Generating Circuit Tests by Exploiting Designed Behavior

This thesis describes two programs for generating tests for digital circuits that exploit several kinds of expert knowledge not used by previous approaches. First, many test generation problems can be solved efficiently using operation relations, a novel representation of circuit behavior that connects internal component operations with directly executable circuit operations. Operation relations can be computed efficiently by searching traces of simulated circuit behavior. Second, experts write test programs rather than test vectors because programs are more readable and compact. Test programs can be constructed automatically by merging program fragments using expert-supplied goal-refinement rules and domain-independent planning techniques

Component unit pricing theory

Building contractors are often commissioned using unit price based contracts. They, nevertheless, often compete on the basis of their overall project bids and yet are paid on the basis of these projectsâ constituent item prices. If a contractor decides these prices by way of applying an uneven mark-up to their estimates of their costs, this is known as unbalanced bidding. This research provides proof and explanation that different item pricing scenarios produce different levels of reward for a contractor, whilst exposing them to different degrees of risk. The theory describes the three identified sources of these rewards as well as provides the first explanation of the risks. It has identified the three types of risk involved and provides a model by which both the rewards as well as these risks can now be measured given any item pricing scenario. The research has included a study of the mainstream microeconomic techniques of Modern Portfolio Theory, Value-at-Risk, as well as Cumulative Prospect Theory that are all suited to making decisions that involve trading-off prospective rewards against risk. These techniques are then incorporated into a model that serves to identify the one item pricing combination that will produce the optimum value of utility as will be best suited to a contractorâs risk profile. The research has included the development of software written especially for this purpose in Java so that this theory could be tested on a hypothetical project. A test produced an improvement of more than 150% on the present-value worth of the contractorâs profit from this project, if they apply this model compared to if they instead price the project in a balanced manner

Extended Duration Simulation and Testing of Cellular and Decellularised Heart Valve Roots

Heart valve disease can affect people of all ages, and can be treated by either valve repair or valve replacement surgery. Currently available replacement heart valves, including mechanical prostheses, bioprostheses, autografts and allografts improve patient survival and quality of life, but have limitations. Key limitations include the risk of immunological reaction and the lack of growth potential and regeneration, which is of particular importance in young patients. To address these limitations, low concentration sodium dodecyl sulphate (SDS) decellularised human aortic, human pulmonary, porcine aortic and porcine pulmonary heart valve roots have been developed. Decellularisation of allografts would potentially reduce the risk of immunological reaction, and the development of a decellularised porcine pulmonary heart valve root would potentially provide an option for right ventricular outflow reconstruction in younger patients who have undergone the Ross Procedure. Before moving to clinical trials, the functional performance of decellularised heart valve roots needs to be pre-clinically assessed appropriately to determine mechanical safety. Whilst there are recommended test methods in place for the in vitro functional performance assessment of newly manufactured and modified surgical replacement heart valves, they need to be optimised or replaced with novel methods suitable for decellularised heart valve roots, due to their time dependent viscoelastic properties. The main aim of this research was to optimise in vitro hydrodynamic and biomechanical performance test methods and develop a novel real time fatigue test method for biological heart valve roots. The secondary aim was to apply the developed in vitro test methods to cellular and decellularised (human and porcine) heart valve roots to evaluate the effect of decellularisation, prior to the decellularised heart valve roots being implanted in patients for clinical trials. In collaboration with NHS Blood and Transplant, Tissue and Eye Services, in vitro biomechanical and hydrodynamic performance of decellularised human aortic and pulmonary heart valve roots was evaluated for the first time in this research. This research determined that the hydrodynamic and functional biomechanical performance of human aortic and pulmonary heart valve roots was not affected by decellularisation treatment. Decellularisation, however, significantly altered some of the directional material properties of pulmonary and aortic heart valve root leaflets. To support clinical translation of decellularised porcine pulmonary heart valve roots, material properties of pulmonary heart valve roots was evaluated following 12 months implantation in sheep. In addition, the effect of the processing steps of cryopreservation and decellularisation on the material properties of porcine pulmonary heart valve roots was investigated. Cryopreservation was shown not to alter the material properties of cellular porcine pulmonary heart valve roots, however, decellularisation did have an effect on the material properties of the porcine pulmonary heart valve root wall. Following 12 months implantation in sheep, the decellularised porcine pulmonary heart valve root wall and leaflets showed a trend for decreasing stiffness and strength; becoming more like the cellular ovine, potentially indicating constructive remodelling. A novel method was developed to investigate the real time fatigue of biological heart valve roots, which was then applied to porcine cellular aortic heart valve roots and porcine decellularised aortic heart valve roots at 120 bpm under physiological cyclic pressures for a maximum of 1.2 million cycles. The results showed no fatigue difference between the cellular and decellularised heart valve roots. Overall, a portfolio of in vitro pre-clinical test methods were developed, optimised and applied to assess the hydrodynamic, biomechanical and fatigue performance of biological heart valve roots including decellularised human and porcine heart valve roots. The in vitro pre-clinical test methods developed in this study will lead to the refinement of in vivo large animal studies and revision of international standards; and the data will help in the development of the next generation of replacement biological heart valve roots, such as decellularised heart valve roots

An integrated approach to testing complex systems

Die steigende Komplexität heutiger Testszenarien für komplexe Systeme erfordert einen ganzheitlichen und offenen Ansatz zur Verwaltung des gesamten Testprozesses. Eine Anwendung klassischer modellbasierter Testansätze, in denen eine präzise und vollständige formale Spezifikation des Systems als Referenz zur automatischen Testfallgenerierung dient, ist in der Praxis nicht möglich.Gründe dafür liegen zum einen im Fehlen einer adäquaten formalen Spezifikation. Komplexe Systeme sind aus verschiedenen Komponenten zusammengesetzt, teils Hardware teils Software und oft auch aus Fremdkomponenten. Dadurch ist es inhärent unrealistisch anzunehmen, dass eine solche formale Spezifikation a priori existiert. Andererseits muss eine ausgereifte Testumgebung die Ausführung von verteilten Testfällen unterstützen, denn die Test-Stimuli und -Beobachtungen können an verschiedenen Teilkomponenten des Systems stattfinden.Diese Arbeit präsentiert einen neuartigen Ansatz für das ganzheitliche Testen komplexer Systeme. Der Ansatz stellt eine 'grobgranulare' Testumgebung zur Verfügung, die mittels einer komponentenbasierten Testfallbeschreibung realisiert ist. Die Basis dafür bildet eine Bibliothek von elementaren, aber intuitiv verständlichen Testfallfragmenten. Die Beziehungen zwischen den Testfallfragmenten sind orthogonal. Dies ermöglicht eine Testbeschreibung und -ausführung, die durch formale Verifikationsmethoden ergänzt wird. Hierdurch können die Testfallbeschreibungsaspekte von Experten des Systems und der verwendeten Testwerkzeuge zu Experten der Systemlogik verschoben werden. Der Ansatz wird durch verschiedene, industrielle Fallstudien in zwei verschiedenen Bereichen illustriert: Computer Telephony Integrations Lösungen und Webbasierte Applikationen. Als Erweiterung des ganzheitlichen Testansatzes wird ein Algorithmus zur a posteriori Generierung approximativer Modelle für komplexe Systeme vorgestellt. Dafür wurde ein bekannter Algorithmus aus dem Maschinellen Lernen an applikationsbedingte Charakteristika angepasst, wie Präfix-Abgeschlossenheit,Input-Determinismus, sowie Unabhängigkeit und Symmetrien zwischen Aktionen. Die resultierenden Modelle können zwar nie exakt sein, in dem Sinne, dass sie das vollständige und korrekte Systemverhalten abbilden. Dennoch können sie von hohem praktischen Nutzen sein, da sie das gesammelte Wissen über das System in einer konsistenten Beschreibungsform repräsentieren.The increasing complexity of today's testing scenarios for complex systems demands an integrated, open, and flexible approach to support the managementof the overall test process. ``Classical'' model-based testing approaches, where a complete and precise formal specification serves as a reference for automatic test generation, are often impractical. Reasons are, on the one hand, the absence of a suitable formal specification. As complex systems are composed of several components, either hardware or software, often pre-built and third party, it is unrealistic to assume that a formal specification exists a priori. On the other hand, a sophisticated test execution environment is needed that can handle distributed test cases. This is because the test actions and observations can take place on different subsystems of the overall system. This thesis presents a novel approach to the integrated testing of complex systems. Our approach offers a coarse grained test environment, realized in terms of a component-based test design on top of a library of elementary but intuitively understandable test case fragments. The relations between the fragments are treated orthogonally, delivering a test design and execution environment enhanced by means of light-weight formal verification methods. In this way we are able to shift the test design issues from total experts of the system and the used test tools to experts of the system's logic only. We illustrate the practical usability of our approach by means of industrial case studies in two different application domains: Computer Telephony Integrated solutions and Web-based applications. As an enhancement of our integrated test approach we provide an algorithm for generating approximate models for complex systems a posteriori. This is done by optimizing a standard machine learning algorithm according to domain-specific structural properties, i.e. properties like prefix-closeness, input-determinism, as well as independency and symmetries of events. The resulting models can never be exact, i.e. reflect the complete and correct behaviour of the considered system. Nevertheless they can be useful in practice, to represent the cumulative knowledge of the system in a consistent description

Testability and redundancy techniques for improved yield and reliability of CMOS VLSI circuits

The research presented in this thesis is concerned with the design of fault-tolerant integrated circuits as a contribution to the design of fault-tolerant systems. The economical manufacture of very large area ICs will necessitate the incorporation of fault-tolerance features which are routinely employed in current high density dynamic random access memories. Furthermore, the growing use of ICs in safety-critical applications and/or hostile environments in addition to the prospect of single-chip systems will mandate the use of fault-tolerance for improved reliability. A fault-tolerant IC must be able to detect and correct all possible faults that may affect its operation. The ability of a chip to detect its own faults is not only necessary for fault-tolerance, but it is also regarded as the ultimate solution to the problem of testing. Off-line periodic testing is selected for this research because it achieves better coverage of physical faults and it requires less extra hardware than on-line error detection techniques. Tests for CMOS stuck-open faults are shown to detect all other faults. Simple test sequence generation procedures for the detection of all faults are derived. The test sequences generated by these procedures produce a trivial output, thereby, greatly simplifying the task of test response analysis. A further advantage of the proposed test generation procedures is that they do not require the enumeration of faults. The implementation of built-in self-test is considered and it is shown that the hardware overhead is comparable to that associated with pseudo-random and pseudo-exhaustive techniques while achieving a much higher fault coverage through-the use of the proposed test generation procedures. The consideration of the problem of testing the test circuitry led to the conclusion that complete test coverage may be achieved if separate chips cooperate in testing each other's untested parts. An alternative approach towards complete test coverage would be to design the test circuitry so that it is as distributed as possible and so that it is tested as it performs its function. Fault correction relies on the provision of spare units and a means of reconfiguring the circuit so that the faulty units are discarded. This raises the question of what is the optimum size of a unit? A mathematical model, linking yield and reliability is therefore developed to answer such a question and also to study the effects of such parameters as the amount of redundancy, the size of the additional circuitry required for testing and reconfiguration, and the effect of periodic testing on reliability. The stringent requirement on the size of the reconfiguration logic is illustrated by the application of the model to a typical example. Another important result concerns the effect of periodic testing on reliability. It is shown that periodic off-line testing can achieve approximately the same level of reliability as on-line testing, even when the time between tests is many hundreds of hours

Vaccination of poultry against highly pathogenic avian influenza - part 1. Available vaccines and vaccination strategies

Several vaccines have been developed against highly pathogenic avian influenza (HPAI), mostly inactivated whole-virus vaccines for chickens. In the EU, one vaccine is authorised in chickens but is not fully efficacious to stop transmission, highlighting the need for vaccines tailored to diverse poultry species and production types. Off-label use of vaccines is possible, but effectiveness varies. Vaccines are usually injectable, a time-consuming process. Mass-application vaccines outside hatcheries remain rare. First vaccination varies from in-ovo to 6 weeks of age. Data about immunity onset and duration in the target species are often unavailable, despite being key for effective planning. Minimising antigenic distance between vaccines and field strains is essential, requiring rapid updates of vaccines to match circulating strains. Generating harmonised vaccine efficacy data showing vaccine ability to reduce transmission is crucial and this ability should be also assessed in field trials. Planning vaccination requires selecting the most adequate vaccine type and vaccination scheme. Emergency protective vaccination is limited to vaccines that are not restricted by species, age or pre-existing vector-immunity, while preventive vaccination should prioritise achieving the highest protection, especially for the most susceptible species in high-risk transmission areas. Model simulations in France, Italy and The Netherlands revealed that (i) duck and turkey farms are more infectious than chickens, (ii) depopulating infected farms only showed limitations in controlling disease spread, while 1-km ring-culling performed better than or similar to emergency preventive ring-vaccination scenarios, although with the highest number of depopulated farms, (iii) preventive vaccination of the most susceptible species in high-risk transmission areas was the best option to minimise the outbreaks' number and duration, (iv) during outbreaks in such areas, emergency protective vaccination in a 3-km radius was more effective than 1- and 10-km radius. Vaccine efficacy should be monitored and complement other surveillance and preventive efforts

Implicación de genes de Pseudomonas simiae PICF7 en endofitismo, control biológico y promoción del crecimiento vegetal

The olive (Olea europaea L.) is the most emblematic tree in the Mediterranean basin, since 98% of its world´s cultivated area is found in this region. The morphological characteristics of this plant, its excellent adaptation to the typical dry and hot summers of this geographical area and the multiple uses of the cultivated and wild olive, make this tree of a huge economic, social and ecological importance. Spain leads the world´s production of olive oil and table olives. However, a/biotic factors such as the presence of pathogens and pests which are difficult to eradicate, erosion and soil loss, and the foreseeable effects of climate change are major threats to the crop. Currently, Verticillium wilt of olive (VWO), a disease caused by the hemibiotrophic fungus Verticillium dahliae Kleb. is considered one of the most devastating diseases affecting olive trees, and constitutes a limiting factor for the olive oil and table olives production. Moreover, recent studies have predicted an increase in temperature and prolonged periods of drought in the Mediterranean basin, which could lead to restrictions in the use of fresh water for irrigation, making it necessary to use saline or reclaimed water. To date, individual control measures employed against VWO have been ineffective. Consequently, integrated control methods combining preventive and palliative measures are considered the best strategy to manage the disease as well as to mitigate the spread of the causal pathogen. Within the integrated management strategy, biological control represents a sustainable and environmentally friendly approach and an alternative to the traditional chemical fungicides. Previous studies have demonstrated that Pseudomonas simiae PICF7 (formerly P. fluorescens PICF7), an indigenous inhabitant of olive roots, is an effective biological control agent (BCA) against VWO. In addition, its ability to promote growth in olive and barley, as well as in the model plant Arabidopsis thaliana, has been demonstrated. So far, strain PICF7 is perhaps the best characterized BCA against VWO. Nevertheless, the mechanisms involved in disease control and its endophytic root colonization ability in several hosts (i.e. olive, barley and wheat) remain largely unknown. Efforts to unravel the keys to the success of strain PICF7 as a BCA have even been made even by studying its interaction with other pathogens. So far, it has only been demonstrated the ability of strain PICF7 to induce systemic resistance to Botrytis cinerea in A. thaliana. Moreover, it is essential that PICF7 and V. dahliae share the same ecological niche (roots, rhizosphere) for the biocontrol of VWO, since an effective suppression of this pathogen has not been observed when both BCA and fungus are spatially separated ("split-root" system). Similarly, no disease control was observed when the phytopathogenic bacterium Pseudomonas savastanoi pv. savastanoi (causal agent of olive knot disease) and PICF7 were inoculated in different parts of the plant (BCA in the roots and pathogen in the stem). However, it was detected a transient reduction of the pathogen population and changes in the morphology of the associated tumors were detected when both microorganisms were applied to the same point (stem). Previous work has also shown that colonization of olive and banana roots by the strain PICF7 results in changes in the expression of genes involved in defensive responses, both locally (roots) and systemically (aerial part). Finally, it has been studied whether bacterial phenotypes traditionally associated with biological control and root colonization (e.g. siderophore production or motility) could be involved in VWO control. Interestingly, the results showed that PICF7 mutants altered in these phenotypes were not compromised in their ability to control the disease. In order to untangle the molecular and genetic bases underlying the effective biocontrol of VWO exerted by strain PICF7, as well as its ability to endophytically colonize olive roots, the present Thesis evaluated the possible contribution of three new phenotypes: 1) biofilm formation, which is traditionally associated with colonization and endophytism; 2) copper tolerance, a metal present in most commercial agricultural antifungals, which could contribute to greater adaptation and survival in soils; and 3) production of phytase, an enzyme involved in phosphorus mobilization and bioavailability, thus promoting plant growth. For this purpose, more than 5.500 Tn5-TcR (Tetracycline-resistance) insertion mutants (transposants) of a pre-existing random transposon insertion mutant bank were screened. Subsequently, for the identification of the Tn5-TcR insertion sites and, consequently, of the altered genes in the selected transposants, nested PCR was used. Finally, the mutants designated as Bfm8 and Bfm9 (unable to form biofilms), Cop1 and Cop33 (altered in copper tolerance) and Phy17 and Phy18 (deficient in phytase activity) were selected to carry out the olive root colonization and VWO biocontrol assays. By using confocal microscopy, it has been shown that all mutants affected in the aforementioned phenotypes were able to superficially colonize olive roots, although analysis of longitudinal sections of these roots revealed that mutants defective in biofilm formation (Bfm8 and Bfm9) did not colonize them internally. None of the studied phenotypes seemed to be involved in the biocontrol ability of PICF7 against VWO, since all selected mutants behaved similar to the wild-type strain PICF7, even though phytase defective mutants (Phy17 and Phy18) were impaired in their ability to antagonize V. dahliae in in vitro assays on one of the culture media used. This fact highlights the importance of contrasting in vitro observations with in planta assays, where the olive-Verticillium-BCA tripartite interaction becomes more complex. This is because there are numerous interactions with and among the remaining plant´s microbiota, as well as with several environmental and pedological factors. Pseudomonas sp. strain PICF6, another effective BCA against VWO, was included in some experiments performed in this Thesis for comparative purposes in order to accomplish some of the proposed objectives. In contrast to strain PICF7, there is virtually no information concerning the control of phytopathogens or the plant growth promotion capabilities of the beneficial strain PICF6, also originating from the olive rhizosphere. To overcome this lack of knowledge, both BCA and the Bfm and Phy mutants of PICF7, were tested in in vitro experiments against several pathogens of great agroeconomic importance in different crops. Strains PICF6 and PICF7, along with all selected mutants derived from the latter, only exhibited the ability to antagonize Verticillium longisporum ELV25, despite the inhibition rates of PICF7 mutants defective in phytase activity being significantly lower. The ability of these rhizobacteria to promote plant growth and colonize oilseed rape roots, host plant of V. longisporum, was also evaluated. Although none of the strains tested promoted the growth of this plant, belonging to the Brassicaceae family, all of them were able to colonize the root surface of oilseed rape seedlings. However, no evidence of endophytic colonization was found. Production of volatile organic compounds (VOC) has been described amongst the possible mechanisms by which beneficial microorganisms are able to inhibit the growth of phytopathogens or promote plant growth. A recent study has shown that strain PICF7 is able to promote plant growth of A. thaliana, not only through direct contact between the BCA and plant roots, but also through the emission of VOC without any physical contact. Nonetheless, it is unknown whether the ability of strains PICF6 and PICF7 to reduce V. dahliae growth could be attributed to this mechanism. In this context, the characterization of PICF6 and PICF7 volatilomes, as well as of the Bfm and Phy mutants, using Gas Chromatography-Mass Spectrometry (GC-MS), allowed the identification of several compounds described in the literature for their antimicrobial potential or plant growth-promoting activity. Conversely, in vitro VOC inhibition assays, using TCVA (Two Clamp VOC Assay) methodology, showed that none of the assayed strains were able to antagonize V. dahliae. Nevertheless, the compounds identified in the volatilome of these rhizobacteria, used individually and at a higher concentration, could represent a new avenue to explore substances with antagonistic capacity against V. dahliae and other relevant pathogens, as well as to evaluate their potential for plant growth promotion. As mentioned above, drought and salinity (abiotic stresses), along with to pathogen infections (biotic stresses), constitute another major threat to olive crop. Several microorganisms have shown high potential to reduce the effects of these stresses in plants. For this reason, the present Thesis has also been focused on studying the role of Pseudomonas sp. PICF6 and P. simiae PICF7 in the alleviation of the symptoms in plants caused by water scarcity of water or by salt accumulation in the soil. Numerous studies have attributed the beneficial effect of certain rhizobacteria on plants subjected to salt or drought stress to the production of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase (ACD). These stresses induce the biosynthesis of ethylene (ET), a phytohormone that, among the numerous processes in which it is involved, causes a negative effect on the plant at high concentrations. ACD catalyzes the conversion of ACC, the immediate precursor of ET, into α-ketobutyrate and ammonia. Therefore, ACD-producing microorganisms contribute to reduce ET levels and, consequently, the stress caused by the accumulation of this phytohormone in the plant. Furthermore, the plant can also develop different defense strategies to mitigate the negative effects caused by this type of stress, including changes in stem water potential (Ψ) and stomatal conductance (gs), or in chlorophyll (Chl), flavonoid (Flv) or proline contents. Experiments were carried out to compare the physiological parameters mentioned above in plants inoculated with an ACD-producing rhizobacteria (Pseudomonas sp. PICF6) and in plants inoculated with a non ACD-producing rhizobacteria (P. simiae PICF7). The results obtained showed that, under the experimental conditions here used, Flv values were influenced by the presence of the bacteria. However, Ψ, gs and the Chl and proline contents remained unchanged upon inoculation of the different strains. Overall, the assayed rhizobacteria, regardless of whether or not they possess ACD activity, did not alleviate the stress produced by drought or salinity in olive plants under the experimental conditions. Finally, as a consequence of the work carried out in this section, it was also demonstrated that the colonization pattern of olive roots by Pseudomonas sp. PICF6 was similar to that observed for PICF7, including its endophytic capacity. In summary, the results obtained in this Thesis allow us to conclude that the three phenotypes of P. simiae PICF7 here examined (phytase activity, biofilm formation and copper tolerance) are not involved in the ability of this BCA to control VWO, although in the particular case of mutants altered in biofilm formation no evidence of endophytic colonization of olive roots was found. Therefore, VWO biocontrol by strain PICF7 does not seem to require inner colonization of this organ. Additinally, strains PICF6 and PICF7 did not show the ability to alleviate symptoms caused by salt or drought stress in olive plants, ruling out the involvement of the ACD activity, at least under the conditions analyzed. Colonization pattern studies for both strains showed that the two olive rhizobacteria superficially colonize the root of this model plant. However, neither PICF6 nor PICF7 promoted the growth of this plant. Yet, the characterization of PICF6 and PICF7 volatilomes allowed for the identification of several VOC with a reported antimicrobial activity. Even though the involvement of VOC in the inhibition of V. dahliae growth in vitro was not observed, the collected information could pave the way for further study of these molecules individually and against different pathogens.El olivo (Olea europaea L.) es el árbol más emblemático de la cuenca mediterránea, concentrándose en esta región el 98% de superficie cultivada de este árbol a nivel mundial. Las características morfológicas de esta planta, su excelente adaptación a los veranos secos y cálidos propios de esta área geográfica, además de los múltiples usos del olivo cultivado y silvestre, hacen que este posea una enorme importancia económica, social y ecológica. España lidera la producción mundial de aceite de oliva y aceituna de mesa. Sin embargo, factores a/bióticos como la presencia de patógenos y plagas de difícil erradicación, la erosión y pérdida de suelo o los previsibles efectos del cambio climático, suponen importantes amenazas para el cultivo. Enfermedades como la verticilosis del olivo (VO), causada por el hongo hemibiotrófico Verticillium dahliae Kleb. y considerada como una de las afecciones más devastadoras del olivo, constituyen un factor limitante para la producción de aceite de oliva y aceitunas de mesa. Asimismo, estudios recientes prevén un aumento de la temperatura y episodios prolongados de sequía en la cuenca mediterránea, lo que podría conducir a restricciones en el uso de agua dulce para irrigación, haciendo necesaria la utilización de agua salada o regenerada y comprometiendo la producción. En lo que respecta a la VO, los diferentes métodos de control de la enfermedad empleados de manera individual han sido ineficaces. Es por ello que el control integrado, combinando medidas preventivas y paliativas de diversa índole, se plantea como la mejor estrategia para manejar la enfermedad y mitigar la dispersión del patógeno que la provoca. Una de las medidas dentro de esta estrategia integrada es el control biológico, herramienta sostenible y respetuosa con el medio ambiente que surge como alternativa a los tradicionales fungicidas químicos. Estudios previos demostraron que Pseudomonas simiae PICF7 (anteriormente P. fluorescens PICF7), bacteria aislada de raíces de olivo (cultivar Picual), es un eficaz agente de control biológico (ACB) frente a la VO. Además, se ha comprobado su capacidad para promover el crecimiento en olivo y cebada, así como en la planta modelo Arabidopsis thaliana. La cepa PICF7 es quizás el ACB mejor caracterizado contra la VO, aunque los mecanismos implicados en el control de la enfermedad y en la capacidad de colonización endofítica de raíces en diversos huéspedes (olivo, cebada y trigo) continúan siendo bastante desconocidos. Las claves del éxito de la cepa PICF7 como ACB se han intentado desvelar incluso estudiando su interacción con otros patógenos. Hasta el momento solo se ha demostrado que induce resistencia sistémica frente a Botrytis cinerea en A. thaliana. Por otra parte, en el caso del control de la VO, es necesario que PICF7 y V. dahliae compartan el mismo nicho ecológico (raíces, rizosfera), pues no se ha observado un control efectivo de este patógeno cuando ACB y hongo se inoculan en raíces separadas en distintos compartimentos (sistema “split-root”). De igual manera, no se observó control cuando la bacteria fitopatógena Pseudomonas savastanoi pv. savastanoi (agente causal de la tuberculosis del olivo) y PICF7 se inoculaban en órganos diferentes de la planta (ACB en las raíces y patógeno en el tallo), pero sí se detectó una reducción transitoria de la población del patógeno y un cambio en la morfología de los tumores cuando ambos microorganismos eran aplicados en el mismo lugar (tallo). Trabajos anteriores también han demostrado que la colonización de las raíces de olivo y platanera por la cepa PICF7 desencadena cambios en la expresión de genes implicados en respuestas defensivas tanto a nivel local (raíces) como sistémico (parte aérea). Finalmente, se ha estudiado si fenotipos bacterianos tradicionalmente asociados con el control biológico y colonización de las raíces (p. ej., producción de sideróforos o motilidad) podían influir en el control de la VO, evidenciándose que mutantes de PICF7 alterados en estos fenotipos no veían mermada su capacidad para controlar la enfermedad. Con el objetivo de desentrañar las bases moleculares y genéticas que subyacen y explican el control efectivo de la VO ejercido por la cepa PICF7, así como su capacidad para colonizar endofíticamente raíces de olivo, en la presente Tesis se evaluó la posible contribución de tres nuevos fenotipos: 1) formación de biopelículas, fenotipo tradicionalmente asociado a colonización y endofitisimo; 2) tolerancia al cobre, metal presente en la mayoría de antifúngicos agrícolas comerciales, que podría contribuir a una mayor adaptación y supervivencia en suelos; y 3) producción de fitasa, enzima que ayuda a incrementar la movilización y disponibilidad del fósforo promoviendo el crecimiento vegetal. Para ello, se llevó a cabo el escrutinio de más de 5.500 mutantes procedentes de una mutateca preexistente generada mediante inserciones al azar del transposón Tn5-TcR, el cual confiere a los transposantes resistencia al antibiótico tetraciclina. Posteriormente, para la identificación de los lugares de inserción de Tn5-TcR y, consecuentemente, de los genes alterados en los transposantes seleccionados, se empleó la técnica de PCR anidada (“nested”-PCR). Finalmente, los mutantes denominados Bfm8 y Bfm9 (incapaces de formar biopelículas), Cop1 y Cop33 (alterados en tolerancia al cobre) y Phy17 y Phy18 (deficientes en actividad fitasa) se seleccionaron para llevar a cabo los ensayos de colonización de la raíz del olivo y biocontrol de la VO. En cuanto a colonización, el uso de microscopía confocal demostró que todos los mutantes afectados en los fenotipos mencionados fueron capaces de colonizar superficialmente las raíces de olivo, aunque el análisis de secciones longitudinales de dichas raíces reveló que los mutantes defectivos en formación de biopelículas (Bfm8 y Bfm9) no las colonizaron internamente. En lo que respecta a capacidad de biocontrol, ningún fenotipo pareció estar involucrado en esta habilidad, ya que todos los mutantes seleccionados controlaron la enfermedad al mismo nivel que la cepa parental PICF7, aun cuando los mutantes defectivos en actividad fitasa (Phy17 y Phy18) vieron mermada su capacidad de antagonizar V. dahliae in vitro en uno de los medios de cultivo empleados. Este hecho pone de manifiesto la crucial importancia de contrastar las observaciones efectuadas in vitro con los ensayos in planta, donde la interacción tripartita olivo-Verticillium-ACB se torna más compleja al entrar en juego las interacciones con y entre el resto de la microbiota de la planta, así como de diversos factores ambientales y pedológicos. La cepa Pseudomonas sp. PICF6, otro ACB eficaz frente a la VO, se incluyó en esta Tesis a efectos comparativos para abordar algunos de los objetivos planteados. Al contrario de lo que ocurre con PICF7, la información sobre esta bacteria beneficiosa originaria de la rizosfera de olivo en lo que respecta al control de fitopatógenos o a la promoción del crecimiento vegetal es prácticamente nula. Para soslayar esta deficiencia, ambos ACB, así como los mutantes Bfm y Phy de PICF7, se enfrentaron en experimentos in vitro a varios patógenos de gran importancia agroeconómica en diferentes cultivos. Las cepas PICF6 y PICF7, así como todos los mutantes derivados de esta última, sólo mostraron capacidad de antagonizar a Verticillium longisporum ELV25, aunque los índices de inhibición de los mutantes PICF7 defectivos en actividad fitasa fueron significativamente inferiores. También se evaluó la capacidad de estas rizobacterias de promover el crecimiento vegetal y de colonizar las raíces de colza, planta huésped de V. longisporum. Aunque ninguna de las cepas ensayadas promovió el crecimiento de esta brasicácea, todas fueron capaces de establecerse superficialmente sobre sus raíces. Sin embargo, no se encontró evidencia de colonización endófita. Entre los posibles mecanismos por los cuales microorganismos beneficiosos son capaces de inhibir el crecimiento de fitopatógenos o promover el crecimiento de las plantas se ha descrito la producción de compuestos orgánicos volátiles (COV). Un estudio reciente ha demostrado que la cepa PICF7 es capaz de promover el crecimiento vegetal de A. thaliana, no solo a través del contacto directo entre el ACB y las raíces de la planta, sino también a través de la emisión de COV sin mediar contacto físico. Sin embargo, se ignora si la capacidad de las cepas PICF6 y PICF7 para reducir el crecimiento de V. dahliae podría deberse a este mecanismo. La caracterización del volatiloma de las rizobacterias PICF6 y PICF7, así como de los mutantes Bfm y Phy de esta última, mediante el uso de Cromatografía de Gases-Espectrometría de Masas (GC-MS), permitió identificar numerosos compuestos descritos en la bibliografía por su potencial actividad antimicrobiana o promotora del crecimiento vegetal. Aun así, los ensayos de inhibición in vitro por COV, en los que se usó la metodología TCVA (de sus siglas en inglés “Two Clamp VOC Assay”), mostraron que ninguna de las cepas analizadas era capaz de antagonizar frente V. dahliae. A pesar de todo, los compuestos identificados en el volatiloma de estas rizobacterias, usados de forma individual y a una concentración mayor, podrían suponer una nueva vía para explorar sustancias con poder antagonista frente a V. dahliae y otros patógenos relevantes, así como para evaluar su potencial en la promoción del crecimiento de plantas. Como se menc

Conservation genomics: speciation of the Neotropical damselfly species Megaloprepus caerulatus – as a model for insect speciation in tropical rainforests

The work presented in this thesis is located at the interface between ecology, evolution and developmental biology. It addresses theories and questions in population biology, phylogeography and speciation as well as methodological approaches for applying Next Generation Sequencing (NGS) data. In the center of this thesis stands the world’s largest extant damselfly, Megaloprepus caerulatus, as a model system for primary rainforests

Creep behaviour of the ternary lead-free solder alloy : Sn-3.8wt.%Ag-0.7wt.%Cu

Electronic equipment is facing the challenge of both miniaturisation and the need to replace lead in interconnections. In service, interconnections generally fail by thermomechanical fatigue, and this behaviour is strongly affected by the creep process. This thesis examines the creep behaviour of a popular lead-free replacement alloy, Sn-3.8wt.%Ag-0.7wt%Cu (Sn-Ag-Cu), in joint and bulk form. Experimental work involved the determination of the creep properties of this alloy at various temperatures, over a range of stresses. Over the regions tested, the creep behaviour is best described by the Norton power law constitutive equation. The stress exponent for bulk Sn-Ag-Cu ranged between 10 and 18 (at 125 to -lOoC respectively) and indicates that a dispersion-strengthened mechanism is dominant in the creep process. The activation energy for creep in the bulk Sn-Ag-Cu is approximately 120kJ/moi and falls in the region similar to that observed for the self-diffusion of tin. In joint form the stress exponent is greater than 10 at high stresses but a change in mechanism is indicated at lower stress where the creep exponent falls to 3. The activation energy for creep in Sn-Ag-Cu when used in joint form is approximately 70kJ/moi and falls in the region similar to that observed for the short-circuit diffusion of tin. Results obtained from the ternary alloy were directly compared to those from Sn- 37wt.%Pb (Sn-Pb) and other prospective lead-free alloys in bulk form. The creep resistance of the ternary lead-free alloy at 75°C is superior to the conventional Sn-Pb alloy and the possible replacement alloys (tin-copper and tin-silver). This superiority is retained when tested at similar homologous temperatures. However, the Sn-3.8Ag- O.7Cu alloy is less ductile but generally possesses strains to failure above 10 percent in comparison to the 25 to 50 percent ductility of Sn-Pb