Mechanical unloading has deleterious effects on the musculoskeletal system and results in significant reductions in bone density, mass, and strength, which do not fully recover even years after returning to weightbearing. For example, the rate of bone loss in microgravity is 10-fold more rapid than the rate of loss seen in elderly Caucasian females, the population group most predisposed to osteoporosis. This raises concern with individuals who are exposed to multiple bed rest periods or crewmembers who make repeated missions. Exercise offers a way to reduce or reverse these effects. Dual-energy X-ray absorptiometry (DXA) densitometry and bone mineral density (BMD) alone are generally insufficient for capturing the complex changes in bone mass, structure, and integrity and not an accurate predictor of fracture risk. Therefore, it is essential to measure the mechanical properties of bone tissue directly using animal models. The hindlimb unloaded (HU) rat model is a well-established ground-based analog for studying bone response to disuse and effects of spaceflight. The current study is one of the very few that has measured longitudinally densitometric and mechanical properties of bone after repeated simulated microgravity and long-term recovery at multiple anatomic sites in skeletally mature rats. The specific aims were to characterize 1) loss and recovery dynamics of bone following a period of unloading, 2) bone response after a second exposure to 28 days of HU, following an initial 28 days of HU and a recovery period equal to twice the duration of initial exposure, and 3) effects of resistance exercise during recovery period following an initial HU exposure and its effects on a subsequent exposure. In general, our data showed that bone response to unloading and recovery is site-specific. More specifically, we found that: 1) the rat proximal tibia metaphysis modeled the loss and discordant recovery dynamics as seen in the International Space Station (ISS) crewmembers proximal femur better than the rat femoral neck; 2) the initial exposure to HU has minimal effect on the subsequent HU exposure, and detrimental effects of the second HU exposure were milder than the initial due to reduced mechanosensitivity of the bone; 3) exercise significantly enhanced recovery following the initial HU exposure, and losses during the second exposure were not affected by exercise in most cases.

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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

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.

Le présent travail de Doctorat porte sur l’adaptation et le déconditionnement cardiovasculaire chez l’astronaute en microgravité réelle prolongée, en microgravité simulée de courte durée (avec et sans contremesures par hypergravité), et en confinement de longue durée. Afin de répondre à cette thématique, 3 études expérimentales sur l’humain ont été réalisées, et de fait, ce présent travail de Doctorat se divise en 3 parties distinctes. La première est la mission « Mars 500 » qui comprend un confinement de 520 jours de 6 sujets-volontaires. La seconde est le projet « Vessel Imaging » qui comprend un vol spatial respectif de 6 mois à bord de la « Station Spatiale Internationale (ISS) » de 10 sujets-astronautes. La troisième est l’étude «Short Time Bed-Rest (STBR)» (12 sujets) qui comprend une courte période de microgravité simulée par alitement prolongé à -6° (5 jours) avec et sans l’utilisation de deux contremesures cardiovasculaires par hypergravité (continue ou intermittente). / This PhD work focuses on astronaut cardiovascular adaptation and deconditioning in real prolonged microgravity, short simulated microgravity (with and without countermeasures) and long-term confinement. To answer to this topic 3 humans experimental studies have been performed, and thus the present PhD work is divided into 3 distinct parts . The first one is the mission « Mars 500 » which consists in 520-days confinement with 6 subjects-volunteers mission. The second is the project « Vessel Imaging » whitch consit in a 6-months spaceflight aboard the « International Space Station » with 10 subjects-astronauts. The third is the « Short Time Bed -Rest (STBR) » study (12 subjects) which consist in a short period of bedrest (-6°, 5 days) with and without the use of two cardiovascular countermeasures by hypergravity (continuous or intermittent).