Economic development capitalizing on brand agriculture : turning development strategy on its head

This paper explores the possibilities of two unique Japanese concepts - the One Village One Product Movement (OVOP) and Michino Eki (or Roadside Stations) - as potential tools for bridging the gap between cities and rural areas through community-driven development. From the viewpoint of spatial economics and endogenous growth theory, this paper considers both OVOP and Michino Eki as rural development strategies of a broader nature based on "brand agriculture." Here, brand agriculture represents a general strategy for community-based rural development that identifies, cultivates and fully utilizes local resources for the development of products or services unique to a certain "village." Selected examples of OVOP and Michino Eki from Japan and developing countries are introduced.

Content Delivery System for Optimal VoD Streaming

The demand of video contents has rapidly increased in the past years as a result of the wide deployment of IPTV and the variety of services offered by the network operators. One of the services that has especially become attractive to the customers is real-time video on demand (VoD) because it offers an immediate streaming of a large variety of video contents. The price that the operators have to pay for this convenience is the increased traffic in the networks, which are becoming more congested due to the higher demand for VoD contents and the increased quality of the videos. As a solution, in this paper we propose a hierarchical network system for VoD content delivery in managed networks, which implements redistribution algorithm and a redirection strategy for optimal content distribution within the network core and optimal streaming to the clients. The system monitors the state of the network and the behavior of the users to estimate the demand for the content items and to take the right decision on the appropriate number of replicas and their best positions in the network. The system's objectives are to distribute replicas of the content items in the network in a way that the most demanded contents will have replicas closer to the clients so that it will optimize the network utilization and will improve the users' experience. It also balances the load between the servers concentrating the traffic to the edges of the network.

A CAD development strategy for the next years

This paper suggests a new strategy to develop CAD applications taking into account some of the most interesting proposals which have recently appeared in the technology development arena. Programming languages, operating systems, user devices, software architecture, user interfaces and user experience are among the elements which are considered for a new development framework. This strategy considers the organizational and architectural aspects of the CAD application together with the development framework. The architectural and organizational aspects are based on the programmed design concept, which can be implemented by means of a three-level software architecture. These levels are the conceptual level based on a declarative language, the mathematical level based on the geometric formulation of the product model and the visual level based on the polyhedral representation of the model as required by the graphic card. The development framework which has been considered is Windows 8. This operating system offers three development environments, one for web pplications (HTML5 + CSS3 + JavaScript), and other for native applications C/C++) and of course yet another for .NET applications (C#, VB, F#, etc.). The use rinterface and user experience for non-web application is described ith XAML (a well known declarative XML language) and the 3D API for games and design applications is DirectX. Additionally, Windows 8 facilitates the use of hybrid solutions, in which native and managed code can interoperate easily. Some of the most remarkable advantages of this strategy are the possibility of targeting both desktop and touch screen devices with the same development framework, the usage of several programming paradigms to apply the most appropriate language to each domain and the multilevel segmentation of developers and designers to facilitate the implementation of an open network of collaborators.

A general strategy to enhance the potency of chimeric transcriptional activators

Efforts to increase the potency of transcriptional activators are generally unsuccessful because poor expression of activators in mammalian cells limits their delivery to target promoters. Here we report that the effectiveness of chimeric activators can be dramatically improved by expressing them as noncovalent tetrameric bundles. Bundled activation domains are much more effective at activating a reporter gene than simple monomeric activators, presumably because, at similar expression levels, up to 4 times as many the activation domains are delivered to the target promoter. These bundled activation domains are also more effective than proteins in which activation domains are tandemly reiterated in the same polypeptide chain, because such proteins are very poorly expressed and therefore not delivered effectively. These observations suggest that there is a threshold number of activation domains that must be bound to a promoter for activation, above which promoter activity is simply a function of the number of activators bound. We show that bundling can be exploited practically to enhance the sensitivity of mammalian two-hybrid assays, enabling detection of weak interactions or those between poorly expressed proteins. Bundling also dramatically improves the performance of a small-molecule-regulated gene expression system when the expression level of regulatory protein is limiting, a situation that may be encountered in gene therapy applications.

Adeno-associated viral-mediated catalase expression suppresses optic neuritis in experimental allergic encephalomyelitis

Suppression of oxidative injury by viral-mediated transfer of the human catalase gene was tested in the optic nerves of animals with experimental allergic encephalomyelitis (EAE). EAE is an inflammatory autoimmune disorder of primary central nervous system demyelination that has been frequently used as an animal model for the human disease multiple sclerosis (MS). The optic nerve is a frequent site of involvement common to both EAE and MS. Recombinant adeno-associated virus containing the human gene for catalase was injected over the right optic nerve heads of SJL/J mice that were simultaneously sensitized for EAE. After 1 month, cell-specific catalase activity, evaluated by quantitation of catalase immunogold, was increased approximately 2-fold each in endothelia, oligodendroglia, astrocytes, and axons of the optic nerve. Effects of catalase on the histologic lesions of EAE were measured by computerized analysis of the myelin sheath area (for demyelination), optic disc area (for optic nerve head swelling), extent of the cellular infiltrate, extravasated serum albumin labeled by immunogold (for blood–brain barrier disruption), and in vivo H2O2 reaction product. Relative to control, contralateral optic nerves injected with the recombinant virus without a therapeutic gene, catalase gene inoculation reduced demyelination by 38%, optic nerve head swelling by 29%, cellular infiltration by 34%, disruption of the blood–brain barrier by 64%, and in vivo levels of H2O2 by 61%. Because the efficacy of potential treatments for MS are usually initially tested in the EAE animal model, this study suggests that catalase gene delivery by using viral vectors may be a therapeutic strategy for suppression of MS.

In vivo gene delivery to the liver using reconstituted chylomicron remnants as a novel nonviral vector

Lipoproteins are emulsion particles that consist of lipids and apolipoproteins. Their natural function is to transport lipids and/or cholesterol to different tissues. We have taken advantage of the hydrophobic interior of these natural emulsions to solubilize DNA. Negatively charged DNA was first complexed with cationic lipids containing a quaternary amine head group. The resulting hydrophobic complex was extracted by chloroform and then incorporated into reconstituted chylomicron remnant particles (≈100 nm in diameter) with an efficiency ≈65%. When injected into the portal vein of mice, there were ≈5 ng of a transgene product (luciferase) produced per mg of liver protein per 100 μg injected DNA. This level of transgene expression was ≈100-fold higher than that of mice injected with naked DNA. However, such a high expression was not found after tail vein injection. Histochemical examination revealed that a large number of parenchymal cells and other types of cells in the liver expressed the transgene. Gene expression in the liver increased with increasing injected dose, and was nearly saturated with 50 μg DNA. At this dose, the expression was kept at high level in the liver for 2 days and then gradually reduced and almost disappeared by 7 days. However, by additional injection at day 7, gene expression in the liver was completely restored. By injection of plasmid DNA encoding human α1-antitrypsin, significant concentrations of hAAT were detected in the serum of injected animals. This is the first nonviral vector that resembles a natural lipoprotein carrier.

Streptavidin facilitates internalization and pulmonary targeting of an anti-endothelial cell antibody (platelet-endothelial cell adhesion molecule 1): A strategy for vascular immunotargeting of drugs

Conjugation of drugs with antibodies to surface endothelial antigens is a potential strategy for drug delivery to endothelium. We studied antibodies to platelet-endothelial adhesion molecule 1 (PECAM-1, a stably expressed endothelial antigen) as carriers for vascular immunotargeting. Although 125I-labeled anti-PECAM bound to endothelial cells in culture, the antibody was poorly internalized by the cells and accumulated poorly after intravenous administration in mice and rats. However, conjugation of biotinylated anti-PECAM (b-anti-PECAM) with streptavidin (SA) markedly stimulated uptake and internalization of anti-PECAM by endothelial cells and by cells expressing PECAM. In addition, conjugation with streptavidin markedly stimulated uptake of 125I-labeled b-anti-PECAM in perfused rat lungs and in the lungs of intact animals after either intravenous or intraarterial injection. The antioxidant enzyme catalase conjugated with b-anti-PECAM/SA bound to endothelial cells in culture, entered the cells, escaped intracellular degradation, and protected the cells against H2O2-induced injury. Anti-PECAM/SA/125I-catalase accumulated in the lungs after intravenous injection or in the perfused rat lungs and protected these lungs against H2O2-induced injury. Thus, modification of a poor carrier antibody with biotin and SA provides an approach for facilitation of antibody-mediated drug targeting. Anti-PECAM/SA is a promising candidate for vascular immunotargeting of bioactive drugs.

Delivery of a stringent dimerizer-regulated gene expression system in a single retroviral vector

Small molecule-regulated transcription has broad utility and would benefit from an easily delivered self-contained regulatory cassette capable of robust, tightly controlled target gene expression. We describe the delivery of a modified dimerizer-regulated gene expression system to cells on a single retrovirus. A transcription factor cassette responsive to the natural product dimerizer rapamycin was optimized for retroviral delivery by fusing a highly potent chimeric activation domain to the rapamycin-binding domain of FKBP-rapamycin-associated protein (FRAP). This improvement led to an increase in both the potency and maximal levels of gene expression induced by rapamycin, or nonimmunosuppressive rapamycin analogs. The modified transcription factor cassette was incorporated along with a target gene into a single rapamycin-responsive retrovirus. Cell pools stably transduced with the single virus system displayed negligible basal expression and gave induction ratios of at least three orders of magnitude in the presence of rapamycin or a nonimmunosuppressive rapamycin analog. Levels of induced gene expression were comparable to those obtained with the constitutive retroviral long terminal repeat and the single virus system performed well in four different mammalian cell lines. Regulation with the dimerizer-responsive retrovirus was tight enough to allow the generation of cell lines displaying inducible expression of the highly toxic diphtheria toxin A chain gene. The ability to deliver the tightly inducible rapamycin system in a single retrovirus should facilitate its use in the study of gene function in a broad range of cell types.

Chronic control of high blood pressure in the spontaneously hypertensive rat by delivery of angiotensin type 1 receptor antisense.

The renin-angiotensin system plays a crucial role in the development and establishment of the hypertensive state in the spontaneously hypertensive (SH) rat. Interruption of this system's activity by pharmacological means results in the lowering of blood pressure (BP) and control of hypertension. However, such means are temporary and require the continuous use of drugs for the control of this pathophysiological state. Our objective in this investigation was to determine if a virally mediated gene-transfer approach using angiotensin type 1 receptor antisense (AT1R-AS) could be used to control hypertension on a long-term basis in the SH rat model of human essential hypertension. Injection of viral particles containing AT1R-AS (LNSV-AT1R-AS) in 5-day-old rats resulted in a lowering of BP exclusively in the SH rat and not in the Wistar Kyoto normotensive control. A maximal anti-hypertensive response of 33 +/- 5 mmHg was observed, was maintained throughout development, and still persisted 3 months after administration of LNSV-AT1R-AS. The lowering of BP was associated with the expression of AT1R-AS transcript and decreases in AT1-receptor in many peripheral angiotensin II target tissues such as mesenteric artery, adrenal gland, heart, and kidney. Attenuation of angiotensin II-stimulated physiological actions such as contraction of aortic rings and increase in BP was also observed in the LNSV-AT1R-AS-treated SH rat. These observations show that a single injection of LNSV-AT1R-AS normalizes BP in the SH rat on a long-term basis. They suggest that such a gene-transfer strategy can be successfully used to control the development of hypertension on a permanent basis.

A European global "security" strategy: offering seven-league boots to become a global actor. Security Policy Brief No. 45, March 2013

In mid-2012 the Foreign Ministers of Italy, Poland, Spain and Sweden took the initiative to launch the debate on a “European Global Strategy” and invited think tanks to set up a dialogue leading to the delivery of a report by May 2013.

E-commerce in Europe: parcel delivery prices in a digital single market. Bruegel Policy Contribution ISSUE 2016/09 May 2016

Highlights • In its Digital Single Market strategy, the European Commission has rightly noted the importance of reducing the price paid for basic cross-border parcel delivery by consumers and by small and medium size retail senders. • The payment flows for cross-border parcel delivery are strikingly similar to those for telecommunications. Comparisons with roaming can be instructive. As with roaming, it is clear that the links between wholesale payments between the national postal operators and retail prices need to be properly understood in order to craft good policy. Another useful lesson is that national postal regulatory authorities are unlikely to address cross-border problems because of limitations in their respective mandates and because they have no incentive to take measures to benefit residents of other countries. • There are also significant differences between roaming and parcel delivery.While high wholesale charges were a major driver of high retail prices for international mobile roaming, the wholesale payments for cross-border parcel delivery appear to be below cost.This implies that it is the ‘spread’ between retail price and thewholesale payment that is inflated, at least for small retail senders and for consumers. • Comprehensive statistics gathering, coordinated at European level, is indispensable.

A Rapid FACS-Based Strategy to Isolate Human Gene Knockin and Knockout Clones

Gene targeting protocols for mammalian cells remain inefficient and labor intensive. Here we describe FASTarget, a rapid, fluorescent cell sorting based strategy to isolate rare gene targeting events in human somatic cells. A fluorescent protein is used as a means for direct selection of targeted clones obviating the need for selection and outgrowth of drug resistant clones. Importantly, the use of a promoter-less, ATG-less construct greatly facilitates the recovery of correctly targeted cells. Using this method we report successful gene targeting in up to 94% of recovered human somatic cell clones. We create functional EYFP-tagged knockin clones in both transformed and non-transformed human somatic cell lines providing a valuable tool for mammalian cell biology. We further demonstrate the use of this technology to create gene knockouts. Using this generally applicable strategy we can recover gene targeted clones within approximately one month from DNA construct delivery to obtaining targeted monoclonal cell lines.

Applications of thermal processing in pharmaceutical drug product development

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz

Papers on business strategy and antitrust.

Item 535

Polymerizable Peptide Monomers for the Targeted and Intracellular Delivery of Cancer Therapeutics

Thesis (Ph.D.)--University of Washington, 2016-06