Background: Respiratory syncytial virus (RSV) is one of the leading causes of acute lower respiratory tract infection and childhood hospitalization worldwide. However, there are currently no vaccines or antivirals available to prevent or treat RSV infections. Of the 11 proteins encoded by RSV’s negative-sense single-stranded RNA genome, the nucleoprotein, phosphoprotein, and large polymerase interact through well characterized domains to form the RNA-dependent RNA polymerase complex. This polymerase complex is essential for viral replication and virulence, which makes it an excellent antiviral target. Previous studies have shown that the nucleoprotein-phosphoprotein interaction of the polymerase complex can be disrupted by synthetic peptides of the last 21 C-terminal (P220-241) or the first 29 N-terminal (P1-29) amino acids of the phosphoprotein. Objective: The Mahony lab has also previously demonstrated that P220-241 conjugated to a maltose binding protein (MBP) and HIV-1 Tat cell penetrating peptide (CPP) could inhibit up to 90% of RSV A replication in vitro. However, the bacterial derived MBP is immunogenic. This study builds on these findings by developing and evaluating the efficacy of a P220-241 peptide mimetic conjugated to human thioredoxin (hTrx) carrier protein and a P1-29 peptide mimetic conjugated to MBP. Methods and Results: Inverse PCR and In-Fusion® cloning was used to clone a hTrx-P220-241 plasmid, which was then expressed as a recombinant protein and purified by affinity chromatography for functional analysis. HTrx-P220-241 was shown to specifically interact with RSV nucleoprotein in a glutathione S-transferase (GST) pull down assays and it could successfully enter into LLC-MK2 cells. However, upon challenge with RSV A, LLC-MK2 cells that were incubated with increasing concentrations of hTrx-P220-241 did not inhibit RSV A replication when assessed by indirect immunofluorescence microscopy. The MBP-P1-29 construct did not exhibit any significant cytotoxicity in LLC-MK2 cells nor BEAS-2B cells. Upon challenge with RSV A, LLC-MK2 cells and BEAS-2B cells pre-treated with MBP-P1-29 demonstrated a dose-dependent inhibition of RSV replication in vitro, with a percent inhibition of infection of 80% and 60% respectively. Furthermore, MBP-P1-29 also reduced the release of infectious progeny virion by up to 74% in LLC-MK2 cells and 34% in BEAS-2B cells. Conclusion: Phosphoprotein peptide mimetics targeting essential nucleoprotein-phosphoprotein interaction are a promising approach in the development of therapeutic treatments for RSV. In this study, a P220-241 peptide mimetic conjugated to a human thioredoxin scaffold protein was not able to inhibit RSV A replication while a P1-29 peptide attached to a maltose binding protein was effective in reducing RSV replication in vitro. Thus, further studies are required to evaluate a P1-29 peptide mimetic against different RSV A and B strains and to find an appropriate human carrier protein to attach it to. / Thesis / Master of Science (MSc) / Respiratory syncytial virus is a respiratory illness that is one of the leading causes of childhood hospitalization worldwide. RSV infects almost all infants at least once by the age of two. It can also repeatedly infect individuals throughout their lives, which puts the elderly and individuals with weak immune, cardiac or pulmonary systems at risk. There are also no approved vaccines or antiviral treatments available to prevent or combat a RSV infection, which highlights the pressing need for the development of new antiviral drugs. This thesis focuses on developing and evaluating the efficacy of two different antiviral peptides, which both target and disrupt the formation of the viral machinery required for the replication of the RSV genome.

Natural killer (NK) cells play a significant role in the detection and destruction of virally infected and tumor cells. The NKR-P1 receptors regulate NK cell function by an alternative missing-self recognition system. Although the NKR-P1 receptors were among the first surface NK receptors identified on rodent NK cells more than 20 years ago, there is still very little known about their biological function and their physiological ligands. Furthermore, no three-dimensional structure of any of the NKR-P1 family receptors has been published so far. To understand the functional architecture of mouse NKR-P1 receptors, we developed a simple and efficient protocol providing large amounts of pure soluble NKR-P1 proteins. The crystal structure of mouse NKR-P1A, determined at 1.7 A resolution, is the first structure of a representative of the NKR-P1 family. Crystal structure is formed by a compact C-type lectin-like domain and an extended loop that participates in domain swapping. A potential role of the swapped loop has been suggested in natural ligand binding by in silico studies. However, chemical cross-linking and H/D exchange in combination with high resolution mass spectrometry revealed this loop in close proximity to the compact core in solution. The discrepancy between the crystal and solution structure...

On développe dans ces travaux un outil numérique permettant de simuler le procédé Friction Stir Spot Welding. Le modèle est basé sur un couplage fluide/solide et permet une description correcte des parties fortement malaxée et solide de la structure. Une approche ALE est utilisée avec un mouvement arbitraire défini de façon que le maillage suive la matière dans la partie solide mais pas dans la partie pâteuse. Ceci permet la simulation de plusieurs tours de l'outil tout en suivant les bords des tôles soudées durant le procédé. Ce modèle numérique s'appuie sur l'élément fini mixte P1+/P1. Ce dernier a été développé avec une formulation température/vitesse/pression en mécanique des fluides (dans le cas d'un écoulement laminaire incompressible et transitoire) et en mécanique des solides dans le cadre des grandes transformations. La transition fluide/solide est effectuée au moyen d'un test explicite sur une température moyenne par élément, l'interface passant alors entre les éléments du maillage. Une procédure d'actualisation de la géométrie associée à l'approche ALE est effectuée à convergence. Ce couplage a été intégré au sein d'une nouvelle option du code SYSWELD. On présente ici une première simulation du procédé Friction Stir Spot Welding. D'autre part, deux montages spécifiques sont proposés pour l'investigation du procédé Friction Stir Spot Welding. Ces deux dispositifs intègrent une démarche de validation globale visant à calibrer la modélisation proposée du procédé. La stratégie expérimentale suivie est détaillée, et des premiers résultats obtenus sur un alliage d'aluminium basique sont présentés.

NK (natural killer) cells, with their ability to identify antigens and extraneous substances, available in the organism through various moleculary receptors, are an important component of the immune system. The NKR-P1A and NKR-P1B proteins belong to the lectin receptors of natural killer cells. Primary ligands of lectin receptors comprise terminal oligosaccharides of glycoproteins on the surface of target (e.g. tumor) cells. The interaction between carbohydrate structures on the surface of antigens and their binding partners on NK receptors is followed by triggering the effector function of NK cells against the targets. The NK cells and NK receptors findings and their interactions with ligands are greatly utilized in the treatment of cancer, viral and autoimmune diseases. Heterologous protein production in the eukaryotic organism brings a lot of advantages. Unlike the prokaryotic organism, the methylotrophic yeast Pichia pastoris has the capability of performing many posttranslational modifications resulting in production of biological active protein molecule. Usually, the P. pastoris expression system disposes of high level protein expression and is also generally regarded as being faster, easier, and less expensive to use than expression systems derived from other eukaryotes. In this thesis, I...

The N-terminal hydrophobic domain of the phage P1 endolysin Lyz was found to facilitate the export of Lyz in a sec-dependent fashion, explaining the ability of Lyz to cause lysis of E.coli in the absence of the P1 holin. The N-terminal domain of Lyz is demonstrated to be both necessary and sufficient not only for export to the membrane but also for release into the periplasm of this endolysin. We propose that this unusual N-terminal domain functions as a "signal arrest- release" (SAR) sequence, which first directs the endolysin to the periplasm in membrane-tethered form and then allows it to be released as a soluble active enzyme in the periplasm. To understand why release from the membrane is required for the physiological expression of the lytic activity of Lyz, we examined the role of its seven cysteine residues in the biogenesis of the active endolysin. The inactive, membrane-tethered and the active, soluble forms of Lyz differ in their pattern of intramolecular disulfide bonding. We conclude that the release of Lyz from the membrane leads to an intramolecular thiol-disulfide bond isomerization causing a dramatic conformational change in the Lyz protein. As a result, an active site cleft that is missing in nascent Lyz is generated in the mature form of the endolysin. Examination of the protein sequences of related bacteriophage endolysins suggests that the presence of an SAR sequence is not unique to Lyz. Studies on holin and antiholin indicated that P1 encodes two holins, LydA and LydC. The antiholin LydB inhibits LydA by binding to it directly on the membrane. All above results demonstrate a new paradigm for control of phage lysis, which is, upon depolarization of the membrane by holin function at a programmed time, endolysin is released from the bilayer leading to the immediate lysis of the host.

Detta arbete tar plats hos Sveriges Radio i Umeå där uppgiften har varit att ta reda på hur processen för att göra ett radioprogram går till. Frågeställningen tar även med två viktiga delar för att ett radioprogram ska bli lyckat. ”Hur gör man en bra intervju?” och ”Hur får man lyssnarna att stanna?”. Programmet som denna rapport behandlar heter Tendens och sänds i P1. Genom att observera reporter Filippa Armstrongs arbete och intervjua henne under arbetets gång så kan denna rapport närgående beskriva alla de steg man tar sig igenom för att göra ett Tendens-program. Syftet med detta arbete är att öka kännedomen om arbetet bakom ett radioprogram och hur de byggs upp för att nå ut till lyssnarna.   Materialet i detta arbete har kommit till genom intervjuer, dagboksförande och litteratursökande. Filippa Armstrong har varit en stor källa till information på en personlig och erfaren nivå, och boken ”Intervju: Konsten att lyssna och fråga” av Jan Krag Jacobsen har gett en stabil vetenskaplig grund att stå på.   Det har varit väldigt lärorikt och roligt att få följa med på denna resa. Att göra ett Tendens-program är ett större projekt än vad många tror och kräver stor fokus och många arbetstimmar.

Thesis (Ph. D.)--University of Virginia, 1999. / Spine title: Adenosine A₁ & A₂[beta] receptors. Includes bibliographical references (p. 122-146). Also available online through Digital Dissertations.

The current study used event-related potentials (ERPs) to investigate how motivational salience in the form of expectation violation influences spatial attention. The medial frontal negativity (MFN) ERP indexes expected value, being negative to unexpected punishments and positive to unexpected rewards. The P1 and N1 ERPs index spatial attention, being larger to stimuli in attended locations. This design attached motivational value to locations by making one visual hemifield economically rewarding (greater probability of a rewarding outcome) and the other punishing (greater probability of a punishing outcome). Keypresses to a dot probe following a reward-signifying stimulus were awarded money if correct, and penalized following a punishment-signifying stimulus if incorrect. We predicted that salience would be attached to visual hemifield, thus the MFN would be most negative to punishing outcomes in the rewarding hemifield and most positive to rewarding outcomes in the punishing hemifield. We also predicted that attention would be allocated to a location where expectation was violated, thus the P1 and N1 ERPs would be larger and RTs (reaction times) faster to dot probes appearing in the same side as an outcome that violated expected value. In a sample of 36 participants, there were no significant effects on the MFN, although the means were in the predicted direction, suggesting a lack of power. Contrary to our hypothesis, keypresses were slower, P1 smaller, and N1 larger to probes opposite the location where an expectation violation occurred. This suggested that expectation violation did not direct attention to a particular location, but produced general interference.

Natural killer (NK) cells are an essential part of immune system, providing self-surveillance of virally infected, stress transformed or cancerous cells. NKR-P1 receptors and their ligands from clec2 gene family represent an alternate missing-self recognition system of NK cells based on interaction of highly related C-type lectin-like receptors. Human NKR-P1 has been described more than twenty years ago but still remains the sole human orthologue of this receptor family, particularly numerous in rodents. On binding to its cognate ligand LLT1, NKR-P1 can relay inhibitory or co-stimulatory signals. Although being interesting targets for their potential role in tumor immune evasion and autoimmunity, nature of their interaction is still unclear. To elucidate the architecture of their interaction, we developed a generally applicable method for recombinant expression of human NKR-P1 and LLT1 and their homologues based on transfection of HEK293S GnTI- cells. Further, we described a stabilizing mutation His176Cys, that enables for expression of highly stable and soluble LLT1. Finally, we have crystallized LLT1 and human NKR-P1 in different glycosylation states both as individuals and in complex. While both structures of LLT1 and NKR-P1 follow the classical C-type lectin-like superfamily fold, contrary to...

No description available.