Doctor of Philosophy / Department of Biology / Mark Ungerer / Elucidating the molecular basis of adaptive phenotypic variation represents a central aim in evolutionary biology. Using the model plant species Arabidopsis thaliana, I studied the intra-specific variation in freezing tolerance among natural accessions across its native range. Considerable variation in freezing tolerance among 71 selected accessions was observed both with and without a prior cold acclimation treatment, suggesting that both differences in cold-acclimation capacity and in intrinsic physiology contribute to this variation. A highly significant positive relationship was observed between freezing tolerance and latitude of origin of these accessions. This clinal pattern of variation is found to be attributable, at least in part, to relaxed purifying selection on CBF/DREB1 genes in the species’ southern range. These CBF/DREB1 genes encode transcriptional activators that play a critical role in the ability of A. thaliana plants to undergo cold acclimation and thereby achieve maximum freezing tolerance. Relative to accessions from northern regions, accessions of A. thaliana from the southern part of their geographic range exhibit significantly higher levels of nonsynonymous polymorphisms in coding regions of CBF/DREB1 genes. Relaxed selection on the CBF/DREB1s in southern accessions also has resulted in mutations in regulatory regions that lead to abrogated expression. These mutations in coding and regulatory regions compromise the function of CBF/DREB1 transcriptional activators during the cold acclimation process, as determined by reductions in rates of induction and maximum levels of expression in the downstream genes they regulate. These mutations could be selective neutral or beneficial in southern accessions depending on whether there is an allocation cost associated with cold acclimation. The fitness benefit and possible allocation cost of cold acclimation was examined in freezing and freezing-free environments using natural accessions exhibiting contrasting abilities of cold acclimation as well as transgenic CBF gene over-expression or knockdown/knockout lines. The extent to which cold acclimation benefits the plant in presence of freezing temperature is revealed, but a cost of cold acclimation wasn’t detected in the absence of freezing temperature under our experimental design, which suggests that these mutations in CBF genes in southern accessions might be neutral to natural selection.

Ice accretion on various surfaces can cause destructive effect of our lives, from cars, aircrafts, to infrastructure, power line, cooling and transportation systems. There are plenty of methods to overcome the icing problems including electrical, thermal and mechanical process to remove already accumulated ice on the surfaces and to reduce the risk of further operation. But all these process required substantial amount of energy and high cost of operation. To save the global energy and to improvement the safety issue in many infrastructure and transportation systems we have to introduce some passive anti-icing coating known as ice-phobic coating to reduce the ice-formation and ice adhesion onto the surface. Ice-phobic coatings mostly devoted to utilizing lotus-leaf-inspired superhydrophobic coatings. These surfaces show promising behavior due to the low contact area between the impacting water droplets and the surface. In this present study we investigate systematically the influence of chemical composition and functionality as well as structure of surfaces on wetting properties and later on icing behavior of surfaces. Robust anti-icing coating has been prepared by using modified silica particles as a particles film. Polymer brushes were synthesized on flat, particle surfaces by using Surface initiated ATRP. We have also investigated the effect of anti-icing behavior on the surfaces by varying surface chemistry and textures by using different sizes of particles. This approach is based on the reducing ice accumulation on the surfaces by reducing contact angle hysteresis. This is achieved by introducing nano to micro structured rough surfaces with varying surface chemistry on different substrates. Freezing and melting dynamics of water has been investigated on different surfaces by water vapour condensation in a high humidity (80%) condition ranging from super hydrophilic to super hydrophobic surfaces below the freezing point of water. Kinetics of frost formation and ice adhesion strength measurements were also performed for all samples. All these experiments were carried out in a custom humidity and temperature controlled chamber. We prepared a superhydrophobic surface by using Poly dimethyl siloxane (PDMS) modified fumed silica which display very low ice-adhesion strength almost 10 times lower than the unmodified surface. Also it has self-cleaning behavior after melting of ice since whole ice layer was folded out from the surface to remove the ice during melting. Systematic investigation of the effect of three parameters as surface energy, surface textures (structure, geometry and roughness) and mechanical properties of polymers (soft and stiff) on icing behavior has also been reported.

It is perhaps paradoxical that many material properties arise from the absence of material rather than the presence of it. For example, the strength, stiffness, and toughness of a concrete are related to its pore structure. Furthermore, the volume, size distribution, and interconnectivity of porosity is important for understanding permeability, diffusivity, and capillary action occurring in concrete, which are necessary for predicting service lives in aggressive environments. This research advances the state-of-the-art of multiscale characterization of cement-based materials, and uses this characterization information to model the material behavior under competing durability concerns. In the first part of this research, a novel method is proposed to characterize the entrained air void system. In the second and third parts of this research, microstructural characterization is used in tandem with mechanical models to investigate the behavior of cementitious materials when exposed to rapid rates of loading and to cyclic freezing and thawing. First, a novel analytical technique is presented which reconstructs the 3D entrained air void distribution in hardened concrete using 2D image analysis. This method proposes a new spacing factor, which is believed to be more sensitive to microstructural changes than the current spacing factor commonly utilized in practiced, and specified in ASTM C457, as a measure of concrete's ability to resist to damage under cyclic freeze/thaw loading. This has the potential to improve economy by improving the quality of petrographic assessment and reducing the need for more expensive and time-consuming freeze/thaw tests, while also promoting the durability of concrete. Second, quantitative measurements of the sizes, shapes, and spatial arrangements of flaws which are through to drive failure at strain rates above 100/s were obtained in order to model mortar subjected to high strain-rate loading (i.e., extremes in load rate). A micromechanics model was used to study the ways in which flaw geometry and flaw interaction govern damage. A key finding suggests that dynamic strength may be multimodal, with larger flaws shifting the dynamic strength upwards into the highest strength failure mode. Third, a robust theoretical approach, based upon poroelasticity, is presented to further validate the utility of the novel spacing factor proposed this research. The model is truly multiscale, using in its formulation pore size data ranging from the nanoscale to the micro-scale, entrained air data from the micro-scale to the millimeter scale, and infers a representative volume element on the centimeter scale. The results provide an underlying physical basis for the performance of the novel spacing factor. Furthermore, the framework could be used as a forensic tool, or as a tool to optimize the entrained air void system against freeze/thaw damage.

This study aims to validate and develop applications for a novel impedance-based process analytical technology for monitoring the attributes of the product during the entire freeze-drying process (from pre-freezing and annealing to primary and then secondary drying). This measurement approach involves the application of foil electrodes, mounted externally to a conventional glass freeze-drying vial, and coupled to a high-impedance analyser. The location of the electrodes on the outside, rather than the inside of the vial, leads to a description of the technology as a through-vial impedance spectroscopy (TV-IS) technique. The principle observation from this approach is the interfacial-polarization process arising from the composite impedance of the glass wall and product interface. For a conventional glass vial (of wall thickness ~ 1 mm and cross sectional diameter ~ 22 mm) it was shown that the process is manifest within the frequency range 101 to 106 Hz, as a single, broad band peak which spans 2-3 decades of the imaginary part spectrum. Features of the interfacial-relaxation process, characterised by the peak amplitude, C″Peak, and peak frequency, fpeak, of the imaginary capacitance spectra and the equivalent circuit elements that model the impedance spectra (i.e. the solution resistance (R) and solution capacitance (C) were monitored along with the product temperature data during the cycle(s), for a variety of surrogate formulations comprising lactose, sucrose, mannitol or maltodextrin solutions, during the freezing, re-heating, annealing and primary drying stages of freeze drying). It was shown that the parameters, fpeak and R, are strongly coupled to each other and change as a function of the temperature of the solution and its phase state, whereas C″Peak is strongly coupled to the amount of ice that remains during the drying process. Both log fpeak and log R have a linear dependence on the temperature of the solution, provided there was no phase change in the solution. The crystallization process (ice onset, solidification and equilibration to shelf temperature) is characterized well by both log fpeak and log R, whereas the parameter R demonstrates most clearly the formation of eutectic crystallization during freezing. In contrast it was the parameter C which was most sensitive to the detection of the glass transition during re-heating. During primary drying, it was shown that C″peak, is dependent on the amount of ice remaining and therefore provides a convenient assessment of the rate of drying and primary drying end point. The impedance changes during annealing provide a mechanistic basis for the modifications in ice structure which result directly in the observed decrease in primary drying times. The principal observation on annealing of a 10% w/v solution of maltodextrin, was the minimal changes in the glass transition (recorded at ~−16 °C) during the re-heating and cooling step (post-annealing). This result alone appears to indicate that a maximum freeze concentration was achieved during first freezing with no further ice being formed on annealing. The phenomenon of devitrification (and the production of more ice, and hence larger ice crystals) was therefore discounted as the mechanism by which annealing impacts the drying time. Having excluded devitrification from the mechanism of annealing enhanced drying, it was then possible to conclude that the decrease in the electrical resistance (that was observed during the annealing hold time) must necessarily result from the simplified structure of the unfrozen fraction and the improved connectivity of ice crystals that may be the consequence of Ostwald ripening. The application of through vial impedance measurement approach provides a non-invasive, real time monitoring of critical process parameters which subsequently leads to an improved understanding of the mechanisms and effects of different parameters, providing a reliable basis for process optimization, along with improved risk management to ensure optimum quality of the formulation and optimization of the freeze drying process.

Micromechanical approach is employed to investigate the transport and freezing within unsaturated porous media. In unsaturated porous media, water film as well as disjoining pressure are introduced in the transport and freezing problems. In the modeling, it is found that, capillary layer along with pore water dominate the transport at high saturation degree (Sr>10%). However, water film will play a significant role in transport at low saturation degree (Sr<10%), and the diffusion coefficient will be lower than 3 to 4 orders of magnitude than that at higher saturation degree. A micromechanical model of freezing in unsaturated porous media is established. Micromechanical model of freezing is more physical based in nature. That is because different from poromechanical model of freezing media in which ice crystal pressure is introduced, the disjoining pressure of unfrozen water film instead of ice crystal pressure is introduced in the micromechanical model of freezing

The pedunculopontine nucleus (PPN) is a reticular collection of neurons at the junction of midbrain and pons. The PPN in animal models appears topographically organised and functionally related to locomotion and arousal. In Parkinson’s disease, the PPN degenerates and is susceptible to abnormal basal ganglia output. In patients with Parkinson’s disease, low frequency PPN stimulation is proposed to improve gait freezing and postural instability. However, the therapeutic mechanisms, optimal clinical application and precise effects on gait and posture of PPN stimulation are unclear. Here, a topographic arrangement of the PPN was supported by local field potential recordings in parkinsonian patients. In the PPN region, beta oscillations were recorded rostrally and alpha oscillations caudally. Alpha oscillations, consistent with their putative role in allocating attention, correlated with gait performance and attenuated with gait freezing. Thus the caudal PPN subregion may be the most relevant target for gait disorders. Accordingly, an unblinded clinical study suggested that stimulation of the caudal PPN subregion was beneficial for gait freezing, postural instability and falls. In a double-blinded study using spatiotemporal gait analysis, caudal PPN stimulation reduced triggered gait freezing, with bilateral stimulation more effective than unilateral. However, akinesia including akinetic gait did not improve with PPN stimulation. Accordingly, dopaminergic medication requirements did not change. Mechanisms underlying gait freezing and PPN stimulation were explored with reaction time experiments. Parkinsonian patients with severe gait freezing and postural instability demonstrated a ‘block’ to pre-programmed movement. This was evidenced by prolonged simple reaction times and the absence of ‘StartReact’, whereby pre-prepared responses are normally accelerated by loud acoustic stimuli. PPN stimulation improved simple reaction time and restored Startreact. The relief of this ‘motor block’ with PPN stimulation may therefore explain the associated improvement in gait freezing and postural instability, as these tend to occur in circumstances requiring triggered, pre-prepared adjustments.

Perennial growers overwintering plant stock require information to assist in deciding which containerized plants are most likely to successfully overwinter. Three studies on container-grown herbaceous perennials were conducted to examine the influence of plant age, soil moisture, and temperature cycling date on cold hardiness. In January, plants were exposed to controlled freezing temperatures of -2, -5, -8, -11, and -14C and then returned to a 3-5C greenhouse. In June, plants were assessed using a visual rating scale of 1-5 (1 = dead, 3-5 = increasing salable quality, varying by cultivar) and dry weights of new growth were determined. Controlled freezing in November and March were also included in the third study. In the first study, two ages of plants were exposed to controlled freezing temperatures in January. For Geranium x cantabrigiense 'Karmina', age had no effect on either rating or dry weight in one study year. In two Sedum 'Matrona' study years, age had no effect on dry weight but ratings were higher for older plants than younger plants in the first year and higher for younger plants than older plants in the second year. In two Leucanthemum x superbum 'Becky' study years, age had an effect on both rating and dry weight which were both generally higher for younger plants than older plants. In the second study, plants were maintained in pots at two different soil moisture levels prior to exposure to controlled freezing temperatures in January. Coreopsis 'Tequila Sunrise' and Carex morrowii 'Ice Dance' showed no effect on either rating or dry weight from soil moisture level. Soil moisture level had no effect on dry weight but ratings were higher for Geranium x cantabrigiense 'Cambridge' “wet” plants and for Heuchera 'Plum Pudding' “dry” plants. Carex laxiculmus 'Hobb' (Bunny Blue™) soil moisture level had an effect where dry weight was higher for “dry” plants. Means at were of salable quality for Geranium and Heuchera at all temperatures and Carex laxiculmus at temperatures above -11C. The effects of soil moisture level on Carex oshimensis were inconclusive. In the third study, during November, January, and March, plants were subjected to temperature cycling treatments prior to exposure to controlled freezing temperatures. Geranium x cantabrigiense 'Cambridge' were more tolerant of both temperature cycling and freezing temperatures in January and an increased number of cycles in November had an advantageous effect. Sedum 'Matrona' were more tolerant of temperature cycling and freezing temperatures in January and an increased number of cycles in March had an advantageous effect. Leucanthemum x superbum 'Becky' were more tolerant of temperature cycling in January in the second year of the study and an increased number of cycles in November had an advantageous effect in the first year and in all months in the second year. Overwintering younger container-grown plants is likely to result in more growth and higher quality following exposure to freezing temperatures. Effects of soil moisture level on overwintering container-grown plant growth and quality are cultivar-specific and a general effect could not be established in these studies. Overwintering container-grown plants are likely to be hardier in January and slight temperature cycles prior to exposure to freezing temperatures generally increase hardiness.

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Cryopreservation in suspension is commonplace for a variety of cell types. However, cryopreservation of adherent cells has achieved limited success. This research aimed to cryopreserve adherent nerve cell networks in vitro in a manner that preserved network morphology and physiology. Successful implementation would enable long term storage of adherent neuronal networks on microelectrode arrays and on-demand access for use in pharmacological and toxicological testing. Based upon morphological assessments, excellent post-thaw preservation was obtained and post-thaw cultures survived in a transitional medium for up to 3.5 hours. However, transitions to native culture medium post-thaw presented difficulties, ultimately resulting in necrosis. A discussion of methods to supplement the current research and increase post-thaw viability is included in the thesis.

The aim of this work is to answer the question of whether the judge's remuneration represent a constitutional guarantee of judicial independence and thus whether they can have a real impact on the judges' decisions. In view of this, the thesis will first deal with the definition of the term "judicial independence" and its possible categorizations. Subsequently, the diploma thesis will focus on Czech legal regulation of judicial remuneration and documentation from important international institutions. The focus of this thesis will be devoted to the analysis of Act No. 236/1995 Sb., o platu a dalších náležitostech spojených s výkonem funkce představitelů státní moci a některých státních orgánů a soudců a poslanců Evropského parlamentu, ve znění pozdějších předpisů (about salary and other elements associated with the performance of State representatives, bodies, judges and members of the European Parliament duties). Specifically looking at case law from the Constitutional Court on the issue of judge's remuneration, in particular with regard to the method of calculating judges' salaries and restrictive interventions in the judiciary, including the removal of additional salaries, the freezing of salaries and changes in the statistical index. The aim of this chapter will be to find out, in particular,...