Molecular cloning and characterization of a conserved nuclear serine(threonine) protein kinase.

Human, Drosophila melanogaster, and Caenorhabditis elegans cDNA clones encoding homologues of a serine(threonine) protein kinase (EC 2.7.1.37) (designated Ndr protein kinase) have been isolated and sequenced. The human and Drosophila cDNAs predict polypeptides of 54 kDa and 52 kDa, respectively, which share approximately 80% amino acid similarity. Northern analysis of human tissues revealed a ubiquitously expressed 3.9-kb transcript. Recombinant GST-Ndr underwent intramolecular autophosphorylation on serine and threonine residues in vitro but failed to transphosphorylate several standard protein kinase substrates. Transfection of the human cDNA into COS-1 cells resulted in the appearance of an intense nuclear staining in cells analyzed by indirect immunofluorescence; deletion mutagenesis identified a short basic peptide, KRKAETWKRNRR, responsible for the nuclear accumulation of Ndr. Thus, Ndr is a conserved and widely expressed nuclear protein kinase. The closest known relative of this previously uncharacterized kinase is Dbf2, a budding yeast protein kinase required for the completion of nuclear division.

Mutagenesis of the putative alpha-helical domain of the Vpr protein of human immunodeficiency virus type 1: effect on stability and virion incorporation.

vpr is one of the auxiliary genes of human immunodeficiency virus type 1 (HIV-1) and is conserved in the related HIV-2/simian immunodeficiency virus lentiviruses. The unique feature of Vpr is that it is the only nonstructural protein incorporated into the virus particle. Secondary structural analysis predicted an amphipathic alpha-helical domain in the amino terminus of Vpr (residues 17-34) which contains five acidic and four leucine residues. To evaluate the role of specific residues of the helical domain for virion incorporation, mutagenesis of this domain was carried out. Substitution of proline for any of the individual acidic residues (Asp-17 and Glu-21, -24, -25, and -29) eliminated the virion incorporation of Vpr and also altered the stability of Vpr in cells. Conservative replacement of glutamic residues of the helical domain with aspartic residues resulted in Vpr characteristic of wild type both in stability and virion incorporation, as did substitution of glutamine for the acidic residues. In contrast, replacement of leucine residues of the helical domain (residues 20, 22, 23, and 26) by alanine eliminated virion incorporation function of Vpr. These data indicate that acidic and hydrophobic residues and the helical structure in this region are critical for the stability of Vpr and its efficient incorporation into virus-like particles.

Crystal structure of an H-2Kb-ovalbumin peptide complex reveals the interplay of primary and secondary anchor positions in the major histocompatibility complex binding groove.

Sequence analysis of peptides naturally presented by major histocompatibility complex (MHC) class I molecules has revealed allele-specific motifs in which the peptide length and the residues observed at certain positions are restricted. Nevertheless, peptides containing the standard motif often fail to bind with high affinity or form physiologically stable complexes. Here we present the crystal structure of a well-characterized antigenic peptide from ovalbumin [OVA-8, ovalbumin-(257-264), SIINFEKL] in complex with the murine MHC class I H-2Kb molecule at 2.5-A resolution. Hydrophobic peptide residues Ile-P2 and Phe-P5 are packed closely together into binding pockets B and C, suggesting that the interplay of peptide anchor (P5) and secondary anchor (P2) residues can couple the preferred sequences at these positions. Comparison with the crystal structures of H-2Kb in complex with peptides VSV-8 (RGYVYQGL) and SEV-9 (FAPGNYPAL), where a Tyr residue is used as the C pocket anchor, reveals that the conserved water molecule that binds into the B pocket and mediates hydrogen bonding from the buried anchor hydroxyl group could not be likewise positioned if the P2 side chain were of significant size. Based on this structural evidence, H-2Kb has at least two submotifs: one with Tyr at P5 (or P6 for nonamer peptides) and a small residue at P2 (i.e., Ala or Gly) and another with Phe at P5 and a medium-sized hydrophobic residue at P2 (i.e., Ile). Deciphering of these secondary submotifs from both crystallographic and immunological studies of MHC peptide binding should increase the accuracy of T-cell epitope prediction.

New multivalent calixarene-based ligands for protein recognition and inhibition

One of the challenges that concerns chemistry is the design of molecules able to modulate protein-protein and protein-ligand interactions, since these are involved in many physiological and pathological processes. The interactions occurring between proteins and their natural counterparts can take place through reciprocal recognition of rather large surface areas, through recognition of single contact points and single residues, through inclusion of the substrates in specific, more or less deep binding sites. In many cases, the design of synthetic molecules able to interfere with the processes involving proteins can benefit from the possibility of exploiting the multivalent effect. Multivalency, widely spread in Nature, consists in the simultaneous formation between two entities (cell-cell, cell-protein, protein-protein) of multiple equivalent ligand-recognition site complexes. In this way the whole interaction results particularly strong and specific. Calixarenes furnish a very interesting scaffold for the preparation of multivalent ligands and in the last years calixarene-based ligands demonstrated their remarkable capability to recognize and inhibit or restore the activity of different proteins, with a high efficiency and selectivity in several recognition phenomena. The relevance and versatility of these ligands is due to the different exposition geometries of the binding units that can be explored exploiting the conformational properties of these macrocycles, the wide variety of functionalities that can be linked to their structure at different distances from the aromatic units and to their intrinsic multivalent nature. With the aim of creating new multivalent systems for protein targeting, the work reported in this thesis regards the synthesis and properties of glycocalix[n]arenes and guanidino calix[4]arenes for different purposes. Firstly, a new bolaamphiphile glycocalix[4]arene in 1,3-alternate geometry, bearing cellobiose, was synthesized for the preparation of targeted drug delivery systems based on liposomes. The formed stable mixed liposomes obtained by mixing the macrocycle with DOPC were shown to be able of exploiting the sugar units emerging from the lipid bilayer to agglutinate Concanavalin A, a lectin specific for glucose. Moreover, always thanks to the presence of the glycocalixarene in the layer, the same liposomes demonstrated through preliminary experiments to be uptaken by cancer cells overexpressing glucose receptors on their exterior surface more efficiently respect to simple DOPC liposomes lacking glucose units in their structure. Then a small library of glycocalix[n]arenes having different valency and geometry was prepared, for the creation of potentially active immunostimulants against Streptococcus pneumoniae, particularly the 19F serotype, one of the most virulent. These synthesized glycocalixarenes bearing β-N-acetylmannosamine as antigenic unit were compared with the natural polysaccharide on the binding to the specific anti-19F human polyclonal antibody, to verify their inhibition potency. Among all, the glycocalixarene based on the conformationally mobile calix[4]arene resulted the more efficient ligand, probably due its major possibility to explore the antibody surface and dispose the antigenic units in a proper arrangement for the interaction process. These results pointed out the importance of how the different multivalent presentation in space of the glycosyl units can influence the recognition phenomena. At last, NMR studies, using particularly 1H-15N HSQC experiments, were performed on selected glycocalix[6]arenes and guanidino calix[4]arenes blocked in the cone geometry, in order to better understand protein-ligand interactions. The glycosylated compounds were studied with Ralstonia solanacearum lectin, in order to better understand the nature of the carbohydrate‐lectin interactions in solution. The series of cationic calixarene was employed with three different acidic proteins: GB1, Fld and alpha synuclein. Particularly GB1 and Fld were observed to interact with all five cationic calix[4]arenes but showing different behaviours and affinities.

Symbol for a Community: The Dickens Opera House and Theaters of the American West

This paper reveals the importance of the Dickens Opera House to the local history of Longmont, Colorado. Through an exploration of pioneer history and of architectural patronage and audience accommodation, this paper illustrates how the Dickens Opera House participated in the construction of cultural identity and civic aspirations of the city of Longmont. Using the Tabor Opera House of Leadville and Wright Opera House of Ouray as framing examples to place the Dickens Opera House within its proper architectural and historical context, I approach the building’s inception, construction, and early years as a way to track the early civic identity of a community through a work of architecture. The Dickens Opera House provided a point for the citizens of Longmont to focus their hopes of success and respectability in a newly formed community. An opera house provided a high-class perception of a town that provided a projection of respectability. Such a construction was built from various sources – the architecture of the building, simply calling the building an ‘opera house’, furnishings in the latest fashions and equipment of the latest technology, and extravagant scenery and curtains. In addition to these outward projections, opera houses also provided a place for community events. It was the location in town that brought people together.

Potential Adverse Effects of the Alaska Natural Gas Pipeline on Arctic Caribou and Proposed Mitigation

The Alaska Natural Gas Pipeline (ANGP) is proposed for construction on the North Slope in 2016. It will be aligned through Arctic caribou habitat and evidence shows that caribou are negatively affected by human development. This Capstone identifies potential adverse affects of ANGP on Arctic caribou using interviews from expert caribou biologists and the 1977 Trans-Alaska Pipeline System (TAPS) as a model. Based on a synthesis of the interviews and TAPS analysis, this capstone proposes and examines a set of seventeen conservation measures to be implemented during construction and operation of ANGP to minimize adverse impacts on caribou herds. These conservation measures can be used as a baseline for future developments on the North Slope to promote caribou herd management.

Water speech: energy, power and inequality in Brazil

The documentary is available in Portuguese at the following link: http://hdl.handle.net/10045/17580

Influence of using slag cement on the microstructure and durability related properties of cement grouts for micropiles

Today, the use of micropiles for different applications has become very common. In Spain, the cement grouts for micropiles are prepared using ordinary Portland cement and w:c ratio 0.5, although the micropiles standards do not restrict the cement type to use, provided that it reaches a certain compressive strength. In this study, the influence of using slag cement on the microstructure and durability related properties of cement grouts for micropiles have been studied until 90 hardening days, compared to an ordinary Portland cement. Finally, slag cement grouts showed good service properties, better than ordinary Portland cement ones.

Blending of industrial waste from different sources as partial substitution of Portland cement in pastes and mortars

Binary and ternary combinations of sewage sludge ash (SSA) with marble dust (MD), fly ash (FA) and rice husk ash (RHA) as replacement in Portland cement pastes, were assessed. Several tests were carried out at different curing ages: thermogravimetry, density, water absorption, ultrasonic pulse velocity and mechanical strengths. Pozzolanic effects of the mineral admixtures, densities similar to control sample and improved absorptions when combining waste materials were identified. In general, the compressive strength reaches or exceeds the cement strength class, and blending SSA, FA and RHA (30% cement replacement) increase of strength by 9%, compared to the control sample, was achieved.

Self-heating and deicing conductive cement. Experimental study and modeling

This research studies the self-heating produced by the application of an electric current to conductive cement pastes with carbonaceous materials. The main parameters studied were: type and percentage of carbonaceous materials, effect of moisture, electrical resistance, power consumption, maximum temperature reached and its evolution and ice melting kinetics are the main parameters studied. A mathematical model is also proposed, which predicts that the degree of heating is adjustable with the applied voltage. Finally, the results have been applied to ensure that cementitious materials studied are feasible to control ice layers in transportation infrastructures.

Targetless image-based method for measuring displacements and strains on concrete surfaces with a consumer camera

Measurement of concrete strain through non-invasive methods is of great importance in civil engineering and structural analysis. Traditional methods use laser speckle and high quality cameras that may result too expensive for many applications. Here we present a method for measuring concrete deformations with a standard reflex camera and image processing for tracking objects in the concretes surface. Two different approaches are presented here. In the first one, on-purpose objects are drawn on the surface, while on the second one we track small defects on the surface due to air bubbles in the hardening process. The method has been tested on a concrete sample under several loading/unloading cycles. A stop-motion sequence of the process has been captured and analyzed. Results have been successfully compared with the values given by a strain gauge. Accuracy of our methods in tracking objects is below 8 μm, in the order of more expensive commercial devices.

Construction of Structures I in Moodle: a new teaching methodology in the Degree of Building Engineer

The subject of Construction of Structures I studies, from a constructive point of view and taking into account current legislation, reinforced concrete structures used in buildings, through the acquisition of knowledge and construction criteria required in the profession of a Technical Architect. The contents acquired in this course are essential for further professional development of technicians and are closely related to many of the subjects taught in the same or other courses of the Degree in Technical Architecture at the University of Alicante. The aim of this paper is to present, analyze and discuss the development of a new methodology proposed in the mentioned subject, as it supposed an important change in the traditional way of teaching Construction and Structures I. In order to incorporate new teaching tools in 2013-2014, the course has been implemented by using a Moodle software tool to promote blended learning with online exercises. Our Moodle community allows collaborative work within an open-source platform where teachers and students share a new and personalized learning environment. Students are easily used to the interface and the platform, value the constant connection with teachers or other fellows and completely agree with the possibility of making questions or share documents 24 hours a day. The proposed methodology consists of lectures and practical classes. In the lectures, the basics of each topic are discussed; class attendance, daily study and conducting scheduled exercises are indispensable. Practical classes allow to consolidate the knowledge gained in theory classes by solving professional exercises and actual construction problems related to structures, that shall be compulsorily delivered online. So, after the correction of the teacher and the subsequent feedback of students, practical exercises ensure lifelong learning of the student, who can download any kind of material at any time (constructive details, practical exercises and even corrected exams). Regarding the general evaluation system, goals achievement is assessed on an ongoing basis (65% of the final mark) along the course through written and graphic evidences in person and online, as well as a individual development of a workbook. In all cases, the acquisition of skills, the ability to synthesize, the capacity of logical and critical thinking are assessed. The other 35 % of the mark is evaluated by a complementary graphic exam. Participation in the computing platform is essential and the student is required to do and present, at least 90% of the practices proposed. Those who do not comply with the practices in each specific date could not be assessed continuously and may only choose the final exam. In conclusion, the subject of Construction of Structures I is essential in the development of the regulated profession of Technical Architect as they are considered, among other professional profiles, as specialists in construction of building structures. The use of a new communication platform and online teaching allows the acquisition of knowledge and constructive approaches in a continuous way, with a more direct and personal monitoring by the teacher that has been highly appreciated by almost 100% of the students. Ultimately, it is important to say that the use of Moodle in this subject is a very interesting tool, which was really well welcome by students in one of the densest and important subjects of the Degree of Technical Architecture.

Can Europe deliver growth? The SAPIR report and beyond. Bruges European Economic Policy (BEEP) Briefing 6/2004

In this critical appraisal of the SAPIR report of July 2003, we choose not to focus on the economic analysis provided in the Sapir Report - where we largely agree - or the analysis of governance questions and design at the EU level. Rather, we concentrate on the assignment, orientation and policy recommendations of the Report with the following question in mind: to what extent does the Report help to revitalize the growth debate in Europe? Unfortunately, the focus of the Report’s recommendations is entirely on the EU level of policy and governance, whereas the motor of growth is very clearly being hindered at the Member State level. The present authors suggest that a number of coordination processes at the EU level are best regarded as ‘dangerous liaisons’which are not really goal-oriented but instead ingeniously seem to serve to protect the actors’ autonomously-decided positions. The Union is trapped in a low-growth equilibrium due to this deceptive construction and because in many policy areas relevant for growth, the EU cannot act without the explicit consent of the Member States, or it simply cannot act at all. Indeed, given the single market and EMU, Europe can only deliver growth at the Member States' level. We exemplify this point in a number of concrete policy areas.

The debt, the stock market and the state-owned enterprises. The sources of chaos on the Chinese financial markets. OSW Commentary NUMBER 201/16.03.2016

The government’s extensive programme for stimulating the economy has enabled China to maintain high economic growth after the global financial crisis in 2008. However, this success has come at the price of a number of negative economic phenomena and the consequences they have had are the major challenge for the government today. The vast programme of investments in infrastructure, construction and fixed assets, which has been the main source of economic growth over the past few years, has caused a rapid increase in China’s debt from 158% of GDP in 2007 to 282% in 2014. Along with the local governments in charge of implementing the programme, the Chinese sector of state-owned enterprises (SOEs) has been heavily burdened by the stimulation policy. The sector’s profitability has fallen, its indebtedness has increased and management problems have been revealed.

Evaluation of safety, design, and operation of shared-use paths : final report /

"HRDS-06/07-07(500)E"--Back cover.