Paulo Silva

Regulation by salt of vacuolar H+-ATPase and H+-pyrophosphatase activities and Na+/H+ exchange

Over the last decades several efforts have been carried out to find out the mechanisms of salt homeostasis in plants and, more recently, to identify genes implicated in salt tolerance, with some plants being successfully genetically engineered to improve resistance to salt. It is well established that the efficient exclusion of Na+ excess from the cytoplasm and vacuolar Na+ accumulation are the most important steps towards the maintenance of ion homeostasis inside the cell. Therefore, the vacuole of plant cells plays a pivotal role in the storage of salt. After the identification of the vacuolar Na+/H+ antiporter Nhx1 in Saccharomyces cerevisiae, the first plant Na+/H+ antiporter, AtNHX1, was isolated from Arabidopsis and its overexpression resulted in plants exhibiting increased salt tolerance. Also, the identification of the plasma membrane Na+/H+ exchanger SOS1 and how it is regulated by a protein kinase SOS2 and a calcium binding protein SOS3 were great achievements towards the understanding of plant salt resistance. Both tonoplast and plasma membrane antiporters exclude Na+ from the cytosol driven by the proton-motive force generated by the plasma membrane H+-ATPase and by the vacuolar membrane H+-ATPase and H+-pyrophosphatase and it has been shown that the activity of these proteins respond to salinity. In this review we focus on the transcriptional and post-transcriptional regulation by salt of tonoplast proton pumps and Na+/H+ exchangers and on the signalling pathways involved in salt sensing.

Activity of tonoplast proton pumps and Na+/H+ exchange in potato cell cultures is modulated by salt

The efficient exclusion of excess Na from the cytoplasm and vacuolar Na(+) accumulation are the main mechanisms for the adaptation of plants to salt stress. This is typically carried out by transmembrane transport proteins that exclude Na(+) from the cytosol in exchange for H(+), a secondary transport process which is energy-dependent and driven by the proton-motive force generated by plasma-membrane and tonoplast proton pumps. Tonoplast enriched-vesicles from control and 150 mM NaCl-tolerant calli lines were used as a model system to study the activity of V-H(+)-PPase and V-H(+)-ATPase and the involvement of Na(+) compartmentalization into the vacuole as a mechanism of salt tolerance in Solanum tuberosum. Both ATP- and pyrophosphate (PP(i))-dependent H(+)-transport were higher in tonoplast vesicles from the salt-tolerant line than in vesicles from control cells. Western blotting of tonoplast proteins confirmed that changes in V-H(+)-PPase activity are correlated with increased protein amount. Conversely, immunodetection of the A-subunit of V-H(+)-ATPase revealed that a mechanism of post-translational regulation is probably involved. Na(+)-dependent dissipation of a pre-established pH gradient was used to measure Na(+)/H(+) exchange in tonoplast vesicles. The initial rates of proton efflux followed Michaelis-Menten kinetics and the V(max) of proton dissipation was 2-fold higher in NaCl-tolerant calli when compared to the control. H(+)-coupled exchange was specific for Na(+) and Li(+) and not for K(+). The increase of both the pH gradient across the tonoplast and the Na(+)/H(+) antiport activity in response to salt strongly suggests that Na(+) sequestration into the vacuole contributes to salt tolerance in potato.

Mannitol transport and mannitol dehydrogenase activities are coordinated in olea europaea under salt and osmotic stresses

The intracellular accumulation of organic compatible solutes functioning as osmoprotectants, such as polyols, is an important response mechanism of several plants to drought and salinity. In Olea europaea a mannitol transport system (OeMaT1) was previously characterized as a key player in plant response to salinity. In the present study, heterotrophic sink models, such as olive cell suspensions and fruit tissues, and source leaves were used for analytical, biochemical and molecular studies. The kinetic parameters of mannitol dehydrogenase (MTD) determined in cells growing in mannitol, at 25°C and pH 9.0, were as follows: K(m), 54.5 mM mannitol; and V(max), 0.47 μmol h⁻¹ mg⁻¹ protein. The corresponding cDNA was cloned and named OeMTD1. OeMTD1 expression was correlated with MTD activity, OeMaT1 expression and carrier-mediated mannitol transport in mannitol- and sucrose-grown cells. Furthermore, sucrose-grown cells displayed only residual OeMTD activity, even though high levels of OeMTD1 transcription were observed. There is evidence that OeMTD is regulated at both transcriptional and post-transcriptional levels. MTD activity and OeMTD1 expression were repressed after Na+, K+ and polyethylene glycol (PEG) treatments, in both mannitol- and sucrose-grown cells. In contrast, salt and drought significantly increased mannitol transport activity and OeMaT1 expression. Taken together, these studies support that olive trees cope with salinity and drought by coordinating mannitol transport with intracellular metabolism.

'Galculator': functional prototype of a Galois-connection based proof assistant

Galculator is the name of the prototype of a proof assistant of a special brand: it is solely based on the algebra of Galois connections. When combined with the pointfree transform and tactics such as the indirect equality principle, Galois connections offer a very powerful, generic device to tackle the complexity of proofs in program verification. The paper describes the architecture of the current Galculator prototype, which is implemented in Haskell in order to steer types as much as possible. The prospect of integrating the Galculator with other proof assistants such as e.g. Coq is also discussed

Constraint-aware Schema Transformation

Data schema transformations occur in the context of software evolution, refactoring, and cross-paradigm data mappings. When constraints exist on the initial schema, these need to be transformed into constraints on the target schema. Moreover, when high-level data types are refined to lower level structures, additional target schema constraints must be introduced to balance the loss of structure and preserve semantics. We introduce an algebraic approach to schema transformation that is constraint-aware in the sense that constraints are preserved from source to target schemas and that new constraints are introduced where needed. Our approach is based on refinement theory and point-free program transformation. Data refinements are modeled as rewrite rules on types that carry point-free predicates as constraints. At each rewrite step, the predicate on the reduct is computed from the predicate on the redex. An additional rewrite system on point-free functions is used to normalize the predicates that are built up along rewrite chains. We implemented our rewrite systems in a type-safe way in the functional programming language Haskell. We demonstrate their application to constraint-aware hierarchical-relational mappings.

Strategic term rewriting and its application to a VDM-SL to SQL conversion

We constructed a tool, called VooDooM, which converts datatypes in Vdm-sl into Sql relational data models. The conversion involves transformation of algebraic types to maps and products, and pointer introduction. The conversion is specified as a theory of refinement by calculation. The implementation technology is strategic term rewriting in Haskell, as supported by the Strafunski bundle. Due to these choices of theory and technology, the road from theory to practise is straightforward.

Logic training through algorithmic problem solving

Although much of mathematics is algorithmic in nature, the skills needed to formulate and solve algorithmic problems do not form an integral part of mathematics education. In particular, logic, which is central to algorithm development, is rarely taught explicitly at preuniversity level, under the justification that it is implicit in mathematics and therefore does not need to be taught as an independent topic. This paper argues in the opposite direction, describing a one-week workshop done at the University of Minho, in Portugal, whose goal was to introduce to high-school students calculational principles and techniques of algorithmic problem solving supported by calculational logic. The workshop resorted to recreational problems to convey the principles and to software tools, the Alloy Analyzer and Netlogo, to animate models.

Hospital database workload and fault forecasting

With the growing importance of hospital information systems, databases became indispensable tools for day-to-day tasks in healthcare units. They store important and confidential information about patients clinical status and about the other hospital services. Thus, they must be permanently available, reliable and at high performance. In many healthcare units, fault tolerant systems are used. They ensure the availability, reliability and disaster recovery of data. However, these mechanisms do not allow the prediction or prevention of faults. In this context, it emerges the necessity of developing a fault forecasting system. The objectives of this paper are monitoring database performance to verify the normal workload for the main database of Centro Hospitalar do Porto and adapt a forecasting model used in medicine into the database context. Based on percentiles it was created a scale to represent the severity of situations. It was observe that the critical workload period is the period between 10:00 am and 12:00 am. Moreover, abnormal situations were detected and it was possible to send alerts and to request assistance.

Compiling CAO: From Cryptographic Specifications to C Implementations

We present a compiler for CAO, an imperative DSL for the cryptographic domain. The tool takes high-level cryptographic algorithm specifications and translates them into C implementations through a series of security-aware transformations and optimizations. The compiler back-end is highly configurable, allowing the targeting of very disparate platforms in terms of memory requirements and computing power.

Type checking cryptography implementations

Cryptographic software development is a challenging field: high performance must be achieved, while ensuring correctness and compliance with low-level security policies. CAO is a domain specific language designed to assist development of cryptographic software. An important feature of this language is the design of a novel type system introducing native types such as predefined sized vectors, matrices and bit strings, residue classes modulo an integer, finite fields and finite field extensions, allowing for extensive static validation of source code. We present the formalisation, validation and implementation of this type system.