Cellular and molecular characterization of an embryonic cell line (BME26) from the tick Rhipicephalus (Boophilus) microplus

The cellular and molecular characteristics of a cell line (BME26) derived from embryos of the cattle tick Rhipicephalus (Boophilus) microplus were studied. The cells contained glycogen inclusions, numerous mitochondria, and vesicles with heterogeneous electron densities dispersed throughout the cytoplasm. Vesicles contained lipids and sequestered palladium meso-porphyrin (Pd-mP) and rhodamine-hemoglobin, suggesting their involvement in the autophagic and endocytic pathways. The cells phagocytosed yeast and expressed genes encoding the antimicrobial peptides (microplusin and defensin). A cDNA library was made and 898 unique mRNA sequences were obtained. Among them, 556 sequences were not significantly similar to any sequence found in public databases. Annotation using Gene Ontology revealed transcripts related to several different functional classes. We identified transcripts involved in immune response such as ferritin, serine proteases, protease inhibitors,. antimicrobial peptides, heat shock protein, glutathione S-transferase, peroxidase, and NADPH oxidase. BME26 cells transfected with a plasmid carrying a red fluorescent protein reporter gene (DsRed2) transiently expressed DsRed2 for up to 5 weeks. We conclude that BME26 can be used to experimentally analyze diverse biological processes that occur in R. (B.) microplus such as the innate immune response to tick-borne pathogens. (C) 2008 Elsevier Ltd. All rights reserved.

T3 rapidly modulates TSH beta mRNA stability and translational rate in the pituitary of hypothyroid rats

Whereas it is well known that T3 inhibits TSH beta gene transcription, its effects on TSH beta mRNA stability and translation have been poorly investigated. This study examined these possibilities, by evaluating the TSH beta transcripts poly(A) tail length, translational rate and binding to cytoskeleton, in pituitaries of thyroidectomized and sham-operated rats treated with T3 or saline, and killed 30 min thereafter. The hypothyroidism induced an increase of TSH beta transcript poly(A) tail, as well as of its content in ribosomes and attachment to cytoskeleton. The hypothyroid rats acutely treated with T3 exhibited a reduction of TSH beta mRNA poly(A) tail length and recruitment to ribosomes, indicating that this treatment decreased the stability and translation rate of TSH beta mRNA. Nevertheless, acute T3 administration to sham-operated rats provoked an increase of TSH beta transcripts binding to ribosomes. These data add new insight to an important role of T3 in rapidly regulating TSH gene expression at posttranscriptional level. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Molecular characterization of Arabidopsis thaliana PUF proteins - binding specificity and target candidates

PUF proteins regulate both stability and translation through sequence-specific binding to the 3` UTR of target mRNA transcripts. Binding is mediated by a conserved PUF domain, which contains eight repeats of approximately 36 amino acids each. Found in all eukaryotes, they have been related to several developmental processes. Analysis of the 25 Arabidopsis Pumilio (APUM) proteins presenting PUF repeats reveals that 12 (APUM-1 to APUM-12) have a PUF domain with 50-75% similarity to the Drosophila PUF domain. Through three-hybrid assays, we show that APUM-1 to APUM-6 can bind specifically to the Nanos response element sequence recognized by Drosophila Pumilio. Using an Arabidopsis RNA library in a three-hybrid screening, we were able to identify an APUM-binding consensus sequence. Computational analysis allowed us to identify the APUM-binding element within the 3` UTR in many Arabidopsis transcripts, even in important mRNAs related to shoot stem cell maintenance. We demonstrate that APUM-1 to APUM-6 are able to bind specifically to APUM-binding elements in the 3` UTR of WUSCHEL, CLAVATA-1, PINHEAD/ZWILLE and FASCIATA-2 transcripts. The results obtained in the present study indicate that the APUM proteins may act as regulators in Arabidopsis through an evolutionarily conserved mechanism, which may open up a new approach for investigating mRNA regulation in plants.

Gene expression profiling reveals molecular marker candidates of laryngeal squamous cell carcinoma

Laryngeal squamous cell carcinoma is very common in head and neck cancer, with high mortality rates and poor prognosis. In this study, we compared expression profiles of clinical samples from 13 larynx tumors and 10 non-neoplastic larynx tissues using a custom-built cDNA microarray containing 331 probes for 284 genes previously identified by informatics analysis of EST databases as markers of head and neck tumors. Thirty-five genes showed statistically significant differences (SNR >= 11.01, p <= 0.001) in the expression between tumor and non-tumor larynx tissue samples. Functional annotation indicated that these genes are involved in cellular processes relevant to the cancer phenotype, such as apoptosis, cell cycle, DNA repair, proteolysis, protease inhibition, signal transduction and transcriptional regulation. Six of the identified transcripts map to intronic regions of protein-coding genes and may comprise non-annotated exons or as yet uncharacterized long ncRNAs with a regulatory role in the gene expression program of larynx tissue. The differential expression of 10 of these genes (ADCY6, AES, AL2SCR3, CRR9, CSTB, DUSP1, MAP3K5, PLAT, UBL1 and ZNF706) was independently confirmed by quantitative real-time RT-PCR. Among these, the CSTB gene product has cysteine protease inhibitor activity that has been associated with an antimetastatic function. Interestingly, CSTB showed a low expression in the tumor samples analyzed (p<0.0001). The set of genes identified here contribute to a better understanding of the molecular basis of larynx cancer, and provide candidate markers for improving diagnosis, prognosis and treatment of this carcinoma.

Global Gene Expression Analysis during Germination in the Chytridiomycete Blastocladiella emersonii

Blastocladiella emersonii is an aquatic fungus of the Chytridiomycete class. During germination, the zoospore, a motile nongrowing cell, goes through a cascade of morphological changes that culminates with its differentiation into the germling cell, capable of coenocytic vegetative growth. Transcriptome analyses of B. emersonii cells were carried out during germination induced under various environmental conditions. Microarray data analyzing 3,563 distinct B. emersonii genes revealed that 26% of them are differentially expressed during germination in nutrient medium at at least one of the time points investigated. Over 500 genes are upregulated during the time course of germination under those conditions, most being related to cell growth, including genes involved in protein biosynthesis, DNA transcription, energetic metabolism, carbohydrate and oligopeptide transport, and cell cycle control. On the other hand, several transcripts stored in the zoospores are downregulated during germination in nutrient medium, such as genes involved in signal transduction, amino acid transport, and chromosome organization. In addition, germination induced in the presence of nutrients was compared with that triggered either by adenine or potassium ions in inorganic salt solution. Several genes involved in cell growth, induced during germination in nutrient medium, do not show increased expression when B. emersonii zoospores germinate in inorganic solution, suggesting that nutrients exert a positive effect on gene transcription. The transcriptome data also revealed that most genes involved in cell signaling show the same expression pattern irrespective of the initial germination stimulus.

The mitochondrial view of Blastocladiella emersonii

The mitochondrial genome of the chytrid Blastocladiella emersonii was sequenced and annotated, revealing the complete set of oxidative phosphorylation genes and tRNAs/rRNAs necessary for the translation process. Phylogenetic reconstructions reinforce the proposal of the new phylum Blastocladiomycota. Evidences of gene duplication due to inserted elements suggest shared susceptibility to gene invasion/exchange between chytrids and zygomycetes. The gene content of B. emersonii is very similar to Allomyces macrogynus but the content of intronic and changeable elements is much lower suggesting a stronger resistance to this kind of exchange. in addition, a total of 401 potential nuclear transcripts encoding mitochondrial proteins were obtained after B. emersonii EST database scanning using Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana data as probes and TargetP tool to find N-terminal mitochondrial signal in translated sequences. (c) 2008 Elsevier B.V. All rights reserved.

Identification of protein-coding and intronic noncoding RNAs down-regulated in clear cell renal carcinoma

The clear cell subtype of renal cell carcinoma (RCC) is the most lethal and prevalent cancer of the urinary system. To investigate the molecular changes associated with malignant transformation in clear cell RCC, the gene expression profiles of matched samples of tumor and adjacent non-neoplastic tissue were obtained from six patients. A custom-built cDNA microarray platform was used, comprising 2292 probes that map to exons of genes and 822 probes for noncoding RNAs mapping to intronic regions. Intronic transcription was detected in all normal and neoplastic renal tissues. A subset of 55 transcripts was significantly down-regulated in clear cell RCC relative to the matched nontumor tissue as determined by a combination of two statistical tests and leave-one-out patient cross-validation. Among the down-regulated transcripts, 49 mapped to untranslated or coding exons and 6 were intronic relative to known exons of protein-coding genes. Lower levels of expression of SIN3B, TRIP3, SYNJ2BP and NDE1 (P<0.02), and of intronic transcripts derived from SND1 and ACTN4 loci (P<0.05), were confirmed in clear cell RCC by Real-time RT-PCR. A subset of 25 transcripts was deregulated in additional six nonclear cell RCC samples, pointing to common transcriptional alterations in RCC irrespective of the histological subtype or differentiation state of the tumor. Our results indicate a novel set of tumor suppressor gene candidates, including noncoding intronic RNAs, which may play a significant role in malignant transformations of normal renal cells. (C) 2008 Wiley-Liss, Inc.

Global Gene Expression Analysis during Sporulation of the Aquatic Fungus Blastocladiella emersonii

The Blastocladiella emersonii life cycle presents a number of drastic biochemical and morphological changes, mainly during two cell differentiation stages: germination and sporulation. To investigate the transcriptional changes taking place during the sporulation phase, which culminates with the production of the zoospores, motile cells responsible for the dispersal of the fungus, microarray experiments were performed. Among the 3,773 distinct genes investigated, a total of 1,207 were classified as differentially expressed, relative to time zero of sporulation, at at least one of the time points analyzed. These results indicate that accurate transcriptional control takes place during sporulation, as well as indicating the necessity for distinct molecular functions throughout this differentiation process. The main functional categories overrepresented among upregulated genes were those involving the microtubule, the cytoskeleton, signal transduction involving Ca(2+), and chromosome organization. On the other hand, protein biosynthesis, central carbon metabolism, and protein degradation were the most represented functional categories among downregulated genes. Gene expression changes were also analyzed in cells sporulating in the presence of subinhibitory concentrations of glucose or tryptophan. Data obtained revealed overexpression of microtubule and cytoskeleton transcripts in the presence of glucose, probably causing the shape and motility problems observed in the zoospores produced under this condition. In contrast, the presence of tryptophan during sporulation led to upregulation of genes involved in oxidative stress, proteolysis, and protein folding. These results indicate that distinct physiological pathways are involved in the inhibition of sporulation due to these two classes of nutrient sources.

Conserved tissue expression signatures of intronic noncoding RNAs transcribed from human and mouse loci

It has been postulated that noncoding RNAs (ncRNAs) are involved in the posttranscriptional control of gene expression, and may have contributed to the emergence of the complex attributes observed in mammalians. We show here that the complement of ncRNAs expressed from intronic regions of the human and mouse genomes comprises at least 78,147 and 39,660 transcriptional units, respectively. To identify conserved intronic sequences expressed in both humans and mice, we used custom-designed human cDNA microarrays to separately interrogate RNA from mouse and human liver, kidney, and prostate tissues. An overlapping tissue expression signature was detected for both species, comprising 198 transcripts; among these, 22 RNAs map to intronic regions with evidence of evolutionary conservation in humans and mice. Transcription of selected human-mouse intronic ncRNAs was confirmed using strand-specific RT-PCR. Altogether, these results support an evolutionarily conserved role of intronic ncRNAs in human and mouse, which are likely to be involved in the fine tuning of gene expression regulation in different mammalian tissues. (C) 2008 Elsevier Inc. All rights reserved.

Värdefull litteratur : Fyra gymnasielärare om samhällsuppdragets etik i förhållande till litteraturen i klassrummet

The ethical aspects of the Swedish Curriculum for the non-compulsory school system, Lpf94, will form the basis of the research in this essay. The ethical aspects constitute fundamental values and goals as well as common tasks that must be carried out by every teacher. Since the school debate focuses a lot on this perspective, which is often mentioned together with the words problematic and contradictory, it will consequently also be the focus of this essay. Here these features are treated in relation to literature in all its forms used by teachers in class, which also represents a part of the school debate. Together ethics and literature form a perspective that lacks research. The goal is to investigate the ethical awareness of teachers in relation to the literature, and find out which ethical decisions are made by teachers and in what ways the ethical aspects of the curriculum is fulfilled. A qualitative method has been used in order to gain results: four upper secondary school teachers/senior high school teachers have been interviewed. The results are presented in interview transcripts which have been analyzed and interpreted in the light of recent studies and research and pedagogical-didactical literature. Accordingly I have been able to draw the conclusions that the ethical features in teaching is not explicit and that there seldom seems to be any conscious ethical reflections in relation to the literature. The ethical aspects are rather invisible and unconscious. However, it is clear that the interviews raise the awareness of the ethical perspective and thus emphasize the growing and developing function of the dialogue. Finally the results have been interpreted from the point of view of the ethical tasks of the teacher in order to make the ethics visible.I detta examensarbete ligger fokus på de etiska aspekterna av samhällsuppdraget i förhållande till den litteratur som lärare använder sig av i klassrumsundervisningen. Eftersom en stor del av skoldebatten handlar om samhällsuppdraget och dess värdegrund utgör också detta perspektiv utgångspunkten för denna undersökning. Målet är att undersöka vilka etiska avvägningar lärare gör i valet av litteratur och på vilket sätt samhällsuppdragets etik blir synlig i lärares arbete. Därmed framkommer också till viss del i vilken omfattning värdegrundsarbetet sker. Genom en kvalitativ undersökningsmetod i form av samtal har fyra gymnasielärare intervjuats. Erhållna resultat har presenterats i form av intervjutranskript som analyserats och tolkats med hjälp av aktuell forskning och pedagogisk-didaktisk litteratur. De slutsatser som kan dras utifrån resultaten är att etiken i lärarnas arbete inte är explicit och att det oftast inte finns en medveten etisk reflektion utifrån litteraturen. Etiken är snarare osynlig och tämligen omedveten. Dock medvetandegörs den under samtalen vilket framhåller dialogens betydelse för utveckling. Vidare framkommer att etiken aldrig är ett mål i sig, utan fungerar mer som medel för att nå andra mål i klassrummet. Slutligen, för att synliggöra etiken, har resultaten tolkats utifrån de etiska krav i samhällsuppdraget som ställs på lärare.

Narration in Swedish pre- and primary school : a recourse for language development and multilingualism

Increased immigration in Europe and worldwide has led to more pre- and primary school students being educated through the medium of a second language, and there is considerable research, much of it coming from Australia, to suggest that in order to cope with this situation, children will need to begin to acquire, from their earliest years in pre-school, a variety of knowledge-based language skills that will be sufficient to carry them through the subject-based education they will encounter in their subsequent schooling. This is particularly important for L2-students who are less likely to meet academic language outside the school. In this paper, based on transcripts of oral interactions in the classroom, it is argued that conversational and story-telling skills, oral and written, provide a rich environment for the development of academic school language, while at the same time promoting and making good use of the cultural diversity that is increasingly a feature of pre-primary and primary classrooms.

Healthcare providers balancing norms and practice : challenges and opportunities in providing contraceptive counselling to young people in Uganda - a qualitative study

BACKGROUND: Pregnancies among young women force girls to compromise education, resulting in low educational attainment with subsequent poverty and vulnerability. A pronounced focus is needed on contraceptive use, pregnancy, and unsafe abortion among young women. OBJECTIVE: This study aims to explore healthcare providers' (HCPs) perceptions and practices regarding contraceptive counselling to young people. DESIGN: We conducted 27 in-depth interviews with doctors and midwives working in seven health facilities in central Uganda. Interviews were open-ended and allowed the participant to speak freely on certain topics. We used a topic guide to cover areas topics of interest focusing on post-abortion care (PAC) but also covering contraceptive counselling. Transcripts were transcribed verbatim and data were analysed using thematic analysis. RESULTS: The main theme, HCPs' ambivalence to providing contraceptive counselling to sexually active young people is based on two sub-themes describing the challenges of contraceptive counselling: A) HCPs echo the societal norms regarding sexual practice among young people, while at the same time our findings B) highlights the opportunities resulting from providers pragmatic approach to contraceptive counselling to young women. Providers expressed a self-identified lack of skill, limited resources, and inadequate support from the health system to successfully provide appropriate services to young people. They felt frustrated with the consultations, especially when meeting young women seeking PAC. CONCLUSIONS: Despite existing policies for young people's sexual and reproductive health in Uganda, HCPs are not sufficiently equipped to provide adequate contraceptive counselling to young people. Instead, HCPs are left in between the negative influence of social norms and their pragmatic approach to address the needs of young people, especially those seeking PAC. We argue that a clear policy supported by a clear strategy with practical guidelines should be implemented alongside in-service training including value clarification and attitude transformation to equip providers to be able to better cater to young people seeking sexual and reproductive health advice.

Inserção de pessoas com deficiência intelectual: um estudo em empresas situadas na região metropolitana de São Paulo

Esta dissertação teve origem no questionamento sobre as reais possibilidades de inclusão das pessoas com deficiência intelectual (PCDI), cada vez mais frequentemente inseridas nas organizações brasileiras. O objetivo principal é analisar a maneira como tem acontecido a inserção de PCDI em cinco grandes empresas situadas na região metropolitana de São Paulo, a partir das perspectivas das pessoas diretamente envolvidas. Isso implica, primeiramente, em uma contextualização das políticas de inserção adotadas pelas organizações participantes. Além disso, a partir da análise dos temas mais abordados pelas pessoas entrevistadas, pretende-se identificar os significados da inserção das pessoas com deficiência intelectual e suas implicações mais relevantes para as pessoas envolvidas: os sujeitos com deficiência intelectual, seus chefes, pares hierárquicos de convivência direta, profissionais de gestão de pessoas e pessoas vinculadas a organizações externas, mas que de alguma forma influenciam ou interferem nas atividades de inserção e inclusão no ambiente organizacional. Como arcabouço teórico foram abordados os conceitos de inclusão, inserção e diversidade nas organizações; preconceito, estigma e demais barreiras atitudinais à inclusão; questões familiares e educacionais; as concepções de deficiência ao longo da história, além de aspectos demográficos e regulatórios relacionados ao tema. A pesquisa foi realizada por meio de entrevistas com baixo grau de estruturação e as transcrições analisadas conforme a abordagem de análise de conteúdo proposta por Bardin (1977), a partir dos pressupostos do interacionismo simbólico estrutural (STRYKER, 2006). A partir da discussão dos temas que emergiram da análise de conteúdo e da contextualização e comparação das políticas de inserção adotadas pelas organizações, pôde-se concluir que é possível incluir as pessoas com deficiência intelectual, tendo em vista as fronteiras organizacionais em que elas se inserem.

Governança e coordenação em arranjos multinível de políticas públicas transversais: assistência social, educação e saúde no Programa Bolsa Família

Este estudo apresenta o desenvolvimento teórico e a aplicação de um modelo voltado à análise de arranjos de governança de políticas públicas que ocorrem quando múltiplas organizações estão envolvidas em lidar com questões complexas. A seguinte pergunta de pesquisa foi proposta: Como opera o conjunto de instrumentos de coordenação empregados na governança de uma política pública transversal? Um estudo de caso exploratório sobre o Programa Bolsa Família foi realizado, especificamente na sua dimensão relacionada à coordenação entre os setores de assistência social, educação e saúde. Os métodos de pesquisa consistiram em análise de conteúdo de documentos e transcrições de entrevistas semiestruturadas com atores-chave do Programa nos três setores de políticas. Os resultados demonstram como diferentes modos de governança coexistem no arranjo multinível estudado, revelando diversos elementos de coordenação cuja operação concomitante resulta em um modo de governança misto. Contribui-se, assim, para o aperfeiçoamento do Programa e para o desenvolvimento teórico, metodológico e aplicado dos conceitos de governança e de coordenação, ficando demonstrado ser possível integrá-los na análise de políticas públicas transversais.

Knowledge disclosure as a weapon in technological change battles: straight shot or backfire?

This work aims at evaluating how effective is knowledge disclosure in attenuating institutional negative reactions caused by uncertainties brought by firms’ new strategies that respond to novel technologies. The empirical setting is from an era of technological ferment, the period of the introduction of the voice over internet protocol (VoIP) in the USA in the early 2000’s. This technology led to the convergence of the wireline telecommu- nications and cable television industries. The Institutional Brokers’ Estimate System (also known as the I/B/E/S system) was used to capture reactions of securities analysts, a revealed important source of institutional pressure on firms’ strategies. For assessing knowledge disclosure, a coding technique and a established content analysis framework were used to quantitatively measure the non-numerical and unstructured data of transcripts of business events occurred at that time. Eventually, several binary response models were tested in order to assess the effect of knowledge disclosure on the probability of institutional positive reactions. The findings are that the odds of favorable institutional reactions increase when a specific kind of knowledge is disclosed. It can be concluded that knowledge disclosure can be considered as a weapon in technological changes situations, attenuating adverse institutional reactions to the companies’ strategies in environments of technological changes.