An investigation of mitochondrial dynamics and networks observed within human undifferentiated and differentiated cell lines
- Authors: Houseman, Pascalené Shannon
- Date: 2018
- Subjects: Mitochondria , Mitochondrial pathology , Degeneration (Pathology) , Mesenchymal stem cells , Neural stem cells , Cell lines , Reactive oxygen species (ROS)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/60687 , vital:27816
- Description: Mitochondrial dynamics refers to a series of constant division and fusion cycles that form interconnecting networks within healthy cells. Reactive oxygen species (ROS) are the byproducts of cellular redox reactions, and, when in excess, have been linked to degenerative diseases and aging. Mesenchymal stem cells (MSCs) require a niche that presents with low levels of ROS; this enables the stem cell to maintain its “sternness”, the stem cell population, as well as the ability to adhere, migrate, and proliferate. If ROS levels increase within the MSC niche, inhibition of cellular adhesion and migration occurs. In contrast, neural stem cells require a niche that presents with a high level of ROS, aiding in their proliferative, self- renewing capacities. Investigations into what constitutes a healthy mitochondrial network versus the disease state of the network are required in order to determine what promotes degeneration and aging within stem cells. It was hypothesized that increased levels of ROS would stunt the ability of MSCs to attach and migrate, and hinder their abilities of proliferation and differentiation. In contrast, neuronal differentiation would present with an increased proliferation. This led to the investigation into the effects of ROS and oxidative stress, and the resulting mitochondrial dynamics, have on undifferentiated and differentiated mesenchymal stem and SH-SY5Y cells. Upon the addition of non-lethal S3I-201 (STAT3 has been linked to a reduction in ROS) to MSCs, an increase in ROS was observed. Higher concentrations of STAT3 inhibitor resulted in a decrease in MSC attachment and proliferation. When exposed to similar conditions, the SH-SY5Y cells underwent an increased proliferation; due to multiple restrictions, they were not used any further within the study. Mitochondrial dynamics were observed using a fusion promoter (M1) and a fission inhibitor (Mdivi-1); the MSCs were dosed with varying concentrations in order to determine the effects that mitochondrial dysfunction may have on the established networks, and cell survival. The mitochondria within MSCs migrated to the extensions of the cell, and displayed an alteration in morphology, or were clustered around the nucleus and/or the lipid deposits. These high density clusters correlated with a high intensity of fluorescence using 2’,7’- dichlorofluorescein diacetate. In conclusion, varying concentrations of ROS have different effects on MSCs in terms of overall maintenance and function; mitochondrial dynamics play an important role in cell survivability and the fate of stem cell differentiation. Further investigation into the mitochondrial dynamics and networks of these cell lines and their differentiated progeny is required.
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- Date Issued: 2018
Analysis of the role of Hsp90 in colon cancer and cancer stem-like cell biology in vitro using a genetically paired cell line model
- Authors: Slater, Cindy
- Date: 2015-04-09
- Subjects: Heat shock proteins , Heat shock protein Hsp90 family , Colon (Anatomy) Cancer Treatment , Cell lines , Cancer stem-like cell , SW480 , SW620
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/480345 , vital:78433
- Description: Colon cancers are commonly associated with mutations or changes in signaling in the Wnt/β-catenin pathway. Cancer treatments, such as chemotherapy and radiation, are challenged by the limited methods of disease detection and the insufficient elimination of contributing factors, such as cancer stem-like cells (CSC), to metastatic disease. CSC are characterised by their ability to survive anchorage-independently and attribute to therapeutic resistance. To examine the biological changes associated with the progression of human colon cancers and the role of Hsp90 in cancer and CSC biology, SW480 and SW620 genetically paired (isogenic) colon cancer cell lines from the same patient were characterised for populations of putative CSC, tumoursphere (TS) forming ability, cell growth, behaviour and response to anti-cancer therapeutics. The SW480 cell line was established from a primary colon adenocarcinoma and the SW620 was established from a lymph node metastasis of the primary cancer one year later. To address the role of Hsp90 in colon cancer, the sensitivity of cells and TS were analysed in response to geldanamycin and novobiocin, and an isoform-specific approach to the targeting of Hsp90α was developed. Flow cytometric analysis of putative CSC by phenotype revealed variable proportions of cells bearing the CD44+/CD133+ surface protein marker, widely used in the identification of colon CSC, in SW480 and SW620 cells. The paired cell lines maintained similar proportions of putative CSC, identified by the expression of the ABCG2 protein (side population; 1 %) and through high aldehyde dehydrogenase activity (ALDEFLUOR; 6 %). SW480 cells demonstrated greater TS forming efficiency than SW620 cells (49.9 and 35.5 %, respectively) and observations of wound-healing showed SW480 cells to be more migratory than SW620 cells. No difference in response to Hsp90 inhibition was observed between paired cell lines, however SW480 TS resisted treatment with geldanamycin. This the first study to report a dose-dependent increase in TS growth in response to novobiocin inhibition of Hsp90, and to demonstrate that that the sensitivity of SW480 and SW620 TS to oxaliplatin, a common drug for the treatment of metastatic colon cancers, was enhanced by novobiocin, providing promise for the elimination of CSC with combined chemotherapeutics. We analysed the Wnt/β-catenin pathway in response to expression of short hairpin RNA (shRNA) against Hsp90α or a control non-targeting shRNA, under the control of a tetracycline-responsive promoter. Hsp90α knockdown contributed to a deregulated stress response, presenting with reduced Hsp27 and β-catenin protein, but corresponded to an increase in the association between Hsp90, β-catenin and Hsp27 in vitro. The reduction of Hsp90α did not influence sensitivity of the colon cancer cells to activators or inhibitors of the Wnt pathway, but rather correlated to reduced TS formation, cell adhesion and spreading, identifying potential therapeutic benefit to the controlled reduction of Hsp90α for the deregulation of colon cancer characteristics. Given that Hsp27 and β-catenin are both involved in cell adhesion, cytoskeletal dynamics and interact directly with each other, we propose a role for targeting Hsp90α in the regulation cell adherence indirectly via reductions in levels of β-catenin and Hsp27, rather than by modifying the transcriptional activity of β-catenin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2015
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- Date Issued: 2015-04-09
A central enrichment-based comparison of two alternative methods of generating transcription factor binding motifs from protein binding microarray data
- Authors: Mahaye, Ntombikayise
- Date: 2013 , 2013-03-13
- Subjects: Transcription factors , Bioinformatics , Protein binding , Protein microarrays , Cell lines
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3890 , http://hdl.handle.net/10962/d1003049 , Transcription factors , Bioinformatics , Protein binding , Protein microarrays , Cell lines
- Description: Characterising transcription factor binding sites (TFBS) is an important problem in bioinformatics, since predicting binding sites has many applications such as predicting gene regulation. ChIP-seq is a powerful in vivo method for generating genome-wide putative binding regions for transcription factors (TFs). CentriMo is an algorithm that measures central enrichment of a motif and has previously been used as motif enrichment analysis (MEA) tool. CentriMo uses the fact that ChIP-seq peak calling methods are likely to be biased towards the centre of the putative binding region, at least in cases where there is direct binding. CentriMo calculates a binomial p-value representing central enrichment, based on the central bias of the binding site with the highest likelihood ratio. In cases where binding is indirect or involves cofactors, a more complex distribution of preferred binding sites may occur but, in many cases, a low CentriMo p-value and low width of maximum enrichment (about 100bp) are strong evidence that the motif in question is the true binding motif. Several other MEA tools have been developed, but they do not consider motif central enrichment. The study investigates the claim made by Zhao and Stormo (2011) that they have identified a simpler method than that used to derive the UniPROBE motif database for creating motifs from protein binding microarray (PBM) data, which they call BEEML-PBM (Binding Energy Estimation by Maximum Likelihood-PBM). To accomplish this, CentriMo is employed on 13 motifs from both motif databases. The results indicate that there is no conclusive difference in the quality of motifs from the original PBM and BEEML-PBM approaches. CentriMo provides an understanding of the mechanisms by which TFs bind to DNA. Out of 13 TFs for which ChIP-seq data is used, BEEML-PBM reports five better motifs and twice it has not had any central enrichment when the best PBM motif does. PBM approach finds seven motifs with better central enrichment. On the other hand, across all variations, the number of examples where PBM is better is not high enough to conclude that it is overall the better approach. Some TFs bind directly to DNA, some indirect or in combination with other TFs. Some of the predicted mechanisms are supported by literature evidence. This study further revealed that the binding specificity of a TF is different in different cell types and development stages. A TF is up-regulated in a cell line where it performs its biological function. The discovery of cell line differences, which has not been done before in any CentriMo study, is interesting and provides reasons to study this further.
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- Date Issued: 2013