Hepatocytes exhibit a regulatory volume decrease (RVD) during hypotonic shock, which comprises loss of intracellular K+ and Cl- accompanied by hyperpolarization of transmembrane potential (Vm) due to an increase in membrane K+ conductance, (GK). To examine hepatocyte K+ homeostasis during RVD, double-barrel, K+-selective microelectrodes were used to measure changes in steady-state intracellular K+ activity (aKi) and Vm during hyposmotic stress. Cell water volume change was evaluated by measuring changes in intracellular tetramethylammonium (TMA+). Liver slices were superfused with modified Krebs physiological salt solution. Hyposmolality (0.8×300 mosm) was created by a 50 m m step-decrease of external sucrose concentration. Hepatocyte Vm hyperpolarized by 19 mV from -27 ± 1 to -46 ± 1 mV and aKidecreased by 14% from 91 ± 4 to 78 ± 4 m m when slices were exposed to hyposmotic stress for 4-5 min. Both Vm and aKireturned to control level after restoring isosmotic solution. In paired measurements, hypotonic stress induced similar changes in Vm and aKiboth control and added ouabain (1 m m) conditions, and these values returned to their control level after the osmotic stress. In another paired measurement, hypotonic shock first induced an 18-mV increase in Vm and a 15% decrease in aKiin control condition. After loading hepatocytes with TMA+, the same hypotonic shock induced a 14-mV increase in Vm and a 14% decrease in aTMAi. This accounted for a 17% increase of intracellular water volume, which was identical to the cell water volume change obtained when aKiwas used as the marker. Nonetheless, hyposmotic stress-induced changes in Vm and aKiwere blocked partly by Ba2+ (2 m m). We conclude that (i) hepatocyte Vm increases and aKidecreases during hypotonic shock; (ii) the changes in hepatocyte Vm and aKiduring and after hypotonic shock are independent of the Na+-K+ pump; (iii) the decrease in aKiduring hypotonic stress results principally from hepatocyte swelling.

The hypothesis that calcium influxes from the extracellular space play an important role in the pathogenesis of irreversible anoxic injury was tested using isolated adult rat myocytes. Myocytes treated with 6 mm amytal and 3 mm iodoacetate and subsequently incubated in either calcium-containing (1.12 mm) or calcium-free media (with or without 1 mm EGTA) developed rigor contracture (cell squaring) and cell death (trypan blue permeability) at the same rate. The rates of cell death in both calcium-containing and calcium-free media were increased by incubation in hypotonic media even though the rates of contracture development remained unaltered. Cells developed osmotic fragility prior to membrane permeability increases. The calcium ionophore, A23187 (10 μm), induced rapid rounding of rod-shaped cells subjected only to mitochondrial inhibition in calcium containing media, confirming its ability to cause an increase in cellular permeability to calcium. However, A23187 did not alter the rates of cell death of totally metabolically inhibited myocytes in either calcium-containing or calcium-free media with EGTA. The results indicate that influxes of calcium are not necessary for the development of irreversible injury in metabolically inhibited, isolated myocytes.

Poor emergence of sorghum [(Sorghum bicolor (L) Moench] affects the stand and potential yields. The major objective of this research was to find correlations among field emergence data and laboratory seed vigor tests. Thirty-two and 30 hybrids of sorghum were planted at three Virginia locations in 1995 and 1996, respectively. Field emergence was subsequently compared with results from laboratory tests that included: 1) standard germination; 2) osmotic-stress using polyethylene glycol 8000 (mw); 3) heat-shock using 2 hr at 50°C stress; 4) electrical conductivity of steep water of germinating seeds; and 5) seed mass. Field emergence of grain sorghum differed among hybrids, years and locations. Mean emergence across years and locations was 67.5%, whereas mean germination in the laboratory was 87.8%. There were interaction between hybrid and location and between hybrid and year. Germination under optimal conditions (standard germination test) and with osmotic or heat-shock stress differed among hybrids. Regression analyses showed a weak correlation between laboratory germination (with or without stress) and field emergence in both years. The fresh weight of seedlings whether from standard germination or stress tests also differed among hybrids in both years, and the associations with field emergence were weakly correlated in 1996. Hybrids showed significant differences in radicle length when grown under laboratory stress in both years following standard germination. There was a weak correlation with field emergence and radicle length following heat-shock treatment in 1996. The measurement of electrical conductivity in the seed steep water showed significant differences among hybrids. A weak correlation with field emergence was seen in 1996. Conductivity values per gram of seed and per cm² of seed area revealed differences among hybrids. The correlation of these parameters with field emergence was higher than conductivity per seed. Seed mass varied among hybrids in both years , but was no correlation between seed mass and emergence. Of the laboratory parameters examined, germination proved to be the most consistent predictor of variations in field emergence of sorghum hybrids. / Ph. D.

<p> Abiotic stresses such as extreme temperatures, drought and high salinity severely compromise plant productivity, and have placed selective pressure for the acquisition of traits enabling plants to adjust to and recover from these unfavorable environmental conditions. Thellungiella salsuginea is a plant that is native to highly saline and semiarid environments and exhibits an exceptional ability to tolerate abiotic stress. In this thesis, I report on laboratory and field studies aimed at identifying traits that allow Thellungiella to tolerate harsh environmental conditions. It was found that Thellungiella accumulates organic solutes in response to abiotic stress. Transcript and metabolite profiling approaches were used to identify metabolic pathways important for the accumulation of compatible organic solutes in Thellungiella in response to sub-optimal environmental conditions. The relative abundance of transcripts encoding enzymes associated with the biosynthesis of compatible organic solutes such as proline or galactinol showed stress-responsive increases in cabinet-grown material and these metabolites were accumulated in salt or drought treated plants, respectively. However, proline and galactinol were found to be of low relative abundance in leaves of field plants. In contrast, several carbohydrates including sucrose, glucose, and fructose made a greater relative contribution to the field plant profiles suggesting that carbohydrates play an important role in plant abiotic stress tolerance during growth under field conditions. The identification of stress-specific metabolic changes can be used to identify important biochemical traits underlying environmental stress tolerance in Thellungiella. This information can be used to improve the tolerance of stress -sensitive crops (including a related crucifer species, canola) that are grown in areas where persistent droughts, saline soils and early or late frosts frequently occur. </p> / Thesis / Doctor of Philosophy (PhD)

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Recovering valuable resources from wastewater will transform wastewater management from a treatment focused to sustainability focused strategy, and creates the need for new technology development. An innovative treatment concept - osmotic bioelectrochemical system (OsBES), which is based on cooperation between bioelectrochemical systems (BES) and forward osmosis (FO), has been introduced and studied in the past few years. An OsBES can accomplish simultaneous treatment of wastewater and recovery of resources such as nutrient, energy, and water (NEW). The cooperation can be accomplished in either an internal (osmotic microbial fuel cells, OsMFC) or external (microbial electrolysis cell-forward osmosis system, MEC-FO) configuration. In OsMFC, higher current generation than regular microbial fuel cell (MFC) was observed, resulting from the lower resistance of FO membrane. The electricity generation in OsMFC could greatly inhibit the reverse salt flux. Besides, ammonium removal was successfully demonstrated in OsMFC, making OsMFCs a promising technology for "NEW recovery" (NEW: nutrient, energy and water). For the external configuration of OsBES, an MEC-FO system was developed. The MEC produced an ammonium bicarbonate draw solute via recovering ammonia from synthetic organic solution, which was then applied in the FO for extracting water from the MEC anode effluent. The system has been advanced with treating landfill leachate. A mathematical model developed for ammonia removal/recovery in BES quantitatively confirmed that the NH4+ ions serve as effective proton shuttles across cation exchange membrane (CEM). / Ph. D.

Joint-head grass (Arthraxon hispidus) is a widespread nonindigenous plant species in the eastern United States. It is observed forming large monodominant patches that impact native and managed grassland systems. With such little understanding of its foundational biology or ecological impacts, leaving land managers desperate for effective control measures to manage A. hispidus invasion. We conducted a series of complementary experiments on six populations of A. hispidus to better understand how environmental factors affect seed germination. Germination is a critical life stage that allows a species to disperse. Freshly harvested seeds germinated effectively within 14 days of imbibition in the dark at 23°C, exhibiting little to no dormancy or influence by light. A pH range of 5 - 10 resulted in ≥ 80% germination, suggesting that pH will not limit colonization in other portions of the United States. Arthaxon hispidus was tolerant to abiotic stressors such as salinity and osmotic potential. The concentration required to limit germination to 50% (LD50) in the populations tested surpassed soil salinity found in the contiguous United States and some tidal systems (Frederick, MD = 354; Lincoln, MO = 354; Williamsburg, VA = 298 mM NaCl). While drought adversely affects A. hispidus germination, the LD50 occurred in moderate to more severe osmotic potentials (Frederick, MD = -0.67; Lincoln, MO = -0.37; Williamsburg, VA = -0.25 MPa) making A. hispidus expansion more likely in wetter years and regions. Constant temperature treatments resulted in germination percentages across a range of temperatures (8 - 37°C), and A. hispidus is well distributed in several major temperature regimes found in the United States. Finally, emergence greatly decreased with burial depth. Emergence occurred at ≥ 43% at 1 - 2 cm, decreasing to 5% at 6 cm, and 0% at 8-cm depths. With adequate soil moisture, a broad range of germination temperatures, and a decreased emergence rate with depth, we believe A. hispidus is unlikely to develop a seed bank. These initial studies on A. hispidus' germination posit a broad range of environmental tolerances; although, it may be limited by other life stages. / Master of Science / Joint-head grass is a non-native invasive plant species commonly found in the eastern United States. It is observed growing in large patches that negatively affect the environment. These effects can include decreasing biodiversity or lowering forage availability which negatively can impact cattle production. To better understand the basic biology of joint-head grass, we decided to harvest seeds to study from several populations in the United States. We tested differences in the populations by examining the different environmental effects on joint-head grass germination. Germination is a critical life stage of invasive plants; therefore, we developed complementary experiments to test the effects of the environment on seed germination. We've determined that germination occurs effectively under the effect of several environmental stressors. Germination occurred under salty (NaCl) conditions which may allow it to establish in tidal systems where brackish water is present. We've also determined that germination occurs effectively ( ≥ 80%) at a pH range from 5 - 10. This will allow joint-head grass to germinate in more basic soils commonly found in the western part of the United States. Further, we tested the moisture requirements for germination to occur, and we've found that it is tolerant to moderate to more severe drought conditions. Joint-head grass is also capable of germinating across a range of temperatures (8-37°C). The United States has well-distributed rainfall and suitable temperatures in large portions of the country. We believe the climate of the United States is well-suited for joint-head grass establishment, and that it may spread more frequently in years with higher precipitation during the growing season. This species potentially poses a threat to both our natural and agricultural systems.

The fate of E. coli O157:H7 and Salmonella spp. during osmotic dehydration of apples was determined at different processing temperatures, times and calcium chloride (CaCl2) concentrations. Apple slices were inoculated to achieve an 8 log CFU/ apple slice concentration of a five strain mixture of E. coli O157:H7 or Salmonella spp. and were soaked in sucrose solutions (60% w/w). In the first study, apple slices were subjected to osmotic dehydration at three different temperatures: 20°C, 45°C and 60°C. In a second study, CaCl₂ was added in the sucrose solution at concentrations of 2%, 4% and 8% to determine its efficacy as an antimicrobial agent. The storage effect of osmotic dehydrated apples on pathogen survival was also tested for seven days at 4°C. Samples were withdrawn at appropriate time intervals, diluted with 0.1% peptone water and surface plated onto recovery media. Recovery of E. coli O157:H7 was compared on Tryptic Soy Agar + 50 ppm nalidixic acid (TSAN) and MacConkey Sorbitol agar (MCS). Recovery of Salmonella was compared on TSAN and XLD agar. There was lower microbial reduction at the lower temperatures tested with approximately 1.0 and 3.0 log CFU/apple slice reduction at 20°C and 45°C, respectively. The population reduction of cells was highest at 60°C, with an approximate five log reduction for both microorganisms (P<0.001). CaCl₂ used as an additive in the osmotic solution, was associated with slightly higher reduction of both E. coli O157:H7 and Salmonella spp. Greater than a 5 log reduction was observed when the combination of CaCl₂ (8%) and 60°C processing temperature was used. During refrigerated storage E. coli O157:H7 and Salmonella decreased by approximately 4.5 log CFU/apple slice, but were still recoverable via direct plating at Day seven. The results of this study show that the survival of E. coli O157:H7 and Salmonella in osmotically dehydrated fruit is influenced by the osmotic processing method used and the level of additive (i.e., CaCl₂) utilized. Parameters associated with decreased survival of pathogens, and therefore, improve product safety, include increasing temperature and time of processing and increasing concentration of CaCl₂. However, E. coli O157:H7 and Salmonella in artificially contaminated apple slices, survived osmotic dehydration processing and subsequent storage under processing and storage parameters of this study. Therefore, processors who produce osmotically dehydrated fruit must consider the potential food safety impact of the osmotic dehydration processes they choose. / Master of Science

Mathematical modelling of cellular osmotic responses to low temperatures is being increasingly used to overcome obstacles in the successful cryopreservation of cells and tissues. Current cryobiological models often contain simplifying assumptions regarding the solution behaviour of the complicated, multisolute intra- and extra-cellular solutions. In order to obtain more accurate predictions of cryobiological outcomes, equations derived from thermodynamic principles that more accurately describe the biological solution behaviour could be used to greatly advance the design of novel cryopreservation protocols. The general hypothesis of this thesis is that the application of the multisolute osmotic virial equation, with mixing rules derived from thermodynamic first principles, to solutions of interest in cryobiology will result in more accurate predictions of the multisolute solution behaviour, which will lead to improved cryobiological modelling and increased understanding of cellular responses to cryopreservation. Specifically, this thesis demonstrates that the osmotic virial coefficients, obtained from single-solute solution data, can be used in the multisolute osmotic virial equation to accurately predict the multisolute solution behaviour, without the need to fit multisolute solution data. The form of the multisolute osmotic virial equation proposed in this thesis was used to predict the solution behaviour of a range of multisolute solutions of interest in cryobiology. The equation commonly used in cryobiology to describe cellular osmotic equilibrium is based on ideal, dilute solution assumptions. In this thesis, a non-ideal osmotic equilibrium equation was derived and, combined with the multisolute osmotic virial equation, used to more accurately predict the osmotic equilibrium of human erythrocytes. The improved equations proposed in this thesis were combined with experimental measurements of the incidence of intracellular ice formation in order to further the understanding of the role of several important cryobiological parameters on the formation of intracellular ice. This thesis work has significantly contributed to the field of cryobiology by substantially improving the accuracy of two key equations used in the modelling of cellular osmotic responses to cryopreservation. The combination of accurate mathematical modelling and results from experiments will allow increased understanding of cellular responses to cryopreservation, leading to the design of novel cryopreservation protocols. / Chemical Engineering and Medical Sciences