CHARACTERIZATION OF SELECTIVE INHIBITION OF ADENYLYL CYCLASE ACTIVITY BY SMALL MOLECULE INHIBITOR NKY80

The nine membrane-bound isoforms of adenylyl cyclase (AC), via synthesis of the signaling molecule cyclic AMP (cAMP), are involved in many isoform specific physiological functions. Decreasing AC5 activity has been shown to have potential therapeutic benefit, including reduced stress on the heart, pain relief, and attenuation of morphine dependence and withdrawal behaviors. However, AC structure is well conserved, and there are currently no isoform selective AC inhibitors in clinical use. P-site inhibitors inhibit AC directly at the catalytic site, but with an uncompetitive or noncompetitive mechanism. Due to this mechanism and nanomolar potency in cell-free systems, attempts at ligand-based drug design of novel AC inhibitors frequently use P-site inhibitors as a starting template. One small molecule inhibitor designed through this process, NKY80, is described as an AC5 selective inhibitor with low micromolar potency in vitro. P-site inhibitors reveal important ligand binding “pockets” in the AC catalytic site, but specific interactions that give NKY80 selectivity are unclear. Identifying and characterizing unique interactions between NKY80 and AC isoforms would significantly aid the development of isoform selective AC inhibitors. I hypothesized that NKY80’s selective inhibition is conferred by AC isoform specific interactions with the compound within the catalytic site. A structure-based virtual screen of the AC catalytic site was used to identify novel small molecule AC inhibitors. Identified novel inhibitors are isoform selective, supporting the catalytic site as a region capable of more potent isoform selective inhibition. Although NKY80 is touted commercially as an AC5 selective inhibitor, its characterization suggests strong inhibition of both AC5 and the closely related AC6. NKY80 was also virtually docked to AC to determine how NKY80 binds to the catalytic site. My results show a difference between NKY80 binding and the conformation of classic P-site inhibitors. The selectivity and notable differences in NKY80 binding to the AC catalytic site suggest a catalytic subregion more flexible in AC5 and AC6 that can be targeted by selective small molecule inhibitors.

NMR structural analysis of cardiac troponin C: Monitoring conformational changes induced by binding calcium, troponin I, and calcium -sensitizing drugs

Contraction of cardiac muscle is regulated through the Ca2+ dependent protein-protein interactions of the troponin complex (Tn). The critical role cardiac troponin C (cTnC) plays as the Ca2+ receptor in this complex makes it an attractive target for positive inotropic compounds. In this study, the ten Met methyl groups in cTnC, [98% 13C ϵ]-Met cTnC, are used as structural markers to monitor conformational changes in cTnC and identify sites of interaction between cTnC and cardiac troponin I (cTnI) responsible for the Ca2+ dependent interactions. In addition the structural consequences that a number of Ca2+-sensitizing compounds have on free cTnC and the cTnC·cTnI complex were characterized. Using heteronuclear NMR experiments and monitoring chemical shift changes in the ten Met methyl 1H-13C correlations in 3Ca2+ cTnC when bound to cTnI revealed an anti-parallel arrangement for the two proteins such that the N-domain of cTnI interacts with the C-domain of cTnC. The large chemical shifts in Mets-81, -120, and -157 identified points of contact between the proteins that include the C-domain hydrophobic surface in cTnC and the A, B, and D helical interface located in the regulatory N-domain of cTnC. TnI association [cTnI(33–80), cTnI(86–211), or cTnI(33–211)] was found also to dramatically reduce flexibility in the D/E central linker of cTnC as monitored by line broadening in the Met 1H- 13C correlations of cTnC induced by a nitroxide spin label, MTSSL, covalently attached to cTnC at Cys 84. TnI association resulted in an extended cTnC that is unlike the compact structure observed for free cTnC. The Met 1H-13C correlations also allowed the binding characteristics of bepridil, TFP, levosimendan, and EMD 57033 to the apo, 2Ca2+, and Ca2+ saturated forms of cTnC to be determined. In addition, the location of drug binding on the 3Ca2+cTnC·cTnI complex was identified for bepridil and TFP. Use of a novel spin-labeled phenothiazine, and detection of isotope filtered NOEs, allowed identification of drug binding sites in the shallow hydrophobic cup in the C-terminal domain, and on two hydrophobic surfaces on N-regulatory domain in free 3Ca2+ cTnC. In contrast, only one N-domain drug binding site exists in 3Ca2+ cTnC·cTnI complex. The methyl groups of Met 45, 60 and 80, which are grouped in a hydrophobic patch near site II in cTnC, showed the greatest change upon titration with bepridil or TFP, suggesting that this is a critical site of drug binding in both free cTnC and when associated with cTnI. The strongest NOEs were seen for Met-60 and -80, which are located on helices C and D, respectively, of Ca2+ binding site II. These results support the conclusion that the small hydrophobic patch which includes Met-45, -60, and -80 constitutes a drug binding site, and that binding drugs to this site will lead to an increase in Ca2+ binding affinity of site II while preserving maximal cTnC activity. Thus, the subregion in cTnC makes a likely target against which to design new and selective Ca2+-sensitizing compounds. ^

Ground beetle (Carabidae) abundance data from north-eastern Australian rainforest, between June 2008 - January 2010, using pitfall traps

Understanding how the environment influences patterns of diversity is vital for effective conservation management, especially in a changing global climate. While assemblage structure and species richness patterns are often correlated with current environmental factors, historical influences may also be considerable, especially for taxa with poor dispersal abilities. Mountain-top regions throughout tropical rainforests can act as important refugia for taxa characterised by low dispersal capacities such as flightless ground beetles (Carabidae), an ecologically significant predatory group. We surveyed flightless ground beetles along elevational gradients in five different subregions within the Australian Wet Tropics World Heritage Area to investigate (1) whether the diversity and composition of flightless ground beetles are elevationally stratified, and, if so, (2) what environmental factors (other than elevation per se) are associated with these patterns. Generalised linear models and model averaging techniques were used to relate patterns of diversity to environmental factors. Unlike most taxonomic groups, flightless ground beetles increased in species richness and abundance with elevation. Additionally, each subregion consisted of distinct assemblages containing a high level of regional endemic species. Species richness was most strongly positively associated with the historical climatic conditions and negatively associated with severity of recent disturbance (treefalls) and current climatic conditions. Assemblage composition was associated with latitude and current and historical climatic conditions. Our results suggest that distributional patterns of flightless ground beetles are not only likely to be associated with factors that change with elevation (current climatic conditions), but also factors that are independent of elevation (recent disturbance and historical climatic conditions). Variation in historical vegetation stability explained both species richness and assemblage composition patterns, probably reflecting the significance of upland refugia at a geographic time scale. These findings are important for conservation management as upland habitats are under threat from climate change.

Estimación de la evapotranspiración real regional a partir de datos satelitales

El objetivo del presente trabajo fue determinar la Evapotranspiración real (ETR) a nivel regional utilizando la información del satélite meteorológico NOAA-AVHRR y comparar los resultados obtenidos con los calculados a partir de un modelo de simulación de balance hídrico. Para la estimación de la ETR se analizaron 30 imágenes que abarcan el oasis Norte de Mendoza. Con la información de los canales C1 (Visible) y C2 (IRC) se obtuvo el índice verde normalizado (NDVI), a través del cual se siguió la evolución anual de la vegetación y con la correspondiente al Infrarrojo térmico (C4 y C5) se calculó la Temperatura de superficie (Ts) por el método Split - Windows Luego se vinculó la Ts calculada por teledetección con la temperatura del aire (Ta), para finalmente calcular la suma acumulada de las diferencias entre Ts y Ta, conocida como SDD (stress degree day) que permite estimar globalmente las características de stress hídrico a nivel regional. Conociendo (Ts-Ta) se estimó la ETR a partir de la radiación neta y de los coeficientes A y B que se estimaron según las características de la cobertura vegetal, aplicando una relación simplificada a partir del balance de energía, desarrollado por Jackson (1977) y Seguin (1983) según la ecuación: ETR = Rn + A -B ( Ts - Ta ) Posteriormente, se incluyó en los cálculos los valores de Emisividad y se hizo variar el coeficiente B de acuerdo a la ocupación del suelo en cada uno de los polígonos en que fue dividida el área de estudio. En la etapa final se compararon estadísticamente los datos de ETR estimados por los distintos métodos con los simulados por el modelo y se obtuvo como conclusión final que: la estimación de la ETR a nivel regional mediante datos satelitales, se adapta muy bien a la mayoría de los casos y es sencilla de calcular, por lo que la metodología desarrollada es fácilmente extrapolable a otros oasis de la región.

Fortalecimiento de la información sobre el aborto legal en mujeres de organizaciones comunitarias y proveedores de salud del primer nivel de atención en la provincia de Sullana, Perú

El objetivo del proyecto es incrementar la información sobre el aborto terapéutico y el protocolo de atención aprobado, en los proveedores de salud. Y fortalecer la exigibilidad del derecho al acceso al aborto legal en mujeres de organizaciones comunitarias.

Redes de innovación en la difusión de las variedades de trigo pan : El caso del sudoeste pampeano

Ante el interés desde distintos ámbitos de mejorar la calidad de los trigos, nos propusimos estudiar la dinámica local de uno de los componentes que la determinan. Con ese fin, efectuamos un trabajo de investigación orientado a caracterizar los mecanismos locales de renovación de las variedades de trigo pan y establecer su articulación en redes con dimensiones territoriales más vastas. Este análisis nos conduce a observar las modalidades de interacción entre actores inscriptos en un territorio dado (comercios locales, asesores técnicos, productores), y las articulaciones con actores frecuentemente distantes (en el sentido geográfico), tales como los centros de mejoramiento genético. Nuestro trabajo de investigación se basa en los aportes teóricos de la 'sociología de la red' o 'de la innovación', efectuados fundamentalmente por los sociólogos Michel Callon y Bruno Latour. El área de trabajo elegida comprende el sudoeste de la provincia de Buenos Aires. Considerando las regiones agro-ecológicas en que tradicionalmente se divide la producción de trigo en el país, este área esta incluida dentro de la subregion V sur. Esta subregion triguera es la más importante del país. La superficie sembrada y la producción representa, en promedio, el 20 del total del país

Redes de innovación en la difusión de las variedades de trigo pan : El caso del sudoeste pampeano

Ante el interés desde distintos ámbitos de mejorar la calidad de los trigos, nos propusimos estudiar la dinámica local de uno de los componentes que la determinan. Con ese fin, efectuamos un trabajo de investigación orientado a caracterizar los mecanismos locales de renovación de las variedades de trigo pan y establecer su articulación en redes con dimensiones territoriales más vastas. Este análisis nos conduce a observar las modalidades de interacción entre actores inscriptos en un territorio dado (comercios locales, asesores técnicos, productores), y las articulaciones con actores frecuentemente distantes (en el sentido geográfico), tales como los centros de mejoramiento genético. Nuestro trabajo de investigación se basa en los aportes teóricos de la 'sociología de la red' o 'de la innovación', efectuados fundamentalmente por los sociólogos Michel Callon y Bruno Latour. El área de trabajo elegida comprende el sudoeste de la provincia de Buenos Aires. Considerando las regiones agro-ecológicas en que tradicionalmente se divide la producción de trigo en el país, este área esta incluida dentro de la subregion V sur. Esta subregion triguera es la más importante del país. La superficie sembrada y la producción representa, en promedio, el 20 del total del país

Redes de innovación en la difusión de las variedades de trigo pan : El caso del sudoeste pampeano

Ante el interés desde distintos ámbitos de mejorar la calidad de los trigos, nos propusimos estudiar la dinámica local de uno de los componentes que la determinan. Con ese fin, efectuamos un trabajo de investigación orientado a caracterizar los mecanismos locales de renovación de las variedades de trigo pan y establecer su articulación en redes con dimensiones territoriales más vastas. Este análisis nos conduce a observar las modalidades de interacción entre actores inscriptos en un territorio dado (comercios locales, asesores técnicos, productores), y las articulaciones con actores frecuentemente distantes (en el sentido geográfico), tales como los centros de mejoramiento genético. Nuestro trabajo de investigación se basa en los aportes teóricos de la 'sociología de la red' o 'de la innovación', efectuados fundamentalmente por los sociólogos Michel Callon y Bruno Latour. El área de trabajo elegida comprende el sudoeste de la provincia de Buenos Aires. Considerando las regiones agro-ecológicas en que tradicionalmente se divide la producción de trigo en el país, este área esta incluida dentro de la subregion V sur. Esta subregion triguera es la más importante del país. La superficie sembrada y la producción representa, en promedio, el 20 del total del país

Thailand's Economic Cooperation with Neighboring Countries and Its Effects on Economic Development within Thailand

Thailand's economic cooperation with neighboring countries, including not only trade and investment but also economic assistance, is tied inseparably to regional development within Thailand. Assistance to develop infrastructure along economic corridors, for example, promotes Thai regional development. This study examines the trade and investment relationships between Thailand and its neighboring countries, as well as related economic policies of Thailand. The study also examines the type of economic assistance being extended, and the resulting regional development taking place. And lastly, the study considers policies for further cooperation by Thailand and the implications this has for Japanese economic cooperation.

Chapter 1 Economic Integration and Industry Location: Theories, Empirical Results, and Implications for CLMV

Trade affects the internal location of industry in two ways: it induces firms to specialize and it expands the set of markets that firms serve. If there are industry-specific external economies, firms in related industries will spatially agglomerate (Hanson 1996a). In the context of economic integration, diminished barriers to trade affect industry location particularly in less developed countries. As described below, regional agreements in North America and Europe have caused frontier regions to expand. These regions, which include border regions and port cities, have advantages over internal regions in terms of access to foreign markets. Since trade liberalization induces many firms in developing countries to participate in production networks and to specialize in labor-intensive activities such as assembling and processing of foreign-made components, their inputs as well as final products need to be carried across borders. Therefore, the best industry location, one that minimizes transport costs, is likely to shift to frontier regions. In East Asia, China has developed rapidly since it opened up to international trade. Simultaneously, a large amount of foreign direct investment (FDI) has been attracted and industry agglomerations have been formed in coastal regions, that is, frontier regions linked to the global market by sea, leaving many internal regions behind. Similarly, Cambodia, Laos, Myanmar, and Vietnam (CLMV) have joined AFTA and/or the WTO and liberalized international trade since the 1990s. Moreover, transport infrastructures such as the East-West Economic Corridor, the Southern Economic Corridor, and the North-South Economic Corridor have been built and narrowed economic distances in the Greater Mekong Subregion (GMS). As a result, frontier regions are likely to increase their location advantages and lure labor-intensive operations from neighboring countries. It is expected that, as has happened in North America and Europe, economic integration in East Asia will significantly affect internal geography in CLMV. In this study, I first review theories relevant to economic integration and industry location within a country. In particular, emphasis is placed on the new economic geography (NEG). Secondly, empirical results for North America and Europe are surveyed since they have preceded East Asia in regional integration and a substantial number of studies have been conducted on these regions. The final section summarizes and discusses implications for internal geography in CLMV.

Ensemble transcript interaction networks: A case study on Alzheimer's disease

Systems biology techniques are a topic of recent interest within the neurological field. Computational intelligence (CI) addresses this holistic perspective by means of consensus or ensemble techniques ultimately capable of uncovering new and relevant findings. In this paper, we propose the application of a CI approach based on ensemble Bayesian network classifiers and multivariate feature subset selection to induce probabilistic dependences that could match or unveil biological relationships. The research focuses on the analysis of high-throughput Alzheimer's disease (AD) transcript profiling. The analysis is conducted from two perspectives. First, we compare the expression profiles of hippocampus subregion entorhinal cortex (EC) samples of AD patients and controls. Second, we use the ensemble approach to study four types of samples: EC and dentate gyrus (DG) samples from both patients and controls. Results disclose transcript interaction networks with remarkable structures and genes not directly related to AD by previous studies. The ensemble is able to identify a variety of transcripts that play key roles in other neurological pathologies. Classical statistical assessment by means of non-parametric tests confirms the relevance of the majority of the transcripts. The ensemble approach pinpoints key metabolic mechanisms that could lead to new findings in the pathogenesis and development of AD

Proyecto de explotación de cantera de calizas del Páramo

Este proyecto tiene por objeto el estudio de la viabilidad, tanto económica como técnica, de la explotación de una cantera de calizas pertenecientes a la formación geológica Calizas del Páramo. El yacimiento explotado es de tipo superficial, con una profundidad media entre 30 y 35 m. La concesión de explotación se localiza en la subcomarca de la Alcarria de Alcalá, al sureste de la Comunidad de Madrid. La caliza extraída es utilizada para fabricación de cemento, micronizados y pinturas, utilizándose el subproducto como árido en diferentes materiales de la construcción, como fabricación de hormigones, aglomerados asfálticos, sub-base para carreteras, caminos y otros usos similares. Desde el punto de vista técnico, la explotación se realiza a cielo abierto por el método de banqueo descendente en profundidad y restauración progresiva, mediante arranque por perforación y voladura, y posterior carga y transporte de materiales por medios mecánicos. El Proyecto de Explotación plantea la extracción de un volumen total de material del orden de 32,85 Mt. Atendiendo a la cifra de reservas explotables y al ritmo de producción anual previsto, el período de explotación contemplado en este Proyecto es del orden de 30 años desde la situación actual. El estudio realizado de los indicadores económicos nos muestra que el proyecto es rentable, con todo ello, parece concluirse que, tanto técnica como económicamente, la explotación es viable. ABSTRACT This project aims to study the economic and technical feasibility for the operation of a limestone quarry belonging to the geological formation of the Calizas del Páramo. The reservoir exploited is superficial type, with an average depth between 30 and 35 m. The mining concession is located in the subregion of the Alcarria de Alcalá, southeast of Madrid. The extracted limestone is used for manufacturing cement, micronized and paintings, using the product as aggregate in different construction materials, such as manufacture of concrete, asphalt mixes, sub-base for roads, paths and other similar uses. From the technical point of view, the operation will be held openpit by the method of depth descending benching and progressive restoration. The operation begins by drilling and blasting, continuing with loading and transport of materials by mechanical methods. The Exploitation Project raises the extraction of a total volume of material of 32.85 Mt approximately Considering the number of exploitable reserves and expected annual production rate, the operating period referred to in this project is of the order of 30 years from the current situation. The study of economic indicators shows that the project is profitable, yet it can be concluded that, both technically and economically, the exploitation of mineral resources is viable.

Genetic analysis of the mouse X inactivation center defines an 80-kb multifunction domain

Dosage compensation in mammals occurs by X inactivation, a silencing mechanism regulated in cis by the X inactivation center (Xic). In response to developmental cues, the Xic orchestrates events of X inactivation, including chromosome counting and choice, initiation, spread, and establishment of silencing. It remains unclear what elements make up the Xic. We previously showed that the Xic is contained within a 450-kb sequence that includes Xist, an RNA-encoding gene required for X inactivation. To characterize the Xic further, we performed deletional analysis across the 450-kb region by yeast-artificial-chromosome fragmentation and phage P1 cloning. We tested Xic deletions for cis inactivation potential by using a transgene (Tg)-based approach and found that an 80-kb subregion also enacted somatic X inactivation on autosomes. Xist RNA coated the autosome but skipped the Xic Tg, raising the possibility that X chromosome domains escape inactivation by excluding Xist RNA binding. The autosomes became late-replicating and hypoacetylated on histone H4. A deletion of the Xist 5′ sequence resulted in the loss of somatic X inactivation without abolishing Xist expression in undifferentiated cells. Thus, Xist expression in undifferentiated cells can be separated genetically from somatic silencing. Analysis of multiple Xic constructs and insertion sites indicated that long-range Xic effects can be generalized to different autosomes, thereby supporting the feasibility of a Tg-based approach for studying X inactivation.

DNase I-hypersensitive sites I and II of the human growth hormone locus control region are a major developmental activator of somatotrope gene expression

High-level expression of the human growth hormone (hGH) gene is limited to somatotrope and lactosomatotrope cells of the anterior pituitary. We previously identified a locus control region (LCR) for the hGH gene composed of four tissue-specific DNase I-hypersensitive sites (HS) located between −14.6 kb and −32 kb 5′ to the hGH transcription start site that is responsible for establishing a physiologically regulated chromatin domain for hGH transgene expression in mouse pituitary. In the present study we demonstrated that the LCR mediates somatotrope and lactosomatotrope restriction on an otherwise weakly and diffusely expressed hGH transgene. The subregion of the LCR containing the two pituitary-specific HS, HSI and HSII (−14.6 to −16.2 kb relative to the hGH promoter and denoted HSI,II), was found to be sufficient for mediating somatotrope and lactosomatotrope restriction, for appropriately timed induction of hGH transgene expression between embryonic days 15.5 and 16.5, and for selective extinction of hGH expression in mature lactotropes. When studied by cell transfection, the HSI,II fragment selectively enhanced transcription in a presomatotrope-derived cell line, although at levels (2- to 3-fold) well below that seen in vivo. The LCR activity of the HSI,II element was therefore localized by scoring transgene expression in fetal founder pituitaries at embryonic day 18.5. The data from these studies indicated that a 404-bp segment of the HSI,II region encodes a critical subset of LCR functions, including the establishment of a productive chromatin environment, cell-specific restriction and enhancement of expression, and appropriately timed induction of the hGH transgene during embryonic development.

Dribble, the Drosophila KRR1p Homologue, Is Involved in rRNA Processing

The Drosophila dribble (dbe) gene encodes a KH domain protein, homologous to yeast KRR1p. Expression of dbe transcripts is ubiquitous during embryogenesis. Overexpressed Dribble protein is localized in the nucleus and in some cell types in a subregion of the nucleolus. Homozygous dbe mutants die at first instar larval stage. Clonal analyses suggest that dbe+ is required for survival of dividing cells. In dbe mutants, a novel rRNA-processing defect is found and accumulation of an abnormal rRNA precursor is detected.