Made available in DSpace on 2014-06-11T19:27:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-01Bitstream added on 2014-06-13T20:56:11Z : No. of bitstreams: 1 maziero_rrd_me_botfmvz.pdf: 928128 bytes, checksum: 359ec822b473e1e1a55462f120819df5 (MD5) / A criopreservação de sêmen equino mostra-se como importante instrumento no ganho genético de rebanhos pela maximização do uso de bons reprodutores. Porém, nos programas de inseminação artificial, a utilização de espermatozóides criopreservados apresenta índices de fertilidade inferiores aos obtidos com sêmen a fresco, constituindo um dos maiores entraves à plena difusão dessa biotecnologia. Assim, o experimento 1 teve como objetivo avaliar o movimento espermático pelo sistema computadorizado (CASA) e a integridade da membrana plasmática por microscopia de epi-fluorescência de espermatozóides equino congelados em palhetas de 0,5 e 0,25 mL, em diferentes taxas de congelação na viabilidade espermática. Para desenvolver o projeto foram utilizadas três metodologias de congelação: o método convencional em caixa de isopor e dois equipamentos automatizados, um deles a máquina Mini Digitcool® (IMV - Technologies - França), que possibilita uma variação de -10ºC a -60ºC por minuto e o outro equipamento TK® 4000C (TK Equipamentos para Congelação) que possibilita uma variação de -10ºC a -40ºC por minuto. Para tanto foram utilizados 3 ejaculados de 4 garanhões de diferentes raças, para cada protocolo de congelação, sendo o protocolo 1: caixa de isopor x máquina TK® 4000C na taxa de -20ºC/min x máquina Mini Digitcool® na taxa de -20ºC/min; protocolo 2: caixa de isopor x máquina TK® 4000C na taxa de -40ºC/min x máquina Mini Digitcool® na taxa de -40ºC/min e protocolo 3: caixa de isopor x máquina TK® 4000C na taxa de -40ºC/min x máquina Mini Digitcool® na taxa de -60ºC/min. Para a análise pós-descongelação as palhetas de 0,5 mL foram descongeladas a 46ºC por 20 segundos e as palhetas de 0,25 mL a 46ºC por 15 segundos. A análise estatística das variáveis estudadas no sêmen congelado/descongelado foi realizada mediante o pacote... / Equine semen cryopreservation comes as an important tool to enhance the herd gene pool by maximizing the use of the top genetic merit stallions. However, the use of frozen semen in the artificial insemination programs presents reduced fertility rates in comparison to those obtained with fresh semen, being one of the biggest hindrances to spread this biotechnology. Thus, Experiment 1 aimed to evaluate the motility pattern using a computer-assisted sperm analysis (CASA) and plasma membrane integrity by epifluorescence microscopy of equine semen that were frozen in 0.5 and 0.25mL straws at different freezing rates and their relationship with sperm viability. Three different methodologies were used: conventional method with isothermic box and two automated systems: the Mini Digitcool® machine (IMV - Technologies - France), which allows a range from -10oC to -60oC per minute, and the TK® 4000C (TK Equipamentos para Congelação, Brazil), that provides a range from -10ºC to -45ºC per minute. Therefore 3 ejaculates for 4 animals were used for each freezing protocol. On protocol 1 we compared isothermic box vs. TK® 4000C machine at a -20ºC/min rate vs. Mini Digitcool® at a -20ºC/min rate; Protocol 2 compared isothermic box vs. TK® 4000C machine at a -40ºC/min rate vs. Mini Digitcool® at a -40ºC/min rate; and Protocol 3 compared isothermic box vs. TK® 4000C machine at a -40ºC/min rate vs. Mini Digitcool® at a -60ºC/min rate. The 0.5mL-straw samples were thawed at 46oC/20 sec whereas samples from the 0.25mL straws were thawed at 46oC/15 sec for post-thawing analysis. Statistical analysis from the frozen-thawed semen evaluated parameters was performed using the statistics software SAS 9.1. At first, it was used the descriptive analysis Box Plot R program. Then, data were analyzed using Proc. MIXED, a variance analysis to investigate the treatment effect (freezing rates and... (Complete abstract click electronic access below)

Nisin is a bacteriocin produced by Lactococcus lactis subsp. lactis, which is active against Gram-positive organisms including bacterial spores. It is not generally active against Gram-negative bacteria, yeasts and fungi. Gram negatives show nisin-sensitivity when their outer membrane permeability is altered by various means, such as treatments with chelators, e.g. EDTA, osmotic shock, heating, freezing, freeze-drying, high- pressure etc. Application of chelators and nisin is effective against Gram-negatives when exogenous nisin is added. Nisin produced in situ and chelators are not an effective combination, since nisin production follows the pH drop caused by sugar fermentation, and this interferes with the sequestering ability of the chelators. Presence of nisin during thermal inactivation of Gram-negatives though is effective. Bacteria become structurally injured during heating showing sensitivity to agents like SDS and deoxycholate and extended detection times by impedimetry. These injured bacteria are inactivated by nisin, with a concomitant reduction of the measured D-values. Low pH and the presence of small amount of chelators enhance the injury and inactivation and reduce D-values further. Gram-negative bacteria injured by chilling and freezing are also sensitive to nisin. The effectiveness of nisin is reduced in a food environment mostly of nisin binding to fat, and food particles. D-values were decreased less or not at all in egg white and liquid whole egg, respectively, and rapid chilling of bacteria attached to chicken skin in presence of nisin did not give the effect seen in laboratory media. Nisin is active against heat-, chill-, and freezing-stressed Gram-negatives only if it is present during the treatments. When the stress factor is removed, the bacteria recover their nisin resistance, implying transient susceptibility to nisin, but not to smaller molecules. This is probably due to rapid reorganisation and restoration of OM permeability damage, rather than biochemical repair. The LPS chain length influences the sensitisation of Gram-negatives to nisin, only in the case of freezing, where the strain with the shorter LPS chain was more sensitive than the wild type. Heat-, and freezing-stressed bacteria lost lipopolysaccharides and increased their cell surface hydrophobicity. This was not seen with chill-stressed bacteria, which were sensitive to nisin though. This indicates that release of LPS is not a prerequisite for nisin sensitivity in Gram-negatives.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第15334号 / 工博第3213号 / 新制||工||1484(附属図書館) / 27812 / 京都大学大学院工学研究科化学工学専攻 / (主査)教授 大嶋 正裕, 教授 田門 肇, 教授 宮原 稔 / 学位規則第4条第1項該当

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. / Applied Science, Faculty of / Chemical and Biological Engineering, Department of / Graduate

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This study tested different concentrations of egg yolk in canine freezing extender void of glycerol, a commonly used cryoprotectant, by examining the motility and morphology throughout the freezing process: initial (baseline after extender added), post-cool (after three hours at 5"C) and post-thaw (after freezing.)  Initial values of pH, osmolarity, motility and morphology were obtained for comparison of the samples.  Spermatozoa from six normal dogs as determined by progressive linear motility > 70% and normal morphology > 60% was used. Semen was collected and pooled for five freezing trials.  The concentrations of egg yolk used in the extender were: 0%, 10%, 20%, 30% and 40%. Assessment of each sample was blinded to the treatments until all results were obtained and statistics had been analyzed. Based on this study a 20% egg yolk concentration is slightly superior to a 30% egg yolk concentration when assessing post-thaw motility, morphology and longevity and significantly superior to a 0%, 10% or 40% egg yolk concentration. The study also showed motility and normal post-cool and post-thaw sperm morphology did not always correlate.  Utilization of 0% and 10% concentrations of egg yolk has negative effects on semen quality as measured by the motility and/or morphology.  Results confirm freezing does not affect secondary sperm abnormalities, abnormalities of the tail and distal section of the middle piece, during cooling or freezing.  Primary abnormalities, abnormalities of the head and midpiece, increased in the 0% extender during cooling and all extenders during freezing. The pH of the extenders before the addition of sperm was significantly different. Once sperm was added to the extenders, there was no longer a significant difference in pH.  There was a positive correlation for both motility and normal morphology percentages post-cool and post-thaw for the extenders with similar osmolarity to the semen. / Master of Science

In the atmosphere, cloud droplets can remain in a supercooled liquid phase at temperatures as low as -40 °C. Above this temperature, cloud droplets freeze via heterogeneous ice nucleation whereby a rare and poorly understood subset of atmospheric particles catalyze the ice phase transition. As the phase state of clouds is critical in determining their radiative properties and lifetime, deficiencies in our understanding of heterogeneous ice nucleation poses a large uncertainty on our efforts to predict human induced global climate change. Experimental challenges in properly simulating particle-induced freezing processes under atmospherically relevant conditions have largely contributed to the absence of a well-established model and parameterizations that accurately predict heterogeneous ice nucleation. Conversely, the sparsity of reliable measurement techniques available struggle to be interpreted by a single consistent theoretical or empirical framework, which results in layers of uncertainty when attempting to extrapolate useful information regarding ice nucleation for use in atmospheric cloud models. In this dissertation a new framework for describing heterogeneous ice nucleation is developed. Starting from classical nucleation theory, the surface of an ice nucleating particle is treated as a continuum of heterogeneous ice nucleating activity and a particle specific distribution of this activity g is derived. It is hypothesized that an individual particle species exhibits a critical surface area. Above this critical area the ice nucleating activity of a particle species can be described by one g distribution, 𝑔, while below it 𝑔 expresses itself expresses externally resulting in particle to particle variability in ice nucleating activity. The framework is supported by cold plate droplet freezing measurements for dust and biological particles in which the total surface area of particle material available is varied. Freezing spectra above a certain surface area are shown to be successfully fitted with 𝑔 while a process of random sampling from 𝑔 can predict the freezing behavior below the identified critical surface area threshold. The framework is then extended to account for droplets composed of multiple particle species and successfully applied to predict the freezing spectra of a mixed proxy for an atmospheric dust-biological particle system. The contact freezing mode of ice nucleation, whereby a particle induces freezing upon collision with a droplet, is thought to be more efficient than particle initiated immersion freezing from within the droplet bulk. However, it has been a decades’ long challenge to accurately measure this ice nucleation mode, since it necessitates reliably measuring the rate at which particles hit a droplet surface combined with direct determination of freezing onset. In an effort to remedy this longstanding deficiency a temperature controlled chilled aerosol optical tweezers capable of stably isolating water droplets in air at subzero temperatures has been designed and implemented. The new temperature controlled system retains the powerful capabilities of traditional aerosol optical tweezers: retrieval of a cavity enhanced Raman spectrum which could be used to accurately determine the size and refractive index of a trapped droplet. With these capabilities, it is estimated that the design can achieve ice supersaturation conditions at the droplet surface. It was also found that a KCl aqueous droplet simultaneously cooling and evaporating exhibited a significantly higher measured refractive index at its surface than when it was held at a steady state temperature. This implies the potential of a “salting out” process. Sensitivity of the cavity enhanced Raman spectrum as well as the visual image of a trapped droplet to dust particle collisions is shown, an important step in measuring collision frequencies of dust particles with a trapped droplet. These results may pave the way for future experiments of the exceptionally poorly understood contact freezing mode of ice nucleation.