1,720,998 research outputs found
Decoding the Regulatory Landscape of Ageing in Musculoskeletal Engineered Tissues Using Genome-Wide DNA Methylation and RNASeq
Get PDFMesenchymal stem cells (MSC) are capable of multipotent differentiation into connective tissues and as such are an attractive source for autologous cell-based regenerative medicine and tissue engineering. Epigenetic mechanisms, like DNA methylation, contribute to the changes in gene expression in ageing. However there was a lack of sufficient knowledge of the role that differential methylation plays during chondrogenic, osteogenic and tenogenic differentiation from ageing MSCs. This study undertook genome level determination of the effects of DNA methylation on expression in engineered tissues from chronologically aged MSCs. We compiled unique DNA methylation signatures from chondrogenic, osteogenic, and tenogenic engineered tissues derived from young; n = 4 (21.8 years ± 2.4 SD) and old; n = 4 (65.5 years±8.3SD) human MSCs donors using the Illumina HumanMethylation 450 Beadchip arrays and compared these to gene expression by RNA sequencing. Unique and common signatures of global DNA methylation were identified. There were 201, 67 and 32 chondrogenic, osteogenic and tenogenic age-related DE protein-coding genes respectively. Findings inferred the nature of the transcript networks was predominantly for ‘cell death and survival’, ‘cell morphology’, and ‘cell growth and proliferation’. Further studies are required to validate if this gene expression effect translates to cell events. Alternative splicing (AS) was dysregulated in ageing with 119, 21 and 9 differential splicing events identified in chondrogenic, osteogenic and tenogenic respectively, and enrichment in genes associated principally with metabolic processes. Gene ontology analysis of differentially methylated loci indicated age-related enrichment for all engineered tissue types in ‘skeletal system morphogenesis’, ‘regulation of cell proliferation’ and ‘regulation of transcription’ suggesting that dynamic epigenetic modifications may occur in genes associated with shared and distinct pathways dependent upon engineered tissue type. An altered phenotype in engineered tissues was observed with ageing at numerous levels. These changes represent novel insights into the ageing process, with implications for stem cell therapies in older patients. In addition we have identified a number of tissue-dependant pathways, which warrant further studies
Deregulation of DNA hydroxymthylases Tet1 and Tet2 compromises skeletal integrity during ageing
No full textEpigenetic modifications have recently been implicated in the development of osteoporosis, particularly Type II osteoporosis associated with ageing. While few genetic links have been reported to date, a host of possible epigenetic and metabolic factors potentially play a role in these bone diseases. One such family of factors is the Ten-Eleven Translocases (TET), a family of DNA hydroxymethylases, have been linked to bone development, with TET1 and 2 proposed to be key factors for both Bone Marrow Stromal Cell (BMSC) replenishment and for driving differentiation of these BMSC into mature functional stromal cell populations. This thesis investigates the effects of Tet1 and Tet2 deregulation on bone development and age-related bone loss, with respect to BMSC proliferation and differentiation. The study uncovered significant reductions to trabecular structure and volume via micro-CT analysis in males at 12- and 52-weeks of age, as well as impaired bone formation rate via histomorphometry. Furthermore, TetDKO animals exhibited reduced osteoblast numbers on the bone front, as well as increased levels of bone marrow adipocytes. Analysis of BMSC in vitro found significant reduction in osteogenesis in Tet1 and 2 double knockout (TetDKO) BMSC, with a shift towards adipogenesis. Changes to proliferation and senescence properties were also observed, with 12-week TetDKO exhibiting increased proliferative potential, with reversal at 52-week where reduced proliferation was observed. RNA sequencing of BMSC of male mice uncovered several differentially expressed genes, and an array of significantly enriched gene set pathways. Notably, factors Pappa2 and Masp1 involved in the bioavailability and regulation of IGF-1 signalling were significantly differentially regulated, leading to reduction in IGF-1 bioavailability in BMSC and differentiated osteoblasts. The results show for the first time, Tet1 and Tet2 regulation of IGF-1 regulatory elements dictates BMSC processes, where deregulation of Tet1 and Tet2 can disrupt this process leading to enhanced bone loss in ageing.Thesis (Ph.D.) -- University of Adelaide, School of Biomedicine, 202
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Stromal precursor cells : purification and the development of bone tissue / Stan Gronthos.
Get PDFBibliography: leaves 152-223.xxiii, 223, [137] leaves, [27] leaves of plates : ill. (chiefly col.) ; 30 cm.Experiments were designed to identify and purify human bone marrow stromal precursor cells by positive immunoselection, based on the cell surface expression of the VCAM-1 and STRO-1 antigens. The data presented demonstrates a hierarchy of bone cell development in vitro.Thesis (Ph.D.)--University of Adelaide, Dept. of Orthopaedics Surgery and Trauma, 199
Reprogramming of human gingival and periodontal ligament fibroblasts to pluripotency with defined factors.
Get PDFBackground: The use of periodontal stem cells with tissue engineering techniques constitutes an attractive strategy for regenerative periodontal therapy. However, technical difficulties of isolating a large quantity of these cells and problem of immune rejection in allogeneic transplantation limit their dental clinical usefulness. Recently, several groups have successfully reprogrammed adult cells to pluripotent cells by retroviral transduction with four genes - Oct3/4, Sox2, Klf4 and cMyc. The resultant induced pluripotent stem (iPS) cells have proliferative and developmental potentials comparable to those of embryonic stem (ES) cells. These cells may represent a good stem cell source for regenerative periodontal therapy. As periodontal tissues are easily accessible, it is hypothesised that periodontal fibroblasts may be an alternative cell source for derivation of iPS cells. Objective: The aim of this study was to generate and characterise iPS cells from human adult gingival fibroblasts and periodontal ligament (PDL) fibroblasts. Methods: Gingival and PDL tissues from around extracted human
adult teeth were collected and digested to obtain single cell suspensions of gingival and PDL fibroblasts. The fibroblasts were lentivirally transduced with mouse receptor for retrovirus mSlc7a1, followed by retroviral transduction with four genes (Oct3/4, Sox2, Klf4 and cMyc). Six days after retroviral transduction, the fibroblasts were re-plated onto mouse embryonic feeders and maintained with daily medium change. At day 25-30, human ES cell-like colonies were harvested for characterisation assays to assess their self-renewal and developmental capacities. Results: Human ES cell-like colonies were observed 25 days after transduction. Cells from these colonies were morphologically similar to human ES cells, expressed ES cell genes assayed by immunostaining and real-time reverse-transcription polymerase chain reaction, showed silencing of exogenous retroviral genes and displayed a normal karyotype. In vitro, these cells formed embryoid bodies with down-regulated expression of ES cell genes and up-regulated expression of ectodermal, mesodermal and endodermal markers. Conclusion: iPS-like cells can be generated from human adult gingival and PDL fibroblasts, and gingival fibroblasts can represent an easily accessible source of cells to derive individual-specific iPS cells for regenerative periodontal therapy.Thesis (M.Phil.) -- University of Adelaide, School of Dentistry, 201
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Role of Eph/ephrin Molecules in Mediating Cross-talk Between Stromal and Neural Populations in the Regulation of Neural Differentiation, Adhesion and Migration
Get PDFThere is substantial evidence from the literature that suggests a close association between the skeletal and nervous systems at the anatomical and physiological level, and this cellular and molecular cross-interaction has been termed the neuro-osteogenic network, critical for skeletal growth, homeostasis, and repair.
The Eph and ephrin molecules are the largest family of receptor tyrosine kinases. These membrane-bound molecules are contact-dependent and predominantly function through cell-to-cell interactions. Forward signalling occurs when the ephrin ligand activates signalling in the Eph expressing cell while reverse signalling can also occur where Eph receptors activate signalling through the ephrin ligand expressing cells. The Eph/ ephrin interactions have known roles independently in the skeletal system regulating bone homeostasis, and skeletal growth and repair, and within the nervous system influencing axon guidance and patterning. Therefore, the roles of Eph/ ephrin mediated cross talk between neural and bone marrow stromal cell (BMSC) populations in regulating neural differentiation, adhesion and migration were investigated.
An in vitro neural differentiation assay was established for the human neuroblastoma line SH-SY5Y which showed upregulated expression of EPHRINA2, EPHRINA5 and EPRHINB1 with the progression of neuronal differentiation. Previous studies in our laboratory have shown that human BMSC highly express EPHA3, EPHA4, EPHB2 and EPHB4 at the gene and protein levels. Therefore, the roles of Eph/ ephrin interacting pairs in mediating cross -communication between BMSC and neurons, respectively, were assessed. These included the stromal expressing Eph receptors with neural expressing ephrin ligands (1) EphA3/A4 with ephrinA2 (2) EphA3/A4 or EphB2 with ephrinA5 and (3) EphB2/B4 with ephrinB1, respectively. To examine the importance of ephrin reverse signalling on neural differentiation, adhesion and migration, soluble EphA3-Fc, EphA4-Fc, EphB2-Fc, EphB4-Fc fusion molecules, were used to activate reverse signalling of the ephrinA2, ephrinA5 and ephrinB1 ligands, respectively. The gene expression of early and mature neuronal and sensory markers in the SH-SY5Y line showed a differential response following activation of the ephrinA and ephrinB ligands during neural induction. However, there was no significant difference in neuronal morphology. The activation of both ephrinA and ephrinB ligands inhibited SH-SY5Y neural adhesion, with reduced neural adhesion in response to EphA4-Fc, EphB2-Fc and EphB4-Fc for two hours, and in response to EphA4-Fc and EphB4-Fc for twenty-four hours. EphB2-Fc and EphB4-Fc stimulation did not exert an effect on neural transwell migration where these molecules were not in direct contact with the neural-like cells. There was no significant difference in neural adhesion, or neural and stromal migration, in response to blocking ephrinB1 on the neural population. To determine functional relevance of the neuro-osteogenic network, the avian embryo dorsal root ganglia (DRG) that innervate the hind limbs, were cultured in vitro in the presence of a range of Eph-Fc molecules. DRG neurite cell bodies within the Eph-Fc occupied spaces was significantly decreased for DRG pooled from the lumbar region while a strong trend of differential DRG neurite cell body number and neurite invasion was observed within the Eph-Fc occupied spaces for DRG from specific lumbar compartments. Future studies would investigate the importance of ephrinA2, ephrinA5 and ephrinB1 mediated cross-communication between the neural and BMSC populations during skeletal and neural development and fracture repair.Thesis (M.Phil) -- University of Adelaide, Adelaide Medical School, 202
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Evaluating the Role of Bone Marrow Microenvironment in the Pathogenesis of Therapy-related Myeloid Neoplasms
Get PDFTherapy-related myeloid neoplasm (tMN), lethal blood cancer developing in long-term cancer survivors, is considered to be direct consequence of cytotoxic therapy-induced DNA damage in haematopoietic stem cells. Despite increased recognition that altered bone marrow stroma can also drive leukemogenesis, the functional biology of tMN bone marrow microenvironment remains unknown. Comprehensive multiomic (transcriptome, DNA damage response, cytokine secretome and functional profiling) characterisation of bone marrow stromal cells from tMN was performed and compared with (i) patients with myeloid neoplasm and a history of another cancer but without cytotoxic exposure, (ii) typical primary myeloid neoplasm and (iii) agematched controls, to decipher bone marrow microenvironmental changes induced by cytotoxics from that age related and myeloid neoplasm induced changes. tMN bone marrow mesenchymal stromal cells exhibited profound senescence with CDKN1A induction, high levels of β-Galactosidase, aberrant morphology, defective proliferation, differentiation and haematopoietic stem cell supportive capacity. Highly senescence phenotype is probably driven by defective DNA repair capacity. Strikingly, despite their dormant state, tMN stromal cells were metabolically highly active with a switch towards glycolysis and secreted multiple pro-inflammatory cytokines indicative of a senescent-secretory phenotype that inhibited adipogenesis. Critically, senolytics not only eliminated dormant cells, but also restored adipogenesis. Finally, sequential patient sampling showed senescence phenotypes are induced within months of cytotoxic exposure, well prior to the onset of secondary cancer. This thesis provides novel insight into role of pro-inflammatory and senescent stromal milieu to tMN pathogenesis and provide a valuable resource for future strategies to delay/prevent the tMN.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 202
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