The interaction between Arabidopsis and its natural downy mildew pathogen, Hyaloperonospora arabidopsidis (Hpa), provides a model for understanding how oomycetes colonize plants. Hpa is a model organism for many highly destructive oomycete pathogens and transcriptomics of this interaction have been well-documented. However, the material in these studies has been derived from infected leaves that contain a mix of pathogen-proximal and pathogen-distal plant cells. The most direct interactions between Arabidopsis and Hyaloperonospora arabidopsidis occur in haustoriated cells- where the pathogen can secrete effectors and acquire nutrients needed for successful colonization and reproduction. These cells are difficult to isolate due to their limited number and ephemeral nature. I have developed a method to isolate the translatome (i.e., mRNAs associated with ribosomes) of pathogen-proximal cells. This method utilizes translating ribosome immuno-purification technology (TRAP), regulated by both pathogen-responsive and tissue-specific promoters, to isolate mRNAs that are being translated in pathogen-proximal cells. Compared to "bulk" transcriptomics of material isolated from homogenized leaves, this method will enrich for transcripts that are differentially expressed, and translated, in pathogen-proximal cells. From this method, RNA was isolated in amount and quality sufficient for sequencing. This sequencing data will enable the discovery of plant genes that may be manipulated by the pathogen to suppress defense responses and extract nutrients. / Ph. D. / The interactions between plants and the pathogens that feed on them are complex and at times difficult to study. Among the many different types of plant pathogens, oomycetes (a class of fungus-like organisms) are especially destructive. Using Arabidopsis and its natural downy mildew pathogen, Hyaloperonospora arabidopsidis (Hpa) as model for understanding how oomycetes colonize plants, I hope to learn more about plant-pathogen interactions. Hpa is a model organism for many highly destructive oomycete pathogens and several aspects of this interaction have been well-documented. However, the material in these studies has been derived from infected leaves that contain a mix of plant cells that are both in direct contact with the pathogen, or from uninfected areas of the plant. The most direct interactions between Arabidopsis and Hpa occur in cells that have been invaginated with a pathogen feeding structure called a haustorium. These cells are difficult to isolate due to their limited number and ephemeral nature. I have developed a method to isolate the translatome (i.e., mRNAs that are being translated by and are associated with ribosomes) of pathogen-proximal cells. This method utilizes translating ribosome immuno-purification technology (TRAP), regulated by both pathogen-responsive and tissue-specific promoters, to isolate mRNAs that are being translated in pathogen-proximal cells. Compared to “bulk” transcriptomics of material isolated from homogenized leaves, this method will enrich for transcripts that are differentially expressed, and translated, in pathogen-proximal cells. From this method, RNA was isolated in amount and quality sufficient for sequencing. This sequencing data will enable the discovery of plant genes that may be manipulated by the pathogen to suppress defense responses and extract nutrients.

Stinkbugs (Pentatomidae) and other agricultural pests such as bark beetles and flea beetles are known to synthesize terpenoids as aggregation pheromones. Knowledge of the genes and enzymes involved in pheromone biosynthesis may allow engineering of pheromone biosynthetic pathways in plants to develop new forms of trap crops and agricultural practices for pest management. The harlequin bug, Murgantia histrionica, a specialist pest on crucifer crops, produces the sesquiterpene, murgantiol, as a male-specific aggregation pheromone. Similarly, the southern green stink bug, Nezara viridula, a generalist pest worldwide on soybean and other crops, releases sesquiterpene cis-/trans-(Z)-α-bisabolene epoxides as male-specific aggregation pheromone. In both species, enzymes called terpene synthases (TPSs) synthesize precursors of the aggregation pheromones, which are sesquipiperitol and (Z)-α-bisabolene as the precursor of murgantiol and cis-/trans-(Z)-α-bisabolene epoxide, respectively. We hypothesized that enzymes in the family of cytochrome P450 monooxygenases are involved in the conversion of these precursors to the final epoxide products. This study investigated the tissue specificity and sequence of these conversions by performing crude enzyme assays with protein extracts from male tissues. Furthermore, candidate P450 genes were selected by RNA-sequencing and co-expression analysis and the corresponding recombinant proteins tested for enzyme activity. To engineer the pheromone biosynthetic enzymes in plants, transient expression of the TPSs of both stink bugs was performed in Nicotiana benthamiana leaves. Both sesquipiperitol and (Z)-α-bisabolene were found to be produced and emitted from inoculated N. benthamiana leaves. Future work will implement stable transformation to engineer murgantiol biosynthesis in crucifer trap crops and develop similar approaches for pheromone engineering of other agricultural pests. / Master of Science / Stinkbugs including the harlequin bug, Murgantia histrionica and southern green stinkbug, Nezara viridula, are major agricultural pests in the US and worldwide. To control these pests with alternative pest management strategies, we have proposed to develop trap crops that emit pheromones to lure the insects away from crop fields. To establish pheromone biosynthesis in plants, we investigated the corresponding enzymatic steps in both stink bugs. We show that terpene synthase enzyme from both stink bugs can be transformed into plants for the engineering of pheromones in trap crops. With identification of P450 genes in pheromone biosynthesis enhanced trap crops can be made.

Trap crops were evaluated for harlequin bug control in broccoli field plots in 1994 and 1995. Mustard and rape prevented low densities of harlequin bug from reaching broccoli, but at high densities the insect moved from the trap plants into the broccoli. This indicates that harlequin bugs that are attracted to trap plants may damage the protected broccoli if their numbers are not prevented from increasing. Harlequin bugs were shown to have two and a partial third generation a year. Trissolcus murgantiae Ashmead and Ooencyrtus johnsoni Howard, were identified as egg parasitoids. Their combined parasitization levels for the two years were 8% and 37%. T. murgantiae accounted for 87% and 96% of the parasitization, respectively.. When 19.6 cm and 11.9 cm broccoli plants were exposed to five densities of harlequin bug adults a negative correlation between plant mortality and insect density was observed (y = 38.00 - 2.32x, r2 = 0.95 and y = 22.17 - 1.17x, r2 = 0.99, respectively). No correlation was observed in broccoli plants 11.9 tall.. Host plants affected harlequin bug development. Nymphs developed faster when reared on rape in comparison with mustard. The preoviposition time for rape reared nymphs was shorter than mustard reared insects. Fecundity and viability of eggs were not different for harlequin bugs reared at different sex ratios. / Master of Science

L'huile de Sésame a un grand intérêt économique et industriel et est utilisée comme ressource biologique riche en protéines, en antioxydants et comme substrat de bioprocédés pour la production d'enzymes, d'antibiotiques ou de biopesticides. Nous avons préparé, par transestérification de l'huile via un ester méthylique, un mélange d'esters de l'acide vanillique (VNA) et obtenu un produit plus hydrosoluble VNA-2, qui a ensuite été encapsulé dans des billes de pectinate de calcium ou de zinc, visant à une libération contrôlée dans le colon. La dissolution des billes a été étudiée aux pH de l'estomac et du colon, et en milieu enzymatique. L'activité antioxydante du principe actif libéré a été évaluée in vitro par des tests DPPH et TRAP et des tests in vivo sont en cours sur le lapin.L'huile de Tamanu, huile végétale non comestible extraite des graines de Calophyllum inophyllum, est utilisée en médecine polynésienne traditionnelle et comme matière première en cosmétique; elle possède des propriétés thérapeutiques: effets antimicrobien, anti-inflammatoire, analgésique, anti-tumoral, cytoprotecteur anti-UV. Sa capacité antioxydante, celle de ses fractions résineuses et de coumarines isolées ont été évaluées par des tests basés sur la réduction de radicaux libres stables (DPPH) ou le piégeage de radicaux libres dérivés de l'oxygène impliqués dans le stress oxydant (TRAP). Les résultats montrent que la résine de Tamanu contient les principales fractions antioxydantes. La molécule la plus active au regard des deux tests est l'Inocalophylline B. Les inophyllums C et E présentent une activité antioxydante modérée vis à vis du test DPPH bien que ne portant pas de fonction hydroxyle / Sesame oil is of great economic and industrial interest. It is used as a protein or antioxidant rich biological resource and as a substrate for bioprocesses for enzymes, antibiotics or biopesticides production. We esterified sesame oil with methanol and then transesterified the resulting mixture with vanillic acid (VNA) in order to get a more water soluble mixture of vanillic esters of its fatty acids (VNA-2). VNA-2 was encapsulated in Ca or Zn pectine beads with the objective of a controlled release in the colon. Beads dissolution was followed in acidic (stomach) and basic (colon) media as well as in enzymatic medium. The antioxidant activity of the liberated substrate was evaluated by in vitro DPPH and TRAP tests and compared to that of the non-encapsulated product. In vivo experiments of drug release in the rabbit are in progress.Tamanu oil is a non-edible vegetable oil extracted from the seeds of Calophyllum inophyllum, widely used in traditional Polynesian medicine and as raw material in cosmetics. It exhibits various therapeutic properties: antimicrobial and anti-inflammatory, analgesic and anti-tumor, cytoprotective anti-UV. Antioxidant capacity of Tamanu oil, its resinous extracts and coumarins isolated from the latter were evaluated using standard tests, based on the reduction of a stable free radical (DPPH) or the scavenging of oxygen-derived radicals involved in oxidative stress. The results show that the resin contains Tamanu oil major antioxidant fractions. The more active molecule in both tests is inocalophylline B. The inophyllums C and E show a moderate antioxidant activity with respect to the DPPH test, while not bearing an hydroxyl group.

Introdução: A terapia com laser de baixa potência (LBP) vem sendo utilizada em Odontologia com diversos objetivos, em especial o de diminuir o tempo de cicatrização de feridas cirúrgicas. Objetivo: O objetivo do presente estudo in vivo foi avaliar qualitativamente e quantitativamente o efeito da irradiação com LBP no processo de remodelação óssea após a extração dentária em ratos jovens. Material e Método: Foram utilizados 60 ratos Wistar, distribuídos aleatoriamente nos seguintes grupos: Grupo Controle (n=30) animais com extração dentária sem aplicação do LBP, e Grupo Experimental (n=30) animais que tiveram extração dentária (Ext) e aplicação do LBP (Ext+LBP) nos três primeiros dias (54 J/cm2 por dia). Os animais foram sacrificados nos períodos de 1, 2, 3, 5, 7 e 10 dias após o procedimento de extração dentária. Neste estudo foram analisados os efeitos da aplicação do laser sobre o reparo alveolar através de microscopia de luz, luz polarizada e imunomarcação. Para isso foram avaliados os seguintes parâmetros: 1) porcentagem de formação óssea no interior do alvéolo; 2) grau de processo inflamatório; 3) grau de amadurecimento do colágeno; 4) imunomarcação para TRAP e RUNX-2. Todos os resultados obtidos foram submetidos à análise estatística através do teste ANOVA e teste de Mann-Whitney U (p<0.05). Resultados: Observou-se que os alvéolos do Grupo Experimental apresentaram processo de reparo mais evoluído quando comparados ao Grupo Controle, caracterizado pela organização mais rápida do coágulo sanguíneo, proliferação de fibroblastos nos restos do ligamento periodontal mais pronunciada, organização do colágeno e formação óssea mais precoce e mais intensa. Conclusão: A utilização do laser de baixa potência acelerou o processo de formação óssea durante as fases iniciais do experimento, embora nos períodos finais não houve diferença no processo de ossificação. / Introduction: Low Power Laser (LPL) therapy has been used in Dentistry to achieve many objectives, particularly to decrease the healing period on wound healing. Objectives: The purpose of this in vivo study was to evaluate qualitatively and quantitatively the irradiation effect with LPL in the bone remodeling process after tooth extraction in young rats. Material and Method: 60 Wistar rats were used, randomly distributed in the following groups: Control Group (n=30) animals with tooth extraction without LPL application, and Experimental Group (n=30) animals with tooth extraction (Ext) and LPL application (Ext+LPL) on the three fist days (54 J/cm2 per day). The animals were euthanized after the end periods of 1, 2, 3, 5, 7 and 10 days after the tooth extraction. This study looked into the effects of laser application on the alveolar repair through light microscopy, polarized light and immunostaining. After that, the following parameters were evaluated: 1) percentage of bone formation inside the dental alveolus; 2) degree of inflammatory process; 3) degree of collagen maturation; 4) immunostaining for TRAP and RUNX-2. All the results obtained were submitted to statistical analysis over the ANOVA and Mann- Whitney (p<0.05) test. Results: It was observed that the alveolus from the Experimental Group presented an improved repair process when compared to the Control Group, characterized by faster blood clot organization, more apparent fibroblasts proliferation on the remnants of the periodontal ligament, earlier and more intense collagen organization and bone generation. Conclusion: The use of Low Power Laser accelerated the bone generation process during the experimental initial period, although there was no difference in the ossification process in the final periods.

Introdução: A terapia com laser de baixa potência (LBP) vem sendo utilizada em Odontologia com diversos objetivos, em especial o de diminuir o tempo de cicatrização de feridas cirúrgicas. Objetivo: O objetivo do presente estudo in vivo foi avaliar qualitativamente e quantitativamente o efeito da irradiação com LBP no processo de remodelação óssea após a extração dentária em ratos jovens. Material e Método: Foram utilizados 60 ratos Wistar, distribuídos aleatoriamente nos seguintes grupos: Grupo Controle (n=30) animais com extração dentária sem aplicação do LBP, e Grupo Experimental (n=30) animais que tiveram extração dentária (Ext) e aplicação do LBP (Ext+LBP) nos três primeiros dias (54 J/cm2 por dia). Os animais foram sacrificados nos períodos de 1, 2, 3, 5, 7 e 10 dias após o procedimento de extração dentária. Neste estudo foram analisados os efeitos da aplicação do laser sobre o reparo alveolar através de microscopia de luz, luz polarizada e imunomarcação. Para isso foram avaliados os seguintes parâmetros: 1) porcentagem de formação óssea no interior do alvéolo; 2) grau de processo inflamatório; 3) grau de amadurecimento do colágeno; 4) imunomarcação para TRAP e RUNX-2. Todos os resultados obtidos foram submetidos à análise estatística através do teste ANOVA e teste de Mann-Whitney U (p<0.05). Resultados: Observou-se que os alvéolos do Grupo Experimental apresentaram processo de reparo mais evoluído quando comparados ao Grupo Controle, caracterizado pela organização mais rápida do coágulo sanguíneo, proliferação de fibroblastos nos restos do ligamento periodontal mais pronunciada, organização do colágeno e formação óssea mais precoce e mais intensa. Conclusão: A utilização do laser de baixa potência acelerou o processo de formação óssea durante as fases iniciais do experimento, embora nos períodos finais não houve diferença no processo de ossificação. / Introduction: Low Power Laser (LPL) therapy has been used in Dentistry to achieve many objectives, particularly to decrease the healing period on wound healing. Objectives: The purpose of this in vivo study was to evaluate qualitatively and quantitatively the irradiation effect with LPL in the bone remodeling process after tooth extraction in young rats. Material and Method: 60 Wistar rats were used, randomly distributed in the following groups: Control Group (n=30) animals with tooth extraction without LPL application, and Experimental Group (n=30) animals with tooth extraction (Ext) and LPL application (Ext+LPL) on the three fist days (54 J/cm2 per day). The animals were euthanized after the end periods of 1, 2, 3, 5, 7 and 10 days after the tooth extraction. This study looked into the effects of laser application on the alveolar repair through light microscopy, polarized light and immunostaining. After that, the following parameters were evaluated: 1) percentage of bone formation inside the dental alveolus; 2) degree of inflammatory process; 3) degree of collagen maturation; 4) immunostaining for TRAP and RUNX-2. All the results obtained were submitted to statistical analysis over the ANOVA and Mann- Whitney (p<0.05) test. Results: It was observed that the alveolus from the Experimental Group presented an improved repair process when compared to the Control Group, characterized by faster blood clot organization, more apparent fibroblasts proliferation on the remnants of the periodontal ligament, earlier and more intense collagen organization and bone generation. Conclusion: The use of Low Power Laser accelerated the bone generation process during the experimental initial period, although there was no difference in the ossification process in the final periods.

Research pertaining to the role of active online internationalization (AOI) in the context of Born Globals has been shown to be an under-researched area of study. This compelled us to explore the possible benefits and challenges that may come from pursuing AOI for Swedish Born Globals. Our theoretical framework combined literature concerning Born Globals and their rapid internationalization with the notion of learning advantages of newness and literature regarding AOI into a theoretical model. Our theoretical investigation displayed a lack of research dealing with the potential challenges of AOI; with the virtuality trap of Yamin and Sinkovics (2006) being the exception. Through abductively analyzing qualitative data collected from four Swedish Born Globals with our theoretical model, we arrived at a number of empirically testable propositions that highlight the effects of using AOI for Swedish Born Globals. Our analysis leads to the conclusion that there are more challenges for Swedish Born Globals pursuing AOI than previously identified by present literature.

The precision of mass measurements in a Penning trap is directly proportional to an ion's charge state and can be increased by using highly charged ions (HCI) from an Electron Beam Ion Trap (EBIT). By bombarding the injected and trapped singly charged ions with an intense electron beam, the charge state of the ions is rapidly increased. To use this method for short-lived isotopes, very high electron beam current densities are required of the TITAN EBIT, built and commissioned at the Max-Planck-Institute for Nuclear Physics in Heidelberg, Germany and transported to TRIUMF for the TITAN on-line facility. This EBIT has produced charge states as high as Kr34+ and Ba54+ with electron beams of up to 500 mA and 27 keV. Once the EBIT is operational at full capacity (5 A, 60 keV), most species can be bred into a He-like configuration within tens of ms. / October 2006

The objective of this research is to design, fabricate, and demonstrate a microfabricated monolithic ion trap for applications in quantum computation and quantum simulation. Most current microfabricated ion trap designs are based on planar-segmented surface electrodes. Although promising scalability to trap arrays containing ten to one hundred ions, these planar designs suffer from the challenges of shallow trap depths, radial asymmetry of the confining potential, and electrode charging resulting from laser interactions with dielectric surfaces. In this research, the design, fabrication, and testing of a monolithic and symmetric two-level ion trap is presented. This ion trap overcomes the challenges of surface-electrode ion traps. Numerical electrostatic simulations show that this symmetric trap produces a deep (1 eV for 171Yb+ ion), radially symmetric RF confinement potential. The trap has an angled through-chip slot that allows back-side ion loading and generous through laser access, while avoiding surface-light scattering and dielectric charging that can corrupt the design control electrode compensating potentials. The geometry of the trap and its dimensions are optimized for trapping long and linear ion chains with equal spacing for use with quantum simulation problems and quantum computation architectures.

This thesis presents results of investigations that have been carried out to understand dynamics in nonlinear Paul trap mass spectrometers. Of the three problems that have been taken up for study in this thesis, the first concerns understanding early/delayed ejection of ions in mass selective boundary ejection experiments. The second looks at the differential resolution observed in forward and reverse scan resonance ejection experiments. The third study explores a coupled nonlinear resonance within the nominally stable region of trap operation. The method of multiple scales has been to elucidate dynamics associated with early and delayed ejection of ions in mass selective ejection experiments in Paul traps. We develop a slow flow equation to approximate the solution of a weakly nonlinear Mathieu equation to describe ion dynamics in the neighborhood of the stability boundary of ideal traps (where the Mathieu parameter qz = qz* = 0.908046). For positive even multipoles in the ion trapping field, in the stable region of trap operation, the phase portrait obtained from the slow flow consists of three fixed points, two of which are saddles and the third is a center. As the qz value of an ion approaches qz*, the saddles approach each other, and a point is reached where all nonzero solutions are unbounded, leading to an observation of early ejection. The phase portraits for negative even multipoles and odd multipoles of either sign are qualitatively similar to each other and display bounded solutions even for qz > qz*, resulting in the observation of delayed ejection associated with a more gentle increase in ion motion amplitudes, a mechanism different from the case of the positive even multipoles. The second study investigates constraints on pre-ejection dynamical states which cause differential resolution in resonance ejection experiments using Paul traps with stretched geometry. Both analytical and numerical computations are carried out to elucidate the role of damping and scan rate in influencing coherence in ion motion associated with the forward and reverse scan. It has been shown that in the forward scan experiments, for a given damping, low scan rates result in coherent motion of ions oof a given mass at the jump point. At this point, the amplitude and phase of ions of a given mass, starting at different initial conditions, become effectively identical. As the scan rate is increased, coherence is destroyed. For a given scan rate, increasing damping introduces coherence in ion motion, while decreasing damping destroys this coherence. In reverse scan experiments, for a given damping, very low scan rates will cause coherent ion motion. Increasing the scan rate destroys this coherence. The effect of damping in reverse scan experiments is qualitatively similar to that in the forward scan experiments, but settling times in the forward scan are shorter, leading to improved coherence and resolution. For mass spectrometrically relevant scan rates and damping values, significantly greater coherence is obtained in the forward scan. In the third study we investigate the weakly coupled and nonlinear Mathieu equations governing ion motion in axial and radial directions in a Paul trap in the neighborhood of a nonlinear resonance point at az* = -0.2313850427 and qz* = 0.9193009931$. Using harmonic balance based approximate averaging up to second order; we obtain a slow flow that, we numerically demonstrate, approximates the actual ion dynamics. We find that the slow flow is Hamiltonian. We study the slow flow numerically with the objective of exploring and displaying some of the possible types of interesting ion motions. In particular, we choose specific but arbitrary parameter values; study the stability of the individual radial and axial motion invariant manifolds; examine the rather large times associated with escape of ions; notice regions in the averaged phase space wherein trajectories do not, in fact, escape; observe apparently chaotic dynamics preceding escape for ions that do escape; and note that trajectories that do not escape appear to be confined to 4-tori. We conclude with some comments on the implications for practical operation of the Paul trap near this resonant point.