Molecular and Functional Characterisation of SBP-box genes: The role of SPL3 during the floral transition of Arabidopsis thaliana

Proper developmental processes require a tight control of spatial and temporal gene regulation, since specific gene and protein expression is a prerequisite of cell differentiation. Transcription factors as well as microRNAs are major components for transcriptional and translational control of gene expression. In Arabidopsis thaliana, one of the plant specific transcription factor families is the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family, which comprises 17 members, that have been shown to play important roles in several developmental processes. A decisive step in plant development is the transition from vegetative to reproductive growth, as it has to happen during favorable conditions to ensure successful reproduction and is a "one-time decision", as this phase change is not reversible in the annual plant Arabidopsis. The MIR156/157-controlled SBP-box transcription factor SPL3 has been shown to play a role during flowering in Arabidopsis, since its constitutive overexpression in a microRNA insensitive form results in early flowering plants, that nevertheless remain photoperiodically sensitive. Moreover, it has been shown that SPL3 binds in vitro to the sequence core motif CGTAC. During this thesis the role of SPL3 during the development to reproductive growth should be elucidated through identification of target genes. The results of this work suggest the floral meristem identity gene FRUITFULL (FUL; AGL8) to be a direct target of SPL3. Expression studies of two transgenes carrying the reportergene GUS in combination with genomic FUL or the FUL promoter region revealed a precocious activation of FUL in cotyledons and leaves in an SPL3OX background. Moreover, these data indicate that both, the binding motifs in the promoter as well as in the FUL first intron, are required for proper activation of FUL. A global expression analysis revealed that sugar metabolism, red light signaling and the circadian clock are affected by overexpression of SPL3. Subsequent analysis of diurnal expression of clock genes as well as of leaf movement in SPL3 overexpressing plants revealed a shortened period of the circadian clock and a precocious activation of so called "evening genes"

Mammalian prion propagation in PrP transgenic Drosophila.

Mammalian prions propagate by template-directed misfolding and aggregation of normal cellular prion related protein PrPC as it converts into disease-associated conformers collectively referred to as PrPSc. Mammalian species may be permissive for prion disease because these hosts have co-evolved specific co-factors that assist PrPC conformational change and prion propagation. We have tested this hypothesis by examining whether faithful prion propagation occurs in the normally PrPC-null invertebrate host Drosophila melanogaster. Ovine PrP transgenic Drosophila exposed at the larval stage to ovine scrapie showed a progressive accumulation of transmissible prions in adult flies. Strikingly, the biological properties of distinct ovine prion strains were maintained during their propagation in Drosophila. Our observations show that the co-factors necessary for strain-specific prion propagation are not unique to mammalian species. Our studies establish Drosophila as a novel host for the study of transmissible mammalian prions.This work was supported by funds from the NC3Rs Project (Grant NC/K000462/1)

Cytosolic PrP can participate in prion-mediated toxicity.

Prion diseases are characterized by a conformational change in the normal host protein PrPC. While the majority of mature PrPC is tethered to the plasma membrane by a glycosylphosphatidylinositol anchor, topological variants of this protein can arise during its biosynthesis. Here we have generated Drosophila transgenic for cytosolic ovine PrP in order to investigate its toxic potential in flies in the absence or presence of exogenous ovine prions. While cytosolic ovine PrP expressed in Drosophila was predominantly detergent insoluble and showed resistance to low concentrations of proteinase K, it was not overtly detrimental to the flies. However, Drosophila transgenic for cytosolic PrP expression exposed to classical or atypical scrapie prion inocula showed a faster decrease in locomotor activity than similar flies exposed to scrapie-free material. The susceptibility to classical scrapie inocula could be assessed in Drosophila transgenic for panneuronal expression of cytosolic PrP, whereas susceptibility to atypical scrapie required ubiquitous PrP expression. Significantly, the toxic phenotype induced by ovine scrapie in cytosolic PrP transgenic Drosophila was transmissible to recipient PrP transgenic flies. These data show that while cytosolic PrP expression does not adversely affect Drosophila, this topological PrP variant can participate in the generation of transmissible scrapie-induced toxicity. These observations also show that PrP transgenic Drosophila are susceptible to classical and atypical scrapie prion strains and highlight the utility of this invertebrate host as a model of mammalian prion disease. Importance: During prion diseases, the host protein PrPC converts into an abnormal conformer, PrPSc, a process coupled to the generation of transmissible prions and neurotoxicity. While PrPC is principally a glycosylphosphatidylinositol-anchored membrane protein, the role of topological variants, such as cytosolic PrP, in prion-mediated toxicity and prion formation is undefined. Here we generated Drosophila transgenic for cytosolic PrP expression in order to investigate its toxic potential in the absence or presence of exogenous prions. Cytosolic ovine PrP expressed in Drosophila was not overtly detrimental to the flies. However, cytosolic PrP transgenic Drosophila exposed to ovine scrapie showed a toxic phenotype absent from similar flies exposed to scrapie-free material. Significantly, the scrapie-induced toxic phenotype in cytosolic transgenic Drosophila was transmissible to recipient PrP transgenic flies. These data show that cytosolic PrP can participate in the generation of transmissible prion-induced toxicity and highlight the utility of Drosophila as a model of mammalian prion disease.This work was supported by funds from the China Scholarship Council and the Cambridge Overseas Trust, which provided Ph.D. studentship support for C.Z. We acknowledge funding support from the NC3Rs.This is the accepted manuscript version. The final version is available from ASM at http://jvi.asm.org/content/88/14/8129.full

Prion-induced neurotoxicity: Possible role for cell cycle activity and DNA damage response.

Protein misfolding neurodegenerative diseases arise through neurotoxicity induced by aggregation of host proteins. These conditions include Alzheimer's disease, Huntington's disease, Parkinson's disease, motor neuron disease, tauopathies and prion diseases. Collectively, these conditions are a challenge to society because of the increasing aged population and through the real threat to human food security by animal prion diseases. It is therefore important to understand the cellular and molecular mechanisms that underlie protein misfolding-induced neurotoxicity as this will form the basis for designing strategies to alleviate their burden. Prion diseases are an important paradigm for neurodegenerative conditions in general since several of these maladies have now been shown to display prion-like phenomena. Increasingly, cell cycle activity and the DNA damage response are recognised as cellular events that participate in the neurotoxic process of various neurodegenerative diseases, and their associated animal models, which suggests they are truly involved in the pathogenic process and are not merely epiphenomena. Here we review the role of cell cycle activity and the DNA damage response in neurodegeneration associated with protein misfolding diseases, and suggest that these events contribute towards prion-induced neurotoxicity. In doing so, we highlight PrP transgenic Drosophila as a tractable model for the genetic analysis of transmissible mammalian prion disease.Supported by The NC3Rs No. NC/K000462/1 (in part).This is the final published version. It first appeared at http://dx.doi.org/10.5501/wjv.v4.i3.188

Tourist safety and security in the Central Region of Ghana -overview and case study

As a result of the sharp rise in the interest for tourism activities, safety and security matters have also become one of the forces causing vicissitudes in the tourism industry in the world. Since tourism sector is contributing greatly to environmental sustainability, tourist safety is also now critical to tourism growth. In the travel and tour experiences, the satisfaction of a tourist is discussed as part of a tourist’s post-procured and consumed assessment of the destination. Review of the three most prominent tourism plans in Ghana was conducted in this study. It was discovered that there was no tourist safety policy and establishment of tourism office in most of the MMDAs. This situation has bred weak governmental administrative linkages and neglect of tourist safety and security at the MMDAs. Three tourist facilities in the Central Region of Ghana were chosen for the review-study. Responses of 550 tourists were reviewed in the study. Chi-square test was used to test whether the feeling of safety at the destination was influenced by their purpose of visit. The lingering thought-provoking question about these visits is, how safe and secured are the tourists and visitors in these facilities? It is recommended that GTA should lead the campaign exercise in these three facilities to produce a digital tourist safety and security documentary about each facility and this can assist and boost the confidence of potential tourists to appraise the safety and security of the destination before traveling

Polymorphisms at amino acid residues 141 and 154 influence conformational variation in ovine PrP

Polymorphisms in ovine PrP at amino acid residues 141 and 154 are associated with susceptibility to ovine prion disease: Leu141Arg154 with classical scrapie and Phe141Arg154 and Leu141His154 with atypical scrapie. Classical scrapie is naturally transmissible between sheep, whereas this may not be the case with atypical scrapie. Critical amino acid residues will determine the range or stability of structural changes within the ovine prion protein or its functional interaction with potential cofactors, during conversion of PrPC to PrPSc in these different forms of scrapie disease. Here we computationally identified that regions of ovine PrP, including those near amino acid residues 141 and 154, displayed more conservation than expected based on local structural environment. Molecular dynamics simulations showed these conserved regions of ovine PrP displayed genotypic differences in conformational repertoire and amino acid side-chain interactions. Significantly, Leu141Arg154 PrP adopted an extended beta sheet arrangement in the N-terminal palindromic region more frequently than the Phe141Arg154 and Leu141His154 variants. We supported these computational observations experimentally using circular dichroism spectroscopy and immunobiochemical studies on ovine recombinant PrP. Collectively, our observations show amino acid residues 141 and 154 influence secondary structure and conformational change in ovine PrP that may correlate with different forms of scrapie.This is the final published version. It is published by Hindawi in BioMed Research International here: http://www.hindawi.com/journals/bmri/2014/372491/

University as coping for dealing with care work of nursing Master's students

OBJECTIVE: To assess the main signs of stress, coping and stressors of nursing Master's students, and the process of writing their dissertation with professional insertion. METHODOLOGICAL PROCEDURES: A explorative qualitative research of thematic analysis was conducted using individual interviews. The convenience sample comprised 18 Master's students and six tutors of a university in the State of São Paulo, in 2004. ANALYSIS OF RESULTS: Although the course was stressful at times, Master's students considered the study as an opportunity to leave care and start teaching. The most satisfying aspect of the post graduation course was to be in an environment where professional issues were reflected upon reinforcing the view of Master degree as an escape and a search for support. The Master's degree was seen by nurses as the legitimization of knowledge to be professionally recognized. That does not occur in care, where they feel neglected, and probably from this context there is this need of "escaping" from the hospital. CONCLUSIONS: For nursing Master's students, the Master's course was less stressful than their professional activities, and it was a way of coping, escaping, and looking for support to deal with care.OBJETIVO: Analisar a relação entre os principais indícios de estresse, coping e estressores em mestrandos enfermeiros e o processo de elaboração da dissertação com sua inserção profissional. PROCEDIMENTOS METODOLÓGICOS: Pesquisa exploratória qualitativa de análise temática utilizando entrevistas individuais. A amostra de conveniência consistiu de 18 mestrandos e seis orientadores de uma universidade do Estado de São Paulo, em 2004. ANÁLISE DOS RESULTADOS: Apesar de o curso apresentar momentos estressantes, os mestrandos consideravam o estudo como oportunidade de abandonar a assistência para vir a lecionar. O aspecto mais prazeroso da pós-graduação foi dispor de um ambiente de reflexão das questões profissionais, reforçando a visão do mestrado como fuga e busca de suporte. O mestrado era visto pelo enfermeiro como fórum de legitimação do saber para conquistar o reconhecimento profissional não encontrado na assistência, onde se sentia desprestigiado, emergindo possivelmente deste contexto sua necessidade de "fugir" do hospital. CONCLUSÕES: Para os mestrandos de enfermagem o mestrado era menos estressante que suas atividades profissionais, consistindo em coping de fuga e busca de suporte para lidar com a prática assistencial