Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisors: C. Buddie Mullins and David T. Allen. Vita. Includes bibliographical references. Also available from UMI.

The aim of the study was to evaluate the effect of ozone (O3) on mature body weight (MBW), feed conversion rate (FCR), mortality rate (MR) and ammonia (NH3) production of broiler chickens (referred to as “broilers” in this thesis) under controlled conditions. Ozone was used for the purifying of air and water in order to eliminate harmful microorganisms. Two trials were conducted over a twelve week production period. In experiment 1, day old broiler chicks were randomly allocated to a control (n=130) and treatment (n=130) group. The treatment groups were exposed to ozone continuously for the entire period of the trial. Both air and water were ozonated on a 24 hours daily basis, while the control group was reared under normal commercial conditions. In experiment 2: day old broiler chicks were randomly allocated to a control (n=123) and a treatment (n=123) group. The treatment group was exposed to ozone for 3 hours per day for the entire period of the trial. Both air and water were ozonated, where the air was treated for 3 hours/day and the water for 1 hour/day during the entire production period. The control group was reared under normal commercial conditions. The results indicate that broilers exposed to continuous O3 (0.1 ppm) conditions had significantly higher (P< 0.001) mortality rates than the control group. The treatment group had a mortality rate of 55.38% compared to the 5.38% for the control group. No significant difference (P= 0. 2468) for MBW was found between the broilers that were reared in the O3 environment compared to the control iii group. Although the control group were on average 200g heavier at the end of the trial. No significant difference (p = 0. 6143) was recorded in FCR between the O3 and control groups. However the FCR of the broilers treated with O3 was lower by 100g than that of the control group. The O3 significantly (P< 0. 0001) reduced the NH3 in the treatment group. When the trial was repeated with limited exposure to an O3 environment there was no significant difference in MBW (P = 0. 0979), FCR (P = 0. 8913) and MR (P = 0. 1108) between the treatment and control groups. However, ammonia levels were lower in the treatment group compared to the control group. When the data was further analyzed comparing the two trials, the results indicated that there was no significant difference (P = 0. 4112) in the MBW for the broilers reared under normal conditions (control group) from experiment 1 and experiment 2, while a significant difference (P = 0. 0002) was recorded between the broilers reared under continuous O3 and regulated O3 conditions from experiment 1 and experiment 2 respectively. When the FCR data was analyzed using the Gompertz equation a significant difference (P = 0.0403) in the FCR of broilers reared in the control group between experiment 1 and experiment 2 was recorded (Table 8). No significant difference (P = 0.3226) in the FCR of broilers raised under O3 conditions of continuous and regulated O3 production were reported between experiment 1 and experiment 2. A highly significant difference (P = 0. 0001) in the MR of broilers reared under ozone conditions was recorded between experiment 1 and experiment 2. In experiment 1, the MR was 55.38% while in experiment 2 the MR was only 8.78%. A significant difference (P = 0.0355) was also recorded in the MR of broilers that iv were raised in the control groups between experiment 1 and experiment 2. The MR for broilers in the control group was 5.38% in experiment 1 and for experiment 2 it was 9.74%. The mortality rate of broilers raised in the control groups conditions increased by approximately 4% points or 81% between experiment 1 and experiment 2. The level (0.1 ppm) of ozone production had a limited effect on the MBW and FCR of broilers, however it is evident that ozone is toxic to broilers when they are exposed to a continuous ozonated environment. Ozone was however effective in controlling ammonia levels.

Sub-acute ozone exposures (doses below the threshold for visible injury) have been found to affect plant growth, stomatal behavior, and the extent of visible injury in response to subsequent acute exposures (doses above the threshold for visible injury). The interrelationships of these effects varied according to the magnitude and timing of the pretreatment dose and the particular species and cultivar tested. When seedlings of bush bean (Phaseolus vulgaris L. cv Pure Gold Wax) were raised from seed in filtered air and then pretreated with 0.02 parts per million (ppm) ozone for six days (six hours per day), beginning at eight days after sowing, susceptibility to acute injury (the extent of visible injury after an acute exposure) was greater than that of controls maintained throughout in filtered air. Seedlings exposed to this low ozone treatment for an additional seven days became less susceptible to acute injury than controls maintained in filtered air. Treatment differences in acute susceptibility were found to correspond to differences in stomatal behavior. At the early stage of increased susceptibility, ozone-p're-treated plants and controls maintained in filtered air had the same stomatal resistance when measured in filtered air, but ozone-pretreated stomata had a reduced tendency, to close in response to an acute ozone exposure (0.4 ppm). At the later stage of decreased acute susceptibility, ozone-pretreated stomata still had this reduced sensitivity to ozone-induced closure, but ozone pretreatment also resulted in an overall increase in stomatal resistance in filtered air, prior to acute exposures. Stomatal entry of ozone may thus provide, in part, an explanation for the differences in susceptibility resulting from these treatments in bean. Exposure of mint cuttings (Mentha arvensis L.) to 0.02 ppm ozone increased their acute susceptibility slightly. The ozone tolerance of mint was approximately six times greater than that of bean seedlings raised in filtered air (based on the dosage required for equivalent degrees of visible injury). In experiments with potted grapevines (Vitis labruscana, Bailey) in open-top field chambers exposed to ambient air (containing "naturally ocurring" ozone) or charcoal-filtered air (containing reduced ozone levels) for an entire growing season, growth was found to be greater in ambient air in two of three cultivars tested. This growth stimulation was greatest in leaves, but occurred also in trunks and roots (both dry weight and fresh weight). Within the three cultivars ('Ives', 'Concord', and 'Delaware') the growth stimulation was directly related to the sensitivity of field-grown vines to ozone injury; the most sensitive cultivar, 'Ives', showed the greatest growth increase in ambient air as compared to filtered air. Only one of the grapevine cultivars. showed any statistically significant effect of chronic exposure to ambient ozone on acute susceptibility. In that case, 'Delaware', the cultivar most resistant to ozone injury in the field, was predisposed to acute injury by chronic pretreatment with low ozone. With grapevines, there were no statistically significant effects of low ozone pretreatment on stomatal behavior, but trends in the data indicated that ozone pretreatment tended to decrease both stomatal resistance when measured in filtered air and closure in response to acute ozone exposures (0.75 ppm). In experiments with grapevines, unlike those with beans, decreased susceptibility appeared to be more closely related to an increase in overall vigor, associated with a lower stomatal resistance in filtered air, rather than to a stomatal mechanism regulating ozone entry into the leaf. Although there were differences between species in the role that stomata play in the response to pretreatment with sub-acute ozone doses, there was the suggestion of a general pattern of increased tendencies toward a protective effect of ozone pretreatment in more ozone-sensitive species (bean versus mint) or cultivars ('Ives' and 'Concord' versus 'Delaware'). / Land and Food Systems, Faculty of / Graduate

Most air pollutant investigations in controlled environments have utilized the conventional "steady state" exposure, yet air pollutant concentrations are rarely static in ambient conditions. Peak concentrations in air pollutant exposures vary in magnitude and occurrence despite equivalent doses and are likely to result in different degrees of injury to plants. The results of experiments involving 5 treatment regimes with a single peak concentration that varied in magnitude and occurrence while treatment doses remained equivalent, confirm this hypothesis and demonstrate that the levels of injury to bush beans (Phaseolus vulgaris)and radish (Raphanus sativus) may vary from 15 to 85% of the leaf area. An absolute injury assessment technique was developed for determining percent necrosis in the 5 treatments, with the same two crop species responding in a similar manner to the various constant dosage regimes. A simple workable model is presented that incorporates stomatal diffusive resistance along with the various exposure components, including cumulative dose, maximum concentration, and the interval of time during which the peak concentration was administered. Preliminary investigations failed to confirm an interaction between ozone and greenhouse whitefly in bush beans. / Land and Food Systems, Faculty of / Graduate

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Le maïs est une plante de culture vivrière commerciale très importante, cultivée partout dans le monde. Sa croissance et les rendements en champ sont affectés par de nombreuses contraintes environnementales. Dans ce travail, l'impact du polluant gazeux phytotoxique, l'ozone, a été étudié sur les feuilles de maïs en utilisant une approche moléculaire et biochimique. Les concentrations en ozone dans la troposphère ont considérablement augmenté dans un passé récent, et actuellement l'ozone affecte clairement les cultures. Un système de fumigation récemment développé à l'INRA a été utilisé pour exposer à l'ozone les maïs en champs de façon contrôlée. Soixante et onze jours après le semis, les plantes ont été soumises à une fumigation d'ozone de 98 ppb.h-1 en moyenne. Les feuilles 10 et 12 ont été récoltées après 20, 35 et 50 jours de traitement. L'expression de trois gènes codant les metacaspases (ZmMCII-1, ZmMCII-2 and ZmMCII-3) ainsi que de leur activité enzymatique globale ont été étudiées lors de la sénescence, et en réponse à l'ozone, dans les feuilles. Pour la première fois, nous avons montré une accumulation significative de l'ARNm des trois metacapases en réponse à l'ozone. Par ailleurs, l'activité enzymatique cellulaire globale des metacaspases est également fortement stimulée dans les deux cas (ozone et sénescence). La surexpression des gènes et la stimulation de l'activité des metacaspases pourraient êtres liés à un ajustement des catabolismes protéiques des feuilles lors de la sénescence et en réponse à l'ozone. Au cours d'autres expériences, les mêmes réponses ont été observées dans le cas de la papaïne-like protéase à cystéine (Mor-CP). De plus, un nouvel inhibiteur des protéases à cysteine, une cystatin a été identifiée dans des feuilles de maïs et a été nommée CC11. L'ADNc complet a été isolé et la protéine recombinante CC11 correspondante a été obtenue et purifiée, à partir d'un système bactérien hétérologue (Vecteur pET30 (EK/LIC) et E. coli BL21 (DE3) plyS). Cette nouvelle cystatine présente une activité inhibitrice optimale vis-à-vis de la papaïne (100%) et très importante (57-80%) vis à vis des protéases à cystéine de feuilles de maïs. Par conséquent, son implication dans la régulation de l'activité des protéases à cystéine en réponse à l'ozone, peut être supposée. Enfin, la production de protéines recombinantes à partir d'ADNc isolés a été réalisée, correspondant aux régions actives de la metacaspase ZmMCII-2 et de la protéase à cystéine Mor-CP. Les anticorps polyclonaux ont été obtenus pour une étude future des événements post-transcriptionels. En conclusion, les protéases à cystéine (caspase-like et papaïne-like) pourraient être utilisées comme paramètres nouveaux pour le criblage de géniteurs dans les programmes d'amélioration du maïs pour la tolérance à l'ozone / Maize is a major crop cultivated all over the world. Its yield and growth is affected by numerous environmental stresses. In the present work, the impact of the gaseous phytotoxic pollutant, ozone, on maize leaves was studied, using a molecular and biochemical approach. Concentrations of ozone in the troposphere have increased dramatically in the recent past and it now negatively affects crops growth and yield. In this work, a recently developed fumigation system was used to expose maize plants in the field. Seventy-one days after sowing, the plants were submitted to ozone fumigation at an average concentration of 98 ppb.h-1. The 10th and 12th leaves were harvested after 20, 35 and 50 days of treatment. Expression of three genes coding caspase-like cysteine proteases “metacaspases” (denoted as ZmMCII-1, ZmMCII-2 and ZmMCII-3) and their global activity was studied in senescent and ozone-exposed maize leaves. For the first time, we observed that mRNA accumulation of the three caspase-like cysteine proteases increased significantly in response to ozone exposure. Moreover, the global activity of metacaspases also increased significantly in both senescencing and ozone-exposed plants. The metacaspases activity and mRNA up-regulation could represent control points for leaf tissues to determine the degree and timing of protein catabolism during senescence and ozone treatment. Protein catabolism was indeed stimulated in response to ozone since increases in the expression of a gene coding an ozone-induced papain-like cysteine protease (Mor-CP) and in global papain-like cysteine proteases activities in leaf tissues were observed. In addition, we identified new cysteine protease inhibitor CC11 “cystatin” in maize leaves. After the recombinant CC11 protein was purified from a bacterial heterologous system (E. coli strain BL21 (DE3) plyS with a pET 30 (EK/LIC) vector), it was shown to be active in vitro against commercial papain (100% inhibition) and against total maize leaf cysteine protease extract (57-80% inhibition). Therefore its involvement in the regulation of cysteine protease activity in response to ozone exposure could be supposed. Production of the recombinant proteins corresponding to the active regions of ZmMCII-2 and Mor-CP was achieved. The corresponding polyclonal antibodies were obtained to study post-transcriptional events in maize leaves, in the future. In conclusion, we have identified the new enzymes, metacaspases and cysteine proteases involved in proteolysis which could be used as novel parameters for screening different maize varieties for improved tolerance to ozone pollution

The accuracy of ozone measurements in the middle atmosphere is of great importance when determining its diurnal variation. A high time resolution of the measured data is desirable and depends on the sensitivity of the receiver used to detect the ozone. This thesis aims to improve the sensitivity of a groundbased 142 GHz microwave radiometer used for measuring atmospheric ozone data. This is done by replacing the previous receiver components with a series of new components and arranging them in different setups for comparison purposes. Mechanics and wiring were changed in order to install the setups along with changes in the optics. Each test setup could be implemented as a first step towards improving the sensitivity of the radiometer. The result show that the optics contribute with an unexpected addition of noise to the measurements and hence the overall performance and improvement of the radiometer could not be determined. Suggestions are made for further work which include improving the optics and performing cryo-measurements.