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Nitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N(2)O. NOR is composed of two subunits: the large one (NorB) is a bundle of 12 transmembrane helices (TMH). It contains a b type heme and a binuclear iron site, which is believed to be the catalytic site, comprising a heme b and a non-hemic iron. The small subunit (NorC) harbors a cytochrome c and is attached to the membrane through a unique TMH. With the aim to perform structural and functional studies of NOR, we have immunized dromedaries with NOR and produced several antibody fragments of the heavy chain (VHHs, also known as nanobodies (TM)). These fragments have been used to develop a faster NOR purification procedure, to proceed to crystallization assays and to analyze the electron transfer of electron donors. BIAcore experiments have revealed that up to three VHHs can bind concomitantly to NOR with affinities in the nanomolar range. This is the first example of the use of VHHs with an integral membrane protein. Our results indicate that VHHs are able to recognize with high affinity distinct epitopes on this class of proteins, and can be used as versatile and valuable tool for purification, functional study and crystallization of integral membrane proteins.

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The characterization of a novel Mo-Fe protein (MorP) associated with a system that responds to Mo in Desulfovibrio alaskensis is reported. Biochemical characterization shows that MorP is a periplasmic homomultimer of high molecular weight (260 +/- 13 kDa) consisting of 16-18 monomers of 15321.1 +/- 0.5 Da. The UV/visible absorption spectrum of the as-isolated protein shows absorption peaks around 280, 320, and 570 nm with extinction coefficients of 18700, 12800, and 5000 M(-1) cm(-1), respectively. Metal content, EXAFS data and DFT calculations support the presence of a Mo-2S-[2Fe-2S]-2S-Mo cluster never reported before. Analysis of the available genomes from Desulfovibrio species shows that the MorP encoding gene is located downstream of a sensor and a regulator gene. This type of gene arrangement, called two component system, is used by the cell to regulate diverse physiological processes in response to changes in environmemtal conditions. Increase of both gene expression and protein production was observed when cells were cultured in the presence of 45 mu M molybdenum. Involvement of this system in Mo tolerance of sulfate reducing bacteria is proposed.

This document describes a work-in-progress development of NGen-VM, a distributed infrastructure that manages execution environments with run-time and programming language support targeting applications developed in the Java programming language, deployed over clusters of many-core computers. For each running application or suite of related applications, a dedicated single-system image will be provided, regardless of the concurrent threads running on a single machine (on several cores) or scattered on different computers. Such system images rely on a single model for concurrency management (Transactional Shared Memory Model), in order fill the gap between the hardware infrastructure of clusters of many-core nodes and the application runtime that is independent from that hardware infrastructure. Interactions between threads in the same tasks will be supported by a Transactional Memory framework that provides the programming language with Atomic and Isolated code regions. Interactions between thread on different machines will also use the Transactional Memory model, but now resorting to a Distributed Shared Memory abstraction.

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An extensive conformational analysis was carried at ab initio and DFT levels of theory on two molecules - methyl 2-azidopropionate (N3CH3CHCOOCH3) and methyl 3-azidopropionate (N3CH2CH2COOCH3). In each case, the lowest energy conformers were characterized and the energy barriers between them were estimated. Ionization energies and vibrational frequencies were also computed, in order to support future spectroscopic studies with ultraviolet photoelectron spectroscopy (UVPES) and matrix isolation infrared spectroscopy (Matrix Isolation FTIR).

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Transactional memory is a new trend in concurrency control that was boosted by the advent of multi-core processors and the near to come many-core processors. It promises the performance of finer grain with the simplicity of coarse grain threading. However, there is a clear absence of software development tools oriented to the transactional memory programming model, which is confirmed by the very small number of related scientific works published until now. This paper describes ongoing work. We propose a very low overhead monitoring framework, developed specifically for monitoring TM computations, that collects the transactional events into a single log file, sorted in a global order. This framework is then used by a visualization tool to display different types of charts from two categories: statistical charts and thread-time space diagrams. These last diagrams are interactive, allowing to identify conflicting transactions. We use the visualization tool to analyse the behavior of two different, but similar, testing applications, illustrating how it can be used to better understand the behavior of these transactional memory applications.

Software Transactional Memory is a concurrency control technique gaining increasing popularity, as it provides high-level concurrency control constructs and eases the development of highly multi-threaded applications. But this easiness comes at the expense of restricting the operations that can be executed within a memory transaction, and operations such as terminal and file I/O are either not allowed or incur in serious performance penalties. Database I/O is another example of operations that usually are not allowed within a memory transaction. This paper proposes to combine memory and database transactions in a single unified model, benefiting from the ACID properties of the database transactions and from the speed of main memory data processing. The new unified model covers, without differentiating, both memory and database operations. Thus, the users are allowed to freely intertwine memory and database accesses within the same transaction, knowing that the memory and database contents will always remain consistent and that the transaction will atomically abort or commit the operations in both memory and database. This approach allows to increase the granularity of the in-memory atomic actions and hence, simplifies the reasoning about them.

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Nitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N2O. NOR is composed of two subunits: the large one (NorB) is a bundle of 12 transmembrane helices (TMH). It contains a b type heme and a binuclear iron site, which is believed to be the catalytic site, comprising a heme b and a non-hemic iron. The small subunit (NorC) harbors a cytochrome c and is attached to the membrane through a unique TMH. With the aim to perform structural and functional studies of NOR, we have immunized dromedaries with NOR and produced several antibody fragments of the heavy chain (VHHs, also known as nanobodies (TM)). These fragments have been used to develop a faster NOR purification procedure, to proceed to crystallization assays and to analyze the electron transfer of electron donors. BIAcore experiments have revealed that up to three VHHs can bind concomitantly to NOR with affinities in the nanomolar range. This is the first example of the use of VHHs with an integral membrane protein. Our results indicate that VHHs are able to recognize with high affinity distinct epitopes on this class of proteins, and can be used as versatile and valuable tool for purification, functional study and crystallization of integral membrane proteins.

The aim of this study was to evaluate the possibility of preparing dexamethasone-loaded starch-based porous matrices in a one-step process. Supercritical phase inversion technique was used to prepare composite scaffolds of dexamethasone and a polymeric blend of starch and poly(l-lactic acid) (SPLA) for tissue engineering purposes. Dexamethasone is used in osteogenic media to direct the differentiation of stem cells towards the osteogenic lineage. Samples with different drug concentrations (5-15 wt.{%} polymer) were prepared at 200 bar and 55 °C. The presence of dexamethasone did not affect the porosity or interconnectivity of the polymeric matrices. Water uptake and degradation studies were also performed on SPLA scaffolds. We conclude that SPLA matrices prepared by supercritical phase inversion have a swelling degree of nearly 90{%} and the material presents a weight loss of ∼25{%} after 21 days in solution. Furthermore, in vitro drug release studies were carried out and the results show that a sustained release of dexamethasone was achieved over 21 days. The fitting of the power law to the experimental data demonstrated that drug release is governed by an anomalous transport, i.e., both the drug diffusion and the swelling of the matrix influence the release of dexamethasone out of the scaffold. The kinetic constant was also determined. This study reports the feasibility of using supercritical fluid technology to process in one step a porous matrix loaded with a pharmaceutical agent for tissue engineering purposes. © 2009 Acta Materialia Inc.

Supercritical fluid technology has proven to be useful for many pharmaceutical applications and is now emerging as an alternative to conventional processes for the preparation of 3D structures and injectable particles suitable to be used in regenerative medicine. A current overview of the basic principles underlying supercritical fluid technology, the state of the art and future potential of this technology are presented.

Supercritical fluid impregnation was tested to prepare a new scaffold loaded with a bioactive compound. Dexamethasone is used in osteogenic media to direct the differentiation of stem cells towards the osteogenic lineage. Dexamethasone was impregnated in chitosan scaffolds at different operating conditions, in order to optimize the impregnation process. Pressure and temperature affect the carbon dioxide density and influence the swelling of the polymer and the drug solubility in the fluid phase, therefore these are two important parameters that were studied in this work. Chitosan sponges prepared by freeze drying were impregnated with the active compound at pressures from 8.0 up to 14.0 MPa and temperatures from 35 up to 55 °C. The effect of the impregnation contact time (3 h and 6 h) was also evaluated. From the experiments performed we can conclude that the yield of impregnation is lower when increasing pressure and temperature. The contact time will mainly influence the amount of drug impregnated in the scaffold and for higher contact times the impregnation yield is also higher. Scanning electron microscopy shows particles of dexamethasone in the bulk of the scaffold, which confirms the feasibility of the supercritical fluid impregnation technology for the preparation of delivery devices. The loading capacity of the scaffolds was determined by spectroscopic analysis and the highest loading was achieved for the experiments performed at 8.0 MPa and 35 °C. Furthermore, in vitro drug release studies were carried out and the results show that dexamethasone was sustainably released. Supercritical fluid impregnation proved to be feasible for the preparation of a drug delivery system for bone tissue engineering purposes. © 2008 Elsevier Ltd. All rights reserved.