Rural electrification revisited : the case of Bihar, India

This study describes the dynamic processes of electrification. Some electrified villages have experienced de-electrification, mainly due to technical issues. Some villages were re-electrified through various efforts. Our econometric exercise indicates that small villages in remote locations tend to not be prioritized in the electrification process. It also finds that the cumulative number of ever-electrified villages is higher among villages having a higher ratio of socially advanced classes. However, some of these experienced de-electrification, rendering ambiguous the impact of village social characteristics on electrification.

Sound of lasers (SOL) - An audiovisual approach to semiconductor laser dynamics

We present an educational software addressed to the students of optical communication courses, for a simple visualization of the basic dynamic processes of semiconductor lasers. The graphic interface allows the user to choose the laser and the modulation parameters and it plots the laser power output and instantaneous frequency versus time. Additionally, the optical frequency variations are numerically shifted into the audible frequency range in order to produce a sound wave from the computer loudspeakers. Using the proposed software, the student can simultaneously see and hear how the laser intensity and frequency change, depending on the modulation and device parameters.

Web based interactive educational software introducing semiconductor laser dynamics: Sound Of Lasers (SOL)

In this work, educational software for intuitive understanding of the basic dynamic processes of semiconductor lasers is presented. The proposed tool is addressed to the students of optical communication courses, encouraging self consolidation of the subjects learned in lectures. The semiconductor laser model is based on the well known rate equations for the carrier density, photon density and optical phase. The direct modulation of the laser is considered with input parameters which can be selected by the user. Different options for the waveform, amplitude and frequency of thpoint. Simulation results are plotted for carrier density and output power versus time. Instantaneous frequency variations of the laser output are numerically shifted to the audible frequency range and sent to the computer loudspeakers. This results in an intuitive description of the “chirp” phenomenon due to amplitude-phase coupling, typical of directly modulated semiconductor lasers. In this way, the student can actually listen to the time resolved spectral content of the laser output. By changing the laser parameters and/or the modulation parameters,consequent variation of the laser output can be appreciated in intuitive manner. The proposed educational tool has been previously implemented by the same authors with locally executable software. In the present manuscript, we extend our previous work to a web based platform, offering improved distribution and allowing its use to the wide audience of the web.

Inteligencia artificial para la predicción y control del acabado superficial en procesos de fresado a alta velocidad

El objetivo principal de esta tesis doctoral es profundizar en el análisis y diseño de un sistema inteligente para la predicción y control del acabado superficial en un proceso de fresado a alta velocidad, basado fundamentalmente en clasificadores Bayesianos, con el prop´osito de desarrollar una metodolog´ıa que facilite el diseño de este tipo de sistemas. El sistema, cuyo propósito es posibilitar la predicción y control de la rugosidad superficial, se compone de un modelo aprendido a partir de datos experimentales con redes Bayesianas, que ayudar´a a comprender los procesos dinámicos involucrados en el mecanizado y las interacciones entre las variables relevantes. Dado que las redes neuronales artificiales son modelos ampliamente utilizados en procesos de corte de materiales, también se incluye un modelo para fresado usándolas, donde se introdujo la geometría y la dureza del material como variables novedosas hasta ahora no estudiadas en este contexto. Por lo tanto, una importante contribución en esta tesis son estos dos modelos para la predicción de la rugosidad superficial, que se comparan con respecto a diferentes aspectos: la influencia de las nuevas variables, los indicadores de evaluación del desempeño, interpretabilidad. Uno de los principales problemas en la modelización con clasificadores Bayesianos es la comprensión de las enormes tablas de probabilidad a posteriori producidas. Introducimos un m´etodo de explicación que genera un conjunto de reglas obtenidas de árboles de decisión. Estos árboles son inducidos a partir de un conjunto de datos simulados generados de las probabilidades a posteriori de la variable clase, calculadas con la red Bayesiana aprendida a partir de un conjunto de datos de entrenamiento. Por último, contribuimos en el campo multiobjetivo en el caso de que algunos de los objetivos no se puedan cuantificar en números reales, sino como funciones en intervalo de valores. Esto ocurre a menudo en aplicaciones de aprendizaje automático, especialmente las basadas en clasificación supervisada. En concreto, se extienden las ideas de dominancia y frontera de Pareto a esta situación. Su aplicación a los estudios de predicción de la rugosidad superficial en el caso de maximizar al mismo tiempo la sensibilidad y la especificidad del clasificador inducido de la red Bayesiana, y no solo maximizar la tasa de clasificación correcta. Los intervalos de estos dos objetivos provienen de un m´etodo de estimación honesta de ambos objetivos, como e.g. validación cruzada en k rodajas o bootstrap.---ABSTRACT---The main objective of this PhD Thesis is to go more deeply into the analysis and design of an intelligent system for surface roughness prediction and control in the end-milling machining process, based fundamentally on Bayesian network classifiers, with the aim of developing a methodology that makes easier the design of this type of systems. The system, whose purpose is to make possible the surface roughness prediction and control, consists of a model learnt from experimental data with the aid of Bayesian networks, that will help to understand the dynamic processes involved in the machining and the interactions among the relevant variables. Since artificial neural networks are models widely used in material cutting proceses, we include also an end-milling model using them, where the geometry and hardness of the piecework are introduced as novel variables not studied so far within this context. Thus, an important contribution in this thesis is these two models for surface roughness prediction, that are then compared with respecto to different aspects: influence of the new variables, performance evaluation metrics, interpretability. One of the main problems with Bayesian classifier-based modelling is the understanding of the enormous posterior probabilitiy tables produced. We introduce an explanation method that generates a set of rules obtained from decision trees. Such trees are induced from a simulated data set generated from the posterior probabilities of the class variable, calculated with the Bayesian network learned from a training data set. Finally, we contribute in the multi-objective field in the case that some of the objectives cannot be quantified as real numbers but as interval-valued functions. This often occurs in machine learning applications, especially those based on supervised classification. Specifically, the dominance and Pareto front ideas are extended to this setting. Its application to the surface roughness prediction studies the case of maximizing simultaneously the sensitivity and specificity of the induced Bayesian network classifier, rather than only maximizing the correct classification rate. Intervals in these two objectives come from a honest estimation method of both objectives, like e.g. k-fold cross-validation or bootstrap.

Active materiality : the agency of matter from the phenomenological perspective = Materialidad activa : la operatividad de la materia desde el punto de vista fenomenológico

The role of matter has remained central to the making and the thinking of architecture, yet many attempts to capture its essence have been trapped in a dialectic tension between form and materiality, between material consistency and immaterial modes of perception, between static states and dynamic processes, between the real and the virtual, thus advancing an increasing awareness of the perplexing complexity of the material world. Within that complexity, the notion of agency – emerging from and within ecological, politico-economic and socio-cultural processes – calls for a reconceptualization of matter, and consequently processes of materialisation, offering a new understanding of context and space, approached as a field of dynamic relationships. In this context, cutting across boundaries between architectural discourse and practice as well as across the vast trans-disciplinary territory, this dissertation aims to illustrate a variety of design methodologies that have derived from the relational approach. More specifically, the intention is to offer new insights into spatial epistemologies embedded within the notion of atmosphere – commonly associated with the so-called New Phenomenology – and to reflect upon its implications for architectural production. In what follows, the intended argumentation has a twofold dimension. First, through a scrutiny of the notion of atmosphere, the aim is to explore ways of thinking and shaping reality through relations, thus acknowledging the aforementioned complexity of the material universe disclosed through human and non-human as well as material and immaterial forces. Secondly, despite the fact that concerns for atmospherics have flourished over the last few decades, the objective is to reveal that the conceptual foundations and procedures for the production of atmosphere might be found beneath the surface of contemporary debates. Hence, in order to unfold and illustrate previously advocated assumptions, an archaeological approach is adopted, tracing a particular projective genealogy, one that builds upon an atmospheric awareness. Accordingly, in tracing such an atmospheric awareness the study explores the notoriously ambiguous nature and the twofold dimension of atmosphere – meteorological and aesthetic – and the heterogeneity of meanings embedded in them. In this context, the notion of atmosphere is presented as parallactic. It transgresses the formal and material boundaries of bodies. It calls for a reevaluation of perceptual experience, opening a new gap that exposes the orthodox space-bodyenvironment relationships to questioning. It offers to architecture an expanded domain in which to manifest itself, defining architectural space as a contingent construction and field of engagement, and presenting matter as a locus of production/performance/action. Consequently, it is such an expanded or relational dimension that constitutes the foundation of what in the context of this study is to be referred to as affective tectonics. Namely, a tectonics that represents processual and experiential multiplicity of convergent time and space, body and environment, the material and the immaterial; a tectonics in which matter neither appears as an inert and passive substance, nor is limited to the traditionally regarded tectonic significance or expressive values, but is presented as an active element charged with inherent potential and vitality. By defining such a relational materialism, the intention is to expand the spectrum of material attributes revealing the intrinsic relationships between the physical properties of materials and their performative, transformative and affective capacities, including effects of interference and haptic dynamics – i.e. protocols of transmission and interaction. The expression that encapsulates its essence is: ACTIVE MATERIALITY RESUMEN El significado de la materia ha estado desde siempre ligado al pensamiento y el quehacer arquitectónico. Sin embargo, muchos intentos de capturar su esencia se han visto sumergidos en una tensión dialéctica entre la forma y la materialidad, entre la consistencia material y los modos inmateriales de la percepción, entre los estados estáticos y los procesos dinámicos, entre lo real y lo virtual, revelando una creciente conciencia de la desconcertante complejidad del mundo material. En esta complejidad, la noción de la operatividad o capacidad agencial– que emerge desde y dentro de los procesos ecológicos, políticos y socio-culturales– requiere de una reconceptualización de la materia y los procesos inherentes a la materialización, ofreciendo una nueva visión del contexto y el espacio, entendidos como un campo relacional dinámico. Oscilando entre el discurso arquitectónico y la práctica arquitectónica, y atravesando un extenso territorio trans-disciplinar, el objetivo de la presente tesis es ilustrar la variedad de metodologías proyectuales que emergieron desde este enfoque relacional. Concretamente, la intención es indagar en las epistemologías espaciales vinculadas a la noción de la atmósfera– generalmente asociada a la llamada Nueva Fenomenología–, reflexionando sobre su impacto en la producción arquitectónica. A continuación, el estudio ofrece una doble línea argumental. Primero, a través del análisis crítico de la noción de atmósfera, el objetivo es explorar maneras de pensar y dar forma a la realidad a través de las relaciones, reconociendo la mencionada complejidad del universo material revelado a través de fuerzas humanas y no-humanas, materiales e inmateriales. Segundo, a pesar de que el interés por las atmósferas ha florecido en las últimas décadas, la intención es demostrar que las bases conceptuales y los protocolos proyectuales de la creación de atmósferas se pueden encontrar bajo la superficie de los debates contemporáneos. Para corroborar e ilustrar estas hipótesis se propone una metodología de carácter arqueológico, trazando una particular genealogía de proyectos– la que se basa en una conciencia atmosférica. Asimismo, al definir esta conciencia atmosférica, el estudio explora tanto la naturaleza notoriamente ambigua y la dimensión dual de la atmósfera– meteorológica y estética–, como la heterogeneidad de significados derivados de ellas. En este contexto, la atmósfera se entiende como un concepto detonante, ya que sobrepasa los limites formales y materiales de los cuerpos, llevando a la re-evaluación de la experiencia perceptiva y abriendo a preguntas la ortodoxa relación espacio- cuerpo-ambiente. En consecuencia, la noción de la atmósfera ofrece a la arquitectura una dimensión expandida donde manifestarse, definiendo el espacio como una construcción contingente, performativa y afectiva, y presentando la materia como locus de producción/ actuación/ acción. Es precisamente esta dimensión expandida relacional la que constituye una base para lo que en el contexto del presente estudio se define como una tectónica afectiva. Es decir, una tectónica que representa una multiplicidad procesual y experiencial derivada de la convergencia entre el tiempo y el espacio, el cuerpo y el entorno, lo material y lo inmaterial; una tectónica en la que la materia no aparece como una substancia pasiva e inerte, ni es limitada al significado considerado tradicionalmente constructivo o a sus valores expresivos, sino que se presenta como elemento activo cargado de un potencial y vitalidad inherentes. A través de la definición de este tipo de materialismo afectivo, la intención es expandir el espectro de los atributos materiales, revelando las relaciones intrínsecas entre las propiedades físicas de los materiales y sus capacidades performativas, transformativas y afectivas, incluyendo efectos de interferencias y dinámicas hápticas– o dicho de otro modo, protocolos de transmisión e interacción. Una expresión que encapsula su esencia vendría a ser: MATERIALIDAD ACTIVA

Modified PNA design and synthesis: a novel approach using Molecular Dynamics and Metadynamics

Gli acidi peptido nucleici sono potenti strumenti utilizzati in ambito biotecnologico per colpire DNA o RNA. PNA contenenti basi o backbone modificati sono attualmente studiati per migliorarne le proprietà in ambito biologico. Bersagliare i micro RNA (anti-miR) è particolarmente interessante nell’ottica di future applicazioni terapeutiche, ma strumenti computazionali che aiutino nel design di nuovi PNA anti-miR non sono stati ancora completamente sviluppati. Le proprietà conformazionali del singolo filamento di PNA (non modificato o recante modificazioni in γ) e dei duplex PNA:RNA e i processi di re-annealing e melting sono stati studiati tramite Dinamica Molecolare e Metadinamica. L’approccio computazionale consolidato, assieme a un programma modificato per la generazione delle strutture dei duplex contenenti PNA, è stato utilizzato per il virtual screening di PNA contenenti basi modificate. Sono state inoltre sintetizzate le unità per l’ottenimento del composto più promettente e una funzione idrolitica da legare al monomero finale.

Deterministic mathematical morphology for CAD/CAM

Purpose – The purpose of this paper is to present a new geometric model based on the mathematical morphology paradigm, specialized to provide determinism to the classic morphological operations. The determinism is needed to model dynamic processes that require an order of application, as is the case for designing and manufacturing objects in CAD/CAM environments. Design/methodology/approach – The basic trajectory-based operation is the basis of the proposed morphological specialization. This operation allows the definition of morphological operators that obtain sequentially ordered sets of points from the boundary of the target objects, inexistent determinism in the classical morphological paradigm. From this basic operation, the complete set of morphological operators is redefined, incorporating the concept of boundary and determinism: trajectory-based erosion and dilation, and other morphological filtering operations. Findings – This new morphological framework allows the definition of complex three-dimensional objects, providing arithmetical support to generating machining trajectories, one of the most complex problems currently occurring in CAD/CAM. Originality/value – The model proposes the integration of the processes of design and manufacture, so that it avoids the problems of accuracy and integrity that present other classic geometric models that divide these processes in two phases. Furthermore, the morphological operative is based on points sets, so the geometric data structures and the operations are intrinsically simple and efficient. Another important value that no excessive computational resources are needed, because only the points in the boundary are processed.

A new boundary-based morphological model

Mathematical morphology addresses the problem of describing shapes in an n-dimensional space using the concepts of set theory. A series of standardized morphological operations are defined, and they are applied to the shapes to transform them using another shape called the structuring element. In an industrial environment, the process of manufacturing a piece is based on the manipulation of a primitive object via contact with a tool that transforms the object progressively to obtain the desired design. The analogy with the morphological operation of erosion is obvious. Nevertheless, few references about the relation between the morphological operations and the process of design and manufacturing can be found. The non-deterministic nature of classic mathematical morphology makes it very difficult to adapt their basic operations to the dynamics of concepts such as the ordered trajectory. A new geometric model is presented, inspired by the classic morphological paradigm, which can define objects and apply morphological operations that transform these objects. The model specializes in classic morphological operations, providing them with the determinism inherent in dynamic processes that require an order of application, as is the case for designing and manufacturing objects in professional computer-aided design and manufacturing (CAD/CAM) environments. The operators are boundary-based so that only the points in the frontier are handled. As a consequence, the process is more efficient and more suitable for use in CAD/CAM systems.

The Emergence of a Network of ‘European Embassies’: Increasing Cooperation between EU Delegations and Member State Diplomatic Missions. EU Diplomacy Paper No. 10, December 2014

Five years after the entry into force of the Treaty of Lisbon and at the end of the first mandate of the High Representative of the Union for Foreign Affairs and Security Policy/Vice-President of the European Commission (HR/VP), this analysis provides an in-depth view of the on-going institutional socialisation between Member State Embassies and EU Delegations. Specifically, it focuses on the Member States’ perceptions of the role of EU Delegations. These perceptions can back up or restrain the EU Delegations in fulfilling their mandate. More precisely, the paper examines to what extent the socialisation between EU Delegations and EU Member State Embassies helps the Delegations to fulfil their mandate in bilateral diplomacy. It argues that EU Delegations are still under dynamic processes of institutional socialisation with the Member States’ Embassies which increasingly accept and expect EU Delegations’ actions. The post-Lisbon context of EU Diplomacy is consolidating a primus inter pares role of Delegations being central hubs coordinating and implementing EU policies on the spot.

Dissolved organic carbon concentrations, amino sugar concentrations and highly significantly correlating FT-ICR mass peak magnitudes obtained from solid phase extracted marine dissolved organic matter during POLARSTERN cruise ANT-XXV/1

Dissolved organic matter (DOM) was extracted with solid phase extraction (SPE) from 137 water samples from different climate zones and different depths along an Eastern Atlantic Ocean transect. The extracts were analyzed with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) with electrospray ionization (ESI). D14C analyses were performed on subsamples of the SPE-DOM. In addition, the amount of dissolved organic carbon was determined for all water and SPE-DOM samples as well as the yield of amino sugars for selected samples. Linear correlations were observed between the magnitudes of 43% of the FT-ICR mass peaks and the extract D14C values. Decreasing SPE-DOM D14C values went along with a shift in the molecular composition to higher average masses (m/z) and lower hydrogen/carbon (H/C) ratios. The correlation was used to model the SPE-DOM D14C distribution for all 137 samples. Based on single mass peaks a degradation index was developed to compare the degradation state of marine SPE-DOM samples analyzed with FT-ICR MS. A correlation between D14C, degradation index, DOC values and amino sugar yield supports that SPE-DOM analyzed with FT-ICR MS reflects trends of bulk DOM. A relative mass peak magnitude ratio was used to compare aged SPE-DOM and fresh SPE-DOM regarding single mass peaks. The magnitude ratios show a continuum of different reactivities for the single compounds. Only few of the compounds present in the FT-ICR mass spectra are expected to be highly degraded in the oldest water masses of the Pacific Ocean. All other compounds should persist partly thermohaline circulation. Prokaryotic (bacterial) production, transformation and accumulation of this very stable DOM occurs probably primarily in the upper ocean. This DOM is an important contribution to very old DOM, showing that production and degradation are dynamic processes.

Diversification economies and specialisation efficiencies in a mixed food and coffee smallholder farming system in Papua New Guinea

Smallholder farming systems in Papua New Guinea are characterised by an integrated set of cash cropping and subsistence food cropping activities. In the Highlands provinces, the subsistence food crop sub-system is dominated by sweet potato production. Coffee dominates the cash cropping sub-system, but a limited number of food crops are also grown for cash sale. The dynamics between sub-systems can influence the scope for complementarity between, and technical efficiency of, their operations, especially in light of the seasonality of demand for household labour and management inputs within the farming system. A crucial element of these dynamic processes is diversification into commercial agricultural production, which can influence factor productivity and the efficiency of crop production where smallholders maintain a strong production base in subsistence foods. In this study we use survey data from households engaged in coffee and food crop production in the Benabena district of Eastern Highlands Province to derive technical efficiency indices for each household over two years. A stochastic input distance function approach is used to establish whether diversification economies exist and whether specialisation in coffee, subsistence food or cash food production significantly influences technical efficiency on the sampled smallholdings. Diversification economics are weakly evident between subsistence food production and both coffee and cash food production, but diseconomies of diversification are discerned between coffee and cash food production. A number of factors are tested for their effects on technical efficiency. Significant technical efficiency gains are made from diversification among broad cropping enterprises.

End-to-end modelling of the HEG shock tunnel

We demonstrate an end-to-end computational model of the HEG shock tunnel as a way to extract more precise test flow conditions and as a way of getting predictions of new operating conditions. For a selection of established operating conditions, the L1d program was used to simulate the one-dimensional gas-dynamic processes within the whole of the facility. The program reproduces the compression tube performance reliably and, with the inclusion of a loss factor near the upstream-end of the compression tube, it provides a good estimate of the equilibrium pressure in the shock-reflection region over the set of six standard operating conditions for HEG.

Editorial overview:new protein production tools for structural biology

Full text: The idea of producing proteins from recombinant DNA hatched almost half a century ago. In his PhD thesis, Peter Lobban foresaw the prospect of inserting foreign DNA (from any source, including mammalian cells) into the genome of a λ phage in order to detect and recover protein products from Escherichia coli [ 1 and 2]. Only a few years later, in 1977, Herbert Boyer and his colleagues succeeded in the first ever expression of a peptide-coding gene in E. coli — they produced recombinant somatostatin [ 3] followed shortly after by human insulin. The field has advanced enormously since those early days and today recombinant proteins have become indispensable in advancing research and development in all fields of the life sciences. Structural biology, in particular, has benefitted tremendously from recombinant protein biotechnology, and an overwhelming proportion of the entries in the Protein Data Bank (PDB) are based on heterologously expressed proteins. Nonetheless, synthesizing, purifying and stabilizing recombinant proteins can still be thoroughly challenging. For example, the soluble proteome is organized to a large part into multicomponent complexes (in humans often comprising ten or more subunits), posing critical challenges for recombinant production. A third of all proteins in cells are located in the membrane, and pose special challenges that require a more bespoke approach. Recent advances may now mean that even these most recalcitrant of proteins could become tenable structural biology targets on a more routine basis. In this special issue, we examine progress in key areas that suggests this is indeed the case. Our first contribution examines the importance of understanding quality control in the host cell during recombinant protein production, and pays particular attention to the synthesis of recombinant membrane proteins. A major challenge faced by any host cell factory is the balance it must strike between its own requirements for growth and the fact that its cellular machinery has essentially been hijacked by an expression construct. In this context, Bill and von der Haar examine emerging insights into the role of the dependent pathways of translation and protein folding in defining high-yielding recombinant membrane protein production experiments for the common prokaryotic and eukaryotic expression hosts. Rather than acting as isolated entities, many membrane proteins form complexes to carry out their functions. To understand their biological mechanisms, it is essential to study the molecular structure of the intact membrane protein assemblies. Recombinant production of membrane protein complexes is still a formidable, at times insurmountable, challenge. In these cases, extraction from natural sources is the only option to prepare samples for structural and functional studies. Zorman and co-workers, in our second contribution, provide an overview of recent advances in the production of multi-subunit membrane protein complexes and highlight recent achievements in membrane protein structural research brought about by state-of-the-art near-atomic resolution cryo-electron microscopy techniques. E. coli has been the dominant host cell for recombinant protein production. Nonetheless, eukaryotic expression systems, including yeasts, insect cells and mammalian cells, are increasingly gaining prominence in the field. The yeast species Pichia pastoris, is a well-established recombinant expression system for a number of applications, including the production of a range of different membrane proteins. Byrne reviews high-resolution structures that have been determined using this methylotroph as an expression host. Although it is not yet clear why P. pastoris is suited to producing such a wide range of membrane proteins, its ease of use and the availability of diverse tools that can be readily implemented in standard bioscience laboratories mean that it is likely to become an increasingly popular option in structural biology pipelines. The contribution by Columbus concludes the membrane protein section of this volume. In her overview of post-expression strategies, Columbus surveys the four most common biochemical approaches for the structural investigation of membrane proteins. Limited proteolysis has successfully aided structure determination of membrane proteins in many cases. Deglycosylation of membrane proteins following production and purification analysis has also facilitated membrane protein structure analysis. Moreover, chemical modifications, such as lysine methylation and cysteine alkylation, have proven their worth to facilitate crystallization of membrane proteins, as well as NMR investigations of membrane protein conformational sampling. Together these approaches have greatly facilitated the structure determination of more than 40 membrane proteins to date. It may be an advantage to produce a target protein in mammalian cells, especially if authentic post-translational modifications such as glycosylation are required for proper activity. Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) 293 cell lines have emerged as excellent hosts for heterologous production. The generation of stable cell-lines is often an aspiration for synthesizing proteins expressed in mammalian cells, in particular if high volumetric yields are to be achieved. In his report, Buessow surveys recent structures of proteins produced using stable mammalian cells and summarizes both well-established and novel approaches to facilitate stable cell-line generation for structural biology applications. The ambition of many biologists is to observe a protein's structure in the native environment of the cell itself. Until recently, this seemed to be more of a dream than a reality. Advances in nuclear magnetic resonance (NMR) spectroscopy techniques, however, have now made possible the observation of mechanistic events at the molecular level of protein structure. Smith and colleagues, in an exciting contribution, review emerging ‘in-cell NMR’ techniques that demonstrate the potential to monitor biological activities by NMR in real time in native physiological environments. A current drawback of NMR as a structure determination tool derives from size limitations of the molecule under investigation and the structures of large proteins and their complexes are therefore typically intractable by NMR. A solution to this challenge is the use of selective isotope labeling of the target protein, which results in a marked reduction of the complexity of NMR spectra and allows dynamic processes even in very large proteins and even ribosomes to be investigated. Kerfah and co-workers introduce methyl-specific isotopic labeling as a molecular tool-box, and review its applications to the solution NMR analysis of large proteins. Tyagi and Lemke next examine single-molecule FRET and crosslinking following the co-translational incorporation of non-canonical amino acids (ncAAs); the goal here is to move beyond static snap-shots of proteins and their complexes and to observe them as dynamic entities. The encoding of ncAAs through codon-suppression technology allows biomolecules to be investigated with diverse structural biology methods. In their article, Tyagi and Lemke discuss these approaches and speculate on the design of improved host organisms for ‘integrative structural biology research’. Our volume concludes with two contributions that resolve particular bottlenecks in the protein structure determination pipeline. The contribution by Crepin and co-workers introduces the concept of polyproteins in contemporary structural biology. Polyproteins are widespread in nature. They represent long polypeptide chains in which individual smaller proteins with different biological function are covalently linked together. Highly specific proteases then tailor the polyprotein into its constituent proteins. Many viruses use polyproteins as a means of organizing their proteome. The concept of polyproteins has now been exploited successfully to produce hitherto inaccessible recombinant protein complexes. For instance, by means of a self-processing synthetic polyprotein, the influenza polymerase, a high-value drug target that had remained elusive for decades, has been produced, and its high-resolution structure determined. In the contribution by Desmyter and co-workers, a further, often imposing, bottleneck in high-resolution protein structure determination is addressed: The requirement to form stable three-dimensional crystal lattices that diffract incident X-ray radiation to high resolution. Nanobodies have proven to be uniquely useful as crystallization chaperones, to coax challenging targets into suitable crystal lattices. Desmyter and co-workers review the generation of nanobodies by immunization, and highlight the application of this powerful technology to the crystallography of important protein specimens including G protein-coupled receptors (GPCRs). Recombinant protein production has come a long way since Peter Lobban's hypothesis in the late 1960s, with recombinant proteins now a dominant force in structural biology. The contributions in this volume showcase an impressive array of inventive approaches that are being developed and implemented, ever increasing the scope of recombinant technology to facilitate the determination of elusive protein structures. Powerful new methods from synthetic biology are further accelerating progress. Structure determination is now reaching into the living cell with the ultimate goal of observing functional molecular architectures in action in their native physiological environment. We anticipate that even the most challenging protein assemblies will be tackled by recombinant technology in the near future.

Modelagem dos efeitos da irradiação luminosa no cérebro de camundongos e rastreamento de neurônios durante experimentos de microscopia de fluorescência

The fluorescent proteins are an essential tool in many fields of biology, since they allow us to watch the development of structures and dynamic processes of cells in living tissue, with the aid of fluorescence microscopy. Optogenectics is another technique that is currently widely used in Neuroscience. In general, this technique allows to activate/deactivate neurons with the radiation of certain wavelengths on the cells that have ion channels sensitive to light, at the same time that can be used with fluorescent proteins. This dissertation has two main objectives. Initially, we study the interaction of light radiation and mice brain tissue to be applied in optogenetic experiments. In this step, we model absorption and scattering effects using mice brain tissue characteristics and Kubelka-Munk theory, for specific wavelengths, as a function of light penetration depth (distance) within the tissue. Furthermore, we model temperature variations using the finite element method to solve Pennes’ bioheat equation, with the aid of COMSOL Multiphysics Modeling Software 4.4, where we simulate protocols of light stimulation tipically used in optogenetics. Subsequently, we develop some computational algorithms to reduce the exposure of neuron cells to the light radiation necessary for the visualization of their emitted fluorescence. At this stage, we describe the image processing techniques developed to be used in fluorescence microscopy to reduce the exposure of the brain samples to continuous light, which is responsible for fluorochrome excitation. The developed techniques are able to track, in real time, a region of interest (ROI) and replace the fluorescence emitted by the cells by a virtual mask, as a result of the overlay of the tracked ROI and the fluorescence information previously stored, preserving cell location, independently of the time exposure to fluorescent light. In summary, this dissertation intends to investigate and describe the effects of light radiation in brain tissue, within the context of Optogenetics, in addition to providing a computational tool to be used in fluorescence microscopy experiments to reduce image bleaching and photodamage due to the intense exposure of fluorescent cells to light radiation.

Geochemical and biogeochemical parameters of Black Sea waters and suspended matter

The monograph focuses on the analysis of data addressing the problem of H2S contamination and oxic-anoxic interface in the Black Sea. Regularities of the fine structure of vertical distribution of oxygen, hydrogen sulfide, biogenic elements, organic substances, suspended matter, and metals of the iron-manganese group in the area of contact of aerobic and anaerobic waters have been revealed. Also effects of biochemical, physico-chemical and dynamic processes on their vertical distribution have been examined. Sulfate reduction in seawater and bottom sediments has been studied. Quantitative estimates of H2S fluxes at the water - bottom sediment and O2-H2S interfaces have been done. Features of H2S oxidation have been studied, its budget in the Black Sea has been calculated. Multiyear spatial-temporal variability of the oxic-anoxic interface has been investigated.