathway results in impaired HSC function and contributes to the development of hematologic malignancies. Activated mTORC1 phosphorylates and subsequently activates ribosomal protein S6 kinase 1 (S6K1). To study the role of S6K1 in hematopoiesis as well as leukemogenesis, we used a genetic model of S6K1 deficient mice (S6K1-/-). We found that loss of S6K1 expression in HSCs results in reduction of absolute HSC number in bone marrow (BM). Following chemotherapy, cycling HSCs undergo apoptosis and quiescent HSCs are required to cycle to regenerate the hematopoietic system. S6K1 regulates the quiescence of HSCs and in the absence of S6K1, mice are more susceptible to repeated myeloablative stress. We also observed that loss of expression as well as gain of expression of S6K1 affects the self-renewal ability of HSCs. Interestingly, when we overexpressed S6K1, it also resulted in reduced self-renewal of HSCs. Next, we assessed the role of S6K1 in the propagation of acute myeloid leukemia (AML). The mixed-lineage leukemia (MLL) gene is required for the maintenance of adult HSCs. Translocations in MLL are detected in approximately 5-10% of adult acute leukemia patients and in approximately 70% of acute leukemias in infants. We expressed MLL-AF9 fusion oncoprotein in WT and S6K1-/- hematopoietic stem and progenitor cells (HSC/Ps) and performed serial transplantation. Upon secondary transplantation, recipients of S6K1 deficient AML cells survived significantly longer compared to controls. In vitro, pharmacological inhibition of S6K1 activity resulted in reduced growth of primary human cells expressing MLL-AF9. Both human and murine HSC/Ps expressing MLL-AF9 showed reduced mTORC1 activity upon inhibition of S6K1 suggesting that loss of S6K1 activity results in reduced Akt-mTORC1 activation both upstream and downstream of mTORC1. Overall, our studies establish a critical role of S6K1 activity in the maintenance of HSC function and in the propagation of leukemia.

Like so many aspects of South African society, teacher education is undergoing change. The researcher studied existing school-based in-service education and training (SBINSET) programmes in the Northern Province and to improve the programmes, proposes Continuous Self-Renewal School-Based INSET (CSR SB INSET). Based on the extensive literature survey, strategies that would improve effective educational transformation are identified. Different concepts of INSET are explained. Components of teacher education are examined as are those factors that influence educational transformation. Advantages and limitations are identified and guidance on how to overcome these are suggested. The aim of this study was to examine INSET towards educational transformation with reference to primary schools in the Northern Province in order to introduce and assess a CSR SBINSET. The study examines the existing SBINSET programmes which are run in the Northern Province. The importance of teachers' continued professional growth is emphasised throughout this study. The researcher views systematic in-service training programmes for teachers as an educational necessity for the continuing professional growth of primary school teachers. An empirical survey has been undertaken to ascertain the effectiveness of CSR SBINSET and the type of delivery strategy that may be implemented.The research considers particular aspects of classroom practice addressed in the programme. The research was guided by the following questions: + Which methods do teachers use in their subject teaching? + What are their approaches to learners and learning? + How do teachers reflect on their teaching? + What strategies do teachers use? This research aims to improve existing SBINSET programmes run in the Northern Province inter alia by observing practising teachers. CSR SBINSET attempts to improve the competencies teachers, lecturers and District Officials, through such means as the presentation of centre-based, school-based and cluster-based workshops. Although the educational authorities should take initiative to provide educational transformation, the following share responsibility in this vital aspect of education: + those in the Department of Education who are responsible for planning; + superintendents of education within districts and regions; + training institutions such as universities, technikons, colleges and private agencies. The importance of and necessity for, continuous professional development of primary school teachers by means of SBINSET programmes, is emphasised. / Educational Studies / D. Ed. (Didactics)

No description available.

The phosphatidylinositol 3' kinase (PI3K) pathway is involved in many cellular processes including cell proliferation, survival, and glucose transport, and is implicated in various disease states such as cancer and diabetes. Though there have been numerous studies dissecting the role of PI3K signaling in different cell types and disease models, the mechanism by which PI3K signaling regulates embryonic stem (ES) cell fate remains unclear. It is believed that in addition to proliferation and tumorigenicity, PI3K activity might also be important for self-renewal of ES cells. Paling et al. (2004) reported that the inhibition of PI3K led to a reduction in the ability of leukemia inhibitory factor (LIF) to maintain self-renewal causing cells to differentiate. Studies in our lab have revealed that ES cells completely lacking GSK-3 remain undifferentiated compared to wildtype ES cells. GSK-3 is negatively regulated by PI3K suggesting that PI3K may play a vital role in maintaining pluripotency in ES cells through GSK-3. By using a modified Flp recombinase system, we expressed activated alleles of PDK-1 and PKB to create stable, isogenic ES cell lines to further study the role of the PI3K signaling pathway in stem cell fate determination. In vitro characterization of the transgenic cell lines revealed a strong tendency towards maintenance of pluripotency, and this phenotype was found to be independent of canonical Wnt signal transduction. To assess growth and differentiation capacity in vivo, the ES cell lines were grown as subcutaneous teratomas. The constitutively active PDK-1 and PKB ES cell lines were able to form all three germ layers when grown in this manner – in contrast to ES cells engineered to lack GSK-3. The resulting PI3K pathway activated cells exhibited a higher growth rate which resulted in large teratomas. In summary, PI3K signaling is sufficient to maintain self-renewal and survival of stem cells. Since this pathway is frequently mutationally activated in cancers, its effect on suppressing differentiation may contribute to its oncogenicity.

The phosphatidylinositol 3' kinase (PI3K) pathway is involved in many cellular processes including cell proliferation, survival, and glucose transport, and is implicated in various disease states such as cancer and diabetes. Though there have been numerous studies dissecting the role of PI3K signaling in different cell types and disease models, the mechanism by which PI3K signaling regulates embryonic stem (ES) cell fate remains unclear. It is believed that in addition to proliferation and tumorigenicity, PI3K activity might also be important for self-renewal of ES cells. Paling et al. (2004) reported that the inhibition of PI3K led to a reduction in the ability of leukemia inhibitory factor (LIF) to maintain self-renewal causing cells to differentiate. Studies in our lab have revealed that ES cells completely lacking GSK-3 remain undifferentiated compared to wildtype ES cells. GSK-3 is negatively regulated by PI3K suggesting that PI3K may play a vital role in maintaining pluripotency in ES cells through GSK-3. By using a modified Flp recombinase system, we expressed activated alleles of PDK-1 and PKB to create stable, isogenic ES cell lines to further study the role of the PI3K signaling pathway in stem cell fate determination. In vitro characterization of the transgenic cell lines revealed a strong tendency towards maintenance of pluripotency, and this phenotype was found to be independent of canonical Wnt signal transduction. To assess growth and differentiation capacity in vivo, the ES cell lines were grown as subcutaneous teratomas. The constitutively active PDK-1 and PKB ES cell lines were able to form all three germ layers when grown in this manner – in contrast to ES cells engineered to lack GSK-3. The resulting PI3K pathway activated cells exhibited a higher growth rate which resulted in large teratomas. In summary, PI3K signaling is sufficient to maintain self-renewal and survival of stem cells. Since this pathway is frequently mutationally activated in cancers, its effect on suppressing differentiation may contribute to its oncogenicity.

No description available.

Like so many aspects of South African society, teacher education is undergoing change. The researcher studied existing school-based in-service education and training (SBINSET) programmes in the Northern Province and to improve the programmes, proposes Continuous Self-Renewal School-Based INSET (CSR SB INSET). Based on the extensive literature survey, strategies that would improve effective educational transformation are identified. Different concepts of INSET are explained. Components of teacher education are examined as are those factors that influence educational transformation. Advantages and limitations are identified and guidance on how to overcome these are suggested. The aim of this study was to examine INSET towards educational transformation with reference to primary schools in the Northern Province in order to introduce and assess a CSR SBINSET. The study examines the existing SBINSET programmes which are run in the Northern Province. The importance of teachers' continued professional growth is emphasised throughout this study. The researcher views systematic in-service training programmes for teachers as an educational necessity for the continuing professional growth of primary school teachers. An empirical survey has been undertaken to ascertain the effectiveness of CSR SBINSET and the type of delivery strategy that may be implemented.The research considers particular aspects of classroom practice addressed in the programme. The research was guided by the following questions: + Which methods do teachers use in their subject teaching? + What are their approaches to learners and learning? + How do teachers reflect on their teaching? + What strategies do teachers use? This research aims to improve existing SBINSET programmes run in the Northern Province inter alia by observing practising teachers. CSR SBINSET attempts to improve the competencies teachers, lecturers and District Officials, through such means as the presentation of centre-based, school-based and cluster-based workshops. Although the educational authorities should take initiative to provide educational transformation, the following share responsibility in this vital aspect of education: + those in the Department of Education who are responsible for planning; + superintendents of education within districts and regions; + training institutions such as universities, technikons, colleges and private agencies. The importance of and necessity for, continuous professional development of primary school teachers by means of SBINSET programmes, is emphasised. / Educational Studies / D. Ed. (Didactics)

(ENGLISH) Hematopoiesis is dependent on the actions of hematopoietic stem cells (HSCs). This process is tightly controlled through a complex array of extrinsic and intrinsic factors. Even though the hematopoiesis seems to be well conserved across the disparate vertebrate animals, erythroid and thrombocytic differentiation have changed during the evolution of mammals. Specifically, adult mammalian red blood cells have the unique feature of being enucleated, and mammalian thrombocytes are not individual cells, but fragments of megakaryocytes, instead. It is likely that these enhancements provided a survival advantage to early mammalian species; however, they also bring up the question of evolutionary origin of these cells that studied using zebrafish (Danio rerio) model. First, it was necessary to generate a toolbox of a recombinant cytokines and optimized culture media that allowed us to manipulate zebrafish hematopoietic cells ex vivo in liquid and clonal cultures. Interestingly, teleost species underwent an extra duplication event during their evolution and as a result, two copies (paralogs) of some of the genes are present in zebrafish. This was also the case for majority of the cytokines from our toolbox and here, we provide functional characterization of these paralogs. Strikingly, our results...

Organoide aus adulten Mäusen sind vielversprechende Modelle für die Nierenforschung. Ihre Charakterisierung wurde jedoch nicht auf ein zufriedenstellendes Niveau gebracht. Hier habe ich ein langfristiges 3D-Maus-Organoid (Tubuloid)-Modell etabliert und charakterisiert, das die Erneuerung und die Reparatur sowie die Architektur und die Funktionalität der adulten tubulären Epithelien rekapituliert. In der Zukunft wird das Modell detaillierte Untersuchungen der Trajektorien selbsterneuernder Zellen sowohl zur teilweisen Wiederherstellung der Niere als auch zur malignen Transformation der Niere ermöglichen. Das klarzellige Nierenzellkarzinom (ccRCC) ist der häufigste und aggressivste Nierenkrebs. Die Inaktivierung des Tumorsuppressorgens Von Hippel-Lindau (VHL) ist der Haupttreiber des ccRCCs. Zuvor hatten wir die Hochregulation der Wnt- und Notch-Signalübertragung in den CXCR4+MET+CD44+-Krebsstammzellen (CSC) aus primären humanen ccRCC-Tumoren identifiziert. Das Blockieren von Wnt und Notch in von Patienten stammenden Xenotransplantaten, Organoiden und nicht-anhaftenden Sphären unter Verwendung von niedermolekularen Inhibitoren beeinträchtigte die Selbsterneuerung der CSC und das Tumorwachstum. Um CSC-gesteuertes humanes ccRCC in genetischen Mausmodellen nachzuahmen, begann ich mit der Erzeugung von zwei Doppelmausmutanten; β-Catenin-GOF; Notch-GOF und Vhl-LOF; β-Catenin-GOF. Sowohl die β-Catenin-GOF; Notch-GOF Mausmutante als auch die Vhl-LOF; β-Catenin-GOF Mausmutante entwickelten innerhalb einiger Monate schwere Krankheitssymptome. Überraschenderweise beobachtete ich weder Tumore oder Tumorvorläuferläsionen noch höhere Zellproliferationsraten in den mutierten Nieren. Weitere Analysen ergaben, dass die Mausmutanten Merkmale chronischer Nierenerkrankung (CKD) aufwiesen. / Adult mouse organoids are promising models for kidney research. However, their characterization has not been pushed forward to a satisfying level. Here, I have generated and characterized a long-term 3D mouse organoid (tubuloid) model, which recapitulates renewal and repair, and the architecture and functionality of the adult tubular epithelia. In the future, the model will allow detailed investigations of trajectories of self-renewing cells towards both the partial recreation and malignant transformation of the kidney. Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive kidney cancer. Inactivation of the Von Hippel-Lindau (VHL) tumor suppressor gene is the major driver of ccRCC. Earlier, we identified the upregulation of Wnt and Notch signaling in CXCR4+MET+CD44+ cancer stem cells (CSCs) from primary human ccRCCs. Blocking Wnt and Notch in patient-derived xenografts, organoids and non-adherent spheres using small-molecule inhibitors impaired self-renewal of CSCs and tumor growth. To mimic CSC-governed human ccRCC in genetic mouse models, I started from the generation of two double mouse mutants; β-catenin-GOF; Notch-GOF and Vhl-LOF; β-catenin-GOF. Surprizingly, I observed neither tumors or tumor precursor lesions nor higher cell proliferation rates in the mutant kidneys. Further analyses revealed that the mutant mice displayed features of chronic kidney disease (CKD). Thus, β-catenin-GOF; Notch-GOF and Vhl-LOF; β-catenin-GOF mouse mutants did not develop kidney tumors under the given experimental conditions.