Recombinant Candida antarctica lipase B was successfullyproduced in the methylotropic yeast Pichia pastoris. Thespecific activities of Candida antarctica lipase B produced inPichia pastoris and commercial Candida antarctica lipase B fromNovozymes were the same. In shake-flask cultivations theexpression levels were about 25 mg L-1. Production levels couldbe increased to 1.5 g L-1, using a fermentor. A model tosimulate growth and oxygen consumption was described. The highcell density growth could be explained by the low maintenancecoefficient of Pichia pastoris. Enrichment of the aeration withoxygen increased the recombinant protein production. The lipasewas also produced as a fusion to a cellulose binding module.The cellulose binding module did not interfere with thespecific activity of the lipase. With this fusion proteincatalytic reactions can be performed in close proximity to acellulose surface. The binding module can also function as anaffinity tag for purification. Establishment of the Candidaantarctica lipase B production system allowed the engineeringof Candida antarctica lipase B variants. Four differentvariants were produced in order to investigate if electrostaticinteractions contributed to enantioselectivity. Theenantioselectivity of two halogenated secondary alcohols wasdoubled for the Ser47Ala variant. Thisimplied thatelectrostatic interactions are important forenantioselectivity. The Trp104His variant showed a decrease inenantioselectivity for all tested substrates. This was causedby an increase in the size of the stereoselectivity pocket.Symmetrical secondary alcohols of different size were used tomap the stereoselectivity pocket. A substituent as large as apropyl or isopropyl could be accommodated in the pocket of theTrp104His variant. In the wild-type lipase thestereoselectivity pocket was estimated to fit an ethyl group.The enzyme variants were subjected to a thermodynamic study, toelucidate changes in the enthalpic and entropic contributionsto enantioselectivity. The enthalpic and entropic contributionschanged for the different lipase variants and werecompensatory. The compensation was not perfect, allowing forchanges in enantioselectivity. In general one can conclude that rational design of newenzyme properties, in order to change the substrateselectivity, is feasible if based on a thorough model ofsubstrate enzyme interactions. <b>Key words:</b>Protein expression, Candida antarctica lipaseB, Pichia pastoris, sitedirected mutagenesis, fermentation,selectivity

Enzymes are gaining increasing importance as catalysts for selective transformations in organic synthetic chemistry. The engineering and design of enzymes is a developing, growing research field that is employed in biocatalysis. In the present thesis, combinatorial protein engineering methods are applied for the development of Candida antarctica lipase A (CALA) variants with broader substrate scope and increased enantioselectivity. Initially, the structure of CALA was deduced by manual modelling and later the structure was established by X-ray crystallography. The elucidation of the structure of CALA revealed several biocatalytically interesting features. With the knowledge derived from the enzyme structure, enzyme variants were produced via iterative saturation mutagenesis (ISM), a powerful protein engineering approach. Several of these variants were highly active and enantioselective towards bulky esters. Furthermore, an extensively combinatorial protein engineering approach was developed and investigated. A CALA variant with a spacious substrate binding pocket that can accommodate an unusually bulky substrate, an ester derivate of the non-steroidal anti-inflammatory drug (S)-ibuprofen, was obtained with this approach. / At the time of the doctoral defence the following paper was unpublished and had a status as follows: Paper nr. 5: Manuscript

In directed forgetting research, participants are instructed to forget information recently learned, and asked instead to remember new information given later. When asked to recall both the to-be-remembered and the to-be-forgotten information, participants successfully exhibit directed forgetting by recalling more to-be-remembered material, than to-be-forgotten material. In the present study, two directed forgetting list method experiments were conducted on undergraduate students of psychology (n = 25; n = 78). The aim of the study was to see if retrieval inhibition between participants with or without traumatic childhood experiences differed, when presented with negative or positive words. All participants were screened for childhood trauma with the CTQ-SF. The participants in the second experiment were additionally screened for dissociation with the DES-II. While Experiment 1, possibly due to small sample size failed to attain a directed forgetting effect, Experiment 2 succeeded. The issue of childhood trauma did not influence the directed forgetting effect.

Mushrooms are believed to have a great potential for production of bioactive metabolites e. g. terpenes, a group of interesting compounds with diverse chemical properties such as antitumour and antibacterial activity. Cop6 is a terpene cyclase isolated from the mushroom Coprinus cinereus that catalyzes the cyclization of farnesyl diphosphate (FPP) to mainly α-cuprenene. In this study gas chromatography combined with mass spectroscopy (GC-MS) is used to analyze the product profile of Cop6 mutants created by PCR based site directed mutagenesis. The goal is to produce trichodiene, the parent hydrocarbon in the biosynthesis of trichothecene antibiotics and mycotoxins. Valine instead of tyrosine in amino acid position 195 resulted in cyclisation of (E)-β-Farnesene and (3Z,6E)-α-Farnesene besides the products of the wild type enzyme. Another mutant with aspartic acid instead of asparagine in position 224 resulted in the synthesis of β-Bisabolene except for α-cuprenene and methionine in position 74 instead of isoleucine killed the activity of the cyclase. Furthermore, an attempt to saturation of position 98 was made, resulting in four mutants. Two of them essentially killed the activity of the cyclase whereas two had minor effect of the product profile compared to the wild type.

Existing literature demonstrates that infants can discriminate between categories of infant-directed (ID) speech based on the speaker’s intended message – that is, infants recognize the difference between comforting and approving ID speech, and treat different utterances from within these two categories similarly. Furthermore, the literature also demonstrates that infants understand many aspects of music and can discriminate between happy and sad music. Building on these findings, the present study investigated whether exposure to happy or sad piano music would systematically affect infants’ preferences for comforting or approving ID speech. Five- to nine-month-old infants’ preferences for comforting or approving ID speech were examined as a function of whether infants were exposed to sad or happy piano music. Seventeen (10 male, 7 female) full-term, healthy infants were included in the study. It was hypothesized that relative to infants exposed to happy music, infants exposed to sad music would demonstrate a stronger desire to hear comforting ID speech. The study employed an infant controlled, preferential looking procedure to test this hypothesis. The results of the study did not statistically support the researchers’ hypotheses. Limitations of the present work and suggestions for future research are discussed.

Aim: This paper is a report on a study that explored the professional development of intensive care unit nurses in journal clubs. Background: Evidence-based practice is important in nursing care (Krom, Batten, & Bautista, 2010). However few nurses feel comfortable using evidence to guide their practice (Pravikoff, Tanner, & Pierce, 2005). Journal clubs are a way to establish science as conversation (Wright, 2004) and foster knowledge translation for evidence-based nursing practice (Goodfellow, 2004). Methods: Monthly journal club meetings were held with the participation of a total of 71 healthcare professionals (65 nurses, 2 physicians, 2 pharmacists, 1 physiotherapist, and 1 respiratory therapist), who worked in two intensive care units of an Ontario hospital. After six months of meetings, 21 individual interviews were conducted with nurses, physicians, pharmacists, and nurse educators. Additional data collection included two focus groups, surveys, a review of staff meeting minutes, and researcher field notes. Findings: Journal clubs provided nurses with incentive to read research articles, improved nurses’ confidence in reading research, created a community of peers who worked collaboratively to improve clinical practice, provided a structure for nurses to reflect-on-practice, and led to reported changes in clinical practice. However, the data suggests that any gains in competence of nurses with the critical appraisal of research articles were probably modest. Barriers to participating in journal clubs and evidence-based practice are also identified. Conclusion: Journal clubs can foster knowledge translation and evidence-based practice through creating a community of practice and by providing nurses with motivation, structure, and confidence to read research articles. However, nurses reported a lack of critical appraisal skills and uncertainty about how to implement evidence into practice. Journal clubs may have a greater impact when implemented alongside other knowledge translation strategies such as working with clinical nurse specialists in order to enhance evidence-based practice.

Context: Self-learning (i.e., students learning independently) and clinical simulation are essential components in contemporary health professions education (HPE). Self-learning is discussed often, yet the concept is seldom the target of rigorous study. Likewise, simulation modalities are abundant, though educational theory that guides their use in HPE remains elusive. Objectives: This dissertation investigates the effects of directed self-guided learning (DSGL) on novice health professions students’ skill acquisition, retention, and transfer in the context of simulation-based education. The objective is to explore how the combination of external direction and student self-guidance influences: students’ cognitive and metacognitive processes, students’ interactions with the learning environment and available resources, and how students learn in different DSGL contexts. Methods: Three research studies used randomized, controlled experimental designs to address five hypotheses. All studies included a performance assessment one-week after the initial practice session that evaluated skill retention and/or skill transfer. Data analysis employed univariate and multivariate analyses of variance and correlational techniques. Results: Regarding students’ cognitive and metacognitive processes, the data show a relation between DSGL and goal-setting. The results suggest that self-guided students benefit when they are directed to set goals related to performance processes, rather than performance outcomes. Regarding the learning environment, when students are directed to practice on simulators that increase progressively in fidelity (i.e., realism) they self-guide their advancement between those simulators effectively and display successful skill transfer. Finally, self-guided students that controlled their learning progression and learning sequence selected the theoretically most appropriate practice schedule (i.e., progressive learning). Students in this latter group seemed able, surprisingly, to direct their own self-guidance. Conclusions: This dissertation adds support to the hypothesis that self-guided students benefit due to their autonomy in controlling practice conditions to meet their own learning needs. Thus, the question of whether or not DSGL is effective, becomes how best to augment the DSGL experience. The instructional design of elements such as goals lists and task structuring (e.g., progressive increases in simulator fidelity) represent techniques that an educator can use to fulfill the role of director in a student’s SGL.

This thesis explores educative ideas such as student choice, student autonomy and student directed learning in an educational environment dominated by a top-down, provincially legislated curriculum. Qualitative data was obtained from the professional stories of two experienced educators teaching in public alternative schools in Toronto, Ontario. Using the theoretical lenses of autonomy, relatedness and competency to filter the collected data reveals that offering students authentic choice in their school day has many benefits. Despite this, our globally entrenched educational paradigm dominates our educational world usurping many alternative models.

Cyanide is acutely toxic to the environment. However, this simple nitrile is used in several industrial applications especially the mining industry. Due to its high affinity to metals, cyanide has been used for years to extract gold and other precious metals from the ore. Cyanide nitrilases are considered for the detoxification of the industrial wastewaters contaminated with cyanide. Their application in cyanide remediation promises cheaper and safer processes compared to chemical detoxification. However, application of these enzymes in industry requires improving their characteristics. The goal of this dissertation is to better understand cyanide nitrilases, in particular the cyanide dihydratase from of Bacillus pumilus and Pseudomonas stutzeri and to improve their activity and stability. The lack of any high resolution structure of these enzymes calls for isolating or screening for mutants showing enhancement in enzyme properties. Described first is a simple and efficient method utilizing in vivo recombination to create recombinant libraries incorporating the products of PCR amplification. This method is useful for generating large pools of randomly mutagenized clones after error-prone PCR mutagenesis. Several parameters were investigated to optimize this technique; length of homology region, vector treatment, induction time and ratio of fragment to vector. Using error-prone PCR for random mutagenesis, several CynDpum mutants were isolated for higher catalysis at pH 7.7. Three point mutations, K93R, D172N and E327K increased the enzyme’s thermostability. The D172N mutation also increased the affinity of the enzyme for its substrate at pH 7.7 suggesting an effect on the active site. However, the A202T mutation located in the dimerization or the A surface rendered the enzyme inactive by destabilizing it. No significant effect on activity at alkaline pH was observed for any of the purified mutants. Lastly, an important region for CynDstut activity was identified in the C-terminus. This same region increased the stability of CynDpum compared to the wild-type enzyme. Also, CynDpum-stut hybrid was found to be highly more stable than CynDpum. This same hybrid exhibited 100% activity at pH9, a pH where the parent enzyme is inactive, and retained 40% of its activity at pH 9.5 making it a true pH tolerant mutant.

This thesis explores educative ideas such as student choice, student autonomy and student directed learning in an educational environment dominated by a top-down, provincially legislated curriculum. Qualitative data was obtained from the professional stories of two experienced educators teaching in public alternative schools in Toronto, Ontario. Using the theoretical lenses of autonomy, relatedness and competency to filter the collected data reveals that offering students authentic choice in their school day has many benefits. Despite this, our globally entrenched educational paradigm dominates our educational world usurping many alternative models.