Selectivity fields: comparative molecular field analysis (CoMFA) of the glycine/NMDA and AMPA receptors

An approach for evaluation of binding selectivity was suggested and exemplified using glycine/NMDA and AMPA receptors. For analyzing the pairwise selectivity, we propose to use the difference between biological activities (expressed as -log Ki) of ligands with respect to different receptor subtypes as a dependent variable for building comparative molecular field analysis (CoMFA) models. The resulting fields (which will be referred to as the "selectivity fields") indicate the ways of increasing selectivity of binding, inhibition, etc. As an example, CoMFA of a set of pyrazolo[1,5-c]quinazolines and triazolo[1,5-c]quinazolines was used for considering the binding selectivity with respect to glycine/NMDA and AMPA receptors. In addition, the mapping of these fields onto the molecular models of the corresponding receptors makes it possible to reveal the reasons for experimentally observed selectivity as well as to suggest additional ways of increasing selectivity.

Autophagic bulk sequestration of cytosolic cargo is independent of LC3, but requires GABARAPs

LC3, a mammalian homologue of yeast Atg8, is assumed to play an important part in bulk sequestration and degradation of cytoplasm (macroautophagy), and is widely used as an indicator of this process. To critically examine its role, we followed the autophagic flux of LC3 in rat hepatocytes during conditions of maximal macroautophagic activity (amino acid depletion), combined with analyses of macroautophagic cargo sequestration, measured as transfer of the cytosolic protein lactate dehydrogenase (LDH) to sedimentable organelles. To accurately determine LC3 turnover we developed a quantitative immunoblotting procedure that corrects for differential immunoreactivity of cytosolic and membrane-associated LC3 forms, and we included cycloheximide to block influx of newly synthesized LC3. As expected, LC3 was initially degraded by the autophagic-lysosomal pathway, but, surprisingly, autophagic LC3-flux ceased after ~2h. In contrast, macroautophagic cargo flux was well maintained, and density gradient analysis showed that sequestered LDH partly accumulated in LC3-free autophagic vacuoles. Hepatocytic macroautophagy could thus proceed independently of LC3. Silencing of either of the two mammalian Atg8 subfamilies in LNCaP prostate cancer cells exposed to macroautophagy-inducing conditions (starvation or the mTOR-inhibitor Torin1) confirmed that macroautophagic sequestration did not require the LC3 subfamily, but, intriguingly, we found the GABARAP subfamily to be essential.

Macroautophagic cargo sequestration assays

Macroautophagy, the process responsible for bulk sequestration and lysosomal degradation of cytoplasm, is often monitored by means of the autophagy-related marker protein LC3. This protein is linked to the phagophoric membrane by lipidation during the final steps of phagophore assembly, and it remains associated with autophagic organelles until it is degraded in the lysosomes. The transfer of LC3 from cytosol to membranes and organelles can be measured by immunoblotting or immunofluorescence microscopy, but these assays provide no information about functional macroautophagic activity, i.e., whether the phagophores are actually engaged in the sequestration of cytoplasmic cargo and enclosing this cargo into sealed autophagosomes. Moreover, accumulating evidence suggest that macroautophagy can proceed independently of LC3. There is therefore a need for alternative methods, preferably effective cargo sequestration assays, which can monitor actual macroautophagic activity. Here, we provide an overview of various approaches that have been used over the last four decades to measure macroautophagic sequestration activity in mammalian cells. Particular emphasis is given to the so-called "LDH sequestration assay", which measures the transfer of the autophagic cargo marker enzyme LDH (lactate dehydrogenase) from the cytosol to autophagic vacuoles. The LDH sequestration assay was originally developed to measure macroautophagic activity in primary rat hepatocytes. Subsequently, it has found use in several other cell types, and in this article we demonstrate a further validation and simplification of the method, and show that it is applicable to several cell lines that are commonly used to study autophagy.

Modulation of intracellular calcium homeostasis blocks autophagosome formation

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress-the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca (2+)-ATPase inhibitor thapsigargin (TG)-potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.

Endosomal signaling and oncogenesis

The endosomal system provides a route whereby nutrients, viruses, and receptors are internalized. During the course of endocytosis, activated receptors can accumulate within endosomal structures and certain signal-transducing molecules can be recruited to endosomal membranes. In the context of signaling and cancer, they provide platforms within the cell from which signals can be potentiated or attenuated. Regulation of the duration of receptor signaling is a pivotal means of refining growth responses in cells. In cancers, this is often considered in terms of mutations that affect receptor tyrosine kinases and maintain them in hyperactivated states of dimerization and/or phosphorylation. However, disruption to the regulatory control exerted by the assembly of protein complexes within the endosomal network can also contribute to disease among which oncogenesis is characterized in part by dysregulated growth, enhanced cell survival, and changes in the expression of markers of differentiation. In this chapter, we will discuss the role of proteins that regulate in endocytosis as tumor suppressors or oncogenes and how changing the fate of internalized receptors and concomitant endosomal signaling can contribute to cancer.

Genome-wide association study for sight-threatening diabetic retinopathy reveals association with genetic variation near the GRB2 gene

AIMS/HYPOTHESIS: Diabetic retinopathy is a serious complication of diabetes mellitus and can lead to blindness. A genetic component, in addition to traditional risk factors, has been well described although strong genetic factors have not yet been identified. Here, we aimed to identify novel genetic risk factors for sight-threatening diabetic retinopathy using a genome-wide association study.

METHODS: Retinopathy was assessed in white Australians with type 2 diabetes mellitus. Genome-wide association analysis was conducted for comparison of cases of sight-threatening diabetic retinopathy (n = 336) with diabetic controls with no retinopathy (n = 508). Top ranking single nucleotide polymorphisms were typed in a type 2 diabetes replication cohort, a type 1 diabetes cohort and an Indian type 2 cohort. A mouse model of proliferative retinopathy was used to assess differential expression of the nearby candidate gene GRB2 by immunohistochemistry and quantitative western blot.

RESULTS: The top ranked variant was rs3805931 with p = 2.66 × 10(-7), but no association was found in the replication cohort. Only rs9896052 (p = 6.55 × 10(-5)) was associated with sight-threatening diabetic retinopathy in both the type 2 (p = 0.035) and the type 1 (p = 0.041) replication cohorts, as well as in the Indian cohort (p = 0.016). The study-wide meta-analysis reached genome-wide significance (p = 4.15 × 10(-8)). The GRB2 gene is located downstream of this variant and a mouse model of retinopathy showed increased GRB2 expression in the retina.

CONCLUSIONS/INTERPRETATION: Genetic variation near GRB2 on chromosome 17q25.1 is associated with sight-threatening diabetic retinopathy. Several genes in this region are promising candidates and in particular GRB2 is upregulated during retinal stress and neovascularisation.

Properties of self-assembled diluted magnetic semiconductor nanostructures

Este trabalho centra-se na investigação da possibilidade de se conseguir um semicondutor magnético diluído (SMD) baseado em ZnO. Foi levado a cabo um estudo detalhado das propriedades magnéticas e estruturais de estruturas de ZnO, nomeadamente nanofios (NFs), nanocristais (NCs) e filmes finos, dopadas com metais de transição (MTs). Foram usadas várias técnicas experimentais para caracterizar estas estruturas, designadamente difracção de raios-X, microscopia electrónica de varrimento, ressonância magnética, SQUID, e medidas de transporte. Foram incorporados substitucionalmente nos sítios do Zn iões de Mn2+ e Co2+ em ambos os NFs e NCs de ZnO. Revelou-se para ambos os iões dopantes, que a incorporação é heterogénea, uma vez que parte do sinal de ressonância paramagnética electrónica (RPE) vem de iões de MTs em ambientes distorcidos ou enriquecidos com MTs. A partir das intensidades relativas dos espectros de RPE e de modificações da superfície, demonstra-se ainda que os NCs exibem uma estrutura core-shell. Os resultados, evidenciam que, com o aumento da concentração de MTs, a dimensão dos NCs diminui e aumentam as distorções da rede. Finalmente, no caso dos NCs dopados com Mn, obteve-se o resultado singular de que a espessura da shell é da ordem de 0.3 nm e de que existe uma acumulação de Mn na mesma. Com o objectivo de esclarecer o papel dos portadores de carga na medição das interacções ferromagnéticas, foram co-dopados filmes de ZnO com Mn e Al ou com Co e Al. Os filmes dopados com Mn, revelaram-se simplesmente paramagnéticos, com os iões de Mn substitucionais nos sítios do Zn. Por outro lado, os filmes dopados com Co exibem ferromagnetismo fraco não intrínseco, provavelmente devido a decomposição spinodal. Foram ainda efectuados estudos comparativos com filmes de ligas de Zn1-xFexO. Como era de esperar, detectaram-se segundas fases de espinela e de óxido de ferro nestas ligas; todas as amostras exibiam curvas de histerese a 300 K. Estes resultados suportam a hipótese de que as segundas fases são responsáveis pelo comportamento magnético observado em muitos sistemas baseados em ZnO. Não se observou nenhuma evidência de ferromagnetismo mediado por portadores de carga. As experiências mostram que a análise de RPE permite demonstrar directamente se e onde estão incorporados os iões de MTs e evidenciam a importância dos efeitos de superfície para dimensões menores que ~15 nm, para as quais se formam estruturas core-shell. As investigações realizadas no âmbito desta tese demonstram que nenhuma das amostras de ZnO estudadas exibiram propriedades de um SMD intrínseco e que, no futuro, são necessários estudos teóricos e experimentais detalhados das interacções de troca entre os iões de MTs e os átomos do ZnO para determinar a origem das propriedades magnéticas observadas.

Radiation-induced defects in quantum-size structures of A3B5 semiconductors

As estruturas quânticas de semicondutores, nomeadamente baseadas em GaAs, têm tido nos últimos vinte anos um claro desenvolvimento. Este desenvolvimento deve-se principalmente ao potencial tecnológico que estas estruturas apresentam. As aplicações espaciais, em ambientes agressivos do ponto de vista do nível de radiação a que os dispositivos estão sujeitos, motivaram todo o desenrolar de estudos na área dos defeitos induzidos pela radiação. As propriedades dos semicondutores e dos dispositivos de semicondutores são altamente influenciadas pela presença de defeitos estruturais, em particular os induzidos pela radiação. As propriedades dos defeitos, os processos de criação e transformação de defeitos devem ser fortemente alterados quando se efectua a transição entre o semicondutor volúmico e as heteroestruturas de baixa dimensão. Este trabalho teve como principal objectivo o estudo de defeitos induzidos pela radiação em estruturas quânticas baseadas em GaAs e InAs. Foram avaliadas as alterações introduzidas pelos defeitos em estruturas de poços quânticos e de pontos quânticos irradiadas com electrões e com protões. A utilização de várias técnicas de espectroscopia óptica, fotoluminescência, excitação de fotoluminescência e fotoluminescência resolvida no tempo, permitiu caracterizar as diferentes estruturas antes e após a irradiação. Foi inequivocamente constatada uma maior resistência à radiação dos pontos quânticos quando comparados com os poços quânticos e os materiais volúmicos. Esta resistência deve-se principalmente a uma maior localização da função de onda dos portadores com o aumento do confinamento dos mesmos. Outra razão provável é a expulsão dos defeitos dos pontos quânticos para a matriz. No entanto, a existência de defeitos na vizinhança dos pontos quânticos promove a fuga dos portadores dos níveis excitados, cujas funções de onda são menos localizadas, provocando um aumento da recombinação nãoradiativa e, consequentemente, uma diminuição da intensidade de luminescência dos dispositivos. O desenvolvimento de um modelo bastante simples para a estatística de portadores fora de equilíbrio permitiu reproduzir os resultados de luminescência em função da temperatura. Os resultados demonstraram que a extinção da luminescência com o aumento da temperatura é determinada por dois factores: a redistribuição dos portadores minoritários entre os pontos quânticos, o poço quântico e as barreiras de GaAs e a diminuição na taxa de recombinação radiativa relacionada com a dependência, na temperatura, do nível de Fermi dos portadores maioritários.

Order and the literary rendering of chaos : children's literature as knowledge, order, and social foundation

Depuis que l'animal humain a conçu un système de technologies pour la pensée abstraite grâce au langage, la guerre contre le monde sauvage est devenu une voie à sens unique vers l'aliénation, la civilisation et la littérature. Le but de ce travail est d'analyser comment les récits civilisationnels donnent une structure à l'expérience par le biais de la ségrégation, de la domestication, de la sélection, et de l'extermination, tandis que les récits sauvages démontrent les possibilités infinies du chaos pour découvrir le monde en toute sa diversité et en lien avec sa communauté de vie. Un des objectifs de cette thèse a été de combler le fossé entre la science et la littérature, et d'examiner l'interdépendance de la fiction et la réalité. Un autre objectif a été de mettre ces récits au cœur d'un dialogue les uns avec les autres, ainsi que de tracer leur expression dans les différentes disciplines et œuvres pour enfants et adultes mais également d’analyser leur manifestations c’est redondant dans la vie réelle. C'est un effort multi-disciplinaires qui se reflète dans la combinaison de méthodes de recherche en anthropologie et en études littéraires. Cette analyse compare et contraste trois livres de fiction pour enfants qui présentent trois différents paradigmes socio-économiques, à savoir, «Winnie-l'Ourson» de Milne qui met en place un monde civilisé monarcho-capitaliste, la trilogie de Nosov sur «les aventures de Neznaika et ses amis» qui présente les défis et les exploits d'une société anarcho-socialiste dans son évolution du primitivisme vers la technologie, et les livres de Moomines de Jansson, qui représentent le chaos, l'anarchie, et l'état sauvage qui contient tout, y compris des épisodes de civilisation. En axant la méthodologie de ma recherche sur la façon dont nous connaissons le monde, j'ai d'abord examiné la construction, la transmission et l'acquisition des connaissances, en particulier à travers la théorie de praxis de Bourdieu et la critique de la civilisation développée dans les études de Zerzan, Ong, et Goody sur les liens entre l'alphabétisation, la dette et l'oppression. Quant à la littérature pour enfants, j'ai choisi trois livres que j’ai connus pendant mon enfance, c'est-à-dire des livres qui sont devenus comme une «langue maternelle» pour moi. En ce sens, ce travail est aussi de «l’anthropologie du champ natif». En outre, j’analyse les prémisses sous-jacentes qui se trouvent non seulement dans les trois livres, mais dans le déroulement des récits de l'état sauvage et de la civilisation dans la vie réelle, des analyses qui paraissent dans cette thèse sous la forme d'extraits d’un journal ethnographique. De même que j’examine la nature de la littérature ainsi que des structures civilisées qui domestiquent le monde au moyen de menaces de mort, je trace aussi la présence de ces récits dans l'expression scientifique (le récit malthusien-darwinien), religieuse, et dans autres expressions culturelles, et réfléchis sur les défis présentés par la théorie anarchiste (Kropotkine) ainsi que par les livres pour enfants écrits du point de vue sauvage, tels que ceux des Moomines.

From Market- to Development Orientation - The Trade Aspect of Food Security and Agriculture

Trade rules are suggested to be one of the reasons for the hunger in the world and environmental damage. As current trade rules encourage market orientation and therefore specialization and industrialization of agriculture, which has as side effects rural hunger and environmental damage, there is room for improvement in the international trade regime. One main finding of Nexus Foundations' work in Geneva is a possible new orientation for agricultural and food markets – an orientation on development, rather than purely on markets. This development orientation consists of several elements from development of soil fertility to local markets and consumer relatedness. Since the Bali Ministerial in 2013, the WTO has set up a four year work programme on the issue of food security related to food reserves. This opens the chance to discuss broader food security issues in the realm of trade negotiations.

Análisis de las tensiones geopolíticas entre Kirguistán y Uzbekistán por el Valle de Fergana y sus recursos energéticos durante el periodo 2001-2010

Las tensiones geopolíticas entre Kirguistán y Uzbekistán por el Valle de Fergana durante el periodo 2001-2010 a partir de un análisis histórico de la formación de la población y la influencia de los diferentes imperios en la región. Adicionalmente, los aspectos relativos a la importancia de los recursos energéticos en el Valle de Fergana como la configuración de las tensiones generadas entre estos dos países, haciendo énfasis en el conflicto étnico latente que se ha generado en la zona. De se utilizarán la teoría constructivista de Alexander Wendt y la teoría de la geopolítica de Yves Lacoste.