Cold hardy Japanese bunching onion (Allium fistulosum L.) was used as a novel model system to examine the role of the apoplast in intracellular freezing avoidance. Since intracellular freezing avoidance is critical to both sensitive and resistant plants, non-acclimated and cold acclimated onion tissue was compared. The large 250 (length) x 50 (width) x 90 µm (thickness) intact single cell epidermal layer system allowed direct observation of the freezing process during freezing and thawing on a single cell basis in live intact tissues in non-acclimated (23/18°C) and acclimated (2 weeks at 12/4°C) plants. Under acclimation treatment, freezing resistance increased from an LT50 of -12°C at zero time to -27°C after two weeks exposure. Ice nucleation was always initiated in the apoplastic space in both non-acclimated and acclimated cells and then penetrated into the intracellular space. We provide direct evidence that lethal intracellular freezing damage was avoided in cold acclimated and CaCl2 treated cells through visible blockage of ice propagation from the apoplastic space to the intracellular region. Subsequent investigation of the apoplastic space revealed cold acclimation increased: a) the insoluble crude cell wall and pectin (galacturonic acid) content, b) total cell wall protein quantity, c) pectin methylesterase activity in the apoplastic space particularly in epidermal cells compared to non-epidermal cells, and d) the levels of un-methylated pectin. The increasing freezing resistance of Japanese bunching onion more depends on the efficiency of PME not only the increase in the amount of apoplast compositions. Collectively, these changes likely led to the observed significant reduction in cell wall permeability. In this way, freezing stress resistance may potentially be increased through avoidance of intracellular freezing by altering key factors related to apoplast permeability and blocking ice propagation across the cell wall.

Abstract The aim of this trial was to evaluate the effect of embryo cryopreservation techniques on the survivability of embryos and fertility following transfer to Boer goat does. The oestrous cycles of 27 mature recipients Boer goat does were synchronised using controlled internal drug release dispensers (CIDR’s) for 16 days. At CIDR removal, does were injected with 300 IU eCG. The recipient does were allocated to 3 groups (n = 9 per group), based on the technique of cryopreservation used for the embryos transferred. The in vivo produced embryos used were at blastocyst stage and surgically collected on day 6 following AI from Boer goat donors superovulated with pFSH. The first group received fresh embryos and served as the control, the second group of does received conventional slow frozen/thawed embryos and the third group received vitrified/thawed embryos. Two blastocysts were transferred per doe. A pregnancy rate of 85.7% (n = 6) was obtained following the transfer of fresh embryos and tended to be better than in does receiving slow frozen and vitrified embryos, (n = 4; 50.0% and n = 3; 37.5% does pregnant, respectively). The overall gestation period recorded for all does was 146.3 ± 3.0 d, with an overall litter size of 1.7 ± 0.5 being recorded. The kidding rate of the recipient does declined to 57.0% (4) and 25.0% (2) for fresh and conventional slow frozen groups, respectively. An embryo survival rate of 35.7% (n = 5) for fresh, 25.0% (n = 4) for conventional slow freezing and 31.3% (n = 5) for vitrification was recorded and was not affected by the number of CL’s present on the respective ovaries at the time of transfer. There was a tendency for more females to be born than males (ratio 1 : 2, male : female) but this could not be related to the cryopreservation technique. Although the pregnancy rate following the transfer of fresh embryos was satisfactory, the embryo survival rate following the transfer of either fresh or cryopreserved embryos tended to be less acceptable. More research is warranted with larger numbers of animals, directed at improving the survivability of embryos following fresh and cryopreserved goat embryo transfer.

Abstract The aim of this trial was to evaluate the effect of embryo cryopreservation techniques on the survivability of embryos and fertility following transfer to Boer goat does. The oestrous cycles of 27 mature recipients Boer goat does were synchronised using controlled internal drug release dispensers (CIDR’s) for 16 days. At CIDR removal, does were injected with 300 IU eCG. The recipient does were allocated to 3 groups (n = 9 per group), based on the technique of cryopreservation used for the embryos transferred. The in vivo produced embryos used were at blastocyst stage and surgically collected on day 6 following AI from Boer goat donors superovulated with pFSH. The first group received fresh embryos and served as the control, the second group of does received conventional slow frozen/thawed embryos and the third group received vitrified/thawed embryos. Two blastocysts were transferred per doe. A pregnancy rate of 85.7% (n = 6) was obtained following the transfer of fresh embryos and tended to be better than in does receiving slow frozen and vitrified embryos, (n = 4; 50.0% and n = 3; 37.5% does pregnant, respectively). The overall gestation period recorded for all does was 146.3 ± 3.0 d, with an overall litter size of 1.7 ± 0.5 being recorded. The kidding rate of the recipient does declined to 57.0% (4) and 25.0% (2) for fresh and conventional slow frozen groups, respectively. An embryo survival rate of 35.7% (n = 5) for fresh, 25.0% (n = 4) for conventional slow freezing and 31.3% (n = 5) for vitrification was recorded and was not affected by the number of CL’s present on the respective ovaries at the time of transfer. There was a tendency for more females to be born than males (ratio 1 : 2, male : female) but this could not be related to the cryopreservation technique. Although the pregnancy rate following the transfer of fresh embryos was satisfactory, the embryo survival rate following the transfer of either fresh or cryopreserved embryos tended to be less acceptable. More research is warranted with larger numbers of animals, directed at improving the survivability of embryos following fresh and cryopreserved goat embryo transfer.

The responses of Chlamydomonas reinhardtii CW15+ (a wall-less mutant) to freezing stress have been examined: Tests following freezing of bulk (0.5ml) samples reveal the existence of an optimum cooling rate f o r preservation of viability, close to l*C min-1. Direct observation of cells during freezing and thawing on a cryo light microscope have allowed different forms of injury to be classified. At suboptimal cooling rates, reduction in surface area during shrinkage can be achieved by severe distortion or by the formation of layered stacks of membrane. Lethal injury is not evident until thawing takes place. The manifestation of injury depends upon the severity of the freezing treatment. The first symptom of injury is membrane blebbing at the cell surface, leading to swelling of the entire cell, followed by collapse. The membrane involved in the swelling might originate i n the mitochondrion and chloroplast envelope. Regions of fusion between membrane of these organelles and the plasma membrane have been observed during rewarming by electron microscopy. Fluorescent markers of the mitochondrial membrane have been detected i n the membrane involved in blebbing. Further damage during slow cooling contributes to rapid lysis on re-expansion. If the freezing treatment is still more severe, osmotic unresponsiveness will result. At supraoptimal cooling rates loss inviability is associated with intra cellular freezing. These responses have been examined in the light of available data regarding the freeze-thaw responses of higher plant protoplasts . The first symptom of damage seen in 'C reinhardtii ie. that of blebbing and massive swelling, is not common to both systems. Thus general rules concerning the responses of plant cells to freezing stresses cannot be made by using either of these protoplasts as a model system, although each may be useful for study of particular aspects of higher plant and algal cell freezing as separate investigations.

No description available.

The incidence of restenosis has been shown to be correlated with the overstretching of the arterial wall during an angioplasty procedure. It has been proposed that slow balloon inflation results in lower intramural stresses, therefore minimizing vascular injury and restenosis rate. The analysis of the biomechanics of the arterial tissue might contribute to understand which factors trigger restenosis. However, few mechanical data are available on human arteries because of the difficulty of testing artery samples often obtained from autopsy while arteries are still considered "fresh". Various solutions mimicking the physiological environment have been used to preserve artery samples from harvesting to testing. In vitro mechanical testing is usually preferred since it is difficult to test arteries in vivo. Uniaxial and biaxial testing has been used to characterize anisotropic materials such as arteries, although methodological aspects are still debated. Several objectives were formulated and analyzed during the making of this thesis. In one study, the effect of deformation rate on the mechanical behavior of arterial tissue was investigated. The effect of several preservation methods, including cryopreservation, on the mechanical properties of porcine thoracic aortas was also analyzed. Finally, the differences in the mechanical behavior between three different types of sample geometry and boundary conditions were compared under uniaxial and equi-biaxial testing. Thoracic aortas were harvested within the day of death of pigs from a local slaughterhouse. Upon arrival, connective tissue was removed from the external wall of the artery. Then the artery was cut open along its length and cut out in rectangular samples for uniaxial testing, and square and cruciform samples for biaxial testing. Samples belonging to the freezing effect study were preserved for two months at -20°C and -80°C in isotonic saline solution, Krebs-Henseleit solution with 1.8 M dimethylsulfoxide, and dipped in liquid nitrogen. Samples belonging to the deformation rate effect study were tested uniaxially and equi-biaxially at deformation rates from 10 to 200 %/s. The uniaxial and biaxial experiments were simulated with the help of an inverse finite element software. The use of inverse modeling to fit the material properties by taking into account the non-uniform stress distribution was demonstrated. A rate-dependent isotropic hyperelastic constitutive equation, derived from the Mooney-Rivlin model, was fitted to the experimental results (i.e. deformation rate study). In the proposed model, one of the material parameters is a linear function of the deformation rate. Overall, inverse finite element simulations using the proposed constitutive relation accurately predict the mechanical properties of the arterial wall. In this thesis, it was found that easier attachment of samples (rectangular and cruciform) is accomplished using clamps rather than hooks. It was also found that the elastic behavior of arteries is nonlinear and non-isotropic when subjected to large deformations. Characterization of the arterial behavior at large deformations over a higherdeformation range was achieved using cruciform samples. The mechanical properties of arteries did not significantly change after preservation of arteries for two months. Under uniaxial and biaxial testing, loading forces were reduced up to 20% when the deformation rate was increased from 10 to 200 %/s, which is the opposite to the behaviour seen in other biological tissues. The differences observed in the mechanical behavior of fresh and thawed samples were not significant, independently of the storing medium or freezing temperature used. The lack of significant differences observed in the freezing study was likely due to the small number of samples tested per storing group. Further studies are required to clarify the impact of cryopreservation on extracellular matrix architecture to help tailor an optimized approach to preserve the mechanical properties of arteries. From the results obtained in the deformation rate study, it is concluded that the stiffness of arteries decreases with an increase in the deformation rate. In addition, the effect of deformation rate was observed to be higher than the effect of anisotropy. The inverse relationship between stiffness and deformation rate raises doubts on the hypothesized relationship between intramural stress, arterial injury, and restenosis.

The need for improved cloud parameterisations in a road surface temperature model is demonstrated. Case studies from early 1994 are used to investigate methods of tracking cloud cover using satellite imagery and upper level geostrophic flow. Two of these studies are included in this thesis. Errors encountered in cloud tracking methods were investigated as well as relationships between cloud height and pixel brightness in satellite imagery. For the first time, a one dimensional energy balance model is developed to investigate the effects of erroneous cloud forecasts on surface temperature. The model is used to determine detailed dependency of surface freezing onset time and minimum temperature on cloud cover. Case studies from the 1995/96 winter in Devon are undertaken to determine effects of differing scenarios of cloud cover change. From each study, an algorithm for predicting road surface temperature is constructed which could be used in future occurrences of the corresponding scenario of the case study. Emphasis is strongly placed on accuracy of predictions of surface freezing onset time and minimum surface temperature. The role o f surface and upper level geostrophic flow, humidity and surface wetness in temperature prediction is also investigated. In selected case studies, mesoscale data are also analysed and compared with observations to determine feasibility of using mesoscale models to predict air temperature. Finally, the algorithms constructed from the 1995/96 studies are tested using case studies from the 1996/97 winter. This winter was significantly different from its preceding one which consequently meant that the algorithm from only one scenario of the 1995/96 winter could be tested. An algorithm is also constructed from a 1996/97 winter case study involving a completely different scenario Recommendations for future research suggest testing of existing algorithms with guidance on additional scenarios.

The incidence of restenosis has been shown to be correlated with the overstretching of the arterial wall during an angioplasty procedure. It has been proposed that slow balloon inflation results in lower intramural stresses, therefore minimizing vascular injury and restenosis rate. The analysis of the biomechanics of the arterial tissue might contribute to understand which factors trigger restenosis. However, few mechanical data are available on human arteries because of the difficulty of testing artery samples often obtained from autopsy while arteries are still considered "fresh". Various solutions mimicking the physiological environment have been used to preserve artery samples from harvesting to testing. In vitro mechanical testing is usually preferred since it is difficult to test arteries in vivo. Uniaxial and biaxial testing has been used to characterize anisotropic materials such as arteries, although methodological aspects are still debated. Several objectives were formulated and analyzed during the making of this thesis. In one study, the effect of deformation rate on the mechanical behavior of arterial tissue was investigated. The effect of several preservation methods, including cryopreservation, on the mechanical properties of porcine thoracic aortas was also analyzed. Finally, the differences in the mechanical behavior between three different types of sample geometry and boundary conditions were compared under uniaxial and equi-biaxial testing. Thoracic aortas were harvested within the day of death of pigs from a local slaughterhouse. Upon arrival, connective tissue was removed from the external wall of the artery. Then the artery was cut open along its length and cut out in rectangular samples for uniaxial testing, and square and cruciform samples for biaxial testing. Samples belonging to the freezing effect study were preserved for two months at -20°C and -80°C in isotonic saline solution, Krebs-Henseleit solution with 1.8 M dimethylsulfoxide, and dipped in liquid nitrogen. Samples belonging to the deformation rate effect study were tested uniaxially and equi-biaxially at deformation rates from 10 to 200 %/s. The uniaxial and biaxial experiments were simulated with the help of an inverse finite element software. The use of inverse modeling to fit the material properties by taking into account the non-uniform stress distribution was demonstrated. A rate-dependent isotropic hyperelastic constitutive equation, derived from the Mooney-Rivlin model, was fitted to the experimental results (i.e. deformation rate study). In the proposed model, one of the material parameters is a linear function of the deformation rate. Overall, inverse finite element simulations using the proposed constitutive relation accurately predict the mechanical properties of the arterial wall. In this thesis, it was found that easier attachment of samples (rectangular and cruciform) is accomplished using clamps rather than hooks. It was also found that the elastic behavior of arteries is nonlinear and non-isotropic when subjected to large deformations. Characterization of the arterial behavior at large deformations over a higherdeformation range was achieved using cruciform samples. The mechanical properties of arteries did not significantly change after preservation of arteries for two months. Under uniaxial and biaxial testing, loading forces were reduced up to 20% when the deformation rate was increased from 10 to 200 %/s, which is the opposite to the behaviour seen in other biological tissues. The differences observed in the mechanical behavior of fresh and thawed samples were not significant, independently of the storing medium or freezing temperature used. The lack of significant differences observed in the freezing study was likely due to the small number of samples tested per storing group. Further studies are required to clarify the impact of cryopreservation on extracellular matrix architecture to help tailor an optimized approach to preserve the mechanical properties of arteries. From the results obtained in the deformation rate study, it is concluded that the stiffness of arteries decreases with an increase in the deformation rate. In addition, the effect of deformation rate was observed to be higher than the effect of anisotropy. The inverse relationship between stiffness and deformation rate raises doubts on the hypothesized relationship between intramural stress, arterial injury, and restenosis.

Thesis (Ph. D.)--University of Minnesota, 1924. / Biographical sketch. "Literature cited": leaves 85-88.

Thesis (Ph. D.)--University of Minnesota, 1924. / Biographical sketch. "Literature cited": leaves 85-88.