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postgraduate thesis: Role of autophagy in vertebrate haematopoiesis and human myelodysplastic syndrome
Title | Role of autophagy in vertebrate haematopoiesis and human myelodysplastic syndrome |
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Authors | |
Issue Date | 2017 |
Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
Citation | Chan, K. [陳國熙]. (2017). Role of autophagy in vertebrate haematopoiesis and human myelodysplastic syndrome. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. |
Abstract | Autophagy is a conserved cellular mechanism essential for degrading expired cytoplasmic constituents and plays a pivotal role in haematopoiesis and haemic malignancies. It is putatively implicated in the pathogenesis of myelodysplastic syndrome because cellular sequelae of deranged autophagy were observed in the patients’ bone marrow cells.
It was found that Atg7 and FIP200, which mediate two different stages of autophagy, were required for haematopoietic stem cell maintenance and differentiation; conditional knockout of these two proteins induced anaemia and atypical myeloproliferation in murine models. However, in the zebrafish model, functional studies of autophagy-related genes are lacking and the precise role of autophagy in haematopoiesis is yet to be clarified.
This study examined the zebrafish homologues, atg7 and atg13, in the context of embryonic haematopoiesis. Spatial and temporal expression of atg7 and atg13 was determined by whole-mount in situ hybridisation and polymerase chain reaction. Knockdown of atg7 and atg13 was conducted by translation-blocking morpholino oligomers. Molecular targeting of the morpholino oligomers was validated by quenching the co-injected fusion fluorescent reporter plasmids. In phenotypic examination, no aberration of primitive and definitive haematopoiesis was induced by atg7-knockdown and atg13-knockdown.
Moreover, two biochemical assays of autophagy, LysoTracker and Autophagy Detection Kit, were evaluated on drug-treated and morphant embryos. LysoTracker staining of autophagy-suppressed embryos displayed significantly reduced fluorescent signals and demonstrated autophagy suppression in morphant embryos. However, Autophagy Detection Kit produced non-specific signals and thus was not recommended for assaying autophagy. Similarly, double-staining of wildtype embryos failed to produce satisfactory co-localisation signals under confocal microscopy.
This study suggests that transient autophagy suppression by morpholino knockdown does not readily affect embryonic haematopoiesis and LysoTracker is a satisfactory autophagy assay. To overcome the limitations encountered in this study, it warrants knockout models for evaluating the long-term effects of autophagy ablation and transgenic fluorescent reporter lines for monitoring autophagy activity in concert with LysoTracker.
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Degree | Master of Research in Medicine |
Subject | Hematopoiesis Myelodysplastic syndromes Autophagic vacuoles |
Dept/Program | Medicine |
Persistent Identifier | http://hdl.handle.net/10722/251321 |
DC Field | Value | Language |
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dc.contributor.author | Chan, Kwok-hei | - |
dc.contributor.author | 陳國熙 | - |
dc.date.accessioned | 2018-02-27T09:53:38Z | - |
dc.date.available | 2018-02-27T09:53:38Z | - |
dc.date.issued | 2017 | - |
dc.identifier.citation | Chan, K. [陳國熙]. (2017). Role of autophagy in vertebrate haematopoiesis and human myelodysplastic syndrome. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. | - |
dc.identifier.uri | http://hdl.handle.net/10722/251321 | - |
dc.description.abstract | Autophagy is a conserved cellular mechanism essential for degrading expired cytoplasmic constituents and plays a pivotal role in haematopoiesis and haemic malignancies. It is putatively implicated in the pathogenesis of myelodysplastic syndrome because cellular sequelae of deranged autophagy were observed in the patients’ bone marrow cells. It was found that Atg7 and FIP200, which mediate two different stages of autophagy, were required for haematopoietic stem cell maintenance and differentiation; conditional knockout of these two proteins induced anaemia and atypical myeloproliferation in murine models. However, in the zebrafish model, functional studies of autophagy-related genes are lacking and the precise role of autophagy in haematopoiesis is yet to be clarified. This study examined the zebrafish homologues, atg7 and atg13, in the context of embryonic haematopoiesis. Spatial and temporal expression of atg7 and atg13 was determined by whole-mount in situ hybridisation and polymerase chain reaction. Knockdown of atg7 and atg13 was conducted by translation-blocking morpholino oligomers. Molecular targeting of the morpholino oligomers was validated by quenching the co-injected fusion fluorescent reporter plasmids. In phenotypic examination, no aberration of primitive and definitive haematopoiesis was induced by atg7-knockdown and atg13-knockdown. Moreover, two biochemical assays of autophagy, LysoTracker and Autophagy Detection Kit, were evaluated on drug-treated and morphant embryos. LysoTracker staining of autophagy-suppressed embryos displayed significantly reduced fluorescent signals and demonstrated autophagy suppression in morphant embryos. However, Autophagy Detection Kit produced non-specific signals and thus was not recommended for assaying autophagy. Similarly, double-staining of wildtype embryos failed to produce satisfactory co-localisation signals under confocal microscopy. This study suggests that transient autophagy suppression by morpholino knockdown does not readily affect embryonic haematopoiesis and LysoTracker is a satisfactory autophagy assay. To overcome the limitations encountered in this study, it warrants knockout models for evaluating the long-term effects of autophagy ablation and transgenic fluorescent reporter lines for monitoring autophagy activity in concert with LysoTracker. | - |
dc.language | eng | - |
dc.publisher | The University of Hong Kong (Pokfulam, Hong Kong) | - |
dc.relation.ispartof | HKU Theses Online (HKUTO) | - |
dc.rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works. | - |
dc.rights | This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. | - |
dc.subject.lcsh | Hematopoiesis | - |
dc.subject.lcsh | Myelodysplastic syndromes | - |
dc.subject.lcsh | Autophagic vacuoles | - |
dc.title | Role of autophagy in vertebrate haematopoiesis and human myelodysplastic syndrome | - |
dc.type | PG_Thesis | - |
dc.description.thesisname | Master of Research in Medicine | - |
dc.description.thesislevel | Master | - |
dc.description.thesisdiscipline | Medicine | - |
dc.description.nature | published_or_final_version | - |
dc.identifier.doi | 10.5353/th_991043983797003414 | - |
dc.date.hkucongregation | 2017 | - |
dc.identifier.mmsid | 991043983797003414 | - |