Several expression systems for recombinant protein production, essentially cells or whole organisms are currently in use today. Recently, research into recombinant protein production revealed a more attractive expression system based on the microalgae, C. reinhardtii, for significant savings in cost and production of correctly folded recombinant proteins. However, protein yield in the microalgae remain very low, non-predictable and whether this was due to limitations in the system was unclear. Using the expression of E. coli β-glucuronidase (gus) in C. reinhardtii chloroplast, the overall aim of the project was to address if the low recombinant gus yield in C. reinhardtii was due to limitations that affect growth and protein production, and if the fluxes for recombinant gus production were suboptimal (limiting). The finding was used to implement a strategy for a more predictable recombinant protein yield in C. reinhardtii. The research involved a range of experiments, analysis, and Flux Balance Analysis (FBA) modelling. The growth of C. reinhardtii cultures were characterized in autotrophic, heterotrophic and mixotrophic conditions to identify factors that limit growth and recombinant gus yields. These factors were availability of light, carbon and nitrogen substrates, pH changes, protein burden and energetic limitation (ATP). The highest biomass was obtained in autotrophic and mixotrophic cultures (>1 g/litre), the lowest biomass was in heterotrophic cultures (~0.4 g/litre). The recombinant gus yields on the basis of dry cell weight were: mixotrophic cultures (0.038%), autotrophic cultures (0.032%), heterotrophic cultures (0.026%). No detectable protein burden was observed for expression of recombinant gus in autotrophic and mixotrophic conditions, but protein burden was significant in heterotrophic condition (15 – 18% reduction in growth rate). A strategy that significantly increased growth and cell productivity (>3 fold) in heterotrophic condition was identified. FBA was used to identify suboptimal amino acid steady state fluxes (bottlenecks) that limited the gus yield. Using FBA modelling, model verifications and corrections, a strategy that significantly increased the yield of recombinant gus in each cell (~2 fold) was identified. Put together, the total increase represents a 6 fold increase in recombinant gus yield. Furthermore, this research presented a framework for identifying, analysing and understanding the effect of the uptake of individual amino acid towards recombinant protein yield.

The research investigation reported herein examined the quality and experience of the functional behavioral assessment (FBA) process as reported by educators working with students with emotional/behavioral disorders (EBD). The data accrued is based on a 30-item survey and participant telephone interviews examined the overall knowledge of the FBA process by educators and their general experience when conducting a FBA, specifically the procedural timeline, types of education professionals involved, and typical outcomes and results. Survey responses indicated two common barriers in the FBA implementation which often inhibit best practice: lack of true collaborative teamwork and insufficient communication among FBA team members. Survey responses also indicated a level of statistical significance from education professionals working in elementary school settings who self-rated positively higher when conducting a FBA than those education professionals working on either a secondary or special campus. Additionally, participant interviews indicated a growing awareness of the advantages of using the FBA as an early intervention process when dealing with challenging behaviors.

A case study of FBAs conducted by school-based teams analyzed 26 complete FBAs from rural LEAs trained by a university-based PBIS project staff. Interviews and observations were more often used than instruments/procedures. School teams initiated FBAs quicker but took longer to complete than did independent consultants. Researchers and practitioners will identify essential FBA components; how FBAs can be analyzed per these components; describe study results; and, discuss implications for research and application.

An overexposure to ultraviolet radiation can cause sunburn and some forms of skin cancer. UV light causes many different photoproducts. The cys-syn cyclobutylpyrimidine dimer (CPD) is the major photoproduct upon UV irradiation. DNA photolyase (PL) is a light-driven flavoprotein that repairs CPD in UV-damaged DNA. This repair process occurs in the presence of blue light through ultrafast photo-induced electron transfer from reduced anionic flavin adenine dinucleotide (FADH¯) to the CPD by an unknown mechanism. Since the excited state flavin transfers an electron to repair the damaged DNA, it is of utmost importance that we understand better the excited state properties of the flavins. In this work the excited state electronic properties of all three-oxidation states of flavin: oxidized form (FAD), semiquinone radical form (FADH•) and reduced anionic form (FADH¯) were studied using Stark spectroscopy and complimented by time dependent density functional theory (TD-DFT) calculations. These results are presented and discussed in Chapter 3 and 4. The difference dipole moments (Δμ) and the difference polarizabilities (Tr(Δα01)) were experimentally determined for first two lowest optically accessible states. The results are discussed in the context of photoreduction of flavins in wider class of flavoprotein blue light photoreceptors and catalytic electron transfer process in DNA repair. In the later part of this thesis (Chapters 5 and 6) the excited state electronic properties of monomeric 2-Aminopurine (2AP), 8-Vinyladenine 8VA were presented. These 8VA, 2AP are examples of fluorescent nucleotide analogues of adenine that can be incorporated into DNA with little perturbation of the normal double-helical structure. The fluorescence of these analogues is quenched when incorporated in double-stranded DNA (dsDNA). The basic mechanism underlying the fluorescence quenching by base stacking of 2AP and 8VA are is not well understood, and thus exploring the excited state electronic structures of these bases is an important first step. We have explored the excited state properties of 2AP and 8VA in frozen LiCl and ethanol solutions using Stark spectroscopy. High-level ab initio and TD-DFT calculations were performed to compliment the experimental results. / Chemistry

The purpose of the study was to further examine the potential efficacy of the Bully Prevention in Positive Behavior Support (BP-PBS) program in addressing bullying in an urban public elementary school with a large and diverse student population, implementing school-wide positive behavior support with relatively low fidelity. Specifically, the study examined the extent to which BP-PBS could decrease target students’ bullying behavior across settings and increase appropriate active response to bullying of students being bullied. Changes in the students’ perceptions and attitudes on school safety were also examined. This study involved a multiple probe design across participants targeting multiple settings for six students (three bullies and three victims) to assess the impact of the BP-PBS intervention and to examine its generalization effects to a non-targeted setting. The results indicated that the BP-PBS intervention was effective in reducing the target students’ bullying maintained by peer and adult attention and increasing active response to bullying. The intervention also increased positive perceptions and attitudes on school safety in students being bullied and the bullies. Changes in student bullying and appropriate and inappropriate responses to bullying were generalized to non-training settings and remained at intervention levels for all participants. Social validity surveys indicated high acceptability of the BP-PBS intervention by teachers.

Practitioners of Forecast-based Action (FbA) argue that a humanitarian response able to utilize forecasts to accurately predict disaster, secure funding, and take action before the onset of a crisis will benefit donors and beneficiaries alike. In search of effective and efficient early-action regimes, a number of major humanitarian actors are developing FbA projects of various designs, predominantly in response to natural disaster and famine. While numerous organizations and institutions have expressed interest in developing FbA mechanisms, the tool has only been applied in a limited capacity to the humanitarian needs generated by armed conflict. This research seeks to understand whether a scalable FbA approach can be developed to stage principled, anticipatory humanitarian action in response to situations in which rigorous evaluations predict the likelihood of imminent armed conflict. The hypothesis is that the application of FbA to armed conflict is possible, but due to the complex political nature of conflict, implementing organizations should try to focus on creating mechanisms managed by humanitarian actors and, in so far as possible, be insulated from outside influence. This research is the first academic work to specifically investigate the application of FbA to armed conflict. Following an extensive review of current FbA mechanisms and conflict early warning practices, this research concludes that a conflict-centered FbA system akin to the automated FbA systems in use today to respond to natural disaster and famine is possible, but that the endeavor presents many practical and conceptual barriers to implementation. In particular, diffuse models such as the Start Fund offer a hopeful glimpse at a type of horizontal, member-driven FbA mechanism that is both highly context-sensitive and relatively insulated from outside influence. Such a design, however, features notable and inherent limitations in its ability to reliably and accurately predict the outbreak of conflict and respond in a manner that minimizes regretful actions.

L'IRES du VHC est organisé en une structure secondaire, complexe et hautement conservée, qui comprend quatre domaines distincts (I-IV). Cette organisation permet la liaison directe du facteur d'initiation eucaryote eIF3 et de la sous-unité 40S du ribosome. Le facteur d'initiation eucaryote eIF3 est un complexe de 13 protéines (~ 800 kDa). Certaines de ses sous-unités montrent une affinité pour l'ARN, et parmi elles deux possèdent des motifs de reconnaissance à l'ARN (MRR) : eIF3b (aa : 185-268) et eIF3g (aa : 239-317). Afin de déterminer l'implication de ces motifs dans l'interaction ARN-protéine, nous avons cloné les domaines contenant ces motifs MRR, ainsi que les protéines entières eIF3b et eIF3g. Nous avons ensuite étudié l'interaction de l'ARN et/ou domaines d'ARN avec le complexe eIF3 et/ou protéines ou domaines protéiques en utilisant la méthode de rétention sur filtre. Nous avons ainsi calculé les différentes constantes apparentes de dissociation entre : eIF3/IRES, eIF3b/IRES et MRR-eIF3b/IRES. Ces résultats montrent que la sous-unité eIF3b se lie directement au domaine III de l'IRES via son motif MRR situé dans le domaine N-terminal. Dans un deuxième temps, nous avons utilisé la spectroscopie RMN pour identifier précisément les acides aminés impliqués dans la reconnaissance de l'ARN viral. En utilisant différentes techniques telles que FBA, RMN et SAXS, il s'avère que le domaine IIId (30 nucléotides) représente le motif minimum impliqué dans l'interaction MRR-eIF3b/IRES. Enfin, la technique de SAXS a été mise à profit pour étudier la structure tridimensionnelle de l'IRES libre en solution.

Cyanobakterien sind meist photoautotroph lebende Prokaryoten, welche nahezu alle Biotope der Welt besiedeln. Sie gehören zu den wichtigsten Produzenten der weltweiten Nahrungskette. Um sich auf den täglichen Wechsel von Tag und Nacht einzustellen, besitzen Cyanobakterien eine innere Uhr, bestehend aus den Proteinen KaiA, KaiB und KaiC, deren biochemische Interaktionen zu einem 24-stündigen Rhythmus von Phosphorylierung und Dephosphorylierung führen. Die circadiane Genexpression im Modellorganismus Synechocystis sp. PCC 6803 habe ich mittels drei verschiedener Zeitserienexperimente untersucht, wobei ich einen genauen Zeitplan der Genaktivierung in einer Tag-Nacht-Umgebung, aber keine selbsterhaltenden Rhythmen entdecken konnte. Allerdings beobachtete ich einen überaus starken Anstieg der ribosomalen RNA in der Dunkelheit. Aufgrund ihrer hohen Wachstumsraten und der geringen Anforderungen an die Umwelt bilden Cyanobakterien eine gute Grundlage für die nachhaltige Erzeugung von Biokraftstoffen, für einen industriellen Einsatz sind aber weitere Optimierung und ein verbessertes Verständnis des Metabolismus von Nöten. Hierfür habe ich die Orthologie von verschiedenen Cyanobakterien sowie die Konservierung von Genen und Stoffwechselwegen untersucht. Mit einer neu entwickelten Methode konnte ich gemeinsam vorkommende Gene identifizieren und zeigen, dass diese Gene häufig an einem gemeinsamen biologischen Prozess beteiligt sind, und damit bisher unbekannte Beziehungen aufdecken. Zusätzlich zu den diskutierten Modulen habe ich den SimilarityViewer entwickelt, ein grafisches Computerprogramm für die Identifizierung von gemeinsam vorkommenden Partnern für jedes beliebige Gen. Des Weiteren habe ich für alle Organismen automatische Rekonstruktionen des Stoffwechsels erstellt und konnte zeigen, dass diese die Synthese von gewünschten Stoffen gut vorhersagen, was hilfreich für zukünftige Forschung am Metabolismus von Cyanobakterien sein wird. / Cyanobacteria are photoautotrophic prokaryotes populating virtually all habitats on the surface of the earth. They are one of the prime producers for the global food chain. To cope with the daily alternation of light and darkness, cyanobacteria harbor a circadian clock consisting of the three proteins KaiA, KaiB, and KaiC, whose biochemical interactions result in a phosphorylation cycle with a period of approximately 24 hours. I conducted three time-series experiments in the model organism Synechocystis sp. PCC 6803, which revealed a tight diurnal schedule of gene activation. However, I could not identify any self-sustained oscillations. On the contrary, I observed strong diurnal accumulation of ribosomal RNAs during dark periods, which challenges common assumptions on the amount of ribosomal RNAs. Due to their high growth rates and low demand on their environment, cyanobacteria emerged as a viable option for sustainable production of biofuels. For an industrialized production, however, optimization of growth and comprehensive knowledge of the cyanobacterial metabolism is inevitable. To address this issue, I analyzed the orthology of multiple cyanobacteria and studied the conservation of genes and metabolic pathways. Systematic analysis of genes shared by similar subsets of organisms indicates high rates of functional relationship in such co-occurring genes. I designed a novel approach to identify modules of co-occurring genes, which exhibit a high degree of functional coherence and reveal unknown functional relationships between genes. Complementing the precomputed modules, I developed the SimilarityViewer, a graphical toolbox that facilitates further analysis of co-occurrence with respect to specific cyanobacterial genes of interest. Simulations of automatically generated metabolic reconstructions revealed the biosynthetic capacities of individual cyanobacterial strains, which will assist future research addressing metabolic engineering of cyanobacteria.