Abstract not available.

The EtOH extracts of the leaves of Margraviaceae, a relatively rare Central American vine for which ethnobotanical reports suggested possible anti-anxiety properties, showed significant anti-anxiety activity in animal models for anxiety. Subsequent bioassay-guided fractionation of these extracts yielded an EtOAc active fraction (f1). Further bioassay-directed chromatography of (f1), led to the isolation of betulinic acid (3) as the bioactive constituent in 0.01% of dry weight. Six known pentacyclic triterpenoids [( 1a), (1b), (2a), (2b), ( 4), (5a)], six known flavonoids [(6), ( 7), (8a), (8b), (9), ( 10)], chondrillasterol (11), linolenic acid (12 ) and a porphyrin type compound (13) were also isolated. When (3) was administered at 0.5 mg per kg (possibly less) in a variety of rat and mouse model assays, the activity of (3) was comparable to that of Valium, the most famous member of the benzodiazepines family. Synthetic derivatives of (3) were prepared and evaluated for anti-anxiety activity. Several of the simple esters appear to have ideal properties as new drug Candidates. In particular, betulinic acid methyl ester or methyl betulinate (3a) exhibited anti-anxiety activity superior to (3). The activity profile of (3a) is such that (3a) can be considered a viable drug candidate. An excellent relay synthesis of (3) from another closely related natural product betulin (14), that is abundantly available in Eastern Ontario, was developed. Radioactive 3H-labelled betulinic acid methyl ester ( 3a″) was also prepared in order to facilitate identification of relevant anti-anxiety receptors and the mechanism of action of the compound. This is important since (3) showed no significant binding to any of the 40 anti-anxiety receptors currently implicated in anxiety. Therefore, it appears to act as an anti-anxiety agent via a new mode of action.* In a second project, the active components of a member of the Piperaceae or Pepper family (P. tuberculatum) from Costa Rica, were isolated and their structures characterized as 5,6-dihydropiperlonguminine (25), 5,6-dihydropiperine (26), piperine ( 27) and piperlonguminine (28). Extracts from this neotropical plant had been previously demonstrated by our biology collaborators, Professor Arnason's group, to be strongly insecticidal towards a variety of pests including mosquitoes, earwigs and white grubs. Moreover, the P. tuberculatum extracts were as effective as the well-documented Asian (P. nigrum) and African ( P. guineense) Piper species. Piperamides (25)--(28) were synthesized in sufficient amounts to allow extensive evaluation of their insecticidal properties. Experiments with these piperamides showed that the tertiary and quaternary mixtures have greater-than-additive toxicity compared to single compounds or binary mixtures. That is, these piperamides synergize each other. Compound (25) was the most acutely toxic in mosquito larvae bioassays. The field trials to date indicate a high potential for the development of an effective, relatively inexpensive botanically based insecticide. Radioactive 3H-labelled piperine (27″ ) was also synthesized for toxicokinetic studies. *Please refer to dissertation for diagrams.

When a substance is above its critical temperature and pressure it is called a supercritical fluid. Supercritical fluids have the extraction power of solvents while possessing the diffusivity of gases. These properties hold exciting prospects for modern residue analysis methods for pesticides and other environmental pollutants. Three new and novel applications for supercritical fluid extraction are presented in this text. A rapid method for extracting petroleum hydrocarbons from soil is presented as well as analysis by gas chromatography-mass spectrometry and supercritical fluid chromatography. The method gives extraction recoveries greater than 90% with total analytical times of less than 1 hour. Two new methods for extraction and analysis of fourth generation insecticides are also presented. Avermectin, a natural product insecticide, is extracted from soil and animal tissue with recoveries over 80% and with no additional clean-up requirement. Azadirachtin, a biorational larvicide, was extracted from soil and insect specimens with recoveries of 70% and better. Determination of some of the properties of modifiers in supercritical carbon dioxide as they relate to the eluotropic series is also discussed. Overall, a modifiers ability to enhance or decrease supercritical chromatographic capacity factors is dependent on the presence of a primary solvent-solute relationship. The presence or absence of such a relationship is independent of modifier presence.

No description available.

No description available.

No description available.

The aim of this study was to investigate two potential beneficial management practices that will reduce the impact of fecal and fertilizers contamination on the environment. The study was conducted in Eastern Ontario on typical small scale agricultural practices. The first part evaluates the impact of a conventional pasture system and an excluding pasture system on surface water quality. The three year study compared water quality endpoints such as: indicator bacteria, pathogens, parasites and nutrients between treatments. Microbial source tracking indicated that livestock was the main source of fecal contamination in the stream. Greater bacteria and nutrient loads were observed in the unrestricted pasture system than the excluding pasture. Moreover, parasite and indicator bacteria concentrations increased after cattle introduction in both systems. The second part compares nitrogen mass balance between managed and conventional subsurface tile drainage. Over two years, nitrogen loads in groundwater, in tile flow, in plants, in soil and denitrification were compared between treatments. Nitrogen was mostly removed from managed tile drainage fields through plant uptake. Compared to nitrogen plant uptake, denitrification, the second greatest nitrogen removing process was 10 times smaller. The denitrification was greater in the unmanaged tile drainage fields. Generally, managed subsurface tile drainage reduces nitrogen mass loads to surface waters and increases nitrogen uptake by plant, which resulted in greater yields.

Surface soil samples from a climosequence were studied with the purpose of relating color, reflectance variations and texture to contents of organic carbon and free iron oxides. Information on the physicochemical properties of the soils were obtained with a fine resolution spectroradiometer, a chromameter and by laboratory analyses. The effect of soil organic carbon and free iron oxides is shown by the varying shape of the soil spectral curves. Both the chromameter and the spectroradiometer detected the varying amounts of organic carbon and free iron oxides in soil. Silt had a positive, highly significant relationship with organic carbon. Clay and silt had a positive highly significant relationship with free iron oxides.

Inaug.-diss.--Leipzig.

The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits, constituting a substantial and increasingly large portion of the total global production of various crops, including cotton, corn, maize and soybeans. Bt toxins are exquisitely selective for targeted pests, typically do not affect off-target insects, and are completely orthogonal to human biology. However, the development of insect resistance to Bt toxins endangers their long-term effectiveness. In this thesis, I describe the development of methodology for the systematic directed evolution of novel Bt toxins to selectively affect resistant insects. Using Phage-Assisted Continuous Evolution (PACE), a previously developed general platform for the continuous directed evolution of biomolecules, I developed a highly sensitized selection for novel protein-protein interactions. This system robustly reported on interactions spanning affinities from low micromolar to picomolar. However, attempts at using this system for the directed evolution of novel protein-protein interactions were largely unsuccessful, presumably as a consequence of low mutagenesis efficiency. To increase the utility of the platform, I sought to enhance the mutagenesis levels afforded by PACE, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency, and narrow mutational spectra. I used a systematic, mechanism-driven approach to create a potent, inducible, broad-spectrum, and vector-based mutagenesis system in E. coli that enhances mutation rates by 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro mutagenesis methods. This system enabled the high-frequency, broad-spectrum mutagenesis of chromosomal, episomal, and viral nucleic acids in vivo, and dramatically enhanced the success of PACE experiments, highlighting the importance of mutagenesis efficiency on selection outcome. Using this enhanced mutagenesis approach and the previously described sensitized selection platform, I was able to evolve variants of the commonly used Bt toxin Cry1Ac that bind toxin binding region of a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not targeted by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (Kd = 11-41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome Bt toxin resistance in insects and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects. In conclusion, these finding offer a novel mechanism of overcoming what is quickly becoming among the largest issue overshadowing the continued success of Bt toxins for pest control and management, and establish a platform for the detection and evolution of a wide array of protein-protein interactions. / Chemical Biology