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.
- Full Text:
- Date Issued: 2018
- 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.
- Full Text:
- Date Issued: 2018
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.
- Full Text:
- Date Issued: 2013
- 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.
- Full Text:
- Date Issued: 2013
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