Enhanced Efficacy of a Codon-Optimized DNA Vaccine Encoding the Glycoprotein Precursor Gene of Lassa Virus in a Guinea Pig Disease Model When Delivered by Dermal Electroporation

vaccine

Karriärvägledning på estetiska programmet - En kvalitativ studie i hur elever på estetiska programmet rustas för det eftergymnasiala studie- och yrkeslivet

Syftet med vårt examensarbete är att undersöka hur och med vilka verktyg vårt urval av vägledare och lärare arbetar med att rusta elever på estetiska programmet för framtida studie- och yrkesliv, i förhållande till elevernas karriärmål/drömmar. Karriär-mål/drömmar som kan rikta sig mot den, enligt Arbetsförmedlingens yrkesprognos, svåretablerade arbetsmarknaden inom kulturbranschen. Vi har genom en kvalitativ metod intervjuat fyra studie- och yrkesvägledare samt två lärare på gymnasieskolans estetiska program. Vi har därefter analyserat resultatet genom relevanta begrepp tagna från kapitlet tidigare forskning samt Social cognitive career theory och begreppen upplevd självförmåga (self-efficacy), personliga mål (personal goals), och förväntade utfall (outcome expectation. Vidare har vi analyserar resultatet utifrån Sociodynamic Counselling: a practical approach to meaning making och begreppen verktyg, att konstruera sin egen framtid (future buildning), empowerment och att konstruera tillsammans (joint action). Vi använder även begreppen utvidga (pushing up), begränsa (cooling out) samt institutionell aktör i syfte att analysera resultatet av vår empiri. Vårt resultat visar på att majoriteten av våra respondenter har ett uppmuntrande förhållningssätt mot eleverna och deras karriärmål/drömmar att arbeta inom kultur-branschen. Det samverkas dock inte kring arbetet med att rusta eleverna för vägarna vidare efter gymnasiet men vägledare och lärare kompletterar varandra omedvetet genom att vägledare rustar dem inför eftergymnasiala studier och lärare inför ett kommande arbetsliv. Sammanfattningsvis har vi genom analysen kommit fram till att vi ser att det kan vara en balansgång för vägledare och lärare att vara uppmuntrande mot elevernas val av framtida karriär och samtidigt få dem att drömma realistiskt. Detta utan att uppfattas som institutionella aktörer som begränsar vad eleverna ser som möjligt att uppnå utifrån vad professionerna själva ser som förnuftigt

Stem-based peptides inhibit both RVFV and VSV.

<p>Peptides were screened for inhibition of the pseudotyped reporter viruses RVF-VSV-luc (A and C) and VSV-luc (B and D). Virus was incubated with peptide RVFV-6, -7, -8, -9, or -10 (A, B) or serial dilutions of RVFV-6, RVFV-10, or the scrambled peptides RVFV-6sc or RVFV-10sc (C, D) prior to infecting a monolayer of Vero E6 cells. Luciferase activity (RLU) was measured approximately 18 h later. Percent inhibition was calculated based on the virus-only controls. Error shown is the standard deviation of the mean. Data are representative of at least 2 experiments.</p

RVFV-6 does not prevent virus from binding to cells.

<p>RVFV-MP12 (A) or EboZ-eGFP (B) was incubated with 50 µM RVFV-6 prior to the addition to a confluent monolayer of Vero E6 cells. After a 1 h adsorption, cells were rinsed with PBS, and RNA was harvested using TRIzol. Real time RT-PCR was conducted in triplicate to quantify the relative amount of viral RNA bound to cells, and results are combined from duplicate experiments. NTC is the no template control, PC (positive control) is RNA purified from either RVFV-MP12 or EboZ-eGFP. Untreated virus was mock treated without peptide. Error shown is the standard deviation of the mean.</p

RVFV-6 binds to cells independent of RVFV GnGc expression.

<p>Vero E6 cells were transfected with a plasmid expressing RVFV GnGc. Forty-eight h later, either the biotin-labeled RVFV-6 peptide or the biotin-labeled RVFV-6sc peptide was added to the cells followed by washing with PBS. Cells were fixed, and peptide binding was identified using an anti-biotin antibody conjugated to Texas Red. Nuclei were stained with DAPI (blue).</p

Molecular hypothesis of RVFV-6 mechanism of action.

<p>Panel A shows the initial stages of the membrane fusion process in bunyaviruses (adapted from <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002430#pntd.0002430-Garry1" target="_blank">[8]</a>). Receptor-binding triggers uptake of virions by endocytosis. Acidification of the endocytic vesicle likely initiates Gn/Gc dissociation. Conformational rearrangements of domains I and II in Gc lead to trimer formation and insertion of the fusion peptide into the endosomal vesicle membrane. Trimer formation exposes the stem binding sites (shown in black) with affinity for the Gc stem and RVFV-6. The main molecular elements involved are: (<i>I</i>) glycoprotein Gc having four main components including [<i>a</i>] domains DI, DII, and DIII shown in green, orange/yellow, and blue, respectively; [<i>b</i>] the fusion loop shown in red; [<i>c</i>] the stem shown as small red cylinders; and [<i>d</i>] the transmembrane domains shown as a magenta cylinder; (<i>II</i>) the glycoprotein Gn, depicted as the receptor-binding protein of bunyaviruses, colored pink; and (<i>III</i>) host-cell receptors, shown in light-blue. Panel B: <i>Zippering action:</i> After acidification of the endocytic vesicle, the stem regions relocate, moving towards the host membrane through a zippering reaction. Docking of the stems into the stem binding sites is an essential step of membrane fusion that leads to opening of a pore or channel. <i>RVFV-6 blocking:</i> RVFV-6 molecules, shown as green cylinders, outcompete the stem fragment and lead to inhibition of fusion by blocking the movement of the native stem. Panel C: Molecular model of the RVFV Gc trimer complex. A molecular surface is used to highlight the three domains of the protein (colored as indicated in A). The stem fragments are shown as a red α-helices docked into the stem binding sites (black surface areas), and stem residues are highlighted using a stick representation.</p

Activation of the viral fusion process is required for RVFV-6 binding to RVFV Gc.

<p>Biotin-conjugated RVFV-6, RVFV-6sc, or no peptide was pre-bound to avidin beads before the addition of RVFV-MP12. Beads were washed to remove unbound virus and treated as indicated with 1) lysis buffer and wash, 2) pH 5.2 treatment followed by lysis buffer and wash, or 3) no pH 5.2 treatment and no lysis buffer. Protein bound to the avidin beads were resolved by SDS-PAGE and probed with the anti-RVFV Gc antibody 4D4. Data represent at least 3 separate experiments.</p

Further characterization of the RVFV stem peptides show broad, non-toxic viral inhibition.

<p>Serial dilutions of peptide were incubated with RVFV-ZH501 (A), EboZ-eGFP (B), or ANDV (C), before infecting a monolayer of Vero E6 cells. Percent inhibition was determined for each virus using virus-only controls. (D) MTT toxicity assay results after overnight incubation of Vero E6 cells with RVFV-6 or RVFV-6sc peptides. Absorbance was measured approximately 18 h after adding the diluted peptide in triplicate. The dashed line in (D) represents the average signal generated by the mock treated control cells. Error shown is the standard deviation of the mean. Data are representative of at least 2 experiments.</p