Heterochromatin und epigenetische Kontrolle der Centromere in Dictyostelium discoideum

Heterochromatin Protein 1 (HP1) is an evolutionarily conserved protein required for formation of a higher-order chromatin structures and epigenetic gene silencing. The objective of the present work was to functionally characterise HP1-like proteins in Dictyostelium discoideum, and to investigate their function in heterochromatin formation and transcriptional gene silencing. The Dictyostelium genome encodes three HP1-like proteins (hcpA, hcpB, hcpC), from which only two, hcpA and hcpB, but not hcpC were found to be expressed during vegetative growth and under developmental conditions. Therefore, hcpC, albeit no obvious pseudogene, was excluded from this study. Both HcpA and HcpB show the characteristic conserved domain structure of HP1 proteins, consisting of an N-terminal chromo domain and a C-terminal chromo shadow domain, which are separated by a hinge. Both proteins show all biochemical activities characteristic for HP1 proteins, such as homo- and heterodimerisation in vitro and in vivo, and DNA binding activtity. HcpA furthermore seems to bind to K9-methylated histone H3 in vitro. The proteins thus appear to be structurally and functionally conserved in Dictyostelium. The proteins display largely identical subnuclear distribution in several minor foci and concentration in one major cluster at the nuclear periphery. The localisation of this cluster adjacent to the nucleus-associated centrosome and its mitotic behaviour strongly suggest that it represents centromeric heterochromatin. Furthermore, it is characterised by histone H3 lysine-9 dimethylation (H3K9me2), which is another hallmark of Dictyostelium heterochromatin. Therefore, one important aspect of the work was to characterise the so-far largely unknown structural organisation of centromeric heterochromatin. The Dictyostelium homologue of inner centromere protein INCENP (DdINCENP), co-localized with both HcpA and H3K9me2 during metaphase, providing further evidence that H3K9me2 and HcpA/B localisation represent centromeric heterochromatin. Chromatin immunoprecipitation (ChIP) showed that two types of high-copy number retrotransposons (DIRS-1 and skipper), which form large irregular arrays at the chromosome ends, which are thought to contain the Dictyostelium centromeres, are characterised by H3K9me2. Neither overexpression of full-length HcpA or HcpB, nor deletion of single Hcp isoforms resulted in changes in retrotransposon transcript levels. However, overexpression of a C-terminally truncated HcpA protein, assumed to display a dominant negative effect, lead to an increase in skipper retrotransposon transcript levels. Furthermore, overexpression of this protein lead to severe growth defects in axenic suspension culture and reduced cell viability. In order to elucidate the proteins functions in centromeric heterochromatin formation, gene knock-outs for both hcpA and hcpB were generated. Both genes could be successfully targeted and disrupted by homologous recombination. Surprisingly, the degree of functional redundancy of the two isoforms was, although not unexpected, very high. Both single knock-out mutants did not show any obvious phenotypes under standard laboratory conditions and only deletion of hcpA resulted in subtle growth phenotypes when grown at low temperature. All attempts to generate a double null mutant failed. However, both endogenous genes could be disrupted in cells in which a rescue construct that ectopically expressed one of the isoforms either with N-terminal 6xHis- or GFP-tag had been introduced. The data imply that the presence of at least one Hcp isoform is essential in Dictyostelium. The lethality of the hcpA/hcpB double mutant thus greatly hampered functional analysis of the two genes. However, the experiment provided genetic evidence that the GFP-HcpA fusion protein, because of its ability to compensate the loss of the endogenous HcpA protein, was a functional protein. The proteins displayed quantitative differences in dimerisation behaviour, which are conferred by the slightly different hinge and chromo shadow domains at the C-termini. Dimerisation preferences in increasing order were HcpA-HcpA << HcpA-HcpB << HcpB-HcpB. Overexpression of GFP-HcpA or a chimeric protein containing the HcpA C-terminus (GFP-HcpBNAC), but not overexpression of GFP-HcpB or GFP-HcpANBC, lead to increased frequencies of anaphase bridges in late mitotic cells, which are thought to be caused by telomere-telomere fusions. Chromatin targeting of the two proteins is achieved by at least two distinct mechanisms. The N-terminal chromo domain and hinge of the proteins are required for targeting to centromeric heterochromatin, while the C-terminal portion encoding the CSD is required for targeting to several other chromatin regions at the nuclear periphery that are characterised by H3K9me2. Targeting to centromeric heterochromatin likely involves direct binding to DNA. The Dictyostelium genome encodes for all subunits of the origin recognition complex (ORC), which is a possible upstream component of HP1 targeting to chromatin. Overexpression of GFP-tagged OrcB, the Dictyostelium Orc2 homologue, showed a distinct nuclear localisation that partially overlapped with the HcpA distribution. Furthermore, GFP-OrcB localized to the centrosome during the entire cell cycle, indicating an involvement in centrosome function. DnmA is the sole DNA methyltransferase in Dictyostelium required for all DNA(cytosine-)methylation. To test for its in vivo activity, two different cell lines were established that ectopically expressed DnmA-myc or DnmA-GFP. It was assumed that overexpression of these proteins might cause an increase in the 5-methyl-cytosine(5-mC)-levels in the genomic DNA due to genomic hypermethylation. Although DnmA-GFP showed preferential localisation in the nucleus, no changes in the 5-mC-levels in the genomic DNA could be detected by capillary electrophoresis.

1st Kassel Student Workshop on Security in Distributed Systems

With this document, we provide a compilation of in-depth discussions on some of the most current security issues in distributed systems. The six contributions have been collected and presented at the 1st Kassel Student Workshop on Security in Distributed Systems (KaSWoSDS’08). We are pleased to present a collection of papers not only shedding light on the theoretical aspects of their topics, but also being accompanied with elaborate practical examples. In Chapter 1, Stephan Opfer discusses Viruses, one of the oldest threats to system security. For years there has been an arms race between virus producers and anti-virus software providers, with no end in sight. Stefan Triller demonstrates how malicious code can be injected in a target process using a buffer overflow in Chapter 2. Websites usually store their data and user information in data bases. Like buffer overflows, the possibilities of performing SQL injection attacks targeting such data bases are left open by unwary programmers. Stephan Scheuermann gives us a deeper insight into the mechanisms behind such attacks in Chapter 3. Cross-site scripting (XSS) is a method to insert malicious code into websites viewed by other users. Michael Blumenstein explains this issue in Chapter 4. Code can be injected in other websites via XSS attacks in order to spy out data of internet users, spoofing subsumes all methods that directly involve taking on a false identity. In Chapter 5, Till Amma shows us different ways how this can be done and how it is prevented. Last but not least, cryptographic methods are used to encode confidential data in a way that even if it got in the wrong hands, the culprits cannot decode it. Over the centuries, many different ciphers have been developed, applied, and finally broken. Ilhan Glogic sketches this history in Chapter 6.

Nuclear organisation and epigenetic regulation of gene expression in Dictyostelium discoideum

A series of vectors for the over-expression of tagged proteins in Dictyostelium were designed, constructed and tested. These vectors allow the addition of an N- or C-terminal tag (GFP, RFP, 3xFLAG, 3xHA, 6xMYC and TAP) with an optimized polylinker sequence and no additional amino acid residues at the N or C terminus. Different selectable markers (Blasticidin and gentamicin) are available as well as an extra chromosomal version; these allow copy number and thus expression level to be controlled, as well as allowing for more options with regard to complementation, co- and super-transformation. Finally, the vectors share standardized cloning sites, allowing a gene of interest to be easily transfered between the different versions of the vectors as experimental requirements evolve. The organisation and dynamics of the Dictyostelium nucleus during the cell cycle was investigated. The centromeric histone H3 (CenH3) variant serves to target the kinetochore to the centromeres and thus ensures correct chromosome segregation during mitosis and meiosis. A number of Dictyostelium histone H3-domain containing proteins as GFP-tagged fusions were expressed and it was found that one of them functions as CenH3 in this species. Like CenH3 from some other species, Dictyostelium CenH3 has an extended N-terminal domain with no similarity to any other known proteins. The targeting domain, comprising α-helix 2 and loop 1 of the histone fold is required for targeting CenH3 to centromeres. Compared to the targeting domain of other known and putative CenH3 species, Dictyostelium CenH3 has a shorter loop 1 region. The localisation of a variety of histone modifications and histone modifying enzymes was examined. Using fluorescence in situ hybridisation (FISH) and CenH3 chromatin-immunoprecipitation (ChIP) it was shown that the six telocentric centromeres contain all of the DIRS-1 and most of the DDT-A and skipper transposons. During interphase the centromeres remain attached to the centrosome resulting in a single CenH3 cluster which also contains the putative histone H3K9 methyltransferase SuvA, H3K9me3 and HP1 (heterochromatin protein 1). Except for the centromere cluster and a number of small foci at the nuclear periphery opposite the centromeres, the rest of the nucleus is largely devoid of transposons and heterochromatin associated histone modifications. At least some of the small foci correspond to the distal telomeres, suggesting that the chromosomes are organised in a Rabl-like manner. It was found that in contrast to metazoans, loading of CenH3 onto Dictyostelium centromeres occurs in late G2 phase. Transformation of Dictyostelium with vectors carrying the G418 resistance cassette typically results in the vector integrating into the genome in one or a few tandem arrays of approximately a hundred copies. In contrast, plasmids containing a Blasticidin resistance cassette integrate as single or a few copies. The behaviour of transgenes in the nucleus was examined by FISH, and it was found that low copy transgenes show apparently random distribution within the nucleus, while transgenes with more than approximately 10 copies cluster at or immediately adjacent to the centromeres in interphase cells regardless of the actual integration site along the chromosome. During mitosis the transgenes show centromere-like behaviour, and ChIP experiments show that transgenes contain the heterochromatin marker H3K9me2 and the centromeric histone variant H3v1. This clustering, and centromere-like behaviour was not observed on extrachromosomal transgenes, nor on a line where the transgene had integrated into the extrachromosomal rDNA palindrome. This suggests that it is the repetitive nature of the transgenes that causes the centromere-like behaviour. A Dictyostelium homolog of DET1, a protein largely restricted to multicellular eukaryotes where it has a role in developmental regulation was identified. As in other species Dictyostelium DET1 is nuclear localised. In ChIP experiments DET1 was found to bind the promoters of a number of developmentally regulated loci. In contrast to other species where it is an essential protein, loss of DET1 is not lethal in Dictyostelium, although viability is greatly reduced. Loss of DET1 results in delayed and abnormal development with enlarged aggregation territories. Mutant slugs displayed apparent cell type patterning with a bias towards pre-stalk cell types.

Producers’ motivation for collective action for kola production and marketing in Cameroon

Collective action has been used as a strategy to improve the benefits of smallholder producers of kola nuts in Cameroon. Despite demonstrated benefits, not all producers are involved in the collective action. The presented study used a modified Technology Acceptance Model (TAM) namely the Collective Action Behaviour model (CAB model) to analyse kola producers’ motivation for collective action activities. Five hypotheses are formulated and tested using data obtained from 185 farmers who are involved in kola production and marketing in theWestern highlands of Cameroon. Results which were generated using Partial Least Squares (PLS) approach for Structural Equation Modelling (SEM) showed that farmers’ intrinsic motivators and ease of use influenced their behavioural intent to join a group marketing activities. The perceived usefulness that was mainly related to the economic benefits of group activities did not influence farmers’ behavioural intent. It is therefore concluded that extension messages and promotional activities targeting collective action need to emphasise the perceived ease of use of involvement and social benefits associated with group activities in order to increase farmers’ participation.

Development and Integration of Biocontrol Products in Branched Broomrape (Phelipanche ramosa) management in Tomato

Parasitic weeds of the genera Striga, Orobanche, and Phelipanche pose a severe problem for agriculture because they are difficult to control and are highly destructive to several crops. The present work was carried out during the period October, 2009 to February, 2012 to evaluate the potential of arbuscular mycorrhizal fungi (AMF) to suppress P. ramosa on tomatoes and to investigate the effects of air-dried powder and aqueous extracts from Euphorbia hirta on germination and haustorium initiation in Phelipanche ramosa. The work was divided into three parts: a survey of the indigenous mycorrhizal flora in Sudan, second, laboratory and greenhouse experiments (conducted in Germany and Sudan) to construct a base for the third part, which was a field trial in Sudan. A survey was performed in 2009 in the White Nile state, Sudan to assess AMF spore densities and root colonization in nine fields planted with 13 different important agricultural crops. In addition, an attempt was made to study the relationship between soil physico-chemical properties and AMF spore density, colonization rate, species richness and other diversity indices. The mean percentage of AMF colonization was 34%, ranging from 19-50%. The spore densities (expressed as per 100 g dry soil) retrieved from the rhizosphere of different crops were relatively high, varying from 344 to 1222 with a mean of 798. There was no correlation between spore densities in soil and root colonization percentage. A total of 45 morphologically classifiable species representing ten genera of AMF were detected with no correlation between the number of species found in a soil sample and the spore density. The most abundant genus was Glomus (20 species). The AMF diversity expressed by the Shannon–Weaver index was highest in sorghum (H\= 2.27) and Jews mallow (H\= 2.13) and lowest in alfalfa (H\= 1.4). With respect to crop species, the genera Glomus and Entrophospora were encountered in almost all crops, except for Entrophospora in alfalfa. Kuklospora was found only in sugarcane and sorghum. The genus Ambispora was recovered only in mint and okra, while mint and onion were the only species on which no Acaulospora was found. The hierarchical cluster analysis based on the similarity among AMF communities with respect to crop species overall showed that species compositions were relatively similar with the highest dissimilarity of about 25% separating three of the mango samples and the four sorghum samples from all other samples. Laboratory experiments studied the influence of root and stem exudates of three tomato varieties infected by three different Glomus species on germination of P. ramosa. Root exudates were collected 21or 42 days after transplanting (DAT) and stem exudates 42 DAT and tested for their effects on germination of P. ramosa seeds in vitro. The tomato varieties studied did not have an effect on either mycorrhizal colonization or Phelipanche germination. Germination in response to exudates from 42 day old mycorrhizal plants was significantly reduced in comparison to non-mycorrhizal controls. Germination of P. ramosa in response to root exudates from 21 day old plants was consistently higher than for 42 day-old plants (F=121.6; P<.0001). Stem diffusates from non-mycorrhizal plants invariably elicited higher germination than diffusates from the corresponding mycorrhizal ones and differences were mostly statistically significant. A series of laboratory experiments was undertaken to investigate the effects of aqueous extracts from Euphorbia hirta on germination, radicle elongation, and haustorium initiation in P. ramosa. P. ramosa seeds conditioned in water and subsequently treated with diluted E. hirta extract (10-25% v/v) displayed considerable germination (47-62%). Increasing extract concentration to 50% or more reduced germination in response to the synthetic germination stimulants GR24 and Nijmegen-1 in a concentration dependent manner. P. ramosa germlings treated with diluted Euphorbia extract (10-75 % v/v) displayed haustorium initiation comparable to 2, 5-Dimethoxy-p-benzoquinon (DMBQ) at 20 µM. Euphorbia extract applied during conditioning reduced haustorium initiation in a concentration dependent manner. E. hirta extract or air-dried powder, applied to soil, induced considerable P. ramosa germination. Pot experiments were undertaken in a glasshouse at the University of Kassel, Germany, to investigate the effects of P. ramosa seed bank on tomato growth parameters. Different Phelipanche seed banks were established by mixing the parasite seeds (0 - 32 mg) with the potting medium in each pot. P. ramosa reduced all tomato growth parameters measured and the reduction progressively increased with seed bank. Root and total dry matter accumulation per tomato plant were most affected. P. ramosa emergence, number of tubercles, and tubercle dry weight increased with the seed bank and were, invariably, maximal with the highest seed bank. Another objective was to determine if different AM fungi differ in their effects on the colonization of tomatoes with P. ramosa and the performance of P. ramosa after colonization. Three AMF species viz. GIomus intraradices, Glomus mosseae and Glomus Sprint® were used in this study. For the infection, P. ramosa seeds (8 mg) were mixed with the top 5 cm soil in each pot. No mycorrhizal colonization was detected in un-inoculated control plants. P. ramosa infested, mycorrhiza inoculated tomato plants had significantly lower AMF colonization compared to plants not infested with P. ramosa. Inoculation with G. intraradices, G. mosseae and Glomus Sprint® reduced the number of emerged P. ramosa plants by 29.3, 45.3 and 62.7% and the number of tubercles by 22.2, 42 and 56.8%, respectively. Mycorrhizal root colonization was positively correlated with number of branches and total dry matter of tomatoes. Field experiments on tomato undertaken in 2010/12 were only partially successful because of insect infestations which resulted in the complete destruction of the second run of the experiment. The effects of the inoculation with AMF, the addition of 10 t ha-1 filter mud (FM), an organic residues from sugar processing and 36 or 72 kg N ha-1 on the infestation of tomatoes with P. ramosa were assessed. In un-inoculated control plants, AMF colonization ranged between 13.4 to 22.1% with no significant differences among FM and N treatments. Adding AMF or FM resulted in a significant increase of branching in the tomato plants with no additive effects. Dry weights were slightly increased through FM application when no N was applied and significantly at 36 kg N ha-1. There was no effect of FM on the time until the first Phelipanche emerged while AMF and N application interacted. Especially AMF inoculation resulted in a tendency to delayed P. ramosa emergence. The marketable yield was extremely low due to the strong fruit infestation with insects mainly whitefly Bemisia tabaci and tomato leaf miner (Tuta absoluta). Tomatoes inoculated with varied mycorrhiza species displayed different response to the insect infestation, as G. intraradices significantly reduced the infestation, while G. mosseae elicited higher insect infestation. The results of the present thesis indicate that there may be a potential of developing management strategies for P. ramosa targeting the pre-attachment stage namely germination and haustorial initiation using plant extracts. However, ways of practical use need to be developed. If such treatments can be combined with AMF inoculation also needs to be investigated. Overall, it will require a systematic approach to develop management tools that are easily applicable and affordable to Sudanese farmers. It is well-known that proper agronomical practices such as the design of an optimum crop rotation in cropping systems, reduced tillage, promotion of cover crops, the introduction of multi-microbial inoculants, and maintenance of proper phosphorus levels are advantageous if the mycorrhiza protection method is exploited against Phelipanche ramosa infestation. Without the knowledge about the biology of the parasitic weeds by the farmers and basic preventive measures such as hygiene and seed quality control no control strategy will be successful, however.

Retargeting of pre-set regions on chromosome for high gene expression in mammalian cells

We have developed a system to hunt and reuse special gene integration sites that allow for high and stable gene expression. A vector, named pRGFP8, was constructed. The plasmid pRGFP8 contains a reporter gene, gfp2 and two extraneous DNA fragments. The gene gfp2 makes it possible to screen the high expression regions on the chromosome. The extraneous DNA fragments can help to create the unique loci on the chromosome and increase the gene targeting frequency by increasing the homology. After transfection into Chinese hamster ovary cells (CHO) cells, the linearized pRGFP8 can integrate into the chromosome of the host cells and form the unique sites. With FACS, 90 millions transfected cells were sorted and the cells with strongest GFP expression were isolated, and then 8 stable high expression GFP CHO cell lines were selected as candidates for the new host cell. Taking the unique site created by pRGFP8 on the chromosome in the new host cells as a targeting locus, the gfp2 gene was replaced with the gene of interest, human ifngamma, by transfecting the targeting plasmid pRIH-IFN. Then using FACS, the cells with the dimmest GFP fluorescence were selected. These cells showed they had strong abilities to produce the protein of interest, IFN-gamma. During the gene targeting experiment, we found there is positive correlation between the fluorescence density of the GFP CHO host cells and the specific production rate of IFN-gamma. This result shows that the strategy in our expression system is correct: the production of the interesting protein increases with the increase fluorescence of the GFP host cells. This system, the new host cell lines and the targeting vector, can be utilized for highly expressing the gene of interest. More importantly, by using FACS, we can fully screen all the transfected cells, which can reduce the chances of losing the best cells.

Identification of plasmodium vivax proteins with potential role in invasion using sequence redundancy reduction and profile hidden markov models

Background: This study describes a bioinformatics approach designed to identify Plasmodium vivax proteins potentially involved in reticulocyte invasion. Specifically, different protein training sets were built and tuned based on different biological parameters, such as experimental evidence of secretion and/or involvement in invasion-related processes. A profile-based sequence method supported by hidden Markov models (HMMs) was then used to build classifiers to search for biologically-related proteins. The transcriptional profile of the P. vivax intra-erythrocyte developmental cycle was then screened using these classifiers. Results: A bioinformatics methodology for identifying potentially secreted P. vivax proteins was designed using sequence redundancy reduction and probabilistic profiles. This methodology led to identifying a set of 45 proteins that are potentially secreted during the P. vivax intra-erythrocyte development cycle and could be involved in cell invasion. Thirteen of the 45 proteins have already been described as vaccine candidates; there is experimental evidence of protein expression for 7 of the 32 remaining ones, while no previous studies of expression, function or immunology have been carried out for the additional 25. Conclusions: The results support the idea that probabilistic techniques like profile HMMs improve similarity searches. Also, different adjustments such as sequence redundancy reduction using Pisces or Cd-Hit allowed data clustering based on rational reproducible measurements. This kind of approach for selecting proteins with specific functions is highly important for supporting large-scale analyses that could aid in the identification of genes encoding potential new target antigens for vaccine development and drug design. The present study has led to targeting 32 proteins for further testing regarding their ability to induce protective immune responses against P. vivax malaria.

A "Trojan horse" strategy to reverse drug-resistance in brain tumors

Los gliomas malignos representan una de las formas más agresivas de los tumores del sistema nervioso central (SNC). De acuerdo con la clasificación de los tumores cerebrales de la Organización Mundial de la Salud (OMS), los astrocitomas han sido categorizados en cuatro grados, determinados por la patología subyacente. Es así como los gliomas malignos (o de alto grado) incluyen el glioma anaplásico (grado III) así como el glioblastoma multiforme (GBM, grado IV),estos últimos los más agresivos con el peor pronóstico (1). El manejo terapéutico de los tumores del SNC se basa en la cirugía, la radioterapia y la quimioterapia, dependiendo de las características del tumor, el estadio clínico y la edad (2),(3), sin embargo ninguno de los tratamientos estándar es completamente seguro y compatible con una calidad de vida aceptable (3), (4). En general, la quimioterapia es la primera opción en los tumores diseminados, como el glioblastoma invasivo y el meduloblastoma de alto riesgo o con metástasis múltiple, pero el pronóstico en estos pacientes es muy pobre (2),(3). Solamente nuevas terapias dirigidas (2) como las terapias anti-angiogénicas (4); o terapias génicas muestran un beneficio real en grupos limitados de pacientes con defectos moleculares específicos conocidos (4). De este modo, se hace necesario el desarrollo de nuevas terapias farmacológicas para atacar los tumores cerebrales. Frente a las terapias los gliomas malignos son con frecuencia quimioresistentes, y esta resistencia parece depender de al menos dos mecanismos: en primer lugar, la pobre penetración de muchas drogas anticáncer a través de la barrera hematoencefálica (BBB: Blood Brain Barrier), la barrera del fluido sangre-cerebroespinal (BCSFB: Blood-cerebrospinal fluid barrier) y la barrera sangre-tumor (BTB: blood-tumor barrier). Dicha resistencia se debe a la interacción de la droga con varios transportadores o bombas de eflujo de droga ABC (ABC: ATP-binding cassette) que se sobre expresan en las células endoteliales o epiteliales de estas barreras. En segundo lugar, estos transportadores de eflujo de drogas ABC propios de las células tumorales confieren un fenotipo conocido como resistencia a multidrogas (MDR: multidrug resistance), el cual es característico de varios tumores sólidos. Este fenotipo también está presente en los tumores del SNC y su papel en gliomas es objeto de investigación (5). Por consiguiente el suministro de medicamentos a través de la BBB es uno de los problemas vitales en los tratamientos de terapia dirigida. Estudios recientes han demostrado que algunas moléculas pequeñas utilizadas en estas terapias son sustratos de la glicoproteína P (Pgp: P-gycoprotein), así como también de otras bombas de eflujo como las proteínas relacionadas con la resistencia a multidrogas (MRPs: multidrug resistance-related proteins (MRPs) o la proteína relacionada con cáncer de seno (BCRP: breast-cancer resistance related protein)) que no permiten que las drogas de este tipo alcancen el tumor (1). Un sustrato de Pgp y BCRP es la DOXOrubicina (DOXO), un fármaco utilizado en la terapia anti cáncer, el cual es muy eficaz para atacar las células del tumor cerebral in vitro, pero con un uso clínico limitado por la poca entrega a través de la barrera hematoencefálica (BBB) y por la resistencia propia de los tumores. Por otra parte las células de BBB y las células del tumor cerebral tienen también proteínas superficiales, como el receptor de la lipoproteína de baja densidad (LDLR), que podría utilizarse como blanco terapéutico en BBB y tumores cerebrales. Es asi como la importancia de este estudio se basa en la generación de estrategias terapéuticas que promuevan el paso de las drogas a través de la barrera hematoencefalica y tumoral, y a su vez, se reconozcan mecanismos celulares que induzcan el incremento en la expresión de los transportadores ABC, de manera que puedan ser utilizados como blancos terapéuticos.Este estudio demostró que el uso de una nueva estrategia basada en el “Caballo de Troya”, donde se combina la droga DOXOrubicina, la cual es introducida dentro de un liposoma, salvaguarda la droga de manera que se evita su reconocimiento por parte de los transportadores ABC tanto de la BBB como de las células del tumor. La construcción del liposoma permitió utilizar el receptor LDLR de las células asegurando la entrada a través de la BBB y hacia las células tumorales a través de un proceso de endocitosis. Este mecanismo fue asociado al uso de estatinas o drogas anticolesterol las cuales favorecieron la expresión de LDLR y disminuyeron la actividad de los transportadores ABC por nitración de los mismos, incrementando la eficiencia de nuestro Caballo de Troya. Por consiguiente demostramos que el uso de una nueva estrategia o formulación denominada ApolipoDOXO más el uso de estatinas favorece la administración de fármacos a través de la BBB, venciendo la resistencia del tumor y reduciendo los efectos colaterales dosis dependiente de la DOXOrubicina. Además esta estrategia del "Caballo de Troya", es un nuevo enfoque terapéutico que puede ser considerado como una nueva estrategia para aumentar la eficacia de diferentes fármacos en varios tumores cerebrales y garantiza una alta eficiencia incluso en un medio hipóxico,característico de las células cancerosas, donde la expresión del transportador Pgp se vió aumentada. Teniendo en cuenta la relación entre algunas vías de señalización reconocidas como moduladores de la actividad de Pgp, este estudio presenta no solo la estrategia del Caballo de Troya, sino también otra propuesta terapéutica relacionada con el uso de Temozolomide más DOXOrubicina. Esta estrategia demostró que el temozolomide logra penetrar la BBB por que interviene en la via de señalización de la Wnt/GSK3/β-catenina, la cual modula la expresión del transportador Pgp. Se demostró que el TMZ disminuye la proteína y el mRNA de Wnt3 permitiendo plantear la hipótesis de que la droga al disminuir la transcripción del gen Wnt3 en células de BBB, incrementa la activación de la vía fosforilando la β-catenina y conduciendo a disminuir la β-catenina nuclear y por tanto su unión al promotor del gen mdr1. Con base en los resultados este estudio permitió el reconocimiento de tres mecanismos básicos relacionados con la expresión de los transportadores ABC y asociados a las estrategias empleadas: el primero fue el uso de las estatinas, el cual condujo a la nitración de los transportadores disminuyendo su actividad por la via del factor de transcripción NFκB; el segundo a partir del uso del temozolomide, el cual metila el gen de Wnt3 reduciendo la actividad de la via de señalización de la la β-catenina, disminuyendo la expresión del transportador Pgp. El tercero consistió en la determinación de la relación entre el eje RhoA/RhoA quinasa como un modulador de la via (no canónica) GSK3/β-catenina. Se demostró que la proteína quinasa RhoA promovió la activación de la proteína PTB1, la cual al fosforilar a GSK3 indujo la fosforilación de la β-catenina, lo cual dio lugar a su destrucción por el proteosoma, evitando su unión al promotor del gen mdr1 y por tanto reduciendo su expresión. En conclusión las estrategias propuestas en este trabajo incrementaron la citotoxicidad de las células tumorales al aumentar la permeabilidad no solo de la barrera hematoencefálica, sino también de la propia barrera tumoral. Igualmente, la estrategia del “Caballo de Troya” podría ser útil para la terapia de otras enfermedades asociadas al sistema nervioso central. Por otra parte estos estudios indican que el reconocimiento de mecanismos asociados a la expresión de los transportadores ABC podría constituir una herramienta clave en el desarrollo de nuevas terapias anticáncer.

Media bias and central bank response. Evidence from the nominal exchange rate behavior in Colombia

Since 1991 Colombia has had a market-determined Peso - US Dollar Nominal Exchange Rate (NER), after more than 20 years of controlled and multiple exchange rates. The behavior (revaluation / devaluation) of the NER is constantly reported in news, editorials and op-eds of major newspapers of the nation with particular attention to revaluation. The uneven reporting of revaluation episodes can be explained by the existence of an interest group particulary affected by revaluation, looking to increase awareness and sympathy for help from public institutions. Using the number of news and op-eds from a major Colombian newspaper, it is shown that there is an over-reporting of revaluation episodes in contrast to devaluation ones. Secondly, using text analysis upon the content of the news, it is also shown that the words devaluation and revaluation are far apart in the distribution of words within the news; and revaluation is highly correlated with words related to: public institutions, exporters and the need of assistance. Finally it is also shown that the probability of the central bank buying US dollars to lessen revaluation effects increases with the number of news; even though the central bank allegedly intervenes in the exchange rate market only to tame volatility or accumulate international reserves.

The simple macroeconomics of North and South in EMU. Bruegel Working Paper 2012/12, 30 July 2012

The euro area today consists of a competitive, moderately leveraged North and an uncompetitive, over-indebted South. Its main macroeconomic challenge is to carry out the adjustment required to restore the competitiveness of its southern part and eliminate its excessive public and private debt burden. This paper investigates the relationship between fiscal and competitiveness adjustment in a stylised model with two countries in a monetary union, North and South. To restore competitiveness, South implements a more restrictive fiscal policy than North. We consider two scenarios. In the first, monetary policy aims at keeping inflation constant in the North. The South therefore needs to deflate to regain competitiveness, which worsens the debt dynamics. In the second, monetary policy aims at keeping inflation constant in the monetary union as a whole. This results in more monetary stimulus, inflation in the North is higher, and this in turn helps the debt dynamics in the South. Our main findings are: •The differential fiscal stance between North and South is what determines real exchange rate changes. South therefore needs to tighten more. There is no escape from relative austerity. •If monetary policy aims at keeping inflation stable in the North and the initial debt is above a certain threshold, debt dynamics are perverse: fiscal retrenchment is self-defeating; •If monetary policy targets average inflation instead, which implies higher inflation in the North, the initial debt threshold above which the debt dynamics become perverse is higher. Accepting more inflation at home is therefore a way for the North to contribute to restoring debt sustainability in the South. •Structural reforms in the South improve the debt dynamics if the initial debt is not too high. Again, targeting average inflation rather than inflation in the North helps strengthen the favourable effects of structural reforms.

The potential to narrow uncertainty in regional climate predictions

Faced by the realities of a changing climate, decision makers in a wide variety of organisations are increasingly seeking quantitative predictions of regional and local climate. An important issue for these decision makers, and for organisations that fund climate research, is what is the potential for climate science to deliver improvements - especially reductions in uncertainty - in such predictions? Uncertainty in climate predictions arises from three distinct sources: internal variability, model uncertainty and scenario uncertainty. Using data from a suite of climate models we separate and quantify these sources. For predictions of changes in surface air temperature on decadal timescales and regional spatial scales, we show that uncertainty for the next few decades is dominated by sources (model uncertainty and internal variability) that are potentially reducible through progress in climate science. Furthermore, we find that model uncertainty is of greater importance than internal variability. Our findings have implications for managing adaptation to a changing climate. Because the costs of adaptation are very large, and greater uncertainty about future climate is likely to be associated with more expensive adaptation, reducing uncertainty in climate predictions is potentially of enormous economic value. We highlight the need for much more work to compare: a) the cost of various degrees of adaptation, given current levels of uncertainty; and b) the cost of new investments in climate science to reduce current levels of uncertainty. Our study also highlights the importance of targeting climate science investments on the most promising opportunities to reduce prediction uncertainty.

An inventory of heavy metals inputs to agricultural soils in England and Wales

An inventory of heavy metal inputs (Zn, Cu, Ni, Pb, Cd, Cr, As and Hg) to agricultural soils in England and Wales in 2000 is presented, accounting for major sources including atmospheric deposition, sewage sludge, livestock manures, inorganic fertilisers and lime, agrochemicals, irrigation water, industrial by-product 'wastes' and composts. Across the whole agricultural land area, atmospheric deposition was the main source of most metals, ranging from 25 to 85% of total inputs. Livestock manures and sewage sludge were also important sources, responsible for an estimated 37-40 and 8-17% of total Zn and Cu inputs, respectively. However, at the individual field scale sewage sludge, livestock manures and industrial wastes could be the major source of many metals where these materials are applied. This work will assist in developing strategies for reducing heavy metal inputs to agricultural land and effectively targeting policies to protect soils from long-term heavy metal accumulation. (C) 2003 Elsevier Science B.V. All rights reserved.

Assessing the discourses and practices of urban regeneration in a growing region

Most research on the discourses and practices of urban regeneration in-the UK has examined case studies located in areas of relative socio-economic distress. Less research has been undertaken on regeneration projects and agendas in areas characterise by strong economic growth. Yet, it is in such places that some of the best examples of the discourses, practices and impacts of contemporary urban regeneration can be. found. In some areas of high demand regeneration projects have used inner urban brownfield sites as locations for new investment. With the New Labour government's urban policy agendas targeting similar forms of regeneration, an examination of completed or on-going schemes is timely and relevant to debates over the direction that policy should take. This paper, drawing on a study of urban regeneration in one of England's, fastest growing towns, Reading in Berkshire, examines the discourses, practices and impacts of redevelopment schemes during the 1990s and 2000s. Reading's experiences have received national attention and have been hailed as a model for other urban areas to follow. The research documents the discursive and concrete aspects of local regeneration and examines the ways in which specific priorities and defined problems have come to dominate agendas. Collectively, the study argues that market-driven objectives come to dominate regeneration agendas, even in areas of strong demand where development agencies wield a relatively high degree of influence. Such regeneration plays a symbolic and practical role in creating new forms of exclusion and interpretations of place. (C) 2003 Elsevier Science Ltd. All rights reserved.

Iron Uptake and Homeostasis in Microorganisms

Preface. Iron is considered to be a minor element employed, in a variety of forms, by nearly all living organisms. In some cases, it is utilised in large quantities, for instance for the formation of magnetosomes within magnetotactic bacteria or during use of iron as a respiratory donor or acceptor by iron oxidising or reducing bacteria. However, in most cases the role of iron is restricted to its use as a cofactor or prosthetic group assisting the biological activity of many different types of protein. The key metabolic processes that are dependent on iron as a cofactor are numerous; they include respiration, light harvesting, nitrogen fixation, the Krebs cycle, redox stress resistance, amino acid synthesis and oxygen transport. Indeed, it is clear that Life in its current form would be impossible in the absence of iron. One of the main reasons for the reliance of Life upon this metal is the ability of iron to exist in multiple redox states, in particular the relatively stable ferrous (Fe2+) and ferric (Fe3+) forms. The availability of these stable oxidation states allows iron to engage in redox reactions over a wide range of midpoint potentials, depending on the coordination environment, making it an extremely adaptable mediator of electron exchange processes. Iron is also one of the most common elements within the Earth’s crust (5% abundance) and thus is considered to have been readily available when Life evolved on our early, anaerobic planet. However, as oxygen accumulated (the ‘Great oxidation event’) within the atmosphere some 2.4 billion years ago, and as the oceans became less acidic, the iron within primordial oceans was converted from its soluble reduced form to its weakly-soluble oxidised ferric form, which precipitated (~1.8 billion years ago) to form the ‘banded iron formations’ (BIFs) observed today in Precambrian sedimentary rocks around the world. These BIFs provide a geological record marking a transition point away from the ancient anaerobic world towards modern aerobic Earth. They also indicate a period over which the bio-availability of iron shifted from abundance to limitation, a condition that extends to the modern day. Thus, it is considered likely that the vast majority of extant organisms face the common problem of securing sufficient iron from their environment – a problem that Life on Earth has had to cope with for some 2 billion years. This struggle for iron is exemplified by the competition for this metal amongst co-habiting microorganisms who resort to stealing (pirating) each others iron supplies! The reliance of micro-organisms upon iron can be disadvantageous to them, and to our innate immune system it represents a chink in the microbial armour, offering an opportunity that can be exploited to ward off pathogenic invaders. In order to infect body tissues and cause disease, pathogens must secure all their iron from the host. To fight such infections, the host specifically withdraws available iron through the action of various iron depleting processes (e.g. the release of lactoferrin and lipocalin-2) – this represents an important strategy in our defence against disease. However, pathogens are frequently able to deploy iron acquisition systems that target host iron sources such as transferrin, lactoferrin and hemoproteins, and thus counteract the iron-withdrawal approaches of the host. Inactivation of such host-targeting iron-uptake systems often attenuates the pathogenicity of the invading microbe, illustrating the importance of ‘the battle for iron’ in the infection process. The role of iron sequestration systems in facilitating microbial infections has been a major driving force in research aimed at unravelling the complexities of microbial iron transport processes. But also, the intricacy of such systems offers a challenge that stimulates the curiosity. One such challenge is to understand how balanced levels of free iron within the cytosol are achieved in a way that avoids toxicity whilst providing sufficient levels for metabolic purposes – this is a requirement that all organisms have to meet. Although the systems involved in achieving this balance can be highly variable amongst different microorganisms, the overall strategy is common. On a coarse level, the homeostatic control of cellular iron is maintained through strict control of the uptake, storage and utilisation of available iron, and is co-ordinated by integrated iron-regulatory networks. However, much yet remains to be discovered concerning the fine details of these different iron regulatory processes. As already indicated, perhaps the most difficult task in maintaining iron homeostasis is simply the procurement of sufficient iron from external sources. The importance of this problem is demonstrated by the plethora of distinct iron transporters often found within a single bacterium, each targeting different forms (complex or redox state) of iron or a different environmental condition. Thus, microbes devote considerable cellular resource to securing iron from their surroundings, reflecting how successful acquisition of iron can be crucial in the competition for survival. The aim of this book is provide the reader with an overview of iron transport processes within a range of microorganisms and to provide an indication of how microbial iron levels are controlled. This aim is promoted through the inclusion of expert reviews on several well studied examples that illustrate the current state of play concerning our comprehension of how iron is translocated into the bacterial (or fungal) cell and how iron homeostasis is controlled within microbes. The first two chapters (1-2) consider the general properties of microbial iron-chelating compounds (known as ‘siderophores’), and the mechanisms used by bacteria to acquire haem and utilise it as an iron source. The following twelve chapters (3-14) focus on specific types of microorganism that are of key interest, covering both an array of pathogens for humans, animals and plants (e.g. species of Bordetella, Shigella, , Erwinia, Vibrio, Aeromonas, Francisella, Campylobacter and Staphylococci, and EHEC) as well as a number of prominent non-pathogens (e.g. the rhizobia, E. coli K-12, Bacteroides spp., cyanobacteria, Bacillus spp. and yeasts). The chapters relay the common themes in microbial iron uptake approaches (e.g. the use of siderophores, TonB-dependent transporters, and ABC transport systems), but also highlight many distinctions (such as use of different types iron regulator and the impact of the presence/absence of a cell wall) in the strategies employed. We hope that those both within and outside the field will find this book useful, stimulating and interesting. We intend that it will provide a source for reference that will assist relevant researchers and provide an entry point for those initiating their studies within this subject. Finally, it is important that we acknowledge and thank wholeheartedly the many contributors who have provided the 14 excellent chapters from which this book is composed. Without their considerable efforts, this book, and the understanding that it relays, would not have been possible. Simon C Andrews and Pierre Cornelis

Integrating drivers influencing the detection of plant pests carried in the international cut flower trade

This paper analyses the cut flower market as an example of an invasion pathway along which species of non-indigenous plant pests can travel to reach new areas. The paper examines the probability of pest detection by assessing information on pest detection and detection effort associated with the import of cut flowers. We test the link between the probability of plant pest arrivals as a precursor to potential invasion, and volume of traded flowers using count data regression models. The analysis is applied to the UK import of specific genera of cut flowers form Kenya between 1996 and 2004. There is a link between pest detection and the Genus of cut flower imported. Hence, pest detection efforts should focus on identifying and targeting those imported plants with a high risk of carrying pest species. For most of the plants studied efforts allocated to inspection have a significant influence on the probabilty of pest detction. However, by better targetting inspection efforts, it is shown that plant inspection effort could be reduced without increasing the risk of pest entry. Similarly, for most of the plants analysed, an increase in volume traded will not necessarily lead to an increase in the number of pests entering the UK. For some species, such as conclude that analysis at the rank of plant Genus is important both to understand the effectiveness of plant pest detection efforts and consequently to manage the risk of introduction of non-indigenous species.