List of Titles

An investigation of mitochondrial dynamics and networks observed within human undifferentiated and differentiated cell lines

- Houseman, Pascalené Shannon

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

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An investigation of the role of mitochondrial STAT3 and modulation of Reactive Oxygen Species in adipocyte differentiation

- Kramer, Adam Hildyard

Authors: Kramer, Adam Hildyard
Date: 2014
Language: English
Type: text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/54632 , vital:26595
Description: Stem cells have the ability to differentiate into a myriad of different cell types. The understanding of the differentiation process is of paramount importance if we are to use these cells in the lab as well as in therapeutics. Here, the levels and localization of the signal transducer and activator of transcription 3 (STAT3), with particular attention focused on the mitochondrial serine 727 phosphorylated form of STAT3 (pSTAT3S727) during differentiation, was investigated. Using the murine preadipocyte progenitor cell line 3T3-L1, as well as adipose derived human mesenchymal stem cells (HMSC-ad) as differentiation models, the relative levels of Reactive Oxygen Species (ROS) and the levels and localization of STAT3 were investigated during the differentiation process. ROS is known to play an important signalling role during differentiation and is well reported during the events of adipogenesis. ROS are generated as a by-product in the Electron Transport Chain (ETC), and it has recently been reported that pSTAT3S727 plays an important role at complex I of the ETC. Various techniques including fluorescence confocal microscopy, flow cytometry and Western blots were utilized to investigate the non-canonical role STAT3 plays during adipogenesis. Mitochondrial isolations were performed to investigate the levels of STAT3 in the mitochondria during differentiation. Further to this, an impedance based real time differentiation assay was developed using the xCELLigence Real Time Cell Analyser to monitor differentiation and the affects various compounds, including a STAT3 inhibitor, have on differentiation. Results indicate that upon induction of differentiation, levels of mitochondrial pSTAT3S727 dramatically decrease and leave the mitochondria. This corresponds to increasing levels of ROS. The canonical active form of STAT3 following phosphorylation on tyrosine 705 (pSTAT3Y705) was found to decrease and lose its nuclear localization. These initial results indicate that STAT3 plays an important non-canonical role in the mitochondria during differentiation.
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Date Issued: 2014

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Development of a 3D bioprinting and standalone bioreactor unit for the production and maintenance of bioscaffolds in vitro

- Hundling, Jethro Daniel

Authors: Hundling, Jethro Daniel
Date: 2021-10-29
Subjects: Bioreactors , Tissue scaffolds , Cell culture , Polyethylene glycol Biotechnology , 3D bioprinting , Poly(ethylene glycol) diacrylate (PEGDA)
Language: English
Type: Master's theses , text
Identifier: http://hdl.handle.net/10962/192063 , vital:45192
Description: The most common method for in vitro cell culture currently is to grow a specific cell type in isolation, in monolayer format, adhered to a 2D culture surface. This brings about many limitations in comparison to in vivo models due to altered cell phenotypes, as caused by the culturing technique itself, and the lack of naturally occurring cell-to-cell interactions. Three dimensional mammalian cell culture technologies have the potential to overcome these limitations, and provide models more representative of natural systems. Unfortunately, the cost and difficulty associated with achieving sustainable and useful 3D mammalian cell culture is still very high, preventing its widespread adoption across scientific platforms. In this research, we investigate the feasibility of developing and producing a visible light-based 3D stereolithographic bioprinter to produce 3D scaffolds for cell culture. Furthermore, we investigate the possibility of developing and implementing a forced perfusion bioreactor system, which would support the produced scaffold and improve longer-term culture conditions. The developed 3D bioprinter, and bioreactor designs were developed and tested alongside Poly (ethylene glycol) diacrylate (PEGDA), a versatile synthetic scaffold material. PEGDA itself was also evaluated for its printability, robustness in culture conditions over time, and its ability to maintain 3D mammalian cell culture. This research showed that both the developed 3D bioprinter, and bioreactor unit were capable of producing and maintaining an easily modifiable PEGDA scaffold, in culture conditions. In addition, the PEGDA formulation developed was shown to allow for the effective and reproducible 3D cell culture conditions over the medium term, with automated media feeding. The research presented here aimed to illustrate a proof of concept that the low-cost development and production of 3D culture scaffold production and maintenance systems was feasible to the scientific research environment. This technology can then be built upon, into a system that would then allow for the broader adoption and investigation of 3D cell culture as a tool within the scientific community. , Thesis (MSc) -- Faculty of Science, Biotechnology Innovation Centre, 2021
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Date Issued: 2021-10-29

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Development of a low-cost bioprinting system for the fabrication of cell-laden sodium alginate hydrogels

- Honiball, John Robert

Authors: Honiball, John Robert
Date: 2018
Subjects: Regenerative medicine , Tissue engineering , Alginates , Colloids , Three-dimensional printing
Language: English
Type: text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/59204 , vital:27470
Description: Bioprinting is a rapidly expanding technology with the ability to fabricate in vitro 3D tissues in a layer-by-layer manner to ultimately produce a living tissue which physiologically resembles native in vivo tissue functionality. Unfortunately, large costs associated with commercially available bioprinters severely limit the amount of people/research groups with access to the technology. Here, we investigated the potential for modifying a commercially available RepRap Prusa iteration 3 (i3) three-dimensional (3D) printer, by replacing the traditional plastic-based print-head with various open-source syringe-housed microextrusion print-head units, such that deposition of composite bioinks consisting of cells, biopolymer scaffolds and/or biomolecules may be achieved at a relatively low cost. Using adipose-derived human mesenchymal stromal stem cells (ad-HMSC) induced for adipogenic differentiation, as well as human umbilical vein endothelial cells (HUVEC), the potential for fabricating vascularised adipose tissue was investigated. The non-toxic, inexpensive algal polysaccharide, sodium alginate, was used to test the printability of the system, as well as for investigating the functionality unmodified sodium alginate has for use as a potential bioink in adipose tissue engineering. Cell viability assays, namely WST-1 and fluorescein diacetate (FDA)/propidium iodide (PI) live/dead cell staining, revealed that ad-HMSC were viable after 7 days of culture. However, viability of HUVEC encapsulated hydrogels revealed significantly lower cell viability. Live/dead cell staining revealed that the modified printing system was able to print ad-HMSC/HUVEC co-cocultures with a large degree of cell viability after 1 day of culture. However, after 7 days of culture, the majority of cells were revealed to be dead. Furthermore, due to the lack of mechanical integrity possessed by alginate in a liquid-like state, printing sodium alginate hydrogels in air consistently resulted in deformation of printed constructs. The newly developed 3D printing technique termed freeform reversible embedding of suspended hydrogels (FRESH) was therefore investigated as a means for achieving 3D spatial control of printed hydrogels using the modified system. Printing cell-free sodium alginate hydrogels within gelatin sacrificial support baths allowed for fabricating constructs in a spatially defined manner. However, overprinting and swelling of alginate hydrogels negatively affected the overall printing accuracy. The present study aimed to pave the way for further system modifications and refinements, such that the ultimate goal of low-cost bioprinting may be achieved. Further optimisation of printing parameters, hydrogel characteristics and sterilisation techniques may allow for fabricating viable, physiologically relevant tissues using the modified system developed.
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Date Issued: 2018

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Modification of Gelatin-Methacrylate, Hyaluronic-Methacrylate and Poly(ethylene) glycol Diacrylate hydrogel bioinks towards the additive manufacturing of articular cartilage

- Barwick, Matthew William

Authors: Barwick, Matthew William
Date: 2021-10
Subjects: Cartilage Diseases , Cartilage Regeneration , Articular cartilage Diseases , Chondrogenesis , Stem cells , Scanning electron microscopy , Fourier transform infrared spectroscopy , Three-dimensional printing , Gelatin-Methacrylate , Hyaluronic-Methacrylate , Poly(ethylene) glycolDiacrylate , Hydrogel bioinks , Real-Time Quantitative Cell Analysis (RTCA) , Bioprinting
Language: English
Type: Master's theses , text
Identifier: http://hdl.handle.net/10962/191181 , vital:45068
Description: Cartilage degradation is most commonly associated with Rheumatoid arthritis and Osteoarthritis, affecting millions of people worldwide. Joint transplants commonly use titanium alloys, which have a shelf life of between 10-15 years. Although the titanium transplant restores partial mobility, side effects such as inflammation, swelling, faulty implants, and metal poisoning in some cases resulting from the transplant. The use of additive manufacturing of articular cartilage sheds new, innovative prospects for joint replacements. This study sets out to formulate and characterize five different hydrogel types towards the additive manufacturing of articular cartilage. Chondrogenic and Adipogenic differentiation was carried out on two separate adipose-mesenchymal stem cell lines A270620-01A, and A311019-02T and validation and efficiency of the differentiation and chondrogenic gene expression was carried out using Alcian Blue stain, Oil Red O stain and Quantitative Reverse Transcription PCR (RT-qPCR). Hydrogel formulation and characterisation of 10 % Gelatin-methacryloyl (GelMA), 10 % Poly (ethylene) glycol diacrylate (PEGDA), 5 % GelMA/5 % PEGDA, 10 % GelMA/0.5 % Hyaluronic Acid Methacrylate (HAMA) and 10 % PEGDA/0.5 % HAMA was carried out through swelling and degradation ratios, surface area and porosity characterisation using Scanning Electron Microscopy (SEM). Hydrogel component and spectroscopic analysis were carried using Real-Time Quantitative Cell Analysis (RTCA) and Fourier-transform Infrared Spectroscopy (FTIR) analysis for each formulated hydrogel's chemical characterisation. Three-dimensional printing (3D) of 10 % PEGDA/0.5 % HAMA and 5 % GelMA/5 % PEGDA was performed using the Zortrax INKSPIRE Resin Ultra-Violet (UV) LCD Desktop 3D Printer. Hydrogel sterility and cell viability were carried out for each hydrogel type using fluorescence microscopy. Both A270620-01A and A311019-02T cell lines showed adipogenic and chondrogenic differentiation ability, with A311019-02T cell line showing greater chondrogenic differentiation of Alcian blue staining. The A270620-01A cell line resulted in a greater collagen gene expression based on the RT-qPCR results. The hydrogel 10 % GelMA showed the greatest swelling ratio of 1260 % in DPBS and 1192 % in DMEM. A significant difference between hydrogel swelling and swelling with Dulbecco's Phosphate Buffered Saline (DPBS) and Dulbecco’s Modified Eagle Medium (DMEM) was observed. The 10 % PEGDA hydrogel had the greatest degradation ratio of 59 % mass remaining, where the 10 % GelMA/0.5 % HAMA showed the least amount of degradation with a mass remaining at 91 %. The 10 % GelMA showed the greatest porosity will the largest pore size of 14 μm in diameter. Hydrogel component and spectroscopic analysis showed no cytotoxic effects for the visible light photoinitiator used to polymerize the hydrogel and no cytotoxic effects for the concentrations used in chondrogenic differentiation. The FTIR analysis showed partial gelatin and hyaluronic acid modification with methacrylic anhydride; however, the distinction between the hybrid hydrogels and single polymer hydrogels could not be made effectively. UV and ethanol washing showed to completely sterilise the hydrogel disks from any contaminants, making them suitable for tissue culture. The cell viability analysis showed the 10 % GelMA/HAMA having the highest cell viability of 77.3 % using 5000 cells/disk and 89.64 % viability using 50 000 cells/disk over a 7-day incubation period. Overall, the combination of two polymers, GelMA and HAMA, has good potential as a 3D hydrogel scaffold towards additive manufacturing of articular cartilage. , Thesis (MSc) -- Faculty of Science, Biotechnology Innovation Centre, 2021
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Date Issued: 2021-10

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The involvement of TRAP1 in the mitochondrial localization of STAT3 in mammalian cells

- Kadye, Rose

Authors: Kadye, Rose
Date: 2014
Language: English
Type: text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/55760 , vital:26731
Description: STAT3 (signal transducer and activator of transcription 3), an oncogene and transcription factor of genes involved in cellular differentiation, proliferation and immune function, that classically localizes in the cytosol and nucleus has also been found in the mitochondria. However, STAT3 does not have a mitochondrial transit peptide, and its mechanism for mitochondrial localization is unknown. Cytosolic Hsp90s chaperone STAT3 to the nucleus therefore we investigated the involvement of the nuclear-encoded mitochondrial Hsp90 molecular chaperone tumor necrosis receptor associated protein 1 (TRAP1) in STAT3’s mitochondrial localization. Using TRAP1 transient over-expression, STAT3 inhibitor S3I- 201 and Hsp90 inhibitor geldanamycin, we demonstrate that TRAP1 and STAT3 co-localize and co-immunoprecipitates in mammalian systems. Taken together with the observation that STAT3 potentially directly interacts with TRAP1, these data suggest that TRAP1 plays a role in the mitochondrial localization of STAT3.
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Date Issued: 2014

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