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This project involves the design and development of a cell stretching bioreactor device that can work in conjunction with a Random Positioning Machine (RPM) apparatus. Microgravity environments, such as in space, have been shown to induce alterations in cellular development due to inadequate mechanical loading of biological tissue. Because of this, long-term spaceflight has led to many health concerns, including osteoporosis and muscle atrophy. Space travel is rare and costly, making this research difficult to conduct, however; techniques to simulate microgravity on Earth can be achieved by using a Random Positioning Machine. This device has been a beneficial tool used to study the effect gravity has on cellular growth, yet certain tissues in the body, such as bone and muscle, require mechanical stress, strain, and mechanical loading to develop properly. Because of this, a device that can induce strain on cells while subjected to microgravity conditions is needed to further improve cellular research for space exploration. The constructed bioreactor consists of 3D printed and custom-made components that can induce uniaxial cyclic strain on cells adhered to an elastic membrane. Validation and testing of the device have shown that this bioreactor is suitable for cellular experimentation to work in conjunction with an RPM to deliver a controlled amount of strain while under microgravity conditions. / Bioengineering

Obesity, a disease characterized by excess adipose tissue (AT), is a growing worldwide epidemic. The Centers for Disease Control and Prevention (CDC), in 2017-2018, reported the prevalence of obesity in adults in the United States was 42.4% . Obesity increases the risk for many other serious health conditions such as type 2 diabetes, cardiovascular diseases, stroke, and some cancers. In individuals with obesity, the hypertrophic expansion of adipocytes, the main cell type within AT, is not matched by new vessel formation, leading to AT hypoxia. As a result, hypoxia inducible factor-1⍺ (HIF-1⍺) accumulates in adipocytes inducing a transcriptional program that upregulates profibrotic genes and biosynthetic enzymes such as lysyl oxidase (LOX) synthesis. This excess synthesis and crosslinking of extracellular matrix (ECM) components cause AT fibrosis. Although fibrosis is a hallmark of obese AT, the role of fibroblasts, cells known to regulate fibrosis in other fibrosis-prone tissues, is not well studied. Adipocytes are mechanoresponsive and affected by different microenvironmental cues, including hypoxia and mechanical (un)loading. Yet, no study has focused on the role of the aforementioned factors on the adipocyte mechanical response, including actin cytoskeletal remodeling. This dissertation aims to develop an in vitro model of healthy/diseased AT to explore the effect of microenvironmental cues on adipocyte function and actin cytoskeletal remodeling. The first aim is to study (1) the crosstalk between fibroblasts and adipocytes in a co-culture model and (2) the effect of hypoxia on the ras homolog gene family member A (RhoA)/Rho-associated coiled-coil kinases (ROCK) mechanical pathway and actin cytoskeletal remodeling in adipocytes. We confirmed that hypoxia creates a diseased phenotype by inhibiting adipocyte maturation and inducing actin stress fiber formation facilitated by myocardin-related transcription factor A (MRTF-A/MKL1) nuclear translocation. The second aim explores the effects of mechanical unloading (simulated microgravity) on key adipocyte functions and actin cytoskeletal remodeling. This study demonstrated that mechanical unloading enhances adipocyte maturation via increased lipogenesis and lipolysis and cortical actin remodeling, which together further enhanced glucose uptake. However, disrupting cortical actin remodeling by using inhibitors or exposure to a high concentration of free fatty acids (FFAs) diminished enhanced adipocyte functions observed in simulated microgravity. Overall, the results of these studies support the importance of microenvironmental cues on adipocyte actin cytoskeletal remodeling. Therefore, targeting mechanical pathways that regulate actin cytoskeletal remodeling can be used to improve adipocyte function and AT metabolism and possibly treat related diseases such as type 2 diabetes and obesity. / Bioengineering

The multifaceted adverse effects of reduced gravity on the skeletal system pose a significant challenge to human spaceflight. There is an interest in investigating any hypothetical differences between partial gravity and microgravity, and in the unmet need to identify countermeasures to both. A hypothesis to be tested is that reduced gravity impairs a variety of osteogenic cell functions, such as proliferation and differentiation, and that these inhibitory effects can be mitigated by nutritional countermeasures or by interrupting signaling pathways that drive undesired osteogenic remodeling. Utilizing the Random Positioning Machine, it is possible to simulate a variety of reduced gravity levels relevant to future manned space missions: Mars, Moon, and Microgravity of the Low Earth Orbit (LEO) environment. In this study, the effects of altered gravity on the physiology and morphology of cultured osteoblasts were investigated, specifically on their proliferation, osteogenic differentiation, and matrix mineralization. In assessing the role of mechanotransduction in microgravity-induced cytoskeletal dysfunction, this thesis also explored whether selective inhibition of specific signaling steps within the Rho-ROCK pathway can be used to modulate the effects of microgravity on osteoblast differentiation and function. Finally, in developing new countermeasures, an investigation was made into the effectiveness of curcumin and carnosic acid, two nutritional antioxidants with pro-osteogenic properties, contrasted with the trace element zinc, as potential alimentary supplements that may mitigate or alleviate the deleterious effects of microgravity. Results showed that short-term (6 days) culture yielded a dose-dependent reduction in proliferation and the enzymatic activity of alkaline phosphatase (ALP), while long-term studies (21 days) showed a distinct dose-dependent inhibition of mineralization. By contrast, expression levels of key osteogenic genes (Alkaline phosphatase, Runt-related Transcription Factor 2, Sparc/osteonectin) exhibited a threshold behavior: gene expression was significantly inhibited when the cells were exposed to Mars-simulating partial gravity, and this was not reduced further when the cells were cultured under simulated Moon or microgravity conditions. My data suggests that impairment of cell function with decreasing simulated gravity levels is graded and that the threshold profile observed for reduced gene expression is distinct from the dose dependence observed for cell proliferation, ALP activity, and mineral deposition. My studies into the gravity-induced re-organization of the cytoskeleton indicate that selective interruption of the Rho-ROCK pathway at ROCK can prevent morphological changes that result in impaired differentiation and mineralization. Further, I found that nutraceuticals partially reversed the inhibitory effects of SMG on ALP activity and promoted osteoblast proliferation and differentiation in the absence of traditional osteogenic media. I further observed a synergistic effect of the intermix of the phytonutrients on ALP activity. Intermixes of phytonutrients may serve as convenient and effective nutritional countermeasures against bone loss in space. / Bioengineering

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The present study investigates vortex phase separator (VPS) technology as a new approach for a liquid amine CO2 removal system. Experimental results obtained using a 99.99% pure CO2 stream and liquid amine with varying concentrations demonstrate the VPS' ability to decrease CO2 volume at its gas outlet. Operating parameters such as CO2 flow rate, relative humidity (RH), and temperature were systematically varied during experimental procedure, as well as working fluid temperature, volume, and flow rate. The subscale design for a VPS with a 3" inner diameter, 3.5" outer diameter, and 3.63" height removed a maximum of 84% of CO2 from a CO2 stream at 3.7 SCFH flow rate, 14°C temperature, and 82% RH, using 100 mL of 100% amine circulated at 1.52 LPM flow rate. The designed VPS also showed to be effective in removing relative humidity of the CO2 stream by up to 26% for the stated parameters. Regeneration of liquid amine in the VPS system is also proposed to allow for continuous CO2 removal. The results obtained in this work characterize the VPS system for CO2 removal in terms of various operating parameters for the gas (CO2) and liquid (liquid amine) phases, as well as provide initial insights into how a VPS sized for stability and applied to CO2 removal performs. This work demonstrates VPS technology as an effective, alternative CO2 removal technology that could be scaled and used to support human exploration of space.

The objectives of this study were (1) to determine the optimum in vitro culture conditions for in vitro fertilized bovine embryos among culture methods and (2) to investigate whether bovine fertilization and embryo development would occur in simulated microgravity conditions in vitro. In the first part of this study, the result showed that the microdrop method was the optimum culture method among groups; however, FBS supplementation did not significantly affect the bovine preimplantation embryo development in vitro. In the second part of this study, the result showed that bovine in vitro fertilization did not occur in simulated microgravity conditions. Moreover, none of the presumptive zygotes and 2-8 cell stage embryos were able to develop to further stages, while embryos cultured in standard microdrop method culture conditions developed normally.

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