Reducing the musculoskeletal deterioration that astronauts experience in microgravity requires countermeasures that can improve the effectiveness of otherwise rigorous and time-expensive exercise regimens in space. The ability of low-intensity vibrations (LIV) to activate force-responsive signaling pathways in cells suggests LIV as a potential countermeasure to improve cell responsiveness to subsequent mechanical challenge. Mechanoresponse of mesenchymal stem cells (MSC), which maintain bone-making osteoblasts, is in part controlled by the “mechanotransducer” protein YAP (Yes-associated protein), which is shuttled into the nucleus in response to cyto-mechanical forces. Here, using YAP nuclear shuttling as a measurement outcome, we tested the effect of 72 h of clinostat-induced simulated microgravity (SMG) and daily LIV application (LIVDT) on the YAP nuclear entry driven by either acute LIV (LIVAT) or Lysophosphohaditic acid (LPA), applied after the 72 h period. We hypothesized that SMG-induced impairment of acute YAP nuclear entry would be alleviated by the daily application of LIVDT. Results showed that while both acute LIVAT and LPA treatments increased nuclear YAP entry by 50 and 87% over the basal levels in SMG-treated MSCs, nuclear YAP levels of all SMG groups were significantly lower than non-SMG controls. LIVDT, applied in parallel to SMG, restored the SMG-driven decrease in basal nuclear YAP to control levels as well as increased the LPA-induced but not LIVAT-induced YAP nuclear entry over SMG only, counterparts. These cell-level observations suggest that daily LIV treatments are a feasible countermeasure for restoring basal nuclear YAP levels and increasing the YAP nuclear shuttling in MSCs under SMG.

The nucleus, central to all cellular activity, relies on both direct mechanical input and its molecular transducers to sense and respond to external stimuli. While it has been shown that isolated nuclei can adapt to applied force ex vivo, the mechanisms governing nuclear mechanoadaptation in response to physiologic forces in vivo remain unclear. To investigate nuclear mechanoadaptation in cells, we developed an atomic force microscopy (AFM) based procedure to probe live nuclei isolated from mesenchymal stem cells (MSCs) following the application of low intensity vibration (LIV) to determine whether nuclear stiffness increases as a result of LIV. Results indicated that isolated nuclei were, on average, 30% softer than nuclei tested within intact MSCs prior to LIV. When the nucleus was isolated following LIV (0.7g, 90Hz, 20min) applied four times (4x) separated by 1h intervals, stiffness of isolated nuclei increased 75% compared to non-LIV controls. LIV-induced nuclear stiffening required functional Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, but was not accompanied by increased levels of the nuclear envelope proteins LaminA/C or Sun-2. While depleting LaminA/C or Sun-1&2 resulted in either a 47% or 39% increased heterochromatin to nuclear area ratio in isolated nuclei, the heterochromatin to nuclear area ratio was decreased by 25% in LIV-treated nuclei compared to controls, indicating LIV-induced changes in the chromatin structure. Overall, our findings indicate that increased apparent cell stiffness in response to exogenous mechanical challenge of MSCs in the form of LIV is in part retained by increased nuclear stiffness and changes in chromatin structure.

A primary component of exercise, mechanical signals, when applied in the form of low intensity vibration (LiV), increases mesenchymal stem cell (MSc) osteogenesis and proliferation. While it is generally accepted that exercise effectively combats the deleterious effects of aging in the musculoskeletal system, how long-term exercise affects stem cell aging, which is typified by reduced proliferative and differentiative capacity, is not well explored. As a first step in understanding the effect of long-term application of mechanical signals on stem cell function, we investigated the effect of LIV during in vitro expansion of MScs. primary MScs were subjected to either a control or to a twice-daily LiV regimen for up to sixty cell passages (P60) under in vitro cell expansion conditions. LIV effects were assessed at both early passage (EP) and late passage (LP). At the end of the experiment, P60 cultures exposed to LIV maintained a 28% increase of cell doubling and a 39% reduction in senescence-associated β-galactosidase activity (p < 0.01) but no changes in telomere lengths and p16 INK4a levels were observed. prolonged culture-associated decreases in osteogenic and adipogenic capacity were partially protected by LiV in both ep and Lp groups (p < 0.05). Mass spectroscopy of late passage MSC indicated a synergistic decrease of actin and microtubule cytoskeleton-associated proteins in both control and LiV groups while LiV induced a recovery of proteins associated with oxidative reductase activity. in summary, our findings show that the application of long-term mechanical challenge (+LiV) during in vitro expansion of MSCs for sixty passages significantly alters MSC proliferation, differentiation and structure. this suggests LiV as a potential tool to investigate the role of physical activity during aging.

The nucleus, central to all cellular activity, relies on both direct mechanical input and its molecular transducers to sense and respond to external stimuli. While it has been shown that isolated nuclei can adapt to force directly ex vivo, nuclear mechanoadaptation in response to physiologic forces in vivo remains unclear. To gain more knowledge regarding nuclear mechanoadaptation in cells, we have developed an atomic force microscopy (AFM) based experimental procedure to isolate live nuclei and specifically test whether nuclear stiffness increases following the application of low intensity vibration (LIV) in mesenchymal stem cells (MSCs). Results indicated that isolated nuclei, on average, were 30% softer than nuclei tested within intact MSCs. When the nucleus was isolated following LIV (0.7g, 90Hz, 20min) applied four times (4x) separated by 1h intervals, stiffness of isolated nuclei increased 75% compared to controls. LIV-induced intact MSC and nuclear stiffening required functional Linker of Nucleoskeleton and Cytoskeleton (LINC) complex but was not accompanied by increased levels of nuclear envelope proteins LaminA/C or Sun-2. Indicating LIV-induced changes in the chromatin structure, while depleting LaminA/C or Sun-1&2 resulted in a 47% and 39% increased heterochromatin to nuclear area ratio in isolated nuclei, ratio of heterochromatin to nuclear area was decreased by 25% in LIV treated nuclei compared to controls. Overall, our findings indicate that increased apparent cell stiffness in response to exogenous mechanical challenge in the form of LIV are in-part retained by increased nuclear stiffness and changes in chromatin structure in MSCs.

27Reducing the bone deterioration that astronauts experience in microgravity requires 28 countermeasures that can improve the effectiveness of rigorous and time-expensive exercise 29 regimens under microgravity. The ability of low intensity vibrations (LIV) to activate force-30 responsive signaling pathways in cells suggests LIV as a potential countermeasure to improve 31 cell responsiveness to subsequent mechanical challenge. Mechanoresponse of mesenchymal 32 stem cells (MSC) that maintains bone-making osteoblasts is in part controlled by the 33 "mechanotransducer" protein YAP (Yes-associated protein) which is shuttled into the nucleus in 34 response cyto-mechanical forces. Here, using YAP nuclear shuttling as a measure of MSC 35 mechanoresponse, we tested the effect of 72 hours of simulated microgravity (SMG) and daily 36 LIV application (LIV DT ) on the YAP nuclear entry driven by either acute LIV (LIV AT ) or 37Lysophosphohaditic acid (LPA), applied at the end of the 72h period. We hypothesized that 38 SMG-induced impairment of acute YAP nuclear entry will be alleviated by daily application of 39 LIV DT . Results showed that while both acute LIV AT and LPA treatments increased nuclear YAP 40 entry by 50% and 87% over the basal levels in SMG-treated MSCs, nuclear YAP levels of all 41 SMG groups were significantly lower than non-SMG controls. Daily dosing of LIV DT , applied in 42 parallel to SMG, restored the SMG-driven decrease in basal nuclear YAP to control levels as 43 well as increasing the LPA-induced but not LIV AT -induced YAP nuclear entry over the non-LIV DT 44 treated, SMG only, counterparts. These cell level observations suggest that utilizing daily LIV DT 45 treatments is a feasible countermeasure for increasing the YAP-mediated anabolic 46 responsiveness of MSCs to subsequent mechanical challenge under SMG. 47 48

The Final Frontier conjures dreams of exploring the great expanse of our solar system, but there is an inherent problem to this vision as space travel negatively impacts the musculoskeletal system. The focus of my research was to study the detrimental effects of radiation and microgravity, two components of space travel, on mesenchymal stem cells through the lens of the yes-associated protein (YAP). Chapter One, discusses our motivation and the goals of our experiments while Chapter Two provides extensive background on the cell type chosen, the known impacts of radiation and microgravity, our model compared with the actual conditions astronauts experience, and a discussion of YAP with its associated pathways. Chapter Three is the manuscript and supplemental, Chapter Four is the conclusion and finally Chapter Five which details some of the other work that I contributed to while at Boise State University.