Endocytosis of prion protein is required for ERK1/2 signaling induced by stress-inducible protein 1

The secreted cochaperone STI1 triggers activation of protein kinase A (PKA) and ERK1/2 signaling by interacting with the cellular prion (PrPC) at the cell surface, resulting in neuroprotection and increased neuritogenesis. Here, we investigated whether STI1 triggers PrPC trafficking and tested whether this process controls PrPC-dependent signaling. We found that STI1, but not a STI1 mutant unable to bind PrPC, induced PrPC endocytosis. STI1-induced signaling did not occur in cells devoid of endogenous PrPC; however, heterologous expression of PrPC reconstituted both PKA and ERK1/2 activation. In contrast, a PrPC mutant lacking endocytic activity was unable to promote ERK1/2 activation induced by STI1, whereas it reconstituted PKA activity in the same condition, suggesting a key role of endocytosis in the former process. The activation of ERK1/2 by STI1 was transient and appeared to depend on the interaction of the two proteins at the cell surface or shortly after internalization. Moreover, inhibition of dynamin activity by expression of a dominant-negative mutant caused the accumulation and colocalization of these proteins at the plasma membrane, suggesting that both proteins use a dynamin-dependent internalization pathway. These results show that PrPC endocytosis is a necessary step to modulate STI1-dependent ERK1/2 signaling involved in neuritogenesis.

Oroidin Inhibits the Activity of the Multidrug Resistance Target Pdr5p from Yeast Plasma Membranes

Oroidin was isolated from the marine sponge Agelas sventres and inhibited the activity and function of Pdr5p, an enzyme responsible for the multidrug resistance phenotype in Saccharomyces cerevisiae. This compound may help in the development of new drugs that reverse this dangerous phenotype of pathogenic yeast and fungi.

Conformational differences between the wild type and V30M mutant transthyretin modulate its binding to genistein: Implications to tetramer stability and ligand-binding

Transthyretin (TTR) is a tetrameric beta-sheet-rich transporter protein directly involved in human amyloid diseases. It was recently found that the isoflavone genistein (GEN) potently inhibits TTR amyloid fibril formation (Green et al., 2005) and is therefore a promising candidate for TTR amyloidosis treatment. Here we used structural and biophysical approaches to characterize genistein binding to the wild type (TTRwt) and to its most frequent amyloidogenic variant, the V30M mutant. In a dose-dependent manner, genistein elicited considerable increases in both mutant and TTRwt stability as demonstrated by high hydrostatic pressure (HHP) and acid-mediated dissociation/denaturation assays. TTR:GEN crystal complexes and isothermal titration calorimetry (ITC) experiments showed that the binding mechanisms of genistein to the TTRwt and to V30M are different and are dependent on apoTTR structure conformations. Furthermore, we could also identify potential allosteric movements caused by genistein binding to the wild type TTR that explains, at least in part, the frequently observed negatively cooperative process between the two sites of TTRwt when binding ligands. These findings show that TTR mutants may present different ligand recognition and therefore are of value in ligand design for inhibiting TTR amyloidosis. (C) 2010 Elsevier Inc. All rights reserved.

RailsTNE : En multimodal informationsplattform i ett interorganisatoriskt nätverk

Att utreda möjligheterna att etablera dataförsörjning riktade mot multimodala transporttjänster är idag av största intresse. Det är också intressant att försöka utnyttja de standarder som tagits fram när en dataförsörjningskedja ska etableras. Detta examensarbete genomfördes på Triona AB i Borlänge. Triona har utvecklat databasen Transport Network Engine (TNE), som idag används för att läsa in data från Nationella VägDataBasen (NVDB) och från skogsnäringen. Målet med arbetet var att ta fram en informationsmodell, som beskriver hur ett transportnät med nätanknutna data, som stödjer multimodala transporter och resor, kan lagras i TNE. En förlängning av målet blev att, utifrån ett givet användarfall, etablera dataförsörjning till TNE och lägga in de nödvändiga datamängderna. TNE bygger på ESRI:s produkter och funktionalitet för hantering av geografisk information och informationsmodell och datahantering i TNE baseras på internationella och svenska standarder. Idag finns all information om järnvägsnätet hos Banverket och det finns ingen koppling mellan Banverkets databas Baninformationssystemet (BIS) och Vägverkets databas NVDB. För att den kopplingen ska kunna ske, krävs att de båda verken kan samarbeta som två stora organisationer i ett väl fungerande nätverk. Ett så kallat interorganisatoriskt nätverk. Det akademiska syftet var att föreslå en arkitekturstrategi som stödjer ett sådant nätverk. Vi upptäckte att arkitekturstrategierna IRM och VBS är uppbyggda för att inrikta sig på enskilda organisationer, och det visade sig svårt att följa en specifik strategi inom ett nätverk med flera organisationer. I vårt användarfall måste nätverket mellan Banverket, Vägverket och Stora Enso samarbeta över organisationsgränserna. Därför behövs en strategi för ett interorganisatoriskt samarbete så att informationen kan struktureras på ett gemensamt sätt. Vi föreslår ett antal idealtypskriterier som vi utgått efter i struktureringen av systemarkitekturen. Den innehåller en informationsmodell för både väg- och järnvägsdata som följer standarden SS 63 70 04. Arbetet avgränsades till att endast innehålla de datamängder som användarfallet kräver. Vi har analyserat KombiTIF-rapporten som är ett avslutat projekt styrt av Banverket på uppdrag av Näringsdepartementet. Rapporten ger ett förslag på en lösning i form av en arena som ska göra data tillgänglig för användning. Vi väljer att se TNE-plattformen som en realisering av denna arena. Enligt vårt förslg så ska TNE-plattformen hämta in det mest grundläggande datat om järnvägsnät och nätanknuten data från BIS och standardiserad information om vägnät och nätanknuten data från NVDB via ett gränssnitt. I framtiden är det möjligt att på samma sätt ta in andra trafiknät såsom Sjöfartsverket, Luftfartsverket och Rikstrafiken. Vi arbetade efter en egen metod som vi tog fram med valda delar från andra metoder; kartläggning, perspektivanalys, verksamhetsanalys, utveckling och utvärdering. Arbetet genomfördes iterativt i de fem olika faserna.

Trätorn : Kan det vara något för svensk vindkraft i skogsmiljö?

Inom ramen för projektet Energi- och miljökompetenscentrum vid Högskolan Dalarna haren kortare förstudie kring tekniken som används av Timber Tower GmbH för högavindkrafttorn i trä gjorts. Huvudsyftet med studien har varit att titta närmare på teknikenoch dess potential för svensk landbaserad vindkraft samt utreda vad detta kan innebära förregionen.Trenden för landbaserad vindkraft i skogsmiljö går mot allt högre torn. Skogsmiljön gör attden marknära turbulensen och vindgradienten blir större än över t.ex. hav vilket gör detmer föredelaktigt att använda högre torn.Tekniken som Timber Tower GmbH använder är att bygga tornet på plats med en koniskhålstomme av plana paneler av korslimmat limträ (som annars främst används vidbyggnation av höga hus med massivträstomme). Tekniken är skyddad av ett flertalpatentansökningar varav minst en är beviljad.Vid en jämförelse mellan trätorn och svetsade ståltorn (som är den dominerandetorntekniken idag) kan det konstateras att trätornstekniken är ekonomiskt intressant.Framförallt blir trätornen mer intressanta i jämförelse med ståltornen vi ökande höjder (>100 m) pga. dess transportfördelar. Vidare bör det nämnas att de torntekniker somanvänds idag vid höjder runt 140 m och högre främst är fackverkstorn i stål ochhybridtorn med en hög bas av förspänd betong och övre delen i stål.Då ett typgodkännande av ett vindkraftverk gäller torn och turbin är det i praktiken alltidturbintillverkaren som prissätter och levererar ett komplett vindkraftverk. Medutgångspunkt i regeringens planeringsram för svensk vindkraftutbyggnad (20 TWh/årlandbaserad vindkraft 2020) kan dock marknadsvärdet för torn uppskattas till 2-3 miljarderkr/år fram till 2020. För trätorn motsvarar detta en årlig potentiell volym på ca 190 000 m3.Slutsatsen i denna korta studie är att det är tekniskt möjligt att designa ett trätorn i rimligadimensioner för en 2-3 MW turbin med navhöjd ca 140 m och att detta sannolikt även ärekonomiskt intressant. Då ett trätorn blir något lättare än motsvarande ståltorn går det åtmindre energi för att göra ett trätorn. Hur mycket mindre är dock osäkert pga. den storaspridningen i siffror för energiåtgång för stålproduktion och i praktiken blir skillnadenäven leverantörsberoende. Eftersom andelen förnyelsebar energi är betydligt större vidtillverkningen av materialet för trätornen kommer trätornet att ge en betydande sänkningpå utsläppt CO2e/kWhel jämfört med motsvarande verk med ståltorn. Ett annat argumentför trätorn är förenklade transporter jämfört med t.ex. svetsade ståltorn eller hybridtornmed prefabricerade betongelement.Om tekniken etableras kan det i förlängningen leda till en regional påverkan i form av ökatlokalt innehåll i vindkraftprojekten t.ex. genom ökade råvaruleveranser (eg. sågat ochtorkat virke), lokal produktion av korslimmat limträ eller exempelvis bildande avspecialiserade montagefirmor. Det största hindret för tekniken just nu är dessmarknadsintroduktion och acceptans samt typgodkännande tillsammans med en för svenskmarknad intressant turbinleverantör. För att få acceptans för tekniken krävs byggande ochutvärdering av minst ett verk med trätorn. En intressant storlek för marknaden är en 2-3MW turbin med navhöjd ca 140 m. Ett sådant verk skulle i så fall få det högstavindkrafttornet i Sverige och sannolikt det högsta vindkrafttornet i trä i världen. Byggandetav ett första verk med trätorn skulle sannolikt även kräva ett behov av stödfinansiering föratt täcka en projektörs ökade risk. Då flera vindkraftetableringar i regionen uppvisar godaproduktionsresultat (med Tavelberget som gott exempel) visar det att regionen ärintressant för introduktion och utvärdering av ny teknologi.

Optimization heuristic solutions, how good can they be? : With empirical applications in location problems

Combinatorial optimization problems, are one of the most important types of problems in operational research. Heuristic and metaheuristics algorithms are widely applied to find a good solution. However, a common problem is that these algorithms do not guarantee that the solution will coincide with the optimum and, hence, many solutions to real world OR-problems are afflicted with an uncertainty about the quality of the solution. The main aim of this thesis is to investigate the usability of statistical bounds to evaluate the quality of heuristic solutions applied to large combinatorial problems. The contributions of this thesis are both methodological and empirical. From a methodological point of view, the usefulness of statistical bounds on p-median problems is thoroughly investigated. The statistical bounds have good performance in providing informative quality assessment under appropriate parameter settings. Also, they outperform the commonly used Lagrangian bounds. It is demonstrated that the statistical bounds are shown to be comparable with the deterministic bounds in quadratic assignment problems. As to empirical research, environment pollution has become a worldwide problem, and transportation can cause a great amount of pollution. A new method for calculating and comparing the CO2-emissions of online and brick-and-mortar retailing is proposed. It leads to the conclusion that online retailing has significantly lesser CO2-emissions. Another problem is that the Swedish regional division is under revision and the border effect to public service accessibility is concerned of both residents and politicians. After analysis, it is shown that borders hinder the optimal location of public services and consequently the highest achievable economic and social utility may not be attained.

Data based Calibration System for Radar used by Vehicle Activated Signs

The accurate measurement of a vehicle’s velocity is an essential feature in adaptive vehicle activated sign systems. Since the velocities of the vehicles are acquired from a continuous wave Doppler radar, the data collection becomes challenging. Data accuracy is sensitive to the calibration of the radar on the road. However, clear methodologies for in-field calibration have not been carefully established. The signs are often installed by subjective judgment which results in measurement errors. This paper develops a calibration method based on mining the data collected and matching individual vehicles travelling between two radars. The data was cleaned and prepared in two ways: cleaning and reconstructing. The results showed that the proposed correction factor derived from the cleaned data corresponded well with the experimental factor done on site. In addition, this proposed factor showed superior performance to the one derived from the reconstructed data.

Inheritance beyond plain heritability : variance-controlling genes in Arabidopsis thaliana

The phenotypic effect of a gene is normally described by the mean-difference between alternative genotypes. A gene may, however, also influence the phenotype by causing a difference in variance between genotypes. Here, we reanalyze a publicly available Arabidopsis thaliana dataset [1] and show that genetic variance heterogeneity appears to be as common as normal additive effects on a genomewide scale. The study also develops theory to estimate the contributions of variance differences between genotypes to the phenotypic variance, and this is used to show that individual loci can explain more than 20% of the phenotypic variance. Two well-studied systems, cellular control of molybdenum level by the ion-transporter MOT1 and flowering-time regulation by the FRI-FLC expression network, and a novel association for Leaf serration are used to illustrate the contribution of major individual loci, expression pathways, and gene-by-environment interactions to the genetic variance heterogeneity.

Limiting glutamate transmission in a Vglut2-expressing subpopulation of the subthalamic nucleus is sufficient to cause hyperlocomotion

The subthalamic nucleus (STN) is a key area of the basal ganglia circuitry regulating movement. We identified a subpopulation of neurons within this structure that coexpresses Vglut2 and Pitx2, and by conditional targeting of this subpopulation we reduced Vglut2 expression levels in the STN by 40%, leaving Pitx2 expression intact. This reduction diminished, yet did not eliminate, glutamatergic transmission in the substantia nigra pars reticulata and entopeduncular nucleus, two major targets of the STN. The knock-out mice displayed hyperlocomotion and decreased latency in the initiation of movement while preserving normal gait and balance. Spatial cognition, social function, and level of impulsive choice also remained undisturbed. Furthermore, these mice showed reduced dopamine transporter binding and slower dopamine clearance in vivo, suggesting that Vglut2-expressing cells in the STN regulate dopaminergic transmission. Our results demonstrate that altering the contribution of a limited population within the STN is sufficient to achieve results similar to STN lesions and high-frequency stimulation, but with fewer side effects.

Increased hippocampal excitability and impaired spatial memory function in mice lacking VGLUT2 selectively in neurons defined by tyrosine hydroxylase promoter activity

Three populations of neurons expressing the vesicular glutamate transporter 2 (Vglut2) were recently described in the A10 area of the mouse midbrain, of which two populations were shown to express the gene encoding, the rate-limiting enzyme for catecholamine synthesis, tyrosine hydroxylase (TH).One of these populations (‘‘TH– Vglut2 Class1’’) also expressed the dopamine transporter (DAT) gene while one did not ("TH–Vglut2 Class2"), and the remaining population did not express TH at all ("TH-Vglut2-only"). TH is known to be expressed by a promoter which shows two phases of activation, a transient one early during embryonal development, and a later one which gives rise to stable endogenous expression of the TH gene. The transient phase is, however, not specific to catecholaminergic neurons, a feature taken to advantage here as it enabled Vglut2 gene targeting within all three A10 populations expressing this gene, thus creating a new conditional knockout. These knockout mice showed impairment in spatial memory function. Electrophysiological analyses revealed a profound alteration of oscillatory activity in the CA3 region of the hippocampus. In addition to identifying a novel role for Vglut2 in hippocampus function, this study points to the need for improved genetic tools for targeting of the diversity of subpopulations of the A10 area

Análise metagenômica da microbiota de ambientes aquáticos do estado do Rio Grande do Norte - Brasil

The screening for genes in metagenomic libraries from soil creates opportunities to explore the enormous genetic and metabolic diversity of microorganisms. Rivers are ecosystems with high biological diversity, but few were examined using the metagenomic approach. With this objective, a metagenomic library was constructed from DNA soil samples collected at three different points along the Jundiaí-river (Rio Grande do Norte-Brazil). The points sampled are from open area, rough terrain and with the direct incidence of sunlight. This library was analyzed functionally and based in sequence. For functional analysis Luria-Bertani solid medium (LB) with NaCl concentration varied from 0.17M to 0.85M was used for functional analysis. Positives clones resistant to hypersaline medium were obtained. The recombinant DNAs were extracted and transformed into Escherichia coli strain DH10B and survival curves were obtained for quantification of abiotic stress resistance. The sequences of clones were obtained and submitted to the BLASTX tool. Some clones were found to hypothetical proteins of microorganisms from both Archaea and Bacteria division. One of the clones showed a complete ORF with high similarity to glucose-6-phosphate isomerase which participates in the synthesis of glycerol pathway and serves as a compatible solute to balance the osmotic pressure inside and outside of cells. Subsequently, in order to identify genes encoding osmolytes or enzymes related halotolerance, environmental DNA samples from the river soil, from the water column of the estuary and ocean were collected and pyrosequenced. Sequences of osmolytes and enzymes of different microorganisms were obtained from the UniProt and used as RefSeqs for homology identification (TBLASTN) in metagenomic databases. The sequences were submitted to HMMER for the functional domains identification. Some enzymes were identified: alpha-trehalose-phosphate synthase, L-ectoina synthase (EctC), transaminase L-2 ,4-diaminobutyric acid (EctB), L-2 ,4-diaminobutyric acetyltransferase (EctA), L-threonine 3 dehydrogenase (sorbitol pathway), glycerol-3-phosphate dehydrogenase, inositol 3-phosphate dehydrogenase, chaperones, L-proline, glycine betaine binding ABC transporter, myo-inositol-1-phosphate synthase protein of proline simportadora / PutP sodium-and trehalose-6-phosphate phosphatase These proteins are commonly related to saline environments, however the identification of them in river environment is justified by the high salt concentration in the soil during prolonged dry seasons this river. Regarding the richness of the microbiota the river substrate has an abundance of halobacteria similar to the sea and more than the estuary. These data confirm the existence of a specialized response against salt stress by microorganisms in the environment of the Jundiaí river

Análise da Expressão de APE1 após Estresse Oxidativo Induzido em Células Proficientes e Deficientes na Via de Reparo por Excisão de Nucleotídeos

Riboflavin is a vitamin very important in aerobic organisms, as a precursor of many coenzymes involved in the electron transporter chain. However, after photosensitization of riboflavin with UV or visible light, it generates reactive oxygen species (ROS), which can oxidize the DNA. The repair of oxidative lesions on DNA occurs through the base excision repair pathway (BER), where APE1 endonuclease plays a central role. On the other hand, the nucleotide excision repair pathway (NER) repairs helix-distorting lesions. Recently, it was described the participation of NERproteins in the repair of oxidative damage and in stimulation of repair function fromAPE1. The aim of this research was to evaluate the cytotoxic effects of photosensitized riboflavin (RF*) in cells proficient and deficient in NER, correlating with APE1 expression. For this propose, the cells were treated with RF* and it was performed the cell viability assay, extraction of whole proteins, cells fractionation, immunoblotting, indirect immunofluorescence and analysis of polymorphisms of BER gens. The results evidenced that cells deficient in XPA and CSB proteins were more sensitive to RF*. However, XPC-deficient cells presented similar resistance to MRC5- SV cells, which is proficient in NER. These results indicate that XPA and CSB proteins have an important role on repair of oxidative lesions induced by RF*. Additionally, it was evidenced that single nucleotide polymorphisms (SNPs) in BER enzymes may influence in sensitivity of NER-deficient cell lines. Concerning the APE1 expression, the results showed that expression of this protein after treatment with RF* only changed in XPC-deficient cells. Though, it was observed that APE1 is recruited and is bound to chromatin in MRC5-SV and XPA cells after treatment with RF*. The results also showed the induction of DNA damage after treatment with RF*, through the analysis of-H2AX, since the treatment promoted an increase of endogenous levels of this phosphorylated protein, which acts signaling double strand-break on DNA. On the other hand, in XPC-deficient cells, regardless of resistance of RF*, the endogenous levels of APE1 are extremely reduced when compared with other cell lines and APE1 is not bound to chromatin after treatment with RF*. These results conclude that RF* was able to induce cell death in NERdeficient cells, where XPA and CSB cells were more sensitive when compared with MRC5-SV and XPC-deficient cells. This last result is potentially very interesting, since XPC-deficient cell line presents low levels of APE1. Additionally, the results evidenced that APE1 protein can be involved in the repair of oxidative damage induced by RF*, because APE1 is recruited and bound strongly to chromatin after treatment.

Treinamento resistido reduz inflamação em músculo esquelético e melhora a sensibilidade à insulina periférica em ratos obesos induzidos por dieta hiperlipídica

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cigarette smoke exposure severely reduces peripheral insulin sensitivity without changing GLUT4 expression in oxidative muscle of Wistar rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

O exercício físico melhora a sensibilidade à insulina de ratos expostos à fumaça de cigarro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)