Effectiveness and Challenges of Three Economic Corridors of the Greater Mekong Sub-region

Since the Greater Mekong Sub-region (GMS) program began in 1992, activities have expanded and flourished. The three economic corridors are composed of the East-West, North-South, and Southern; these are the most important parts of the flagship program. This article presents an evaluation of these economic corridors and their challenges in accordance with the regional distribution of population and income, population pyramids of member countries, and trade relations of member economies.

Algoritmos de distribución de cargas de proceso en computación con membranas

Con el surgir de los problemas irresolubles de forma eficiente en tiempo polinomial en base al dato de entrada, surge la Computación Natural como alternativa a la computación clásica. En esta disciplina se trata de o bien utilizar la naturaleza como base de cómputo o bien, simular su comportamiento para obtener mejores soluciones a los problemas que los encontrados por la computación clásica. Dentro de la computación natural, y como una representación a nivel celular, surge la Computación con Membranas. La primera abstracción de las membranas que se encuentran en las células, da como resultado los P sistemas de transición. Estos sistemas, que podrían ser implementados en medios biológicos o electrónicos, son la base de estudio de esta Tesis. En primer lugar, se estudian las implementaciones que se han realizado, con el fin de centrarse en las implementaciones distribuidas, que son las que pueden aprovechar las características intrínsecas de paralelismo y no determinismo. Tras un correcto estudio del estado actual de las distintas etapas que engloban a la evolución del sistema, se concluye con que las distribuciones que buscan un equilibrio entre las dos etapas (aplicación y comunicación), son las que mejores resultados presentan. Para definir estas distribuciones, es necesario definir completamente el sistema, y cada una de las partes que influyen en su transición. Además de los trabajos de otros investigadores, y junto a ellos, se realizan variaciones a los proxies y arquitecturas de distribución, para tener completamente definidos el comportamiento dinámico de los P sistemas. A partir del conocimiento estático –configuración inicial– del P sistema, se pueden realizar distribuciones de membranas en los procesadores de un clúster para obtener buenos tiempos de evolución, con el fin de que la computación del P sistema sea realizada en el menor tiempo posible. Para realizar estas distribuciones, hay que tener presente las arquitecturas –o forma de conexión– de los procesadores del clúster. La existencia de 4 arquitecturas, hace que el proceso de distribución sea dependiente de la arquitectura a utilizar, y por tanto, aunque con significativas semejanzas, los algoritmos de distribución deben ser realizados también 4 veces. Aunque los propulsores de las arquitecturas han estudiado el tiempo óptimo de cada arquitectura, la inexistencia de distribuciones para estas arquitecturas ha llevado a que en esta Tesis se probaran las 4, hasta que sea posible determinar que en la práctica, ocurre lo mismo que en los estudios teóricos. Para realizar la distribución, no existe ningún algoritmo determinista que consiga una distribución que satisfaga las necesidades de la arquitectura para cualquier P sistema. Por ello, debido a la complejidad de dicho problema, se propone el uso de metaheurísticas de Computación Natural. En primer lugar, se propone utilizar Algoritmos Genéticos, ya que es posible realizar alguna distribución, y basada en la premisa de que con la evolución, los individuos mejoran, con la evolución de dichos algoritmos, las distribuciones también mejorarán obteniéndose tiempos cercanos al óptimo teórico. Para las arquitecturas que preservan la topología arbórea del P sistema, han sido necesarias realizar nuevas representaciones, y nuevos algoritmos de cruzamiento y mutación. A partir de un estudio más detallado de las membranas y las comunicaciones entre procesadores, se ha comprobado que los tiempos totales que se han utilizado para la distribución pueden ser mejorados e individualizados para cada membrana. Así, se han probado los mismos algoritmos, obteniendo otras distribuciones que mejoran los tiempos. De igual forma, se han planteado el uso de Optimización por Enjambres de Partículas y Evolución Gramatical con reescritura de gramáticas (variante de Evolución Gramatical que se presenta en esta Tesis), para resolver el mismo cometido, obteniendo otro tipo de distribuciones, y pudiendo realizar una comparativa de las arquitecturas. Por último, el uso de estimadores para el tiempo de aplicación y comunicación, y las variaciones en la topología de árbol de membranas que pueden producirse de forma no determinista con la evolución del P sistema, hace que se deba de monitorizar el mismo, y en caso necesario, realizar redistribuciones de membranas en procesadores, para seguir obteniendo tiempos de evolución razonables. Se explica, cómo, cuándo y dónde se deben realizar estas modificaciones y redistribuciones; y cómo es posible realizar este recálculo. Abstract Natural Computing is becoming a useful alternative to classical computational models since it its able to solve, in an efficient way, hard problems in polynomial time. This discipline is based on biological behaviour of living organisms, using nature as a basis of computation or simulating nature behaviour to obtain better solutions to problems solved by the classical computational models. Membrane Computing is a sub discipline of Natural Computing in which only the cellular representation and behaviour of nature is taken into account. Transition P Systems are the first abstract representation of membranes belonging to cells. These systems, which can be implemented in biological organisms or in electronic devices, are the main topic studied in this thesis. Implementations developed in this field so far have been studied, just to focus on distributed implementations. Such distributions are really important since they can exploit the intrinsic parallelism and non-determinism behaviour of living cells, only membranes in this case study. After a detailed survey of the current state of the art of membranes evolution and proposed algorithms, this work concludes that best results are obtained using an equal assignment of communication and rules application inside the Transition P System architecture. In order to define such optimal distribution, it is necessary to fully define the system, and each one of the elements that influence in its transition. Some changes have been made in the work of other authors: load distribution architectures, proxies definition, etc., in order to completely define the dynamic behaviour of the Transition P System. Starting from the static representation –initial configuration– of the Transition P System, distributions of membranes in several physical processors of a cluster is algorithmically done in order to get a better performance of evolution so that the computational complexity of the Transition P System is done in less time as possible. To build these distributions, the cluster architecture –or connection links– must be considered. The existence of 4 architectures, makes that the process of distribution depends on the chosen architecture, and therefore, although with significant similarities, the distribution algorithms must be implemented 4 times. Authors who proposed such architectures have studied the optimal time of each one. The non existence of membrane distributions for these architectures has led us to implement a dynamic distribution for the 4. Simulations performed in this work fix with the theoretical studies. There is not any deterministic algorithm that gets a distribution that meets the needs of the architecture for any Transition P System. Therefore, due to the complexity of the problem, the use of meta-heuristics of Natural Computing is proposed. First, Genetic Algorithm heuristic is proposed since it is possible to make a distribution based on the premise that along with evolution the individuals improve, and with the improvement of these individuals, also distributions enhance, obtaining complexity times close to theoretical optimum time. For architectures that preserve the tree topology of the Transition P System, it has been necessary to make new representations of individuals and new algorithms of crossover and mutation operations. From a more detailed study of the membranes and the communications among processors, it has been proof that the total time used for the distribution can be improved and individualized for each membrane. Thus, the same algorithms have been tested, obtaining other distributions that improve the complexity time. In the same way, using Particle Swarm Optimization and Grammatical Evolution by rewriting grammars (Grammatical Evolution variant presented in this thesis), to solve the same distribution task. New types of distributions have been obtained, and a comparison of such genetic and particle architectures has been done. Finally, the use of estimators for the time of rules application and communication, and variations in tree topology of membranes that can occur in a non-deterministic way with evolution of the Transition P System, has been done to monitor the system, and if necessary, perform a membrane redistribution on processors to obtain reasonable evolution time. How, when and where to make these changes and redistributions, and how it can perform this recalculation, is explained.

Distribution of indole-3-acetic acid in Petunia hybrida shoot tip cuttings and relationship between auxin transport, carbohydrate metabolism and adventitious root formation.

To determine the contribution of polar auxin transport (PAT) to auxin accumulation and to adventitious root (AR) formation in the stem base of Petunia hybrida shoot tip cuttings, the level of indole-3-acetic acid (IAA) was monitored in non-treated cuttings and cuttings treated with the auxin transport blocker naphthylphthalamic acid (NPA) and was complemented with precise anatomical studies. The temporal course of carbohydrates, amino acids and activities of controlling enzymes was also investigated. Analysis of initial spatial IAA distribution in the cuttings revealed that approximately 40 and 10% of the total IAA pool was present in the leaves and the stem base as rooting zone, respectively. A negative correlation existed between leaf size and IAA concentration. After excision of cuttings, IAA showed an early increase in the stem base with two peaks at 2 and 24h post excision and, thereafter, a decline to low levels. This was mirrored by the expression pattern of the auxin-responsive GH3 gene. NPA treatment completely suppressed the 24-h peak of IAA and severely inhibited root formation. It also reduced activities of cell wall and vacuolar invertases in the early phase of AR formation and inhibited the rise of activities of glucose-6-phosphate dehydrogenase and phosphofructokinase during later stages. We propose a model in which spontaneous AR formation in Petunia cuttings is dependent on PAT and on the resulting 24-h peak of IAA in the rooting zone, where it induces early cellular events and also stimulates sink establishment. Subsequent root development stimulates glycolysis and the pentosephosphate pathway

Water for small-scale biogas digesters in sub-Saharan Africa

Funded by UK Natural Environment Research Council ESPA project. Grant Number: NE/K010441/1 Afri-Flame

Developmental disorder associated with increased cellular nucleotidase activity

Four unrelated patients are described with a syndrome that included developmental delay, seizures, ataxia, recurrent infections, severe language deficit, and an unusual behavioral phenotype characterized by hyperactivity, short attention span, and poor social interaction. These manifestations appeared within the first few years of life. Each patient displayed abnormalities on EEG. No unusual metabolites were found in plasma or urine, and metabolic testing was normal except for persistent hypouricosuria. Investigation of purine and pyrimidine metabolism in cultured fibroblasts derived from these patients showed normal incorporation of purine bases into nucleotides but decreased incorporation of uridine. De novo synthesis of purines and cellular phosphoribosyl pyrophosphate content also were moderately decreased. The distribution of incorporated purines and pyrimidines did not reveal a pattern suggestive of a deficient enzyme activity. Assay of individual enzymes in fibroblast lysates showed no deficiencies. However, the activity of cytosolic 5′-nucleotidase was elevated 6- to 10-fold. Based on the possibility that the observed increased catabolic activity and decreased pyrimidine salvage might be causing a deficiency of pyrimidine nucleotides, the patients were treated with oral pyrimidine nucleoside or nucleotide compounds. All patients showed remarkable improvement in speech and behavior as well as decreased seizure activity and frequency of infections. A double-blind placebo trial was undertaken to ascertain the efficacy of this supplementation regimen. Upon replacement of the supplements with placebo, all patients showed rapid regression to their pretreatment states. These observations suggest that increased nucleotide catabolism is related to the symptoms of these patients, and that the effects of this increased catabolism are reversed by administration of uridine.

Direct Visualization of the Human Estrogen Receptor α Reveals a Role for Ligand in the Nuclear Distribution of the Receptor

The human estrogen receptor α (ER α) has been tagged at its amino terminus with the S65T variant of the green fluorescent protein (GFP), allowing subcellular trafficking and localization to be observed in living cells by fluorescence microscopy. The tagged receptor, GFP-ER, is functional as a ligand-dependent transcription factor, responds to both agonist and antagonist ligands, and can associate with the nuclear matrix. Its cellular localization was analyzed in four human breast cancer epithelial cell lines, two ER+ (MCF7 and T47D) and two ER− (MDA-MB-231 and MDA-MB-435A), under a variety of ligand conditions. In all cell lines, GFP-ER is observed only in the nucleus in the absence of ligand. Upon the addition of agonist or antagonist ligand, a dramatic redistribution of GFP-ER from a reticular to punctate pattern occurs within the nucleus. In addition, the full antagonist ICI 182780 alters the nucleocytoplasmic compartmentalization of the receptor and causes partial accumulation in the cytoplasm in a process requiring continued protein synthesis. GFP-ER localization varies between cells, despite being cultured and treated in a similar manner. Analysis of the nuclear fluorescence intensity for variation in its frequency distribution helped establish localization patterns characteristic of cell line and ligand. During the course of this study, localization of GFP-ER to the nucleolar region is observed for ER− but not ER+ human breast cancer epithelial cell lines. Finally, our work provides a visual description of the “unoccupied” and ligand-bound receptor and is discussed in the context of the role of ligand in modulating receptor activity.

Differential Distribution of Dynamin Isoforms in Mammalian Cells

Dynamins are 100-kDa GTPases that are essential for clathrin-coated vesicle formation during receptor-mediated endocytosis. To date, three different dynamin genes have been identified, with each gene expressing at least four different alternatively spliced forms. Currently, it is unclear whether these different dynamin gene products perform distinct or redundant cellular functions. Therefore, the focus of this study was to identify additional spliced variants of dynamin from rat tissues and to define the distribution of the dynamin family members in a cultured rat epithelial cell model (Clone 9 cells). After long-distance reverse transcription (RT)-PCR of mRNA from different rat tissues, the full-length cDNAs encoding the different dynamin isoforms were sequenced and revealed four additional spliced variants for dynamin I and nine for dynamin III. Thus, in rat tissues there are a total of at least 25 different mRNAs produced from the three dynamin genes. Subsequently, we generated stably transfected Clone 9 cells expressing full-length cDNAs of six different spliced forms tagged with green fluorescent protein. Confocal or fluorescence microscopy of these transfected cells revealed that many of the dynamin proteins associate with distinct membrane compartments, which include clathrin-coated pits at the plasma membrane and the Golgi apparatus, and several undefined vesicle populations. These results indicate that the dynamin family is more extensive than was originally predicted and suggest that the different dynamin proteins are localized to distinct cytoplasmic or membrane compartments.

Phosphorylated Human Keratinocyte Ornithine Decarboxylase Is Preferentially Associated with Insoluble Cellular Proteins

Ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis, is highly regulated by many trophic stimuli, and changes in its levels and organization correlate with cytoskeletal changes in normal human epidermal keratinocytes (NHEK). NHEK ODC exhibits a filamentous perinuclear/nuclear localization that becomes more diffuse under conditions that alter actin architecture. We have thus asked whether ODC colocalizes with a component of the NHEK cytoskeleton. Confocal immunofluorescence showed that ODC distribution in NHEK was primarily perinuclear; upon disruption of the actin cytoskeleton with cytochalasin D, ODC distribution was diffuse. The ODC distribution in untreated NHEK overlapped with that of keratin in the perinuclear but not cytoplasmic area; after treatment with cytochalasin D, overlap between staining for ODC and for keratin was extensive. No significant overlap with actin and minimal overlap with tubulin filament systems were observed. Subcellular fractionation by sequential homogenizations and centrifugations of NHEK lysates or detergent and salt extractions of NHEK in situ revealed that ODC protein and activity were detectable in both soluble and insoluble fractions, with mechanical disruption causing additional solubilization of ODC activity (three- to sevenfold above controls). Fractionation and ODC immunoprecipitation from [32P]orthophosphate-labeled NHEK lysates showed that a phosphorylated form of ODC was present in the insoluble fractions. Taken together, these data suggest that two pools of ODC exist in NHEK. The first is the previously described soluble pool, and the second is enriched in phospho-ODC and associated with insoluble cellular material that by immunohistochemistry appears to be organized in conjunction with the keratin cytoskeleton.

Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X2-green fluorescent protein receptors

ATP-gated P2X2 receptors are widely expressed in neurons, but the cellular effects of receptor activation are unclear. We engineered functional green fluorescent protein (GFP)-tagged P2X2 receptors and expressed them in embryonic hippocampal neurons, and report an approach to determining functional and total receptor pool sizes in living cells. ATP application to dendrites caused receptor redistribution and the formation of varicose hot spots of higher P2X2-GFP receptor density. Redistribution in dendrites was accompanied by an activation-dependent enhancement of the ATP-evoked current. Substate-specific mutant T18A P2X2-GFP receptors showed no redistribution or activation-dependent enhancement of the ATP-evoked current. Thus fluorescent P2X2-GFP receptors function normally, can be quantified, and reveal the dynamics of P2X2 receptor distribution on the seconds time scale.

Cellular and subcellular localization of the vasopressin- regulated urea transporter in rat kidney.

The renal urea transporter (RUT) is responsible for urea accumulation in the renal medulla, and consequently plays a central role in the urinary concentrating mechanism. To study its cellular and subcellular localization, we prepared affinity-purified, peptide-derived polyclonal antibodies against rat RUT based on the cloned cDNA sequence. Immunoblots using membrane fractions from rat renal inner medulla revealed a solitary 97-kDa band. Immunocytochemistry demonstrated RUT labeling of the apical and subapical regions of inner medullary collecting duct (IMCD) cells, with no labeling of outer medullary or cortical collecting ducts. Immunoelectron microscopy directly demonstrated labeling of the apical plasma membrane and of subapical intracellular vesicles of IMCD cells, but no labeling of the basolateral plasma membrane. Immunoblots demonstrated RUT labeling in both plasma membrane and intracellular vesicle-enriched membrane fractions from inner medulla, a subcellular distribution similar to that of the vasopressin-regulated water channel, aquaporin-2. In the outer medulla, RUT labeling was seen in terminal portions of short-loop descending thin limbs. Aside from IMCD and descending thin limbs, no other structures were labeled in the kidney. These results suggest that: (i) the RUT provides the apical pathway for rapid, vasopressin-regulated urea transport in the IMCD, (ii) collecting duct urea transport may be increased by vasopressin by stimulation of trafficking of RUT-containing vesicles to the apical plasma membrane, and (iii) the rat urea transporter may provide a pathway for urea entry into the descending limbs of short-loop nephrons.

On signal sequence polymorphisms and diseases of distribution.

We report a previously unappreciated property of the signals that target organelle-specific proteins to their subcellular sites of action. Such targeting sequences are shown to be polymorphic. We discovered this polymorphism when we cloned the mitochondrial manganese-containing superoxide dismutase from cell lines of normal individuals and patients with genetic diseases of premature aging and compared their sequences to each other and to those previously reported. The polymorphism consists of a single nucleotide change in the region of the DNA that encodes the signal sequence such that either an alanine or valine is present. Subsequently, eight cell lines were analyzed and all three possible combinations of the two signal sequences were observed. Such signal sequence polymorphisms could result in diseases of distribution, where essential proteins are not properly targeted, thereby leading to absolute or relative deficiencies of critical enzymes within specific cellular compartments. Progeria and related syndromes may be diseases of distribution.

Detection of sub-8-nm movements of kinesin by high-resolution optical-trap microscopy.

Kinesin is a molecular motor that transports organelles along microtubules. This enzyme has two identical 7-nm-long motor domains, which it uses to move between consecutive tubulin binding sites spaced 8 nm apart along a microtubular protofilament. The molecular mechanism of this movement, which remains to be elucidated, may be common to all families of motor proteins. In this study, a high-resolution optical-trap microscope was used to measure directly the magnitude of abrupt displacements produced by a single kinesin molecule transporting a microscopic bead. The distribution of magnitudes reveals that kinesin not only undergoes discrete 8-nm movements, in agreement with previous work [Svoboda, K., Schmidt, C. F., Schnapp, B. J. & Block, S.M. (1993) Nature (London) 365, 721-727], but also frequently exhibits smaller movements of about 5 nm. A possible explanation for these unexpected smaller movements is that kinesin's movement from one dimer to the next along a protofilament involves at least two distinct events in the mechanical cycle.

Contrasting roles for Myc and Mad proteins in cellular growth and differentiation.

The positive effects of Myc on cellular growth and gene expression are antagonized by activities of another member of the Myc superfamily, Mad. Characterization of the mouse homolog of human mad on the structural level revealed that domains shown previously to be required in the human protein for anti-Myc repression, sequence-specific DNA-binding activity, and dimerization with its partner Max are highly conserved. Conservation is also evident on the biological level in that both human and mouse mad can antagonize the ability of c-myc to cooperate with ras in the malignant transformation of cultured cells. An analysis of c-myc and mad gene expression in the developing mouse showed contrasting patterns with respect to tissue distribution and developmental stage. Regional differences in expression were more striking on the cellular level, particularly in the mouse and human gastrointestinal system, wherein c-Myc protein was readily detected in immature proliferating cells at the base of the colonic crypts, while Mad protein distribution was restricted to the postmitotic differentiated cells in the apex of the crypts. An increasing gradient of Mad was also evident in the more differentiated subcorneal layers of the stratified squamous epithelium of the skin. Together, these observations support the view that both downregulation of Myc and accumulation of Mad may be necessary for progression of precursor cells to a growth-arrested, terminally differentiated state.

Chromosomal insertion of foreign (adenovirus type 12, plasmid, or bacteriophage lambda) DNA is associated with enhanced methylation of cellular DNA segments.

Insertion of foreign DNA into an established mammalian genome can extensively alter the patterns of cellular DNA methylation. Adenovirus type 12 (Ad12)-transformed hamster cells, Ad12-induced hamster tumor cells, or hamster cells carrying integrated DNA of bacteriophage lambda were used as model systems. DNA methylation levels were examined by cleaving cellular DNA with Hpa II, Msp I, or Hha I, followed by Southern blot hybridization with 32P-labeled, randomly selected cellular DNA probes. For several, but not all, cellular DNA segments investigated, extensive increases in DNA methylation were found in comparison with the methylation patterns in BHK21 or primary Syrian hamster cells. In eight different Ad12-induced hamster tumors, moderate increases in DNA methylation were seen. Increased methylation of cellular genes was also documented in two hamster cell lines with integrated Ad12 DNA without the Ad12-transformed phenotype, in one cloned BHK21 cell line with integrated plasmid DNA, and in at least three cloned BHK21 cell lines with integrated lambda DNA. By fluorescent in situ hybridization, the cellular hybridization probes were located to different hamster chromosomes. The endogenous intracisternal A particle genomes showed a striking distribution on many hamster chromosomes, frequently on their short arms. When BHK21 hamster cells were abortively infected with Ad12, increases in cellular DNA methylation were not seen. Thus, Ad12 early gene products were not directly involved in increasing cellular DNA methylation. We attribute the alterations in cellular DNA methylation, at least in part, to the insertion of foreign DNA. Can alterations in the methylation profiles of hamster cellular DNA contribute to the generation of the oncogenic phenotype?