Idag används 1:1 datorer, d.v.s. en dator för varje elev, i svenska skolor. Detta ämne är intressant att undersöka i vårt växande globala digitalsamhälle där kommuner och skolor strävar efter att erbjuda en hög tillgänglighet och kompetens till eleverna. Syftet med mitt arbete är att belysa 1:1 datoranvändning, samt om det förekommer skillnader mellan manliga och kvinnliga elevers användning. Det empiriska materialet har samlats in genom enkäter och intervjuer. Detta har bearbetats och därefter analyserats enligt tre kategorier: datorns användning, kommunikation, och samarbete. Resultatet visar att samtliga deltagare i undersökningen är överlag positiva till projektet, både lärare och elever anser att det är lättare att ta kontakt med varandra och skolarbetet underlättas. Det som upplevts som negativt är viss bristande uppmärksamhet under lektionerna pga. sociala medier. Elevernas användarkunskap skiljer sig inte åt könsmässigt, däremot visar undersökningen skillnader i användning efter intresseområden.

Submitted by Gizele Lima (gizele.lima@inpa.gov.br) on 2016-09-22T14:31:54Z No. of bitstreams: 2 Dissertação Dayane.pdf: 20358939 bytes, checksum: d0ddbe8f452b69b381be809c7baeff64 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-22T14:31:54Z (GMT). No. of bitstreams: 2 Dissertação Dayane.pdf: 20358939 bytes, checksum: d0ddbe8f452b69b381be809c7baeff64 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-06-29 / Fundação de Amparo à Pesquisa do Estado do Amazonas - FAPEAM / Saguinus is the largest and most complex genus of the subfamily Callitrichinae, with 23 species. They are distributed from Southern of Central America to northern of South America. Saguinus bicolor have very limited geographic distribuition, affected by demographic expansion of the city Manaus. In contrast, Saguinus midas have largest geographic distribuition among the Saguinus. They share the same characteristics general and overlap the north of Manaus. Cytogenetics studies with Saguinus described a karyotypic macrostructure conserved, with 2n=46 and patterns of similar bands. However, mapping studies of repetitive sequence are incipient. Repetitive sequence in tandem: telomere and rDNA; and repetitive sequence dispersed include the transposable elements were searched in the work. Analysis were made on S. midas and two populations of S. bicolor. The classical cytogenetics confirmed macrostructure of 2n=46, but differed in morphology of chromosomes, classified into: 8 metacentrics; 10 submetacentrics; 10 subtelocentrics and 6 acrocentrics. The patterns bands were similar, but showed variations among individuals of the same species. The G-bands patterns suggest the fourth pair as cytogenetic markers that show differences among two species and identify natural hybrids in contact zone. The NOR’s were detected in pairs 17 and 18, agreeing with the localization of sequences of rDNA 18S in region pericentromeric of long arms of chromosomes 17, 18 and 19, located in heterochomatic region. LINE–1 was found in regions: euchromatics – having an impact on the organization and function of genome, and; heterochromatics - particularly in centromeric heterochromatin. Accumulation in sex chromosomes are associated with inactivation of one chromosome X in females to promote the gene silencing and ensure gene dosage of sex pair when compared with male. It is possible to observe his presence in regions of negatives G-bands (light bands) implying that deposition in genome of S. bicolor and S. midas is recent in evolutionary time. Differences of sinalization of LINE-1 among populations of S. bicolor were detected, possibly due to isolation the two populations. / Saguinus é o maior e mais complexo gênero da subfamília Callitrichinae, com 23 espécies. Eles estão distribuídos do sul da América Central ao norte da América do Sul. Saguinus bicolor possui uma distribuição geográfica muito limitada, afetada pela expansão demográfica da cidade de Manaus. Ao contrário, Saguinus midas possui a maior distribuição geográfica dentre os Saguinus. São próximas filogeneticamente, compartilham das mesmas características gerais e se sobrepõem ao norte de Manaus. Estudos citogenéticos dos Saguinus descrevem uma macroestrutura cariotípica conservada, com 2n=46 e padrões de bandas similares. Porém, estudos com mapeamento de sequências repetitivas são incipientes. Sequências repetidas em tandem: telômericas e DNAr, e; sequências repetitivas dispersas que englobam os elementos transponíveis são pesquisadas neste trabalho. Análises citogenéticas foram feitas em S. midas e duas populações de S. bicolor. A citogenética clássica confirmou a macroestrutura das duas espécies em 2n=46, porém diferiu na morfologia dos cromossomos quando comparadas com estudos anteriores, sendo aqui, classificados em: 8 M; 10 SM; 20 ST e 6 A. O padrão de banda G apresentou variações entre as espécies, sugerindo o quarto par como marcador citogenético que diferenciaria as duas espécies e identificaria híbridos naturais de 1a geração, em zona de contato. As RON’s foram detectadas nos pares 17 e 18, sendo confirmadas pela localização da sequência de DNAr 18S na região pericentromérica dos braços longos nos cromossomos 17, 18 e 19, localizada em regiões próximas as heterocromatinas. A sequência LINE-1 foi encontrada nas regiões: eucromáticas – podendo influenciar na organização e função do genoma, e; heterocromáticas - particularmente na heterocromatina centromérica. O acúmulo de LINE-1 nos cromossomos sexuais está relacionado com a inativação de um dos cromossomos X nas fêmeas garantindo a dosagem gênica do par sexual quando comparado com os machos. A presença de LINE- 1 nas regiões de bandas G negativas (bandas claras) indica que sua deposição no genoma de S. bicolor e S. midas é recente no tempo evolutivo. Diferenças de sinalização do LINE-1 entre populações de S. bicolor foram detectadas, possivelmente devido ao isolamento das duas populações.

An important step in the production of infectious HIV-1 particles is maturation of the virus core. This process is completed by cleavage of the capsid (CA) domain of Gag, from its precursor, CA-SP1, by the viral protease. Large deletions in stem-loop 1 (SL1) in the 5' untranslated region (UTR) of HIV-1 genomic RNA (gRNA) delay CA-SP1 processing. SL1 harbours the dimerisation initiation site (DIS) palindrome suggesting that efficient Gag processing may be linked to gRNA dimerization as shown in HIV-2. However, a dimerisation mutant with normal Gag processing was identified. Gag processing defects are hallmarks of late domain mutants, and SL1 mutation was found to result in reduced virus release. HIV-1 hijacks the host's endosomal complexes required for transport (ESCRT) pathway to enable budding. An ESCRT-associated protein, ALIX, is known to be capable of binding to the nucleocapsid (NC) domain of Gag using lipids or RNA as a 'bridge' in vitro. It was hypothesised that SL1 mutation disrupts an RNA-dependent interaction that occurs during virus assembly. Consistent with this, an intact SL1 was found to be required for efficient ALIX function. Increasing the abundance of gRNA in the cell by expressing it in trans accelerated CA-SP1 processing in a manner that required ALIX's binding motif in p6. Gag processing could also be accelerated by introducing previously identified compensatory mutations into the SP1 and NC domains of Gag, in a manner reminiscent of the actions of maturation inhibitor resistance mutations. The effects of the compensatory mutations were also dependent on intact late domain motifs. These data suggest that gRNA is involved in regulating virus budding and maturation through interaction with ALIX. A model is proposed whereby the packaging signal (psi) region of gRNA acts as a bridge between Gag and ALIX, acting as a checkpoint mechanism to promote Gag processing and optimise release of virions that have successfully packaged gRNA.

Parallèlement au rôle central du récepteur aux androgènes, l’environnement tumoral immédiat exerce aussi une action majeure sur la progression du cancer de la prostate. L’hypoxie locale, par le biais de régulations multiples, serait impliquée dans la migration cellulaire et la dissémination tumorale. L’objectif de ma thèse a été d’identifier de nouvelles cibles thérapeutiques et de nouveaux marqueurs pronostiques pour ces cancers. J’ai tout d’abord participé à l’identification d’un partenaire du récepteur aux androgènes, la protéine CAD, enzyme clé de la synthèse des pyrimidines. Parallèlement, nous nous sommes intéressés au rôle d’un anti-angiogénique endogène dans la progression du cancer de la prostate, la thrombospondine-1 (TSP-1). Bien que l’activité anti angiogénique soit souvent considérée comme anti-tumorale, nous avons mis en évidence le caractère protumoral de la TSP-1, par son action promigratoire sur les cellules tumorales. Ces travaux m’ont conduits à étudier l’axe CXCR4/SDF-1, régulé en partie par l’hypoxie et stimulant la migration cellulaire. Nous avons montré sur tissu humain que CXCR4 était exprimé principalement au niveau du front tumoral des cancers localisés et localement avancés, et que son expression était associée à une transition épithélio-mésenchymateuse. SDF-1 était surexprimée selon un gradient croissant allant du centre des tumeurs vers le tissu péritumoral distant, exerçant possiblement un chimiotactisme sur les cellules du front tumoral. La surexpression de CXCR4 au front tumoral ainsi que le gradient de SDF1 étaient associés au pronostic. / In addition to the pivotal role of the androgen receptor, the immediate tumor microenvironment plays an essential role in prostate cancer progression. Local hypoxia, through multiple regulation mechanisms, may be implicated in the migration of tumor cells and dissemination. The aim of my thesis was to identify new therapeutic targets and prognostic markers for these cancers. I first participated in the identification of a new partner of the androgen receptor, the protein CAD that is a key enzyme of pyrimidine synthesis. We also studied the role of the endogenous anti-angiogenic thrombospondin-1 (TSP-1) in prostate cancer progression. Although anti-angiogenic activity is usually matched with tumor inhibition, we showed that TSP-1 exerted protumoral effects by stimulating cell migration. These observations led me to study the hypoxia-induced CXCR4/SDF-1 axis that is known to promote cell migration through the extra-cellular matrix. We showed on human tissue that CXCR4 was overexpressed in the tumor front of localized and locally advanced prostate cancers, and that its expression was associated with an epithelial mesenchymal transition. An increasing gradient of SDF-1 was observed from the tumor center to the distant peritumoral tissue, potentially attracting CXCR4-expressing tumor cells of the tumor front. CXCR4 overexpression in the tumor front, as well as SDF1 gradient, were associated with prognosis.

O HIV-1 é o agente etiológico da síndrome da imunodeficiência adquirida (AIDS) e o HTLV-I da leucemia/linfoma de célula T no adulto (ATL) e da paraparesia espástica tropical ou mielopatia associada ao HTLV (HAM/TSP), principalmente. Ambos são retrovírus com genoma RNA e possuem o gene gag que codifica as proteínas p24 (HIV-1 e HTLV-1) e p19 (HTLV-1) que formam o capsídeo e a matriz do vírus, respectivamente, e o gene env que codifica as proteínas gp41 e gp120 (HIV-1) e gp21 e gp46 (HTLV-1) que compõem o envelope viral. Os primeiros anticorpos produzidos nas infecções por ambos os vírus são destinados a essas proteínas e os diferentes testes diagnósticos disponíveis no mercado usam uma combinação dessas proteínas virais. O diagnóstico precoce é de extrema importância para o controle da epidemia, tratamento dos indivíduos e planejamento dos gastos com saúde pública. Os kits diagnósticos usados em laboratórios clínicos, bancos de sangue e hospitais brasileiros para o diagnóstico destas viroses são na sua maioria de empresas estrangeiras e o Brasil despende milhares de reais importando esses materiais. No Brasil, há a necessidade e incentivo para a produção de sistemas de diagnóstico com tecnologia nacional. Neste trabalho, os genes das proteínas p24, gp41 e gp120 do HIV-1 e p19 do HTLV-1 foram clonados com sucesso em diferentes vetores e em diferentes linhagens de E. coli, porém essas proteínas não foram expressas. As proteínas gp21, p24 e gp46 do HTLV-1 foram produzidas em bactérias BL21(DE3) com vetor pET28a(+). Essas três proteínas foram solubilizadas dos corpos de inclusão, purificadas por IMAC e identificadas pelas técnicas de Western Blotting e por espectrometria de massas. As proteínas recombinantes gp21, p24 e gp46 foram reconhecidas pelos soros de indivíduos com HTLV-1 e não foram reconhecidas por soros de indivíduos com HIV-1 e saudáveis, o que confere a elas especificidade e grande potencial diagnóstico. Os resultados deste trabalho são os primeiros passos para atingir o objetivo maior de produzir todas as sete proteínas em maior escala e, por fim, chegar a produção de um kit diagnóstico sensível, específico e barato com tecnologia nacional, diminuindo os gastos com a importação destes produtos e fomentando a indústria biotecnológica nacional. / HIV-1 is the etiologic agent of acquired immunodeficiency syndrome (AIDS) and HTLV-I is the cause of adult T-cell leukemia/lymphoma (ATL) and HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP). Both are retroviruses with RNA genome and possess the gene gag and env. The gag gene encodes for p24 protein (HTLV-1 and HIV-1) and p19 (HTLV-1) forming the viral capsid and matrix, respectively, and the env gene encodes for proteins gp120 and gp41 (HIV-1) and gp21 and gp46 (HTLV-1) making the viral envelope. The first antibodies produced in infections by both viruses are against these proteins and the various diagnostic tests on the market use a combination of those viral proteins. Early diagnosis is extremely important to control the epidemia, treatment of individuals and planning of public health expenditures. The diagnostic kits used in clinical laboratories, blood banks and in Brazilian hospitals for the diagnosis of these viruses are mostly from foreign companies. Brazil spends thousands of reais importing these materials. In Brazil, there is a need and incentive for the production of diagnostic systems with national technology. In this study, the genes of p24, gp41 and gp120 of HIV-1 and p19 of HTLV-1 have been successfully cloned in different vectors and different strains of E. coli, but these proteins were not expressed. The proteins gp21, gp46 and p24 of HTLV-1 were produced in bacteria BL21 (DE3) with vector pET28a (+). These three proteins were solubilized from inclusion bodies, purified by IMAC and identified by Western blotting techniques and mass spectrometry. The recombinant proteins gp21, p24 and gp46 were recognized by sera from patients with HTLV-1 and were not recognized by sera from individuals with HIV-1 and healthy people, which gives them great specificity and diagnostic potential. These results are the first steps to achieve the ultimate goal of producing all seven proteins on a larger scale and finally get the production of a diagnostic kit sensitive, specific and cheap with national technology, reducing spending on imports of these products and fostering the national biotechnology industry.

Cyclooxygenases (COXs) are of important therapeutic value as they are the target site of aspirin-like drugs. Here I report nine new COX-1 splice variants in chapter 1, which I characterized with regard to heme-binding and other properties. Inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) are co-inducible in many tissues following mitogenic and proinflammatory stimulation. In chapter 2, I investigate the physical and enzymatic properties of human COX-2 and iNOS and demonstrate that, despite reports to the contrary by another laboratory, they do not interact. The only reported COX-1 splice variant to exhibit cyclooxygenase activity has been isolated from dog brain and is termed COX-3. It contains an in-frame insertion of intron 1. However the existence of human COX-3 remains questionable since intron 1 is out of frame. Two putative in-frame human COX-3 isozymes, COX-1b2 and COX-1b3, (herein designated as COX-3-72 and COX-3-50) have been reported in the literature, but only one of them, COX-3-72, has been characterized. In chapter 3, COX-3-50 and COX-3-72 are reported to be over-expressed and determined to be active cyclooxygenases. COX-3-72 and, to a greater extent, COX-3-50, were stimulated by rofecoxib at physiological concentrations. A similar rofecoxib-stimulated COX activity is observed in quiescent A549 cells. Immunoblot and immunoprecipitation analysis suggest that human platelet and potentially A549 cells, contain a COX-3-50 like protein. Lonicera japonica is used as an anti-inflammatory treatment in traditional Chinese medicine. Its working mechanism is not well known. In chapter 4, I report that extracts from this herb inhibit COX-2 by three mechanisms: direct inhibition, transcriptional and post-transcriptional down regulation. COX-1 and COX-2 are similar to each other in their crystallographic structures. One of the most striking differences is that there are eight amino acids immediately following the signal peptide in COX-1 which are not found in COX-2. The function of this sequence is unknown. In chapter 5, I found that deletion of these amino acids decreased COX-1 Vmax by approximately 4-fold, but had little effect on other properties of the enzyme. Selecting bacteria transformed with recombinant plasmids is a laborious step in gene cloning experiments. This selection process is even more tedious when large numbers of clones need to be screened. In appendix I, I describe an ultra fast plasmid screening method. This new method was frequently used in the experiments performed in chapters 2-6.

PGE2 plays an important role in liver inflammation and carcinogenesis. Its metabolism is regulated by a cascade of reactions catalyzed by enzymes including COX-1/2, mPGES-1/2, 15-PGDH. Among these regulators, mPGES-1 is a cytokine-inducible enzyme mainly responsible for catalyzing terminal synthesis of PGE2, 15-PGDH catalyzes the oxidation of PGE2 to 15-keto-PGE2. In this context, we exogenously expressed mPGES-1 or 15-PGDH genes in mice hepatocytes to constitute a physiological condition ideal for evaluating PGE2 and its metabolites function in liver pathogenesis. In the first part, we developed transgenic mice with targeted expression of mPGES-1 in the liver and assessed the response of the transgenic mice to Fas-induced hepatocyte apoptosis and acute liver injury. Compared to wild type mice, the mPGES-1 Tg mice showed less liver hemorrhage, lower serum alanine transaminase and aspartate transaminase levels, less hepatic necrosis/apoptosis, and lower levels of caspase activation after intraperitoneal injection of the anti-Fas antibody Jo2. Western blotting analyses revealed increased expression and activation of the serine/threonine kinase Akt and associated anti-apoptotic molecules in the liver tissues of Jo2-treated mPGES-1 Tg mice. Pretreatment with the mPGES-1 inhibitor (MF63) or the Akt inhibitor (Akt inhibitor V) restored the susceptibility of the mPGES-1 Tg mice to Fas-induced liver injury. Our findings provide novel evidence that mPGES-1 prevents Fas-induced liver injury through activation of Akt and related signaling. This finding is consistent with previous reports of the anti-apoptotic and pro-proliferative role of PGE2. Our results suggest that induction of mPGES-1 or treatment with PGE2 may represent a potential therapeutic strategy for the prevention and treatment of Fas-associated liver injuries. In the second part, we generated transgenic mice with targeted expression of 15-PGDH in the liver and the animals were subjected to LPS/GalN-induced acute liver inflammation and injury. Compared to the wild type mice, the 15-PGDH Tg mice showed lower levels of alanine aminotransferase and aspartate aminotransferase, less liver tissue damage, less hepatic apoptosis/necrosis, less macrophage activation, and lower inflammatory cytokine production. In Kupffer cell cultures, treatment with 15-keto-PGE2 or the conditioned medium (CM) from 15-PGDH Tg hepatocyes inhibited LPS-induced cytokine production. Both 15-keto-PGE2 and the CM from15-PGDH Tg hepatocyes also up-regulated the expression of PPAR-γ downstream genes in Kupffer cells. In cultured hepatocytes, 15-keto-PGE2 treatment or 15-PGDH overexpression did not influence TNF-α-induced hepatocyte apoptosis. These findings suggest that 15-PGDH protects against LPS/GalN-induced liver injury and the effect is mediated via 15-keto-PGE2, which activates PPAR-γ in Kupffer cells and thus inhibits their ability to produce inflammatory cytokines. Accordingly, we observed that the PPAR-γ antagonist, GW9662, reversed the effect of 15-keto-PGE2 in Kupffer cell in vitro and restored the susceptibility of 15-PGDH Tg mice to LPS/GalN-induced acute liver injury in vivo. Our findings not only support the pro-inflammatory role of PGE2, but also reveal a novel anti-inflammatory role of 15-keto-PGE2. The data suggest that induction of 15-PGDH expression or utilization of a 15-keto-PGE2 analog may be therapeutic for treatment of endotoxin-associated liver inflammation/injury. Consistent with a pro-carcinogenic role for PGE2, overexpression mPGES-1 enhances growth of either HCC or cholangiocarcinoma cells, while overexpression 15-PGDH inhibits tumor cell growth in vitro. In the third part, we use a pharmacological method to induce 15-PGDH in cholangiocarcinoma tumor cells to inhibit PGE2 production. Our results indicated that treatment of human cholangiocarcinoma cells (CCLP1 and TFK-1) with ω-3 PUFA (DHA) or transfection of these cells with the Fat-1 gene (encoding Caenorhabditis elegans desaturase which converts ω-6 PUFA to ω-3 PUFA) significantly increased 15-PGDH protein level in cholangiocarcinoma cell lines. Human cholangiocarcinoma cells treated with DHA or transfected with a Fat-1 expression vector showed reduction of miRNA26a and miRNA26b (both miRNAs target 15-PGDH mRNA thus inhibiting 15-PGDH translation). Consistent with these findings, we observed that overexpression of miR26a or miR26b decreased 15-PGDH protein, reversed ω-3 PUFA-induced accumulation of 15-PGDH protein, and prevented ω-3 PUFA-induced inhibition of cholangiocarcinoma cell growth. Knockdown of 15-PGDH also attenuated ω-3 PUFA-induced inhibition of tumor cell growth. We observed that ω-3 PUFA suppressed miRNA26a and miRNA26b by inhibiting c-myc, a transcription factor that co-regulates a gene cluster comprised of miR-26a/b and carboxy-terminal domain RNA polymerase II polypeptide A small phosphatases (CTDSPs). Accordingly, overexpression of c-myc enhanced the expression of miRNA26a/b and prevented ω-3 PUFA-induced inhibition of tumor cell growth. Taken together, our results support a pro-tumorigenic role for PGE2, and suggest induction of 15-PGDH as potential way for the prevention and treatment of human cholangiocarcinoma. / 1 / LU YAO

Die antiapoptotischen Effekte der Pim-1 Kinase im Rahmen der ischämischen und Desfluran-induzierten Postkonditionierung / Antiapopiotic Effects of ishemic- and Desflurane-induced postconditioning are mediated by Pim-1 Kinase

Zugl.: Duisburg, Essen, Universiẗat, Diss., 2007.

Achieving agreement about whether and how to control greenhouse gas emissions would be difficult enough even if the consequences were fully known. Unfortunately, choices must be made in the face of great uncertainty, about both likely climate effects and the costs of control. Because several of the greenhouse gases have residence times of decades to centuries, any economic and environmental consequences are for practical purposes irreversible on those time scales. On the other hand, the commitment of resources to emissions control also has an irreversible aspect: investment foregone leaves a permanent legacy of reduced human welfare. Neither of the extreme positions, to take urgent action now or do nothing awaiting firm evidence, is a constructive response to the climate threat. Responsible treatment of this issue leads to a difficult position somewhere in between. / Includes bibliographical references (p. 32-34). / Abstract in HTML and technical report in HTML and PDF available on the Massachusetts Institute of Technology Joint Program on the Science and Policy of Global Change website (http://mit.edu/globalchange/www/).