A timescale for diatom evolution based on four molecular markers: reassessment of ghost lineages and major steps defining diatom evolution.

ABSTRACT. – Phylogenies and molecular clocks of the diatoms have largely been inferred from SSU rDNA sequences. A new phylogeny of diatoms was estimated using four gene markers SSU and LSU rDNA rbcL and psbA (total 4352 bp) with 42 diatom species. The four gene trees analysed with a maximum likelihood (ML) and Baysian (BI) analysis recovered a monophyletic origin of the new diatom classes with high bootstrap support, which has been controversial with single gene markers using single outgroups and alignments that do not take secondary structure of the SSU gene into account. The divergence time of the classes were calculated from a ML tree in the MultliDiv Time program using a Bayesian estimation allowing for simultaneous constraints from the fossil record and varying rates of molecular evolution of different branches in the phylogenetic tree. These divergence times are generally in agreement with those proposed by other clocks using single genes with the exception that the pennates appear much earlier and suggest a longer Cretaceous fossil record that has yet to be sampled. Ghost lineages (i.e. the discrepancy between first appearance (FA) and molecular clock age of origin from an extant taxon) were revealed in the pennate lineage, whereas those ghost lineages in the centric lineages previously reported by others are reviewed and referred to earlier literature.

Delivery of nucleic acids for cancer gene therapy: overcoming extra- and intra-cellular barriers

The therapeutic potential of cancer gene therapy has been limited by the difficulty of delivering genetic material to target sites. Various biological and molecular barriers exist which need to be overcome before effective nonviral delivery systems can be applied successfully in oncology. Herein, various barriers are described and strategies to circumvent such obstacles are discussed, considering both the extracellular and intracellular setting. Development of multifunctional delivery systems holds much promise for the progression of gene delivery, and a growing body of evidence supports this approach involving rational design of vectors, with a unique molecular architecture. In addition, the potential application of composite gene delivery platforms is highlighted which may provide an alternative delivery strategy to traditional systemic administration.

Conditions for Teaching and Learning in Religious Education (RE) : Perspectives of Teachers and Pupils at the Beginning of the 6th Grade in Sweden

In this article empirical findings from interviews with teachers of three classes of 12-year-old pupils are presented, together with questionnaire-responses from these 54 pupils. The interviews focus on teaching aims for Religious Education (RE), a subject that in Sweden, besides dealing with religion, also explores other kinds of beliefs, ethics and life questions. In the questionnaire the pupils are asked to solve four RE tasks with content that is central from a Swedish curriculum perspective. The research involves pupils at the beginning of the sixth grade and the purpose of this article is to look at the teachers’ aims and the pupils’ responses, and consider what these may indicate about conditions for teaching and learning RE in these classes. The findings show that the perspectives of the pupils at the beginning of the sixth grade seem to be rather far from the expectations of the RE syllabus. The pupils’ statements are rather vague with regard to religion as a phenomenon and there are few examples of pupils interpreting religious symbols in a way that is useful in further analysis. While existential and ethical plots, messages and point of views are comparatively easy to describe, it is harder to express multiple perspectives, reasons, comparisons and questions. A problem for the teachers in developing the perspectives of their pupils is that they find it hard to say what kind of general difficulties pupils have in RE, a fact that makes it hard to direct the teaching. Another challenge is that the teachers’ RE-aims are rather overarching and primarily related to fostering fundamental values. What improves the conditions for teaching and learning is the teachers’ concern for the pupils and their relationships with the teacher and with each other, a factor which is of vital importance for learning and which can also be used as a specific teaching method in subject matter education.

Algebro-Geometric Aspects of the Classical Yang-Baxter Equation

In this thesis we consider algebro-geometric aspects of the Classical Yang-Baxter Equation and the Generalised Classical Yang-Baxter Equation. In chapter one we present a method to construct solutions of the Generalised Classical Yang-Baxter Equation starting with certain sheaves of Lie algebras on algebraic curves. Furthermore we discuss a criterion to check unitarity of such solutions. In chapter two we consider the special class of solutions coming from sheaves of traceless endomorphisms of simple vector bundles on the nodal cubic curve. These solutions are quasi-trigonometric and we describe how they fit into the classification scheme of such solutions. Moreover, we describe a concrete formula for these solutions. In the third and final chapter we show that any unitary, rational solution of the Classical Yang-Baxter Equation can be obtained via the method of chapter one applied to a sheaf of Lie algebras on the cuspidal cubic curve.

Exact Solution of Bayes and Minimax Change-Detection Problems

The challenge of detecting a change in the distribution of data is a sequential decision problem that is relevant to many engineering solutions, including quality control and machine and process monitoring. This dissertation develops techniques for exact solution of change-detection problems with discrete time and discrete observations. Change-detection problems are classified as Bayes or minimax based on the availability of information on the change-time distribution. A Bayes optimal solution uses prior information about the distribution of the change time to minimize the expected cost, whereas a minimax optimal solution minimizes the cost under the worst-case change-time distribution. Both types of problems are addressed. The most important result of the dissertation is the development of a polynomial-time algorithm for the solution of important classes of Markov Bayes change-detection problems. Existing techniques for epsilon-exact solution of partially observable Markov decision processes have complexity exponential in the number of observation symbols. A new algorithm, called constellation induction, exploits the concavity and Lipschitz continuity of the value function, and has complexity polynomial in the number of observation symbols. It is shown that change-detection problems with a geometric change-time distribution and identically- and independently-distributed observations before and after the change are solvable in polynomial time. Also, change-detection problems on hidden Markov models with a fixed number of recurrent states are solvable in polynomial time. A detailed implementation and analysis of the constellation-induction algorithm are provided. Exact solution methods are also established for several types of minimax change-detection problems. Finite-horizon problems with arbitrary observation distributions are modeled as extensive-form games and solved using linear programs. Infinite-horizon problems with linear penalty for detection delay and identically- and independently-distributed observations can be solved in polynomial time via epsilon-optimal parameterization of a cumulative-sum procedure. Finally, the properties of policies for change-detection problems are described and analyzed. Simple classes of formal languages are shown to be sufficient for epsilon-exact solution of change-detection problems, and methods for finding minimally sized policy representations are described.

Aprendizaje basado en problemas, ABP : una propuesta para transformar la enseñanza-aprendizaje de las aplicaciones de la trigonometría en la solución de triángulos en el grado 10º

El estudio tuvo como propósito determinar la efectividad relativa del ABP, comparado con el método tradicional para desarrollar habilidades de resolución de problemas en el aprendizaje de las aplicaciones de la solución de triángulos en el grado 10º de la Institución Educativa El Progreso, de El Carmen de Viboral, Antioquia. La enseñanza-aprendizaje de las matemáticas sustentadas con la estrategia didáctica Aprendizaje Basado en Problemas permite a los estudiantes y docentes aproximarse al conocimiento de una manera similar a como lo hacen los científicos; el primer paso es una situación de duda, perplejidad del estudiante provocada por la Situación Problema planteada por el docente, el segundo un momento de “sugerencias” en las que la mente salta hacia adelante en busca de una posible solución (Dewey, 1933, p. 102). El tercer paso “intelectualización” de la dificultad que se ha percibido para convertirlo en un problema que debe solucionarse (Dewey, 1933, p. 103). La cuarta es “la idea conductora o hipótesis”, las cuales se basan en la formulación de explicaciones sugeridas o soluciones posibles (Dewey, 1933, p. 104). El quinto paso sería el “razonamiento”, consiste en la elaboración racional de una idea que se va desarrollando de acuerdo a las habilidades de cada persona (Dewey, 1933, p. 105). El paso final es la “comprobación de hipótesis” en situaciones reales. Este proceso se evidenció a través de cuatro Situaciones-Problema enfocadas desde un contexto auténtico “la remodelación del parque principal de El Carmen de Viboral” con el objetivo de motivar a los estudiantes para el aprendizaje de algunos conceptos matemáticos y el desarrollo de habilidades de resolución de problemas. La metodología de la investigación fue un diseño cuasi-experimental con grupo experimental compuesto por 38 estudiantes del grado 10º2 y grupo control con 37 estudiantes del grado 10º1. Se empleó como técnica de recolección de la información una prueba pre-test antes del tratamiento y una prueba post-test que se aplicó después del tratamiento a ambos grupos; se aplicó también una escala de satisfacción de los estudiantes con la metodología tradicional en ambos grupos y una escala de satisfacción con la estrategia didáctica ABP sólo al grupo experimental; la observación directa, y el portafolio que evidenciaba todas las construcciones de los estudiantes. La aplicación de la estrategia didáctica experimental se aplicó durante 4 meses, con una intensidad horaria de cuatro horas semanales, tiempo durante el cual se implementaron las cuatro Situaciones-Problema. Se concluyó entre otros aspectos que el 86,5% de los estudiantes encuentran las clases de matemáticas como interesantes, contextualizadas, aplicables y significativas, mientras que antes del tratamiento sólo el 44,4% se encontraba satisfecho con las clases de matemáticas, con una diferencia en cambio de actitud de 42,1% frente a las clases de matemáticas con la metodología tradicional. En el análisis comparativo de adquisición de competencias específicas se demuestra que el grupo experimental demostró ser matemáticamente más competente con respecto al grupo control en todas las competencias evaluadas: capacidad de modelación, inductiva, comunicativa y habilidad procedimental. Además, el proyecto de investigación tuvo un valor agregado: 10 estudiantes tuvieron la oportunidad de conocer más sobre su cultura ceramista mediante el diseño y construcción de mosaicos que los ofreció la casa de la cultura en forma gratuita.

Developmental programming of polycystic ovary syndrome (PCOS): prenatal androgens establish pancreatic islet α/β cell ratio and subsequent insulin secretion

Exogenous androgenic steroids applied to pregnant sheep programmes a PCOS-like phenotype in female offspring. Via ultrasound guidance we applied steroids directly to ovine fetuses at d62 and d82 of gestation, and examined fetal (day 90 gestation) and postnatal (11 months old) pancreatic structure and function. Of three classes of steroid agonists applied (androgen - Testosterone propionate (TP), estrogen - Diethystilbesterol (DES) and glucocorticoid - Dexamethasone (DEX)), only androgens (TP) caused altered pancreatic development. Beta cell numbers were significantly elevated in prenatally androgenised female fetuses (P=0.03) (to approximately the higher numbers found in male fetuses), whereas alpha cell counts were unaffected, precipitating decreased alpha:beta cell ratios in the developing fetal pancreas (P=0.001), sustained into adolescence (P=0.0004). In adolescence basal insulin secretion was significantly higher in female offspring from androgen-excess pregnancies (P=0.045), and an exaggerated, hyperinsulinaemic response to glucose challenge (P=0.0007) observed, whereas prenatal DES or DEX treatment had no effects upon insulin secretion. Postnatal insulin secretion correlated with beta cell numbers (P=0.03). We conclude that the pancreas is a primary locus of androgenic stimulation during development, giving rise to postnatal offspring whose pancreas secreted excess insulin due to excess beta cells in the presence of a normal number of alpha cells.

Determinants of self-medication with NSAIDs in a Portuguese community pharmacy

Background: Non-steroid anti-inflammatory drugs (NSAIDs) are a widely used therapeutic group in the world, and particularly in the Portuguese population. Objective: To compare NSAID’s use by prescription and self-medication acquisition and to determine the pattern of indication of NSAIDs, their usage profile and possible implications for patients’ safety. Methods: A cross-sectional design was used where individuals presenting at a community pharmacy requesting NSAIDs during the study period (one month) were invited to answer a face-to-face interview where socio-demographic characteristics, the indication pattern and previous experience of side effects were assessed. A follow-up interview was performed one week later to assess the incidence of adverse effects. The study was ethically approved. Results: A sample of 130 NSAIDs users was recruited, comprising mostly women (n=87; 66.9%), actively employed (n=77; 59.2%) and presenting a mean age of 49.5 years old (SD=20.49). An equal proportion of individuals acquired NSAIDs by self-medication and with medical prescription (n=65; 50%). Over 4/5 of patients (n=57; 87.7%) acquiring NSAIDs without a prescription were self-medicated by their own initiative, and only 10.8% (n=7) had been advised by the pharmacist. The most commonly acquired active substances were ibuprofen and diclofenac. Self-medicated users more frequently resorted to topical NSAIDs following short term treatments. The major underlying condition motivating NSAIDs sought were musculoskeletal disorders (45.0%), regardless of the regimen. An important proportion of prevalent users of NSAIDs reported previous experience of adverse effects (11.3%). One week after initiating NSAID therapy, a small proportion of patients reported incidence of adverse effects. Conclusion: Self-medication with NSAIDs is sought for numerous medical conditions. Reported adverse effects (prevalent and incident) confirm the need for a more rational use of NSAIDs and ongoing pharmacovigilance.

Strong shift equivalence, algebraic K-theory, and isolating zero-dimensional dynamics on manifolds

We study the relations of shift equivalence and strong shift equivalence for matrices over a ring $\mathcal{R}$, and establish a connection between these relations and algebraic K-theory. We utilize this connection to obtain results in two areas where the shift and strong shift equivalence relations play an important role: the study of finite group extensions of shifts of finite type, and the Generalized Spectral Conjectures of Boyle and Handelman for nonnegative matrices over subrings of the real numbers. We show the refinement of the shift equivalence class of a matrix $A$ over a ring $\mathcal{R}$ by strong shift equivalence classes over the ring is classified by a quotient $NK_{1}(\mathcal{R}) / E(A,\mathcal{R})$ of the algebraic K-group $NK_{1}(\calR)$. We use the K-theory of non-commutative localizations to show that in certain cases the subgroup $E(A,\mathcal{R})$ must vanish, including the case $A$ is invertible over $\mathcal{R}$. We use the K-theory connection to clarify the structure of algebraic invariants for finite group extensions of shifts of finite type. In particular, we give a strong negative answer to a question of Parry, who asked whether the dynamical zeta function determines up to finitely many topological conjugacy classes the extensions by $G$ of a fixed mixing shift of finite type. We apply the K-theory connection to prove the equivalence of a strong and weak form of the Generalized Spectral Conjecture of Boyle and Handelman for primitive matrices over subrings of $\mathbb{R}$. We construct explicit matrices whose class in the algebraic K-group $NK_{1}(\mathcal{R})$ is non-zero for certain rings $\mathcal{R}$ motivated by applications. We study the possible dynamics of the restriction of a homeomorphism of a compact manifold to an isolated zero-dimensional set. We prove that for $n \ge 3$ every compact zero-dimensional system can arise as an isolated invariant set for a homeomorphism of a compact $n$-manifold. In dimension two, we provide obstructions and examples.

Microfluidic platforms for the characterization of in meso membrane protein crystallization

Membrane proteins, which reside in the membranes of cells, play a critical role in many important biological processes including cellular signaling, immune response, and material and energy transduction. Because of their key role in maintaining the environment within cells and facilitating intercellular interactions, understanding the function of these proteins is of tremendous medical and biochemical significance. Indeed, the malfunction of membrane proteins has been linked to numerous diseases including diabetes, cirrhosis of the liver, cystic fibrosis, cancer, Alzheimer's disease, hypertension, epilepsy, cataracts, tubulopathy, leukodystrophy, Leigh syndrome, anemia, sensorineural deafness, and hypertrophic cardiomyopathy.1-3 However, the structure of many of these proteins and the changes in their structure that lead to disease-related malfunctions are not well understood. Additionally, at least 60% of the pharmaceuticals currently available are thought to target membrane proteins, despite the fact that their exact mode of operation is not known.4-6 Developing a detailed understanding of the function of a protein is achieved by coupling biochemical experiments with knowledge of the structure of the protein. Currently the most common method for obtaining three-dimensional structure information is X-ray crystallography. However, no a priori methods are currently available to predict crystallization conditions for a given protein.7-14 This limitation is currently overcome by screening a large number of possible combinations of precipitants, buffer, salt, and pH conditions to identify conditions that are conducive to crystal nucleation and growth.7,9,11,15-24 Unfortunately, these screening efforts are often limited by difficulties associated with quantity and purity of available protein samples. While the two most significant bottlenecks for protein structure determination in general are the (i) obtaining sufficient quantities of high quality protein samples and (ii) growing high quality protein crystals that are suitable for X-ray structure determination,7,20,21,23,25-47 membrane proteins present additional challenges. For crystallization it is necessary to extract the membrane proteins from the cellular membrane. However, this process often leads to denaturation. In fact, membrane proteins have proven to be so difficult to crystallize that of the more than 66,000 structures deposited in the Protein Data Bank,48 less than 1% are for membrane proteins, with even fewer present at high resolution (< 2Å)4,6,49 and only a handful are human membrane proteins.49 A variety of strategies including detergent solubilization50-53 and the use of artificial membrane-like environments have been developed to circumvent this challenge.43,53-55 In recent years, the use of a lipidic mesophase as a medium for crystallizing membrane proteins has been demonstrated to increase success for a wide range of membrane proteins, including human receptor proteins.54,56-62 This in meso method for membrane protein crystallization, however, is still by no means routine due to challenges related to sample preparation at sub-microliter volumes and to crystal harvesting and X-ray data collection. This dissertation presents various aspects of the development of a microfluidic platform to enable high throughput in meso membrane protein crystallization at a level beyond the capabilities of current technologies. Microfluidic platforms for protein crystallization and other lab-on-a-chip applications have been well demonstrated.9,63-66 These integrated chips provide fine control over transport phenomena and the ability to perform high throughput analyses via highly integrated fluid networks. However, the development of microfluidic platforms for in meso protein crystallization required the development of strategies to cope with extremely viscous and non-Newtonian fluids. A theoretical treatment of highly viscous fluids in microfluidic devices is presented in Chapter 3, followed by the application of these strategies for the development of a microfluidic mixer capable of preparing a mesophase sample for in meso crystallization at a scale of less than 20 nL in Chapter 4. This approach was validated with the successful on chip in meso crystallization of the membrane protein bacteriorhodopsin. In summary, this is the first report of a microfluidic platform capable of performing in meso crystallization on-chip, representing a 1000x reduction in the scale at which mesophase trials can be prepared. Once protein crystals have formed, they are typically harvested from the droplet they were grown in and mounted for crystallographic analysis. Despite the high throughput automation present in nearly all other aspects of protein structure determination, the harvesting and mounting of crystals is still largely a manual process. Furthermore, during mounting the fragile protein crystals can potentially be damaged, both from physical and environmental shock. To circumvent these challenges an X-ray transparent microfluidic device architecture was developed to couple the benefits of scale, integration, and precise fluid control with the ability to perform in situ X-ray analysis (Chapter 5). This approach was validated successfully by crystallization and subsequent on-chip analysis of the soluble proteins lysozyme, thaumatin, and ribonuclease A and will be extended to microfluidic platforms for in meso membrane protein crystallization. The ability to perform in situ X-ray analysis was shown to provide extremely high quality diffraction data, in part as a result of not being affected by damage due to physical handling of the crystals. As part of the work described in this thesis, a variety of data collection strategies for in situ data analysis were also tested, including merging of small slices of data from a large number of crystals grown on a single chip, to allow for diffraction analysis at biologically relevant temperatures. While such strategies have been applied previously,57,59,61,67 they are potentially challenging when applied via traditional methods due to the need to grow and then mount a large number of crystals with minimal crystal-to-crystal variability. The integrated nature of microfluidic platforms easily enables the generation of a large number of reproducible crystallization trials. This, coupled with in situ analysis capabilities has the potential of being able to acquire high resolution structural data of proteins at biologically relevant conditions for which only small crystals, or crystals which are adversely affected by standard cryocooling techniques, could be obtained (Chapters 5 and 6). While the main focus of protein crystallography is to obtain three-dimensional protein structures, the results of typical experiments provide only a static picture of the protein. The use of polychromatic or Laue X-ray diffraction methods enables the collection of time resolved structural information. These experiments are very sensitive to crystal quality, however, and often suffer from severe radiation damage due to the intense polychromatic X-ray beams. Here, as before, the ability to perform in situ X-ray analysis on many small protein crystals within a microfluidic crystallization platform has the potential to overcome these challenges. An automated method for collecting a "single-shot" of data from a large number of crystals was developed in collaboration with the BioCARS team at the Advanced Photon Source at Argonne National Laboratory (Chapter 6). The work described in this thesis shows that, even more so than for traditional structure determination efforts, the ability to grow and analyze a large number of high quality crystals is critical to enable time resolved structural studies of novel proteins. In addition to enabling X-ray crystallography experiments, the development of X-ray transparent microfluidic platforms also has tremendous potential to answer other scientific questions, such as unraveling the mechanism of in meso crystallization. For instance, the lipidic mesophases utilized during in meso membrane protein crystallization can be characterized by small angle X-ray diffraction analysis. Coupling in situ analysis with microfluidic platforms capable of preparing these difficult mesophase samples at very small volumes has tremendous potential to enable the high throughput analysis of these systems on a scale that is not reasonably achievable using conventional sample preparation strategies (Chapter 7). In collaboration with the LS-CAT team at the Advanced Photon Source, an experimental station for small angle X-ray analysis coupled with the high quality visualization capabilities needed to target specific microfluidic samples on a highly integrated chip is under development. Characterizing the phase behavior of these mesophase systems and the effects of various additives present in crystallization trials is key for developing an understanding of how in meso crystallization occurs. A long term goal of these studies is to enable the rational design of in meso crystallization experiments so as to avoid or limit the need for high throughput screening efforts. In summary, this thesis describes the development of microfluidic platforms for protein crystallization with in situ analysis capabilities. Coupling the ability to perform in situ analysis with the small scale, fine control, and the high throughput nature of microfluidic platforms has tremendous potential to enable a new generation of crystallographic studies and facilitate the structure determination of important biological targets. The development of platforms for in meso membrane protein crystallization is particularly significant because they enable the preparation of highly viscous mixtures at a previously unachievable scale. Work in these areas is ongoing and has tremendous potential to improve not only current the methods of protein crystallization and crystallography, but also to enhance our knowledge of the structure and function of proteins which could have a significant scientific and medical impact on society as a whole. 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Down scaling to regional assessment of greenhouse gas emissions to enable consistency in accounting for emissions reduction projects and national inventory accounts for northern beef production in Australia

This paper explores the effect of using regional data for livestock attributes on estimation of greenhouse gas (GHG) emissions for the northern beef industry in Australia, compared with using state/territory-wide values, as currently used in Australia’s national GHG inventory report. Regional GHG emissions associated with beef production are reported for 21 defined agricultural statistical regions within state/territory jurisdictions. A management scenario for reduced emissions that could qualify as an Emissions Reduction Fund (ERF) project was used to illustrate the effect of regional level model parameters on estimated abatement levels. Using regional parameters, instead of state level parameters, for liveweight (LW), LW gain and proportion of cows lactating and an expanded number of livestock classes, gives a 5.2% reduction in estimated emissions (range +12% to –34% across regions). Estimated GHG emissions intensity (emissions per kilogram of LW sold) varied across the regions by up to 2.5-fold, ranging from 10.5 kg CO2-e kg–1 LW sold for Darling Downs, Queensland, through to 25.8 kg CO2-e kg–1 LW sold for the Pindan and North Kimberley, Western Australia. This range was driven by differences in production efficiency, reproduction rate, growth rate and survival. This suggests that some regions in northern Australia are likely to have substantial opportunities for GHG abatement and higher livestock income. However, this must be coupled with the availability of management activities that can be implemented to improve production efficiency; wet season phosphorus (P) supplementation being one such practice. An ERF case study comparison showed that P supplementation of a typical-sized herd produced an estimated reduction of 622 t CO2-e year–1, or 7%, compared with a non-P supplemented herd. However, the different model parameters used by the National Inventory Report and ERF project means that there was an anomaly between the herd emissions for project cattle excised from the national accounts (13 479 t CO2-e year–1) and the baseline herd emissions estimated for the ERF project (8 896 t CO2-e year–1) before P supplementation was implemented. Regionalising livestock model parameters in both ERF projects and the national accounts offers the attraction of being able to more easily and accurately reflect emissions savings from this type of emissions reduction project in Australia’s national GHG accounts.

Twenty years of Hendra virus: laboratory submission trends and risk factors for infection in horses

SUMMARY Hendra virus (HeV) was first described in 1994 in an outbreak of acute and highly lethal disease in horses and humans in Australia. Equine cases continue to be diagnosed periodically, yet the predisposing factors for infection remain unclear. We undertook an analysis of equine submissions tested for HeV by the Queensland government veterinary reference laboratory over a 20-year period to identify and investigate any patterns. We found a marked increase in testing from July 2008, primarily reflecting a broadening of the HeV clinical case definition. Peaks in submissions for testing, and visitations to the Government HeV website, were associated with reported equine incidents. Significantly differing between-year HeV detection rates in north and south Queensland suggest a fundamental difference in risk exposure between the two regions. The statistical association between HeV detection and stockhorse type may suggest that husbandry is a more important risk determinant than breed per se. The detection of HeV in horses with neither neurological nor respiratory signs poses a risk management challenge for attending veterinarians and laboratory staff, reinforcing animal health authority recommendations that appropriate risk management strategies be employed for all sick horses, and by anyone handling sick horses or associated biological samples.

Applications of functional nucleic acids in imaging, sensing and drug delivery and investigations of DNAzyme-metal interactions

Functional nucleic acids (FNA), including nucleic acids catalysts (ribozymes and DNAzymes) and ligands (aptamers), have been discovered in nature or isolated in a laboratory through a process called in vitro selection. They are nucleic acids with functions similar to protein enzymes or antibodies. They have been developed into sensors with high sensitivity and selectivity; it is realized by converting the reaction catalyzed by a DNAzyme/ribozyme or the binding event of an aptamer to a fluorescent, colorimetric or electrochemical signal. While a number of studies have been reported for in vitro sensing using DNAzymes or aptamers, there are few reports on in vivo sensing or imaging. MRI is a non-invasive imaging technique; smart MRI contrast agents were synthesized for molecular imaging purposes. However, their rational design remains a challenge due to the difficulty to predict molecular interactions. Chapter 2 focuses on rational design of smart T1-weighted MRI contrast agents with high specificity based on DNAzymes and aptamers. It was realized by changing the molecular weight of the gadolinium conjugated DNA strand with the analytes, which lead to analyte-specific water proton relaxation responses and contrast changes on an MRI image. The designs are general; the high selectivity of FNA was retained. Most FNA-based fluorescent sensors require covalent labeling of fluorophore/quencher to FNAs, which incurrs extra expenses and could interfere the function of FNAs. Chapter 3 describes a new sensor design avoiding the covalent labeling of fluorophore and quencher. The fluorescence of malachite green (MG) was regulated by the presence of adenosine. Conjugate of aptamers of MG and adenosine and a bridge strand were annealed in a solution containing MG. The MG aptamer did not bind MG because of its hybridization to the bridge strand, resulting in low fluorescence signal of MG. The hybridization was weakened in the presence of adenosine, leading to the binding of MG to its aptamer and a fluorescence increase. The sensor has comparable detection limit (20 micromolar) and specificity to its labeled derivatives. Enzymatic activity of most DNAzymes requires metal cations. The research on the metal-DNAzyme interaction is of interest and challenge to scientists because of the lack of structural information. Chapters 4 presents the research on the characterization of the interaction between a Cu2+-dependent DNAzyme and Cu2+. Electron paramagnetic resonance (EPR) and UV-Vis spectroscopy were used to probe the binding of Cu2+ to the DNAzyme; circular dichroism was used to probe the conformational change of the DNAzyme induced by Cu2+. It was proposed that the conformational change by the Cu2+ binding is important for the activity of the DNAzyme. Chapter 5 reports the dependence of the activity of 8-17 DNAzyme on the presence of both Pb2+ and other metal cations including Zn2+, Cd2+ and Mg2+. It was discovered that presence of those metal cations can be cooperative or inhibitive to 8-17 activity. It is hypothesized that the 8-17 DNAzyme had multiple binding sites for metal cations based on the results. Cisplatin is effective killing tumor cells, but with significant side effects, which can be minimized by its targeted delivery. Chapter 6 focuses on the effort to functionalize liposomes encapsulating cisplatin by an aptamer that selectively bind nucleolin, an overexpressed protein by breast cancer cells. The study proved the selective cytotoxicity to breast cancer cells of the aptamer-functionalized liposome.

Carbon-carbon bond formation methods for the preparation of shape-persistent architectures

Carbon-rich, conjugated organic scaffolding is a popular basis for functional materials, especially for electronic and photonic applications. However, synthetic methods for generating these types of materials lack diversity and, in many cases, efficiency; the insistence of investigators focusing on the properties of the end product, rather than the process in which it was created, has led to the current state of the relatively homogeneous synthetic chemistry of functional organic materials. Because of this, there is plenty of room for improvement at the most basic level. Problems endemic to the preparation of carbon-rich scaffolding can, in many cases, be solved with modern advances in synthetic methodology. We seek to apply this synthesis-focused paradigm to solve problems in the preparation of carbon-rich scaffolds. Herein, the development and utilization of three methodologies: iridium-catalyzed arene C-H borylation; zinc- mediated alkynylations; and Lewis acid promoted Mo nitride-alkyne metathesis, are presented as improvements for the preparation of carbon-rich architectures. In addition, X-ray crystallographic analysis of two classes of compounds are presented. First, an analysis of carbazole-containing arylene ethynylene macrocycles showcases the significance of alkyl chain identity on solid-state morphology. Second, a class of rigid zwitterionic metal-organic compounds display an unusual propensity to crystallize in the absence of inversion symmetry. Hirshfeld surface analysis of these crystalline materials demonstrates that subtle intermolecular interactions are responsible for the overall packing motifs in this class of compounds.

Recommended reporting standards for test accuracy studies of infectious diseases of finfish, amphibians, molluscs and crustaceans: the STRADAS-aquatic checklist

Complete and transparent reporting of key elements of diagnostic accuracy studies for infectious diseases in cultured and wild aquatic animals benefits end-users of these tests, enabling the rational design of surveillance programs, the assessment of test results from clinical cases and comparisons of diagnostic test performance. Based on deficiencies in the Standards for Reporting of Diagnostic Accuracy (STARD) guidelines identified in a prior finfish study (Gardner et al. 2014), we adapted the Standards for Reporting of Animal Diagnostic Accuracy Studies—paratuberculosis (STRADAS-paraTB) checklist of 25 reporting items to increase their relevance to finfish, amphibians, molluscs, and crustaceans and provided examples and explanations for each item. The checklist, known as STRADAS-aquatic, was developed and refined by an expert group of 14 transdisciplinary scientists with experience in test evaluation studies using field and experimental samples, in operation of reference laboratories for aquatic animal pathogens, and in development of international aquatic animal health policy. The main changes to the STRADAS-paraTB checklist were to nomenclature related to the species, the addition of guidelines for experimental challenge studies, and the designation of some items as relevant only to experimental studies and ante-mortem tests. We believe that adoption of these guidelines will improve reporting of primary studies of test accuracy for aquatic animal diseases and facilitate assessment of their fitness-for-purpose. Given the importance of diagnostic tests to underpin the Sanitary and Phytosanitary agreement of the World Trade Organization, the principles outlined in this paper should be applied to other World Organisation for Animal Health (OIE)-relevant species.