The Function and Regulation of Photobodies in Phytochrome Signaling

Light is a critical environmental signal that regulates every phase of the plant life cycle, from germination to floral initiation. Of the many light receptors in the model plant Arabidopsis thaliana, the red- and far-red light-sensing phytochromes (phys) are arguably the best studied, but the earliest events in the phy signaling pathway remain poorly understood. One of the earliest phy signaling events is the translocation of photoactivated phys from the cytoplasm to the nucleus, where they localize to subnuclear foci termed photobodies; in continuous light, photobody localization correlates closely with the light-dependent inhibition of embryonic stem growth. Despite a growing body of evidence supporting the biological significance of photobodies in light signaling, photobodies have also been shown to be dispensable for seedling growth inhibition in continuous light, so their physiological importance remains controversial; additionally, the molecular components that are required for phy localization to photobodies are largely unknown. The overall goal of my dissertation research was to gain insight into the early steps of phy signaling by further defining the role of photobodies in this process and identifying additional intragenic and extragenic requirements for phy localization to photobodies.

Even though the domain structure of phys has been extensively studied, not all of the intramolecular requirements for phy localization to photobodies are known. Previous studies have shown that the entire C-terminus of phys is both necessary and sufficient for their localization to photobodies. However, the importance of the individual subdomains of the C-terminus is still unclear. For example a truncation lacking part of the most C-terminal domain, the histidine kinase-related domain (HKRD), can still localize to small photobodies in the light and behaves like a weak allele. However, a point mutation within the HKRD renders the entire molecule completely inactive. To resolve this discrepancy, I explored the hypothesis that this point mutation might impair the dimerization of the HKRD; dimerization has been shown to occur via the C-terminus of phy and is required for more efficient signaling. I show that this point mutation impairs nuclear localization of phy as well as its subnuclear localization to photobodies. Additionally, yeast-two-hybrid analysis shows that the wild-type HKRD can homodimerize but that the HKRD containing the point mutation fails to dimerize with both itself and with wild-type HKRD. These results demonstrate that dimerization of the HKRD is required for both nuclear and photobody localization of phy.

Studies of seedlings grown in diurnal conditions show that photoactivated phy can persist into darkness to repress seedling growth; a seedling's growth rate is therefore fastest at the end of the night. To test the idea that photobodies could be involved in regulating seedling growth in the dark, I compared the growth of two transgenic Arabidopsis lines, one in which phy can localize to photobodies (PBG), and one in which it cannot (NGB). Despite these differences in photobody morphology, both lines are capable of transducing light signals and inhibiting seedling growth in continuous light. After the transition from red light to darkness, the PBG line was able to repress seedling growth, as well as the accumulation of the growth-promoting, light-labile transcription factor PHYTOCHROME INTERACTING FACTOR 3 (PIF3), for eighteen hours, and this correlated perfectly with the presence of photobodies. Reducing the amount of active phy by either reducing the light intensity or adding a phy-inactivating far-red pulse prior to darkness led to faster accumulation of PIF3 and earlier seedling growth. In contrast, the NGB line accumulated PIF3 even in the light, and seedling growth was only repressed for six hours; this behavior was similar in NGB regardless of the light treatment. These results suggest that photobodies are required for the degradation of PIF3 and for the prolonged stabilization of active phy in darkness. They also support the hypothesis that photobody localization of phys could serve as an instructive cue during the light-to-dark transition, thereby fine-tuning light-dependent responses in darkness.

In addition to determining an intragenic requirement for photobody localization and further exploring the significance of photobodies in phy signaling, I wanted to identify extragenic regulators of photobody localization. A recent study identified one such factor, HEMERA (HMR); hmr mutants do not form large photobodies, and they are tall and albino in the light. To identify other components in the HMR-mediated branch of the phy signaling pathway, I performed a forward genetic screen for suppressors of a weak hmr allele. Surprisingly, the first three mutants isolated from the screen were alleles of the same novel gene, SON OF HEMERA (SOH). The soh mutations rescue all of the phenotypes associated with the weak hmr allele, and they do so in an allele-specific manner, suggesting a direct interaction between SOH and HMR. Null soh alleles, which were isolated in an independent, tall, albino screen, are defective in photobody localization, demonstrating that SOH is an extragenic regulator of phy localization to photobodies that works in the same genetic pathway as HMR.

In this work, I show that dimerization of the HKRD is required for both the nuclear and photobody localization of phy. I also demonstrate a tight correlation between photobody localization and PIF3 degradation, further establishing the significance of photobodies in phy signaling. Finally, I identify a novel gene, SON OF HEMERA, whose product is necessary for phy localization to photobodies in the light, thereby isolating a new extragenic determinant of photobody localization. These results are among the first to focus exclusively on one of the earliest cellular responses to light - photobody localization of phys - and they promise to open up new avenues into the study of a poorly understood facet of the phy signaling pathway.

Spatial and temporal scales of neuronal correlation in visual area V4.

The spiking activity of nearby cortical neurons is correlated on both short and long time scales. Understanding this shared variability in firing patterns is critical for appreciating the representation of sensory stimuli in ensembles of neurons, the coincident influences of neurons on common targets, and the functional implications of microcircuitry. Our knowledge about neuronal correlations, however, derives largely from experiments that used different recording methods, analysis techniques, and cortical regions. Here we studied the structure of neuronal correlation in area V4 of alert macaques using recording and analysis procedures designed to match those used previously in primary visual cortex (V1), the major input to V4. We found that the spatial and temporal properties of correlations in V4 were remarkably similar to those of V1, with two notable differences: correlated variability in V4 was approximately one-third the magnitude of that in V1 and synchrony in V4 was less temporally precise than in V1. In both areas, spontaneous activity (measured during fixation while viewing a blank screen) was approximately twice as correlated as visual-evoked activity. The results provide a foundation for understanding how the structure of neuronal correlation differs among brain regions and stages in cortical processing and suggest that it is likely governed by features of neuronal circuits that are shared across the visual cortex.

Improving Indwelling Glucose Sensor Performance: Porous, Dexamethasone-Releasing Coatings that Modulate the Foreign Body Response

Inflammation and the formation of an avascular fibrous capsule have been identified as the key factors controlling the wound healing associated failure of implantable glucose sensors. Our aim is to guide advantageous tissue remodeling around implanted sensor leads by the temporal release of dexamethasone (Dex), a potent anti-inflammatory agent, in combination with the presentation of a stable textured surface.

First, Dex-releasing polyurethane porous coatings of controlled pore size and thickness were fabricated using salt-leaching/gas-foaming technique. Porosity, pore size, thickness, drug release kinetics, drug loading amount, and drug bioactivity were evaluated. In vitro sensor functionality test were performed to determine if Dex-releasing porous coatings interfered with sensor performance (increased signal attenuation and/or response times) compared to bare sensors. Drug release from coatings monitored over two weeks presented an initial fast release followed by a slower release. Total release from coatings was highly dependent on initial drug loading amount. Functional in vitro testing of glucose sensors deployed with porous coatings against glucose standards demonstrated that highly porous coatings minimally affected signal strength and response rate. Bioactivity of the released drug was determined by monitoring Dex-mediated, dose-dependent apoptosis of human peripheral blood derived monocytes in culture.

The tissue modifying effects of Dex-releasing porous coatings were accessed by fully implanting Tygon® tubing in the subcutaneous space of healthy and diabetic rats. Based on encouraging results from these studies, we deployed Dex-releasing porous coatings from the tips of functional sensors in both diabetic and healthy rats. We evaluated if the tissue modifying effects translated into accurate, maintainable and reliable sensor signals in the long-term. Sensor functionality was accessed by continuously monitoring glucose levels and performing acute glucose challenges at specified time points.

Sensors treated with porous Dex-releasing coatings showed diminished inflammation and enhanced vascularization of the tissue surrounding the implants in healthy rats. Functional sensors with Dex-releasing porous coatings showed enhanced sensor sensitivity over a 21-day period when compared to controls. Enhanced sensor sensitivity was accompanied with an increase in sensor signal lag and MARD score. These results indicated that Dex-loaded porous coatings were able to elicit a favorable tissue response, and that such tissue microenvironment could be conducive towards extending the performance window of glucose sensors in vivo.

The diabetic pilot animal study showed differences in wound healing patters between healthy and diabetic subjects. Diabetic rats showed lower levels of inflammation and vascularization of the tissue surrounding implants when compared to their healthy counterparts. Also, functional sensors treated with Dex-releasing porous coatings did not show enhanced sensor sensitivity over a 21-day period. Moreover, increased in sensor signal lag and MARD scores were present in porous coated sensors regardless of Dex-loading when compared to bare implants. These results suggest that the altered wound healing patterns presented in diabetic tissues may lead to premature sensor failure when compared to sensors implanted in healthy rats.

Dietary inference from upper and lower molar morphology in platyrrhine primates.

The correlation between diet and dental topography is of importance to paleontologists seeking to diagnose ecological adaptations in extinct taxa. Although the subject is well represented in the literature, few studies directly compare methods or evaluate dietary signals conveyed by both upper and lower molars. Here, we address this gap in our knowledge by comparing the efficacy of three measures of functional morphology for classifying an ecologically diverse sample of thirteen medium- to large-bodied platyrrhines by diet category (e.g., folivore, frugivore, hard object feeder). We used Shearing Quotient (SQ), an index derived from linear measurements of molar cutting edges and two indices of crown surface topography, Occlusal Relief (OR) and Relief Index (RFI). Using SQ, OR, and RFI, individuals were then classified by dietary category using Discriminate Function Analysis. Both upper and lower molar variables produce high classification rates in assigning individuals to diet categories, but lower molars are consistently more successful. SQs yield the highest classification rates. RFI and OR generally perform above chance. Upper molar RFI has a success rate below the level of chance. Adding molar length enhances the discriminatory power for all variables. We conclude that upper molar SQs are useful for dietary reconstruction, especially when combined with body size information. Additionally, we find that among our sample of platyrrhines, SQ remains the strongest predictor of diet, while RFI is less useful at signaling dietary differences in absence of body size information. The study demonstrates new ways for inferring the diets of extinct platyrrhine primates when both upper and lower molars are available, or, for taxa known only from upper molars. The techniques are useful in reconstructing diet in stem representatives of anthropoid clade, who share key aspects of molar morphology with extant platyrrhines.

Sea Urchin Body Plan Development and Evolution: An Integrative Transcriptomic Approach

My dissertation work integrates comparative transcriptomics and functional analyses to investigate gene expression changes underlying two significant aspects of sea urchin evolution and development: the dramatic developmental changes associated with an ecologically significant shift in life history strategy and the development of the unusual radial body plan of adult sea urchins.

In Chapter 2, I investigate evolutionary changes in gene expression underlying the switch from feeding (planktotrophic) to nonfeeding (lecithotrophic) development in sea urchins. In order to identify these changes, I used Illumina RNA-seq to measure expression dynamics across 7 developmental stages in three sea urchin species: the lecithotroph Heliocidaris erythrogramma, the closely related planktotroph Heliocidaris tuberculata, and an outgroup planktotroph Lytechinus variegatus. My analyses draw on a well-characterized developmental gene regulatory network (GRN) in sea urchins to understand how the ancestral planktotrophic developmental program was altered during the evolution of lecithotrophic development. My results suggest that changes in gene expression profiles occurred more frequently across the transcriptome during the evolution of lecithotrophy than during the persistence of planktotrophy. These changes were even more pronounced within the GRN than across the transcriptome as a whole, and occurred in each network territory (skeletogenic, endomesoderm and ectoderm). I found evidence for both conservation and divergence of regulatory interactions in the network, as well as significant changes in the expression of genes with known roles in larval skeletogenesis, which is dramatically altered in lecithotrophs. I further explored network dynamics between species using coexpression analyses, which allowed me to identify novel players likely involved in sea urchin neurogenesis and endoderm patterning.

In Chapter 3, I investigate developmental changes in gene expression underlying radial body plan development and metamorphosis in H. erythrogramma. Using Illumina RNA-seq, I measured gene expression profiles across larval, metamorphic, and post-metamorphic life cycle phases. My results present a high-resolution view of gene expression dynamics during the complex transition from pre- to post-metamorphic development and suggest that distinct sets of regulatory and effector proteins are used during different life history phases.

Collectively, my investigations provide an important foundation for future, empirical studies to investigate the functional role of gene expression change in the evolution of developmental differences between species and also for the generation of the unusual radial body plan of sea urchins.

A multi-scale atomistic-continuum modelling of crack propagation in a two-dimensional macroscopic plate

A novel multi-scale seamless model of brittle-crack propagation is proposed and applied to the simulation of fracture growth in a two-dimensional Ag plate with macroscopic dimensions. The model represents the crack propagation at the macroscopic scale as the drift-diffusion motion of the crack tip alone. The diffusive motion is associated with the crack-tip coordinates in the position space, and reflects the oscillations observed in the crack velocity following its critical value. The model couples the crack dynamics at the macroscales and nanoscales via an intermediate mesoscale continuum. The finite-element method is employed to make the transition from the macroscale to the nanoscale by computing the continuum-based displacements of the atoms at the boundary of an atomic lattice embedded within the plate and surrounding the tip. Molecular dynamics (MD) simulation then drives the crack tip forward, producing the tip critical velocity and its diffusion constant. These are then used in the Ito stochastic calculus to make the reverse transition from the nanoscale back to the macroscale. The MD-level modelling is based on the use of a many-body potential. The model successfully reproduces the crack-velocity oscillations, roughening transitions of the crack surfaces, as well as the macroscopic crack trajectory. The implications for a 3-D modelling are discussed.

Multiscale numerical modelling of crack propagation in two-dimensional metal plate

A novel multiscale model of brittle crack propagation in an Ag plate with macroscopic dimensions has been developed. The model represents crack propagation as stochastic drift-diffusion motion of the crack tip atom through the material, and couples the dynamics across three different length scales. It integrates the nanomechanics of bond rupture at the crack tip with the displacement and stress field equations of continuum based fracture theories. The finite element method is employed to obtain the continuum based displacement and stress fields over the macroscopic plate, and these are then used to drive the crack tip forward at the atomic level using the molecular dynamics simulation method based on many-body interatomic potentials. The linkage from the nanoscopic scale back to the macroscopic scale is established via the Ito stochastic calculus, the stochastic differential equation of which advances the tip to a new position on the macroscopic scale using the crack velocity and diffusion constant obtained on the nanoscale. Well known crack characteristics, such as the roughening transitions of the crack surfaces, crack velocity oscillations, as well as the macroscopic crack trajectories, are obtained.

Molecular dynamics simulation of fractures using an N-body potential

Molecular dynamics has been employed to model the fracture of a two dimensional triangular atomic lattice. The N-body Sutton-Chen potential developed for fcc metals and its extended version (Rafii-Tabar and Sutton) for fcc random binary alloys were used for the interatomic interactions. It is shown that at low temperatures cleavage fractures can occur in both an elemental metal and an alloy. At elevated temperatures the nucleation of dislocations is shown to cause a brittle-to-ductile transition. For the brittle crack propagation in the elemental metal, crack propagation speeds have been computed for different stress rates, and a crack instability found to exist as the speed reaches a critical value of about 32% of the Rayleigh wave speed. For the random alloy, we find that the dislocation movement can be affected by the distorted lattice.

The use of evacuation modelling techniques in the design of very large transport aircraft and blended wing body aircraft

Very Large Transport Aircraft (VLTA) pose considerable challenges to designers, operators and certification authorities. Questions concerning seating arrangement, nature and design of recreational space, the number, design and location of internal staircases, the number of cabin crew required and the nature of the cabin crew emergency procedures are just some of the issues that need to be addressed. Other more radical concepts such as blended wing body (BWB) design, involving one or two decks with possibly four or more aisles offer even greater challenges. Can the largest exits currently available cope with passenger flow arising from four or five aisles? Do we need to consider new concepts in exit design? Should the main aisles be made wider to accommodate more passengers? In this paper we demonstrate how computer based evacuation models can be used to investigate these issues through examination of staircase evacuation procedures for VLTA and aisle/exit configuration for BWB cabin layouts.

Correlation of solder paste rheology with computational simulations of the stencil printing process

Soldering technologies continue to evolve to meet the demands of the continuous miniaturisation of electronic products, particularly in the area of solder paste formulations used in the reflow soldering of surface mount devices. Stencil printing continues to be a leading process used for the deposition of solder paste onto printed circuit boards (PCBs) in the volume production of electronic assemblies, despite problems in achieving a consistent print quality at an ultra-fine pitch. In order to eliminate these defects a good understanding of the processes involved in printing is important. Computational simulations may complement experimental print trials and paste characterisation studies, and provide an extra dimension to the understanding of the process. The characteristics and flow properties of solder pastes depend primarily on their chemical and physical composition and good material property data is essential for meaningful results to be obtained by computational simulation.This paper describes paste characterisation and computational simulation studies that have been undertaken through the collaboration of the School of Aeronautical, Mechanical and Manufacturing Engineering at Salford University and the Centre for Numerical Modelling and Process Analysis at the University of Greenwich. The rheological profile of two different paste formulations (lead and lead-free) for sub 100 micron flip-chip devices are tested and applied to computational simulations of their flow behaviour during the printing process.

Solid phase velocity measurements utilising electrostatic sensors and cross correlation signal processing

Gas-solids two phase systems are widely employed within process plant in the form of pneumatic conveyors, dust extraction systems and solid fuel injection systems. The measurement of solids phase velocity therefore has wide potential application in flow monitoring and, in conjunction with density measurement instrumentation, solids mass flow rate measurement. Historically, a number of authors have detailed possible measurement techniques, and some have published limited test results. It is, however, apparent that none of these technologies have found wide application in industry. Solids phase velocity measurements were undertaken using real time cross correlation of signals from two electrostatic sensors spaced axially along a pipeline conveying pulverised coal (PF). Details of the measurement equipment, the pilot scale test rig and the test results are presented.

A regional chemostratigraphically-defined correlation framework for the late Triassic TAG-I Formation in Blocks 402 and 405a, Algeria

The Triassic Argilo-Gréseux Inférieur Formation (TAG-I) is one of the principal hydrocarbon reservoirs in the Berkine Basin of Algeria. Sedimentological studies have shown that it exhibits marked spatial and temporal facies variations on both a local field scale and a regional basinal scale. This variability, combined with a lack of diagnostic flora and fauna, makes regional correlation within the unit difficult. In turn, the lack of a consistent regional stratigraphic framework hampers the comparison of the various correlation schemes devised by operators in the basin. Contrasting the TAG-I in Blocks 402 and 405a exemplifies the problems encountered when attempting regionally to define a correlation framework for the interval. Between these two blocks, a distance of approximately 200 km, there are marked changes in the style of deposition from sand-dominated, proximal fluvial systems in the SW (Block 405a, MLN, MLC, KMD and MLNW fields) to a more distal, more clay-prone system in the NE (Block 402, ROD/BRSE/BSFN, SFNE and BSF fields). A chemostratigraphic study of the TAG-I in these two blocks has allowed a four-fold correlation framework to be defined, where each chemostratigraphic package has distinctive geochemical features. Chemostratigraphic Package 10, the oldest unit, lies above the Hercynian Unconformity, but beneath a geochemically identifiable hiatal surface. Chemostratigraphic Package 20 lies above the hiatal surface but is separated from the overlying packages by a mineralogical change identifiable in both claystone and sandstone geochemistry. Chemostratigraphic Packages 30 and 40 are chemically somewhat similar, but are separated by a regional event interpreted as a period of dolocrete and lacustrine development. By combining the geochemical differentiation of the units and recognition of their stratal boundaries, it is possible to define a correlation for the TAG-I between Blocks 402 and 405a. The proposed correlation between the two blocks suggests that the northern parts of Block 405a may have been occupied by a spur or subsidiary channel from the main SW–NE-trending fluvial system, resulting in one of the chemically defined packages being demonstrably absent in the MLNW, MLN, KMD and MLC fields when compared with the other areas of the study.

New genus and two new species of the family Ethmolaimidae (Nematoda: Chromadorida), found in two different cold-seep environments

This study describes a new genus Dystomanema gen. nov. with two new species, D. cadizensis sp. nov. and D. brandtae sp. nov. within the family Ethmolaimidae, subfamily Neotonchinae, based on specimens from two low-activity cold-seep environments at distant geographical locations. The new genus was first identified in samples from the Darwin mud volcano (1100 m depth) in the Gulf of Cadiz and later on also found in samples from a low-activity seep in the Larsen B embayment (820m depth) off the eastern Antarctic Peninsula. Until now, the family Ethmolaimidae contained nine genera: Ethmolaimus and Paraethmolaimus in the subfamily Ethmolaiminae, and Comesa, Filitonchoides, Filitonchus, Gomphionchus, Gomphionema, Nannolaimus, and Neothonchus in the subfamily Neotonchinae. The most important family characteristics are: an annulated cuticle bearing transverse rows of dots, cephalic sensilla arrangement of 6+6+4, a spiral amphid, an oesophagus with muscular posterior bulb, paired gonads and males with cup-shaped precloacal supplements. The new genus resembles Comesa and Neotonchus, but is typified by a ventrally displaced oral opening with three very small teeth that are easily overlooked. D. cadizensis gen. nov. sp. nov. is characterized by the 1401-2123 mu m long body; cuticle transversally striated with fine punctation; head conical; low lips; amphid spiralled 3 turns, oral opening ventrally displaced, male with outstretched testes; spicules of equal size; gubernaculum plate-like and ten to twelve conspicuous cup-shaped precloacal supplements with external longitudinal articulated flange. D. brandtae gen. nov. sp. nov. can be distinguished by the 2438-3280 mu m long body; cuticle transversally striated with fine punctuation; head conical; low lips; amphid spiraled 3+ turns; oral opening ventrally displaced; male with anterior testes outstretched and posterior one smaller and reflexed; spicules of equal size; gubernaculum plate-like and twenty conspicuous cup-shaped precloacal supplements with external longitudinal articulated flange. Notes on the ecology and habitat of the new genus are provided in light of its discovery in cold-seep environments.

A comparison of two zooplankton time series data collected in the Gulf of Maine

Interannual and seasonal trends of zooplankton abundance and species composition were compared between the Bongo net and Continuous Plankton Recorder (CPR) time series in the Gulf of Maine. Data from 5799 Bongo and 3118 CPR samples were compared from the years 1978–2006. The two programs use different sampling methods, with the Bongo time series composed of bimonthly vertically integrated samples from locations throughout the region, while the CPR was towed monthly at 10 m depth on a transect that bisects the region. It was found that there was a significant correlation between the interannual (r = 0.67, P < 0.01) and seasonal (r = 0.95, P < 0.01) variability of total zooplankton counts. Abundance rankings of individual taxa were highly correlated and temporal trends of dominant copepods were similar between samplers. Multivariate analysis also showed that both time series equally detected major shifts in community structure through time. However, absolute abundance levels were higher in the Bongo and temporal patterns for many of the less abundant taxa groups were not similar between the two devices. The different mesh sizes of the samplers probably caused some of the discrepancies; but diel migration patterns, damage to soft bodied animals and avoidance of the small CPR aperture by some taxa likely contributed to the catch differences between the two devices. Nonetheless, Bongo data presented here confirm the previously published patterns found in the CPR data set, and both show that the abundance increase of the 1990s has been followed by average to below average levels from 2002 to 06.

Simulation of Dynamic Operation of a Single-Cylinder Two-Stroke Engine

Presented is a study that expands the body of knowledge on the effect of in-cycle speed fluctuations on performance of small engines. It uses the engine and drivetrain models developed previously by Callahan, et al. (1) to examine a variety of engines. The predicted performance changes due to drivetrain effects are shown in each case, and conclusions are drawn from those results. The single-cylinder, high performance four-stroke engine showed significant changes in predicted performance compared to the prediction with zero speed fluctuation in the model. Measured speed fluctuations from a firing Yamaha YZ426 engine were applied to the simulation in addition to data from a simple free mass model. Both methods predicted similar changes in performance. The multiple-cylinder, high performance two-stroke engine also showed significant changes in performance depending on the firing configuration. With both engines, the change in performance diminished with increasing mean engine speed. The low output, single-cylinder two-stroke engine simulation showed only a negligible change in performance, even with high amplitude speed fluctuations. Because the torque versus engine speed characteristic for the engine was so flat, this was expected. The cross-charged, multi-cylinder two-stroke engine also showed only a negligible change in performance. In this case, the combination of a relatively high inertia rotating assembly and the multiple cylinder firing events within the revolution smoothing the torque pulsations reduced the speed fluctuation amplitude itself.