The influenza viral ribonucleoprotein complexes (vRNPs) are responsible for viral RNA synthesis. Each vRNP is comprised of one vRNA segment, the viral RNA dependent RNA polymerase complex (RdRP), and multiple copies of nucleoprotein (NP). NP serves as scaffold in formation of vRNPs, but also regulates vRNP activity. The N-terminus of NP contains a nonconventional nuclear localization signal (NLS1) essential for initial vRNP nuclear import, but also interacts with host RNA helicases to enhance viral RNA replication in the nucleus. NP contains at least one additional NLS sequence, with bioinformatics revealing a third NLS in some NP proteins. Published yeast-two hybrid results indicate that the first 20 amino acids of NP can sufficiently bind with cellular protein UAP56. Suggesting the interaction of NP-UAP56 can be a major mechanism of how NP involve in viral replication. Thus, to examine the role of the N-terminus of NP aside from its vRNP nuclear localization activity N-terminal 20 amino acid deletion mutants with or without the addition of the conventional NLS from SV-40 T-antigen were constructed, termed del20NLS-NP and del20-NP. Nuclear localization of vRNPs with these constructs was assessed by GFP expression and western blotting. All these constructs exhibit nuclear localization, consistent with NLS1 being utilized for vRNP localization but not NP localization and vRNP formation in the nucleus. Furthermore, qPCR results demonstrated decreased vRNA synthesis activity, exacerbated as the vRNA template is lengthened in both plasmids, consistent with a lack of interaction with host RNA helicases. Interestingly, del20-NP vRNP activity is less severe than del20NLS-NP, suggesting perturbations of the N-terminus disrupt vRNP activity. To narrow down the region responsible for vRNA expression defect, del10-NP was constructed. GFP expression displayed similar activity between del10-NP and WT-NP with del20-NP showing a severe defection, suggesting NP amino acids 11-20 might be the major region responsible for the vRNA synthesis defect. However, sucrose density gradient results do not support the published interaction between NP and UAP56 in 293T cells. These results support the N-terminal region, potentially amino acids 11-20 of NP, is playing the important role in efficient viral gene expression during virus replication especially as vRNA template lengthen, and that the NLS1 of NP is not essential for NP/vRNP nuclear localization in our reconstituted vRNP assay.

A raiva é uma antropozoonose e causa a morte de 55 mil pessoas anualmente no mundo. Neste trabalho foi utilizado um sistema genético derivado do Vírus da Floresta de Semliki (SFV) carregando RNA para expressão gênica da glicoproteína do vírus da raiva (RVGP) e da proteína verde fluorescente (GFP), a título de controle do experimento. Lotes de partículas virais de SFV-RVGP e SFV-GFP foram obtidos em células BHK-21 e quantificados através de qRT-PCR. Foram realizados ensaios de infecção tanto com vírus ativado e não ativado em duas linhagens de macrófagos murinos IC-21 e J774A-1. A expressão da proteína RVGP foi avaliada pelo teste de Imunofluorescência Indireta (IFI) e a expressão da proteína GFP analisada por Microscopia de Fluorescência e Citometria de Fluxo em ambas as linhagens. A entrada do vírus SFV em células de mamíferos ocorre após 2 horas da infeção e de acordo com o experimento no qual analisamos a titulação viral do sobrenadante celular, indicaram que até as 72 horas pós infecção a titulação viral se manteve com o mesmo valor inicial, sugerindo que não houve entrada dos vírus na célula por transdução ou por fagocitose. / Rabies is a anthropozoonosis and causes the death of 55,000 people worldwide each year. In this work we used a genetic system derived from the Semliki forest virus (SFV) carrying RNA for gene expression of the rabies virus glycoprotein (RVGP) and green fluorescent protein (GFP), by way of experiment control. Plots of viral particles of SFV-RVGP and SFV-GFP were obtained in BHK-21 cells and quantified by qRT-PCR. Infection assays were performed both with activated and not activated in two virus strains of murine macrophages IC-21 and J774A-1. The expression of RVGP protein was assessed by indirect immunofluorescence test (IFI) and the expression of GFP protein analyzed by fluorescence microscopy and flow cytometry in both strains. The SFV entry of viruses into mammalian cells occurs after 2 hours of infection and according to the experiment in which we analyzed the viral titer of the cell supernatant showed that by 72 hours post-infection viral titer remained at the same initial value, suggesting no entry of virus into the cell by transduction or by phagocytosis.

A raiva é uma antropozoonose e causa a morte de 55 mil pessoas anualmente no mundo. Neste trabalho foi utilizado um sistema genético derivado do Vírus da Floresta de Semliki (SFV) carregando RNA para expressão gênica da glicoproteína do vírus da raiva (RVGP) e da proteína verde fluorescente (GFP), a título de controle do experimento. Lotes de partículas virais de SFV-RVGP e SFV-GFP foram obtidos em células BHK-21 e quantificados através de qRT-PCR. Foram realizados ensaios de infecção tanto com vírus ativado e não ativado em duas linhagens de macrófagos murinos IC-21 e J774A-1. A expressão da proteína RVGP foi avaliada pelo teste de Imunofluorescência Indireta (IFI) e a expressão da proteína GFP analisada por Microscopia de Fluorescência e Citometria de Fluxo em ambas as linhagens. A entrada do vírus SFV em células de mamíferos ocorre após 2 horas da infeção e de acordo com o experimento no qual analisamos a titulação viral do sobrenadante celular, indicaram que até as 72 horas pós infecção a titulação viral se manteve com o mesmo valor inicial, sugerindo que não houve entrada dos vírus na célula por transdução ou por fagocitose. / Rabies is a anthropozoonosis and causes the death of 55,000 people worldwide each year. In this work we used a genetic system derived from the Semliki forest virus (SFV) carrying RNA for gene expression of the rabies virus glycoprotein (RVGP) and green fluorescent protein (GFP), by way of experiment control. Plots of viral particles of SFV-RVGP and SFV-GFP were obtained in BHK-21 cells and quantified by qRT-PCR. Infection assays were performed both with activated and not activated in two virus strains of murine macrophages IC-21 and J774A-1. The expression of RVGP protein was assessed by indirect immunofluorescence test (IFI) and the expression of GFP protein analyzed by fluorescence microscopy and flow cytometry in both strains. The SFV entry of viruses into mammalian cells occurs after 2 hours of infection and according to the experiment in which we analyzed the viral titer of the cell supernatant showed that by 72 hours post-infection viral titer remained at the same initial value, suggesting no entry of virus into the cell by transduction or by phagocytosis.

In Acute Myocardial Infarction (AMI), inflammation is a prerequisite for healing but it can paradoxically extend tissue injury; hence it needs to be modulated. Here, we investigated the role of the pro resolving GPCR FPR2/ALX and its agonist Annexin A1 (AnxA1) in AMI using mice lacking of the Fpr2/3 genes and with an in-frame GFP gene ‘knocked-in’. We developed protocols aimed to determine GFP expression as an indication of Fpr2 gene activity. Also, the Left Anterior Descending Coronary Artery of male Fpr2/3 KO and littermate controls (WT) was occluded for 30min and re-opened for 90min. At the end tissue injury and inflammatory response were studied. A significant proportion of Fpr2/3 KO perished during the procedure. The rest survived up to 90 min and exhibited a larger infarct size, with higher troponin I and inflammation markers (KC, TNFα) than WT animals. At the end of reperfusion, Fpr2/3 KO displayed an unbalanced production of pro and anti-inflammatory lipids (higher PGE2, PGI2, LTB4 and attenuated PGA1, RvD2, LXA4) and a deregulated activation of the cardioprotective IL-6/JAK/STAT3 signalling. Administration of AnxA1 afforded cardioprotection (reduction of infarct size; Troponin I, Caspase3 activity and TNFα) in WT but not in Fpr2/3 KO. A parallel in vitro investigation on the functional FPR2/ALX domains required by AnxA1 and other agonists was also conducted. HEK-293 cells transfected with FPR1, FPR2/ALX and FPR1/FPR2 chimeric receptor were used and calcium flux, 4 pERK and gene modulation analysed. AnxA1 required the N-terminus and the II and III extracellular loops of FPR2/ALX to evoke canonical responses. SAA interacted/activated the I and the II extracellular loops of FPR2/ALX, whereas the compound 43 suffices the I extracellular loop. In summary, the FPR2/AnxA1 pathway exerts a protective role in AMI. AnxA1 mimetic that activated selective FPR2/ALX domains can be synthetize to prevent tissue damage caused by AMI.

Grass family is most important family in plant kingdom due to intensive usage of crops in agriculture. To date, molecular biology researches on grass family have had limitations because of inappropriate characteristics of barley and wheat to conduct experiments on them. Brachypodium distachyon that belongs to grass family has recently emerged as a model organism for crops. It shares common characteristics for a model plant due to its small genome, small physical plant size, a short lifecycle, and less demanding growth requirements / as other model organisms / Arabidopsis thaliana, Oryza sativa, and Zea mays (Draper et al. 2001). Especially after appreciating, the genetic distance of O. sativa to grasses (Garvin et al. 2008), it become a key organism to understand complicated genomic organization of agriculturally valuable grasses. Virus-induced gene silencing (VIGS) is one of the revolutionary methods allowing a rapid and effective loss of a gene function through RNA interference (Holzberg et al. 2002 / Liu et al. 2008). Barley stripe mosaic virus (BSMV) is still the most effective vector used in monocot gene silencing. It has a tripartite RNA genome having a wide range of infection ability for monocots including barley, oat, wheat, and maize as host (Holzberg et al. 2002 / Scofield 2005). In this thesis, Phytoene desaturase (PDS) gene of Brachypodium distachyon was silenced via BSMV mediated VIGS. Additionally, with Green fluorescence protein (GFP) bearing BSMV transcripts, GFP expression was observed under fluorescent microscope. To our knowledge, this is the first report demonstrating a VIGS via BSMV in Brachypodium distachyon. The success of virus induced gene silencing method in Brachypodium distachyon, will be a new convenient tool for evaluating functions of crop genes in this model organism.