SACM - United Kingdom
Permanent URI for this collectionhttps://drepo.sdl.edu.sa/handle/20.500.14154/9667
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Item Restricted Investigating the role of Gfi1s/1b transcription factors in the HSC-derived thrombocyte lineage(University of Nottingham, 2023-03-27) Jaddah, Mariam; Gering, MartinBlood formation, haematopoiesis, during development occurs in multiple waves, and it starts from mesodermal cells which give rise to primitive wave blood cells and endothelial cells (ECs) which become hemogenic endothelium (HE). Haematopoietic stem cells (HSCs) emerge from HE in the ventral wall of the dorsal aorta (vDA). Growth factor independence 1aa (gfi1aa) is expressed in HE, and in our lab, we found that HSC formation is not affected in the gfi1aa mutant. However, there was upregulation of its paralogue expression, gfi1ab, in the ventral wall of the dorsal aorta (vDA) in the absence of Gfi1aa, suggesting a functional redundancy between Gfi1aa and Gfi1ab. Here, zebrafish embryos that were double homozygous for the gfi1aa and gfi1ab mutants were used to determine whether HSC emergence is affected in the absence of both proteins. Whole mount in situ hybridisation experiments revealed that haematopoietic stem cells (HSCs) were still able to emerge from the ventral wall of the dorsal aorta (vDA), but the number of definitive blood cells that seed the subsequent haematopoietic tissues was severely reduced. The reduction in Hematopoietic stem and progenitor cells (HSPCs) was confirmed using the CD41: GFP+ line which expresses low levels of GFP in definitive progenitors and increasing levels as they turn into thrombocytes. Transgenic embryos that were homozygous for mutant gfi1aa and gfi1ab were found to carry fewer GFP+ cells, suggesting a lower number of emerging hematopoietic stem and progenitor cells (HSPCs). In the gfi1aa single mutant and gfi1aa/ab double mutant, gfi1b was not expressed in the vDA of these embryos at any point during the emergence of hematopoietic stem and progenitor cells (HSPCs), unlike gfi1ab. Further loss of Gfi1b did not abrogate definitive HSPCs formation but expanded the myb+ blood cell population in the caudal hematopoietic tissues, CHT, when depleted in combination with Gfi1aa. In addition, using the CD41: GFP+ line with the third homologs mutant embryos, gfi1b, thrombocytes failed to enter circulation. Data from the adult kidney marrow of the gfi1b mutant showed that the fraction of CD41+ cells was significantly increased, while peripheral blood examination revealed thrombocytopenia. These data suggest that thrombocytes are held back into the kidney marrow. Further analysis of kidney marrow (KM) using gata1 and cd41 double transgenic fish revealed the accumulation of transcriptionally misprogrammed mature thrombocytes. In addition, thrombocytes in embryos and adults were functionally impaired in gfi1b qmc554 hom mutant carriers. To gain a deeper understanding of the molecular programming of Gfi1b-depleted thrombocytes, bulk RNA-sequencing analysis was performed. RNA-Seq data have shown upregulation of adhesion, endothelial, and globin expression genes and downregulation of cytoskeleton gene expression in Gfi1b depleted thrombocytes and thrombocyte progenitors.15 0Item Restricted Exploring the Role of Prostaglandin G/H Synthase Signalling in Chemoresistance in Acute Myeloid Leukaemia(University of Liverpool, 2024-03-05) Alshammari, Abdullah Mohammad D; Woolley, John; MacEwan, DaveAntioxidant signalling is demonstrated to be important for leukemic stem cell (LSC) and haematopoietic stem cell (HSC) function. HSCs, which are found in the bone marrow, are found to be more quiescent under hypoxic conditions. Reduced cell division creates problems for these cells, with the majority of cells using this process to address oxidative damaged DNA and proteins. In this situation, HSCs must approach oxidative damage management in other ways. Prostaglandins improve mechanisms to protect from oxidative stresses through causing reductions in reactive oxygen species (ROS). Prostaglandin G/H synthases (PTGS1/2) play the main role in catalysing synthesis of prostaglandins. Research has demonstrated that PGE2, produced through PTGS1/2 activity, assists HSCs in surviving, proliferating and homing within their niche. Similarly, the data provided in this study points to a protective function of PTGS1/2 signal pathways for acute myeloid leukaemia. PTGS genes were investigated for their association with outcomes in AML, in an approach combining biochemistry, bioinformatics, molecular biology and cellular biology. Analysis of bioinformatics databases aimed to assess how PTGS1/2 was expressed in AML and the effects of this expression on outcomes including overall survival. An efficacy assessment was made for PTGS1 inhibitors (SC560, Tenidap) for AML cell lines. Flow cytometry was used to evaluate the cell cycle and apoptosis. Lentiviral PTGS1/2 over-expression was used for U937 and HL-60 in order to assess how these genes act in cell survival, proliferative activity and resistance to drugs. Finally, PTGS1/2 was analysed in terms of promotion of immunosuppression in acute myeloid leukaemia. Investigation of freely accessible bioinformatics databases demonstrated increased PTGS1 expression in the HSC, which reduced in cells committed to a lineage for myeloid progenitor cells. This was not found for PTGS2. There is a notable association between increased expression of PTGS1 and lower overall survival in data on AML (TCGA, Verhaak), with none seen for PTGS2. Findings in vitro show that inhibiting PTGS1 (SC560, Tenidap) leads to decreased growth of cells, arrests the cell cycle and elevates apoptosis. Overexpressed PTGS1 leads to higher WNT signalling for AML cell lines, as well as raising PGE2 secretions and reversing PTGS1 inhibitor impacts. Other effects of increased PTGS1 expression include resistance to cytarabine, as linked with lower generation of ROS. It is notable that overexpression of PTGS1 and PTGS2 lead to significant differences in transcriptome alterations in AML, which could explain the varied patient outcomes with PTGS1/2 overexpression.18 0