The Sensor Network Workbench: Towards Functional Specification, Verification and Deployment of Constrained Distributed Systems

As the commoditization of sensing, actuation and communication hardware increases, so does the potential for dynamically tasked sense and respond networked systems (i.e., Sensor Networks or SNs) to replace existing disjoint and inflexible special-purpose deployments (closed-circuit security video, anti-theft sensors, etc.). While various solutions have emerged to many individual SN-centric challenges (e.g., power management, communication protocols, role assignment), perhaps the largest remaining obstacle to widespread SN deployment is that those who wish to deploy, utilize, and maintain a programmable Sensor Network lack the programming and systems expertise to do so. The contributions of this thesis centers on the design, development and deployment of the SN Workbench (snBench). snBench embodies an accessible, modular programming platform coupled with a flexible and extensible run-time system that, together, support the entire life-cycle of distributed sensory services. As it is impossible to find a one-size-fits-all programming interface, this work advocates the use of tiered layers of abstraction that enable a variety of high-level, domain specific languages to be compiled to a common (thin-waist) tasking language; this common tasking language is statically verified and can be subsequently re-translated, if needed, for execution on a wide variety of hardware platforms. snBench provides: (1) a common sensory tasking language (Instruction Set Architecture) powerful enough to express complex SN services, yet simple enough to be executed by highly constrained resources with soft, real-time constraints, (2) a prototype high-level language (and corresponding compiler) to illustrate the utility of the common tasking language and the tiered programming approach in this domain, (3) an execution environment and a run-time support infrastructure that abstract a collection of heterogeneous resources into a single virtual Sensor Network, tasked via this common tasking language, and (4) novel formal methods (i.e., static analysis techniques) that verify safety properties and infer implicit resource constraints to facilitate resource allocation for new services. This thesis presents these components in detail, as well as two specific case-studies: the use of snBench to integrate physical and wireless network security, and the use of snBench as the foundation for semester-long student projects in a graduate-level Software Engineering course.

Learning to Teach and its Implications for the Continuum of Teacher Education: A Nine Country Cross-national Study.

The purpose of this Report is to inform discussions, policy formulation, and strategic planning on teacher education in Ireland. The research gives priority to initial teacher education (ITE) and induction, their interface, and implications for the continuum of teacher education, including continuing professional development (CPD). The study involved a two-pronged approach: a narrative review of recent and relevant literature and a cross-national review of teacher education policies in nine countries, namely, Ireland, Northern Ireland, Scotland, England, Finland, USA, Poland, Singapore and New Zealand. Adopting a broad, balanced and comprehensive understanding of the role of the contemporary teacher, it provides a framework for developing quality teacher education in Ireland. The Report incorporates exemplars of good practice and notes their implementation challenges for the Irish context.  Chapter One provides a framework for conceptualising quality teacher education and the continuum. Key features that emerge from the literature are discussed: teachers¿ practice, quality teaching, the professional life-cycle, teacher learning and relationships. With more specific reference to the continuum, Chapter Two overviews initial teacher education, induction, learning outcomes and accreditation in the selected countries, including Ireland. Key features of policy in the various countries are summarised. Individual country profiles, incorporating descriptions of socio-political, teaching and teacher education contexts, are further detailed in Appendix A. Chapter Three analyses relevant literature on initial teacher education, induction, learning outcomes/professional standards and accreditation. Along with previous chapters it provides the basis for recommendations for teacher education that are presented in Chapter Four. Chapter Four draws together the findings emerging from the cross-national review in terms of the contemporary context of teacher education in Ireland and identifies key challenges and possible lines of policy development as well as recommendations for the Teaching Council and other teacher education stakeholders. Each generation has an opportunity to provide the vision and resources for renewing teacher education in light of ambitious social, economic and educational aspirations to meet perceived societal and education challenges (as occurred in the 1970s). Despite the publication of two key reviews of initial teacher education a number of years ago, there is considerable scope for further reform of teacher education. However, significant changes have occurred to teacher education course provision and content over the last 100 years. In this report, we have stressed the need for, and called for investment in, greater system and programme coherence, mentoring to support assisted practice, knowledge integration, critical reflective practice, inquiry and the development of vibrant partnerships between higher education institutions and schools as the basis for teacher education reform across the continuum. This Executive Summary presents the Report¿s context, key findings and recommendations emerging from the analysis.  

Mutational and molecular analyses of lactococcal bacteriophages TP901-1 and Tuc2009

Bacteriophages belonging to the Siphoviridae family represent viruses with a noncontractile tail that function as extremely efficient bacterium-infecting nanomachines. The Siphoviridae phages TP901-1 and Tuc2009 infect Lactococcus lactis, and both belong to the so-called P335 species. As P335 phages are typically capable of a lytic and lysogenic life cycle, a number of molecular tools are available to analyse their virions. This doctoral thesis describes mutational and molecular analyses of TP901-1 and Tuc2009, with emphasis on the role of their tail-associated structural proteins. Several novel and intriguing findings discovered during the course of this study on the nature of Siphoviridae phages furthers a basic molecular understanding of their virions, and the role of their virion proteins, during the initial stages of infection. While Siphoviridae virions represent complex quaternary structures of multiple proteins and subunits thereof, mutagenic analysis represents an efficient mechanism to discretely characterize the function of individual proteins, and constituent amino acids, in the assembly of the phage structure and their biological function. However, as always, more research is required to delve deeper into the mechanisms by which phages commence infection. This is important to advance our understanding of this intricate process and to facilitate application of such findings to manipulate phage infections. On the one hand, we may want to prevent phages from infecting starter cultures used in the dairy industry, while on the other hand it may be desirable to optimize viral infection for the application of phages as bacterial parasites and therapeutic agents.

Aspects of the biology of the parasite Bonamia ostreae with a view to gaining a greater understanding of how to alleviate its impact on the European flat oyster, Ostrea edulis.

The parasite Bonamia ostreae has decimated Ostrea edulis stocks throughout Europe. The complete life cycle and means of transmission of the parasite remains unknown. The methods used to diagnose B. ostreae were examined to determine sensitivity and reproducibility. Two methods, with fixed protocols, should be used for the accurate detection of infection within a sample. A 13-month study of two stocks of O. edulis with varying periods of exposure to B. ostreae, was undertaken to determine if varying lengths of exposure would translate into observations of differing susceptibility. Oyster stocks can maintain themselves over extended periods of time in B. ostreae endemic areas. To identify a well performing spat stock, which could be used to repopulate beds within the region, hatchery bred spat from three stocks found in the North sea were placed on a B. ostreae infected bed and screened for growth, mortality and prevalence of infection. Local environmental factors may influence oyster performance, with local stocks better adapted to these conditions. Sediment and macroinvertebrate species were screened to investigate mechanisms by which B. ostreae may be maintaining itself on oyster beds. Mytilus edulis was positive, indicating that B. ostreae may use incidental carriers as a method of maintaining itself. The ability of oyster larvae to pick up infection from the surrounding environment was investigated by collecting larvae from brooding oysters from different areas. Larvae may acquire the pathogen from the water column during the process of filter feeding by the brooding adult, even when the parents themselves are uninfected. A study was undertaken to elucidate the activity of the parasite during the initial stage of infection, when it cannot be detected within the host. A naïve stock screened negative for infection throughout the trial, using heart imprints and PCR yet B. ostreae was detected by in-situ hybridisation.

Feasibility of combined wind-wave energy platforms

The European Union has set out an ambitious 20% target for renewable energy use by 2020. It is expected that this will be met mainly by wind energy. Looking towards 2050, reductions in greenhouse gas emissions of 80-95% are to be sought. Given the issues securing this target in the transport and agriculture sectors, it may only be possible to achieve this target if the power sector is carbon neutral well in advance of 2050. This has permitted the vast expansion of offshore renewables, wind, wave and tidal energy. Offshore wind has undergone rapid development in recent years however faces significant challenges up to 2020 to ensure commercial viability without the need for government subsidies. Wave energy is still in the very early stages of development so as yet there has been no commercial roll out. As both of these technologies are to face similar challenges in ensuring they are a viable alternative power generation method to fossil fuels, capitalising on the synergies is potentially a significant cost saving initiative. The advent of hybrid solutions in a variety of configurations is the subject of this thesis. A singular wind-wave energy platform embodies all the attributes of a hybrid system, including sharing space, transmission infrastructure, O&M activities and a platform/foundation. This configuration is the subject of this thesis, and it is found that an OWC Array platform with multi-MegaWatt wind turbines is a technically feasible, and potentially an economically feasible solution in the long term. Methods of design and analysis adopted in this thesis include numerical and physical modelling of power performance, structural analysis, fabrication cost modelling, simplified project economic modelling and time domain reliability modelling of a 210MW hybrid farm. The application of these design and analysis methods has resulted in a hybrid solution capable of producing energy at a cost between €0.22/kWh and €0.31/kWh depending on the source of funding for the project. Further optimisation through detailed design is expected to lower this further. This thesis develops new and existing methods of design and analysis of wind and wave energy devices. This streamlines the process of early stage development, while adhering to the widely adopted Concept Development Protocol, to develop a technically and economically feasible, combined wind-wave energy hybrid solution.

Grooming and hygienic behaviours of the honey bee (Apis mellifera) toward the parasitic mite Varroa destructor

Despite a multitude of environmental stressors, the Varroa mite is still regarded as the greatest cause of honey bee mortality in its invaded range. Breeding honey bees that are resistant to the mite is an important area of research. This thesis aimed to gain a better understanding of the grooming and hygienic behaviours of Russian honey bees (RHB). The effect of a break in the synchrony of a mite’s life cycle on reproductive success was tested through brood inoculation experiments. Mites released by hygienic behaviour and forced to enter a new cell are less likely to lay male offspring. Through laboratory cage assays it was found that daughter mites are more susceptible to grooming behaviour. A new method of marking Varroa mites was developed which would enable a single cohort of mites to be followed after inoculation. A strong brood removal trait was noticed in RHB colonies, therefore they were tested for Varroa sensitive hygienic (VSH) behaviour. RHB demonstrated levels of VSH as high as the USDA line bred specifically for this behaviour. In addition the same QTL found to be responsible for the trait in VSH bees, was associated with VSH in RHB stock. Previous work showed that the ratio of older mites to total trapped mites (O/T) in the debris of honey bee colonies demonstrated the strongest association with colony infestation. This research showed that O/T is associated with VSH and brood removal behaviour. In addition, bees that displayed high levels of VSH in this study were also more likely to spend a longer amount of time grooming in laboratory assays. This indicates that both grooming and hygienic behaviours play important roles in the resistance of RHB stock. Their likelihood to be expressed by other stocks is discussed and recommendations for further research are provided.

Transitions: From novice participation to expertise, pre-school 'matters' in an Irish pre-school community

This in depth, qualitative, participant observer study tracks children's transition experiences from novice to experienced membership of their pre-school community. It also considers adult roles in mediating this process in the context of the recent introduction of a universal free-pre-school year for children growing up in Ireland. Participation and the space to negotiate a participatory identity is understood in this study as a key element of positive experiences of early years transitions, within pre-school and beyond. The underlying theoretical framework is socio-cultural. This approach shifts from a scientific positivist view of thinking and learning as an individual inside the head process and asserts the historical, social, cultural as well as the situated context of learning and meaning making All participants, including myself as researcher, are recognised, explored and valued as embedded in the cultural context studied. In a sense, this approach tilts the worlds being observed through participation in them and reflects them in new light. The aim is to interpret and reflect the multiple realities constructed in this context rather than seek a truth out there waiting to be found. Special efforts are made to be invited in to and acknowledge children's expertise in the cultural worlds they negotiate with peers and adults in pre-school. The aim is to better understand what children may find motivating, interesting or problematic as they interpret reproduce and transform meaning within their play and learning worlds. My aim is for an honest rendering of the voices of stakeholders in pre-school communities from teachers, parents, and policy makers to children themselves. It makes visible constraints; potentials and possibilities within everyday Irish pre-school practices in the situated context studied as well as the broader societal, legislative and macro policy influences it reflects. Casting light on the taken for granted opens the possibility of adaptation or transformation. Transition itself can act as a tool to meet the changing needs of children on their developmental pathways across the life cycle

Micro and nanostructured impedance sensors for biological and biomedical applications

This thesis work covered the fabrication and characterisation of impedance sensors for biological applications aiming in particular to the cytotoxicity monitoring of cultured cells exposed to different kind of chemical compounds and drugs and to the identification of different types of biological tissue (fat, muscles, nerves) using a sensor fabricated on the tip of a commercially available needle during peripheral nerve block procedures. Gold impedance electrodes have been successfully fabricated for impedance measurement on cells cultured on the electrode surface which was modified with the fabrication of gold nanopillars. These nanostructures have a height of 60nm or 100nm and they have highly ordered layout as they are fabricated through the e-beam technique. The fabrication of the threedimensional structures on the interdigitated electrodes was supposed to improve the sensitivity of the ECIS (electric cell-substrate impedance sensing) measurement while monitoring the cytotoxicity effects of two different drugs (Antrodia Camphorata extract and Nicotine) on three different cell lines (HeLa, A549 and BALBc 3T3) cultured on the impedance devices and change the morphology of the cells growing on the nanostructured electrodes. The fabrication of the nanostructures was achieved combining techniques like UV lithography, metal lift-off, evaporation and e-beam lithography techniques. The electrodes were packaged using a pressure sensitive, medical grade adhesive double-sided tape. The electrodes were then characterised with the aid of AFM and SEM imaging which confirmed the success of the fabrication processes showing the nanopillars fabricated with the right layout and dimensions figures. The introduction of nanopillars on the impedance electrodes, however, did not improve much the sensitivity of the assay with the exception of tests carried out with Nicotine. HeLa and A549 cells appeared to grow in a different way on the two surfaces, while no differences where noticed on the BALBc 3T3 cells. Impedance measurements obtained with the dead cells on the negative control electrodes or the test electrodes with the drugs can be compared to those done on the electrodes containing just media in the tested volume (as no cells are attached and cover the electrode surface). The impedance figures recorded using these electrodes were between 1.5kΩ and 2.5 kΩ, while the figures recorded on confluent cell layers range between 4kΩ and 5.5kΩ with peaks of almost 7 kΩ if there was more than one layer of cells growing on each other. There was then a very clear separation between the values of living cell compared to the dead ones which was almost 2.5 - 3kΩ. In this way it was very easy to determine whether the drugs affected the cells normal life cycle on not. However, little or no differences were noticed in the impedance analysis carried out on the two different kinds of electrodes using cultured cells. An increase of sensitivity was noticed only in a couple of experiments carried out on A549 cells growing on the nanostructured electrodes and exposed to different concentration of a solution containing Nicotine. More experiments to achieve a higher number of statistical evidences will be needed to prove these findings with an absolute confidence. The smart needle project aimed to reduce the limitations of the Electrical Nerve Stimulation (ENS) and the Ultra Sound Guided peripheral nerve block techniques giving the clinicians an additional tool for performing correctly the peripheral nerve block. Bioimpedance, as measured at the needle tip, provides additional information on needle tip location, thereby facilitating detection of intraneural needle placement. Using the needle as a precision instrument and guidance tool may provide additional information as to needle tip location and enhance safety in regional anaesthesia. In the time analysis, with the frequency fixed at 10kHz and the samples kept at 12°C, the approximate range for muscle bioimpedance was 203 – 616 Ω, the approximate bioimpedance range for fat was 5.02 - 17.8 kΩ and the approximate range for connective tissue was 790 Ω – 1.55 kΩ. While when the samples were heated at 37°C and measured again at 10kHz, the approximate bioimpedance range for muscle was 100-175Ω. The approximate bioimpedance range of fat was 627 Ω - 3.2 kΩ and the range for connective tissue was 221-540Ω. In the experiments done on the fresh slaughtered lamb carcass, replicating a scenario close to the real application, the impedance values recorded for fat were around 17 kΩ, for muscle and lean tissue around 1.3 kΩ while the nervous structures had an impedance value of 2.9 kΩ. With the data collected during this research, it was possible to conclude that measurements of bioimpedance at the needle tip location can give valuable information to the clinicians performing a peripheral nerve block procedure as the separation (in terms of impedance figures) was very marked between the different type of tissues. It is then feasible to use an impedance electrode fabricated on the needle tip to differentiate several tissues from the nerve tissue. Currently, several different methods are being studied to fabricate an impedance electrode on the surface of a commercially available needle used for the peripheral nerve block procedure.

Morphological and genomic characterization of Filobasidiella depauperata: a homothallic sibling species of the pathogenic cryptococcus species complex.

The fungal species Cryptococcus neoformans and Cryptococcus gattii cause respiratory and neurological disease in animals and humans following inhalation of basidiospores or desiccated yeast cells from the environment. Sexual reproduction in C. neoformans and C. gattii is controlled by a bipolar system in which a single mating type locus (MAT) specifies compatibility. These two species are dimorphic, growing as yeast in the asexual stage, and producing hyphae, basidia, and basidiospores during the sexual stage. In contrast, Filobasidiella depauperata, one of the closest related species, grows exclusively as hyphae and it is found in association with decaying insects. Examination of two available strains of F. depauperata showed that the life cycle of this fungal species shares features associated with the unisexual or same-sex mating cycle in C. neoformans. Therefore, F. depauperata may represent a homothallic and possibly an obligately sexual fungal species. RAPD genotyping of 39 randomly isolated progeny from isolate CBS7855 revealed a new genotype pattern in one of the isolated basidiospores progeny, therefore suggesting that the homothallic cycle in F. depauperata could lead to the emergence of new genotypes. Phylogenetic analyses of genes linked to MAT in C. neoformans indicated that two of these genes in F. depauperata, MYO2 and STE20, appear to form a monophyletic clade with the MATa alleles of C. neoformans and C. gattii, and thus these genes may have been recruited to the MAT locus before F. depauperata diverged. Furthermore, the ancestral MATa locus may have undergone accelerated evolution prior to the divergence of the pathogenic Cryptococcus species since several of the genes linked to the MATa locus appear to have a higher number of changes and substitutions than their MATalpha counterparts. Synteny analyses between C. neoformans and F. depauperata showed that genomic regions on other chromosomes displayed conserved gene order. In contrast, the genes linked to the MAT locus of C. neoformans showed a higher number of chromosomal translocations in the genome of F. depauperata. We therefore propose that chromosomal rearrangements appear to be a major force driving speciation and sexual divergence in these closely related pathogenic and saprobic species.

The distribution of sales revenues from pharmaceutical innovation.

OBJECTIVE: This report updates our earlier work on the returns to pharmaceutical research and development (R&D) in the US (1980 to 1984), which showed that the returns distributions are highly skewed. It evaluates a more recent cohort of new drug introductions in the US (1988 to 1992) and examines how the returns distribution is emerging for drugs with life cycles concentrated in the 1990s versus the 1980s. DESIGN AND SETTING: Methods were described in detail in our earlier reports. The current sample included 110 new drug entities (including 28 orphan drugs), and sales data were obtained for the period 1988 to 1998, which represented between 7 and 11 years of sales for the drugs included. 20 years was chosen as the expected market life for this cohort, and a 2-step procedure was used to project future sales for the drugs--during the period until patent expiry and then beyond patent expiry until the 20-year time-horizon was completed. Thus, the values in the first half of the life cycle are essentially based on realised sales, while those in the second half are projected using information on patent expiry and other inputs. MAIN OUTCOME MEASURES AND RESULTS: Peak annual sales for the top decile of drugs introduced between 1988 and 1992 in the US amounted to almost $US1.1 billion compared with peak sales of less than $US175 million (1992 values) for the mean compound. In particular, the top decile accounted for 56% of overall sales revenue. Although the sales distributions were skewed in both our earlier and current analysis, the top decile in the later time-period exhibited more rapid rates of growth after launch, a peak that was more than 50% greater in real terms than for the 1980 to 1984 cohort, and a faster rate of expected decline in sales after patent expiry. One factor contributing to the distribution of sales revenues becoming more skewed over time is the orphan drug phenomenon (i.e. most of the orphan drugs are concentrated at the bottom of the distribution). CONCLUSION: The distribution of sales revenues for new drug compounds is highly skewed in nature. In this regard, the top decile of new drugs accounts for more than half of the total sales generated by the 1988 to 1992 cohort analysed. Furthermore, the distribution of sales revenues for this cohort is more skewed than that of the 1980 to 1984 cohort we analysed in previous research.

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.

Sensitive and precise quantification of insulin-like mRNA expression in Caenorhabditis elegans.

Insulin-like signaling regulates developmental arrest, stress resistance and lifespan in the nematode Caenorhabditis elegans. However, the genome encodes 40 insulin-like peptides, and the regulation and function of individual peptides is largely uncharacterized. We used the nCounter platform to measure mRNA expression of all 40 insulin-like peptides as well as the insulin-like receptor daf-2, its transcriptional effector daf-16, and the daf-16 target gene sod-3. We validated the platform using 53 RNA samples previously characterized by high density oligonucleotide microarray analysis. For this set of genes and the standard nCounter protocol, sensitivity and precision were comparable between the two platforms. We optimized conditions of the nCounter assay by varying the mass of total RNA used for hybridization, thereby increasing sensitivity up to 50-fold and reducing the median coefficient of variation as much as 4-fold. We used deletion mutants to demonstrate specificity of the assay, and we used optimized conditions to assay insulin-like gene expression throughout the C. elegans life cycle. We detected expression for nearly all insulin-like genes and find that they are expressed in a variety of distinct patterns suggesting complexity of regulation and specificity of function. We identified insulin-like genes that are specifically expressed during developmental arrest, larval development, adulthood and embryogenesis. These results demonstrate that the nCounter platform provides a powerful approach to analyzing insulin-like gene expression dynamics, and they suggest hypotheses about the function of individual insulin-like genes.

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.

The Physarum polycephalum Genome Reveals Extensive Use of Prokaryotic Two-Component and Metazoan-Type Tyrosine Kinase Signaling.

Physarum polycephalum is a well-studied microbial eukaryote with unique experimental attributes relative to other experimental model organisms. It has a sophisticated life cycle with several distinct stages including amoebal, flagellated, and plasmodial cells. It is unusual in switching between open and closed mitosis according to specific life-cycle stages. Here we present the analysis of the genome of this enigmatic and important model organism and compare it with closely related species. The genome is littered with simple and complex repeats and the coding regions are frequently interrupted by introns with a mean size of 100 bases. Complemented with extensive transcriptome data, we define approximately 31,000 gene loci, providing unexpected insights into early eukaryote evolution. We describe extensive use of histidine kinase-based two-component systems and tyrosine kinase signaling, the presence of bacterial and plant type photoreceptors (phytochromes, cryptochrome, and phototropin) and of plant-type pentatricopeptide repeat proteins, as well as metabolic pathways, and a cell cycle control system typically found in more complex eukaryotes. Our analysis characterizes P. polycephalum as a prototypical eukaryote with features attributed to the last common ancestor of Amorphea, that is, the Amoebozoa and Opisthokonts. Specifically, the presence of tyrosine kinases in Acanthamoeba and Physarum as representatives of two distantly related subdivisions of Amoebozoa argues against the later emergence of tyrosine kinase signaling in the opisthokont lineage and also against the acquisition by horizontal gene transfer.

Multiphysics modelling for electronics design

The future of many companies will depend to a large extent on their ability to initiate techniques that bring schedules, performance, tests, support, production, life-cycle-costs, reliability prediction and quality control into the earliest stages of the product creation process. Important questions for an engineer who is responsible for the quality of electronic parts such as printed circuit boards (PCBs) during design, production, assembly and after-sales support are: What is the impact of temperature? What is the impact of this temperature on the stress produced in the components? What is the electromagnetic compatibility (EMC) associated with such a design? At present, thermal, stress and EMC calculations are undertaken using different software tools that each require model build and meshing. This leads to a large investment in time, and hence cost, to undertake each of these simulations. This paper discusses the progression towards a fully integrated software environment, based on a common data model and user interface, having the capability to predict temperature, stress and EMC fields in a coupled manner. Such a modelling environment used early within the design stage of an electronic product will provide engineers with fast solutions to questions regarding thermal, stress and EMC issues. The paper concentrates on recent developments in creating such an integrated modeling environment with preliminary results from the analyses conducted. Further research into the thermal and stress related aspects of the paper is being conducted under a nationally funded project, while their application in reliability prediction will be addressed in a new European project called PROFIT.