Muscle atrophy is a serious side effect seen with extended time in space. Proteolytic degradation of specific muscle proteins leads to smaller, weaker muscles that are structurally more susceptible to damage. Calpains are proteases that specifically degrade target proteins of the myofibril, and have been implicated in many types of muscle atrophy. Calpain activity is regulated by a combination of activation by calcium and inhibition by calpastatin, its endogenous inhibitor. This dissertation describes the generation of a skeletal muscle-specific, doxycycline (Dox) controlled, calpastatin over-expression system in transgenic mice to regulate calpain activity. A dual construct system, the transactivator line utilizes an optimized tet-on system and a modified muscle creatine kinase promoter to create muscle specific expression of a tet transactivator. The second transgenic line, consisting of a bi-directional promoter centered on a tet responder element controlling both a luciferase reporter gene and a tagged calpastatin, is transcriptionally silent until activated by a dox induced transactivator molecule. Compound hemizygous mice showed high level, Dox dependent, muscle-specific overexpression of luciferase and transgenic calpastatin, demonstrating the effectiveness and flexibility of the tet-on system to provide a tightly regulated overexpression system in adult skeletal muscle. Consistent overexpression of calpastatin was hard to maintain, however, and not all of the proposed experiments could be achieved. Additional studies compared the degradation of hindlimb suspended mouse muscle proteins (weightlessness model) to those of ground control muscle proteins briefly incubated in Ca²⁺ (to initiate calpain degradation). Four proteins known as targets for calpain degradation were selected for analysis. Degradation responses of myofibrillar proteins titin, nebulin, and troponin T in hindlimb suspension clearly mimicked that seen with the calcium incubations. The cytoskeletal calpain target protein, desmin, however did not respond the same to both treatments showing moderate degradation with calcium and no degradation with hindlimb suspension. These data suggest that myofibrillar calpain target proteins, but not necessarily cytoskeletal proteins, are rapidly targeted for degradation in hindlimb suspension in a manner similar to that induced by calcium, implicating calpain as a mediator of this degradation.

A complex set of developmental events transform early primordia within endocardial cushions into the atrioventricular (AV) valves and septa of the mature heart. Endocardial cushions are composed of an inner endocardial layer, and outer myocardial layer, and an acellular extracellular matrix rich layer separating these two layers. A subset of endocardial cells within cushions transform into mesenchymal cells that migrate into the extracellular matrix and differentiate into mature valve and septum tissue. This cell transformation process coupled with asymmetrical cell proliferation within endocardial cushions remodels simple early endocardial cushions into elongated valves. Investigation into molecular mechanisms involved in valve and septum morphogenesis centered on a family of growth factors, called Wnts, due to previous reports showing involvement of this growth factor family in similar developmental processes. Expression analysis of Wnt growth factors, Wnt receptors, and soluble Wnt inhibitors was performed in search of Wnt signaling components with restricted expression in AV endocardial cushions. Initial RT-PCR analysis demonstrated expression of several Wnts (Wnt-5a, Wnt-6, Wnt-9a, Wnt-10a and Wnt-11), Wnt receptors (Fz-1, Fz-3, Fz-4, and Fz-6) and the soluble Wnt antagonist Frzb in early endocardial cushions. Spatial and temporal expression analysis revealed restricted endocardial cushion expression of Wnt-9a and Frzb. Wnt-9a is expressed in the endocardial cell layer while Frzb is expressed in the endocardium and transformed mesenchyme of AV endocardial cushions. Overexpression of Wnt-9a in endocardial cushions increases cell proliferation resulting in enlarged hypercellular endocardial cushions. Overexpression of a truncated mutant form of Wnt-9a, which acts in a dominant negative manner, increases programmed cell death in endocardial cushions. Overexpression of Wnt-9a also induces β-catenin responsive transcription in AV canals consistent with Wnt-9a signaling in a canonical Wnt/β-catenin pathway. Wnt-9a induced increases in cell proliferation are inhibited by overexpression of Frzb. Spatial expression patterns suggest that Frzb delineates a zone of Wnt-9a induced cell proliferation near the endocardial cell layer to promote endocardial cushion outgrowth and remodeling into mature valve leaflets.

Populations of simultaneously recorded hippocampal place cells were studied in three tasks that required shuttling between a variably placed box and a reward location predicted by fixed or variably located landmarks. Groups of place cells fired at fixed locations relative to the behaviorally relevant landmarks or to the absolute spatial framework of the environment. The initial segment of the journeys was represented by a sequence of cells that fired at fixed distances with respect to the landmark of origin, behind the animal, irrespective of where that landmark was located. The final segments of the journeys were represented by cells that fired at fixed locations relative to the landmark of destination. We propose that on the initial parts of the journey, the spatial coordinate represented by each cell was updated predominantly by path integration. When approaching the landmark of destination, the coordinates were updated and corrected by visual landmarks (or other sensory cues). The intermediate zone was characterized by a transition from the reference frame of the origin to the reference frame of the destination. Small distortion of the environment led to a smooth, continuous transition, while a large distortion led to abrupt transitions. These observations suggest that in the hippocampus, place can be represented in multiple spatial reference frames established by behaviorally relevant landmarks. Further experiments showed that these landmarks, or their sensory properties, are not represented explicitly by place cells, because cells did not maintain their landmark-related firing in a similar task, performed in a different environment.

Restoration of blood flow to the ischemic myocardium is the most effective approach to limit necrosis. However, it appears that the act of reperfusion causes additional tissue damage, and this condition is known as myocardial reperfusion injury. It is well established that leukocytes contribute to additional myocyte and vascular injury during reperfusion, and that this injury occurs within the first minutes of flow restoration. A great deal is known about the leukocyte contribution to myocardial reperfusion injury. However, the mechanisms under which leukocytes accumulate in the coronary microcirculation during early reperfusion are unclear. The purpose of these studies was to identify the mechanisms of leukocyte accumulation in the coronary microcirculation during early reperfusion following ischemia. Using direct visualization techniques, it was observed that leukocyte accumulation in coronary capillaries and venules during the first minutes of reperfusion differs with respect to the magnitude and persistence of accumulation. During reperfusion, significant leukostasis in coronary capillaries occurs in the absence of blood activation, and is significantly enhanced when the blood is activated and when the reperfusion blood flow is moderately reduced. Also, the results indicate that postischemic capillary leukostasis can be attenuated by increasing leukocyte deformability and by inhibition of P- and L-selectin adhesion molecules. In contrast, leukocyte adhesion to the coronary venules during reperfusion is not significantly increased when the blood is not activated and returned at full flow. However, leukocyte adhesion to the venules is enhanced when the blood is activated or when reperfusion blood flow is severely reduced. In addition, under conditions of low reperfusion blood flow, leukocyte adhesion to the venules is attenuated by P- and L-selectin adhesion molecule inhibition. In these experiments, leukocytes remained in the capillaries longer than they remained in the venules during reperfusion. These results indicate that the degree to which leukocyte accumulation occurs in postischemic capillaries and venules is dependent upon the conditions of reperfusion. The results of this study also suggest that attempts to limit early myocardial leukocyte-mediated reperfusion injury should consider leukocyte deformability, the state of activation of the leukocyte, reperfusion blood flow, and selectin-mediated adhesion events.

Objectives were to determine the effects of feeding dry-rolled (DR) vs steam-flaked (SF) sorghum grain and degree of processing (flake density, FD) of corn and sorghum grain on site and extent of starch digestion and post-absorptive metabolism of energy-yielding nutrients in steers fed 77% grain. The design for each trial was a randomized block. Seven steers (400 kg) with duodenal and ileal cannulas were used to determine corn and sorghum starch digestibilities (Cr ratio, 3-d collection). Steers fed SF versus DR sorghum increased starch digestibility in the rumen (23%; P < .01), total tract (2.3%; P < .01), and in the small intestine (6%; P < .01, as percentage of starch entering duodenum). Decreasing flake density of SF sorghum grain increased linearly (P < .05) starch digestion (percentage of intake) in the rumen and total tract, and diminished linearly (P < .05) starch digestibilities post-ruminally and in the small intestine. Similar responses in starch digestibilities occurred by lowering FD of SF corn. Percent dietary corn or sorghum starch digestibility in the large intestine was less than 2% of intake. Feeding SF compared to DR sorghum did not alter net absorption and uptake or release of energy-yielding nutrient across SPL tissues. As expected, net absorption of glucose across portal-drained viscera (PDV) was negative (-.60 mol/d). Incrementally decreasing FD of SF sorghum linearly increased net PDV absorption of lactate (P =.04), glucose synthesis by the liver (P =.03), and SPL output of glucose (P < .01) and L-lactate (P =.03). Net propionate PDV absorption (P =.18), hepatic uptake (P =.21), and SPL output (P =.15) tended to be increased with lower FD. Increasing degree of grain processing, by incrementally decreasing FD, linearly increased ruminal and total tract starch digestibilities and net absorption of glucose precursors (propionate and L-lactate), resulting in increased hepatic synthesis and greater output of glucose from the gut and liver to the rest of the body. Based on these changes, the optimum FD for SF sorghum grain was 283 g/L (SF22).

Three experiments were conducted to determine the effects of bST on lactational performance throughout several stages of lactation, emphasizing advanced lactation and summer heat stress conditions. In the first experiment, a preliminary trial was conducted using seven Holstein cows averaging 424 DIM. Injections of bST increased milk, fat and protein yields, but did not affect milk composition. In the second trial, 24 cows in advanced lactation (292 DIM) with breeding problems were assigned to one of the three treatments in a randomized block design. Treatments were: (1) CON; low energy diet, without bST injection; (2) LED; low energy diet, injected with bST; and (3) HED; high energy diet, injected with bST. Cows showed a large response to bST in yields of milk, FCM, fat and protein, as well as on efficiency of feed conversion to milk. Energy density in the diet of cows receiving bST did not significantly affect any measured variable. In the second experiment, 76 lactating Holstein cows were distributed in three trials according to stage of lactation. Six intervals of DIM were considered: very early, early, mid, late, advanced, and extended lactations. A favorable response in milk yield to bST injections was observed in mid, late, advanced and extended lactations, while only a tendency was observed in early, but not in very early lactation cows. In the third experiment, 32 Holstein cows averaging 195 DIM were placed on four treatments to determine the effect of bST and evaporative cooling on lactational performance during summer heat stress. Milk yield was significantly increased by bST injections and tended to be higher for cooled cows. Fat and protein yields were increased by bST, but not by cooling. Non-esterified fatty acids in blood serum were higher in cows injected with bST, suggesting greater fat mobilization to support the increased milk production. Rectal temperatures and respiration rates were decreased by evaporative cooling, but not affected by bST injections.

Grasshoppers, like most other organisms, require sterols for the production of cellular membranes and, like other animals, as precursors to steroid hormones. However, grasshoppers, like other insects, cannot synthesize sterols de novo and must acquire these essential nutrients from their diets. Survival and performance of five grasshopper species, representing three phylogenetic lineages and with different host preferences, were compared on artificial diets that varied in sterol type. Sterols used were: cholesterol (Δ⁵) sitosterol (Δ⁵) stigmasterol (Δ⁵ʼ²²) spinasterol (Δ⁷ʼ²²) and lathosterol (Δ⁷); these are the most common plant sterols. For all species studied, none survived to the adult stage on stigmasterol, spinasterol or lathosterol. Performance on the cholesterol and sitosterol diets were similar for most species. This study suggests that grasshoppers, as a group, are extremely limited with respect to the sterols they can use. Grasshoppers, however, are highly polyphagous insects, often feeding on many different host-plants. Some of these host-plants contain unsuitable sterols. The effect of sterol mixtures on survival and performance was examined by rearing the grasshopper, Schistocerca americana, on artificial diets containing sitosterol (suitable) and spinasterol (unsuitable) presented either alone or in combination. Results indicated that spinasterol, even in the presence of high levels of a sitosterol, could prevent development to the adult stage. Biochemical analysis of grasshopper tissues revealed that dietary sterols with Δ⁷ and/or Δ²²-configurations could not be metabolized to cholesterol. Unmetabolized dietary sterols tended to accumulate in the midgut tissues. Grasshoppers are known to regulate feeding in response to some nutrients in relation to need. To determine if grasshoppers could regulate intake with respect to different sterols, a series of behavioral assays was performed. The grasshopper S. americana was observed feeding on artificial diets with different sterols, in some cases up to seven hours. Results suggest that S. americana developed aversion responses to diets that contained unsuitable dietary sterols (Δ⁷ and/or Δ²²) Additional experiments suggested that the aversion response developed through a combination of post-ingestive feedbacks and associative learning. Taken together, these results suggest that sterol metabolic limitations may partially determine host-plant affiliations in grasshoppers.

The differentiation of the gonads begins at mid-gestation when sex differences are first identified histologically. Prior to gonadal differentiaton, male and female embryos are phenotypically indistinguishable. After gonadal differentiation, males are identified by the presence of testes and females by the presence of ovaries. The Sry gene located on the Y chromosome has been identified as the testis determining gene. Evidence of Sry's role as the testis determining gene being both necessary and sufficient was proven by the development of testes in XX female mice transgenic for Sry. Transcription of Sry, thought to be critical for testicular differentiation, is hypothesized to be limited to the male gonad at the time of gonadal differentiation. We will demonstrate that transcription of Sry is not limited to the time of gonadal differentiation, but is detectable throughout the preimplantation period. Therefore, detection of transcription of Sry may be indicative of sex determining events occurring prior to gonadal differentiation in the preimplantation mouse embryo. This dissertation addresses the expression and function of Sry in the mouse preimplantation embryo. Furthermore, this dissertation addresses the sex dimorphism of a faster growth rate in male versus female embryos and a hypothesized relationship between growth rate and gonadal differentiation. Our hypotheses are that sex determining events occur prior to gonadal differentiation in the preimplantation embryo and/or that Sry has additional developmental roles in embryogenesis. Furthermore, the faster growth of males predisposes the XY gonad to differentiate into a testis. My conclusions are: (1) That Sry transcription is not limited to the time of gonadal differentiation, thus sex determining events may occur earlier. This is based on my findings that Sry mRNA is detectable throughout the preimplantation period and that at the blastocyst stage the linear Sry transcript is found on polyribosomes. In addition, my findings indicate that the faster growth of the male preimplantation embryo may contribute to the male gonad differentiating into a testis. (2) That Sry or Sry-like Sox genes play a developmental role in ovarian differentiation. This is based on my creation of female mice transgenic for an antisense oriented Sry transgene developing supernumerary ovaries.

This investigation was designed to examine speech production, the motion of the chest wall, and breathing-related perceptions under a condition of heightened respiratory drive. Ten healthy young men were studied during spontaneous breathing and during speaking in three gas conditions: room air, air delivered from a pressurized tank, and a gas mixture high in carbon dioxide (7% CO₂) delivered from a pressurized tank. Magnetometers that transduced diameter changes of the rib cage and abdomen were used to study chest wall motion. Subjects also reported their breathing-related perceptions. Results indicate that chest wall behavior during spontaneous breathing and speaking did not differ between room-air and tank-air conditions, but differed substantially in the high-CO₂ condition. In the high-CO₂ condition, spontaneous breathing and speaking usually were characterized by larger lung volumes, larger rib cage volumes, higher breathing rates, longer expiratory times, and higher expiratory flows than in the two air conditions. Further, speaking in high-CO₂ was characterized by shorter speech duration, fewer syllables per breath group, and greater average lung volume expenditure per syllable compared to speaking in air. In high-CO₂, subjects reported a range of breathing-related percepts including "breathlessness," "effortful breathing," and "gasping for air." Without exception, speaking in high-CO₂ was judged by the subjects to be more difficult than breathing in high-CO₂.

A model of the human head and neck that incorporates active and passive muscles is presented for use in the analysis of non-impact loading in high "g" environments. The inclusion of the active muscles is shown to improve the accuracy of the model response to impulse loading conditions, especially during the interval that begins approximately 120 ms after the application of the impulse. The active muscles have the capability of activating partially and in different combinations. The parameters defining the force-length and force-velocity relationships used for the active muscles are based on recent experimental data for neck muscles, including: muscle length, sarcomere length, and physiological cross-sectional area. The parameters, which fall within a large range of input selections, are applied to the fifteen muscle pairs of the neck. The model is implemented in MADYMO using lumped parameters and Hill muscles. A comparison of simulation results with experimental data, generated by the Naval Biodynamics Laboratory, shows excellent agreement for a flexion/rebound 15 g impulsive load and a 7 g lateral load. A model incorporating the head and neck with active muscles is implemented with a body of a 50th percentile male anthropomorphic test device. This model is subjected to the 15 g flexion impulsive load and also shows excellent agreement with the experimental data.