Long duration spaceflight missions out of lower earth orbit, back to the lunar surface, or possibly to Mars highlight the importance of preserving muscle mass and function. Muscle atrophy occurs within days of exposure to microgravity and prevailing thought is that a primary mechanism for muscle atrophy is a reduction in skeletal muscle protein synthesis. This dissertation examines the ability of skeletal muscle to recover muscle protein synthesis with slight perturbation, such as ambulatory reloading during disuse as well as partial loading, similar to body mass seen on the moon or Mars. We use traditional precursor-product labeling to measure protein synthesis, but use a relatively novel tracer, deuterium oxide, in order to make cumulative measures of protein synthesis over 24 h. The overarching goal of this dissertation is to define the response of skeletal muscle protein synthesis to different loading parameters in order to better understand the contribution of protein synthesis to skeletal muscle mass during disuse. In the first study, we demonstrate that muscle atrophy during 5 days of hindlimb unloading is in part due to a decrease in protein synthesis. We also highlight the ability of skeletal muscle to adapt by allowing two 1 h ambulatory reloading sessions on days 2 and 4. Although this countermeasure is able to rescue protein synthesis in soleus and gastrocnemius, it is unable attenuate any losses in muscle mass. In the second study, we compare partial weight loading to traditional hindlimb unloading. Weight bearing of 1/3 or 1/6 body weight is able to attenuate losses in muscle mass seen with unloading. Protein synthesis is maintained after 21 days of the experimental protocol, suggesting that protein synthesis is responsive to load and is likely not the only mechanism for determining muscle mass. In the final study, the effects of < 1 Gy x-ray exposure and partial weight suspension are measured to better understand the complex space environment, which includes a wide variety of radiation. Surprisingly, we found no effects of radiation on muscle protein synthesis in 1 G or partial loading. Targeting only protein synthesis may not be enough of a stimulus as evidenced by the data in this dissertation. Future plans should use a multiple-systems approach to counteract atrophy by increasing protein synthesis to maintain/elevate muscle mass during periods when it is otherwise compromised.

Iroquois (Irx) homeobox genes have important and redundant functions during embryogenesis. Irx3/5 double knock out (Irx3/5 KO) mouse embryos exhibit severe hindlimb phenotypes. In these mutant hindlimbs, digit 1 and tibia are absent, moreover femur and pelvis are hypoplastic. Here, we demonstrate that Irx3/5 are expressed in the hindlimb field prior to limb bud initiation, and are required at this early stage for the pattern formation along the anteroposterior axis. Their early function is involved in prepatterning and positioning the Shh expression domain. In addition, Irx3/5 KO mutant hindlimb buds have a mild outgrowth defect and increased cell death at early stages of limb development, which may explain the small hindlimb bud size in these mutant embryos. To examine whether Irx3/5-expressing cells are the origin of lost and affected structures in Irx3/5 KO mutant hindlimbs, targeting vectors with Cre genes inserted into the Irx5 locus have been generated.

No description available.

Rats that undergo hindlimb unloading (HU) as a simulation for space flight experience bone changes similar to astronauts in microgravity. The purpose of this research was to assess whether an exercise countermeasure would be effective in preventing or mitigating bone degradation during HU. Controlled electrical muscle stimulation was applied to the lower left hindlimb to simulate resistive exercise. Adult 6-mo. old male rats were assigned to 3 groups of 12 each: hindlimb unloaded (HU), aging cage control (CC), and baseline (BL). The CC group was pair-fed to match the nutritional intake of HU animals during the 28 days of the study. The left leg was exercised 3 days a week for the duration of the study, with the unexercised right leg serving as a contra-lateral control. Mechanical tests were conducted to assess the strength of cancellous bone in the proximal tibia metaphysis. Although isolated specimens of cancellous bone are not feasible, reduced platen compression (RPC) was employed to directly load only the cancellous core region of each specimen. There was no significant difference in ultimate stress or elastic modulus between BL, CC, and HU-Ex (exercised). However, HU-Ex results were dramatically and significantly higher than HU-No Ex (contra-lateral unexercised control) for both ultimate stress (68%) and elastic modulus (81%). It is also notable that ultimate stress was 32% higher (but not statistically significant) for HU-Ex compared to CC. The total bone mineral density in the tibial metaphysis was significantly larger, 11%, in the HUEx compared to the HU-No Ex group's values. The results clearly demonstrate the efficacy of the exercise protocol in preventing the substantial mechanical deterioration induced by HU.

Blood vessel growth is mediated by angiogenesis, which is defined as the formation of new blood vessel out of existing vessels, as well as vasculogenesis, a process that circulating progenitor cells contributes to adult neovascularization. Cilostazol, a commercially available drug holding antiplatelet and vasodilating effects, increases intracellular cyclic adenosine monophosphate (cAMP) levels through inhibiting the activity of phosphodiesterase 3. Interestingly, this chemical compound has a lot of cellular effects. In current work, we demonstrated that cilostazol promoted proliferation and migration of human umbilical cord vein endothelial cells (HUVECs), enhanced in-vitro vascular tube formation, and increased releasing of cAMP and NO from them. Furthermore, cilostazol activated eNOS and PI3-K/Akt signaling pathways. We also examined the angiogenic and vasculogenic effects of cilostazol in a murine hindlimb ischemia model. Our data showed that cilostazol enhanced angiogenesis and vasculogenesis with resultant flow recovery after murine hindlimb ischemia partly mediated by promoting mobilization of bone marrow-derived stem cells into circulation and increasing in situ expression of some proteins involved in angiogenesis. In addition, cilostazol significant increased colony forming unit of human endothelial progenitor cells. These results are unique and clinically significant with potential in translational therapy. According to our report, further preclinical and clinical studies of cilostazol on the other ischemic situations such as myocardial infarction will be justified.

Iroquois (Irx) homeobox genes have important and redundant functions during embryogenesis. Irx3/5 double knock out (Irx3/5 KO) mouse embryos exhibit severe hindlimb phenotypes. In these mutant hindlimbs, digit 1 and tibia are absent, moreover femur and pelvis are hypoplastic. Here, we demonstrate that Irx3/5 are expressed in the hindlimb field prior to limb bud initiation, and are required at this early stage for the pattern formation along the anteroposterior axis. Their early function is involved in prepatterning and positioning the Shh expression domain. In addition, Irx3/5 KO mutant hindlimb buds have a mild outgrowth defect and increased cell death at early stages of limb development, which may explain the small hindlimb bud size in these mutant embryos. To examine whether Irx3/5-expressing cells are the origin of lost and affected structures in Irx3/5 KO mutant hindlimbs, targeting vectors with Cre genes inserted into the Irx5 locus have been generated.

Thesis (Ph. D.)--Northeastern University, 2008. / Title from title page (viewed Aug. 4, 2009) Graduate School of Arts and Sciences, Dept. of Biology. Includes bibliographical references.

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第19278号 / 人健博第30号 / 新制||人健||3(附属図書館) / 32280 / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 市橋 則明, 教授 三谷 章, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 141-150). Also available on the Internet.

Reduction of cardiac myocyte loss and repair of the vasculature post myocardial infarction are important therapeutic goals because the potential for intrinsic repair is limited. Preclinical and limited clinical data support the possibility that bone marrow-derived mesenchymal stem cells may be a suitable cell type for cellular therapy. The goal of this research was to determine the effectiveness of using MSCs from aged mice in cellular therapy for the treatment of AMI. The central hypothesis for this research was that therapeutic potential of mesenchymal stem cells decreases with age. This research utilized global gene expression analysis to investigate molecular differences in MSCs harvested from three different age groups of mice. Microarray analysis was performed to investigate changes in gene expression with respect to aging. Furthermore, both in vitro and in vivo experiments were completed to analyze the functional and molecular characteristics of the MSCs. The data identified age-related defects in mouse MSCs as well as determined the molecular basis for these deficiencies. This study indicates that MSCs from 26m mice are severely deficient in the induction of angiogenesis and cardiac repair due to defective paracrine factor secretion caused by decreased expression of growth factor/cytokine genes. Hypoxia attenuates the deficiency in the aged mice, whereas in young mice low oxygen promotes secretion of paracrine growth factors. It was determined a dysfunction in HIF-1 alpha signaling was present in MSCs from 26m mice and is regulated by the PI3K/Akt signaling in MSCs. Furthermore, two novel and important and novel aspects of this study were the discovery that cell cycle regulation gene expression decreases with age and MSCs have increased insulin resistance with age. Increased insulin resistance in this cell type with aging is likely to have profound effects on the clinical outcomes of using these cells therapeutically. Likewise, loss of cell cycle regulation during proliferation could also lead to undesirable clinical effects. Gaining insight to the repair potential of these cells with respect to age will help to better define future trials of autologous stem cells not only for heart disease but for all of the many applications proposed for these cells.