Phospholipases and the network of auxin signal transduction with ABP1 and TIR1 as two receptors: a comprehensive and provocative model

Three types of phospholipases, phospholipase D, secreted phospholipase A2, and patatin-related phospholipase A (pPLA) have functions in auxin signal transduction. Potential linkage to auxin receptors ABP1 or TIR1, their rapid activation or post-translational activation mechanisms, and downstream functions regulated by these phospholipases is reviewed and discussed. Only for pPLA all aspects are known at least to some detail. Evidence is gathered that all these signal reactions are located in the cytosol and seem to merge on regulation of PIN-catalyzed auxin efflux transport proteins. As a consequence, auxin concentration in the nucleus is also affected and this regulates the E3 activity of this auxin receptor. We showed that ABP1, PIN2, and pPLA, all outside the nucleus, have an impact on regulation of auxin-induced genes within 30 min. We propose that regulation of PIN protein activities and of auxin efflux transport are the means to coordinate ABP1 and TIR1 activity and that no physical contact between components of the ABP1-triggered cytosolic pathways and TIR1-triggered nuclear pathways of signaling is necessary to perform this.

Undersampled Critical Branching Processes on Small-World and Random Networks Fail to Reproduce the Statistics of Spike Avalanches

The power-law size distributions obtained experimentally for neuronal avalanches are an important evidence of criticality in the brain. This evidence is supported by the fact that a critical branching process exhibits the same exponent t~3=2. Models at criticality have been employed to mimic avalanche propagation and explain the statistics observed experimentally. However, a crucial aspect of neuronal recordings has been almost completely neglected in the models: undersampling. While in a typical multielectrode array hundreds of neurons are recorded, in the same area of neuronal tissue tens of thousands of neurons can be found. Here we investigate the consequences of undersampling in models with three different topologies (two-dimensional, small-world and random network) and three different dynamical regimes (subcritical, critical and supercritical). We found that undersampling modifies avalanche size distributions, extinguishing the power laws observed in critical systems. Distributions from subcritical systems are also modified, but the shape of the undersampled distributions is more similar to that of a fully sampled system. Undersampled supercritical systems can recover the general characteristics of the fully sampled version, provided that enough neurons are measured. Undersampling in two-dimensional and small-world networks leads to similar effects, while the random network is insensitive to sampling density due to the lack of a well-defined neighborhood. We conjecture that neuronal avalanches recorded from local field potentials avoid undersampling effects due to the nature of this signal, but the same does not hold for spike avalanches. We conclude that undersampled branching-process-like models in these topologies fail to reproduce the statistics of spike avalanches.

Heat transfer performance investigation of nanofluids flow in pipe

Different types of base fluids, such as water, engine oil, kerosene, ethanol, methanol, ethylene glycol etc. are usually used to increase the heat transfer performance in many engineering applications. But these conventional heat transfer fluids have often several limitations. One of those major limitations is that the thermal conductivity of each of these base fluids is very low and this results a lower heat transfer rate in thermal engineering systems. Such limitation also affects the performance of different equipments used in different heat transfer process industries. To overcome such an important drawback, researchers over the years have considered a new generation heat transfer fluid, simply known as nanofluid with higher thermal conductivity. This new generation heat transfer fluid is a mixture of nanometre-size particles and different base fluids. Different researchers suggest that adding spherical or cylindrical shape of uniform/non-uniform nanoparticles into a base fluid can remarkably increase the thermal conductivity of nanofluid. Such augmentation of thermal conductivity could play a more significant role in enhancing the heat transfer rate than that of the base fluid. Nanoparticles diameters used in nanofluid are usually considered to be less than or equal to 100 nm and the nanoparticles concentration usually varies from 5% to 10%. Different researchers mentioned that the smaller nanoparticles concentration with size diameter of 100 nm could enhance the heat transfer rate more significantly compared to that of base fluids. But it is not obvious what effect it will have on the heat transfer performance when nanofluids contain small size nanoparticles of less than 100 nm with different concentrations. Besides, the effect of static and moving nanoparticles on the heat transfer of nanofluid is not known too. The idea of moving nanoparticles brings the effect of Brownian motion of nanoparticles on the heat transfer. The aim of this work is, therefore, to investigate the heat transfer performance of nanofluid using a combination of smaller size of nanoparticles with different concentrations considering the Brownian motion of nanoparticles. A horizontal pipe has been considered as a physical system within which the above mentioned nanofluid performances are investigated under transition to turbulent flow conditions. Three different types of numerical models, such as single phase model, Eulerian-Eulerian multi-phase mixture model and Eulerian-Lagrangian discrete phase model have been used while investigating the performance of nanofluids. The most commonly used model is single phase model which is based on the assumption that nanofluids behave like a conventional fluid. The other two models are used when the interaction between solid and fluid particles is considered. However, two different phases, such as fluid and solid phases is also considered in the Eulerian-Eulerian multi-phase mixture model. Thus, these phases create a fluid-solid mixture. But, two phases in the Eulerian-Lagrangian discrete phase model are independent. One of them is a solid phase and the other one is a fluid phase. In addition, RANS (Reynolds Average Navier Stokes) based Standard κ-ω and SST κ-ω transitional models have been used for the simulation of transitional flow. While the RANS based Standard κ-ϵ, Realizable κ-ϵ and RNG κ-ϵ turbulent models are used for the simulation of turbulent flow. Hydrodynamic as well as temperature behaviour of transition to turbulent flows of nanofluids through the horizontal pipe is studied under a uniform heat flux boundary condition applied to the wall with temperature dependent thermo-physical properties for both water and nanofluids. Numerical results characterising the performances of velocity and temperature fields are presented in terms of velocity and temperature contours, turbulent kinetic energy contours, surface temperature, local and average Nusselt numbers, Darcy friction factor, thermal performance factor and total entropy generation. New correlations are also proposed for the calculation of average Nusselt number for both the single and multi-phase models. Result reveals that the combination of small size of nanoparticles and higher nanoparticles concentrations with the Brownian motion of nanoparticles shows higher heat transfer enhancement and thermal performance factor than those of water. Literature suggests that the use of nanofluids flow in an inclined pipe at transition to turbulent regimes has been ignored despite its significance in real-life applications. Therefore, a particular investigation has been carried out in this thesis with a view to understand the heat transfer behaviour and performance of an inclined pipe under transition flow condition. It is found that the heat transfer rate decreases with the increase of a pipe inclination angle. Also, a higher heat transfer rate is found for a horizontal pipe under forced convection than that of an inclined pipe under mixed convection.

Development of dendritic cells in the intestine

The intestinal tract is exposed to a large variety of antigens such as food proteins, commensal bacteria and pathogens and contains one of the largest arms of the immune system. The intestinal immune system has to discriminate between harmless and harmful antigens, inducing tolerance to harmless antigens and active immunity towards pathogens and other harmful materials. Dendritic cells (DC) in the mucosal lamina propria (LP) are central to this process, as they sample bacteria from the local environment and constitutively migrate to the draining mesenteric lymph nodes (MLN), where they present antigen to naïve T cells in order to direct an appropriate immune response. Despite their crucial role, understanding the function and phenotype of LP DC has been hampered by the fact that they share phenotypic markers with macrophages (mφ), which are the dominant population of mononuclear phagocyte (MP) in the LP. Recent work in our own and other laboratories has established gating strategies and phenotyping panels that allow precise discrimination between intestinal DC and mφ using the mφ specific markers CD64 and F4/80. In this way four bona fide DC subsets with distinct functions have been identified in adult LP based on their expression of CD11b and CD103 and a major aim of my project was to understand how these subsets might develop in the neonatal intestine. At the beginning of my PhD, the laboratory had used these new methods to show that signal regulatory protein α (SIRPα), an inhibitory receptor expressed by myeloid cells, was expressed by mφ and most DC in the intestine, except for those expressing CD103 alone. In addition, mice carrying a non-signalling mutation in SIRPα (SIRPα mt) had a selective reduction in CD103+CD11b+ DC, a subset which is unique to the intestinal LP. This was the basis for the initial experiments of my project, described in Chapter 3, where I investigated if the phenotype in SIRPα mt mice was intrinsic to haematopoietic cells or not. To explore this, I generated bone marrow (BM) chimeric mice by reconstituting irradiated WT mice with SIRPα mt BM, or SIRPα mt animals with WT BM. These experiments suggested that the defect in CD103+CD11b+ DC was not replicated in DC derived from BM of SIRPα origin. However as this seemed inconsistent with other data, I considered the possibility that 18 the phenotype may have been lost with age, as the BM chimeric mice were considerably older than those used in the original studies of SIRPα function. However a comparison of DC subsets in the intestine of WT and SIRPα mt mice as they aged provided no conclusive evidence to support this idea. As these experiments did show age-dependent effects on DC subsets, in Chapter 4, I went on to investigate how the DC populations appeared in the intestine and other tissues in the neonatal period. These experiments showed there were few CD103+CD11b+ DC present in the LP and migratory DC compartment of the MLN in the neonate and that as this population gradually increased in proportion with age, there was a reciprocal decrease in the relative proportion of CD103-CD11b+ DC. Interestingly, most of the changes in DC numbers in the intestine were found during the second or third week of life when the weaning process began. To validate my findings that there were few CD103+CD11b+ DC in the neonate and that this was not merely an absence of CD103 upregulation, I examined the expression of CD101 and Trem-1, markers that other work in the laboratory had suggested were specific to the CD103+CD11b+ DC lineage. My work showed that CD101 and Trem-1 were co- expressed by most CD103+CD11b+ DC in small intestine (SI) LP, as well as a small subset of CD103-CD11b+ DC in this tissue. Interestingly, Trem-1 was highly specific to the SI LP and migratory DC in the MLN, but absent from the colon and other tissues. CD101 expression was also only found on CD11b+ DC, but showed a less restricted pattern of distribution, being found in several tissues as well as the SI LP. The relative timing of their development suggested there might be a relationship between CD103+CD11b+ and CD103-CD11b+ DC and this was supported by microarray analysis. I hypothesised that the CD103-CD11b+ DC that co-expressed CD101 and Trem-1 may be the cells that developed into CD103+CD11b+ DC. To investigate this I analysed how CD101 and Trem-1 expression changed with age amongst the DC subsets in SI LP, colonic LP (CLP) and MLN. The proportion of CD101+Trem-1+ cells increased amongst CD103+CD11b+ DC in the SI LP and MLN with age, while amongst CD103+CD11b+ DC in the CLP this decreased. This was not the same in CD103-CD11b+ DC, where CD101 and Trem-1 expression was more varied with age in all tissues. CD101 and Trem-1 were not expressed to any great extent on CD103+CD11b- or CD103-CD11b- DC. The phenotypic development of the 19 intestinal DC subsets was paralleled by the gradual upregulation of CD103 expression, while the production of retinoic acid (RA), as assessed by the AldefluorTM assay, was low early in life and did not attain adult levels until after weaning. Thus DC in the neonatal intestine take some time to acquire the adult pattern of phenotypic subsets and are functionally immature compared with their adult counterparts. In Chapter 5, I used CD101 and Trem-1 to explore the ontogeny of intestinal DC subsets in CCR2-/- and SIRPα mt mice, both of which have selective defects in one particular group of DC. The selective defect seen amongst CD103+CD11b+ DC in adult SIRPα mt mice was more profound in mice at D7 and D14 of age, indicating that it may be intrinsic to this population and not highly dependent on environmental factors that change after birth. The expression of CD101 and Trem-1 by both CD103+CD11b+ and CD103-CD11b+ DC was reduced in SIRPα mt mice, again indicating that this entire lineage was affected by the lack of SIRPα signalling. However there was also a generalised defect in the numbers of all DC subsets in many tissues from early in life, suggesting there was compromised development, recruitment or survival of DC in the absence of SIRPα signalling. In contrast to the findings in SIRPα mt mice, more CD103+CD11b+ DC co-expressed CD101 and Trem-1 in CCR2-/- mice, while there were no differences in the expression of these molecules amongst CD103-CD11b+ DC. This may suggest that CCR2+ CD103-CD11b+ DC are not the cells that express CD101 and Trem-1 that are predicted to be the direct precursors of CD103+CD11b+ DC. I also examined the expression of DC growth factor receptors on DC subsets from mice of different ages, but no clear age or subset- related patterns of the expression of mRNA for Csf2ra, Irf4, Tgfbr1 and Rara could be observed. Next, I investigated whether Trem-1 played any role in DC development. Preliminary experiments in Trem-1-/- mice show no differences between any of the DC subsets, nor were there any selective effects on individual subsets when DC development from Trem-1-/- KO and WT BM was compared in competitive chimeras. However these experiments were difficult to interpret due to viability problems and because I found an unexpected defect in the ability of Trem-1-/- BM to generate all DC, irrespective of whether they expressed Trem-1 or not. 20 The final experiments I carried out were to examine the role of the microbiota in driving the differentiation of intestinal DC subsets, based on the hypothesis that this could be one of the environmental factors that might influence events in the developing intestine. To this end I performed experiments in both antibiotic treated and germ free adult mice, both of which showed no significant phenotypic differences amongst any of the DC subsets. However the study of germ free mice was compromised by recent contamination of the colony and may not be the conclusive answer. Together the data in this thesis have shown that the population of CD103+CD11b+ DC, which is unique to the intestine, is not present at birth. These cells gradually increase in frequency over time and as this occurs there is a reciprocal decrease in the frequency of CD103-CD11b+ DC. Along with other results, this leads to the idea that there may be a linear developmental pathway from CD103-CD11b+ DC to CD103+CD11b+ DC that is driven by non-microbial factors that are located preferentially in the small intestine. My project indicates that markers such as CD101 and Trem-1 may assist the dissection of this process and highlights the importance of the neonatal period for these events.

Rôle des protéoglycanes à héparane sulfate dans le transfert de gène des cellules CHO et HEK293

La possibilité de programmer une cellule dans le but de produire une protéine d’intérêt est apparue au début des années 1970 avec l’essor du génie génétique. Environ dix années plus tard, l’insuline issue de la plateforme de production microbienne Escherichia coli, fut la première protéine recombinante (r-protéine) humaine commercialisée. Les défis associés à la production de r-protéines plus complexes et glycosylées ont amené l’industrie biopharmaceutique à développer des systèmes d’expression en cellules de mammifères. Ces derniers permettent d’obtenir des protéines humaines correctement repliées et de ce fait, biologiquement actives. Afin de transférer le gène d’intérêt dans les cellules de mammifères, le polyéthylènimine (PEI) est certainement un des vecteurs synthétiques le plus utilisé en raison de son efficacité, mais aussi sa simplicité d’élaboration, son faible coût et sa stabilité en solution qui facilite son utilisation. Il est donc largement employé dans le contexte de la production de r-protéines à grande échelle et fait l’objet d’intenses recherches dans le domaine de la thérapie génique non virale. Le PEI est capable de condenser efficacement l’ADN plasmidique (vecteur d’expression contenant le gène d’intérêt) pour former des complexes de petites tailles appelés polyplexes. Ces derniers doivent contourner plusieurs étapes limitantes afin de délivrer le gène d’intérêt au noyau de la cellule hôte. Dans les conditions optimales du transfert de gène par le PEI, les polyplexes arborent une charge positive nette interagissant de manière électrostatique avec les protéoglycanes à héparane sulfate (HSPG) qui décorent la surface cellulaire. On observe deux familles d’HSPG exprimés en abondance à la surface des cellules de mammifères : les syndécanes (4 membres, SDC1-4) et les glypicanes (6 membres, GPC1-6). Si l’implication des HSPG dans l’attachement cellulaire des polyplexes est aujourd’hui largement acceptée, leur rôle individuel vis-à-vis de cet attachement et des étapes subséquentes du transfert de gène reste à confirmer. Après avoir optimisées les conditions de transfection des cellules de mammifères CHO et HEK293 dans le but de produire des r-protéines secrétées, nous avons entrepris des cinétiques de capture, d’internalisation des polyplexes et aussi d’expression du transgène afin de mieux comprendre le processus de transfert de gène. Nous avons pu observer des différences au niveau de ces paramètres de transfection dépendamment du système d’expression et des caractéristiques structurelles du PEI utilisé. Ces résultats présentés sous forme d’articles scientifiques constituent une base solide de l’enchaînement dans le temps des évènements essentiels à une transfection efficace des cellules CHO et HEK293 par le PEI. Chaque type cellulaire possède un profil d’expression des HSPG qui lui est propre, ces derniers étant plus ou moins permissifs au transfert de gène. En effet, une étude menée dans notre laboratoire montre que les SDC1 et SDC2 ont des rôles opposés vis-à-vis du transfert de gène. Alors que tous deux sont capables de lier les polyplexes, l’expression de SDC1 permet leur internalisation contrairement à l’expression de SDC2 qui l’inhibe. De plus, lorsque le SDC1 est exprimé à la surface des cellules HEK293, l’efficacité de transfection est augmentée de douze pourcents. En utilisant la capacité de SDC1 à induire l’internalisation des polyplexes, nous avons étudié le trafic intracellulaire des complexes SDC1 / polyplexes dans les cellules HEK293. De plus, nos observations suggèrent une nouvelle voie par laquelle les polyplexes pourraient atteindre efficacement le noyau cellulaire. Dans le contexte du transfert de gène, les HSPG sont essentiellement étudiés dans leur globalité. S’il est vrai que le rôle des syndécanes dans ce contexte est le sujet de quelques études, celui des glypicanes est inexploré. Grâce à une série de traitements chimiques et enzymatiques visant une approche « perte de fonction », l’importance de la sulfatation comme modification post-traductionnelle, l’effet des chaînes d’héparanes sulfates mais aussi des glypicanes sur l’attachement, l’internalisation des polyplexes, et l’expression du transgène ont été étudiés dans les cellules CHO et HEK293. L’ensemble de nos observations indique clairement que le rôle des HSPG dans le transfert de gène devrait être investigué individuellement plutôt que collectivement. En effet, le rôle spécifique de chaque membre des HSPG sur la capture des polyplexes et leur permissivité à l’expression génique demeure encore inconnu. En exprimant de manière transitoire chaque membre des syndécanes et glypicanes à la surface des cellules CHO, nous avons déterminé leur effet inhibiteur ou activateur sur la capture des polyplexes sans pouvoir conclure quant à l’effet de cette surexpression sur l’efficacité de transfection. Par contre, lorsqu’ils sont présents dans le milieu de culture, le domaine extracellulaire des HSPG réduit l’efficacité de transfection des cellules CHO sans induire la dissociation des polyplexes. Curieusement, lorsque chaque HSPG est exprimé de manière stable dans les cellules CHO, seulement une légère modulation de l’expression du transgène a pu être observée. Ces travaux ont contribué à la compréhension des mécanismes d'action du vecteur polycationique polyéthylènimine et à préciser le rôle des protéoglycanes à héparane sulfate dans le transfert de gène des cellules CHO et HEK293.

Filmes de nanopartículas de TiO2 e óxido de grafeno para fotocatálise

Dissertação (mestrado)—Universidade de Brasília, Instituto de Química, Programa de Pós-Graduação em Química, 2016.

Processing of laser Doppler flowmetry signals from healthy subjects and patients with varicose veins: information categorization approach based on intrinsic mode functions and entropy computation

International audience

Enhancement of non-viral transfection efficiency with nuclear localization signal peptides

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Theoretical Rationalization of the Singlet–Triplet Gap in OLEDs Materials: Impact of Charge-Transfer Character

New materials for OLED applications with low singlet–triplet energy splitting have been recently synthesized in order to allow for the conversion of triplet into singlet excitons (emitting light) via a Thermally Activated Delayed Fluorescence (TADF) process, which involves excited-states with a non-negligible amount of Charge-Transfer (CT). The accurate modeling of these states with Time-Dependent Density Functional Theory (TD-DFT), the most used method so far because of the favorable trade-off between accuracy and computational cost, is however particularly challenging. We carefully address this issue here by considering materials with small (high) singlet–triplet gap acting as emitter (host) in OLEDs and by comparing the accuracy of TD-DFT and the corresponding Tamm-Dancoff Approximation (TDA), which is found to greatly reduce error bars with respect to experiments thanks to better estimates for the lowest singlet–triplet transition. Finally, we quantitatively correlate the singlet–triplet splitting values with the extent of CT, using for it a simple metric extracted from calculations with double-hybrid functionals, that might be applied in further molecular engineering studies.

Strategy for succession in family owned small businesses as a wicked problem to be tamed

Contemporary strategic-planning processes don’t help family businesses cope with some of the big problems they face. Owner managers admit that they are confronted with issues, such as those associated with succession and inter-generational transfer that cannot be resolved merely by gathering additional data, defining issues more clearly, or breaking them down into small problems. Preparing for succession is often put off or ignored, many planning techniques don’t generate fresh ideas and implementing solutions is often fraught with political peril. This paper presents a framework to explore the idea of wicked problems, its relevance to succession planning in family businesses and its implications for practice and policy. A wicked problem has many and varied elements, and is complex as well as challenging. These problems are different to hard but ordinary problems, which people can solve in a finite time period by applying standard techniques. In this paper the authors argue that the wicked problem of family business succession requires a different approach to strategy, founded on social planning processes to engage multiple stakeholders and reconcile family/business interests to foster a joint commitment to possible ways of resolution. This requires academics and practitioners to re-frame traditional business strategic planning processes to achieve more sustainable family business futures.

MODELING OF TRANSFER PATH FOR DETERMINATION OF COMBUSTION AND NOISE METRICS ON DIESEL ENGINES

Determination of combustion metrics for a diesel engine has the potential of providing feedback for closed-loop combustion phasing control to meet current and upcoming emission and fuel consumption regulations. This thesis focused on the estimation of combustion metrics including start of combustion (SOC), crank angle location of 50% cumulative heat release (CA50), peak pressure crank angle location (PPCL), and peak pressure amplitude (PPA), peak apparent heat release rate crank angle location (PACL), mean absolute pressure error (MAPE), and peak apparent heat release rate amplitude (PAA). In-cylinder pressure has been used in the laboratory as the primary mechanism for characterization of combustion rates and more recently in-cylinder pressure has been used in series production vehicles for feedback control. However, the intrusive measurement with the in-cylinder pressure sensor is expensive and requires special mounting process and engine structure modification. As an alternative method, this work investigated block mounted accelerometers to estimate combustion metrics in a 9L I6 diesel engine. So the transfer path between the accelerometer signal and the in-cylinder pressure signal needs to be modeled. Depending on the transfer path, the in-cylinder pressure signal and the combustion metrics can be accurately estimated - recovered from accelerometer signals. The method and applicability for determining the transfer path is critical in utilizing an accelerometer(s) for feedback. Single-input single-output (SISO) frequency response function (FRF) is the most common transfer path model; however, it is shown here to have low robustness for varying engine operating conditions. This thesis examines mechanisms to improve the robustness of FRF for combustion metrics estimation. First, an adaptation process based on the particle swarm optimization algorithm was developed and added to the single-input single-output model. Second, a multiple-input single-output (MISO) FRF model coupled with principal component analysis and an offset compensation process was investigated and applied. Improvement of the FRF robustness was achieved based on these two approaches. Furthermore a neural network as a nonlinear model of the transfer path between the accelerometer signal and the apparent heat release rate was also investigated. Transfer path between the acoustical emissions and the in-cylinder pressure signal was also investigated in this dissertation on a high pressure common rail (HPCR) 1.9L TDI diesel engine. The acoustical emissions are an important factor in the powertrain development process. In this part of the research a transfer path was developed between the two and then used to predict the engine noise level with the measured in-cylinder pressure as the input. Three methods for transfer path modeling were applied and the method based on the cepstral smoothing technique led to the most accurate results with averaged estimation errors of 2 dBA and a root mean square error of 1.5dBA. Finally, a linear model for engine noise level estimation was proposed with the in-cylinder pressure signal and the engine speed as components.

Direct Write Fabrication of Waveguides and Interconnects for Optical Printed Wiring Boards

Current copper based circuit technology is becoming a limiting factor in high speed data transfer applications as processors are improving at a faster rate than are developments to increase on board data transfer. One solution is to utilize optical waveguide technology to overcome these bandwidth and loss restrictions. The use of this technology virtually eliminates the heat and cross-talk loss seen in copper circuitry, while also operating at a higher bandwidth. Transitioning current fabrication techniques from small scale laboratory environments to large scale manufacturing presents significant challenges. Optical-to-electrical connections and out-of-plane coupling are significant hurdles in the advancement of optical interconnects. The main goals of this research are the development of direct write material deposition and patterning tools for the fabrication of waveguide systems on large substrates, and the development of out-of-plane coupler components compatible with standard fiber optic cabling. Combining these elements with standard printed circuit boards allows for the fabrication of fully functional optical-electrical-printed-wiring-boards (OEPWBs). A direct dispense tool was designed, assembled, and characterized for the repeatable dispensing of blanket waveguide layers over a range of thicknesses (25-225 µm), eliminating waste material and affording the ability to utilize large substrates. This tool was used to directly dispense multimode waveguide cores which required no UV definition or development. These cores had circular cross sections and were comparable in optical performance to lithographically fabricated square waveguides. Laser direct writing is a non-contact process that allows for the dynamic UV patterning of waveguide material on large substrates, eliminating the need for high resolution masks. A laser direct write tool was designed, assembled, and characterized for direct write patterning waveguides that were comparable in quality to those produced using standard lithographic practices (0.047 dB/cm loss for laser written waveguides compared to 0.043 dB/cm for lithographic waveguides). Straight waveguides, and waveguide turns were patterned at multimode and single mode sizes, and the process was characterized and documented. Support structures such as angled reflectors and vertical posts were produced, showing the versatility of the laser direct write tool. Commercially available components were implanted into the optical layer for out-of-plane routing of the optical signals. These devices featured spherical lenses on the input and output sides of a total internal reflection (TIR) mirror, as well as alignment pins compatible with standard MT design. Fully functional OEPWBs were fabricated featuring input and output out-of-plane optical signal routing with total optical losses not exceeding 10 dB. These prototypes survived thermal cycling (-40°C to 85°C) and humidity exposure (95±4% humidity), showing minimal degradation in optical performance. Operational failure occurred after environmental aging life testing at 110°C for 216 hours.

Nitrogen removal and sustainability of vertical flow constructed wetlands for small scale wastewater treatment

The challenge for wastewater professionals is to design and operate treatment processes that support human well being and are environmentally sensitive throughout the life-cycle. This research focuses on one technology for small-scale wastewater treatment: the vertical flow constructed wetland (VFCW), which is herein investigated for the capacity to remove ammonium and nitrate nitrogen from wastewater. Hydraulic regime and presence/absence of vegetation are the basis for a three-phase bench scale experiment to determine oxygen transfer and nitrogen fate in VFCWs. Results show that 90% NH4+-N removal is achieved in aerobic downflow columns, 60% NO3--N removal occurs in anaerobic upflow columns, and 60% removal of total nitrogen can be achieved in downflow-upflow in-series. The experimental results are studied further using a variably saturated flow and reactive transport model, which allows a mechanistic explanation of the fate and transport of oxygen and nitrogen. The model clarifies the mechanisms of oxygen transport and nitrogen consumption, and clarifies the need for readily biodegradable COD for denitrification. A VFCW is then compared to a horizontal flow constructed wetland (HFCW) for life cycle environmental impacts. High areal emissions of greenhouse gases from VFCWs compared to HFCWs are the driver for the study. The assessment shows that because a VFCW is only 25% of the volume of an HFCW designed for the same treatment quality, the VFCW has only 25-30% of HFCW impacts over 12 impact categories and 3 damage categories. Results show that impacts could be reduced by design improvements. Design recommendations are downflow wetlands for nitrification, upflow wetlands for denitrification, series wetlands for total nitrogen removal, hydraulic load of 142 L/m2d, 30 cm downflow wetland depth, 1.0 m upflow wetland depth, recycle, vegetation and medium-grained sand. These improvements will optimize nitrogen removal, minimize gaseous emissions, and reduce wetland material requirements, thus reducing environmental impact without sacrificing wastewater treatment quality.

Bayesian Estimation of Small Proportions Using Binomial Group Test

Group testing has long been considered as a safe and sensible relative to one-at-a-time testing in applications where the prevalence rate p is small. In this thesis, we applied Bayes approach to estimate p using Beta-type prior distribution. First, we showed two Bayes estimators of p from prior on p derived from two different loss functions. Second, we presented two more Bayes estimators of p from prior on π according to two loss functions. We also displayed credible and HPD interval for p. In addition, we did intensive numerical studies. All results showed that the Bayes estimator was preferred over the usual maximum likelihood estimator (MLE) for small p. We also presented the optimal β for different p, m, and k.

Pseudomonas Aeruginosa AmpR Transcriptional Regulatory Network

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAO∆ampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors.