The sustained delivery of antisense oligodeoxynucleotides using biodegradable polymer microspheres

Antisense technology is a novel drug discovery method, which provides an essential tool for directly using gene sequence information to rationally design specific inhibitions of mRNA, to treat a wide range of diseases. The efficacy of naked oligodeoxynucleotides (ODNs) is relatively short lived due to rapid degradation in vivo. The entrapment of ODNs within biodegradable sustained-release delivery systems may improve ODN stability and reduce dose required for efficacy. Biodegradable polymer microspheres were evaluated as delivery devices for ODNs and ribozymes. Poly(lactide-co-glycolide) polymers were used due to their biocompatibility and non toxic degradation products. Microspheres were prepared using a double emulsion-deposition method and the formulations characterised. In vitro release profiles were characterised by an initial burst effect during the first 48 hours of release followed by a more sustained release. The release profiles were influenced by microsphere size, copolymer molecular weight, copolymer ratio, ODN loading, ODN length, and ODN chemistry. The serum stability of ODNs was significantly improved when entrapped within polymer microspheres. The cellular association of ODNs entrapped within small spheres (1-2μm) was improved by approximately 20-fold in A431 carcinoma cells compared with free ODNs. Fluorescence microscopy studies showed a more diffuse subcellular distribution when delivered as a microsphere formulation compared with free ODNs, which exhibited the characteristic punctate periplasmic distribution. For in vivo evaluation, polymer microspheres containing fluorescently-labelled ODNs were stereo-taxically administered to the neostriatum of the rat brain. Free ODN resulted in a punctate cellular distribution after 24 hours. In comparison ODN delivered using polymer microspheres were intensely visible in cells 48 hours post administration, and fluorescence appeared to be diffuse covering both cytosolic and nuclear regions. Whole-body autoradiography was also used to evaluate the biodistribution of free tritium labelled ODN and ODN entrapped microspheres, following subcutaneous administration to Balb-C mice. Polymer entrapped ODN gave a similar biodistribution to free ODN. Free ODN was distributed within 24 hours, whereas polymer released ODN was observed still presented in organs and at the site of administration seven days post administration.

Fibre-grating sensors for the measurement of physiological pulsations

Mechanical physiological pulsations are movements of a body surface incited by the movements of muscles in organs inside the body. Here we demonstrate the use of long-period grating sensors in the detection of cardio-vascular pulsations (CVP), in particular apex and carotid pulsations. To calibrate the sensors, we use a mechanical tool designed specifically to measure the sensor response to a localized perturbation at different grating curvatures as working points. From the data we infer the amplitude of the CVP. Together with the electrophysiological signals, the CVP signals obtained from the sensors can provide significant information on heart function which is inaccessible to the electrocardiogram. The low cost and easy handling of the fibre sensors increase their prospects to become the sensors of choice for novel diagnostic devices. © 2013 The Royal Swedish Academy of Sciences.

Peru’s Shining Path: Recent Dynamics and Future Prospects

Although soundly defeated in the early to mid-1990s, Shining Path (Sendero Luminoso) did not disappear. Over the past five years, it has reemerged in a substantially different form, with both a military and a political component. The organization, once again coordinated by jailed leader Abimael Guzmán Reynoso, has eschewed shorter-term military objectives in favor of a longer-term strategy of slowly rebuilding popular support and establishing a party within the Peruvian political system. In so doing, it has also moved from extreme Maoist ideological rigidity to a more pragmatic, though perhaps only tactical, approach. Financial support is derived once again from cocaine production and trafficking in the Upper Huallaga Valley (UHV). Although there may be some individual exceptions, Shining Path is not a narcoterrorist organization. At the same time, Sendero is still very small, in no way a threat to the Peruvian state, and divided. The Lima-based political organization and the military wing in the UHV continue to follow Guzmán‟s leadership, while the Apurímac-Ene Valleys (VRAE) group remains committed to the armed struggle. The Government of Peru (GOP) response to date, both military and civilian, has been inadequate. VRAE military operations are hampered by poor leadership, ill-trained troops, and an outdated strategy. VRAE development resources have been cut, and the long-promised paved road remains in the planning stage. Without significant GOP adjustments, Shining Path is likely to continue to grow.

Selective and specific activation of Rab5 during endocytosis of receptor tyrosine kinases

The Rab family of proteins are low molecular weight GTPases that have the ability to switch between GTP- (active) and GDP- (inactive) bound form, and in that sense act as molecular switches. Through distinct localization on various vesicles and organelles and by cycling through GTP/GDP bound forms, Rabs are able to recruit and activate numerous effector proteins, both spatially and temporally, and hence behave as key regulators of trafficking in both endocytic and biosynhtetic pathways. The Rab5 protein has been shown to regulate transport from plasma membrane to the early endosome as well as activate signaling pathways from the early endosome. This dissertation focused on understanding Rab5 activation via endocytosis of receptor tyrosine kinases (RTKs). First, tyrosine kinase activity of RTKs was linked to endosome fusion by demonstrating that tyrosine kinase inhibitors block endosome fusion and activation of Rab5, and a constitutively active form of Rab5 is able to rescue endosome fusion. However, depending on how much ligand is available at the cell surface, the receptor-ligand complexes can be internalized via a number of distinct pathways. Similarly, Rab5 was activated in a ligand-dependent concentration dependent manner via clathrin- and caveolin-mediated pathways, as well as a pathway independent of both. However, overexpression Rabex-5, a nucleotide exchange factor for Rab5, is able to rescue activation even when all of the pathways of EGF-receptor internalization were blocked. Next, the three naturally occurring splice variants of Rabex-5 selectively activated Rab5. Lastly, Rabex-5 inhibits differentiation of 3T3-L1 and PC12 cells through 1) degradation of signaling endosome via Rab5-dependent fusion with the early endosome, 2) and inhibition of signaling cascade via ubiquitination of Ras through the ZnF domain at the N-terminus of Rabex-5. In conclusion, these data shed light on complexity of the endosomal trafficking system where tyrosine kinase activity of the receptor is able to affect endosome fusion; how different endocytic pathways affect activation of one of the key regulators of early endocytic events; and how selective activation of Rab5 via Rabex-5 can control adipogenesis and neurogenesis.

Comércio e cidade: processos e formas espaciais em Pau dos Ferros/RN

This wor k problematizou - if the relationship between trade and city, with a view to considering the role of this activity in urban dynamics of Pau dos Ferros, from the decade of 1980. From this perspective, characterized the city from a historical point of view, em phasizing the commercial dynamics in the urban context, regarding the number of establishments, typologies, specializations; identified the vectors of modernization of commercial activity in the city; it was recognized from the dissemination of trade in ur ban space, the processes and the space forms associated with this dynamic. The methodology involved bibliographic research, documentary research in organs, such as the Instituto Brasileiro de Geografia e Estatística (IBGE), Serviço Brasileiro de Apoio a Mi cro e Pequenas Empresas (SEBRAE), Câmara de Dirigentes Lojistas (CDL) of Pau dos Ferros and Municipal Government, as well as field research. The results of the survey indicate that the City of Pau dos Ferros, from a historical point of view, had its origin linked to trade and has the center as the site of the genesis of this activity, because the largest number of establishments, typologies and specializations are there; the commercial activity is marked by the coexistence between traditional ways and moder n, these being allocated in the central area of the city, for example, supermarkets and specialty stores; and the dissemination of trade in the urban area of Pau dos Ferros revealed the center of the city and the Avenue Independence as the places where the y are more expressive, the processes and forms associated with this activity.

Bioengineering strategies to improve functional qualities of nisin

The abuse of antibiotics and the emergence of multi-drug resistant bacterial strains have created the need to explore alternative methods of controlling microbial pathogens. The bacteriocin family of antimicrobial peptides has been proposed as one such alternative to classic antibiotics. Nisin A belongs to the subgroup of bacteriocins called the lantibiotics, which contain several unusual amino acids as a consequence of enzyme-mediated post-translational modifications. As nisin is produced by generally regarded as safe (GRAS) microorganisms, it could potentially be applied in a clinical setting. However, as lantibiotics are naturally produced in such small quantities, this can hinder their industrial potential. In order to overcome this, several approaches can be utilised. For example, given the gene encoded nature of lantibiotics, genetic engineering approaches can be implemented in order to yield variants with enhanced properties. Here, the use of mutagenesis-based strategies was employed to obtain a derivative of nisin with enhanced bioactivity in vitro. Investigations with purified peptide highlighted the enhanced specific activity of this variant, nisin M21V, against food-borne Listeria monocytogenes strains. Furthermore, this specific enhanced bioactivity was evident in a mouse model of listeriosis. Reductions in bioluminescence and microbial counts in organs from infected mice were observed following treatment with nisin M21V compared to that of wild-type nisin A. Peptide bioengineering approaches were also implemented to obtain additional novel derivatives of nisin. The generation of “S5X” and “S33X” banks (representing a change of natural serines at positions 5 and 33 to all possible alternative residues) by a combination of site-saturation and site-directed mutagenesis led to the identification of several derivatives exhibiting improved stability. This allowed the rational design of variants with enhanced stability compared to that of wild type nisin. Another means of tackling issues associated with lantibiotic yield is to combine lantibiotics with other antimicrobials. This could circumvent the need for enhanced production while also reducing concentrations of the peptide antimicrobials. We observed that combinations of nisin variants and low levels of plant essential oils (thymol, carvacrol, trans-cinnamaldehyde) significantly controlled Gram negative foodborne pathogens in in vitro assays compared to nisin A-essential oil combinations. This enhanced control was also evident in model food systems. Nisin variants used in conjunction with carvacrol significantly reduced numbers of E. coli O157:H7 in apple juice while a commercial nisin preparation used in combination with citric acid significantly controlled C. sakazakii in infant milk formula. It is noteworthy that while nisin is generally associated with Gram positive targets, upon combination with plant essential oils the spectrum of inhibition was broadened to Gram negative targets.

El Norte de Africa y el Sahel y la Estrategia Global de Seguridad de la Unión Europea

El objetivo del artículo es exponer una adecuada y necesaria redefinición de la política de la UE hacia el Norte de África y el Sahel, tanto en términos geográficos como en términos estratégicos de acuerdo con lo expuesto en la nueva Estrategia Global Europea. En este sentido, se revisarán los postulados y la evolución de la antigua Estrategia Europea de Seguridad (EES) y su consiguiente Política de Vecindad (ENP), en comparación con la recién lanzada Estrategia. La nueva Estrategia es concebida como una guía para el desarrollo de la política exterior y de seguridad que la UE ha de llevar a cabo, inter alia, en su periferia. Lamentablemente su concepción de esta zona regional resulta anclada en el pasado.

Identificação do transcrito CYP1A no peixe Phalloceros caudimaculatus e resposta para a exposição a beta-naftoflavona e amostras ambientais de sedimento

Contaminantes orgânicos, como os hidrocarbonetos policíclicos aromáticos (HPAs), podem atingir corpos da água e possuem potencial para causar efeitos tóxicos em organismos. A exposição aos HPAs causa indução nos níveis de citocromo P450 1A (CYP1A) em peixes, e portanto, é utilizado como um biomarcador de contaminação ambiental. O guarú Phalloceros caudimaculatus ocorre naturalmente em ambientes aquáticos dulcícolas e mixohalinos na América do Sul. O presente estudo identificou a sequência nucleotídica do transcrito CYP1A de P. caudimaculatus, que codifica uma proteína com 521 aminoácidos, e que apresenta 91% e 70% de identidade com CYP1A de killifish e paulistinha, respectivamente. A partir desta sequência foi possível realizar a avaliação dos níveis de mRNA de CYP1A deste peixe por RTq-PCR. Foi realizada uma caracterização de sua indução órgão- e tempo-dependente frente a exposição ao HPA beta-naftoflavona (BNF) e ao elutriato preparado a partir de sedimento de dois corpos da água possivelmente contaminados com HPAs. Foi constatado um aumento significativo nos níveis de mRNA de CYP1A em fígado, brânquia, intestino, cérebro, nadadeira anal de macho adultos e em alevinos na primeira hora de exposição a 1 µM de BNF, em relação ao grupo controle. O rim e as nadadeiras caudal e dorsal apresentaram indução de CYP1A após duas horas de exposição ao BNF. As maiores induções nos peixes dos grupos expostos ao BNF em relação ao controle foram de 176 no rim e 122 vezes no cérebro, observadas respectivamente após 8 e 48 horas de exposição. Os níveis de mRNA de CYP1A nos órgãos e tecidos de alevino, mantiveramse induzidos pela exposição ao BNF até o final das 96 horas de exposição. A exposição dos peixes ao elutriato produzido a partir dos sedimentos coletados em dois locais potencialmente contaminados causou indução do CYP1A no fígado de 22 e 122 vezes em relação ao controle. Os resultados demonstram que a indução de CYP1A em Phalloceros caudimaculatus ocorre em tempos curtos de exposição, além da variação de acordo com o tempo de exposição e com o órgão analisado. Além disso, foi demonstrado que tecidos externos também podem ser utilizados para tais análises e que o elutriato feito a partir de sedimento de locais que recebem descargas de contaminantes podem causar indução de CYP1A nos organismos.

La protección a las víctimas de trata de personas en Brasil

La trata de personas es una acción ilegal presente en todomundo, incluso en Brasil. El Protocolo de las Naciones Unidascontra la Trata de Personas fue ratificado por Brasil en 2004. Apesar de esto, aún la evolución normativa del país no ha sidosignificativa. Este artículo busca analizar la protección normativaexistente y futura a las víctimas, especialmente extranjeras, detrata de personas en Brasil. Así, inicialmente se presenta lo que secomprende como trata de personas, con base en el Protocolo delas Naciones Unidas para prevenir, reprimir y sancionar la trata depersonas, especialmente mujeres y niños, y son apuntadas algunasconsideraciones sobre la realidad de la trata de personas en elmundo y en Brasil. En la secuencia son presentadas las medidasde protección a las víctimas, y previstas en el Protocolo de lasNaciones Unidas. Por último, son enumeradas y analizadas lasacciones normativas existentes y los proyectos de ley que puedencambiar esta realidad. La conclusión del estudio indica que Brasilaún tiene una débil protección para las víctimas extranjeras detrata de personas, pero hay una perspectiva optimista de mejoríacon los proyectos de ley que tramitan en el Congreso Nacional.

Estudo sócio-ecológico sobre tráfico de seres humanos para fins de exploração laboral na região do Alentejo : análise espacial e estatística

O presente relatório, inserido no Mestrado em Gestão do Território, Área de Especialização em Deteção Remota e Sistemas de Informação Geográfica, lecionado pelo Departamento de Geografia e Planeamento Regional da Faculdade de Ciências Sociais e Humanas da Universidade Nova de Lisboa, pretende descrever o trabalho desenvolvido pelo mestrando enquanto estagiário no Observatório do Tráfico de Seres Humanos (OTSH). O relatório está estruturado em três capítulos distintos. No primeiro capítulo é realizada uma abordagem teórica sobre o Tráfico de Seres Humanos e a distinção entre o mesmo com o Auxílio à Imigração Ilegal. Neste, é também feita uma pequena referência à problemática dos novos fluxos de refugiados/migrantes que, no momento da realização do mesmo, constituem uma questão bastante complexa sobretudo ao nível europeu. No segundo capítulo é realizada uma caracterização da área de estudo, assim como a descrição dos dados utilizados e a metodologia aplicada no mesmo. No terceiro capítulo são apresentados os resultados finais do estudo e a cartografia de síntese que sustenta os mesmos. Para a realização deste estudo recorreu-se a uma análise multicritério em SIG para prever a localização de áreas de maior suscetibilidade de ocorrência de novos casos relativos ao crime do tráfico de seres humanos para exploração laboral na agricultura, na região do Alentejo (distritos de Beja, Évora e Portalegre), através do recurso a dados estatísticos disponibilizados tanto pelo OTSH, como por outras entidades. A metodologia apresentada integra um SIG baseado num modelo raster com o Analytical Hierarchy Process (AHP). Através da realização deste estudo, a importância dos SIG como ferramenta no auxílio ao processo de tomada de decisão, pôde ser testada, conjuntamente com o processo metodológico AHP, através dos resultados apresentados. Com um possível desenvolvimento deste modelo analítico, pretende-se que o mesmo seja adaptável a outras regiões e em última instância, outros tipos de exploração e/ou tráfico.

Selective and Specific Activation of Rab5 during Endocytosis of Receptor Tyrosine Kinases

The Rab family of proteins are low molecular weight GTPases that have the ability to switch between GTP- (active) and GDP- (inactive) bound form, and in that sense act as molecular switches. Through distinct localization on various vesicles and organelles and by cycling through GTP/GDP bound forms, Rabs are able to recruit and activate numerous effector proteins, both spatially and temporally, and hence behave as key regulators of trafficking in both endocytic and biosynhtetic pathways. The Rab5 protein has been shown to regulate transport from plasma membrane to the early endosome as well as activate signaling pathways from the early endosome. This dissertation focused on understanding Rab5 activation via endocytosis of receptor tyrosine kinases (RTKs). First, tyrosine kinase activity of RTKs was linked to endosome fusion by demonstrating that tyrosine kinase inhibitors block endosome fusion and activation of Rab5, and a constitutively active form of Rab5 is able to rescue endosome fusion. However, depending on how much ligand is available at the cell surface, the receptor-ligand complexes can be internalized via a number of distinct pathways. Similarly, Rab5 was activated in a ligand-dependent concentration dependent manner via clathrin- and caveolin-mediated pathways, as well as a pathway independent of both. However, overexpression Rabex-5, a nucleotide exchange factor for Rab5, is able to rescue activation even when all of the pathways of EGF-receptor internalization were blocked. Next, the three naturally occurring splice variants of Rabex-5 selectively activated Rab5. Lastly, Rabex-5 inhibits differentiation of 3T3-L1 and PC12 cells through 1) degradation of signaling endosome via Rab5-dependent fusion with the early endosome, 2) and inhibition of signaling cascade via ubiquitination of Ras through the ZnF domain at the N-terminus of Rabex-5. In conclusion, these data shed light on complexity of the endosomal trafficking system where tyrosine kinase activity of the receptor is able to affect endosome fusion; how different endocytic pathways affect activation of one of the key regulators of early endocytic events; and how selective activation of Rab5 via Rabex-5 can control adipogenesis and neurogenesis.

El rol de las redes de apoyo transnacional en la evolución del régimen de trata de personas en la región del mekong, 2004-2015

Con el fin de la unipolaridad no sólo se fortalecieron mecanismos de gobernanza global como los Regímenes Internacionales, sino también se fortalecieron actores no estatales. A pesar de la importancia que tomaron estos dos elementos aún no existe una teoría que explique exhaustivamente la relación que existe entre ellos. Es por lo anterior que, la investigación busca responder de qué manera el rol de las Redes de Apoyo Transnacional ha incidido en la evolución del régimen de tráfico de personas en la Región del Mekong. Asimismo tiene como objetivo comprender las relación entre el Régimen y las Redes de Apoyo Transnacional a través de la formulación de un caso de estudio basado en metodologías cualitativas, específicamente, en el análisis teórico-constructivista y el análisis de contenido de documentos producidos por actores estatales y no estatales.

Retos en el cumplimiento del protocolo de Palermo. Estudio de caso: trata de personas con fines de explotación sexual en Colombia

El presente estudio de caso tiene como objetivo analizar los retos frente al cumplimiento del Protocolo de Palermo, en materia de explotación sexual, en el Sistema Institucional Colombiano, durante el período comprendido entre los años 2003 y 2014. De manera preliminar se indica que dichos retos son político-jurídicos en cuanto a la manera en la que se cumplen los tratados internacionales, las obligaciones derivadas de ellos y quiénes tienen competencia para desarrollar los mandatos contenidos en los mismos. Igualmente existen retos institucionales y organizacionales pues Colombia carece de una estructura organizacional clara y la coordinación inter-agencial es escasa en materia de trata. En este sentido, esta investigación tiene un enfoque multidisciplinar, puesto que combina aspectos propios tanto del Derecho Internacional Público como de las Relaciones Internacionales. Para ello, se hará un análisis cualitativo por medio del análisis de datos y de literatura académica respecto a la trata de personas en Colombia, con el fin de lograr comprender con mayor precisión el panorama actual del país respecto al flagelo.

Retos en la implementación de las normas internacionales al interior de los estados. estudio de caso: la región del Mekong y el protocolo de Palermo contra la trata de personas. (2007-2014)

El presente estudio de caso tiene como principal objetivo el de analizar la manera como las características sociopolíticas de los Estados del Mekong, específicamente en el caso de Camboya y Myanmar, dificultan la implementación de las normas enunciadas en el Protocolo de las Naciones Unidas para Prevenir, Reprimir y Sancionar la Trata de Personas, Especialmente Mujeres y Niños, también conocido como el Protocolo de Palermo. En este sentido, se parte de las características principales del Protocolo y de la manera como el tráfico de personas se presenta en el Mekong para posteriormente analizar la forma como la corrupción, la impunidad y la desigualdad de género representan retos sociopolíticos que obstruyen la implementación de los mandatos internacionales enmarcados en este instrumento

Colombia como referente en la lucha contra las drogas : perspectivas de la cooperación con África Occidental

Este estudio de caso se realiza con el objetivo de analizar cómo la cooperación entre Colombia y África occidental en la lucha contra el tráfico de drogas repercute en la imagen del Estado colombiano como referente en esfuerzos antinarcóticos desde la periferia. En consecuencia, se busca conocer la forma en la cual los acuerdos bilaterales interinstitucionales, la participación en foros y la creación de una agenda internacional de lucha contra las drogas para un escenario nacional transformado, configuran la imagen del Estado colombiano. Para tal objetivo, el trabajo se desarrollará a través de los conceptos de identidad de Alexander Wendt, periferia de Mohammed Ayoob y Cooperación Sur-Sur de la Organización de Naciones Unidas y la Agencia Presidencial de Cooperación Internacional de Colombia.