Partial characterization of the cloned dihydrofolate reductase gene of Saccharomyces cerevisiae

The cloned dihydrofolate reductase gene of Saccharomyces cerevisiae (DFR 1) is expressed in Escherichia coli. Bacterial strain JF1754 transformed with plasmids containing DFR 1 is at least 5X more resistant to inhibition by the folate antagonist trimethoprim. Expression of yeast DFR 1 in E. coli suggests it is likely that the gene lacks intervening sequences. The 1.8 kbp DNA fragment encoding yeast dhfr activity probably has its own promotor, as the gene is expressed in both orientations in E. coli. Expression of the yeast dhfr gene cloned into M13 viral vectors allowed positive selection of DFR 1 - M13 bacterial transfectants in medium supplemented with trimethoprim. A series of nested deletions generated by nuclease Bal 31 digestion and by restriction endonuclease cleavage of plasmids containing DFR 1 physically mapped the gene to a 930 bp region between the Pst 1 and Sal 1 cut sites. This is consistent with the 21,000 molecular weight attributed to yeast dhfr in previous reports. From preliminary DNA sequence analysis of the dhfr DNA fragment the 3' terminus of DFR 1 was assigned to a position 27 nucleotides from the Eco Rl cut site on the Bam Hi - Eco Rl DNA segment. Several putative yeast transcription termination consensus sequences were identified 3' to the opal stop codon. DFR 1 is expressed in yeast and it confers resistance to the antifolate methotrexate when the gene is present in 2 - 10 copies per cell. Plasmid-dependent resistance to methotrexate is also observed in a rad 6 background although the effect is somewhat less than that conferred to wild-type or rad 18 cells. Integration of DFR 1 into the yeast genome showed an intermediate sensitivity to folate antagonists. This may suggest a gene dosage effect. No change in petite induction in these yeast strains was observed in transformed cells containing yeast dhfr plasmids. The sensitivity of rad 6 , rad 18 and wild-type cell populations to trimethoprim were unaffected by the presence of DFR 1 in transformants. Moreover, trimethoprim did not induce petites in any strain tested, which normally results if dhfr is inhibited by other antifolates such as methotrexate. This may suggest that the dhfr enzyme is not the only possible target of trimethoprim in yeast. rad 6 mutants showed a very low level of spontaneous petite formation. Methotrexate failed to induce respiratory deficient mutants in this strain which suggested that rad 6 might be an obligate grande. However, ethidium bromide induced petites to a level approximately 50% of that exhibited by wild-type and rad 18 strains.

The impact of tobacco control policies on university students' smoking in Ontario

Objective. Despite steady declines in the prevalence of tobacco use among Canadians, young adult tobacco use has remained stubbornly high over the past two decades (CTUMS, 2005a). Currently in Ontario, young adults have the highest proportion of smokers of all age cohorts at 26%. A growing body of evidence shows that smoking restrictions and other tobacco control policies can reduce tobacco use and consumption among adults and deter initiation among youth; whether young adult university students' smoking participation is influenced by community smoking restrictions, campus tobacco control policies or both remains an empirical question. The purpose of this study is to examine the relationship among current smoking status of students on university campuses across Ontario and various tobacco control policies, 3including clean air bylaws of students' home towns, clean air by-laws of the community where the university is situated, and campus policies. Methods. Two data sets were used. The 200512006 Tobacco Use in a Representative Sample of Post-Secondary Students data set provides information about the tobacco use of 10,600 students from 23 universities and colleges across Ontario. Data screening for this study reduced the sample to 5,114 17-to-24 year old undergraduate students from nine universities. The second data set is researcher-generated and includes information about strength and duration of, and students' exposure to home town, local and campus tobacco control policies. Municipal by-laws (of students' home towns and university towns) were categorized as weak, moderate or strong based on criteria set out in the Ontario Municipal By-law Report; campus policies were categorized in a roughly parallel fashion. Durations of municipal and campus policies were calculated; and length of students' exposure to the policies was estimated (all in months). Multinomial logistic regression analyses were used to examine the relationship between students' current smoking status (daily, less-than-daily, never-smokers) and the following policy measures: strength of, duration of, and students' exposure to campus policy; strength of, duration of, and students' exposure to the by-law in the university town; and, strength of, duration of, and students' exposure to the by-law in the home town they grew up in. Sociodemographic variables were controlled for. Results. Among the Ontario university students surveyed, 7.0% currently use tobacco daily and 15.4% use tobacco less-than-daily. The proportions of students experiencing strong tobacco control policies in their home town, the community in which their university is located and at their current university were 33.9%,64.1 %, and 31.3% respectively. However, 13.7% of students attended a university that had a weak campus policy. Multinomial logistic regressions suggested current smoking status was associated with university town by-law strength, home town by-law strength and the strength of the campus tobacco control policy. In the fmal model, after controlling for sociodemographic factors, a strong by-law in the university town and a strong by-law in students' home town were associated with reduced odds of being both a less-than-daily (OR = 0.64, 95%CI: 0.48-0.86; OR = 0.80, 95%CI: 0.66-0.95) and daily smoker (OR = 0.59, 95%CI: 0.39-0.89; OR = 0.76, 95%CI: 0.58-0.99), while a weak campus tobacco control policy was associated with higher odds of being a daily smoker (OR = 2.08, 95%CI: 1.31-3.30) (but unrelated to less-than-daily smoking). Longer exposure to the municipal by-law (OR = 0.93; 95%CI: 0.90-0.96) was also related to smoking status. Conclusions. Students' smoking prevalence was associated with the strength of the restrictions in university, and with campus-specific tobacco control policies. Lessthan- daily smoking was not as strongly associated with policy measures as daily smoking was. University campuses may wish to adopt more progressive campus policies and support clean air restrictions in the broader community. More research is needed to determine the direction of influence between tobacco control policies and students' smoking.

The nesting biology of Ceratina (Hymenoptera: Apidae) in the Niagara Region : new species, nest site selection and parasitism

One of the most common bee genera in the Niagara Region, the genus Ceratina (Hymenoptera: Apidae) is composed of four species, C. dupla, C. calcarata, the very rare C. strenua, and a previously unknown species provisionally named C. near dupla. The primary goal of this thesis was to investigate how these closely related species coexist with one another in the Niagara ~ee community. The first necessary step was to describe and compare the nesting biologies and life histories of the three most common species, C. dupla, C. calcarata and the new C. near dupla, which was conducted in 2008 via nest collections and pan trapping. Ceratina dupla and C. calcarata were common, each comprising 49% of the population, while C. near dupla was rare, comprising only 2% of the population. Ceratina dupla and C. near dupla both nested more commonly in teasel (Dipsacus sp.) in the sun, occasionally in raspberry (Rubus sp.) in the shade, and never in shady sumac (Rhus sp.), while C. calcarata nested most commonly in raspberry and sumac (shaded) and occasionally in teasel (sunny). Ceratina near dupla differed from both C. dupla and C. calcarata in that it appeared to be partially bivoltine, with some females founding nests very early and then again very late in the season. To examine the interactions and possible competition for nests that may be taking place between C. dupla and C. calcarata, a nest choice experiment was conducted in 2009. This experiment allowed both species to choose among twigs from all three substrates in the sun and in the shade. I then compared the results from 2008 (where bees chose from what was available), to where they nested when given all options (2009 experiment). Both C. dupla and C. calcarata had the same preferences for microhabitat and nest substrate in 2009, that being raspberry and sumac twigs in the sun. As that microhabitat and nest substrate combination is extremely rare in nature, both species must make a choice. In nature Ceratina dupla nests more often in the preferred microhabitat (sun), while C. calcarata nests in the preferred substrate (raspberry). Nesting in the shade also leads to smaller clutch sizes, higher parasitism and lower numbers of live brood in C. calcarata, suggesting that C. dupla may be outcompeting C. calcarata for the sunny nesting sites. The development and host preferences of Ceratina parasitoids were also examined. Ceratina species in Niagara were parasitized by no less than eight species of arthropod. Six of these were wasps from the superfamily Chalcidoidea (Hymenoptera), one was a wasp from the family Ichneumonidae (Hymenoptera) and one was a physogastric mite from the family Pyemotidae (Acari). Parasites shared a wide range of developmental strategies, from ichneumonid larvae that needed to consume multiple Ceratina immatures to complete development, to the species from the Eulophidae (Baryscapus) and Encyrtidae (Coelopencyrtus), in which multiple individuals completed development inside a single Ceratina host. Biological data on parasitoids is scarce in the scientific literature, and this Chapter documents these interactions for future research.

The relationship between body mass index and breast cancer recurrence/progression and breast cancer-specific death

The aim of this study was to describe the nonlinear association between body mass index (BMI) and breast cancer outcomes and to determine whether BMI improves prediction of outcomes. A cohort of906 breast cancer patients diagnosed at Henry Ford Health System, Detroit (1985-1990) were studied. The median follow-up was 10 years. Multivariate logistic regression was used to model breast cancer recurrence/progression and breast cancer-specific death. Restricted cubic splines were used to model nonlinear effects. Receiver operator characteristic areas under the curves (ROC AUC) were used to evaluate prediction. BMI was nonlinearly associated with recurrence/progression and death (p= 0.0230 and 0.0101). Probability of outcomes increased with increase or decrease ofBMI away from 25. BMI splines were suggestive of improved prediction of death. The ROC AUCs for nested models with and without BMI were 0.8424 and 0.8331 (p= 0.08). I f causally associated, modifying patients BMI towards 25 may improve outcomes.

Using a Bayesian model for bankruptcy prediction : a comparative approach

The purpose of this study is to examine the impact of the choice of cut-off points, sampling procedures, and the business cycle on the accuracy of bankruptcy prediction models. Misclassification can result in erroneous predictions leading to prohibitive costs to firms, investors and the economy. To test the impact of the choice of cut-off points and sampling procedures, three bankruptcy prediction models are assessed- Bayesian, Hazard and Mixed Logit. A salient feature of the study is that the analysis includes both parametric and nonparametric bankruptcy prediction models. A sample of firms from Lynn M. LoPucki Bankruptcy Research Database in the U. S. was used to evaluate the relative performance of the three models. The choice of a cut-off point and sampling procedures were found to affect the rankings of the various models. In general, the results indicate that the empirical cut-off point estimated from the training sample resulted in the lowest misclassification costs for all three models. Although the Hazard and Mixed Logit models resulted in lower costs of misclassification in the randomly selected samples, the Mixed Logit model did not perform as well across varying business-cycles. In general, the Hazard model has the highest predictive power. However, the higher predictive power of the Bayesian model, when the ratio of the cost of Type I errors to the cost of Type II errors is high, is relatively consistent across all sampling methods. Such an advantage of the Bayesian model may make it more attractive in the current economic environment. This study extends recent research comparing the performance of bankruptcy prediction models by identifying under what conditions a model performs better. It also allays a range of user groups, including auditors, shareholders, employees, suppliers, rating agencies, and creditors' concerns with respect to assessing failure risk.

Physical Activity and Fitness in Children with Developmental Disorder

Introduction: Developmental coordination disorder (DCD) is a prevalent condition characterized by poor motor proficiency that interferes with a child‟s activities of daily living. Children with DCD often experience compromised health-related fitness components such as cardiorespiratory fitness (CRF). Purpose: To better understand the physical activity and fitness characteristics of children with probable DCD (pDCD), with a particular focus on CRF. Specifically: (1) to present a synopsis of current literature; (2) to determine the longitudinal trajectories of CRF; (3) to compare the submaximal CRF of children with and without pDCD. Methods: A comprehensive, systematic literature review was conducted of the recent available data on fitness and physical activity and pDCD (Chapter 2). This review provided the background for the other two studies included in this thesis. In Chapter 3, a prospective cohort design was used to assess how CRF in children with pDCD changes over time (56 months) relative to a group of typically developing controls. Using a nested-case control design, 63 subjects with pDCD and 63 matched controls from the larger sample were recruited to participate in the lab-based component of the study (Chapter 4). In this investigation CRF was examined using the oxygen cost of work (VO2) during an incremental test on a cycle ergometer. Results: The literature review showed that fitness parameters, including CRF and physical activity levels, were consistently reduced in children with pDCD. Chapter 3 demonstrated that the difference in CRF between children with pDCD and typically developing children is substantial, and that it tends to increase over time. Results from VO2 assessments showed that children with pDCD utilized more oxygen to sustain the same submaximal workloads compared to typically developing children. Conclusions: Findings from this thesis have made several important contributions to our understanding of children with pDCD. Since differences in CRF between children with and without pDCD tend to worsen over time, this adds to the argument that interventions intended to improve CRF may be appropriate for children with motor difficulties. This thesis also presented the first evidence suggesting that DCD involves higher energy expenditure, and could help explain why children with pDCD perform poorly on tasks requiring CRF.