Towards reverse engineering of Photosystem II: Synergistic Computational and Experimental Approaches

ABSTRACT Photosystem II (PSII) of oxygenic photosynthesis has the unique ability to photochemically oxidize water, extracting electrons from water to result in the evolution of oxygen gas while depositing these electrons to the rest of the photosynthetic machinery which in turn reduces CO2 to carbohydrate molecules acting as fuel for the cell. Unfortunately, native PSII is unstable and not suitable to be used in industrial applications. Consequently, there is a need to reverse-engineer the water oxidation photochemical reactions of PSII using solution-stable proteins. But what does it take to reverse-engineer PSII’s reactions? PSII has the pigment with the highest oxidation potential in nature known as P680. The high oxidation of P680 is in fact the driving force for water oxidation. P680 is made up of a chlorophyll a dimer embedded inside the relatively hydrophobic transmembrane environment of PSII. In this thesis, the electrostatic factors contributing to the high oxidation potential of P680 are described. PSII oxidizes water in a specialized metal cluster known as the Oxygen Evolving Complex (OEC). The pathways that water can take to enter the relatively hydrophobic region of PSII are described as well. A previous attempt to reverse engineer PSII’s reactions using the protein scaffold of E. coli’s Bacterioferritin (BFR) existed. The oxidation potential of the pigment used for the BFR ‘reaction centre’ was measured and the protein effects calculated in a similar fashion to how P680 potentials were calculated in PSII. The BFR-RC’s pigment oxidation potential was found to be 0.57 V, too low to oxidize water or tyrosine like PSII. We suggest that the observed tyrosine oxidation in BRF-RC could be driven by the ZnCe6 di-cation. In order to increase the efficiency of iii tyrosine oxidation, and ultimately oxidize water, the first potential of ZnCe6 would have to attain a value in excess of 0.8 V. The results were used to develop a second generation of BFR-RC using a high oxidation pigment. The hypervalent phosphorous porphyrin forms a radical pair that can be observed using Transient Electron Paramagnetic Resonance (TR-EPR). Finally, the results from this thesis are discussed in light of the development of solar fuel producing systems.

Human Endogenous Retrovirus (HERV) Insertional Polymorphisms

Human endogenous retroviruses (HERVs) are the result of ancient germ cell infections of human germ cells by exogenous retroviruses. HERVs belong to the long terminal repeat (LTR) group of retrotransposons that comprise ~8% of the human genome. The majority of the HERVs documented have been truncated and/or incurred lethal mutations and no longer encode functional genes; however a very small number of HERVs seem to maintain functional in making new copies by retrotranspositon as suggested by the identification of a handful of polymorphic HERV insertions in human populations. The objectives of this study were to identify novel insertion of HERVs via analysis of personal genomic data and survey the polymorphism levels of new and known HERV insertions in the human genome. Specifically, this study involves the experimental validation of polymorphic HERV insertion candidates predicted by personal genome-based computation prediction and survey the polymorphism level within the human population based on a set of 30 diverse human DNA samples. Based on computational analysis of a limited number of personal genome sequences, PCR genotyping aided in the identification of 15 dimorphic, 2 trimorphic and 5 fixed full-length HERV-K insertions not previously investigated. These results suggest that the proliferation rate of HERVKs, perhaps also other ERVs, in the human genome may be much higher than we previously appreciated and the recently inserted HERVs exhibit a high level of instability. Throughout this study we have observed the frequent presence of additional forms of genotypes for these HERV insertions, and we propose for the first time the establishment of new genotype reporting nomenclature to reflect all possible combinations of the pre-integration site, solo-LTR and full-length HERV alleles.

Our future in geriatrics : an examination of the knowledge, attitudes and career choices of physical therapy students in Ontario : a pretest-posttest study /

There continues to be a shortage of health professionals interested in providing care for the older adult. Part of the problem seems to stem from the negative perceptions of geriatrics as a clinical speciality. This study examines the knowledge, attitudes and career decisions of physical therapy students in Ontario before and after an educational intervention. Surveys were conducted with 144 physical therapy students from five universities before and after their geriatrics course in order to measure their knowledge, attitudes and interest in working with older adults. The incoming class of physical therapy students (n = 1 86) acted as control subjects for the study. The Revised Palmore Facts On Aging Quiz measured the students' knowledge of aging (Miller & Dodder, 1980). The Revised Tuckman-Lorge (Axelrod & Eisdorfer, 1961) and the Kogan Old People Scales (Kogan, 1961) were used to examine attitude. An environmental scale was developed based on the work of Snape (1986) to measure the impact of the working conditions on the students' career choices. A 10-point Likert-type scale based on the work of Michlelutte & Diseker (1985) was modified and used to measure career interest in working with the elderly. On independent sample t-tests, positive attitudes were related to the demographic characteristic of gender; ethnicity was negatively related; and marital status was found to be unrelated to attitude (fi<.05). Having a relationship with an older adult and taking courses in gerontology were also found to be positively related to attitude (fi<.05). Results on a betweensubjects design which compared students before and after the course found that knowledge scores improved from pretest to posttest (fi<.05). In general, attitude scores improved from T1 to T2 on both measurement tools (b<.05). The environmental and vocational interest scales yielded statistically significant differences between the control and experimental groups during the intervention period (p<.05). The results of this research indicated that knowledge and attitudes improve after an educational intervention; however, there was little impact on the students' overall career decisions. Further research is indicated to examine the complex relationship between attitude and behaviour and its impact on students' career choices. In addition, the impact of geriatric clinical environment on students' attitudes and career decisions needs to be further explored.

What happens in the first 200 ms of word reading : ERP studies on visual word recognition with top-down and bottom-up approaches /

In this thesis, three main questions were addressed using event-related potentials (ERPs): (1) the timing of lexical semantic access, (2) the influence of "top-down" processes on visual word processing, and (3) the influence of "bottom-up" factors on visual word processing. The timing of lexical semantic access was investigated in two studies using different designs. In Study 1,14 participants completed two tasks: a standard lexical decision (LD) task which required a word/nonword decision to each target stimulus, and a semantically primed version (LS) of it using the same category of words (e.g., animal) within each block following which participants made a category judgment. In Study 2, another 12 participants performed a standard semantic priming task, where target stimulus words (e.g., nurse) could be either semantically related or unrelated to their primes (e.g., doctor, tree) but the order of presentation was randomized. We found evidence in both ERP studies that lexical semantic access might occur early within the first 200 ms (at about 170 ms for Study 1 and at about 160 ms for Study 2). Our results were consistent with more recent ERP and eye-tracking studies and are in contrast with the traditional research focus on the N400 component. "Top-down" processes, such as a person's expectation and strategic decisions, were possible in Study 1 because of the blocked design, but they were not for Study 2 with a randomized design. Comparing results from two studies, we found that visual word processing could be affected by a person's expectation and the effect occurred early at a sensory/perceptual stage: a semantic task effect in the PI component at about 100 ms in the ERP was found in Study 1 , but not in Study 2. Furthermore, we found that such "top-down" influence on visual word processing might be mediated through separate mechanisms depending on whether the stimulus was a word or a nonword. "Bottom-up" factors involve inherent characteristics of particular words, such as bigram frequency (the total frequency of two-letter combinations of a word), word frequency (the frequency of the written form of a word), and neighborhood density (the number of words that can be generated by changing one letter of an original word or nonword). A bigram frequency effect was found when comparing the results from Studies 1 and 2, but it was examined more closely in Study 3. Fourteen participants performed a similar standard lexical decision task but the words and nonwords were selected systematically to provide a greater range in the aforementioned factors. As a result, a total of 18 word conditions were created with 18 nonword conditions matched on neighborhood density and neighborhood frequency. Using multiple regression analyses, we foimd that the PI amplitude was significantly related to bigram frequency for both words and nonwords, consistent with results from Studies 1 and 2. In addition, word frequency and neighborhood frequency were also able to influence the PI amplitude separately for words and for nonwords and there appeared to be a spatial dissociation between the two effects: for words, the word frequency effect in PI was found at the left electrode site; for nonwords, the neighborhood frequency effect in PI was fovind at the right elecfrode site. The implications of otir findings are discussed.

Exploratory study : exploring the effectiveness of a literacy-based intervention with children with language impairments

Research points clearly to the need for all concerned stakeholders to adopt a preventative approach while intervening with children who are at-risk for future reading disabilities. Research has indicated also that a particular sub-group of children at-risk for reading impairments include preschool children with language impairments (Catts, 1993). Preschool children with language impairments may have difficulties with emergent literacy skills - important prerequisite skills necessary for successful formal reading. Only in the past decade have researchers begun to study the effects of emergent literacy intervention on preschool children with language impairments. As such, the current study continues this investigation of how to effectively implement an emergent literacy therapy aimed at supporting preschool children with language impairments. In addition to this, the current study explores emergent literacy intervention within an applied clinical setting. The setting, presents a host of methodological and theoretical challenges - challenges that will advance the field of understanding children within naturalistic settings. This exploratory study included thirty-eight participants who were recruited from Speech Services Niagara, a local preschool speech and language program. Using a between-group pre- and posttest design, this study compared two intervention approaches - an experimental emergent literacy intervention and a traditional language intervention. The experimental intervention was adopted from Read It Again! (Justice, McGinty, Beckman, & Kilday, 2006) and the traditional language intervention was based on the traditional models of language therapy typically used in preschool speech and language models across Ontario. 5 Results indicated that the emergent literacy intervention was superior to the ,t..3>~, ~\., ;./h traditional language therapy in improving the children's alphabet knowledge, print and word awareness and phonological awareness. Moreover, results revealed that children with more severe language impairments require greater support and more explicit instruction than children with moderate language impairments. Another important finding indicated that the effects of the preschool emergent literacy intervention used in this study may not be sustainable as children enter grade one. The implications of this study point to the need to support preschool children with language impairments with intensive emergent literacy intervention that extends beyond preschool into formal educational settings.

Characterisation and optimisation of the flavour of health-promoting, plantderived bitterants in functional beverages.

Flavour is a combination of taste, odour, and chemesthetic sensations. Close associations exist between these sensory modalities, and thus, the overall flavour of a food or beverage product can change when the intensity of one or more of these sensations is altered. Strategies to modify flavour are often utilized by the food industry, and are central to the engineering of new and reformulated products. For functional food and beverages, flavour modification is particularly important, as fortifying agents can elicit high levels of less than desirable sensations, such as bitterness and astringency. The application of various flavour modifying strategies can decrease the perceived intensity of these sensations, and in tum, improve the sensory profile of the product. This collection of studies describes the sensory characteristics of experimental functional beverages fortified with trans-resveratrol, (+)-catechin, and/or caffeine, and examines the impact of novel flavour modifying strategies on the perceived flavour of these beverages. In the first study, results demonstrate that the flavour profile of Cabemet Sauvignon wines fortified with 20 mglL and 200 mg/L of trans-resveratrol is not perceived as different compared to control wine (0 mglL). However, Riesling wine fortified with 200 mg/L is perceived as significantly higher in bitterness compared to 20 mglL and control. For some functional food formulations, alternative strategies for flavour modification are needed. Traditional methods, such as the addition of sucrose and sodium chloride, may decrease the perceived 'healthiness' of a product, and thus, may be sub-optimal. In a second study, high and low concentrations of five different bitter inhibiting compounds - 'bitter blockers' - (B-cyclodextrin, homoeridictyol sodium salt, carboxymethylcellulose - low viscosity, zinc sulfate, magnesium sulfate) were tested for their efficacy towards decreasing the bitterness of high and low concentrations of caffeine and (+)catechin - two health-relevant, plant-derived bitterants. B-cyclodextrin and homoeridictyol sodium salt were the most effective blockers at decreasing (+ )-catechin and caffeine, respectively. In addition to bitter blockers, additional flavour modifying strategies, either alone or in combination - may also be successful in functional food formulations. Both sucrose and rebaudioside A - a plant-derived sweetener - were effective at decreasing the bitterness of (+)catechin. When added to (+)-catechin along with B-cyc1odextrin, both sweeteners provided the most effective decrease in bitterness compared to binary, ternary, or quaternary mixtures of (+)catechin together with bitter blockers, sweeteners, andlor odourants. The perceived intensity of sensations elicited by sweeteners and odourants was not affected by the addition of bitter blockers, and thus, their impact within these complex matrices is minimal. In addition, withinmodal (taste-taste) compared to cross-modal (taste-odour) sensory interactions were more effective at decreasing the bitterness of (+ )-catechin. Overall, results from these studies demonstrate that certain novel, alternative flavour modifying approaches may be successful towards lowering the bitterness and astringency elicited by (+ )-catechin and caffeine in aqueous solutions.

Fit For Action: A Comparative Case Study of the Implementation of an Adaptive Fitness and Conditioning Program for Moderate Functioning Teens and Transition Age Youth with ASD

The purpose of my research was to develop and refine pedagogic approaches, and establish fitness baselines to adapt fitness and conditioning programs for Moderate-functioning ASD individuals. I conducted a seven-week study with two teens and two trainers. The trainers implemented individualized fitness and conditioning programs that I developed. I conducted pre and post fitness baselines for each teen, a pre and post study interview with the trainers, and recorded semi-structured observations during each session. I used multi-level, within-case and across case analyses, working inductively and deductively. My findings indicated that fundamental movement concepts can be used to establish fitness baselines and develop individualized fitness programs. I tracked and evaluated progressions and improvements using conventional measurements applied to unconventional movements. This process contributed to understanding and making relevant modifications to activities as effective pedagogic strategies for my trainers. Further research should investigate fitness and conditioning programs with lower functioning ASD individuals.