The 21-dehydroxylation of corticosteroids: evidence for an enol intermediate and the hydroxylation of steroids by fungal cyto-chrome P450: evidence for a stepwise mechanism

Two enzyme mechanisms were examined: the 21-dehydroxylation of corticosteroids by the anaerobe Eubacterium l en tum, and the hydroxylation of steroids by fungal cytochrome P450. Deuterium labelling techniques were used to study the enzymic dehydroxylation. Corticosteroids doubly labelled (2H) at the C-21 position were incubated with a culture of Eubacterium lentum. It was found that t he enzymic dehydroxylation proceeded with the loss of one 2H f rom C-21 per molecule of substrate. The kinetic isotope ef fect f or the reaction was found to be k~kD = 2. 28. These results suggest that enzyme/substr ate binding in this case may proceed via t he enol form of the substrate. Also , it appears that this binding is, at least in part, the rate determining step of t he reaction. The hydroxylation of steroids by fungal cytochrome P450 was examined by means of a product study. Steroids with a double bond at the A8 (9), ~( lO ), or ~ (ll) position were synthesized. These steroids were then incubated with fungal strains known to use a cytochrome P450 monooxygenase to hydroxylate at positions allylic to these doubl e bonds. The products formed in these incubations indicated that the double bonds had migrated during allylic hydroxylat ion. This suggests that a carbon centred radical or ion may be an intermediate i n the cytochrome P450 cat alytic cycle.

Synthesis of 4-hydroxycinnamic amides of di-, tri-, and tetraamines : potential insect toxins /

The monoconjugates of phenolic acids (i.e. coumaric acid) with polyamines such as spermidine and spermine are strikingly similar to some toxins from spiders and predatory wasps. Many plants contain phenolic acid polyamine conjugates and there is some reliable information supporting their roles as plant defense chemicals. Eleven monoacylated compounds of diamines, triamines, tetraamines and oxa-polyamine amines were prepared in three to seven steps: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32. The synthesis proceeds through stepwise construction of the polyamine backbone (as in 62 and 72), followed by protection and deprotection steps of the amino functions. Desymmetrization of readily available and prepared symmetrical polyamines is a key step in the synthesis. The protecting groups employed were tert-butoxycarbonyl (BOC) and trifluoroacetyl (TFA) group which were removed under different conditions: acid and base respectively. Deprotection and refunctionalization of the polyamine reagent demonstrated the versatility of these systems for N-acylation.

The effects of yeast inoculation rate and acclimatization to juice on icewine fermentation /

Icewine is an intensely sweet, unique dessert wine fennented from the juice of grapes that have frozen naturally on the vine. The juice pressed from the frozen grapes is highly concentrated, ranging from a minimum of 35° Brix to approximately 42° Brix. Often Icewine fennentations are sluggish, taking months to reach the desired ethanol level, and sometimes become stuck. In 6 addition, Icewines have high levels of volatile acidity. At present, there is no routine method of yeast inoculation for fennenting Icewine. This project investigated two yeast inoculum levels, 0.2 gIL and 0.5 gIL. The fennentation kinetics of inoculating these yeast levels directly into the sterile Icewine juice or conditioning the cells to the high sugar levels using a step wise acclimatization procedure were also compared. The effect of adding GO-FERM, a yeast nutrient, was also assessed. In the sterile fennentations, yeast inoculated at 0.2 gIL stopped fennenting before the required ethanol level was achieved, producing only 7.8% (v/v) and 8.1 % (v/v) ethanol for the direct and conditioned inoculations, respectively. At 0.5 gIL, the stepwise conditioned cells fennented the most sugar, producing 12.2% (v/v) ethanol, whereas the direct inoculum produced 10.5% (v/v) ethanol. The addition of the yeast nutrient GO-FERM increased the rate of biomass accumulation, but reduced the ethanol concentration in wines fennented at 0.5 gIL. There was no significant difference in acetic acid concentration in the final wines across all treatments. Fennentations using unfiltered Icewine juice at the 0.5 gIL inoculum level were also compared to see if the effects of yeast acclimatization and micronutrient addition had the same impact on fennentation kinetics and yeast metabolite production as observed in the sterile-filtered juice fennentations. In addition, a full descriptive analysis of the finished wines was carried out to further assess the impact of yeast inoculation method on Icewine sensory quality. At 0.5 gIL, the stepwise conditioned cells fennented the most sugar, producing 11.5% (v/v) ethanol, whereas the direct inoculum produced 10.0% (v/v) ethanol. The addition of the yeast nutrient GO-FERM increased the peak viable cell numbers, but reduced the ethanol concentration in wines fennented at 0.5 gIL. There was a significant difference 7 in acetic acid concentration in the final wines across all treatments and all treatments affected the sensory profiles of the final wines. Wines produced by direct inoculation were described by grape and raisin aromas and butter flavour. The addition of GO-FERM to the direct inoculation treatment shifted the aroma/flavour profiles to more orange flavour and aroma, and a sweet taste profile. StepWise acclimatizing the cells resulted in wines described more by peach and terpene aroma. The addition of GO-FERM shifted the profile to pineapple and alcohol aromas as well as alcohol flavour. Overall, these results indicate that the addition of GO-FERM and yeast acclimatization shortened the length of fermentation and impacted the sensory profiles of the resultant wines.

Amplification of the DFR1 gene in Saccaromyces cerevisiae

A new system was employed to study amplification of t,he DHF'R gene DFB,1 ) in Sa<,:;charoillYCB§. .Q~~Yi...S!i<;1~. . This system consists of a series of yeast strains containing a casset,te which encodes t he yeast, D..ERl gene ttghtly linked tjO a f usion of the yeast 1EU2. regulat,ory region wi tJ1 the LAQZ str ctural gene from E. cO.1-1 (,) . M. Clement , unpubl i,::;hed) . Th's casset;t e was shown t.o be integrat,ed int o a unj que chromosomal l ocati on in each strain . Yeast cells were se l ected for MTX-resistance and overproduction of ~ galac t osi d se ( B-gal ). Since the inserted DF'Rl and ~ACZ genes are independently regulated, it was thought that cel l s with this phenotype probably contain e d ampl if ications of the cassette. A lar ge variat ion in the f requn y o f MTX-resistance was found between the di ff e r ent str ains. These freqlen c ~ es r anged from about 2 x 10 - 7 fo r a population of cells containing the cassette integrated at, the BI J2.l gene in t,he middle of the long arm of chromosome V, to about 5 x 10-4 for a strain with the cassette i nserted in the r DNA cluster Abo It 85% of the MTX- res i stcmt iso l ates examined showed enhanced B·-gal act i v ity rel a t ive t o the parental strain . For the ma jorit y of strains, the mean B- gal activity in drug-r sistant clones was about 3 times that o f the parent following a single se l ect i on step . I n con t r ast, primary MTX-resistant derivat~ves of cells with the cassette inserted 3 at the rDNA cluster showed inc r eases in B- gal activity ranging from 9 - 14 f old r elative to the parent. Analysis of the latte r s train by Southe rn hybr idization indicated that the cassette was inde e d amplified several fold in MTX-re sistant derivatives. A sing l e strain, in which the cassette was inserted at the !lEA;], loc u.s , was used to examine in more detai 1 , the parameters affecting DFRl gene amplificat~ion in yeast . The mean B- gal activity in drug-resistant derivatives of this strain could be increased from 3 to 6 or 7 fold relative to the parent, by stepwise sel ection using increasing MTX concentrations. B-gal overproduction was found to be un stable in all primary and highly -resistant isolates examined. There was no indication, h owever, of a decrease i n growth r a t e in MTX-res i s tant cells which overproduced B - gal.

Personality type and self-directed learning

This correlational study was designed to investigate the relationship between self-directed learning and personality type. A sample of 133 graduate and undergraduate education students completed the MBTI and the SDLRS. Two hypotheses were examined: (a) scores on the intuitive scale will account for a significant amount of the variance in the prediction of selfdirected learning readiness and, (b) scores on the introverted scale will account for a significant amount of the variance in self-directed learning readiness. Stepwise multiple regression analyses indicated that psychological type accounts for 28% of the variance in self-directed learning. Support for the first hypothesis was found with 15% of the variance in selfdirected learning accounted for by intuition. The second hypothesis was not supported. Introversion accounted for 13% of the variance but in a negative manner. Results of this study indicate that personality type does influence the ability of the learner to be self-directed in studies. These findings add another dimension for the adult educator to consider when attempting to develop self-directedness in learners.

Test of Hockman and Oldham's job characteristics model in a post-secondary educational setting

One hundred and seventy-two subj ects participated in this quantitative, correlational survey which tested Hackman and Oldham's Job Characteristics Model in an educational setting. Subjects were Teaching Masters, Chairmen and Deans from an Ontario community college. The data were collected via mailed questionnaire, on all variables of the model. Several reliable, valid instruments were used to test the variables. Data analysis through Pearson correlation and stepwise multiple regression analyses revealed that core job characteristics predicted certain critical psychological states and that these critical psychological states, in turn were able to predict various personal and work outcomes but not absenteeism. The context variable, Satisfaction with Co-workers, was the only consistent moderating variable between core characteristics and critical psychological states; however, individual employee differences did moderate the relationship between critical psychological states and all of the personal and work outcomes except Internal Work Motivation. Two other moderator variables, Satisfaction with Context and Growth Need Strength, demonstrated an ability to predict the outcome General Job Satisfaction. The research suggests that this model may be used for job design and redesign purposes within the community college setting.

The impact of meaningfulness of and responsibility for work on the relationships among work values, organizational commitment, and job satisfaction for part- time faculty in a community college

One hundred and thirty four subjects participated in this survey. Quantitative data were obtained and correlational analyses were used to test a model to study the relationships among the achievement of work values and organizational commitment and job satisfaction and to identify the moderating effects of the meaningfulness of work and responsibility for work on these relationships. Part-time faculty in the Faculty of Continuing Education of a community college were mailed a questionnaire on all the variables of the model. Several reliable, valid instruments were used to test the variables. Data analysis through Pearson correlation and stepwise multiple regression analyses revealed that the achievement of the work values of recognition and satisfaction with promotions did predict organizational commitment and job satisfaction, although the moderating effects of the meaningfulness of work and responsibility for work was not supported in this study. This study suggests that the revised model may be used for determining the relationships between the achievement of work values and organizational commitment and job satisfaction in a community college setting.

Work values and the job characteristics - job outcome relationship

The importance and achievement of work values were found to be significant variables in the job characteristics-job outcome relationship when investigated with a sample of 117 correctional nurses. Data collected via mailed questionnaires were analyzed using Pearson correlation coefficients and stepwise multiple regression. The results indicated that the achievement of intrinsic work values was found to mediate the relationship between job characteristics and internal work motivation and satisfaction, adding considerable explained variance to each of the job outcomes. In addition, the importance of the same intrinsic work values moderated the overall relationship as well as the relationship between job characteristics and achievement of work values. The importance of work values did not, however, moderate the achievement of work values-job outcome relationship as predicted. The practical implications for correctional administrators and nurses, as well as implications for further research were discussed.

Predictors of primary health care utilization by former residents of institutions in Ontario

For years institutionalization has been the primary method of service delivery for persons with developmental disabilities (DD). However, in Ontario the last institution was closed on March 31, 2009 with former residents now residing in small, communitybased homes. This study investigated potential predictors of primary health care utilization by former residents. Several indirect measures were employed to gather information from 60 participants on their age, health status, adaptive functioning level, problem behaviour, mental health status and, total psychotropic medication use. A direct measure was used to gather primary health care utilization information, which served as the dependent variable. A stepwise linear regression failed to reveal significant predictors of health care utilization. The data were subsequently dichotomized and the outcomes of a logistic regression analysis indicated that mental health status, psychotropic medication use and, an interaction between mental health status and health status significantly predicted higher primary health care usage.

(A) Optimization of solid body phase synthesis of RNA using the Cpep chemistry. (B) Synthesis of BODIPY labeled oligonucleotides

(A) Solid phase synthesis of oligonucleotides are well documented and are extensively studied as the demands continue to rise with the development of antisense, anti-gene, RNA interference, and aptamers. Although synthesis of RNA sequences faces many challenges, most notably the choice of the 2' -hydroxy protecting group, modified 2' -O-Cpep protected ribonucleotides were synthesized as alternitive building blocks. Altering phosphitylation procedures to incorporate 3' -N,N-diethyl phosphoramidites enhanced the overall reactivity, thus, increased the coupling efficiency without loss of integrety. Furthermore, technical optimizations of solid phase synthesis cycles were carried out to allow for successful synthesis of a homo UIO sequences with a stepwise coupling efficiency reaching 99% and a final yield of 91 %. (B) Over the past few decades, dipyrrometheneboron difluoride (BODIPY) has gained recognition as one of the most versatile fluorophores. Currently, BODIPY labeling of oligonucleotides are carried out post-synthetically and to date, there lacks a method that allows for direct incorporation of BODIPY into oligonucleotides during solid phase synthesis. Therefore, synthesis of BODIPY derived phosphoramidites will provide an alternative method in obtaining fluorescently labelled oligonucleotides. A method for the synthesis and incorporation of the BODIPY analogues into oligonucleotides by phosphoramidite chemistry-based solid phase DNA synthesis is reported here. Using this approach, BODIPY-labeled TlO homopolymer and ISIS 5132 were successfully synthesized.

Aspects of spatial and habitat ecology of multiple Anopheles species (Diptera: Culicidae): malaria vectors in the highlands and foothills of Ecuador

The resurgence of malaria in highland regions of Africa, Oceania and recently in South America underlines the importance of the study of the ecology of highland mosquito vectors of malaria. Since the incidence of malaria is limited by the distribution of its vectors, the purpose of this PhD thesis was to examine aspects of the ecology of Anopheles mosquitoes in the Andes of Ecuador, South America. A historical literature and archival data review (Chapter 2) indicated that Anopheles pseudopunctipennis transmitted malaria in highland valleys of Ecuador prior to 1950, although it was eliminated through habitat removal and the use of chemical insecticides. Other anopheline species were previously limited to low-altitude regions, except in a few unconfirmed cases. A thorough larval collection effort (n=438 attempted collection sites) in all road-accessible parts of Ecuador except for the lowland Amazon basin was undertaken between 2008 - 2010 (Chapter 3). Larvae were identified morphologically and using molecular techniques (mitochondrial COl gene), and distribution maps indicated that all five species collected (Anopheles albimanus, An. pseudopunctipennis, Anopheles punctimacula, Anopheles oswaldoi s.l. and Anopheles eiseni) were more widespread throughout highland regions than previously recorded during the 1940s, with higher maximum altitudes for all except An. pseudopunctipennis (1541 m, 1930 m, 1906 m, 1233 m and 1873 m, respectively). During larval collections, to characterize species-specific larval habitat, a variety of abiotic and biotic habitat parameters were measured and compared between species-present and species-absent sites using chi-square tests and stepwise binary logistic regression analyses (Chapter 4). An. albimanus was significantly associated with permanent pools with sand substrates and An. pseudopunctipennis with gravel and boulder substrates. Both species were significantly associated with floating cyanobacterial mats and warmer temperatures, which may limit their presence in cooler highland regions. Anopheles punctimacula was collected more often than expected from algae-free, shaded pools with higher-than-average calculated dissolved oxygen. Anopheles oswaldoi s.l., the species occurring on the Amazonian side of the Andes, was associated with permanent, anthropogenic habitats such as roadside ditches and ponds. To address the hypothesis that human land use change is responsible for the emergence of multiple highland Anopheles species by creating larval habitat, common land uses in the western Andes were surveyed for standing water and potential larval habitat suitability (Chapter 5). Rivers and road edges provided large amounts of potentially suitable anopheline habitat in the western Andes, while cattle pasture also created potentially suitable habitat in irrigation canals and watering ponds. Other common land uses surveyed (banana farms, sugarcane plantations, mixed tree plantations, and empty lots) were usually established on steep slopes and had very little standing water present. Using distribution and larval habitat data, a GIS-based larval habitat distribution model for the common western species was constructed in ArcGIS v.l 0 (ESRI 2010) using derived data layers from field measurements and other sources (Chapter 6). The additive model predicted 76.4 - 97.9% of the field-observed collection localities of An. albimanus, An. pseudopunctipennis and An. punctimacula, although it could not accurately distinguish between species-absent and speciespresent sites due to its coarse scale. The model predicted distributional expansion and/or shift of one or more anopheline species into the following highland valleys with climate warming: Mira/Chota, Imbabura province, Tumbaco, Pichincha province, Pallatanga and Sibambe, Chimborazo province, and Yungilla, Azuay province. These valleys may serve as targeted sites of future monitoring to prevent highland epidemics of malaria. The human perceptions of malaria and mosquitoes in relation to land management practices were assessed through an interview-based survey (n=262) in both highlands and lowlands, of male and female land owners and managers of five property types (Chapter 7). Although respondents had a strong understanding of where the disease occurs in their own country and of the basic relationship among standing water, mosquitoes and malaria, about half of respondents in potential risk areas denied the current possibility of malaria infection on their own property. As well, about half of respondents with potential anopheline larval habitat did not report its presence, likely due to a highly specific definition of suitable mosquito habitat. Most respondents who are considered at risk of malaria currently use at least one type of mosquito bite prevention, most commonly bed nets. In conclusion, this interdisciplinary thesis examines the occurrence of Anopheles species in the lowland transition area and highlands in Ecuador, from a historic, geographic, ecological and sociological perspective.