Structure-function analysis of human Integrator subunit-4

Structure-function analysis of human Integrator subunit 4 Anupama Sataluri Advisor: Eric. J. Wagner, Ph.D. Uridine-rich small nuclear RNAs (U snRNA) are RNA Polymerase-II (RNAPII) transcripts that are ubiquitously expressed and are known to be essential for gene expression. snRNAs play a key role in mRNA splicing and in histone mRNA expression. Inaccurate snRNA biosynthesis can lead to diseases related to defective splicing and histone mRNA expression. Although the 3′ end formation mechanism and processing machinery of other RNAPII transcripts such as mRNA has been well studied, the mechanism of snRNA 3′ end processing has remained a mystery until the recent discovery of the machinery that mediates this process. In 2005, a complex of 14 subunits (the Integrator complex) associated with RNA Polymerase-II was discovered. The 14subunits were annotated Integrator 1-14 based on their size. The subunits of this complex together were found to facilitate 3′ end processing of snRNA. Identification of the Integrator complex propelled research in the direction of understanding the events of snRNA 3’end processing. Recent studies from our lab confirmed that Integrator subunit (IntS) 9 and 11 together perform the endonucleolytic cleavage of the nascent snRNA 3′ end to generate mature snRNA. However, the role of other members of the Integrator complex remains elusive. Current research in our lab is focused on deciphering the role of each subunit within the Integrator complex This work specifically focuses on elucidating the role of human Integrator subunit 4 (IntS4) and understanding how it facilitates the overall function of the complex. IntS4 has structural similarity with a protein called “Symplekin”, which is part of the mRNA 3’end processing machinery. Symplekin has been thoroughly researched in recent years and structure-function correlation studies in the context of mRNA 3’end processing have reported a scaffold function for Symplekin due to the presence of HEAT repeat motifs in its N-terminus. Based upon the structural similarity between IntS4 and Symplekin, we hypothesized that Integrator subunit 4 may be behaving as a Symplekin-like scaffold molecule that facilitates the interaction between other members of the Integrator Complex. To answer this question, the two important goals of this study were to: 1) identify the region of IntS4, which is important for snRNA 3′ end processing and 2) determine binding partners of IntS4 which promote its function as a scaffold. IntS4 structurally consists of a highly conserved N-terminus with 8 HEAT repeats, followed by a nonconserved C- terminus. A series of siRNA resistant N and C-terminus deletion constructs as well as specific point mutants within its N-terminal HEAT repeats were generated for human IntS4 and, utilizing a snRNA transcriptional readthrough GFP-reporter assay, we tested their ability to rescue misprocessing. This assay revealed a possible scaffold like property of IntS4. To probe IntS4 for interaction partners, we performed co-immunoprecipitation on nuclear extracts of IntS4 expressing stable cell lines and identified IntS3 and IntS5 among other Integrator subunits to be binding partners which facilitate the scaffold like function of hIntS4. These findings have established a critical role for IntS4 in snRNA 3′ end processing, identified that both its N and C termini are essential for its function, and mapped putative interaction domains with other Integrator subunits.

Role of Neurogranin in the regulation of calcium binding to Calmodulin

Role of Neurogranin in the regulation of calcium binding to Calmodulin Anuja Chandrasekar, B.S Advisor: M. Neal Waxham, Ph.D The overall goal of my project was to gain a quantitative understanding of how the interaction between two proteins neurogranin (RC3) and calmodulin (CaM) alters a fundamental property of CaM. CaM, has been extensively studied for more than four decades due to its seminal role in almost all biological functions as a calcium signal transducer. Calcium signals in cardiac and neuronal cells are exquisitely precise and enable activation of some processes while down-regulating others. CaM, with its four calcium binding sites, serves as a central component of calcium signaling in these cells. It is aided in this role as a regulatory hub that differentially activates targets in response to a calcium flux by proteins that alter its calcium binding properties. Neurogranin, also known as RC3, is a member of a family of small neuronal IQ (SNIQ) domain proteins that was originally thought to play a ‘capacitive’ role by sequestering CaM until a calcium influx of sufficient intensity arrived. However, based on earlier work in our lab on neurogranin, we believe that this protein plays a more nuanced role in neurons than simply acting as a CaM buffer. We believe that neurogranin is one of the proteins which, by altering the kinetics of calcium binding allow CaM to decode a variety of signals with fine precision. To quantify the interaction between CaM, neurogranin and calcium, I used biophysical techniques and computational simulations. From my results, I conclude that neurogranin finely regulates the proportion of calcium-saturated CaM and thereby directs CaM’s target specificity.

Use of Echogenic Immunoliposomes for Delivery of both Drug and Stem Cells for Inhibition of Atheroma Progression

Use of Echogenic Immunoliposomes for Delivery of both Drug and Stem Cells for Inhibition of Atheroma Progression By Ali K. Naji B.S. Advisor: Dr. Melvin E. Klegerman PhD Background and significance: Echogenic liposomes can be used as drug and cell delivery vehicles that reduce atheroma progression. Vascular endothelial growth factor (VEGF) is a signal protein that induces vasculogenesis and angiogenesis. VEGF functionally induces migration and proliferation of endothelial cells and increases intracellular vascular permeability. VEGF activates angiogenic transduction factors through VEGF tyrosine kinase domains in high-affinity receptors of endothelial cells. Bevacizumab is a humanized monoclonal antibody specific for VEGF-A which was developed as an anti-tumor agent. Often, anti-VEGF agents result in regression of existing microvessels, inhibiting tumor growth and possibly causing tumor shrinkage with time. During atheroma progression neovasculation in the arterial adventitia is mediated by VEGF. Therefore, bevacizumab may be effective in inhibiting atheroma progression. Stem cells show an ability to inhibit atheroma progression. We have previously demonstrated that monocyte derived CD-34+ stem cells that can be delivered to atheroma by bifunctional-ELIP ( BF-ELIP) targeted to Intercellular Adhesion Molecule-1 (ICAM-1) and CD-34. Adhesion molecules such as ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) are expressed by endothelial cells under inflammatory conditions. Ultrasound enhanced liposomal targeting provides a method for stem cell delivery into atheroma and encapsulated drug release. This project is designed to examine the ability of echogenic liposomes to deliver bevacizumab and stem cells to inhibit atheroma progression and neovasculation with and without ultrasound in vitro and optimize the ultrasound parameters for delivery of bevacizumab and stem cells to atheroma. V Hypotheses: Previous studies showed that endothelial cell VEGF expression may relate to atherosclerosis progression and atheroma formation in the cardiovascular system. Bevacizumab-loaded ELIP will inhibit endothelial cell VEGF expression in vitro. Bevacizumab activity can be enhanced by pulsed Doppler ultrasound treatment of BEV-ELIP. I will also test the hypothesis that the transwell culture system can serve as an in vitro model for study of US-enhanced targeted delivery of stem cells to atheroma. Monocyte preparations will serve as a source of CD34+ stem cells. Specific Aims: Induce VEGF expression using PKA and PKC activation factors to endothelial cell cultures and use western blot and ELISA techniques to detect the expressed VEGF.  Characterize the relationship between endothelial cell proliferation and VEGF expression to develop a specific EC culture based system to demonstrate BEV-ELIP activity as an anti-VEGF agent. Design a cell-based assay for in vitro assessment of ultrasound-enhanced bevacizumab release from echogenic liposomes.  Demonstrate ultrasound delivery enhancement of stem cells by applying different types of liposomes on transwell EC culture using fluorescently labeled monocytes and detect the effect on migration and attachment rate of these echogenic liposomes with and without ultrasound in vitro.

Hesiod And The Didactic Double

Un rasgo inusual de la estrategia didáctica de Hesíodo en Trabajos y Días es la inclusión de su hermano Perses, como destinatario. Si existe o no una base histórica para la disputa financiera entre hermanos, tal como aparece representada en el poema, resulta menos interesante que la posición que tal situación (posiblemente ficcional) transmite. Por la figura del consejero, Hesíodo sostiene, naturalmente, una actitud diferente hacia un hermano, que la que quisiera si su aconsejado fuera un hijo, un futuro rey, o un estudiante -roles que son más típicos en las tradiciones didácticas de la literatura universal. Este artículo explora las tensiones, resonancias míticas y ambigüedades inherentes a la elección de la figura del hermano como recipiendario del consejo y concluye que esta particular configuración didáctica provee una más abierta-conclusiva y aceptable entrada a través de la cual cualquier audiencia puede interactuar con la tradición de sabiduría atesorada en el verso hesiódico.

Hesiod And The Didactic Double

Un rasgo inusual de la estrategia didáctica de Hesíodo en Trabajos y Días es la inclusión de su hermano Perses, como destinatario. Si existe o no una base histórica para la disputa financiera entre hermanos, tal como aparece representada en el poema, resulta menos interesante que la posición que tal situación (posiblemente ficcional) transmite. Por la figura del consejero, Hesíodo sostiene, naturalmente, una actitud diferente hacia un hermano, que la que quisiera si su aconsejado fuera un hijo, un futuro rey, o un estudiante -roles que son más típicos en las tradiciones didácticas de la literatura universal. Este artículo explora las tensiones, resonancias míticas y ambigüedades inherentes a la elección de la figura del hermano como recipiendario del consejo y concluye que esta particular configuración didáctica provee una más abierta-conclusiva y aceptable entrada a través de la cual cualquier audiencia puede interactuar con la tradición de sabiduría atesorada en el verso hesiódico.

Hesiod And The Didactic Double

Un rasgo inusual de la estrategia didáctica de Hesíodo en Trabajos y Días es la inclusión de su hermano Perses, como destinatario. Si existe o no una base histórica para la disputa financiera entre hermanos, tal como aparece representada en el poema, resulta menos interesante que la posición que tal situación (posiblemente ficcional) transmite. Por la figura del consejero, Hesíodo sostiene, naturalmente, una actitud diferente hacia un hermano, que la que quisiera si su aconsejado fuera un hijo, un futuro rey, o un estudiante -roles que son más típicos en las tradiciones didácticas de la literatura universal. Este artículo explora las tensiones, resonancias míticas y ambigüedades inherentes a la elección de la figura del hermano como recipiendario del consejo y concluye que esta particular configuración didáctica provee una más abierta-conclusiva y aceptable entrada a través de la cual cualquier audiencia puede interactuar con la tradición de sabiduría atesorada en el verso hesiódico.

Comparison between SAM and Thermoflow for Linear Fresnel Solar Power Plants design

System Advisor Model is a software tool develped by National Renewable Laboratory (NREL), Department Of Energy, USA to design Solar Power Plants.

SolarPaces 2013: Innovations on direct steam generation in linear fresnel collectors

Direct Steam Generation (DSG) in Linear Fresnel (LF) solar collectors is being consolidated as a feasible technology for Concentrating Solar Power (CSP) plants. The competitiveness of this technology relies on the following main features: water as heat transfer fluid (HTF) in Solar Field (SF), obtaining high superheated steam temperatures and pressures at turbine inlet (500ºC and 90 bar), no heat tracing required to avoid HTF freezing, no HTF degradation, no environmental impacts, any heat exchanger between SF and Balance Of Plant (BOP), and low cost installation and maintenance. Regarding to LF solar collectors, were recently developed as an alternative to Parabolic Trough Collector (PTC) technology. The main advantages of LF are: the reduced collector manufacturing cost and maintenance, linear mirrors shapes versus parabolic mirror, fixed receiver pipes (no ball joints reducing leaking for high pressures), lower susceptibility to wind damages, and light supporting structures allowing reduced driving devices. Companies as Novatec, Areva, Solar Euromed, etc., are investing in LF DSG technology and constructing different pilot plants to demonstrate the benefits and feasibility of this solution for defined locations and conditions (Puerto Errado 1 and 2 in Murcia Spain, Lidellin Newcastle Australia, Kogran Creek in South West Queensland Australia, Kimberlina in Bakersfield California USA, Llo Solar in Pyrénées France,Dhursar in India,etc). There are several critical decisions that must be taken in order to obtain a compromise and optimization between plant performance, cost, and durability. Some of these decisions go through the SF design: proper thermodynamic operational parameters, receiver material selection for high pressures, phase separators and recirculation pumps number and location, pipes distribution to reduce the amount of tubes (reducing possible leaks points and transient time, etc.), etc. Attending to these aspects, the correct design parameters selection and its correct assessment are the main target for designing DSG LF power plants. For this purpose in the recent few years some commercial software tools were developed to simulatesolar thermal power plants, the most focused on LF DSG design are Thermoflex and System Advisor Model (SAM). Once the simulation tool is selected,it is made the study of the proposed SFconfiguration that constitutes the main innovation of this work, and also a comparison with one of the most typical state-of-the-art configuration. The transient analysis must be simulated with high detail level, mainly in the BOP during start up, shut down, stand by, and partial loads are crucial, to obtain the annual plant performance. An innovative SF configurationwas proposed and analyzed to improve plant performance. Finally it was demonstrated thermal inertia and BOP regulation mode are critical points in low sun irradiation day plant behavior, impacting in annual performance depending on power plant location.

Managing Soil Organic Carbon: A Farm Perspective

Farming practices that lead to declining returns and inputs of carbon to soils pose a threat to key soil functions. The EU FP 7 interdisciplinary project Smart SOIL is using scientific testing and modeling to identify management practices that can optimize soil carbon storage and crop productivity. A consultation with advisors and policymakers in six European case study regions seeks to identify barriers to, and incentives for, uptake of such practices. Results from preliminary interviews are reported. Overall advisor and farmer awareness of management practices specifically directed towards soil carbon. is low. Most production- related decisions are taken in the short term, but managing soil carbon needs a long- term approach. Key barriers to uptake of practices include: perceived scientifi c uncertainty about the effi cacy of practices; lack of real life ?best practice? examples to show farmers; diffi culty in demonstrating the positive effects of soil carbon management practices and economic benefi ts over a long time scale; and advisors being unable to provide suitable advice due to inadequate information or training. Most farmers are unconvinced of the economic benefi ts of practices for managing soil carbon. Incentives are therefore needed, either as subsidies or as evidence of the cost effectiveness of practices. All new measures and advice should be integrated into existing programmes to avoid a fragmented policy approach.

Modelización del efecto de la pendiente de la vía sobre el consumo de vehículos turismo

El presente trabajo tiene por objetivo generar una metodología validada que permita predecir el consumo de vehículos turismo circulando en cualquier tramo de vía a partir del perfil orográfico y del diagrama velocidad-tiempo. Para la generación de la metodología, se ha realizado un modelo de simulación con el programa ADVISOR que permite calcular el consumo de combustible para un determinado recorrido en el que se tiene en cuenta el perfil orográfico. Este modelo fue validado con datos reales medidos con equipos on-board y se usó para calcular el consumo de combustible diferencial debido al efecto de la pendiente de la vía, al poderse simular con y sin pendiente. Se realizaron múltiples simulaciones de recorridos con velocidad máxima variable con el fin de obtener un número significativo de datos. Con los resultados de las diferentes simulaciones, se realizó un estudio estadístico, para determinar las variables influyentes y se generó una función estadística (Ecuación de Consumo Estimado – ECE) que permite calcular el consumo de combustible debido a la pendiente de la vía, conociendo el consumo del vehículo en carretera llana (sin pendiente). Esta función estadística generada (ECE), se validó con datos reales medidos en tráfico real. Con el fin de darle generalidad y aplicabilidad a la función generada, y teniendo en cuenta que el consumo de combustible en carretera llana no está siempre disponible, se ha calculado el consumo de combustible sin pendiente utilizando la metodología Copert 4, metodología oficial desarrollada por la Agencia de Medio Ambiente de Europa (EEA) para la estimación de emisiones y consumo de combustible que está basada en datos experimentales pero que no tiene en cuenta la pendiente de la vía. La Ecuación de Consumo Estimado (ECE) aplicada a los consumos calculados por la metodología Copert 4, se valida también usando datos reales medidos en tráfico real y se comprueba que esta función se ajusta considerablemente bien a la realidad, con un error en el consumo acumulado frente al del ensayo real de un 1% y una correlación con el consumo instantáneo del ensayo real de 0,93. Se concluye, que la Función de Consumo Estimado (ECE), permite predecir el efecto de la pendiente sobre el consumo de combustible de un vehículo turismo en tráfico real con un error menor del 1%. Abstract This projects aims to develop a validated methodology that enables to predict cars consumption while circulating at any kind of road section based on its orographic outline and the speed-time diagram. In order to develop this methodology, a simulation model has been performed with the programme ADVISOR, that allows fuel consumption calculation for an specific route in which the orographic outline is considered. This model was validated by real data measured with an on-board equipment and it was used to calculate the differential fuel consumption caused by the effect of the slope on the road, as it was possible to simulate with or without slope. Many simulations were run with routes with variable maximum speed, aiming to obtain a significant amount of data. An statistical study was carried out with the results of those simulations with the purpose to determine the influential variables and an statistical function ( Estimated Consumption Equation – ECE) that enables fuel consumption calculation due to the road’s slope when the consumption of a vehicle on horizontal road (without any slope) is known. This statistical function (ECE) was validated by real data measured in real traffic conditions. With the purpose to generalise the function and increase its applicability, considering that the consumption of a vehicle on horizontal road is not always available, the nonslope fuel consumption has been calculated through Copert 4 methodology, which is the official methodology developed by the European Environmental Agency (EEA) for emissions and fuel consumption calculation based on experimental data, but without taking into consideration the road’s slope. The Estimated Consumption Equation (ECE) applied to the consumption calculated through Copert 4 methodology is also validated using real data measured in real traffic conditions. It was verified that this function considerably adjusts to reality, with an error on the accumulated consumption compared to the real test of 1% and a correlation with the real test immediate fuel consumption of 0,93. It is concluded that the Estimated Consumption Equation (ECE) enables to predict the effect of the slope on the fuel consumption of a car in real traffic conditions with an error less than 1%.

Linked-data based domain-specific sentiment lexicons

In this paper we present a dataset componsed of domain-specific sentiment lexicons in six languages for two domains. We used existing collections of reviews from Trip Advisor, Amazon, the Stanford Network Analysis Project and the OpinRank Review Dataset. We use an RDF model based on the lemon and Marl formats to represent the lexicons. We describe the methodology that we applied to generate the domain-specific lexicons and we provide access information to our datasets.

Realizing Sustained School Improvement

Author: Jeanice Kerr Swift Title: REALIZING SUSTAINED SCHOOL IMPROVEMENT Advisor: Dr. Kent Seidel and Dr. Linda Brookhart Degree Date: June, 2011 Developing effective methods for improving America's schools is dependent upon coordinated work of practitioners and researchers. School improvement proves a complicated, confusing and most often troubled process; one characterized by the unprecedented challenges of dramatic societal shift, increasing levels of student need, and ongoing involvement from political and governmental influences. The drive to discover better ways to consistently improve schools is fueled by urgency for dramatic results. This research examines both the macro and the micro levels of the school improvement process and illuminates the need for an intentional new way of thinking and leading. At the macro level, the study examined the need for implementing a systems approach to improve America's schools. The study explored a single school's improvement journey. This work aligned individual and group perceptions of staff members alongside student achievement outcome data framed against the findings in the literature. The study rendered themes from within the reflected experiences of participant educators; the work pointed to the need for an improved social technology, the importance of teams, and shared leadership in the orchestrating of successful school improvement processes, particularly the essential roles of collective listening, learning, and leading to realize transformed outcomes for schools.

Amino Acid Residues Implicated in the Interaction of Melanocortin Ligands and their Receptors: A Study of MC2R Selectivity

Author: Kristopher D. Veo Title: Amino acid residues implicated in the interaction of Melanocortin ligands and their receptors: A study of MC2R selectivity Advisor: Dr. Robert M. Dores Degree Date: August 2009 ABSTRACT Melanocortin receptor ligand selectivity has been a question not easily answered. The inability to functionally express melanocortin 2 receptor (MC2R) has inhibited the study of why MC2R is only stimulated by ACTH, a melanocortin hormone. With the recent discovery of the MC2R accessory protein (MRAP), creating a heterologous system is now feasible. Using a general cell line like CHO-K1 cells, which do not express endogenous MCRs, we were able to create a heterologous expression system and test the selectivity of MC2R using analog variants of ACTH(1-24). Our results indicate an amino acid requirement in the C-terminal portion of ACTH(1-24) for activation, which supports the 2-step method of activation hypothesized for MC2R. This site, the tetra basic cleavage site, when altered does not stimulate cAMP production and does not compete with ACTH(1-24) for binding. We also demonstrate the potential for a non-mammalian MC2R system in cloning full length Silurana tropicalis MC2R and completed localization studies with this system with MRAP using CHO-K1 cells.

The Impact of Shyness on Loneliness, Social Anxiety, and School Liking in Late Childhood

Author: Charity M. Walker Title: THE IMPACT OF SHYNESS ON LONELINESS, SOCIAL ANXIETY, AND SCHOOL LIKING IN LATE CHILDHOOD Advisor: Maria T. Riva, Ph.D. Degree Date: August 2011 ABSTRACT Shyness is associated with several emotional, social, and academic problems. While there are multiple difficulties that often accompany shyness, there appear to be some factors that can moderate negative effects of shyness. Research has demonstrated that certain parenting factors affect the adjustment of shy children in early childhood, but there is minimal research illuminating the effect of parenting factors in older age groups. The first purpose of this study was to examine relationships between shyness and loneliness, social anxiety, and school liking. The second purpose was to investigate whether the quality of the relationship between a parent and a 10- to 15-year-olds child influences the amount of loneliness or social anxiety a shy child experiences or how the child feels about school. Parent-child dyads served as participants and were recruited from public and private middle schools and church youth groups in Colorado and Indiana. Child participants completed several self-report surveys regarding their relationship with a parent, shyness, loneliness, social anxiety, and their attitude toward school. Parents completed a survey about their relationship with their child and responded to questions related to their perceptions of their child's shyness. Data was analyzed with a series of correlation and regression analyses. Greater degrees of self-reported shyness were found to be associated with higher levels of loneliness and social anxiety and less positive feelings about school. Due to a problem with multicollinearity during data analysis, this study was not able to explore the effect of the parent-child relationship quality on the associations between shyness and adjustment factors. Overall, these findings imply that shyness remains an important issue as children approach adolescence. Further research is needed to continue learning about the potential importance of parent-child interactions in reducing maladjustment for shy children during late childhood.