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Development of recombinant proteins for selection, immobilization and expansion of stem cellsXu, Yin, School of Biotechnology & Biomolecular Science, UNSW January 2007 (has links)
Cellulose binding domains (CBDs) are found in cellulolytic microorganisms as discrete domains in free cellulases or as cellulosomes, which are extracellular multi-enzyme complexes. CBDs bind to cellulose and help the catalytic domains to access cellulose substrates. CBDs are used as affinity tags for immobilizing cells, proteins or molecules on cellulose matrices. They can also be used in protein engineering to alter protein expression and solubilities. Cohesins and dockerins are domains exclusive to cellulosomes. They interact with high affinity and the interactions are Ca2+-dependent. Chelation of Ca2+ causes irreversible conformational change to dockerins thus disrupting the interactions. The first aim of this project was to validate a putative CBD from endoglucanase EngD of C. cellulovorans, to test its effect on solubility as a fusion in chimeras. The second aim was to use chimeric proteins containing CBD, cohesin, dockerin and LG to establish a system for efficient cell immobilization, expansion and harvest in hollow cellulose fibres. A putative CBD from an enzyme with its natural linker (PTCBDengD), a CBD from a scaffoldin (CBDcbpA), three cohesin domains from different strains (Cip7, Coh6 and CipC1) and an antibody binding protein (LG) were used to construct various chimeric and fusion proteins. The two CBDs were fused to different cohesins and LG respectively and the chimeras? solubility was analyzed. The results showed that fusing with CBDcbpA did not significantly help to increase the solubility of the insoluble domain Coh6 and it even greatly reduced the solubility of the soluble domain CipC1. In contrast, PTCBDengD fusion increased the solubility of Coh6 by three fold and it did not alter the solubility of soluble protein/ domains. These results suggested that PTCBDengD may be a better domain to use as a fusion tag for expression and other biotechnology applications. Cellulose binding specificity of PTCBDengD and its chimeric proteins were tested and SDS-PAGE analysis results clearly demonstrated that PTCBDengD and its chimeras specifically bound to crystalline cellulose Avicel and non-crystalline cellulose Cuprophan. The results confirmed that PTCBDengD is a true CBD. Chimeric protein pairs CBDcbpA-Cip7/ LG-Doc and Cip7-PTCBDengD/ LG-Doc were used to build the scaffold on Cuprophan hollow cellulose fibre for reversible cell immobilization studies. Cell adhesion assay results showed that the double-chimera systems efficiently immobilize cells onto Cuprophan hollow fibre. Dissociation of LG-Doc from amorphous cellulose Cuprophan-bound CBDcbpA-Cip7 by EDTA treatment resulted in decrease of cell binding by almost 90%; however, re-association of LG-Doc after EDTA dissociation only achieved 50% efficiency of cell immobilization. Dot blot and SDS-PAGE analysis showed that dissociation/ re-association of LG-Doc to Cip7-PTCBDengD could be decreased in was interfered by the presence of cellulose. Preliminary results indicated that crystalline cellulose Avicel may improve dissociation/ re-association efficiency. In conclusion, studies on recombinant proteins validated CBDengD's specific affinity to cellulose and its solubilizing effect on its fusion partner. Chimera pairs CBDcbpA-Cip7/ LG-Doc and Cip7-PTCBDengD/ LG-Doc are effective in cell immobilization. However, optimization is required to develop recyclable protein scaffolding and complexes on cellulosic matrices.
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Functional studies of transcription factors GATA-1, Fli-1 and FOG-1 in Megakaryocyte development.Pan, Shu, St. George Clinical School, UNSW January 2007 (has links)
Transcription factors GATA-1, Fli-1 and FOG-1 are essential proteins for normal megakaryopoiesis, however, the detailed analyses of their functions within developmental stages of megakaryopoiesis are lacking. In my thesis, over expression of gene in target cells was adopted as the main strategy to study the biological functions of these proteins, therefore, an efficient gene delivery method was first developed by using retrovirus.This approach was then utilized to over express GATA-1, Fli-1 and FOG-1 in murine leukemia M1 cells and mouse hematopoietic stem cells (HSCs), and their effects on different developmental stages of megakaryopoiesis were investigated. In the transduced M1 cells, enforced expression of GATA-1 and Fli-1 was found to induce the megakaryocytic development, which was associated with the formation of megakaryocyte (Mk) and the increased expression of Mk specific genes c-Mpl and GPIX. In the transduced mouse HSCs, it was found that the expression of endogenous GATA-1, Fli-1 and FOG-1 was up-regulated throughout Mk differentiation; enforced expression of these transcription factors led to the significantly enhanced Mk development. Megakaryocytes over expressing GATA-1, Fli-1 and FOG-1 were characterized by the increased expression of various Mk-specific genes including GPIX, c-Mpl, platelet factor 4 (PF4), acetylcholinesterase (AChE) and NF-E2, an important transcription factor for terminal megakaryopoiesis; however, GATA-1, Fli-1 and FOG-1 displayed the different abilities in promoting the proliferation of hematopoietic cells and MK differentiation, as well as regulating other transcription factors involved in hematopoiesis. To further elucidate the role of the functional domains of Fli-1, various mutants of Fli-1 were also over expressed in mouse HSCs. The results demonstrated that first, the combination of the activation domain of Fli-1 and its Ets domain is required for early megakaryopoiesis but not sufficient for terminal megakaryopoiesis; second, DNA binding of Fli-1 was not the only requirement for early Mk enhancement, moreover, the interaction between Fli-1 and GATA-1 through the Ets domain and the resultant transcriptional synergy was the essential determinant for Fli?1 ability in Mk development. Taken together, the studies presented in this thesis provided strong in vitro evidence that GATA-1, Fli-1 and FOG-1 indeed play the critical roles in normal megakaryopoiesis.
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Embryonic stem cell research and the metaphysics of identityCopland, Paul S, n/a January 2007 (has links)
Embryonic stem cell research has the potential to revolutionise both the practice of medicine and our understanding of the human body. Although the usual technical and financial limitations of research apply, perhaps the greatest obstacle to the progress of this research at the present time is the ethical concerns surrounding the destruction of early human embryos. The established debate over the ethical significance of the early embryo has thus taken on renewed importance.
Within biology stem cell research has begun to overturn some long held assumptions about the roles of genes and cellular interaction in development. Building on recent advances in stem cell biology I develop a concept of Form that neatly captures what it is to be individuals like us in biological terms. Form not only defines a biological individual that exists across time regardless of changes in its physical constituents but also provides the biological foundation for our higher mental properties and our identity as persons.
At the heart of the embryo debate is confusion over what human individuals are and therefore when they began. Defining when we began as the ethically significant individuals that we are now is the key to the embryo debate. Our metaphysics of identity is thus crucial to understanding the moral significance of the embryo.
Compared to alternative understandings of identity within the debate surrounding the embryo Form provides compelling reasons why the very early embryo, at the stage that embryonic stem cells are derived, lacks any right to life or associated ethical significance. The derivation of embryonic stem cells is thus found to be ethically permissible.
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Histone gene "knock-out" in mouse embryonic stem cells / by Varaporn Thonglairoam.Thonglairoam, Varaporn January 1994 (has links)
Bibliography: leaves 113-126. / v, 126, [113] leaves, [10] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Studies the biological significance of the mouse Listone variant H2A.Z. Describes the isolation and characterisation of H2A.Z genomic clones from different mouse genomic libraries; H2A.Z gene targeting in mouse E14 embryonic stem cells; and an attempt to generatae ES cell lines and mice which lack the functional H2A.Z protein to investigate H2A.Z function in vitro and in vivo. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1995?
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Haemopoiesis, leukaemia & imatinib: c-fms, a novel target for small molecule inhibitor therapy.Dewar, Andrea L. January 2004 (has links)
Understanding the factors that regulate the growth and differentiation of haemopoietic stem cells (HSC) remains a major challenge. In this study, the proliferation and differentiation of CD34+ cells from normal donors and chronic myeloid leukaemia (CML) patients was compared. The proliferation and entry of CML cells into the cell cycle was decreased relative to cells from normal donors, and greater heterogeneity in the phenotype of CML cells at the initiation of culture was observed. Analysis of phenotype concomitant with cell division also demonstrated that the differentiation of normal CD34+ cells was consistent between donors, while marked variability was observed in the differentiation of CD34+ cells from CML patients. This included expression of CD13, CD33, CD38 and HLA-DR, which were linked to cell division in normal but not CML cells. The tyrosine kinase inhibitor, imatinib, is a novel drug displaying promising results in the treatment of CML by specifically inhibiting the growth of leukaemic cells. To examine whether myelosuppression observed in patients treated with imatinib may arise from inhibition of normal haemopoiesis, imatinib was added to colony assays established using cells from normal bone marrow. Suppression of monocyte/macrophage growth, but not that of eosinophils or neutrophils, was observed at therapeutic concentrations of imatinib. Inhibition of monocytic differentiation to macrophages was also observed and was associated with decreased functional capacity such as altered antigen uptake, production of proinflammatory cytokines and stimulation of responder cells. The specific suppression of monocyte/macrophage differentiation and function was not due to blockade of tyrosine kinases known to be inhibited by imatinib and was consistent with an inhibition of the M-CSF/c-fms signalling pathway. This hypothesis was tested using a cell line that was dependent on M-CSF for growth and survival. Cell proliferation and phosphorylation of c-fms were inhibited at an IC50 of 1.9μM and 1.4μM imatinib respectively and this was not attributable to decreased c-fms expression. These important findings therefore identify c-fms as a further target of imatinib, and suggest that imatinib should be considered for treatment of diseases where c-fms is implicated. This includes breast and ovarian cancer and inflammatory conditions such as rheumatoid arthritis. Potential side effects resulting from imatinib treatment must also be considered. / Thesis (Ph.D.)--School of Medicine, 2004.
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Effects of bacterial toxins on the proliferation, osteogenic differentiation and toll-like receptor expressions of human mesenchymal stromal cells /Mo, Fung-ying, Irene. January 2006 (has links)
Thesis (M. Phil.)--University of Hong Kong, 2007. / Also available online.
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Cell and gene therapies for diabetes exploration of novel therapeutic approaches /Li, Hua, January 2006 (has links)
Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.
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Effects of bacterial toxins on the proliferation, osteogenic differentiation and toll-like receptor expressions of human mesenchymal stromal cellsMo, Fung-ying, Irene. January 2006 (has links)
Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.
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Prion Protein is Expressed on Long-term Repopulating Hematopoietic Stem Cells and is Necessary for their Self-renewalLodish, Harvey F., Zhang, Cheng Cheng, Steele, Andrew D., Lindquist, Susan L. 01 1900 (has links)
We show that the prion protein (PrP) is expressed on the surface of bone marrow cell populations enriched in long-term repopulating hematopoietic stem cells. Affinity purification of the PrP-positive and PrP-negative fractions from these populations, followed by competitive reconstitution assays, show that all long-term repopulating hematopoietic stem cells express PrP. Hematopoietic stem cells from PrP null bone marrow exhibit impaired self-renewal in serial competitive transplantation experiments, and premature exhaustion when exposed to cell cycle-specific myelotoxic injury. Therefore, PrP is a novel marker for hematopoietic stem cells and regulates their self-renewal. / Singapore-MIT Alliance (SMA)
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The role of cultured chondrocytes and mesenchymal stem cells in the repair of acute articular cartilage injuriesSecretan, Charles Coleman 06 1900 (has links)
Osteoarthritis (OA) is a disease that has significant individual, social, and economic impact worldwide. Although many etiologies lead to the eventual development of OA, one potentially treatable cause is the acute articular cartilage (AC) injury. These injuries are common and have a poor inherent healing capacity, leading to the formation of OA. In an effort to repair AC injuries several treatment strategies have been developed but none have proven completely successful.
Studies examining AC tissue-engineering strategies have suggested that those with the most potential for success involve the introduction of autogenous or allogenous cells to the site of injury. These strategies are designed to encourage creation of a matrix with the appropriate characteristics of normal AC. However, development of a completely successful repair method has proven difficult because the biomechanical properties of normal AC are not easy to replicate, a cell source with the appropriate functional characteristics has not been optimized, and the problem of effective incorporation of a repair construct into the host tissue remains unresolved.
In an effort to more fully understand the cartilage repair process, this work first focused on the development and utilization of an in vitro human explant model of AC to study the ability of seeded human chondrocytes to integrate into an AC defect. Further work elucidated the gene expression patterns of cultured adult human chondrocytes and human mesenchymal stem cell (MSC)-derived chondrocytes.
Results from this work determined that cultured human chondrocytes were able to adhere to articular cartilage defects in a viable in vitro explant model and produce a matrix containing collagen type II. However, further work with the in vitro expanded chondrocytes revealed that these cells have increased expression of collagen type I which promotes the formation of a less durable fibrocartilagenous tissue. This unfavorable expression persisted despite placing the chondrocytes in an environment favoring a chondrocytic phenotype. Further work with MSC-derived chondrocytes demonstrated a similar and unfavorable production of collagen type I. This work represented an important first step towards a treatment for acute AC lesions but it is clear that further work to optimize the culture microenvironment is still required. / Experimental Surgery
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